Stable and Thin-Polymer-Based Modification of Neurovascular Stents with 2-Methacryloyloxyethyl Phosphorylcholine Polymer for Antithrombogenicity.

The advent of intracranial stents has revolutionized the endovascular treatment of cerebral aneurysms. The utilization of stents has rendered numerous cerebral aneurysm amenable to endovascular treatment, thereby obviating the need for otherwise invasive open surgical options. Stent placement has become a mainstream approach because of its safety and efficacy. However, further improvements are required for clinically approved devices to avoid the frequent occurrence of thrombotic complications. Therefore, controlling the thrombotic complications associated with the use of devices is of significant importance. Our group has developed a unique stent coated with a 2-methacryloyloxyethyl phosphorylcholine (MPC)-based polymer. In this study, the surface characteristics of the polymer coating were verified using X-ray photoelectron spectroscopy and atomic force microscopy. Subsequently, the antithrombotic properties of the coating were evaluated by measuring platelet count and thrombin-antithrombin complex levels of whole human blood after 3 h of incubation in a Chandler loop model. Scanning electron microscopy was utilized to examine thrombus formation on the stent surface. We observed that MPC polymer-coated stents significantly reduced thrombus formation as compared to bare stents and several clinically approved devices. Finally, the coated stents were further analyzed by implanting them in the internal thoracic arteries of pigs. Angiographic imaging and histopathological examinations that were performed one week after implantation revealed that the vascular lumen was well maintained and coated stents were integrated within the vascular endothelium without inducing adverse effects. Thus, we demonstrated the efficacy of MPC polymer coating as a viable strategy for avoiding the thrombotic risks associated with neurovascular stents.

Extracellular vesicle-liposome hybrids via membrane fusion using cell-penetrating peptide-conjugated lipids.

Extracellular vesicles (EVs) are natural carriers for intercellular communication within the human body. Mimicking and utilizing EVs by combining them with artificial nanocarriers such as liposomes for drug delivery has garnered considerable attention. However, current technologies for manipulating EVs to facilitate their fusion with liposomes are limited; the existing technique of polyethylene glycol (PEG)-induced fusion is highly inefficient for fusion. In our previous study, we demonstrated that membrane fusion could be induced by Tat peptide (YGRKKRRQRRR)-conjugated poly(ethylene glycol)-phospholipids (Tat-PEG-lipids), in which the Tat peptide and lipid domain facilitate membrane attachment and subsequent fusion between cells and liposomes. This approach is promising for forming EV and liposomal hybrids. In this study, we aim to fuse EVs and liposomes using Tat-PEG-lipids. We isolated and characterized EVs derived from HEK293T cell culture medium and treated a mixture of EVs and liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol (1:1, molar ratio), with Tat-PEG-lipids with different lipid chain lengths. Here, we used nonanoyl (C9), dodecanoyl (C12), and myristoyl (C14) groups as lipid anchors with 5 kDa PEG chains. Dynamic light scattering analysis revealed a large increase in the apparent size of mixture of EVs and liposomes by adding Tat-PEG-lipids (especially C14, C12, followed by C9). Fluorescence resonance energy transfer, confocal laser scanning microscopy, and transmission electron microscopy, used to analyze the reaction process, revealed that the membrane fusion occurred between EVs and liposomes but not their aggregates. The short lipid domain of Tat-PEG-lipids effectively induced membrane fusion and the formation of hybrid EVs and liposomes. Thus, Tat-PEG-lipids (C9 and C12) could be promising candidates for inducing membrane fusion to fabricate EV-liposome hybrids.

Activation of cellular antioxidative stress and migration activities by purified components from immortalized stem cells from human exfoliated deciduous teeth.

