Shobha Regmi

Development of Bioactive PEGylated Nanostructured Platforms for Sequential Delivery of Doxorubicin and Imatinib to Overcome Drug Resistance in Metastatic Tumors

Metastasis of cancers accounts for almost all cancer-related deaths. In this study, we report a PEGylated nanostructured platform for coadministration of doxorubicin (DOX) and imatinib (IMT) intended to effectively inhibit metastatic tumors. The DOX and IMT coloaded nanostructured system (DOX/IMT-N) is characterized by an excellent encapsulation potential for both drugs and shows sequential and sustained drug release in vitro. DOX/IMT-N significantly inhibited the in vitro proliferation of MDA-MB-231 and SK-MEL-28 cells. The inhibitory effect on in vitro proliferation of the cells was significantly greater than the effect of free DOX, DOX/IMT cocktail, or the nanostructured system housing DOX only (DOX-N). DOX/IMT-N remarkably enhanced cellular drug uptake, resulting in enhanced apoptosis, caused by significant increases in the expression levels of apoptotic marker proteins. Intravenous administration of DOX/IMT-N to MBA-MB-231 xenograft tumor-bearing mice resulted in significantly improved inhibition of tumor progression compared to that with DOX, DOX/IMT, or DOX-N. Therefore, the nanostructured DOX/IMT-N system could potentially aid in overcoming drug resistance in metastatic tumors and improve the effectiveness of metastatic tumor therapeutics.

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.

Engineered islet cell clusters transplanted into subcutaneous space are superior to pancreatic islets in diabetes

An alternative route for pancreatic islet transplantation is the subcutaneous space; however, inadequate vascularization in the subcutaneous space limits the availability of oxygen and nutrients to the subcutaneously transplanted islets, which leads to the development of a necrotic core in the islets, thereby causing islet dysfunction. Thus, we aimed to prevent the early apoptosis of pancreatic islets after transplantation into subcutaneous space by preparing islet clusters of appropriate size. We prepared fully functional islet cell clusters (ICCs) by using the hanging‐drop technique. We optimized the size of ICCs on the basis of viability and functionality after culture in an hypoxic environment. We transplanted ICCs into the subcutaneous space of diabetic mice and evaluated the viability of the islets at the transplantation site. In an hypoxic environment, ICCs exhibited improved viability and functionality compared with control islets. ICCs, upon transplantation into the hypoxic subcutaneous space of diabetic mice, showed better glycemic control compared with control islets. Live/dead imaging of the islets after retrieval from the transplanted area revealed significantly reduced apoptosis in ICCs. Transplantation of ICCs may be an attractive strategy to prevent islet cell apoptosis that results from nonimmune‐mediated physiologic stress at the transplantation site.—Pathak, S., Regmi, S., Gupta, B., Pham, T. T., Yong, C. S., Kim, J. O., Yook, S., Kim, J.‐R., Park, M. H., Bae, Y. K., Jeong, J.‐H. Engineered islet cell clusters transplanted into subcutaneous space are superior to pancreatic islets in diabetes. FASEB J. 31, 5111–5121 (2017). www.fasebj.org

Folate receptor-targeted hybrid lipid-core nanocapsules for sequential delivery of doxorubicin and tanespimycin

When exposed to cancer cells, cytotoxic drugs such as doxorubicin (DOX) can lead to the induction of heat shock protein 90 (Hsp90), a molecular chaperone associated with a number of cancer-related client proteins, and result in cell survival. Co-administration of DOX with tanespimycin (TNP), an Hsp90 inhibitor, can sensitize the cancer cells to the cytotoxic effects of DOX. The effect of such a combination has been found to depend on the schedule of administration. Sequential administration of DOX and TNP has been linked to highly synergistic combination effects. Therefore, we aimed to develop folate-receptor targeted hybrid lipid-core nanocapsules comprising a hybrid lipid core lodging TNP and a polymeric corona lodging DOX (F-DTN). These nanocarriers were capable of delivering DOX and TNP sequentially, which was well demonstrated by an in vitro release study. The in vitro release profiles displayed pH-dependent and sustained release features. F-DTN exhibited excellent morphological characteristics with highly monodispersed particles. In vitro tests with F-DTN in MCF-7 cell line demonstrated exceptional cytotoxicity, with high cellular uptake and apoptosis. These findings were appreciably more assertive than tests with free individual drugs (DOX, TNP), free drug combination (DOX/TNP), or non-folate receptor-targeted hybrid lipid-core nanocapsules (DTN). In vivo pharmacokinetic study revealed noticeable enhancement of bioavailability and plasma circulation time of the drugs when encapsulated in the carrier system. Therefore, hybrid lipid-core nanocapsules have the potential to be utilized for application in folate receptor-targeted combination chemotherapy.

