Patent Application
20170112760
The present invention is directed to a cell delivery system for the delivery of materials including nucleic acids across a biological barrier and methods of use thereof.
Transdermal delivery offers advantages over conventional oral and parenteral administration, including prevention of drug degradation in the stomach, avoidance of first pass liver metabolism, possibility of improved bioavailability etc and for parenteral administration poor compliance with injections. However, at present there are only a handful of drugs offered for transdermal patch delivery. This is due to the excellent barrier function of the skin, which is accomplished almost entirely by the outermost 10-15 microns of tissue, the stratum corneum. Thus, to date the transdermal delivery and subsequent intracellular delivery of materials, such as nucleic acids, across a biological barrier such as the stratum corneum (SC) is problematic achieving only moderate success and the many different approaches encountering significant problems.
For example, the delivery of a vaccine across a biological barrier is problematic. Vaccination consists of stimulating the immune system with an infectious agent, or components of an infectious agent, modified in such a manner that no harm or disease is caused, but ensuring that when the host is confronted with that infectious agent, the immune system can adequately neutralize it before it causes any ill effect. Conventionally, vaccination has been effected by one of two approaches: either introducing specific antigens against which the immune system reacts directly; or introducing live attenuated infectious agents that replicate within the host without causing disease, synthesize the antigens that subsequently prime the immune system.
Due to safety issues and deleterious side effects, traditional pathogenic vaccines are almost obsolete. Vaccine development now focuses on vaccines developed from purified subunits, recombinant proteins or synthetic peptides. However, although these vaccines have an excellent safety profile, their immunogenic potency is significantly compromised with the induction of only an antibody response. To overcome this and to activate the cell-mediated immune response primarily through cytotoxic T lymphocytes (CTLs), DNA vaccination is now utilised in which plasmid DNA encodes for a particular antigen for a disease.
DNA vaccination is a radical new approach and involves the direct introduction into appropriate tissues of a plasmid containing the DNA sequence encoding the antigen(s) against which an immune response is sought, and relies on the in situ production of the target antigen. This approach offers a number of potential advantages over traditional approaches, including the stimulation of both B- and T-cell responses, improved vaccine stability, the absence of any infectious agent and the relative ease of large-scale manufacture.
However, in order for the desired antigen to be expressed the plasmid DNA must be delivered to the nucleus of cells to enable the production of the protein antigen in the cytoplasm of the host cell. In addition the DNA must then be delivered to the antigen presenting cell (APC's) via the MHC I pathway to result in an effective cell-mediated immune response. One of the advantages of using a DNA vaccine is that they can evoke a long term immune response without the need for adjuvants or repeated injections. Furthermore, in addition to a prophylactic response, DNA vaccines can also be administered to treat a pre-existing infection. Additionally, DNA vaccines are inexpensive to manufacture, heat stable and easy to store.
Despite these advantages, at present there is no highly effective delivery system to transport the DNA to the APC's to evoke the desired immune response. Current developments often employ physical methods such as intramuscular injection of ‘naked DNA’, coating of gold nanoparticles with DNA and using a gene gun or jet injectors. However, the use of these physical methods require large quantities of DNA and can damage the DNA being delivered and do not overcome the intracellular barriers for delivery of the DNA to the nucleus.
At present there are 3 licenced DNA vaccines (West Nile virus in Horses, DNA vaccine Apex®-IHN and ONCEPT™) commercially available and these utilise physical methods for delivery. For example, ONCEPT™ was developed with licensed delivery technology from Vical involving a physical Needle Free Injection Therapy System (NFITS) that forces macromolecules through the skin. All of these vaccines use physical forces to deliver the DNA and do not take account of the critical intercellular barriers in their systems.
Alternative delivery means, including microneedle technology has been used to deliver DNA either by coating metal microneedles or inserting DNA into hollow microneedles. However, these delivery methods have been found to be unsuccessful due to resultant low transfection rates. Although, DNA delivery to the skin is generally effective, this microneedle technology does not provide an effective delivery system to transport the DNA intracellularly to the nucleus.
In the extracellular barrier described above, several biological barriers exist intracellularly. Upon systemic administration the delivery vector must not be degraded in the circulation and must be able to extravasate to surround tissues. Again stability is necessary in the extracellular matrix and the fibrous network of proteins must be navigated. Even when reaching the target tissue cellular entry must be achieved and this is dependent upon charge and size of the particle to be delivered. When foreign particles are endocytosed they become trapped in the endosome which is degraded into a lysozyme. Therefore endosomal escape is critical for successful delivery to the cytoplasm. However several studies have shown that the uptake of DNA into the cytoplasm does not correlate with efficient gene delivery and this is perhaps because the most important barrier is the one to the nucleus. If the final destination site is the nucleus then an active transport system is required otherwise entry into the nucleus is a chance effect during cellular division when the nuclear membrane dissolves. Translocation to the nucleus is dependent on the presence of basic amino acids known as a nuclear localisation signal. The nuclear localisation signal binds to the importin alpha protein which has an importin beta binding domain. The importin beta binding domain then recruits and binds importin beta which will transport the whole complex through the nuclear pore channel through the transient association and disassociation of the phenyalanine-glycine repeats (
To overcome some of these problems, as disclosed in WO 2009/040548, a transdermal delivery means was developed comprising a microprotrusion-based device for the delivery of beneficial substances across or into the skin. WO 2009/040548 discloses the use of a swellable polymer composition in the microprotrusion array. The microprotrusion array is used in the delivery of an active agent transdermally, that is through the stratum corneum (SC). WO 2009/040548 is a general teaching outlining many potential active agents including beneficial substances such as a drug, a nutrient or a cosmetic agent. WO 2009/040548 defines the term drug to include ‘beneficial substances’ for the treatment or prophylaxis of disease, for example, drug substances, substances that may improve the general health of the skin, for example, vitamins and minerals, and substances that may improve the aesthetic appearance of the skin, for example, by reducing the appearance of wrinkles or improving the degree of hydration of the skin. However, the examples only disclose active agents such as fluorescent photosensitiser drug meso-Tetra (N-methyl-4-pyridyl) porphine tetra tosylate (MW 1363.6 Da) (TMP), 5-aminolevulinc acid (ALA, MW 167 Da), bovine serum albumin (BSA). Furthermore, WO 2009/040548 only enables the transport of these beneficial substances across the SC.
The present invention aims to overcome at least some of these problems to provide a more effective delivery technology for nucleic acids and other agents both transdermally and subsequently intracellularly.
According to a first aspect of the invention, there is provided a cell delivery system comprising
According to a second aspect of the invention, there is provided the cell delivery system of the invention for use in inducing an immune response against an antigen in a subject in need thereof. In this manner, the cell delivery system ideally comprises nucleic acid including plasmid DNA which encodes an antigen and the cell delivery system induces an immune response in a host against the antigen. Alternatively, the cell delivery system comprises an other agent such as a negatively charged or hydrophilic compound.
According to a third aspect of the invention, there is provided the cell delivery of the invention for use in gene therapy in a subject in need thereof. In this manner, ideally the cell delivery system is designed to deliver a nucleic acid encoding a functional gene or protein which is deficient or mutated in the subject. Alternatively, the cell delivery system is designed to deliver an inhibitory nucleic acid such as RNA including an siRNA/shRNA/miRNA molecule. Alternatively, the cell delivery system is designed to deliver an other agent such as a negatively charged or hydrophilic compound.
According to a fourth aspect of the invention, there is provided the cell delivery system of the invention for use in the treatment and/or prophylaxis of cancer in a subject in need thereof. In this manner, ideally the cell delivery system is designed to deliver a nucleic acid encoding a functional gene or protein which is deficient or mutated in the subject. Alternatively, the cell delivery system is designed to deliver an inhibitory nucleic acid such as RNA including an siRNA/shRNA/miRNA molecule. Still alternatively, the cell delivery system is designed to deliver an other agent such as a negatively charged or hydrophilic compound.
According to a fifth aspect of the invention, there is provided a method of inducing an immune response against an antigen in a subject comprising the administration of the cell delivery system of the invention to a subject in need thereof comprising the steps of
According to a sixth aspect of the invention, there is provided a method for the treatment or prophylaxis of an infection or cancer comprising the administration of the cell delivery system of the invention to a subject in need thereof comprising the steps of
According to a seventh aspect of the invention, there is provided the use of the cell delivery system of the invention for the administration of the material to a cell or subject in need thereof.
According to an eighth aspect of the invention, there is provided a composition or nanoparticle comprising the amphipathic cell penetrating peptide of the invention and a DNA vaccine. Essentially, the nanoparticles are formed from the DNA vaccine complexed with an amphipathic cell penetrating peptide. Preferably, the DNA vaccine comprises plasmid DNA encoding an antigen for a disease. In this manner, the composition or nanoparticle acts as antigen and elicits an immune response against the antigen in a patient in need thereof. Accordingly. the cell delivery system of the invention comprises a material which itself comprises the composition or nanoparticle as defined herein.
In this specification, the term “dissolvable” covers agents which are dissolvable in liquid, such as water and interstitial fluid. The microprotrusion array dissolves at a rate determined by the polymer used. In this manner on application to the skin, the microprotrusion array can initially increase in volume (swell) and then dissolve.
In this specification, the term “swellable” covers agents which swell or imbibe liquid, such as biological fluid when in contact with interstitial fluid for example. The microprotrusion array of the invention can initially increase in volume (swell) and then dissolve.
It will be understood that the polymer of the invention is a polymer that swells and/or dissolves in the presence of water and have sufficient mechanical strength to function as microprotrusions that can puncture the stratum corneum barrier. Ideally, the polymer should be non-toxic when used in vitro or in vivo.
In this specification, it will be understood the terms “complexed” and “condensing” are interchangeable. As such, the nanoparticle is formed when the nucleic acid or other agent is complexed with or condensed with the amphipathic cell penetrating peptide of the invention.
In this specification, the term “loaded with” relates to a microprotrusion array which encapsulates or incorporates the material comprising the amphipathic cell penetrating peptide of the invention, such as nanoparticles. In this manner, the microprotrusion array contains in its polymer matrix the material/nanoparticles of the invention. For example, the microprotrusion array is ideally prepared from a solution of the polymer which is mixed with or loaded with the material/nanoparticles of the invention, placed in a mould and dried to result in a microprotrusion array supported by a polymer substrate or base element. It will be understood that the nanoparticles of the invention may be freeze-dried or spray-dried before they are mixed the polymer solution to form the microneedle array. In this manner, the microprotrusion array comprises a base element and a plurality of microprotrusions which project from the base element. The microprotrusions and the base element may comprise the same or different polymer. For example, both the base element polymer and the microprotrusion polymer may be dissolvable. Alternatively, only the microprotrusion polymer may be dissolvable. Additionally, one or both of the plurality of microprotrusions and the base element may comprise the material/nanoparticles.
The present invention is directed to a cell delivery system comprising
Advantageously, the present invention overcomes the physical and biological barriers encountered in conventional nucleic acid delivery methods to facilitate both transdermal, intradermal and intracellular delivery of the material. This is enabled by the unique combination of a microprotrusion array loaded with material comprising or consisting of nanoparticles comprising an amphipathic cell penetrating peptide encapsulated nucleic acid, or other agent, including a protein or drug.
Advantageously, the microprotrusion array facilitates transport of the nanoparticles across the first biological barrier, for example the skin e.g. the subcutaneous layer (SC). Then, as the microprotrusion array is composed of a swellable and/or dissolvable polymer composition it dissolves upon contact with interstitial fluid enabling release of the material comprising or consisting of nanoparticles into the extracellular space. The nanoparticles then enables the transport of the nucleic acid, or other agent such as a negatively charged or hydrophilic compound, including a protein or drug, across cell membranes, out of the endosomes and to the nucleus ideally for presentation to the antigen presenting cells. Thus, the use of swellable and/or dissolvable polymer microprotrusion array combined with the nanoparticles ensures that the nanoparticles and its contents reach the high APC population in the skin cells to evoke maximum effect. We have shown that this specific combination of microprotrusion array/microneedle with nanoparticle provides unexpectedly superior results compared to microneedle delivery alone or nanoparticle delivery alone.
In the present invention, ideally the nanoparticles are formed from a nucleic acid or other agent complexed with an amphipathic cell penetrating peptide. The amphipathic cell penetrating peptide has been designed to condense the nucleic acid or other agent into nanoparticles, protect the nucleic acid or other agent from degradation and facilitate cellular entry through natural endocytosis because they are <100 nm and have a positive charge. The cell penetrating peptide of the invention had improved or equivalent cell penetration activity compared to conventional transfection reagents.
Ideally, nanoparticles have a N:P ratio from for example 0.5 to 12, preferably from 0.5 to 6. N:P ratio indicates the ratio of peptide (N) to the nucleic acid (P) or other agent. We have found that these N:P ratios maximise transfection efficiency in dendritic cells in-vivo. Other N:P ratios may also be contemplated.
After the microprotrusion array has facilitated transport of the nanoparticles across the biological barrier (SC) to the extracellular space, the drop in pH changes the conformation of the amphipathic cell penetrating peptide so that it disrupts the endosome and gets into the cytoplasm. The presence of arginine in particular in the peptide also facilitates active nuclear transport. This means that there is a greater chance of the nucleic acid reaching the nucleus, and when the nucleic acid is a DNA vaccine more antigen will be produced to give a more potent immune response.
Advantageously, the present inventors have found that nanoparticles can be loaded into the microprotrusion arrays without compromising the microprotrusion structure which is required for cell penetration for example.
Furthermore, the inventors have shown that nucleic acid (e.g. DNA) from the nanoparticles was released from the microprotrusion arrays across a biological barrier, namely skin. The integrity of the nucleic acid (e.g. DNA) was not compromised following an extended incubation period.
Importantly, in-vivo studies have shown expression of nanoparticle nucleic acid (e.g. DNA) illustrating the functionality of this technology. These advantages are clearly illustrated in
According to a preferred embodiment, the nucleic acid is a DNA vaccine, preferably plasmid DNA encoding an antigen for a disease. In this manner, the cell delivery system induces an immune response in a host against the antigen (see for example
According to an alternative embodiment, the nucleic acid is suitable for gene therapy, preferably in the form of DNA or RNA including mRNA, miRNA or siRNA. For example, the cell delivery system of the invention delivers a nucleic acid encoding a functional gene or protein which is deficient or mutated in the subject. Alternatively, the cell delivery system may deliver an inhibitory nucleic acid such as RNA including an siRNA/shRNA/miRNA molecule.
According to yet another embodiment, the other agent is a negatively charged or hydrophilic compound, such as a protein, drug or active agent. preferably a phosphate or lipophilic based drug, more preferably a bisphosphonate drug or gold.
According to one aspect of the invention, there is provided the cell delivery system for use in inducing an immune response (i.e. inducing a prophylactic effect) against an antigen in a subject in need thereof. In this embodiment, the material/nanoparticle comprises a DNA vaccine, preferably plasmid DNA encoding an antigen for a disease which when expressed induces an immune response against the antigen. The inventors have shown that the cell delivery system of the invention can be used to deliver a DNA vaccine to have both a prophylactic/immunization (e.g.
According to another aspect of the invention, there is provided the cell delivery system of the invention for use in gene therapy in a subject in need thereof. In this embodiment the cell delivery system is designed to deliver a nucleic acid encoding a gene which is deficient or mutated in the subject. Alternatively, the cell delivery system is designed to deliver an inhibitory nucleic acid such as RNA including an siRNA/shRNA/miRNA molecule.
According to another aspect of the invention, there is provided a cell delivery system of the invention for use in the treatment and/or prophylaxis of cancer in a subject in need thereof. In this manner, the cell delivery system is designed to deliver a nucleic acid encoding a gene which is deficient or mutated in the subject (therapeutic effect). Alternatively, the cell delivery system is designed to deliver an inhibitory nucleic acid such as RNA including an siRNA/shRNA/miRNA molecule.
According to yet another aspect of the invention, there is provided method of inducing an immune response against an antigen in a subject comprising the administration of the cell delivery system of the invention to a subject in need thereof comprising the steps of
In a preferred embodiment, the material/nanoparticle comprises a DNA vaccine, preferably plasmid DNA encoding an antigen for a disease which when expressed induces an immune response against the antigen. It will be understood that the nanoparticle may comprise any nucleic acid or other agent as defined previously.
According to yet another aspect of the invention, there is provided a method for the treatment and/or prophylaxis of an infection or cancer comprising the administration of the cell delivery system of the invention to a subject in need thereof comprising the steps of
In a preferred embodiment, the cell delivery system is designed to deliver a nucleic acid encoding a gene which is deficient or mutated in the subject. Alternatively, the cell delivery system is designed to deliver an inhibitory nucleic acid such as RNA including an siRNA/shRNA/miRNA molecule. It will be understood that the cell delivery system is designed to deliver any nucleic acid or other agent as defined previously.
According to another aspect of the invention, there is provided the use of the cell delivery system according to any of the preceding claims for the transdermal, intradermal and/or intracellular administration of the material to a subject in need thereof. The inventors have shown that transdermal/intradermal administration of the cell delivery system of the invention is superior than intramuscular administration of the same nanoparticles (see e.g.
Further details on each component of the cell delivery system of the invention are expanded on below.
Ideally, the microprotrusion array is prepared from a solution of a polymer which is mixed with or loaded with the nanoparticles of the invention, placed in a mould and dried to result in a microprotrusion array supported by a polymer substrate or base element. The nanoparticles may be freeze-dried or spray-dried before being loaded into the polymer solution.
Ideally, the microprotrusion array comprises a base element and a plurality of microprotrusions which project from the base element. The base element and a plurality of microprotrusions may be made from the same or different polymer materials. base element and a plurality of microprotrusions may both comprise nanoparticles of the invention or only one of these elements may comprise nanoparticles.
As disclosed in WO 2009/040548, the microprotrusions are composed of swellable and/or dissolvable polymers which can puncture the stratum corneum of mammalian skin without breaking upon insertion into the skin and can be used for efficient delivery of active substances through the stratum corneum without many of the problems associated with the use of conventional solid microneedles.
Any polymer which can penetrate the stratum corneum of the skin and which swells in the presence of liquid may be used.
Ideally, the microprotrusion array polymer is dissolvable in the natural cellular, interstitial environment. For example, FDA-approved hydrogel materials can be utilised to form the microprotrusion and such hydrogel materials can be inexpensive and biocompatible.
The material dose which can be provided by a microprotrusion may not necessarily limited by how much can be loaded into a microprotrusion, as the material could be contained in an attached material reservoir attached to the upper surface of the microprotrusion array.
It will be understood that the swelling of the microprotrusions on entry to the skin has a number of advantages over conventional microneedle arrays or indeed sugar microneedles. For example, where the microprotrusion array is used, the increased surface area of microprotrusion in contact with the epidermal layer underneath the stratum corneum resulting from the swelling of the microprotrusions enables enhanced delivery of a material to the epidermal layer underneath the stratum corneum. In particular embodiments arrays of swellable polymeric microprotrusions can absorb moisture upon insertion into the skin and swell to form continuous aqueous channels between the external environment and the dermal microcirculation, thus forming an ‘aqueous bridge’ across the lipophilic stratum corneum barrier. Such channels do not have the tendency to block on positioning of the array, in contrast to conventional silicon based microneedle devices having channels therein. Optionally, the microprotrusions can release the material from every point on the surface of the microprotrusion further minimising blockage of the microprotrusion by tissue. Optionally a hydrogel microprotrusion array could be integrated with a material reservoir to give a rapid bolus dose, achieving a therapeutic plasma level, followed by controlled, prolonged delivery to maintain this level. Optionally, swollen hydrogel materials can contain >70%, for example >80%, such as >90% water. By having a high water content, material diffusion is facilitated, as there will be less chance of impedance of material movement due to collision with polymer chains. In addition, water allows passage of ions and polar substances and facilitates electroosmotic flow under a potential gradient. Thus, conduction of charged and/or polar substances and fluid moving by electro-osmotic flow is possible.
