Patent Grant
9717688
The present invention relates to ceramic particles for encapsulation and controlled release of biological entities.
Required features of a biomolecule delivery system are to maintain the biomolecule structural integrity during encapsulation, storage and release, and to have a mechanism for enabling suitable release kinetics.
One common application is that of protein drug delivery. The present technology in protein drug delivery applications is largely polymer-based. There are several disadvantages with polymer-based systems as encapsulants for biological entities such as proteins:
WO 01/62232 (Barbé and Bartlett) refers to the incorporation of biological active materials into ceramic encapsulants, however, the chemistry described in the patent is not ideal for encapsulation and release of larger biomolecules. The short-chain alcohols released on hydrolysis of the silicon alkoxide precursors used to form the silica spheres are known to denature protein molecules, leading to significant loss of biological activity. In addition, the sol-gel reactions are usually conducted in presence of an acid or base catalyst, resulting in pHs incompatible with most biological molecules. Also, proteins range in size up to about 3000 kDa, and may exceed 10 nm diameter. The micropores formed in acidic conditions are commonly too small to allow release of molecules of this size, although the mesopores formed under basic conditions are larger and may enable release of small proteins. Ideally a system is required in which the pH can be maintained within the typical physiological range of ˜5-8, conditions which are not suitable for catalysing the hydrolysis of silicon alkoxides.
JP5 261274 (Lion Corp.) describes a process for encapsulating biomolecules in a ceramic matrix. However the particles made by the patented process are not designed for controlled release of the biomolecules. In addition, the process exposes the biomolecules to harsh conditions such as extremes of pH and high shear which may denature or otherwise harm sensitive biomolecules, in particular proteins. Further, the rapid flocculation used in the process is likely to lead to very broad and uncontrolled particle size distributions.
There is therefore a need for a delivery system for biological entities which displays desirable release kinetics and is capable of maintaining the structural integrity of the biological entity during encapsulation, storage and release.
It is the object of the present invention to overcome or substantially ameliorate at least one of the above disadvantages. It is another object to at least partially meet the above need.
In a first aspect of the invention there is provided a process for releasably encapsulating a biological entity comprising:
In the step of forming an emulsion, a first emulsion may be formed from the non-polar solvent, a surfactant and the precursor material, and the biological entity combined with the first emulsion, or a first emulsion may be formed from the non-polar solvent, a surfactant and the biological entity, and the precursor material combined with that emulsion, or the biological entity may be combined with the precursor material and the resulting mixture combined with the non-polar solvent and surfactant to form the emulsion, or some other order of addition may be employed. Alternatively the step of forming an emulsion may comprise combining the surfactant and the non-polar solvent with an aqueous solution, optionally an acidic aqueous solution, to form a first emulsion, and combining the first emulsion with the precursor material and the biological entity to form the emulsion. The first emulsion may be for example a microemulsion, or a small droplet size emulsion.
Thus the present invention provides a process for releasably encapsulating a biological entity comprising:
The process may additionally comprise the step of adjusting the pH of the first emulsion to a pH appropriate for the biological entity in question, i.e. to a pH that will not degrade or denature the biological entity or to a pH that will not cause it to deteriorate. The pH may be adjusted to a pH at which the biological entity remains active, e.g. biologically active. That pH may be between about 1 and about 11, or between about 2 and 11, 3 and 11, 4 and 11, 5 and 11, 6 and 11, 7 and 11, 1 and 9, 1 and 7, 1 and 5, 2 and 10, 2 and 8, 2 and 7, 3 and 9, 3 and 7, 3 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 5 and 8, 5 and 7, 5 and 8.5, 10 and 11, 5 and 7, 8 and 10, 8.5 and 10, 9 and 11 or 8.5 and 11, for example about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5 or 11. Adjusting the pH may be performed before step d).
Step a) of the process may comprise:
Alternatively step a) may comprise:
As another alternative, step a) may comprise:
The precursor may comprise a ceramic precursor. It may comprise colloidal silica or some other ceramic precursor, or may be a mixture of two or more different ceramic precursors. The biological entity may comprise a protein. It may comprise an enzyme. The process may additionally comprise adding a gelation aid before step b). The gelation aid may comprise a salt (e.g. sodium chloride) and/or a water soluble polymer. The gelation aid may be added in solution, optionally in aqueous solution. The process may comprise at least partially separating the particles from the non-polar phase. The step of at least partially separating may comprise the step of combining the emulsion comprising the particles with a precipitating solvent so as to precipitate the particles, said precipitating solvent being miscible with the non-polar solvent. A suitable precipitating solvent may be acetone, ethanol or a mixture of these. The precipitating solvent may be chosen so as to not denature or otherwise damage the biological entity. The step of at least partially separating may additionally or alternatively comprise centrifuging the emulsion comprising the particles. After the step of at least partially separating, the particles may be dried.
In an embodiment the process comprises:
In another embodiment the process comprises:
In another embodiment the process comprises:
In another embodiment the process comprises:
In another embodiment of the first aspect, there is provided a process for releasably encapsulating a biological entity comprising:
The step of combining the precursor material, the surfactant and the non-polar solvent may comprise combining the surfactant and the non-polar solvent (e.g. dissolving the surfactant in the non-polar solvent) and then adding the precursor.
The precursor material may be a solution, a suspension, a dispersion, a sol or an emulsion, and may be capable of forming the particles. It may be polar. It may be aqueous. The step of forming particles may comprise the steps of:
The step of forming particles may comprise destabilizing and/or gelling and/or aggregating the precursor material. The precursor material may comprise water, and may be an aqueous solution, suspension, dispersion, emulsion or sol. It may comprise a ceramic precursor material (i.e. a precursor to a ceramic material). The ceramic precursor material may comprise a metal oxide precursor material, for example a water soluble salt of a metal oxo anion. The metal oxo anion may be for example silicate, aluminate, titanate, zirconate or some other oxo anion. The ceramic precursor may comprise a silica precursor material such as colloidal silica or silica sol or an alkoxysilane (e.g. a tetraalkoxysilane such as tetramethylsilane) or a metal silicate (e.g. sodium silicate) or a mixture of any two or more of these. It may comprise any hydrous metal oxide which is capable of forming a stable colloidal dispersion. The oxide may be an oxide of a Group 2 to 4 element, including transition elements and lanthanides. The precursor material may comprise primary particles, and the primary particles may be between about 5 and about 500 nm in diameter, or between about 5 and about 100 or about 5 and about 50 nm. It may comprise a mixture of different sized primary particles, and the different sized primary particles may be combined before the step of combining the precursor material with the solution of surfactant. The precursor material may be alkaline, and may have a pH between about 9 and about 12, or it may be acidic, and may have a pH between about 0.5 and about 3.5 or between about 3.5 and about 5.5, or it may have some other pH. The surfactant may be anionic, cationic, non-ionic or zwitterionic, and may be soluble in the non-polar solvent. The emulsion may be a water-in-oil (WO) emulsion. It may have a droplet size between about 0.01 and about 500 microns. The sufficient time for the emulsion droplets to form the particles may be between about 1 minute and 24 hours, or between about 1 and 12 hours. During the formation of the particles from the emulsion droplets, the emulsion may be stirred, swirled or otherwise agitated.
The step of combining may comprise stirring, shaking, mixing, swirling or agitating. It may comprise combining the precursor material with a solution of the surfactant in the non-polar solvent. The step of adding the biological entity may be conducted at low shear. The low shear may be sufficiently low to avoid harming, for example denaturing, the biological entity. The biological entity may be added in solution or in suspension. The biological entity may be a biomolecule, and may be for example a protein, a peptide, an antibody, an enzyme, a polysaccharide, a DNA or RNA strand or fragment, or some other biomolecule.
