Buddhadev Layek

Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells

Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack of stability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles of amphiphilic polymers) are considerably more stable compared to liposomes; however, they also demonstrate a slow release for the encapsulated contents, limiting their efficacy as a drug-delivery tool. As a solution, we prepared and characterized echogenic polymersomes, which are programmed to release the encapsulated drugs rapidly when incubated with cytosolic concentrations of glutathione. These vesicles encapsulated air bubbles inside and efficiently reflected diagnostic-frequency ultrasound. Folate-targeted polymersomes showed an enhanced uptake by breast and pancreatic-cancer cells in a monolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with the anticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. With further improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offering a triggered release as well as diagnostic ultrasound imaging.

Amino Acid Grafted Chitosan for High Performance Gene Delivery: Comparison of Amino Acid Hydrophobicity on Vector and Polyplex Characteristics

To develop a safe, effective, and biocompatible gene delivery vector, a series of hydrophobic amino acid grafted chitosan (AGC) derivatives were synthesized by carbodiimide mediated coupling reaction. Chemical characterization of AGC polymers was performed by NMR and elemental analysis. AGC polymers demonstrated excellent blood compatibility and cell viability, as evaluated by hemolysis and MTT assay, respectively. AGC polymers could effectively bind and condense plasmid DNA (pDNA) to form polyplexes in the size range of 161-263 nm and possessed net cationic charge. The resultant polyplex showed 3.4-5.4-fold greater cellular uptake and 13-30-fold higher transfection efficiency in HEK 293 cells as compared to unmodified chitosan. Moreover, both cellular uptake and transfection efficiency improved with the increasing amino acid hydrophobicity. Hydrophobic amino acid substitution contributed to the enhance pDNA release at cytosolic pH. These data demonstrate AGC polymers as a promising novel nonviral gene delivery vector.

Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of rhein in human plasma: Application to a pharmacokinetic study

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of rhein with 100 microL human plasma using celecoxib as an internal standard (IS). The API-4,000 Q-Trap LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray ionization technique. The assay procedure involved extraction of rhein and IS from human plasma with acetonitrile, which yielded consistent recoveries of 36.01 and 65.85% for rhein and IS, respectively. The total chromatographic run time was 5.0 min and the elution of rhein and IS occurred at approximately 1.60 and 3.96 min, respectively. The resolution of peaks was achieved with 0.01 m ammonium acetate (pH 6.0):acetonitrile:methanol (30:58:12, v/v) on an Inertsil ODS-3 column. The method was proved to be accurate and precise at a linearity range of 0.005-5.00 microg/mL with a correlation coefficient (r) of >or=0.995. The lower limit of quantitation was 0.005 microg/mL. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. Rhein was found to be stable in the battery of stability studies. The application of the assay to pre-clinical pharmacokinetic studies confirmed the utility of the assay to derive pharmacokinetic parameters.

Cell Penetrating Peptide Conjugated Chitosan for Enhanced Delivery of Nucleic Acid

Gene therapy is an emerging therapeutic strategy for the cure or treatment of a spectrum of genetic disorders. Nevertheless, advances in gene therapy are immensely reliant upon design of an efficient gene carrier that can deliver genetic cargoes into the desired cell populations. Among various nonviral gene delivery systems, chitosan-based carriers have gained increasing attention because of their high cationic charge density, excellent biocompatibility, nearly nonexistent cytotoxicity, negligible immune response, and ideal ability to undergo chemical conjugation. However, a major shortcoming of chitosan-based carriers is their poor cellular uptake, leading to inadequate transfection efficiency. The intrinsic feature of cell penetrating peptides (CPPs) for transporting diverse cargoes into multiple cell and tissue types in a safe manner suggests that they can be conjugated to chitosan for improving its transfection efficiency. In this review, we briefly discuss CPPs and their classification, and also the major mechanisms contributing to the cellular uptake of CPPs and cargo conjugates. We also discuss immense improvements for the delivery of nucleic acids using CPP-conjugated chitosan-based carriers with special emphasis on plasmid DNA and small interfering RNA.

