The cornea is consisted with continuous and smooth epithelial cells in the center and limbal stem cells in the edge. Their transparency plays important role in maintaining our vision. Any deficiency in cornea epithelial cells (CECs) or limbal stem cells (LSCs) will lead to transparency lost and blindness. In our study, we successfully developed a protocol for cell culture to expand LSCs from human donors and animal tissues. Enabling us to generate a homogenous cell population to study key factors involved in LSCs fate determination and CEC differentiation. Through the genome-wide gene expression analysis, we found WNT7A and PAX6 were highly involved in LSCs and CECs, which gave us a insight of these two genes regulation for limbal stem cell fate determination.

Nanoparticle drug delivery has revolutionized the way we think of disease treatment over the last decade. The encapsulation of drugs into nanoparticles has led to better bioavailability, longer circulation times and an extended therapeutic window, and fewer off target effects than free drug administration. Nanoparticles are able to be tailored to specific applications through their size, shape, and surface design. Nanoparticles are just beginning to see clinical translation and FDA approval. Recently, significant efforts have been put into creating biomimetic targeting, particularly utilizing cell membrane coatings. Cell membrane can be used as a biomaterial to “cloak” nanoparticles, endowing them with the surface properties of the parent cell. Each different cell type in the body has a distinct surface structure with lipids, proteins, and receptors perfectly tailored to its purpose and location. Some of these proteins, such as CD47 or the selectins, have well known purposes like immune evasion or specific receptor targeting respectively. Additionally, there are yet

undescribed and uncharacterized surface moieties on cells whose properties can be retained by using the entire cell membrane as a biomaterial. By cloaking nanoparticles in cell membrane, they retain many of the properties of the original cell type. We show that this allows for new biointerfacing abilities and a highly specific drug delivery vehicle. This new technology also promises future clinical translation,as the materials are inherently biocompatible. Herein, we will discuss engineered nanoparticle platforms that utilize this biomimetic cell membrane coating technology to improve the delivery of drugs, and additionally the detoxification and removal of pathogens. The biomimetic techniques developed during this PhD range from novel formulations of classical small molecule targeting for cancer therapy, to new methods of utilizing a fusion of natural cell membranes to create custom tailored targeting. These improvements to the field of targeted drug delivery will hopefully lead to better use of drugs and treatment of disease, and a higher level of tailoring ability available to engineers designing future platforms.

The nanodelivery of biomolecules offers several benefits compared with use of the same compounds in their free form. First, payload entrapment and protection by a nanoparticle matrix minimizes the chance of interference caused by degradative agents and nonspecific cellular interactions. This helps to prolong circulation half-life and enhances the biological stability of the payload, both of which are crucial for maximizing its bioactivity. Second, owing to the relatively small size of nanocarriers, the encapsulated payloads can more tunably localize and accumulate at specific sites via common administration routes. For example, the intravenous administration of nanocarriers enables efficient bloodstream distribution and sustained tissue accumulation, whereas intra-articular injection of nanocarriers results in fast local accumulation at joint. Furthermore, intrinsic capability of nanocarriers can greatly enhance the nanodelivery efficacy. For instance, self-dissociation property of some materials at certain pH condition can facilitate the fast release of the biomolecule at local sites. By leveraging proper materials design, nanoparticulate platforms can be synthesized with specific targeting functionality and controllable release to greatly improve biomolecule payload bioavailability and ensure bioactivity at minimal dosages of the active ingredient.

The use of cell membrane coatings to camouflage existing synthetic nanomaterials is an effective biomimetic method for nanoparticle functionalization. The membrane-coated nanoparticles fabricated using such platform technology exhibit cell-mimicking properties that enable them to excel at in vivo applications. For example, red blood cell membrane coatings can greatly prolong circulation within the bloodstream, whereas platelet membrane coatings enable targeted delivery to bacteria, cancer, and damaged vasculature. It was also demonstrated that nanoparticles functionalized with white blood cell membrane can be used as nanoscale decoys to absorb and neutralize inflammatory cytokines, with potential applications for autoimmune disorders and sepsis treatment. Overall, cell membrane coatings can be derived from any type of cell, enabling researchers a wide range of options for adding functionality and creating synergies with nanoparticle-based biomolecule delivery.