The particles may be mesoporous, and may have an average pore size between about 2 and about 50 nm diameter. They may comprise aggregates each of which comprises a plurality of primary particles. The particles may have a mean particle size between about 0.05 and about 500 microns, or between about 0.05 and about 100 microns, or between about 0.5 and about 50 microns. The particles may have a broad, intermediate or narrow particle size distribution.
The process may additionally comprise one or more of the following steps:
The gelation aid may be in sufficient amount to aid formation of spherical particles. It may be added in solution, and the solution may also comprise the biological entity. The gelation aid may be a water soluble salt, for example sodium chloride. Alternatively it may be some other material, for example a water soluble polymer such as hydroxymethylcellulose or hydroxypropylcellulose. The gelation aid may be added in solution, for example aqueous solution, in a concentration of between about 0.1 and 40% w/v. If the gelation aid is a salt, the solution of the salt may be between about 0.5 and 6M. The step of at least partially separating may comprise centrifuging, filtering, microfiltering, sedimenting or some other suitable method. The step of washing may comprise washing with a washing solvent, which may be an organic solvent, for example the non-polar solvent or a solvent miscible with the non-polar solvent, or it may be a polar solvent, for example a polar organic solvent, water or an aqueous solution. The step of washing may be repeated and each repeat may use the same or a different washing solvent. Each washing step may be followed by a step of at least partially separating the particles from the washing solvent, for example by filtering or centrifuging. The step of drying may comprise exposing the particles to a stream of gas, for example air, oxygen, nitrogen, carbon dioxide or some other gas that does not damage or denature the biological entity. The step of drying may be conducted at ambient temperature or at a different temperature that does not damage or denature the biological entity. It may additionally or alternatively comprise applying a vacuum to, or freeze drying, the biological entity.
In an embodiment there is provided a process for releasably encapsulating a biomolecule comprising:
The step of adjusting the pH of the emulsion droplets may be performed before, at the same time as or after the step of adding the biological entity. The soluble salt, if added, may be added before, at the same time as or after adding the biological entity.
In another embodiment there is provided a process for releasably encapsulating a biomolecule comprising:
In another embodiment there is provided a process for releasably encapsulating a biomolecule comprising:
In a second aspect of the invention there is provided a particle comprising a releasable biological entity, said particle having an average pore size between about 2 and 50 nm diameter and a mean particle size between about 0.05 and about 500 microns or between about 0.05 and about 100 microns. There is also provided a plurality of said particles. The biological entity may be distributed substantially homogeneously through the particle, or may be distributed inhomogeneously therethrough. The particle may be such that the biological entity is biologically active following release from the particle. For example, if the biological entity is an enzyme, the enzyme may retain its enzymatic activity following release from the particle. The biological entity may retain at least about 50% of its activity prior to encapsulation following release from the particle, or at least about 60, 70, 80 or 90% of its activity.
The particle may be made by a process comprising:
It may be made by the process of the first aspect of the invention.
The particle may comprise an aggregate of primary particles between about 5 and 500 nm in diameter. The particle may have the biological entity releasably encapsulated therein and/or thereon. The particle and the primary particles may comprise a ceramic and may comprise a metal oxide, for example silica, zirconia, alumina, titania or a mixture of any two or more of these, or may comprise a mixed metal oxide of any two or more of silicon, titanium, zirconium and aluminium. The particle may be mesoporous, and may have an average pore size between about 2 and 50 nm diameter. It may comprise an aggregate which comprises a plurality of primary particles. The particle may have a mean particle size between about 0.05 and about 500 microns, or between about 0.5 and about 50 microns. The biological entity may be a biomolecule, and may be for example a protein, a peptide, an antibody, an enzyme, a polysaccharide, a DNA or RNA strand or fragment, or some other biomolecule.
In a third aspect of the invention there is provided a ceramic particle having a biological entity releasably encapsulated therein and/or thereon, said ceramic particle being made by the process of the first aspect of the invention.
In a fourth aspect of the invention there is provided a method for delivering a biological entity to a patient comprising administering to the patient one or more particles according to the second or the third aspect of the invention. The method may be for the purposes of treating a condition, such as a disease, in the patient, whereby the biological entity is indicated in treating the condition. The biological entity may be indicated for treatment of the condition. The administering may be by injection, for example intravenous, intramuscular, subdermal or some other type of injection or may be by means of pulmonary, nasal, oral or transdermal delivery or some other suitable delivery method. The method may comprise suspending the one or more particles in a clinically acceptable carrier, said carrier being suitable for injection or for pulmonary, nasal, oral or transdermal delivery.
In a fifth aspect of the invention there is provided a method for delivering a biological entity to a liquid comprising exposing the liquid to one or more particles according to the second or the third aspect of the invention. In this aspect the biological entity may be for example an enzyme. The biological entity may catalyse a reaction in the liquid.
In a sixth aspect of the invention there is provided the use of a particle, or particles, according to the second or third aspect of the invention for delivering the biological entity to either a patient or a liquid. The use may be for the purpose of treating a condition in the patient, or for catalyzing a reaction in the liquid or for some other purpose. The use may comprise controlled release of the biological entity to the patient or to the liquid.
In a seventh aspect of the invention there is provided the use of a particle, or particles, according to the second or third aspect of the invention for the manufacture of a medicament for the treatment of a condition in a patient. The biological entity may be indicated for said treatment. The patient may be an animal or human patient, and the animal may be a mammal, a primate a bird or some other animal.
In an eighth aspect of the invention there is provided a medicament for treating a condition in a patient, said medicament comprising a particle according to the present invention, or a plurality of said particles, wherein the biological entity of said particle or particles, is indicated for treatment of the condition. The medicament may also comprise one or more clinically acceptable carriers and/or adjuvants. It may be suitable for injection into the patient, or for pulmonary, nasal, oral or transdermal delivery to the patient. The carriers and/or adjuvants may be suitable for injection into the patient, or for pulmonary, nasal, oral or transdermal delivery.
A preferred form of the present invention will now be described by way of example with reference to the accompanying drawings wherein:
The present invention relates to the encapsulation and release of biological entities (for example biomolecules and/or microorganisms) in particles. In one example, the particles comprise sol-gel silica derived from aqueous colloidal silica and/or a silicate salt solution. Near neutral pH and the absence of organic chemicals are relatively benign conditions which may assist in retaining the native structure of the biological entity. In the case where a silica sol is used as precursor, the gel may be mesoporous as a result of aggregation of silica primary particles, the size of which determines the pore dimension, thus enabling tailoring of the gel porosity. This control of porosity provides the potential for controlled release applications, if the particle size and shape can also be controlled. By aggregating protein-doped colloidal silica inside a water-in-oil emulsion, the inventors have produced controlled size spheres which may be used for controlled release of biological entities such as biomolecules (e.g. proteins). The process of the present invention may produce particles in which the biological entity is substantially homogeneously distributed through the particle. This may facilitate controlled release of the biological entity.
