Nanomodulator-Mediated Restructuring of Adipose Tissue Immune Microenvironments for Antiobesity Treatment.

In obesity, the interactions between proinflammatory macrophages and adipocytes in white adipose tissues are known to play a crucial role in disease progression by providing inflammatory microenvironments. Here, we report that the functional nanoparticle-mediated modulation of crosstalk between adipocytes and macrophages can remodel adipocyte immune microenvironments. As a functional nanomodulator, we designed antivascular cell adhesion molecule (VCAM)-1 antibody-conjugated and amlexanox-loaded polydopamine nanoparticles (VAPN). Amlexanox was used as a model drug to increase energy expenditure. Compared to nanoparticles lacking antibody modification or amlexanox, VAPN showed significantly greater binding to VCAM-1-expressing adipocytes and lowered the interaction of adipocytes with macrophages. In high fat diet-fed mice, repeated subcutaneous administration of VAPN increased the populations of beige adipocytes and ameliorated inflammation in white adipose tissues. Moreover, the localized application of VAPN in vivo exerted a systemic metabolic effect and reduced metabolic disorders, including insulin tolerance and liver steatosis. These findings suggested that VAPN had potential to modulate the immune microenvironments of adipose tissues for the immunologic treatment of obesity. Although we used amlexanox as a model drug and anti-VCAM-1 antibody in VAPN, the concept of immune nanomodulators can be widely applied to the immunological treatment of obesity.

RNA Nanomedicine: Delivery Strategies and Applications.

Delivery of RNA using nanomaterials has emerged as a new modality to expand therapeutic applications in biomedical research. However, the delivery of RNA presents unique challenges due to its susceptibility to degradation and the requirement for efficient intracellular delivery. The integration of nanotechnologies with RNA delivery has addressed many of these challenges. In this review, we discuss different strategies employed in the design and development of nanomaterials for RNA delivery. We also highlight recent advances in the pharmaceutical applications of RNA delivered via nanomaterials. Various nanomaterials, such as lipids, polymers, peptides, nucleic acids, and inorganic nanomaterials, have been utilized for delivering functional RNAs, including messenger RNA (mRNA), small interfering RNA, single guide RNA, and microRNA. Furthermore, the utilization of nanomaterials has expanded the applications of functional RNA as active pharmaceutical ingredients. For instance, the delivery of antigen-encoding mRNA using nanomaterials enables the transient expression of vaccine antigens, leading to immunogenicity and prevention against infectious diseases. Additionally, nanomaterial-mediated RNA delivery has been investigated for engineering cells to express exogenous functional proteins. Nanomaterials have also been employed for co-delivering single guide RNA and mRNA to facilitate gene editing of genetic diseases. Apart from the progress made in RNA medicine, we discuss the current challenges and future directions in this field.

Enzyme-responsive macrocyclic metal complexes for biomedical imaging.

Metal chelator-based contrast agents are used as tumor navigators for cancer diagnosis. Although approved metal chelators show excellent contrast performance in magnetic resonance imaging (MRI), large doses are required for cancer diagnoses due to rapid clearance and nonspecific accumulation throughout the body, which can compromise safety. The present study describes an enzyme-responsive metal delivery system, in which enzyme overexpressed in the tumor microenvironment selectively activates the tumor uptake of gadolinium (Gd). Gd was loaded into enzyme-responsive macrocyclam (ErMC) modified with a PEGylated enzyme-cleavable peptide resulting in Gd@ErMC. The PEGylated shell layer protected Gd@ErMC from nonspecific binding in the blood, increasing the half-life of the contrast agent. Specific cleavage of the PEGylated shell layer by the enzyme selectively liberated Gd from Gd@ErMC at the tumor site. Evaluation of the in vivo distribution of Gd@ErMC in tumor-bearing mice by MRI and positron emission tomography (PET) showed that Gd@ErMC had an extended half-life and was highly specific. Histological and serological analysis of Gd@ErMC-treated mice showed that this agent was safe. This novel enzyme-responsive contrast agent delivery system shows promise as specific theranostic agent for MR-guided radiotherapy.

