Phospholipid Polymer Research Articles

High-sensitivity measurement of immunoglobulin G (IgG) using HRP-IgG-phospholipid polymer conjugate

This report describes the high-sensitivity measurements of immunoglobulin (IgG) with the help of a phospholipid polymer conjugate, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer-IgG-horseradish peroxidase (HRP) conjugate. The phospholipid polymer, poly(MPC-co-2-aminoethyl methacrylate) (PMAE), was synthesized. To prepare the 1-step conjugate, IgG and PMAE were chemically modified with HRP at the same time. To prepare the 2-step conjugate, PMAE was first chemically modified with HRP, and then IgG was chemically modified. The activities of 1-step and 2-step conjugates significantly decreased compared with that of a conventional IgG conjugated with HRP (conventional conjugate) (p 0.05). The detection sensitivity of the 1-step and 2-step conjugates was also compared with that of conventional conjugate. When 0.5 µg/mL anti(goat IgG)IgG was used as an analyte, compared with a conventional conjugate, the values of Δ absorbance of the 1-step and 2-step conjugates were 4.8 and 9.8 times respectively. These results demonstrate that high-sensitivity measurements are possible using HRP-IgG-PMAE conjugate.

The Noninvasive Treatment for Sentinel Lymph Node Metastasis by Photodynamic Therapy Using Phospholipid Polymer as a Nanotransporter of Verteporfin.

Aim. The usefulness of photodynamic therapy (PDT) for treating sentinel lymph node (SLN) metastasis was evaluated. Materials and Methods. Verteporfin, a hydrophobic photosensitizer, forms a soluble aggregate with poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB). The concentrations of verteporfin were determined by measuring the fluorescence emitted at 700 nm. Seven days after the inoculation of A431 cells at the forearm of BALB/c nude mice, PMB-verteporfin was injected at dorsum manus and 75 J of light energy was delivered for 1 minute. Fifty-three mice were randomly assigned to the combination of PMB-verteporfin injection and light exposure, light exposure alone, PMB-verteporfin injection alone, and no treatment groups. Ten days after PDT, brachial lymph nodes, which were considered as SLNs, were harvested and evaluated. Results. The concentration of verteporfin in SLN was significantly higher than other organs. The combination of PMB-verteporfin injection and light exposure group significantly reduced the SLN metastasis (13%) comparing with no treatment group (52%), light exposure alone group (57%), and PMB-verteporfin injection alone group (46%). Conclusions. These data suggested that PDT using PMB as a nanotransporter of verteporfin could be a minimally invasive treatment of SLN metastasis in breast cancer and represent a potential alternative procedure to SLNB.

Simultaneous patterning of proteins and cells through bioconjugation with photoreactable phospholipid polymers

Photoreactable bioconjugated macromolecule composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) and 2-(4-azidobenzamido)ethyl methacrylate was synthesized to develop simultaneously patterned surface of proteins and cells.

Clinical safety and wear resistance of the phospholipid polymer-grafted highly cross-linked polyethylene liner.

To reduce the production of wear particles and subsequent aseptic loosening, we created a human articular cartilage-mimicked surface for a highly cross-linked polyethylene liner, whose surface grafted layer consisted of a biocompatible phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine). Although our previous in vitro findings showed that poly(2-methacryloyloxyethyl phosphorylcholine)-grafted particles were biologically inert and caused no subsequent bone resorptive responses, and poly(2-methacryloyloxyethyl phosphorylcholine) grafting markedly decreased wear in hip joint simulator tests, the clinical safety, and in vivo wear resistance of poly(2-methacryloyloxyethyl phosphorylcholine)-grafted highly cross-linked polyethylene liners remained open to question. Therefore, in the present study, we evaluated clinical and radiographic outcomes of poly(2-methacryloyloxyethyl phosphorylcholine)-grafted highly cross-linked polyethylene liners 5 years subsequent to total hip replacement in 68 consecutive patients. No reoperation was required for any reason, and no adverse events were associated with the implanted liners. The average Harris Hip Score increased from 38.6 preoperatively to 96.5 5 years postoperatively, and health-related quality of life, as indicated by the Short Form 36 Health Survey, improved. Radiographic analyses showed no periprosthetic osteolysis or implant migration. Between 1 and 5 years postoperatively, the mean steady-state wear rate was 0.002 mm/year, which represented a marked reduction relative to other highly cross-linked polyethylene liners, and appeared to be unaffected by patient-related or surgical factors. Although longer follow up is required, poly(2-methacryloyloxyethyl phosphorylcholine)-grafted highly cross-linked polyethylene liners improved mid-term clinical outcomes. The clinical safety and wear-resistance results are encouraging with respect to the improvement of long-term clinical outcomes with poly(2-methacryloyloxyethyl phosphorylcholine)-grafted highly cross-linked polyethylene liners. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2007-2016, 2017.