Important considerations for selecting suitable solvent/surfactant mixtures for use in the present invention are to minimise disruption to the biological entity and to avoid materials which could interact significantly with the colloidal nanoparticles. The non-polar solvent may have a melting point below the temperature at which the biological entity decomposes, denatures or deteriorates. That temperature will depend on the nature of the biological entity. The melting point may be below about 60° C., or below about 55, 50, 45, 40 or 35° C. Suitable solvents which may be used include alkanes, for example long chain alkanes. The alkanes may be linear, branched or cyclic. They may have between about 5 and 24 carbon atoms, or between about 10 and 24, 20 and 24, 5 and 20, 5 and 10 or 10 and 20 carbon atoms, and may have about 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22 or 24 carbon atoms. The solvent may be a mixture of different compounds, for example a mixture of different alkanes. Solvents which may be used include dodecane, kerosene, n-hexane, cyclohexane and toluene. Other solvents that may be used include halogenated solvents. The solvent may be a low polarity solvent and commonly is a solvent for surfactant. The solvent should not denature the biological entity or otherwise cause it to deteriorate or decompose. It should not react with the biological entity under conditions pertaining during the process of the present invention. The solvent may be chosen in order to have low cost.
Surfactants containing sufficiently long polyethoxy (—O—CH2—CH2—) chains (such as Brij52) have been found to prevent formation of silica spheres. The inventors hypothesize that this may be due to hydrogen bonding to the primary particle surface, thereby providing a steric barrier which prevents aggregation and gelation. Suitable surfactants for use in the present invention may be anionic, cationic, non-ionic or zwitterionic, and may for example include sorbitan esters such as Span 20 and sulfosuccinates such as Aerosol OT. Non-ionic surfactants or ionic surfactants with the same charge sign (i.e. positive or negative) as the colloidal particles at the pH of gelation are preferred. Thus when the particles are formed at low pH (e.g. less than about pH 8) it is commonly advisable to avoid surfactants having long polyethoxy (—O—CH2—CH2—) chains. When particles are formed at higher pH (e.g. above about pH8), certain surfactants having polyoxyethylene chains have been found to produce suitable particles. The pH of gelation may depend on the nature of the precursor material.
The precursor material may be a silica sol or colloidal silica and may additionally or alternatively comprise a water soluble salt of a metal oxo anion. The water soluble salt may be a silicate, for example an alkali silicate such as sodium silicate, or may be a zirconate or some other suitable ceramic precursor (i.e. precursor to a ceramic material). A suitable precursor material is Bindzil 30/360 (Eka Chemicals), a colloidal silica which has primary particles around 9 nm, and forms a bulk gel within several hours on lowering the pH from 10 to 6. Other brands of colloidal silica of similar size such as Snowtex ST-40 (Nissan Chemicals) are also suitable. Ludox SM-30 (Grace Davison) may also be used, however it contains a biocide and thus may be unsuitable for some applications of the invention. Precursor materials may have primary particles between about 5 and 500 nm in diameter, or between about 5 and 250, 5 and 100, 5 and 50, 5 and 40, 5 and 30, 5 and 20, 10 and 100, 20 and 100, 10 and 30, 10 and 20, 100 and 500, 100 and 250, 250 and 500 or 50 and 250 nm in diameter, and may have primary particles of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or 500 nm in diameter. A mixture of precursor materials having different sized primary particles may be used. Other precursor materials include aluminates, zirconates, titanates, other metal oxo anions, and mixtures of these.
The process of the invention comprises the step of combining a precursor material and a solution of a surfactant in a non-polar solvent to form an emulsion. The emulsion may be a water-in-oil (W/O) emulsion. It may have a droplet size between about 0.05 and 500 microns, or between about 0.05 and 250 microns, 0.05 and 100 microns, 0.05 and 50 microns, 0.05 and 25 microns, 0.05 and 10 microns, 0.05 and 5 microns, 0.05 and 2 microns, 0.05 and 1 micron, 0.05 and 0.5 microns, 0.1 and 50 microns, 0.5 and 50 microns, 1 and 50 microns, 10 and 50 microns, 25 and 50 microns, 1 and 20 microns, 1 and 10 microns, 1 and 5 microns, 100 and 500 microns, 5 and 500 microns, 250 and 500 microns, 1 and 250 microns, 1 and 100 microns, 1 and 50 microns, and 20 microns, 0.1 and 100 microns, 0.1 and 10 microns or 1 and 2 microns, and may have a droplet size about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 microns.
The ratio of surfactant to non-polar solvent may be between about 5 and 30%, or between about 5 and 20, 5 and 15, 5 and 10, 10 and 30, 15 and 30 or 10 and 20%, and may be about 5, 10, 15, 20, 25 or 30% or a w/w or w/v basis. The amount of total water present (which determines the amount of precursor material added) may be between about 2:1 and 10:1 as a mole ratio of water:surfactant, or between about 5:1 and 10.1 or between about 2:1 and 5:1 or between about 3:1 and 7:1, and may be about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1 as a mole ratio of water:surfactant. The amount of biomolecule added may be dependent on the solubility of the biomolecule in aqueous solution. It may for example be about 20 mg per g of silica. The amount of biomolecule may be between about 1 and 50 mg/g of silica, and may be between about 1 and 20, 1 and 10, 1 and 5, 5 and 50, 10 and 50, 25 and 50, 10 and 40 or 10 and 30 mg/g, and may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mg/g silica, or may be some other amount, depending at least in part on the nature of the biological entity and/or the desired release profile thereof.
In the process of the invention, the precursor material is converted into particles having the biological material therein and/or thereon. The particles may be porous and may have pores of average diameter between about 1 and 50 nm [Note from Chris: both the silicate and the ZrTiO have exhibit micropores but still do release small proteins their average pore size is between 1-2 nm. I think it is therefore better to use average pore size and start >1 nm), or between about 2 and 20, 2 and 10, 2 and 5, 5 and 20, 10 and 20, 20 and 50, 10 and 40, 5 and 30 or 5 and 10 nm, and may have pore diameters about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 nm. The particles may have a diameter (e.g. mean diameter) between about 0.05 and 500 microns, or between about 0.05 and 250 microns, 0.05 and 100 microns, 0.05 and 50 microns, 0.05 and 25 microns, 0.05 and 10 microns, 0.05 and 5 microns, 0.05 and 2 microns, 0.05 and 1 micron, 0.05 and 0.5 microns, 0.1 and 50 microns, 0.5 and 50 microns, 1 and 50 microns, 10 and 50 microns, 25 and 50 microns, 1 and 20 microns, 1 and 10 microns, 1 and 5 microns, 100 and 500 microns, 50 and 500 microns, 250 and 500 microns, 1 and 250 microns, 1 and 100 microns, 1 and 50 microns, 1 and 20 microns, 0.1 and 100 microns, 0.1 and 10 microns or 1 and 2 microns, and may have a diameter about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 microns.