Lysyl oxidase-responsive anchoring nanoparticles for modulation of the tumor immune microenvironment.

In the tumor microenvironment, lysyl oxidase (LOX) is known to play a key role in stabilizing the tumor extracellular matrix. Here, we designed LOX-responsive nanoparticles to interact with the collagen matrix of the tumor microenvironment. Collagen-coated and imiquimod-loaded polydopamine nanoparticles (CPN/IQ) could form crosslinked structures with the collagen matrix via LOX. In vitro, anchoring of CPN/IQ nanoparticles was observed with LOX-secreting CT26 cells, but this was blocked by a LOX inhibitor. In CT26 tumor-bearing mice, co-administration of nanoparticles plus the LOX inhibitor did not significantly alter the antitumor efficacy among nanoparticles. In the absence of the LOX inhibitor, however, a single administration of CPN/IQ could provide sustained responsiveness to near-infrared irradiation and ablation of primary tumors. In the primary tumor microenvironment, CPN/IQ lowered the Treg cell population but increased the cytotoxic CD3+CD8+ T cell population. In splenic dendritic cells, CPN/IQ treatment significantly increased the CD11c+CD86+ and CD11c+CD80+ cell populations. In a CT26 distant tumor-rechallenge model, CPN/IQ treatment increased the cytotoxic CD3+CD8+ T cell population and provided 100% survival of mice until 64 days. This study indicates the feasibility of tumor immune microenvironment modulation using LOX-responsive size-transforming nanoparticles. Although we tested the concept in a CT26 cell-derived tumor model, the concept of LOX-responsive collagen matrix- anchoring nanoparticles may be broadly applied to other tumor tissues with LOX-rich tumor microenvironments.

External cold atmospheric plasma-responsive on-site hydrogel for remodeling tumor immune microenvironment.

Although immunotherapy has recently emerged as a promising anti-tumor approach, it remains limited by the immunosuppressive tumor microenvironment. Cold atmospheric plasma irradiation can generate reactive oxygen species and trigger the presentation of tumor-associated antigens. Here, we exploited cold atmospheric plasma for on-site hydrogel application in the tumor environment, aiming to facilitate the sustainable uptake of tumor-associated antigens and nanoadjuvants by dendritic cells. Hyaluronic acid-tyramine conjugate was intratumorally injected as a liquid and formed an on-site hydrogel under irradiation with cold atmospheric plasma. Intratumoral delivery of hyaluronic acid-tyramine conjugate with transforming growth factor ß-blocking nanoadjuvant (TLN) followed by cold atmospheric plasma irradiation yielded a micro-network of TLN-loaded hydrogel (TLN@CHG). In vivo intratumoral injection of TLN@CHG promoted the activation of dendritic cells and more effectively increased the proportion of CD4 T cells and CD8 T cells in the tumor microenvironment, compared to the groups receiving TLN or hydrogel alone. Moreover, in CT26 tumor model mice, cold atmospheric plasma-induced TLN@CHG therapy ablated the primary tumor and provided 100% survival among mice rechallenged with CT26 cells. Taken together, our findings suggest that an on-site hydrogel-based micro-network of TLN has the potential to remodel the tumor immune microenvironment. Although we used TLN in this study, the concept could be extended to support the sustained action of other nanoadjuvants in a hydrogel micro-network.

Nanomaterials for antigen-specific immune tolerance therapy.

