Nanoporous electroporation needle for localized intracellular delivery in deep tissues.

The exogenous control of intracellular drug delivery has been shown to improve the overall efficacy of therapies by reducing nonspecific off-target toxicity. However, achieving a precise on-demand dosage of a drug in deep tissues with minimal damage is still a challenge. In this study, we report an electric-pulse-driven nanopore-electroporation (nEP) system for the localized intracellular delivery of a model agent in deep tissues. Compared with conventional bulk electroporation, in vitro nEP achieved better transfection efficiency (>60%) with a high cell recovery rate (>95%) under a nontoxic low electroporation condition (40 V). Furthermore, in vivo nEP using a nanopore needle electrode with a side drug-releasing compartment offered better control over the dosage release, time, and location of propidium iodide, which was used as a model agent for intracellular delivery. In a pilot study using experimental animals, the nEP system exhibited two times higher transfection efficiency of propidium iodide in the thigh muscle tissue, while minimizing tissue damage (<20%) compared to that of bulk electroporation. This tissue-penetrating nEP platform can provide localized, safe, and effective intracellular delivery of diverse therapeutics into deep tissues in a controlled manner.

Injectable Self-Crosslinkable Thiolated Hyaluronic Acid for Stem Cell Therapy of Atopic Dermatitis.

Stem cell therapies offer great promise in regenerative medicine to reinstate the normal function of diseased tissue, thereby avoiding the need for replacement. In stem cell therapies, damaged cells are replaced or restored by regulating inflammation and the immune system. However, the low survival rate and local retention of transplanted cells pose a significant challenge. In this study, injectable self-crosslinkable hydrogels using thiol-functionalized hyaluronic acid (HA-SH) were developed to improve the efficacy of mesenchymal stem cells (MSCs) for treating atopic dermatitis (AD)-related inflammatory lesions. The gelation kinetics and mechanical properties of HA-SH hydrogels were easily tuned by varying the concentration of the polymer in the precursor solution before injection. The MSC-laden HA-SH hydrogels exhibited high cell viability (>80%) for 1 week and good in vivo biocompatibility after implantation beneath the mouse skin. Moreover, the MSC-laden HA-SH hydrogel showed increased expression of anti-inflammatory cytokines, which can alleviate the immune response. In an AD animal model, a reduction in epidermal thickness and mast cell infiltration was achieved by applying a self-crosslinkable HA-SH solution including MSCs. This HA-based injectable hydrogel represents a potential carrier of stem cells, and its strong immunomodulation capabilities can be utilized for treating inflammation-related diseases.

Enhanced Cellular Cryopreservation by Biopolymer-Associated Suppression of RhoA/ROCK Signaling Pathway.

With increasing demands on long-term storage of cells, cryopreservation of cells is gaining more importance in cell-based research and applications. Dimethyl sulfoxide (DMSO) is a commonly used chemical cryoprotectant, providing increased cell survival during the freezing process. However, its use is limited in clinical applications due to its low biocompatibility above cryogenic temperatures. Herein, we present a new approach for reducing the use of DMSO in cryopreservation by using biodegradable hyaluronic acids (HAs). By adding HAs into cryoprotectant media containing a low concentration of DMSO, higher cell viability and cell proliferation rate were observed upon thawing after cryopreservation. The HA-supplemented cryopreservation media did not reduce the size of the ice crystal, which significantly influenced cell viability during cell freezing, but decreased the Ras homolog family member A (RhoA)/Rho-associated protein kinase (ROCK) signaling pathway related to apoptosis. The cell-interactive cryoprotectants containing HA can be applied to the development of a new cryoprotectant that reduces the adverse effect of DMSO.

Sodium/glucose Co-Transporter 2 Inhibitor, Empagliflozin, Alleviated Transient Expression of SGLT2 after Myocardial Infarction.

BACKGROUND AND OBJECTIVES: Large clinical studies of sodium/glucose cotransporter 2 (SGLT2) inhibitors have shown a significant beneficial effect on heart failure-associated hospitalization and cardiovascular events. As SGLT2 is known to be absent in heart cells, improved cardiovascular outcomes are thought to be accounted for by the indirect effects of the drug. We sought to confirm whether such benefits were mediated through SGLT2 expressed in the heart using myocardial infarction (MI) model. METHODS: Mice pre-treated with empagliflozin (EMPA), an SGLT2 inhibitor, showed a significantly reduced infarct size compared with the vehicle group three days post-MI. Interestingly, we confirmed SGLT2 localized in the infarct zone. The sequential changes of SGLT2 expression after MI were also evaluated. RESULTS: One day after MI, SGLT2 transiently appeared in the ischemic areas in the vehicle group and increased until 72 hours. The appearance of SGLT2 was delayed and less in amount compared with the vehicle group. Additionally, there was a significant difference in metabolites, including glucose and amino acids in the ¹H nuclear magnetic resonance analysis between groups. CONCLUSIONS: Our work demonstrates that SGLT2 is transiently expressed in heart tissue early after MI and EMPA may directly operate on SGLT2 to facilitate metabolic substrates shifts.

