Protective Topical Dual-Sided Nanofibrous Hemostatic Dressing Using Mussel and Silk Proteins with Multifunctionality of Hemostasis and Anti-Bacterial Infiltration.

Topical hemostatic agents are preferred for application to sensitive bleeding sites because of their immediate locoregional effects with less tissue damage. However, the majority of commercial hemostatic agents fail to provide stable tissue adhesion to bleeding wounds or act as physical barriers against contaminants. Hence, it has become necessary to investigate biologically favorable materials that can be applied and left within the body post-surgery. In this study, a dual-sided nanofibrous dressing for topical hemostasis is electrospun using a combination of two protein materials: bioengineered mussel adhesive protein (MAP) and silk fibroin (SF). The wound-adhesive inner layer is fabricated using dihydroxyphenylalanine (DOPA)-containing MAP, which promotes blood clotting by aggregation of hemocytes and activation of platelets. The anti-adhesive outer layer is composed of alcohol-treated hydrophobic SF, which has excellent spinnability and mechanical strength for fabrication. Because both proteins are fully biodegradable in vivo and biocompatible, the dressing would be suitable to be left in the body. Through in vivo evaluation using a rat liver damage model, significantly reduced clotting time and blood loss are confirmed, successfully demonstrating that the proposed dual-sided nanofibrous dressing has the right properties and characteristics as a topical hemostatic agent having dual functionality of hemostasis and physical protection.

A potent immunotoxin targeting fibroblast activation protein for treatment of breast cancer in mice.

Fibroblast activation protein (FAP) is highly expressed in the tumor-associated fibroblasts (TAFs) of most human epithelial cancers. FAP plays a critical role in tumorigenesis and cancer progression, which makes it a promising target for novel anticancer therapy. However, mere abrogation of FAP enzymatic activity by small molecules is not very effective in inhibiting tumor growth. In this study, we have evaluated a novel immune-based approach to specifically deplete FAP-expressing TAFs in a mouse 4T1 metastatic breast cancer model. Depletion of FAP-positive stromal cells by FAP-targeting immunotoxin αFAP-PE38 altered levels of various growth factors, cytokines, chemokines and matrix metalloproteinases, decreased the recruitment of tumor-infiltrating immune cells in the tumor microenvironment and suppressed tumor growth. In addition, combined treatment with αFAP-PE38 and paclitaxel potently inhibited tumor growth in vivo. Our findings highlight the potential use of immunotoxin αFAP-PE38 to deplete FAP-expressing TAFs and thus provide a rationale for the use of this immunotoxin in cancer therapy.

Clickable protein nanocapsules for targeted delivery of recombinant p53 protein.

Encapsulating anticancer protein therapeutics in nanocarriers is an attractive option to minimize active drug destruction, increase local accumulation at the disease site, and decrease side effects to other tissues. Tumor-specific ligands can further facilitate targeting the nanocarriers to tumor cells and reduce nonspecific cellular internalization. Rationally designed non-covalent protein nanocapsules incorporating copper-free "click chemistry" moieties, polyethylene glycol (PEG) units, redox-sensitive cross-linker, and tumor-specific targeting ligands were synthesized to selectively deliver intracellular protein therapeutics into tumor cells via receptor-mediated endocytosis. These nanocapsules can be conjugated to different targeting ligands of choice, such as anti-Her2 antibody single-chain variable fragment (scFv) and luteinizing hormone releasing hormone (LHRH) peptide, resulting in specific and efficient accumulation within tumor cells overexpressing corresponding receptors. LHRH-conjugated nanocapsules selectively delivered recombinant human tumor suppressor protein p53 and its tumor-selective supervariant into targeted tumor cells, which led to reactivation of p53-mediated apoptosis. Our results validate a general approach for targeted protein delivery into tumor cells using cellular-responsive nanocarriers, opening up new opportunities for the development of intracellular protein-based anticancer treatment.

In situ modulation of dendritic cells by injectable thermosensitive hydrogels for cancer vaccines in mice.

