WO2014064710A1 - A process for the prepartion of non-viral vector for delivery of nucleic acids by mucosal route - Google Patents

Abstract

The invention relates to a process for preparation of non-viral vector for delivery of nucleic acids by mucosal route comprising the steps' of (a) synthesis of pDNA loaded calcium phosphate nanoparticles using reverse micro-emulsion method having n- Hexane as oil phase and water as the aqueous phase; (b) addition of excess water to make total volume of water to adjust the molar ratio of water to AOT at 10 before stirriung both the micro-emulsions; (c) taking 1.36M of anhydrous calcium chloride and 0.35 M di-sodium hydrogen phosphate in two separate micro-emulsion system as the precursors; (d) adding 3μg of pDNA of interested into each system followed by continuous stirring for 12 hours; (e) mixing the micro- emulsion with di-sodium hydrogen phosphate to the micro-emulsion with calcium chloride at slow rate with continuous stirring at 4°C; (f) keeping the mixture, as obtained in step (e), undisturbed at low temperature under continuous stirring for 24 hours; (g) removing n- Hexane by using Bucci-evaporator and dissolving the resulting mass of AOT in 10 ml of absolute ethanol (99.9%) by vortexing; (h) centrifuging the solution for half an hour at 800 rpm at 4°C in a cold centrifuge; (i) washing the pelleted nanoparticles with absolute alcohol three times; (j) dispersing the pelleted nanoparticles in double distilled water at 4°C by vortexing to secure clear dispersion; and (k) dialyzing the dispersion, as obtained in step (j), in cold room using 14 kD dialysis membrane bag and followed finally for coating over nanoparticles with the polymers.

Description

A PROCESS FOR THE PREPARTION OF NON-VIRAL VECTOR FOR DELIVERY OF NUCLEIC ACIDS BY MUCOSAL ROUTE

FIELD OF INVENTION:

The present invention relates to a process for preparation of non- viral vector for delivery of nucleic acids by mucosal route, in particular to novel nucleic acid entrapped calcium phosphate core- shell particles, method of forming the polyelectrolyte shell on the core particles, method of using them as non viral gene delivery vector for delivery optimized for the oral route and delivery of pH sensitive biomolecules, more specially delivering the core-shell calcium phosphate particles may be used for delivering therapeutic DNA, DNA or RNA vaccines, as well as deliver DNA or RNA sequences that inhibit or silence gene expression by utilizing the core-shell calcium phosphate particles.

BACKGROUND OF INVENTION:

Nanometer scale calcium phosphate particles have been proposed for use as carrier particles, as supports for biologically active molecules, such as proteins and nucleic acids. The nanoparticles disclosed in most of the publications and patents are core particles of calcium phosphate with entrapped or adsorbed nucleic acid. Shell formation has been limited to carrier stabilization by polyethylene glycol or copolymers of polyethylene glycol. None of the papers/ patents have described the formation of a polyelectrolyte shell consisting of polyanion and polycation on core calcium phosphate particles. Neither have the papers/ patents described the role of the shell in the protection of the core particle and bioactive molecule on exposure to extremes of pH and enzymes in gastrointestinal tract Or on nasal, buccal and pulmonary mucosa.

The generally accepted method for carrier mediated delivery of nucleic acid, protein and drug through the mucosal route has been by the use of mucoadhesive natural or synthetic polymers. Such polymers include chitosan, sodium alginate, hyaluronic acid, polyacrylic acid, polymethylmethacrylate or copolymers thereof. There has been suggestion in literature of the synthesis of calcium phosphate nanoparticles encapsulating pDNA and highly efficient in vitro transfection using the same.

According to Roy et al. (2003), nanoparticles of calcium phosphate encapsulating plasmid DNA (pDNA) of size 100-120 nm in diameter were prepared. The maximum loading of pDNA and its release from nanoparticles were studied using gel electrophoresis. The time dependent size measurement of these particles demonstrated that these particles show strong aggregational behavior in aqueous dispersion. Calcium phosphate nanoparticles were found to be dissolved even in low acidic buffer (pH 5.0) ⁽ releasing the pDNA, which suggested that DNA release from these particles in the endosomal compartment was possible. In vitro transfection efficiency of these calcium phosphate nanoparticles was found to be higher than that of the commercial transfecting reagent Poly feet.

He et al. (2002) have reported mucosal delivery of calcium phosphate nanoparticles carrying herpes simplex virus type 2 (HSV- 2) glycoprotein. The particles, synthesized by co-precipitation in aqueous medium were of 1.2 μπι and used as vaccine adjuvant. Vaginal and nasal delivery was carried out with significant effect reported in eliciting both mucosal and systemic immunity.

Kakizawa et al. (2004) synthesized block copolymer-coated calcium phosphate nanoparticles sensing intracellular environment for oligodeoxynucleotide and siRNA delivery. The organic-inorganic hybrid nanoparticles entrapping oligodeoxynucleotide (ODN) or siRNA were prepared through the self- associating phenomenon of the block copolymer, poly(ethylene glycol)-blockpoly(aspartic acid) (PEG-PAA), with calcium phosphate. The nanoparticles have diameters in the range of several hundreds of nanometers depending on the PEG-PAA concentration and revealed excellent colloidal stability due to the steric repulsion effect of the PEG layer surrounding the calcium phosphate core.

Bisht et al. (2005) documents the preparation of plasmid DNA encapsulated in calcium phosphate nanoparticles of size 100- 200nm in diameter. The study relates to the process of synthesis and plasmid DNA encapsulation by co-precipitation and stabilization of the nanoparticles in aqueous medium. XRD studies of these nanoparticles showed them to be crystalline in nature having hydroxyapatite structure. The nanoparticles show high transfection efficiency in vitro in comparison to standard transfection agent lipofectamine.

Kakizawa et al. (2006) have reported the novel preparation route of organic-inorganic hybrid nanocarriers entrapping siRNA based on the self-assembly of the block aniomer, poly (ethylene glycol) -block-poly (methacrylic acid), with calcium phosphate crystals. The nanocarriers have diameters in the range of several hundreds of nanometers and revealed excellent colloidal stability due to the steric stabilization effect of the PEG palisade. The biological activity of siRNA loaded in nanocarriers was assessed using HEK-293 cells stably expressing luciferase gene, showing the remarkably high gene silencing-efficacy without the use of any adjuvant molecules such as chloroquin. Further advantage of the system is the serum tolerability, which is a critical issue for in vivo application.

