An injectable subcutaneous colon-specific immune niche for the treatment of ulcerative colitis.

As a chronic autoinflammatory condition, ulcerative colitis is often managed via systemic immunosuppressants. Here we show, in three mouse models of established ulcerative colitis, that a subcutaneously injected colon-specific immunosuppressive niche consisting of colon epithelial cells, decellularized colon extracellular matrix and nanofibres functionalized with programmed death-ligand 1, CD86, a peptide mimic of transforming growth factor-beta 1, and the immunosuppressive small-molecule leflunomide, induced intestinal immunotolerance and reduced inflammation in the animals' lower gastrointestinal tract. The bioengineered colon-specific niche triggered autoreactive T cell anergy and polarized pro-inflammatory macrophages via multiple immunosuppressive pathways, and prevented the infiltration of immune cells into the colon's lamina propria, promoting the recovery of epithelial damage. The bioengineered niche also prevented colitis-associated colorectal cancer and eliminated immune-related colitis triggered by kinase inhibitors and immune checkpoint blockade.

Immune Checkpoint Ligand Bioengineered Schwann Cells as Antigen-Specific Therapy for Experimental Autoimmune Encephalomyelitis.

Failure to establish immune tolerance leads to the development of autoimmune disease. The ability to regulate autoreactive T cells without inducing systemic immunosuppression represents a major challenge in the development of new strategies to treat autoimmune disease. Here, a translational method for bioengineering programmed death-ligand 1 (PD-L1)- and cluster of differentiation 86 (CD86)-functionalized mouse Schwann cells (SCs) to prevent and ameliorate multiple sclerosis (MS) in established mouse models of chronic and relapsing-remitting experimental autoimmune encephalomyelitis (EAE) is described. It is shown that the intravenous (i.v.) administration of immune checkpoint ligand functionalized mouse SCs modifies the course of disease and ameliorates EAE. Further, it is found that such bioengineered mouse SCs inhibit the differentiation of myelin-specific helper T cells into pathogenic T helper type-1 (Th 1) and type-17 (Th 17) cells, promote the development of tolerogenic myelin-specific regulatory T (Treg ) cells, and resolve inflammatory central nervous system microenvironments without inducing systemic immunosuppression.

In Vivo Bioengineering of Beta Cells with Immune Checkpoint Ligand as a Treatment for Early-Onset Type 1 Diabetes Mellitus.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by autoreactive T cells targeting the insulin-producing beta (ß) cells. Despite advances in insulin therapy, T1DM still leads to high morbidity and mortality in patients. A key focus of T1DM research has been to identify strategies that re-establish self-tolerance and suppress ongoing autoimmunity. Here, we describe a strategy that utilizes pretargeting and glycochemistry to bioengineer ß cells in situ to induce ß-cell-specific tolerance. We hypothesized that ß-cell-targeted Ac4ManNAz-encapsulated nanoparticles deliver and establish ß cells with high levels of surface reactive azide groups. We further theorized that administration of a dibenzylcyclooctyne (DBCO)-functionalized programmed death-ligand 1 immunoglobulin fusion protein (PD-L1-Ig) can be readily conjugated to the surface of native ß cells. Using nonobese diabetic (NOD) mice, we demonstrated that our strategy effectively and selectively conjugates PD-L1 onto ß cells through bioorthogonal stain-promoted azide-alkyne cycloaddition. We also showed that the in vivo functionalized ß cells simultaneously present islet-specific antigen and PD-L1 to the engaged T cells, reversing early onset T1DM by reducing IFN-gamma expressing cytotoxic toxic T cells and inducing antigen-specific tolerance.

Immune Checkpoint-Bioengineered Beta Cell Vaccine Reverses Early-Onset Type 1 Diabetes.

Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease that results from autoreactive T cells destroying insulin-producing pancreatic beta (ß) cells. The development of T1DM is associated with the deficiency of co-inhibitory immune checkpoint ligands (e.g., PD-L1, CD86, and Gal-9) in ß cells. Here, a new translational approach based on metabolic glycoengineering and bioorthogonal click chemistry, which bioengineers ß cells with co-inhibitory immune checkpoint molecules that induce antigen-specific immunotolerance and reverse early-onset hyperglycemia is reported. To achieve this goal, a subcutaneous injectable acellular pancreatic extracellular matrix platform for localizing the bioengineered ß cells while creating a pancreas-like immunogenic microenvironment, in which the autoreactive T cells can interface with the ß cells, is devised.

