Anupama Mittal

Efficacy of gemcitabine conjugated and miRNA-205 complexed micelles for treatment of advanced pancreatic cancer.

Clinical effectiveness of gemcitabine in pancreatic cancer is hindered due to its rapid plasma metabolism and development of chemo-resistance. We have previously delineated the significant role of miRNAs in mediating the growth and proliferation of cancer stem cells (CSCs) which in turn result in chemo-resistance, invasion and metastasis. Here, we designed self-assembling, gemcitabine conjugated cationic copolymers for co-delivery of a tumor suppressor miRNA-205 (miR-205) and evaluated their in vivo efficacy in a pancreatic cancer ectopic tumor model developed using gemcitabine resistant MIA PaCa-2(R) cells. Combination formulations showed mean a particle size of <100 nm and gemcitabine payload of >10% w/w, exhibited miRNA complexation at N/P ratio of 4/1, sustained release of gemcitabine for >10 days, transfection efficiency of >90%, extended miRNA and drug stability in serum. Functional assays in gemcitabine resistant MIA PaCa-2(R) and CAPAN-1(R) pancreatic cancer cells revealed that the combination formulations effectively reversed chemo-resistance, invasion and migration. In pancreatic tumor model, the combination formulation treated group showed significant inhibition of tumor growth. Immuno-hiostochemical analysis revealed decreased tumor cell proliferation with increased apoptosis in the animals treated with miR-205 and gemcitabine combination.

Self-assembling, amphiphilic polymer-gemcitabine conjugate shows enhanced antitumor efficacy against human pancreatic adenocarcinoma.

The therapeutic efficacy of gemcitabine is severely compromised due to its rapid plasma metabolism. Moreover, its hydrophilicity poses a challenge for its efficient entrapment in nanosized delivery systems and to provide a sustained release profile. In this study, gemcitabine was covalently conjugated to poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate) (PEG-PCC) which could self-assemble into micelles of 23.6 nm. These micelles afforded protection to gemcitabine from plasma metabolism as evident by negligible amount of gemcitabine and its metabolite dFdU detected in the plasma after 24 h. A controlled release of gemcitabine from the micelles was observed with 53.89% drug release in 10 days in the presence of protease enzyme Cathepsin B. Gemcitabine conjugated micelles were cytotoxic, showed internalization, and induced cell apoptosis in MIA PaCa-2 and L3.6pl pancreatic cancer cell lines. These micelles efficiently inhibited tumor growth when injected intravenously into MIA PaCa-2 cell derived xenograft tumor bearing NSG mice at a dose of 40 mg/kg in terms of reduced tumor volume and tumor weight (0.38 g vs 0.58 g). TUNEL assay revealed that gemcitabine conjugated micelles induced a much higher extent of apoptosis in the tumor tissues compared to free gemcitabine. In conclusion, gemcitabine conjugated micelles were able to enhance the drug payload, protect it from rapid plasma metabolism, and provide a sustained release and showed enhanced antitumor activity, and thus have the potential to provide a better therapeutic alternative for treating pancreatic cancer.

miRNAs in pancreatic cancer: therapeutic potential, delivery challenges and strategies.

Pancreatic ductal adenocarcinoma (PDAC) is a severe pancreatic malignancy and is predicted to victimize 1.5% of men and women during their lifetime (Cancer statistics: SEER stat fact sheet, National Cancer Institute, 2014). miRNAs have emerged as a promising prognostic, diagnostic and therapeutic tool to fight against pancreatic cancer. miRNAs could modulate gene expression by imperfect base-pairing with target mRNA and hence provide means to fine-tune multiple genes simultaneously and alter various signaling pathways associated with the disease. This exceptional miRNA feature has provided a paradigm shift from the conventional one drug one target concept to one drug multiple target theory. However, in vivo miRNA delivery is not fully realized due to challenges posed by this special class of therapeutic molecules, which involves thorough understanding of the biogenesis and physicochemical properties of miRNA and delivery carriers along with the pathophysiology of the PDAC. This review highlights the delivery strategies of miRNA modulators (mimic/inhibitor) in cancer with special emphasis on PDAC since successful delivery of miRNA in vivo constitutes the major challenge in clinical translation of this promising class of therapeutics.