US 20060051424 Al on "COMPOSITIONS OF ORAL GENE THERAPY AND METHODS OF USING SAME" relates to nanoparticle compositions comprising a cationic biopolymer comprising of biologically active molecules which are susceptible to degradation in the gastrointestinal tract. The invention further provides compositions and methods for the oral administration for gene therapy.

Sokolova et al. (2006) documents the formation of coated multi- shell calcium phosphate nanoparticles for cell transfection. Their technique involved the formation of core calcium phosphate nanoparticle coated with DNA. They reported considerable efficiency of transfection and colloidal stabilisation by adding another layer of calcium phosphate on the surface, thereby incorporating DNA into the particle. The transfection efficiency of EGFP-encoding DNA tested with different cell lines (T-HUVEC, HeLa, and LTK) was significantly higher than that of simple DNA-coated calcium phosphate nanoparticles.

US 20060216494 Al on "ORGANIC-INORGANIC NANOCOMPOSITE COATINGS FOR IMPLANT MATERIALS AND METHODS OF PREPARATION THEREOF" focuses on the methods for making coatings that consist of sequentially adsorbed polyelectrolyte film inter-grown with calcium phosphate crystals. The successions of positively and negatively charged monolayers comprising biocompatible polyelectrolytes, preferably polyaminoacids.

Sokolova et al. (2007) Calcium phosphate nanoparticles were prepared by precipitation from water and were then functionalized by DNA. These particles were taken up by living cells and functioned as gene transfer agents, i.e., transfecting agent. By adding the red- fluorescing marker tetramethylrhodamine isothiocyanate-bovine serum albumin (TRITC-BSA) to the nanoparticles, their pathway into the cell and within the cell was followed by fluorescence microscopy. A clear correlation between the uptake of nanoparticles and the efficiency of transfection was found. Aggregates of DNA/TRITC-BSA alone were not able to enter the cells, i.e., the inorganic nanoparticles were necessary as a carrier through the cell membrane.

Paul and Sharma (2008 and 2010) have reported the delivery of proteins by oral route using calcium phosphate nanoparticles.

Xuechao et al. (2008) conducted a study using calcium phosphate nanoparticles to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stem cells (STRO- 1). Nanoparticles incorporating pDNA (plasmid enhanced green fluorescent protein-BMP2) were prepared in reverse micelles and showed diameter of approximately lOOnm.

Ramachandra et al. (2009) report the synthesis of PEGylated calcium phosphate nanoparticles for oral insulin delivery. WO 2009/ 135190 on "THERAPEUTIC CALCIUM PHOSPHATE PARTICLES AND METHODS OF MAKING AND USING SAME" describes therapeutic calcium phosphate particles and methods of making and using the same. The invention provides novel calcium phosphate nanoparticles suitable for efficient encapsulation of biologically active molecules.

Joyappa et al. (2009) have reported the use of calcium phosphate nanoparticles that provide safe and easily manufactured vaccine adjuvant and delivery system for DNA vaccines. In the study FMDV "O" P1-3CD DNA vaccine was encapsulated in calcium phosphate nanoparticles of size 50-100 nm diameters. In vitro transfection efficiency of these calcium phosphate nanoparticles was found to be as good as commercial transfecting . reagent lipofectamine. In vivo analysis of the calcium phosphate nanoparticle P1-3CD (CaPNPl-3CD) FMDV "O" vaccine in mice and guinea pigs could induce significant cell mediated and humoral immune response. Also, immunized mice and guinea pigs were protected against the challenge virus.

Kovtun et al. (2009) reported the formation of single-shell and multi-shell calcium phosphate nanoparticles prepared and functionalized with DNA and siRNA. The expression of enhanced green fluorescing protein (eGFP) could be induced (by using pcDNA3-EGFP) or silenced (by using siRNA). The single-shell nanoparticles were prepared by rapid mixing of aqueous solutions of calcium nitrate and diammonium hydrogen phosphate. The multi- shell nanoparticles were produced by adding further layers of calcium phosphate and DNA to protect DNA from the intracellular degradation by endonucleases. The size of the nanoparticles according to dynamic light scattering and electron microscopy was up to 100 nm with a zeta potential around -30 mV.

US 20107651694 on "THERAPEUTIC CALCIUM PHOSPHATE PARTICLES AND METHODS OF MAKING AND USING SAME" reports a therapeutic calcium phosphate particles and methods of making and using same. The present invention provides calcium phosphate nanoparticles encapsulated with biologically active macromolecules. The particles may be used as carriers of biologically active macromolecule. Li et al. (2010) reported the formation of biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery. A lipid coated calcium phosphate (LCP) nanoparticle (NP) formulation was developed for efficient delivery of small interfering RNA (siRNA) to a xenograft tumor model by intravenous administration. The LCP NP was further modified by post-insertion of polyethylene glycol (PEG) with or without anisamide, a sigma-1 receptor ligahd for systemic administration. Luciferase siRNA was used to evaluate the gene silencing effect in H-460 cells which were stably transduced with a luciferase gene. The anisamide modified LCP NP silenced about 70% and 50% of luciferase activity for the tumor cells in culture and those grown in a xenograft model, respectively. The untargeted NP showed a very low silencing effect.

WO 2010/068359 on "SURFACE-TREATED CALCIUM PHOSPHATE PARTICLES SUITABLE FOR ORAL CARE AND DENTAL COMPOSITIONS" discloses surface -treated calcium phosphate particles suitable for oral care and dental compositions. Calcium phosphate particles are described comprising a surface treatment wherein the surface treatment comprises at least one sugar alcohol, at least one glycerophosphoric acid compound, or mixture thereof.

US 20110039947 Al on "SURFACE-MODIFIED NANOPARTICLES" describes a composition that comprises surface- modified nanoparticles of at least one metal phosphate. The nanoparticles bear on at least a portion of their surfaces, a surface modification comprising at least one organosilane surface modifier comprising at least one organic moiety comprising at least about six carbon atoms.

Giger et al. (2011) have proposed a versatile, surfactant-free method to stabilize calcium phosphate-DNA nanoparticles based on the use of poly (ethylene glycol) -functionalized bisphosphonate. The strength of the interaction between the bisphosphonate and the calcium phosphate enabled the formation of stable, but bioresorbable particles of around 200 nm, which exhibited physical stability over several days. Additionally, the nanoparticles revealed good and sustained ability to transfect cells while displaying low toxicity.

