CD39 delineates chimeric antigen receptor regulatory T cell subsets with distinct cytotoxic & regulatory functions against human islets.

Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases, including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs, most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets, may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes, a human ß cell line and human islet ß cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet ß cells. In exploring subsets and mechanisms that may explain this pattern, we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39- CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly, ß cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased ß cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet ß cells in the setting of CAR immunotherapy. In summary, introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction.

Endogenous stem cell mobilization and localized immunosuppression synergistically ameliorate DSS-induced Colitis in mice.

BACKGROUND: Stem cell therapy is a promising alternative for inflammatory diseases and tissue injury treatment. Exogenous delivery of mesenchymal stem cells is associated with instant blood-mediated inflammatory reactions, mechanical stress during administration, and replicative senescence or change in phenotype during long-term culture in vitro. In this study, we aimed to mobilize endogenous hematopoietic stem cells (HSCs) using AMD-3100 and provide local immune suppression using FK506, an immunosuppressive drug, for the treatment of inflammatory bowel diseases. METHODS: Reactive oxygen species (ROS)-responsive FK506-loaded thioketal microspheres were prepared by emulsification solvent-evaporation method. Thioketal vehicle based FK506 microspheres and AMD3100 were co-administered into male C57BL6/J mice with dextran sulfate sodium (DSS) induced colitis. The effect of FK506-loaded thioketal microspheres in colitis mice were evaluated using disease severity index, myeloperoxidase activity, histology, flow cytometry, and gene expression by qRT-PCR. RESULTS: The delivery of AMD-3100 enhanced mobilization of HSCs from the bone marrow into the inflamed colon of mice. Furthermore, targeted oral delivery of FK506 in an inflamed colon inhibited the immune activation in the colon. In the DSS-induced colitis mouse model, the combination of AMD-3100 and FK506-loaded thioketal microspheres ameliorated the disease, decreased immune cell infiltration and activation, and improved body weight, colon length, and epithelial healing process. CONCLUSION: This study shows that the significant increase in the percentage of mobilized hematopoietic stem cells in the combination therapy of AMD and oral FK506 microspheres may contribute to a synergistic therapeutic effect. Thus, low-dose local delivery of FK506 combined with AMD3100 could be a promising alternative treatment for inflammatory bowel diseases.

Hypoxia-Mediated ROS Amplification Triggers Mitochondria-Mediated Apoptotic Cell Death via PD-L1/ROS-Responsive, Dual-Targeted, Drug-Laden Thioketal Nanoparticles.

Amalgamation of the reactive oxygen species (ROS)-responsive stimulus with nanoparticles has gained considerable interest owing to their high tumor specificity. Hypoxia plays a pivotal role in the acceleration of intracellular ROS production. Herein, we report the construction of a cancer cell (PD-L1)- and ROS-responsive, dual-targeted, temozolomide (TMZ)-laden nanosystem which offers a better anticancer effect in a hypoxic tumor microenvironment. A dual-targeted system boosted permeation in the cancer cells. Hypoxic conditions elevating the high ROS level accelerated the in situ release of TMZ from anti-PD-L1-TKNPs. Hyperaccumulated ROS engendered from TMZ caused oxidative damage leading to mitochondria-mediated apoptosis. TMZ fabricated in the multifunctional nanosystem (anti-PD-L1-TMZ-TKNPs) provided excellent tumor accumulation and retarded tumor growth under in vivo conditions. The elevated apoptosis effect with the activation of an apoptotic marker, DNA double-strand breakage marker, and downregulation of the angiogenesis marker in the tumor tissue following treatment with anti-PD-L1-TMZ-TKNPs exerts robust anticancer effect. Collectively, the nanoconstruct offers deep tumor permeation and high drug release and broadens the application of the ROS-responsive nanosystem for a successful anticancer effect.

Identification of Novel FNIN2 and FNIN3 Fibronectin-Derived Peptides That Promote Cell Adhesion, Proliferation and Differentiation in Primary Cells and Stem Cells.