Microprotrusions can be fabricated from any suitable swellable and/or dissolvable polymer, which in its dry state is hard and brittle to allow penetration of the stratum corneum, but then which, upon taking up moisture swells to allow diffusion of therapeutic active agents. The polymers of the invention swell and/or dissolve in the viable skin layers, where interstitial fluid is present.
The polymers that may be used in the present invention include, but are not limited to the following poly(vinylalcohol), poly(vinylpyrrolidone), poly(hydroxyethylmethacrylate) and derivatives thereof, poly(methylvinylether/maleic acid) and derivatives thereof, poly(methylvinylether/maleic anhydride) and derivatives thereof, poly(acrylic acid), poly(caprolactone, hydroxyethylcellulose and derivatives thereof, poly(ethyleneglycol) and derivatives thereof, hyaluronic acid, chitosan and carbohydrate materials (eg galactose, fructose etc.) and derivatives thereof. For in-vivo or in-vitro use the polymer chosen should be non-cytotoxic.
According to a preferred embodiment of the invention, the swellable polymer composition used in the microprotrusion array is polyvinylpyrrolidone (PVP). PVP is fully licensed and FDA-approved PVP is a water-soluble and biodegradable polymer which eliminates the risk of leaving biohazardous sharp waste in the skin. PVP is also a low cost material which provides for ease of fabrication in the micro-moulding process and enable the mass production of the arrays. We have found that when PVP is used the presence of the amphipathic cell penetrating peptide is essential for the protection of the nucleic acid or other agent within the nanoparticles to prevent the PVP interacting with the nucleic acid and having a detrimental effect.
According to another preferred embodiment of the invention, the polymer composition used in the microprotrusion array is poly(vinylalcohol) (PVA). This is a dissolvable polymer.
Whilst both PVP and PVA are suitable for in-vivo usage we have found that PVA is less toxic to cells and in some applications may be preferable.
Polymers, such as PVP and PVA, may be used with a molecular weight of less than approximately 400 KDa. Preferably, the polymers, such as PVP and PVA, with a molecular weight less than 60 KDa may be used. More preferably, the polymers, such as PVP and PVA, with a molecular weight less than 15 KDa may be used. Even more preferably, the polymers, such as PVP and PVA, with a molecular weight less than 10 KDa, typically from 8-10 KDa, may be used. Ideally, PVA with a molecular weight ranging from 9-23 KDa may be used, preferably 9-10 KDa PVA and 13-23 KDa PVA.
Optionally, from 15 to 40% w/w, preferably from 20 to 30% w/w, polymer may be used at varying molecular weights.
We have found that both PVP and PVA are preferred polymers which retain height on introduction of the nanoparticles and are strong enough to penetrate the skin and withstand forces of approximately 15 Newtons cm−2. Furthermore, the nucleic acid/DNA or other agent within the nanoparticles retain their integrity and functionality within the microneedles.
In particular embodiments, the polymers of the microprotrusions are crosslinked, either physically, chemically or both. The microprotrusion array can comprise groups of microprotrusions wherein a first group comprises at least one different cross-linker to at least a second group.
In particular embodiments the microprotrusions may not be crosslinked and will dissolve following an initial swelling phase upon puncturing the stratum corneum and coming into contact with skin moisture. In this case, the material can be released into the skin at a rate determined by the rate of dissolution of the microprotrusions. The rate of dissolution of particular microprotrusions is dependent on their physicochemical properties which can be tailored to suit a given application or desired rate of material release.
Combinations of non-crosslinked, lightly crosslinked and extensively crosslinked microprotrusions can be combined in a single device so as to deliver a bolus dose of the material achieving a therapeutic plasma level, followed by controlled delivery to maintain this level. This strategy can be successfully employed whether the material is contained in the microprotrusions and base element or in an attached reservoir.
In further embodiments, the base element and microprotrusions may contain in their matrix, defined quantities of one or more water soluble excipients. Upon insertion into skin these excipients will dissolve leaving pores behind in the matrix of the base element and microprotrusions. This can enhance the rate of release, which can be further controlled by changing the excipient, its concentration and/or its particle size. Suitable excipients include, but are not limited to glucose, dextrose, dextran sulfate, sodium chloride and potassium chloride, sodium carbonate, sodium hydroxide, sodium hydrogen carbonate or other water soluble excipients known in the art. Other excipients include conventional pharmaceutical disintegrants used in solid dosage forms, including for example cross-carmellose or crospovidone.
As noted above, in order to be of use in transdermal delivery arrays of microprotrusions must be capable of creating openings in the stratum corneum barrier through which beneficial substances can move. Thus, the force of insertion is less than the force required to fracture the microprotrusions.
Suitably, the microprotrusions do not fracture when a pressure of insertion of less than 5.0 N cm−2, for example less than 3.0 N cm−2, such as less than 0.5 N cm−2 is exerted on the microprotrusions along their length.
A microprotrusion can be any suitable size and shape for use in an array to puncture the stratum corneum. The microprotrusions are designed to pierce and optionally cross the stratum corneum. Suitably, the height of the microprotrusions can be altered so as to allow penetration into the upper epidermis, as far as the deep epidermis or even the upper dermis, but not allowing penetration deep enough into the skin to cause bleeding. In one embodiment, the microprotrusions are conical in shape with a circular base which tapers to a point at a height of the microprotrusion above the base.
In embodiments of the microprotrusion array the microprotrusions can be in the range of 1 μm to 3000 μm in height. For example, the microprotrusions can have heights in the range 50 μm to 400 μm, for example 50 to 100 μm. Suitably, in embodiments of the arrays of the invention, microprotrusions can have a width, e.g. diameter in the case of microprotrusions of circular cross-section diameter of 1-500 μm at their base. In one embodiment microprotrusions of and for use in the invention can have a diameter in the range 50-300 μm, for example 100-200 μm. In another embodiment, the microprotrusion of the invention may be of a diameter in the range of 1 μm to 50 μm, for example in the range 20-50 μm.
The apical separation distance between each of the individual microprotusions in an array can be modified to ensure penetration of the skin while having a sufficiently small separation distance to provide high transdermal transport rates. In embodiments of the device the range of apical separation distances between microprotrusions can be in the in the range 50-1000 μm, such as 100-300 μm, for example 100-200 μm. This allows a compromise to be achieved between efficient penetration of the stratum corneum and enhanced delivery of therapeutic active agents or passage of interstitial fluid or components thereof.
It will be apparent to those skilled in the art that the microprotrusions of the invention can take any reasonable shape, including, but not limited to, microneedles, cones, rods and/or pillars. As such, the microprotrusions may have the same diameter at the tip as at the base or may taper in diameter in the direction base to tip. The microprotrusions may have at least one sharp edge and may be sharp at the tips. The microprotrusions may be solid, have a hollow bore down at least one longitudinal axis at an angle to the base element and extending to the first side of the base element, they may be porous, or may have at least one channel running down at least one outer surface from tip to base element.
In use, the microprotrusions may be inserted into the skin by gentle applied pressure or by using a specially-designed mechanical applicator applying a pre-defined force. An additional device may be used to reduce the elasticity of skin by stretching, pinching or pulling the surface of the skin so as to facilitate insertion of the microprotrusions. This latter function could be usefully combined with the function of the applicator to produce a single integrated device for insertion of a microprotrusion array.
The material contained in the microprotrusions themselves will be rapidly released upon swelling, initially as a burst release due to material at the surface of the microprotrusions. The subsequent extent of release will be determined by crosslink density and the physicochemical properties of the material. Release of material from the drug reservoir will occur more slowly at first as a result of the time required to swell the microprotrusions up as far as the material reservoir, subsequent partitioning of the material into the swollen microprotrusions and diffusion of the material through the swollen matrix.
A number of applicators for microneedle based delivery are known in the art. For example, US20046743211 describes methods and devices for limiting the elasticity of skin by means of stretching, pulling or pinching the skin, so as to present a more rigid, less deformable surface in the area to which microneedle-array-based transdermal drug delivery systems are applied. US 20060200069 describes a spring-loaded impact applicator for the application of coated microprojection arrays to the skin. A further application known in the art is the Alza Macroflux® device which is applied to skin using a specially-designed spring-loaded applicator (Alza Corporation, 2007).
Manufacture of Microprotrusion Array Loaded with Material
Microprotrusions composed of polymers known to form hydrogels can be manufactured by any such methods known in the art. For example, they can be prepared by a micromoulding technique using a master template, such as a microprotrusion array made from one or more of a wide variety of materials, including for example, but not limited to; silicon, metal polymeric material. Master templates can be prepared by a number of methods, including, but not limited to, electrochemical etching, deep plasma etching of silicon, electroplating, wet etch processes, micromoulding, microembossing, “thread-forming” methods and by the use of repetitive sequential deposition and selective x-ray irradiation of radiosensitive polymers to yield solid microprotrusion arrays.
Micromoulds can be prepared by coating the master template with a liquid monomer or polymer which is then cured and the master template removed to leave a mould containing the detail of the master template. In the micromoulding technique, a liquid monomer, with or without initiator and/or crosslinking agent is placed in the mould, which is filled by means of gravitational flow, application of vacuum or centrifugal forces, by application of pressure or by injection moulding. The monomer may then be cured in the mould by means of heat or application of irradiation (for example, light, UV radiation, x-rays) and the formed microprotrusion array, which is an exact replicate of the master template is removed.
Alternatively, a solution of a polymer with or without crosslinking agent can be placed in the mould, which is filled by means of gravitational flow, application of vacuum or centrifugal forces, by application of pressure or by injection moulding. The solvent can then be evaporated to leave behind a dried microprotrusion array, which is an exact replicate of the master template, and can then be removed from the mould. The solvents that can be used include, but are not limited to, water, acetone, dichloromethane, ether, diethylether, ethyl acetate. Other suitable solvents will be obvious to one skilled in the art. Micromoulds can also be produced without the need for master templates by, for example, micromachining methods and also other methods that will be obvious to those skilled in the art.
For example, in one embodiment, the microprotrusion arrays may be prepared using micromoulds prepared using a method in which the shape of the desired microprotrusions are drilled into a suitable mould material, for example using a laser and the moulds are then filled using techniques known in the art or as described herein.
Microprotrusions composed of polymers known to form hydrogels can also be manufactured using a “self-moulding” method. In this method, the polymeric material is first made into a thin film using techniques well known in the art, including for example, but not limited to, casting, extrusion and moulding. The material may, or may not be crosslinked before the “self moulding” process. In this process, the thin film is placed on a previously-prepared microprotrusion array and heated. Plastic deformation due to gravity causes the polymeric film to deform and, upon hardening, create the desired microprojection structure.
Microprotrusions with a hollow bore can be manufactured by using moulds prepared from hollow master templates or suitably altering the micromachining methods or other methods used to prepare solid microprotrusions. Hollow bores can also be drilled mechanically or by laser into formed microprotrusions. Microprotrusions which have at least one channel running down at least one outer surface from tip to base element can also be produced by suitable modification of the method used to prepare solid microprotrusions. Such alterations will be obvious to those skilled in the art. Channels can also be drilled mechanically or by laser into formed microprotrusions.
Microprotrusions composed of polymers known to form hydrogels can also be manufactured using a “thread forming” method whereby a polymer solution spread on a flat surface has its surface contacted by a projection which is then moved upwards quickly forming a series of polymer “threads”, which then dry to form microprotrusions.
In all of the above methods, substances to be incorporated into the microprotrusions themselves (e.g., material/nanoparticles/freeze-dried or spray-dried nanoparticles/pore forming agents, enzymes etc.) can be added into the liquid monomer or polymer solution during the manufacturing process. Alternatively, such substances can be imbibed from their solution state in a solution used to swell the formed microprotrusion arrays and dried thereafter or the formed arrays can be dipped into a solution containing the agent of interest or sprayed with a solution containing the agent of interest. Solvents used to make these solutions include water, acetone, dichloromethane, ether, diethylether, ethyl acetate. Other suitable solvents will be obvious to those skilled in the art, as will the processes used to dry the microprotrusion arrays. If the microprotrusions and/or base elements are to be made adhesive, the formed arrays can be dipped into a solution containing an adhesive agent or sprayed with a solution containing an adhesive agent. The adhesive agents used can be a pressure sensitive adhesive or a bioadhesive. These substances are well known and will be obvious to those skilled in the art.
Alternatively, the substances to be incorporated into the microprotrusions themselves (e.g., material/nanoparticles/pore forming agents, enzymes etc.) are freeze-dried or spray-dried prior to incorporation within the swellable and/or dissolvable polymer composition. They are then reconstituted using one or more of water, trehalose and/or PVP.
The base element on which the microprotrusions are formed can be varied in thickness by suitable modification of the method of manufacture, including, for example, but not limited to increasing the quantity of liquid monomer or polymer solution used in the manufacturing process. In this way the barrier to diffusion/transport of therapeutic active agents and/or analytes of interest can be controlled so as to achieve, for example rapid delivery or sampling or sustained release. Where therapeutic active agent(s) is/are to be contained within the matrix of the microprotrusions and base element, the thickness of the base element can usefully be increased so as it functions as a fully integrated reservoir.
According to a preferred embodiment of the invention, the microprotrusion array is prepared from a solution of the polymer which is mixed with or loaded with the material of the invention, placed in a mould and dried to result in a microprotrusion array support by a polymer substrate or baseplate.
It will be understood that the material/nanoparticles may be loaded into the plurality of microprotrusions alone; or may be loaded into both the baseplate and the plurality of microprotusions microneedles; or may be loaded into the baseplate alone.
In an alternative embodiment, the microprotrusion array may be made from a swellable or dissolving polymer composition and the material/nanoparticles may be added separately as a reservoir in the form of an attached patch, semi-solid gel or liquid. In a preferred embodiment, the material is a nanoparticle comprising a nucleic acid complexed with a cell penetrating peptide.
Ideally the microprotrusion array of the invention is in the form of a patch. The patch is formed for transdermal delivery to facilitate transdermal and/or intradermal delivery of the material within the microprotrusion array to the cell and/or subject. Such a patch may adapted to adhere to the skin or cell surface and as explained below may comprise a backing layer with adhesive to adhere to the skin or cell surface or the microprotrusion array may itself adhere to the skin or cell surface.
The size of the patch will dictate the number of microprotrusions present in the array, which in turn dictates the amount of nucleic acid present in the nanoparticle comprising material that may be loaded into the microprotrusion array.
For example, we have found that a 1 cm2 patch may be loaded with approximately 20 μg of DNA. Our initial experiments have found that a suitable average dosage may be approximately 10-50 μg DNA/cm2, preferably 20 μg DNA/cm2.
In order to increase the amount of nucleic acid present in the nanoparticle comprising material that may be loaded into the microprotrusion array, the nanoparticles may be freeze-dried or spray-dried. We have found that up to 500 μg of freeze-dried nanoparticles per 1×1 cm patch can be loaded into the microneedles (i.e. 500 DNA/cm2). Ideally, 100 μg of freeze-dried nanoparticles per 1×1 cm patch can be loaded into the microneedles (ie. 100 DNA/cm2).
We have found that DNA delivery is approximately 60% of the total DNA loaded into the microneedles after 5 mins and this increases to approximately 90% of the total DNA loaded into the microneedles after 24 hours.
Thus, advantageously the freeze-drying of the nanoparticles increased the amount of nanoparticles in the microneedles and also increased the percentage DNA delivery after administration.
One aspect of the present invention is directed to a transdermal drug delivery system for delivering a material) to a biological interface. As described above, such a system can comprise a base element and plurality of microprotrusions formed thereon. In particular embodiments said base element can have a first side and a second side; and said plurality of microprotrusions comprise a plurality of elements which project from the second side of said base element at an angle. In one embodiment with respect to said base element said angle is in the range 45° to 90°, for example in the range 70° to 90°. In a particular embodiment, said angle is about 90°. In particular embodiments of the device, said base element and plurality of microprotrusions can be formed of polymeric materials known to form hydrogels upon absorption of moisture. Suitably, in preferred embodiments, in use, upon insertion into skin the polymeric materials of said microprotrusions and base element can absorb moisture and increase in size to form swollen hydrogels; wherein the material can diffuse through said swollen base element and swollen hydrogel microprotrusions. In particular embodiments, the material can be provided from a reservoir; wherein said reservoir can be attached to the first side of the base element. In particular embodiments of the device, the reservoir can be a material dispersed in a suitable matrix material, for example a suitable adhesive or non-adhesive polymer matrix, or a material-containing reservoir.
In an alternative embodiment of a transdermal delivery device of the invention, an attached reservoir is not present. In such embodiments, the material may be contained within the swellable polymer composition of said base element and/or plurality of microprotrusions. Said substances can be either dissolved in the swellable polymer composition or suspended in particulate form. Upon insertion into skin and swelling of the microprotrusions, the material can be released into the skin at a rate determined by the degree of crosslinking of the microprotrusions and the material itself.
A backing layer with an adhesive border extending beyond the area of the base element of the microprotrusions may be used to keep microprotrusion-based devices in place on the skin surface for protracted periods of time, for example up to or greater than 72 hours. The surface of a base element of and, optionally, the microprotrusions themselves, may be coated with an adhesive material, so as to promote retention at the site of application.
In particular embodiments, the material can be chemically bonded to the polymer(s) making up the microprotrusions and base elements. In this case, the material can be released upon insertion into the skin by; dissolution of the microprotrusions, hydrolysis, enzymatic or spontaneous non-catalysed breakage of the bonds holding it to the polymer(s). The rate of material release can thus be determined by the rate of reaction/bond breakage.
In an alternative embodiment, the polymeric composition of the microprotrusions and/or base elements can be adjusted such that it can be stimulus-responsive. For example, local changes in pH or temperature can alter the properties (eg ability to swell upon imbibing moisture) of the microprotrusions and base elements, such that a change in the rate of delivery of material occurs. Alternatively, an external stimulus, such as light illumination, can be used to affect a change in the properties of the microprotrusions and base elements, such that a change in the rate of delivery of the material occurs.
In embodiments of arrays of the invention, the polymeric composition of the microprotrusions and base elements can be adjusted such that the surface properties of the device are altered, becoming more hydrophilic, lipophilic, anionic or cationic in character.
Another aspect of the present invention is directed to an iontophoretic transdermal drug delivery system for delivering a material to a biological interface. Such a system can comprise a cell delivery device of the invention. In specific embodiments the material can be provided from a reservoir. Said reservoir can be a matrix-type reservoir or a material-containing reservoir. In such embodiments the device may further comprise a first electrode and a second electrode at a location different to said first electrode, both electrodes being proximal to said reservoir, a power source, electronic controller and central processing circuit. Application of a potential difference between the electrodes facilitates delivery of the material from said reservoir into the skin by iontophoresis or electroosmotic flow through said swollen base element and microprotrusions.
The present invention is directed to the transdermal, intradermal and intracellular transport of a material across a biological barrier.
According to the invention the material comprises or consists of a composition or nanoparticles formed from a nucleic acid or other agent complexed or condensed with the amphipathic cell penetrating peptide of the invention. In this specification, it will be understood the terms “complexed” and “condensing” are interchangeable.
