Preparation and Characterization of Acrylic and Methacrylic Phospholipid-Mimetic Polymer Hydrogels and Their Applications in Optical Tissue Clearing.

The 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers are mimetic to phospholipids, being widely used as biocompatible polymers. In our previous study, MPC polymer hydrogels proved more effective for optical tissue clearing compared to acrylamide (AAm) polymer hydrogels. In the present study, 2-acryloyloxyethyl phosphorylcholine (APC) was synthesized and employed to create hydrogels for a comparative analysis with methacrylic MPC-based hydrogels. APC, an acrylic monomer, was copolymerized with AAm in a similar reactivity. In contrast, MPC, as a methacrylic monomer, demonstrated higher copolymerization reactivity than AAm, leading to a spontaneously delayed two-step polymerization behavior. This suggests that the polymer sequences and network structures became heterogeneous when both methacrylic and acrylic monomers, as well as crosslinkers, were present in the copolymerization system. The molecular weight of the APC polymers was considerably smaller than that of the MPC polymers due to the formation of mid-chain radicals and subsequent ß-scission during polymerization. The swelling ratios in water and strain sweep profiles of hydrogels prepared using acrylic and methacrylic compounds differed from those of hydrogels prepared using only acrylic compounds. This implies that copolymerization reactivity influences the polymer network structures and crosslinking density in addition to the copolymer composition. APC-based hydrogels are effective for the optical clearing of tumor tissues and are applicable to both passive and electrophoretic methods.

Mechanical detection of interactions between proteins related to intermediate filament and transcriptional regulation in living cells.

Intermediate filaments (IF) bind to various proteins and regulate cell function in the cytoplasm. Recently, IFs were found to regulate gene expression by acting as capture scaffolds for transcription-related proteins and preventing their translocation into the nucleus. To reveal such transcriptional regulatory mechanisms controlled by IFs, a method to analyze the interaction between IFs and transcription-related proteins is necessary. Although there are many methods to observe interactions in living cells, it is still challenging to measure protein-protein interactions in living cells in their unmodified and native state. In this study, we utilized a nanoneedle that can access the cytosol by insertion into the cell. Modification of antibody recognizing transcription-related proteins allows the needle to detect mechanical force required to unbind the interaction between antibody and target proteins interacting with IFs during retraction of the needle from the cell. We focused on IF vimentin, a marker of epithelial-mesenchymal transition, to mechanically detect transcription-related proteins trapped by vimentin filaments. Prohibitin 2 (PHB2), a transcription-related factor, was selected as the candidate vimentin-binding protein. We conducted mechanical detection of PHB2 using atomic force microscopy and anti-PHB2 antibody-modified nanoneedles in vimentin-expressing mouse breast cancer and vimentin-knockout (VKO) cells. Significantly larger unbinding forces were detected in the vimentin-expressing cells than in the VKO cells. The results demonstrate that this method is useful for in-cell mechanical detection of IF-binding proteins.

Photoinduced immobilization of 2-methacryloyloxyethyl phosphorylcholine polymers with different molecular architectures on a poly(ether ether ketone) surface.

Poly(ether ether ketone) (PEEK) has seen increasing use in biomedical fields as a replacement for metal implants. Accordingly, the surface functionalities of PEEK are important for the development of medical devices. We have focused on the application of photoinduced reactions in PEEK to immobilize a functional polymer via radical generation on the surface, which can react with hydrocarbon groups. In this study, we used zwitterionic copolymers comprising 2-methacryloyloxyethyl phosphorylcholine (MPC) units and n-butyl methacrylate (BMA) units with various molecular architectures for surface modification. A random copolymer (poly(MPC-co-BMA) (r-PMB)), an AB-type diblock copolymer (di-PMB), and an ABA-type triblock copolymer (tri-PMB) (A segment: poly(BMA); B segment: poly(MPC)) were synthesized with the same monomer compositions. All PMBs were successfully immobilized on the PEEK surface via UV irradiation after the dip-coating process, regardless of their molecular structure. In this reaction, the alkyl group of the BMA unit functioned as a photoreactive site on the PEEK surface. This indicates that the molecular structure differences affect the surface properties. For example, compared to r-PMB and tri-PMB, di-PMB-modified surfaces exhibited an extremely low water contact angle of approximately 10°. The findings of this study demonstrate that this surface functionalization method does not require a low-molecular-weight compound, such as an initiator, and can be applied to the surface of inert PEEK through a simple photoreaction under room temperature, atmospheric pressure, and dry state conditions.

