Enhanced Caspase-Mediated Abrogation of Autophagy by Temozolomide-Loaded and Panitumumab-Conjugated Poly(lactic-co-glycolic acid) Nanoparticles in Epidermal Growth Factor Receptor Overexpressing Glioblastoma Cells.

The mechanism of cell death has attracted a great deal of research interest in the design of antitumor therapy in recent days. Several attempts have been carried out in this direction and in our study also, we studied this phenomenon with the design of panitumumab (PmAb)-conjugated and temozolomide (TMZ)-loaded poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs), termed PmAb-TMZ-PLGA-NPs. First, PmAb was functionalized on the surface of TMZ-PLGA-NPs using ethyl(dimethylaminopropyl)carbodiimide (EDC)-N-hydroxysuccinimide (NHS) chemistry. Targeted PLGA-NPs significantly enhanced the cellular uptake of nanoparticles in the U-87 MG cell line as a result of the high epidermal growth factor receptor (EGFR) expression, compared to the LN229 cell line. Our study demonstrated that following the treatment of PmAb-TMZ-PLGA-NPs, a more pronounced anticancer effect was noticed in comparison with free TMZ and TMZ-PLGA-NPs. Further, a more pronounced cytotoxic effect of PmAb-TMZ-PLGA-NPs was observed in the high EGFR-overexpressed glioblastoma multiforme (GBM) model (U-87 MG) cell line compared to the low EGFR GBM model (LN229). Our study demonstrated that the treatment of PmAb-TMZ-PLGA-NPs in GBM tried to adopt the autophagic pathway of the cell survival mechanism with the elevated level of autophagic marker (Beclin-1 and LC3B) at 24 h time point, thereby suppressing the expression of caspase-9 and PARP. However, at the 48 h time point, the elevated expression of caspase-9 and PARP with the downregulation of Beclin-1 and LC3B, following the treatment of PmAb-TMZ-PLGA-NPs in the GBM model, suggested that apoptotic cell death was superior over autophagic cell survival. It was also noteworthy the activation of caspase-9 was correlated with the continuous overproduction of reactive oxygen species up to a 48 h time point after the treatment of PmAb-TMZ-PLGA-NPs. This result sheds light on the biological effect of targeted chemotherapy and illustrates that PmAb-TMZ-PLGA-NPs could be applied for EGFR-overexpressed different cancer models.

Dual-Receptor-Targeted (DRT) Radiation Nanomedicine Labeled with 177Lu Is More Potent for Killing Human Breast Cancer Cells That Coexpress HER2 and EGFR Than Single-Receptor-Targeted (SRT) Radiation Nanomedicines.