US 20110236685 Al on "THERAPEUTIC CALCIUM PHOSPHATE PARTICLES AND METHODS OF MANUFACTURE AND USE" highlights methods of making novel calcium phosphate nanoparticles, methods of using them as vaccine adjuvants, as cores, as carriers of biologically active material. The core particles may have a surface modifying agent and /or biologically active material, such as antigenic material or natural immunoenhancing factor, polynucleotide material, or therapeutic proteins or peptides, partially coating the particle or impregnated therein. The core particles have a diameter between about 300nm and about 4000nm, more particularly between about 300nm and about 2000nm, and even more particularly between about 300nm and about lOOOnm, are substantially spherical in shape, and have a substantially smooth surface.

Pittella et al. (2011) have presented a hybrid nanocarrier system composed of calcium phosphate comprising the block copolymer of poly (ethylene glycol) (PEG) and charge-conversional polymer (CCP) as a siRNA vehicle. In these nanoparticles, the calcium phosphate forms a stable core to incorporate polyanions, siRNA and PEG-CCP. The synthesized PEG-CCP is proposed as a non-toxic endosomal escaping unit, which induces endosomal membrane de stabilization by the produced polycation through degradation of the flanking cis-aconitylamide of CCP in acidic endosomes. The nanoparticles prepared by mixing of each component was confirmed to possess excellent siRNA-loading efficiency (~80% of dose), and to present relatively homogenous spherical shape and small size, with negligible cytotoxicity. The nanoparticles efficiently induced vascular endothelial growth factor (VEGF) mRNA knockdown (-80%) in pancreatic cancer cells (PanC- 1)·

Nanometer scale calcium phosphate particles have been proposed for use as carrier. Particles, as supports for biologically active molecules, such as nucleic acids and proteins as disclosed in U.S 6,355,271. The particles disclosed in the above-referenced patents although are for in vivo delivery, the delivery route cited is the parenteral route. In the present patent proposal the route for local and systemic delivery is cited as the mucosal route. None of these patents and publications disclose the in vivo delivery of calcium phosphatenanoparticles by the mucosal route, as mechanisms for therapeutic nucleic acids, as delivery mechanisms for nucleotide sequences that inhibit gene expression, or DNA vaccines.

Therefore, an important need remains for calcium phosphate core particles with an appropriate shell that can be effectively used as carriers through mucosal routes, for in vivo delivery of therapeutic nucleic acids as supports and matrices for release of polynucleotide material (DNA or RNA) encoding therapeutic or immunogenic polypeptides. There is also a need for particles that can be used to deliver small inhibitory RNA, which is double stranded RNA that can be used to silence or inhibit gene expression.

Although there are a number of local gastrointestinal tract and systemic diseases that could benefit from protein-based therapies, oral or rectally administered protein drugs are rapidly degraded by the proteolytic enzymes. In addition, in certain cases, such as the administration of non-humanized antibodies, there is development of an immune response that limits chronic use. Local production of protein drugs using a gene therapy strategy could provide an efficient alternative to oral and rectal administration. Efficient oral gene therapy provides an opportunity for the sustained of therapeutic protein at the disease site in the gastrointestinal tract or at a site where maximum systemic absorption can occur due to low proteolytic activity.

The oral route is ideal for gene therapy of gastrointestinal disorders, and oral vaccination purposes, as it allows easy and rapid access to the site of action upon administration. Although very appealing from a patient compliance perspective, oral formulations have to overcome anatomical (mucus and epithelial layer) and physiological barriers (varying pH, degradative enzymes) of the gastrointestinal tract to become successful in drug delivery. These difficulties become even more pronounced when attempting gene delivery, as the DNA molecule has to reach the nucleus of cells for efficient transfection. Polymeric nano-and microparticle technologies have been used for oral drugs, including a number of protein therapeutics. First they are more stable in the gastrointestinal tract than other colloidal carriers, such as liposomes, and can protect encapsulated drugs from gastrointestinal environment. Second, the use of various polymeric materials enable the modulation of physicochemical characteristics (e.g. hydrophobicity, zeta potential), drug release properties (e.g. delayed, prolonged, triggered), and biological behaviour (e.g. targeting, bioadhesion, improved cellular uptake) of NPs. Finally, the particle surface can be modified by adsorption or chemical grafting of certain molecules such as poly (ethylene glycol) (PEG), poloxamers, and bioactive molecules (lectins, invasins ...). Moreover, their submicron size and their large specific surface area favour their absorption compared to larger carriers. Consequently, it has already been extensively shown that nano- encapsulation of peptides and protein colloidal particles protect them against the harsh environment of the gastrointestinal tract, and enhance their transmucosal transport. For gene therapy, there are few reported cases of success.

The first instance of proper oral gene delivery was reported in 1997. In this pivotal study, polyanhydride microspheres were used to orally administer plasmid DNA encoding Pgalactosidase into rats. Encapsulation of plasmids within .the microspheres allowed a greater expression of the gene in both the small intestine and the liver. The increased expression may be attributed to the delayed release of DNA due to a slow dissolution of sphere matrix, increased DNA uptake into cells due to physical contact of microspheres with target cells and resistance against degradation proffered by complexation of DNA with the microsphere matrix as noted for other types of microspheres.

The pioneering study reporting oral plasmid DNA administration using polymeric NPs was put forth by Roy et al. in 1999. In this study, chitosan NPs in the size range of 100 - 200 nm were prepared by the salting-out technique with the plasmid DNA (pArah2), which encodes for the peanut allergen Arah2. The NPs were orally administered to mice, and the serum and fecal levels of IgG or IgA were measured periodically. High levels of anti-arah2 IgG were observed in the titer of the group which was fed with low molecular weight chitosan NPs complexed with the plasmid DNA, compared with other groups, which were dose with high molecular weight chitosan NPs. The mice from all groups were challenged with crude peanut extracts 4 weeks after the booster dose and positive antibody response were detected in groups immunized with DNA- containing NPs. These results suggest that chitosan-plasmid DNA NPs delivered through the oral route can modify the immune system in mice and protect against food allergen-induced hypersensitivity.

Kai and Ochiya successfully used an N-acetyl derivative of chitosan to form polyplexes with plasmid DNA containing two genes - one encoding for β-galactosidase and the other for murine cytokine the IL- 10. These polyplexes were delivered via the oral route by incorporating them in rodent feed. Five days after administration, qualitative and quantitative transfection efficiency with the DNA- containing formulations was confirmed by testing for β-galactosidase expression with X-gal staining and also quantifying the production of IL- 10 by an ELISA, respectively. Both the upper and lower intestines were transfected with the formulated particles, with the colon showing the highest level of IL- 10 expression among all of the test organs.

