M1-polarized macrophage-derived cellular nanovesicle-coated lipid nanoparticles for enhanced cancer treatment through hybridization of gene therapy and cancer immunotherapy.

Optimum genetic delivery for modulating target genes to diseased tissue is a major obstacle for profitable gene therapy. Lipid nanoparticles (LNPs), considered a prospective vehicle for nucleic acid delivery, have demonstrated efficacy in human use during the COVID-19 pandemic. This study introduces a novel biomaterial-based platform, M1-polarized macrophage-derived cellular nanovesicle-coated LNPs (M1-C-LNPs), specifically engineered for a combined gene-immunotherapy approach against solid tumor. The dual-function system of M1-C-LNPs encapsulates Bcl2-targeting siRNA within LNPs and immune-modulating cytokines within M1 macrophage-derived cellular nanovesicles (M1-NVs), effectively facilitating apoptosis in cancer cells without impacting T and NK cells, which activate the intratumoral immune response to promote granule-mediating killing for solid tumor eradication. Enhanced retention within tumor was observed upon intratumoral administration of M1-C-LNPs, owing to the presence of adhesion molecules on M1-NVs, thereby contributing to superior tumor growth inhibition. These findings represent a promising strategy for the development of targeted and effective nanoparticle-based cancer genetic-immunotherapy, with significant implications for advancing biomaterial use in cancer therapeutics.

The TOX-RAGE axis mediates inflammatory activation and lung injury in severe pulmonary infectious diseases.

Thymocyte selection-associated high-mobility group box (TOX) is a transcription factor that is crucial for T cell exhaustion during chronic antigenic stimulation, but its role in inflammation is poorly understood. Here, we report that TOX extracellularly mediates drastic inflammation upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by binding to the cell surface receptor for advanced glycation end-products (RAGE). In various diseases, including COVID-19, TOX release was highly detectable in association with disease severity, contributing to lung fibroproliferative acute respiratory distress syndrome (ARDS). Recombinant TOX-induced blood vessel rupture, similar to a clinical signature in patients experiencing a cytokine storm, further exacerbating respiratory function impairment. In contrast, disruption of TOX function by a neutralizing antibody and genetic removal of RAGE diminished TOX-mediated deleterious effects. Altogether, our results suggest an insight into TOX function as an inflammatory mediator and propose the TOX-RAGE axis as a potential target for treating severe patients with pulmonary infection and mitigating lung fibroproliferative ARDS.

Enhanced Postsurgical Cancer Treatment Using Methacrylated Glycol Chitosan Hydrogel for Sustained DNA/Doxorubicin Delivery and Immunotherapy.

Background: Cancer recurrence and metastasis are major contributors to treatment failure following tumor resection surgery. We developed a novel implantable drug delivery system utilizing glycol chitosan to address these issues. Glycol chitosan is a natural adjuvant, inducing dendritic cell activation to promote T helper 1 cell immune responses, macrophage activation, and cytokine production. Effective antigen production by dendritic cells initiates T-cell-mediated immune responses, aiding tumor growth control. Methods: In this study, we fabricated multifunctional methacrylated glycol chitosan (MGC) hydrogels with extended release of DNA/doxorubicin (DOX) complex for cancer immunotherapy. We constructed the resection model of breast cancer to verify the anticancer effects of MGC hydrogel with DNA/DOX complex. Results: This study demonstrated the potential of MGC hydrogel with extended release of DNA/DOX complex for local and efficient cancer therapy. The MGC hydrogel was implanted directly into the surgical site after tumor resection, activating tumor-related immune cells both locally and over a prolonged period of time through immune-reactive molecules. Conclusions: The MGC hydrogel effectively suppressed tumor recurrence and metastasis while enhancing immunotherapeutic efficacy and minimizing side effects. This biomaterial-based drug delivery system, combined with cancer immunotherapy, can substantial improve treatment outcomes and patient prognosis.

Secured delivery of basic fibroblast growth factor using human serum albumin-based protein nanoparticles for enhanced wound healing and regeneration.