Resistance to HER2-targeted therapies in breast cancer (BC) is associated in some cases with an increased expression of epidermal growth factor receptors (EGFR). We describe a dual-receptor-targeted (DRT) radiation nanomedicine for local intratumoral (i.t.) treatment of BC composed of 15 nm sized gold nanoparticles (AuNPs) modified with trastuzumab (TmAb) to target HER2 and panitumumab (PmAb) to target EGFR. The AuNPs were modified with poly(ethylene glycol) (PEG3k) linked to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelators to complex the ß-particle emitter, 177Lu. Our aim was to compare the properties of these DRT-AuNP-177Lu with single-receptor-targeted (SRT)-TmAb-AuNP-177Lu or PmAb-AuNP-177Lu or nontargeted (NT)-AuNP-177Lu using human BC cells that expressed HER2, EGFR, or both receptors. To construct these radiation nanomedicines, PEG5K was linked to TmAb or PmAb, while PEG3k was linked to DOTA. These polymers were conjugated to AuNP via two Au-thiol bonds using a terminal lipoic acid (LA) group on the polymers. NT-AuNP-177Lu were constructed without modification with TmAb or PmAb. MDA-MB-231-H2N, MDA-MB-468, and BT-474 human BC cells were designated as HER2mod/EGFRmod, EGFRhigh/HER2neg, and HER2high/EGFRlow, respectively, based on the expression of these receptors. Specific binding to HER2 and/or EGFR was assessed by incubating BC cells with DRT-AuNP-177Lu or TmAb-AuNP-177Lu or PmAb-AuNP-177Lu, or NT-AuNP-177Lu in the absence or presence of an excess of TmAb or PmAb or both competitors. Binding and internalization of AuNP by BC cells were assessed by dark-field microscopy. Cell fractionation studies were conducted to quantify AuNP-177Lu bound and internalized. The cytotoxicity of DRT-AuNP-177Lu was determined in clonogenic survival (CS) assays after an exposure of 5 × 105 BC cells to 3 MBq (1.4 × 1012 AuNP) for 16 h and then seeding and culturing the cells for 7-15 days. CS was compared to exposure to TmAb-AuNP-177Lu and PmAb-AuNP-177Lu or NT-AuNP-177Lu. The absorbed doses to the nucleus in these CS assays were estimated. DRT-AuNP-177Lu were specifically bound by BC cells that expressed HER2 or EGFR or both receptors. In contrast, SRT-TmAb-AuNP-177Lu and PmAb-AuNP-177Lu were bound and internalized only by BC cells that expressed HER2 or EGFR, respectively. NT-AuNP-177Lu exhibited very low binding to BC cells. DRT-AuNP-177Lu and SRT-TmAb-AuNP-177Lu or PmAb-AuNP-177Lu were internalized by BC cells in accordance with the receptor expression. Importantly, DRT-AuNP-177Lu were more potent in vitro than PmAb-AuNP-177Lu for killing MDA-MB-231-H2N cells that coexpress HER2 and EGFR (CS = 18.8 ± 1.0 vs 51.5 ± 10.4%; P = 0.006). Furthermore, DRT-AuNP-177Lu were more potent for killing BT-474 cells with high HER2 but low EGFR expression than TmAb-AuNP-177Lu (CS = 8.9 ± 3.3 vs 20.7 ± 2.4%; P = 0.007) or PmAb-AuNP-177Lu (CS = 63.9 ± 1.7%; P < 0.0001). Even for MDA-MB-468 cells that overexpress EGFR but have negligible HER2, DRT-AuNP-177Lu were more potent for cell killing than PmAb-AuNP-177Lu (CS = 3.2 ± 3.0 vs 7.5 ± 1.8%; P = 0.001) or TmAb-AuNP-177Lu (63.2 ± 3.2%; P = 0.0002). All targeted forms of AuNP-177Lu were more cytotoxic to BC cells than those of NT-AuNP-177Lu. High absorbed doses (36-119 Gy) were deposited in the nucleus of BC cells by DRT-AuNP-177Lu. We conclude that a DRT radiation nanomedicine is more potent for killing BC cells that coexpress HER2 and EGFR than SRT radiation nanomedicines. These results are promising for further evaluation of these DRT-AuNP-177Lu in vivo for the local radiation treatment of human BC tumors that coexpress HER2 and EGFR in mice following i.t. injection, especially tumors that are resistant to HER2-targeted therapies.

Doxorubicin and Anti-PD-L1 Antibody Conjugated Gold Nanoparticles for Colorectal Cancer Photochemotherapy.

Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide. The prognosis and overall survival of CRC are known to be significantly correlated with the overexpression of PD-L1. Since combination therapies can significantly improve therapeutic efficacy, we constructed doxorubicin (DOX) conjugated and anti-PD-L1 targeting gold nanoparticles (PD-L1-AuNP-DOX) for the targeted chemo-photothermal therapy of CRC. DOX and anti-PD-L1 antibody were conjugated to the α-terminal end group of lipoic acid polyethylene glycol N-hydroxysuccinimide (LA-PEG-NHS) using an amide linkage, and PD-L1-AuNP-DOX was constructed by linking LA-PEG-DOX, LA-PEG-PD-L1, and a short PEG chain on the surface of AuNP using thiol-Au covalent bonds. Physicochemical characterizations and biological studies of PD-L1-AuNP-DOX were performed in the presence of near-infrared (NIR) irradiation (biologic studies were conducted using cellular uptake, apoptosis, and cell cycle assays in CT-26 cells). PD-L1-AuNP-DOX (40.0 ± 3.1 nm) was successfully constructed and facilitated the efficient intracellular uptake of DOX as evidenced by pronounced apoptotic effects (66.0%) in CT-26 cells. PD-L1-AuNP-DOX treatment plus NIR irradiation significantly and synergistically suppressed the in vitro proliferation of CT-26 cells by increasing apoptosis and cell cycle arrest. The study demonstrates that PD-L1-AuNP-DOX in combination with synergistic targeted chemo-photothermal therapy has a considerable potential for the treatment of localized CRC.

