A modular approach to enhancing cell membrane-coated nanoparticle functionality using genetic engineering.

Since their initial development, cell membrane-coated nanoparticles (CNPs) have become increasingly popular in the biomedical field. Despite their inherent versatility and ability to enable complex biological applications, there is considerable interest in augmenting the performance of CNPs through the introduction of additional functionalities. Here we demonstrate a genetic-engineering-based modular approach to CNP functionalization that can encompass a wide range of ligands onto the nanoparticle surface. The cell membrane coating is engineered to express a SpyCatcher membrane anchor that can readily form a covalent bond with any moiety modified with SpyTag. To demonstrate the broad utility of this technique, three unique targeted CNP formulations are generated using different classes of targeting ligands, including a designed ankyrin repeat protein, an affibody and a single-chain variable fragment. In vitro, the modified nanoparticles exhibit enhanced affinity towards cell lines overexpressing the cognate receptors for each ligand. When formulated with a chemotherapeutic payload, the modularly functionalized nanoparticles display strong targeting ability and growth suppression in a murine tumour xenograft model of ovarian cancer. Our data suggest genetic engineering offers a feasible approach for accelerating the development of multifunctional CNPs for a broad range of biomedical applications.

Extending the In Vivo Residence Time of Macrophage Membrane-Coated Nanoparticles through Genetic Modification.

Nanoparticles coated with natural cell membranes have emerged as a promising class of biomimetic nanomedicine with significant clinical potential. Among them, macrophage membrane-coated nanoparticles hold particular appeal due to their versatility in drug delivery and biological neutralization applications. This study employs a genetic engineering approach to enhance their in vivo residence times, aiming to further improve their performance. Specifically, macrophages are engineered to express proline-alanine-serine (PAS) peptide chains, which provide additional protection against opsonization and phagocytosis. The resulting modified nanoparticles demonstrate prolonged residence times when administered intravenously or introduced intratracheally, surpassing those coated with the wild-type membrane. The longer residence times also contribute to enhanced nanoparticle efficacy in inhibiting inflammatory cytokines in mouse models of lipopolysaccharide-induced lung injury and sublethal endotoxemia, respectively. This study underscores the effectiveness of genetic modification in extending the in vivo residence times of macrophage membrane-coated nanoparticles. This approach can be readily extended to modify other cell membrane-coated nanoparticles toward more favorable biomedical applications.

Malaria Biomimetic for Tumor Targeted Drug Delivery.

Malaria infected erythrocytes utilize the parasite protein VAR2CSA to bind to a unique presentation of chondroitin sulfate (CS) for their placenta specific tropism. Interestingly, many cancers express a similar form of CS, thereby termed oncofetal CS (ofCS). The distinctive tropism of malaria infected erythrocytes and the identification of oncofetal CS, therefore, represent potentially potent tools for cancer targeting. Here we describe an intriguing drug delivery platform that effectively mimics infected erythrocytes and their specificity for ofCS. We used a lipid catcher-tag conjugation system for the functionalization of erythrocyte membrane-coated drug carriers with recombinant VAR2CSA (rVAR2). We show that these malaria mimicking erythrocyte nanoparticles (MMENPs) loaded with docetaxel (DTX) specifically target and kill melanoma cells in vitro. We further demonstrate effective targeting and therapeutic efficacy in a xenografted melanoma model. These data thus provide a proof of concept for the use of a malaria biomimetic for tumor targeted drug delivery. Given the broad presentation of ofCS found across various types of malignancies, this biomimetic may therefore show potential as a broadly targeted cancer therapy against multiple tumor indications.

Outer Membrane Vesicle-Coated Nanoparticle Vaccine Protects Against Acinetobacter baumannii Pneumonia and Sepsis.

The highly multidrug-resistant (MDR) Gram-negative bacterial pathogen Acinetobacter baumannii is a top global health priority where an effective vaccine could protect susceptible populations and limit resistance acquisition. Outer membrane vesicles (OMVs) shed from Gram-negative bacteria are enriched with virulence factors and membrane lipids but heterogeneous in size and cargo. We report a vaccine platform combining precise and replicable nanoparticle technology with immunogenic A. baumannii OMVs (Ab-OMVs). Gold nanoparticle cores coated with Ab-OMVs (Ab-NPs) induced robust IgG titers in rabbits that enhanced human neutrophil opsonophagocytic killing and passively protected against lethal A. baumannii sepsis in mice. Active Ab-NP immunization in mice protected against sepsis and pneumonia, accompanied by B cell recruitment to draining lymph nodes, activation of dendritic cell markers, improved splenic neutrophil responses, and mitigation of proinflammatory cytokine storm. Nanoparticles are an efficient and efficacious platform for OMV vaccine delivery against A. baumannii and perhaps other high-priority MDR pathogens.