Integration of porosity and bio-functionalization to form a 3D scaffold: cell culture studies and in vitro degradation

In this study, porous poly(lactide-co-glycolide) (PLGA) (50/50) microspheres have been fabricated by the gas-foaming technique using ammonium bicarbonate as a gas-foaming agent. Microspheres of different porosities have been formulated by varying the concentration of the gas-foaming agent (0%, 5%, 10% and 15% w/v). These microspheres were characterized for particle size, porosity and average pore size, morphology, water uptake ratio and surface area and it was found that the porosity, pore size and surface area increased on increasing the concentration of the gas-foaming agent. Further, the effect of porosity on degradation behavior was evaluated over a 12 week period by measuring changes in mass, pH, molecular weight and morphology. Porosity was found to have an inverse relationship with degradation rate. To render the surface of the microspheres biomimetic, peptide P-15 was coupled to the surface of these microspheres. In vitro cell viability, proliferation and morphological evaluation were carried out on these microsphere scaffolds using MG-63 cell line to study the effect of the porosity and pore size of scaffolds and to evaluate the effect of P-15 on cell growth on porous scaffolds. MTT assay, actin, alizarin staining and SEM revealed the potential of biomimetic porous PLGA (50/50) microspheres as scaffolds for tissue engineering. As shown in graphical representation, an attempt has been made to correlate the cell behavior on the scaffolds (growth, proliferation and cell death) with the concurrent degradation of the porous microsphere scaffold as a function of time.

Nanocarrier-based co-delivery of small molecules and siRNA/miRNA for treatment of cancer

Aberrant gene expression can trigger several vital molecular events that not only result in carcinogenesis but also cause chemoresistance, metastasis and relapse. Gene-based therapies using siRNA/miRNA have been suggested as new treatment method to improve the current regimen. Although these agents can restore the normal molecular cascade thereby resensitizing the cancer cells, delivering a standard regimen (either subsequently or simultaneously) is necessary to achieve the therapeutic benefit. However, co-delivery using a single carrier could give an additional advantage of similar biodistribution profile of the loaded agents. While much research has been carried out in this field in recent years, challenges involved in designing combination formulations including efficient coloading, stability, appropriate biodistribution and target specificity have hampered their clinical translation. This article highlights current aspects of nano-carriers used for co-delivery of small molecules and genes to treat cancer.

Cytomodulin-functionalized porous PLGA particulate scaffolds respond better to cell migration, actin production and wound healing in rodent model.

In the present study, porous PLGA microparticulate scaffolds (PMS_P), surface‐hydrolysed scaffolds (PMS_Hyd) and cytomodulin‐coupled scaffolds (PMS_CM) were prepared and characterized. After coupling the particles with cytomodulin, the size was reduced from 334 µm (span 0.53) to 278 µm due to hydrolysis, and contact angle also decreased from 70.87 ± 8.56 to 31.43 ± 7.43, indicating an increase in hydrophilicity. Surface roughness and pore density increased, along with an increase in surface area from 9.59 ± 0.36 to 16.82 ± 0.064 m2/g after attaching the biomolecule CM onto the PLGA particles. In vitro cell culture experiments on human dermal fibroblasts (HDFs) were performed for 21 days, in which MTT assay indicated two‐fold higher cell proliferation on PMS_Hyd than on PMS_CM; however, cell distribution, cell spreading and actin production were significantly higher on PMS_CM than on other scaffolds. Migration of cells from PMS_CM to a 2D plate was gradual but the migrated cells attained early confluence, indicating the preservation of normal cellular functions. In a full‐thickness wound mouse model, PMS_CM exhibited 80% wound closure within 2 weeks. Further, at the end of week 3, the inflammatory cell count in the PMS_CM group was reduced to one‐third of the control group, while in PMS_P and PMS_Hyd the extent of inflammation was much higher and more severe. In the case of PMS_CM, abundant fibroblast proliferation, early formation of the scar tissue, eschar formation and inward movement of the wound margins (a zipper‐like movement) towards the deeper layers of the skin suggested advanced wound healing. Cytomodulin‐coupled scaffolds ensured better cell spreading and migration and thus enabled rapid wound healing (see Supporting information, Figure S1). Copyright

Structural modifications in polymeric micelles to impart multifunctionality for improved drug delivery

Polymeric micelles are macromolecular nanoconstructs which are formed by self-assembly of synthetic amphiphilic block copolymers. These copolymers could be chemically modified to expand their functionality and hence obtain a multifunctional micelle which could serve several functions simultaneously, for example, long circulation time along with active targeting, smart polymeric micelles providing on-demand drug release for example, pH responsive micelles, redox- and light-sensitive micelles, charge-conversion micelles and core/shell cross-linked micelles. Additionally, micelles could be tailored to carry a contrast agent or siRNA/miRNA along with the drug for greater clinical benefit. The focus of the current commentary would be to highlight such chemical modifications which impart multifunctionality to a single carrier and discuss challenges involved in clinical translation of these multifunctional micelles.

