Yin Fun Poon

A photopolymerized antimicrobial hydrogel coating derived from epsilon-poly-L-lysine.

Hydrogels made from epsilon-poly-l-lysine-graft-methacrylamide (EPL-MA) have been found to have impressive wide spectrum antimicrobial activity against both bacteria (specifically Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus) and fungi (specifically Candida albicans and Fusarium solani). The EPL-MA hydrogel also possesses in vitro biocompatibility and EPL-MA solution is relatively non-hemolytic: the concentration needed for onset of human red blood cell (hRBC) hemolysis is 12,500 μg/mL so that the selectivity for the pathogenic microorganisms over hRBCs is 230-1560. Further, EPL-MA hydrogel can be conveniently ultraviolet-immobilized onto plasma-treated plastic surfaces to form thin highly adherent antimicrobial hydrogel coatings for medical devices and implants.

Cationic peptidopolysaccharides show excellent broad-spectrum antimicrobial activities and high selectivity.

The ongoing emergence of drug resistance in pathogens calls for continuing development of novel antimicrobial molecules. [ 1–3 ] Cationic antimicrobial peptides (AMPs) and their synthetic analogues, which target the pathogen cytoplasmic membrane, demonstrate a promising approach to lower the probability of development of pathogen resistance. [ 4 , 5 ] The microbial cytoplasmic membrane is surrounded by cell wall, a barrier which must be penetrated by all effective antimicrobial molecules. [ 6 , 7 ] However, the structural affi nity of antimicrobial molecules with the microbial cell wall has generally not been considered in previous designs of cationic antimicrobial polymers. Peptidoglycan is a common component of the bacteria cell wall, a feature absent from animal cells, so that peptidoglycanmimicry may be exploited to achieve high antimicrobial activity with low hemolytic activity. [ 8 ] Here, we report a new class of antimicrobial polymers based on cationic peptidopoly saccharides that mimic the peptidoglycan structure and that show excellent antimicrobial activity and high selectivity. The optimum tested peptidopolysaccharide, specifi cally a copoly mer of chitosan and polylysine (CSg -K 16 ), is effective against clinically signifi cant Gram-negative and Gram-positive bacteria and fungi with low minimum inhibitory concentrations (5–20 μ g mL − 1 , or 0.2–0.9 μ M), high selectivity ( > 5000–10 000) and low toxicity to mammalian cells. Our preliminary results of low/non secretion of tumor necrosis factorα by cultured macro phages when challenged with CSg -K 16 also suggests that the compound stimulates little or no infl ammatory response. The cationic charge of our peptidopolysaccharides causes them to target the anionic microbial cell envelope, and their structural affi nity with microbial cell wall constituents promotes their penetration of the cell wall to reach the cytoplasmic membrane, where the peptidopolysaccharide acts as an effective membrane disruptor. The combination of these features results in excellent antimicrobial activity and selectivity. This class of antimicrobial

Regulating orientation and phenotype of primary vascular smooth muscle cells by biodegradable films patterned with arrays of microchannels and discontinuous microwalls.

Vascular smooth muscle cells (vSMCs) cultured in vitro are known to exhibit phenotype hyperplasticity. This plasticity is potentially very useful in tissue engineering of blood vessels. The synthetic phenotype is necessary for cell proliferation on the tissue scaffold but the cells must ultimately assume a quiescent, contractile phenotype for normal vascular function. In vitro control of vSMC phenotype has been challenging. This study shows that microchannel scaffolds with discontinuous walls can support primary vSMC proliferation and, when the cells reach confluence inside the channels, transform the cell phenotype towards greater contractility and promote cell alignment. A thorough time-resolved study was undertaken to characterize the expression of the contractile proteins alpha-actin, calponin, myosin heavy chain (MHC) and smoothelin as a function of time and initial cell density on microchannel scaffolds. The results consistently indicate that primary vSMCs cultured on the microchannel substrate substantially align parallel to the microwalls, become more elongated and significantly increase their expression of contractile proteins only when the cells reach confluence. MHC immunostaining was visible in the micropatterned cells after confluence but not in flat substrate cells or non-confluent micropatterned cells, which further verifies the increased contractility of the confluent channel-constrained vSMCs. The higher total amount of deposited elastin and collagen in confluent flat cultures than in confluent micropatterned cultures also provides confirmation of the higher contractility of the channel-constrained cells. These results establish that our microchanneled film can trigger the switch of primary vSMCs from a proliferative state to a more contractile phenotype at confluence.

