Patent Application
20080160096
The present invention relates to the preparation of nanoparticles from biopolymers such as polycations and polyanions via an ionotropic gellation process, for the purpose of encapsulating nucleic acid such as therapeutic DNA, RNA, siRNA, antisense oligonucleotides (AS-ON) etc., to achieve specific and intracellular delivery of such compounds and provide means of gene therapy.
There is an increasing demand for nanodevices, which are capable of carrying drugs and other therapeutic agents such as nucleic acids to tissue or cells. Recently many drugs have been discovered, which show good efficacy in treatment of cancer or other diseases, however, because of their serious side effects, healthy tissues and organs are affected. The targeted delivery of drugs and chemotherapies using nanodevices offers protection to healthy body segments and also allows dosage reduction. The present nanodevices with their sandwich-like structure are able to protect the active ingredient carried and their surface is designed to avoid immune reactions.
Gene therapy means the transferring of genetic material into specific cells of a patient to treat genetic diseases such as hemophilia, muscular dystrophy, cystic fibrosis, cardiovascular and neurological conditions, infectious diseases or cancer by replacing the errant genes, altering gene expression, producing cytotoxic proteins/pro-drug activating enzymes to stop cell proliferation, or vaccinating against viruses by the above means.
A critical barrier to clinical gene therapy is that an efficient and safe delivery vehicle remains to be discovered. Non-viral gene delivery vectors are superior to viral vectors (recombinant viruses) by providing improved safety, flexibility and facile manufacturing. Such polymer- or lipid-based vectors (usually polycations) electrostatically bind DNA/RNA and condense it into nano-sized particles (polyplexes or lipoplexes), protect the genes from degradation and mediate cellular entry.
Viral vectors allow a (i) high transfection rate and a (ii) rapid transcription of the foreign material inserted in the viral genome. However (i) safety issues have been raised following the death of a patient during a clinical trial, (ii) only small sequences of DNA can be inserted into the virus genome and (iii) large-scale production may be difficult. Finally, toxicity, immune and inflammatory responses can occur and insertional mutagenesis and oncogenic effects have also been observed in vivo.
Non-viral systems—synthetic and natural polycations offer (i) low immunogenicity, (ii) relatively large sequences may be condensed in small particles, (iii) good protection to DNA, (iv) easy manufacture, and may be modified to target specific cells and/or diseases. However several problems such as toxicity, lack of biodegradability, low yield of gene transfection, biocompatibility and in particular, low transfection efficiency need to be solved prior to practical use features in shuttling genes into cells.
Design criteria for non-viral vectors are as follows. Such systems have to protect nucleic acids from degradation, enable packaging of large DNA plasmids, provide for easy administration, support serum stability and targetability to specific cell types. Simplicity of fabrication and inexpensive synthesis and facile purification are also desired. They have to be robust/stable, facilitate internalization into cells, promote endolysosomal escape of the load, achive nuclear transport and efficient unpackaging for the function of the nucleic acids to be manifested. Infection of non-dividing cells is important in tissue therapy. Further important requirements include general safety, non-toxicity, non- or low immunogenicity and non-pathogenicity (Pack et al., 2005; Tiera et al., 2006; Barron-Peppas et al., 2007; Huang, 2005; Hetzline et al., 2004).
There is an increasing demand for nanodevices, which are capable of targeting drugs to tissue or cells. Many drugs have been discovered, which show good efficacy in treatment of cancer or other diseases, however, because of their serious side effects, healthy tissues and organs are affected. The targeted delivery of drugs and chemotherapies using nanodevices offers protection to healthy body segments and also allows dosage reduction.
