The synthesis, characterization and biocompatibility of poly(ester urethane)/polyhedral oligomeric silesquioxane nanocomposites

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<ul><li><p>omo</p><p>a,*</p><p>ippi,, MS</p><p>5 May 2009Accepted 19 May 2009Available online 27 May 2009</p><p>Keywords:Poly(ester urethane)sPolyhedral oligomeric silesquioxanesBiocompatibility and cytotoxicity</p><p>reactive polyhedral oligomeric silsesquioxanes (POSS) as a result oftheir unique physical characteristics [46] previously not possiblebecause of classical physical laws [7]. POSS, as a nanoscale hollowcage structure of Si, with the bioactivity and other physical prop-erties of porous Si [8,9], should have similar biocompatibility and</p><p>applicable. For example, it is well known that blood vessel devel-opment is a complex process that requires endothelial cell prolif-eration, differentiation, and assembly into the tube-like structures.This process critically depends on the interaction between endo-thelial cells and extracellular matrix (ECM) that supports the cellgrowth. Although much work in the eld uses natural biopolymersfor basic research, the unavailability and complexity of the naturalECM materials signicantly limit their potential for biomedicalapplication. Consequently, there is an increasing need to develop</p><p>* Corresponding author. Tel.: 1 601 266 5596.</p><p>Contents lists availab</p><p>Polym</p><p>els</p><p>Polymer 50 (2009) 57495757E-mail address: joshua.otaigbe@usm.edu (J.U. Otaigbe).One motivation for the present study is from the work onnanostructured polymer blends previously reported by Otaigbe andco-workers [13]. In that work, the feasibility of making nano-structured polymer blends directly from in situ simultaneouspolymerization and compatibilization of reactive cyclic monomersin the presence of a compatible molten polymer was demonstrated[13]. In this nanostructured polymer blend the scale of dispersionof the one polymer phase in the other was </p></li><li><p>improved biocompatibility, biodegradation rate, and thermo-mechanical properties. This study may stimulate a better under-standing of the proposed rational synthesis of biodegradable andbiocompatible polyurethane materials with improved propertiesand biological function not now possible, making them widelyapplicable. Our data also provide a quantitative experimental basisfor future detailed structure/property studies of the relatively newhybrid PEU/POSS nanocomposite lms aimed at the prediction of</p><p>er 50 (2009) 57495757biocompatible synthetic polymers as substitutes for the naturalECM materials. A prerequisite for any biomedical application ofsynthetic polymers is their biocompatibility with the targetedtissues and organs, in addition to their basic chemical andmechanical properties. In a previous paper [17], we showed thatpure PEU does not have apparent toxicity on three cell types tested,endothelial cells, epithelial cells and stem cells, indicating its broadcompatibility with different cells. In the current paper, we inves-tigated the effects of POSS content on the structure and biocom-patibility of PEU/POSS nanocomposite as a critical prerequisite forthe potential application of the PEU/POSS nanocomposite inmanufacturing biomedical devices, such as materials for stents andarticial vessels in cardiovascular tissue engineering.