Iron Oxide Nanoparticle-Incorporated Mesenchymal Stem Cells for Alzheimer's Disease Treatment.

Alzheimer's disease (AD) is a neurodegenerative disease with multifactorial pathogenesis. However, most current therapeutic approaches for AD target a single pathophysiological mechanism, generally resulting in unsatisfactory therapeutic outcomes. Recently, mesenchymal stem cell (MSC) therapy, which targets multiple pathological mechanisms of AD, has been explored as a novel treatment. However, the low brain retention efficiency of administered MSCs limits their therapeutic efficacy. In addition, autologous MSCs from AD patients may have poor therapeutic abilities. Here, we overcome these limitations by developing iron oxide nanoparticle (IONP)-incorporated human Wharton's jelly-derived MSCs (MSC-IONPs). IONPs promote therapeutic molecule expression in MSCs. Following intracerebroventricular injection, MSC-IONPs showed a higher brain retention efficiency under magnetic guidance. This potentiates the therapeutic efficacy of MSCs in murine models of AD. Furthermore, human Wharton's jelly-derived allogeneic MSCs may exhibit higher therapeutic abilities than those of autologous MSCs in aged AD patients. This strategy may pave the way for developing MSC therapies for AD.

Senolytic Therapy for Cerebral Ischemia-Reperfusion Injury.

Ischemic stroke is one of the leading causes of death, and even timely treatment can result in severe disabilities. Reperfusion of the ischemic stroke region and restoration of the blood supply often lead to a series of cellular and biochemical consequences, including generation of reactive oxygen species (ROS), expression of inflammatory cytokines, inflammation, and cerebral cell damage, which is collectively called cerebral ischemia-reperfusion (IR) injury. Since ROS and inflammatory cytokines are involved in cerebral IR injury, injury could involve cellular senescence. Thus, we investigated whether senolytic therapy that eliminates senescent cells could be an effective treatment for cerebral IR injury. To determine whether IR induces neural cell senescence in vitro, astrocytes were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). OGD/R induced astrocyte senescence and senescent cells in OGD/R-injured astrocytes were effectively eliminated in vitro by ABT263, a senolytic agent. IR in rats with intraluminal middle cerebral artery occlusion induced cellular senescence in the ischemic region. The senescent cells in IR-injured rats were effectively eliminated by intravenous injections of ABT263. Importantly, ABT263 treatment significantly reduced the infarct volume and improved neurological function in behavioral tests. This study demonstrated, for the first time, that senolytic therapy has therapeutic potential for cerebral IR injury.

Engineered Nanoparticles for Enhanced Antitumoral Synergy Between Macrophages and T Cells in the Tumor Microenvironment.

T cells and macrophages have the potential to collaborate to eliminate tumor cells efficiently. Macrophages can eliminate tumor cells through phagocytosis and subsequently activate T cells by presenting tumor antigens. The activated T cells, in turn, can kill tumor cells and redirect tumor-associated macrophages toward an antitumoral M1 phenotype. However, checkpoint molecules expressed on tumor cells impede the collaborative action of these immune cells. Meanwhile, monotherapy with a single immune checkpoint inhibitor (ICI) for either macrophages or T cells yields suboptimal efficacy in cancer patients. To address this challenge, here a nanoparticle capable of efficiently delivering dual ICIs to tumors for both macrophages and T cells is developed. These programmed cell death protein 1 (PD-1)-transfected macrophage membrane-derived nanoparticles (PMMNPs) can target tumors and provide signal-regulatory protein alpha and PD-1 to block CD47 and programmed cell death-ligand 1 (PD-L1), respectively, on tumor cells. PMMNPs enhance macrophage-mediated cancer cell phagocytosis and antigen presentation, promote T cell activation, and induce the reprogramming of macrophages toward an antitumoral phenotype. In syngeneic tumor-bearing mice, PMMNPs demonstrate superior therapeutic efficacy compared to nanoparticles delivering single ICIs and non-targeted delivery of anti-CD47 and anti-PD-L1 antibodies. PMMNPs capable of augmenting the antitumoral interplay between macrophages and T cells may offer a promising avenue for cancer immunotherapy.

