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Microtubule binding agents such as paclitaxel and vincristine have activity in metastatic melanoma. However, even responsive tumors develop resistance, highlighting the need to investigate new drug molecules. Here, we showed that a new compound, CH-2-102, developed by our group, has high anti-tumor efficacy in human and murine melanoma cells. We confirmed that CH-2-102 robustly suppresses the microtubule polymerization process by directly interacting with the colchicine binding site. Our results unveil that CH-2-102 suppresses microtubule polymerization and subsequently induces G2 phase cell arrest as one of the possible mechanisms. Notably, CH-2-102 maintains its efficacy even in the paclitaxel resistance melanoma cells due to different binding sites and a non-Pgp substrate. We developed a pH-responsive drug-polymer Schiff bases linker for high drug loading into nanoparticles (NPs). Our CH-2-102 conjugated NPs induced tumor regression more effectively than Abraxane® (Nab-paclitaxel, N-PTX), free drug, and non-sensitive NPs in B16-F10 cell-derived lung metastasis mouse model. Furthermore, our results suggest that the formulation has a high impact on the in vivo efficacy of the drug and warrants further investigation in other cancers, particularly taxane resistant. In conclusion, the microtubule polymerization inhibitor CH-2-102 conjugated pH-responsive NPs induce tumor regression in lung metastasis melanoma mice, suggesting it may be an effective strategy for treating metastatic melanoma.

Type 2 diabetes mellitus (T2DM) associated non-alcoholic fatty liver disease (NAFLD) is the fourth-leading cause of death. Hyperglycemia induces various complications, including nephropathy, cirrhosis and eventually hepatocellular carcinoma (HCC). There are several etiological factors leading to liver disease development, which involve insulin resistance and oxidative stress. Free fatty acid (FFA) accumulation in the liver exerts oxidative and endoplasmic reticulum (ER) stresses. Hepatocyte injury induces release of inflammatory cytokines from Kupffer cells (KCs), which are responsible for activating hepatic stellate cells (HSCs). In this review, we will discuss various molecular targets for treating chronic liver diseases, including homeostasis of FFA, lipid metabolism, and decrease in hepatocyte apoptosis, role of growth factors, and regulation of epithelial-to-mesenchymal transition (EMT) and HSC activation. This review will also critically assess different strategies to enhance drug delivery to different cell types. Targeting nanocarriers to specific liver cell types have the potential to increase efficacy and suppress off-target effects.

Melanoma is the most aggressive type of skin cancer, which readily metastasizes through lymph nodes to the lungs, liver, and brain. Since the repeated administration of most chemotherapeutic drugs develops chemoresistance and severe systemic toxicities, herein we synthesized 2-(4-hydroxy-1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl) methanone (abbreviated as QW-296), a novel tubulin destabilizing agent with little susceptible to transporter-mediated drug resistance. QW-296 disturbed the microtubule dynamics at the nanomolar concentration in A375 and B16F10 melanoma cells. QW-296 binding to colchicine-binding site on tubulin protein was confirmed by molecular modeling and tubulin polymerization assay. QW-296 significantly inhibited A375 and B16F10 cell proliferation, induced G2/M cell cycle arrest and led to apoptosis and cell death. To improve its aqueous solubility, QW-296 was encapsulated into methoxy poly(ethyleneglycol)-b-poly(carbonate-co-lactide) [mPEG-b-P(CB-co-LA)] polymeric nanoparticles by solvent evaporation, with the mean particle size of 122.0 ± 2.28 nm and drug loading of 3.70% (w/w). Systemic administration of QW-296 loaded nanoparticles into C57/BL6 albino mice bearing lung metastatic melanoma at the dose of 20 mg/kg 4 times a week for 1.5 weeks resulted in significant tumor regression and prolonged mouse median survival without significant change in mouse body weight. In conclusion, QW-296 loaded nanoparticles have the potential to treat metastatic melanoma.

Castration-resistant prostate cancer that has become resistant to docetaxel (DTX) represents one of the greatest clinical challenges in the management of this malignancy. There is an urgent need to develop novel therapeutic agents to overcome chemoresistance and improve the overall survival of patients. We have designed a novel microtubule destabilizer (2-(4-hydroxy-1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (QW-296) and combined it with a newly synthesized hedgehog (Hh) signaling pathway inhibitor 2-chloro-N(1)-[4-chloro-3-(2-pyridinyl)phenyl]-N(4),N(4)- bis(2-pyridinylmethyl)-1,4-benzenedicarboxamide (MDB5) to treat taxane-resistant (TXR) prostate cancer. The combination of QW-296 and MDB5 exhibited stronger anticancer activity toward DU145-TXR and PC3-TXR cells and suppressed tumor colony formation when compared with single-drug treatment. Because these drugs are hydrophobic, we synthesized the mPEG-p(TMC-MBC) [methoxy-poly(ethylene glycol)-block-poly(trimethylene carbonate-co-2-methyl-2-benzoxycarbonyl-propylene carbonate)] copolymer, which could self-assemble into micelles with loading capacities of 8.13% ± 0.75% and 9.12% ± 0.69% for QW-296 and MDB5, respectively. Further, these micelles provided controlled the respective drug release of 58% and 42% release of QW-296 and MDB5 within 24 hours when dialyzed against PBS (pH 7.4). We established an orthotopic prostate tumor in nude mice using stably luciferase expressing PC3-TXR cells. There was maximum tumor growth inhibition in the group treated with the combination therapy of QW-296 and MDB5 in micelles compared with their monotherapies or combination therapy formulated in cosolvent. The overall findings suggest that combination therapy with QW-296 and MDB5 has great clinical potential to treat TXR prostate cancer, and copolymer mPEG-p(TMC-MBC) could serve as an effective delivery vehicle to boost therapeutic efficacy in vivo.

Non-alcoholic fatty liver disease (NAFLD) is a cluster of several liver diseases like hepatic steatosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), liver fibrosis, and cirrhosis which may eventually progress to liver carcinoma. One of the primary key factors associated with the development and pathogenesis of NAFLD is diabetes mellitus. The present review emphasizes on diabetes-associated development of liver fibrosis and its treatment using different lipid nanoparticles such as stable nucleic acid lipid nanoparticles, liposomes, solid lipid nanoparticles, nanostructured lipid carriers, self-nanoemulsifying drug delivery systems, and conjugates including phospholipid, fatty acid and steroid-based. We have comprehensively described the various pathological and molecular events linking effects of elevated free fatty acid levels, insulin resistance, and diabetes with the pathogenesis of liver fibrosis. Various passive and active targeting strategies explored for targeting hepatic stellate cells, a key target in liver fibrosis, have also been discussed in detail in this review.

Instability, poor cellular uptake and unfavorable pharmacokinetics and biodistribution of many therapeutic molecules require modification in their physicochemical properties. The conjugation of these APIs with fatty acids has demonstrated an enhancement in their lipophilicity and stability. The improvement in the formulations that resulted from the conjugation of a drug with a fatty acid includes increased half-life, enhanced cellular uptake and retention, targeted tumor delivery, reduced chemoresistance in cancer, and improved blood-brain-barrier (BBB) penetration. In this review, various therapeutic molecules, including small molecules, peptides and oligonucleotides, that have been conjugated with fatty acid have been thoroughly discussed along with various conjugation strategies. The application of nano-system based delivery is gaining a lot of attention due to its ability to provide controlled drug release, targeting and reducing the extent of side effects. This review also covers various nano-carriers that have been utilized for the delivery of fatty acid drug conjugates. The enhanced lipophilicity of the drug-fatty acid conjugate has shown to enhance the affinity of the drug towards these carriers, thereby increasing the entrapment efficiency and formulation performance.