Advantageously, the peptide of the invention condenses the nucleic acid or other agent, preferably a negatively charged or hydrophilic compound.
The other agent is preferably a negatively charged or hydrophilic compound, including a protein, drug or active agent. In this embodiment, nanoparticles may also be formed from a negatively charged or hydrophilic compound complexed with an amphipathic cell penetrating peptide. These negatively charged or hydrophilic compound include but are not limited to any phosphate or lipophilic based drug, preferably a bisphosphonate drug and gold for example. These are described in more detail below.
Advantageously, we have found that the nanoparticles of the invention, once administered transdermally through the microprotrusion array, facilitates intracellular transport and results in the nuclear localisation of the nucleic acids to cells, both in-vitro or in-vivo. This gives the amphipathic cell penetrating peptide of the invention a distinct advantage over conventional delivery systems which do not achieve nuclear localisation.
Advantageously, we have found that the claimed amphipathic cell penetrating peptide can create nanoparticles with a size less than 150 nm or even 100 nm with nucleic acids or other agents. This facilitates transport of these agents across cell membranes, out of the endosomes and to the nucleus. We have found that these nanoparticles are stable in serum and over a temperature range of 4 to 37° C.
According to one embodiment, the amphipathic cell penetrating peptide of the invention is complexed with a nucleic acid, preferably DNA, mRNA. miRNA or siRNA, to form discrete spherical nanoparticles, each nanoparticle with a diameter less than approximately 150 nm, preferably less than or equal to 100 nm.
This delivery system is applicable across a wide range of nucleic acids, including DNA, RNA, mRNA, miRNA, siRNA and/or shRNA, and other agents, preferably small molecule agents, such as proteins, drugs or other active agents.
For example, the nucleic acid may be a DNA vaccine in the form of plasmid DNA. Ideally, the DNA vaccine targets cancer, such as cervical, breast or prostate cancer. Other cancers may also be targeted.
For example, the plasmid DNA may provide protection against herpes simplex virus (HPV), namely, HPV-16 E6, HPV-16 E7 and HPV-16 E6.E7, which cause cervical cancer. For prostate cancer, DNA coding for the tumour associated antigens (TAAs) Prostatic Antigen Phosphatase (PAP), Prostate Specific Antigen (PSA) and Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) could be utilized in this system. While TAAs for breast cancer could use DNA coding for HER-2/neu or the membrane associated glycoprotein (MUC-1).
Alternatively, the nucleic acid may be miRNA, siRNA or shRNA and may inhibit the expression of a disease causing gene, including cancer causing genes. In this embodiment, the nanoparticles comprise the claimed amphipathic cell penetrating peptide and siRNA, and hence act as a siRNA transfection agent. The inventors have shown, with siRNA, there is a much higher level of cellular entry compared to commercially available transfection reagents e.g. Oligofectamine®. In-vivo tests have shown that successful gene delivery following systemic injection into the bloodstream. Importantly, repeated injection of the nanoparticles does not illicit a significant immune response, either adaptive (IgG or IgM) or inflammatory (IL-6, II-1b). Furthermore, the inventors have shown that the there is no neutralisation of the claimed amphipathic cell penetrating peptide following systemic delivery. This is another major advantage of the nanoparticles of the invention.
According to another embodiment, the amphipathic cell penetrating peptide of the invention is complexed with a nucleic used in gene therapy. The nucleic acid may encode a functional, therapeutic gene to replace a mutated gene. Alternatively, the nucleic acid may correct a mutation or encodes a therapeutic protein drug. In this manner, the nanoparticles of the invention may be used as adjuvant gene therapy treatment administered optionally prior to conventional treatments.
According to a general aspect of the invention, the cell penetrating peptide of the invention had improved or equivalent cell penetration activity compared to conventional transfection reagents. The cell penetrating peptide of the invention acts as a transfection reagent and enables intracellular delivery, ideally to the nucleus of the subject.
In this specification and particularly the examples, the amphipathic cell penetrating peptide of the invention may optionally be referred to as the “RALA peptide”.
According to a general aspect of the invention, the amphipathic cell penetrating peptide comprises or consists of an amphipathic cell penetrating peptide less than approximately 50 amino acid residues comprising at least 6 arginine residues (R), at least 12 Alanine Residues (A), at least 6 leucine residues (L), optionally at least one cysteine residue (C) and at least two but no more than three glutamic acids (E). Optionally,
According to a preferred embodiment, the amphipathic cell penetrating peptide comprises or consists of an amphipathic cell penetrating peptide less than approximately 50 amino acid residues comprising at least 6 arginine residues (R), at least 12 Alanine Residues (A), at least 6 leucine residues (L), optionally at least one cysteine residue (C) and at least two but no more than three glutamic acids (E) wherein
We have found that the presence of arginine (R) residues in the amphipathic cell penetrating peptide is essential. Ensuring an even distribution of arginine (R) residues along the length of the peptide facilitates delivery of the peptide across a cell membrane by condensing the negatively charged compound or nucleic acid through electrostatic interactions. The presence of arginine (R) enables nanoparticles less than 20 nm to form and ensures a positive zeta potential which enables internalisation into the cell. We have also found that the presence of arginine (R) residues also enhances nuclear localisation.
The ratio of the positively charged amino acid residues arginine (R) to negatively charged amino acid is also important because this is necessary to condense the payload into nanoparticles through electrostatic interactions. It is generally accepted that a nanoparticle less than <200 nm will be small enough to cross the cell membrane. In addition, the ratio of positively charged residues ensures an overall positively charged nanoparticle which has two main advantages. Firstly, that the particles will not aggregate and repel each other which aids in systemic delivery otherwise embolisms could occur. Secondly, as the cell membrane is negatively charged, nanoparticles that are either neutral or mildly positively charged will not enter the cell.
Finally, the peptide has a greater proportion of hydrophobic residues than hydrophilic residues (see table below) because this enables an amphipathic helical conformation and when the pH lowers in the endosome it is likely that RALA undergoes a conformational change to a mixture of alpha helix and random coil. This conformational change exposes the hydrophobic residues that can then fuse and destabilize the endosomal membrane enabling release to the cytosol. Having more hydrophobic residues increases the extent of membrane destabilisation.
The peptide of the invention has improved cell penetration activity compared to, for example, KALA (see table below) for DNA delivery and conventional transfection reagents such as Oligofectamine® for siRNA delivery. Advantageously, the peptide of the invention is less toxic than another conventional transfection reagent such as, for example, Lipofectamine 2000®.
According to a preferred embodiment of the invention, the arginine (R) residues are evenly distributed at every third and/or fourth amino acid position along the entire length of the peptide.
According to another preferred embodiment of the invention, the amount of hydrophilic amino acid residues in the peptide should not exceed approximately 40% or 37% and the ratio of hydrophilic amino acid residues to hydrophobic amino acid residues ratio at pH 7 is from 30:67 to 40:60, preferably 30:70 to 37:63.
According to another embodiment of the invention, the peptide comprises less than approximately 40 amino acid residues. Optionally, the peptide comprises 35, 34, 33, 32, 31, 30 amino acid residues, preferably 30, 29, 28, 27, 26, 25, 24 or 23 amino acid residues.
Ideally the amphipathic cell penetrating comprises at least 17, 18, 19, 20, 21, 22, 23, 24, 25, preferably at least 24 amino acids. According to a preferred embodiment, the peptide of the invention comprises at least 24 amino acids.
Ideally, the peptide comprises the consensus sequence EARLARALARALAR (SEQ ID No. 15).
Optionally, the peptide may comprise the consensus sequences EARLARALARALAR and/or LARALARALRA (SEQ ID No. 16) as highlighted in the preferred sequences according to the invention listed below:
Ideally, the present invention provides a peptide comprising the amino acid sequence
or a sequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identical, wherein
Preferably, the peptide comprises or consists of one of the following amino acid sequences:
The table below provides further details of the several different examples amino acid sequences of preferred amphipathic cell penetrating peptides listed above.
A most preferred sequence comprises/consists of the amino acid sequence WEARLARALARALARHLARALARALRACEA (herein referred to as “RALA”) (SEQ ID No. 1).
It will be understood, in this specification “RALA” is a generic term referring to the RALA sequence (SEQ ID No.1) or other similar sequences, including but not limited to SEQ ID Nos. 2 to 7, which also fall within the scope of the invention.
The invention also encompasses sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity or sequence homology with SEQ ID Nos. 1 to 7.
Advantageously, the amphipathic cell penetrating peptides of the invention consists of arginine/alanine/leucine/alanine repeats that result in a specifically tailored hydrophobic and hydrophilic region facilitating interaction with the lipid bilayers enabling transport of the peptide across cellular membranes. As stated above, the presence of arginine (R) residues is an essential feature of the claimed peptide. There are two main advantages of using arginine. Firstly, arginine has consistently been shown to be the optimal amino acid for condensing DNA with arginine rich sequences binding in milliseconds. Secondly, arginine rich sequences based on the Rev sequence have the capacity to actively transport DNA into the nucleus of cells via the importin pathway.
In addition, there must be at least 2, but no more than 3, glutamate residues (E) to ensure pH-dependent solubility and protonation which facilitates endosomal disruption.
The present invention is also directed to modified peptides or peptide derivatives.
Optionally, the peptide according to any of the preceding claims is coupled or conjugated to a polyethylene glycol (PEG) molecule, such as RALA-PEG. Preferably, coupling takes place at the C-terminus of the peptide. The presence of the PEG molecule is advantageous because it increases circulation time of the peptide in vivo and provides for an enhanced permeation and retention effect of the peptide.
Alternatively, the peptide of the invention may comprise a cell targeting motif, preferably a motif which confers specificity to metastatic cell lines, conjugated to the N-terminus of the peptide through or via a spacer sequence. Ideally, the spacer sequence is an alpha helical spacer.
In this manner, the cell targeting motif may be the metastatic prostate cancer targeting peptide TMTP-1 (NVVRQ) and the spacer may be an alphahelical concatemeric spacer, preferably comprising 1 or more, preferably, 2, 3, or 4 repeats of the sequence EAAAK.
Advantageously, we have found that the claimed amphipathic cell penetrating peptide (RALA and similar sequences) or modified peptide/peptide derivative facilitates nuclear localisation. This gives the amphipathic cell penetrating peptide of the invention a distinct advantage over conventional non-viral and viral delivery systems. Surprisingly, the claimed amphipathic cell penetrating peptide has also been shown to form nanoparticles after 5 mins and be stable up to 48 hours at room temperature. The peptides of the present invention have been found to be stable as nanoparticles up to 5, 6 and 15 days after delivery.
Advantageously, we have found that the claimed amphipathic cell penetrating peptide can create nanoparticles with a size less than 150 nm or even 100 nm with nucleic acids or other agents. This facilitates transport of these agents across cell membranes, out of the endosomes and to the nucleus. We have found that these nanoparticles are stable in serum and over a temperature range of 4 to 37° C. These nanoparticles may be used as cell delivery systems themselves or together with a microprotrusion array of the invention for delivery of nucleic acids or other agents across cell membranes and/or nuclear localisation.
Accordingly, the peptide as defined above presents a viable alternative in the field of gene delivery and may be used as a transfection agent for siRNA.
According to this embodiment, the claimed amphipathic cell penetrating peptide may also be used in DNA gene therapy. Confocal imaging has clearly shown delivery of Cy3 labelled DNA to the nucleus of prostate cancer cells. This provides the opportunity for the delivery of any nucleic acid to a cell in vivo, in which the nucleic acid may be utilised for gene therapy.
The nucleic acid may encode a functional, therapeutic gene to replace a mutated gene. Alternatively, the nucleic acid may correct a mutation or encodes a therapeutic protein drug. In this manner, the nanoparticles of the invention may be used as adjuvant gene therapy treatment administered optionally prior to conventional treatments.
According to another embodiment of the invention, the nucleic acid may be DNA in the form of an iNOS (inducible nitric oxide synthase) plasmid DNA under control of a tumour specific promoter. The iNOS plasmid DNA may be condensed with or complexed with the peptide of the invention to form nanoparticles and delivered as nanoparticles in-vivo. This results in the inducible production of nitric oxide in-vivo which is detrimental to tumour metastasis. In this manner, the nanoparticles or cell delivery system of the invention comprise the claimed amphipathic cell penetrating peptide and iNOS plasmid DNA.
According to one embodiment the tumour specific promoter is the human osteocalcin (hOC) promoter. It will be understood that the hOC promoter is specific to ovarian, breast and prostate cancers and although the peptide of the invention will deliver to all tissues the use of this promoter will ensure transcriptional targeting and expression of the desired gene only in the tumours. However, other known promoters may be used which will be dependent on differential expression in tumour tissue. Examples include the osteopontin promoter known to be overexpressed in breast cancer, the prostate specific membrane antigen promoter for prostate cancer or radiation inducible promoters such as WAF1 or CARG. Both WAF1 and CARG have the added advantage of also being activated in hypoxic regions such as those found in the centre of tumours.
According to another embodiment of the invention, the tumour specific promoter may be a prostate specific promoter, such as the prostate membrane specific antigen promoter (PSMA).
The amphipathic cell penetrating peptide of the invention may also be used to deliver hOC-iNOS (inducible nitric oxide synthase) systemically in vivo to any tumour model that has been shown to metastasise to bone. In this manner iNOS plasmid DNA may be condensed with the peptide of the invention and delivered as nanoparticles in-vivo. Advantageously, the RALA/hOC-iNOS nanoparticles may be administered in tandem with the current recommended chemotherapy regimen of docetaxel. For those with bone metastases docetaxel remains the standard front-line treatment but increasingly many patients develop resistance to this drug. This new combination therapy provides an alternative strategy for treating bone metastases.
Alternatively, promoters specific for cardiovasculature may be used to increase the levels of iNOS to dilate blood vessels. One potential administration method includes the application of the nanoparticles as a coating for stents. A major unresolved issue following percutaneous transluminal coronary angioplasty (PTCA) is the physical injury to the blood vessel wall, which leads to vessel re-occlusion, i.e. restenosis. The endothelial denudation associated with this injury is accompanied by varying degrees of medial disruption and is followed by an inappropriate response-to-injury of vascular smooth muscle. Therefore using smooth muscle cell (SMC) (e.g. SM22 alpha promoters) promoters to drive expression of the iNOS transgene will confer tissue specific targeting at the site of injury either with or without stents.
The material of the invention, may comprise nanoparticles formed from other agents, preferably small molecule agents complexed with the amphipathic cell penetrating peptide. For example, the agents may comprise proteins or a therapeutic agent or drug.
These include but are not limited to any phosphate or lipophilic based drug, preferably a bisphosphonate drug and gold. Bisphosphonate drugs are characterised by a very low bioavailability, rapid excretion from the body, harsh side effects and poor patient compliance. Improving upon the delivery of this drug to where it is needed there provides a significant impact on patient health. As a lipophilic drug, bisphosphonates cannot cross the cell membrane to effect the therapy. Therefore there is a need for an effective delivery system to encapsulate the bisphosphonates and improve cellular entry and bioavailability in vivo.
The therapeutic agent may be a phosphate based drug, preferably a bisphosphonate drug including alendronate, etidronate, zolendrate or any other nitrogen or non-nitrogen based bisphosphonate drug. Bisphosphonate drugs have low bioavailability which can advantageously be enhanced when complexed with the peptide of the present invention. The cell delivery system of the invention will improve the bioavailability of a phosphate based drug, preferably a bisphosphonate drug. The cell penetrating peptide of the invention may be used for the condensation and delivery of the nitrogen bisphosphonate, Alendronate. N-BP nanoparticles were formed with sizes less than 100 nm and an overall positive charge facilitating cellular entry. The alendronate nanoparticles were spherical, uniform and did not aggregate as evidenced by TEM. More importantly, when the alendronate loaded nanoparticles were added to prostate cancer cells in vitro there was significantly greater cytotoxicity at lower concentrations compared to the alendronate only treated cells. Thus, the delivery system of the invention provides significant promise for improving the delivery and bioavailability of bisphosphonates patients with osteoporosis and cancer.
An alternative use involves the improvement of the delivery of gold particles. The effectiveness of many radiotherapy treatment plans are limited by normal tissue toxicity. Using gold nanoparticles (GNPs) can increase the therapeutic benefit by radiosensitising cancer cells. However, whenever these gold nanoparticles are delivered the majority remain trapped within the endosome creating an inhomogeneous distribution and limiting their full potential. We have found that when the GNPs are wrapped with the peptide of the invention there is a significant increase in endosomal escape which facilitates a marked increase in therapeutic efficacy.
According to another aspect of the invention, there is provided a transdermal patch comprising the cell delivery system according to the invention and a pharmaceutically acceptable excipient. In this manner the cell delivery system of the invention is adapted for transdermal delivery.
According to another important aspect of the invention there is provided freeze-dried or spray-dried nanoparticles. Standard/conventional freeze-drying and spray-drying techniques may be used. We have found that these nanoparticles are stable after lyophilisation with no reduction in transfection efficacy. They remain as discrete nanoparticles when reconstituted or rehydrated. Advantageously, one or more of water or trehalose may be used to reconstitute the freeze-dried or spray-dried nanoparticles. We have also found that PVP alone or in combination with trehalose may be used to reconstitute the freeze-dried or spray-dried nanoparticles. One the advantages of freeze-drying the nanoparticles is that significantly more nanoparticles can be loaded into the microprotrusion array/microneedles. We have found that up to 500 μg of nanoparticles per 1×1 cm patch can be loaded into the microneedles. Ideally, 100 μg of nanoparticles per 1×1 cm patch can be loaded into the microneedles.
The present invention will now be described with reference to the following non-limiting figures and examples.
No immunoreactivity was observed compared to the controls. Data are the mean of three independent experiments+/−S.E.
Following preparation of RALA/pORF-mIL4 nanoparticles N:P 10 they were characterised over a range of temperatures (4-37° C.) and following incubation at room temperature for up to 6 h using the Malvern Zetasizer NanoZS with DTS software. The measurements are reported as mean±SEM, (n=3).
The following peptide (called “RALA” herein) was synthesised commercially in accordance with conventional techniques with the amino acid sequence
RALA arrives in a lyophilised form and is reconstituted with molecular grade water to a desired concentration, aliquotted out and stored at −20° C. until further use. An aliquot is then taken as needed and defrosted on ice.
DNA was complexed with either the RALA peptide at various N:P ratios (the molar ratio of positively charged nitrogen atoms to negatively charged phosphates in DNA). As the number of positive side-groups in a protein side chain depends upon the sequence, different proteins will have differing numbers of positive charges per unit mass. In order to calculate this, the following equation was used:
NP=M
protein
/M
DNA
C
NP
Where M protein is the mass of a protein, M DNA is the mass of DNA and C NP is the N:P constant. The N:P constant is the ratio of the protein's side chain positive charge density to the DNAs backbone density, with the charge density being the charge of a substance divided by its molecular mass. For the protein, lysine, arginine and histidine side groups are counted. For the DNA the average mass of one single base pair, and the charge of the phosphate group are used. For RALA an N:P ratio of 1 is 1.45 μg of RALA: 1 μg of DNA.
