Effect of functionalization of polymeric nanoparticles incorporated with whole attenuated rabies virus antigen on sustained release and efficacy

AbstractNanovaccines introduced a new dimension to prevent or cure diseases in an efficient and sustained manner. Various polymers have been used for the drug delivery to increase the therapeutic value with minimal side effects. Thus the present study incorporates both nanotechnology and polymers for the drug delivery. Poly(d,l-lactic-co-glycolic acid)-b-poly(ethylene glycol) was incorporated with the rabies whole attenuated viral antigen using double emulsion (W/O/W) method and characterized by Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Chitosan-PEG nanoparticles incorporated with the rabies whole attenuated virus antigen (CS-PEG NP-RV Ag.) were prepared using Ionic Gelation method. The CS-PEG NP-RV Ag. was surface modified with biocompatible polymers such as Acacia, Bovine Serum Albumin (BSA), Casein, Ovalbumin and Starch by Ionic Gelation method. The morphology was confirmed by SEM and Transmission Electron Microscopy (TEM). The surface modification was confirmed by Fourier Transform Infrared Spectroscopy (FTIR), Zeta potential. The size distribution of CS-PEG-RV Ag. and surface modified CS-PEG-RV Ag. by respective biocompatible polymers was assessed by Zetasizer. Release profile of both stabilized nanoparticles was carried out by modified centrifugal ultrafiltration method which showed the sustained release pattern of the Rabies Ag. Immune stimulation under in-vitro condition was studied using rosette assay and phagocytosis assay. In-vitro toxicity using human blood and genotoxicity using human blood DNA was also studied to assess the toxicity of the nanoformulations. The results of these studies infer that PLGA-b-PEG nanoparticles, CS-PEG and surface modified CS-PEG nanoparticles may be an efficient nanocarrier for the RV Ag. to elicit immune response sustainably with negligible toxic effect to the human system

Evaluation of cystamine-modified hyaluronic acid/chitosan polyplex as retinal gene vector

A successful gene therapy approach can prevent or treat congenital and acquired diseases. However, there is still no ideal non-viral vector for gene delivery in a safe and timely manner. In this report the anionic polymer hyaluronic acid (HA) was investigated as a potential vector for gene therapy. Due to its intrinsic characteristics it constitutes an excellent candidate to deliver therapeutic genes, pending the modification of its surface charge

Cell-penetrating chitosan/doxorubicin/TAT conjugates for efficient cancer therapy

In this study, a cell-penetrating peptide, the transactivating transcriptional factor (TAT) domain from HIV, was linked to a chitosan/doxorubicin (chitosan/DOX) conjugate to form a chitosan/DOX/TAT hybrid. The synthesized chitosan/DOX/TAT conjugate showed a different intracellular distribution pattern from a conjugate without TAT. Unlike both free DOX and the conjugate without TAT, the chitosan/DOX/TAT conjugate was capable of efficient cell entry. The chitosan/DOX/TAT conjugate was found to be highly cytotoxic, with an IC 50 value of approximately 480 nM, 2 times less than that of chitosan/DOX (980 nM). The chitosan/DOX/TAT provided decreases in tumor volume of 77.4 and 57.5% compared to free DOX and chitosan/DOX, respectively, in tumor-bearing mice. Therefore, this study suggests that TAT-mediated chitosan/DOX conjugate delivery is effective in slowing tumor growth.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/83474/1/25578_ftp.pd

Oligopeptide-mediated gene transfer into mouse corneal endothelial cells: expression, design optimization, uptake mechanism and nuclear localization

Gene transfer to the corneal endothelium has potential in preventing corneal transplant rejection. In this study, we transfected mouse corneal endothelial cells (MCEC) with a class of novel arginine-rich oligopeptides. The peptides featured a tri-block design and mediated reporter gene expression in MCEC more efficiently than the commercial polyethylenimine standard. The functionality of each block was demonstrated to critically influence the performance of the peptide. Results from confocal imaging and flow cytometry then showed that energy-dependent endocytosis was the dominant form of uptake and multiple pathways were involved. Additionally, uptake was strongly dependent on interactions with cell-surface heparan sulphate. Fluorescence resonance energy transfer studies revealed that the peptide/DNA entered cells as an associated complex and some will have dissociated by 8.5 h. Large-scale accumulation of uncondensed DNA within the nucleus can also be observed by 26 h. Finally, as a proof of biological relevance, we transfected MCEC with plasmids encoding for the functional indoleamine 2,3-dioxygenase (IDO) enzyme. We then demonstrated that the expressed IDO could catalyse the degradation of l-tryptophan, which in turn suppressed the growth of CD4+ T-cells in a proliferation assay