</p><p>In the past, incorporation of silicon into polymers has been inthe form of siloxane, which in itself is noncompliant as a result ofelasticity mismatch of the composite constituents [18,19]. Thephilosophy of the present study is to explore the feasibility of analternative approach of incorporating very small amounts of siliconin the form of special nanoscale reactive POSS molecules as anintegral part of polyurethane chain segments which wouldpreserve the favorable properties (e.g. improved stiffness, strength,thermal stability and biocompatibility) of the hybrid polyurethane/POSS nanodispersions and compliance of the resulting solid-statehybrid polyurethane/POSS nanocomposite while signicantlylowering its surface tension properties [20,21]. The resultingpolyurethane/POSS nanocomposite materials are expected topossess improved properties such as lower surface tension [20] andlower bacterial adhesion than that of current synthetic biomaterials(e.g., PTFE) used in biomedical devices [22]. POSS is made directlyfrom silicones, silanes and a limited number can even be preparedfrom silica. As structurally well-dened silicon-based molecules,they can be reactively compounded and/or nonreactively blendedinto a wide variety of functionalities suitable for polymerization,grafting, surface bonding, and other transformations [2326]. POSSsiliconoxygen framework (SiO1.5) is intermediate between that ofa silicone (SiO) and silica (SiO2). This imparts excellent oxidativestability and thermal resistance. The carbon R groups (organic)located on the silicon atoms aid in the solubilization and compa-tibilization of the POSS cagewith organic resins, biological systems,and surfaces. The overall three-dimensional nature of POSS nano-structures is nearly equivalent in size, 0.73 nm (ave.1.5 nm) tomost polymer dimensions. Thus, when incorporated into a polymerat a molecular level, POSS can effectively control chain motion,glass transition and other physical properties. Structural control atthis length-scale has not previously been possible, yet it is at thenanoscopic length scale that a wide number of physical propertiesoriginate [27]. Therefore the high surface area of POSS allows it toreadily interact with a large number of polymer chains at relativelylow loadings (from 1 to 10 wt%) [20,2730].</p><p>The current work described in this article is part of a long-rangeresearch program that envisions that the behavior of these inter-esting biodegradable and biocompatible PEU/POSS nanocompositematerials is governed by the method of nano-reinforcement, thenano-interface, the synthetic process utilized, its microstructuraleffects, and the interaction between the PEU and POSS components.The present study focuses on preparing and characterizing biode-gradable and biocompatible poly(ester-urethane)/POSS (PEU/POSS)nanocomposites through homogeneous solution polymerization.The PEU/POSS nanocomposite lms will be obtained from meltprocessing of the as-synthesized PEU/POSS nanocompositeswithout using processing aids or modiers (lubricants) that cannegatively impact surface properties such as biological activity andantibacterial surface action of the resulting nanocomposite mate-rials. A special reactive diol-POSS was used to obtain new PEU/POSS</p><p>W. Wang et al. / Polym5750nanocomposites by using a highly efcient catalyst, resulting intheir properties and biological function, increasing our level ofunderstanding of the behavior of these systems and other similarpolymers. Therefore, it is likely that increased attention by otherresearchers will be focused on these materials in the future.</p><p>2. Materials and methods</p><p>2.1. Materials</p><p>Poly(3-caprolactone) diol (TONE Polyol 5249) was purchasedfrom Dow Chemical Company and the methylenediphenyl diiso-cyanate (MDI), toluene, dibutyltin dilaurate and 1,4-butanediolwere obtained from SigmaAldrich. 1,2-Propanediolisobutyl POSS(AL0130) was donated by Hybrid Plastics, Hattiesburg, MS. Phos-phate Buffer Solution (1 M, pH 7.4) was purchased from SigmaAldrich Company. The polyol and MDI were dried in a vacuum ovenat 40 C for 24 h prior to use.