Movement of a Quantum Dot Covered with Cytocompatible and pH-Responsible Phospholipid Polymer Chains under a Cellular Environment.

Quantum dots (QDs) were functionalized with well-defined polymer chains having both cytocompatibility and pH-responsiveness to monitor the movement of nanoparticles in a cellular environment with changing local pH. We used a triblock-type water-soluble polymer composed of three segments: (1) a pH-responsive poly[2-(N,N-diethylamino)ethyl methacrylate; DEAEMA] segment, (2) a poly[ω-(p-nitrophenyloxycarbonyl oligo(ethylene glycol)) methacrylate; MEONP] segment bearing an active ester group to react with an amino compound, and (3) a cytocompatible poly[(2-methacryloyloxyethyl phosphorylcholine; MPC) segment. Moreover, hydrophobic and carboxyl groups were attached as terminals of the polymer chain. The triblock-type polymer was attached to the QD surface through a hydrophobic layer, which was covered with the QD by hydrophobic interaction. This produced hybrid QD particles (QD/MPC polymer nanoparticles). The QD/MPC polymer nanoparticles had good water-dispersion ability after the modification. A fluorescence resonance energy transfer (FRET) phenomenon between QD and fluorescence dye (Alexa) was clearly observed at pH 7.4 and 9.0 when a fluorescence dye was reacted with the poly(MEONP) segment of the polymer. However, the efficiency decreased at pH 5.0. This was due to a change in the distance between the QD and the fluorescence dye in response to the protonation degree of the poly(DEAEMA) segment. The permeability of QD/MPC polymer nanoparticles through the cell membrane was enhanced by reacting the cell-penetrating peptide, octaarginine (R8), to the carboxyl group at the end of the polymer. The R8-QD/MPC polymer/Alexa nanoparticles attached onto the HeLa cell membrane surface within 15 min after they were added to the cell culture. This attachment initiated nanoparticle penetration of the cell membrane by endocytosis. The nanoparticles could be followed continuously as they moved in the cell culture. The change in the FRET index was determined during this process. Use of the R8-QD/MPC polymer/Alexa nanoparticle enabled us to determine nanoparticle location, based on the surrounding local pH. We concluded that QDs, modified with a cytocompatible and pH-responsible MPC polymer, provide a new imaging and transport tool in cell-based science and engineering.

Preventive effects of a phospholipid polymer coating on PMMA on biofilm formation by oral streptococci

The regulation of biofilm formation on dental materials such as denture bases is key to oral health. Recently, a biocompatible phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) coating, was reported to inhibit sucrose-dependent biofilm formation by Streptococcus mutans, a cariogenic bacterium, on the surface of poly(methyl methacrylate) (PMMA) denture bases. However, S. mutans is a minor component of the oral microbiome and does not play an important role in biofilm formation in the absence of sucrose. Other, more predominant oral streptococci must play an indispensable role in sucrose-independent biofilm formation. In the present study, the effect of PMB coating on PMMA was evaluated using various oral streptococci that are known to be initial colonizers during biofilm formation on tooth surfaces. PMB coating on PMMA drastically reduced sucrose-dependent tight biofilm formation by two cariogenic bacteria (S. mutans and Streptococcus sobrinus), among seven tested oral streptococci, as described previously [N. Takahashi, F. Iwasa, Y. Inoue, H. Morisaki, K. Ishihara, K. Baba, J. Prosthet. Dent. 112 (2014) 194–203]. Streptococci other than S. mutans and S. sobrinus did not exhibit tight biofilm formation even in the presence of sucrose. On the other hand, all seven species of oral streptococci exhibited distinctly reduced glucose-dependent soft biofilm retention on PMB-coated PMMA. We conclude that PMB coating on PMMA surfaces inhibits biofilm attachment by initial colonizer oral streptococci, even in the absence of sucrose, indicating that PMB coating may help maintain clean conditions on PMMA surfaces in the oral cavity.