Local Radiation Treatment of HER2-Positive Breast Cancer Using Trastuzumab-Modified Gold Nanoparticles Labeled with 177Lu.

PURPOSE: To compare the effectiveness of trastuzumab-modified gold nanoparticles (AuNP) labeled with 177Lu (trastuzumab-AuNP-177Lu) targeted to HER2 with non-targeted AuNP-177Lu for killing HER2-overexpressing breast cancer (BC) cells in vitro and inhibiting tumor growth in vivo following intratumoral (i.t.) injection. METHODS: AuNP (30 nm) were modified with polyethylene glycol (PEG) polymers linked to trastuzumab or to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelators to complex 177Lu. The binding and internalization of trastuzumab-AuNP-177Lu in HER2-positive SK-BR-3, BT-474 and MDA-MB-361 human BC cells were studied. Clonogenic survival and DNA double-strand breaks (DSBs) were measured after exposure of SK-BR-3 or MDA-MB-361 cells to trastuzumab-AuNP-177Lu or AuNP-177Lu. NOD/SCID mice with s.c. MDA-MB-361 tumor xenografts were treated by i.t. injection of 3 MBq (0.15 mg) of trastuzumab-AuNP-177Lu, AuNP-177Lu or normal saline. Tumor growth was measured over 16 days and normal tissue toxicity evaluated. RESULTS: Trastuzumab-AuNP-177Lu was bound and internalized by HER2 positive BC cells (KD = 7.6 ± 2.0 nM). Trastuzumab-AuNP-177Lu was 42.9 and 2.6-fold more effective than AuNP-177Lu at decreasing the clonogenic survival of SK-BR-3 (1.3 × 106 HER2/cell) and MDA-MB-361 (5.1 × 105 HER2/cell) cells, respectively, exposed overnight to these agents (1.5 nM; 20 MBq/mg Au). Under the same treatment conditions, 10-fold and 2.8-fold more DNA DSBs were observed in SK-BR-3 and MDA-MB-361 cells, respectively, exposed to trastuzumab-AuNP-177Lu than AuNP-177Lu. Trastuzumab-AuNP-177Lu was 1.8-fold more effective at inhibiting tumor growth than AuNP-177Lu. No or minimal normal tissue toxicity was observed for trastuzumab-AuNP-177Lu or AuNP-177Lu treatments. CONCLUSION: Trastuzumab-AuNP-177Lu enables an efficient local radiation treatment of HER2-positive BC.

Stability and Biodistribution of Thiol-Functionalized and (177)Lu-Labeled Metal Chelating Polymers Bound to Gold Nanoparticles.

We are studying a novel radiation nanomedicine approach to treatment of breast cancer using 30 nm gold nanoparticles (AuNP) modified with polyethylene glycol (PEG) metal-chelating polymers (MCP) that incorporate 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelators for complexing the ß-particle emitter, (177)Lu. Our objective was to compare the stability of AuNP conjugated to MCP via a single thiol [DOTA-PEG-ortho-pyridyl disulfide (OPSS)], a dithiol [DOTA-PEG-lipoic acid (LA)] or multithiol end-group [PEG-pGlu(DOTA)8-LA4] and determine the elimination and biodistribution of these (177)Lu-labeled MCP-AuNP in mice. Stability to aggregation in the presence of thiol-containing dithiothreitol (DTT), L-cysteine or glutathione was assessed and dissociation of (177)Lu-MCP from AuNP in human plasma measured. Elimination of radioactivity from the body of athymic mice and excretion into the urine and feces was measured up to 168 h post-intravenous (i.v.) injection of (177)Lu-MCP-AuNP and normal tissue uptake was determined. ICP-AES was used to quantify Au in the liver and spleen and these were compared to (177)Lu. Our results showed that PEG-pGlu(DOTA)8-LA4-AuNP were more stable to aggregation in vitro than DOTA-PEG-LA-AuNP and both forms of AuNP were more stable to thiol challenge than DOTA-PEG-OPSS-AuNP. PEG-pGlu((177)Lu-DOTA)8-LA4 was the most stable in plasma. Whole body elimination of (177)Lu was most rapid for mice injected with (177)Lu-DOTA-PEG-OPSS-AuNP. Urinary excretion accounted for >90% of eliminated (177)Lu. All (177)Lu-MCP-AuNP accumulated in the liver and spleen. Liver uptake was lowest for PEG-pGlu((177)Lu-DOTA)8-LA4-AuNP but these AuNP exhibited the greatest spleen uptake. There were differences in Au and (177)Lu in the liver for PEG-pGlu((177)Lu-DOTA)8-LA4-AuNP. These differences were not correlated with in vitro stability of the (177)Lu-MCP-AuNP. We conclude that conjugation of AuNP with PEG-pGlu((177)Lu-DOTA)8-LA4 via a multithiol functional group provided the greatest stability in vitro and lowest liver uptake in vivo and is, therefore, the most promising for constructing (177)Lu-MCP-AuNP for radiation treatment of breast cancer.