In one of the study with the use of chitosan NPs for oral gene delivery by Chen et al. mEpo was transfected to the intestinal epithelium of mice. Chitosan NPs containing plasmid DNA encoding for erythropoietin have been orally administered to mice along with other appropriate control formulations; erythropoietin gene expression was evaluated every 2 days by measuring the haematocrit of the mice. Mice that received chitosan NPs encapsulated with mEpo showed a 15% increase in haematocrit over the control formulations, indicating successful transfection of the mEpo gene across the intestinal epithelium and systemic absorption of the erythropoietin for pharmacological activity. These results suggest that chitosan NPs were able to protect mEpofrom degradation by DNases and, hence, there is a possibility of using them as gene delivery vehicles via the oral route, where the protein drug is absorbed for systemic therapeutic activity.

In another study showing oral absorption of locally produced protein drug, NPs prepared from cationic biopolymers, including chitin, chitosan and their derivatives, were used as carriers for the oral administration of a therapeutic gene. In this case, the NPs were encapsulated with plasmid DNA encoding for human coagulation Factor IX, which is absent in hemophilia. Human Factor IX was detected in the systemic circulation of the mice within 3 days, following oral delivery of the plasmid DNA-containing NPs and declined after 14 days. The investigators also demonstrated the bioactivity of the Factor IX transgene product in Factor IX knockout mice. Hemophilia B is an X-linked bleeding disorder caused by a mutation in the Factor IX gene. After orally feeding Factor IX transgene-loaded NPs to the knockout mice, the blood clotting time was reduced from 3.5 min to 1.3 min, which was comparable with a clotting time of 1 min observed with normal mice. This study elegantly shows that locally produced protein drug in the gastrointestinal tract can be systemically absorbed and shows therapeutic activity.

In 1997, Jones et al. first reported on the use of polymeric microparticle formulations made from PLGA for delivery of DNA vaccine. They used plasmid DNA expressing the insect protein luciferase under the transcriptional control of the human cytomegalovirus immediate-early promoter. The DNA-containing formulation was administered orally and by intra-peritoneal injection. Upon comparison of serum Ig levels after both routes of administration, they found that intra-peritoneal route elicited good serum IgG and IgM responses and a modest IgA response; the oral route was able to elicit a good serum response of all three immunoglobulins. The same group also reported on the in vivo therapeutic potential of oral DNA vaccine using PLGA microparticle technology. They used plasmid DNA encoding for the rotavirus coat protein called VP6. After administration of just a single dose of DNA vaccine formulation in Balb/c mice, a protective immune response was observed in terms of serum antibodies, as well as intestinal IgA production, resulting in stimulation of both systemic and humoral immunity. Animals showed adequate protection from challenge with homologous rotavirus after 12 weeks post- immunization.

In 2004, Chang et al, investigated the feasibility of using nonionic polymeric micelles of poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) (PEO-PPO-PEO) as a carrier for oral DNA delivery in vivo. Duodenal penetration of DNA/ PEO-PPO-PEO polymeric micelles was evaluated in vitro by calculating the apparent permeability coefficient. The results showed a dose-independent penetration rate of (5.75 ±0.37) 3 1025 cm/ sec at low DNA concentrations (0.026-0.26 x / μ\), but a decrease to (2.89 ±0.37) 3 1025 cm/ sec at a concentration of 1.3 μg/μl. After oral administration of six doses at 8-hr intervals, the highest expression of transferred gene lacZ was seen 48 hr. after administration of the first dose, with gene expression detected in the villi, crypts, and goblet cells of the duodenum and in the crypt cells of the stomach. Reporter gene activity was seen in the duodenum, stomach, and liver.

In 2005, He et al. reported on the use of PLGA microsphere encapsulating DNA vaccine for induction of immunity against hepatitis B after a single oral dose. They obtained the gene for hepatitis B virus antigen from the serum of a patient positive for hepatitis B virus infection. Oral administration of PLGA-pDNA microparticles in Balb/c mice induced a long-lasting and stable, antigen- specific antibody response in the systemic delivery in the gastrointestinal tract, for the local production of proteins or for systemic absorption and therapeutic effect. In addition to the above mentioned reports, Mittal et al. investigated the effect of the route of administration and co-administration of bovine adenovirus t pe-3 (BAdV3) DNA vaccine, along with biodegradable alginate microspheres, on systemic and mucosal immune responses in mice. Antibody titer results showed that systemic routes were able to induce a better response, but low levels were observed at the mucosal sites. In contrast, the mucosal routes showed high levels of induction at the mucosal sites and low levels of systemic induction. In 2005, lyophilised preparation of chitosan-DNA microparticle was prepared using coacervation process and investigated the stability of pDNA in this complex. The complex was able to protect the pDNA from nuclease degradation. The release of the pDNA from the microparticles was studies in simulated gastric juices, simulated intestinal medium and acidic PBS (pH 4.5) buffer o

at 37 C and released pDNA was assayed spectrophotometrically. In the gastric and acidic PBS mediums, pDNA was released from chitosan microparticles in shorter periods than in the intestinal medium, and released amounts was high (approximately 85 and 100%). In the intestinal medium, the released pDNA amount was lower (approximately 30%) than the acidic medium. In vitro release of pDNA from the microparticles was dependent on pH, as the pH of the release medium increased, release profile decreased, and vice versa which was mainly due to differences in release profiles were caused by different solubilities of chitosan in acidic and basic pH of mediums.

Martien R et al. gave a breakthrough idea that stability of cationic polymer/ pDNA NPs toward salts and enzymatic fluid could be reduced by using enzyme inhibitors (ATA). Chitosan enzyme inhibitor conjugate was generated. This complex was the coacervated with pDNA and it was found that chitosan-ATA/pDNA NPs were significantly more stable than unmodified chitosan/ pDNA NPs. Apart from improved stability, chitosan- ATA /pD A NPs showed a 2.6 fold higher transfection rate than the rest in the Caco-2 cell line. The same group synthesized and evaluated a thiolated chitosan to improve the efficacy of oral gene delivery systems. Due to stability towards nucleases the transfection rate of thiolated chitosan/ pDNA NPs was five-fold higher than that of unmodified chitosan/ pDNA NPs.