Herein, we discuss biomimetic nanodelivery of several biomolecules by novel cell-membrane coated nanoparticles. The biomimetic delivery techniques developed from novel formulations encased of liquid perfluorocarbon for oxygen delivery, to new methods composed of metal-organic framework for small-interfering RNA and enzyme delivery. Specifically, red blood cell membrane-coated oxygen-loaded perfluorocarbon nanoemulsions can act as a promising candidate of next-generation blood substitute with tremendous physiological stability, prolonged blood circulation and excellent biocompatibility, which may have the potential to address a critical need in the clinic. On the other hand, platelet membrane-coated small-interfering RNA-loaded metal-organic framework nanoparticles present high silencing efficiency against multiple target genes, which could be used to expand the applicability of gene therapy across a range of disease-relevant applications. Moreover, macrophage membrane-coated enzyme-loaded metal-organic framework nanoparticles show excellent local retention and cytokine neutralization, thus can synergize with the loaded enzyme for degradation of cognate substrate and alleviation of relevant disease. Taking together, these biomimetic approaches by novel cell membrane-coated nanoparticles will hopefully lead to better use of the biomolecules and treatment of the diseases, and a higher level of tailoring ability available to engineers designing future platforms.

Nanofibers have shown promising results as an excellent drug delivery platform and a wound healing medium due to their fibrous morphology and high porosity. They are also a highly versatile nanosystem with customizable material composition and surface structure. Recent emergence of the cell membrane cloaking technology and its detoxification capability enabled a new way of nanofiber functionalization. The endotoxin and cytokine neutralizing properties of mouse J774 macrophage cell membrane coated nanoparticles (MϕNP) have previously been reported, but the nature of free nanoparticles prohibits local cytokine neutralization. Herein, we introduce a triple-action nanofiber-nanoparticle hybrid system that functions both as a drug delivery and a MϕNP retention platform. This project explored attaching MϕNP onto dual-drug loaded polycaprolactone (PCL) nanofiber surface. Our final nanoparticle-nanofiber hybrid system attenuates inflammation in three directions: curcumin reduces inflammation through modulating cytokine transcription; ciprofloxacin provides powerful anti-microbial effects; the attached MϕNPs show improved cytokine neutralization. The hybrid system serves as a new way to integrate traditional drug-delivery nano-platforms with the innovative biomimetic membrane toxin-neutralizing nanoparticles.

Antibiotic resistance has become an undeniable burden on global health as we move further into the twenty-first century. It is predicted that drug-resistant infections could lead to an annual mortality rate of 10 million people by 2050 and a cumulative cost of up to 100 trillion USD on the world’s economy. These unsettling projections have necessitated the exploration of new and more effective ways to manage bacterial infection. This dissertation focuses on novel strategies for addressing this pressing challenge via nanomedicine, particularly the use of natural cell-derived membrane to enhance the biointerfacing of synthetic nanomaterials. The resulting membrane- cloaked platforms exhibit unique, cell-specific properties that can be leveraged for antibacterial therapy. Additionally, novel nano/micromotors are further exploited to design new biomimetic therapeutic modalities capable of active movement.

The first part of this thesis will focus on novel antibiotic delivery systems, including targeted delivery and active delivery platforms. For targeted antibiotic delivery, the native function of the source cells, particularly their natural adhesion property was exploit by the natural cell membranes-coated nanoparticles. Meanwhile, the second delivery platform involves mobile micromotor which are acid-powered and enable active delivery. Not only can the propulsion of antibiotic-loaded motors in gastric media effectively deliver the drug payload, but the motor-based therapy can also rapidly neutralize gastric acid without the need of proton pump inhibitor.

the second portion of this dissertation will focus on the exploitation of biomim- icking nanoplatforms as countermeasures against pathologic moieties for the abroga- tion of bacterial infection. This is demonstrated through a range of bacterial virulence including diarrhea-causing cholera toxin, Shiga toxin, and systemic endotoxin. Ul- timately, cell membrane-coated technology has the potential to greatly impact the landscape of nanomedicine and contribute to the management of bacterial infections in the future.

