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Among the various receptors, asialoglycoprotein receptors and mannose receptors are the most promising for gene targeting because they exhibit high affinity and are rapidly internalized. Wide variety of macrophages (including Kupffer cells) express mannose specific membrane receptors, which internalize glycoprotein bearing mannose residue via clathrin-coated vesicles, to allow a delivery into the endosomal system. In gene delivery systems, the mannosylation of cationic polymers, such as mannosylated polylysine and pei, were used to achieve the delivery of gene to macrophages 82,. Mannosylated chitosan for plasmid dna has also been reported earlier for macrophage targeting. Although chitosan has been studies for more than a decade as a gene vector for dna, odn, and sirna 54, 96, so for to our knowledge, there is no study that has been carried out to investigate the use of mannosylated chitosan nanoparticles to determine. In the present study, we formulate mannosylated lmw-water soluble chitosan formulations by self-assembled method, characterized for gel retardation assay, particle size, particles shape, and particles surface morphology and zeta potential, complexing capacity, protection ability, and mchodn nps stability. Cytotoxicity studies, transfection efficiency, and antisense assay were also assessed for developed nanoparticles formulations. Materials and Methods The phosphorothioate modified as odn used in this study, tju-2755, designed to target the 3-utr of the primary rna transcript of tnf-, was synthesized by sigma Aldrich. The sequence of tju-2755 is 5-tgatccactcccccctccact-3; random sequence of odn is 5-cctccactgctacctcacctc-3; fitc-odn was also received from Sigma Aldrich.
It is a suitable candidate for gene delivery system due to its ability to spontaneously form interpolyelectrolyte stable complexes with genetic material (ODNs or dna) as a result of cooperative electrostatic henna interactions between the positive amino groups of chitosan and the negative phosphate groups. Consequently, several groups have conducted studies using chitosan/dna nanoparticles, including use of galactosylated chitosan 60, galactosylated (PVP) 61, mannosylated chitosan/dna nanoparticles 62, trimethylated chitosan oligomers 63, n-dodecylated chitosan 64, deoxycholic acid modified chitosan 65, galactosylated chitosan/odn vector 54, or ligand attached chitosans for targeting cell. However, chitosan was difficult to solubilize in water and was dissolved in acidic solution. Low molecular weight water soluble chitosan has been employed, which is highly water soluble and forms complex with plasmid dna/odn at physiological pH 54, 67,. They can display high shiseido transfection efficiency along with the higher plasmid dna/odn loading, protection against the nuclease degradation, and being less toxic 54, 69,.
Ligands are molecular extension that pave fate of delivery system for active targeting and increase overall therapeutic potential. Thus the receptor mediated gene targeting is a promising approach to obtain cell-selective gene transfection. A number of receptor mediated gene delivery systems have been developed to introduce the foreign dna/odn into specific cell types. Ligands currently being investigated include galactose 71 80, mannose 66, 81 85, lactose 86, transferrin 87, 88, and epidermal growth factor. Active targeting using receptor mediated interaction has been effective in gene delivery.
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Among the gene delivery vectors, nonviral vectors are preferred due to its ease of synthesis, low immunogenicity, and unrestricted gene materials size in addition to potential benefits in terms of safety. Nanoparticles (NPs) were introduced as nonviral gene vectors to solve issues related to odns delivery. Other carriers such as microspheres, matrices, complexes with polycations, and starburst dendrimers have been recently investigated 10,. The major advantage of using nanocarriers resides in the possibility of conjugating ligands to them as to direct them to a desired site for localized delivery in cells, tissue, or organ. Odns in complex state with nanocarriers avail protection against nuclease degradation 38,.
Further, their cell uptake could be increased as carrier-odns complexes are taken up through active endocytosis process. Recently, biodegradable nanoparticles have been studied as potential inert and biocompatible carriers for genetic materials, for example, polylactide polyethylene glycol (pla-peg cationic polystyrene spongelike alginate, polyalkylcyanoacrylate, poly isohexylcyanoacrylate or poly isobutylcyanoacrylate, and albumin chitosan nanoparticles 36,. Among the large number of cationic polymers described, chitosan is shown to be an effective vector because of condensing and delivered dna, sirna in vitro and in vivo 52, 53, and odn in vitro. It is a natural positively charged polymer that can be utilized for preparation ofnanoparticulate carriers which represents a novel strategy for the safe and efficient delivery of gene. It has been extensively examined for its potential in the development of controlled release drug delivery formulation due to its unique polymeric cationic characteristic gel forming and film forming properties 55,. Chitosan has beneficial qualities such as low toxicity, low immunogenicity, excellent biodegradability, and biocompatibility 57,.
