Engineered probiotics with sustained release of interleukin-2 for the treatment of inflammatory bowel disease after oral delivery.

Inflammatory bowel disease (IBD) is a kind of auto-immune disease characterized by disrupted intestinal barrier and mucosal epithelium, imbalanced gut microbiome and deregulated immune responses. Therefore, the restoration of immune equilibrium and gut microbiota could potentially serve as a hopeful approach for treating IBD. Herein, the oral probiotic Escherichia coli Nissle 1917 (ECN) was genetically engineered to express secretable interleukin-2 (IL-2), a kind of immunomodulatory agent, for the treatment of IBD. In our design, probiotic itself has the ability to regulate the gut microenvironment and IL-2 at low dose could selectively promote the generation of regulatory T cells to elicit tolerogenic immune responses. To improve the bioavailability of ECN expressing IL-2 (ECN-IL2) in the gastrointestinal tract, enteric coating Eudragit L100-55 was used to coat ECN-IL2, achieving significantly enhanced accumulation of engineered probiotics in the intestine. More importantly, L100-55 coated ECN-IL2 could effectively activated Treg cells to regulate innate immune responses and gut microbiota, thereby relieve inflammation and repair the colon epithelial barrier in dextran sodium sulfate (DSS) induced IBD. Therefore, genetically and chemically modified probiotics with excellent biocompatibility and efficiency in regulating intestinal microflora and intestinal inflammation show great potential for IBD treatment in the future.

Non-invasive transdermal delivery of biomacromolecules with fluorocarbon-modified chitosan for melanoma immunotherapy and viral vaccines.

Transdermal drug delivery has been regarded as an alternative to oral delivery and subcutaneous injection. However, needleless transdermal delivery of biomacromolecules remains a challenge. Herein, a transdermal delivery platform based on biocompatible fluorocarbon modified chitosan (FCS) is developed to achieve highly efficient non-invasive delivery of biomacromolecules including antibodies and antigens. The formed nanocomplexes exhibits effective transdermal penetration ability via both intercellular and transappendageal routes. Non-invasive transdermal delivery of immune checkpoint blockade antibodies induces stronger immune responses for melanoma in female mice and reduces systemic toxicity compared to intravenous injection. Moreover, transdermal delivery of a SARS-CoV-2 vaccine in female mice results in comparable humoral immunity as well as improved cellular immunity and immune memory compared to that achieved with subcutaneous vaccine injection. Additionally, FCS-based protein delivery systems demonstrate transdermal ability for rabbit and porcine skins. Thus, FCS-based transdermal delivery systems may provide a compelling opportunity to overcome the skin barrier for efficient transdermal delivery of bio-therapeutics.

Minimalist Nanovaccine with Optimized Amphiphilic Copolymers for Cancer Immunotherapy.

Cancer vaccines with the ability to elicit tumor-specific immune responses have attracted significant interest in cancer immunotherapy. A key challenge for effective cancer vaccines is the spatiotemporal codelivery of antigens and adjuvants. Herein, we synthesized a copolymer library containing nine poly(ethylene glycol) methyl ether methacrylate-co-butyl methacrylate-co-2-(azepan-1-yl)ethyl methacrylate (PEGMA-co-BMA-co-C7AMA) graft copolymers with designed proportions of different components to regulate their properties. Among these polymers, C-25, with a C7AMA:BMA ratio at 1.5:1 and PEG wt % of 25%, was screened as the most effective nanovaccine carrier with enhanced ability to induce mouse bone marrow-derived dendritic cell (BMDC) maturation. Additionally, RNA-sequencing (RNA-Seq) analysis revealed that C-25 could activate dendritic cells (DCs) through multisignaling pathways to trigger potent immune effects. Then, the screened C-25 was used to encapsulate the model peptide antigen, OVA257-280, to form nanovaccine C-25/OVA257-280. It was found that the C-25/OVA257-280 nanovaccine could effectively facilitate DC maturation and antigen cross-presentation without any other additional adjuvant and exhibited excellent prophylactic efficacy in the B16F10-OVA tumor model. Moreover, in combination with antiprogrammed cell death protein-ligand 1 (anti-PD-L1), the C-25/OVA257-280 nanovaccine could significantly delay the growth of pre-existing tumors. Therefore, this work developed a minimalist nanovaccine with a simple formulation and high efficiency in activating tumor-specific immune responses, showing great potential for further application in cancer immunotherapy.