The DNA/siRNA was diluted in molecular grade water to 200 μg/ml. 1 μg of DNA was added to a 1.5 ml eppendorf centrifuge tube. For 1 μg of DNA the final volume was 50 μl. The appropriate volume of protein to use to make the desired N:P ratio was added to a separate tube and the volume made up to 50 μl with molecular grade water. The 50 μl solution containing the protein was added to the 50 μl containing the DNA. The molecular grade water was added to the DNA before the protein. The tube was flicked five times in order to mix the content. The complexes were allowed to incubate for 30 minutes at room temperature prior to use. The results are shown in
RALA/DNA complexes were prepared at N:P ratios 1-15. Following incubation at room temperature for 30 minutes, 30 μL of the samples (corresponding to 0.6 μg of DNA) were electrophoresed through a 1% agarose gel containing 0.5 μg/mL ethidium bromide (EtBr) (Sigma, UK) to visualize DNA. A current of 80 V was applied for 1 h and the gel imaged using a Multispectrum Bioimaging System (UVP, UK). The purpose of this assay is to determine which N:P ratio/s neutralise the DNA. The assay works upon the principle that when complexes are formed with an excess positive charge DNA remains in the wells or migrates up the gel, hence, no DNA band will be visible following gel electrophoresis. However, DNA alone or complexed to give a net negative charge will migrate down the gel (
In order to obtain particle size and charge distributions the mean hydrodynamic particle size measurements RALA complexes were performed using Dynamic Light Scattering (DLS). Dynamic Light Scattering is based upon the principle that when particles are illuminated with a laser, due to Brownian motion there will be scattering of the light. The intensity of the scattered light fluctuates as a result of this Brownian motion caused by bombardment of the particles by solvent molecules. A correlation curve reflecting the decay rate is generated based on fluctuations of the scattered light where a slower correlation decay rate represents a slower moving particle. Based on the Stokes-Einstein equation larger particles move more slowly and, thus, the correlation function can be used to determine the size distribution of the particles. dynamic light scattering (DLS) was used.
Surface charge measurements of the RALA nanoparticles were determined by Laser Doppler Velocimetry. The zeta potential of the particles was measured using disposable foltable zeta cuvettes. Zeta cuvettes for the measurement of zeta potential were first washed with 70% ethanol, followed by two rinses with double distilled H2O prior to loading the sample. Enough diluted sample used for size measurement was used for determination of zeta potential.
The nanoparticles were made up at an appropriate range of N:P ratios with at least using 2 μg of DNA in each sample. Nanoparticles were analysed using either and analysis was completed on either the Zetasizer-HS3000 (Malvern Instruments) or the Zetasizer-Nano instrument with DTS software (Malvern Instruments, UK). Zetasizer-Nano (Malvern Instruments) (
This assay is designed to illustrate the stability of RALA complexes to indicate the optimal time period for nanoparticle formation. Following incubation at room temperature for 30 min the mean hydrodynamic size and zeta potential were measured using the Malvern Zetasizer NanoZS with DTS software at 15 or 30 min intervals over a period of 360 min. Size and zeta potential are reported as mean±SEM, n=3, where n represents the number of independent batches prepared for measurement (
This assay determines the stability of the nanoparticles over a range of temperatures. Following preparation of the nanoparticles by incubation at room temperature for 30 min the mean hydrodynamic size and zeta potential were measured over a temperature range of 4-37° C. in 4° C. intervals using the Malvern Zetasizer NanoZS with DTS software. The sample was allowed to equilibrate at each temperature for 120 sec before measurements were taken in triplicate. Results are reported as mean±SEM, n=3, where n represents the number of independent batches prepared for measurement (
In order to determine the stability of the RALA nanoparticles when exposed to serum the following procedure was carried out. Six replicates of the complexes at NP ratios 5, 10 and 15 were made. Each N:P ratio was split into 3 aliquots or in the case of RALA 18 aliquots. 10% foetal calf serum was added to 12 of the aliquots. The 18 aliquots were incubated at 37° C. Every 55 min SDS (sodium dodecyl sulphate (Sigma, UK)) was added to one of aliquots containing serum for each N:P ratio which were then incubated for a further 5 min. For RALA the stability was assessed over a 6 h time course. Loading dye (Ficoll (Sigma, UK), Tris-HCl, bromophenol blue (Sigma, UK) in ddH2O) was added to all the aliquots prior to loading onto an ethidium bromide prestained 0.8% agarose-TAE gel. A current of 80V was applied for 1 h and the gel was visualised using a Multispectrum Bioimaging System (UVP, UK). (
In an attempt to confirm the results obtained by DLS and obtain additional information about the structure of the nanoparticles Transmission Electron Microscopy was employed. The RALA complexes were prepared as perf or standard conditions and 5 μl was pipetted onto formvar coated copper grids (Agar Scientific, UK) and allowed to air dry overnight. Subsequently samples were stained with 5% aqueous 5% uranyl acetate for 5 minutes and allowed to dry overnight before visualisation. The nanoparticles were imaged using JEOL 100CXII transmission electron microscope at an accelerating voltage of 80 kV (
700 μl of RALA-pEGFP-N1 nanoparticles were subject to freezing for 1 h at −40° C. This was followed by primary drying at −40° C. and 60 mTorr for 24 h. This was followed by the secondary drying program; 3 h at −35° C. and 120 mTorr, 3 h at −30° C. and 190 mTorr, 3 h at −25° C. and 190 mTorr and 6 h at 20° C. (
Transfection of ZR-75-1 & PC-3 Cells in 96 Well Plates with the RALA Nanoparticles
In order to test the RALA in vitro, small scale transfections were performed carried out. 5×104 cells were seeded onto each well of a 96 well plate and the cells incubated under with complete medium standard conditions for 48 hours. The medium was subsequently removed from the plates and 100 μl of transfection medium (Optimem Invitrogen, UK) was added to each well. Cells were incubated for 2 hours at 37° C. and 5% CO2 standard conditions. In the meanwhile complexes were made up using 1 μg of plasmid DNA with the RALA vector and added to the cells when the two hours had passed. 100 μl of the each N:P ratio were added to each well of the cells. Cells were then incubated for a further 4 hours under standard conditions and the medium with RPMI-1640 supplemented with +10% FCS. (
ZR-75-1 & PC-3 cells that were transfected with RALA/pEGFP-N1 complexes were trypsinised and washed twice with 2% formaldehyde in phosphate buffered saline. The expression of green fluorescent protein was measured by flow cytometry using FACS calibur system (BD Bioscience, UK). The data was analysed using the Flo-Jo software program and fluorescent intensity is reported at 4% gating. (
Cell viability was evaluated by manual counting of the viable adherent cells using a haemocytometer as described in. PC-3 prostate cancer cells were seeded in a 96-well flat-bottom tissue culture plate at a density of 1×104 cells per well and incubated in complete culture medium for 24 h. Two hours prior to transfection the cells were conditioned in OptiMEM serum-free medium (Invitrogen, UK) optimised for transfection. Cells were treated with solutions of BP to achieve a final exposure concentration of 5 μM to 1 mM. RALA/BP nanoparticles were prepared using a mass ratio of 10:1 such that the final concentration of BP per well was in the range 5 μM to 75 μM. Cells were incubated at 37° C. with 5% CO2 for 6 h before medium was replaced with completed culture medium and left to incubate for 72 h. Following incubation the cells were trypsinised and counted. Cell viability was expressed as a percentage of the untreated control where the untreated control is considered to be 100% viable. Dose-response curves were obtained for free BP and RALA/BP allowing determination of EC50 values for each. EC50 values refer to the concentration that induces a response halfway between the baseline and the maximum plateau obtained (
The WST-1 assay is a colorimetric assay that can analyse the number of viable cells present and hence, indicate the toxicity of complexes added to cells in vitro. The assay is based on the cleavage of tetrazolium salts that are added to the culture medium. The stable tetrazolium salt WST-1 is cleaved to a soluble formazan by a cellular mechanism that occurs primarily at the cell surface. This WST-1 cleavage is dependent on the glycolytic production of NAD(P)H in viable cells, therefore, the amount of formazan dye formed directly correlates to the number of metabolically active cells in the culture.
Cells were transfected and the complete medium was discarded at a range of time points and replaced with 100 μL Opti-MEM with 10% WST-1 reagent (Roche, UK). Cells were incubated for 2 h under standard cell culture conditions. Subsequently the plates were shaken for 1 min and absorbance measured at 450 nm on an EL808 96-well plate reader (Biotek, USA). The measured absorbance values are expressed as a percentage of the control where the control is defined as 100% viable (
ZR-75-1 or PC-3 cells were trypsinised until they had detached and 8 ml of medium was added per flask. The cell suspension was transferred into 20 ml universal tubes. The cells were and centrifuged for 5 minutes at 80 g. Cells were resuspended in RPMI+10% FCS and counted using a Coulter Counter (Beckman Coulter, UK). Cells were subsequently centrifuged as before, and resuspended at 108 cells per ml in PBS before being diluted 1: in 1 in matrigel (BD Biosciences, UK). The matrigel cell suspension was loaded into syringes and kept on ice until implantation. Matrigel was only required for the ZR-75-1 cells. Balb-C SCID mice were anaesthetised with isofluorane (Abbott, UK) and the rear dorsum was shaved. Subsequently the skin on the rear dorsum was pinched between forefinger and thumb and 5×106 cells (100 μl) were injected intradermally using with a 26 G needle (BD Biosciences, UK) at the prepared site. Mice were observed while recovering from the anaesthesia and then subsequently returned to their box (
The length (L), width (W) and depth (D) of the tumour was measured using vernier with calipers. Subsequently the volume of the tumour was estimated by using the equation, V=πLWD/6, an approximation of V=4/3πr3.
Mice were anaesthetised with isofluorane and a 26 G needle (BD Biosciences, UK) was inserted bevel side down into the tumour. 100 μl of the nanoparticle treatment was injected slowly before rotating the needle and removing very slowly. For the multiple dose regimen used in this study a ‘round the clock’ system of injections was used. Recovery of mice from anaesthesia was monitored (47,52).
Mice were placed into a heat box at 36° C. for 5 minutes or until both of the tail veins were clearly visible. They were then moved into a heavy brass restrainer and injected with 50-100 μl of treatment into the tail vein with an insulin syringe (BD Biosciences, UK) equipped with 28 G needle. Mice were then replaced into the cage and monitored for signs of suffering associated with the injection. Mice found to be suffering or dying were euthanized by a schedule one protocol. (
For the harvesting of blood and intraperitoneal macrophages, cervical dislocation was the preferred method of euthanasia. Cardiac puncture was performed using a 21 G gauge needle (BD Biosciences, UK). The needle was placed horizontally slightly to the left side of the sternum to go up through the diaphragm. The needle was then withdrawn very slowly until 500 μl of blood was collected and placed in an eppendorf. The eppendorf was then stored at room temperature with an open lid to facilitate coagulation. After 30 min the eppendorfs were centrifuged at 2000 rpm for 10 min. The supernatant containing the serum was carefully decanted and placed into a clean eppendorf and stored at −20° C. until further use. When harvesting intraperitoneal macrophages an incision was made and the peritoneal cavity was flushed out with 30% sucrose (Sigma, UK) solution. The macrophages were stored at 4° C. until they could be cultured (
Western Blots with In Vitro and In Vivo Samples
Organs were homogenised and lysed in RIPA overnight. The samples were centrifuged at 5000 g for 10 minutes and the supernatant transferred to a fresh eppendorf tube. The lysate was diluted 1:2 in laemmli buffer, boiled for 10 minutes and loaded onto a Bis-Tris gel. Cells were put directly into laemmli buffer. The gel was run at 120V till the dye reached the bottom. The gel was and transferred into a western cassette. The protein was subsequently transferred for 2.5 hours at 25V onto a nitrocellulose membrane (Amersham, Biosciences, UK). Protein transfer was visualised by staining with Ponceau stain (Sigma, UK). The membrane was then subsequently incubated with primary antibody in blocking solution (PBS (Invitrogen, UK), 0.1% Tween (Sigma, UK), Skimmed milk (Merck, Germany)). Subsequently the membrane was then rinsed twice within Tween-PBS and once within PBS before being incubated in secondary antibody for 1.5 hours. The membrane was then was rinsed again, twice with Tween-PBS and once within PBS before the application of Immobilon reagent (Millipore, UK). Western blots were quantified using imageJ software (
Female C57/BL6 mice (5-6 weeks old) were treated with one of;
Mice receiving DNA received 10 μg total. Nanoparticles were formulated with an N:P ratio of 10. Mice receiving RALA alone received an amount of vector equivalent to that received in the RALA/DNA group. Treatments were administered by tail vein injection performed over a three week period. There was 15 mice per treatment group, with 5 mice per time point. All animals received the relevant treatment on Day 0. Following 7 days, five mice from each group were sacrificed and blood from each will be isolated by cardiac puncture. Serum was isolated, serum from the five mice per group was pooled, heat-inactivated at 56° C. for 30-60 min, and serially diluted in Opti-MEM to produce serum concentrations of 10% v/v, 1% v/v and 0.1% v/v, plus a 0% control.
To these serum dilutions, fresh RALA/DNA nanoparticles (as above) were added at a DNA concentration of 1 μg/200 μl (the standard concentration for RALA/DNA transfection in 96 well plate format), and incubated at 37° C. for 1 h. This pre-incubated mix was then transferred to ZR-75-1 breast cancer cells previously seeded in 96 well plates (104 cells/well) on Day 6, and transfection was performed in the usual manner. Transfection of the GFP construct was assessed by FACS analysis after 24 h.
On Day 7, the remaining 10 mice received a second administration of the appropriate treatment. On Day 14, five mice left the experiment and were treated as above, while the remaining five mice per group received a final administration of the appropriate treatment, and on Day 21, followed by the previously outlined treatment (
These assays were performed on the serum collected from immunocompetent C57/BL6 mice following either 1, 2, or 3 intravenous injection with the RALA/pEGFP-N1 nanoparticles. IgG, IgM, 11-12, IL-6, and TNF-6, ELISAs were performed using the ENZO ELISA Kits in accordance with the recommended protocol (
Nunc Maxisorp ELISA plates were coated with RALA-pEGFP nanoparticles equivalent to 1 μg DNA per well. The wells were subsequently blocked with PBS/5% BSA. Wells were probed for 1 h with sera from mice diluted (1:500) in PBS/0.5% BSA at room temperature. (NB the sera came from the mice treated in the vector neutralisaiton assay). The wells were washed with PBS/0.5% Tween 20 and then probed for 30 min with HRP-conjugated anti-mouse secondary antibody. Wells were then washed again and probed with TMB substrate for 30 min. Colour development was measured at 450 nm with a reference wavelength of 550 nm (
5000 ZR-75-1 breast cancer or PC-3 prostate cancer cells were grown on cover slips and transfected with Cy3 labelled RALA/pEGFP (lacks the promoter contained in the construct used in the neutralisation assay) or fluorescent siRNA. Confocal microscopy was used to determine subcellular localisation of RALA/Cy3-pEGFP nanoparticles (
5 nm phosphorylated gold nanoparticles were incubated with RALA peptide at a ratio of approximately 1:10 for 30 mins before being added to MDA-MB-231 breast cancer cells for 24 hours. The MDA-MB-231 cells (5000) had been seeded onto a coverslip. After 24 h the cells were fixed with 50% methanol and 50% acetone and sent to Cytoviva (Auburn, Ala.) for imaging (
Cells seeded in multiwell plates (6 or 24 well) were transfected with various amounts of pDNA (CMV/iNOS or hOC/iNOS) complexed with RALA at N:P 10 for 6 h, following which, transfection complexes were removed, and cells returned to normal growth medium (Minimum Essential Medium—MEM). After 48 h, 70 μl aliquots of conditioned MEM were assayed for their total nitrate (an indirect indicator of nitric oxide content) content using a Nitric Oxide Quantitation kit (Active Motif) following the manufacturer's instructions. A standard curve (using 0-35 μM sodium nitrate) was constructed and used to quantify nitrate content in sample wells of the assay plate. After incubation of standards and unknown samples with nitrate reductase and co-factors, Greiss reagents A and B were added to wells, and after a 20 min incubation to allow colour development, the absorbance of each well at 540 nm was determined (
PC-3s grown in T25 tissue culture flasks were starved of serum by Opti-MEM incubation for 2 h before transfection with 10 μg of pDNA (CMV/iNOS, hOC/iNOS or CMV/GFP) for 6 h. Following transfection, media were replaced with MEM, and the cells incubated overnight. The next day, cells were trypsinised, resuspended in growth medium, enumerated, and plated in triplicate into 6 well plates (200 or 500 cells per well). Plates were incubated for 14 days to allow clonogenic growth, following which, medium was aspirated, colonies were stained with crystal violet and counted manually. Percentage cell survival was calculated by comparison with untransfected cells (
Female Balb/c SCID mice (5-8 weeks old) were inoculated via the left cardiac ventricle with 2×105 MDA-MB-231-luc2 breast cancer cells that express firefly luciferase. Mice then received an intraperitoneal injection of 200 μl D-luciferin (15 mg/ml) and were imaged (following 10 min) using IVIS imaging; successful left ventricular delivery was confirmed by whole body luminescence immediately following intracardiac delivery. Mice possessing luminescence limited to the thoracic cavity were sacrificed at this point. Remaining successfully inoculated mice were randomly assigned to one of four treatment groups (water, RALA only, RALA-CMV/iNOS or RALA-hOC/iNOS), and received five treatments twice weekly commencing two days post inoculation. Gene therapy mice received 10 μg pDNA complexed with RALA at N:P 10, RALA only mice received the corresponding amount of RALA dissolved with water; treatments were of 100 μl, and were delivered via the tail vein. Mice were routinely imaged twice weekly as described above, were observed daily by experienced animal husbandry experts, and body mass was monitored as an indicator of general health. A loss of 20% of original body mass was considered indicative of poor health of the mice, and this combined with a moribund appearance was determined to be a humane experimental end point (
The effects of Runx2 knockdown on cell proliferation were evaluated at different time-points following transfection with RALA/Runx2 siRNA nanoparticles. Nanoparticles were prepared such that the final concentration of Runx2 siRNA was 100 nM and based on a N:P ratio of 12. Two Silencer Select Runx2 siRNAs were used and a Silencer Select non-coding siRNA (Invitrogen, UK). Cells were serum starved for 2 h prior to transfection. Transfections were carried out with both RALA peptide and Oligofectamine for a duration of 4 h in serum-free RPMI 1640 before RPMI 1640 containing 30% FCS was added to achieve a final FCS concentration of 10%. After 24, 48 and 72 h cells were detached using 2× trypsin and subsequently neutralised with RPMI 1640 containing 10% FCS. Cells were counted manually using a haemocytometer as described in 3.2.11.2 and the cell viability determined based on the assumption of a 100% viability of the untreated cells. Results are reported as mean±SEM, n=3, where n represents the number of independent batches prepared for analysis (
To assess the ability of RALA/Runx2 siRNA nanoparticles to successfully inhibit Runx2 protein expression a range of siRNA concentrations and time-points following transfection were evaluated by Western blotting. PC-3 prostate cancer cells were seeded at a density of 150,000 cells per well in a 12-well plate. Transfections were initially carried out with various amounts of two types of Silencer Select Runx2 siRNA and Silencer Select non-targeting control siRNA such that the final siRNA concentration in the well was 50, 100 or 200 nM. Transfection was for 4 h followed by 48 h incubation. Following optimisation of the concentration the optimal time following transfection was determined using 100 nM concentrations. Cells were washed with ice-cold tris buffered saline (TBS) and lysed in a direct lysis buffer supplemented with MG-132 (Calbiochem, UK) and protease inhibitor cocktail (Roche, UK) (Appendix 1). Lysed samples were stored at −20° C. until required. Samples were run on 8% acrylamide gels at 100 V for 15 min followed by 150 V until the dye front reached the bottom of the gel in a tris-glycine running buffer. Subsequently the protein was transferred to PVDF membranes at 200 mA for 90 min in a tris-glycine transfer buffer. Membranes were blocked for up to 1 h in 2% blocking solution before leaving in primary antibody overnight at 4° C. with rocking. Runx2 primary antibody (MBL International, Woburn, Mass.) was used at a concentration of 1:200 and β-actin (Abcam, UK) at a concentration of 1:5000. Membranes were washed in TBS-tween (TBS-T) for 30 min before applying anti-mouse secondary antibody at 1:5000 for 1 h at room temperature. Membranes were washed vigorously in TBS-T for 30 min before developing. The chemiluminescent used for Runx2 protein was Thermo Scientific SuperSignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, Mass.) and for β-actin Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, Mass.) (
Studies with the RAT Peptide
RAT was synthesised from a commercial company and is a fusogenic, consisting of RALA with an alphahelical concatemeric spacer, (EAAAK)4, and the TMTP1 (NVVRQ) metastatic prostate cancer targeting peptide (
A pegylated version of RALA has been synthesised (
RALA nanoparticles were prepared using desalted peptide in MOPS buffer at 50° C. to give a concentration of 50 μg/ml of DNA. PLGA and a series of PLA-PEG block copolymers were synthesized with various PEG chain length and LA/EG ratio (PLA10-PEG2; PLA25-PEG5; PLA50-PEG5) and formulated into composite nanoparticles (diameter <200 n m and PDI <0.2000) containing the RNPs. 100 μl of RALA nanoparticles was added to 0.5 ml 4% w/v copolymeric polymeric solution in dichloromethane under vortex and probe sonicated (120 Sonic Dismembrator with 3 mm probe, Fisher Scientific, USA) for 60 seconds at 50% of amplitude. This water-in-oil (w/o) emulsion was added to 2.5 ml of 5% w/v PVA solution in distilled water under vortex and probe sonicated as before in an ice bath for 2 minutes. The resultant emulsion was stirred overnight to form the composite nanoparticles. These were collected by centrifugation at 30,000 g for 30 min (3K30, Sigma Centrifuge, UK) and washed twice with distilled water, before suspending in 1 ml 5% w/v trehalose in water and were freeze-dried (Advantage, VirTis, Gardiner, N.Y., USA). TEM (JEOL JEM1400 transmission electron microscope at an accelerating voltage of 80 kV) was performed by loading samples onto a copper grid (Formvar/Carbon 200 mesh, Agar scientific). Osmium tetraoxide was incorporated by adding it to the organic phase during preparation of the composite nanoparticles.