On-site fabrication of injectable 131I-labeled microgels for local radiotherapy.

Nuclear medicine is a routine but essential clinical option for diagnostic imaging and disease treatment. Encapsulating radioisotopes in injectable biodegradable hydrogels is ideal for localizing radiation sources to target tissues or organs to achieve long-term, low-dose radiotherapy. However, difficulties in the on-site production of radioactive gels upon treatment and the unpredictable radiation level at the target region are major obstacles to their clinical use. In this study, we bypassed these limitations by developing locally injectable hydrogel microparticles based on 131I-labeled photo-crosslinkable hyaluronic acid (HA) and a microfluidic high-throughput droplet generator. This approach enabled rapid on-site production of injectable, radioactive, biodegradable (IRB) HA microgels, thus allowing their immediate therapeutic application with improved local retention and predictable radioactivity. We demonstrated the clinical utility of this comprehensive approach by preparing IRB HA microgels within 15 min and localizing them to the target tissue (rat muscle) with minimal off-target biodistribution and in vivo radioactivity that extended beyond 3 weeks.

Formyl Peptide Receptor 2 Activation Ameliorates Dermal Fibrosis and Inflammation in Bleomycin-Induced Scleroderma.

Systemic sclerosis is a profibrotic autoimmune disease mediated by the dysregulation of extracellular matrix synthesis. Formyl peptide receptor 2 (Fpr2) is a G protein-coupled receptor that modulates inflammation and host defense by regulating the activation of inflammatory cells, such as macrophages. However, the role of Fpr2 in the development and therapy of scleroderma is still unclear. The present study was conducted to investigate the effects of Fpr2 activation in the treatment of scleroderma fibrosis. We found that intradermal administration of WKYMVm, an Fpr2-specific agonist, alleviated bleomycin-induced scleroderma fibrosis in mice and decreased dermal thickness in scleroderma skin. WKYMVm-treated scleroderma skin tissues displayed reduced numbers of myofibroblasts expressing α-smooth muscle actin, Vimentin, and phosphorylated SMAD3. WKYMVm treatment attenuated macrophage infiltration in scleroderma skin and reduced the number of M2 macrophages. The therapeutic effects of WKYMVm in scleroderma-associated fibrosis and inflammation were completely abrogated in Fpr2 knockout mice. Moreover, WKYMVm treatment reduced the serum levels of inflammatory cytokines, such as tumor necrosis factor-α, and interferon-γ, in the scleroderma model of wild-type mice but not in Fpr2 knockout mice. These results suggest that WKYMVm-induced activation of Fpr2 leads to alleviation of fibrosis by stimulating immune resolution in systemic sclerosis.

Role of stem cell mobilization in the treatment of ischemic diseases.

Stem cell mobilization plays important roles in the treatment of severe ischemic diseases, including myocardial infarction, limb ischemia, ischemic stroke, and acute kidney injury. Stem cell mobilization refers to the egress of heterogeneous stem cells residing in the bone marrow into the peripheral blood. In the clinic, granulocyte colony-stimulating factor (G-CSF) is the drug most commonly used to induce stem cell mobilization. Plerixafor, a direct antagonist of CXCR4, is also frequently used alone or in combination with G-CSF to mobilize stem cells. The molecular mechanisms by which G-CSF induces stem cell mobilization are well characterized. Briefly, G-CSF activates neutrophils in the bone marrow, which then release proteolytic enzymes, such as neutrophil elastase, cathepsin G, and matrix metalloproteinase 9, which cleave a variety of molecules responsible for stem cell retention in the bone marrow, including CXCL12, VCAM-1, and SCF. Subsequently, stem cells are released from the bone marrow into the peripheral blood. The released stem cells can be collected and used in autologous or allogeneic transplantation. To identify better conditions for stem cell mobilization in the treatment of acute and chronic ischemic diseases, several preclinical and clinical studies have been conducted over the past decade on various mobilizing agents. In this paper, we are going to review methods that induce mobilization of stem cells from the bone marrow and introduce the application of stem cell mobilization to therapy of ischemic diseases.

Role of CXCR2 in the Ac-PGP-Induced Mobilization of Circulating Angiogenic Cells and its Therapeutic Implications.