Spatiotemporal control of neutrophil fate to tune inflammation and repair for myocardial infarction therapy.

Neutrophils are critical mediators of both the initiation and resolution of inflammation after myocardial infarction (MI). Overexuberant neutrophil signaling after MI exacerbates cardiomyocyte apoptosis and cardiac remodeling while neutrophil apoptosis at the injury site promotes macrophage polarization toward a pro-resolving phenotype. Here, we describe a nanoparticle that provides spatiotemporal control over neutrophil fate to both stymie MI pathogenesis and promote healing. Intravenous injection of roscovitine/catalase-loaded poly(lactic-co-glycolic acid) nanoparticles after MI leads to nanoparticle uptake by circulating neutrophils migrating to the infarcted heart. Activated neutrophils at the infarcted heart generate reactive oxygen species, triggering intracellular release of roscovitine, a cyclin-dependent kinase inhibitor, from the nanoparticles, thereby inducing neutrophil apoptosis. Timely apoptosis of activated neutrophils at the infarcted heart limits neutrophil-driven inflammation, promotes macrophage polarization toward a pro-resolving phenotype, and preserves heart function. Modulating neutrophil fate to tune both inflammatory and reparatory processes may be an effective strategy to treat MI.

A Personalized Cancer Nanovaccine that Enhances T-Cell Responses and Efficacy Through Dual Interactions with Dendritic Cells and T Cells.

Conventional approaches to developing therapeutic cancer vaccines that primarily activate tumor-specific T cells via dendritic cells (DCs) often demonstrate limited efficacy due to the suboptimal activation of these T cells. To address this limitation, here a therapeutic cancer nanovaccine is developed that enhances T cell responses by interacting with both DCs and T cells. The nanovaccine is based on a cancer cell membrane nanoparticle (CCM-MPLA) that utilizes monophosphoryl lipid A (MPLA) as an adjuvant. To allow direct interaction between the nanovaccine and tumor-specific T cells, anti-CD28 antibodies (aCD28) are conjugated onto CCM-MPLA, resulting in CCM-MPLA-aCD28. This nanovaccine activates tumor-specific CD8+ T cells in both the presence and absence of DCs. Compared with nanovaccines that interact with either DCs (CCM-MPLA) or T cells (CCM-aCD28), CCM-MPLA-aCD28 induces more potent responses of tumor-specific CD8+ T cells and exhibits a higher antitumor efficacy in tumor-bearing mice. No differences in T cell activation efficiency and therapeutic efficacy are observed between CCM-MPLA and CCM-aCD28. This approach may lead to the development of effective personalized therapeutic cancer vaccines prepared from autologous cancer cells.

Senescent cancer cell-derived nanovesicle as a personalized therapeutic cancer vaccine.

The development of therapeutic cancer vaccines (TCVs) that provide clinical benefits is challenging mainly due to difficulties in identifying immunogenic tumor antigens and effectively inducing antitumor immunity. Furthermore, there is an urgent need for personalized TCVs because only a limited number of tumor antigens are shared among cancer patients. Several autologous nanovaccines that do not require the identification of immunogenic tumor antigens have been proposed as personalized TCVs. However, these nanovaccines generally require exogenous adjuvants (e.g., Toll-like receptor agonists) to improve vaccine immunogenicity, which raises safety concerns. Here, we present senescent cancer cell-derived nanovesicle (SCCNV) as a personalized TCV that provides patient-specific tumor antigens and improved vaccine immunogenicity without the use of exogenous adjuvants. SCCNVs are prepared by inducing senescence in cancer cells ex vivo and subsequently extruding the senescent cancer cells through nanoporous membranes. In the clinical setting, SCCNVs can be prepared from autologous cancer cells from the blood of liquid tumor patients or from tumors surgically removed from solid cancer patients. SCCNVs also contain interferon-γ and tumor necrosis factor-α, which are expressed during senescence. These endogenous cytokines act as adjuvants and enhance vaccine immunogenicity, avoiding the need for exogenous adjuvants. Intradermally injected SCCNVs effectively activate dendritic cells and tumor-specific T cells and inhibit primary and metastatic tumor growth and tumor recurrence. SCCNV therapy showed an efficacy similar to that of immune checkpoint blockade (ICB) therapy and synergized with ICB. SCCNVs, which can be prepared using a simple and facile procedure, show potential as personalized TCVs.

