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Figure S1 showed the effect of miR-34a on prostate cancer cell viability. Figure S2 characterized the PEG-PCD polymer. Figure S3 evaluated polymer stability. Figure S4 determined organ toxicity of our formulation.

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.

Successful treatment of pancreatic cancer remains a challenge due to desmoplasia, development of chemoresistance, and systemic toxicity. Herein, we synthesized (6-(3-hydroxy-4-methoxylphenyl)pyridin-2-yl) (3,4,5-trimethoxyphenyl)methanone (CH-3-8), a novel microtubule polymerization inhibitor with little susceptible to transporter-mediated chemoresistance. CH-3-8 binding to the colchicine-binding site in tubulin protein was confirmed by tubulin polymerization assay and molecular modeling. CH-3-8 disrupted microtubule dynamics at the nanomolar concentration in MIA PaCa-2 and PANC-1 pancreatic cancer cell lines. CH-3-8 significantly inhibited the proliferation of these cells, induced G2/M cell cycle arrest, and led to apoptosis. CH-3-8 is hydrophobic with an aqueous solubility of 0.97 ± 0.16 μg/mL at pH 7.4. We further conjugated it with dodecanol through diglycolate linker to increase hydrophobicity and thus loading in lipid-based delivery systems. Hence, we encapsulated CH-3-8 lipid conjugate (LDC) into methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol) (mPEG-b-PCC-g-DC) polymeric nanoparticles (NPs) by solvent evaporation, resulting in a mean particle size of 125.6 ± 2.3 nm and drug loading of 10 ± 1.0% (w/w) while the same polymer could only load 1.6 ± 0.4 (w/w) CH-3-8 using the same method. Systemic administration of 6 doses of CH-3-8 and LDC loaded NPs at the dose of 20 mg/kg into orthotopic pancreatic tumor-bearing NSG mice every alternate day resulted in significant tumor regression. Systemic toxicity was negligible, as evidenced by histological evaluations. In conclusion, CH-3-8 LDC loaded NPs have the potential to improve outcomes of pancreatic cancer by overcoming transporter-mediated chemoresistance and reducing systemic toxicity.

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.

Rationale: Hedgehog (Hh) pathway plays an essential role in liver fibrosis by promoting the proliferation of hepatic stellate cells (HSCs) by enhancing their metabolism via yes-associated protein 1 (YAP1). Despite the presence of several inhibitors, Hh signaling cannot be controlled exclusively due to their poor efficacy and the lack of a suitable delivery system to the injury site. Therefore, it is rationale to develop new potent Hh inhibitors and suitable delivery carriers. Methods: Based on the structure and activity of Hh inhibitor GDC-0449, we replaced its sulfonamide group with two methylpyridine-2yl at amide nitrogen to synthesize MDB5. We compared the Hh pathway inhibition and anti-fibrotic effect of MDB5 with GDC-0449 in vitro. Next, we developed MDB5 loaded micelles using our methoxy poly(ethylene glycol)-blockpoly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol (PEG-PCC-g-DC) copolymer and characterized for physicochemical properties. We evaluated the therapeutic efficacy of MDB5 loaded micelles in common bile duct ligation (CBDL) induced liver fibrosis, mouse model. We also determined the intrahepatic distribution of fluorescently labeled micelles after MDB5 treatment. Results: Our results show that MDB5 was more potent in inhibiting Hh pathway components and HSC proliferation in vitro. We successfully developed MDB5 loaded micelles with particle size of 40 ± 10 nm and drug loading up to 10% w/w. MDB5 loaded micelles at the dose of 10 mg/kg were well tolerated by mice, without visible sign of toxicity. The serum enzyme activities elevated by CBDL was significantly decreased by MDB5 loaded micelles compared to GDC-0449 loaded micelles. MDB5 loaded micelles further decreased collagen deposition, HSC activation, and Hh activity and its target genes in the liver. MDB5 loaded micelles also prevented liver sinusoidal endothelial capillarization (LSEC) and therefore restored perfusion between blood and liver cells. Conclusions: Our study provides evidence that MDB5 was more potent in inhibiting Hh pathway in HSC-T6 cells and showed better hepatoprotection in CBDL mice compared to GDC-0449.