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.

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.

Hyaluronic acid-capped compact silica-supported mesoporous titania nanoparticles for ligand-directed delivery of doxorubicin

Mesoporous titania nanoparticles (MTN), owing to their high surface area to volume ratio and tunable pore sizes, appear capable of delivering sizable amounts of drug payloads, and hence, show considerable promise as drug delivery candidates in cancer therapy. We designed silica-supported MTN (MTNst) coated with hyaluronic acid (HA) to effectively deliver doxorubicin (DOX) for breast cancer therapy. The HA coating served a dual purpose of stabilizing the payload in the carriers as well as actively targeting the nanodevices to CD44 receptors. The so-formed HA-coated MTNst carrying DOX (HA/DOX-MTNst) had spheroid particles with a considerable drug-loading capacity and showed significantly superior in vitro cytotoxicity against MDA-MB-231 cells as compared to free DOX. HA/DOX-MTNst markedly improved the cellular uptake of DOX in an apparently CD44 receptor-dependent manner, and increased the number of apoptotic cells as compared to free DOX. These nanoplatforms accumulated in large quantities in the tumors of MDA-MB-231 xenograft tumor-bearing mice, where they significantly enhanced the inhibition of tumor growth compared to that observed with free DOX with no signs of acute toxicity. Based on these excellent results, we deduced that HA/DOX-MTNst could be successfully used for targeted breast cancer therapy. STATEMENT OF SIGNIFICANCE: This is the first study to use silica-supported mesoporous titania nanoparticles (MTNst) for doxorubicin (DOX) delivery to treat breast cancer, which exhibited effective and enhanced in vitro and in vivo apoptosis and tumor growth inhibition. Solid silica was used to support the mesoporous TiO2 resulting in MTNst, which efficiently incorporated a high DOX payload. The hyaluronic acid (HA) coating over the MTNst surface served a dual purpose of first, stabilizing DOX inside the MTNst (capping agent), and second, directing the nanoplatform device to CD44 receptors that are highly expressed in MDA-MB-231 cells (targeting ligand). The NPs exhibited highly efficacious in vitro tumor-cell killing and excellent in vivo tumor regression, highlighting the enormous promise of this system for breast cancer therapy.

Delivery of Pancreatic Islets and Single Dose Local Immune Suppression Into Subcutaneous Space Using Injectable Hydrogel Provides Indefinite Survival of the Graft in Mouse Model of Diabetes

Introduction Pancreatic islet transplantation is a promising technique to treat type 1 diabetes. Long-term survival of the graft is required for a successful islet transplantation. Repeated use of immunosuppressive drugs after organ/cell transplantation often leads to severe adverse effects including nephrotoxicity, hepatotoxicity, and opportunistic infections. Thus, development of a local immunosuppression protocol is necessary to improve the islet graft survival in clinics. Materials and Methods Pancreatic islets from Sprague-Dawley rats were transplanted into the subcutaneous space of B6 mice using injectable hydrogel. Three major groups were prepared for transplantation. (1) Islets transplanted FK506-loaded poly(lactic-co-glycolic acid) microspheres (10 mg/kg), (2) Islets transplanted with clodronate liposomes (6.25 mg/kg), and (3) Islet transplanted with the combination of both immune suppressants. The suspension of islets and immune suppressants in Matrigel was then injected into the subcutaneous space over the flanks of streptozocin-induced diabetic mice. Results Islets transplanted without any immunosuppression were rejected within two weeks. In contrast, the islets transplanted with the single immunosuppressive regimen of FK506 or clodronate improved survival rate compared with that of the control mice. More interestingly, the graft transplanted using the combination of both immune suppressants survived indefinitely. Immunological studies revealed that the immunosuppressive cocktail inhibited the proliferation of immune cells residing at the peripheral lymph nodes. Interestingly, the systemic immune system of the transplanted mice remained unaffected. Furthermore, histochemical analysis revealed the intact morphology of the islets at the transplanted site when codelivered with the immunosuppressant. Discussion Antigen presenting cells and T-cells orchestrate the immune rejection cascade. Macrophage depletion by the liposomal clodronate and the inhibition of T-cell activation by FK506 completely blocked the immune rejection cascade in the immune competent mice. The inhibition of immune stimulation in the peripheral lymph nodes improved the islet grafted into the subcutaneous space. Thus, the use of local immune suppression is an effective approach to enhance the survival of the transplanted islets. Conclusion We developed a protocol for the local codelivery of pancreatic islets and immune suppressive agents. Indefinite graft survival was obtained with the use of macrophage depleting agent and T-cell inhibitor. The single dose of local immune suppression during transplantation may avoid toxic effects associated with a long-term use of immune suppressive agents in clinics. National Research Foundation of Korea (NRF) Grant nos: 2015R1A5A2009124 and 2017R1D1A1B03027831. Korea Health Industry Development Institute (KHIDI) Grant no: HI16C1767.