Type 1 diabetes (T1D) is a chronic autoimmune disease caused by proinflammatory T cell infiltration associated cytokine secretion and reactive oxygen species (ROS) production. In this study, our objective was to determine whether co-delivery of sunitinib, a member of tyrosine kinase inhibitor (TKI) family, and siRNA against Alox15 (siAlox15) could reverse the new-onset of T1D in non-obese diabetic (NOD)/ShiLtJ female mice which spontaneously develop diabetes. A cationic copolymer methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (mPEG-b-PCC-g-DC-g-TEPA) was used to encapsulate sunitinib with 8.32-9.39% loading efficacy and form complex with siAlox15 at the N/P ratio of 16:1. These micellar formulations exhibited sustained release of sunitinib for around 50% at 24 h and protected siAlox15 from serum degradation for at least 10 h. Further, both sunitinib and siAlox15 inhibited human peripheral blood mononuclear cell (PBMC) proliferation and activation by downregulating the phosphorylation of Akt, platelet-derived growth factor receptor (PDGFR) at protein levels and Alox15 in mRNA levels, respectively. Besides, siAlox15 treatment reversed the cytokine induced ROS upregulation in rat INS-1E β cells. Finally, systemic administration of these micelles carrying sunitinib and siAlox15 efficiently reversed T1D in NOD mice as indicated by decreased blood glucose concentration to below 250 mg/dL. The mice showed fast response to high glucose which was determined by intraperitoneal glucose tolerance test and promoted serum insulin production. Tissue staining indicated that sunitinib and siAlox15 combination could inhibit T cell infiltration to mouse islets, thereby promoting islet survival and function. In summary, our results demonstrated that combination therapy of sunitinib and siAlox15 could be a novel, reliable and safe approach for patients who were recently diagnosed with T1D.

Hedgehog (Hh) signaling is involved in the initiation and progression of various cancers and is essential for embryonic and postnatal development. This pathway remains in the quiescent state in adult tissues but gets activated upon inflammation and injuries. Inhibition of Hh signaling pathway using natural and synthetic compounds has provided an attractive approach for treating cancer and inflammatory diseases. While the majority of Hh pathway inhibitors target the transmembrane protein Smoothened (SMO), some small molecules that target the signaling cascade downstream of SMO are of particular interest. Substantial efforts are being made to develop new molecules targeting various components of the Hh signaling pathway. Here, we have discussed the discovery of small molecules as Hh inhibitors from the diverse chemical background. Also, some of the recently identified natural products have been included as a separate section. Extensive structure-activity relationship (SAR) of each chemical class is the focus of this review. Also, clinically advanced molecules are discussed from the last 5 to 7 years. Nanomedicine-based delivery approaches for Hh pathway inhibitors are also discussed concisely.

miR-34a is a master tumor suppressor playing a key role in the several signaling mechanisms involved in cancer. However, its delivery to the cancer cells is the bottleneck in its clinical translation. Herein we report cationic amphiphilic copolymers grafted with cholesterol (chol), N, N-dimethyldipropylenetriamine (cation chain) and 4-(2-aminoethyl)morpholine (morph) for miR-34a delivery. The copolymer interacts with miR-34a at low N/P ratios (∼2/1) to form nanoplexes of size ∼108 nm and a zeta potential ∼ +39 mV. In vitro studies in 4T1 and MCF-7 cells indicated efficient transfection efficiency. The intracellular colocalization suggested that the copolymer effectively transported the FAM labeled siRNA into the cytoplasm within 2 h and escaped from the endo-/lysosomal environment. The developed miR-34a nanoplexes inhibited the breast cancer cell growth as confirmed by MTT assay wherein 28% and 34% cancer cell viability was observed in 4T1 and MCF-7 cells, respectively. Further, miR-34a nanoplexes possess immense potential to induce apoptosis in both cell lines.

Treatment of prostate cancer with paclitaxel often fails due to the development of chemoresistance caused by downregulation of the tumor suppressor gene miR-34a. In this study, we demonstrate that codelivery of paclitaxel and 2'-hydroxy-2,4,4',5,6'-pentamethoxychalcone (termed rubone) drives upregulation of miR-34a and chemosensitizes paclitaxel-resistant prostate cancer cells, killing both cancer stem–like cells (CSC) and bulk tumor cells. Rubone upregulated miR-34a and reversed its downstream target genes in DU145-TXR and PC3-TXR cells. Paclitaxel and rubone combination therapy inhibited tumor cell growth, migration, and CSC population growth. We synthesized poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol; PEG-PCD) to prepare micelles. The drug-loading capacities were 9.70% ± 0.10% and 5.34% ± 0.02% for paclitaxel and rubone, respectively, controlling a drug release of 60.20% ± 2.67% and 60.62% ± 4.35% release of paclitaxel and rubone at 24 hours. Delivery of miR-34a and rubone decreased PC3-TXR cell viability with increasing paclitaxel concentration. Coincubation with a miR-34a inhibitor diminished the effect of rubone. Paclitaxel IC50 in PC3 and PC3-TXR cells was 55.6 and 2,580 nmol/L, respectively, but decreased to 49.8 and 93.2 nmol/L when treated in combination with rubone, demonstrating a reversal of paclitaxel resistance by rubone. Systemic administration of micelles carrying paclitaxel and rubone inhibited orthotopic prostate tumor growth in nude mice, compared with monotherapy, by reversing the expression of miR-34a, SIRT1, cyclin D1, and E-cadherin. In summary, our results showed how rubone acts as an efficient small-molecule modulator of miR-34a to reverse chemoresistance and further enhance the therapeutic efficacy of paclitaxel in paclitaxel-resistant prostate cancer. Cancer Res; 77(12); 3244–54. ©2017 AACR.

Hepatocellular carcinoma (HCC), also called malignant hepatoma, is one of the deadliest cancers due to its complexities, reoccurrence after surgical resection, metastasis and heterogeneity. Incidence and mortality of HCC are increasing in Western countries and are expected to rise as a consequence of the obesity epidemic. Multiple factors trigger the initiation and progression of HCC including chronic alcohol consumption, viral hepatitis B and C infection, metabolic disorders and age. Although Sorafenib is the only FDA approved drug for the treatment of HCC, numerous treatment modalities such as transcatheter arterial chemoembolization/transarterial chemoembolization (TACE), radiotherapy, locoregional therapy and chemotherapy have been tested in the clinics. Polymeric nanoparticles, liposomes, and micelles carrying small molecules, proteins, peptides and nucleic acids have attracted great attention for the treatment of various cancers including HCC. Herein, we discuss the pathogenesis of HCC in relation to its various recent treatment methodologies using nanodelivery of monoclonal antibodies (mAbs), small molecules, miRNAs and peptides. Synopsis of recent clinical trials of mAbs and peptide drugs has been presented with a broad overview of the pathogenesis of the disease and treatment efficacy.

Ineffective drug delivery and poor prognosis are two major challenges in the treatment of pancreatic ductal adenocarcinoma (PDAC). While there is significant downregulation of tumor suppressor microRNA-34a (miR-34a), which targets many oncogenes related to proliferation, apoptosis, and invasion, high expression level of Polo-like kinase 1 (PLK1) is closely associated with short survival rates of pancreatic cancer patients. Therefore, the objective is to codeliver miR-34a mimic and small molecule PLK1 inhibitor volasertib (BI6727) using poly(ethylene glycol)–poly[aspartamidoethyl(p-boronobenzyl)diethylammonium bromide] (PEG-B-PAEBEA). This polymer could self-assemble into micelles of ~100 nm with 10% drug loading of volasertib and form a complex with miR-34a at the N/P ratio of 18 and higher. Combination treatment of volasertib and miR-34a displayed the synergistic effect and superior antiproliferative activity along with an enhanced G(2)/M phase arrest and suppression of colony formation, leading to cell death due to potential c-myc targeting therapeutics. Orthotopic pancreatic tumor bearing NSG mice were scanned for fluorescence by IVIS after systemic administration of micelles encapsulating volasertib and miR-34a at doses of 5 and 1 mg/kg, respectively. Cy5.5 concentration in plasma and major organs was determined by measuring fluorescence intensity. There was significant reduction in tumor volume, and histological examination of major organs suggested negligible systemic toxicity. In conclusion, PEG-B-PAEBEA micelles carrying volasertib and miR-34a mimic have the potential to treat pancreatic cancer.