Preparation of a thermo-responsive drug carrier consisting of a biocompatible triblock copolymer and fullerene.

A triblock copolymer (PEG-b-PUEM-b-PMPC; EUM) comprising poly(ethylene glycol) (PEG), thermo-responsive poly(2-ureidoethyl methacrylate) (PUEM), and poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) blocks was synthesized via controlled radical polymerization. PEG and PMPC blocks exhibit hydrophilicity and biocompatibility. The PUEM block exhibits an upper critical solution temperature (UCST). PMPC can dissolve hydrophobic fullerenes in water to form a complex by grinding PMPC and fullerene powders. Fullerene-C70 (C70) and EUM were ground in a mortar and phosphate-buffered saline (PBS) was added to synthesize a water-soluble complex (C70/EUM). C70/EUM has a core-shell-corona structure, whose core is a complex of C70 and PMPC, the shell is PUEM, and corona is PEG. The maximum C70 concentration dissolved in PBS was 0.313 g L-1 at an EUM concentration of 2 g L-1. The C70/EUM hydrodynamic radius (Rh) was 34 nm in PBS at 10 °C, which increased due to the PUEM block's UCST phase transition with increasing temperature, and Rh attained a constant value of 38 nm above 36 °C. An anticancer drug, doxorubicin, was encapsulated in the PUEM shell by hydrophobic interactions in C70/EUM at room temperature, which can be released by heating. The generation of singlet oxygen (1O2) from C70/EUM upon visible-light irradiation was confirmed using the singlet oxygen sensor green indicator. Water-soluble C70/EUM may be used as a carrier that releases encapsulated drugs when heated and as a photosensitizer for photodynamic therapy.

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.

Transepithelial delivery of insulin conjugated with phospholipid-mimicking polymers via biomembrane fusion-mediated transcellular pathways.

Epithelial barriers that seal cell gaps by forming tight junctions to prevent the free permeation of nutrients, electrolytes, and drugs, are essential for maintaining homeostasis in multicellular organisms. The development of nanocarriers that can permeate epithelial tissues without compromising barrier function is key for establishing a safe and efficient drug delivery system (DDS). Previously, we have demonstrated that a water-soluble phospholipid-mimicking random copolymer, poly(2-methacryloyloxyethyl phosphorylcholine30-random-n­butyl methacrylate70) (PMB30W), enters the cytoplasm of live cells by passive diffusion manners, without damaging the cell membranes. The internalization mechanism was confirmed to be amphiphilicity-induced membrane fusion. In the present study, we demonstrated energy-independent permeation of PMB30W through the model epithelial barriers of Madin-Darby canine kidney (MDCK) cell monolayers in vitro. The polymer penetrated epithelial MDCK monolayers via transcellular pathways without breaching the barrier functions. This was confirmed by our unique assay that can monitor the leakage of the proton as the smallest indicator across the epithelial barriers. Moreover, energy-independent transepithelial permeation was achieved when insulin was chemically conjugated with the phospholipid-mimicking nanocarrier. The bioactivity of insulin as a growth factor was found to be maintained even after translocation. These fundamental findings may aid the establishment of transepithelial DDS with advanced drug efficiency and safety. STATEMENT OF SIGNIFICANCE: A nanocarrier that can freely permeate epithelial tissues without compromising barrier function is key for successful DDS. Existing strategies mainly rely on paracellular transport associated with tight junction breakdown or transcellular transport via transporter recognition-mediated active uptake. These approaches raise concerns about efficiency and safety. In this study, we performed non-endocytic permeation of phospholipid-mimicking polymers through the model epithelial barriers in vitro. The polymer penetrated via transcytotic pathways without breaching the barriers of biomembrane and tight junction. Moreover, transepithelial permeation occurred when insulin was covalently attached to the nanocarrier. The bioactivity of insulin was maintained even after translocation. The biomimetic design of nanocarrier may realize safe and efficient transepithelial DDS.