Attempts to develop cell-based cancer vaccines have shown limited efficacy, partly because transplanted dendritic cells (DCs) do not survive long enough to reach the lymph nodes. The development of biomaterials capable of modulating DCs in situ to enhance antigen uptake and presentation has emerged as a novel method toward developing more efficient cancer vaccines. Here, we propose a two-step hybrid strategy to produce a more robust cell-based cancer vaccine in situ. First, a significant number of DCs are recruited to an injectable thermosensitive mPEG-PLGA hydrogel through sustained release of chemoattractants, in particular, granulocyte-macrophage colony-stimulating factor (GM-CSF). Then, these resident DCs can be loaded with cancer antigens through the use of viral or nonviral vectors. We demonstrate that GM-CSF-releasing mPEG-PLGA hydrogels successfully recruit and house DCs and macrophages, allowing the subsequent introduction of antigens by vectors to activate the resident cells, thus, initiating antigen presentation and triggering immune response. Moreover, this two-step hybrid strategy generates a high level of tumor-specific immunity, as demonstrated in both prophylactic and therapeutic models of murine melanoma. This injectable thermosensitive hydrogel shows great promise as an adjuvant for cancer vaccines, potentially providing a new approach for cell therapies through in situ modulation of cells.

Site-specific modification of adeno-associated viruses via a genetically engineered aldehyde tag.

As a consequence of their well-defined nanostructure and intrinsic bioactive functionality, virus-based nanoparticles have shown promise for mediating gene delivery. Adeno-associated virus (AAV) nanoparticles, which possess an excellent safety profile and therapeutic potential, hold potential for use in human gene therapy. However, because of their native tropisms, the applicability of AAV nanoparticles is often limited to restricted ranges of cells or tissues. Thus, retargeting AAV particles to the desired cell populations has continued to be a major research focus in many gene therapy applications. In this study, a general strategy is reported for nanoparticle targeting. This involves the site-specific modification of AAV type 2 (AAV2) by genetically incorporating a short peptide, in this case an aldehyde tag, in the viral capsid. Such a tag can be exploited for site-specific attachment of targeting molecules and allows for further introduction of targeting antibodies or ligands. It is shown that this modification neither affects the level of infectious viral titer nor intracellular trafficking properties. Furthermore, the site-specific conjugation of targeting antibodies could significantly enhance viral transduction to those target cells that have otherwise exhibited very low permissiveness to AAV2 infection. This method also allows the functional incorporation of RGD peptides onto AAV2 for enhanced delivery with implications for cancer gene therapy.

Visible light-crosslinkable tyramine-conjugated alginate-based microgel bioink for multiple cell-laden 3D artificial organ.

While the natural carbohydrate alginate has enabled effective three-dimensional (3D) extrusion bioprinting, it still suffers from some issues such as low printability and resolution and limited cellular function due to ionic crosslinking dependency. Here, we prepared a harmless visible light-based photocrosslinkable alginate by chemically bonding tyrosine-like residues onto alginate chains to propose a new microgel manufacturing system for the development of 3D-printed bioinks. The photocrosslinkable tyramine-conjugated alginate microgel achieved both higher cell viability and printing resolution compared to the bulk gel form. This alginate-based jammed granular microgel bioink showed excellent 3D bioprinting ability with maintained structural stability. As a biocompatible material, the developed multiple cell-loaded photocrosslinkable alginate-based microgel bioink provided excellent proliferation and migration abilities of laden living cells, providing an effective strategy to construct implantable functional artificial organ structures for 3D bioprinting-based tissue engineering.

Gene editing of human embryonic stem cells via an engineered baculoviral vector carrying zinc-finger nucleases.

Human embryonic stem (hES) cells are renewable cell sources that have potential applications in regenerative medicine. The development of technologies to produce permanent and site-specific genome modifications is in demand to achieve future medical implementation of hES cells. We report herein that a baculoviral vector (BV) system carrying zinc-finger nucleases (ZFNs) can successfully modify the hES cell genome. BV-mediated transient expression of ZFNs specifically disrupted the CCR5 locus in transduced cells and the modified cells exhibited resistance to HIV-1 transduction. To convert the BV to a gene targeting vector, a DNA donor template and ZFNs were incorporated into the vector. These hybrid vectors yielded permanent site-specific gene addition in both immortalized human cell lines (10%) and hES cells (5%). Modified hES cells were both karyotypically normal and pluripotent. These results suggest that this baculoviral delivery system can be engineered for site-specific genetic manipulation in hES cells.

Glycan chip based on structure-switchable DNA linker for on-chip biosynthesis of cancer-associated complex glycans.

