Simvastatin-Loaded Polymeric Nanoparticles: Targeting Inflammatory Macrophages for Local Adipose Tissue Browning in Obesity Treatment.

Obesity is defined as chronic, low-grade inflammation within specific tissues. Given the escalating prevalence of obesity among individuals of all ages, obesity has reached epidemic proportions, posing an important public health challenge. Despite significant advancements in treating obesity, conventional approaches remain largely ineffective or involve severe side effects, thus underscoring the pressing need to explore and develop treatment approaches. Targeted and local immunomodulation using nanoparticles (NPs) can influence fat production and utilization processes. Statins, known for their anti-inflammatory properties, show the potential for mitigating obesity-related inflammation. A localized delivery option offers several advantages over oral and parenteral delivery methods. Here, we developed simvastatin (Sim) encapsulated within PLGA NPs (Sim-NP) for localized delivery of Sim to adipose tissues (ATs) for immunomodulation to treat obesity. In vitro experiments revealed the strong anti-inflammatory effects of Sim-NPs, which resulted in enhanced modulation of macrophage (MΦ) polarization and induction of AT browning. We then extended our investigation to an in vivo mouse model of high-fat-diet (HFD)-induced obesity. Sim-NP administration led to the controlled release of Sim within AT, directly impacting MΦ activity and inducing AT browning while inducing weight loss. Our findings demonstrated that Sim-NP administration effectively inhibited the progression of obesity-related inflammation, controlled white fat production, and enhanced AT modulation. These results highlight the potential of Sim-NP as a potent nanotherapy for treating obesity by modulating the immune system.

Chaos-Assisted Production of Micro-Architected Spheres (CAPAS).

Hydrogel droplets with inner compartments are valuable in various fields, including tissue engineering. A droplet-based biofabrication method is presented for the chaos-assisted production of architected spheres (CAPAS) for the rapid generation of multilayered hydrogel spheres (ranging from 0.6 to 3.5 mm in diameter) at high-throughput rates (up to 2000 spheres per min). This method is based on the use of chaotic advection generated by a Kenics static mixer (KSM) nozzle. The configuration of the KSM (i.e., the number of mixing elements) determines the number of compartments within the sphere. Sphere size is adjusted by flow rate, printhead outlet diameter, polymer concentration (sodium alginate or gelatin-methacryloyl (GelMA)), and crosslinking bath composition. This versatile system operates in dripping and jetting modes, preserving multilayered architecture in both modes. Proof-of-concept experiments with breast cancer (MDA-MB-231), human dermal fibroblast (HDF), and murine myoblast (C2C12) lines show over 80% cell viability immediately post-fabrication, maintained over extended culture (14 or 30 days). CAPAS is used to create a breast cancer model with cancer-tissue-like and healthy-tissue-like micro-niches to test paclitaxel doses. It is envisioned that CAPAS will enable high-throughput fabrication of hydrogel spheres for tissue engineering, chemical engineering, and material sciences applications.

Dissolvable Immunomodulatory Microneedles for Treatment of Skin Wounds.

Sustained inflammation can halt or delay wound healing, and macrophages play a central role in wound healing. Inflammatory macrophages are responsible for the removal of pathogens, debris, and neutrophils, while anti-inflammatory macrophages stimulate various regenerative processes. Recombinant human Proteoglycan 4 (rhPRG4) is shown to modulate macrophage polarization and to prevent fibrosis and scarring in ear wound healing. Here, dissolvable microneedle arrays (MNAs) carrying rhPRG4 are engineered for the treatment of skin wounds. The in vitro experiments suggest that rhPRG4 modulates the inflammatory function of bone marrow-derived macrophages. Degradable and detachable microneedles are developed from gelatin methacryloyl (GelMA) attach to a dissolvable gelatin backing. The developed MNAs are able to deliver a high dose of rhPRG4 through the dissolution of the gelatin backing post-injury, while the GelMA microneedles sustain rhPRG4 bioavailability over the course of treatment. In vivo results in a murine model of full-thickness wounds with impaired healing confirm a decrease in inflammatory biomarkers such as TNF-α and IL-6, and an increase in angiogenesis and collagen deposition. Collectively, these results demonstrate rhPRG4-incorporating MNA is a promising platform in skin wound healing applications.