Polymeric microsphere-facilitated site-specific delivery of quercetin prevents senescence of pancreatic islets in vivo and improves transplantation outcomes in mouse model of diabetes

Attenuation of senescence progression may be attractive way to preserve the functionality of pancreatic islets (PI) after transplantation. In this study, we developed a model for in vitro induction of premature senescence in rat PI and showed the effectiveness of quercetin (QU) to prevent the senescence. To provide targeted-delivery of QU to the PI after transplantation, we prepared the hybrid clusters (HC) of islet single cells (ISC) and QU-loaded polymeric microspheres (QU; ∼7.55 ng HC-1). Long-term culture of the HC revealed reduced levels of reactive oxygen species and decreased expression of senescence-associated beta galactosidase, Rb, p53, p16, and p21 compared to that of the control islets. Transplantation of HC into subcutaneous space of the immune-deficient mice produced better glycemic control compared to the control islets or the ICC-transplanted mice. SA-β-Gal staining of the in vivo transplanted HC sample showed lower intensity compared to that of the control islets or the islet cell clusters. Thus, in situ delivery of therapeutic agent may be a promising approach to improve therapeutic outcomes in cell therapy. STATEMENT OF SIGNIFICANCE In this study, we aimed to improve outcomes in islet transplantation using in situ delivery of quercetin to pancreatic islets, using polymeric microspheres. We prepared prolonged release-type microspheres and constructed hybrid clusters of pancreatic islets and the microspheres using hanging drop method. The presence of quercetin in the cellular microenvironment attenuated the progression of senescence in the pancreatic islets in a long-term in vitro culture. Moreover, transplantation of the hybrid clusters in the diabetic mice produced better glycemic control compared to that of the control islets. In addition, quercetin delayed the progression of senescence in the pancreatic islets after in vivo transplantation. Thus, local delivery of antioxidants like quercetin may be an attractive way to improve outcomes in cell therapy.

Polyamino Acid Layer-by-Layer (LbL) Constructed Silica-Supported Mesoporous Titania Nanocarriers for Stimuli-Responsive Delivery of microRNA 708 and Paclitaxel for Combined Chemotherapy

Cellular Fas-associated protein with death domain-like interleukin-1β-converting enzyme-inhibitory protein (c-FLIP), often strongly expressed in numerous cancers, plays a pivotal role in thwarting apoptosis and inducing chemotherapy resistance in cancer. An integrated approach combining chemotherapy with suppression of c-FLIP levels could prove paramount in the treatment of cancers with c-FLIP overexpression. In this study, we utilized a polymeric layer-by-layer (LbL) assembly of silica-supported mesoporous titania nanoparticles (MTNst) to co-deliver paclitaxel (PTX) and microRNA 708 (miR708) for simultaneous chemotherapy and c-FLIP suppression in colorectal carcinoma. The resulting LbL miR708/PTX-MTNst showed dose-dependent cytotoxicity in HCT-116 and DLD-1 colorectal carcinoma cell lines, which was remarkably superior to that of free PTX or LbL PTX-MTNst. LbL miR708/PTX-MTNst strongly inhibited c-FLIP expression and resulted in increased expression of proapoptotic proteins. In DLD-1 xenograft tumor-bearing mice, the nanoparticles accumulated in the tumor, resulting in remarkable tumor regression, with the PTX and miR708-loaded nanoparticles showing significantly greater inhibitory effects than the free PTX or PTX-loaded nanoparticles. Immunohistochemical analyses of the tumors further confirmed the remarkable apoptotic and antiproliferative effects of the nanoparticles, whereas organ histology reinforced the biocompatibility of the system. Therefore, the LbL miR708/PTX-MTNst system, owing to its ability to deliver both chemotherapeutic drug and inhibitory miRNA to the tumor site, shows great potential to treat colorectal carcinoma in clinical settings.