Sometimes called biomimetic nanoparticles, or cell-mimicking nanoparticles, nanosponges consist of a polymer nanoparticle wrapped in membranes derived from diverse cell types, from red blood cells (RBCs) to macrophages. Because they retain the receptors and membrane structures of natural cells, nanosponges can be used as decoys to prevent pathogens and their harmful products from meddling with the real deal. While nanosponges are not ready for clinical use, the growing list of applications for the technology-from dampening overactive immune responses to shuttling drugs to specific tissues and cells-has set the stage for taking these particles from the laboratory bench to the hospital bedside.
What Are Nanosponges and How Are They Made?
Cellular nanosponges are nanoparticles sheathed in naturally derived cell membranes. They are part of the burgeoning field of nanomedicine-the use of materials and devices at the molecular scale to advance health and fight disease. Nanoparticles are generated from various materials (e.g., lipids, silver, gold and natural or synthetic polymers) and have diverse biomedical applications, including drug delivery and cell labeling and imaging, among others.
How does one make a nanosponge? In essence, scientists start with a human host cell and "use a combination of physical methodologies to remove the intracellular contents," said Dr. Liangfang Zhang, the scientific founder of Cellics Therapeutics (a company that uses cellular nanoparticle technology to treat and prevent diseases) during ASM Microbe 2022. "Then, we collect the plasma membrane from the cell and coat it onto a biodegradable, biocompatible nanoparticle surface."
For example, to generate RBC-derived nanosponges, researchers first lyse the cells by placing them in a hypotonic solution. These "RBC ghosts" (i.e., empty membranes) are then broken up via sonication to create membrane vesicles. Nanoparticles generated from poly(lactic-co-glycolic acid)-PLGA, a biodegradable polymer approved for clinical use by the U.S. Food and Drug Administration (FDA)-are mixed with the vesicle soup. The purpose of the PLGA particles is to provide structural support for the cell membranes. Accordingly, the vesicle-polymer mixture is mechanically extruded through a porous membrane, the force of which pushes the PLGA particles into the RBC membrane blebs.
Zhang, who invented nanosponge technology and has played an instrumental role in its development, highlighted that a single natural cell can be broken down into thousands of vesicles. This is attractive from a manufacturing standpoint, as it allows scientists to generate a hefty sum of nanosponges from a relatively small number of natural cells.
To that end, must nanosponges be generated from an individual's own cells for them to be tolerated and effective? Not according to Zhang. He noted that, for its RBC nanosponges, Cellics uses type O RBCs that are compatible with a majority of the population. The company is also developing a master cell line to make macrophage nanosponges that lack surface molecules, which could trigger an immune response, thus ensuring their tolerability and broad applicability within the population.
An added benefit: cellular nanosponges can be suspended in solution or lyophilized (dried out) into a powder, making them stable for long-term storage. Moreover, each nanosponge "has only 2 components-the cell membrane and a biodegradable polymer," Zhang said. "When it gets into the body [via injection], the polymer will degrade into monomers, and eventually metabolize into water and carbon dioxide, leaving nothing toxic in the body."
How Are Nanosponges Used?
The potential uses of nanosponges are limited only by the diversity of receptors on a cell's surface-and even that can change with a little genetic engineering. With that in mind, scientists are beginning to understand just how versatile nanosponges can be.
Preventing Viral Infections
When viruses invade the body, they attach to host cells and hijack the internal machinery to replicate. The goal of antiviral compounds is to thwart infection by, among other mechanisms, binding to the host cell (or virus) to block viral entry and replication. However, it can be challenging to develop antivirals that inhibit viral replication but leave normal cell processes unaffected.
This is where nanosponges show great potential: rather than interfering with natural cells, they pretend to be them. That is, because nanosponges look like host cells, viruses erroneously bind receptors on their surface. Once bound, the viral particles are stuck, and thus unable to infect real cells. For example, Zhang and his colleagues found that, after incubation with nanosponges generated from human alveolar epithelial cells and macrophages (i.e., immune cells that phagocytose foreign materials, including pathogens), SARS-CoV-2, the virus that causes COVID-19, was unable to infect natural cells.
In another study, scientists generated cellular nanoparticles using human monocytes (i.e., cells that differentiate into macrophages and dendritic cells which, in turn, phagocytose pathogens, among other functions). They found that the monocyte-derived "nanodecoys" inhibited SARS-CoV-2 replication and infection. Such inhibition can be enhanced by increasing the amount of heparin (a cellular receptor SARS-CoV-2 uses to enter cells, in addition to ACE2) on the nanosponge surface. And it's not just SARS-CoV-2. Zhang's team showed that T-cell-mimicking nanosponges could neutralize HIV-1, thus protecting natural T cells from infection and showcasing the potential of nanosponges to combat diverse viruses.