Co-delivery of etoposide and cisplatin in dual-drug loaded nanoparticles synergistically improves chemoradiotherapy in non-small cell lung cancer models.

Chemoradiotherapy with cisplatin and etoposide is a curative management regimen for both small and non-small cell lung cancers. While the treatment regimen is effective, it also has a high toxicity profile. One potential strategy to improve the therapeutic ratio of chemoradiation is to utilize nanotherapeutics. Nanoparticle formulation of cisplatin and etoposide, however, is challenging due to the significant mismatch in chemical properties of cisplatin and etoposide. Herein we report the formulation of a polymeric nanoparticle formulation of cisplatin and etoposide using a prodrug approach. We synthesized a hydrophobic platinum prodrug, which was then co-delivered with etoposide using a nanoparticle. Using mouse models of lung cancer, we demonstrated that dual-drug loaded nanoparticles are significantly more effective than small molecule chemotherapy in chemoradiotherapy. These results support further investigation of nanoparticle-based drug formulations of combination chemotherapies and the use of nanotherapeutics in chemoradiotherapy. STATEMENT OF SIGNIFICANCE: The treatment of lung cancer often involves a combination of chemotherapy and radiation. While it can be effective, it also has a high toxicity profile. Preferential delivery of chemotherapeutics to the tumor while avoiding normal tissue would improve efficacy and lower toxicity. While this is challenging with conventional drug delivery technologies, nanotechnology offers a unique opportunity. In this study, we have engineered nanoparticles that are loaded with combination chemotherapeutics and showed such nanotherapeutics are more effective and less toxic than free chemotherapeutics in chemoradiotherapy. Our work highlights the importance and potential of nanoformulations of combination chemotherapy in chemoradiotherapy and cancer treatment. This approach can be translated clinically and it can have a significant impact on cancer treatment.

Trispecific natural killer cell nanoengagers for targeted chemoimmunotherapy.

Activation of the innate immune system and natural killer (NK) cells has been a key effort in cancer immunotherapy research. Here, we report a nanoparticle-based trispecific NK cell engager (nano-TriNKE) platform that can target epidermal growth factor receptor (EGFR)-overexpressing tumors and promote the recruitment and activation of NK cells to eradicate these cancer cells. Moreover, the nanoengagers can deliver cytotoxic chemotherapeutics to further improve their therapeutic efficacy. We have demonstrated that effective NK cell activation can be achieved by the spatiotemporal coactivation of CD16 and 4-1BB stimulatory molecules on NK cells with nanoengagers, and the nanoengagers are more effective than free antibodies. We also show that biological targeting, either through radiotherapy or EGFR, is critical to the therapeutic effects of nanoengagers. Last, EGFR-targeted nanoengagers can augment both NK-activating agents and chemotherapy (epirubicin) as highly effective anticancer agents, providing robust chemoimmunotherapy.

Pretargeted delivery of PI3K/mTOR small-molecule inhibitor-loaded nanoparticles for treatment of non-Hodgkin's lymphoma.

Overactivation of the PI3K/mTOR signaling has been identified in non-Hodgkin's lymphoma. BEZ235 is an effective dual PI3K/mTOR inhibitor, but it was withdrawn from early-phase clinical trials owing to poor solubility and on-target/off-tumor toxicity. Here, we developed a nanoparticle (NP)-based pretargeted system for the therapeutic delivery of BEZ235 to CD20- and HLA-DR-expressing lymphoma cells for targeted therapy. The pretargeted system is composed of dibenzocyclooctyne-functionalized anti-CD20 and anti-Lym1 antibodies as the tumor-targeting components and azide-functionalized BEZ235-encapsulated NPs as the effector drug carrier. Using lymphoma cell lines with different CD20 and HLA-DR antigen densities as examples, we demonstrate that the dual antibody pretargeted strategy effectively raises the number of NPs retained on the target tumor cells and improves the in vitro and in vivo antitumor activity of BEZ235 through the inhibition of the PI3K/mTOR pathway. Our data demonstrate that the NP-based pretargeted system improves the therapeutic window of small-molecule kinase inhibitor.

High-Performance Concurrent Chemo-Immuno-Radiotherapy for the Treatment of Hematologic Cancer through Selective High-Affinity Ligand Antibody Mimic-Functionalized Doxorubicin-Encapsulated Nanoparticles.