Cell penetrating peptide conjugated polymeric micelles as a high performance versatile nonviral gene carrier

The major goal of this study is to design, synthesize, and evaluate linoleic acid and penetratin dual-functionalized chitosan (CS-Lin-Pen) as a nonviral gene carrier. The amphiphilic CS-Lin-Pen self-assembles to form cationic micelles in an aqueous environment. These polymeric micelles exhibited excellent hemocompatibility and cell viability, as evaluated by in vitro hemolysis and MTT assay, respectively. When CS-Lin-Pen micelles were added to plasmid DNA (pDNA) solution, the electrostatic interaction between the cationic micelles and anionic pDNA led to the formation of stable CS-Lin-Pen/pDNA polyplexes with ~100 nm in size. The resultant polyplexes demonstrated ~5-fold higher cellular uptake as compared to unmodified chitosan. Furthermore, CS-Lin-Pen micelles showed efficient protection of pDNA from DNase I attack and exhibited ~34-40-fold higher transfection in comparison with unmodified chitosan in HEK 293, CHO, and HeLa cells. These findings illustrate that the CS-Lin-Pen micelles could be exploited as a potential nonviral vector for efficient gene therapy.

Hexanoic acid and polyethylene glycol double grafted amphiphilic chitosan for enhanced gene delivery: Influence of hydrophobic and hydrophilic substitution degree

Gene therapy holds immense potential as a future therapeutic strategy for the treatment of numerous genetic diseases which are incurable to date. Nevertheless, safe and efficient gene delivery remains the most challenging aspects of gene therapy. To overcome this difficulty a series of hexanoic acid (HA) and monomethoxy poly(ethylene glycol) (mPEG) double grafted chitosan-based (HPC) nanomicelles were developed as nonviral gene carrier. HPC polymers with various HA and mPEG substitution degrees were synthesized, and their chemical structures were confirmed by (1)H NMR spectroscopy. HPC nanomicelles exhibited excellent blood compatibility and cell viability, as demonstrated by in vitro hemolysis and MTT assay, respectively. The cationic HPC nanomicelles retained the plasmid DNA (pDNA) binding capacity of chitosan and formed stable HPC/pDNA polyplexes with diameters below 200 nm. Both hydrophobic and hydrophilic substitution resulted in suppressed nonspecific protein adsorption on HPC/pDNA polyplexes and increased pDNA dissociation. However, resistance against DNase I degradation was enhanced by HA conjugation while being inhibited by mPEG substitution. Amphiphilic modification resulted in 3-4.5-fold higher cellular uptake in human embryonic kidney 293 cells (HEK 293) mainly through clathrin-mediated pathway. The optimal HPC/pDNA polyplexes displayed 50-fold and 1.2-fold higher gene transfection compared to unmodified chitosan and Fugene, respectively, in HEK 293 cells. Moreover, both the cellular uptake and in vitro transfection study suggested a clear dependence of gene expression on the extent of HA and mPEG substitution. These findings demonstrate that amphiphilic HPC nanomicelles with the proper combination of HA and mPEG substitution could be used as a promising gene carrier for efficient gene therapy.

APC targeted micelle for enhanced intradermal delivery of hepatitis B DNA vaccine.

Chronic hepatitis B is a serious liver disease and puts people at high risk of death from cirrhosis and liver cancer. Although DNA vaccination has been emerged as a potential immunotherapeutic strategy for the treatment of chronic hepatitis B, the efficiencies were not adequate in clinical trials. Here we describe the design, synthesis, and evaluation of mannosylated phenylalanine grafted chitosan (Man-CS-Phe) as a DNA delivery vector for direct transfection of antigen presenting cells to improve cellular and humoral immunity to plasmid-coded antigen. The cationic Man-CS-Phe micelles condense plasmid DNA into nanoscale polyplexes and provide efficient protection of complexed DNA from nuclease degradation. The mannose receptor-mediated enhanced cell uptake and high in vitro transfection efficiency of the polyplexes were demonstrated in RAW 264.7 and DC 2.4 cells using GFP-expressing plasmid DNA. Furthermore, intradermal immunization of BALB/c mice indicated that hepatitis B DNA vaccine/Man-CS-Phe polyplexes not only induced multi-fold higher serum antibody titer in comparison to all other formulations including FuGENE HD, but also significantly stimulated T-cell proliferation and skewed T helper toward Th1 polarization. These results illustrate that the Man-CS-Phe can serve as a promising DNA delivery vector to harness both cellular and humoral arms of immune system.