On-chip glycan biosynthesis is an effective strategy for preparing useful complex glycan sources and for preparing glycan-involved applications simultaneously. However, current methods have some limitations when analyzing biosynthesized glycans and optimizing enzymatic reactions, which could result in undefined glycan structures on a surface, leading to unequal and unreliable results. In this work, a glycan chip is developed by introducing a pH-responsive i-motif DNA linker to control the immobilization and isolation of glycans on chip surfaces in a pH-dependent manner. On-chip enzymatic glycosylations are optimized for uniform biosynthesis of cancer-associated Globo H hexasaccharide and its related complex glycans through stepwise quantitative analyses of isolated products from the surface. Successful interaction analyses of the anti-Globo H antibody and MCF-7 breast cancer cells with on-chip biosynthesized Globo H-related glycans demonstrate the feasibility of the structure-switchable DNA linker-based glycan chip platform for on-chip complex glycan biosynthesis and glycan-involved applications.

Adhesive protein-based angiogenesis-mimicking spatiotemporal sequential release of angiogenic factors for functional regenerative medicine.

Damaged vascular structures after critical diseases are difficult to completely restore to their original conditions without specific treatments. Thus, therapeutic angiogenesis has been spotlighted as an attractive strategy. However, effective strategies for mimicking angiogenic processes in the body have not yet been developed. In the present work, we developed a bioengineered mussel adhesive protein (MAP)-based novel therapeutic angiogenesis platform capable of spatiotemporally releasing angiogenic growth factors to target disease sites with high viscosity and strong adhesiveness in a mucus-containing environment with curvature. Polycationic MAP formed complex coacervate liquid microdroplets with polyanionic hyaluronic acid and subsequently gelated into microparticles. Platelet-derived growth factor (PDGF), which is a late-phase angiogenic factor, was efficiently encapsulated during the process of coacervate microparticle formation. These PDGF-loaded microparticles were blended with vascular endothelial growth factor (VEGF), which is the initial-phase angiogenic factor, in MAP-based pregel solution and finally crosslinked in situ into a hydrogel at the desired site. The microparticle-based angiogenic-molecule spatiotemporal sequential (MASS) release platform showed good adhesion and underwater durability, and its elasticity was close to that of target tissue. Using two in vivo critical models, i.e., full-thickness excisional wound and myocardial infarction models, the MASS release platform was evaluated for its in vivo feasibility as an angiogenesis-inducing platform and demonstrated effective angiogenesis as well as functional regenerative efficacy. Based on these superior physicochemical characteristics, the developed MASS release platform could be successfully applied in many biomedical practices as a waterproof bioadhesive with the capability for the spatiotemporal delivery of angiogenic molecules in the treatment of ischemic diseases.

Tunicate-Inspired Photoactivatable Proteinic Nanobombs for Tumor-Adhesive Multimodal Therapy.

Near-IR (NIR) light-responsive multimodal nanotherapeutics have been proposed to achieve improved therapeutic efficacy and high specificity in cancer therapy. However, their clinical application is still elusive due to poor biometabolization and short retention at the target site. Here, innovative photoactivatable vanadium-doped adhesive proteinic nanoparticles (NPs) capable of allowing biological photoabsorption and NIR-responsive anticancer therapeutic effects to realize trimodal photothermal-gas-chemo-therapy treatments in a highly biocompatible, site-specific manner are proposed. The photoactivatable tumor-adhesive proteinic NPs can enable efficient photothermal conversion via tunicate-inspired catechol-vanadium complexes as well as prolonged tumor retention by virtue of mussel protein-driven distinctive adhesiveness. The incorporation of a thermo-sensitive nitric oxide donor and doxorubicin into the photoactivatable adhesive proteinic NPs leads to synergistic anticancer therapeutic effects as a result of photothermal-triggered "bomb-like" multimodal actions. Thus, this protein-based phototherapeutic tumor-adhesive NPs have great potential as a spatiotemporally controllable therapeutic system to accomplish effective therapeutic implications for the complete ablation of cancer.

Double-layered adhesive microneedle bandage based on biofunctionalized mussel protein for cardiac tissue regeneration.

Heart failure following myocardial infarction (MI), the primary cause of mortality worldwide, is the consequence of cardiomyocyte death or dysfunction. Clinical efforts involving the delivery of growth factors (GFs) and stem cells with the aim of regenerating cardiomyocytes for the recovery of structural and functional integrity have largely failed to deliver, mainly due to short half-lives and rapid clearance in in vivo environments. In this work, we selected and genetically fused four biofunctional peptides possessing angiogenic potential, originating from extracellular matrix proteins and GFs, to bioengineered mussel adhesive protein (MAP). We found that MAPs fused with vascular endothelial growth factor (VEGF)-derived peptide and fibronectin-derived RGD peptide significantly promoted the proliferation and migration of endothelial cells in vitro. Based on these characteristics, we fabricated advanced double-layered adhesive microneedle bandages (DL-AMNBs) consisting of a biofunctional MAP-based root and a regenerated silk fibroin (SF)-based tip, allowing homogeneous distribution of the regenerative factor via swellable microneedles. Our developed DL-AMNB system clearly demonstrated better preservation of cardiac muscle and regenerative effects on heart remodeling in a rat MI model, which might be attributed to the prolonged retention of therapeutic peptides as well as secure adhesion between the patch and host myocardium by MAP-inherent strong underwater adhesiveness.