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.

Active ingredients from Chinese medicine (AIFCM) are emerging as a class of anticancer agents for treating hepatocellular carcinoma (HCC). AIFCM have unique advantages over traditional chemotherapeutic drugs, such as their multi-target, multi-level, and coordinated intervention effects against HCC. Recent researches demonstrated their potentials in inhibiting HCC initiation, cell proliferation, angiogenesis, and metastasis. With the advantages of nano-carriers including better bioavailability, significant features of passive or active targeting and stimulus-responsive drug release. Formulation of AIFCM and nano-carriers shows great potential in promoting anti-HCC therapeutic development. Here, we critically assess nano-carriers for delivery and targeting of AIFCM for HCC therapy. First, we offer an overview of major pathogenic factors to induce HCC, including virus infection, alcoholic liver disease, mycotoxin, obesity, and type 2 diabetes. Second, the mechanisms of AIFCM for HCC therapy comprising gene regulation and transduction of different molecular pathways are discussed. Third, significant achievements of AIFCM-encapsulated nano-carriers and AIFCM-modified nano-carriers against HCC are presented. Finally, we provide concluding remarks and future perspectives of AIFCM for HCC therapy, hoping to offer some insight on how to improve AIFCM-based HCC therapy.

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.

MicroRNAs (miRNAs) are noncoding small RNAs that regulate various pathophysiological cellular processes. Here, we report that expression of the miR-378 family was significantly induced by metabolic inflammatory inducers, a high-fructose diet, and inflammatory cytokine tumor necrosis factor-α. Hepatic miRNA profiling revealed that expression of miR-378a was highly upregulated, which, in turn, targeted the 3′-untranslated region of PPARα mRNA, impaired mitochondrial fatty acid β-oxidation, and induced mitochondrial and endoplasmic reticulum stress. More importantly, the upregulated miR-378a can directly bind to and activate the double-strand RNA (dsRNA)–dependent protein kinase R (PKR) to sustain the metabolic stress. In vivo, genetic depletion of miR-378a prevented PKR activation and ameliorated inflammatory stress and insulin resistance. Counterbalancing the upregulated miR-378a using nanoparticles encapsulated with an anti-miR-378a oligonucleotide restored PPARα activity, inhibited PKR activation and ER stress, and improved insulin sensitivity in fructose-fed mice. Our study delineated a novel mechanism of miR-378a in the pathogenesis of metabolic inflammation and insulin resistance through targeting metabolic signaling at both mRNA (e.g., PPARα) and protein (e.g., PKR) molecules. This novel finding of functional interaction between miRNAs (e.g., miR-378a) and cellular RNA binding proteins (e.g., PKR) is biologically significant because it greatly broadens the potential targets of miRNAs in cellular pathophysiological processes.

MicroRNAs (miRNAs) are noncoding small RNAs that regulate various pathophysiological cellular processes. Here we reported that expression of the miR-378 family was significantly induced by metabolic inflammatory inducers, a high-fructose diet, and inflammatory cytokine TNFa. Hepatic miRNA profiling revealed that expression of miR-378a was highly upregulated which, in turn, targeted the 3’-UTR of PPARa mRNA, impaired mitochondrial fatty acid b-oxidation and induced mitochondrial and ER stress. More importantly, the upregulated miR-378a can directly bind to and activate the dsRNA-dependent protein kinase R (PKR) to sustain the metabolic stress. In vivo, genetic depletion of miR-378a prevented PKR activation, ameliorated inflammatory stress and insulin resistance. Counterbalancing the upregulated miR-378a using nanoparticles encapsulated with an anti-miR-378a oligonucleotide restored PPARa activity, inhibited PKR activation and ER stress, and improved insulin sensitivity in the fructose-fed mice. Conclusion: Our study delineated a novel mechanism of miRNA-378a in the pathogenesis of metabolic inflammation and insulin resistance through targeting metabolic signaling at both mRNA (e.g., PPARa) and protein (e.g., PKR) molecules. This novel finding of functional interaction between miRNAs (e.g., miR-378a) and cellular RNA binding protein(s) (e.g., PKR) is biologically significant as it greatly broadens the potential targets of miRNAs in cellular pathophysiological processes.