Diffusion-Induced Hydrophilic Conversion of Polydimethylsiloxane/Block-Type Phospholipid Polymer Hybrid Substrate for Temporal Cell-Adhesive Surface.

In this study, diffusion-induced hydrophobic-hydrophilic conversion of the surface of the cross-linked polydimethylsiloxane (PDMS) substrate was realized by employing a simple swelling-deswelling process of PDMS substrate in a block-type polymer solution with the aim of development of a temporal cell-adhesive substrate. The ABA block-type polymer composed of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) segment and PDMS segment with over 70% of dimethylsiloxane unit composition could be successfully incorporated in the PDMS substrate during the swelling-deswelling process to prepare the PDMS/phospholipid block copolymer hybrid substrates. During the aging process of the PDMS substrate for 4 days in aqueous medium, its surface property changed gradually from hydrophobic to hydrophilic. X-ray photoelectron spectroscopy and atomic force microscopy data provided strong evidence that the time-dependent hydrophilic conversion of the PDMS/block-type phospholipid polymer hybrid substrate was induced by the diffusion of the hydrophilic PMPC segment in the block-type polymer to be tethered on the substrate. During the hydrophilic conversion process, surface-adsorbed fibronectin was gradually desorbed from the substrate surface, and this resulted in successful detachment of two-dimensional connected cell crowds.

In situ growth of a C-terminal interferon-alpha conjugate of a phospholipid polymer that outperforms PEGASYS in cancer therapy

Conjugating therapeutic proteins and peptides to poly(ethylene glycol) (PEG) can improve their pharmacokinetics and therapeutic potential. However, PEGylation suffers from non-specific conjugation, low yield and immunogenicity. Herein we report a new and general methodology to synthesize a protein-polymer conjugate with site-specificity, high yield and activity, long circulation half-life and excellent therapeutic efficacy. A phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), was grown solely from the C-terminus of interferon-alpha to form a site-specific (C-terminal) and stoichiometric (1:1) PMPC conjugate of interferon-alpha in high yield. Notably, the PMPC conjugate showed 194- and 158-fold increases in systemic exposure and tumor uptake as compared with interferon-alpha, respectively. The in vitro antiproliferative bioactivity of the PMPC conjugate was 8.7-fold higher than that of PEGylated interferon-alpha (PEGASYS). In a murine cancer model, the PMPC conjugate completely inhibited tumor growth and cured 75% mice, whereas at the same dose, no mice treated with interferon-alpha or PEGASYS survived. We believe that this new approach to synthesize C-terminal protein conjugates of PMPC may be applicable to a large subset of protein and peptide drugs, thereby providing a general platform for the development of next-generation protein therapeutics.

Development and evaluation of vitamin E d-α-tocopheryl polyethylene glycol 1000 succinate-mixed polymeric phospholipid micelles of berberine as an anticancer nanopharmaceutical.

Berberine (Brb) is an active alkaloid occurring in various common plant species, with well-recognized potential for cancer therapy. Brb not only augments the efficacy of antineoplastic chemotherapy and radiotherapy but also exhibits direct antimitotic and proapoptotic actions, along with distinct antiangiogenic and antimetastatic activities in a variety of tumors. Despite its low systemic toxicity, several pharmaceutical challenges limit the application of Brb in cancer therapy (ie, extremely low solubility and permeability, very poor pharmacokinetics (PKs), and oral bioavailability). Among lipid-based nanocarriers investigated recently for Brb, stealth amphiphilic micelles of polymeric phospholipid conjugates were studied here as a promising strategy to improve Brb delivery to tumors. Specifically, physicochemically stable micelles made of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG-PE) mixed with d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) (PEG-succinate ester of vitamin E), in a 3:1 M ratio, increased Brb solubilization by 300%. Our PEG-PE/TPGS-mixed micelles firmly retained the incorporated Brb, displaying extended-release profile in simulated media, with up to 30-fold projected improvement in simulated PKs of Brb. Owing to the markedly better uptake of Brb-containing mixed micelles in vitro, our Brb-mixed micelles nanoformulation significantly amplified apoptosis and overall cytotoxic effectiveness against monolayer and spheroid cultures of human prostate carcinomas (16- to 18-fold lower half-maximal inhibitory concentration values in PC3 and LNPaC, respectively), compared to free Brb. Mixed PEG-PE/TPGS micelles represent a promising delivery platform for the sparingly soluble anticancer agent, Brb, encouraging further pharmaceutical development of this drug for cancer therapy.