In 2006, Yan Li et al. designed Chitosan NPs as gene therapy vector via GI tract and tested the in vitro and in vivo transfection efficiency. The group used three types of chitosan as NPs entrapping pDNA encoding GFP for both in vitro and in vivo transfection experiments. Quaternized chitosan had better transfection ef icacy than the other two. They also determined as the time dependent intensity of the protein expression which increased as the time period increased. When the mass ratio of the TMCO-60%: pDNA was 3.2: 1, the GFP expression in the alimentary tract mucosa was the highest, which was consistent with the in vitro transfection result.

Gene Delivery using non-viral vector via oral route suffers a setback majorly due to prevailing extreme physiological conditions. One such consideration is for differential pH conditions, due to which the non-viral vectors tends to bring non-specific results. Chitosan which has been used as one such cationic non-viral suffers a serious setback due to the dissolution of the polymer as lower pH which thereby release the pDNA in the stomach. Hence, most of the scientist mixed the non-viral vector of Chitosan with the mice feed and even in some other experiments; the non- viral vectors were directly injected into the intestinal region. The extreme acidic conditions within the stomach not only degrade the nanoparticles/ carriers but also cause the non-specific uptake of the gene.

Calcium Phosphate (CaPi) shows good transfection efficiency and also has good biocompatibility in-vivo. The dissolution of the NPs at lower pH of the stomach has been one hurdle. Hence, we designed a carrier system comprising of CaPi which is layered by polyelectrolyte namely pAA and Chitosan based on the electrostatic interaction between the ceramic nanoparticles and the polymers. pAA which is anionic have tendency to get hydrolyzed in low pH which causes the polymer to shrink. The compaction brought about by the shrinkage prevents the entry of any degradative enzyme thereby providing protection for the nanoparticle which is in the core of the pAA layer. Use of Chitosan as a layer is to provide high mucoadhesivity to CaPi/pDNA NPs. But at lower pH, Chitosan has the tendency to dissolve, hence a final layer of pAA is provided.

OBJECTS OF THE INVENTION;

The main object of the invention is to design a nano- particulate carrier of genetic material by the mucosal route. Another object of this invention is to develop a delivery vehicle for nucleic acid to withstand the harsh environment of- the gastrointestinal tract.

An additional object of this invention is to keep to provide nanoparticle surface mucoadhesivity.

Yet another object of this invention is to ensure nanoparticle uptake and gene expression.

A further object of this invention is to use of polyelectrolyte coating over the nanoparticles for enhancing the transfection efficiency of the non-viral vectors but also for protecting the vectors against degradation in the harsh, extreme physiological conditions of stomach/ intestine in gastrointestinal tract.

An additional object of this invention is to propose the possibility of local and systemic gene therapy by mucosal delivery through ease of target accessibility thereby enhancing patient compliance.

A further additional object of the invention is manufacture a new product using core nanoparticle comprising of calcium phosphate entrapping nucleic acid.

Yet another addition object of the invention is to enhance the transfection efficiency by the mucosal route using these surface coated calcium phosphate nanoparticles.

The foregoing has outlined some of the pertinent objects of the invention. These objects should be construed to be merely illustrative of some of the more prominent features and applications of the intended invention. Many other beneficial results can be attained by applying the disclosed invention in a different manner or modifying the invention within the scope of disclosure. Accordingly, other objects and a full understanding of the invention and the detailed description of the preferred embodiment in addition to the scope of the invention are to be defined by the claims taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

Further objects and advantages of this invention will be more apparent from the ensuing description when read in conjunction with the accompanying drawings wherein:

Figure 1 : A schematic representation of a procedure for the synthesis of core calcium phosphate nanoparticles co-precipitated with plasmid DNA and coated with three layers of polyelectrolyte.

Figure 2 : The transmission electron photomicrographs and schematic diagram of the core calcium phosphate nanoparticles co-precipitated with plasmid DNA and the sequentially layered core nanoparticles.

Figure 2A : Core nanoparticles with plasmid DNA.

Figure 2B : A representation of polyacrylic acid layered over core particles.

Figure 2C : Core particles layered with polyacrylic acid and chitosan.

Core shell particles having shell of polyacrylic acid, chitosan and polyacrylic layered on core particles.

Infrared Spectra of the core calcium phosphate nanoparticles coprecipitated with plasmid DNA and sequentially layered core nanoparticles.

Core nanoparticles with plasmid DNA.

A representation of polyacrylic acid layered over core particles. Core particles layered with polyacrylic acid and chitosan.

Core shell particles having shell of polyacrylic acid, chitosan and polyacrylic layered on core particles.

Photographic representation of agarose gel electrophoresis of the core calcium phosphate nanoparticles co-precipitated with plasmid DNA and sequentially layered core nanoparticles recorded by gel documentation system.

Photographic representation of agarose gel electrophoresis of the core calcium phosphate nanoparticles co-precipitated with plasmid DNA and sequentially layered core nanoparticles.

Photographic representation of agarose gel electrophoresis of the core calcium phosphate nanoparticles co-precipitated with plasmid DNA and sequentially layered core nanoparticles treated with pH 8 solutions.

Dynamic Light Scattering graph of the core calcium phosphate nanoparticles co-precipitated with plasmid DNA and sequential layering with polyelectrolytes of the core nanoparticles.

Core nanoparticles with plasmid DNA.

Representation of polyacrylic acid layered over core particles.

Core particles layered with polyacrylic acid and chitosan.

Core shell particles having shell of polyacrylic acid, chitosan and polyacrylic layered on core particles. Figure 6 : Graphical representation of pH dependent size variations of the sequentially layered core particles.

Figure 6A,B,C: Size of the layered core particles at pH 5. Figure

6B, 6C and 6D is a representation of size of layered core particles at pH 6 7 and 8 respectively.

Figure 7 : Graphical representation of zeta potential of the core calcium phosphate nanoparticles co- precipitated with plasmid DNA and sequential layering with polyelectrolytes of the core nanoparticles.

Figure 7 A : Core nanoparticles with plasmid DNA.

Polyacrylic acid layered over core particles.

Core particles layered with polyacrylic acid and chitosan.

Core shell particles having shell of polyacrylic acid, chitosan and polyacrylic layered on core particles.

A series of photomicrographs of fluorescing HEK- 293 cells transfected with core calcium phosphate nanoparticles co-precipitated with plasmid DNA and sequentially layered core nanoparticles.