BACKGROUND: Basic fibroblast growth factor (bFGF) is one of the critical components accelerating angiogenesis and tissue regeneration by promoting the migration of dermal fibroblasts and endothelial cells associated with matrix formation and remodeling in wound healing process. However, clinical applications of bFGF are substantially limited by its unstable nature due to rapid decomposition under physiological microenvironment. RESULTS: In this study, we present the bFGF-loaded human serum albumin nanoparticles (HSA-bFGF NPs) as a means of enhanced stability and sustained release platform during tissue regeneration. Spherical shape of the HSA-bFGF NPs with uniform size distribution (polydispersity index < 0.2) is obtained via a simple desolvation and crosslinking process. The HSA-bFGF NPs securely load and release the intact soluble bFGF proteins, thereby significantly enhancing the proliferation and migration activity of human dermal fibroblasts. Myofibroblast-related genes and proteins were also significantly down-regulated, indicating decrease in risk of scar formation. Furthermore, wound healing is accelerated while achieving a highly organized extracellular matrix and enhanced angiogenesis in vivo. CONCLUSION: Consequently, the HSA-bFGF NPs are suggested not only as a delivery vehicle but also as a protein stabilizer for effective wound healing and tissue regeneration.

Enhancement of anti-tumor activity in melanoma using arginine deiminase fused with 30Kc19α protein.

Arginine deiminase (ADI) is a microbial-derived enzyme which catalyzes the conversion of L-arginine into L-citrulline. ADI originating from Mycoplasma has been reported to present anti-tumor activity against arginine-auxotrophic tumors, including melanoma. Melanoma cells are sensitive to arginine depletion due to reduced expression of argininosuccinate synthase 1 (ASS1), a key enzyme for arginine biosynthesis. However, clinical applications of recombinant ADI for melanoma treatment present some limitations. Since recombinant ADI is not human-derived, it shows instability, proteolytic degradation, and antigenicity in human serum. In addition, there is a problem of drug resistance issue due to the intracellular expression of once-silenced ASS1. Moreover, recombinant ADI proteins are mainly expressed as inclusion body forms in Escherichia coli and require a time-consuming refolding process to turn them back into active form. Herein, we propose fusion of recombinant ADI from Mycoplasma hominis and 30Kc19α, a cell-penetrating protein which also increases stability and soluble expression of cargo proteins, to overcome these problems. We inserted matrix metalloproteinase-2 cleavable linker between ADI and 30Kc19α to increase enzyme activity in melanoma cells. Compared to ADI, ADI-LK-30Kc19α showed enhanced solubility, stability, and cell penetration. The fusion protein demonstrated selective cytotoxicity and reduced drug resistance in melanoma cells, thus would be a promising strategy for the improved efficacy in melanoma treatment. KEY POINTS: • Fusion of ADI with 30Kc19α enhances soluble expression and productivity of recombinant ADI in E. coli • 30Kc19α protects ADI from the proteolytic degradation by shielding effect, helping ADI to remain active • Intracellular delivery of ADI by 30Kc19α overcomes ADI resistance in melanoma cells by degrading intracellularly expressed arginine.

Enhanced anti-cancer effect using MMP-responsive L-asparaginase fused with cell-penetrating 30Kc19 protein.

As the acute lymphoblastic leukaemia (ALL) develops, expression of L-asparaginase (ASNase) protein is known to decrease. Therefore, deficiency of the ASNase protein would be regarded as one of the significant indications of the ALL. For the treatment of ALL, recombinant ASNase protein derived from bacterial origin is used which causes cytotoxicity by deprivation of Asn. However, short half-life of the protein is an obstacle for medical use. In order to overcome this limit, recombinant ASNase was fused to 30Kc19 with protein-stabilizing and cell-penetrating properties. As the 30Kc19 protein may induce steric hindrance, we further added a PLGLAG linker sequence (LK) between the ASNase and 30Kc19. The treatment of ASNase-LK-30Kc19 fusion protein demonstrated enhanced stability, cell-penetrating property, and anti-cancer activity. Intracellular delivery of both the non-cleaved and cleaved forms of the protein were observed, suggesting that ASNase acted both internally and externally, performing high anti-cancer activity by effective depletion of intracellular Asn. Additionally, ASNase-LK-30Kc19 showed high selectivity towards cancer cells. In terms of the dosage, releasable ASNase from ASNase-LK-30Kc19 reached the same half-maximal inhibitory concentration at a concentration five times lower than non-releasable ASNase-30Kc19. Altogether, the findings suggest that this fusion approach has potential applications in the treatment of ALL.