Transmucosal Delivery of Nasal Nanovaccines Enhancing Mucosal and Systemic Immunity.

Intranasal vaccines can induce protective immune responses at the mucosa surface entrance, preventing the invasion of respiratory pathogens. However, the nasal barrier remains a major challenge in the development of intranasal vaccines. Herein, a transmucosal nanovaccine based on cationic fluorocarbon modified chitosan (FCS) is developed to induce mucosal immunity. In our system, FCS can self-assemble with the model antigen ovalbumin and TLR9 agonist CpG, effectively promoting the maturation and cross-presentation of dendritic cells. More importantly, it can enhance the production of secretory immunoglobin A (sIgA) at mucosal surfaces for those intranasally vaccinated mice, which in the meantime showed effective production of immunoglobulin G (IgG) systemically. As a proof-of-concept study, such a mucosal vaccine inhibits ovalbumin-expressing B16-OVA melanoma, especially its lung metastases. Our work presents a unique intranasal delivery system to deliver antigen across mucosal epithelia and promote mucosal and systemic immunity.

Antibacterial Fusobacterium nucleatum-Mimicking Nanomedicine to Selectively Eliminate Tumor-Colonized Bacteria and Enhance Immunotherapy Against Colorectal Cancer.

Clinical evidence indicates that tumor-colonizing bacteria can be closely related to the tumor development and therapeutic responses. Selectively eliminating bacteria within tumors may be an attractive approach to enhance cancer treatment without additional side effects. Herein, it is found that, owing to the high affinity between the membrane protein Fap-2 on Fusobacterium nucleatum and d-galactose-ß (1-3)-N-acetyl-d-galactosamine (Gal-GalNAc) overexpressed on colorectal tumor cells, F. nucleatum can colonize in colorectal tumors, as evidenced by both clinical samples and animal tumor models. Notably, F. nucleatum colonized in colorectal tumors can lead to an immunosuppressive tumor microenvironment, greatly reducing their responses to immune checkpoint blockade (ICB) therapy. Inspired by this finding, an F. nucleatum-mimetic nanomedicine is designed by fusing F. nucleatum cytoplasmic membrane (FM) with Colistin-loaded liposomes to achieve selective killing of tumor-colonizing F. nucleatum without affecting gut microbes. As a result, the therapeutic responses of F. nucleatum-colonized tumors to ICB therapies can be successfully restored, as demonstrated in an F. nucleatum-infected subcutaneous CT-26 tumor model, chemically induced spontaneous colorectal cancer models, and MC-38 tumor model. In summary, this work presents an F. nucleatum-mimicking nanomedicine that can selectively eliminate tumor-colonized bacteria, which is promising for enhancing the responses of cancer immunotherapy against F. nucleatum-colonized colorectal cancer.

Inhaled Lipid Nanoparticles Alleviate Established Pulmonary Fibrosis.

Pulmonary fibrosis, a sequela of lung injury resulting from severe infection such as severe acute respiratory syndrome-like coronavirus (SARS-CoV-2) infection, is a kind of life-threatening lung disease with limited therapeutic options. Herein, inhalable liposomes encapsulating metformin, a first-line antidiabetic drug that has been reported to effectively reverse pulmonary fibrosis by modulating multiple metabolic pathways, and nintedanib, a well-known antifibrotic drug that has been widely used in the clinic, are developed for pulmonary fibrosis treatment. The composition of liposomes made of neutral, cationic or anionic lipids, and poly(ethylene glycol) (PEG) is optimized by evaluating their retention in the lung after inhalation. Neutral liposomes with suitable PEG shielding are found to be ideal delivery carriers for metformin and nintedanib with significantly prolonged retention in the lung. Moreover, repeated noninvasive aerosol inhalation delivery of metformin and nintedanib loaded liposomes can effectively diminish the development of fibrosis and improve pulmonary function in bleomycin-induced pulmonary fibrosis by promoting myofibroblast deactivation and apoptosis, inhibiting transforming growth factor 1 (TGFß1) action, suppressing collagen formation, and inducing lipogenic differentiation. Therefore, this work presents a versatile platform with promising clinical translation potential for the noninvasive inhalation delivery of drugs for respiratory disease treatment.

Oral Delivery of Therapeutic Antibodies with a Transmucosal Polymeric Carrier.

