Mesenchymal Stem Cell Capping on ECM-Anchored Caspase Inhibitor-Loaded PLGA Microspheres for Intraperitoneal Injection in DSS-Induced Murine Colitis.

Mesenchymal stem cells (MSCs) are considered as a promising alternative for the treatment of various inflammatory disorders. However, poor viability and engraftment of MSCs after transplantation are major hurdles in mesenchymal stem cell therapy. Extracellular matrix (ECM)-coated scaffolds provide better cell attachment and mechanical support for MSCs after transplantation. A single-step method for ECM functionalization on poly(lactic-co-glycolic acid) (PLGA) microspheres using a novel compound, dopamine-conjugated poly(ethylene-alt-maleic acid), as a stabilizer during the preparation of microspheres is reported. The dopamine molecules on the surface of microspheres provide active sites for the conjugation of ECM in an aqueous solution. The results reveal that the viability of MSCs improves when they are coated over the ECM-functionalized PLGA microspheres (eMs). In addition, the incorporation of a broad-spectrum caspase inhibitor (IDN6556) into the eMs synergistically increases the viability of MSCs under in vitro conditions. Intraperitoneal injection of the MSC-microsphere hybrid alleviates experimental colitis in a murine model via inhibiting Th1 and Th17 differentiation of CD4+ T cells in colon-draining mesenteric lymph nodes. Therefore, drug-loaded ECM-coated surfaces may be considered as attractive tools for improving viability, proliferation, and functionality of MSCs following transplantation.

Preparation of High-Payload, Prolonged-Release Biodegradable Poly(lactic-co-glycolic acid)-Based Tacrolimus Microspheres Using the Single-Jet Electrospray Method.

Tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres (TAC-PLGA-M) can be administered for the long-term survival of transplanted organs due to their immunosuppressive activity. The purpose of our study was to optimize the parameters of the electrospray method, and to prepare TAC-PLGA-M with a high payload and desirable release properties. TAC-PLGA-M were prepared using the electrospray method. In vitro characterization and evaluation were performed using scanning electron microscopy, X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier-transform infrared spectroscopy. Drug-loading efficiency was greater than 80% in all formulations with a maximum loading capacity of 16.81±0.37%. XRD and DSC studies suggested that the drug was incorporated in an amorphous state or was molecularly dispersed in the microspheres. The in vitro release study showed prolonged release patterns. TAC-PLGA-M with enhanced drug loading and prolonged-release patterns were successfully prepared using the electrospray method.

Local release of NECA (5'-(N-ethylcarboxamido)adenosine) from implantable polymeric sheets for enhanced islet revascularization in extrahepatic transplantation site.

Clinical intraportal pancreatic islet infusion is popular for treating type I diabetes. However, multiple doses of islets and anti-rejection protocols are needed to compensate for early large cell losses post-infusion due to the harsh hepatic environment. Thus, extrahepatic sites are utilized to enable efficient islet engraftment and reduce islet mass. Here, we reported an effective islet revascularization protocol that was based on the co-implantation of islet/fibrin gel construct with poly(lactic-co-glycolic) acid sheet releasing NECA (5'-(N-ethylcarboxamido) adenosine; a potent agonist of adenosine) into mouse epididymal fat pad. Thin, flexible sheets (d = 4 mm) prepared by simple casting exhibited sustained NECA release for up to 21 days, which effectively improved early islet engraftment with a median diabetic reversal time of 18.5 days. Western blotting revealed the facilitative effect of NECA on VEGF expression from islets in vitro and from grafts in vivo. In addition, NECA directly promoted the angiogenic activities of islet-derived endothelial cells by enhancing their proliferation and vessel-like tube formation. As a result, neovasculatures were effectively formed in the engrafted islet vicinity, as evidenced by vasculature imaging and immunofluorescence. Taken together, we suggest NECA-releasing PLGA sheets offer a safe and effective drug delivery system that enhances islet engraftment while reducing islet mass at extrahepatic sites for clinical relevance.

Immunoisolation of pancreatic islets via thin-layer surface modification.