Impairment of immune tolerance might cause autologous tissue damage or overactive immune response against non-pathogenic molecules. Although autoimmune disease and allergy have complicated pathologies, the current strategies have mainly focused on symptom amelioration or systemic immunosuppression which can lead to fatal adverse events. The induction of antigen-specific immune tolerance may provide therapeutic benefits to autoimmune disease and allergic response, while reducing nonspecific immune adverse responses. Diverse nanomaterials have been studied to induce antigen-specific immune tolerance therapy. This review will cover the immunological background of antigen-specific tolerance, clinical importance of antigen-specific immune tolerance, and nanomaterials designed for autoimmune and allergic diseases. As nanomaterials for modulating immune tolerances, lipid-based nanoparticles, polymeric nanoparticles, and biological carriers have been covered. Strategies to provide antigen-specific immune tolerance have been addressed. Finally, current challenges and perspectives of nanomaterials for antigen-specific immune tolerance therapy will be discussed.

Tolerogenic Nanovaccine for Prevention and Treatment of Autoimmune Encephalomyelitis.

Herein, a tolerogenic nanovaccine is developed and tested on an animal model of multiple sclerosis. The nanovaccine is constructed to deliver the self-antigen, myelin oligodendrocyte glycoprotein (MOG) peptide, and dexamethasone on an abatacept-modified polydopamine core nanoparticle (AbaLDPN-MOG). AbaLDPN-MOG can target dendritic cells and undergo endocytosis followed by trafficking to lysosomes. AbaLDPN-MOG blocks the interaction between CD80/CD86 and CD28 in antigen-presenting cells and T cells, leading to decreased interferon gamma secretion. The subcutaneous administration of AbaLDPN-MOG to mice yields significant biodistribution to lymph nodes and, in experimental-autoimmune encephalomyelitis (EAE) model mice, increases the integrity of the myelin basic sheath and minimizes the infiltration of immune cells. EAE mice are treated with AbaLDPN-MOG before or after injection of the autoantigen, MOG. Preimmunization of AbaLDPN-MOG before the injection of MOG completely blocks the development of clinical symptoms. Early treatment with AbaLDPN-MOG at three days after injection of MOG also completely blocks the development of symptoms. Notably, treatment of EAE symptom-developed mice with AbaLDPN-MOG significantly alleviates the symptoms, indicating that the nanovaccine has therapeutic effects. Although AbaLDPN is used for MOG peptide delivery in the EAE model, the concept of AbaLDPN can be widely applied for the prevention and alleviation of other autoimmune diseases.

Lipid nanoparticle-mediated CRISPR/Cas9 gene editing and metabolic engineering for anticancer immunotherapy.

Metabolic engineering of the tumor microenvironment has emerged as a new strategy. Lactate dehydrogenase A (LDHA) is a prominent target for metabolic engineering. Here, we designed a cationic lipid nanoparticle formulation for LDHA gene editing. The plasmid DNA delivery efficiency of our lipid nanoparticle formulations was screened by testing the fluorescence of lipid nanoparticles complexed to plasmid DNA encoding green fluorescence protein (GFP). The delivery efficiency was affected by the ratios of three components: a cationic lipid, cholesterol or its derivative, and a fusogenic lipid. The lipid nanoparticle designated formulation F3 was complexed to plasmid DNA co-encoding CRISPR-associated protein 9 and LDHA-specific sgRNA, yielding the lipoplex, pCas9-sgLDHA/F3. The lipoplex including GFP-encoding plasmid DNA provided gene editing in HeLa-GFP cells. Treatment of B16F10 tumor cells with pCas9-sgLDHA/F3 yielded editing of the LDHA gene and increased the pH of the culture medium. pCas9-sgLDHA/F3 treatment activated the interferon-gamma and granzyme production of T cells in culture. In vivo, combining pCas9-sgLDHA/F3 with immune checkpoint-inhibiting anti-PD-L1 antibody provided a synergistic antitumor effect and prolonged the survival of tumor model mice. This study suggests that combining metabolic engineering of the tumor microenvironment with immune checkpoint inhibition could be a valuable antitumor strategy.

Blood-declustering excretable metal clusters assembled in DNA matrix.