Embolization of Vascular Malformations via In Situ Photocrosslinking of Mechanically Reinforced Alginate Microfibers using an Optical-Fiber-Integrated Microfluidic Device.

Embolization, which is a minimally invasive endovascular treatment, is a safe and effective procedure for treating vascular malformations (e.g., aneurysms). Hydrogel microfibers obtained via spatiotemporally controllable in situ photocrosslinking exhibit great potential for embolizing aneurysms. However, this process is challenging because of the absence of biocompatible and morphologically stable hydrogels and the difficulty in continuously spinning the microfibers via in situ photocrosslinking in extreme endovascular environments such as those involving a tortuous geometry and high absorbance. A double-crosslinked alginate-based hydrogel with tantalum nanopowder (DAT) that exploits the synergistic effect of covalent crosslinking by visible-light irradiation and ionic crosslinking using Ca2+ , which is present in the blood, is developed in this study. Furthermore, an effective strategy to design and produce an optical-fiber-integrated microfluidic device (OFI-MD) that can continuously spin hydrogel microfibers via in situ photocrosslinking in extreme endovascular environments is proposed. As an embolic material, DAT exhibits promising characteristics such as radiopacity, nondissociation, nonswelling, and constant mechanical strength in blood, in addition to excellent cyto- and hemo-compatibilities. Using OFI-MD to spin DAT microfibers continuously can help fill aneurysms safely, uniformly, and completely within the endovascular simulator without generating microscopic fragments, which demonstrates its potential as an effective embolization strategy.

Sutureless Transplantation of Amniotic Membrane Using a Visible Light-Curable Protein Bioadhesive for Ocular Surface Reconstruction.

The conjunctiva is a thin mucous membrane of the eye. Pterygium, a commonly appearing disease on the ocular surface, requires surgery to excise the conjunctiva to prevent visual deterioration. Recently, transplantation of the amniotic membrane (AM), which is the innermost membrane of the placenta, has been highlighted as an efficient method to cure conjunctiva defects because of its advantages of no side effects compared to mitomycin C treatment and not leaving additional scars on donor site compared to conjunctival autografting. However, to minimize additional damage to the ocular surface by suturing, AM transplantation (AMT) needs to be simplified by using a less invasive, time-saving method. In this work, a visible light-curable protein bioadhesive (named FixLight) for efficient sutureless AMT is applied. FixLight, which is based on bioengineered mussel adhesive protein (MAP), is easily applied between damaged ocular surfaces and transplanted AM, and rapidly cured by harmless blue light activation. Through in vivo evaluation using a rabbit model, the authors demonstrated that FixLight enabled facile, fast, and strong attachment of AM on sclera and promoted ocular surface reconstruction with good biocompatibility. Thus, FixLight can be successfully used as a promising clinical bioadhesive in opthalmological surgeries that require sutureless and rapid operation.

Preclinical evaluation of a regenerative immiscible bioglue for vesico-vaginal fistula.

Currently, there are no clinically available tissue adhesives that work effectively in the fluid-rich and highly dynamic environments of the human body, such as the urinary system. This is especially relevant to the management of vesico-vaginal fistula, and developing a high-performance tissue adhesive for this purpose could vastly expand urologists' surgical repertoire and dramatically reduce patient discomfort. Herein, we developed a water-immiscible mussel protein-based bioadhesive (imWIMBA) with significantly improved properties in all clinical respects, allowing it to achieve rapid and strong underwater adhesion with tunable rheological properties. We evaluated in vivo potential of imWIMBA for sealing thermally injured fistula tracts between the bladder and vagina. Importantly, the use of imWIMBA in the presence of prolonged bladder drainage resulted in perfect closure of the vesico-vaginal fistula in operated pigs. Thus, imWIMBA could be successfully used for many surgical applications and improve treatment efficacy when combined with conventional surgical methods. STATEMENT OF SIGNIFICANCE: Vesico-vaginal fistula (VVF) is an abnormal opening between the bladder and the vagina, which is a stigmatized disease in many developing countries. Leakage of urine into internal organs can induce serious complications and delay wound repair. Conventional VVF treatment requires skillful suturing to provide a tension-free and watertight closure. In addition, there is no clinically approved surgical glue that works in wet and highly dynamic environments such as the urinary system. In this work, for potential clinical VVF closure and regeneration, we developed an advanced immiscible mussel protein-based bioglue with fast, strong, wet adhesion and tunable rheological properties. This regenerative immiscible bioglue could be successfully used for sealing fistulas and further diverse surgical applications as an adjuvant for conventional suture methods.