Although stem cell-based regenerative medicine has been extensively studied, it remains difficult to reconstruct three dimensional tissues and organs in combination with vascular systems in vitro. One clinically successful therapy is transplantation of mesenchymal stem cells (MSC) into patients with graft versus host disease. However, transplanted cells are immediately damaged and destroyed because of innate immune reactions provoked by thrombogenic inflammation, and patients need to take immunosuppressive drugs for the immunological regulation of allogeneic cells. This reduces the benefits of stem cell transplantation. Therefore, alternative therapies are more realistic options for clinical use. In this study, we aimed to take advantage of the therapeutic efficacy of MSC and use multiple cytokines released from MSC, that is, stem cells from human exfoliated deciduous teeth (SHEDs). Here, we purified components from conditioned media of immortalized SHED (IM-SHED-CM) and evaluated the activities of intracellular dehydrogenase, cell migration, and antioxidative stress by studying the cells. The immortalization of SHED could make the stable supply of CM possible. We found that the fractionated component of 50-100 kD from IM-SHED-CM had higher efficacy than the original IM-SHED-CM in terms of intracellular dehydrogenase and cell migration in which intracellular signal transduction was activated via receptor tyrosine kinases, and the glutathione peroxidase and reductase system was highly active. Although antioxidative stress activities in the fractionated component of 50-100 kD had slightly lower than that of original IM-SHE-CM, the fraction still had the activity. Thus, the use of fractionated components of 50-100 kD from IM-SHED-CM could be an alternative choice for MSC transplantation because the purified components from CM could maintain the effect of cytokines from SHED.

Stent coating containing a charged silane coupling agent that regulates protein adsorption to confer antithrombotic and cell-adhesion properties.

The evolution of endovascular therapies, particularly in the field of intracranial aneurysm treatment, has been truly remarkable and is characterized by the development of various stents. However, ischemic complications related to thrombosis or downstream emboli pose a challenge for the broader clinical application of such stents. Despite advancements in surface modification technologies, an ideal coating that fulfills all the desired requirements, including anti-thrombogenicity and swift endothelialization, has not been available. To address these issues, we investigated a new coating comprising 3-aminopropyltriethoxysilane (APTES) with both anti-thrombogenic and cell-adhesion properties. We assessed the anti-thrombogenic property of the coating using an in vitro blood loop model by evaluating the platelet count and the level of the thrombin-antithrombin (TAT) complex, and investigating thrombus formation on the surface using scanning electron microscopy (SEM). We then assessed endothelial cell adhesion on the metal surfaces. In vitro blood tests revealed that, compared to a bare stent, the coating significantly inhibited platelet reduction and thrombus formation; more human serum albumin spontaneously adhered to the coated surface to block thrombogenic activation in the blood. Cell adhesion tests also indicated a significant increase in the number of cells adhering to the APTES-coated surfaces compared to the numbers adhering to either the bare stent or the stent coated with an anti-fouling phospholipid polymer. Finally, we performed an in vivo safety test by implanting coated stents into the internal thoracic arteries and ascending pharyngeal arteries of minipigs, and subsequently assessing the health status and vessel patency of the arteries by angiography over the course of 1 week. We found that there were no adverse effects on the pigs and the vascular lumens of their vessels were well maintained in the group with APTES-coated stents. Therefore, our new coating exhibited both high anti-thrombogenicity and cell-adhesion properties, which fulfill the requirements of an implantable stent.

Regulation of the innate immune system by fragmented heparin-conjugated lipids on lipid bilayered membranes in vitro.

Surface modification with heparin is a powerful biomaterial coating strategy that protects against innate immunity activation since heparin is a part of the proteoglycan heparan sulfate on cell surfaces in the body. We studied the heparinization of cellular and material surfaces via lipid conjugation to a heparin-binding peptide. In the present study, we synthesized fragmented heparin (fHep)-conjugated phospholipids and studied their regulation of the innate immune system on a lipid bilayered surface using liposomes. Liposomes have versatile applications, such as drug-delivery systems, due to their ability to carry a wide range of molecules. Owing to their morphological similarity to cell membranes, they can also be used to mimic a simple cell-membrane to study protein-lipid interactions. We investigated the interaction of complement-regulators, factor H and C4b-binding protein (C4BP), as well as the coagulation inhibitor antithrombin (AT), with fHep-lipids on the liposomal surface. Herein, we studied the ability of fHep-lipids to recruit factor H, C4BP, and AT using a quartz crystal microbalance with dissipation monitoring. With dynamic light scattering, we demonstrated that liposomes could be modified with fHep-lipids and were stable up to 60 days at 4 °C. Using a capillary western blot-based method (Wes), we showed that fHep-liposomes could recruit factor H in a model system using purified proteins and assist in the degradation of the active complement protein C3b to iC3b. Furthermore, we found that fHep-liposomes could recruit factor H and AT from human plasma. Therefore, the use of fHep-lipids could be a potential coating for liposomes and cell surfaces to regulate the immune system on the lipid surface.