We report polymeric DNA-supported gold clusters that achieve interparticle plasmon-coupling, generate immunotherapeutic effects at the tumor tissue, but decluster in the bloodstream. As immunostimulating DNA, we used polyCpG DNA, which could act as a supporting matrix for metal clusters, enabling the clusters to decluster in the bloodstream. We constructed polyCpG-supported gold nanoclusters (AuPCN). For comparison with AuPCN, monomer CpG-bound gold nanoparticles (AuMC) were used. Unlike AuMC, AuPCN showed an interparticle plasmon-coupling effect and a higher light-to heat conversion efficiency. In the serum, AuPCN declustered to subunits. The CT26 tumor rechallenge of mice pretreated with AuPCN(+NIR) was followed by 0% tumor recurrence and 100% survival for up to 80 days. Compared with other groups, AuPCN(+NIR)-treated mice revealed greater cytotoxic T cell-infiltration in distant tumors and higher memory T cells in the lymph nodes. Until 7 days post-dose, the urinary excretion of Au was observed in the AuPCN-treated group, but not in the Au nanoparticle-treated mice. Although we used gold clusters and concatemeric immunostimulatory CpG as components of AuPCN, the concept of declustering in the bloodstream can be applied to design other functional DNA scaffold-based metal clusters with reduced concerns for long-term retention in the body.

Sequential Nano-Penetrators of Capillarized Liver Sinusoids and Extracellular Matrix Barriers for Liver Fibrosis Therapy.

During liver fibrogenesis, liver sinusoidal capillarization and extracellular matrix (ECM) deposition construct dual pathological barriers to drug delivery. Upon capillarization, the vanished fenestrae in liver sinusoidal endothelial cells (LSECs) significantly hinder substance exchange between blood and liver cells, while excessive ECM further hinders the delivery of nanocarriers to activated hepatic stellate cells (HSCs). Herein, an efficient nanodrug delivery system was constructed to sequentially break through the capillarized LSEC barrier and the deposited ECM barrier. For the first barrier, LSEC-targeting and fenestrae-repairing nanoparticles (named HA-NPs/SMV) were designed on the basis of the modification with hyaluronic acid and the loading of simvastatin (SMV). For the second barrier, collagenase I and vitamin A codecorated nanoparticles with collagen-ablating and HSC-targeting functions (named CV-NPs/siCol1α1) were prepared to deliver siCol1α1 with the goal of inhibiting collagen generation and HSC activation. Our in vivo results showed that upon encountering the capillarized LSEC barrier, HA-NPs/SMV rapidly released SMV and exerted a fenestrae-repairing function, which allowed more CV-NPs/siCol1α1 to enter the space of Disse to degrade deposited collagen and finally to achieve higher accumulation in activated HSCs. Scanning electronic microscopy images showed the recovery of liver sinusoids, and analysis of liver tissue sections demonstrated that HA-NPs/SMV and CV-NPs/siCol1α1 had a synergetic effect. Our pathological barrier-normalization strategy provides an antifibrotic therapeutic regimen.

DNA-cloaked nanoparticles for tumor microenvironment-responsive activation.

Although progress has been made in developing tumor microenvironment-responsive delivery systems, the list of cargo-releasing stimuli remains limited. In this study, we report DNA nanothread-cloaked nanoparticles for reactive oxygen species (ROS)-rich tumor microenvironment-responsive delivery systems. ROS is well known to strongly induce DNA fragmentation via oxidative stress. As a model anticancer drug, hydrophobic omacetaxine was entrapped in branched cyclam ligand-modified nanoparticles (BNP). DNA nanothreads were prepared by rolling-circle amplification and complexed to BNP, yielding DNA nanothread-cloaked BNP (DBNP). DBNP was unmasked by DNA nanothread-degrading ROS and culture supernatants of LNCaP cells. The size and zeta potential of DBNP were changed by ROS. In ROShigh LNCaP cells, but not in ROSlow fibroblast cells, the uptake of DBNP was higher than that of other nanoparticles. Molecular imaging revealed that DBNP exhibited greater distribution to tumor tissues, compared to other nanoparticles. Ex vivo mass spectrometry-based imaging showed that omacetaxine metabolites were distributed in tumor tissues of mice treated with DBNP. Intravenous administration of DBNP reduced the tumor volume by 80% compared to untreated tumors. Profiling showed that omacetaxine treatment altered the transcriptional profile. These results collectively support the feasibility of using polymerized DNA-masked nanoparticles for selective activation in the ROS-rich tumor microenvironment.