As shown in
Particle formation between DNA and RALA was studied by gel retardation assays and dynamic light scattering. It was found that RALA fully condensed DNA at N:P ratios above 4 (
Additionally, serum stability of particles at N:Ps of 5, 10 and 15 showed that the nanoparticles are stable in the presence of 10% serum and dissociate in 1% SDS revealing that the integrity of the DNA remains intact (
ZR-75-1 cells were transfected with RALA/pEGFP-N1 nanoparticles. Epifluorescence microscopy showed a high transfection efficacy of ZR-75-1 cells, when transfected with RALA/pEGFP at N:P of 10 with and without chloroquine. Chloroquine is a known endosomal disrupter and will increase transfection if the nanoparticles are inefficient endosome disrupters. At N:P 10 this is clearly not the case. Flow cytometry was then used to further analyse the effect of N:P on transfection efficacy and revealed an optimal transfection efficacy of around 30% between N:P ratios 8-12. More importantly though the WST-1 cell viability assay revealed minimal toxicity of the nanoparticles over a range of N:P ratios. Cell viability was 90% for N:P 4 and 80% at N:P 10. Indeed when cellular proliferation was examined there was significant difference between lipofectamine 2000 and RALA/pEGFP-N1 transfected cells (
To determine if RALA/pEGFP-N1 nanoparticles are significantly more efficient at eliciting cellular transfection in comparison to KALA/pEGFP-N1 nanoparticles a transfection experiment with both peptide-based nanoparticles was carried out in parallel (
Confocal microscopy also confirmed successful transfection with a time course revealing diffuse pattern of distribution of nanoparticles that focus into distinct foci with increasing duration of transfection (
As RALA/pEGFP-N1 nanoparticles transfect cells efficiently and are non-toxic, it was decided to use these nanoparticles as a model of a potentially therapeutic peptide based polyplex. It is well know that a major problem with gene therapy protocols is storage as both peptide and DNA degrade if stored in aqueous solutions at room temperature for prolonged periods of time. As such, the nanoparticles were lyophylised with a range of concentrations of trehalose as a lyoprotectant. Transfections, as well as serum stability assays were performed before and after freeze-drying. Serum stability assays were performed on all formulations up to 6 h. All formulations were found to be as stable upon incubation with 10% serum as the fresh particles without trehalose (
Overall these results highlight the stability of RALA/pEGFP-N1 nanoparticles as well as the ease with which dried formulations can be stored, even without lyoprotection. These data indicate that the RALA could be lyophilised, stored and reconstituted prior to administration without losing activity.
As RALA has proven highly effective in vitro, the next logical step would be to test its transfection efficacy and distribution and most importantly, bio-compatability in vivo. As such, ZR-75-1 tumour bearing BALB/C-SCID mice were injected intravenously with 50 μl of N:P 10 RALA/pEGFP-N1 or RALA/phOCMetLuc nanoparticles carrying a total of 10 μg of plasmid DNA per dose. Western blots showed transfection in all organs with the pEGFP-N1 carrying nanoparticles and in the tumour, surrounding tissue and liver with phOCMetLuc nanoparticles (
In order to determine whether the RALA based nanoparticles would be safe for repeated administration, immunocompetent C57/BL6 mice were treated once a week with either 50 μl of PBS, PEI, RALA, pEGFP-N1, PEI/pEGFP-N1 or RALA/pEGFP-N1 for 3 weeks. In each instance the dose of plasmid DNA delivered was 10 μg. Blood was collected via cardiac puncture and ELISA's were performed for IgGs, IgMs, TNFα, IL6 and IL1β, alongside a Greiss test for increased nitric oxide concentrations. No morbidity or visible immune response was seen upon inspection of the live animals. ELISAs for interleukins yielded no statistically significant differences between groups of treatments (
Furthermore multiple injections of the RALA nanoparticles did not evoke neutralising antibodies that would prevent RALA from delivering its payload. FACS analysis of PC3 and ZR-75-1 cells indicated that transfection of both cell types was hampered by the presence of 10% serum, but this occurred with the FBS controls as well eliminating the activation of an immune response (
Transfection of PC-3 and MDA-MB-231 with plasmid iNOS constructs complexed with RALA evoked nitric oxide production (as determined by total nitrate content of growth media—an indirect method of nitric oxide quantification). PC-3s and MDA MB-231s transfected with the inducible hOC/iNOS plasmid produced significantly more nitrates than were present in control (P=0.038 and 0.048 respectively), and those transfected with the constitutively active CMV/iNOS also produced levels of nitrates considerably higher than seen in control. Nitrate content of media of cells transfected with green fluorescent protein constructs under the control of the same promoters were consistent with control (
Transfection of PC-3s with hOC/iNOS complexed with RALA prior to clonogenic assay resulted in significantly lower clonogenic survival compared to control (P=0.004). Transfection of the same cells with CMV/iNOS resulted in a roughly similar loss of clonogenic survival (0.69±0.08 vs 0.61±0.03), while transfection with CMV/GFP did not affect clonogenic survival of PC-3s (surviving fraction of 1.01±0.11) (
Metastatic deposits were established in female BALB/c SCID mice by inoculation with 2×105 MDA-MB-231-D3H1 that express luciferase via the left ventricle of the heart. Metastatic development was monitored routinely by IVIS imaging of bioluminescence (
To confirm that Runx2 protein expression could successfully be knocked down using the RALA, PC-3 prostate cancer cells were transfected and the cell lysate collected for Western blotting. Two types of Runx2 siRNA were used as well as a non-targeting scrambled siRNA. Furthermore, Oligofectamine was used as a positive control for comparison. Initially the concentration of siRNA required to achieve knockdown was assessed followed by the optimal incubation time post-transfection. Densitometry of the Western blots using Image J software enabled the degree of knockdown of protein expression to be quantified by assuming the scrambled control siRNA results in 0% knockdown.
RALA peptide was able to achieve comparable levels of knockdown to the commercial RNA transfection reagent, Oligofectamine. Analysis of the transfection profile of RALA and Oligofectamine using fluorescent siRNA showed a peak in transfection immediately after transfection with RALA but it took 24 h to reach a peak with Oligofectamine.
To determine the effects of Runx2 knockdown on prostate cancer cell proliferation, PC-3 prostate cancer cells were transfected with 100 nM Runx2_1, Runx2_2 or non-targeting scrambled siRNA using RALA or Oligofectamine as a positive control. Where RALA was used nanoparticles were prepared at N:P 12 and Oligofectamine was used as per the manufacturer's guidelines. Cells were trypsinised and counted using a haemocytometer at 24, 48 and 72 h following the 4 h transfection. Untreated cells were assumed to have 100% viability and the percentage viability for all other treatments was based on this.
Cell viability was significantly lower with Runx2_1 compared to Runx2_2 24 h following transfection with RALA peptide (p=0.0376). However, no significant difference between the two siRNAs is seen at any other timepoint or following delivery using Oligofectamine (p>0.05) as determined by two-way ANOVA. Furthermore, there is no significant difference in cell viability following transfection of Runx2_1 and Runx2_2 across the timepoints studied up to 72 h (p>0.05) when determined by two-way ANOVA. RALA/Runx2_1 siRNA nanoparticles resulted in a significant reduction in cell viability when compared to RALA/scrambled siRNA nanoparticles at each of the 24, 48 and 72 h timepoints evaluated (p<0.001, 0.05 and 0.01 respectively). Similar results were found with RALA/Runx2 siRNA nanoparticles (p<0.01, 0.01 and 0.001 respectively). These results were consistent with the positive control, Oligofectamine, which also resulted in a significant decrease in cell viability compared to the scrambled control with Runx2_1 (p<0.001, 0.01 and 0.001 at 24, 48 and 72 h respectively) and Runx2_2 (p<0.01, 0.05 and 0.01 at 24, 48 and 72 h respectively). Overall, knockdown of Runx2 protein expression results in a reduction in cell viability of approximately 30% over 72 h (
Tumours were grown on the rear dorsum of BALB-C SCID mice until the volume reached approximately 150 mm3 before intratumoural treatment with either RALA/Runx2 siRNA nanoparticles, Runx2 siRNA only or RALA/scrambled siRNA nanoparticles commenced. Runx2_1 and Runx2_2 siRNA were pooled for the purposes of in vivo analysis as neither was found to be significantly better in achieving Runx2 knockdown. Dosing was once weekly until tumour quadrupling defined the endpoint of the experiment. Control tumours grew rapidly with all tumours quadrupling in volume within 16 days of the start of treatment (average 15 days). RALA/scrambled siRNA nanoparticle treatment mice follow a similar rate of growth as the untreated. The rate of growth is also similar for Runx2 siRNA treated mice until after the second treatment; following this the tumours grow at a slower rate than the untreated and RALA/scrambled siRNA groups. In mice treated with RALA/Runx2 siRNA nanoparticles, tumours grow at a slower rate than all other groups until the point of tumour volume quadrupling (
In order to assess the effectiveness of RALA as a delivery agent for optimisation of the antitumour effects of BPs, PC-3 prostate cancer cells were either treated with free BP or transfected with RALA/BP nanoparticles at a range of concentrations for 6 h and then incubated for 72 h before evaluating cell viability. Cell viability was analysed by cell counting using a haemocytometer. EC50 values were determined using the dose-response curves generated from this cell viability data. The EC50 of alendronate was reduced from 100.3 μM to 17.6 μM when delivered in a RALA nanoparticle, a potentiation factor of 5.7 (
Tumours were grown on the rear dorsum of BALB-C SCID mice until the volume reached approximately 100 mm3 before intratumoural treatment with RALA/alendronate, alendronate or RALA commenced. Dosing was thrice weekly until tumour quadrupling defined the endpoint of the experiment. It can be seen clearly that RALA only had no significant effect on tumour growth (p=0.0792) while alendronate and RALA/alendronate show high statistical significance when compared to the untreated control (p<0.0001 and p=0.0004 respectively) (
RAT was synthesized (
A serum incubation study was used to determine if RAT/pEGFP-N1 nanoparticles were stable over a 6 h time period with and without the presence of foetal calf serum (
The specificity of the RAT peptide was assessed using a targeting inhibition study (
TEM also confirmed the presence of the RALA nanoparticles inside the composite nanoparticles (
In summary, the results presented show that RALA is efficient, stable, safe and a viable delivery vehicle for iNOS DNA, RUNX2 siRNA and bisphosphonate anti-cancer therapeutics.
The physical properties of the RALA/pEGFP-N1 nanoparticles have been analysed and their efficacy as a transfection agent demonstrated both in vitro and in vivo. RALA was found to form stable complexes with pEGFP-N1 and facilitate the transfection of ZR-75-1 cells. Gel retardations show that complexes are formed at N:P ratios as low N:P 1, but full complexation is not seen until N:P 4, which is comparable with KALA and ppTG peptides [Rittner et al. 2002]. The RALA/pEGFP-N1 complexes cannot be defined as nanoparticles until N:P 4, as their size at N:P ratios 2 and 3 was in the micrometer range. At ratios of N:P 4 and above, RALA forms nanoparticles with pEGFP-N1 with a positive charge of 30 mV. This is in agreement with the counter-ion condensation theory, which states that particle sizes of charged complexes should be lower than those of uncharged particles, as electrostatic repulsion should prevent aggregation [de Smedt et al. 2000, Bagwe et al. 2006].
Given that at the N:P ratios which yield the highest transfection efficacy, the particles have a positive surface charge and a mean diameter below 100 nm, it is possible that they bind to the negatively charged cell surface proteoglycans non-specifically and are subsequently taken up into the endosomes.
With respect to transfection efficiency, the use of arginine in the RALA peptide has two distinct advantages; firstly arginine has consistently been shown to be the optimal amino acid for condensing DNA with arginine rich sequences binding in milliseconds (Murray et al 2001). Secondly arginine rich sequences based on the Rev sequence have the capacity to actively transport DNA into the nucleus of cells via the importin pathway (Malim et al 1989). This gives RALA a distinct advantage over conventional peptide delivery systems.
We have also shown that the RALA/pEGFP-N1 nanoparticles are not strongly cytotoxic, causing only a 20% reduction in cell viability in transfected cell monolayers. Perhaps the most important result is the confirmation of in vivo activity of the nanoparticles following systemic administration. High levels of delivery to the lungs were seen when a plasmid expressing luciferase was delivered to mice using the ppTG-1 peptide, but the liver was not examined [Rittner et al. 2002]. When fluorescently labelled siRNA was delivered with the MPG-8 peptide, it was observed in the majority of organs with high levels in the lungs and liver [Crombez et al. 2009]. No morbidity or mortality of animals was observed following treatment in the experiments described in this work, although this has not always been the case with peptide based gene delivery agents (Rittner et al. (2002) reported the death of several mice when delivering the plasmid systemically with the ppTG1 peptide.
In addition, RALA does not appear to cause a significant immune response upon repeated administration beyond the inflammation associated with tissue damage caused by the needle at the site of injection. There is also no neutralization of RALA following repeated administration. Furthermore, RALA appears to shield naked DNA from generating an adaptive immune response and does not cause an antibody response on its own. This is an encouraging result given that peptides are often used as vaccines because they share homology with viral and tumour proteins and produce a high antigenic response [Yang et al. 2009, Rodriguez and Grubman 2009]. As such, it might be expected that RALA, a peptide that is analogous to viral fusion proteins, might likewise be highly immunogenic. It appears, that as RALA uses a simple highly repetitive, artificially designed sequence that is not common in nature, its immunogenicity is low.
Part of the effectiveness of RALA as a transfection agent is probably related to its ability to protect DNA or siRNA from a hostile environments. The complexation of RALA to plasmid DNA forms nanoparticles that protect DNA from, freeze-drying and degradation in serum. While the ability to protect the cargo from degradation by serum has a bearing on transfection efficacy, the ability to act as a lyoprotectant has implications for further formulation related issues that surround transfection agents. The logistics behind supplying gene medicine to clinics are complicated by the lack of stability of most prospective vectors. Since viral vectors are notoriously difficult to store and non-viral vectors usually require lyoprotectants, which alter the final formulation, before they can be successfully freeze-dried, it is promising to see that RALA/pEGFP-N1 nanoparticles retain activity following reconstitution after lyophylisation.
RALA has also been shown to successfully condense and form nanoparticles with a range of bisphosphonates, siRNA and is an excellent tool for local delivery. It has also been used for the systemic delivery of the iNOS therapeutic to metastatic deposits of cancer with an excellent response. This indicates a wide range of applications for this peptide delivery system.
The following peptide sequences based on RALA (WEARLARALARALARHLARALARALRACEA) were also prepared using conventional commercial techniques as expanded on in Example 1.
The table below shows the key characteristics of RALA (WEARLARALARALARHLARALARALRACEA) derivative Peptides in ZR-75-1 breast cancer cells.
The results in terms of transfection efficiency in ZR-75-1 cells are shown above. Peptides 1-5 successfully condensed the DNA into nanoparticles less than 100 nm. The exception being peptide 6, where the smallest nanoparticle measured was 308 nm. It can also be deduced that the highest transfection efficiency was with peptide 2 at 55% and as the hydrophilic ratios increase up to 40% the surface charge of the nanoparticle decreases. Furthermore the addition of glutamic residues reduces transfection efficiency as evidenced by peptide 3 and peptide 6. Nevertheless all sequences have potential as delivery vehicles for nucleic acids and hydrophilic compounds.
A 22mer WEARLARALARALARHLRACEA was also tested but was unable to condense DNA into nanoparticles and transfect cells and was therefor deemed unsuccessful.
Aqueous 30% stock solution of Gantrez® AN-139 poly(methylvinylether/maleic acid), (PMVE/MA) was prepared using 30 g of poly(methylvinylether/maleic anhydride), (PMVE/MAH) (ISP Corp. Ltd., Guildford, UK) which was added to 70 mL ice-cooled water and stirred vigorously to ensure complete wetting and prevention of aggregation. The mixture was then heated and maintained between 95° C. and 100° C. until a clear solution was formed. Upon cooling, the blend was then readjusted to the final concentration of 30% w/w by addition of an appropriate amount of deionised water.
Aqueous 30% stock solution of PVA (Polyvinyl alcohol), (Sigma, UK), was prepared using 30 g of PVA which was added to 70 mL ice-cooled water and stirred vigorously to ensure complete wetting and prevention of aggregation. The mixture was then heated and maintained between 95° C. and 100° C. until a clear solution was formed. Upon cooling, the blend was then readjusted to the final concentration of 30% w/w by addition of an appropriate amount of deionised water.
Aqueous 40% stock solution of PVP (Polyvinylpyrrolidone), (Sigma, UK), was prepared using 40 g of PVP which was added to ice-cooled water and stirred vigorously to ensure complete wetting and prevention of aggregation. The mixture was then heated and maintained between 95° C. and 100° C. until a clear solution was formed. Upon cooling, the blend was then readjusted to the final concentration of 40% w/w by addition of an appropriate amount of deionised water.