Hydrogels based on dual curable chitosan-graft-polyethylene glycol-graft-methacrylate: application to layer-by-layer cell encapsulation.

Ultraviolet (UV) photo-cross-linkable hydrogels have been commonly used for three-dimensional (3D) encapsulation of cells. Previous UV cross-linkable hydrogels have employed one-shot hardening of mixtures of hydrogels and cells. Here we propose an alternative method of making hydrogel-encapsulated cell constructs through layer by layer (LBL) buildup of alternating layers of cells and hydrogel. The LBL method potentially permits better spatial control of different cell types and control of cell orientation. Each hydrogel layer must be hardened before deposition of the next layer of cells. A UV-curable gel precursor that can also be gelled at physiological temperature is desirable to avoid repeated UV exposure of cells after deposition of each successive hydrogel layer. We designed, synthesized, and applied such a precursor, dual-curable-both thermoresponsive and UV-curable-chitosan-graft-polyethylene glycol-graft-methacrylate (CEGx-MA) copolymer (x is the PEG molecular weight in Daltons). We found that CEG350-MA copolymer solutions (5 wt % polymer) formed physical gels at approximately 37 degrees C and could be further photopolymerized to form thermally stable dual-cured hydrogels. This material was applied to the creation of a two-layer LBL smooth muscle cell (SMC)/hydrogel construct using temperature elevation to approximately 37 degrees C to gel each hydrogel layer. The physically gelled two-layered hydrogel/cell construct was finally exposed to a single UV shot to improve its mechanical properties and render it thermally stable. CEG350-MA solution and gel are nontoxic to SMCs. Cells remained mostly viable when they were encapsulated inside both physically gelled and dual-cured CEG350-MA and suffered little damage from the single brief UV exposure. The combination of LBL tissue engineering with a dual curable hydrogel precursor such as CEG350-MA permits the buildup of viable thick and complex tissues in a stable, biocompatible, and biodegradable matrix.

Addition of β-Malic Acid-Containing Poly(ethylene glycol) Dimethacrylate To Form Biodegradable and Biocompatible Hydrogels

Poly(malic acid) is water-soluble, functionalizable, and biodegradable, making it attractive as a precursor of hydrogels for biomedical applications. However, homopoly(malic acid), with pK(1/2) of 4.3, is too acidic for biocompatibility. To overcome the acidity, we have synthesized beta-malic acid-containing poly(ethylene glycol) dimethacrylate (PEGMAc) with pK(a) of 5.02. Solutions of methacrylated O-carboxymethylchitosan (OCMCS), PEGMAc, and poly(ethylene glycol) diacrylate (PEGDA; 7:7:86 and 6:20:74 (w/w/w)) in water (80%) have near neutral pHs (6.8-6.9). These solutions form firm hydrogels when photopolymerized. These are referred to as O7-PEGMAc7-B86 and O6-PEGMAc20-B74 (where the numerals refer to the weight content of each component, O is OCMCS and B is PEGDA added to make blend). The carboxyl groups in PEGMAc permit the surface grafting of hydrogels with Arg-Gly-Asp (RGD). The cytocompatibilities of smooth muscle cells (SMCs) on RGD-grafted hydrogels were studied. From the tetrazolium salt reduction assay, O6-PEGMAc20-B74 was found to have significantly better 10th day cytocompatibility compared to hydrogels containing lower or no PEGMAc. These gels degrade upon hydrolysis releasing malic acid, PEG and OCMCS. The increased cell compatibility of O6-PEGMAc20-B74 is possibly due to increased surface RGD content and near neutral pH even during biodegradation. Our novel PEGMAc-modified blends are a promising functionalizable biodegradable hydrogel precursor providing improved cell proliferation.

Synthesis and characterization of functionalized biodegradable poly(DL-lactide-co-RS-β-malic acid)