Injectable hydrogels for personalized cancer immunotherapies.

The field of cancer immunotherapy has shown significant growth, and researchers are now focusing on effective strategies to enhance and prolong local immunomodulation. Injectable hydrogels (IHs) have emerged as versatile platforms for encapsulating and controlling the release of small molecules and cells, drawing significant attention for their potential to enhance antitumor immune responses while inhibiting metastasis and recurrence. IHs delivering natural killer (NK) cells, T cells, and antigen-presenting cells (APCs) offer a viable method for treating cancer. Indeed, it can bypass the extracellular matrix and gradually release small molecules or cells into the tumor microenvironment, thereby boosting immune responses against cancer cells. This review provides an overview of the recent advancements in cancer immunotherapy using IHs for delivering NK cells, T cells, APCs, chemoimmunotherapy, radio-immunotherapy, and photothermal-immunotherapy. First, we introduce IHs as a delivery matrix, then summarize their applications for the local delivery of small molecules and immune cells to elicit robust anticancer immune responses. Additionally, we discuss recent progress in IHs systems used for local combination therapy, including chemoimmunotherapy, radio-immunotherapy, photothermal-immunotherapy, photodynamic-immunotherapy, and gene-immunotherapy. By comprehensively examining the utilization of IHs in cancer immunotherapy, this review aims to highlight the potential of IHs as effective carriers for immunotherapy delivery, facilitating the development of innovative strategies for cancer treatment. In addition, we demonstrate that using hydrogel-based platforms for the targeted delivery of immune cells, such as NK cells, T cells, and dendritic cells (DCs), has remarkable potential in cancer therapy. These innovative approaches have yielded substantial reductions in tumor growth, showcasing the ability of hydrogels to enhance the efficacy of immune-based treatments. STATEMENT OF SIGNIFICANCE: As cancer immunotherapy continues to expand, the mode of therapeutic agent delivery becomes increasingly critical. This review spotlights the forward-looking progress of IHs, emphasizing their potential to revolutionize localized immunotherapy delivery. By efficiently encapsulating and controlling the release of essential immune components such as T cells, NK cells, APCs, and various therapeutic agents, IHs offer a pioneering pathway to amplify immune reactions, moderate metastasis, and reduce recurrence. Their adaptability further shines when considering their role in emerging combination therapies, including chemoimmunotherapy, radio-immunotherapy, and photothermal-immunotherapy. Understanding IHs' significance in cancer therapy is essential, suggesting a shift in cancer treatment dynamics and heralding a novel period of focused, enduring, and powerful therapeutic strategies.

Cancer Immunotherapy via Targeting Cancer Stem Cells Using Vaccine Nanodiscs.

Cancer stem cells (CSCs) proliferate extensively and drive tumor metastasis and recurrence. CSCs have been identified in over 20 cancer types to date, but it remains unknown how to target and eliminate CSCs in vivo. Aldehyde dehydrogenase (ALDH) is a marker that has been used extensively for isolating CSCs. Here we present a novel approach to target and reduce the frequency of ALDHhigh CSCs by vaccination against ALDH. We have identified ALDH1-A1 and ALDH1-A3 epitopes from CSCs and developed synthetic high-density lipoprotein nanodiscs for vaccination against ALDHhigh CSCs. Nanodiscs increased antigen trafficking to lymph nodes and generated robust ALDH-specific T cell responses. Nanodisc vaccination against ALDHhigh CSCs combined with anti-PD-L1 therapy exerted potent antitumor efficacy and prolonged animal survival in multiple murine models. Overall, this is the first demonstration of a simple nanovaccine strategy against CSCs and may lead to new avenues for cancer immunotherapy against CSCs.

Synthetic High-density Lipoprotein Nanodiscs for Personalized Immunotherapy Against Gliomas.