Therapeutic proteins are playing increasingly important roles in treating numerous types of diseases. However, oral administration of proteins, especially large ones (e.g., antibodies), remains a great challenge due to their difficulties in penetrating intestinal barriers. Herein, fluorocarbon-modified chitosan (FCS) is developed for efficient oral delivery of different therapeutic proteins, in particular large ones such as immune checkpoint blockade antibodies. In our design, therapeutic proteins are mixed with FCS to form nanoparticles, lyophilized with appropriate excipients, and then filled into enteric capsules for oral administration. It has been found that FCS could promote transmucosal delivery of its cargo protein via inducing transitory rearrangement of tight junction associated proteins between intestinal epithelial cells and subsequently release free proteins into blood circulation. It is shown that at a 5-fold dose oral delivery of anti-programmed cell death protein-1 (αPD1) or its combination with anti-cytotoxic T-lymphocyte antigen 4 (αCTLA4) using this method could achieve comparable antitumor therapeutic responses to that achieved by intravenous injection of corresponding free antibodies in various types of tumor models and, more excitingly, result in significantly reduced immune-related adverse events. Our work successfully demonstrates the enhanced oral delivery of antibody drugs to achieve systemic therapeutic responses and may revolutionize the future clinical usage of protein therapeutics.

Eyedrop-based macromolecular ophthalmic drug delivery for ocular fundus disease treatment.

Therapeutic antibodies are extensively used to treat fundus diseases by intravitreal injection, as eyedrop formulation has been rather challenging due to the presence of ocular barriers. Here, an innovative penetrating carrier was developed for antibody delivery in eyedrop formulations. We found that fluorocarbon-modified chitosan (FCS) would self-assemble with proteins to form nanocomplexes, which could effectively pass across the complicated ocular structure to reach the posterior eye segments in both mice and rabbits. In a choroidal melanoma-bearing mouse model, eyedrops containing FCS/anti-PDL1 could induce stronger antitumor immune responses than those triggered by intravenous injection of anti-PDL1. Moreover, in choroidal neovascularization-bearing mouse and rabbit models, FCS/anti-VEGFA eyedrops effectively inhibited vascular proliferation, achieving comparable therapeutic responses to those observed with intravitreal injection of anti-VEGFA. Our work presents an effective delivery carrier to treat fundus diseases using eyedrop of therapeutic proteins, which may enable at-home treatment of many eye diseases with great patient compliance.

Vitamin C supramolecular hydrogel for enhanced cancer immunotherapy.

Vitamin C (VitC) has shown great promise to promote cancer immunotherapy, however, its high hydrophilicity makes it quickly excreted, leading to limited therapeutic efficiency even with frequent high-dose administration. Herein, we provide a pioneering report about the employment of VitC amphiphile self-assembled nanofiber hydrogels for enhanced cancer immunotherapy. Specifically, driven by hydrogen bonding and hydrophobic interactions, the synthesized VitC amphiphile, consisting of a hydrophilic VitC headgroup and a hydrophobic alkyl chain, could self-assemble into an injectable nanofiber hydrogel with self-healing properties. The formed VitC hydrogel not only serves as a reservoir for VitC but also acts as an effective delivery platform for stimulator of interferon genes (STING) agonist-4 (SA). Interestingly, the VitC hydrogel itself exhibits antitumor effects by upregulating genes related to interferon (IFN) signaling, apoptotic signaling and viral recognition and defense. Moreover, the SA-encapsulated VitC hydrogel (SA@VitC hydrogel) synergistically activated the immune system to inhibit the progression of both local and abscopal tumors.

Supramolecular biomaterials for enhanced cancer immunotherapy.

Cancer immunotherapy has achieved promising clinical results. However, many limitations associated with current cancer immunotherapy still exist, including low response rates and severe adverse effects in patients. Engineering biomaterials for the delivery of immunotherapeutic reagents has been suggested to be an effective strategy to improve cancer immunotherapy. Among different biomaterials, supramolecular biomaterials with flexible and versatile structures and functions have exhibited unparalleled advantages in promoting cancer immunotherapy. In recent years, various supramolecular formulations have been extensively explored as immunotherapeutic delivery platforms due to their high cargo-loading capacity/feasibility, facile immunization function, and excellent biocompatibility, which make them possible candidates for modular and personalized cancer immunotherapy. These nanoarchitectures with unique topologies possess distinguishing advantages in cancer immunotherapy, incarnating a structure-property relationship. Based on extensive state-of-the-art research, this minireview highlights recent advances in supramolecular biomaterials for cancer immunotherapy and discusses the possible mechanisms underlying how supramolecular biomaterials promote the development of cancer immunotherapy together with their potential for clinical translation.