</p><p>2.2. Synthesis of poly(ester urethane)/POSS nanocomposites</p><p>A 100 ml round-bottomed three-necked ask equipped witha magnetic stirrer was used as a reaction vessel for the polymeri-zation reaction whose temperature was controlled by usinga constant temperature oil bath. Desired amounts of poly(3-capro-lactone) diol, POSS diol, toluene, and methylenediphenyl diisocya-nate were added to the ask (see Table 1). The ask was thenimmersed in the oil bath maintained at 60 C and its contentsstirred under a nitrogen atmosphere. After the solid contents dis-solved completely, dibutyltin dilaurate (1 wt% of the total weight)was added to the reaction system. A relatively high concentration ofdibutyltin dilaurate was necessary due to the low reactivity of thePOSS diol used. After 3 h, 1,4-butanediol was added to the ask aschain extender. The overall molar ratio of hydroxyl to isocyanatefunctional groups is 1:1.05. The reaction was allowed to continuefor another 5 h while maintaining the temperature at 60 C. Thereaction product was precipitated into ethanol and dried ina vacuum oven at 40 C for 48 h. Subsequently, the dried productswere compression molded at 143 C into test specimens withdesired shapes for the following measurements except the cellculture culture and cytotoxicity analysis. Note that thin lms thatwere prepared via solution casting were used for the cell cultureand cytotoxicity analysis.</p><p>2.3. NMR spectroscopy</p><p>Liquid phase 1H NMR measurements were performed at roomtemperature on the PEU/POSS nanocomposite using a Mercury 300</p><p>Table 1The feed ratio of the samples and their molecular weights.</p><p>Sample PCL diol (g) POSS diol (g) MDI (g) BDO (g) Mw 104g/mol</p><p>Mn 104g/mol</p><p>PEU 10 0 4.33 1 3.51 1.67PEU/1.5%POSS 9.75 0.25 4.37 1 4.02 1.74PEU/3%POSS 9.5 0.5 4.41 1 3.38 1.77PEU/4.5%POSS 9.25 0.75 4.45 1 3.68 1.80</p><p>PEU/6%POSS 9 1 4.49 1 4.27 1.84</p></li><li><p>spectrometer operating at 300 MHz with (methyl sulfoxide)-d6 assolvent, following standard procedures.</p><p>2.4. X-ray diffraction</p><p>The crystallinity of the PEU/POSS nanocomposite was evaluatedusing X-ray diffraction (XRD) analysis. A Rigaku Ultima III powderdiffractometer was used, employing Cu Ka radiation (at 40 kV and44 mA). Data were collected over the range of 2q 335 at a rateof 1/min.</p><p>2.5. DSC and TGA measurements</p><p>Differential scanning calorimetry (DSC) was carried out on thesamples over a temperature range of 80 C to 250 C using a TAInstruments (TAQ100) operating under a nitrogen atmosphere. TheDSC heating or cooling rate was 10 C/min. The midpoint of thetransition zonewas taken as the glass transition temperature (Tg) ofthe sample. Thermogravimetric analysis (TGA) tests were con-ducted on the samples using a PerkinElmer (Pyris 1 TGA) equip-ment operating from 50 C to 800 C at a heating rate of 10 C/minand under a nitrogen atmosphere. Thermal decompositiontemperature was dened as the temperature corresponding to themaximum rate of weight loss.</p><p>2.6. Static and dynamic mechanical measurements</p><p>method. The tests were performed in triplicate to give mean valuesreported in this paper.</p><p>The temperature dependencies of the storage moduli E0 andmechanical damping (tan d) of the compression molded rectan-gular test specimens (20 51 mm3) were determined usinga PYRIS Diamond Dynamic Mechanical Analyzer (DMA) operatingat temperatures ranging from 80 C to 150 C at a heating rate of3 C/min, a frequency of 1 Hz, and a linear strain amplitude of 10%.</p><p>2.7. Degradation in buffer solution</p><p>Degradation tests on the molded samples were conducted ina buffer solution of pH 7.4 at a degradation temperature of 37 Cfollowing our previously reported procedures [17].</p><p>2.8. Cell culture and cytotoxicity analysis</p><p>Three types of cells were used in this study; human umbilicalvein endothelial cells (HUVECs), mouse embryonic stem cells(mESCs) and human KB cells (an epithelial cell line derived froma human carcinoma of the nasopharynx). The cell culture condi-tions have been described previously [17]. Similar results wereobtained from the three types of cells tested. The data presented inthis study were obtained from experiments using KB cells. Micro-scopic coverglasses were coated with PEU or PEU/POSS thin lms,which served as cell growth matrices. For cell proliferation