In recent years, a major rise in the demand for biotherapeutic drugs has centered on enhancing the quality and efficacy of cell culture and developing new cell culture techniques. Here, we report fibronectin (FN) derived, novel peptides fibronectin-based intergrin binding peptide (FNIN)2 (18-mer) and FNIN3 (20-mer) which promote cell adhesion proliferation, and the differentiation of primary cells and stem cells. FNIN2 and 3 were designed based on the in silico interaction studies between FN and its receptors (integrin α5ß1, αvß3, and αIIbß3). Analysis of the proliferation of seventeen-cell types showed that the effects of FNINs depend on their concentration and the existence of expressed integrins. Significant rhodamine-labeled FNIN2 fluorescence on the membranes of HeLa, HepG2, A498, and Du145 cells confirmed physical binding. Double coating with FNIN2 or 3 after polymerized dopamine (pDa) or polymerized tannic acid (pTA) precoating increased HBEpIC cell proliferation by 30-40 percent, suggesting FNINs potently affect primary cells. Furthermore, the proliferation of C2C12 myoblasts and human mesenchymal stem cells (MSCs) treated with FNINs was significantly increased in 2D/3D culture. FNINs also promoted MSC differentiation into osteoblasts. The results of this study offer a new approach to the production of core materials (e.g., cell culture medium components, scaffolds) for cell culture.

Heterospheroid formation improves therapeutic efficacy of mesenchymal stem cells in murine colitis through immunomodulation and epithelial regeneration.

Tissue repairing capacity and immunomodulatory effects of mesenchymal stem cells (MSCs) have been extensively utilized for treating various inflammatory disorders; however, inconsistent efficacy and therapeutic outcomes due to low survival rate after transplantation often restrain their clinical potential. To overcome these limitations, 3-dimensional culture (3D-culture) was established to augment stemness and paracrine functions of MSCs, although hypoxic stress at the core often leads to unexpected cell death. Thus, we designed a novel strategy to improve the microenvironment of MSCs by creating heterospheroids (HS) consisting of MSCs and quercetin (QUR)-loaded microspheres (MSCHS), to achieve local drug delivery to the cells. Notably, MSCHS exhibited resistance for senescence-associated phenotype and oxidative stress-induced apoptosis compared to 3D-cultured MSCs (MSC3D), as well as to 2D-cultured cells (MSC2D) in vitro. In a murine model of colitis, MSC3D and MSCHS exhibited enhanced anti-inflammatory impact than MSC2Dvia attenuating neutrophil infiltration and regulating helper T cell (Th) polarization into Th1 and Th17 cells. Interestingly, MSCHS provided better therapeutic outcomes compared to MSC3D, partially due to their enhanced survival capacity in vivo. Moreover, we found that MSC-derived paracrine factor, prostaglandin E2 (PGE2), can directly drive the epithelial regeneration process by inducing specialized tissue-repairing cell generation using the intestinal organoid culture. Importantly, MSC3D and MSCHS displayed an outstanding regeneration-inducing potency compared to MSC2D owing to their superior PGE2 secretion. Taken together, we suggest a convergent strategy of MSCHS formation with reactive oxygen species (ROS) scavenger, QUR, which can maximize the inflammation-attenuating and tissue-repairing capacity of MSCs, as well as the engraftment efficiency after transplantation.

Photoimmunotherapy with cetuximab-conjugated gold nanorods reduces drug resistance in triple negative breast cancer spheroids with enhanced infiltration of tumor-associated macrophages.