Toxin threats are everywhere and underlie various diseases. Biological neutralization represents a strategy of deploying therapeutic agents to bind with toxin threats and neutralize their bioactivity. Despite their therapeutic potential, traditional neutralization platforms rely on targeting the molecular structure of individual toxic agents. This approach has been thwarted by the enormous structural diversity of toxic agents and yields narrow-spectrum neutralization with limited therapeutic efficacy. Recently, a novel class of biomimetic nanomedicine, known as “cellular nanosponges”, has emerged as a compelling neutralization platform. Composed of synthetic nanoparticle cores cloaked with natural cell membranes, cellular nanosponges mimic the host cells and can act as cell decoys to intercept and neutralize toxic agents. This neutralization strategy shifts the focus away from the distinct structures of toxic agents to their shared interactions with the host cells, thereby overcoming their structural diversity and enabling broad-spectrum neutralization. The first chapter of this dissertation reviews the recent progress in using cellular nanosponges for neutralization against bacterial toxins, chemical toxicants, inflammatory cytokines, pathological antibodies, and viruses. Despite their broad-spectrum neutralization capabilities, these nanosponges typically possess an inert core and rely solely on toxin binding with the membrane for neutralization. The second chapter presents a novel cellular nanosponge design featuring a functional core loaded with toxin-binding proteins. This design enables concurrent toxin binding by the membrane and core, leading to enhanced neutralization. Besides toxin-binding proteins, cellular nanosponges can encapsulate toxin-degrading enzymes to attain continuous toxin degradation and boost neutralization capacity. Such continuous toxin degradation can be accelerated by increasing the membrane permeability of cellular nanosponges. The third chapter presents membrane cholesterol depletion as a facile and versatile approach to enhance the membrane permeability of cellular nanosponges and accelerate toxin neutralization. Overall, this dissertation provides a framework for the rational design of protein-loaded cellular nanosponges for biological neutralization. This work is expected to inspire more innovations in the design of cellular nanosponges to address unsolved medical problems.

Biomimetic technology takes cues from nature’s elegant engineering and applies these principles to novel therapeutics. By endowing nanoparticle and microparticle scaffolds with complex, multivalent functionalities, biomimicry allows us to enhance the potency of drug molecules and vaccines without sacrificing patient safety. In particular, membrane coated nanoparticles, derived from native extracellular and cellular membranes present in nature, have demonstrated tremendous success treating and protecting against various diseases.

The first chapter of this dissertation explores biomimicry and its applications in nanomedicine in the context of bacteria. The remaining chapters of this dissertation apply these design principles to disease states. Through novel biomimetic designs, dosage requirements for effective treatment and vaccination are significantly reduced. Chapter two focuses on the design, fabrication, and application of extracellular membrane coated nanoparticles to treat lung disease. By endowing anti-inflammatory nanoparticulate cores with lung-tropic breast cancer exosome membrane, lung inflammation can be successfully managed with a lower therapeutic dose. The third chapter of this dissertation is dedicated to biomimetic vaccination against anthrax utilizing a known anthrax virulence mechanism. Macrophage membrane cloaked nanoparticles containing a potent adjuvant are further functionalized with an anthrax toxin to enable antigen-adjuvant co-delivery and single, low-dose vaccine protection.

Overall, the rational design principles presented in this dissertation serve as a framework to develop a wide range of highly efficient, low-dose biomimetic nanocarriers to address unmet clinical needs.