0.5 g of polymer gel was poured into a silicon mould. To ensure the polymer matrix reached the tips of the MN mould, the moulds were centrifuged at 3000 rpm for 15 min. Following centrifugation, the arrays were dried at room temperature for 48 h. Upon hardening, the arrays were released from the mould by carefully peeling it away. Arrays are shown in
Agarose Gel Analysis of Nanoparticle Release from Polymeric Solutions
RALA/pEGFP-N1 complexes at N:P ratio 10 were prepared at room temperature and incubated at room temperature for 30 min. Following this incubation 50 mg of polymeric stock solution was added to the complexes and incubated at room temperature for 30 min. Subsequently, SDS (Sigma, UK) was added (10%) to the eppendorfs to decomplex DNA from the peptide. Following incubation, 30 μL of the samples (corresponding to 0.6 μg of DNA) were electrophoresed through a 1% agarose gel containing 0.5 μg/mL EtBr to visualize DNA mobility. A current of 80 V was applied for 1 h and the gel imaged using a Multispectrum Bioimaging System (UVP, UK). This experiment was repeated with proteinase K as the NP lysing agent. Results illustrated in
Standard Curve for the Determination of DNA Concentration Following Release from RALA/DNA NPs
RALA/pEGFP-N1 NPs were analysed through fluorescence detection with Quant-iT™ Picogreen® Reagent (Invitrogen, UK). Quant-iT™ Picogreen® Reagent is a fluorescent nucleic acid stain for quantitating double-stranded DNA in solution. Upon addition to the solution the reagent binds to the double stranded DNA and it's fluorescence intensity increases several hundred fold, the fluorescence intensity of the resulting Picogreen/DNA complex is directly proportional to the amount of DNA in the sample.
For determination of DNA detection using this method following release from RALA/pEGFP-N1 NPs a representative standard curve was used. A solution of RALA/pEGFP-N1 NPs were made and subsequently diluted with Tris 10 mM to produce a range of NP solutions of known concentrations. 50 μL of these solutions were then pipetted into a 96-well plate and 50 μL of 0.1 mg/mL Proteinase K (Sigma, UK) subsequently added and samples incubated at 37° C. for 30 min. Quant-iT™ Picogreen® Reagent was then added to the samples and the samples analysed by excitation at 480 nm and the fluorescence emission intensity measured at 520 nm using a Synergy 2 Multi-Mode Microplate Reader (BioTek Instruments Inc, UK). Results are shown in
Quantification of NP Release from Polymer Matrices
RALA/pEGFP-N1 N:P 10 NPs containing 1 μg DNA were incorporated into the stock solutions of the polymeric matrices to form 20% polymeric solutions. These NP/polymer mixtures were incubated at room temperature for 1 h and subsequently dissolved in 1 mL Tris buffer (10 mM) for 1 h. 50 μL samples of these solutions were then pipetted into a 96-well plate and 50 μL of 0.1 mg/mL Proteinase K (Sigma, UK) subsequently added and samples incubated at 37° C. for 30 min. Quant-iT™ Picogreen® Reagent was then added to the samples and the samples analysed using a Synergy 2 Multi-Mode Microplate Reader (BioTek Instruments Inc, UK). Results are shown in
Determination of pDNA Secondary Structure in the Presence of PMVE/MA by Circular Dichroism
To examine the secondary structure of pDNA after incorporation into PMVE/MA samples of PMVE/MA only, pLux only and PMVE/MA-pLux were made. The samples were then dissolved in 2 mL PBS so the final concentration of pLux in solution was 50 μg/mL. CD spectra were obtained with Jasco J-185 spectopolarimeter equipped with a temperature controller. CD spectra were collected at 20° C. using a 1 cm quartz cell over the wavelength range of 240-350 nm. Results are shown in
NCTC-929 fibroblast cells were seeded at a density of 30,000 cells per well onto 96-well tissue culture plates (VWR, UK) for 24 h prior to the assay. Media was then supplemented with 0, 5, 10 or 20 mg/mL of either 20% PVA, 20% PVP or 20% PMVE/MA and incubated for 6 h under standard cell culture. Following this incubation 10% WST-1 reagent (Roche, UK) was added to the cell media and the cells were incubated for a further 2 h. Subsequently the plates were shaken for 1 min and absorbance measured at 450 nm on an EL808 96-well plate reader (BioTek Instruments Inc, UK). The measured absorbance values are expressed as a percentage of the control (untreated cells) where the control is defined as 100% viable. Results are shown in
To determine the axial forces (i.e. the force applied parallel to the MN vertical axis) necessary for mechanical fracture of the MNs, the TA-XT2 Texture Analyser (Stable Microsystems, U.K) was employed. MN arrays of 3×3 MNs were used. The arrays were attached to the moveable cylindrical probe of the Texture Analyser using double-sided adhesive tape. An axial compression load was applied to the MN arrays to deduce the changes that occur to the structure of the MNs upon force application. The test station pressed the MN arrays against a flat aluminium block of dimensions 9.2×5.2 mm at a rate 0.5 mm per sec with defined forces of 0.05, 0.1, 0.2, 0.3 and 0.4 N/needle for 30 s. Before and after fracture testing, 3 MNs of each array were examined by a digital microscope (GE-5 USB Digital Microscope) under magnification 180× to determine the height of the MNs after testing. The MN height was measured using the ruler function of the microscope software so the percentage reduction in the MN height could be calculated. Results shown in
RALA/pEGFP-N1 loaded MNs were prepared using the micromoulding process, MNs manufactured from aqueous blends of 20% PVP encapsulating RALA/pEGFP-N1 NPs were prepared by diluting the 40% stock solution 50:50 with NP solution. 0.2 g of the polymeric gel containing the RALA/pEGFP-N1 NPs was weighed into the moulds and centrifuged at 3000 rpm for 10 min to ensure the MN cavities were filled. A further 0.3 g of 20% PVP polymer was added to the moulds to form the baseplate to which the microneedles are attached and centrifuged again at 3000 rpm for 10 min. The arrays were left to dry at room temperature and after 48 h, were manually released from the moulds and the polymeric side walls removed using a heated scalpel. Each MN array was either composed of 9 (3×3) or 361 (19×19) needles perpendicular to the baseplate depending on the mould used for fabrication. The MNs were of conical shape, 600 μm high with base width of 300 μm and 300 μm interspacing.
RALA/pEGFP-N1 NP loaded 20% PVP microneedle arrays were fabricated and mounted onto metal stubs with double sided carbon tape and sputter coated with gold and allowed to dry overnight. Arrays were visualised using a Jeol JSM-840A scanning microscope (Jeol, UK). Images shown in
20% PVP polymer loaded with RALA/pEGFP-N1 N:P 10 NPs containing 1 μg DNA were prepared and incubated at (A) 20° C., 46% relative humidity (RH) for 0, 1, 5, 3 or 7 days. At each time point the polymeric formulations were dissolved in 50 μL distilled water (Gibco, UK) and separated into 25 μL samples, to which 10% SDS was added to one sample to decomplex the NPs present in solution. 30 μL samples of each solution were then electrophoresed through a 1% agarose gel containing 0.5 μg/mL ethidium bromide (EtBr) (Sigma, UK) to visualize DNA. A current of 80 V was applied for 1 h and the gel imaged using a Multispectrum Bioimaging System (UVP, UK). Results are shown in
Determination of Functionality of RALA/pEGFP-N1 N:P Ratio 10 NPs Encapsulated within 20% PVP Matrix Up to 7 Days
20% PVP polymers loaded with either pEGFP-N1 or RALA/pEGFP-N1 N:P 10 NPs containing 1 μg DNA were prepared and incubated at room temperature for 1 h and 7 days. Following these incubations the polymeric formulations were dissolved in 200 μL PBS for 1 h.
NCTC-929 cells were prepared for transfection by seeding at a density of 30,000 cells per well onto 96-well tissue culture plates (VWR, UK) for 24 h prior to transfection. Cells were conditioned for 2 h in Opti-MEM serum free media (Gibco, UK) which was then supplemented with 100 μL of polymer/NP solution. Following incubation for 6 h the media was removed and replaced with serum supplemented culture media. Cells were imaged using the Nikon Eclipse TE300 inverted microscope with epifluorescence attachment (Nikon, USA) and images captured using a Nikon DXM1200 digital camera (Nikon, USA) using a ×200 magnification 24 h post transfection. Images are displayed in
MNs were manufactured from aqueous blends of 20% w/w PVP encapsulating pDNA and RALA/pDNA NPs were prepared by diluting the stock solution of 40% PVP 50:50 with the appropriate amount of pDNA/NP solution. When fabricating MNs loaded with concentrated RALA/pDNA NPs the RALA and pDNA were combined initially and incubated at room temperature for 30 min before incorporation into the PVP matrix.
25 mg of the polymeric solution containing the DNA or NPs was weighed into the moulds and centrifuged at 3000 rpm for 5 mins, this was repeated twice to ensure the microneedle cavities were filled. A further 0.5 g of 20% PVP solution was added to the moulds to form the baseplate to which the microneedles are attached and centrifuged again at 3000 rpm for 10 min. The arrays were left to dry at room temperature and after 48 h, were manually released from the moulds and the polymeric side walls removed using a heated scalpel. Each MN array was composed of 361 (19×19) needles perpendicular to the baseplate.
Quantification of RALA/pEGFP-N1 N:P Ratio 10 NPs Encapsulated within the MN Array
In order to determine the quantity of DNA present in the MNs themselves the MNs were fabricated containing RALA/pEGFP-N1 NPs. The needles were sheared from the baseplate and both components of the array dissolved in 4 mL 10 mM Tris buffer for 1 h. 50 μL samples of these solutions were then pipetted into a 96-well plate and 50 μL of 0.1 mg/mL Proteinase K (Sigma, UK) subsequently added and samples incubated at 37° C. for 30 min. Quant-iT™ Picogreen® Reagent was then added to the samples and the samples analysed. Results are detailed in Table 1.
Evaluation of RALA/pEGFP-N1 Nanoparticle Release from 20% PVP MNs Across Neonatal Porcine Skin
Neonatal porcine skin was obtained from stillborn piglets and immediately (<24 hours after birth) excised, trimmed to a thickness of 300±50 μm using dermatome and frozen in liquid nitrogen vapour. Skin was then stored in aluminium foil at −20° C. until further use. Shaved skin samples were mounted on the receptor compartment with stratum corneum (SC) side of the skin exposed to ambient conditions and dermal side in contact with the release medium. 20% PVP MN arrays containing concentrated RALA/pDNA NPs were pressed into the porcine skin using a syringe plunger to ensure insertion of the MNs into the SC. Samples were withdrawn from the receptor compartment at pre-determined time intervals and the volume taken was replaced by the same volume of fresh receptor medium to maintain constant conditions. 50 μL samples of these solutions were then pipetted into a 96-well plate and 50 μL of 0.1 mg/mL Proteinase K (Sigma, UK) subsequently added and samples incubated at 37° C. for 30 min. Quant-iT™ Picogreen® Reagent was then added to the samples and the samples analysed. Results are shown in
Optical Coherence Tomographic Assessment of MN Penetration into Full Thickness Neonatal Porcine Skin
Optical coherence tomography (OCT) was used to determine the penetration characteristics of 19×19 20% PVP MN arrays loaded with RALA/μLux NPs following insertion into excised full thickness neonatal porcine skin using either spring-activated applicator or manually using gentle thumb pressure. Neonatal full thickness porcine skin was prepared and equilibrated in PBS for 30 min at 37° C. to restore conditions resembling the in vivo state. The skin was then placed onto a sheet of dental wax for support with the SC side facing towards the environment. MN arrays were inserted into the skin using an applicator, at forces of 8 N, 11 N and 16 N. To use the applicator, firstly the required spring was loaded into the piston shaft. The flat base of the piston was then pushed up towards the piston shaft until it locked into place. This applicator could then be activated by simply pressing a release button, which drives the piston towards the target surface. This process was repeated three times to ensure proper MNs insertion into the skin. The skin was immediately viewed using the OCT scanner and images were analysed using Image J software.
To investigate the insertion of the MNs using gentle thump pressure, the skin was prepared as described previously and the MN array inserted into the full thickness porcine skin by applying gentle thumb pressure against the array for 30 sec. The skin was immediately viewed using OCT Scanner and images were analysed using Image J software. Results are shown in
Confocal Microscopy of Murine Ears Following Application of MN Arrays Containing Concentrated Cy-3 Labelled pOVA and Cy-3 Labelled RALA/pOVA NPs
pOVA was fluorescently labelled with the Cy-3 fluorophore (Mirus, USA) according to the manufacturer's instructions. Briefly, 100 μg pOVA was labelled with 20 μL Cy-3 Label IT Reagent at 37° C. for 1 h. The labelled DNA was then concentrated using ethanol precipitation to produce Cy-3 labelled pOVA at a concentration of 5 μg/μL. This DNA was then used to form to form 20% PVP MN arrays containing either Cy-3 pOVA or Cy-3 RALA/pOVA NPs.
25 mg of the polymeric solution containing the Cy-3 labelled pOVA or RALA/pOVA NPs was weighed into the moulds and centrifuged at 3000 rpm for 5 mins, this was repeated twice to ensure the microneedle cavities were filled. A further 0.5 g of 20% PVP solution was added to the moulds to form the baseplate to which the microneedles are attached and centrifuged again at 3000 rpm for 10 min. The arrays were left to dry at room temperature and after 48 h, were manually released from the moulds and the polymeric side walls removed using a heated scalpel. Each MN array was composed of 361 (19×19) needles perpendicular to the baseplate.
The MNs were applied to the mouse ear for 1 h, then the animals were sacrificed. Following harvesting of ear tissue from sacrificed animals the tissue was stored in 4% formaldehyde solution overnight. Ear tissue was then mounted into a microscope slide (VWR, UK) using 100% glycerol (Sigma, UK) and imaged using a TSC SP5-Leica Microsystems confocal microscope (Leica, UK). Images were analysed using LAS AF Lite Software (Leica, UK). Images shown in
Prior to application of the MN arrays the mice were anaesthetized via intraperitoneal (i.p.) injection of Rompun and Ketaset. The dorsal ear skin of the mice was wetted with 10 μL of water and the MN arrays manually inserted by holding in place for 5 min into both ears of each animal. In order to keep the MN arrays in place micropore tape was used to secure the arrays to the ear tissue. MN arrays were removed 24 h following application.
Following harvesting of the organs from sacrificed animals they were subsequently placed in a 6-well plate (VWR, UK) bathed in D-Luciferin Potassium Salt (PerkinElmer, UK) in PBS (15 mg/mL) for 10 min. The organs were then transferred to a 24-well plate (VWR, UK) and imaged using the Xenogen IVIS 200 Imaging System (PerkinElmer, UK). Images were analysed using Living Image® 3.2 software (Leica, UK). Results shown in
Flow Cytometric Analysis of Harvested Auricular Lymph Nodes Following Immunisation with RALA/pOVA NPs Via MN Application
Animals were sacrificed 10 days post immunization and the auricular lymph nodes were harvested and collected into small petri dishes (VWR, UK) with 1 ml of RPMI media (Gibco, UK) and manually dissociated by compression through a nylon membrane. The resulting cell suspensions were centrifuged at 500 g for 5 mins and the cell pellet resuspended in 1 mL PBS. The cells were then stained with SIINFEKL/H-2 Kb pentamers conjugated to APC for 20 mins in accordance with the manufacturers instructions (Pro-Immune Limited, UK). The cells were then stained using fluorochrome-conjugated antibodies for CD8 and B220 (BD Biosciences and eBioscience, UK) to determine the T and B-cell populations respectively. Data was collected on FACS Canto II (BD Biosciences) and analyzed using FlowJo software (Tree Star). Results are shown in
Microneedles loaded with RALA/HPV-16 E6 or RALA/HPV-16 E7 N:P 10 NPs containing 1 μg DNA were prepared and left 48 h to dry. Microneedles were incubated either at 4° C., 35% relative humidity (RH) or 20° C., 40% RH, or 20° C., 86% RH for 7, 14, or 21 days. At each time point microneedles were dissolved in 500 μL distilled water (Gibco, UK) and separated into 250 μL samples, to which proteinase K (0.5 mg/mL) was added (10%) to one sample to decomplex the NPs present in solution. 30 μL samples of each solution were then electrophoresed through a 1% agarose gel containing 0.5 μg/mL ethidium bromide (EtBr) (Sigma, UK) to visualize DNA. A current of 80 V was applied for 1 h and the gel imaged using a Multispectrum Bioimaging System (UVP, UK). Results are shown in
Determination of Functionality of RALA/HPV-16 E6, RALA/HPV-16 E7, and RALA/HPV-16 E6/E7 N:P Ratio 10 NPs Encapsulated within 20% PVP Matrix
20% PVP polymers loaded with either HPV-16 E6, HPV-16 E7, HPV-16 E6/E7, RALA/HPV-16 E6, RALA/HPV-16 E7 or RALA/HPV-16 E6/E7 NPs containing 1 μg DNA were prepared and incubated at room temperature for 48 h to dry. Following incubation, microneedles were dissolved in 200 μL PBS for 1 h. NCTC-929 cells were prepared for transfection by seeding at a density of 30,000 cells per well onto 96-well tissue culture plates (VWR, UK) for 24 h prior to transfection. Cells were conditioned for 2 h in Opti-MEM serum free media (Gibco, UK) which was then supplemented with 100 μL of polymer/NP solution. Following incubation for 6 h the media was removed and replaced with serum supplemented culture media. After 24 h, cells were harvested with Laemelli buffer (Sigma, UK). Cell lysates were electrophoreised through a SDS-PAGE gel and transferred onto nitro-cellulose membrane according to standard procedures. The membrane was probed with both HPV-16 E6 and E7 antibodies and developed using chemiluminesce kit (Millipore, UK) according to the manufacturer's instructions. Results shown in
MNs loaded with pDNA encoding the tdTomato fluorophore were applied to a hairless area of skin on the dorsum of C57BL/6 mice for 24 h as described previously. 4 days post MN application the animals were sacrificed and the draining lymph nodes harvested and enzymatic degradation performed with Collagenase, Type IV (Gibco, Cat no: 17104-019). Using sharp scissors the lymph nodes were cut for 10 min until completely liquefied. Using RPMI media (5 ml) the cells were washed to the bottom of a 15 ml falcon tube and warmed to 37° C. 170 μl of collagenase (30 mg/ml) was added to the 5 ml and the cells pipetted vigorously for 20 mins, another 170 μl of collagenase was added and pipetting continued for another 10 mins. The cell suspension was then filtered through a 100 μm mesh filter into a clean 15 ml falcon tube. The tube and mesh were then rinsed with another 2 ml RPMI and centrifuged at 600 rpm, 4° C. for 10 min. The cells were then resuspended in 1 ml PBS and transferred to flow tubes. The falcon was rinsed with a further 1 ml PBS which was also transferred to the corresponding flow tubes followed centrifuged again. The cells were then stained in a two-step process. Step 1: MHC class-II stain i.e. 1 ml PBS, 1 μl MHC class-II biotin antibody (eBioscience, Cat no: 13-5321-82) and 20 μl MHC class-II antibody (BD Pharmingen, Cat no: 556999) for 20 mins on ice followed by step 2: CD11c and Streptavidin mix i.e. 1 ml PBS, 2 μl CD11c antibody (eBioscience, cat no: 51-0114-82) and 1 μl Streptavidin-PEcy7 antibody (eBioscience, cat no: 25-4317-82) and incubated on ice for a further 20 mins. The cells were resuspended in 200 μl PBS and analysed by flow analysis on the FACS Canto II and using FlowJo software (
Determination of Circulating HPV-16 E6/E7 IgG Antibody Levels Generated Following Immunisation with Plasmid DNA Expressing HPV-16 E6/E7 Antigens
C57BL/6 mice (n=4) were immunized 3 times, at fortnightly intervals. Each immunization involved delivering 50 μg plasmid DNA encoding HPV-16 E6/E7 antigens±g pla via i.m. and MN delivery. Circulating levels of HPV-16 E6/E7 IgG antibodies were determined by ELISA analysis of serum collected 10 days post 2nd and 3rd immunisations. 96-microwell plate was coated with 100 μl (0.5 mg/ml) HPV-16 E7/E6 peptides incubated at 4° C. overnight. The wells were then blocked with PBS containing 20% fetal bovine serum and incubated at 4° C. for 16 h. Serum samples diluted in PBS (1:100) were added and incubated at 37° C. for 2 h. The plate is incubated with a 1:2000 dilution of a goat antimouse IgG HRP-conjugated antibody at room temperature for 1 h. Subsequently an enzyme substrate (OPD, Sigma) was added for colour development. Immunoreactivity is detected with an ELISA plate reader at a wavelength of 450 nm. Quantification IgG was performed using Easy titer IgG assay kit (Thermo scientific, UK) (
Determination of Generation of HPV-16 E6/E7-Specific Cytotoxic T Cells Following Immunisation with Plasmid DNA Expressing HPV-16 E6/E7 Antigens
Spleens were harvested from immunised C57BL/6 mice 10 days post 3rd immunisation. Each immunization involved delivering 50 μg plasmid DNA encoding HPV-16 E6/E7 antigens±RALA via i.m. and MN delivery. Spleens are removed aseptically, homogenised and resuspended in red blood cell (RBC) lysis buffer to remove RBCs. Following RBC lysis, isolated splenocytes from the same group are pooled and re-suspended in RPMI 1640 medium (TC-1 medium) and counted. T cells (used as the effecter cells) were co-cultured in RPMI-1640 medium containing irradiated TC-1 cells (104 per well) (used as the target cells) in 24-well plate. Media was supplemented with 20 units of interleukin-2 (Peprotech) and incubated under standard tissue culture conditions (37° C., 5% CO2) for 6 days. Dead T cells were removed by centrifugation with Percoll solution (Amersham Biosciences). Viable T cells are seeded with non-irradiated TC-1 cells in the ratios of 5:1 and 10:1 in an assay medium (1% BSA medium) in triplicates and incubated under standard tissue culture conditions (37° C., 5% CO2) for 5 h. Supernatant was harvested and cytotoxicity determined using cytotoxicity detection kit (LDH) (Roche) according to manufacturers protocol. The colour change was detected by plate reader analysis at a wavelength of 450 nm and the cytotoxicity calculated by the following equation:
“High control”=the total LDH released from the target cells, after lyzing TC-1 cells with 1% Triton X-100 in assay medium.