Bone Graft Biomineral Complex Coderived from Marine Biocalcification and Biosilicification.

Bone graft materials have been mainly developed based on inorganic materials, including calcium phosphate. However, these graft materials usually act as osteoconductive rather than osteoinductive scaffolds. To improve bone reconstruction, a combination of several materials has been proposed. However, there are still no alternatives that can completely replace the existing animal-derived bone graft materials. In this work, a marine-inspired biomineral complex was suggested as a potential bone graft material. The proposed biosilicified coccolithophore-derived coccoliths using bioengineered mussel adhesive proteins show osteopromotive ability through the synergistic effects of osteoconductivity from calcium carbonate and osteoinductivity from silica. Its possibility of use as a bone substitute was determined by evaluating the in vitro osteogenic behaviors of multipotent mesenchymal stem cells and in vivo bone regeneration in a rat calvarial defect model. Therefore, the marine-inspired biomineral complex developed in this study could be successfully used for bone tissue engineering.

Targeting lentiviral vector to specific cell types through surface displayed single chain antibody and fusogenic molecule.

BACKGROUND: Viral delivery remains one of the most commonly used techniques today in the field of gene therapy. However, one of the remaining hurdles is the off-targeting effect of viral delivery. To overcome this obstacle, we recently developed a method to incorporate an antibody and a fusogenic molecule (FM) as two distinct molecules into the lentiviral surface. In this report, we expand this strategy to utilize a single chain antibody (SCAb) for targeted transduction. RESULTS: Two versions of the SCAb were generated to pair with our various engineered FMs by linking the heavy chain and the light chain variable domains of the anti-CD20 antibody (alphaCD20) via a GS linker and fusing them to the hinge-CH2-CH3 region of human IgG. The resulting protein was fused to either a HLA-A2 transmembrane domain or a VSVG transmembrane domain for anchoring purpose. Lentiviral vectors generated with either version of the SCAb and a selected FM were then characterized for binding and fusion activities in CD20-expressing cells. CONCLUSION: Certain combinations of the SCAb with various FMs could result in an increase in viral transduction. This two-molecule lentiviral vector system design allows for parallel optimization of the SCAb and FMs to improve targeted gene delivery.

Protein nanocapsule weaved with enzymatically degradable polymeric network.

Target proteins can be functionally encapsulated using a cocoon-like polymeric nanocapsule formed by interfacial polymerization. The nanocapsule is cross-linked by peptides that can be proteolyzed by proteases upon which the protein cargo is released. The protease-mediated degradation process can be controlled in a spatiotemporal fashion through modification of the peptide cross-linker with photolabile moieties. We demonstrate the utility of this approach through the cytoplasmic delivery of the apoptosis inducing caspase-3 to cancer cells.

Body temperature-activated protein-based injectable adhesive hydrogel incorporated with decellularized adipose extracellular matrix for tissue-specific regenerative stem cell therapy.