Biomimetic polyelectrolyte coating of stem cells suppresses thrombotic activation and enhances its survival and function.

Mesenchymal stem cells (MSCs) therapy is a promising approach for treating inflammatory diseases due to their immunosuppressive and tissue repair characteristics. However, allogenic transplantation of MSCs induces thrombotic complications in some patients which limits its potential for clinical translation. To address this challenge, we have exploited the bioactivity of heparin, a well-known anticoagulant and immunosuppressive polysaccharide that is widely used in clinics. We have developed a smart layer-by-layer (LbL) coating strategy using gelatin and heparin polymers exploiting their overall positive and negative charges that enabled efficient complexation with the MSCs' glycocalyx. The stable coating of MSCs suppressed complement attack and mitigated thrombotic activation as demonstrated in human whole blood. Gratifyingly, the MSC coating retained its immunosuppressive properties and differentiation potential when exposed to inflammatory conditions and differentiation factors. We believe the simple coating procedure of MSCs will increase allogenic tolerance and circumvent the major challenge of MSCs transplantation.

Surface modulation of extracellular vesicles with cell-penetrating peptide-conjugated lipids for improvement of intracellular delivery to endothelial cells.

Exosomes (diameter 30-200 nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293 T cell cultures. Tat conjugated PEG-lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exosome surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5 kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluorescence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes.

Therapeutic regulation of complement activation in extracorporeal circuits and intravascular treatments with special reference to the alternative pathway amplification loop.

A number of clinical treatment modalities involve contact between blood and biomaterials: these include extracorporeal circuits such as hemodialysis, cardiopulmonary bypass, plasmapheresis, and intravascular treatments. Common side effects arising from these treatments are caused by activation of the cascade systems of the blood. Many of these side effects are mediated via the complement system, including thromboinflammatory reactions and rejection of implants. Depending on the composition of the materials, complement activation is triggered via all the activation pathways but is by far mostly driven by the alternative pathway amplification loop. On biomaterial surfaces the alternative pathway amplification is totally unregulated and leads under optimal conditions to deposition of complement fragments, mostly C3b, on the surface leading to a total masking of the underlying surface. In this review, we discuss the mechanism of the complement activation, clinical consequences of the activation, and potential strategies for therapeutic regulation of the activation, using hemodialysis as demonstrator.

Impact of spontaneous liposome modification with phospholipid polymer-lipid conjugates on protein interactions.

Liposome surface coating has been studied to avoid the immunological responses caused by the complement system, and alternative materials to poly(ethylene glycol) (PEG) have been explored recently since the production of anti-PEG IgM antibodies has been found in humans. We previously reported a liposome coating with poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC))-conjugated lipids (PMPC-lipids) and demonstrated its protective effect on blood protein interactions. Here, we attempted to modify the liposome surface by exogenously adding PMPC-lipids, which were spontaneously incorporated into the outer membrane via hydrophobic interactions. The polymerization degree of the PMPC segment was regulated from 10 to 100. The incorporated ratio of PMPC-lipid increased with a decrease in the degree of PMPC polymerization. Due to surface modification with PMPC-lipids, increase in the length of the PMPC-chain increased the size of the liposomes. The modified liposomes were kept stable for 14 d in terms of their size, polydispersity, and surface properties, where approximately 70% of PMPC-lipids were incorporated into the liposome surface. We demonstrated that liposome surface modification with PMPC-lipids can inhibit protein adsorption when exposed to serum, regardless of the degree of polymerization of PMPC. In addition, the PMPC-lipid modified surface was not recognized by the anti-PEG IgM antibody, whereas PEG-lipid was recognized by the antibody. Thus, we successfully fabricated an inert liposome surface via spontaneous modification with PMPC-lipids, where only the outer bilayer surface was modified. This technique can be available for full loading of water-soluble active pharmaceutical ingredient inside the modified liposome.