Two factors governing the choice of target particle size are:
The inventors have found that addition of a gelation aid to the emulsion may promote the formation of spherical particles in the case of silica. The gelation aid may be a salt, or it may be some other material, for example a water soluble polymer such as hydroxymethylcellulose or hydroxypropylcellulose. The gelation aid may be added in solution, for example aqueous solution, in a concentration between about 0.1 and 40% w/w or w/v, or between about 0.1 and 20, 0.1 and 10, 0.1 and 5, 0.1 and 1, 0.5 and 40, 1 and 40, 5 and 40, 10 and 40, 20 and 40, 1 and 20 or 5 and 20% w/w or w/v, and may be added in a solution with concentration of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40% w/w or w/v. The gelation aid may be water-soluble, and may be added in solution, for example in aqueous solution. If the gelation aid is a salt, the solution may be between about 0.5 and 6M in the salt, and may be between about 0.5 and 3, about 0.5 and 1, about 1 and 6, about 3 and 6, about 0.5 and 2 or about 1 and 2M, and may be about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 or 6M in the salt. Inclusion of potassium dihydrogen phosphate (0.0037-0.015 M) with the gelation aid serves to keep the pH in the range 5-7. The concentration of potassium dihydrogen phosphate may be in the range 0.0037-0.01M, 0.0037-0.005M, 0.005-0.015M, 0.01-0.015M or 0.005-0.01M, and may be about 0.0037, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014 or 0.015M. Other buffer solutions may also be used. The gelation aid may be added before, during, after addition of the biological entity, or together with the biological entity. The amount of gelation aid added varies depending on the nature of the precursor and the aid employed, but in the case of colloidal silica, typically ranges between about 1:10 and 1:200, or between about 1:10 and 1:20 as a mass ratio of gelation aid:silica. The amount of gelation aid may be between about 1:10 and 1:100, 1:10 and 1:50, 1:10 and 1:20, 1:20 and 1:100, 1:50 and 1:100, 1:100 and 1:200, 1:100 and 1:150, 1:150 and 1:200, 1:20 and 1:0, 1:20 and 1:50, 1:10 and 1:15, 1:15 and 1:20 or 1:13 and 1:17, and may be about 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:20, 1:140, 1:160, 1:180 or 1:200 as a mass ratio of gelation aid:silica.
It may be necessary to wait for some time for particles to both form and age so that they may be washed and dried without damage. It may be between about 1 minute and 24 hours, or between about 10 minutes and 24 hours or between about 30 minutes and 24 hours or between about 0.5 and 12 hours or 0.5 and 6 or 1 and 24 or 6 and 24 or 12 and 24 or 1 and 12 or 2 and 8 or 4 and 8 hours or between about 1 and 60 minutes or between about 1 and 30, 1 and 10, 1 and 5, 5 and 60, 5 and 30 or 15 and 30 minutes, and may be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes or about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20 or 24 hours. During the period of waiting, the emulsion may be stirred, swirled or otherwise agitated, or it may be allowed to rest unagitated.
After the particles have formed and aged, they may be washed. They may be at least partially separated from the non-polar solvent before washing. The step of at least partially separating may comprise centrifuging, filtering, microfiltering, sedimenting or some other suitable method or a combination of any two or more of these methods. The step of washing may be repeated and may be conducted with different washing solvents in some or all of the repetitions. Washing solvents that may be used include the non-polar solvent, water, alcohols (for example methanol, ethanol, propanol, isopropanol, butanol, isobutanol, depending on the sensitivity of the biological entity), alkanes, halogenated alkanes, ketones, esters and other common solvents, or mixtures of these. The washing may comprise immersing the particles in the washing solvent, and may comprise agitating (for example swirling, shaking, stirring) the washing solvent with the particles immersed therein, and/or may comprise passing the washing solvent through the particles, for example by filtration. It may additionally comprise at least partially separating the particles from the washing solvent.
The particles may be dried, for example in a stream of gas and/or by heating and/or applying a vacuum. The temperature at which the particles are dried may depend on the nature of the biological entity, and should be below the temperature at which the biological entity may be damaged or denatured. The temperature may be for example between about 15 and 50° C., or between about 15 and 40, 15 and 30, 15 and 20, 20 and 50, 30 and 50, or 20 and 40° C., and may be about 15, 20, 25, 30, 35, 40, 45 or 50° C. Alternatively the particles may be freeze-dried. The temperature for freeze-drying may be about −80° C. (Note from Chris that's correct our freeze dryer operates at −84 C. The overall range should be from −196 C i.e. the temperature of liquid N2 to −30). It may be less than about −30° C., or less than about −40, −50, −60, −70 or 80° C. It may be between about −30 and about −100, or between about −30 and −80, −30 and −60, −50 and −100, −50 and −80 or −70 and −90° C., and may be about −30, −40, −50, −60, −70, −80, −90 or −100° C. The pressure for freeze-drying may for example be between about 10 and about 200 millitorr, or between about 1 (Two stage oil pump typically go down to 1 mTorr) and 150, 10 and 100, 10 and 50, 50 and 200, 100 and 200, 150 and 200, 100 and 150, 50 and 100 or 120 and 170 millitorr, and may be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 millitorr. The pressure during the drying may be between 0 and 1 atmosphere, or between 0.01 and 1, 0.1 and 1, 0.5 and 1, 0.01 and 0.5, 0.01 and 0.1 or 0.1 and 0.5 atmospheres, and may be about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 atmosphere, or may be at some other suitable pressure.
The particles of the present invention may be used for delivering the biological entity which is encapsulated therein or thereon. The biological entity may be a therapeutic substance, and may be delivered to a patient by administering the particles to the patient. This may be for the purpose of treating a condition for which the biological entity is indicated. The condition may be a disease. Examples of conditions for which biological entities may be indicated include autoimmune diseases, which may be treated by monodonal antibodies, and cancers, the treatment of which may involve the activation of a prodrug by an enzyme. The administration may be for example by injection of a suspension of the particles in a fluid, or it may be orally, pulmonarily, or by some other route. The patient may be a vertebrate, and the vertebrate may be a mammal, a marsupial or a reptile. The mammal may be a primate or non-human primate or other non-human mammal. The mammal may be selected from the group consisting of human, non-human primate, equine, murine, bovine, leporine, ovine, caprine, feline and canine. The mammal may be selected from a human, horse, cattle, cow, ox, buffalo, sheep, dog, cat, goat, llama, rabbit, ape, monkey and a camel, for example.
The particles of the present invention may also be used to deliver the biological entity to a liquid, for example a reaction mixture. The biological entity may be for example a catalytic substance such as an enzyme, and the particles may be used to deliver the biological entity to a reaction mixture to be catalysed by the catalytic substance. An example is the incorporation of particles comprising a protein-cleaving enzyme such as subtilisin, into a powdered laundry detergent, for subsequent release on dispersal of the detergent. A second example is the incorporation of enzymes commonly used for oral hygiene purposes, such as glucose oxidase, and/or lactoperoxidase, into particles which may be incorporated into toothpaste.
Encapsulation of the biological entity in the particles of the present invention may protect the biological entity from harmful environmental conditions, such as high shear, and thereby provide for easier handling or extended life of the biological entity. The encapsulation may also provide for controlled release of the biological entity, whereby the biological entity is delivered at a controlled rate to a patient or a liquid. The rate may be controlled by controlling the pore size of the particles.
Gelation Process
The inventors have found that gelation occurs spontaneously when colloidal silica is dispersed in a surfactant solution, and particles are formed and aged subsequently in a few minutes. The pH of the initial colloidal suspension is typically about pH 10. It is possible to reduce the pH of the colloidal suspension before addition to the surfactant solution, but there is a limiting pH range (about 7.5-10, depending on the colloidal solution/surfactant/acid employed) over which spherical particles may be formed.
The surfactant used in this process may be for example: NP-5, AOT, Span20, Span40, Span60, Span80, etc. Colloids which may be used include: Ludox SM-30, Ludox HS-40, Bindzil 30/360, Bindzil 15/500, Snowtex 40, Snowtex UP, etc. Preferably, the colloidal particles should be less than about 30 nm in diameter although somewhat larger particles may be used. Colloidal silica and surfactant concentration may be broad, and the solvent may be selected from a range of non-polar solvents.
Properties of Colloidal Silica
Colloidal silica suspensions are made by dispersing negatively charged, amorphous silica particles in water. The particles are generally spherical in shape. OH− ions exist at the surface of the particles with an electric double layer formed by alkali ions. Stabilization is achieved by the repulsion between the negatively charged particles. Perturbation of the charge balance causes aggregation, resulting in high viscosity and/or gelation of the suspension. Colloidal silica may be destabilised by pH change or addition of salts, electrolytes, organic solvents, or surfactants.