Targeting tumor-associated macrophages with mannosylated nanotherapeutics delivering TLR7/8 agonist enhances cancer immunotherapy.

Tumor-associated macrophages (TAMs) constitute 50-80% of stromal cells in most solid tumors with high mortality and poor prognosis. Tumor-infiltrating dendritic cells (TIDCs) and TAMs are key components mediating immune responses within the tumor microenvironment (TME). Considering their refractory properties, simultaneous remodeling of TAMs and TIDCs is a potential strategy of boosting tumor immunity and restoring immunosurveillance. In this study, mannose-decorated poly(lactic-co-glycolic acid) nanoparticles loading with R848 (Man-pD-PLGA-NP@R848) were prepared to dually target TAMs and TIDCs for efficient tumor immunotherapy. The three-dimensional (3D) cell culture model can simulate tumor growth as influenced by the TME and its 3D structural arrangement. Consequently, cancer spheroids enriched with tumor-associated macrophages (TAMs) were fabricated to assess the therapeutic effectiveness of Man-pD-PLGA-NP@R848. In the TME, Man-pD-PLGA-NP@R848 targeted both TAMs and TIDCs in a mannose receptor-mediated manner. Subsequently, Man-pD-PLGA-NP@R848 released R848 to activate Toll-like receptors 7 and 8, following dual-reprograming of TIDCs and TAMs. Man-pD-PLGA-NP@R848 could uniquely reprogram TAMs into antitumoral phenotypes, decrease angiogenesis, reprogram the immunosuppressive TME from "cold tumor" into "hot tumor", with high CD4+ and CD8+ T cell infiltration, and consequently hinder tumor development in B16F10 tumor-bearing mice. Therefore, dual-reprograming of TIDCs and TAMs with the Man-pD-PLGA-NP@R848 is a promising cancer immunotherapy strategy.

Fabrication of stem cell heterospheroids with sustained-release chitosan and poly(lactic-co-glycolic acid) microspheres to guide cell fate toward chondrogenic differentiation.

Mesenchymal stem cell (MSC)-based therapies show great potential in treating various diseases. However, control of the fate of injected cells needs to be improved. In this work, we developed an efficient methodology for modulating chondrogenic differentiation of MSCs. We fabricated heterospheroids with two sustained-release depots, a quaternized chitosan microsphere (QCS-MP) and a poly (lactic-co-glycolic acid) microsphere (PLGA-MP). The results show that heterospheroids composed of 1 × 104 to 5 × 104 MSCs formed rapidly during incubation in methylcellulose medium and maintained high cell viability in long-term culture. The MPs were uniformly distributed in the heterospheroids, as shown by confocal laser scanning microscopy. Incorporation of transforming growth factor beta 3 into QCS-MPs and of dexamethasone into PLGA-MPs significantly promoted the expression of chondrogenic genes and high accumulation of glycosaminoglycan in heterospheroids. Changes in crucial metabolites in the dual drug depot-engineered heterospheroids were also evaluated using 1H NMR-based metabolomics analysis to verify their successful chondrogenic differentiation. Our heterospheroid fabrication platform could be used in tissue engineering to study the effects of various therapeutic agents on stem cell fate.

Effects of surface camouflaged islet transplantation on pathophysiological progression in a db/db type 2 diabetic mouse model.