Caproic acid grafted chitosan cationic nanocomplexes for enhanced gene delivery: Effect of degree of substitution

This work was designed to investigate the effect of the degree of substitutions of caproic acid on plasmid DNA (pDNA) binding, cellular uptake, biocompatibility, and transfection efficiency of caproic acid grafted chitosan (CGC). The CGC with three substitution degrees (CGC-5, CGC-15, and CGC-25) were synthesized by coupling caproic acid with chitosan. Chemical characterization of graft polymers was performed using FTIR, NMR, and elemental analysis. The CGC polymers showed good pDNA condensing capacity and efficient protection of pDNA from DNase I. The nanosized CGC/pDNA polyplexes exhibited well-defined spherical shapes and stability in serum. Isothermal titration calorimetry demonstrated reduction in CGC-pDNA binding constant with increase in the degree of caproic acid substitution. Caproic acid substitution resulted in 2-7-fold higher cellular uptake in HEK 293 cells mainly via the clathrin-mediated pathway without affecting biocompatibility. In vitro transfection study suggested a dependence of transfection efficiency on the variability of caproic acid substitution. The CGC-15 polymer exhibited 31-fold and 1.33-fold higher gene expression compared to chitosan and the marketed non-viral vector FuGENEHD, respectively. These finding suggests that the CGC-15 graft polymer is a promising non-viral gene delivery vector due to its superior transfection efficiency and biocompatibility.

N-hexanoyl, N-octanoyl and N-decanoyl chitosans: Binding affinity, cell uptake, and transfection.

Low transfection efficiency of chitosan limits its use as a non-viral vector for practical purposes. This study was designed to investigate the effect of fatty acyl chain length on physicochemical properties, pDNA binding affinity, cell uptake, and in vitro transfection efficiency of N-acyl chitosan (NAC). NAC polymers were synthesized by carbodiimide mediated coupling reaction of chitosan with n-hexanoic, n-octanoic, and n-decanoic acid, respectively. These NAC polymers effectively condensed pDNA resulting in the size range of 220-342 nm with net positive charge. NAC polymers also showed good pDNA binding capacity, high protection of pDNA from nuclease degradation and excellent biocompatibility. Transfection efficiency of chitosan, in HEK 293 cells, was enhanced 15-25-fold after coupling with fatty acid and increased with a decrease in fatty acyl chain length of NAC. Thus, the present study demonstrates that the NAC polymers hold great potential as novel non-viral gene delivery vector.

Tamoxifen Citrate Encapsulated Sustained Release Liposomes: Preparation and Evaluation of Physicochemical Properties

The present study was designed for the development of a stable sustained release liposomal drug delivery system for tamoxifen citrate (TC) using soya phosphatidylcholine (SPC), cholesterol (CH) and span 20 as main ingredients. Liposomes were prepared by formation of thin lipid film followed by hydration. The mean vesicle diameter was found to be 203.5 ± 19.5 nm with 21% of the liposomal population having average diameter below 76.72 ± 6.7 nm. There was a good vesicular distribution with the polydispersity index of 0.442 ± 0.03. The maximum loading of drug was determined to be 53.60% of the initial amount that is 34.58 μg of drug per mg of lipid. Amongst the different storage conditions, liposomes stored at 2–8°C were found to be most stable and only 4% of the drug was lost over the storage period of 5 weeks. In vitro release studies of liposomes showed that 50% of drug was released within 3 hours (h) whereas 95% drug was released in 30 h. This indicates the usefulness of the liposomal delivery system for sustaining the in vitro release of tamoxifen citrate.