Neutralizing Bacterial Toxins
Nanosponges are also useful in the context of bacterial infections. Indeed, many bacteria secrete pore-forming toxins that lyse and kill host cells; according to Zhang, there are over 80 families of pore-forming toxins. However, they all have 1 thing in common: their receptors are on the cell membrane. Nanosponges, with their diverse repertoire of surface receptors, broadly bind to and clean up toxins before they cause cellular damage. This can "affect bacterial colonization, proliferation and also facilitate the immune system to clear the pathogen," Zhang explained.
For example, RBC-derived nanosponges sequester streptolysin O, a pore-forming toxin secreted by Group A Streptococcus, and block its ability to kill macrophages and neutrophils. These immune cells are then free to deal with the microbial threat. RBC-derived nanosponges can also inhibit hemolysis (i.e., the rupturing of RBCs) induced by alpha-hemolysin secreted by methicillin-resistant Staphylococcus aureus and listeriolysin O from Listeria monocytogenes, among other toxins.
Notably, unlike antibiotics, nanosponges don't place selective pressure on a pathogen. They are effective regardless of the drug resistance profile of the microbe. As such, combining antimicrobials with nanosponges could be a viable option for "disarming" a pathogen while killing it through immune and drug-associated mechanisms.
Moreover, Zhang noted, by fishing out bacterial toxin antigens with nanosponges, "the antigens are now safe. Because they are bound to the cell membrane already, they have finished their task." The toxin-bound nanosponges temporarily circulate within the bloodstream, then are eventually broken down. As a result, toxin-coated nanosponges could be used as multivalent vaccines. For instance, vaccination with macrophage-derived nanosponges coated with virulence factors from Pseudomonas aeruginosa led to "rapid and long-lasting immunity" against pneumonia in immunodeficient mice.
Tempering Immune Responses
Infection is a complicated dance between a pathogen and the host immune system. Immune cells detect and directly combat invading microbes, while also releasing cytokines and other factors to regulate the response. Sometimes, these factors do more harm than good. Zhang pointed to sepsis as an example: infection by a pathogen stimulates macrophages to secrete cytokines, which activate more macrophages and other immune cells, ultimately leading to a "cytokine storm," organ failure and possibly death.
Nanosponges to the rescue! Zhang described how macrophage-derived nanosponges are "good for sepsis. Because of all these natural cellular receptors on the macrophage, they can be used to adsorb and neutralize all the corresponding parties in the body." That is, by acting as macrophage decoys, nanosponges can bind and neutralize bacterial endotoxins that trigger the sepsis-associated inflammatory cascade, while also sucking up cytokines that perpetuate the damaging response. The idea, according to Zhang, is not to paralyze the immune response, but merely to dampen it enough to prevent bodily harm. For this reason, nanosponges are also being explored to treat autoimmune diseases, which are characterized by overactive, self-directed immune responses.
Delivering Drugs Throughout the Body
Nanosponges are naturally empty-but they don't have to be. They can also be loaded with therapeutics to be shuttled throughout the body. A recent study showed that macrophage-derived nanosponges loaded with lopinavir, a model antiviral drug with in vitro activity against SARS-CoV-2, could alleviate inflammation and reduce tissue viral loads in a mouse model of coronavirus infection. Because nanosponges maintain the signaling structures of natural cells, they can also deliver drugs to specific body regions. Indeed, Zhang and colleagues created nanosponges from cells genetically engineered to express a protein, VLA-4, that binds closely to VCAM-1, a molecule commonly upregulated by inflamed endothelial cells. The nanosponges showed improved delivery of the anti-inflammatory drug, dexamathsone, to inflamed tissue in a mouse model of lung inflammation. The results illustrate that, by capitalizing on natural ligand-target interactions, nanosponges can be programmed to deliver therapeutic payloads to defined cells and tissues.
The Clinical Future of Nanosponges
For all their promise, Zhang stated that cellular nanosponges are "nowhere close to entering the market." Most studies have taken place in vitro and in animal models. Furthermore, there are challenges associated with scaling the production of nanosponges to a level needed for clinical trials and human use. For example, the "unique biological and interfacial properties" of cell membranes means creating batch-to-batch consistency can be difficult. That nanosponges are a non-traditional therapy further complicates matters "because when something is non-traditional, it means there is no existing pathway or established methodology," Zhang noted. He described the difficulty of developing in vitro quality control assays and determining where the therapy fits in the regulatory framework of the FDA.
Still, despite these challenges, nanosponges have started on the path toward clinical use. The FDA recently approved the Investigational New Drug (IND) application from Cellics for its drug product candidate CTI-005, Human Red Blood Cell Nanosponges. The company plans to launch a clinical phase 1b/2a trial that investigates the safety and potential efficacy of the product in people hospitalized with methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-sensitive Staphylococcus aureus (MSSA) pneumonia, with other products (e.g., macrophage nanosponges for inflammatory bowel disease and sepsis) in the works.
Research in this article was presented at ASM Microbe, the annual meeting of the American Society for Microbiology, held June 9-13, 2022, in Washington, D.C.