Redox-responsive nanomedicine of miR-519c and gemcitabine effectively inhibits pancreatic tumor growth in mice., Desmoplastic and hypoxic pancreatic cancer microenvironment induces aberrant expression of miRNAs and hypoxia-inducible factor-1α (HIF-1α) responsible for gemcitabine (GEM) resistance. We demonstrated that miR-519c was down-regulated in pancreatic cancer and transfection of miR-519c in GEM-resistant pancreatic cancer cells inhibited HIF-1α level under hypoxia. We synthesized redox-sensitive mPEG-co-P(Asp)-g-DC-g-S-S-GEM polymer, with GEM payload of 14% (w/w) and 90% GEM release upon incubation with l-glutathione. We synthesized mPEG-co-P(Asp)-g-TEPA-g-DC for complex formation with miRNA. Chemical modification of miR-519c with 2′-O-methyl phosphorothioate (OMe-PS) at 3′ end enhanced its stability and activity without being immunogenic. Epidermal growth factor receptor targeting peptide GE11 decoration increased tumor accumulation of micelles after systemic administration and significantly inhibited orthotopic desmoplastic pancreatic cancer growth in NSG mice by down-regulating HIF-1α and genes responsible for glucose uptake and cancer cell metabolism. Our multifunctional nanomedicine of GEM and OMe-PS–miR-519c offers a novel therapeutic strategy to treat desmoplasia and hypoxia-induced chemoresistance in pancreatic cancer.

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.

Medulloblastoma (MB) is a clinically challenging, childhood brain tumor with a diverse genetic makeup and differential miRNA profile. Aiming to identify deregulated miRNAs in MB, the miRNA expression profile of human MB samples was compared to that of normal cerebellar tissues. As a result, 8 upregulated and 64 downregulated miRNAs were identified in MB samples. Although various algorithms have been developed to predict the interaction between miRNA-mRNA pairs, the complexity and fidelity of miRNA-mRNA remain a concern. Therefore, to identify the signatures of miRNA-mRNA interactions essential for MB pathogenesis, miRNA profiling, RNA sequencing, and ingenuity pathway analysis (IPA) were performed in the same primary human MB samples. Further, when miR-217 was inhibited, a significant upregulation of predicted target genes SIRT1, ROBO1, FOXO3, and SMAD7 in HDMB03 cells was observed, confirming the validity of our approach. Functional analysis revealed that the inhibition of miR-217 in HDMB03 cells suppresses colony formation, migration, invasion, promoted apoptosis, and arrested cell population in S phase, indicating that manipulation of miR-217 may have a therapeutic potential for MB patients. Therefore, our study provides an essential platform for future investigations of specific miRNAs responsible for MB pathogenesis. Keywords: medulloblastoma, miRNA profiling, RNA-seq, miRNA-mRNA correlation, miR-217