Non-Hodgkin lymphoma is one of the most common types of cancer. Relapsed and refractory diseases are still common and remain significant challenges as the majority of these patients eventually succumb to the disease. Herein, we report a translatable concurrent chemo-immuno-radiotherapy (CIRT) strategy that utilizes fully synthetic antibody mimic Selective High-Affinity Ligand (SHAL)-functionalized doxorubicin-encapsulated nanoparticles (Dox NPs) for the treatment of human leukocyte antigen-D related (HLA-DR) antigen-overexpressed tumors. We demonstrated that our tailor-made antibody mimic-functionalized NPs bound selectively to different HLA-DR-overexpressed human lymphoma cells, cross-linked the cell surface HLA-DR, and triggered the internalization of NPs. In addition to the direct cytotoxic effect by Dox, the internalized NPs then released the encapsulated Dox and upregulated the HLA-DR expression of the surviving cells, which further augmented immunogenic cell death (ICD). The released Dox not only promotes ICD but also sensitizes the cancer cells to irradiation by inducing cell cycle arrest and preventing the repair of DNA damage. In vivo biodistribution and toxicity studies confirm that the targeted NPs enhanced tumor uptake and reduced systemic toxicities of Dox. Our comprehensive in vivo anticancer efficacy studies using lymphoma xenograft tumor models show that the antibody-mimic functional NPs effectively inhibit tumor growth and sensitize the cancer cells for concurrent CIRT treatment without incurring significant side effects. With an appropriate treatment schedule, the SHAL-functionalized Dox NPs enhanced the cell killing efficiency of radiotherapy by more than 100% and eradicated more than 80% of the lymphoma tumors.

Nanoparticle co-delivery of wortmannin and cisplatin synergistically enhances chemoradiotherapy and reverses platinum resistance in ovarian cancer models.

Most ovarian cancer patients respond well to initial platinum-based chemotherapy. However, within a year, many patients experience disease recurrence with a platinum resistant phenotype that responds poorly to second line chemotherapies. As a result, new strategies to address platinum resistant ovarian cancer (PROC) are needed. Herein, we report that NP co-delivery of cisplatin (CP) and wortmannin (Wtmn), a DNA repair inhibitor, synergistically enhances chemoradiotherapy (CRT) and reverses CP resistance in PROC. We encapsulated this regimen in FDA approved poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) NPs to reduce systemic side effects, enhance cellular CP uptake, improve Wtmn stability, and increase therapeutic efficacy. Treatment of platinum-sensitive ovarian cancer (PSOC) and PROC murine models with these dual-drug loaded NPs (DNPs) significantly reduced tumor burden versus treatment with combinations of free drugs or single-drug loaded NPs (SNPs). These results support further investigation of this NP-based, synergistic drug regimen as a means to combat PROC in the clinic.

Bespoke Pretargeted Nanoradioimmunotherapy for the Treatment of Non-Hodgkin Lymphoma.

Non-Hodgkin lymphoma (NHL) is one of the most common types of hematologic malignancies. Pretargeted radioimmunotherapy (PRIT), the sequential administration of a bispecific antibody-based primary tumor-targeting component followed by a radionucleotide-labeled treatment effector, has been developed to improve the treatment efficacy and to reduce the side effects of conventional RIT. Despite the preclinical success of PRIT, clinical trials revealed that the immunogenicity of the bispecific antibody as well as the presence of competing endogenous effector molecules often compromised the treatment. One strategy to improve PRIT is to utilize bio-orthogonal ligation reactions to minimize immunogenicity and improve targeting. Herein, we report a translatable pretargeted nanoradioimmunotherapy strategy for the treatment of NHL. This pretargeting system is composed of a dibenzylcyclooctyne (DBCO)-functionalized anti-CD20 antibody (α-CD20) tumor-targeting component and an azide- and yttrium-90-(90Y) dual-functionalized dendrimer. The physicochemical properties of both pretargeting components have been extensively studied. We demonstrated that an optimized dual-functionalized dendrimer can undergo rapid strain-promoted azide-alkyne cycloaddition with the DBCO-functionalized α-CD20 at the physiological conditions. The treatment effector in our pretargeting system can not only selectively deliver radionucleotides to the target tumor cells but also increase the complement-dependent cytotoxicity of α-CD20 and thus enhance the antitumor effects, as justified by comprehensive in vitro and in vivo studies in mouse NHL xenograft and disseminated models.