Cleavage-Responsive Biofactory T Cells Suppress Infectious Diseases-Associated Hypercytokinemia.

Severe infectious diseases, such as coronavirus disease 2019 (COVID-19), can induce hypercytokinemia and multiple organ failure. In spite of the growing demand for peptide therapeutics against infectious diseases, current small molecule-based strategies still require frequent administration due to limited half-life and enzymatic digestion in blood. To overcome this challenge, a strategy to continuously express multi-level therapeutic peptide drugs on the surface of immune cells, is established. Here, chimeric T cells stably expressing therapeutic peptides are presented for treatment of severe infectious diseases. Using lentiviral system, T cells are engineered to express multi-level therapeutic peptides with matrix metallopeptidases- (MMP-) and tumor necrosis factor alpha converting enzyme- (TACE-) responsive cleavage sites on the surface. The enzymatic cleavage releases γ-carboxyglutamic acid of protein C (PC-Gla) domain and thrombin receptor agonist peptide (TRAP), which activate endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1), respectively. These chimeric T cells prevent vascular damage in tissue-engineered blood vessel and suppress hypercytokinemia and lung tissue damages in vivo, demonstrating promise for use of engineered T cells against sepsis and other infectious-related diseases.

Nanocomplex-Mediated In Vivo Programming to Chimeric Antigen Receptor-M1 Macrophages for Cancer Therapy.

Chimeric antigen receptor-T (CAR-T) cell immunotherapy has shown impressive clinical outcomes for hematologic malignancies. However, its broader applications are challenged due to its complex ex vivo cell-manufacturing procedures and low therapeutic efficacy against solid tumors. The limited therapeutic effects are partially due to limited CAR-T cell infiltration to solid tumors and inactivation of CAR-T cells by the immunosuppressive tumor microenvironment. Here, a facile approach is presented to in vivo program macrophages, which can intrinsically penetrate solid tumors, into CAR-M1 macrophages displaying enhanced cancer-directed phagocytosis and anti-tumor activity. In vivo injected nanocomplexes of macrophage-targeting nanocarriers and CAR-interferon-γ-encoding plasmid DNA induce CAR-M1 macrophages that are capable of CAR-mediated cancer phagocytosis, anti-tumor immunomodulation, and inhibition of solid tumor growth. Together, this study describes an off-the-shelf CAR-macrophage therapy that is effective for solid tumors and avoids the complex and costly processes of ex vivo CAR-cell manufacturing.

Evaluation of the anti-oxidative and ROS scavenging properties of biomaterials coated with epigallocatechin gallate for tissue engineering.

In tissue engineering, excessively generated reactive oxygen species (ROS) during biomaterial implantation or cell transplantation is a one of major causes of diminishing therapeutic effects. In this study, we prepared biomaterial surfaces coated with antioxidant epigallocatechin gallate (EGCG) and metal ions, and evaluated their anti-oxidative and ROS scavenging properties. We revealed that EGCG-coating on polycaprolactone (PCL) film surface increased hydrophilicity and anti-oxidative properties as a function of total phenol content (TPC) potentially due to the increase in phenolic -OH and π-electrons from structural maintenance and directly removed the hydrogen peroxide (H2O2) by resonance-stabilization. Furthermore, EGCG-coated PCL film increased attachment, spreading area, and viability of human adipose-derived stem cells (hADSCs) against H2O2 treatment while stimulated the cellular signaling to reduce apoptotic gene and enhance anti-oxidative enzyme expression. Further, we applied EGCG coating on the surface of poly-L-lactic acid (PLLA) fibers. Spheroids incorporating EGCG-coated PLLA fibers were able to maintain their shape and showed improved viability and anti-oxidative activities in response to H2O2-induced oxidative stress than control spheroids. Therefore, metal-phenolic network (MPN) coating of EGCG is a suitable method to impart the anti-oxidative properties to biomaterials by evaluating the structural properties and biological effects.