A new, bioactive, antibacterial‐eluting, composite graft for infection‐free wound healing

The current work focuses on the in vivo performance of a newly developed injectable composite graft in infected full‐thickness wounds. The composite graft was composed of bioactive porous Poly dl‐lactide‐co‐glycolide scaffolds, antibiotic gentamicin, and crosslinked gelatin as carrier gel. Treated infected wounds exhibited a faster wound closure, rapid weight gain, lower neutrophil count, higher breaking strength, and 100 times lesser microbial count (102 colony forming units/g in infected treated vs. 104 colony forming units/g in infected control group) in comparison with infected control group 28 days post treatment. During healing, collagen production was more in the treated groups at day 7 than controls and thereafter gradually reduced to normal levels. Histology revealed a mature scar tissue formation, fibroblast proliferation, epidermal resurfacing, and collagen deposition in reticular alignment similar to normal healthy skin in treated wounds. Further, the plasma concentration of gentamicin was 35–45 μg/mL during the initial 12 hours and reduced to 1 μg/mL in 24 hours, which indicated safe levels of the antibiotic drug during healing. These results clearly indicate a faster, infection‐free, and safe after treatment with the developed graft.

Lipid-polymer hybrid nanocarriers for delivering cancer therapeutics

ABSTRACT Cancer remained a major cause of death providing diversified challenges in terms of treatment including non‐specific toxicity, chemoresistance and relapse. Nanotechnology‐ based delivery systems grabbed tremendous attention for delivering cancer therapeutics as they provide benefits including controlled drug release, improved biological half‐life, reduced toxicity and targeted delivery. Majority of the nanocarriers consists of either a polymer or a lipid component along with other excipients to stabilize the colloidal system. Lipid‐based systems provide advantages like better entrapment efficiency, scalability and low‐ cost raw materials, however, suffer from limitations including instability, a burst release of the drug, and limited surface functionalization. On the other hand, polymeric systems provide an excellent diversity of chemical modifications, stability, controlled release, however limited drug loading capacities and scale up limit their use. Hybrid nanocarriers consisting of lipid and polymer were able to overcome some of these disadvantages while retaining the advantages of both the systems. Designing a stable lipid‐polymer hybrid system requires a thorough understanding of the material properties and their behavior in in vitro and in vivo environments. This review highlights the current status and future prospects of lipid‐polymer hybrid systems with a particular focus on cancer nanotherapeutics. Graphical abstract Figure. No caption available.

Effective cellular internalization, cell cycle arrest and improved pharmacokinetics of Tamoxifen by cholesterol based lipopolymeric nanoparticles

&NA; The present study aims at the development of cholesterol based lipopolymeric nanoparticles for improved entrapment, better cell penetration and improved pharmacokinetics of Tamoxifen (TMX). Self‐assembling cholesterol grafted lipopolymer, mPEG‐b‐(CB‐{g‐chol}‐co‐LA) was synthesized from poly(ethyleneglycol)‐block‐2‐methyl‐2‐carboxyl‐propylenecarboxylic acid‐co‐poly (L‐lactide) [mPEG‐b‐(CB‐{g‐COOH}‐co‐LA)] copolymer followed by carbodiimide coupling for attaching cholesterol. Lipopolymeric nanoparticles were prepared using o/w solvent evaporation technique, which were subsequently characterized to determine its particle size, entrapment efficiency, release pattern and compared with mPEG‐PLA nanoparticles. Further, in order to assess the in vitro efficacy, cytotoxicity studies, uptake, apoptosis assay and cell cycle analysis were performed in breast cancer cell lines (MCF‐7 and 4T1). Finally, the pharmacokinetic profile of TMX loaded mPEG‐b‐(CB‐{g‐chol}‐co‐LA) lipopolymeric nanoparticles was also performed. TMX loaded lipopolymeric nanoparticles of particle size 151.25 ± 3.74 (PDI 0.123) and entrapment efficiency of 73.62 ± 3.08% were formulated. The haemolytic index, protein binding and in vitro drug release of the optimized nanoparticles were found to be comparable to that of the TMX loaded mPEG‐PLA nanoparticles. Lipopolymeric nanoparticles demonstrated improved IC50 values in breast cancer cells (22.2 &mgr;M in 4T1; 18.8 &mgr;M in MCF‐7) than free TMX (27.6 &mgr;M and 23.5 &mgr;M respectively) and higher uptake efficiency. At IC50 values, TMX loaded lipopolymeric nanoparticles induced apoptosis and cell cycle arrest (G0/G1 phase) to similar extent as that of free drug. Pharmacokinetic studies indicated ˜2.5‐fold increase in the half‐life (t1/2) (p < 0.001) and ˜2.7‐fold (p < 0.001) increase in the mean residence time (MRT) of TMX following incorporation into lipopolymeric nanoparticles. Thus, mPEG‐b‐(CB‐{g‐chol}‐co‐LA) lipopolymeric nanoparticles offer a more promising approach for delivery of Tamoxifen in breast cancer by improving drug internalization and prolonging the mean residence time of the drug indicating possibility of dose reduction and hence bypassing the adverse effects of TMX therapy. Graphical abstract Figure. No caption available.