Rationale: Successful treatment of pancreatic cancer remains a challenge due to desmoplasia and prevalence of KRAS mutation. While hedgehog (Hh) ligand levels are upregulated in pancreatic cancer cells and contribute to desmoplasia, there is significant downregulation of tumor suppressor let-7b, which targets mutant KRAS, C-MYC and several other genes involved in pancreatic cancer progression, invasion, and metastasis. We recently explored combination therapy of GDC-0449 (Hh inhibitor) and let-7b mimic using poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (PEG-b-PCC-g-DC-g-TEPA) micelles in pancreatic tumor mouse model. Here, our objective was to determine the biodistribution (BD), pharmacokinetics (PK), therapeutic efficacy and toxicity of this micellar formulation. Methods: We determined the PK of micelles encapsulating Cy5.5-let-7b and GDC-0449 following intravenous injection in orthotopic pancreatic tumor-bearing NSG mice at doses of 2 mg/kg and 10 mg/kg, respectively. Mice were scanned for fluorescence by IVIS to determine the biodistribution of Cy5.5-let-7b at the whole-body level, and its concentration in plasma and major organs was determined by measuring fluorescence using a fluorimeter and by real-time RT-PCR. GDC-0449 concentration was determined by LC/MS/MS. Therapeutic efficacy and toxicity of the micellar formulation of let-7b and GDC-0449 was also determined after two weeks of treatment. Results: The use of a micellar formulation markedly prolonged the elimination half-life (t1/2, e) of Cy5.5-let-7b in plasma from 0.49 ± 0.19 h to 2.65 ± 0.46 h and increased the area-under-the-curve (AUC 0-∞ ) by 7-fold, while t1/2,e and AUC 0-∞ of GDC-0449 were increased by 1.78-fold and 3.2-fold, respectively. The micelles significantly decreased the clearance of both encapsulated let-7b mimic and GDC-0449 compared to the emulsion formulation. Compared to the emulsion counterpart, the micellar formulation elevated the delivery of Cy5.5-let-7b and GDC-0449 to the orthotopic pancreatic tumor tissue by 7.8- and 4.2-fold, respectively. Furthermore, there was a significant reduction in tumor volume and negligible systemic toxicity as evident by hematological parameters and histological evaluation. Conclusion: PEG-b-PCC-g-DC-g-TEPA micelles carrying GDC-0449 and let-7b mimic have great potential to improve drug delivery for pancreatic cancer treatment.

Chemoresistance and inadequate therapeutics transport across the blood brain barrier (BBB) remain the major barriers to treating medulloblastoma (MB). Hedgehog (Hh) and IGF/PI3K pathways regulate tumor cell proliferation and resistance in MB. Current Hh inhibitors are effective initially to treat SHH-MB but acquire resistance. Herein, we showed that Hh inhibitor MDB5 and BRD4/PI3K dual inhibitor SF2523 synergistically inhibited the proliferation of DAOY and HD-MB03 cells when used in combination. Treatment of these MB cells with the combination of MDB5 and SF2523 significantly decreased colony formation and expression of MYCN, p-AKT, and cyclin D1 but significantly increased in Bax expression, compared to individual drugs. We used our previously reported copolymer mPEG-b-PCC-g-DC copolymer, which showed 8.7 ± 1.0 and 6.5 ± 0.1% loading for MDB5 and SF2523 when formulated into nanoparticles (NPs). There was sustained drug release from NPs, wherein 100% of MDB5 was released in 50 h, but only 60% of SF2523 was released in 80 h. Targeted NPs prepared by mixing 30:70 ratio of COG-133-PEG-b-PBC and mPEG-b-PCC-g-DC copolymer delivered a significantly higher drug concentration in the cerebellum at 6 and 24h after intravenous injection into orthotopic SHH-MB tumor-bearing NSG mice . Moreover, systemic administration of COG-133-NPs loaded with MDB5 and SF2523 resulted in decreased tumor burden compared to non-targeted drug-loaded NPs, without any hepatic toxicity. In conclusion, our nanomedicine of MDB5 and SF2523 offers a novel therapeutic strategy to treat chemoresistant MB.

Designing grafted biodegradable polymers with tailored multi-functional properties is one of the most researched fields with extensive biomedical applications . Among many biodegradable polymers, polycarbonates have gained much attention due to their ease of synthesis, high drug loading, and excellent biocompatibility profiles. Among various monomers , 2,2-bis(hydroxymethyl) propionic acid ( bis-MPA ) derived cyclic carbonate monomers have been extensively explored in terms of their synthesis as well as their polymerization. Since the late 90s, significant advancements have been made in the design of bis-MPA derived cyclic carbonate monomers as well as in their reaction schemes. Currently, bis-MPA derived polycarbonates have taken a form of an entire platform with a multitude of applications, the latest being in the field of nanotechnology, targeted drug, and nucleic acid delivery. The present review outlines an up to date developments that have taken place in the last two decades in the design, synthesis, and biomedical applications of bis-MPA derived cyclic carbonates and their (co)polymers.

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.