Enhanced stability of NADH/dehydrogenase mixture system by water-soluble phospholipid polymers

To maintain activity in a coenzyme/enzyme mixture system, such as β-nicotinamide adenine dinucleotide (NADH)/dehydrogenase, the water-soluble 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers as an additive were synthesized and investigated for their stabilizing function. The inhibitor for the NADH/dehydrogenase reaction was spontaneously formed when the NADH was stored in the dehydrogenase solution. Therefore, we hypothesized that if the additive polymer could interact with an inhibitor without any adverse effect on the dehydrogenase, the activity in the NADH/dehydrogenase mixture could be maintained. We selected lactose dehydrogenase (LDH) as the enzyme, and the NADH was dissolved and incubated at 37°C in the LDH solution containing the polymers. The phospholipid polymers used in this study were poly(MPC) (PMPC), poly(MPC-co-3-trimethylammonium-2-hydroxypropyl methacrylate chloride) (PMQ) and poly[MPC-co-potassium 3-methacryloyloxypropyl sulfonate (MSO3)] (PMMSO3). The poly(MSO3) was used as a reference. For the PMQ and PMSO3 aqueous solutions, the activity of the NADH/LDH mixture system decreased with incubation time as the same level or lower than that in the Tris buffered solution in the absence of the polymers. However, for the poly(MPC-co-MSO3) (PMMSO3) aqueous solution, the activity of the NADH/LDH mixed system was six times higher than that in the buffered solution even after a 3-days incubation. The LDH activity was 1.5-1.8 times higher in the presence of the PMMSO3 compared with that in the PMSO3 solution. The mixture of two polymers, poly(MPC) and poly(MSO3), did not produce any stabilization. Thus, both the MPC and MSO3 units in the polymer chain had important and cooperative effects for stabilizing the NADH/LDH mixture.

Sitting Phases of Polymerizable Amphiphiles for Controlled Functionalization of Layered Materials.

Precisely tailoring surface chemistry of layered materials is a growing need for fields ranging from electronics to biology. For many applications, the need for noncovalently adsorbed ligands to simultaneously control interactions with a nonpolar substrate and a polar solvent is a particular challenge. However, biology routinely addresses a similar challenge in the context of the lipid bilayer. While conventional standing phases of phospholipids (such as those found in a bilayer) would not provide spatially ordered interactions with the substrate, here we demonstrate formation of a sitting phase of polymerizable phospholipids, in which the two alkyl chains extend along the surface and the two ionizable functionalities (a phosphate and an amine) sit adjacent to the substrate and project into the solvent, respectively. Interfacial ordering and polymerization are assessed by high-resolution scanning probe measurements. Water contact angle titrations demonstrate interfacial pKa shifts for the lipid phosphate but not for the amine, supporting localization of the phosphate near the nonpolar graphite surface.

Photo-assisted generation of phospholipid polymer substrates for regiospecific protein conjugation and control of cell adhesion