Ceria-vesicle nanohybrid therapeutic for modulation of innate and adaptive immunity in a collagen-induced arthritis model.

Commencing with the breakdown of immune tolerance, multiple pathogenic factors, including synovial inflammation and harmful cytokines, are conjointly involved in the progression of rheumatoid arthritis. Intervening to mitigate some of these factors can bring a short-term therapeutic effect, but other unresolved factors will continue to aggravate the disease. Here we developed a ceria nanoparticle-immobilized mesenchymal stem cell nanovesicle hybrid system to address multiple factors in rheumatoid arthritis. Each component of this nanohybrid works individually and also synergistically, resulting in comprehensive treatment. Alleviation of inflammation and modulation of the tissue environment into an immunotolerant-favourable state are combined to recover the immune system by bridging innate and adaptive immunity. The therapy is shown to successfully treat and prevent rheumatoid arthritis by relieving the main symptoms and also by restoring the immune system through the induction of regulatory T cells in a mouse model of collagen-induced arthritis.

A Therapeutic Nanovaccine that Generates Anti-Amyloid Antibodies and Amyloid-specific Regulatory T Cells for Alzheimer's Disease.

Alzheimer's disease (AD), the most common cause of dementia, is a complex condition characterized by multiple pathophysiological mechanisms including amyloid-ß (Aß) plaque accumulation and neuroinflammation in the brain. The current immunotherapy approaches, such as anti-Aß monoclonal antibody (mAb) therapy, Aß vaccines, and adoptive regulatory T (Treg) cell transfer, target a single pathophysiological mechanism, which may lead to unsatisfactory therapeutic efficacy. Furthermore, Aß vaccines often induce T helper 1 (Th1) cell-mediated inflammatory responses. Here, a nanovaccine composed of lipid nanoparticles loaded with Aß peptides and rapamycin is developed, which targets multiple pathophysiological mechanisms, exhibits the combined effects of anti-Aß antibody therapy and adoptive Aß-specific Treg cell transfer, and can overcome the limitations of current immunotherapy approaches for AD. The Nanovaccine effectively delivers rapamycin and Aß peptides to dendritic cells, produces both anti-Aß antibodies and Aß-specific Treg cells, removes Aß plaques in the brain, alleviates neuroinflammation, prevents Th1 cell-mediated excessive immune responses, and inhibits cognitive impairment in mice. The nanovaccine shows higher efficacy in cognitive recovery than an Aß vaccine. Unlike anti-Aß mAb therapy and adoptive Treg cell transfer, both of which require complicated and costly manufacturing processes, the nanovaccine is easy-to-prepare and cost-effective. The nanovaccines can represent a novel treatment option for AD.

Nanovesicle-Mediated Targeted Delivery of Immune Checkpoint Blockades to Potentiate Therapeutic Efficacy and Prevent Side Effects.

Despite the clinically proven efficacies of immune checkpoint blockades, including anti-cytotoxic T lymphocyte-associated protein 4 antibody (αCTLA-4), the low response rate and immune-related adverse events (irAEs) in cancer patients represent major drawbacks of the therapy. These drawbacks of αCTLA-4 therapy are mainly due to the suboptimal activation of tumor-specific cytotoxic T lymphocytes (CTLs) and the systemic nonspecific activation of T cells. To overcome such drawbacks, αCTLA-4 is delivered by dendritic cell-derived nanovesicles presenting tumor antigens (DCNV-TAs) that exclusively interact with tumor-specific T cells, leading to selective activation of tumor-specific CTLs. Compared to conventional αCTLA-4 therapy, treatment with αCTLA-4-conjugated DCNV-TAs significantly inhibits tumor growth and reduces irAEs in syngeneic tumor-bearing mice. This study demonstrates that the spatiotemporal presentation of both αCTLA-4 and tumor antigens enables selective activation of tumor-specific T cells and potentiates the antitumor efficacy of αCTLA-4 without inducing systemic irAEs.