The completion of human genome project, decrease in the sequencing cost, and correlation of genome sequencing data with specific diseases led to the exponential rise in the nucleic acid-based therapeutic approaches. In the third annual Biopharmaceutical Research and Development Symposium (BRDS) held at the Center for Drug Discovery and Lozier Center for Pharmacy Sciences and Education at the University of Nebraska Medical Center (UNMC), we highlighted the remarkable features of the nucleic acid-based nanomedicines, their significance, NIH funding opportunities on nanomedicines and gene therapy research, challenges and opportunities in the clinical translation of nucleic acids into therapeutics, and the role of intellectual property (IP) in drug discovery and development.

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.

Precision medicine is promising for treating human diseases, as it focuses on tailoring drugs to a patient’s genes, environment, and lifestyle. The need for personalized medicines has opened the doors for turning nucleic acids into therapeutics. Although gene therapy has the potential to treat and cure genetic and acquired diseases, it needs to overcome certain obstacles before creating the overall prescription drugs. Recent advancement in the life science has helped to understand the effective manipulation and delivery of genome-engineering tools better. The use of sequence-specific nucleases allows genetic changes in human cells to be easily made with higher efficiency and precision than before. Nanotechnology has made rapid advancement in the field of drug delivery, but the delivery of nucleic acids presents unique challenges. Also, designing efficient and short time-consuming genome-editing tools with negligible off-target effects are in high demand for precision medicine. In the fourth annual Biopharmaceutical Research and Development Symposium (BRDS) held at the University of Nebraska Medical Center (UNMC) on September 7–8, 2017, we covered different facets of developing tools for precision medicine for therapeutic and diagnosis of genetic disorders.

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.

AIM This study evaluated the influence of CYP2C19 polymorphisms on the pharmacokinetics of nelfinavir and its metabolite M8 in patients with pancreatic cancer. METHODS Nelfinavir was administered orally to patients for over 10 days. The plasma concentrations of nelfinavir and M8 were measured by HPLC. The genotypes of CYP2C19*1, CYP2C19*2 and CYP2C19*3 were determined by the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS Pharmacokinetic profiles of nelfinavir and M8 were characterized by wide interindividual variability. The mean Cmax of nelfinavir in CYP2C19*1/*1 patients was 3.89 ± 0.40 (n = 3) and 5.12 ± 0.41 (n = 30) µg ml(-1) , while that of CYP2C19*1/*2 patients was 3.60 (n = 1) and 6.14 ± 0.31 (n = 5) µg ml(-1) at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. For the M8 metabolite, the mean Cmax of CYP2C19*1/*1 patients was 1.06 ± 0.06 (n = 3) and 1.58 ± 0.27 (n = 30) µg ml(-1) , while those of CYP2C19*1/*2 patients were 1.01 (n = 1) and 1.23 ± 0.15 (n = 5) µg ml(-1) at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. The area under the plasma concentration-time curve (AUC(0,12 h)) values of nelfinavir for CYP2C19*1/*1 patients were 28.90 ± 1.27 and 38.90 ± 4.99 µg ml(-1) ·h and for CYP2C19*1/*2 patients, AUC(0,12 h) was 28.20 (n = 1) and 40.22 ± 3.17 (n = 5) µg ml(-1) ·h at the doses of 625 and 1250 mg nelfinavir twice daily, respectively. The Cmax of nelfinavir was significantly higher (P

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.

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

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.

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.

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.