Novel photo-reactive phospholipid polymers were synthesized for use in the preparation of nonfouling surfaces with protein conjugation capacity. Poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-ran-N-methacryloyl-(l)-tyrosinemethylester (MAT)] (P(MPC/MAT)) was synthesized by conventional radical polymerization, with the MAT units capable of being oxidized by 254nm UV irradiation. Because of this photo-oxidation, active species such as catechol and quinone were alternately generated in the copolymer. A silicon wafer was subjected to surface modification through spin coating of P(MPC/MAT) from an aqueous solution for use as a model substrate. The surface was then irradiated several times with UV light. The thickness of the polymer layers formed on the Si wafers was influenced by various parameters such as polymer concentration, UV irradiation time, and composition of the MAT units in P(MPC/MAT). Oxidized MAT units were advantageous not only for polymer adhesion to a solid surface but also for protein conjugation with the adhered polymers. The amount of protein immobilized on UV-irradiated P(MPC/MAT) was dependent on the composition of the MAT units in the polymer. Furthermore, it was confirmed that protein immobilization on the polymer occurred through the oxidized MAT units because the protein adsorption was significantly reduced upon blocking these units through pretreatment with glycine. Conjugation of regiospecific protein could also be achieved through the use of a photomask. In addition, nonspecific protein adsorption was reduced on the non-irradiated regions whose surface was covered with physisorbed P(MPC/MAT). Therefore, P(MPC/MAT) can be used in the preparation of nonfouling substrates, which enable micrometer-sized manipulation of proteins through photo-irradiation. Function of proteins immobilized on MPC copolymers was also confirmed by cell adhesion test. As such, photo-reactive MPC copolymers are suitable for performing controlled protein conjugation and preparing polymer–protein hybrid platforms for use in biomedical and diagnostic devices. Statement of SignificanceNovel photo-reactive phospholipid polymers have been synthesized for immobilization on solid surfaces and regiospecific protein conjugation. Tyrosine residues embedded in 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymers could be photo-oxidized, resulting in polymers able to form layers on a solid surface and conjugate with proteins. Moreover, nonspecific biofouling on the surface significantly reduced when the oxidized tyrosine units in the polymer layers were blocked. Upon UV irradiation through a photomask, the UV-exposed tyrosine units were selectively oxidized, forming the only specific regions in which protein conjugation could occur.

Cell membrane-permeable and cytocompatible phospholipid polymer nanoprobes conjugated with molecular beacons

Event Abstract Back to Event Cell membrane-permeable and cytocompatible phospholipid polymer nanoprobes conjugated with molecular beacons Xiaojie Lin1, Tomohiro Konno2 and Kazuhiko Ishihara1, 2 1 the University of Tokyo, Department of Materials Engineering, Japan 2 the University of Tokyo, Department of Bioengineering, Japan Introduction: Real-time monitoring the dynamic distribution of mRNA in living cells could aid in the early detection of cell pathogenesis, accurate clinical diagnoses and effective treatments. To overcome the limitations of traditional methods, a noninvasive probe with a high degree of sensitivity as well as high spatial and temporal resolution is required. Amphiphilic 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers contain extremely hydrophilic phosphorylcholine groups and have plasma membrane-inspired structure can penetrate into living cell through a simple diffusion process. Molecular beacons (MBs) with DNA hairpin structure that are widely used as fluorescent probes for the detection of complementary intra- and extracellular molecules in real time. Highly permeable MPC polymers conjugated with MBs could be used to rapidly label biomolecules and allow the extended monitoring of intracellular events. Materials and Methods: The water-soluble amphiphilic phospholipid polymer, poly[MPC-co-n-butyl methacrylate (BMA)-co-N-succinimidyloxycarbonyl tetra(ethylene glycol) methacrylate (PENHS)] (PMBS), was synthesized through conventional radical polymerization method (Figure 1)[1]. PMBS was conjugated with MBs to form nanoprobes by a chemical reaction between ester group of N-hydroxysuccinimide and amine group of the MBs. The morphology of PMBS in serum-free DMEM was determined by measurement of surface tension of polymer solution. Cellular uptake of the nanoprobes by the HeLa cells was observed with a laser scanning confocal microscopy. The single-stranded DNA-binding proteins and target mRNA fragment were used to evaluate the stability and specificity of nanoprobe, respectively. Results and Discussion: PMBS polymer with higher composition of hydrophobic BMA unit can strongly reduce the surface tension of serum-free DMEM, which remained constant at concentrations greater than 0.5 mg/mL. This result indicates that the PMBS361 (composition, MPC: BMA: PENHS = 3: 6: 1) formed stable polymer aggregates at concentrations above 0.5 mg/mL. Laser scanning confocal microscopy results show that the red fluorescence of Cy3 molecules from PMBS361-MB nanoprobe was detected in cell cytoplasm after 2 h, colocalizing well with MitoTracker, while fluorescence of other probe can not be observed inside cell (Figure 2). Thus, aggregate formation is considered to be a predominant factor contributing to the ability of these probes to penetrate the cell membrane. Conjugation of PMBS to MB can significantly increase the stability without causing any damage to the specificity of MB[1],[2]. Conclusion: Cell-membrane-permeable and cytocompatible nanoprobes composed of PMBS and MBs were synthesized successfully as a noninvasive tool for monitoring intracellular biomolecules in living cells. The nanoprobes have high target specificity and resistance to nonspecific degradation by surrounding proteins, and they effectively penetrated the cytoplasm, permitting the real time visualization of mRNA in HeLa cells. Thus, membrane-penetrating, amphiphilic, phospholipid-based polymers can be combined with nano/sub-nano-scale oligonucleotide MBs to generate highly sensitive nanoprobes that can be used to deepen our understanding of basic cellular processes and could also be applied toward the early detection, accurate clinical diagnosis, and effective treatment of diseases in the future. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Nanomedicine Molecular Science” (No. 2306) from Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Development of blood carrier's material by using the phospholipid polymer with polyolefin