Immunostimulatory DNA Hydrogel Enhances Protective Efficacy of Nanotoxoids against Bacterial Infection.

While vaccines have been highly successful in protecting against various infections, there are still many high-priority pathogens for which there are no clinically approved formulations. To overcome this challenge, researchers have explored the use of nanoparticulate strategies for more effective antigen delivery to the immune system. Along these lines, nanotoxoids are a promising biomimetic platform that leverages cell membrane coating technology to safely deliver otherwise toxic bacterial antigens in their native form for antivirulence vaccination. Here, in order to further boost their immunogenicity, nanotoxoids formulated against staphylococcal α-hemolysin are embedded into a DNA-based hydrogel with immunostimulatory CpG motifs. The resulting nanoparticle-hydrogel composite is injectable and improves the in vivo delivery of vaccine antigens while simultaneously stimulating nearby immune cells. This leads to elevated antibody production and stronger antigen-specific cellular immune responses. In murine models of pneumonia and skin infection caused by methicillin-resistant Staphylococcus aureus, mice vaccinated with the hybrid vaccine formulation are well-protected. This work highlights the benefits of combining nanoparticulate antigen delivery systems with immunostimulatory hydrogels into a single platform, and the approach can be readily generalized to a wide range of infectious diseases.

Genetically engineered cellular nanoparticles for biomedical applications.

In recent years, nanoparticles derived from cellular membranes have been increasingly explored for the prevention and treatment of human disease. With their flexible design and ability to interface effectively with the surrounding environment, these biomimetic nanoparticles can outperform their traditional synthetic counterparts. As their popularity has increased, researchers have developed novel ways to modify the nanoparticle surface to introduce new or enhanced capabilities. Moving beyond naturally occurring materials derived from wild-type cells, genetic manipulation has proven to be a robust and flexible method by which nanoformulations with augmented functionalities can be generated. In this review, an overview of genetic engineering approaches to express novel surface proteins is provided, followed by a discussion on the various biomedical applications of genetically modified cellular nanoparticles.

Single Low-Dose Nanovaccine for Long-Term Protection against Anthrax Toxins.

Anthrax infections caused by Bacillus anthracis are an ongoing bioterrorism and livestock threat worldwide. Current approaches for management, including extended passive antibody transfusion, antibiotics, and prophylactic vaccination, are often cumbersome and associated with low patient compliance. Here, we report on the development of an adjuvanted nanotoxoid vaccine based on macrophage membrane-coated nanoparticles bound with anthrax toxins. This design leverages the natural binding interaction of protective antigen, a key anthrax toxin, with macrophages. In a murine model, a single low-dose vaccination with the nanotoxoids generates long-lasting immunity that protects against subsequent challenge with anthrax toxins. Overall, this work provides a new approach to address the ongoing threat of anthrax outbreaks and bioterrorism by taking advantage of an emerging biomimetic nanotechnology.

mRNA nanomedicine: Design and recent applications.

The rational design and application of mRNA-based medicine have recently yielded some key successes in the clinical management of human diseases. mRNA technology allows for the facile and direct production of proteins in vivo, thus circumventing the need for lengthy drug development cycles and complex production workflows. As such, mRNA formulations can significantly improve upon the biological therapies that have become commonplace in modern medicine. Despite its many advantages, mRNA is inherently fragile and has specific delivery requirements. Leveraging the engineering flexibility of nanobiotechnology, mRNA payloads can be incorporated into nanoformulations such that they do not invoke unwanted immune responses, are targeted to tissues of interest, and can be delivered to the cytosol, resulting in improved safety while enhancing bioactivity. With the rapidly evolving landscape of nanomedicine, novel technologies that are under development have the potential to further improve the clinical utility of mRNA medicine. This review covers the design principles relevant to engineering mRNA-based nanomedicine platforms. It also details the current research on mRNA nanoformulations for addressing viral infections, cancers, and genetic diseases. Given the trends in the field, future mRNA-based nanomedicines have the potential to change how many types of diseases are managed in the clinic.

Nanotoxoid vaccination protects against opportunistic bacterial infections arising from immunodeficiency.

The rise in nosocomial infections caused by multidrug-resistant pathogens is a major public health concern. Patients taking immunosuppressants or chemotherapeutics are naturally more susceptible to infections. Thus, strategies for protecting immunodeficient individuals from infections are of great importance. Here, we investigate the effectiveness of a biomimetic nanotoxoid vaccine in defending animals with immunodeficiency against Pseudomonas aeruginosa. The nanotoxoids use a macrophage membrane coating to sequester and safely present bacterial virulence factors that would otherwise be too toxic to administer. Vaccination with the nanoformulation results in rapid and long-lasting immunity, protecting against lethal infections despite severe immunodeficiency. The nanovaccine can be administered through multiple routes and is effective in both pneumonia and septicemia models of infection. Mechanistically, protection is mediated by neutrophils and pathogen-specific antibodies. Overall, nanotoxoid vaccination is an attractive strategy to protect vulnerable patients and could help to mitigate the threat posed by antibiotic-resistant superbugs.