Pulmonary drug delivery is a non-invasive and effective route for local or systemic drug administration. Despite several products in the market, the mechanism of drug absorption from the lungs is not well understood. An in vitro model for aerosol deposition and transport across epithelia that uses particle deposition may be a good predictor of and help understand in vivo drug disposition. The objective of this study was to examine the uptake of HFA fluticasone (Flovent HFA) particles at various stages of the Next Generation Impactor (NGI) by human Calu-3 cell line derived from human bronchial respiratory epithelial cell monolayer. Particles were directly deposited on Calu-3 cells incorporated onto stages 3, 5, and 7 of the NGI at the air-liquid interface (ALI). We modified the NGI apparatus to allow particle deposition directly on cells and determined the in vitro deposition characteristics using modified NGI. Particles of different size ranges showed different in vitro epithelial transport rates. This study highlights the need to develop in vitro test systems to determine the deposition of aerosol particles on cell monolayers by simultaneously considering aerodynamic properties.

There is a direct correlation between increase in the number of cancer stem cells CSCs and chemoresistance that impedes successful chemotherapy. Synergistic therapy by targeting both bulk tumor cells and CSCs has shown promise in reversing chemoresistance and treating resistant prostate cancer. Herein, we demonstrated the fabrication of a pH and glutathione (GSH) sensitive nanocarrier for co-delivery of docetaxel (DTX) and rubone (RUB), a miR-34 activator for targeting CSCs, for the treatment of taxane resistant (TXR) prostate cancer. DTX loaded P-RUB (DTX/P-RUB) micelles were prepared by encapsulating DTX into pH responsive diisopropylaminoethanol (DIPAE) and GSH responsive RUB prodrug conjugated polycarbonate based micelles. The self-assembled DTX/P-RUB micelles displayed good stability in vitro and could efficiently target to tumors by enhanced permeability and retention (EPR) effect. After endocytosis by tumor cells, the micelles underwent expansion and disassembly due to the protonation of DIPAE and GSH induced cleavage of disulfide bond in acidic endocytic vesicles, resulting in fast release of DTX and RUB. The released RUB then upregulated the intracellular miR-34a, which then affected the expression of proteins involved in chemoresistance, thus sensitizing the tumor cells towards DTX and further leading to significant inhibition of TXR tumor progression. Thus, DTX/P-RUB micelles have the potential to treat TXR prostate cancer. By taking advantage of this dual responsive strategy, the successful delivery of many other hydrophobic drugs can be achieved for cancer treatment.

Trans-differentiation of quiescent hepatic stellate cells (HSCs) into active myofibroblasts secretes excess amounts of extracellular matrix (ECM) proteins. miR-29b1 has the potential to treat liver fibrosis, because it targets several profibrotic genes. We previously demonstrated that miR-29b1 and the hedgehog (Hh) pathway inhibitor GDC-0449 could, together, inhibit the activation of HSCs and ECM production in common bile-duct-ligated (CBDL) mice. Herein, we determined the effect of chemical modifications of miR-29b1 on its stability, immunogenicity, and Argonaute-2 ( Ago2 ) loading in vitro , after modifying its antisense strand with phosphorothioate (PS-miR-29b1), 2′-O-methyl-phosphorothioate (OMe-miR-29b1), locked nucleic acid (LNA-miR-29b1), and N,N’-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (ZEN-miR-29b1). Chemical modifications significantly improved stability of miR-29b1 in 50% FBS. Among all the modified miRNAs tested, OMe-PS-miR-29b1 showed the highest stability with low immunogenicity, without the loss of efficacy in vitro . Therefore, OMe-PS-miR-29b1 was complexed with poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylenecarbonate-graft-dodecanol-graft-tetraethylenepentamine (mPEG-b-PCC-g-DC-g-TEPA) cationic micelles, and anti-fibrotic efficacy was evaluated in CBDL mice. There was a significant improvement in liver histology and decrease in the levels of injury markers. Further, mRNA/protein levels of collagen, α-SMA , and TIMP-1 were significantly lower for the OMe-PS-miR-29b1-loaded micelles compared to miR-29b1-loaded micelles. In conclusion, micellar delivery of OMe-PS-miR-29b1 is a promising strategy to treat liver fibrosis.

T cell immunity plays a vital role in pathogen infections. MicroRNA (miRNAs) are small, single-stranded noncoding RNAs that regulate T cell immunity by targeting key transcriptional factors, signaling proteins, and cytokines associated with T cell activation, differentiation, and function. The dysregulation of miRNA expression in T cells may lead to specific immune responses and can provide new therapeutic opportunities against various infectious diseases. Here, we summarize recent studies that focus on the roles of miRNAs in T cell immunity and highlight miRNA functions in prevalent infectious diseases. Additionally, we also provide insights into the functions of extracellular vesicle miRNAs and attempt to delineate the mechanism of miRNA sorting into extracellular vesicles and their immunomodulatory functions. Moreover, methodologies and strategies for miRNA delivery against infectious diseases are summarized. Finally, potential strategies for miRNA-based therapies are proposed.

The objective of this study was to design GE11 peptide (YHWYGYTPQNVI) linked micelles of poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-gemcitabine-graft-dodecanol (PEG-b-PCC-g-GEM-g-DC) for enhanced stability and target specificity of gemcitabine (GEM) to EGFR-positive pancreatic cancer cells. GE11-PEG-PCD/mPEG-b-PCC-g-GEM-g-DC mixed micelles showed EGFR-dependent enhanced cellular uptake, and cytotoxicity as compared to scrambled peptide HW12-PEG-PCD/mPEG-b-PCC-g-GEM-g-DC mixed micelles and unmodified mPEG-b-PCC-g-GEM-g-DC micelles. Importantly, GE11-linked mixed micelles preferentially accumulated in orthotopic pancreatic tumor and tumor vasculature at 24 h post systemic administration. GE11-linked mixed micelles inhibited orthotopic pancreatic tumor growth compared to HW12-linked mixed micelles, unmodified mPEG-b-PCC-g-GEM-g-DC micelles, and free GEM formulations. Tumor growth inhibition was mediated by apoptosis of tumor cells and endothelial cells as determined by immunohistochemical staining. In summary, GE11-linked mixed micelles is a promising approach to treat EGFR overexpressing cancers.

Phototherapy (PT), a light activated treatment modality, is a potential therapeutic option for the treatment of melanoma. In spite of the excellent safety profile and absorption in the near infrared (NIR) range, clinical potential of indocyanine green (ICG) as PT is limited by its short half-life and inefficient tumor accumulation. In this study, we have covalently conjugated ICG-NH2 to the pendant carboxyl groups of poly (ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate) (PEG-PCC) copolymer using carbodiimide coupling, which self-assembled into micelles with a particle size of 30-50 nm and high ICG loading. These ICG conjugated micelles exhibited significant in vitro photodynamic cytotoxicity. Use of sodium azide and NIR radiate on at 4 °C revealed photodynamic and photothermal as mechanism of cytotoxicity of ICG solution and ICG conjugated micelles, respectively. In vivo NIR imaging demonstrated that ICG conjugated micelles prolonged its circulation and increased tumor accumulation through enhanced permeability and retention (EPR) effect. Enhanced tumor accumulation improved therapeutic efficacy with complete tumor regression in NIR irradiated ICG conjugated micelles compared to ICG solution and control in a A375 human melanoma tumor model in athymic nude mice. These results suggest that ICG conjugated micelles can be potentially utilized for PT and imaging of melanoma.