Adipose tissue engineering represents a valuable alternative for reconstructive and cosmetic applications to restore soft tissue loss. Herein, for the development of a tissue-engineered adipose substitute, we designed an injectable thermoresponsive tissue adhesive hydrogel by grafting bioengineered mussel adhesive protein (MAP) with poly(N-isopropylacrylamide) (PNIPAM) and incorporating decellularized adipose tissue (DAT) powder as a biochemical cue. The body temperature-activated PNIPAM-grafted MAP (MAP-PNIPAM) hydrogel showed 3.2-times higher water retention ability, high porosity, and 8.4-times stronger tissue adhesive properties compared to the PNIPAM gel alone with pore collapse. Moreover, we found that the introduction of 5 wt% DAT powder had adipo-inductive and adipo-conductive effects, which might be due to the provision of biochemical substrates enriched in collagen and laminin for cell-cell and cell-matrix interactions. In vivo subcutaneous injection of the adipose-derived stem cell-laden DAT-incorporated MAP-PNIPAM hydrogel further demonstrated better volume maintenance, angiogenesis, and lipid accumulation than control injectable alginate gel or DAT powder only. Collectively, our injectable body temperature-activated tissue adhesive MAP-PNIPAM hydrogel system with a decellularized extracellular matrix source can be utilized as a promising alternative for tissue-specific regenerative stem cell therapy. STATEMENT OF SIGNIFICANCE: For adipose tissue engineering, we designed an injectable body temperature-activated adhesive hydrogel by grafting bioengineered mussel adhesive protein (MAP) with poly(N-isopropylacrylamide) (PNIPAM) and incorporating adipose-derived stem cells (ASCs) and decellularized adipose tissue (DAT) powder as regenerative cell and ECM sources. PNIPAM has been widely used for cell sheet engineering, but not for cell carriers due to its dramatic thermal contractive properties. By conjugation with hydrophilic MAP, water retention ability and tissue adhesiveness of the scaffold increased by a factor of 3.2- and 8.4-fold, respectively, which are highly required for survival of the transplanted cells and interfacial integration with host tissues. In vivo performance demonstrated that ASCs/DAT powder-laden MAP-PNIPAM hydrogel achieved better volume maintenance, neovascularization, and adipogenesis than control injectable groups.

Stability-Controllable Self-Immobilization of Carbonic Anhydrase Fused with a Silica-Binding Tag onto Diatom Biosilica for Enzymatic CO2 Capture and Utilization.

Exploiting carbonic anhydrase (CA), an enzyme that catalyzes the hydration of CO2, is a powerful route for eco-friendly and cost-effective carbon capture and utilization. For successful industrial applications, the stability and reusability of CA should be improved, which necessitates enzyme immobilization. Herein, the ribosomal protein L2 (Si-tag) from Escherichia coli was utilized for the immobilization of CA onto diatom biosilica, a promising renewable support material. The Si-tag was redesigned (L2NC) and genetically fused to CA from the marine bacterium Hydrogenovibrio marinus (hmCA). One-step self-immobilization of hmCA-L2NC onto diatom biosilica by simple mixing was successfully achieved via Si-tag-mediated strong binding, showing multilayer adsorption with a maximal loading of 1.4 wt %. The immobilized enzyme showed high reusability and no enzyme leakage even under high temperature conditions. The activity of hmCA-L2NC was inversely proportional to the enzyme loading, while the stability was directly proportional to the enzyme loading. This discovered activity-stability trade-off phenomenon could be attributed to macromolecular crowding on the highly dense surface of the enzyme-immobilized biosilica. Collectively, our system not only facilitates the stability-controllable self-immobilization of enzyme via Si-tag on a diatom biosilica support for the robust, facile, and green construction of stable biocatalysts, but is also a unique model for studying the macromolecular crowding effect on surface-immobilized enzymes.

Reusability Comparison of Melt-Blown vs Nanofiber Face Mask Filters for Use in the Coronavirus Pandemic.

Shortage of face masks is a current critical concern since the emergence of coronavirus-2 or SARS-CoV-2 (COVID-19). In this work, we compared the melt-blown (MB) filter, which is commonly used for the N95 face mask, with nanofiber (NF) filter, which is gradually used as an effective mask filter, to evaluate their reusability. Extensive characterizations were performed repeatedly to evaluate some performance parameters, which include filtration efficiency, airflow rate, and surface and morphological properties, after two types of cleaning treatments. In the first cleaning type, samples were dipped in 75% ethanol for a predetermined duration. In the second cleaning type, 75% ethanol was sprayed on samples. It was found that filtration efficiency of MB filter was significantly dropped after treatment with ethanol, while the NF filter exhibited consistent high filtration efficiency regardless of cleaning types. In addition, the NF filter showed better cytocompatibility than the MB filter, demonstrating its harmlessness on the human body. Regardless of ethanol treatments, surfaces of both filter types maintained hydrophobicity, which can sufficiently prevent wetting by moisture and saliva splash to prohibit not only pathogen transmission but also bacterial growth inside. On the basis of these comparative evaluations, the wider use of the NF filter for face mask applications is highly recommended, and it can be reused multiple times with robust filtration efficiency. It would be greatly helpful to solve the current shortage issue of face masks and significantly improve safety for front line fighters against coronavirus disease.