The influence of each of those factors on the gelation time depends on both the characteristic of the sol and the parameter inducing destabilisation. For example, the higher the concentration or the smaller the particle size, the greater the effect of pH on the gelation time, i.e. the shorter the gelation time. However, the gelation time differs with the kind of acid used for pH adjustment. Organic acids commonly provide better stability in terms of gelation time, depending on the SiO2 concentration and particle size. For example, under the same SiO2 and acid concentrations, acetic acid leads to slower gelation than a strong acid such as HCl. This may be due to the fact that less H+ is released from a weak acid, thus diminishing the reaction between H+ and —O− on particle surface.
Synthetic Procedure
An example of a generalised synthesis procedure for forming particles at pH about 10 is outlined in
0.2 mol/L surfactant solution in 50 mL non-polar organic solvent was prepared. 2.16 mL colloidal silica (pH=9 or above), containing a biological entity, e.g. a protein, was then added at ambient temperature. After stirring for about 10 minutes, 40 mL polar solvent was added to destabilise and dilute the emulsion. The resulting particles were then filtered off, and then rinsed with solvent. The particles were then dried at room temperature.
In a particular example, NP-5 and cyclohexane were used to prepare the surfactant solution, and the colloidal silica was Ludox SM-30. Acetone was used as the polar solvent acetone to destabilize and dilute the emulsion, and to wash particles.
Alternatively, particles containing the biological entity may be centrifuged at 2000 rpm for 3 minutes to remove them from the emulsion, and the particles may then be washed by benign solvents such as kerosene and/or n-hexane. Particles may be dried at room temperature under flowing nitrogen.
Possible Mechanism for Gelation:
In colloidal solution, there are two main forces: the van der Waals force (FVDW) and the electrostatic force (FEL). Total force (FTOT) is the sum of FVDW and FEL (according to DLVO theory). In a colloidal solution containing large polymers, one more force exists, the depletion force, FOS. Under this circumstance, the total force FTOT=FVDW+FEL+FOS. The stability of the colloid may be destroyed by rearranging these forces by changing pH, adding salts or introducing surface active agents, etc.
In a thermodynamically stable microemulsion (W/O) (which typically occurs when the surfactant has an HLB between about 10 and about 13), the interfacial tension is very low. When a colloidal solution is mixed with a microemulsion, the local interfacial force may drive the colloidal silica to aggregate in the water droplets to form large particles. One important factor in this aggregation is the colloid concentration. It is thought that the mechanism for aggregation is as follows. As the colloid suspension is added into surfactant solvent mixture, a hydrophilic domain containing water and colloid is formed. Some of the water molecules will interact with the hydrophilic heads of surfactant molecules forming a hydration layer at the liquid-liquid interface. This results in a number of water molecules being adsorbed and trapped, decreasing the amount of free water in the pool, and thus the concentration of colloid in the water pool is artificially increased, leading to gelation of the colloid. Many parameters can influence the formation of the spherical microparticles: silica concentration in the colloid, surfactant concentration, and water to surfactant molar ratio, amongst others.
To form particles according to this process, the surfactant may need to have a medium strength molecular interaction between its polar head and the water pool. This molecular interaction may be characterised by the surfactant footprint (A), which may be calculated by dividing the surface area of the water droplet surface (π*d2, where d is the water pool diameter) by the surfactant aggregation number (N).
A=(π*d2)/N
Using values from the literature, the footprint was calculated for the range of surfactants used. The results are listed below.
A medium interaction corresponds to a footprint between about 1 and about 5 nm2 per molecule, which corresponds to about 10−2 surfactant molecules per 10 nm2. Any surfactant with footprint value less than about 1 nm2 per molecule (e.g. Brij 30) could form an extremely stable microemulsion with small size water pools. Hence, only submicron spherical particles may be produced. An emulsion system with bigger footprint (>about 5 nm2 per molecule) may not be suitable for forming spherical particles by this process.
Another hypothesis is that the oxyethylene units in the surfactant molecule may play a role in the gelation of primary particles to form submicron particles. The oxyethylene units, which form the polar head of the surfactant molecule, may interact with the particle surface by hydrogen bonding, thus influencing the interaction between the silica particle surface and water, which may control the coalescence process. This may explain why at low pH, where the number ratio between (—OH) and (—O−) is higher and hence the hydrogen bonding is stronger, the coalescence of primary colloid is not favoured, and no microparticles are produced.
The above assumption may only be satisfied for microemulsions, i.e. when the surfactant has an HLB from about 10 to about 13. For surfactants with HLB less than about 9, it is necessary to understand the mechanism of particle formation in a different way. It is widely acknowledged that materials with an HLB value in the range of 3-9 are suitable as emulsifiers for water-in-oil type emulsions or as a wetting agent. All the Span surfactants used have the same hydrophilic head but different lipophilic tails, and their HLB value is between 4.3 for Span 80 and 8.6 for Span 20. Hence a W/O type emulsion is produced by these surfactants. A proposed mechanism is as follows. When colloidal silica aqueous suspension is introduced, it is likely to penetrate through the liquid-liquid interface and form a hydrophilic domain (water droplets), in which the interfacial force disturbs the forces stabilising the colloid. As a result, the colloidal solution gels to form large spheres. A possible explanation for the observation that particles formed by Span 20 are much smoother than those formed by Span 80 is that the HLB of Span 80 is so small that interfacial force is very strong. Once the colloidal suspension encounters the surfactant solution, the gelation rate of colloidal silica is fast, consequently, smaller particles are initially produced. Consequently rough surfaced microparticles are formed via fusion and fission processes of water droplets.
Effect of Surfactant Type:
Surfactants which been tested are listed in the table below. Solutions of 0.2 mol/L surfactant in cyclohexane solution were prepared. For the Triton X-114, NP-9, and Triton X-100 systems, 0.2 mol/L cosurfactant (1-pentanol) was added to promote emulsion stability. Tween and Span surfactants produced unstable emulsions. The procedure was according to typical synthesis process described in
Spherical microparticles were formed using NP-5, AOT, and the different Span surfactants. The particles formed by Brij 30 and NP-6 appeared to be aggregated. All other systems produced irregular shaped products.
The structures of the surfactants, which lead to formation of spherical microparticles, are listed in
Effect of Surfactant Concentration:
NP-5 was selected as the surfactant to investigate the surfactant concentration effect on particle morphology. The surfactant concentration was varied from 0.05 mol/L to 0.5 mol/L. The corresponding SEM images are displayed in
Effect of Emulsion Solvent:
Using the typical synthetic procedure outlined in
In “Effect of reaction condition and solvent on the size and morphology of silica powder prepared by an emulsion technique”, W-Kyu Part, et al., Korean J. Ceram., 6, 229-235 (2000), it was demonstrated that the droplet size in the emulsion, and hence the silica gel particulate size, could be significantly influenced by the steric effect of the organic solvent. In order to confirm this, the authors used octane isomers of various structures with the same chemical formula, and a series of CnH2n+2 alkanes to produce emulsions. The average size of particles in the octane isomers and alkane group series decreased with increasing chain lengths, as expected. The average size obtained from iso-octane was 64 μm and that of octane was 46 μm. The average size of the silica gel powder decreased gradually from 75 μm to 28 μm with increasing chain length. The particle sizes obtained from use of n-hexane, n-heptane, n-octane, nonane, and n-decane were 75, 51, 46, 44, and 28 μm, respectively. These figures are consistent with the present results.