To investigate the inhibition effects of pancreatic islet transplantation on the progression of obese type 2 diabetes, we analyzed the effects of surface camouflaged islet transplantation on delaying the disease progression in a db/db diabetic mouse model. Surface camouflaged islets using 6-arm-PEG-catechol were transplanted in db/db diabetic mice. The fat accumulation and toxicity in the liver, the expansion of islets in the pancreas, and the size change of abdominal adipocyte were analyzed. In addition, the blood glucose control, insulin levels and immunohistochemical staining of recovered tissues were analyzed after transplantation. Then co-administration of anti-CD154 monoclonal antibody and Tacrolimus (IT group) deterred the pathophysiological progression of obese type 2 diabetes. At day 3 of transplantation, the serum insulin concentration of IT group was increased compared to the db/db diabetic mice group. The immunohistochemical studies demonstrated that the mass of 6-arm-PEG-catechol grafted islet was preserved in the transplantation site for 14 days. Surface modification using 6-arm-PEG-catechol effectively inhibited the immune cell infiltration and activation of host immune cells when immunosuppressive drug was given to the db/db type 2 diabetes mice. Therefore, 6-arm-PEG-catechol grafted islets effectively restored the insulin secretion in islet recipients and prevented the disease progression in type 2 diabetes.

Engineered therapeutic proteins for sustained-release drug delivery systems.

Proteins play a vital role in diverse biological processes in the human body, and protein therapeutics have been applied to treat different diseases such as cancers, genetic disorders, autoimmunity, and inflammation. Protein therapeutics have demonstrated their advantages, such as specific pharmaceutical effects, low toxicity, and strong solubility. However, several disadvantages arise in clinical applications, including short half-life, immunogenicity, and low permeation, leading to reduced drug effectiveness. The structure of protein therapeutics can be modified to increase molecular size, leading to prolonged stability and increased plasma half-life. Notably, the controlled-release delivery systems for the sustained release of protein drugs and preserving the stability of cargo proteins are envisioned as a potential approach to overcome these challenges. In this review, we summarize recent research progress related to structural modifications (PEGylation, glycosylation, poly amino acid modification, and molecular biology-based strategies) and promising long-term delivery systems, such as polymer-based systems (injectable gel/implants, microparticles, nanoparticles, micro/nanogels, functional polymers), lipid-based systems (liposomes, solid lipid nanoparticles, nanostructured lipid carriers), and inorganic nanoparticles exploited for protein therapeutics. STATEMENT OF SIGNIFICANCE: In this review, we highlight recent advances concerning modifying proteins directly to enhance their stability and functionality and discuss state-of-the-art methods for the delivery and controlled long-term release of active protein therapeutics to their target site. In terms of drug modifications, four widely used strategies, including PEGylation, poly amino acid modification, glycosylation, and genetic, are discussed. As for drug delivery systems, we emphasize recent progress relating to polymer-based systems, lipid-based systems developed, and inorganic nanoparticles for protein sustained-release delivery. This review points out the areas requiring focused research attention before the full potential of protein therapeutics for human health and disease can be realized.

Dual receptor specific nanoparticles targeting EGFR and PD-L1 for enhanced delivery of docetaxel in cancer therapy.

Dual-receptor targeted (DRT) nanoparticles which contain two distinct targeting agents may exhibit higher cell selectivity, cellular uptake, and cytotoxicity toward cancer cells than single-ligand targeted nanoparticle systems without additional functionality. The purpose of this study is to prepare DRT poly(lactic-co-glycolic acid) (PLGA) nanoparticles for targeting the delivery of docetaxel (DTX) to the EGFR and PD-L1 receptor positive cancer cells such as human glioblastoma multiform (U87-MG) and human non-small cell lung cancer (A549) cell lines. Anti-EGFR and anti-PD-L1 antibody were decorated on DTX loaded PLGA nanoparticles to prepare DRT-DTX-PLGA via. single emulsion solvent evaporation method. Physicochemical characterizations of DRT-DTX-PLGA, such as particle size, zeta-potential, morphology, and in vitro DTX release were also evaluated. The average particle size of DRT-DTX-PLGA was 124.2 ± 1.1 nm with spherical and smooth morphology. In the cellular uptake study, the DRT-DTX-PLGA endocytosed by the U87-MG and A549 cells was single ligand targeting nanoparticle. From the in vitro cell cytotoxicity, and apoptosis studies, we reported that DRT-DTX-PLGA exhibited high cytotoxicity and enhanced the apoptotic cell compared to the single ligand-targeted nanoparticle. The dual receptor mediated endocytosis of DRT-DTX-PLGA showed a high binding affinity effect that leads to high intracellular DTX concentration and exhibited high cytotoxic properties. Thus, DRT nanoparticles have the potential to improve cancer therapy by providing selectivity over single-ligand-targeted nanoparticles.