Physicochemical and biological characterization of chitosan-microRNA nanocomplexes for gene delivery to MCF-7 breast cancer cells

Cancer gene therapy requires the design of non-viral vectors that carry genetic material and selectively deliver it with minimal toxicity. Non-viral vectors based on cationic natural polymers can form electrostatic complexes with negatively-charged polynucleotides such as microRNAs (miRNAs). Here we investigated the physicochemical/biophysical properties of chitosan–hsa-miRNA-145 (CS–miRNA) nanocomplexes and the biological responses of MCF-7 breast cancer cells cultured in vitro. Self-assembled CS–miRNA nanocomplexes were produced with a range of (+/−) charge ratios (from 0.6 to 8) using chitosans with various degrees of acetylation and molecular weight. The Z-average particle diameter of the complexes was <200 nm. The surface charge increased with increasing amount of chitosan. We observed that chitosan induces the base-stacking of miRNA in a concentration dependent manner. Surface plasmon resonance spectroscopy shows that complexes formed by low degree of acetylation chitosans are highly stable, regardless of the molecular weight. We found no evidence that these complexes were cytotoxic towards MCF-7 cells. Furthermore, CS–miRNA nanocomplexes with degree of acetylation 12% and 29% were biologically active, showing successful downregulation of target mRNA expression in MCF-7 cells. Our data, therefore, shows that CS–miRNA complexes offer a promising non-viral platform for breast cancer gene therapy

Chitosan-Graft-Branched Polyethylenimine Copolymers: Influence of Degree of Grafting on Transfection Behavior

BACKGROUND: Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI(x)) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI(x) derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEI(x) series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI(x) copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI(2.7%) was as effective as and less cytotoxic than the gold standard 25 kDa bPEI. CONCLUSIONS/SIGNIFICANCE: This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI(x) copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity

In vitro and in vivo mRNA delivery using lipid-enveloped pHresponsive polymer nanoparticles

Biodegradable core−shell structured nanoparticles with a poly(β-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell were developed for in vivo mRNA delivery with a view toward delivery of mRNA-based vaccines. The pH-responsive PBAE component was chosen to promote endosome disruption, while the lipid surface layer was selected to minimize toxicity of the polycation core. Messenger RNA was efficiently adsorbed via electrostatic interactions onto the surface of these net positively charged nanoparticles. In vitro, mRNA-loaded particle uptake by dendritic cells led to mRNA delivery into the cytosol with low cytotoxicity, followed by translation of the encoded protein in these difficult-to-transfect cells at a frequency of 30%. Particles loaded with mRNA administered intranasally (i.n.) in mice led to the expression of the reporter protein luciferase in vivo as soon as 6 h after administration, a time point when naked mRNA given i.n. showed no expression. At later time points, luciferase expression was detected in naked mRNA-treated mice, but this group showed a wide variation in levels of transfection, compared to particle-treated mice. This system may thus be promising for noninvasive delivery of mRNA-based vaccines.United States. Dept. of Defense (Institute for Soldier Nanotechnology, contract W911NF-07-D-0004)Ragon Institute of MGH, MIT and HarvardSingapore. Agency for Science, Technology and ResearchHoward Hughes Medical Institute (Investigator

Marine Polysaccharides in Pharmaceutical Applications: An Overview

The enormous variety of polysaccharides that can be extracted from marine plants and animal organisms or produced by marine bacteria means that the field of marine polysaccharides is constantly evolving. Recent advances in biological techniques allow high levels of polysaccharides of interest to be produced in vitro. Biotechnology is a powerful tool to obtain polysaccharides from a variety of micro-organisms, by controlling the growth conditions in a bioreactor while tailoring the production of biologically active compounds. Following an overview of the current knowledge on marine polysaccharides, with special attention to potential pharmaceutical applications and to more recent progress on the discovering of new polysaccharides with biological appealing characteristics, this review will focus on possible strategies for chemical or physical modification aimed to tailor the final properties of interest

Functionalization of chitosan via atom transfer radical polymerization for gene delivery

10.1002/adfm.201000177Advanced Functional Materials20183106-3116AFMD