Cell membrane coating technology is a technique that strips plasma membrane from intact cells and faithfully endows the biological functionalities onto inanimate materials. For immune modulation, this elegant strategy enables an effortless approach to design and synthesize multivalent vaccines against a multitude of different diseases. In combination with the unique attributes of nanotechnology, cell membrane coating technology can drastically improve the efficacy and safety of conventional drug formulations. The first chapter of this dissertation will explore the advantages of nanoparticles and how those distinctive properties can be leveraged for the application of biomimetic nanovaccines for personalized therapies.

The second part of this dissertation will focus on the design of cell membrane-coated nanovaccines fabricated with materials sourced from pathogens. Through this process, the immune system is trained directly against the infectious agents and can immediately identify the threat upon encounter. Examples of this vaccination approach for a parasite and a multidrug-resistant bacteria will be elaborated. Along the lines of pathogenic diseases, the third section of this dissertation will examine a toxin-detainment approach for antivirulence vaccination. Host cell membrane is utilized to capture and attenuate the toxicity of bacterial virulence factors before the entire package is safely introduce to the host for immune priming. Rather than eliciting immunity towards the actual bacteria, antivirulence vaccination improve patient outcomes by removing the tools pathogens exploit for survival.

The last portion of the dissertation will switch over to therapeutic vaccines for cancer therapy. In the first half, leukemic cell membrane is employed to educate host immune system to recognize lingering malignant cells following chemotherapy. In the second half, a micromotor is utilized along with bacteria-derived outer membrane vesicles for the physical disruption of solid tumors. The immunostimulatory membrane vesicles help recruit local immune cells to process the destroyed tumor cells for downstream immune activation. Ultimately, a potent immune response personalized for the treated tumor type is induced. By harnessing the ideas in this dissertation as a framework, a plethora of novel vaccines can be easily developed with cell membrane coating technology to address other areas of unmet clinical needs.

Nanomedicine is a flourishing scientific field involving the design and control of matter on the nanometer scale for therapeutic use. One emerging avenue within the nanomedicine field is biodetoxification-based therapy, in which nano-sized structures are used to capture and retain biotoxins that would otherwise attack host cells, cause cellular damage, and trigger damage-associated signaling pathways to propagate diseases. Particularly, inflammation is a biological process that involves complex signaling networks and disease-specific cellular responses, posing significant challenge for designing medicine with high specificity and potency. To this end, the design flexibility of nanoparticle size and surface modification, and unique particle-cell interaction at the nanoscale can lead to novel and efficacious routes for anti-inflammatory therapy. Herein, we discuss the new generations of cell membrane-coated nanoparticles specifically tailored for biodetoxification-based anti-inflammatory therapy. Firstly, recent advances in neutralizing inflammatory cytokine will be discussed. The second portion of this dissertation will present neutrophil membrane-coated nanoparticle (neutrophil-NP) for therapeutic treatment of inflammatory arthritis. Neutrophil-NPs targeted and penetrated into the inflamed tissue, and effectively neutralized inflammatory cytokines. The third portion will cover the development of macrophage membrane-coated nanoparticle for neutralization of bacterial endotoxin and inflammatory cytokines, and management of bacterial sepsis. Finally, we will focus on the design of a unique ‘lure and kill’ nanoparticle for effective inhibition of phospholipase A2 (PLA2). The cell membrane works synergistically with a PLA2 attractant to ‘lure’ PLA2 for attack, then the PLA2 inhibitor in the cell membrane further ‘kills’ the enzyme. With effective inhibition of venomous PLA2, these nanoparticles further demonstrated strong inhibitory activity against PLA2 and attenuation of the inflammatory cascade during acute pancreatitis progression. This dissertation will serve as a paradigm for rational design of cell membrane-coated nanoparticles for therapeutic treatment of inflammatory disorders. By tapping into the biological challenges associated with anti-inflammatory therapy, cell membrane-coated nanoparticles can be tailored towards its designated biological target. By harnessing the design flexibility, this nanotechnology holds great potential to be developed into a class of drug-free anti-inflammatory nanomedicine that will be extraordinarily valuable for biomedical researchers and clinicians alike.