PURPOSE: Gliomas are brain tumors with dismal prognoses. The standard-of-care treatments for gliomas include surgical resection, radiation, and temozolomide administration; however, they have been ineffective in providing significant increases in median survival. Antigen-specific cancer vaccines and immune checkpoint blockade may provide promising immunotherapeutic approaches for gliomas. EXPERIMENTAL DESIGN: We have developed immunotherapy delivery vehicles based on synthetic high-density lipoprotein (sHDL) loaded with CpG, a Toll-like receptor-9 agonist, and tumor-specific neoantigens to target gliomas and elicit immune-mediated tumor regression. RESULTS: We demonstrate that vaccination with neoantigen peptide-sHDL/CpG cocktail in combination with anti-PD-L1 immune checkpoint blocker elicits robust neoantigen-specific T-cell responses against GL261 cells and eliminated established orthotopic GL261 glioma in 33% of mice. Mice remained tumor free upon tumor cell rechallenge in the contralateral hemisphere, indicating the development of immunologic memory. Moreover, in a genetically engineered murine model of orthotopic mutant IDH1 (mIDH1) glioma, sHDL vaccination with mIDH1 neoantigen eliminated glioma in 30% of animals and significantly extended the animal survival, demonstrating the versatility of our approach in multiple glioma models. CONCLUSIONS: Overall, our strategy provides a general roadmap for combination immunotherapy against gliomas and other cancer types.

Self-Antigens Displayed on Liposomal Nanoparticles above a Threshold of Epitope Density Elicit Class-Switched Autoreactive Antibodies Independent of T Cell Help.

Epitope density has a profound impact on B cell responses to particulate Ags, the molecular mechanisms of which remain to be explored. To dissect the role of epitope density in this process, we have synthesized a series of liposomal particles, similar to the size of viruses, that display a model self-antigen peptide at defined surface densities. Immunization of C57BL/6J mice using these particles elicited both IgM and class-switched IgG1, IgG2b, and IgG3 autoreactive Abs that depended on the epitope density. In C57BL/6 gene knockout mice lacking either functional TCRs or MHC class II molecules on B cells, the liposomal particles also elicited IgM, IgG1, IgG2b, and IgG3 responses that were comparable in magnitudes to wild-type mice, suggesting that this B cell response was independent of cognate T cell help. Notably, the titer of the IgG in wild-type animals could be increased by more than 200-fold upon replacement of liposomes with bacteriophage Qß virus-like particles that displayed the same self-antigen peptide at comparable epitope densities. This enhancement was lost almost completely in gene knockout mice lacking either TCRs or MHC class II molecules on B cells. In conclusion, epitope density above a threshold on particulate Ags can serve as a stand-alone signal to trigger secretion of autoreactive and class-switched IgG in vivo in the absence of cognate T cell help or any adjuvants. The extraordinary immunogenicity of Qß viral-like particles relies, in large part, on their ability to effectively recruit T cell help after B cell activation.

Dual TLR agonist nanodiscs as a strong adjuvant system for vaccines and immunotherapy.

Recent studies have shown that certain combinations of Toll-like receptor (TLR) agonists can induce synergistic immune activation. However, it remains challenging to achieve such robust responses in vivo in a manner that is effective, facile, and amenable for clinical translation. Here, we show that MPLA, a TLR4 agonist, and CpG, a TLR9 agonist, can be efficiently co-loaded into synthetic high-density lipoprotein nanodiscs, forming a potent adjuvant system (ND-MPLA/CpG) that can be readily combined with a variety of subunit antigens, including proteins and peptides. ND-MPLA/CpG significantly enhanced activation of dendritic cells, compared with free dual adjuvants or nanodiscs delivering a single TLR agonist. Importantly, mice immunized with physical mixtures of protein antigens ND-MPLA/CpG generated strong humoral responses, including induction of IgG responses against protein convertase subtilisin/kexin 9 (PCSK9), leading to 17-30% reduction of the total plasma cholesterol levels. Moreover, ND-MPLA/CpG exerted strong anti-tumor efficacy in multiple murine tumor models. Compared with free adjuvants, ND-MPLA/CpG admixed with ovalbumin markedly improved antigen-specific CD8+ T cell responses by 8-fold and promoted regression of B16F10-OVA melanoma (P < 0.0001). Furthermore, ND-MPLA/CpG admixed with E7 peptide antigen elicited ~20% E7-specific CD8+ T cell responses and achieved complete regression of established TC-1 tumors in all treated animals. Taken together, our work highlights the simplicity, versatility, and potency of dual TLR agonist nanodiscs for applications in vaccines and cancer immunotherapy.