Magnetic resonance imaging of pancreatic islets using tissue-adhesive particles containing iron oxide nanoparticles.

Post-transplantation tracking of pancreatic islets is a prerequisite for advancing cell therapy to treat type 1 diabetes. Magnetic resonance imaging (MRI) has emerged as a safe and non-invasive technique for visualizing cells in clinical applications. In this study, we proposed a novel MRI contrast agent formulation by encapsulating iron oxide nanoparticles (IONPs) in poly(lactic-co-glycolic acid) (PLGA) particles functionalized with a tissue adhesive polydopamine (PD) layer (IONP-PLGA-PD MS). Intriguingly, our particles facilitated efficient and robust labeling through a one-step process, allowing for the incorporation of a substantial amount of IONPs without detrimental impacts on the viability and functionality of pancreatic islets. The MRI signals emanating from islets labeled using our particles were found to be stable over 30 days in vitro and 60 days when transplanted under kidney capsules of diabetic mice. These results suggest that our approach provides a potential platform for monitoring the fate of pancreatic islets after transplantation.

T-cell engaging poly(lactic-co-glycolic acid) nanoparticles as a modular platform to induce a potent cytotoxic immunogenic response against PD-L1 overexpressing cancer.

Bispecific nanoparticles (NPs) are conjugated with two antibodies that enhance T cell cytotoxicity by sequentially targeting CD3 and tumor-specific proteins. This interaction redirects T cells to specific tumor antigens and activates them to lyse tumor cells by blocking two different signaling pathways simultaneously. This study developed NP-based bispecific T-cell engagers (nanoBiTEs), which are R848-loaded bispecific poly(lactic-co-glycolic acid) NPs decorated with anti-CD3 antibody targeting T cells and anti-PD-L1 antibody targeting PD-L1 ligands (bis-R848-PLGA-NPs). Bis-R848-PLGA-NPs enhance the immunogenic response in destroying cancer cells by restoring the T cell effector functions. These interactions allow T cells to come in close proximity to the tumor cells. Finally, the release of R848 from PLGA-NPs activates dendritic cells, enhancing T cell activation. In vitro results show maximum internalization of bis-R848-PLGA-NPs in SK-OV3 and B16F10 cell lines, attributed to high PD-L1 expression in both cells. Furthermore, bis-R848-PLGA-NPs-treated CD8+ T cells exhibit a significantly increased total amount of CD8+/CD25+, CD8+/CD69+, and cytokine expression that leads to the robust inhibition of PD-L1 expressed cancer cells. Additionally, tumor growth is significantly inhibited by bis-R848-PLGA-NPs in the B16F10 xenograft mouse model and significantly enhanced intratumoral infiltration of CD4+ and CD8+ T cells, as well as tumor-infiltrated cytokines.

The impact of locally-delivered tacrolimus-releasing microspheres and polyethylene glycol-based islet surface modification on xenogeneic islet survival.

Pancreatic islet replacement therapy is an advanced choice for severe cases of type I diabetes. Nevertheless, extensive host immune response toward islet grafts remains a huge challenge for long-term graft function, and a lack of islet donors further increases the difficulties associated with upscaling this therapy. Mounting evidence suggests local delivery of immunosuppressive agents provides a feasible means of enhancing graft-protection. Among many immunosuppressants, tacrolimus (FK506) is one of the most potent interleukin-2 (IL-2)-mediated T-cell proliferation blockers. Here, we reported the effect of locally-delivered FK506-releasing PLGA microspheres (FK506-M) combined with polyethylene glycol (PEG)-based islet surface modification on xenogeneic islet survival in C57BL/6 mouse model. FK506-M was prepared using an emulsion method to a particle size of 10-40 µm and released FK506 over 40 days in vitro. Around 80% of the initial dose of FK506-M stably localized near transplanted islets, as observed under a bioimaging instrument and by immunofluorescence staining of islet grafts. Interestingly, FK506-M at very low-doses (equivalent to 150 to 2400 ng FK506 per recipient) was found to inhibit the infiltration of immune cells into grafts and reduce serum IL-1ß levels, thereby improving graft survival times dose-dependently. The PEGylation of islets alone was not enough to protect islets from early rejection. However, combined treatment with FK506-M additively prolonged xenograft survival. In conclusion, this study describes a safe, effective approach for translating a systemic exposure-free local drug delivery into clinical trials of islet transplantation.