Islet transplantation is an alternative method of replacing exogenous insulin to treat type 1 diabetes. However, transplantation of allo- or xenograft islets causes the activation of host's immune reaction, which leads to the failure of the transplanted grafts. Immunosuppressive-sparing strategies have been introduced to avoid adverse effects associated with a long-term use of the immunosuppressive drugs. In this regard, macro/microencapsulation, surface camouflage, and surface modification with immune-privileged cells have been performed to protect the transplanted islets against instant blood-mediated inflammatory reactions or immune reactions. However, the increased size of the encapsulated islets after transplantation leads to insufficient oxygen and nutrients for the islets, causing most of them to undergo apoptosis. Therefore, recent studies have aimed at reducing the capsule thickness while maintaining immunoprotective ability of encapsulated islets. In this review, we discuss several techniques of thin-layer surface coating of pancreatic islets using a variety of polymers, therapeutic agents (TA), TA-loaded nano or microparticles, and living cells.

Inflammation-triggered local drug release ameliorates colitis by inhibiting dendritic cell migration and Th1/Th17 differentiation.

Enteric-coated formulations using Eudragit® polymers have been extensively used for delivering drugs to the lower gastrointestinal tract. However, these drug-delivery systems cannot accurately deliver the therapeutic cargoes to colon because of early degradation of the polymers at alkaline pH of the small intestine. Here, we describe a precise method of delivering drugs to inflammation sites in colon using an oral drug delivery system. Tacrolimus (FK506)-loaded microspheres were prepared using a thioketal-based polymer that releases drug in response to reactive oxygen species (ROS), which are abundantly produced at the sites of inflammation in acute colitis. Orally-administered FK506-loaded thioketal microspheres (FK506-TKM) led to a substantial accumulation of FK506 in inflamed colon and effectively alleviated dextran-sulfate sodium (DSS)-induced murine colitis. At the molecular level, FK506-TKM significantly inhibited infiltration of CD4+ and CD8+ T lymphocytes in colon and differentiation of CD4+ T cells into Th1 and Th17 cells in colon-draining mesenteric lymph nodes via restricting dendritic cell migration from colon. Our findings indicate orally-administered thioketal-based drug delivery system as a promising means of treating acute inflammatory bowel diseases.

Tissue adhesive FK506-loaded polymeric nanoparticles for multi-layered nano-shielding of pancreatic islets to enhance xenograft survival in a diabetic mouse model.

This study aims to develop a novel surface modification technology to prolong the survival time of pancreatic islets in a xenogenic transplantation model, using 3,4-dihydroxyphenethylamine (DOPA) conjugated poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) nanoparticles (DOPA-NPs) carrying immunosuppressant FK506 (FK506/DOPA-NPs). The functionalized DOPA-NPs formed a versatile coating layer for antigen camouflage without interfering the viability and functionality of islets. The coating layer effectively preserved the morphology and viability of islets in a co-culture condition with xenogenic lymphocytes for 7 days. Interestingly, the mean survival time of islets coated with FK506/DOPA-NPs was significantly higher as compared with that of islets coated with DOPA-NPs (without FK506) and control. This study demonstrated that the combination of surface camouflage and localized low dose of immunosuppressant could be an effective approach in prolonging the survival of transplanted islets. This newly developed platform might be useful for immobilizing various types of small molecules on therapeutic cells and biomaterial surface to improve the therapeutic efficacy in cell therapy and regenerative medicine.

PEGylated thermosensitive lipid-coated hollow gold nanoshells for effective combinational chemo-photothermal therapy of pancreatic cancer.

Pancreatic cancer has extremely poor prognosis with an 85% mortality rate that results from aggressive and asymptomatic growth, high metastatic potential, and rapid development of resistance to already ineffective chemotherapy. In this study, plasmonic hollow gold nanoshells (GNS) coated with PEGylated thermosensitive lipids were prepared as an efficient platform to ratiometrically co-deliver two drugs, bortezomib and gemcitabine (GNS-L/GB), for combinational chemotherapy and photothermal therapy of pancreatic cancer. Bortezomib was loaded within the lipid bilayers, while gemcitabine was loaded into the hydrophilic interior of the porous GNS via an ammonium sulfate-driven pH gradient method. Physicochemical characterizations and biological studies of GNS-L/GB were performed, with the latter using cytotoxicity assays, cellular uptake and apoptosis assays, live/dead assays, and western blot analysis of pancreatic cancer cell lines (MIA PaCa-2 and PANC-1). The nanoshells showed remotely controllable drug release when exposed to near-infrared laser for site-specific delivery. GNS-L/GB showed synergistic cytotoxicity and improved internalization by cancer cells. High-powered near-infrared continuous wave laser (λ=808nm) effectively killed cancer cells via the photothermal effect of GNS-L/GB, irrespective of cell type in a power density-, time-, and GNS dose-dependent manner. These results suggest that this method can provide a novel approach to achieve synergistic combinational chemotherapy and photothermal therapy, even with resistant pancreatic cancer.