Inhalable nanocatchers for SARS-CoV-2 inhibition.

The global coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2), presents an urgent health crisis. More recently, an increasing number of mutated strains of SARS-CoV-2 have been identified globally. Such mutations, especially those on the spike glycoprotein to render its higher binding affinity to human angiotensin-converting enzyme II (hACE2) receptors, not only resulted in higher transmission of SARS-CoV-2 but also raised serious concerns regarding the efficacies of vaccines against mutated viruses. Since ACE2 is the virus-binding protein on human cells regardless of viral mutations, we design hACE2-containing nanocatchers (NCs) as the competitor with host cells for virus binding to protect cells from SARS-CoV-2 infection. The hACE2-containing NCs, derived from the cellular membrane of genetically engineered cells stably expressing hACE2, exhibited excellent neutralization ability against pseudoviruses of both wild-type SARS-CoV-2 and the D614G variant. To prevent SARS-CoV-2 infections in the lung, the most vulnerable organ for COVID-19, we develop an inhalable formulation by mixing hACE2-containing NCs with mucoadhesive excipient hyaluronic acid, the latter of which could significantly prolong the retention of NCs in the lung after inhalation. Excitingly, inhalation of our formulation could lead to potent pseudovirus inhibition ability in hACE2-expressing mouse model, without imposing any appreciable side effects. Importantly, our inhalable hACE2-containing NCs in the lyophilized formulation would allow long-term storage, facilitating their future clinical use. Thus, this work may provide an alternative tactic to inhibit SARS-CoV-2 infections even with different mutations, exhibiting great potential for treatment of the ongoing COVID-19 epidemic.

Nanoparticle-Mediated Delivery of Inhaled Immunotherapeutics for Treating Lung Metastasis.

Despite the critical breakthrough achieved by immune checkpoint blockade (ICB), the clinical benefits are usually restricted by inefficient infiltration of immune cells and immune-associated adverse effects. Noninvasive aerosol inhalation, as a definitive procedure for treatment of respiratory diseases, for ICB immunotherapy against lung metastasis, has not been realized to the best knowledge. Herein, an inhaled immunotherapeutic chitosan (CS)-antibody complex is developed for immunotherapy against lung cancer. In this system, CS is used as a carrier to assemble with anti-programmed cell death protein ligand 1 (aPD-L1) to enable efficient transmucosal delivery. Moreover, CS exhibits adjuvant effects to drive potent immune responses via activating the cyclic-di-GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. Interestingly, repeated inhalation of CS/aPD-L1 complex can effectively activate the immune system by promoting the infiltration of different immune cells especially CD8+ T cells around tumor lesions, and finally prolongs the survival of mice to 60 days. Thus, the work presents a unique aerosol inhalation delivery system for ICB antibody, which is promising for immunotherapy against lung metastasis without the concern of systemic toxicity.

Physiological and transcriptional response to heat stress in heat-resistant and heat-sensitive maize (Zea mays L.) inbred lines at seedling stage.

To understand the molecular and physiological mechanism underlying the heat stress in maize, transcriptional and physiological response to heat stress in the heat-resistant Huangzaosi (HZS) and heat-sensitive Lv-9-Kuan (L9K) inbred lines at seedling stage were analyzed and compared at seedling stage. Our results indicated that MDA content of the two inbred lines increased significantly under heat stress; the values of MDA in L9K was significantly higher than that in HZS. The level of SOD, CAT, and POD enzyme activities in HZS was higher than those in L9K for both the heat-treated group and controls. The values of Fv/Fm, qP, and ФPSII reduced by heat stress in L9K were higher than the respective values in HZS. RNA-seq data showed that heat stress induced more heat stress-related genes in HZS (257 heat stress-related genes) than in L9K (224 heat stress-related genes). GO and KEGG enrichment analyses indicated that HZS and L9K changed their physiological and biochemical mechanisms in response to heat stress through different molecular mechanisms. Weighted Gene Co-expression Network Analysis showed that HZS might obtain stronger heat resistance than L9K through a unique transcriptional regulatory network. Our findings provide insights into the molecular networks that mediate the tolerance of maize heat stress and also help us to mine key heat stress-related genes.