A Robust Method to Store Complement C3 With Superior Ability to Maintain the Native Structure and Function of the Protein.

Complement components have a reputation to be very labile. One of the reasons for this is the spontaneous hydrolysis of the internal thioester that is found in both C3 and C4 (but not in C5). Despite the fact that ≈20,000 papers have been published on human C3 there is still no reliable method to store the protein without generating C3(H2O), a fact that may have affected studies of the conformation and function of C3, including recent studies on intracellular C3(H2O). The aim of this work was to define the conditions for storage of native C3 and to introduce a robust method that makes C3 almost resistant to the generation of C3(H2O). Here, we precipitated native C3 at the isoelectric point in low ionic strength buffer before freezing the protein at -80°C. The formation of C3(H2O) was determined using cation exchange chromatography and the hemolytic activity of the different C3 preparations was determined using a hemolytic assay for the classical pathway. We show that freezing native C3 in the precipitated form is the best method to avoid loss of function and generation of C3(H2O). By contrast, the most efficient way to consistently generate C3(H2O) was to incubate native C3 in a buffer at pH 11.0. We conclude that we have defined the optimal storage conditions for storing and maintaining the function of native C3 without generating C3(H2O) and also the conditions for consistently generating C3(H2O).

Enhancement of intercellular interaction between iPSC-derived neural progenitor cells and activated endothelial cells using cell surface modification with functional oligopeptides.

Cell-based therapy has been used to treat stroke related disorders, which have no treatment options available 4.5 hours after onset. Although the administration of tissue plasminogen activator and mechanical thrombectomy are potent treatments, their clinical implementation is limited within the available time. Here, we aimed to use induced pluripotent stem cell-derived neural progenitor cells (NPCs) for stroke treatment with higher delivery efficiency in stroke areas, which will improve the therapeutic effect. E-selectin binding oligopeptide (Esbp) was conjugated with poly(ethylene glycol)-conjugated-lipid (Esbp-PEG-lipid) with different molecular weights of PEG (5 and 40 kDa) for cell surface modification. Then, we optimized the cell surface modification of NPCs by studying cell-binding ability onto the model surfaces of stroke areas, such as recombinant E-selectin-immobilized surfaces and TNF-α activated endothelium. As a result, the cell surface modification of NPCs with Esbp-PEG-lipid was found to induce specific intercellular interactions with the activated endothelium through the binding of Esbp with E-selectin. Additionally, the shorter PEG spacer was suitable for intercellular interactions. Thus, our technique shows potential for use in cell therapy with enhanced cell accumulation in infarct areas.

Induction of mesenchymal stem cell differentiation by co-culturing with mature cells in double-layered 2-methacryloyloxyethyl phosphorylcholine polymer hydrogel matrices.