Intravenous Administration of Dehydroxymethylepoxyquinomicin With Polymer Enhances the Inhibition of Pancreatic Carcinoma Growth in Mice.

BACKGROUND/AIM: Pancreatic cancer, which exhibits resistance to cytotoxic and molecular targeted drugs, has an extremely poor prognosis. Nuclear factor-κB (NF-κB) is constitutively activated in many pancreatic cancer cases. Although the NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) has exhibited anti-cancer effects in pancreatic cancer models, its poor solubility limits its use to intraperitoneal administration. MATERIALS AND METHODS: Poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) forms stable polymer aggregates with DHMEQ. The stability of DHMEQ aggregated with PMB in the human blood was measured by high-performance liquid chromatography-mass spectrometry (HPLC-MS) ex vivo. Anti-pancreatic cancer effects in AsPC-1 and MIA PaCa-2 pancreatic cancer cells were evaluated by cell growth inhibition assay in vitro and tumor growth inhibition assay in vivo. RESULTS: DHMEQ aggregated with PMB (PMB-DHMEQ) remained detectable after 60 min of incubation in the human blood, whereas DHMEQ aggregated with carboxymethyl cellulose (CMC-DHMEQ) was barely detectable. PMB-DHMEQ significantly inhibited AsPC-1 and MIA PaCa-2 cell growth in vitro compared to CMC-DHMEQ. Intravenous administration of PMB-DHMEQ reduced the tumor volume and liver metastasis compared to untreated or CMC-DHMEQ-treated mice. CONCLUSION: Aggregation with PMB improved the solubility of DHMEQ, and effectively inhibited pancreatic cancer cell growth both in vitro and in vivo.

Impact of REDV peptide density and its linker structure on the capture, movement, and adhesion of flowing endothelial progenitor cells in microfluidic devices.

Ligand-immobilization to stents and vascular grafts is expected to promote endothelialization by capturing flowing endothelial progenitor cells (EPCs). However, the optimized ligand density and linker structure have not been fully elucidated. Here, we report that flowing EPCs were selectively captured by the REDV peptide conjugated with a short linker. The microchannel surface was modified with the REDV peptide via Gly-Gly-Gly (G3), (Gly-Gly-Gly)3 (G9), and diethylene glycol (diEG) linkers, and the moving velocity and captured ratio were evaluated. On the unmodified microchannels, the moving velocity of the cells exhibited a unimodal distribution similar to the liquid flow. The velocity of the endothelial cells and EPCs on the peptide-immobilized surface indicated a bimodal distribution, and approximately 20 to 30% of cells moved slower than the liquid flow, suggesting that the cells were captured and rolled on the surface. When the immobilized ligand density was lower than 1 molecule/nm2, selective cell capture was observed only in REDV with G3 and diEG linkers, but not in G9 linkers. An in silico study revealed that the G9 linker tends to form a bent structure, and the REDV peptide is oriented to the substrate side. These results indicated that REDV captured the flowing EPC in a sequence-specific manner, and that the short linker was more adequate.

Control of Cell-Substrate Binding Related to Cell Proliferation Cycle Status Using a Cytocompatible Phospholipid Polymer Bearing Phenylboronic Acid Groups.