Stem Cell-Derived Exosomes and Nanovesicles: Promotion of Cell Proliferation, Migration, and Anti-Senescence for Treatment of Wound Damage and Skin Ageing.

Extracellular vesicles (EVs), such as exosomes, are nano-sized vesicles derived from endocytic membranes and contain biomolecules such as proteins, lipids, RNAs, and DNAs for the transfer of signals to recipient cells, playing significant roles in cell-to-cell communication. Discovery of exosomes has attracted attention for possible use as next generation therapies in clinical applications; however, several studies suggest that cells secrete exosomes that perform as mediators in the tumor niche and play several roles in tumorigenesis, angiogenesis, and metastasis. Recently, stem cell-derived exosomes have been suggested as a desirable source for regenerative medicine due to their roles in the promotion of angiogenesis via migratory and proliferative mechanisms. This review is aimed at demonstrating the present knowledge of stem cell-derived exosomes and cell-engineered nanovesicles (CNVs) as proliferative, migratory, and anti-senescent therapeutic biomaterial for use in tissue regeneration; wound healing and anti-ageing are explained. We conclude this review by discussing the future perspectives of stem cell-derived exosomes and CNVs as a platform in therapeutic strategies for treatment of wound damage and skin aging.

COVID-19-activated SREBP2 disturbs cholesterol biosynthesis and leads to cytokine storm.

Sterol regulatory element binding protein-2 (SREBP-2) is activated by cytokines or pathogen, such as virus or bacteria, but its association with diminished cholesterol levels in COVID-19 patients is unknown. Here, we evaluated SREBP-2 activation in peripheral blood mononuclear cells of COVID-19 patients and verified the function of SREBP-2 in COVID-19. Intriguingly, we report the first observation of SREBP-2 C-terminal fragment in COVID-19 patients' blood and propose SREBP-2 C-terminal fragment as an indicator for determining severity. We confirmed that SREBP-2-induced cholesterol biosynthesis was suppressed by Sestrin-1 and PCSK9 expression, while the SREBP-2-induced inflammatory responses was upregulated in COVID-19 ICU patients. Using an infectious disease mouse model, inhibitors of SREBP-2 and NF-κB suppressed cytokine storms caused by viral infection and prevented pulmonary damages. These results collectively suggest that SREBP-2 can serve as an indicator for severity diagnosis and therapeutic target for preventing cytokine storm and lung damage in severe COVID-19 patients.

Protein-Based Nanoparticles as Drug Delivery Systems.

Nanoparticles have been extensively used as carriers for the delivery of chemicals and biomolecular drugs, such as anticancer drugs and therapeutic proteins. Natural biomolecules, such as proteins, are an attractive alternative to synthetic polymers commonly used in nanoparticle formulation because of their safety. In general, protein nanoparticles offer many advantages, such as biocompatibility and biodegradability. Moreover, the preparation of protein nanoparticles and the corresponding encapsulation process involved mild conditions without the use of toxic chemicals or organic solvents. Protein nanoparticles can be generated using proteins, such as fibroins, albumin, gelatin, gliadine, legumin, 30Kc19, lipoprotein, and ferritin proteins, and are prepared through emulsion, electrospray, and desolvation methods. This review introduces the proteins used and methods used in generating protein nanoparticles and compares the corresponding advantages and disadvantages of each.