The purpose of this study to develop targeted liposomes of MDB5 and miR-29b combination for medulloblastoma (MB) treatment. Due to high heterogeneity and diverse genetic make-up, MB treatment is a challenge. Aberrant activation of hedgehog (Hh) pathway regulates cell growth, cancer stem cell (CSC) proliferation, and tumorigenicity in sonic Hh subgroup of MB. Further, Hh signaling regulates yes-associated protein 1 (YAP1) and glutaminolysis for energy need of tumor cells. GDC-0449 a smoothened receptors (SMO) antagonist treats (SHH)-MB initially, but resistance develops after repeated administration. Cholesterol potentiates Hh pathway independent of SHH by binding directly to the SMO. On the other hand, our RNA-seq and miRNA profiling confirmed downregulation of tumor suppressor miR-29b-3p in human MB samples. miR-29b control genes of apoptosis; p53, MDM2, AKT2, and cholesterol biosynthesis including HMGCR and SREBP1. We recently synthesized a potent GDC-0449 analog MDB5 (SMO inhibitors), which binds strongly to various Smo-mutants compared to GDC-0449. We found that a combination of MDB5 and miR-29b can treat MB synergistically. When we modified miR-29b backbone with 2-O’-methoxyethyl phosphorothioate (OMe-PS-miR-29b-3p), it showed enhanced stability in 50% serum. We designed a cationic lipid MDC2 for liposomal delivery of MDB5 and miR-29b1 simultaneously. Liposomes were prepared using film hydration technique and characterized for particle size and zeta potential, drug loading and release profile, miRNA complexation, stability, and transfection efficiency. These liposomes incorporated 88% MDB5 when the drug to phospholipid molar ratio was kept at 5%. Complete complexation was observed at N/P ratio of 3:1. These liposomes when loaded with green fluorescent protein (GFP) siRNA, resulted in 70% of signal silencing into DAOY MB cells (Shh driven). Liposomal MDB5 had higher cytotoxicity in DAOY cells compared to GDC-0449. Further, MDB5 and OMe-PS-miR-29b loaded liposomes resulted in higher, cytotoxicity and % of apoptosis, G0-G1 phase arrest, and decreased target gene expression in DAOY cells. Combination of drugs to simultaneously target multiple pathways related to Hh, glutaminolysis, cholesterol biosynthesis, and apoptosis has the potential to treat MB. Successful completion of this study will provide a nanomedicine for treating SHH-MB and other brain tumors. Citation Format: Virender Kumar, Bharti Sethi, Vinod Kumar, Timothy R. Mcguire, Don W. Coulter, Ram I. Mahato. Targeting vismodegib-resistant medulloblastoma using novel hedgehog pathway inhibitor and miR-29b mimic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 15.

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.

Therapeutic efficacy of gemcitabine (GEM) is severely limited due to its rapid metabolism by enzymatic deamination in vivo. We recently determined its therapeutic efficacy before (F-GEM) and after conjugation to poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate) (mPEG-b-PCC-g-GEM-g-DC, abbreviated as P-GEM) in subcutaneous and orthotopic pancreatic tumor bearing mice. In this study, pharmacokinetic (PK) parameters and biodistribution profiles of F-GEM and P-GEM were determined after intravenous injection into orthotopic pancreatic tumor bearing NSG mice. To assess the short-term toxicity, the levels of hematological, hepatic, and renal injury markers were measured after 24 h postadministration into these mice. P-GEM was distributed to all the major organs, with higher accumulation in the liver, spleen, and tumor compared to F-GEM. Area under the curve (AUC), elimination half-life (t1/2), and mean residence time (MRT) of P-GEM treated group were significantly higher compared to those of F-GEM treated group: 246,425 ± 1605 vs 83,591 ± 1844 ng/mL × h as AUC, 5.77 ± 2.02 vs 1.99 ± 0.09 h as t1/2, and 4.45 ± 0.15 vs 1.12 ± 0.13 h as MRT. Further, P-GEM exhibited negligible systemic toxicity as evidenced by almost similar alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values for both P-GEM and F-GEM. These results suggest that P-GEM protects GEM from degradation and provides sustained drug release, resulting in enhanced GEM delivery to the tumor by more than 2.5-fold compared to F-GEM. Hence, P-GEM is a promising gemcitabine conjugated polymeric micelle 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.

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.