“Low control”=the natural release of LDH from the target cells, which is obtained by adding TC-1 cells only in the assay medium.
“T-cell control”=use to measure the natural release of LDH from T cells was obtained by adding the different ratios of T cells only in the assay medium (
Determination of Interferon-Gamma Secretion from Splenocytes Restimulated with E6/E7-Expressing TC-1 Cells Ex Vivo
Spleens are removed aseptically, homogenised and resuspended in red blood cell (RBC) lysis buffer to remove RBCs. Following RBC lysis, isolated splenocytes from the same group are pooled and re-suspended in RPMI 1640 medium (TC-1 medium) and counted. T cells (used as the effecter cells) were co-cultured in RPMI-1640 medium containing irradiated TC-1 cells (104 per well) (used as the target cells) in a ratio of 10:1, and media supplemented with 20 units of interleukin-2 (Peprotech) in 24-well plates. The cells were cultured in standard tissue culture conditions (37° C., 5% CO2) for 4 days, then media was harvested for ELISA analysis of interferon-gamma (IFN-γ) (PeproTech, Cat no: 900-K98) (
For ELISA assay, the capture antibody was diluted with PBS to a concentration of 1.0 μg/ml. and immediately added (100 μl) to each ELISA plate well. The plate was sealed and incubated overnight at room temperature. Following washing of the excess capture antibody from the wells 300 μl of blocking buffer was added to each well and Incubated for 1 h at room temperature. The harvested cell media was added to the prepared ELISA plate in triplicate and incubated at room temperature for 2 h. The detection antibody was diluted to a concentration of 0.25 μg/ml, and added 100 μl per well. Plate was incubated at room temperature for 2 h. This was followed by further washing, then Avidin Peroxidase (diluted 1:2000) was added and plate was incubated for 30 min at room temperature. ABTS substrate was added to each well, and incubated at room temperature for colour development. The plate was read using ELISA plate reader at 405 nm.
Determination of Efficacy of Prophylactic Immunisation with Plasmid DNA Expressing HPV-16 E6/E7±RALA Against Establishment of Tumour Following Implantation of TC-1 Cells In Vivo
C57BL/6 mice (n=9) were immunised 3 times, at fortnightly intervals. Each immunization involved delivering 100 μg plasmid DNA encoding HPV-16 E6/E7 antigens±RALA via i.m. and MN delivery. One week post 3rd immunisation mice were challenged with 1×105 E6/E7-expressing TC-1 cells per mouse via intradermal implantation on the dorsum The mice were monitored for evidence of tumour growth by palpation and tumour growth measured three times per week (
Determination of Efficacy of Therapeutic Immunisation with Plasmid DNA Expressing HPV-16 E6/E7±RALA Against Growth of Established TC-1 Tumour
C57BL/6 mice (n=3) were implanted subcutaneously with 1×105 E6/E7-expressing TC-1 cells per mouse, When tumour volume reached 50 mm3 the mice were immunised 3 times, at weekly intervals. Each immunization involved delivering 100 μg plasmid DNA encoding HPV-16 E6/E7 antigens±RALA via i.m. and MN delivery. The mice were monitored for evidence of tumour growth by palpation and measurement three times per week (
RALA/pHPV-16 E6/E7 (N:P ratio of 6) nanoparticles were freeze-dried using Advantage, VirTis freeze dryer and 5% w/v trehalose was used as cryoprotectant. MN arrays were formulated using 3 polymers, 360 kDa PVP, 58 kDa PVP and 9-10 kDa PVA, to contain RALA/pHPV-16 E6/E7 (N:P ratio of 6) nanoparticles encapsulating either 50 or 100 μg DNA. MN arrays were applied to the dorsal side of C57BL/6 mice ears for 5 min or 24 h followed by removal of the array and quantification of the HPV-16 E6/E7 DNA remaining in the array by Quant-iT™ PicoGreen® dsDNA quantification (Life Technologies, UK). Delivery of DNA from MNs formulated to contain 36 μg DNA (as used in previous in vivo studies) was also performed as a comparison. Following application of the MN arrays for (A) 5 min or (B) 24 h, the remaining array was removed and subsequently dissolved in 5 mL Tris buffer (10 mM) for 1 h. 50 μL samples of these solutions were then pipetted into a 96-well plate and 50 μL of 1 mg/mL Proteinase K subsequently added and samples incubated at 37° C. for 2 h. Quant-iT™ Picogreen® Reagent was then added to the samples and the samples analysed by excitation at 480 nm and the fluorescence emission intensity measured at 520 nm using a Synergy 2 Multi-Mode Microplate Reader (BioTek Instruments Inc, UK) (
Fabrication of MN Arrays from PVP and PVA Polymers of a Range of Molecular Weights Loaded with RALA/pDNA Nanoparticles
50% w/w 360 kDa PVP, 13-23 kDa PVA and 9-10 kDa PVA stock solutions were manufactured by thoroughly mixing 5 g of lyophilised polymer with 5 g of refrigerated double distilled molecular grade water (Invitrogen, UK). Stock solution was then heated to 80° C. and mixed hourly until a homogenous, clear polymeric solution was formed. 75% w/w 58 kDa PVP stock solution was produced by thoroughly mixing 7 g of PVP powder with 3 g of refrigerated double distilled molecular grade water.
20% w/w MNs (360 kDa PVP, 13-23 kDa and 9-10 kDa PVA) and 30% w/w MNs (58 kDa PVP) containing RALA/pDNA were fabricated by mixing 50% w/w (360 kDa PVP, 13-23 kDa and 9-10 kDa PVA) or 75% w/w (58 kDa PVP) polymer solutions with RALA/pDNA solution at a ratio of 2:3. 25 mg of polymer-RALA/pDNA solution was then weighed into silicon moulds and centrifuged at 4000 rpm for 10 min to ensure complete filling of MN cavities. For fabrication of 360 kDa PVP, 13-23 kDa and 9-10 kDa PVA MN arrays, following centrifugation 0.5 g of 20% w/w polymer stock was weighed onto moulds to form a baseplate attached to the RALA/pDNA-loaded MNs and moulds were centrifuged again at 4000 rpm for 10 min. MN arrays were incubated at room temperature for 48 h for solidification and then peeled carefully from the mould. For fabrication of 58 kDa PVP MN arrays, 0.2 g of 30% w/w 58 kDa PVP solution was weighed into moulds which were centrifuged again as above and left to solidify by incubation at room temperature for 24 h. Subsequent to this incubation, 0.5 g of 20% 360 kDa PVP was weighed into moulds which were centrifuged again as detailed above. MN arrays were incubated at room temperature for 48 h to solidify and then peeled carefully from the mould (
Measurement of MNs, Fabricated from PVP and PVA Polymers of a Range of Molecular Weights, Percentage Height Reduction Following Application of an Axial Force
MN arrays with 361 (19×19) needles were fabricated as detailed above, imaged and MN height measured prior to compression using a light microscope at ×35 magnification. MN arrays were then adhered to the movable probe of the TA-XT2 Texture Analyser (Stable Microsystems, UK) with double-sided sticky tape and a compression force of 45 N (0.125 N/needle) was then applied uniformly to the needles against a flat aluminium block. Following compression, MNs were re-imaged and measured using a light microscope at ×35 magnification. Percentage height reduction was calculated as the difference in MN height following compression divided by the original height×100 (
Optical Coherence Tomographic Analysis of MN Penetration Mouse Ears Following Fabrication from PVP and PVA Polymers of a Range of Molecular Weights
20% w/w 360 kDa PVP, 13-23 kDa PVA, 9-10 kDa PVA and 30% w/w 58 kDa PVP MN arrays with 19×19 needles were fabricated as previously described. Murine ears were equilibrated in PBS for 30 min at 37° C. prior to insertion of MNs. Following equilibration the skin was placed on a sheet of dental wax with the epidermis facing externally. MNs were pressed into skin using the movable probe of the TA-XT2 Texture Analyser applying a range of forces (Stable Microsystems, UK) (10 N, 20 N, 30 N and 40 N). Following application of MNs, the skin was analysed using the optical coherence tomography (OCT) scanner. Images were then analysed and MN penetration depth measured using Image J software (
Fibroblast NCTC-929 and dendritic DC 2.4 cell lines were seeded in a 96-well plate at densities of 10,000 and 17,500 cells/well respectively. Cells were left to adhere overnight and the following day media was supplemented with polymer at concentrations of 0-40 mg/mL. Following 24 h incubation under standard tissue culture conditions, 10% MTS reagent (CellTiter 96 AQeous One Solution Reagent, Promega, UK) was added per well and cells were incubated for a further 2 h. Subsequently absorbance at 490 nm was measured using a EL808 96-well plate reader (Biotek Instruments Inc, UK). Measured absorbance values are expressed as a percentage of the absorbance of untreated control cells, where the control represents 100% viability (
Quantification of RALA/pDNA Release from PVP and PVA Polymers of a Range of Molecular Weights
250 mg 20% w/w polymeric gels, 20% 360 kDa PVP, 20% 13-23 kDa PVA, 20% 9-10 kDa PVA and 30% w/w 58 kDa PVP loaded with 10 μg pDNA were fabricated by mixing 50% w/w (360 kDa PVP, 13-23 kDa and 9-10 kDa PVA) or 75% w/w (58 kDa PVP) polymer solutions with RALA/pDNA solution [N:P ratios (0-10)] at a ratio of 2:3. Following solidification of gels by incubation at room temperature for 48 h, gels were dissolved in 10 mM Tris buffer pH 8.0 for 1 h with stirring. Following dissolution, 50 μL samples of solution were transferred in triplicate to wells in a black 96 well plate. Samples were then incubated with 50 μL 0.2 mg/ml Proteinase K for 120 min at 37° C. Samples were then incubated at room temperature for 30 min with Quanti-iT Picogreen™ reagent and fluorescent emission at 520 nm was quantified using a Synergy 2 Multi-Mode Microplate Reader (BioTek Instruments Inc, UK) following excitation at 480 nm. The fluorescence of samples was used to determine concentration of pDNA in the solution using a standard curve as detailed previously and total pDNA release was subsequently calculated (
Quantification of RALA/pDNA Release from MNs Fabricated from PVP and PVA Polymers of a Range of Molecular Weights
20% w/w 19×19 MNs incorporating RALA/pDNA were fabricated as previously described. Arrays were produced with solutions of RALA/pDNA N:P ratio 10 encapsulating 32 μg pDNA. The sidewalls were removed from arrays using a heated scalpel. Sidewalls and the baseplate and needles were then dissolved in 10 mM Tris buffer pH 8.0 for 1 h with stirring. Following dissolution, 50 μL samples were transferred in triplicate to wells in a black 96 well plate. Samples were then incubated with 50 μL 0.2 mg/ml Proteinase K for 120 min at 37° C. Samples were further incubated at room temperature for 30 min with Quanti-iT Picogreen™ reagent and fluorescent emission at 520 nm was quantified using a Synergy 2 Multi-Mode Microplate Reader (BioTek Instruments Inc, UK) following excitation at 480 nm. The fluorescence of samples was used to determine concentration of DNA in the solution using a standard curve as detailed previously and total DNA release was subsequently calculated (
Agarose Gel Analysis of pDNA Integrity Following Incorporation into PVP and PVA Polymers of a Range of Molecular Weights
250 mg 20% w/w polymer gels (360 kDa PVP, 13-23 kDa PVA and 9-10 kDa PVA) and 30% w/w 58 kDa PVP gels loaded with 30 μg pLux were fabricated by mixing 50% w/w (360 kDa PVP, 13-23 kDa and 9-10 kDa PVA) or 75% w/w (58 kDa PVP) polymer solutions with RALA/pDNA solution (N:P ratios 0 and 6) at a ratio of 2:3. Gels were incubated at room temperature for either 0 or 7 days to assess pDNA stability. Gels were then dissolved in 10 mM Tris buffer pH 8.0 for 1 h with stirring. Following dissolution, two 35 μL samples of solution were transferred into 0.5 mL eppendorfs. Samples were incubated with 35 μL 10 mM Tris buffer pH 8.0, or, 35 μL 2 mg/ml Proteinase K for 120 min at 37° C. Following incubation, 30 μL samples were transferred to 0.5 mL eppendorfs and mixed with 5 μl 5× Nucleic Acid Loading Buffer (Biorad, UK). Subsequently, 20 μl samples were loaded onto 1% agarose gels containing 0.2 μg/mL Ethidium Bromide (EtBr) as a DNA intercalating agent. Samples were electrophoresed at 100 V for 1 h in 1×TAE buffer and then visualised under UV light using a Multispectrum Bioimaging System (UVP, UK) (
In Vitro Cell Transfection with RALA/pEGFP-N1 Complexes Following Release from PVP and PVA Polymers of a Range of Molecular Weights
250 mg 20% w/w polymer gels (360 kDa PVP, 13-23 kDa PVA and 9-10 kDa PVA) and 30% w/w 58 kDa PVP gels incorporating 20 μg pEGFP-N1 were fabricated by mixing 50% w/w (360 kDa PVP, 13-23 kDa and 9-10 kDa PVA) or 75% w/w (58 kDa PVP) polymer solutions with RALA/pEGFP-N1 solution (N:P ratios 0-12) at a ratio of 2:3. Fibroblast NCTC-929 and macrophage RAW 264.7 cell lines were seeded in a 24-well plate at densities of 100,000 and 140,000 cells/well respectively. The following morning cell media was removed and cells were incubated for 2 h with serum-free Opti-MEM media (Life Technologies, UK). Polymeric gels were placed 1 mL of Opti-MEM media and incubated at 37° C. for 1 h to allow dissolution. Following incubation, cells were treated for 4 h with 250 μL of Opti-MEM media containing RALA/pEGFP-N1 complexes (N:P ratio 0-12) released from dissolved polymers. Following transfection, RALA/pEGFP-N1 complexes were removed, cells were washed with PBS and placed in normal media and incubated at 37° C. and with 5% CO2.
Fluorescent Microscopy of Cells Transfected Following Nanoparticle Release from PVP and PVA Polymers of a Range of Molecular Weights
48 h post transfection, GFP reporter-gene expression was visualised by imaging cells at ×10 magnification under epifluorescence using the EVOS FL Cell Imaging System (Life Technologies).
Flow Cytometric Analysis of GFP Expression Following RALA/pGFP-N1 Nanoparticle Release from PVP and PVA Polymers of a Range of Molecular Weights
48 h post transfection, cells were washed with PBS and trypsinised with 0.5% Trypsin (Life Technologies, UK) for 5 min. Harvested cells were centrifuged at 1500 rpm for 10 min. Supernatant was removed and the cell pellet resuspended in 500 μL 2% Paraformaldehyde (Sigma, UK). Cells were stored at 4° C. until analysis of GFP expression using the FACS caliber system (BD Biosciences, UK). Data was analysed using Cyflogic software. Fluorescent intensity is reported at 4% gating (
Fabrication of MNs from PMVE/MA, PVA and PVP
Three polymeric matrices were investigated as the potential structural polymer for the manufacture of the dissolving MN arrays, Gantrez® AN-139 poly(methylvinylether/maleic acid), (PMVE/MA), Polyvinyl alcohol (PVA) and Polyvinylpyrrolidone (PVP).
Agarose Gel Analysis of Nanoparticle Release from Polymeric Solutions
The stability of pEGFP-N1 and RALA/pEGFP-N1 NPs within the polymeric matrices under investigation was evaluated 24 h post fabrication by dissolving the polymer/nanoparticle formulations is 300 μL water and loading samples onto an agarose gel for electrophoresis. As illustrated in
In contrast, pEGFP-N1 and RALA/pEGFP-N1 incorporated into the PMVE/MA polymer were not visible on the agarose gel. It is possible to see that the wells of the agarose gel have been degraded following electrophoresis, suggesting the polymer adversely affects this method of analysis and thus, it is not possible to determine the stability of these DNA complexes within this polymer through agarose analysis.
This experiment was repeated as illustrated in
Samples were incubated for 30 min with 20% proteinase K (0.1 mg/mL) and loaded onto a 1% agarose gel. Analysis on the gel indicates that the RALA peptide is cleaved by proteinase K thus releasing the DNA into solution and enabling it to travel down the gel without causing degradation of the encapsulated DNA.
Fluorescent Detection of DNA Released from RALA NPs
The fluorescence intensity of the Picogreen® reagent is directly proportional to the quantity of free′ or ‘naked’ DNA present in solution as chelation of the reagent with DNA causes a 1000-fold increase in fluorescence.
Conversely, when the NPs were incubated with proteinase K for 30 min prior to the addition of Picogreen® reagent, fluorescence intensity increases proportionally to the free′ DNA content of the solution as indicated by
Quantification of NP Release from Polymer Matrices
RALA/pEGFP-N1 nanoparticles, N:P ratio 10 containing 1 μg pEGFP-N1 were incorporated into polymer matrices to produce 20% PVA, 20% PVP and 20% PMVE/MA as described previously. Following dissolution of these polymers in 10 mM Tris buffer, proteinase K was added to lyse the NPs for 1 h and the resulting released DNA was then quantified through addition of Picogreen® reagent and subsequent fluorescence detection using a EL808 96-well plate reader (Biotek, UK). The same protocol was carried out for NPs in solution without the presence of polymer and so quantification of released DNA from these complexes, in terms of fluorescence is regarded as 100% release.