Development of blood carrier's material by using the phospholipid polymer with polyolefin

Controlled photo-immobilization of proteins on phospholipid polymer surfaces

Event Abstract Back to Event Controlled photo-immobilization of proteins on phospholipid polymer surfaces Masako Tanaka1* and Yasuhiko Iwasaki2, 3* 1 Kansai university, Graduate School of Science and Engineering, Japan 2 Kansai university, Faculty of Chemistry, Materials and Bioengineering, Japan 3 Kansai university, ORDIST, Japan Introduction: Control of protein adsorption on synthetic materials is still important issue for creation of biomedical materials. It is well known that 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers can reduce nonspecific protein adsorption. By adding ligands to MPC polymer surfaces, specific molecular interaction between the surface and proteins or cells could be regulated [1]. In the present study, the photo-reactive MPC polymers bearing tyrosine residues have been newly synthesized. The coating layer of MPC copolymers having tyrosine residues was formed on the solid substrates via UV-irradiation through the binding between oxidized tyrosine residues [2] and the substrate. The nonfouling property on the substrates was improved by the surface modification. Moreover, the protein immobilization through oxidized tyrosine [3] on the MPC copolymers having high composition of tyrosine residues have been performed. Material and Method: Poly(MPC) (PMPC) and MPC copolymers with N-methacryloyl-(L)-tyrosine methylestr(MAT) (P(MPC/MAT)) were synthesized by conventional radical polymerization. The aqueous solution containing 1wt% P(MPC/MAT) was coated on the silicon wafers using a spin coater. The specimens were irradiated UV light (254 nm) for several periods and rinsed with methanol and water. Surface analysis for P(MPC/MAT)-immobilized surfaces was performed using X-ray photoelectron spectroscope (XPS) and ellipsometer. P(MPC/MAT)-immobilized specimens were exposed to 0.45 g/dL fluorescein isothiocyanate (FITC)-labeled bovine albumin in PBS for 30 min, and rinsed with PBS and water. The sample was dried in an argon stream and observed using a fluorescent microscope. Results: The molar fraction of MPC of P(MPC/MAT) was coincident with that in copolymers. Oxidation of tyrosine units via UV light irradiation was monitored using UV-visible spectrophotometer. Fig. 1 shows the thickness of polymer layers immobilized on Si wafers. When the UV irradiation time was adjusted at 60 min, the thickness of P(MPC/MAT) coated on Si wafers was increased with an increase in the concentration of the polymer solution and the molar fraction of MAT. Fig. 2 shows a fluorescence micrograph for protein microarray generated on P(MPC/MAT)70/30. UV light was irradiated on P(MPC/MAT)70/30 coated surface was through a mesh for transmission electron microscopy. The proteins attached on the specific regions whose surface were exposed to UV light. Discussion: Considering from the layer thickness and phosphorus composition of surface analysis, MAT of P(MPC/MAT) is dominant unit to regulate the feature of polymer layers. Oxidized MAT units showed an advantage not only for the polymer adhesion to a solid surface but also protein conjugation with the adherent polymer. The amount of proteins immobilized on oxidized P(MPC/MAT)70/30 increased with an increase in the thickness of the polymer layer. It has been also clarified that conjugation of proteins on P(MPC/MAT) was selectively occurred through the oxidized MAT units. Conclusion: The photo-reactive MPC polymers have been newly designed to generate biofunctional interface. Tyrosine residues incorporated MPC copolymers could be photooxidized and resulted polymers showed functions to bind on solid surfaces and conjugate with proteins. The activity of site-specifically immobilized proteins on MPC polymer surface was well preserved. Moreover, nonspecific biofouling on the surface well reduced. The photo-reactive MPC polymers will be useful for surface preparation of biomedical and diagnostic devices. A part of this work was supported by AMED Strategic Promotion of Innovative Research and Development