Intracellular Delivery of Recombinant RUNX2 Facilitated by Cell-Penetrating Protein for the Osteogenic Differentiation of hMSCs.

Human mesenchymal stem cells (hMSCs) are a commonly used cell source for cell therapy and tissue engineering because of their easy accessibility and multipotency. Runt-related transcription factor 2 (RUNX2) is a master regulator of the osteogenic commitment of hMSCs. Either recombinant plasmid delivery or viral transduction has been utilized to activate RUNX2 gene expression for effective hMSC differentiation. In this study, recombinant RUNX2 fused with cell-penetrating 30Kc19α protein (30Kc19α-RUNX2) was delivered into hMSCs for osteogenic commitment. Fusion of recombinant RUNX2 with 30Kc19α resulted in successful delivery of the protein into cells and enhanced soluble expression of the protein. Intracellular delivery of the 30Kc19α-RUNX2 fusion protein enhanced the osteogenic differentiation of hMSCs in vitro. 30Kc19α-RUNX2 treatment resulted in increased ALP accumulation and elevated calcium deposition. Finally, implantation of hMSCs treated with 30Kc19α-RUNX2 showed osteogenesis via cell delivery into the subcutaneous tissue and bone regeneration in a cranial defect mouse model. Therefore, we suggest that 30Kc19α-RUNX2, an osteoinductive recombinant protein, is an efficient tool for bone tissue engineering.

α-Galactosidase delivery using 30Kc19-human serum albumin nanoparticles for effective treatment of Fabry disease.

Fabry disease is a genetic lysosomal storage disease caused by deficiency of α-galactosidase, the enzyme-degrading neutral glycosphingolipid that is transported to lysosome. Glycosphingolipid accumulation by this disease causes multi-organ dysfunction and premature death of the patient. Currently, enzyme replacement therapy (ERT) using recombinant α-galactosidase is the only treatment available for Fabry disease. To maximize the efficacy of treatment, enhancement of cellular delivery and enzyme stability is a challenge in ERT using α-galactosidase. In this study, protein nanoparticles using human serum albumin (HSA) and 30Kc19 protein, originating from silkworm, were used to enhance the delivery and intracellular α-galactosidase stability. 30Kc19-HSA nanoparticles loaded with the α-galactosidase were formed by desolvation method. 30Kc19-HSA nanoparticles had a uniform spherical shape and were well dispersed in cell culture media. 30Kc19-HSA nanoparticles had negligible toxicity to human cells. The nanoparticles exhibited enhanced cellular uptake and intracellular stability of delivered α-galactosidase in human foreskin fibroblast. Additionally, they showed enhanced globotriaosylceramide degradation in Fabry patients' fibroblasts. It is expected that 30Kc19-HSA protein nanoparticles could be used as an effective tool for efficient delivery and enhanced stability of drugs.

Stabilization of cellular mitochondrial enzyme complex and sialyltransferase activity through supplementation of 30Kc19 protein.

In previous studies, 30Kc19, a lipoprotein in silkworm hemolymph, enhanced productivity and glycosylation by expression of a 30Kc19 gene or supplementation with a recombinant 30Kc19 protein. Additionally, 30Kc19 exhibited enzyme-stabilizing and cell-penetrating abilities in vitro. In this study, we hypothesized that supplemented 30Kc19 penetrated into the cell and enhanced the stability of the cellular enzyme. We investigated this using in vitro and cellular assessments. The activity of sialyltransferase (ST) and isolated mitochondrial complex I/III was enhanced with 30Kc19 in dose-dependent manner while initial reaction rate was unchanged, suggesting that 30Kc19 enhanced enzyme stability rather than specific activity. For intracellular enzyme activity assessment, ST activity inside erythropoietin (EPO)-producing Chinese hamster ovary (CHO) cells increased more than 25 % and mitochondrial complex II activity in HeLa cells increased more than 50 % with 30Kc19. The increase in intracellular ST activity resulted in an increase in sialic acid content of glycoproteins produced in CHO cells supplemented with 30Kc19. Similarly, enhanced mitochondrial complex activity increased mitochondrial membrane potential and ATP production in HeLa cells with 30Kc19 by over 50 %. Because 30Kc19 stabilized intracellular enzymes for glycosylation and enhanced protein productivity with supplementation in the culture medium, we expect that 30Kc19 can be a valuable tool for effective industrial recombinant protein production.