Clinical translation of tubulin inhibitors for treating melanoma is limited by multidrug efflux transporters, poor aqueous solubility, and dose-limiting peripheral toxicities. Tubulin inhibitors with efficacy in taxane-resistant cancers are promising drug candidates and can be used as single agent or in conjunction with other chemotherapy. Systemic therapy of such a novel tubulin inhibitor, 2-(1H-indol-5-yl)thiazol-4-yl)3,4,5-trimethoxyphenyl methanone (abbreviated as LY293), is limited by its poor aqueous solubility. The objective of this study was to design a polymeric nanocarrier for systemic administration of LY293 to improve tumor accumulation and reduce side effects of tubulin inhibitor in a lung metastasis melanoma mouse model. Methoxy polyethylene glycol-b-poly(carbonate-co-lactide) (mPEG-b-P(CB-co-LA)) random copolymer was synthesized and characterized by ¹H NMR and gel permeation chromatography (GPC). Polymeric nanoparticles were formulated using oil/water (o/w) emulsification method with a mean particle size of 150 nm and loading efficiency of 7.40%. Treatment with LY293-loaded nanoparticles effectively inhibited the proliferation of melanoma cells in vitro and exhibited concentration-dependent cell cycle arrest in G2/M phase. Mitotic arrest activated the intrinsic apoptotic machinery by increasing the cellular levels of cleaved poly ADP ribose polymerase (PARP) and fraction of sub-G1 cells. In vivo, LY293-loaded nanoparticles significantly inhibited the proliferation of highly aggressive metastasized melanoma in a syngeneic lung metastasis melanoma mouse model without toxicity to vital organs. In conclusion, we have designed a promising polymeric nanocarrier for systemic delivery of LY293 for treating metastatic melanoma while minimizing the toxicity associated with the administration of cosolvents.

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.

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.

The aim of this study was to determine whether co-administration of hedgehog (Hh) pathway inhibitor cyclopamine (CYP) and microtubule stabilizer docetaxel (DTX) as polymer-drug conjugates, methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylenecarbonate-graft-dodecanol-graft-cyclopamine) (P-CYP) and methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-docetaxel) (P-DTX) could synergistically inhibit orthotopic pancreatic tumor growth in NSG mice. P-DTX and P-CYP were synthesized from mPEG-b-PCC through carbodiimide coupling reaction and characterized by 1H–NMR. The micelles were prepared by film hydration and particle size was measured by dynamic light scattering (DLS). Cytotoxicity, apoptosis and cell cycle analysis of P-DTX and P-CYP were evaluated in MIA PaCa-2 cells. In vivo efficacy of P-DTX and P-CYP were evaluated in NSG mice bearing MIA PaCa-2 cells derived orthotopic pancreatic tumor. P-CYP and P-DTX self-assembled into micelles of

Non-alcoholic fatty liver disease (NAFLD) increases the risk of various liver injuries, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis, and ultimately hepatocellular carcinoma (HCC). Ample evidence has suggested that aberrant expression of microRNAs (miRNAs) is functionally involved in the activation of cellular stress, inflammation and fibrogenesis in hepatic cells, including hepatocytes, Kupffer and hepatic stellate cells (HSCs), at different pathological stages of NAFLD and liver fibrosis. Here, we overview recent findings on the potential role of miRNAs in the pathogenesis of NAFLD, including lipotoxicity, oxidative stress, metabolic inflammation and fibrogenesis. We critically assess the literatures on both human subjects and animal models of NAFLD and liver fibrosis with miRNA dysregulation and their mechanisms of actions in liver damage. We further highlight the potential use of miRNA mimics or antimiRNAs as therapeutic approaches for the prevention and treatment of NAFLD and liver fibrosis.

Gemcitabine (GEM), a first-line chemotherapy for pancreatic cancer undergoes rapid metabolism and develops chemoresistance after repeated administration. We previously demonstrated that the combination of GEM and miR-205 provides an effective therapeutic strategy to sensitize GEM-resistant pancreatic cancer cells. Since epidermal growth factor receptor (EGFR) is overexpressed in pancreatic cancer cells, in this study, we aimed to deliver mixed micelles containing GEM and miR-205 decorated with EGFR-targeting cetuximab (C225) monoclonal antibody for targeted therapy. Cetuximab C225 was conjugated to malemido-poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graf t-dodecanol (C225–PEG-PCD) to prepare mixed micelles with mPEG-b-PCC-g-GEM-g-DC-g-TEPA for targeted codelivery of GEM and miR-205. This mixed micelle formulation showed a significant enhancement in EGFR-mediated cellular uptake in GEM-resistant MIA PaCa-2(R) cells. Further, an enhanced tumor accumulation of C225-micelles conjugated with near-infrared fluorescent Cy7.5 dye and Dy677-labeled miR-205 in orthotopic pancreatic tumor bearing NSG mice was evident after systemic administration. In addition, inhibition of tumor growth was also observed with increased apoptosis and reduced EMT after treatment with C225-micelles containing GEM and miR-205. Therefore, we believe that the targeted delivery of GEM and miR-205 in combination could be a novel strategy for treating advanced pancreatic cancer.

Medulloblastoma (MB) is the most common childhood brain tumor, which occurs in the posterior fossa. MB tumors are highly heterogeneous and have diverse genetic make-ups, with differential microRNA (miRNA) expression profiles and variable prognoses. MB can be classified into four subgroups, each with different origins, pathogenesis, and potential therapeutic targets. miRNA and small-molecule targeted therapies have emerged as a potential new therapeutic paradigm in MB treatment. However, the development of chemoresistance due to surviving cancer stem cells and dysregulation of miRNAs remains a challenge. Combination therapies using multiple drugs and miRNAs could be effective approaches. In this review we discuss various MB subtypes, barriers, and novel therapeutic options which may be less toxic than current standard treatments.

Treatment of pancreatic cancer with gemcitabine (GEM) is limited due to its rapid plasma metabolism and development of chemoresistance. MicroRNA (miRNA) regulates cancer stem cell (CSC) maintenance and induces chemoresistance in cancer cells. In this study, we observed differential downregulation of miR-205 (miR-205-5p) in human pancreatic cancer tissues and cells. Compared to GEM-sensitive MIA PaCa-2 cells, miR-205 was highly downregulated in GEM-resistant MIA PaCa-2R cells. Lentivirus-mediated overexpression of miR-205 inhibits MIA PaCa-2R cell proliferation after GEM-treatment. Further investigation confirmed that miR-205 alone significantly reduces the proliferation of CSCs and tumor growth in mouse models. However, miR-205 in combination with GEM was more efficient in reducing the proliferation of CSCs and 3D spheroids. Moreover, miR-205 overexpressing MIA PaCa-2R cells induced orthotopic tumor growth was significantly inhibited after intravenous administration of GEM-conjugated methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate)-graft-gemcitabine-graft-dodecanol (mPEG-b-PCC-g-GEM-g-DC) (mPEG-b-PCC-g-GEM-g-DC) polymeric micelles. Also, a reduction in CSCs, EMT and chemoresistance markers was observed in miR-205 overexpressing MIA PaCa-2R cells. Immunohistochemical analysis of orthotopic tumors showed a decrease in drug resistance protein caveolin-1 and cell proliferation marker Ki-67 in combination treatment. Overall, our findings suggest that miR-205 resensitizes GEM-resistant pancreatic cancer cells to GEM and acts as a tumor suppressor miRNA.