Co-delivery of paclitaxel and cisplatin with biocompatible PLGA-PEG nanoparticles enhances chemoradiotherapy in non-small cell lung cancer models.

Chemoradiotherapy (CRT) with paclitaxel (PTX) and cisplatin (CP) is part of the standard of care for patients with locally advanced non-small cell lung cancer (NSCLC). Despite the high treatment intensity, many patients still develop local recurrence after treatment. Thus, there is a strong need to further improve CRT for lung cancer. One strategy is to co-deliver cytotoxic chemotherapy agents using biocompatible nanoparticles (NPs) which can limit off-target tissue toxicity and improve therapeutic efficacy. Herein, we report the development of dual-drug loaded nanoformulations that improve the efficacy of CRT for NSCLC by co-encapsulation of cisplatin (CP) and PTX in PLGA-PEG NPs. Mice bearing NSCLC xenografts given the dual-drug loaded NPs during CRT showed greater inhibition of tumor growth than free drug combinations or combinations of single-drug loaded NPs. These results indicate that using a NP co-delivery strategy for this common CRT regimen may improve clinical responses in NSCLC patients.

Folate-targeted pH-responsive calcium zoledronate nanoscale metal-organic frameworks: Turning a bone antiresorptive agent into an anticancer therapeutic.

Zoledronate (Zol) is a third-generation bisphosphonate that is widely used as an anti-resorptive agent for the treatment of cancer bone metastasis. While there is preclinical data indicating that bisphosphonates such as Zol have direct cytotoxic effects on cancer cells, such effect has not been firmly established in the clinical setting. This is likely due to the rapid absorption of bisphosphonates by the skeleton after intravenous (i.v.) administration. Herein, we report the reformulation of Zol using nanotechnology and evaluation of this novel nanoscale metal-organic frameworks (nMOFs) formulation of Zol as an anticancer agent. The nMOF formulation is comprised of a calcium zoledronate (CaZol) core and a polyethylene glycol (PEG) surface. To preferentially deliver CaZol nMOFs to tumors as well as facilitate cellular uptake of Zol, we incorporated folate (Fol)-targeted ligands on the nMOFs. The folate receptor (FR) is known to be overexpressed in several tumor types, including head-and-neck, prostate, and non-small cell lung cancers. We demonstrated that these targeted CaZol nMOFs possess excellent chemical and colloidal stability in physiological conditions. The release of encapsulated Zol from the nMOFs occurs in the mid-endosomes during nMOF endocytosis. In vitro toxicity studies demonstrated that Fol-targeted CaZol nMOFs are more efficient than small molecule Zol in inhibiting cell proliferation and inducing apoptosis in FR-overexpressing H460 non-small cell lung and PC3 prostate cancer cells. Our findings were further validated in vivo using mouse xenograft models of H460 and PC3. We demonstrated that Fol-targeted CaZol nMOFs are effective anticancer agents and increase the direct antitumor activity of Zol by 80-85% in vivo through inhibition of tumor neovasculature, and inhibiting cell proliferation and inducing apoptosis.

Direct Observation of Early-Stage High-Dose Radiotherapy-Induced Vascular Injury via Basement Membrane-Targeting Nanoparticles.

Collagen IV-targeting peptide-conjugated basement membrane-targeting nanoparticles are successfully engineered to identify early-stage blood vessel injury induced by high-dose radiotherapy.

Improving Cancer Chemoradiotherapy Treatment by Dual Controlled Release of Wortmannin and Docetaxel in Polymeric Nanoparticles.

Combining molecularly targeted agents and chemotherapeutics is an emerging strategy in cancer treatment. We engineered sub-50 nm diameter diblock copolymer nanoparticles (NPs) that can sequentially release wortmannin (Wtmn, a cell signaling inhibitor) and docetaxel (Dtxl, genotoxic anticancer agent) to cancer cells. These NPs were studied in chemoradiotherapy, an important cancer treatment paradigm, in the preclinical setting. We demonstrated that Wtmn enhanced the therapeutic efficacy of Dtxl and increased the efficiency of radiotherapy (XRT) in H460 lung cancer and PC3 prostate cells in culture. Importantly, we showed that NPs containing both Wtmn and Dtxl release the drugs in a desirable sequential fashion to maximize therapeutic efficacy in comparison to administering each drug alone. An in vivo toxicity study in a murine model validated that NPs containing both Dtxl and Wtmn do not have a high toxicity profile. Lastly, we demonstrated that Dtxl/Wtmn-coencapsulated NPs are more efficient than each single-drug-loaded NPs or a combination of both single-drug-loaded NPs in chemoradiotherapy using xenograft models. Histopathological studies and correlative studies support that the improved therapeutic efficacy is through changes in signaling pathways and increased tumor cell apoptosis. Our findings suggest that our nanoparticle system led to a dynamic rewiring of cellular apoptotic pathways and thus improve the therapeutic efficiency.