To provide high-quality cellular raw materials for cell engineering and pharmaceutical engineering, a polymer substrate is prepared for cell separation focusing on the cell proliferation cycle. There are many types of sugar chains on cell membranes, which function as signaling molecules to control interactions with the exterior of the cell; their abundance changes during the cell-proliferation cycle. In this study, a phenylboronic acid group, which has affinity for sugar chains, is introduced into a polymer containing a phosphorylcholine group that does not induce cell activation. On the surface of this polymer, human promyelocytic leukemia cells can adhere. The adhesion rate is increased by pretreating the substrate with an alkaline solution. Moreover, cell adhesion is dependent on the sugar additive in the culture medium. Therefore, cell adhesion is governed by reactions between the sugar chain on the cell membrane and the phenylboronic acid groups on the substrate. It is revealed that the adhesion rate changes depending on the expression level of sugar chains related to the cell-proliferation cycle. Based on this, it may be proposed a cell proliferation cycle-specific separation process using the polymer substrate based on cell adhesion depending on sugar chain density.

Anticancer Activity of Cell-Penetrating Redox Phospholipid Polymers.

Redox-active molecules are promising anticancer compounds because cancer cells are vulnerable to oxidative stress. Anticancer drugs are often incorporated into synthetic polymers to improve water solubility, stability, and retention in the body. Most conventional redox-active polymers are regarded as stimuli-responsive polymers, which induce the release of anticancer drugs in response to the surrounding redox environment. Here, we prepared redox phospholipid polymers composed of 2-methacryloyloxyethyl phosphorylcholine units and ferrocene or quinone units as anticancer redox polymers. Redox phospholipid polymers can disturb the intracellular redox state owing to their redox activity and cell membrane permeability. We observed that the redox potential of the polymers affected the reactivity with intracellular redox species and O2, resulting in a different impact on the viability of human cancer and normal cells. Notably, the polymer with moderate reactivity with the intracellular redox species and O2 was shown to suppress the viability of the cancer cells selectively.

Promotion of cell membrane fusion by cell-cell attachment through cell surface modification with functional peptide-PEG-lipids.

Cell fusion is a fundamental event in various biological processes and has been applied to a number of biotechnologies. However, cell fusion efficiency is still low and strongly depends on cell lines and skills, though some improvements have been made. Our hypothesis is that two distinct cell membranes need to be brought together for cell membrane fusion, which is important for mimicking cell fusion in vitro. Here, we aimed to improve the homogeneous and heterogeneous cell fusion efficiency using a cell-cell attachment technique. We modified cellular membranes with two distinctive poly(ethylene glycol)-lipids (PEG-lipids) carrying oligopeptide, three repeated units of the EIAALEK and KIAALKE sequences (fuE3 and fuK3, respectively), which induce cell-cell attachment. The ratio and area of cell-cell attachment can be controlled through surface modification with fuE3-and fuK3-PEG-lipids by changing the number of each incorporated peptide. By combining this technique with the PEG-induced method, the cell fusion efficiency was significantly improved for homogeneous and heterogeneous cell fusion compared to conventional PEG-induced methods. For homogeneous CCRF-CEM cell fusion, the efficiency increased up to 64% from the 8.4% with the PEG-induced method. In addition, for heterogeneous cell fusion of myeloma cells and splenocytes, the efficiency increased up to 18% from almost zero. Thus, cell membrane fusion could be promoted effectively between closely contacted cell membranes induced by the cell-cell attachment technique.

Identification of Metal-Binding Peptides and Their Conjugation onto Nanoparticles of Superparamagnetic Iron Oxides and Liposomes.

Metallic materials are used for clinical medical devices such as vascular stents and coils to treat both ischemic and hemorrhagic vascular diseases. An antiplatelet drug is required to avoid thromboembolic complication until metallic surface is covered with a neo-endothelial cell layer. It is important to identify endothelial cell coverage on the metallic surface. However, it is difficult since there are no selective ligands. Here, we used the phage display method to identify peptide ligands that had high affinity for the metallic surface of Ni-Ti stents, Pt-W coils, and Co-Cr stents. The binding assay using fluorescence labeling revealed that several synthetic peptides could bind onto those surfaces. We also chose some oligopeptides for the conjugation onto superparamagnetic iron oxide (SPIO) nanoparticles and liposome-encapsulating SPIO nanoparticles and studied their ability to bind to the stent and coils. By SEM and fluorophotometry, we found that those modified SPIOs and liposomes were selectively bound onto those surfaces. In addition, both treated stents and coils could be detected by magnetic resonance imaging due to the magnetic artifact through the SPIOs and liposomes that were immobilized onto the surface. Thus, we identified metal-binding peptides which may enable to stop antiplatelet therapy after vascular stenting or coiling.