Hedgehog (Hh) pathway is involved in epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) maintenance resulting in tumor progression. GDC-0449, an inhibitor of Hh pathway component smoothened (Smo) has shown promise in the treatment of various cancers including pancreatic cancer. However, the emergence of resistance during GDC-0449 treatment with numerous side effects limits its use. Therefore, here we report the design, synthesis and evaluation of novel GDC-0449 analogs using N-[3-(2-pyridinyl) phenyl] benzamide scaffold. Cell-based screening followed by molecular simulation revealed 2-chloro-N1-[4-chloro-3-(2-pyridinyl)phenyl]-N4,N4-bis(2-pyridinylmethyl)-1,4-benzenedicarboxamide (MDB5) as most potent analog, binding with an extra interactions in seven-transmembrane (7-TM) domain of Smo due to an additional 2-pyridylmethyl group than GDC-0449. Moreover, MDB5 was more efficient in inhibiting Hh pathway components as measured by Gli-1 and Shh at transcriptional and translational levels. Additionally, a significant reduction of ALDH1, CD44 and Oct-3/4, key markers of pancreatic CSC was observed when MIA PaCa-2 cells were treated with MDB5 compared to GDC-0449. In a pancreatic tumor mouse model, MDB5 containing nanoparticles treated group showed significant inhibition of tumor growth without loss in body weight. These evidence highlight the enhanced Hh pathway inhibition and anticancer properties of MDB5 leaving a platform for mono and/or combination therapy.

Liver fibrosis is a pathological condition originating from liver damage that leads to excess accumulation of extracellular matrix (ECM) proteins in the liver. Viral infection, chronic injury, local inflammatory responses and oxidative stress are the major factors contributing to the onset and progression of liver fibrosis. Multiple cell types and various growth factors and inflammatory cytokines are involved in the induction and progression of this disease. Various strategies currently being tried to attenuate liver fibrosis include the inhibition of HSC activation or induction of their apoptosis, reduction of collagen production and deposition, decrease in inflammation, and liver transplantation. Liver fibrosis treatment approaches are mainly based on small drug molecules, antibodies, oligonucleotides (ODNs), siRNA and miRNAs. MicroRNAs (miRNA or miR) are endogenous noncoding RNA of ~22 nucleotides that regulate gene expression at post transcription level. There are several miRNAs having aberrant expressions and play a key role in the pathogenesis of liver fibrosis. Single miRNA can target multiple mRNAs, and we can predict its targets based on seed region pairing, thermodynamic stability of pairing and species conservation. For in vivo delivery, we need some additional chemical modification in their structure, and suitable delivery systems like micelles, liposomes and conjugation with targeting or stabilizing the moiety. Here, we discuss the role of miRNAs in fibrogenesis and current approaches of utilizing these miRNAs for treating liver fibrosis.

Our objective was to synthesize LHRH-conjugated amphiphilic copolymer for micellar delivery of CBDIV17, a novel antiandrogen for treating prostate cancer.LHRH-PEG-b-p(CB-co-LA) was synthesized by opening polymerization of carbonate (CB), lactide (LA), and HOOC-PEG-OH followed by conjugation with LHRH analogue. Bicalutamide analogue CBIDV17 loaded micelles were formulated by film hydration method, and characterized for critical micelle concentration (CMC), drug loading and in vitro drug release. Formulations were tested on LNCaP and C4-2 cells for cellular uptake, induction of apoptosis, viability and dowregulation of androgen receptor (AR). In vivo studies were performed in ectopic tumor bearing athymic nude mice after tail vein injection at a dose of 10 mg/kg. Tumor volume and body weight were measured for 25 days followed by immunohistochemistry (IHC) of tumor samples for Ki-67, caspase-3, and prostate specific antigen (PSA).HOOC-PEG-b-p(CB-co-LA) and LHRH-PEG-b-p(CB-co-LA) were characterized by (1)HNMR and used for preparing micelles, which had a mean particle size of 75.60 ± 2.25 and 72.64 ± 1.15 nm, respectively and CBDIV17 loading of 4.6% w/w. LHRH conjugated micelles showed higher cellular uptake, cytotoxicity, and apoptosis in LNCaP and C4-2 cells compared to non-targeted micelles. CBDIV17 loaded LHRH micelles more efficiently inhibited the proliferation and induced apoptosis of tumor cells according to Ki-67, caspase-3, and PSA expression. There was significant inhibition of tumor growth with the treatment of CBDIV17 loaded LHRH-conjugated micelles.These results demonstrated that LHRH-b-PEG-p(CB-co-LA) micelles have the potential for targeted delivery of CBDIV17 to treat advanced prostate cancer.

Prostate cancer is the second leading cause of cancer related death in American men. Androgen deprivation therapy (ADT) is used to treat patients with aggressive prostate cancers. After androgen deprivation therapy, prostate cancers slowly progress to an androgen-independent status. Taxanes (e.g., docetaxel) are used as standard treatments for androgen-independent prostate cancers. However, these chemotherapeutic agents will eventually become ineffective due to the development of drug resistance. A microRNA (miRNA) is a small noncoding RNA molecule, which can regulate gene expression at the post-transcription level. miRNAs elicit their effects by binding to the 3′-untranslated region (3′-UTR) of their target mRNAs, leading to the inhibition of translation or the degradation of the mRNAs. miRNAs have received increasing attention as targets for cancer therapy, as they can target multiple signaling pathways related to tumor progression, metastasis, invasion, and chemoresistance. Emerging evidence suggests that aberrant expression of miRNAs can lead to the development of resistant prostate cancers. Here, we discuss the roles of miRNAs in the development of resistant prostate cancers and their involvement in various drug resistant mechanisms including androgen signaling, apoptosis avoidance, multiple drug resistance (MDR) transporters, epithelialmesenchymal transition (EMT), and cancer stem cells (CSCs). In addition, we also discuss strategies for treating resistant prostate cancers by targeting specific miRNAs. Different delivery strategies are also discussed with focus on those that have been successfully used in human clinical trials.

Human islet transplantation can be a permanent treatment of type 1 diabetes if the immune rejection and primary nonfunction (PNF) of transplanted islet grafts were properly addressed. In this study, we determined whether cotransplantation of human bone marrow-derived mesenchymal stem cells (hBMSCs) could prevent immune rejection and improve human islet transplantation in a humanized NOD scid gamma (NSG) mouse model. Human immunity was rebuilt and maintained in NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NSG) mice up to 13 weeks after intraperitoneal injection of mature human peripheral blood mononuclear cells (PBMCs). The blood glucose control and the levels of serum insulin and c-peptide clearly indicated a better outcome of islet transplantation when islets were cotransplanted with hBMSCs. hBMSCs actively interacted with interleukin-10 (IL-10)-producing CD14+ monocytes to suppress the proliferation and activation of T cells in the PBMC/hBMSC coculture and prevent the T cell recruitment into the transplantation site. hBMSCs also increased the percentage of immunosuppressive regulatory T cells (Tregs) and prevented the cytokine-induced loss-of-function of human islets. Taken together, our studies demonstrated that transplantation of islets with hBMSCs is a promising strategy to improve the outcome of human islet transplantation.

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.

Melanoma is the most aggressive type of skin cancer. It is highly metastatic, migrating through lymph nodes to distant sites of the body, especially to lungs, liver and brain. Systemic chemotherapy remains the mainstay of treatment; however, the development of multidrug resistance (MDR) restricts the efficacy of current chemotherapeutic drugs. We synthesized a series of microtubule destabilizers, substituted methoxybenzoyl-ary-thiazole (SMART) compounds, which inhibited tubulin polymerization and effectively circumvented MDR. Due to poor water solubility of SMART compounds, co-solvent delivery is required for their systemic administration, which is usually associated with hepatotoxicity, nephrotoxicity and hemolysis. To solve this problem and also to increase circulation time, we synthesized a new SMART analogue, SMART-OH, and its polymer-drug conjugate, methoxy-poly (ethylene glycol)-block-poly (2-methyl-2-carboxyl-propylene carbonate-graft-SMART-graft-dodecanol) (abbreviated as P-SMART), with 14.3±2.8% drug payload of SMART-OH. Similar to its parent drug, P-SMART showed significant anticancer activity against melanoma cells in cytotoxicity, colony formation, and cell invasion studies. In addition, P-SMART treatment led to cell cycle arrest at G2/M phase and cell accumulation in sub-G1 phase. We established a model of metastatic melanoma to the lung in C57/BL6 albino mice to determine in vivo efficacy of P-SMART and SMART-OH at the dose of 20mg/kg. P-SMART treatment resulted in significant inhibition of tumor growth and prolonged mouse median survival. In conclusion, P-SMART, a novel polymer-microtubule destabilizer conjugate, has the potential to treat metastatic melanoma.