On-demand delivery of protein drug from 3D-printed implants.

Here, we constructed 3D-printed multiunit implants to enable remote light-controlled protein drug delivery in a spatiotemporal manner. Multiunit implants were designed to be 3D printed using polycaprolactone, lauric acid, and melanin as a matrix, and a polycaprolactone scaffold as a multiunit divider. As a model drug, insulin was loaded to each unit of the implant. The 3D printing yielded a rectangular matrix with multiunit sectors segregated by polycaprolactone lanes. Irradiation with near infrared light (NIR) triggered controlled release of insulin from the irradiated locus: Upon NIR irradiation, heat generated from the melanin melted the polycaprolactone/lauric acid matrix to release insulin from the scaffold. In the absence of melanin in the matrix, the implant did not show NIR-responsive insulin release. When lauric acid was absent from the matrix, the NIR-irradiated unit did not undergo dismantling. When the insulin-loaded multiunit implant was applied to a mouse diabetic model and irradiated with NIR, repetitive insulin release resulted in an efficient decrease of the blood glucose level over multiple days. Together, these results suggest that 3D printing technology-based multi-dosing of insulin on demand can enable convenient treatment of diabetes through external NIR irradiation, potentially avoiding the pain and discomfort of repeated insulin injections.

In vivo fate and intracellular trafficking of vaccine delivery systems.

With the pandemic of severe acute respiratory syndrome coronavirus 2, vaccine delivery systems emerged as a core technology for global public health. Given that antigen processing takes place inside the cell, the intracellular delivery and trafficking of a vaccine antigen will contribute to vaccine efficiency. Investigations focusing on the in vivo behavior and intracellular transport of vaccines have improved our understanding of the mechanisms relevant to vaccine delivery systems and facilitated the design of novel potent vaccine platforms. In this review, we cover the intracellular trafficking and in vivo fate of vaccines administered via various routes and delivery systems. To improve immune responses, researchers have used various strategies to modulate vaccine platforms and intracellular trafficking. In addition to progress in vaccine trafficking studies, the challenges and future perspectives for designing next-generation vaccines are discussed.

DNA-based artificial dendritic cells for in situ cytotoxic T cell stimulation and immunotherapy.

In immunotherapy, ex vivo stimulation of T cells requires significant resources and effort. Here, we report artificial dendritic cell-mimicking DNA microflowers (DM) for programming T cell stimulation in situ. To mimic dendritic cells, DNA-based artificial dendritic microflowers were constructed, surface-coated with polydopamine, and further modified with anti-CD3 and anti-CD28 antibodies to yield antibody-modified DM (DM-A). The porous structure of DM-A allowed entrapment of the T cell-stimulating cytokine, ineterleukin-2, yielding interleukin-2-loaded DM-A (DM-AI). For comparison, polystyrene microparticles coated with polydopamine and modified with anti-CD3 and anti-CD28 antibodies (PS-A) were used. Compared to PS-A, DM-AI showed significantly greater contact with T cell surfaces. DM-AI provided the highest ex vivo expansion of cytotoxic T cells. Local injection of DM-AI to tumor tissues induced the recruitment of T cells and expansion of cytotoxic T cells in tumor microenvironments. Unlike the other groups, model animals injected with DM-AI did not exhibit growth of primary tumors. Treatment of mice with DM-AI also protected against growth of a rechallenged distant tumor, and thus prevented tumor recurrence in this model. DM-AI has great potential for programmed stimulation of CD8+ T cells. This concept could be broadly extended for the programming of specific T cell stimulation profiles.