Tumor-associated macrophages (TAM) constitute up to 50-80% of stromal cells in breast cancer (BC), and are correlated with poor prognosis. As epidermal growth factor receptor (EGFR) is overexpressed in 60-80% of patients with triple negative breast cancer (TNBC), photoimmunotherapy (PIT) with cetuximab-targeted gold nanorods (CTX-AuNR) is an attractive therapeutic strategy for TNBC. The 3D cell culture model can mimic drug resistance conferred by the tumor microenvironment and its 3D organization; therefore, TAM and non-TAM embedded TNBC spheroids were constructed to evaluate the therapeutic efficacy of CTX-AuNR plus near infrared (NIR) irradiation. Cytotoxicity, reactive oxygen species (ROS) generation, and protein expression were compared in TNBC (± TAM) spheroids. The IC50 values of doxorubicin (DOX) in TAM-embedded TNBC spheroids were significantly higher than those in TNBC spheroids, demonstrating drug resistance, which could be explained by activation of IL-10/IL-10 receptor/STAT3/Bcl-2 signaling. However, 3D in vitro and in vivo results demonstrated that the efficacy of CTX-AuNR plus NIR irradiation was not significantly different in (± TAM) embedded TNBC cells. By enhancing ROS generation, CTX-AuNR plus NIR irradiation reprogrammed TAM polarization to the M1 anti-tumor phenotype, as indicated by macrophage mannose receptor (MMR) downregulation. Thus, CTX-AuNR plus NIR can serve as a potent PIT strategy for treating EGFR-overexpressing TNBC cells.

Particulate-Based Single-Dose Local Immunosuppressive Regimen for Inducing Tolerogenic Dendritic Cells in Xenogeneic Islet Transplantation.

Recent studies emphasize on developing immune tolerance by an interim administration of various immunosuppressive drugs. In this study, a robust protocol is reported for local immunomodulation using a single-dose of FK506 microspheres and clodronate liposomes (mFK+CLO) in a xenogeneic model of islet transplantation. Surprisingly, the single-dose treatment with mFK+CLO induce tolerance to the islet xenograft. The recipient mice display tolerogenic dendritic cells (tDCs) with decreased antigen presenting ability and T cell activation capacity. Furthermore, a reduced percentage of CD4+ and CD8+ T cells and an impaired differentiation of naïve CD4+ T cells into interferon-γ producing Th1 and interleukin-17 producing Th17 cells are observed. In addition, the immunosuppressive protocol leads to the generation of Foxp3+ regulatory T cells (Tregs) which are required for the long-term graft survival. The enhanced generation of tDCs and Tregs by the single treatment of mFK+CLO cause xenograft tolerance, suggesting a possible clinical strategy which may pave the way towards improving therapeutic outcomes of clinical islet transplantation.

Enhanced viability and function of mesenchymal stromal cell spheroids is mediated via autophagy induction.

Mesenchymal stromal cells (MSCs) have received attention as promising therapeutic agents for the treatment of various diseases. However, poor post-transplantation viability is a major hurdle in MSC-based therapy, despite encouraging results in many inflammatory disorders. Recently, three dimensional (3D)-cultured MSCs (MSC3D) were shown to have higher cell survival and enhanced anti-inflammatory effects, although the underlying mechanisms have not yet been elucidated. In this study, we investigated the molecular mechanisms by which MSC3D gain the potential for enhanced cell viability. Herein, we found that macroautophagy/autophagy was highly induced and ROS production was suppressed in MSC3D as compared to 2D-cultured MSCs (MSC2D). Interestingly, inhibition of autophagy induction caused decreased cell viability and increased apoptotic activity in MSC3D. Furthermore, modulation of ROS production was closely related to the survival and apoptosis of MSC3D. We also observed that HMOX1 (heme oxygenase 1) was significantly up-regulated in MSC3D. In addition, gene silencing of HMOX1 caused upregulation of ROS production and suppression of the genes related to autophagy. Moreover, inhibition of HIF1A (hypoxia inducible factor 1 subunit alpha) caused suppression of HMOX1 expression in MSC3D, indicating that the HIF1A-HMOX1 axis plays a crucial role in the modulation of ROS production and autophagy induction in MSC3D. Finally, the critical role of autophagy induction on improved therapeutic effects of MSC3D was further verified in dextran sulfate sodium (DSS)-induced murine colitis. Taken together, these results indicated that autophagy activation and modulation of ROS production mediated via the HIF1A-HMOX1 axis play pivotal roles in enhancing the viability of MSC3D.Abbreviations: 3D: three dimensional; 3MA: 3 methlyadenine; AMPK: AMP-activated protein kinase; Baf A1: bafilomycin A1; CFSE: carboxyfluorescein succinimidyl ester; CoCl2: cobalt chloride; CoPP: cobalt protoporphyrin; DSS: dextran sulfate sodium; ECM: extracellular matrix; FOXO3/FOXO3A: forkhead box O3; HIF1A: hypoxia inducible factor 1 subunit alpha; HMOX1/HO-1: heme oxygenase 1; HSCs: hematopoietic stem cells; IL1A/IL-1α: interleukin 1 alpha; IL1B/IL-1ß: interleukin 1 beta; IL8: interleukin 8; KEAP1: kelch like ECH associated protein 1; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; MSC2D: 2D-cultured MSCs; MSC3D: 3D-cultured MSCs; MSCs: mesenchymal stromal cells; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; PGE2: prostaglandin E2; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PINK1: PTEN induced kinase 1; ROS: reactive oxygen species; siRNA: small interfering RNA; SIRT1: sirtuin 1; SOD2: superoxide dismutase 2; SQSTM1/p62: sequestosome 1; TGFB/TGF-ß: transforming growth factor beta.