Combination of two antithrombogenic methodologies for preventing thrombus formation on a poly(ether ether ketone) substrate.

To enhance the total antithrombogenicity of poly(ether ether ketone) (PEEK), we examined a combination of two methodologies for the suppression of activation in both the platelet and coagulation systems. A random copolymer (PMT) composed of a zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC) unit and a cationic 2-methacryloyloxyethyl trimethylammonium chloride (TMAEMA) unit was grafted onto the PEEK surface by photoinduced self-initiated graft polymerization of the PEEK substrate (PMTx-g-PEEK). Then, negatively charged heparin was immobilized by ionic binding with TMAEMA units (Hep/PMTx-g-PEEK). The TMAEMA unit composition on grafted PMT altered the surface ζ-potentials of the PEEK substrates. Amounts of immobilized heparin depended on the ζ-potential. The concentration of heparin became constant on the sample surface where the TMAEMA unit composition was 30% or more, and was approximately 2.0 µg/cm2. The Hep/PMTx-g-PEEK with a TMAEMA unit composition of 50% showed not only decreased platelet adhesion, but also a 4-fold extension of the blood coagulation time of the poly(MPC)-g-PEEK substrate. The poly(MPC) layer could inhibit platelet adhesion and activation, resulting in surface antithrombogenic properties. Additionally, heparin released from the Hep/PMTx-g-PEEK prevented activation of the coagulation system in whole blood. Therefore, the combination of these antithrombogenic methodologies was promising for prolonging the blood coagulation period of the materials.

Cell-Membrane Permeable Redox Phospholipid Polymers Induce Apoptosis in MDA-MB-231 Human Breast Cancer Cells.

Induction of oxidative stress is an effective approach to causing apoptotic death of cancer cells. Since oxidative stress is generally caused by an intracellular redox imbalance, altering the intracellular redox is a promising strategy toward the growth suppression of cancer cells. Here, we attempted to induce apoptosis in MDA-MB-231 human breast cancer cells by adding a cell-membrane permeable redox phospholipid polymer that can alter the intracellular redox. We found that apoptosis and the deactivation of oxidative phosphorylation were induced in the MDA-MB-231 cells in the presence of the oxidized form of the redox polymer. Remarkably, such phenomena were not observed in the presence of the reduced form of the redox polymer that cannot intercept metabolic electrons. These results indicate that the redox polymer that mediates extracellular electron transfer (EET) generates oxidative stress, leading to the apoptosis of the cancer cells.

Translocation Mechanisms of Cell-Penetrating Polymers Identified by Induced Proton Dynamics.

Unlike the majority of nanomaterials designed for cellular uptake via endocytic pathways, some of the functional nanoparticles and nanospheres directly enter the cytoplasm without overt biomembrane injuries. Previously, we have shown that a water-soluble nanoaggregate composed of amphiphilic random copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butyl methacrylate (BMA), poly(MPC- random-BMA) (PMB), passes live cell membranes in an endocytosis-free manner. Yet, details in its translocation mechanism remain elusive due to the lack of proper analytical methods. To understand this phenomenon experimentally, we elaborated the original pH perturbation assay that is extremely sensitive to the pore formation on cell membranes. The ultimate sensitivity originates from the detection of the smallest indicator H+ (H3O+) passed through the molecularly sized transmembrane pores upon challenge by exogenous reagents. We revealed that water-soluble PMB at the 30 mol % MPC unit (i.e., PMB30W) penetrated into the cytosol of model mammalian cells without any proton leaks, in contrast to conventional cell-penetrating peptides, TAT and R8 as well as the surfactant, Triton X-100. While exposure of PMB30W permeabilized cytoplasmic lactate dehydrogenase out of the cells, indicating the alteration of cell membrane polarity by partitioning of amphiphilic PMB30W into the lipid bilayers. Nevertheless, the biomembrane alterations by PMB30W did not exhibit cytotoxicity. In summary, elucidating translocation mechanisms by proton dynamics will guide the design of nanomaterials with controlled permeabilization to cell membranes for bioengineering applications.