A three-dimensional assemblage of gingiva-derived mesenchymal stem cells and NO-releasing microspheres for improved differentiation.

Stem cell therapy is an attractive approach to bone tissue regeneration. Nitric oxide (NO) has been reported to facilitate osteogenic differentiation of stem cells. To enhance osteogenic differentiation of gingiva-derived mesenchymal stem cells (GMSCs), we designed a method for in situ delivery of exogenous NO to these cells. A NO donor, polyethylenimine/NONOate, was incorporated into poly(lactic-co-glycolic acid) microspheres to deliver NO to the cells for an extended period of time under in vitro culture conditions. A hybrid aggregate of GMSCs and NO-releasing microspheres was prepared by the hanging drop technique. Confocal microscopy revealed homogeneous arrangement of the stem cells and microspheres in heterospheroids. Western blot analysis and live-dead imaging showed no significant change in cell viability. Importantly, the in situ delivery of NO within the heterospheroids enhanced osteogenic differentiation indicated by a 1.2-fold increase in alkaline phosphatase activity and an approximately 10% increase in alizarin red staining. In addition, a low dose of NO promoted proliferation of the GMSCs in this 3D system. Thus, delivery of the NO-releasing microsphers to induce differentiation of stem cells within this three dimensional system may be one of possible strategies to direct differentiation of a stem cell-based therapeutic agent toward a specific lineage.

Potential differentiation ability of gingiva originated human mesenchymal stem cell in the presence of tacrolimus.

The aim of the present study is to evaluate the potential differentiation ability of gingiva originated human mesenchymal stem cell in the presence of tacrolimus. Tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres were prepared using electrospraying technique. In vitro release study of tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres was performed in phosphate-buffered saline (pH 7.4). Gingiva-derived stem cells were isolated and incubated with tacrolimus or tacrolimus-loaded microspheres. Release study of the microspheres revealed prolonged release profiles of tacrolimus without any significant initial burst release. The microsphere itself did not affect the morphology of the mesenchymal stem cells, and cell morphology was retained after incubation with microspheres loaded with tacrolimus at 1 µg/mL to 10 µg/mL. Cultures grown in the presence of microspheres loaded with tacrolimus at 1 µg/mL showed the highest mineralization. Alkaline phosphatase activity increased with an increase in incubation time. The highest expression of pSmad1/5 was achieved in the group receiving tacrolimus 0.1 µg/mL every third day, and the highest expression of osteocalcin was achieved in the group receiving 1 µg/mL every third day. Biodegradable poly(lactic-co-glycolic acid)-based microspheres loaded with tacrolimus promoted mineralization. Microspheres loaded with tacrolimus may be applied for increased osteoblastic differentiation.

Multilayer-Coated Liquid Crystalline Nanoparticles for Effective Sorafenib Delivery to Hepatocellular Carcinoma.

Hepatocellular carcinoma is one of the most common cancers in adults and develops due to activation of oncogenes and inactivation of tumor suppressor genes. Sorafenib (SF) is a U.S. Food and Drug Administration (FDA) approved drug for the treatment of hepatocellular carcinoma. However, its clinical use is limited by its poor aqueous solubility and undesirable side effects. Monoolein-based liquid crystalline nanoparticles (LCN) are self-assembled structures that have been determined as promising drug-delivery vehicles. Therefore, the main aim of this study was to prepare layer-by-layer (LbL) polymer-assembled SF-loaded LCNs (LbL-LCN/SF) for effective delivery of SF to hepatocellular carcinoma. Results revealed that LbL-LCN/SF presented optimum particle size (∼165 nm) and polydispersity index (PDI, ∼0.14) with appropriate polymer layer assembly confirmed by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Furthermore, LbL-LCN/SF effectively controlled burst release and exhibited pH-sensitive release of SF, thereby increasing drug release in the acidic microenvironment of tumor cells. Compared to free SF and bare LCN, the hemolytic activity of LbL-LCN/SF was significantly reduced (p<0.01). Interestingly, LbL-LCN/SF was more cytotoxic to HepG2 cells than the free drug was. Additionally, high cellular uptake and greater apoptotic effects of LbL-LCN/SF in HepG2 cells indicates superior antitumor effects. Therefore, LbL-LCN/SF is a potentially effective formulation for hepatocellular carcinoma.