A Senolytic-Eluting Coronary Stent for the Prevention of In-Stent Restenosis.

The vast majority of drug-eluting stents (DES) elute either sirolimus or one of its analogues. While limus drugs stymie vascular smooth muscle cell (VSMC) proliferation to prevent in-stent restenosis, their antiproliferative nature is indiscriminate and limits healing of the endothelium in stented vessels, increasing the risk of late-stent thrombosis. Oxidative stress, which is associated with vascular injury from stent implantation, can induce VSMCs to undergo senescence, and senescent VSMCs can produce pro-inflammatory cytokines capable of inducing proliferation of neighboring nonsenescent VSMCs. We explored the potential of senolytic therapy, which involves the selective elimination of senescent cells, in the form of a senolytic-eluting stent (SES) for interventional cardiology. Oxidative stress was modeled in vitro by exposing VSMCs to H2O2, and H2O2-mediated senescence was evaluated by cytochemical staining of senescence-associated ß-galactosidase activity and qRT-PCR. Quiescent VSMCs were then treated with the conditioned medium (CM) of H2O2-treated VSMCs. Proliferative effects of CM were analyzed by staining for proliferating cell nuclear antigen. Senolytic effects of the first-generation senolytic ABT263 were observed in vitro, and the effects of ABT263 on endothelial cells were also investigated through an in vitro re-endothelialization assay. SESs were prepared by dip coating. Iliofemoral arteries of hypercholesteremic rabbits were implanted with SES, everolimus-eluting stents (EESs), or bare-metal stents (BMSs), and the area of stenosis was measured 4 weeks post-implantation using optical coherence tomography. We found that a portion of H2O2-treated VSMCs underwent senescence, and that CM of H2O2-treated senescent VSMCs triggered the proliferation of quiescent VSMCs. ABT263 reverted H2O2-mediated senescence and the proliferative capacity of senescent VSMC CM. Unlike everolimus, ABT263 did not affect endothelial cell migration and/or proliferation. SES, but not EES, significantly reduced stenosis area in vivo compared with bare-metal stents (BMSs). This study shows the potential of SES as an alternative to current forms of DES.

Local Delivery of Senolytic Drug Inhibits Intervertebral Disc Degeneration and Restores Intervertebral Disc Structure.

Intervertebral disc (IVD) degeneration (IVDD) is a leading cause of chronic low back pain. There is a strong clinical demand for more effective treatments for IVDD as conventional treatments provide only symptomatic relief rather than arresting IVDD progression. This study shows that senolytic therapy with local drug delivery can inhibit IVDD and restore IVD integrity. ABT263, a senolytic drug, is loaded in poly(lactic-co-glycolic acid) nanoparticles (PLGA-ABT) and intradiscally administered into injury-induced IVDD rat models. The single intradiscal injection of PLGA-ABT may enable local delivery of the drug to avascular IVD, prevention of potential systemic toxicity caused by systemic administration of senolytic drug, and morbidity caused by repetitive injections of free drug into the IVD. The strategy results in the selective elimination of senescent cells from the degenerative IVD, reduces expressions of pro-inflammatory cytokines and matrix proteases in the IVD, inhibits progression of IVDD, and even restores the IVD structure. This study demonstrates for the first time that local delivery of senolytic drug can effectively treat senescence-associated IVDD. This approach can be extended to treat other types of senescence-associated degenerative diseases.

T-Cell-Derived Nanovesicles for Cancer Immunotherapy.