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.

Tregs and Mixed Chimerism as Approaches for Tolerance Induction in Islet Transplantation.

Pancreatic islet transplantation is a promising method for the treatment of type 1 and type 3 diabetes whereby replacement of islets may be curative. However, long-term treatment with immunosuppressive drugs (ISDs) remains essential for islet graft survival. Current ISD regimens carry significant side-effects for transplant recipients, and are also toxic to the transplanted islets. Pre-clinical efforts to induce immune tolerance to islet allografts identify ways in which the recipient immune system may be reeducated to induce a sustained transplant tolerance and even overcome autoimmune islet destruction. The goal of these efforts is to induce tolerance to transplanted islets with minimal to no long-term immunosuppression. Two most promising cell-based therapeutic strategies for inducing immune tolerance include T regulatory cells (Tregs) and donor and recipient hematopoietic mixed chimerism. Here, we review preclinical studies which utilize Tregs for tolerance induction in islet transplantation. We also review myeloablative and non-myeloablative hematopoietic stem cell transplantation (HSCT) strategies in preclinical and clinical studies to induce sustained mixed chimerism and allograft tolerance, in particular in islet transplantation. Since Tregs play a critical role in the establishment of mixed chimerism, it follows that the combination of Treg and HSCT may be synergistic. Since the success of the Edmonton protocol, the feasibility of clinical islet transplantation has been established and nascent clinical trials testing immune tolerance strategies using Tregs and/or hematopoietic mixed chimerism are underway or being formulated.

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.

Intraportally delivered stem cell spheroids localize in the liver and protect hepatocytes against GalN/LPS-induced fulminant hepatic toxicity.

BACKGROUND: Systemic inflammatory response syndrome (SIRS) is common in severe fulminant hepatic failure (FHF) and has a high mortality rate (20-50%) due to irreversible cerebral edema or sepsis. Stem cell-based treatment has emerged as a promising alternative therapeutic strategy to prolong the survival of patients suffering from FHF via the inhibition of SIRS due to their immunomodulatory effects. METHODS: 3D spheroids of adipose-derived mesenchymal stem cells (3D-ADSC) were prepared by the hanging drop method. The efficacy of the 3D-ADSC to rescue FHF was evaluated in a D-galactosamine/lipopolysaccharide (GalN/LPS)-induced mouse model of FHF via intraportal transplantation of the spheroids. RESULTS: Intraportally delivered 3D-ADSC better engrafted and localized into the damaged livers compared to 2D-cultured adipose-derived mesenchymal stem cells (2D-ADSC). Transplantation of 3D-ADSC rescued 50% of mice from FHF-induced lethality, whereas only 20% of mice survived when 2D-ADSC were transplanted. The improved transplantation outcomes correlated with the enhanced immunomodulatory effect of 3D-ADSC in the liver microenvironment. CONCLUSION: The study shows that the transplantation of optimized 3D-ADSC can efficiently ameliorate GalN/LPS-induced FHF due to improved viability, resistance to exogenous ROS, and enhanced immunomodulatory effects of 3D-ADSC.