Toward Antibiofouling PVDF Membranes.

Membranes for biologically and biomedically related applications must be bioinert, that is, resist biofouling by proteins, human cells, bacteria, algae, etc. Hydrophobic materials such as polysulfone, polypropylene, or poly(vinylidene fluoride) (PVDF) are often chosen as matrix materials but their hydrophobicity make them prone to biofouling, which in turn limits their application in biological/biomedical fields. Here, we designed PVDF-based membranes by precipitation from the vapor phase and zwitterionized them in situ to reduce their propensity to biofouling. To achieve this goal, we used a copolymer containing phosphorylcholine groups. An in-depth physicochemical characterization revealed not only the controlled presence of the copolymer in the membrane but also that bicontinuous membranes could be formed. Membrane hydrophilicity was greatly improved, resulting in the mitigation of a variety of biofoulants: the attachment of Stenotrophomonas maltophilia, Streptococcus mutans, and platelets was reduced by 99.9, 99.9, and 98.9%, respectively. Besides, despite incubation in a plasma platelet-poor medium, rich in plasma proteins, a flux recovery ratio of 75% could be measured while it was only 40% with a hydrophilic commercial membrane of similar structure and physical properties. Similarly, the zwitterionic membrane severely mitigated biofouling by microalgae during their harvesting. All in all, the material/process combination presented in this work leads to antibiofouling porous membranes with a large span of potential biomedically and biologically related applications.

Modification of human MSC surface with oligopeptide-PEG-lipids for selective binding to activated endothelium.

Promising cell therapies using mesenchymal stem cells (MSCs) is proposed for stroke patients. Therefore, we aimed to efficiently accumulate human MSC (hMSC) to damaged brain area to improve the therapeutic effect using poly(ethylene glycol) (PEG)-conjugated phospholipid (PEG-lipid) carrying an oligopeptide as a ligand, specific for E-selectin which is upregulated on activated endothelial cells under hypoxia-like stroke. Here we synthesized E-selectin-binding oligopeptide (ES-bp) conjugated with PEG spacer having different molecular weights from 1 to 40 kDa. We found that ES-bp can be immobilized onto the hMSC surface through PEG-lipid without influence on cell growth and differentiation into adipocytes and osteocytes, respectively. It is also possible to control the immobilization of ES-bp on hMSC surface (<108 ES-bp per cell). Immobilized ES-bp can be continuously immobilized at the outside of cell membrane when PEG-lipids with PEG 5 and 40 kDa were used. In addition, the modified hMSC can specifically attach onto E-selectin-immobilized surface as a model surface of activated endothelium in human blood, indicating the sufficient number of immobilized ES-bp onto hMSC. Thus, this technique is one of the candidates for hMSC accumulation to cerebral infarction area. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1779-1792, 2019.

Short-term evaluation of thromboresistance of a poly(ether ether ketone) (PEEK) mechanical heart valve with poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-grafted surface in a porcine aortic valve replacement model.

Improved thromboresistance of mechanical valves is desired to decrease the risk of thromboembolism and thrombosis and reduce the dosage of anticoagulation with a vitamin K antagonist (e.g., warfarin). For several mechanical valves, design-related features are responsible for their improved thromboresistance. However, it remains unclear whether material-related features provide a practical level of thromboresistance to mechanical valves. Here, we studied the effect of a bileaflet valve made of poly(ether ether ketone) (PEEK) with a poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-grafted surface (PEEK-g-PMPC). PMPC is a well-known thromboresistant polymeric material. A short-term (<26 h) porcine aortic valve replacement model using neither an anticoagulant nor an antiplatelet agent showed that the PEEK-g-PMPC valve opened and closed normally with an allowable transvalvular gradient. Unlike an untreated PEEK valve, no thrombus formed on the PEEK-g-PMPC valves on gross anatomy examination in addition to the absence of traveled thrombi in the kidney and lung tissues. Material (PEEK-g-PMPC)-related thromboresistance appeared to decrease the risk of thromboembolism and thrombosis for patients with mechanical valves. However, thromboresistance of the PEEK-g-PMPC valve requires improvement because fibrous fouling was still observed on the leaflet. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1052-1063, 2019.