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.

Polydopamine-tailored paclitaxel-loaded polymeric microspheres with adhered NIR-controllable gold nanoparticles for chemo-phototherapy of pancreatic cancer.

Chemotherapeutic drugs often used as a first-line treatment of pancreatic cancer (PC) exhibit challenges due to resistance development, lack of selectivity, and tumor heterogeneity. Currently, combination chemo-photothermal therapy is known to enhance the therapeutic efficacy of chemotherapeutic drugs in PC. In this study, we develop adherent gold nanoparticles (GNPs) and paclitaxel (PTX)-loaded PLGA microspheres for the treatment of PC. Polydopamine (pD) was used as a linker to adhere GNPs to the surface of PLGA-Ms and characterized using TEM. Short-term cytotoxicity of GNPs-pD-PTX-PLGA-Ms with or without NIR treatment was evaluated using CCK-8 assays. ROS and western blot assay were performed to determine the intensity of ROS following the treatment of GNPs-pD-PTX-PLGA-Ms with or without NIR in Panc-1 cell line. Successful adhesion of GNPs on the microspheres was confirmed by TEM. CCK-8 assay revealed that GNPs-pD-PTX-PLGA-Ms with NIR showed three-fold higher cytotoxicity, compared to the group without NIR. Furthermore, ROS and western blot assay suggest that GNPs-pD-PTX-PLGA-Ms with NIR showed more ROS generation, followed by downregulation of the expression levels of antioxidant enzyme (SOD2 and CATALASE). These results suggest that the GNPs-pD-PTX-PLGA-Ms in combination with NIR irradiation can provide a synergistic chemo-photothermal therapy for the treatment of PC.

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.

Engineering "cell-particle hybrids" of pancreatic islets and bioadhesive FK506-loaded polymeric microspheres for local immunomodulation in xenogeneic islet transplantation.

Host immune response remains an obstacle in cell-replacement therapy for treating type I diabetes. Long-term systemic immunosuppression results in suboptimal efficacy and adverse reactions. Thus, "cell-particle hybrids" of pancreatic islets and tissue-adhesive, polydopamine-coated, FK506-loaded biodegradable microspheres (PD-FK506-MS) were developed to locally modulate the immune response at the transplantation site. Coating of FK506-MS with PD enabled the rapid formation of stable cell-particle hybrids without significant changes in islet viability and functionality. Extremely low quantities of FK506 (approximately 600 ng per recipient) sustainably released from cell-particle hybrids effectively prolonged survival of xenogeneic islet graft. Interestingly, FK506 exhibited extended bioavailability in the grafts but was undetectable in systemic circulation and other tissues. Moreover, mRNA expression of inflammatory cytokines was significantly inhibited in the PD-FK506-MS-containing grafts but not in lymphoid organs. This study presents a promising platform that facilitates the translation of local immunomodulation towards an effective strategy with improved safety profiles for treating type I diabetes.

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.

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.

Surface modification of pancreatic islets using heparin-DOPA conjugate and anti-CD154 mAb for the prolonged survival of intrahepatic transplanted islets in a xenograft model.

This study proposes a combination method of using 3,4-dihydorxy-l-phenylalanine (DOPA) conjugated heparin (heparin-DOPA) and a low dose of anti-CD154 monoclonal antibody (MR-1) treatment to improve the survival time of intrahepatic islet xenograft. To inhibit instant blood mediated inflammatory reactions, heparin-DOPA was directly grafted to the pancreatic islet surface. The surface coverage of heparin-DOPA, the viability and functionality of heparin-DOPA grafted islets were evaluated. In addition, the combined effect of grafted heparin-DOPA and a low dose of MR-1 (a T-cell targeting immunosuppressive drug) on the survival of islet was evaluated in a xenograft model. Both unmodified islets and heparin-DOPA grafted islets were completely rejected within 2 weeks after intraportal transplantation. However, when 0.1 mg/mouse of MR-1 was administered (at day 0, 2, 4, 6 of transplantation) to 11 mice that had heparin-DOPA grafted islets transplanted to, seven out of the recipients maintained normoglycemia over 60 days. Therefore, we propose that a developed combinatory immunoprotection protocol of surface modification of pancreatic islets using heparin-DOPA with a low dose of MR-1 can be effective in prolonging the survival rate of transplanted islets in a xenograft model.