The effects of differentiated cells on stem cell differentiation were analyzed via co-culturing using a cell-encapsulated double-layered hydrogel system. As a polymer hydrogel matrix, a water-soluble zwitterionic polymer having both a 2-methacryloyloxyethyl phosphorylcholine unit and a p-vinylphenylboronic acid unit (PMBV), was complexed spontaneously with poly(vinyl alcohol) (PVA) under mild cell culture conditions. The creep modulus of the hydrogel was controlled by changing the composition of the polymer in the solution. Mouse mesenchymal stem cells (MSCs), C3H10T1/2 cells, were encapsulated into PMBV/PVA hydrogels and cultured. In the PMBV/PVA hydrogel with a lower creep modulus (0.40 kPa), proliferation of C3H10T1/2 cells occurred, and the formation of cell aggregates was observed. On the other hand, a higher creep modulus (1.7 kPa) of the hydrogel matrix prevented cell proliferation. Culturing C3H10T1/2 cells encapsulated in the PMBV/PVA hydrogel in the presence of bone morphogenetic protein-2 increased the activity of intracellular alkaline phosphatase (ALP). This indicated that C3H10T1/2 cells differentiated into mature osteoblasts. When the C3H10T1/2 cells encapsulated in the PMBV/PVA hydrogel were cultured in combination with the mature osteoblasts in the hydrogel by a close contacting double-layered hydrogel structure, higher ALP activity was observed compared with the cells cultured separately. It was considered that the differentiation of C3H10T1/2 cells in the hydrogel layer was induced by cytokines diffused from mature osteoblasts encapsulated in another hydrogel layer. It could be concluded that the PMBV/PVA hydrogel system provides a good way to observe the effects of the surrounding cells on cell function in three-dimensional culture.

Effect of liposome surface modification with water-soluble phospholipid polymer chain-conjugated lipids on interaction with human plasma proteins.

Alternative liposome surface coatings for PEGylation to evade the immune system, particularly the complement system, have garnered significant interest. We previously reported poly(2-methacryloyloxyethyl phosphorylcholine) (MPC)-based lipids (PMPC-lipids) and investigated the surface modification of liposomes. In this study, we synthesize PMPC-lipids with polymerization degrees of 10 (MPC10-lipid), 20 (MPC20-lipid), 50 (MPC50-lipid), and 100 (MPC100-lipid), and coated liposomes with 1, 5, or 10 mol% PMPC-lipids (PMPC-liposomes). Non-modified and PEGylated liposomes are used as controls. We investigate the liposome size, surface charge, polydispersity index, and adsorption of plasma proteins to the liposomes post incubation in human plasma containing N,N,N',N'-ethylenediamine tetraacetic acid (EDTA) or lepirudin by some methods such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and automated capillary western blot, with emphasis on the binding of complement protein C3. It is shown that the coating of liposome PMPC-lipids can suppress protein adsorption more effectively with an increase in the molecular weight and molar ratio (1-10 mol%). Apolipoprotein A-I is detected on PMPC-liposomes with a higher molecular weight and higher molar ratio of PMPC-lipids, whereas α2-macroglobulin is detected on non-modified, PEGylated, and PMPC-liposomes with a shorter polymer chain. In addition, a correlation is shown among the PMPC molecular weight, molar ratio, and C3 binding. The MPC10-lipid cannot inhibit C3 binding efficiently, whereas surface modifications with 10 mol% MPC20-lipid and 5 mol% and 10 mol% MPC50-lipid suppress both total protein and C3 binding. Hence, liposome modification with PMPC-lipids can be a possible strategy for avoiding complement activation.

Current status of ischemic stroke treatment: From thrombolysis to potential regenerative medicine.

Ischemic stroke is a major cause of death and disability worldwide and is expected to increase in the future with the aging population. Currently, there are no clinically available treatments for damage sustained during an ischemic stroke, but much research is being conducted in this area. In this review, we will introduce current ischemic stroke treatments along with their limitations, as well as research on potential short and long-term future treatments. There are advantages and disadvantages in these potential treatments, but our understanding of these methods and their effectiveness in clinical trials are improving. We are confident that some future treatments introduced in this review will become commonly used in clinical settings in the future.

Preparation of Magnetic Hydrogel Microparticles with Cationic Surfaces and Their Cell-Assembling Performance.