pH-Responsive Polyion Complex Vesicle with Polyphosphobetaine Shells.

When a bioactive molecule is taken into cells by endocytosis, it is sometimes unable to escape from the lysosomes, resulting in inefficient drug release. We prepared pH-responsive polyion complex (PIC) vesicles that collapse under acidic conditions such as those inside a lysosome. Furthermore, under acidic conditions, cationic polymer was released from the PIC vesicles to break the lysosome membranes. Diblock copolymers (P20M167 and P20A190) consisting of water-soluble zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) block and cationic or anionic blocks were synthesized via reversible addition-fragmentation chain transfer (RAFT) radical polymerization. Poly(3-(methacrylamidopropyl) trimethylammonium chloride) (PMAPTAC) and poly(sodium 6-acrylamidohexanoate) (PAaH) were used as the cationic and anionic blocks, respectively. The pendant hexanoate groups in the PAaH block are ionized in basic water and in phosphate buffered saline (PBS), while the hexanoate groups are protonated in acidic water. In basic water, PIC vesicles were formed from a charge neutralized mixture of oppositely charged diblock copolymers. At the interface of PIC vesicle and water exists biocompatible PMPC shells. Under acidic conditions, the PIC vesicles collapsed, because the charge balance shifted due to protonation of the PAaH block. After collapse of the PIC vesicles, P20A190 formed micelles composed of protonated PAaH core and PMPC shells, while P20M167 was released as unimers. PIC vesicles can encapsulate hydrophilic nonionic guest molecules into their hollow core. Under acidic conditions, the PIC vesicles can release the guest molecules and P20M167. The cationic P20M167 can break the lysosome membrane to efficiently release the guest molecules from the lysosomes to the cytoplasm.

Initial Cell Adhesion onto a Phospholipid Polymer Brush Surface Modified with a Terminal Cell Adhesion Peptide.

Dynamic changes in the properties of adsorbed protein layers at material surfaces make it difficult to analyze a cell adhesion behavior. Adhesion is affected by the ligand molecules in the adsorbed protein layers on the material's surface. This study aimed to quantitatively analyze the initial cell adhesion onto a polymeric surface modified with immobilized cell adhesion molecules with a well-defined structure. Peptides containing an arginine-glycine-aspartic acid (RGD) sequence were introduced at almost all the termini of the grafted poly(2-methacryloyloxyethyl phosphorylcholine) [poly(MPC)] chains using a click reaction at a highly protein-resistant poly(MPC) brush layer. Thus, the surface could bind to the cell membrane proteins only through the immobilized RGD. Furthermore, the degree of polymerization of the grafted poly(MPC) chains could control the hydrated poly(MPC) brush layer softness, as determined by measuring the dissipation energy loss using a quartz crystal microbalance. At the initial stage of cell adhesion, the density of cells adhering to the RGD-immobilized poly(MPC) brush layers did not depend on the poly(MPC) brush layer softness. However, spreading of the adherent cells was inhibited on the RGD-immobilized poly(MPC) brush layers with a higher softness. Hence, the results suggested that the layer softness did not affect the binding number between the RGD and cell membrane protein during initial cell adhesion; however, the intracellular signaling triggered by the RGD-receptor interaction was inhibited. The poly(MPC) brush surface carrying immobilized cell adhesion molecules has the potential to analyze precisely the effect of the properties of cell adhesion molecules on initial cell adhesion.