cRGD grafted siRNA nano-constructs for chemosensitization of gemcitabine hydrochloride in lung cancer treatment.

PURPOSE: The aim of present investigation was to effectively deliver ribonucleotide reductase subunit 1 (RRM1) targeted siRNA and assess chemo-sensitization of lung cancer cells against Gemcitabine hydrochloride. It was hypothesised that effective and selective delivery of RRM1 siRNA will help in the treatment of lung cancer chemotherapy using Gemcitabine hydrochloride by reducing drug dose and thereby, reduces dose related toxicity of Gemcitabine hydrochloride. METHODS: In this investigation, cRGD grafted siRNA nano-constructs were developed for efficient and targeted intracellular delivery of siRNA. Developed formulations were characterized for gel retardation assay, particle size, zeta potential, cryo transmission electron microscopy, serum stability, in vitro cytotoxicity, qualitative and quantitative cell uptake, gene expression, and chemo-sensitization. RESULTS: Complete complexation of siRNA with cRGD grafted nano-constructs was found at N/P ratio of 2.0. Naked siRNA was found to degrade within 6 h in presence of 50% serum while nano-constructs protected the complexed siRNA even after 24 h. RRM1 level significantly reduced when siRNA was delivered in nano-construct form as compared to naked siRNA. Pre-exposure of RRM1 siRNA decreased the IC50 value of Gemcitabine hydrochloride 5 folds in A-549 cells compared to Gemcitabine hydrochloride alone. CONCLUSION: These results suggest the application of present siRNA delivery strategy to potentiate the chemotherapeutic effect by means of chemosensitization which may be utilized for effective and thorough remission of lung cancer.

Development and characterization of siRNA lipoplexes: Effect of different lipids, in vitro evaluation in cancerous cell lines and in vivo toxicity study.

Cationic liposomes have long been used as non-viral vectors for small interfering RNA (siRNA) delivery but are associated with high toxicity, less transfection efficiency, and in vivo instability. In this investigation, we have developed siRNA targeted to RRM1 that is responsible for development of resistance to gemcitabine in cancer cells. Effect of different lipid compositions has been evaluated on formation of stable and less toxic lipoplexes. Optimized cationic lipoplex (D2CH) system was comprised of dioleoyl-trimethylammoniumpropane (DOTAP), dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), hydrogenated soya phosphocholine (HSPC), cholesterol, and methoxy(polyethyleneglycol)2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG2000-DSPE). D2CH lipoplexes have shown particle size (147.5 ± 2.89 nm) and zeta potential (12.26 ± 0.54 mV) characteristics essential for their in vivo use. In vitro cytotoxicity study has shown low toxicity of developed lipoplexes as compared with lipofectamine-2000 up to N/P ratio as high as 7.5. Cell uptake studies and gene expression studies have confirmed intracellular availability of siRNA. In addition, developed lipoplexes also showed ~3 times less hemolytic potential as compared with DOTAP/DOPE lipoplexes at lipid concentration of 5 mg/mL. Lipoplexes also maintained particle size less than 200 nm on exposure to high electrolyte concentration and showed >70% siRNA retention in presence of serum showing siRNA protection conferred by lipoplexes. Furthermore, in vivo acute toxicity studies in mice showed that formulation was non-toxic up to a dosage of 0.75 mg of siRNA/kg as lipoplexes and 300 mg lipid/kg as blank liposomes indicating tolerability of lipoplexes at a dose much higher than required for therapeutic use. Promising results of this study warrant further investigation of developed siRNA lipoplexes for cancer treatment.

Mucosal immunization: a review of strategies and challenges.

The vast majority of pathogens enter the human body via the mucosal surfaces of the gastrointestinal, respiratory, and urogenital tracts, where they initiate mucosal infections that lead to systemic infections. Despite strong evidence that a good mucosal immune response can effectively prevent systemic infection too, only a few mucosal vaccines are available due to their low efficiency. Most current immunization techniques involve systemic injection, but they are ineffective to induce immunization at a mucosal site. It is a great challenge to target a mucosal compartment that can induce protective immunity at mucosal sites as well as systemic sites. A better understanding of cellular and molecular factors involved in the regulation of mucosal immunity will aid in the design of safer mucosal vaccines that elicit the desired protective immunity against infectious diseases such as HIV. The development of mucosal vaccines, whether for prevention of infectious diseases or for immunotherapy, requires antigen delivery and adjuvant systems that can effectively present vaccine or immunotherapeutic antigens to the mucosal sites. In this review, we examine the mechanism of mucosal protection, induction of mucosal immune response, types of vaccines, current status of marketed vaccines, and novel strategies for protection against infections and for treatment of inflammatory disorders. Additionally, we offer perspectives on future challenges and research directions.

cRGD grafted liposomes containing inorganic nano-precipitate complexed siRNA for intracellular delivery in cancer cells.