In another reference: “Solvent Effects on Copper Nanoparticle Growth Behaviour in AOT Reverse Micelle Systems”, J. P. Cason, et al., J. Phys. Chem. B, 105, 2297-2302, (2001), the copper particle growth was found to be significantly faster in isooctane solvent than in cyclohexane solvent. This reference stated that cyclohexane was able to support a slightly larger terminal particle size than isooctane. This dependence is due to the fact that cyclohexane is able to pack into the micelle tails and effectively solvate the surfactant tails, whereas the bulky nature of isooctane does not allow it to solvate the tails as readily.
Effect of Colloid Concentration:
Varying amounts of Ludox SM-30 were added to 50 ml of emulsion containing 10 mmol NP-5 to produce silica microparticles. Results are shown in the table below, and SEM images are shown in
From
Effect of Colloid pH:
By contrast, if the pH of Ludox SM-30 was reduced using acetic acid, most particles were spherical if the pH was above 9 (
Effect of Colloid Type:
The typical synthesis procedure was followed using addition of 1.62 mL of various colloidal silicas, with results as listed in the table below. The corresponding SEM/TEM images are shown in
Colloid Properties and Corresponding Products for Different Colloidal Silicas.
Particle Size Distribution:
The size distribution of the particles produced may be determined by light scattering (e.g. using a Malvern Mastersizer 2000). Size distributions of silica particles produced using the typical synthesis, with the amount of Ludox SM-30 varying from 1.62 mL to 5.40 mL, are shown in
In order to reduce the particle size distribution, more energy may be supplied to the system. This may be achieved using more rapid stirring, or shear-mixing, for example.
Encapsulation of Proteins
Certain proteins may be encapsulated at high pH, depending on their pKa. Alkaline phosphatase has a pKa of 9.5 and a procedure for encapsulating this enzyme while retaining full enzymatic activity is given in Example 1 (see below). However, the majority of enzymes have optimum activity around neutral pH. It is possible to reduce the pH of the colloidal precursor before addition to the surfactant solution as described above, and a method for encapsulating alpha-chymotrypsin, subtilisin and alkaline phosphatase in colloid reduced to pH=7.5 is given in Example 2 (see below). However, the inventors have found that doped particles may be formed by addition of an aqueous precursor to a surfactant solution to form an emulsion, followed by addition of acid to reduce the pH to a suitable value, and subsequent addition of the biological entity. Although particles are formed quickly after addition at pH 10, the inventors hypothesize that the particles are not fully dense immediately after formation, and consequently that proteins or other biological entities may be able to infuse into the particles as they age in the water droplets of the emulsion. Typically, the pH in the water droplet has been reduced to 6.0 before addition of the protein. Using this method, enzymes of varying sizes, alpha-chymotrypsin (˜25 kDa), subtilisin (˜27 kDa), alkaline phosphatase (˜160 kDa), and urease (˜480 kDa), have been encapsulated. The surfactant used for most of this encapsulation (ie reduction of pH to 6.0 inside the emulsion) has been Span20, although AOT has also been used, with similar particles being formed in both cases. A typical SEM image of particles formed using this method with Span 20 is shown in
A mechanism for adjusting the release rates of alpha-chymotrypsin, alkaline phosphatase and urease from such particles was investigated in Example 3 (see below), involving the use of different-sized colloidal precursors to influence the average pore size of the particles. Example 4 (see below) also describes the use of different sized colloidal solutions to control the amount of subtilisin released. The distribution of ferritin in a microparticle has been examined in Example 5 (see below), using cross-sectional TEM to map the location of the ferritin molecules. The effect of the encapsulation process on the activity of subtilisin has been examined in Example 6 (see below). A study of the storage stability of subtilisin and alkaline phosphatase has been described in Example 7 (see below). The encapsulation of enzymes in alternative ceramic (i.e. other than silica) matrices has been described in Example 8 (see below).
0.5 mL of 0.5 mol/L nitric add was added to 10 mL of Bindzil 30/360 to give a pH of 9.7. Alkaline phosphatase (8 mg dissolved in 400 μl of buffer at pH=9.5) was mixed with 2.5 mL of the above colloidal silica suspension, then added with stirring to a Span 20 solution (0.2 mol/L) in 50 mL kerosene. After stirring for about 10 minutes, particles were separated by centrifugation at 2000 rpm for 3 minutes. The resulting particles were washed once with kerosene, followed by three washes with hexane (using the centrifuge to separate the supernatant from the solid after each wash) and then dried at room temperature under flowing nitrogen and then stored in a freezer.
Alkaline phosphatase was encapsulated with a loading of approximately 0.6% (by weight) protein (see method below for protein content determination). The enzyme activity was measured immediately after drying and was found to actually be higher than that of free enzyme in solution. This indicates that the encapsulation process as described did not denature the protein to any significant degree.
Protein Content Determination
Protein content of microparticles, and quantification of protein released from microparticles was determined using the Bicinchoninic Acid (BCA) Assay as follows:
Standard Assay:
Reagent A: Sodium Bicinchoninate (0.1 g), Na2CO3.2H2O (2 g), sodium tartrate (dihydrate) (0.16 g), NaOH (0.4 g), NaHCO3 (0.95 g), made up to 100 mL. If necessary adjust pH to 11.25 using NaOH.
Reagent B: CuSO4.5H2O (0.4 g), made up to 10 mL
Standard Working Reagent (SWR)=100 volumes of reagent A+2 volumes of reagent B.
Method:
Quantification of Protein in Microparticles:
Protein containing microparticles (20 mg) were suspended in phosphate buffered saline (PBS solution) (400 μL), and the suspension ultrasonicated for 5 minutes. A sample of the suspension (50 μL) is taken in triplicate, and combined with SWR (1 mL) and incubated at 60° C. for 60 minutes. The sample is centrifuged at 3000 rpm for 5 seconds, and the absorbance of the solution is measured at 562 nm, and compared to that of a series of standards at 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL.
Quantification of Protein Released from Microparticles:
Protein containing microparticles (100 mg) were suspended in PBS solution (2 mL), and agitated. At time points required the suspension was centrifuged, and a sample (50 μL) removed. The samples from each time point were combined with SWR (1 mL) and incubated at 60° C. for 60 minutes. The sample absorbance was measured at 562 nm, and compared to that of a series of standards at 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL.
alpha-chymotrypsin, subtilisin and alkaline phosphatase were encapsulated into particles formed from Bindzil 30/360 using the following method; 4.5 g of Span20 was dissolved in 30 ml of kerosene with stirring. 1.25 ml of Bindzil 30/360 was mixed with 158 ml of 1M HCl to reduce the pH from 10 to 7.5. 4 mg of protein was dissolved in 200 μl of water, and then dispersed with stirring into the Bindzil solution. The precursor solution containing the protein was then added to the emulsion. After stirring for several minutes, 72 μl of 1 M HCl was added to further reduce the pH to 6.0. Finally, 175 μl of salt solution 2 (see Example 6 for composition) was added to the emulsion. After 6 hours stirring, the emulsions were centrifuged and the solids washed once with kerosene, then twice with hexane, then dried overnight.