Fibroblast activation protein activated antifibrotic peptide delivery attenuates fibrosis in mouse models of liver fibrosis.

In liver fibrosis, activated hepatic stellate cells are known to overexpress fibroblast activation protein. Here we report a targeted antifibrotic peptide-delivery system in which fibroblast activation protein, which is overexpressed in fibrotic regions of the liver, liberates the antifibrotic peptide melittin by cleaving a fibroblast activation protein-specific site in the peptide. The promelittin peptide is linked to pegylated and maleimide-functionalized liposomes, resulting in promelittin-modified liposomes. The promelittin-modified liposomes were effective in reducing the viability of activated hepatic stellate cells but not that of control cells. In three types of liver fibrosis mouse models, intravenously administered promelittin-modified liposomes significantly reduces fibrotic regions. In addition, in the bile duct ligation mouse model promelittin-modified liposome-treatment increases overall survival. Although this peptide-delivery concept was tested for liver fibrosis, it can potentially be adapted to other fibrotic diseases.

Nanotherapeutics for immune network modulation in tumor microenvironments.

Immunotherapy has shown promise in cancer treatment, and is thus drawing increasing interest in this field. While the standard chemotherapy- and/or radiotherapy-based cancer treatments aim to directly kill cancer cells, immunotherapy uses host immune cell surveillance to fight cancer. In the tumor environment, there is a close relationship between tumor cells and the adjacent immune cells, which are largely suppressed by cancer-related regulation of immune checkpoints, immune-suppressive cytokines, and metabolic factors. The immune modulators currently approved for cancer treatment remain limited by issues with dose tolerance and insufficient efficacy. Researchers have developed and tested various nano-delivery systems with the goal of improving the treatment outcome of these drugs. By encapsulating immune modulators in particles and directing their tissue accumulation, some such systems have decreased immune-related toxicity while sharpening the antitumor response. Surface-ligand modification of nanoparticles has allowed drugs to be delivered to specific immune cells types. Researchers have also studied strategies for depleting or reprogramming the immune-suppressive cells to recover the immune environment. Combining a nanomaterial with an external stimulus has been used to induce immunogenic cell death; this favors the inflammatory environment found in tumor tissues to promote antitumor immunity. The present review covers the most recent strategies aimed at modulating the tumor immune environment, and discusses the challenges and future perspectives in developing nanoparticles for cancer immunotherapy.

In vivo therapeutic genome editing via CRISPR/Cas9 magnetoplexes for myocardial infarction.

CRISPR/Cas9-mediated gene-editing technology has gained attention as a new therapeutic method for intractable diseases. However, the use of CRISPR/Cas9 for cardiac conditions such as myocardial infarction remains challenging due to technical and biological barriers, particularly difficulties in delivering the system and targeting genes in the heart. In the present study, we demonstrated the in vivo efficacy of the CRISPR/Cas9 magnetoplexes system for therapeutic genome editing in myocardial infarction. First, we developed CRISPR/Cas9 magnetoplexes that magnetically guided CRISPR/Cas9 system to the heart for efficient in vivo therapeutic gene targeting during heart failures. We then demonstrated that the in vivo gene targeting of miR34a via these CRISPR/Cas9 magnetoplexes in a mouse model of myocardial infarction significantly improved cardiac repair and regeneration to facilitate improvements in cardiac function. These results indicated that CRISPR/Cas9 magnetoplexes represent an effective in vivo therapeutic gene-targeting platform in the myocardial infarction of heart, and that this strategy may be applicable for the treatment of a broad range of cardiac failures.