Bacterial membrane vesicles for vaccine applications.

Vaccines have been highly successful in the management of many diseases. However, there are still numerous illnesses, both infectious and noncommunicable, for which there are no clinically approved vaccine formulations. While there are unique difficulties that must be overcome in the case of each specific disease, there are also a number of common challenges that have to be addressed for effective vaccine development. In recent years, bacterial membrane vesicles (BMVs) have received increased attention as a potent and versatile vaccine platform. BMVs are inherently immunostimulatory and are able to activate both innate and adaptive immune responses. Additionally, BMVs can be readily taken up and processed by immune cells due to their nanoscale size. Finally, BMVs can be modified in a variety of ways, including by genetic engineering, cargo loading, and nanoparticle coating, in order to create multifunctional platforms that can be leveraged against different diseases. Here, an overview of the interactions between BMVs and immune cells is provided, followed by discussion on the applications of BMV vaccine nanotechnology against bacterial infections, viral infections, and cancers.

Virus-Mimicking Cell Membrane-Coated Nanoparticles for Cytosolic Delivery of mRNA.

Effective endosomal escape after cellular uptake represents a major challenge in the field of nanodelivery, as the majority of drug payloads must localize to subcellular compartments other than the endosomes in order to exert activity. In nature, viruses can readily deliver their genetic material to the cytosol of host cells by triggering membrane fusion after endocytosis. For the influenza A virus, the hemagglutinin (HA) protein found on its surface fuses the viral envelope with the surrounding membrane at endosomal pH values. Biomimetic nanoparticles capable of endosomal escape were fabricated using a membrane coating derived from cells engineered to express HA on their surface. When evaluated in vitro, these virus-mimicking nanoparticles were able to deliver an mRNA payload to the cytosolic compartment of target cells, resulting in the successful expression of the encoded protein. When the mRNA-loaded nanoparticles were administered in vivo, protein expression levels were significantly increased in both local and systemic delivery scenarios. We therefore conclude that utilizing genetic engineering approaches to express viral fusion proteins on the surface of cell membrane-coated nanoparticles is a viable strategy for modulating the intracellular localization of encapsulated cargoes.

Engineering of stimuli-responsive self-assembled biomimetic nanoparticles.

Nanoparticle-based therapeutics have the potential to change the paradigm of how we approach the diagnosis and treatment of human disease. Employing naturally derived cell membranes as a surface coating has created a powerful new approach by which nanoparticles can be functionalized towards a wide range of biomedical applications. By using membranes derived from different cell sources, the resulting nanoparticles inherit properties that can make them well-suited for a variety of tasks. In recent years, stimuli-responsive platforms with the ability to release payloads on demand have received increasing attention due to their improved delivery, reduced side effects, and precision targeting. Nanoformulations have been developed to respond to external stimuli such as magnetic fields, ultrasound, and radiation, as well as local stimuli such as pH gradients, redox potentials, and other chemical conditions. Here, an overview of the novel cell membrane coating platform is provided, followed by a discussion of stimuli-responsive platforms that leverage this technology.

Physical Disruption of Solid Tumors by Immunostimulatory Microrobots Enhances Antitumor Immunity.

The combination of immunotherapy with other forms of treatment is an emerging strategy for boosting antitumor responses. By combining multiple modes of action, these combinatorial therapies can improve clinical outcomes through unique synergisms. Here, a microrobot-based strategy that integrates tumor tissue disruption with biological stimulation is shown for cancer immunotherapy. The microrobot is fabricated by loading bacterial outer membrane vesicles onto a self-propelling micromotor, which can react with water to generate a propulsion force. When administered intratumorally to a solid tumor, the disruption of the local tumor tissue coupled with the delivery of an immunostimulatory payload leads to complete tumor regression. Additionally, treatment of the primary tumor results in the simultaneous education of the host immune system, enabling it to control the growth of distant tumors. Overall, this work introduces a distinct application of microrobots in cancer immunotherapy and offers an attractive strategy for amplifying cancer treatment efficacy when combined with conventional therapies.

Nanotechnology for virus treatment.