100 mg of the sample was dispersed in 2 ml of PBS in the case of subtilisin and alpha-chymotrypsin, and in ethanolamine buffer (pH=9.5) in the case of alkaline phosphatase. At the specified time points, the sample was spun down at 10,000 rpm for 10 seconds, and 50 μl removed. The protein content was determined as described in Example 1, and the release curves calculated.
alpha-chymotrypsin, alkaline phosphatase and urease were encapsulated into particles formed from Bindzil 30/360 by using the following method:
A flow diagram describing the particle synthesis is shown in
To increase the size of the pores in the silica, a mixture of Bindzil 30/360 and Snowtex ZL was employed. Snowtex ZL consists of 70-100 nm colloidal particles, considerably larger than the 9 ml colloidal particles in Bindzil 30/360. alpha-chymotrypsin, alkaline phosphatase and urease were encapsulated into particles formed from a mixture of Bindzil 30/360 and Snowtex ZL by the following method:
18 g of Span20 was dissolved in 120 mL kerosene with stirring. 1.5 mL of Bindzil 30/360 was mixed with 1.5 mL of Snowtex-ZL, and added to the emulsion with stirring. 30 mg of protein was dissolved in a solution of 0.31 mL 1M HCl, and 0.422 ml of concentrated salt solution (concentrated by a factor of 4), and added to the emulsion. After six hours, the solids were removed by centrifugation, washed with kerosene and iso-propanol, and dried overnight. Release rates were measured as described in Example 1, and are shown in
Preparation of particles using both Bindzil 30/360 and Bindzil 15/500 was conducted using the method outlined in Example 4, but without the addition of salt solution. The products consisted largely of spherical particles, with a small component of non-spherical material. Particle size and porosity measurements indicated that the size and internal microstructure of the particles were virtually identical to those made using salt solution. Comparison of particles made with and without salt suggested that there are two main advantages in adding salt. The first is to reduce the proportion of non-spherical material. The second is that the addition of salt results in a higher yield of encapsulated protein. In the case of alkaline phosphatase as the biological entity, omitting the step of adding salt solution resulted in a 40% reduction in the protein loading (from 1.5 wt % to 1.1 wt % for the same initial amounts of aqueous colloid and enzyme solution). Salt solution may be more important when gelling colloidal solutions such as Snowtex-40, which comprises larger primary particles (10-20 nm). However, particularly in situations where the presence of salt might to cause problems, it may be omitted.
Salt solution composition in 200 mL (50 mL for concentrated salt solution):
0.1 g KH2PO4
0.2 g NH4Cl
0.21 g Na2SO4
0.223 g CaCl2
1.2 g sodium lactate
0.06 g sodium citrate
4.1 g NaCl
1.973 g MgCl2.6H2O
Release rate measurements of subtilisin from particles made using Bindzil 30/360 indicated that most of the protein release occurred within 1 hour of immersion of the powder in 0.02M PBS solution. One possible method for reducing the pore size is to use a smaller colloid as a silica precursor. Bindzil 30/360 comprises 9 nm silica particles, 30 wt % in solution. Bindzil 15/500 comprises of 6 nm silica particles, 15 wt % in solution. There is a small difference in the amount of acid required to reduce the pH to 6.0 (0.115 mL 1M HCl per mL of Bindzil 15/500, compared to 0.183 mL per mL of Bindzil 30/360). The porosity of this product is discussed below.
Alternatively sodium silicate solution may be used as a precursor instead of colloidal silica. Spherical particles were produced by first preparing an emulsion containing 9 g Span20, 60 mL kerosene, and 1 mL 4M HCl. Addition of 1 ml of sodium silicate solution (27%) resulted in the formation of spherical particles in the size range about 1-100 micron. However, this preparation process is not suitable for encapsulation of protein due to the extreme pH encountered. Reduction of the pH of sodium silicate solution results in immediate precipitation. In order to reduce the pH, it is necessary to dilute the sodium silicate solution, and reduce the sodium content using an ion exchange resin.
Example Preparation of Deionised Silicate Solution:
33 mL of sodium silicate solution (27%) was diluted to 99 mL with distilled water. 34.5 g of Duolite cation exchange resin (H+ form) was added with stirring to reduce the pH to 11.45. The duolite resin was removed by filtration, and 31.16 g of fresh resin added to reduce the pH to 9.8.
20 g Span 20 was dissolved with stirring in 135 mL kerosene. 6 ml of the silicate solution at pH=9.8 was added and stirred for several minutes to disperse in the surfactant mixture. 1 mL of 1 M HCl was added and the emulsion left to stir. After 6 hours, the solid was removed by centrifugation, and washed using kerosene, and ethanol (×2). The average pore size of a freeze-dried sample is compared with those of other colloidal precursors in the table below.
Encapsulation of Subtilisin:
4.5 g of Span 20 was dissolved in 30 mL kerosene, and stirred to dissolve. 1.25 mL of either Bindzil 15/500 or Bindzil 30/360 was added to the mixture to form an emulsion. The emulsion was acidified with 1 M HCl (144 μL for Bindzil 15/500, and 198 μL for Bindzil 30/360), followed by addition of 10 mg subtilisin in 200 μl of water and 98 μl of salt solution 4. The reaction was stirred for 5 hours. Particles were isolated by centrifugation, washed with kerosene and twice with cyclohexane and dried under a stream of nitrogen to give a pale white powder.
Subtilisin was encapsulated in silicate particles by the following method:
18 g Span 20 was dissolved in 120 mL kerosene with stirring. A solution of 8 mg of subtilisin in 200 μl of water was added to the surfactant solution, followed by addition of 4 ml of the deionised silicate, prepared as described above. 0.67 mL of 1M HCl was added to reduce the pH to 7.2. The solution was stirred for 6 hours, then the solid removed by centrifugation. The particles were washed once with kerosene, then twice with hexane (centrifuging to remove the supernatant after each wash) and dried overnight.
Particle Porosity
Nitrogen adsorption data has been modelled using Density Functional Theory (DFT), which describes the behaviour of gas adsorption on a molecular level, and is appropriate for modelling a wide range of pore sizes. Cylindrically shaped pores were assumed. Average pore sizes are tabulated for a variety of silica precursors, are tabulated below. The average pore size appears to be determined by the size of the initial colloidal particle and the pH of gelation. Unless otherwise specified, the surfactant used in the preparation was Span20.
The inventors considered the possibility that a protein may tend to remain in the interfacial region of an emulsion rather than in the interior of a water droplet, due to the presence of hydrophobic regions in the protein molecule. It was considered that this orientational effect may have resulted in the protein being encapsulated in the outer shell of the microparticle forming inside the emulsion droplet. In order to investigate the distribution of encapsulated protein throughout the body of the particle, silica particles were doped with ferritin, which contains an iron core and thus should be easily detectable by Transmission Electron Microscopy (TEM).
Preparation Details:
Span 20 (1.8 g) was dissolved in kerosene (12 mL). Ferritin solution (˜100 mg/mL, 126 μL) was mixed with Bindzil 30/360 (300 μL). This mixture was then added to the surfactant solution dropwise, with stirring at 500 rpm. HCl (1 M, 480 μL) and a concentrated salt solution were mixed. 91 μL of this solution was added to the emulsion. The emulsion was left stirring for 2.5 hours, at which time solid material appeared on the bottom of the reaction vessel. The mixture was centrifuged (2000 rpm, 3 minutes) and the solid was washed once with kerosene and twice with isopropanol. The solid material was dried under flowing nitrogen. The final powder was an ‘ochre’ colour, indicating the successful encapsulation of ferritin within the particles.
Mapping of Protein Distribution in Particle
Particles were imbedded in resin and 80 nm thin sections were cut using a 30° Diatome diamond knife on a Leica Ultracut UCT ultramicrotome and applied to holey carbon coated copper grids.
Protein activity post-release is clearly an important issue for the use of the particles of the present invention. In an attempt to identify which components of the total assay could be responsible for any loss in activity, assays were performed using both alpha-chymotrypsin and subtilisin. The compositions of the various salt solutions used in these assays are given below. All solutions were made up to a volume of 50 ml with deionised water.