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These include inhibition of transcription, inhibition of splicing, and inhibition of mrna maturation. Among them, activation of rnase h is by far one of the most recognized theories accepted by many researchers. After entering cells, odns hybridize to wrinkled the target mrna and form a sense-antisense rna dna duplex. For therapeutic application, antisense technology promises greater advantages over drugs currently on the market by offering new types of drugs that are easy to design and have a very high molecular target selectivity and efficacy, inhibiting a specific gene expression, and an expected low toxicity. Vitravene, is only odn based therapeutic (antisense odn) approved by fda and several others are in Phase 3 clinical trials. However, odns therapeutics have been associated with multiple obstacles, that is, poor physiological stability and cellular uptake 11, 12, inadequate intracellular odns concentration 13, 14, inability to target specific cell 14, 15, lack of tissue specificity 16, and nuclease degradation 14,. Thus, intracellular delivery and therapeutic applications in diseases of odn-based therapeutics have been compromised. Although various chemical modifications of odns can be used to overcome these problems 18, they offer other limitations such as low binding affinity, nonsequences specific biological effects, toxicity, and acute homeostatic changes after in vivo administration. Therefore, development of effective delivery systems that are capable of protecting and efficiently deliver odns intracellularly in target cells español becomes essential to exploit the promising applications offered by successful odn delivery.
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Oligonucleotides based therapeutic techniques have attracted much attention because they provide a rational way to design antisense oligonucleotides (As-odn decoy oligonucleotides that bind to dna-binding regulatory proteins, and sirna/mirna that suppress specific gene expressions. Odns with base sequences complementary to a specific rna have offered the sportschema exciting potential to selectively modulate the expression of an individual gene and, thus, possess the potential for activity in the treatment of viral infections or cancer. Odns include a new generation of antiviral, antitumoral, and anti-inflammatory agents, in various categories. As-odns are anionic, large molecular weight (510 kda 1030 nucleotide bases sequence of short single strands of dna; rna their analogs molecules designed to modulate gene expression 5 and possessing high hydrophilicity. For antisense applications, odns interact and form a duplex with their complementary target mRNAs or pre-mRNAs through Watson-Crick base pairing in a sequence-specific manner and then inhibit their translation 7,. Therefore the genetic code in the rna cannot be read, which consequently inhibits production of the disease-causing protein. At the molecular level, several mechanisms of antisense actions have been proposed for antisense drugs.
Mannosylated chitosan odn nanoparticles (mchodn nps) make were formulated by self-assembled method using various ratio (moles of amine groups of mch to phosphate moieties of odns) and characterized for gel retardation assay, physicochemical characteristics, cytotoxicity and transfection efficiency, and antisense assay. Complete complexation of mch/odn was achieved at charge ratio of 1:1 and above. On increasing the ratio of mch/odn, particle size of the nps decreased whereas zeta potential (ZV) increased. Mchodn nps displayed much higher transfection efficiency into raw 264.7 cells (bears mannose receptors) than Hela cells and no significant toxicity was observed at all mch concentrations. Antisense assay revealed that reduction in lipopolysaccharide (LPS) induced serum tnf- is due to antisense activity of tju-2755 odn (sequence complementary to 3-utr of tnf-). These results suggest that mchodn nps are acceptable choice to improve transfection efficiency in vitro and in vivo. Introduction, gene therapy is a potential approach to cure or prevent diseases through gene expression before the transcription stage. It includes approaches based on utilization of deoxyribonucleic acid (DNAs oligodeoxyribonucleotides (ODNs small interference rnas (sirna and so forth for treatment and mitigation of diseases.
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1Maharishi markandeshwar College of Pharmacy, maharishi markandeshwar University, mullana Ambala, haryana 133207, India 2Department of Pharmaceutical Sciences,. Hari singh gour Vishwa vidyala, sagar, madhya pradesh 470003, India. Copyright 2014 gyati Shilakari Asthana. This is an open access article distributed under the. Creative commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The therapeutic potential of antisense oligonucleotides (asodn) is primarily dependent upon its safe and efficient delivery to specific cells overcoming degradation and maximizing cellular uptake in vivo. The present study focuses on designing mannosylated low molecular weight (LMW) chitosan nanoconstructs for safe odns delivery by macrophage targeting. Mannose groups were coupled with lmw chitosan and characterized spectroscopically.