Design and manufacture of 3D cell culture plate for mass production of cell-spheroids.

A 3D cell culture is preferred to 2D cell culture since it allows cells to grow in all directions in vitro, similar to how they would in vivo. 3D cell culture plates currently used in tissue engineering research have limited access to control the geometry. Furthermore, 3D cell culture plate manufacturing methods are relatively complex, time-consuming, labor-intensive, and expensive. Therefore, a design and manufacturing method, which has relatively low cost, high throughput, and high size flexibility, is proposed. Cell culture plate was fabricated by computer aided design and manufacturing software using polydimethylsiloxane as a plate constituent. With the successfully-developed 3D cell culture plate, the morphology and viability of the cultured mesenchymal stem cells were tested.The mesenchymal stem cells seeded on the newly-fabricated 3D cell culture plate aggregated to form 3D spheroids within 24 h of incubation and well-maintained their viability. Thus, due to the capacity of mass production of the cell spheroids with a desired cell viability, the newly-fabricated plate has a great promise to prepare 3D cell spheroids for experimental as well as clinical applications.

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.

Mesenchymal stem cell therapy for the treatment of inflammatory diseases: Challenges, opportunities, and future perspectives.

Mesenchymal stem cells (MSCs) are promising alternative agents for the treatment of inflammatory disorders due to their immunomodulatory functions, and several clinical trials on MSC-based products are currently being conducted. In this review, we discuss recent progress made on the use of MSCs as immunomodulatory agents, developmental challenges posed by MSC-based therapy, and the strategies being used to overcome these challenges. In this context, current understanding of the mechanisms responsible for MSC interactions with the immune system and the molecular responses of MSCs to inflammatory signals are discussed. The immunosuppressive activities of MSCs are initiated by cell-to-cell contact and the release of immuno-regulatory molecules. By doing so, MSCs can inhibit the proliferation and function of T cells, natural killer cells, B cells, and dendritic cells, and can also increase the proliferation of regulatory T cells. However, various problems, such as low transplanted cell viability, poor homing and engraftment into injured tissues, MSC heterogeneity, and lack of adequate information on optimum MSC doses impede clinical applications. On the other hand, it has been shown that the immunomodulatory activities and viabilities of MSCs might be enhanced by 3D-cultured systems, genetic modifications, preconditioning, and targeted-delivery.

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.

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.

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-1ß-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.

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-ß-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.

Paclitaxel and Erlotinib-co-loaded Solid Lipid Core Nanocapsules: Assessment of Physicochemical Characteristics and Cytotoxicity in Non-small Cell Lung Cancer.

PURPOSE: Lung cancer is the leading cause of cancer-related deaths. The aim of this study was to design solid lipid core nanocapsules (SLCN) comprising a solid lipid core and a PEGylated polymeric corona for paclitaxel (PTX) and erlotinib (ERL) co-delivery to non-small cell lung cancer (NSCLC), and evaluate their physicochemical characteristics and in vitro activity in NCI-H23 cells. METHODS: PTX/ERL-SLCN were prepared by nanoprecipitation and sonication and physicochemically characterized by dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, X-ray diffraction, and Fourier-transform infrared spectroscopy. In vitro release profiles at pH 7.4 and pH 5.0 were studied and analyzed. In vitro cytotoxicity and cellular uptake and apoptosis assays were performed in NCI-H23 cells. RESULTS: PTX/ERL-SLCN exhibited appropriately-sized spherical particles with a high payload. Both PTX and ERL showed pH-dependent and sustained release in vitro profiles. PTX/ERL-SLCN demonstrated concentration- and time-dependent uptake by NCI-H23 cells and caused dose-dependent cytotoxicity in the cells, which was remarkably greater than that of not only the free individual drugs but also the free drug cocktail. Moreover, well-defined early and late apoptosis were observed with clearly visible signs of apoptotic nuclei. CONCLUSION: PTX/ERL-SLCN could be employed as an optimal approach for combination chemotherapy of NSCLC.