HEK-293 cells transfected with core particles of calcium phosphate co-precipitated with plasmid DNA.

HEK-293 cells transfected with core particles layered with first coating of polyacrylic acid. Figure 8C : HEK-293 cells transfected with core particles layered with second ^x coating of chitosan over polyacrylic acid.

Figure 8D : HEK-293 cells transfected with core particles layered with second coating of chitosan over polyacrylic acid.

Figure 9 : Confocal photomicrographs of HEK-293 cells transfected with commerically available Polyfectamin as positive control.

Figure 9A : Cell transfected with core calcium phosphate nanoparticles co-precipitated with green flourescent plasmid.

Figure 9B,C : Cells transfected with core particles layered with polyacrylic acid and with final layered core particles respectively.

Figure 10 : A series of photomicrographs of fluorescing cells in the small intestine of mouse.

Figure 10A : Cells transfected with core particles of calcium phosphate co-precipitated with plasmid DNA.

Figure 10B : Cells transfected with core particles layered with first coating of polyacrylic acid. Figure IOC : Cells transfected with core particles layered with second coating of chitosan over polyacrylic acid. Figure lOD : Cells transfected with core particles layered with third coating of polyacrylic acid over chitosan surface layer.

Figure 11 : Confocal photomicrographs of fluorescing cells in the small intestine (transverse section) of mouse 72 hours after following oral delivery of core calcium phosphate nanoparticles co-precipitated with plasmid DNA and sequentially layered core nanoparticles.

Figure 11A Cells transfected with core particles of calcium phosphate co-precipitated with plasmid DNA.

Figure 11B : Cells transfected with core particles layered with sequentially layered core nanoparticles co- precipitated with GFP.

While the invention is described in conjunction with the illustrated embodiments, it is understood that it is not intended to limit the invention to such embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents may be included within the spirit and scope of the invention disclosure as defined by the claims.

STATEMENT OF THE INVENTION:

According to the invention, there is provided a process for preparation of non-viral vector for delivery of nucleic acids by mucosal route comprising the steps of (a) synthesis of pDNA loaded calcium phosphate nanoparticles using reverse micro-emulsion method having n-Hexane as oil phase and water as the aqueous phase; (b) addition of excess water to make total volume of water to adjust the molar ratio of water to AOT at 10 before stirriung both the micro-emulsions; (c) taking 1.36M of anhydrous calcium chloride and 0.35 M di-sodium hydrogen phosphate in two separate micro- emulsion system as the precursors; (d) adding 3μg of pDNA of interested into each system followed by continuous stirring for 12 hours; (e) mixing the micro-emulsion with di-sodium hydrogen phosphate to the micro-emulsion with calcium chloride at slow rate with continuous stirring at 4°C; (f) keeping the mixture, as obtained in step (e), undisturbed at low temperature under continuous stirring for 24 hours; (g) removing n-Hexane by using Bucci- evaporator and dissolving the resulting mass of AOT in 10 ml of absolute ethanol (99.9%) by vortexing; (h) centrifuging the solution for half an hour at 800 rpm at 4°C in a cold centrifuge; (i) washing the pelleted nanoparticles with absolute alcohol three times; (j) dispersing the pelleted nanoparticles in double distilled water at 4°C by vortexing to secure clear dispersion; and (k) dialyzing the dispersion, as obtained in step (j), in cold room using 14 kD dialysis membrane bag and followed finally for coating over nanoparticles with the polymers.

DETAILED DESCRIPTION OF THE INVENTION:

At the outset of the description, which follows, it is to be understood that the ensuing description only illustrate a particular form of the invention. However, such a particular form is only an exemplary embodiment and the teachings of the invention are not intended to be taken restrictively.

For the purpose of promoting an understanding of the principles of the invention, reference is now to be made to the embodiments illustrated and the specific language would be used to describe the same. It is nevertheless to be understood that no limitations of the scope of the invention is hereby intended, such alterations and further modifications in the illustrated bag and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

The embodiments are described with reference to the drawings in which like parts are referred to by like numerals. These embodiments are for illustrative purpose only and it should be noted that invention is not limited to the embodiments illustrated in the drawings. Certain details, e.g. manufacturing / assembly details, have been omitted since they are not necessary to understand product functioning.

As used herein and subsequently in the claims, the singular form 'a', 'an', and 'the' includes plural reference unless the context clearly indicates otherwise. The present invention relates to the manufacture of a new product using core nanoparticle comprising of calcium phosphate entrapping nucleic acid. The core nanoparticle has been used for in vitro transfection and has demonstrated high transfection efficiency. The shell is formed of three monolayers of polyelectrolyte, the first and third layers comprised of a polyanion, and second layer of a polycation. The shell formed has mucoadhesivity as the polyelectrolytes forming the shell are mucoadhesive in nature.

The polyelectrolyte coating protects the calcium phosphate nanoparticles and the nucleic acid from degradation in high as well as low environmental pH, exposure to en2ymes and other adverse conditions during delivery in gastrointestinal tract and uptake by the mucosal route. At low pH the polyanion layers do not imbibe water and thus prevent the uptake of water or salts through the shell. This protects the core from degradation as hydroxyapatite nanocrystals are known to degrade at acidic pH. At high pH the polycation will prevent the movement of water or salts through the shell. Thus the core particle remains protected till it is taken up by the cells of the mucous membrane.

The layers of anionic and cationic polyelectrolytes will also ensure protection to nanoparticles at the stage of transcytosis through mucosal layer of epithelium. In addition the multilayered coating will ensure cell uptake by endocytosis and the process of endosomolysis. The coating will enable the attachment of cell specific ligands for both site specific gene delivery in vivo, and/ or enhanced uptake through mucosa.

The present invention provides a method for making a product for efficient delivery of genetic material through any mucosal route. The process of the invention is related to the formation of polyelectrolyte coating or shell on the core particle consisting of genetic material entrapped in calcium phosphate nanoparticles. The polyelectrolyte shell is formed to protect the core nanoparticles and the genetic material from degradation in high or low pH, on exposure to enzymes and other adverse conditions during delivery to and uptake by the cells of the mucous membrane. The polyelectrolyte shell is designed to provide mucoadhesivity and functional groups for the attachment of ligand molecules. This modification is required for both site specific gene delivery in vivo and/ or enhanced uptake through mucosa.