Circulating angiogenic cells (CACs) have been implicated in the repair of ischemic tissues, and their mobilization from bone marrow is known to be regulated by the activations of chemokine receptors, including CXCR2 and CXCR4. This study was conducted to investigate the role of N-acetylated proline-glycine-proline (Ac-PGP; a collagen-derived chemotactic tripeptide) on CAC mobilization and its therapeutic potential for the treatment of peripheral artery diseases. Ac-PGP was administered daily to a murine hind limb ischemia model, and the effects of Ac-PGP on blood perfusion and CAC mobilization (Sca1+ Flk1+ cells) into peripheral blood were assessed. Intramuscular administration of Ac-PGP significantly improved ischemic limb perfusion and increased limb salvage rate by increasing blood vessel formation, whereas Ac-PGP-induced blood perfusion and angiogenesis in ischemic limbs were not observed in CXCR2-knockout mice. In addition, Ac-PGP-induced CAC mobilization was found to occur in wild-type mice but not in CXCR2-knockout mice. Transplantation of bone marrow from green fluorescent protein (GFP) transgenic mice to wild-type mice showed bone marrow-derived cells homed to ischemic limbs after Ac-PGP administration and that GFP-positive cells contributed to the formation of ILB4-positive capillaries and α smooth muscle actin (α-SMA)-positive arteries. These results suggest CXCR2 activation in bone marrow after Ac-PGP administration improves blood perfusion and reduces tissue necrosis by inducing CAC mobilization. These findings suggest a new pharmaceutical basis for the treatment of critical limb ischemia. Stem Cells Translational Medicine 2019;8:236&246.

Role of Notch1 in the arterial specification and angiogenic potential of mouse embryonic stem cell-derived endothelial cells.

BACKGROUND: Endothelial cells have been shown to mediate angiogenesis in ischemic injury sites and contribute to the repair of damaged tissues. However, the treatment of ischemic disease requires a significant number of endothelial cells, which are difficult to isolate from patients. Embryonic stem cells have been considered a potential source of therapeutic cells due to their unlimited self-renewal and pluripotent properties. With regard to vascular development, Notch1 has been established as a key regulator of the specification of arterial endothelial cells. METHODS: Using a doxycycline-induced expression system of the intracellular domain of Notch1, we explored the role of Notch1 in the differentiation of embryonic stem cells to arterial endothelial cells. The therapeutic effect of the arterial endothelial cells was investigated in a murine hindlimb ischemia model. The blood perfusion rate in the ischemic limb was determined by laser Doppler perfusion imaging, and vasculogenesis was quantified using immunocytochemistry. RESULTS: Induced expression of the intracellular domain of Notch1 increased the levels of endothelial markers, such as CD31 and VE-cadherin, in differentiated endothelial cells. Induction of intracellular domain of Notch1 stimulated expression of the arterial-type endothelial cell markers (Nrp1 and Ephrin B2), but not the venous-type endothelial cell markers (Nrp2 and Coup-TFII). In addition, overexpression of intracellular domain of Notch1 resulted in increased expression of CXCR4, a chemokine receptor involved in vascular development. Induction of intracellular domain of Notch1 increased endothelial tube formation and migration of differentiated endothelial cells. Intramuscular administration of Notch1-induced arterial endothelial cells was more effective than administration of the control endothelial cells in restoring the blood flow in an ischemic hindlimb mouse model. Transplantation of Notch1-induced arterial endothelial cells augmented the number of blood vessels and incorporation of endothelial cells into newly formed blood vessels. CONCLUSIONS: These results suggest that Notch1 promotes endothelial maturation and arterial specification during the differentiation of embryonic stem cells to endothelial cells and increases the angiogenic potential of endothelial cells.

Atrial natriuretic peptide accelerates human endothelial progenitor cell-stimulated cutaneous wound healing and angiogenesis.

Atrial natriuretic peptide (ANP) is a powerful vasodilating peptide secreted by cardiac muscle cells, and endothelial progenitor cells (EPCs) have been reported to stimulate cutaneous wound healing by mediating angiogenesis. To determine whether ANP can promote the EPC-mediated repair of injured tissues, we examined the effects of ANP on the angiogenic properties of EPCs and on cutaneous wound healing. In vitro, ANP treatment enhanced the migration, proliferation, and endothelial tube-forming abilities of EPCs. Furthermore, small interfering RNA-mediated silencing of natriuretic peptide receptor-1, which is a receptor for ANP, abrogated ANP-induced migration, tube formation, and proliferation of EPCs. In a murine cutaneous wound model, administration of either ANP or EPCs had no significant effect on cutaneous wound healing or angiogenesis in vivo, whereas the coadministration of ANP and EPCs synergistically potentiated wound healing and angiogenesis. In addition, ANP promoted the survival and incorporation of transplanted EPCs into newly formed blood vessels in wounds. These results suggest ANP accelerates EPC-mediated cutaneous wound healing by promoting the angiogenic properties and survival of transplanted EPCs.