To determine the pharmacokinetic parameters and biodistribution of GDC-0449 loaded polymeric micelles after systemic administration into common bile duct ligation (CBDL) induced liver fibrotic mice. We used GDC-0449 encapsulated methoxy poly (ethylene glycol)-block-poly (2-methyl-2-carboxyl-propylene carbonate)-graft-dodecanol (PEG-PCD) non-targeted polymeric micelles for GDC-0449 delivery to normal and liver fibrotic mice. To maximize GDC-0449 delivery to hepatic stellate cells (HSCs), mixed micelles formulations with 10, 20 and 30% w/w mannose-6-phosphate (M6P)-conjugated micelles were administered to normal and liver fibrotic mice for targeting M6P/IGF-IIR overexpressed on activated HSCs and biodistribution of GDC-0449 was determined at 30 and 120 min post systemic administration. GDC-0449 distributed to all major organs after systemic administration of drug loaded micelles, with higher accumulation in the liver of both normal and fibrotic mice. The plasma concentration versus time profiles suggest rapid clearance of GDC-0449 after systemic administration of drug loaded micelles in both normal and fibrotic mice, with similar plasma clearance (CL), area under the curve (AUCint) and volume of distribution at steady state (Vss). However, there is significant increase in GDC-0449 accumulation in the liver when M6P-conjugated mixed micelles were injected, with the highest GDC-0449 concentration in the liver with mixed micelles carrying 30% M6P-conjugated polymer. HSCs accounted for 14.19% of GDC-0449 accumulation for M6P-targeted micelles in fibrotic mice compared to 5.62% of non-targeted micelles in the liver uptake study. M6P-conjugated GDC-0449 loaded mixed micelles may be used as a potential drug delivery vehicle for treating liver fibrosis.

Human bone marrow mesenchymal stem cells (hBMSCs) and their exosomes can suppress immune reaction and deliver small RNAs. Thus, they may improve islet transplantation by delivering small RNAs for promoting islet function and inhibiting immune rejection. Here, we proposed an hBMSC and its exosome-based therapy to overcome immune rejection and poor islet function, both of which hinder the success of islet transplantation. We found overexpressed siFas and anti-miR-375 in plasmid encoding shFas and anti-miR-375 transfected hBMSC-derived exosomes, which silenced Fas and miR-375 of human islets and improved their viability and function against inflammatory cytokines. This plasmid transfected hBMSCs downregulated Fas and miR-375 of human islets in a humanized NOD scid gamma (NSG) mouse model, whose immune reaction was inhibited by injecting hBMSC and peripheral blood mononuclear cell (PBMC) co-cultured exosomes. These exosomes suppressed immune reaction by inhibiting PBMC proliferation and enhancing regulatory T cell (Treg) function. Collectively, our studies elucidated the mechanisms of RNA delivery from hBMSCs to human islets and the immunosuppressive effect of hBMSC and peripheral blood mononuclear cell co-cultured exosomes for improving islet transplantation.

Repeated treatments with chemotherapeutic agent(s) fail due to cancer stem cells (CSCs) and chemoresistance regulated by microRNAs (miRNA) whose expression alters owing to dysfunctional signaling pathways including Hedgehog (Hh) signaling. We previously demonstrated the combination of Hh inhibitor cyclopamine (CYP) and paclitaxel (PTX) effectively inhibit PTX-resistant cells and side population, a cell fraction rich in CSCs. In this study, we synthesized mPEG-b-PCC-g-PTX-g-DC (P-PTX) and mPEG-b-PCC-g-CYP-g-DC (P-CYP) polymer-drug conjugates, which they self-assembled into micelles. The combination of P-PTX and P-CYP alleviated PTX resistance and suppressed tumor colony formation. Further, combination therapy inhibited Hh signaling and up-regulated tumor suppressor miRNAs. We established orthotopic prostate tumor in nude mice and there was significant tumor growth inhibition in the group treated with the combination therapy of P-PTX and P-CYP compared with monotherapy. In conclusion, this combination therapy of P-PTX and P-CYP has the potential to treat chemoresistant prostate cancer.

Hedgehog (Hh) signaling plays an important role in the development and metastasis of pancreatic ductal adenocarcinoma (PDAC). Although gemcitabine (GEM) has been used as a first-line therapy for PDAC, its rapid metabolism and short plasma half-life restrict its use as a single chemotherapy. Combination therapy with more than one drugs is a promising approach for treating cancer. Herein, we report the use of methoxy poly(ethyleneglycol)-block-ploy(2-methyl-2-carboxyl-propylenecarbonate)-graft-dodecanol (mPEG-b-PCC-g-DC) copolymer for conjugating GEM and encapsulating an Hh inhibitor, Vismodegib (GDC-0449) into its hydrophobic core for treating PDAC. Our objective was to determine whether the mixed micelle formulation of these two drugs could show better response in inhibiting Hh signaling pathway and restraining the proliferation and metastasis of pancreatic cancer. The in vivo stability of GEM significantly increased after conjugation, which resulted in its increased antitumor efficacy. Almost 80 % of encapsulated GDC-0449 and 19 % conjugated GEM were released in vitro at pH 5.5 in 48 h in a sustained manner. The invasion, migration and colony forming features of MIA PaCa-2 cells were significantly inhibited by micelle mixture carrying GEM and GDC-0449. Remarkable increase in PARP cleavage and Bax proved increased apoptosis by this combination formulation compared to individual micelles. This combination therapy efficiently inhibited tumor growth, increased apoptosis and reduced Hh ligands PTCH-1, Gli1, and lowered EMT-activator ZEB-1 when injected to athymic nude mice bearing subcutaneous tumor generated using MIA PaCa-2 cells compared to monotherapy as observed from immunohistochemical analysis. In conclusion, micelle mixtures carrying GEM and GDC-0449 have the potential to treat pancreatic cancer.

limiting the chance for com-plete excision of these tumors. Furthermore, prolonged treatment with chemotherapyresultsinmultidrugresistance.Inrecentyears,scientistsandphysicianshaveexpandedsignificant efforts to explain the underlying mechanism of chemoresistance forimproving both duration of survival and quality of life. The highly complex natureof drug resistance renders current single agent or multiple drug combinations ineffec-tive, leading to poor patient survival making pancreatic cancer one of the deadliestmalignancies. To date, a variety of distinct and disparate molecular mechanismshave been suggested including the role of ATP binding cassette (ABC)-transporters,increased DNA repair and dysregulation of the apoptotic machinery, enhanced cyto-plasmic inactivation of drug metabolites and overexpression of oncogenes or down-regulation of tumor suppressor genes. Despite these advances, our understanding ofchemoresistance in pancreatic cancer is fragmentary and incomplete. Recent studiesin our lab and by other groups

Overexpression of the androgen receptor (AR) and anti-apoptotic genes including X-linked inhibitor of apoptosis protein (XIAP) provide tumors with a proliferative advantage. Therefore, our objective was to determine whether novel antiandrogen (CBDIV17) and XIAP inhibitor based combination therapy can treat advanced prostate cancer.CBDIV17 and embelin-6g were synthesized and their effect on cell proliferation, apoptosis, cell cycle and AR and XIAP gene silencing determined.CBDIV17 was more potent than bicalutamide and inhibited proliferation of C4-2 and LNCaP cells, IC(50) for CBDIV17 was ≈ 12 μM and ≈ 21 μM in LNCaP and C4-2 cells, respectively, whereas bicalutamide had IC(50) of ≈ 46 μM in LNCaP cells and minimal effect in C4-2 cells. CBDIV17 induced apoptosis more effectively compared to bicalutamide and significantly inhibited DNA replication. Combination of CBDIV17 and embelin resulted in supra-additive antiproliferative and apoptotic effects. Embelin downregulated AR expression and decreased androgen-mediated AR phosphorylation at Ser(81). These hydrophobic drugs were solubilized using micelles prepared with polyethylene glycol-b-poly (carbonate-co-lactide) (PEG-b-p(CB-co-LA)) copolymer. Combination therapy inhibited prostate tumor growth more effectively compared to control or monotherapy in vivo.Our results demonstrated that CBDIV17 in combination with embelin can potentially treat advanced prostate cancer.