Cationic magnetic hydrogel microparticles with high retention on cell surfaces were prepared using a two-step procedure. Using these magnetic hydrogel microparticles, cells were clustered with each other, and cell aggregates were prepared effectively. Cross-linked poly(vinyl alcohol) (PVA) hydrogel microparticles containing iron oxide nanoparticles were prepared. The diameter of the microparticles was in the range of 200-500 nm. Water-soluble cationic polymers containing both trimethyl ammonium (TMA) groups and phenylboronic acid (PBA) groups were synthesized for the surface modification of the microparticles. To regulate the composition, electrically neutral phosphorylcholine groups were introduced into the polymer. Covalent bonds were formed between the hydroxy groups of PVA microparticles and PBA groups in the polymer. The surface zeta potential of the microparticles reflected the composition of the TMA groups. The particles responded to an external magnetic field and clustered rapidly. Microparticles were adsorbed on the floating cell surface and induced cell aggregation quickly when a magnetic field was applied. Under the most effective conditions, the diameter of the cell aggregates increased to approximately 1 mm after 30 min. Denser cell aggregates were formed by the synergistic effects of the magnetic field and the properties of the microparticles. The formed cell aggregates continued to grow for more than 4 days under an applied magnetic field, indicating that the ability of the cells in the aggregate to proliferate was well maintained.

Induction of Spontaneous Liposome Adsorption by Exogenous Surface Modification with Cell-Penetrating Peptide-Conjugated Lipids.

The use of amphiphilic molecules such as poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) enables incorporation into liposome surfaces by exogenous addition as a result of the self-assembly with lipids. This technique can be applicable for manipulation of both liposomes and cells. In this study, we aimed to characterize Tat peptide (YGRKKRRQRRR)-conjugated PEG-lipids when used to exogenously surface modify liposomes (size: ca. 100 nm). We earlier reported that cells, which were surface modified with Tat peptides conjugated to PEG-lipids could attach spontaneously to material surfaces without any chemical modification. Here, we synthesized different types of Tat-PEG-lipids by combining PEG of different molecular weights (5 and 40 kDa) with different lipids with three acyl chains (myristoyl, palmitoyl, and stearoyl, respectively) and then studied the spontaneous adsorption of modified liposomes onto a substrate surface induced by the different Tat-PEG-lipids. The amount of adsorbed liposomes strongly depended on the number of incorporated Tat-PEG-lipid moieties: a decrease in both the PEG and the acyl chain lengths led to adsorption of higher amounts of liposomes. Furthermore, when a collagenase-cleavable amino acid sequence was inserted between the Tat sequence and the PEG segment, adsorbed liposomes could be harvested from the substrate by collagenase treatment with no difference in desorption efficiency between the different Tat-PEG-lipids. Thus, Tat-PEG-lipid can be a suitable tool for the manipulation of liposomes and cells.

Synthesis of poly(2-methacryloyloxyethyl phosphorylcholine)-conjugated lipids and their characterization and surface properties of modified liposomes for protein interactions.

Poly(ethylene glycol) (PEG) is frequently used for liposomal surface modification. However, as PEGylated liposomes are cleared rapidly from circulation upon repeated injections, substitutes of PEG are being sought. We focused on a water-soluble polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units, and synthesized poly(MPC) (PMPC)-conjugated lipid (PMPC-lipid) with degrees of MPC polymerization ranging from 10 to 100 (calculated molecular weight: 3 to 30 kDa). In addition, lipids with three different alkyl chains, myristoyl, palmitoyl, and stearoyl, were applied for liposomal surface coating. We studied the interactions of PMPC-lipids with plasma albumin, human complement protein C3 and fibrinogen using a quartz crystal microbalance with energy dissipation, and found that adsorption of albumin, C3 and fibrinogen could be suppressed by coating with PMPC-lipids. In particular, the effect was more pronounced for PMPC chains with higher molecular weight. We evaluated the size, polydispersity index, surface charge, and membrane fluidity of the PMPC-lipid-modified liposomes. We found that the effect of the coating on the dispersion stability was maintained over a long period (98 days). Furthermore, we also demonstrated that the anti-PEG antibody did not interact with PMPC-lipids. Thus, our findings suggest that PMPC-lipids can be used for liposomal coating.