The pDNA loaded Calcium Phosphate Nanoparticles are synthesized using reverse micro-emulsion method wherein n-Hexane is oil phase and water as the aqueous phase. Before stirring both the micro-emulsions, excess water was added to make total volume of water, to adjust Wo, i.e. the molar ratio of water to AOT at 10. 1.36M of anhydrous Calcium Chloride and 0.35M di-Sodium Hydrogen Phosphate is used as the precursors taken in 2 separate micro- emulsion system. 3μg of pDNA of interested added into each systems and kept under continuous stirring for 12 hours. Ones, optically clear, micro-emulsion with di- Sodium Hydrogen Phosphate is added to micro-emulsion with Calcium Chloride at a slow rate with o

contmuous stirring at 4 C. The system is kept undisturbed at low temperature under continuous stirring for another 24 hours. Development of translucency indicates the nanoparticle formation in the aqueous core of the micro-emulsion droplets. N-Hexane is then removed by using Bucci- Evaporator and the resulting solid mass of AOT dissolved in 10 ml of absolute ethanol (99.9%) by vortexing. The solutions were centrifuged for half an hour at 8000 rpm at 4°C in a cold centrifuge. The pelleted nanoparticles were washed with absolute ethanol three times. Finally, the pelleted nanoparticles were dispersed in double distilled water at 4°C by vortexing to give clear dispersion. This nanoparticle dispersion is dialysed in cold room using 14kD dialysis membrane bag.

The major principle followed for coating over the nanoparticles is the electrostatic interactions between the nanoparticles and the polymers. 1ml of sample was taken, followed with addition of 1ml 0.01M pre-formed pAA in pH 8 phosphate buffer is added at extremely slow rate (1 drop/4 minutes) while under constant stirring. Once done, the mixture is incubated under stirring for 3 hours followed by 2 hours dialysis using a 14kD dialysis membrane bag. For 2^nd layering, ΙΟΟμΙ of 0.1% Chitosan of low molecular weight (1 lOkD) in 6% acetic acid was added to pAA layered CaPi NPs at slow rate and stirred for 2 hours with a dialysis at the end of the procedure. To render high positive charge to the Chitosan, the polymer is dissolved in 6% acetic acid and the pH of this acidic solution is increased to 5 using 5.2μ1 of liquor ammonia. The Chitosan layered CaPi/pDNA NPs are finally coated with pAA which is dispersed in phosphate buffer of pH 8 following the similar procedure. The coated nanoparticles are dialyzed at 4°C over night and later dispersed in neutral pH.

The uncoated and coated CaPi/pDNA NPs were initially characterized for the size and polydispersit using transmission electron microscopy. The size of uncoated nanoparticles were in the range of 60-65nm with no aggregation implying high stability of the

St

nanoparticles (Figure 2A). The 1 layered coated nanoparticles gave a slightly larger size ranging between 100- 120nm (Figure 2B). Since

HOkD Chitosan was used for 2^nd layer coating, an electron dense core of nanoparticles and a less electron dense layer was visible and the size ranged to 350nm (Figure 2C). Due to high positive charge of rd

Chitosan, the interaction of pAA of the 3 layer was high which caused the Chitosan to compact between adjacent layers of pAA. On rd closer observation of the TEM image for the 3 layered nanoparticles, an electron dense layers sandwiched the less electron dense Chitosan layer (Figure 2D).

The lyophilized nanoparticles were characterized for Fourier- Transform Infra-Red Spectroscopy and the interpretation of the spectrum for each samples revealed excellent layering/ masking of the preceding layer. Figure 3A is a spectrum for pDNA wherein peaks 3426.66 cm 1750 cm ¹ and 1227 cm ¹ corresponds to the heterocyclic NH2 group of the base pair stretching, sugar-base pair stretching, asymmetric PO2 stretching respectively present in the pDNA molecule. On coating these particles with pAA, the peaks corresponding to those of pDNA gets masked off and new peaks

(Figure 3B) at 3370 cm^"1, 1723 cm^"1, 1300-1000 cm^"1 are observed which correlates to O-H stretching, C=O absorption of Carbonyl and C-O stretching respectively. Similarly, Chitosan coating effectively masks the peaks those of pAA revealing new peaks, namely at 3500 cm \ 1593 cm 1406 cm ^Xand 1099 cm ¹ which coincides with NH2 stretch/ O-H stretch, NH2 scissoring/ NH2 and N-H wagging, CH2/CH3 deformation and C-N stretch present in Chitosan (Figure 3C). A similar change is observed when pAA is layered over Chitosan (Figure 3D). pDNA loaded CaPi NPs were synthesized and these NPs layered by polyelectrolyte in-order to protect the NPs against the harsh environment of GI Tract. In order to test the efficiency of the encapsulation of pDNA within the NPs, the naked and coated NPs were subjected to 1% agarose gel electrophoresis (Figure 4A). The free pDNA moved freely to its usual position (Lane 3), pDNA loaded within the matrix of the CaPi NPs hardly showed any movement and remained within the well (lane 5). Similar results were observed with the coated NPs (lane 6, 7 and 8). The efficiency of the layering and the protection provided by them against was confirmed by subjecting the naked and coated NPs in pH 4 and 8 buffers respectively for 4 hours at 37°C. These samples were then run on 1% agarose gel. From the Figure 4B, it is apparent that the naked CaPi NPs degraded releasing the pDNA which moved to its usual position (lane 5), whereas the NPs who were had coating over them were effective in withstanding the pH differences and proving its high effectiveness in protecting the pDNA (lane 6, 7 and 8). On the other hand, pH 8 did not show any observable change in the phenomena of the naked or the coated NPs (Figure 4C).

The size of the nanoparticles formed was found to be dependent on wo values (i.e. the molar ratio of water to AOT). The mean size distribution of CaPi NPs while dispersed in microemulsion at wo = 10 was in the range of 60-80 nm. Figure 5 is a representative size distribution profile of naked CaPi NPS encapsulating pDNA (A) and coated CaPi NPs encapsulating pDNA with pAA, pAA+Chitosan, pAA+Chitosan+pAA (B, C and D respectively). The size of naked nanoparticle in aqueous dispersion was measured to 144nm and that of the coated nanoparticles varied between 280nm to 405nm.