The present invention relates to the preparation of nanoparticles from biopolymers such as polycations and polyanions via an ionotropic gellation process, for the purpose of encapsulating nucleic acid such as therapeutic DNA, RNA, antisense oligonucleotides (AS-ON), small interfering RNA molecules (siRNA), triple helix forming oligonucleotides (TFO) etc., to achieve specific and intracellular delivery of such compounds to provide means of gene therapy. In the preferred embodiment, polycation (PC) is complexed with DNA and coated with polyanion (PA), via ion-ion interactions (
For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:
Macromolecules with ionizable functional groups such as carboxyl, amino, etc., in an aqueous medium form polycations and polyanions. Under specific conditions polycations and polyanions form nanoparticles by ion-ion interactions. The formation of nanoparticles requires specific reaction parameters, otherwise flocculation and precipitation occurs. However, once the nanoparticles were formed at specific pH and salt concentration, the nanosystem is stable.
Ion-ion interaction can be performed between the functional groups of polyions, and the ratio of original polyions and the order of mixing can affect the particle structure and morphology. The linear polyelectrolyte chains can collapse in compact globules or can extend to coil conformations depending on the pH. The conformation of polymers is an important factor. The final formation of globular nanoparticles is dependent upon interactions between polyelectrolytes. Core-shell or sandwich like morphology can be obtained by varying the ratio of original polyions, the pH and the order of mixing.
The size of nanoparticles depends on the pH of the solution. The hydrodynamic diameter of nanoparticles increases by increasing the pH.
Surface charge of nanoparticles reveals the sequence of polyion addition. At lower pH, positively charged nanoparticles can be found, independently of the ratio of polyions or order of mixing. By increasing the pH, negatively charged nanoparticles are formed. The ratio of charged free functional groups determines the charge extent of nanoparticles, which depends on the pH and the ratio of functional groups.
The hydrodynamic diameter and the stability of nanoparticles were investigated in KCl solution. It was found that the hydrodynamic diameters decreased with increasing salt concentration, but the stability of the aqueous solutions was independent of the salt concentration.
The stability of the aqueous solution and the size of nanoparticles depend on the original concentration of polyions. The hydrodynamic diameter of nanoparticles increases with increasing the original concentration of polyions. The stability of the aqueous solution decreases with increasing the original concentrations, and precipitation can be observed in some cases of mixing at high concentration of original polyions.
PAA with Mw=200 kDa and poly(2-methacryloxyethyltrimethylammonium bromide) were dissolved in water at a concentration of 1 mg/ml. The pH value of solutions was adjusted to pH 3 by 0.10 mol/dm3 sodium hydroxide. The solution of PAMM was added to the solution of PAA with gentle stirring. After 1 hour the pH was increased to 7 resulting in a stable nanosystem with particle size of 50 to 350 nm measured by laser light scattering method.
The size of nanoparticles is variable in a range of 10-1,000 nm by using polymers with different molecular weight. Also the particle size increased at higher pH due to the repulsion of negative charges.
CHIT with Mv=320 kDa and PGA with Mw=1.3 MDa were dissolved in distilled water. The concentration was varied in the range 0.1 mg/ml-2.0 mg/ml. The pH value of the solutions was adjusted to pH=3 with 0.10 mol/dm3 hydrochloric acid. The ratio of polyelectrolyte and the order of mixing were modulated. After 1 hour mixing, the pH was increased with 0.1 M sodium hydroxide solution resulting in stable nanosystems. The hydrodynamic diameter of nanoparticles was in the range of 40-480 nm at pH 3, and at pH 7 was 470-1300 nm measured by laser light scattering method. There was some precipitation at higher pH caused by flocculation and coagulation.
The size of nanoparticles can be varied by using polymers with different molecular weights.
CHIT with Mv=320 kDa and HYAL with Mw=2.5 MDa were dissolved in water. The concentration of CHIT was varied in the range 0.1 mg/ml-1.0 mg/ml, and of HYAL 0.04-0.2 mg/ml. The pH value of solutions was adjusted to pH 3 with 0.10 mol/dm3 hydrochloric acid. The ratio of polyelectrolyte and the order of mixing were modulated. After 1 hour mixing the pH was increased with 0.1 M sodium hydroxide solution resulting in stable nanosystems. The hydrodynamic diameter of nanoparticles was in the range of 130-350 nm at pH 3, and was higher than 600 nm at pH 7 as measured by laser light scattering. There was some precipitation at higher pH caused by flocculation and coagulation.