In the past decade, small RNAs including small interfering RNA (siRNA) and microRNA (miRNA) have attracted intense interest due to their unique roles in fundamental biological processes by regulating the stabilities and functions of mRNAs. As illustrated in Fig. 1, the numbers of publications related to siRNA and miRNA have increased dramatically in recent years. Although miRNA was discovered earlier than siRNA, its importance was not fully realized as siRNA from 2000 to 2005. However, while the siRNA publications reached a plateau after 2007, the number of publications on miRNA has increased exponentially since 2005. siRNAs are artificial double-stranded RNAs of 21–23 nt in length, while miRNAs are endogenous singlestranded RNAs of 21–25 nt. Both siRNA and miRNA are non-coding RNAs that post-transcriptionally suppress gene expressions by binding to their complementary mRNAs. The major difference between siRNA and miRNA is that each siRNA only specifically targets a single mRNA and induces its degradation, while a single miRNA targets hundreds of mRNAs either by blocking protein translation or destabilizing the mRNA molecules. This difference comes from the fact that siRNA always forms a perfect match to its complementary mRNA, while miRNA only forms imperfect matches to its multiple mRNA targets (1–3). This theme issue focuses on recent advances in the biological and therapeutic applications of siRNA and miRNA. There are six articles in this issue highlighting research from studying gene functions to delivery to therapeutic application of siRNA. Since its discovery, siRNA has been widely applied as a powerful tool to study genetic functions of a specific gene. To further expand the applications of siRNA in gene function studies, Friedman et al. describe a novel light-activated RNA interference (RNAi) strategy by incorporating photolabile groups (di-methoxy nitro phenyl ethyl or DMNPE) to multiple double-stranded precursors of siRNA (dsRNA). Phosphorothioate linkages are also incorporated to increase the serum stability of the dsRNA. This strategy allows the control of the spacing, timing, and amount of gene expressions by adjusting light exposure. A successful light-controllable RNAi is particularly useful for the study of developmental biology or targeted delivery to specific sites in the body. Another promising application of siRNA is the treatment of various diseases by silencing their key modulators. Currently, there are 13 ongoing clinical trials using siRNA for numerous diseases. A review by Schaffer et al. discusses basic action mechanisms and therapeutic applications of RNAi in antiviral therapy. Design considerations, delivery strategies, computational insight, and areas for future improvement are described. Another article by Nemunaitis and colleagues summarizes the potentials, challenges, and current status of small RNA-based cancer therapies. As an example of developing siRNA therapy, Cheng’s group explore the potential therapeutic application of PCBP2 siRNA in treating alcoholic liver fibrosis which is characK. Cheng (*) Division of Pharmaceutical Sciences, School of Pharmacy University of Missouri—Kansas City 2464 Charlotte Street, HSB 5250 Kansas City, Missouri 64108-2718, USA e-mail: chengkun@umkc.edu

The use of adult stem cells for therapeutic purposes has met with great success in recent years. Among several types of adult stem cells, mesenchymal stem cells (MSCs) derived from bone marrow (BM) and other sources have gained popularity for basic research and clinical applications because of their therapeutic potential in treating a variety of diseases. Because of their tissue regeneration potential and immune modulation effect, MSCs were recently used as cell-based therapy to promote revascularization, increase pancreatic β-cell proliferation, and avoid allograft rejection in islet transplantation. Taking advantage of the recent progress in gene therapy, genetically modified MSCs can further enhance and expand the therapeutic benefit of primary MSCs while retaining their stem-cell-like properties. This review aims to gain a thorough understanding of the current obstacles to successful islet transplantation and discusses the potential role of primary MSCs before or after genetic modification in islet transplantation.

Our objective was to synthesize an amphiphilic diblock copolymer for micellar delivery of rapamycin. Poly(ethylene glycol)-block-poly(2-methyl-2-benzoxycarbonyl-propylene carbonate) (PEG-b-PBC) with different hydrophobic core lengths were synthesized from methoxy poly(ethylene glycol) and 2-methyl-2-benzoxycarbonyl-propylene carbonate through ring-opening polymerization using 1,8-diazabicycloundec-7-ene as a catalyst. The critical micelle concentration of PEG-b-PBC was around 10 −8 M and depends on the hydrophobic core length. Rapamycin was effectively incorporated into micelles and drug loading increased with increasing hydrophobic core length, with maximal drug loading of 10% (w/w, drug/polymer), drug loading efficiency of about 85%, and mean particle size of around 70 nm. The drug release profile was also dependent on the hydrophobic core length and the drug release from PEG 114 -b-PBC 30 micelles was the slowest. We also determined the toxicity of rapamycin micelles on insulinoma (INS-1E) β-cells and human islets. Encapsulation of rapamycin into PEG-b-PBC micelles reduced its toxicity. Biodistribution of rapamycin-loaded PEG-b-PBC micelles was determined after systemic administration into mice. Rapamycin-loaded PEG-b-PBC micelles showed little difference in pharmacokinetics and biodistribution characteristics in mice compared with rapamycin carrying nanosuspension. In conclusion, rapamycin formulated with PEG-b-PBC micelles showed significantly reduced toxicity on INS-1E β-cells and human islets, but had similar biodistribution profiles as those of nanosuspensions.

To evaluate the efficacy of human bone marrow-derived mesenchymal stem cells (hBMSCs) as gene delivery vehicles to simultaneously express human hepatocyte growth factor (HGF) and interleukin 1 receptor antagonist (IL-1Ra) to improve the outcome of islet transplantation.Morphology and islet-binding affinity of hBMSCs were checked by microscope. The expression of target genes and endogenous genes was determined by ELISA. Protection of islets by hBMSCs was evaluated in vitro by Calcein-AM/Propidium Iodide staining and in vivo by allogeneic islet transplantation study. Function and revascularization of islets was evaluated by immune fluorescence study.Non-donor-specific hBMSCs showed strong binding affinity to human islets and protected viability and function. Transduction of hBMSCs with adenovirus encoding human HGF and human IL-1Ra (Adv-hHGF-hIL-1Ra) prior to co-culturing with islets further protected from apoptotic cell death, helped maintain 3D structures and morphology, and enhanced insulin secretion. Transplantation of human islets reconstituted with Adv-hHGF-hIL-1Ra transduced hBMSCs under the kidney capsule of streptozotocin-induced diabetic non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice reversed diabetes by reducing blood glucose levels to ≤ 200 mg/dL for up to 15 weeks and reduced the number of islets required to achieving normoglycemia. Blood glucose levels of mice transplanted with islets alone reversed to ≥ 500 mg/dL 4 weeks post-transplantation.Results indentified hBMSCs as effective gene delivery vehicles to improve the outcome of islet transplantation.