Development of effective vector for intracellular delivery of siRNA has always been a challenge due to its hydrophilicity, net negative surface charge and sensitivity against nucleases in biological milieu. The present investigation was aimed to develop a novel non-viral liposomal carrier for siRNA delivery. Nano-precipitate of calcium phosphate was entrapped in liposomes composed of a neutral lipid (DPPC), a fusogenic lipid (DOPE), a PEGylated lipid (DSPE-mPEG2000) and cholesterol. siRNA was made permeable through liposomal bilayer and complexed to calcium phosphate precipitates inside the liposomes. siRNA entrapped liposomes were further grafted with cRGD to achieve targeting potential against cancer cells. More than 80% of siRNA was entrapped inside the liposomes having average particle size below 150nm. Cryo-transmission electron microscopy revealed the intra-liposomal calcium phosphate precipitation and unilamellar morphology of prepared liposomes. The viability of A549 lung cancer cells was significantly higher after treatment with siRNA entrapped liposomes as compared to Lipofectamine2000 complexed siRNA. Fluorescent intensity in lung carcinoma cells was significantly higher after exposure to fluorescent siRNA entrapped liposomes than with Lipofectamine2000, which were confirmed by both confocal microscopy and flow cytometry. Live imaging by confocal microscopy ascertained the targeting efficacy of cRGD grafted liposomes compared to naked siRNA and non-grafted liposomes. Developed liposomal formulation showed effective protection of siRNA against serum nucleases along with less haemolytic potential and excellent stability against electrolyte induced flocculation. At 5nM concentration gene expression of target protein was reduced up to 24.1±3.4% while Lipofectamine2000 reduced expression level up to 26.35±1.55%. In vivo toxicity in mice suggested admirable safety profile for developed lipid based delivery vector. These results advocate that prepared liposomal system would be of high value for intracellular delivery of siRNA.

Protein functionalized tramadol-loaded PLGA nanoparticles: preparation, optimization, stability and pharmacodynamic studies.

Poly (d,l-lactide-co-glycolide acid) (PLGA) Nanoparticles (NPs) with sustained drug release and enhanced circulation time presents widely explored non-invasive approach for drug delivery to brain. However, blood-brain barrier (BBB) limits the drug delivery to brain. This can be overcome by anchoring endogenous ligand like Transferrin (Tf) and Lactoferrin (Lf) on the surface of NPs, allowing efficient brain delivery via receptor-mediated endocytosis. The aim of the present investigation was preparation, optimization, characterization and comparative evaluation of targeting efficiency of Tf- vs. Lf-conjugated NPs. Tramadol-loaded PLGA NPs were prepared by nanoprecipitation techniques and optimized using 3(3) factorial design. The effect of polymer concentration, stabilizer concentration and organic:aqueous phase ratio were evaluated on particle size (PS) and entrapment efficiency (EE). The formulation was optimized based on desirability for lower PS (<150 nm) and higher EE (>70%). Optimized PLGA NPs were conjugated with Tf and Lf, characterized and evaluated for stability study. Pharmacodynamic study was performed in rat after intravenous administration. The optimized formulation had 100 mg of PLGA, 1% polyvinyl alcohol (PVA) and 1:2 acetone:water ratio. The Lf and Tf conjugation to PLGA NPs was estimated to 186 Tf and 185 Lf molecules per NPs. Lyophilization was optimized at 1:2 ratio of NPs:trehalose. The NPs were found stable for 6 months at refrigerated condition. Pharmacodynamic study demonstrated enhanced efficacy of ligand-conjugated NPs against unconjugated NPs. Conjugated NPs demonstrated significantly higher pharmacological effect over a period of 24 h. Furthermore Lf functionalized NPs exhibited better antinociceptive effect as compared to Tf functionalized NPs.

Formulation and development of taste masked fast-disintegrating tablets (FDTs) of Chlorpheniramine maleate using ion-exchange resins.

CONTEXT: Masking of bitter taste of drug for better patient compliance. OBJECTIVE: The objective of this research was to mask the bitter taste of Chlorpheniramine maleate using cation exchange resins. MATERIALS AND METHODS: Different cation exchange resins were used for taste masking. The drug resin complexes (DRC) were prepared by batch process. Complexes of ion-exchange resin and Chlorpheniramine maleate were prepared by taking drug: resin ratios 1:1, 1:2, 1:3 and 1:4 (w/w). The optimum drug:resin ratio and the time required for maximum complexation was determined. The drug resinates were evaluated for the drug content, taste, drug release, FTIR, DSC and X-ray diffraction (PXRD). RESULTS AND DISCUSSION: The X-ray diffraction study confirmed the monomolecularity of entrapped drug in the resin beads. The taste evaluation depicted the successful taste masking of Chlorpheniramine maleate with DRCs. Fast disintegrating tablets (FDTs) were developed depending upon percent complexation, release study at salivary and gastric pH, taste evaluation; Chlorpheniramine maleate: Indion-234 complex of ratio 1:2 was used to develop and formulate FDTs. The drug release of 94.77% in 30 min was observed from FDTs. CONCLUSION: The Effective taste masking can be obtained from DRC that can be formulated as FDTs for better patient compliance.

Patents review in siRNA delivery for pulmonary disorders.

siRNA inhibits protein expression by degrading complementary mRNA sequence and hence, it is widely applicable for the treatment of various diseases where single or multiple gene knock down is necessary. Due to the severity and lethality of pulmonary diseases, siRNA has been focused for improved health in these diseases. Pulmonary accumulation of siRNA can be achieved by different means like intranasal or inhalation administration or intratracheal route which is mainly utilized for in vivo animal studies. However, various pulmonary obstacles and intracellular barriers for siRNA transport challenge this novel therapeutic moiety. Researchers have utilized different viral and non-viral delivery vectors for intracellular delivery of siRNA to knock down target mRNA. The promise of RNA interference, mediated by siRNAs, has revolutionized the prospects for modulating gene expression as a way to achieve therapeutic aims in disease treatment. This review focuses on patents describing the siRNA delivery either in naked form or along with a single/multiple delivery vectors. Many inventors have shown promising results for pulmonary utilization of siRNA and more concentration on delivery system may make this genomic approach available to the clinics soon.