Self assembled dual responsive micelles stabilized with protein for co-delivery of drug and siRNA in cancer therapy.

Design of safe and efficient vehicles for the combinatorial delivery of drugs and genetic agents is an emerging requisite for achieving enhanced therapeutic effect in cancer. Even though several nanoplatforms have been explored for the co-delivery of drugs and genetic materials the translation of these systems to clinical phase is still a challenge, mainly due to tedious synthesis procedures, lack of serum stability, inefficient scalability etc. Here in, we report development of reduction and pH sensitive polymeric graft of low molecular weight poly (styrene -alt -maleic anhydride) and evaluation of its efficacy in co-delivering drug and siRNA. The polymer was modified with suitable components, which could help in overcoming various systemic and cellular barriers for successful co-delivery of drugs and nucleic acids to cancer cells, using simple chemical reactions. The polymeric derivative could easily self assemble in water to form smooth, spherical micellar structures, indicating their scalability. Doxorubicin and PLK-1 siRNA were selected as model drug and nucleic acid, respectively. Doxorubicin could be loaded in the self assembling micelles with an optimum loading content of ∼8.6% w/w and efficient siRNA complexation was achieved with polymer/siRNA weight ratios >40. The polyplexes were stabilized in physiological saline by coating with bovine serum albumin (BSA). Stable drug loaded nanoplexes, for clinical administration, could be easily formulated by gently dispersing them in physiological saline containing appropriate amount of albumin. Drug release from the nanoplexes was significantly enhanced at low pH (5) and in the presence of 10 mM glutathione (GSH) showing their dual stimuli sensitive nature. In vitro cell proliferation assay and in vivo tumor regression study have shown synergistic effect of the drug loaded nanoplexes in inhibiting cancer cell proliferation. Facile synthesis steps, scalability and ease of formulation depict excellent clinical translation potential of the proposed nanosystem.

Combinatorial delivery of superparamagnetic iron oxide nanoparticles (γFe2O3) and doxorubicin using folate conjugated redox sensitive multiblock polymeric nanocarriers for enhancing the chemotherapeutic efficacy in cancer cells.

Redox sensitive, folate conjugated multiblock polymeric system of (-PLGA-PEG-PLGA-urethane-ss-) demonstrated self-assembly into stable nanoplatforms. The polymeric nanocarriers were encapsulated with doxorubicin and highly crystalline γFe2O3 superparamagnetic iron oxide nanoparticles (SPIONs), for co-delivery of the same to cancer cells, with average particle size of ~170nm and zeta potential of ~-33mV. Furthermore, the designed formulation was evaluated for protein adsorption, hemo-cytocompatibility and stability. Glutathione (GSH) induced redox sensitivity of the nanocarriers was depicted by ~4.47 fold increase in drug release in the presence of 10mM GSH. In vitro cellular uptake studies of the designed nanocarriers showed synergistic cytotoxic effect in folate overexpressing cells (HeLa and MDA-MB-231), after subjecting the cells to radio frequency (RF) induced hyperthermia (~43°C). Negligible effect of the combinatorial therapy was observed in normal cells (L929). The developed polymeric system depicted facile synthesis, reproducibility and potential for achieving combinatorial and targeted delivery of drug and SPIONs to cancer cells. This combinatorial approach can help in achieving better therapeutic effect with minimal side effects of chemotherapy.

Folic acid and trastuzumab conjugated redox responsive random multiblock copolymeric nanocarriers for breast cancer therapy: In-vitro and in-vivo studies.

The study represents synthesis, characterization and biological evaluation of redox responsive polymeric nanoparticles based on random multiblock copolymer for doxorubicin delivery in breast cancer. The random multiblock copolymer was synthesized via ring opening polymerization of lactide with polyethylene glycol to form triblock copolymer followed by isomerization polymerization of the triblock copolymer and 2-hydroxyethyl disulfide with the help of hexamethylene diisocynate in presence of dibutyltin dilaurate as a catalyst. Folic acid was conjugated to hydroxyl group from the multiblock polymer through DCC-NHS coupling. High drug loading content of ∼22% was achieved in the polymeric nanoparticles with size range of ∼110nm and polyethylene glycol fraction of ∼18% in the multiblock copolymer. Drug release profile confirmed the redox responsive behavior of polymeric nanoparticles with ∼72% drug release at pH 5.5 in presence of 10mM GSH as compared to ∼18% drug release at pH 7.4. In vitro cellular uptake studies showed ∼22% cellular uptake with dual (folic acid and trastuzumab) conjugated polymeric nanoparticles as compared to non-targeted polymeric nanoparticles. Fluorescence activated cell sorting (FACS) studies demonstrated higher apoptosis (∼80%) as compared to non-conjugated polymeric nanoparticles (20%) in MCF-7 cell line. In vivo studies showed 91% tumor regression in Ehrlich ascites tumor (EAT) as compared to free doxorubicin treated mice without showing any significant toxicity. Thus, it is envisaged that these redox responsive polymeric nanocarriers act as Trojan horses in cancer therapeutics.