assay,</p><p>HO</p><p>S O C N R N C O O C N *</p><p>P</p><p>OCO</p><p>O</p><p>W. Wang et al. / Polymer 50 (2009) 57495757 5751Static mechanical tensile stressstrain measurements wereperformed on the samples using a Material Testing System AllianceRT/10 and a MTS Testworks 4 computer software package forautomatic control of test sequences and data acquisition andanalysis. Dumbbell-shaped test specimens (with an effective cross-section of 4 0.7 mm2) were cut from the compression moldedPEU/POSS sheets and tested at room temperature using a crossheadspeed of 20 mm/min according to the ASTM D882-88 standard</p><p>* RHN C</p><p>OPCL O C</p><p>ONH</p><p>R NH</p><p>CO</p><p>O POS</p><p>m</p><p>OCN RHN C</p><p>OO PCL O C</p><p>ONH</p><p>RHN C</p><p>OO</p><p>m</p><p>RHNHNC</p><p>O*</p><p>HO-POSS-OH</p><p>R NCOOCN + PCLOHScheme 1. The synthesis route of poly(esOCNH2C NCO</p><p>R NCOOCNn</p><p>p</p><p>OxKB cells were grown on the coverglasses in 24-well cell culturedishes at a density of 5103/ml. The cells were cultured in RPMImedium that contained 10% fetal bovine serum. After incubation for48 h at 37 C in a humidied incubator (5% CO2, 95% air), the cellswere xed and stained with 1% toluidine blue as previouslydescribed [17]. Cells were washed extensively with water andexamined under an Olympusmicroscopewith a phase contrast lensand photographed with a Cannon digital camera. To quantitativelydetermine cell number, toluidine blue was extracted with 2%</p><p>OH</p><p>O H H O O H</p><p>OSS O CO</p><p>NH</p><p>R NCOn</p><p>R NCONCO+</p><p>H POSS OHOH+ter urethane)/POSS nanocomposites.</p></li><li><p>2q 7.1, respectively. When the POSS content was increased to6 wt%, three peaks at 2q 7.1, 19.7 and 21.3 were observed. Theseresults indicate that low POSS concentrations (3 wt%) decreasethe crystallinity of the PEU soft segment and consequently improvethe compatibility between the soft and hard segments of the PEU.In addition, the results are consistent with the formation of POSSnanocrystals in the PEU hard segment (especially at high POSS</p><p>10 9 8 7 6 5 4 3 2 1 0</p><p>PEU/3% POSS</p><p>PEU</p><p>POSS</p><p>ppm</p><p>Fig. 1. NMR spectra of PEU, POSS and PEU/POSS nanocomposites.</p><p>5 10 15 20 25 30 35 40</p><p>30000</p><p>y</p><p>PCL</p><p>2 ()</p><p>4 6 8 101000</p><p>2000</p><p>3000</p><p>4000PEU/6% POSS</p><p>In</p><p>ten</p><p>sity</p><p>2</p><p>Fig. 2. XRD spectra of PEU and PEU/POSS nanocomposites. The bottom gure is</p><p>er 50 (2009) 57495757sodium dodecyl sulfate. The absorbance at 630 nm, which corre-lates with cell number, was measured with a microtiter platereader. For cell adhesion analysis, 1104 KB cells were seeded ondifferent matrices in 24-well cell culture dishes. The cells wereincubated for 2 h and then xed. The number of attached cells wasdetermined by the same method as described for cell proliferationassay.</p><p>2.9. Microscopic analysis of PEU/POSS matrix structure and cellmatrix interaction</p><p>To further analyze the cell morphology and their interactionwith PEU or PEU/POSS matrices, cells were double stained with 1%toluidine blue and 10 mM 40,6-diamidino-2-phenylindole (DAPI,a DNA binding dye). The nuclei stained with DAPI emit bright bluecolor under a uorescent microscope. The cells were examinedunder an Olympus uorescence microscope (BX60, UplanFl 100/1.30 oil lens) with lters set for the examination of DAPI staining.The images were photographed with a micropublisher digitalcamera (Qimaging). To visualize the auto-uorescence of thepolymer lms, the samples were examined under a Nikon uo-rescence microscope (Eclipse 80i, Plan Fluor 10X/0.75 DIC M/N2lens) with lters set for the examination of uorescein iso-thiocyanate (FITC). The images were processed using Image-ProPlus software.</p><p>3. Results and discussion</p><p>3.1. The synthesis of PEU/POSS nanocomposites</p><p>Five samples of PEU/POSS nanocomposites with different POSScontent were synthesized a...</p></li></ul>