Mohan Rathi

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.

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.

Development and Characterization of siRNA Lipoplexes: Effect of Different Lipids, In Vitro Evaluation in Cancerous Cell Lines and In Vivo Toxicity Study

ABSTRACTCationic 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.

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 33 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.

Gene Delivery Using Physical Methods

Gene delivery using nonviral methods is an exciting research area that has been explored for intracellular transgene expression in gene therapy. Nonviral gene delivery is achieved using both physical and chemical methods for safe and efficient transgene expression in target cells. The various physical methods used for gene delivery are naked DNA injection, electroporation, hydrodynamic gene therapy, particle bombardment with a gene gun, microinjection, sonoporation/ultrasound, iontophoresis, magnetofection, laser beam gene transduction, and impalefection. These methods involve the use of external forces for local delivery of naked DNA. This chapter summarizes the basic principles, mechanisms, and protocols of various physical methods used in gene transfer and compares their advantages and limitations. Moreover, the technical aspects for optimum transgene expression with desired cell targetability and minimum toxicity at the desired efficiency, kinetics, in vivo feasibility, and reproducibility of all physical methods have been discussed and compared. Current research in the area of nonviral gene delivery using physical methods has demonstrated their safety and efficacy.

Abstract 2063: Improved sensitivity and in vitro efficacy of RGD grafted PEGylated gemcitabine liposomes in RRM1 siRNA pretreated cancer cells

Aim of the study was to prepare and characterize RGD grafted PEGylated liposomes of gemcitabine (PLGs) and to evaluate its cellular uptake, in vitro anti-proliferative activity and apoptotic effect in siRNA pretreated lung cancer cells. PLGs were prepared by thin film hydration method and optimized for particle size, zeta potential and entrapment efficiency. Functionalization of liposomes was done by coupling reaction between DSPE mPEG and cRGD peptide by maleimide based reaction. Similarly, RGD grafted RRM1 siRNA liposomes were also formulated and evaluated. MTT assay was done to determine IC50 values of RGD grafted PLGs in A549 and H1299 cancer cells which were pre-treated with RGD grafted RRM1 siRNA loaded liposomes at a concentration of 50 pM of siRNA for gene silencing. DNA content analysis was done by flow cytometry using rhodamine in A549. The mode of cell death at different time and concentration was determined by FITC-AnnexineV assay in A549 cells and confocal microscopy was performed to assess the potential of RGD grafting on cellular uptake. RGD conjugated and unconjugated PLGs were found Nano sized and had negative zeta potential with entrapment of 65%. H1299 cell line showed more amount of viable cells after 48 hr as compared to A549 cells in RGD conjugated PLGs. siRNA pre-treated PLGs exposed cells showed significantly less IC50 values as compared to cells without siRNA pretreatment and non-grafted liposomes. The results showed that the RGD conjugated liposomes at the concentration of 7nM showed 46% G1 phase arrest in siRNA pretreated cells as compared to 22% G1 phase arrest without prior siRNA treatment at 16hr. Two types of mode of cell death were found during the FITC-Annexine V assay. At 24 hr, the treatment with RGD grafted PLGs resulted in 17% & 4.4% necrotic & apoptotic cell death respectively. While at equivalent drug concentration, the PLGs and drug solution showed 5.3% & 32.2% and 4.2% & 29.6% necrotic & apoptotic cell death respectively. Furthermore, the apoptosis was found to be time and concentration dependent. Results substantiate the sensitization effect by pre-exposure of siRNA in liposomal forms at Pico molar concentration along-with phagocytosis as mechanism of uptake of RGD-grafted liposomes. To conclude, prior silencing of the resistance imparting gene can manifest the effect of therapy by conferring improved sensitivity in cancer cell lines. The effect can further be augmented by employing receptor targeting peptides such as RGD. Hence, Nano-constructs of chemotherapeutic drugs conjugated with RGD can effectively target lung cancer cells and pretreatment of RRM1 siRNA can probably reduce the limitation of drug resistance associated with lung cancer chemotherapy. Citation Format: Rohan A. Lalani, Priyanka Bhatt, Mohan Rathi, Ambikanandan Misra. Improved sensitivity and in vitro efficacy of RGD grafted PEGylated gemcitabine liposomes in RRM1 siRNA pretreated cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2063.