Although T-cell therapy is a remarkable breakthrough in cancer immunotherapy, the therapeutic efficacy is limited for solid tumors. A major cause of the low efficacy is T-cell exhaustion by immunosuppressive mechanisms of solid tumors, which are mainly mediated by programmed death-ligand 1 (PD-L1) and transforming growth factor-beta (TGF-ß). Herein, T-cell-derived nanovesicles (TCNVs) produced by the serial extrusion of cytotoxic T cells through membranes with micro-/nanosized pores that inhibit T-cell exhaustion and exhibit antitumoral activity maintained in the immunosuppressive tumor microenvironment (TME) are presented. TCNVs, which have programmed cell death protein 1 and TGF-ß receptor on their surface, block PD-L1 on cancer cells and scavenge TGF-ß in the immunosuppressive TME, thereby preventing cytotoxic-T-cell exhaustion. In addition, TCNVs directly kill cancer cells via granzyme B delivery. TCNVs successfully suppress tumor growth in syngeneic-solid-tumor-bearing mice. Taken together, TCNV offers an effective cancer immunotherapy strategy to overcome the tumor's immunosuppressive mechanisms.

Multilayered Cell Sheets of Cardiac Reprogrammed Cells for the Evaluation of Drug Cytotoxicity.

BACKGROUND: Various cell-culture systems have been used to evaluate drug toxicity in vitro. However, factors that affect cytotoxicity outcomes in drug toxicity evaluation systems remain elusive. In this study, we used multilayered sheets of cardiac-mimetic cells, which were reprogrammed from human fibroblasts, to investigate the effects of the layer number on drug cytotoxicity outcomes. METHODS: Cell sheets of cardiac-mimetic cells were fabricated by reprogramming of human fibroblasts into cardiac-mimetic cells via coculture with cardiac cells and electric stimulation, as previously described. Double-layered cell sheets were prepared by stacking the cell sheets. The mono- and double-layered cell sheets were treated with 5-fluorouracil (5-FU), an anticancer drug, in vitro. Subsequently, apoptosis and lipid peroxidation were analyzed. Furthermore, effects of cardiac-mimetic cell density on cytotoxicity outcomes were evaluated by culturing cells in monolayer at various cell densities. RESULTS: The double-layered cell sheets exhibited lower cytotoxicity in terms of apoptosis and lipid peroxidation than the mono-layered sheets at the same 5-FU dose. In addition, the double-layered cell sheets showed better preservation of mitochondrial function and plasma membrane integrity than the monolayer sheets. The lower cytotoxicity outcomes in the double-layered cell sheets may be due to the higher intercellular interactions, as the cytotoxicity of 5-FU decreased with cell density in monolayer cultures of cardiac-mimetic cells. CONCLUSION: The layer number of cardiac-mimetic cell sheets affects drug cytotoxicity outcomes in drug toxicity tests. The in vitro cellular configuration that more closely mimics the in vivo configuration in the evaluation systems seems to exhibit lower cytotoxicity in response to drug.

Nanocomplex-Mediated In Vivo Programming to Chimeric Antigen Receptor-M1 Macrophages for Cancer Therapy.

Chimeric antigen receptor-T (CAR-T) cell immunotherapy has shown impressive clinical outcomes for hematologic malignancies. However, its broader applications are challenged due to its complex ex vivo cell-manufacturing procedures and low therapeutic efficacy against solid tumors. The limited therapeutic effects are partially due to limited CAR-T cell infiltration to solid tumors and inactivation of CAR-T cells by the immunosuppressive tumor microenvironment. Here, a facile approach is presented to in vivo program macrophages, which can intrinsically penetrate solid tumors, into CAR-M1 macrophages displaying enhanced cancer-directed phagocytosis and anti-tumor activity. In vivo injected nanocomplexes of macrophage-targeting nanocarriers and CAR-interferon-γ-encoding plasmid DNA induce CAR-M1 macrophages that are capable of CAR-mediated cancer phagocytosis, anti-tumor immunomodulation, and inhibition of solid tumor growth. Together, this study describes an off-the-shelf CAR-macrophage therapy that is effective for solid tumors and avoids the complex and costly processes of ex vivo CAR-cell manufacturing.