Our objective was to synthesize and evaluate lactic acid- and carbonate-based biodegradable core- and core-co- rona crosslinkable copolymers for anticancer drug delivery. Methoxy poly(ethylene glycol)-b-poly(carbonate-co-lactide-co- 5-methyl-5-allyloxycarbonyl-1,3-dioxane-2-one) (mPEG-b-P(CB- co-LA-co-MAC)) and methoxy poly(ethylene glycol)-b-poly(acry- loyl carbonate)-b-poly(carbonate-co-lactide) (mPEG-b-PMAC-b- P(CB-co-LA)) copolymers were synthesized by ring-opening polymerization of LA, CB, and MAC using mPEG as an macroi- nitiator and 1,8-diazabicycloundec-7-ene as a catalyst. These amphiphilic copolymers which exhibited low polydispersity and critical micelle concentration values (0.8-1 mg/L) were used to prepare micelles with or without drug and stabilized by crosslinking via radical polymerization of double bonds introduced in the core and interface to improve stability. mPEG114-b-P(CB8-co-LA35-co-MAC2.5) had a higher drug encap- sulation efficiency (78.72% 6 0.15%) compared to mPEG114-b- PMAC2.5-b-P(CB9-co-LA39) (20.29% 6 0.11%). 1 H NMR and IR spectroscopy confirmed successful crosslinking (� 70%) while light scattering and transmission electron microscopy were used to determine micelle size and morphology. Crosslinked micelles demonstrated enhanced stability against extensive dilution with aqueous solvents and in the presence of physio- logical simulating serum concentration. Furthermore, bicaluta- mide-loaded crosslinked micelles were more potent compared to non-crosslinked micelles in inhibiting LNCaP cell prolifera- tion irrespective of polymer type. Finally, these results suggest crosslinked micelles to be promising drug delivery vehicles for chemotherapy. V C 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 000: 000-000, 2012

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.

Background Islet transplantation has the potential for treating type I diabetes; however, its widespread clinical application is limited by the massive apoptotic cell death and poor revascularization of transplanted islet grafts. Methods We constructed a surface-modified adenoviral vector with RGD (Arg-Gly-Asp) sequences encoding human X-linked inhibitor of apoptosis and hepatocyte growth factor (RGD-Adv-hHGF-hXIAP). In vitro transgene expression in human islets was determined by enzyme-liniked immunosorbent assay. RGD-Adv-hHGF-hXIAP-transduced human islets were transplanted under the kidney capsule of streptozotocin-induced diabetic NOD/SCID mice. The blood glucose levels of mice were measured weekly. The kidneys bearing islets were isolated at the end of the experiment and subjected to immunofluorescence staining. Results The transduction efficiency on human islets was significantly improved using RGD-modified adenovirus. HGF and XIAP gene expressions were dose-dependent after viral transduction. When exposed to a cocktail of inflammatory cytokines, RGD-Adv-hHGF-hXIAP-transduced human islets showed decreased caspase 3 activity and reduced apoptotic cell death. Prolonged normoglycemic control could be achieved by transplanting RGD-Adv-hHGF-hXIAP-transduced human islets. Immunofluorescence staining of kidney sections bearing RGD-Adv-hHGF-hXIAP-transduced islets was positive for insulin and von Willebrand factor (vWF) at 200 days after transplantation. Conclusions These results indicated that ex vivo transduction of islets with RGD-Adv-hHGF-hXIAP decreased apoptotic islet cell death and improved islet revascularization, and eventually might improve the outcome of human islet transplantation. Copyright © 2011 John Wiley & Sons, Ltd.

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.

Liver fibrosis is a consequence of chronic liver disorders which lead to the accumulation of extracellular matrix (ECM). Particularly, there is an increased accumulation of collagen in the fibrotic liver. We have therefore used a triplex forming oligonucleotide (TFO) against the type α 1 (I) collagen and evaluated, whether it can attenuate liver fibrosis induced by common bile duct ligation (CBDL) in rats. There was a significant decrease in hydroxyproline levels and Masson’s trichrome staining for collagen in TFO-treated CBDL groups compared to non-treated CBDL group. There was over expression of type α1(I) collagen, α-smooth muscle actin (α-SMA) and TGF-β1 expression in the CBDL group compared to TFO-treated CBDL group. Also, the serum alanine transaminase (ALT) and aspartate transaminase (AST) concentrations were less in the TFO treated group compared to non-treated CBDL group. There was also less neutrophils accumulation in TFO treated CBDL group assayed by myeloperoxidase (MPO) assay. These results suggests that TFO can be used to downregulate type 1 collagen gene expression and can alleviate liver fibrosis induced by common bile duct ligation.