Dimerization of 30Kc19 protein in the presence of amphiphilic moiety and importance of Cys-57 during cell penetration.

Recently, the recombinant 30Kc19 protein, originating from silkworm hemolymph of Bombyx mori has attracted attention due to its cell-penetrating property and potential application as a protein delivery system. However, this observation of penetration across cell membrane has raised questions concerning the interaction of the protein-lipid bilayer. Here, we report a dimerization propensity of the 30Kc19 protein in the presence of amphiphilic moieties; sodium dodecyl sulfate (SDS) or phospholipid. Native PAGE showed that the 30Kc19 monomer formed a dimer when SDS or phospholipid was present. In the glutathione-S-transferase (GST) pull-down assay, supplementation of the 30Kc19 protein to mammalian cell culture medium showed dimerization and penetration; due to phospholipids at the cell membrane, the main components of the lipid bilayer. Mutagenesis was performed, and penetration was observed by 30Kc19 C76A and not 30Kc19 C57A, which meant that the presence of cysteine at position 57 (Cys-57) is involved in dimerization of the 30Kc19 at the cell membrane during penetration. We anticipate application of the native 30Kc19 protein with high cell-penetrating efficiency for delivery of cargos to various cell types. The intracellular cargo delivery using the 30Kc19 protein is a non-virus derived (e.g. TAT) delivery method, which can open up new approaches for the delivery of therapeutics in bioindustries, such as pharma- and cosmeceuticals.

Microfluidic bead-based sensing platform for monitoring kinase activity.

Protein kinases control cellular functions by regulating protein phosphorylation. Monitoring protein kinase activity is essential for medical diagnosis and drug screening. Here, we present a novel microfluidic device for performing simple and versatile protein kinase assays, which utilizes a microbead-based chemosensor. An automatic mix-and-measure technique was achieved using integrated pneumatic valves. After mixing each reagent for the kinase assay, the mixture was transferred to the sensing chamber. Then, phosphorylated and fluorescence-labeled peptides were captured and detected by the chemosensor. A fluorescence signal was observed depending on the presence of the kinase. Furthermore, activities of various kinases in the cell lysate and the inhibitory effect of specific chemicals on the kinases were monitored. These results indicate that chemosensor-based microfluidic chips can be developed as a versatile kinase assay system.

Enzyme delivery using the 30Kc19 protein and human serum albumin nanoparticles.

Nanoparticles have been widely used for delivering various chemical and biomolecular drugs, such as anti-cancer drugs and therapeutic proteins. Among nanoparticles, protein nanoparticles have advantages of non-cytotoxicity and biodegradability. In this study, a recombinant 30Kc19 protein was applied to human serum albumin (HSA) nanoparticles to enhance cellular uptake and stability of a nanoparticle cargo enzyme. The 30Kc19 protein, which originates from silkworm, has cell-penetrating and enzyme-stabilizing abilities. Therefore, 30Kc19-HSA nanoparticles were expected to enhance cellular uptake and stability of an enzyme loaded on the nanoparticles. Here, nanoparticles loaded with ß-galactosidase were prepared using the desolvation method. The 30Kc19-HSA nanoparticles were uniformly spherical in shape, dispersed evenly in phosphate buffered saline and cell culture media, and released ß-galactosidase in a sustained manner. The 30Kc19-HSA nanoparticles had negligible toxicity to animal cells and exhibited enhanced cellular uptake and intracellular stability of ß-galactosidase in HeLa and HEK293 cells when compared with those of HSA nanoparticles. These results suggest that 30Kc19-HSA protein nanoparticles could be used as a versatile tool for drug delivery to various cells.