The size of nanoparticles can be varied by using polymers with different molecular weights.
CHIT with Mv=320 kDa and ALGA with Mv=30 kDa were dissolved in water. The concentration of CHIT was varied in the range 0.1 mg/ml-1 mg/ml, and of ALGA 0.04-0.2 mg/ml. The pH value of solutions was adjusted to pH=3 by 0.10 mol/dm3 hydrochloric acid. The ratio of polyelectrolyte and the order of mixing were modulated. After 1 hour mixing the pH was increased by 0.1 M sodium hydroxide solution resulting stable nanosystems at a pH=3. There was some precipitation at higher pH caused by flocculation and coagulation.
The size of nanoparticles can be varied by using polymers with different molecular weights.
Chitosan was partially modified by betaine. The modification was performed by using the carbodiimide technique. CHIT was dissolved in hydrochloric acid media. Betaine was dissolved in water and then adjusted to pH 6.5 with 0.1 M sodium hydroxide solution. Water soluble carbodiimide was added to the betaine solution and the reaction was stirred for 30 min and subsequently mixed with the chitosan solution.
The modified CHIT and PGA with a Mw=1.3 MDa were dissolved in water. The concentration was varied in the range 0.1 mg/ml-2.0 mg/ml. The pH values of the solutions was adjusted to pH=3 with 0.10 mol/dm3 hydrochloric acid. The ratio of polyelectrolyte and the order of mixing were modulated. After 1 hour mixing the pH was increased by 0.1 M sodium hydroxide solution resulting in stable nanosystems. There was some precipitation at higher pH caused by flocculation and coagulation.
The size of nanoparticles can be varied by using polymers with different molecular weight.
To assess the suitability of CHIT/PGA nanosystem for intracellular delivery of bioactive compounds including nucleic acids, first additional components were incorporated to allow e.g. cancer cell specific targeting and detection of cellular uptake. As a targeting moiety, the vitamin folic acid (FA) was chosen, which has a high affinity for folate receptors (FAR) which are overexpressed in a number of epithelial and myeloid cancer cells.
FA was conjugated to poly-γ-glutamic acid (MW 1.3 MDa, GPC) using water soluble carbodiimide. After the dropwise addition of EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, 8 mg in 1 ml dd. Water) to the γ-PGA solution (50 ml, 1 mg/ml, pH 6.5), the reaction mixture was stirred at room temperature for 30 min. FA (12 mg in DMSO) was added and stirred at room temperature for 24 h. The γ-PGA-FA conjugate was purified by dialysis and the number of FA molecules per γ-PGA was estimated by UV-VIS absorption spectroscopy (λmax1 368 nm, ε9120; λmax2 283 nm, ε25100). This showed that an average of 7 FA molecules was attached to one PGA molecules by this method.
Low molecular weight chitosan (MW 320,000 Da, as determined by viscosity measurements, and with a degree of deacetylation of 88%) solution (10 ml, 1 mg/ml in water, solubilised with HCl and pH adjusted to 6.5 with NaOH) was mixed with an aliquot of fluorescein isothiocyanate (FITC, 1 mg/ml in DMSO, 250 μl) and the reaction mixture was stirred at room temperature for 24 h. Fluorescein-labelled chitosan (CHIT-FITC) was purified by dialysis against water (MW cutoff 10 000 Da, 3 days) and characterised by UV-VIS spectrophotometry, which showed that 71 fluorescein molecules were attached per chitosan molecules by this method.
Stable self-assembled polyelectrolytes were developed via an ionotropic gelation process between the folated γ-PGA and the fluorescently labelled chitosan linear chains. When an equal volume of aqueous γ-PGA-FA (0.3 mg/ml, pH 9.0) and CHIT-FITC (0.3 mg/ml, pH 4.0) were mixed under continuous stirring, an opaque colloidal system was formed (75% transmittance at λ500 nm, pH 7.4), which remained stable at room temperature for several weeks at physiological pH. The presence of individual nanoparticles was confirmed and their size distribution characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM) and dynamic light scattering (DLS) as described before. The analyses demonstrated that the CHIT-FITC/γ-PGA-FA nanosystem consists of spherical particles with a smooth surface both in aqueous environment and in a dried state (
CHIT with Mv=320 kDa was dissolved in water at pH=3. An aqueous solution of DNA with Mw=32 kDa and with specific sequence was added. A stable nanosystem was formed. In the second step, PGA with Mw=1.2 MDa was added to cover the residual surface. The sandwich-like composite nanodevice containing the DNA molecules was stable at pH=7 and the NaCl concentration was 0.1 g/dm3.