Previously, we successfully conjugated galactosylated poly(ethylene glycol) (Gal-PEG) to oligonucleotides (ODNs) via an acid labile ester linker (Zhu et al., Bioconjugate Chem. 2008, 19, 290-8). In this study, sense strands of siRNA were conjugated to Gal-PEG and mannose 6-phosphate poly(ethylene glycol) (M6P-PEG) for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells (HSCs), respectively. These siRNA conjugates were purified by ion exchange chromatography and verified by gel retardation assay. To evaluate their RNAi functions, the validated siRNA duplexes targeting firefly luciferase and transforming growth factor beta 1 (TGF-β1) mRNA were conjugated to Gal-PEG and M6P-PEG, and their gene silencing efficiencies were determined after transfection into HepG2 and HSC-T6 cells. The disulfide bond between PEG and siRNA was cleaved by dithiothreitol, leading to the release of intact siRNA. Both Gal-PEG-siRNA and M6P-PEG-siRNA conjugates could silence luciferase gene expression by about 40% without any transfection reagents, while the gene silencing effects reached more than 98% with the help of cationic liposomes at the same dose. Conjugation of TGF-β1 siRNA with Gal-PEG and M6P-PEG could silence endogenous TGF-β1 gene expression as well. In conclusion, these siRNA conjugates have the potential for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells for efficient gene silencing in vivo.

A novel microtubule destabilizer, substituted methoxybenzoyl-ary-thiazole (SMART)-100, was synthesized, which showed good anticancer activity in HepG2 cells. SMART-100 was able to circumvent multidrug resistance (MDR) and effectively inhibited the growth of cell lines that overexpress P-glycoprotein (P-gp). SMART-100 inhibited P-gp activity, which may be responsible for its ability to overcome MDR. Since SMART-100 is poorly soluble in water, it was formulated in polyethylene-b-poly(D,L-lactide) (PEG-PLA) micelles. The solubility of SMART-100 was increased by more than 1.1x10(5) folds. SMART-100 loaded PEG-PLA micelles could effectively inhibit HepG2 cell growth and arrest cell cycle progression at G2/M phase, followed by appearance of a sub-G1 phase, which is indicative of cell apoptosis. Increased Caspase-3 activity was also observed when HepG2 cells were treated with SMART-100. The anticancer activity of SMART-100 loaded PEG-PLA micelles was also evaluated on luciferase expressing C4-2-Luc cell lines by IVIS imaging. Our results suggest that SMART-100 has the potential to treat resistant cancers.

Bicalutamide is the most widely used non-steroidal antiandrogen for treating early stage prostate cancer, but suffers variable oral absorption due to its limited aqueous solubility. Thus, our objective was to synthesize novel biodegradable copolymers for the systemic micellar delivery of bicalutamide. Flory–Huggins interaction parameter ( χ FH ) was used to assess compatibility between bicalutamide and poly( l -lactide) or poly(carbonate-co-lactide) polymer pairs. Polyethylene glycol-b-poly(carbonate-co-lactide) [PEG-b-P(CB-co-LA)] copolymers were synthesized and characterized by NMR and gel permeation chromatography. These micelles had average diameter of 100 nm and had a smooth surface and distinct spherical shape. Drug loading studies revealed that adding the carbonate monomer could increase bicalutamide loading. Among the series, drug loading of micelles formulated with PEG-b-P(CB-co-LA) copolymer containing 20 mol% carbonate was about four-fold higher than PEG-b-PLLA and aqueous solubility of bicalutamide increased from 5 to 4000 μg/mL. CMC values for PEG-b-P(CB-co-LA) copolymers was up to 10-fold lower than those of PEG-b-PLLA. In vitro release experiments showed PEG-b-P(CB-co-LA) copolymers to be more efficient in sustaining the release of bicalutamide compared to PEG-b-PLLA. Bicalutamide-loaded PEG-b-P(CB-co-LA) micelles showed significant inhibition of LNCaP cell growth in a dose-dependent manner which was similar to the methanol solution of free drug.

Gene therapy for ischemic diseases has been developed with various growth factors and anti-apoptotic genes. However, non-specific expression of therapeutic genes may induce deleterious side effects such as tumor formation. Hypoxia-specific regulatory systems can be used to regulate transgene expression in hypoxic tissues, in which gene expression is induced in ischemic tissues, but reduced in normal tissues by transcriptional, translational or post-translational regulation. Since hypoxia-inducible factor 1 (HIF-1) activates transcription of genes in hypoxic tissues, it can play an important role in the prevention of myocardial and cerebral ischemia. Hypoxia-specific promoters including HIF-1 binding sites have been used for transcriptional regulation of therapeutic genes. Also, hypoxia-specific untranslated regions (UTRs) and oxygen dependent degradation (ODD) domains have been investigated for translational and post-translational regulations, respectively. Hypoxia-specific gene expression systems have been applied to various ischemic disease models, including ischemic myocardium, stroke, and injured spinal cord. This review examines the current status and future challenges of hypoxia-specific systems for safe and effective gene therapy of ischemic diseases.

Ex vivo gene transfer can improve the outcome of islet transplantation for treating type I diabetes. Earlier we have shown coexpression of human vascular endothelial growth factor (hVEGF) and human interleukin-1 receptor antagonist (hIL-1Ra) after transfection of plasmid DNA encoding these two genes. Due to poor transfection efficiency of plasmid DNA and the better known islet transduction efficiency of adenoviral (Adv) vectors, in this study, we constructed Adv-hVEGF-hIL-1Ra by cloning hVEGF and hIL-1Ra coding sequences and polyA signal under separate cytomegalovirus (CMV) promoters in Adenoquick plasmid (Ad 13.1). There was dose and time dependent expression of these genes after transduction of Adv-hVEGF-hIL1Ra into human islets. The mRNA expression of hVEGF and hIL-1Ra was more than 100 times higher than that of the nontransduced and bipartite plasmid transfected control islets. Transduced islets were viable as evidenced by insulin release upon glucose challenge. Coexpression of hVEGF and hIL-1Ra by islets showed decrease in caspase-3 activity and apoptosis induced by a cocktail of inflammatory cytokines such as TNF-alpha, IL-1beta and IFN-gamma. Compared to nontreated or Adv-LacZ transduced islets, transduction of islets with Adv-hVEGF-hIL-1Ra prior to transplantation under the kidney capsules of diabetic NOD-SCID mice reduced the blood glucose levels, and increased serum insulin and c-peptide levels. Immunohistochemical staining of the islet bearing kidney sections at day 20 after transplantation was positive for human insulin, hVEGF and von Willebrand factor. These results indicate that the bipartite Adv vector efficiently expresses both growth factor and antiapoptotic genes, decreases apoptosis and improves the outcome of islet transplantation.

Ex vivo gene therapy can improve the outcome of islet transplantation for treating type I diabetes. Hepatocyte growth factor (HGF) increases beta-cell proliferation and promotes revascularization of islets, while interleukin-1 receptor antagonist (hIL-1Ra) inhibits islet cell apoptosis.We constructed Adv-hHGF-hIL-1Ra by cloning hHGF and hIL-1Ra coding sequences and polyA signal under separate CMV promoters in Adenoquick plasmid.There was dose and time dependent expression of these genes after transduction of Adv-hHGF-hIL-1Ra into human islets. Compared to un-transduced islets, hHGF and hIL-1Ra gene expression at protein levels was more than 60 and 40 times higher at 1,000 MOI, respectively. Transduced islets were viable after incubation with the cocktail of TNF-alpha, IL-1beta and IFN-gamma, as evidenced by insulin release in response to glucose concentration. Co-expression of hHGF and hIL-1Ra led to significant decrease in caspase-3 induced by the cytokines. Compared to un-transduced islets, transduction of islets with Adv-hHGF-hIL-1Ra at 1,000 MOI prior to transplantation under the kidney capsules of streptozotocin-induced-diabetic NOD-SCID mice reduced blood glucose levels, and increased serum insulin and c-peptide levels.Transduction of islets with Adv-hHGF-hIL-1Ra efficiently expresses both growth factor and antiapoptotic genes, decreases caspase-3 and improves the outcome of islet transplantation.