The continued emergence of novel viruses poses a significant threat to global health. Uncontrolled outbreaks can result in pandemics that have the potential to overburden our healthcare and economic systems. While vaccination is a conventional modality that can be employed to promote herd immunity, antiviral vaccines can only be applied prophylactically and do little to help patients who have already contracted viral infections. During the early stages of a disease outbreak when vaccines are unavailable, therapeutic antiviral drugs can be used as a stopgap solution. However, these treatments do not always work against emerging viral strains and can be accompanied by adverse effects that sometimes outweigh the benefits. Nanotechnology has the potential to overcome many of the challenges facing current antiviral therapies. For example, nanodelivery vehicles can be employed to drastically improve the pharmacokinetic profile of antiviral drugs while reducing their systemic toxicity. Other unique nanomaterials can be leveraged for their virucidal or virus-neutralizing properties. In this review, we discuss recent developments in antiviral nanotherapeutics and provide a perspective on the application of nanotechnology to the SARS-CoV-2 outbreak and future virus pandemics.

Engineered Cell-Membrane-Coated Nanoparticles Directly Present Tumor Antigens to Promote Anticancer Immunity.

The recent success of immunotherapies has highlighted the power of leveraging the immune system in the fight against cancer. In order for most immune-based therapies to succeed, T cell subsets with the correct tumor-targeting specificities must be mobilized. When such specificities are lacking, providing the immune system with tumor antigen material for processing and presentation is a common strategy for stimulating antigen-specific T cell populations. While straightforward in principle, experience has shown that manipulation of the antigen presentation process can be incredibly complex, necessitating sophisticated strategies that are difficult to translate. Herein, the design of a biomimetic nanoparticle platform is reported that can be used to directly stimulate T cells without the need for professional antigen-presenting cells. The nanoparticles are fabricated using a cell membrane coating derived from cancer cells engineered to express a co-stimulatory marker. Combined with the peptide epitopes naturally presented on the membrane surface, the final formulation contains the necessary signals to promote tumor antigen-specific immune responses, priming T cells that can be used to control tumor growth. The reported approach represents an emerging strategy that can be used to develop multiantigenic, personalized cancer immunotherapies.

Nanoparticle-hydrogel superstructures for biomedical applications.

The incorporation of nanoparticles into hydrogels yields novel superstructures that have become increasingly popular in biomedical research. Each component of these nanoparticle-hydrogel superstructures can be easily modified, resulting in platforms that are highly tunable and inherently multifunctional. The advantages of the nanoparticle and hydrogel constituents can be synergistically combined, enabling these superstructures to excel in scenarios where employing each component separately may have suboptimal outcomes. In this review, the synthesis and fabrication of different nanoparticle-hydrogel superstructures are discussed, followed by an overview of their use in a range of applications, including drug delivery, detoxification, immune modulation, and tissue engineering. Overall, these platforms hold significant clinical potential, and it is envisioned that future development along these lines will lead to unique solutions for addressing areas of pressing medical need.

T-Cell-Mimicking Nanoparticles Can Neutralize HIV Infectivity.

To improve human immunodeficiency virus (HIV) treatment and prevention, therapeutic strategies that can provide effective and broad-spectrum neutralization against viral infection are highly desirable. Inspired by recent advances of cell-membrane coating technology, herein, plasma membranes of CD4+ T cells are collected and coated onto polymeric cores. The resulting T-cell-membrane-coated nanoparticles (denoted as "TNPs") inherit T cell surface antigens critical for HIV binding, such as CD4 receptor and CCR5 or CXCR4 coreceptors. The TNPs act as decoys for viral attack and neutralize HIV by diverting the viruses away from their intended host targets. This decoy strategy, which simulates host cell functions for viral neutralization rather than directly suppressing viral replication machinery, has the potential to overcome HIV genetic diversity while not eliciting high selective pressure. In this study, it is demonstrated that TNPs selectively bind with gp120, a key envelope glycoprotein of HIV, and inhibit gp120-induced killing of bystander CD4+ T cells. Furthermore, when added to HIV viruses, TNPs effectively neutralize the viral infection of peripheral mononuclear blood cells and human-monocyte-derived macrophages in a dose-dependent manner. Overall, by leveraging natural T cell functions, TNPs show great potential as a new therapeutic agent against HIV infection.

Biomimetic Targeting of Nanoparticles to Immune Cell Subsets via Cognate Antigen Interactions.

Within the body, cellular recognition is mediated in large part by receptor-ligand interactions that result from the surface marker expression of the participant cells. In the case of immune cells, these interactions can be highly specific, enabling them to carry out their protective functions in fighting off infection and malignancy. In this work, we demonstrate the biomimetic targeting of antigen-specific immune cell populations by using nanoparticles functionalized with natural membrane derived from cells expressing the cognate antigen. Using red blood cell (RBC)-specific B cells as a model target, it is shown that RBC membrane-coated nanoparticles exhibit enhanced affinity compared with control nanoparticles. The concept is further demonstrated using murine models of alloimmunity and autoimmunity, where B cells elicited against RBCs can be positively labeled using the biomimetic nanoparticles. This strategy for antigen-specific immune cell targeting may have utility for the detection and treatment of various autoimmune conditions, and it may additionally have implications for the prevention of immune cell malignancies.