Nanoparticles were formed from CHIT, DNA and PGA by a general method represented in
CHIT with Mv=320 kDa was labelled with FITC as described in Example 6. PGA with Mw=1.2 MDa was conjugated with folic acid (FA) as described in Example 6. Single stranded DNA consisting of 20 nucleotides and with a specific sequence was labelled at the 3′ end with Cy3 fluorescent dye (DNA-Cy3). CHIT-FITC was dissolved in water at a concentration of 0.3 mg/ml and at pH 4. DNA-Cy3 was dissolved in water at a concentration of 0.6 mg/ml and at pH 7.4. PGA-FA was dissolved in water at a concentration of 0.3 mg/ml and at pH 9.5. Nanoparticles were formed by either mixing 1 ml of CHIT-FITC solution pre-combined with 50 μl DNA-Cy3 solution and 1 ml of PGA-FA solution or by mixing 1 ml of CHIT-FITC solution and 1 ml of PGA-FA solution pre-combined with 50 μl DNA-Cy3 solution.
The nanosystems were characterised by dynamic light scattering (DLS) or atomic force microscopy (AFM). The results showed that both types of nanoparticles (which differ in the order in which components were mixed) had an effective diameter of 75-77 nm in aqueous environment (by DLS with a distribution between 46-148 nm) or up to 31 nm in a dried state (by AFM) as shown in
Nanoparticles from Example 8 were tested for intracellular delivery of single stranded oligonucleotides into the human cervical cancer cell line, HeLa, by laser scanning confocal microscopy (LSCM). Cells were cultured in RPMI 1640 medium (Sigma, St. Louis, Mo.) supplemented with 10% fetal bovine serum (Fisher Chemicals, Fairlawn, N.J.). Cells were grown at 37° C. in a humidified atmosphere of 5% CO2 (v/v) in air. All experiments were performed on cells in the exponential growth phase. A culture of the cells were incubated with the nanoparticles for up to 30 min and the internalisation of the oligonucleotide (DNA-Cy3) as well as the CHIT-FITC was observed. The experiments clearly showed that the DNA was delivered into the cytoplasm of HeLa cells (
Accordingly, a chitosan/poly-γ-glutamic acid-based self-assembling nanoparticulate system as a delivery platform for nucleic acids is provided. The two main components of this polycationic-polyanionic gel-type composite nanosystem are renewable as chitosan is derived from chitin of crustacean shell by alkaline deacetylation while γ-PGA is also easily obtained from Bacillus sp. ferments, where it is produced as slime. CS and γ-PGA are known to be fully biocompatible, biodegradable and likely non-immunogenic and also failed to display any toxicity in our cellular and in vivo studies. Also, the two polymers have a wide range of biomedical applications in separate or in combination. Additional advantages of the CS/γ-PGA nanosystem as nanocarrier are that (i) its self-assembling nature provides simple preparation without resorting to chemical cross-linking, organic solvents, or other toxic additives (ii) the use of degraded polymers facilitate particle size control, and that (iii) surface charge and functionality of NPs are conveniently tunable by varying component mixing ratios.
Thus, it will be seen that the advantages set forth above, and those made apparent from the foregoing description, are efficiently attained and since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.
It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween. Now that the invention has been described,
This application claims priority on U.S. Application Ser. No. 60/833,672 entitled “POLYMERIC NANOPARTICLES BY ION-ION INTERACTIONS” filed Jul. 27, 2006, the disclosures of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60833672 | Jul 2006 | US |