Near-infrared light-triggered irreversible aggregation of poly(oligo(ethylene glycol) methacrylate)-stabilised polypyrrole nanoparticles under biologically relevant conditions.

We report the use of near-infrared (NIR) radiation to trigger the irreversible flocculation of poly(oligo(ethylene glycol) methacrylate)-stabilised polypyrrole nanoparticles in physiological buffer.

Anti-biofouling conducting polymer nanoparticles as a label-free optical contrast agent for high resolution subsurface biomedical imaging.

Optical coherence tomography (OCT) is a modern high resolution subsurface medical imaging technique. Herein we describe: (i) the synthesis of a thiophene-functionalized oligo(ethylene glycol) methacrylate (OEGMA)-based statistical copolymer, denoted poly(2TMOI-OEGMA); (ii) the preparation of sterically-stabilized polypyrrole (PPy) nanoparticles of approximately 60 nm diameter; (iii) the evaluation of these nanoparticles as a NIR-absorbing optical contrast agent for high-resolution OCT imaging. We show that poly(2TMOI-OEGMA)-stabilized PPy nanoparticles exhibit similar optical properties to poly(vinyl alcohol) (PVA)-stabilized PPy nanoparticles of comparable size prepared using commercially available PVA. Spectroscopic measurements and Mie calculations indicate that both types of PPy nanoparticles strongly absorb NIR radiation above 1000 nm, suggesting their potential use as OCT contrast agents. In vitro OCT studies indicate that both types of PPy nanoparticles reduce NIR backscattering within homogeneous intralipid tissue phantoms, offering almost identical contrast performance in this medium. However, PVA-stabilized PPy nanoparticles became colloidally unstable when dispersed in physiological buffer and immersed in a solid biotissue phantom and hence failed to generate a strong contrast effect. In contrast, the poly(2TMOI-OEGMA)-stabilized PPy nanoparticles remained well-dispersed and hence exhibited a strong rapid onset contrast effect within the biotissue phantom under identical physiological conditions. Ex vivo studies performed on excised chicken and porcine skin tissue demonstrated that topical administration of a low concentration of poly(2TMOI-OEGMA)-stabilized PPy nanoparticles rapidly enhances OCT image contrast in both cases, allowing key tissue features to be readily identified.

Heterocoagulation as a facile route to prepare stable serum albumin-nanoparticle conjugates for biomedical applications: synthetic protocols and mechanistic insights.

There is increasing interest in using serum albumin, the most abundant plasma protein, as a stabilizing agent in the context of nanomedicine. Using poly(vinyl amine)-stabilized polypyrrole nanoparticles as an example, we report a facile generic route to prepare serum albumin-nanoparticle conjugates via heterocoagulation. Time-resolved dynamic light scattering (DLS), disk centrifuge photosedimentometry (DCP), and circular dichroism (CD) spectroscopy studies confirm that bovine serum albumin (BSA) adsorbs rapidly onto the cationic poly(vinyl amine)-stabilized polypyrrole nanoparticles and suggest that the initial well-defined protein coronal is subsequently cross-linked via thiol-disulfide exchange. These BSA-nanoparticle conjugates were further characterized by X-ray photoelectron spectroscopy (XPS), aqueous electrophoresis, field emission scanning electron microscopy (FE SEM), and transmission electron microscopy (TEM). They exhibit excellent long-term colloidal stability under physiological conditions without further purification, suggesting strong irreversible adsorption by the BSA. Protein adsorption appears to be co-operative and both thermodynamic and mechanistic aspects were examined via aqueous electrophoresis, DCP, and DLS studies.

Polypyrrole nanoparticles: a potential optical coherence tomography contrast agent for cancer imaging.

A near-infrared (NIR) absorbing contrast agent based on polypyrrole nanoparticles is described. Quantitative optical coherence tomography studies on tissue phantoms and Mie scattering calculations indicate their potential application for early-stage cancer diagnosis.