Cytocompatible magnetic polymer nanoparticle covered with phospholipid polymer brush layer for in situ analysis of time-dependent cell functions

Cytocompatible magnetic polymer nanoparticle covered with phospholipid polymer brush layer for in situ analysis of time-dependent cell functions

Surface functionalization on biomaterials by precise fabrication of the phospholipid polymer brush layer

Surface functionalization on biomaterials by precise fabrication of the phospholipid polymer brush layer

Novel photoreactive phospholipid polymers for preparing a stable biocompatible surface with a smart process

Event Abstract Back to Event Novel photoreactive phospholipid polymers for preparing a stable biocompatible surface with a smart process Tomozumi Noda1*, Masaru Matsuda1*, Nobuyuki Sakamoto1*, Shigeru Kitano1*, Yuuki Inoue2*, Kyoko Fukazawa2* and Kazuhiko Ishihara2* 1 NOF Corporation, Life Sciences Products Division, Japan 2 The University of Tokyo, Department of Materials Engineering, School of Engineering, Japan Introduction: 2-Methacryloyloxyethyl phosphorylcholine (MPC) [1] polymers have been widely applied to some medical devices, including implantable blood pumps, cardiovascular stents and other medical goods. Stabilization of the MPC polymer layer on the substrate is essential issue to solve to have conventional surface treatment. In this study, in order to react the MPC polymer layer to the substrate, we have newly synthesized photoreactive MPC polymers to modify the plastic surface, and evaluated fundamental properties regarding biocompatibility, that is, protein adsorption and cell adhesion. By using these photoreactive MPC polymers, surface treatment is conducted very easy way, such as, coating of the MPC polymer by solvent dipping and after that irradiation light. Materials and Methods: Two types of photoreactive MPC polymers bearing n-butyl methacrylate (BMA) and benzophenone group or phenylazide group were synthesized in a conventional free-radical copolymerization. The presence of benzophenone group (7.20-8.00 ppm ) or phenylazide group (7.10-7.35 ppm and 7.90-8.25ppm) was confirmed by Proton NMR. Each MPC polymer was used for surface modification on various substrates, such as polyethylene and polystyrene, by dip or cast coating, and following the photoirradiation of UV light at 254nm for 7 min. After photoirradiation, all substrates were soaked in ethanol more than 16 h to evaluate reactivity of MPC polymers. The hydrophilic characteristics of the substrates modified with photoreactive MPC polymers were evaluated contact angle measurement by air under water. Then the 96-well polystyrene plates treated with photoreactive MPC polymers were used for examining protein adsorption using peroxidase conjugated antibody and cell adhesion of NIH3T3 cells. Result: Novel photoreactive MPC polymers possessing MPC unit, BMA unit and photoreactive groups were successfully synthesized. The monomer unit fraction of the MPC unit was 60 mol %, BMA unit was 30 mol % and photoreactive group was about 10 mol %. The photoreactive groups were confirmed by 1H-NMR. These polymers can be dissolved in alcohol. Regardless of the kind of the MPC polymers, the treatment resulted in high contact angles of air on the surfaces under water (100 o to 140 o). In addition, protein adsorption and cell adhesion were suppressed. On the other hand, those of the substrates without photoirradiation were equivalent to non-treated substrates in all examinations. Discussion: The substrates treated with the MPC polymers coating and photoirradiation were demonstrated good hydrophilicity even after washing process with ethanol. At this substrate, we observed reduced protein adsorption and cell adhesion. These are one of the fundamental characteristics for making blood-contacting medical devices and implantable devices. These results were due to formation of covalent bonds between the MPC polymers and the substrates and stable surface modification layer was constructed. Conclusion: We developed a smart process to obtain stable and biocompatible surface using photoreactive MPC polymers. The surfaces of various substrates modified with the photoreactive MPC polymer showed super-hydrophilic character, reduction of protein adsorption and inhibition of cell adhesion. Also, the photoreaction with the MPC polymer will be applied to treat a microspace of the substrate. Thus, this excellent anti-biofouling nature will be important to make biocompatible medical devices and biodevices. This research was supported by S-innovation from Japan Agency for Medical Research and Development (AMED)