The release of pEGFP-N1 from RALA/pEGFP-N1 NPs in the 20% PVP and 20% PVA formulations was determined to be 83.3% and 83.7% respectively as illustrated in
Determination of pDNA Secondary Structure in the Presence of PMVE/MA by Circular Dichroism
To further elucidate the interaction between PMVE/MA and pDNA CD was carried out. For nucleic acids, the position, polarity and intensity of the CD peaks are functions of the base-stacking interactions and helicity of the DNA. Therefore, analysing intermolecular complexes formed with DNA via CD presents an excellent indicator of changes to the secondary structure of the DNA.
It is possible to see in
WST-1 cell viability assay was carried out using the NCTC-929 fibroblast cell line.
In contrast, following incubation in the presence of PMVE/MA the cells exhibited significant toxicity and with a subsequent decease in cell viability. Cells were incubated in media containing concentrations of 0, 5, 10 and 20 mg/mL of 20% PMVE/MA and the resulting percentage cell viability after 6 h were 100%, 41.29%, 16.95% and 10.4% respectively indicating that even the lowest concentration of 5 mg/mL resulted in significant toxicity.
Axial fracture force tests were performed in order to determine the mechanical strength of the polymeric MNs fabricated from 20% PVA and 20% PVP. All MNs were visually inspected before and after testing and all MNs were originally 600 μm in height. In the first stage of this experiment, in order to select the most mechanically robust material, an axial compression force of 0.05 N/needle was exerted on MNs fabricated from the two polymer matrices. The percentage decrease in the height of MNs for 20% PVA MNs was 26.4% and 15.8% for 20% PVP MNs suggesting that the 20% PVP MNs are more mechanically robust than MNs fabricated from 20% PVA.
In order to obtain further insight into the behaviour of these MNs under axial loads, additional fracture force studies were performed. Forces ranging from 0.05 to 0.4 N/needle were applied to the MNs and percentage changes in their height were recorded as illustrated in
The percentage reduction in the height of 20% PVA and 20% PVP MNs under increasing axial forces revealed that MNs exhibited progressive deformation without dramatic breakage at any point i.e. the MN protrusions did not break from the backing plate of the array. Within the range of forces applied, MN height decreased with increasing force exerted. For example, the mean percentage height reduction for arrays fabricated from 20% PVP was 15.8, 21.6, 29.4, 36.6 and 46.2% when the applied forces were 0.05, 0.10, 0.20, 0.30 and 0.40 N/needle respectively. Reductions in height over the same range of forces from MNs fabricated from 20% PVA were 26.4, 33.4, 45.2, 53.2 and 63.1% respectively which are significantly different to those detected with the PVP matrix at all forces investigated.
MN images displayed in
Short-Term Stability Study of RALA/pEGFP-N1 N:P Ratio 10 NPs Encapsulated within 20% PVP Matrix Up to 7 Days
To evaluate the storage stability of RALA/pEGFP-N1 nanoparticles N:P ratio 10 encapsulated within 20% PVP matrix these complexes were stored in temperature controlled environments of 20.0±1.0° C. and exposed to relative humidities (RH) of 46% to represent bench-top conditions in the laboratory and 45.0±1.0° C. and exposed to RH of 75% for up to 7 days representing a warmer climate and humidity encountered in the outdoor environment.
The stability studies revealed that under both conditions investigated the RALA/pEGFP-N1 nanoparticles were still intact following incubation for 0, 1, 3, 5 and 7 days investigated as indicated by an absence of DNA running through the gel in the lanes labelled ‘NPs’ in
Short-Term Stability Study of the Functionality of RALA/pEGFP-N1 N:P Ratio 10 NPs Encapsulated within 20% PVP Matrix Up to 7 Days
To evaluate the storage stability of RALA/pEGFP-N1 nanoparticles (N:P 10) encapsulated within the 20% PVP matrix these complexes were stored at room temperature for 7 days. As illustrated by
A further transfection study was carried out to investigate the GFP expression following incorporation of naked pEGFP-N1 within the 20% PVP matrix as illustrated in
Quantification of DNA Encapsulated in Tips and Baseplate of MN Arrays Loaded with RALA/μLux N:P 10 NPs Containing 36 μg DNA
Concentrated NPs were formulated and incorporated into the PVP matrix for MN manufacture. As such, not all of this DNA will be present in the MN tips of the array due to their small capacity (approx. 5 mg). In order to determine the quantity of DNA present in the MNs themselves and the baseplate of the array a quantification assay was used. The MNs were sheared from the baseplate and both components of the array dissolved in 4 mL 10 mM Tris buffer. The amount of DNA present was then assessed using the Picogreen® assay.
As detailed in Table 1 the MNs contained 9.4 μg DNA in the MNs and 17.5 μg in the baseplate of the array suggesting that the rest of the DNA has been removed from the array when cutting off the sidewalls of the array. This suggests that 74.7% of the NPs loaded into the array is still present. Therefore manufacture of the MN array with concentrated NPs is a more efficient method of MN fabrication compared to that described in Chapter 4 where less that 20% of the DNA content originally loaded into the array was present following manufacture.
A large proportion, 64.8%, of the DNA still present in the array resides in the baseplate of the array rather than the MN tips, however, this is expected due to the small volume of polymer capable of being loaded into the MN tips.
Quantification of DNA Encapsulation within MN Arrays Loaded with 36 μg RALA/pEGFP-N1 NPs.
MN arrays were fabricated from 20% PVP and loaded with RALA/pEGFP-N1 N:P 10 NPs containing 36 μg DNA. Following manufacture of the array as described in section 2.2.8 the needles were sheared off the array using a scalpel and dissolved in 0.5 mL 20 mM Tris buffer, pH8 and the remaining baseplate also dissolved in 0.5 mL 20 mM Tris buffer, pH8. 50 μL volumes of the solutions were then pipetted into a black 96-well plate and 50 μL 0.1 mg/mL proteinase K added and the plate incubated for 1 h at 37° C. Subsequently, 50 μL picogreen reagent is added and the plate incubated for a further 30 min. The plate was then shaken for 1 min and absorbance measured at 450 nm on an EL808 96-well plate reader (Biotek, USA).
Ex Vivo Release Profile of RALA/μLux N:P 10 NPs Released from 20% PVP MN Arrays Across Neonatal Porcine Skin
The release of RALA/μLux (N:P 10) NPs from 20% PVP MN arrays which contain approximately 27 μg was investigated through a release profile across neonatal porcine skin, 300 μm in thickness.
Detectable amounts of NPs were present in the receptor compartment of the apparatus following 5 min (Illustrated in
Determination of Force Required to Insert RALA/μLux NP-Loaded 20% PVP MN Array into Full Thickness Neonatal Porcine Skin Via OCT
Having ascertained that RALA/DNA NPs can be incorporated into and subsequently released from MNs fabricated from 20% PVP without dissociation, or loss of functionality it is necessary to determine if the MNs are of sufficient strength to penetrate full thickness neonatal porcine skin and what force is required for efficient breach of the SC and penetration into the dermis.
The OCT images in
Confocal Microscopic Study of NP Release into Mouse Ear Tissue Following Insertion MN Loaded with Cy-3 Labelled RALA/pOVA NPs
To further analyse the dissolution of the 20% PVP MN array in vivo and the release of RALA/DNA NPs a study was carried out to visualise the NP distribution within the mouse ear tissue following MN application. pOVA was fluorescently labelled with the Cy-3 fluorophore and then used to form RALA/pOVA NPs which were subsequently encapsulated to form 20% PVP MN arrays. The array was applied using manual force to a mouse ear in vivo and 1 h following application the animal was sacrificed and the ear removed for confocal analysis which is illustrated in
This study confirms that the delivery of RALA/DNA NPs via a 20% PVP MN array results in protein expression in vivo. Although expression has only been detected in the liver and kidneys of each animal it is possible that there are lower levels of protein expression in other organs and tissues which are lower than the limit of detection of the IVIS system employed in this study.
Analysis of OVA-Specific CD8+ T-Cells Detected 10 Days Post Microneedle Immunization with pOVA and RALA/pOVA Nanoparticles
A number of research groups developing MN delivery systems have utilised it as a means to deliver nucleic acids for vaccination purposes. In order to determine if the RALA/pOVA NP-loaded MN arrays developed in this study elicited antigen expression and a subsequent CD8+ T-cell response to the protein expressed, MNs containing either pOVA or RALA/pOVA NPs were fabricated. C57BL/6 mice were immunized with these arrays and 10 days post immunization sacrificed, the auricular lymph nodes harvested and stained for the OVA-specific CD8+ surface receptor followed by antibody staining for CD8+ and B220 and then analysed using flow cytometry.
Agarose Gel Determination of the Stability of RALA/HPV-16 E6 and RALA/HPV-16 E7 NPs Encapsulated within 20% PVP Matrix Up to 21 Days
To evaluate the storage stability of RALA/HPV-16 E6 and RALA/HPV-16 E7 nanoparticles N:P ratio 10 encapsulated within 20% PVP matrix these complexes were stored in temperature controlled environments of 4° C., 35% relative humidity (RH), 20° C., 40% RH, or 20° C., 86% RH for 7, 14 and 21 days.
The stability studies revealed that under all conditions investigated the RALA/HPV-16 E6 and RALA/HPV-16 E7 nanoparticles were still intact following incubation for 7, 14 and 21 days investigated as indicated by an absence of DNA running through the gel in the lanes labelled ‘NP’ in
Determination of Functionality of RALA/HPV-16 E6, RALA/HPV-16 E7, and RALA/HPV-16 E6/E7 N:P Ratio 10 NPs Encapsulated within 20% PVP Matrix
To evaluate the functionality of RALA/HPV-16 E6, RALA/HPV-16 E7 and RALA/HPV-16 E6/E7 N:P ratio 10 NPs encapsulated within 20% PVP matrix the polymers were dissolved in PBS and the solution used to transfect NCTC-929 fibroblast cells. Transfection efficacy was determined via western blot analysis as illustrated in
As illustrated in
Determination of Circulating HPV-16 E6/E7 IgG Antibody Levels Generated Following Immunisation with Plasmid DNA Expressing HPV-16 E6/E7 Antigens
As illustrated in
Determination of Generation of HPV-16 E6/E7-Specific Cytotoxic T Cells Following Immunisation with Plasmid DNA Expressing HPV-16 E6/E7 Antigens
As illustrated in
Determination of Interferon-Gamma Secretion from Splenocytes Restimulated with E6/E7-Expressing TC-1 Cells Ex Vivo
Determination of Efficacy of Prophylactic Immunisation with Plasmid DNA Expressing HPV-16 E6/E7±RALA Against Establishment of Tumour Following Implantation of TC-1 Cells In Vivo
The data displayed in
Determination of Efficacy of Therapeutic Immunisation with Plasmid DNA Expressing HPV-16 E6/E7±RALA Against Growth of Established TC-1 Tumour
A therapeutic study was also performed to determine whether immunization can induce therapeutic antitumor immunity, i.e. if a pre-established tumour could be treated by immunisation. As illustrated in
It's possible to see in
Measurement of MNs, Fabricated from PVP and PVA Polymers of a Range of Molecular Weights, Percentage Height Reduction Following Application of an Axial Force
All polymers formed strong, sharp needles that were a replica of the silicon mould (
Optical Coherence Tomographic Analysis of MN Penetration into Mouse Ears Following Fabrication from PVP and PVA Polymers of a Range of Molecular Weights
19×19 arrays were fabricated using 20% w/w 360 kDa PVP, 20% 13-23 kDa PVA, 20% 9-10 kDa PVA and 30% w/w 58 kDa polymer stock solutions. To determine whether MNs had sufficient strength to penetrate the SC arrays were applied into mouse ears using the TA-XT2 Texture Analyser (10-40 N) for 30 sec. Following application, skin was immediately analysed using the optical coherence tomography (OCT) scanner to determine needle penetration depth, images were analysed and measured using Image J software (
The affect of polymers on cell viability of the fibroblast NCTC-929 and DC 2.4 dendritic cell lines was assessed by MTS assay. It was determined that following 24 h incubation 360 kDa and 58 kDa PVP caused significant cellular toxicity from the lowest polymer concentration analysed (10 mg/mL) to NCTC-929 and DC 2.4 cell lines. However, 13-23 kDa and 9-10 kDa PVA were not found to cause significant toxicity to either cell line at any concentration investigated indicating they would be suitable to develop further as an in vivo delivery system.
Quantification of RALA/pDNA Release from PVP and PVA Polymers of a Range of Molecular Weights
To determine the effect of polymer and N:P ratio on pDNA release, gels were formulated with 10 μg loading of pDNA by diluting concentrated polymer with RALA/pDNA (N:P ratios 0-10) aqueous solutions. The quantity of pDNA released was then assessed using a standard curve following incubation with Quanti-iT Picogreen™ reagent and Proteinase K (
Arrays were formulated with a 32 μg loading of pDNA per MN array by diluting concentrated polymer with RALA/pDNA (N:P ratio 10) aqueous solution. Not all of the pDNA loaded into the MN array shall be present in the baseplate and needles of the array and therefore available for delivery across the SC. Therefore, to determine the quantity of pDNA loaded in the baseplate and MN projections of arrays, potentially available for delivery, the sidewalls of arrays were removed with a heated scalpel to allow separate quantification. The baseplate and MN projections of the array and the sidewalls were dissolved in 10 mM Tris buffer pH 8. The quantity of pDNA released was then assessed as by Picogreen assay, described previously. pDNA release from the baseplate and microneedles of 9-10 kDa PVA arrays (17.74 μg) was found to be significantly greater than from release from 13-23 kDa PVA (p=0.0497), 58 kDa PVP (p=0.0376) and 360 kDa PVP arrays (p=0.0023) as illustrated in
Agarose Gel Analysis of pDNA Integrity Following Incorporation into PVP and PVA Polymers of a Range of Molecular Weights
As illustrated in
In Vitro Cell Transfection Efficacy of RALA/pEGFP-N1 Complexes Following Release from PVP and PVA Polymers of a Range of Molecular Weights
Microscopic analysis of cells 48 h post transfection to detect fluorescent reporter-gene expression (
Flow cytometric analysis of GFP reporter-gene expression demonstrates that RALA is necessary to achieve transfection in vitro. The most efficient transfection in fibroblast NCTC-929 cells was achieved with RALA/pEGFP-N1 nanoparticles (N:P ratio 12) released from 13-23 kDa PVA gels (43.687%). The percentage transfection was similar following release of nanoparticles at the same ratio from 9-10 kDa PVA gels (43.347%) (p<0.05). However, transfection of cells with nanoparticles at the same N:P ratio was significantly lower following release from 360 kDa (9.817%) and 58 kDa (28.623%) PVP gels (p=0.0009 and 0.017 respectively). The maximum transfection achieved in RAW 246.7 cells was with RALA/pEGFP-N1 nanoparticles (N:P ratio 12) released from 9-10 kDa PVA gels (18.783%). The percentage transfection of achieved was lower with RALA/pEGFP-N1 nanoparticles at the same N:P ratio released from 13-23 kDa PVA (14.280%, p=0.2582) and 58 kDa PVP (11.753%, p=0.1414) gels and significantly lower following release from 360 kDa PVP gels (4.973%, p=0.0333). These results indicate that the transfection efficacy achieved is cell-line dependent but in all cases, the inclusion of the RALA peptide significantly increased reporter-gene expression in all cases.
The aim of this research was the development of a polymeric MN array using a mechanically robust polymeric matrix suitable for low-cost manufacture of the arrays that will not compromise the transfection efficacy of the bioactive RALA/DNA cargo. The fabricated 20% PVP arrays have proven to be mechanically strong at room temperature for insertion into full thickness neonatal porcine skin and mouse ear tissue indicating they are viable devices for insertion into human skin clinically. It was also shown that the NP-loaded arrays remain stable following short-term storage and manufacture under ambient conditions suggesting these devices circumvent the need for ‘cold chain’ storage.
Additionally, the combination of the novel amphipathic delivery RALA/DNA NPs and the dissolving polymeric MN array resulted in the production of a delivery device capable of eliciting gene delivery and resultant protein production in vivo. Furthermore, it has been proven that when DNA encoding a model antigen is delivered in this manner an antigen-specific CD8+ T-cell response is elicited, suggesting this delivery system had potential to not only transform the field of gene therapy but more specifically, DNA vaccination. Further investigation has determined that RALA/pDNA nanoparticles delivered intradermally using this delivery system are primarily taken up by skin-resident DCs which subsequently travel to the skin-draining lymph nodes for antigen-presentation to the lymph-resident T cell populations.
Following these encouraging results it was hypothesised the delivery system could be utilised for the delivery of plasmids to elicit protection against herpes simplex virus (HPV) causing cervical cancer, namely, HPV-16 E6, HPV-16 E7 and HPV-16 E6.E7. As such, it has been established that MN arrays encapsulating RALA/HPV-16 E6, RALA/HPV-16 E7 and RALA/HPV-16 E6/E7 NPs are stable following storage under adverse conditions for prolonged periods of time and the functionality of the NPs following encapsulating within the PVP matrix. Thus, further studies investigating the efficacy of this delivery system at eliciting protection against HPV-16 E6/E7-expressing tumours was demonstrated using both a prophylactic and therapeutic vaccination regimen. The positive results from both of these studies indicate that i) this delivery platform may be used to protect non-infected patients against establishment of an E6/E7-expressing tumour and ii) this delivery platform is capable of inhibiting progression of pre-established E6/E7-expressing tumours and can cause a reduction in tumour burden.
Moreover, investigation into further optimisation of the delivery system has demonstrated that a range of non-cytotoxic polymeric matrices may be used for fabrication of the dissolving nanoparticle-loaded MN arrays. PVP and PVA polymers, formulated at a range of molecular weights have demonstrated capabilities to fabricate robust MNs and release functional nanoparticles in vitro. Additionally, loading efficacy and subsequent delivery of the RALA/DNA cargo can be improved by freeze-drying the nanoparticles prior to incorporation with the polymeric matrices. These advances in formulation indicate that this nanoparticle-loaded dissolving MN delivery system may be capable of producing even more promising results in vivo.
The focus of this research was on developing a suitable delivery vehicle for the intracellular delivery of DNA and then incorporating these complexes into dissolvable MNs to facilitate non-invasive delivery of the DNA cargo in vivo. The RALA peptide has been demonstrated as an efficient delivery vehicle for pDNA both in vitro and in vivo, overcoming both the extracellular and intracellular barriers against gene expression as demonstrated by its superior transfection profile when compared to ‘naked’ DNA delivery. Furthermore, the formulation methods for PVP MN fabrication employed in this study are straightforward and avoid complex and time-consuming coating processes such as those described in the literature for the manufacture of similar delivery systems. Moreover, the polymer excipients used are cheap, non-toxic and can be processed at room temperature. Importantly, the MNs dissolve rapidly upon insertion into the skin and consequently the MN arrays cannot be reused following removal from a patient and there is no requirement for specific disposal arrangements. All these advantages suggest that the NP-loaded dissolvable MN arrays fabricated from PVP using laser-engineered moulds have vast potential for clinical use.
All documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the spirit of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1410270.1 | Jun 2014 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/062834 | 6/9/2015 | WO | 00 |