Pluronic Micelle-Mediated Tissue Factor Silencing Enhances Hemocompatibility, Stemness, Differentiation Potential, and Paracrine Signaling of Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) evoke great excitement for treating different human diseases due to their ability to home inflamed tissues, suppress inflammation, and promote tissue regeneration. Despite great promises, clinical trial results are disappointing as allotransplantation of MSCs trigger thrombotic activity and are damaged by the complement system, compromising their survival and function. To overcome this, a new strategy is presented by the silencing of tissue factor (TF), a transmembrane protein that mediates procoagulant activity. Novel Pluronic-based micelles are designed with the pendant pyridyl disulfide group, which are used to conjugate TF-targeting siRNA by the thiol-exchange reaction. This nanocarrier design effectively delivered the payload to MSCs resulting in ∼72% TF knockdown (KD) without significant cytotoxicity. Hematological evaluation of MSCs and TF-KD MSCs in an ex vivo human whole blood model revealed a significant reduction in an instant-blood-mediated-inflammatory reaction as evidenced by reduced platelet aggregation (93% of free platelets in the TF-KD group, compared to 22% in untreated bone marrow-derived MSCs) and thrombin-antithrombin complex formation. Effective TF silencing induced higher MSC differentiation in osteogenic and adipogenic media and showed stronger paracrine suppression of proinflammatory cytokines in macrophages and higher stimulation in the presence of endotoxins. Thus, TF silencing can produce functional cells with higher fidelity, efficacy, and functions.

Exogenous Cell Surface Modification with Cell Penetrating Peptide-Conjugated Lipids Causes Spontaneous Cell Adhesion.

The technique of cell patterning on a substrate is of great importance for platforms in cell-based assays. Chemical treatment of the substrate is commonly performed for cell patterning using cationic polymers, extracellular matrices, and antibodies. However, cell patterning could be easier if there is an approach to immobilize cells without treating the substrate surface. We previously reported that cell adhesion could be induced by the modification of the cellular surface with a cell-penetrating peptide (CPP)-conjugated poly(ethylene glycol)-phospholipid (CPP-PEG-lipid). This approach does not require chemical modification of the substrate surface, such as polystyrene or glass, and can be used for the cell patterning of floating cells. Here, we aimed to study the mechanism of induced cell adhesion using a representative CPP, Tat peptide (Tat-PEG-lipid). We found that cell adhesion was induced via electrostatic interactions between the Tat peptide and the substrate surface, which could be induced more efficiently by increasing the molecular weight of PEG together with CPPs but not with cationic peptides. The excluded volume effect between neighboring PEG chains could stretch the cell shape better than PEG with lower molecular weight, allowing the cell to spread firmly. In addition, Tat-PEG-lipid did not activate actin filament formation and did not influence the expression of focal adhesion kinase. Thus, the induced cell adhesion by CPP-PEG-lipid did not affect internal cell signaling.

Harnessing hyaluronic acid-based nanoparticles for combination therapy: A novel approach for suppressing systemic inflammation and to promote antitumor macrophage polarization.

Anti-inflammatory drugs such as dexamethasone (DEX) are commonly administered to cancer patients along with anticancer drugs, however, the effect of DEX on human cancers is poorly understood. In this article, we have tailored self-assembled nanoparticles derived from hyaluronic acid (HA) wherein, anti-inflammatory DEX was used as a hydrophobic moiety for inducing amphiphilicity. The HA-DEX micelles were subsequently loaded with chemotherapeutic agent, doxorubicin (DOX) (HA-DEX-DOX) and was utilized to deliver drug cargo to human cancer cells expressing different levels of CD44 receptors. We found that DEX suppressed the cytotoxicity of DOX in HCT116, while it synergistically enhanced cytotoxicity in MCF-7 cells. When we tested DOX and HA-DEX-DOX in an ex-vivo human whole blood, we found activation of complement and the coagulation cascade in one group of donors. Encapsulation of DOX within the nanoparticle core eliminated such deleterious side-effects. The HA-DEX-DOX also polarized bone-marrow-derived anti-inflammatory M2 macrophages, to pro-inflammatory M1 phenotype with the upregulation of the cytokines TNF-α, iNOS and IL-1ß.