Novel tissue-binding phospholipid polymer hydrogels for mucosal tissue regeneration

Novel tissue-binding phospholipid polymer hydrogels for mucosal tissue regeneration

High fatigue and wear resistance of phospholipid polymer grafted cross-linked polyethylene with anti-oxidant reagent

Event Abstract Back to Event High fatigue and wear resistance of phospholipid polymer grafted cross-linked polyethylene with anti-oxidant reagent Masayuki Kyomoto1, 2, 3, Toru Moro2, 4, Shihori Yamane1, 2, 3, Kenichi Saiga2, 3, Kenichi Watanabe2, 3, Sakae Tanaka4 and Kazuhiko Ishihara1 1 the University of Tokyo, Department of Materials Engineering, Japan 2 The University of Tokyo, Division of Science for Joint Reconstruction, Japan 3 KYOCERA Medical Corporation, Research Department, Japan 4 The University of Tokyo, Sensory & Motor System Medicine, Japan Introduction: Osteolysis caused by wear particles from polyethylene (PE) is a serious complication in the total hip arthroplasty (THA). To reduce wear, we have developed an articular cartilage-inspired technology (Aquala®) for surface modification with poly(2-methacryloyloxyethyl phosphorylcholine [MPC]) (PMPC) grafting. In natural synovial joints, fluid thin-film lubrication by the hydrated layer of cartilage is essential for the smooth motion of joints. Based on the biomimetic concept, a nanometer-scale layer of PMPC was formed on a cross-linked PE (CLPE) surface to better reproduce the ideal hydrophilicity and lubricity of the physiological joint surface. However, wear is only one of several important indicators of the clinical performance of acetabular liners. Oxidative degradation and fatigue fracture have been considered a potential limiting factor. The incorporation of the antioxidant vitamin E (VE) has been proposed recently to prevent the oxidation. Additionally, the fatigue fracture can be potentially avoided by using the VE. Our ultimate goal is to obtain not only high wear resistance but also high oxidative and fatigue resistances for life-long orthopedic bearings. We asked in this study: Will the hydration lubrication characteristics of the PMPC grafting layer and the oxidative stability of VE blended substrate affect fatigue fracture and wear resistance of CLPE liner under an accelerative aging condition? Materials and Methods: PE blended with 0.1 mass% VE was irradiated with gamma-rays (100 kGy) and annealed at 120°C for 12 h for cross-linking (HD-CLPE(VE)). HD-CLPE(VE) coated with benzophenone were immersed in a 0.5 mol/L aqueous MPC solution. The photoinduced graft polymerization on the HD-CLPE(VE) surface was carried out under ultraviolet irradiation of 5 mW/cm2 at 60°C for 90 min (PMPC-grafted HD-CLPE(VE)). All samples were then sterilized by a 25-kGy dose of gamma-rays under N2 gas. The obtained samples were accelerative aged according to the ASTM F2003. Impact-to-wear tests (ASTM F732) were conducted using a Pin-on-disk testing machine. The simulated wear test (ISO14242-3) was performed using a hip simulator. The wear particles isolated from the lubricant after wear test was observed by scanning electron microscopy. Results: In the impact-to-wear test, the PMPC-grafted surface characteristics did not appear to affect the impact fatigue resistance regardless of aging (Fig. 1B&C). Even after impact loads of 2.0 × 106 cycles, we did not observe either mechanical fracture or delamination in the sliding or backside surfaces of all groups. After 1.0 × 107 cycles of the hip simulator test, the PMPC-grafted HD-CLPE(VE) were found to show significantly low and stable wear compared with untreated HD-CLPE(VE) even after accelerative aging (Fig. 2). Substantially fewer wear particles isolated from lubricants were found for both PMPC-grafted HD-CLPE(VE) than for untreated HD-CLPE(VE). Discussion: The PMPC-grafted HD-CLPE(VE) provided high wear resistance. The PMPC-grafted layer leads to a significant reduction in the sliding friction between the surfaces because PMPC grafting causes formation of water thin films that can act as extremely efficient lubricants. The advantage of PMPC-grafting will be brought regardless of substrate characteristics. Conclusion: The PMPC-grafted HD-CLPE(VE) provides high fatigue and wear resistance even under an accelerative aging condition for life-long orthopedic bearings.