Click modified amphiphilic graft copolymeric micelles of poly(styrene-alt-maleic anhydride) for combinatorial delivery of doxorubicin and plk-1 siRNA in cancer therapy.

The anti-apoptotic defense mechanism of cancer cells poses a major hurdle which makes chemotherapy less effective. Combinatorial delivery of drugs and siRNAs targeting anti-apoptotic proteins is a vital means for improving therapeutic effects. The present study aims at designing a suitable carrier which can effectively co-deliver doxorubicin and plk1 siRNA to tumor cells. Low molecular weight poly(styrene-alt-maleic anhydride) was chemically modified via a click reaction to obtain a cationic amphiphilic polymer for the co-delivery of therapeutic agents. Short glycol chains were utilized as linker molecules for grafting which in turn imparted a stealth nature and minimized plasma protein adsorption to the polymeric surface. Isonicotinic acid was grafted to the polymer due to its ability to penetrate the endolysosomal membrane and arginine-lysine conjugates were embedded for complexing siRNA. The polymer was able to self-assemble in to smooth, spherical micellar structures with a CMC of ∼3 µg mL-1. The particle size of the micelles was ∼14-30 nm as depicted using TEM and FESEM. Atomic force microscopic analysis showed an average height of ∼12 nm for the polymeric micelles. An optimum doxorubicin loading of ∼9% w/w was achieved with the micelles using a dialysis method. Effective complexation of siRNA occurred above a polymer/siRNA weight ratio of 10 without any significant change in the particle size. Doxorubicin and fluorescent labeled siRNA loaded micelles exhibited excellent co-localization within the cytoplasm of MCF-7 cells. The synergistic effect of the active agents in inhibiting tumor cell proliferation was depicted using an MTT assay and visualized using calcein/propidium iodide staining of the treated cells. Co-administration of doxorubicin and plk1 siRNA in EAT tumor bearing Swiss albino mice using the cationic micelles significantly enhanced the antitumor efficacy.

Eugenol nanocapsule for enhanced therapeutic activity against periodontal infections.

Eugenol is a godsend to dental care due to its analgesic, local anesthetic, and anti-inflammatory and antibacterial effects. The aim of the present research work was to prepare, characterize and evaluate eugenol-loaded nanocapsules (NCs) against periodontal infections. Eugenol-loaded polycaprolactone (PCL) NCs were prepared by solvent displacement method. The nanometric size of the prepared NCs was confirmed by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The in vitro drug release was found to follow a biphasic pattern and followed Michaelis-Menten like model. The percentage cell viability values near to 100 in the cell viability assay indicated that the NCs are not cytotoxic. In the in vivo studies, the eugenol NC group displayed significant difference in the continuity of epithelium of the interdental papilla in comparison to the untreated, pure eugenol and placebo groups. The in vivo performance of the eugenol-loaded NCs using ligature-induced periodontitis model in rats indicated that eugenol-loaded NCs could prevent septal bone resorption in periodontitis. On the basis of our research findings it could be concluded that eugenol-loaded PCL NCs could serve as a novel colloidal drug delivery system for enhanced therapeutic activity of eugenol in the treatment of periodontal infections.

Lopinavir loaded solid lipid nanoparticles (SLN) for intestinal lymphatic targeting.

The poor orally available lopinavir was successfully encapsulated in glyceryl behenate based solid lipid nanoparticles (Lo-SLN) for its ultimate use to target intestinal lymphatic vessels in combined chemotherapy-the so-called Highly Active Anti-Retroviral Therapy (HAART). SLN with mean particle size of 230 nm (polydispersity index, PDI<0.27) and surface electrical charge of approx. -27mV, were produced by hot homogenization process followed by ultrasonication. Particles were characterized using differential scanning calorimetry (DSC), wide angle X-ray scattering (WAXS) and atomic force microscopy (AFM) to confirm their solid character and the homogeneous distribution of drug within the lipid matrix. In vitro release studies at pH 6.8 phosphate buffer (PBS) and at pH 1.2 HCl 0.1N showed a slow release in both media. From the intestinal lymphatic transport study it became evident that SLN increased the cumulative percentage dose of lopinavir secreted into the lymph, which was 4.91-fold higher when compared with a conventional drug solution in methyl cellulose 0.5% (w/v) as suspending agent (Lo-MC). The percentage bioavailability was significantly enhanced. The AUC for the Lo-SLN was 2.13-fold higher than that obtained for the Lo-MC of similar concentration. The accelerated stability studies showed that there was no significant change in the mean particle size and PDI after storage at 25±2°C/60±5% RH. The shelf life of optimized formulation was assessed based on the remained drug content in the stabilized formulation and was shown to be 21.46 months.