Alkaline phosphatase, with a pKa of 9.5, is significantly more stable than most enzymes at higher pH. This enzyme was used to test the effect on activity of encapsulating at pH about 10, and to relate this to the activity of enzymes submitted to the encapsulation procedure at pH 6.
Further kinetic studies of enzymes released from particles according to the present invention are described below.
Synthesis Details:
Precursors for the samples containing HPC were prepared by dissolving HPC into Bindzil 30/360 at two different concentrations, corresponding to 2 mg and 5 mg of HPC respectively per mL of Bindzil 30/360. In the case of the third sample, the precursor consisted of a mixture of 0.625 mL Bindzil 30/360 and 1.875 mL of deionised silicate solution, prepared as described in Example 4.
For each sample, 9 g Span20 was dissolved in 60 ml of kerosene. 2.5 ml of the precursor solutions described above were added with stirring. The pH was reduced to 6.0 inside the emulsion by addition of 0.46 mL of 1 M HCl. A solution containing 8 mg of subtilisin in 200 μl of water was then added, followed by addition of 0.35 mL of salt solution 1 (described above). The particles were isolated using centrifugation and washed with kerosene and hexane before drying.
The storage stability of enzymes encapsulated in microparticles is an important consideration. The majority of proteins require long term storage at temperatures below 0° C. The structural viability of microparticles formed using the process described below has been examined during a freeze-thaw process. It was initially suspected that the expansion of the water content of the particles during the freezing process may lead to an increased rate of broken or cracked particles, reducing their viability for long term storage.
Although the structural integrity of the overall microparticle is important, the viability of the protein stored within the matrix of the microparticles was also examined.
Subtilisin, a serine protease, is robust and stable in a wide variety of chemical environments. However, being a protease enzyme makes it self destructive, thereby reducing its storage stability over long periods. It is significant that the protein may be kept freeze-dried in the freezer for long periods of time, whereas the activity of the enzyme was clearly diminished inside microparticles under the same conditions. This suggests that the environment inside the microparticles may be essentially quasi-aqueous. This suggestion was tested by preparing two subtilisin doped samples as described below, and freeze-drying one. Both samples were then stored in a freezer. After two days storage, the activity of the freeze-dried sample was three times higher than the undried sample, and was essentially unchanged after 9 days storage. This confirms that, although the material appears a dry solid, the amount of water present could be problematic in the case of protease enzymes, and samples should be freeze-dried before storage. Conversely, as can be seen from
Synthesis Details:
18 g of Span 20 was dissolved in 120 mL kerosene. 5 mL of Bindzil 30/360 was added with stirring. The pH was lowered to 6.0 by addition of 0.915 mL 1M HCl. A solution of 16 mg of subtilisin in 400 μl water was added to the emulsion, followed by 0.39 mL of salt solution 1. The particles were isolated using centrifugation and washed with kerosene and hexane before drying.
Two alternative ceramic matrices have been investigated. The first, alumina, was prepared from alumina sol as described below. Alumina sol was prepared by hydrolysis of aluminium sec-butoxide in water, using a water:alkoxide ratio of 10:1, and reaction temperature of 75° C. The mixture was stirred for 30 minutes and the temperature raised to 81° C. to remove the alcohol produced. Nitric acid was then added at a H+:alkoxide molar ratio of 0.07:1 and the solution stirred for one hour at 81° C. The mixture was then sealed and stored at 80° C. to complete peptisation. Light scattering indicated that the mean colloid size was 9 nm. The sol was concentrated by rotary evaporation to a concentration of 10 wt % alumina.
9 g of Span20 was dissolved in 60 mL kerosene. 2 mL of 10 wt % alumina sol was added to the Span20/kerosene mixture, with stirring at 500 rpm. A 0.05 mL aliquot taken from a 50 mL aqueous solution containing 0.1 g KH2PO4, 6.69 g NaCl, and 1.3 g CaCl2 was added. Stirring was continued for five hours, and then the mixture was centrifuged at 2000 rpm to remove the solid, which was washed once with kerosene, then twice with ethanol, before drying. An optical micrograph (
A zirconotitanate sol was prepared using a 1:1 (mol) mixture of zirconium tetrabutoxide (ZBT) and titanium tetrabutoxide (TBT). Acetic acid (5:1 (mol) acetic acid:(Ti+Zr)) was added to slow down the hydrolysis of ZBT and TBT, followed by addition of water (25:1 (mol) H2O:(Ti+Zr)). PCS measurements indicate that the sol consisted of 28 nm colloidal particles. The pH of the sol was 3.0. 2.5 mL of the above sol was added to a solution of 9 g Span 20 in 60 mL of kerosene. After stirring for one hour, the solid was removed by centrifugation, and washed using kerosene and ethanol. Spherical microparticles were observed by optical microscopy.
As an example of encapsulating a biomolecule in the zirconotitanate particles, bromelain (a proteinase derived from pineapples) was chosen because of its relatively small size (˜28 kDa) and stability in acidic conditions. The release curve is shown in
Synthesis Details:
9 g Span 20 was dissolved in 60 ml kerosene. 8 mg of bromelaine was partially dissolved in 200 μl water. The sample was centrifuged to remove undissolved protein, before addition to 2.5 mL of the zirconotitanate sol, prepared as described above. The sol/protein mixture was dispersed with stirring into the surfactant solution, and stirred for 6 hours. The solid was removed by centrifugation, and was washed with kerosene once, and twice with hexane, using centrifugation to remove the supernatant after each wash.
By comparison with polymeric systems, use of a ceramic encapsulant as described in the present invention offers the following advantages:
Additionally, the ceramic system has intrinsic features which make it attractive for application to protein drug delivery, as follows:
The process of the present invention was developed in order to extend the controlled release technology detailed in Barbé and Bartlett, WO 01/62232 (2001) from release of small molecules such as drugs to release of larger biomolecules, such as proteins (including enzymes), polypeptides, and DNA/RNA fragments. The process is based on the use of a solvent/surfactant emulsion system to form spherical silica particles, but uses chemistry which is more suited to proteins and other biological entities. A suitable precursor material which may be used in the invention is a commercial silica colloid, or mixture of colloids, with optional addition of sodium silicate solution to further control the particle pore size. Use of aqueous based silica precursor contributes to overcoming two problems. Firstly, proteins are typically denatured by the alcohols produced in the hydrolysis of silicon alkoxides, which is avoided by use of the present system. Secondly, use of aqueous silica gel precursor results in a mesoporous product with pores in a suitable size range for release of proteins, which may range in size from 1-15 nm. Although aqueous based silica precursors are preferred because of their low cost and ease of preparation, they may be substituted if required (e.g. for the purpose of protection of the payload in base) with other aqueous based ceramic precursors such as titanates, zirconates or aluminates.
Possible applications for the technology described in the present invention include
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004907219 | Dec 2004 | AU | national |
This application is a divisional of U.S. application Ser. No. 11/721,728, which was filed on Sep. 10, 2007, which in turn is a national stage entry of International Application No. PCT/AU2005/001915, which was filed on Dec. 20, 2005, which in turn claims priority to Australian Application No. 2004907219, which was filed on Dec. 20, 2004, wherein the entireties of said patent applications are incorporated herein by reference.
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| Number | Date | Country | |
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| 20150147401 A1 | May 2015 | US |
| Number | Date | Country | |
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| Parent | 11721728 | US | |
| Child | 14612739 | US |