The effective layering of the NPs was carried out by manipulating the charges over the surface of the naked and coated NPs. In order to prove the hypothesis, pH dependent size variation of the 3^rd layered nanoparticles were carried out. The coated pDNA loaded CaPi nanoparticles were incubated differential pH conditions varying from pH 5 to pH 8. The nanoparticles incubated at pH 5, revealed a size of 287nm which gradually increased to 357 nm, 402 nm and finally 521nm on incubation of the nanoparticles in pH 6, pH 7 and lastly pH 8 respectively. (Figure 6)

This gradual decrease in size /compaction of the polymer at lower pH which is similar to the characteristic condition of the stomach helps in the prevention of nanoparticles from acidic degradation.

The surface charge of CaPi NPS and that of the coated nanoparticles were determined by measuring the zeta potential in neutral pH. The pH dependent zeta potential as shown in Figure 7 indicated that the particles were positively charged in neutral aqueous buffer (A) and that of the pAA coated particles were negatively charged (B). Chitosan coating over the pAA coated nanoparticles shifted it charges from negative towards positive (C). The final layering of the nanoparticles showed positive charge as the pAA was not able to neutralize the charges of the Chitosan (D). in-vitro data:

The commercially available transfecting agents, though highly efficient, most are known to show a little extent of toxicity. Calcium Phosphate has long been known as one of the good competitors for transfecting as the positive charge of the di-cationic Calcium, compacts and packs the pDNA based on the electrostatic interactions. Calcium Phosphate nanoparticles are has also been used in this aspect and have shown to good transfection efficiency.

Efforts were successful to determine a high transfection efficiency of these CaPi/pDNA nanoparticles on in-vitro testing which were comparable to the ones of achieved by using commercially available lipofectamin. Both uncoated and coated CaPi/pDNA has shown good transfection. HEK-293 cell line was used for the in-vitro studies. The cells were grown in the complete DMEM medium with 5% CO2 supply. Ones, the cells reached sub-confluent stage, the transfection was carried with the commercially available kit and the test samples for time period of 24 hours. It was recommend to change the medium was changed after 8 hours to minus off the cytotoxic effects caused due to lipofectamin. The test was used under normal condition without change of medium. After the experimental time period, the cells were was with O. IM phosphate buffer saline and fixed using 4% paraformaldehyde. The slides were prepared and observed under the confocal microscope. (Figure 8) in-vivo data:

Ones, the nanoparticles were proved as good transfecting agents in vitro, these nanoparticles were also tested for transfection in vivo. Albino mice of 8- 10g were used for the in-vivo experiments. The mice were kept under fasting for 12 hours prior to the experiments. About 750 μΐ of the nanoparticles loaded with pDNA (total pDNA concentration used was nearly 50 g) was fed to the mice using a feeding canuala tube though the oral route.

3 days after feeding the nanoparticles, the mice were sacrificed and the intestine was surgically removed. The intestine was thoroughly washed with saline solution. The microscopic slides with both horizontal and vertical sections were prepared for confocal imaging.

References:

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The invention can be utilized for the delivery of genetic material by non-parenteral routes such as the mucosal routes which include nasal, buccal, pulmonary and oral. The invention can be used for DNA / RNA vaccination or DNA / RNA therapy through these routes. The DNA / RNA can be localized in the mucosa, sub mucosa, the Peyer's patch or delivered in vivo through the mucous membrane. The present invention can simplify the delivery of nucleic acid for therapeutic or prophylactic purpose, making it easily applicable. Enhanced transfection efficiency by the mucosal route can be achieved using these surface coated calcium phosphate nanoparticles.

All documents cited in the description are incorporated herein by reference. The present invention is not to be limited in scope by the specific embodiments and examples which are intended as illustration of a number of aspects of the scope of this invention. Those skilled in the art will know or to be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein.

It is to be noted that the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant embodiments employing the concepts and features of this invention are intended to be within the scope of the present invention, which will be further set forth under the claims.

Claims

WE CLAIM

1. A Process for preparation of non-viral vector, for delivery of nucleic acids by mucosal route comprising the steps of: a) synthesis of pDNA loaded calcium phosphate nanoparticles using reverse micro-emulsion method having n-Hexane as oil phase and water as the aqueous phase; b) addition of excess water to make total volume of water to adjust the molar ratio of water to AOT at 10 before stirring both the micro-emulsions; c) taking 1.36M of anhydrous calcium chloride and 0.35 M di- sodium hydrogen phosphate in two separate micro- emulsion system as the precursors; d) adding 3pg of pDNA of interest into each system followed by continuous stirring for 12 hours; e) mixing the micro-emulsion with di- sodium hydrogen phosphate to the micro-emulsion with calcium chloride at slow rate with continuous stirring at 4°C; f) keeping the mixture, as obtained in step (e), undisturbed at low temperature under continuous stirring for 24 hours; g) removing n-Hexane by using Bucci-evaporator and dissolving the resulting mass of AOT in 10 ml of absolute ethanol (99.9%) by vortexing; h) centrifuging the solution for half an hour at 8000 rpm at 4°C in a cold centrifuge; i) washing the pelleted nanoparticles with absolute alcohol three times; j) dispersing the pelleted nanoparticles in double distilled water at 4°C by vortexing to secure clear dispersion; k) dialyzing the dispersion, as obtained in step (j), in cold room using 14 kD dialysis membrane bag and followed finally by coati.ng over nanoparticles with the polymers.

2. A Process for preparation of non- viral vector as claimed in claim 1 wherein coating over nanoparticles with polymers comprises of: a) addition of 1 ml of 0.01M pre-formed pAA in pH 8 phosphate buffer to equal volume of sample at extremely slow rate (1 drop/ 4 minutes) under constant stirring; b) incubation under stirring for 3 hours followed by 2 hours dialysis using 14 kD dialysis memberane; c) adding ΙΟΟμΙ of 0.1% Chitosan of low molecular weight (HOkD) in 6% acetic acid to pAA^ layered CaPi nanoparticles at slow rate with continuous stirring for 2 hours followed with a dialysis; d) pH of the dispersion obtained in step (c) is increased to 5 using 5.2μ1 of liquor ammonia to render high positive charge to chitosan; e) coating CaPi/pDNA nanoparticles with 1 ml of 0.01M preformed pAA in pH 8 phosphate buffer to equal volume of sample at extremely slow rate (1 drop/4minutes) under constant stirring; and finally f) incubation under stirring for 3 hours followed by 2 hours dialysis using 14 kD dialysis membrane.

3. A Process for preparation of non-viral vector, for delivery of nucleic acids by mucosal route as claimed in preceding claims as substantially described herein with reference to the accompanying drawings and examples.

Dated : 16^th day of January, 2013