Amorphous poly(DL-lactide-co-RS-beta-malic acid) (PDLLMAc) was synthesized by hydrogenolysis of poly(DL-lactide-co-RS-beta-malolactonate) (PDLLMA), which was obtained from the ring-opening polymerization of DL-lactide (DLLA) and RS-beta-benzyl malolactonate (MA) using stannous octoate as the catalyst. The amount of malolactonate (MA) in the feeding dose was varied from 0 to 8.0 mol %. The copolymers were characterized by 1H NMR, FTIR, GPC, and DSC. The tensile properties and water uptake of the copolymers were measured. The protective benzyl groups in PDLLMA were completely removed in hydrogenolysis to produce PDLLMAc. The molecular weight (M(n)) of the copolymers decreased with increasing MA content. However, with low feed MA content of 0.6 and 1.0%, high molecular weight PDLLMAc with M(n) of 63 and 35 kDa, respectively, were obtained; these copolymers exhibited good tensile yield stress and modulus of 17-23 MPa and 1.1-1.4 GPa, which are comparable to PDLLA homopolymer. The corresponding protected PDLLMA have tensile yield stress/modulus of 2.0-2.4 MPa and 11-42 MPa. The malic acid comonomer in PDLLMAc significantly improves the tensile strength and modulus compared to the protected PDLLMA. Further, the functionalizable PDLLMAc (with 0.6 mol % feed MA) was grafted with bioactive RGD peptide. The culture of primary umbilical artery smooth muscle cells was investigated. Methylthiazoletetrazolium results showed that both the RGD- and COOH-functionalized (0.6 mol %) PDLLMAc copolymers were significantly more biocompatible than the control PDLLA and could potentially be employed as tissue engineering scaffolds.

Aligned 3D human aortic smooth muscle tissue via layer by layer technique inside microchannels with novel combination of collagen and oxidized alginate hydrogel

Tissue engineering of the small diameter blood vessel medial layer has been challenging. Recreation of the circumferentially aligned multilayer smooth muscle tissue has been one of the major technical difficulties. Some research has utilized cyclic stress to align smooth muscle cells (SMCs) but due to the long time conditioning needed, it was not possible to use primary human cells because of expeditious senescence occurred . We demonstrate rapid buildup of a homogeneous relatively thick (30-40 μm) aligned smooth muscle tissue via layer by layer (LBL) technique within microchannels and a soft cell-adhesive hydrogel. Using a microchannelled scaffold with gapped microwalls, two layers of primary human SMCs separated by an interlayer hydrogel were cultured to confluence within the microchannels. The SMCs aligned along the microchannels because of the physically constraining microwalls. A novel double layered gel consisting of a mixture of pristine and oxidized alginate hydrogel coated with collagen was designed to place between each layer of cells, leading to a thicker tissue in a shorter time. The SMCs penetrated the soft thin interlayer hydrogel within 6 days of seeding of the 2nd cell layer so that the entire construct became more or less homogeneously populated by the SMCs. The unique LBL technique applied within the micropatterned scaffold using a soft cell-adhesive gel interlayer allows rapid growth and confluence of SMCs on 2D surface but at the same time aligns the cells and builds up multiple layers into a 3D tissue. This pseudo-3D buildup method avoids the typical steric resistance of hydrogel embedding.

Polymer removal from electronic grade single-walled carbon nanotubes after gel electrophoresis

Semiconducting single-walled carbon nanotubes (s-SWNTs) are attractive candidates for next-generation printable semiconductors. However, all current synthesis methods produce s-SWNTs which are co-mingled with metallic (m-) SWNTs. Agarose gel electrophoresis has been reported to be an effective technique for the separation of s-SWNTs from m-SWNTs but removal of the agarose gel after separation has proved to be non-trivial. To remove agarose and the organic dispersing agent, specifically chondroitin sulfate in this work, from sorted s-SWNTs obtained by agarose gel electrophoresis, we employ the multi-step process involving a chlorosulfonic acid (HSO3Cl) wash, a base wash and thermal annealing. Herein, we report the detailed analysis of the effects of the various steps for gel removal from SWNTs by Fourier transform infrared (FTIR) spectroscopy, thermal gravimetric analysis (TGA), FTIR-TGA, scanning electron microscopy, atomic force microscopy and Raman spectroscopy. The polymer-contaminated s-SWNTs were dissolved in HSO3Cl, then selectively precipitated in a large excess of water, then washed with a base (NaOH) and finally thermally annealed. A detailed analysis confirmed that the final annealed samples contained almost no residual polymers. Field effect transistors were also fabricated from the annealed s-SWNTs and they showed good performance metrics with on/off ratio and mobility in the ∼102 to 106 and ∼2.5–9.5 cm2 V−1 s−1 ranges, respectively. Our method of gel electrophoresis and chlorosulfonic acid treatment produces clean and defect-free tubes which may be used for electronic applications.

Direct intermolecular force measurements between functional groups and individual metallic or semiconducting single-walled carbon nanotubes.

Many electronic applications of single-walled carbon nanotubes (SWNTs) require electronic homogeneity in order to maximally exploit their outstanding properties. Non-covalent separation is attractive as it is scalable and results in minimal alteration of nanotube properties. However, fundamental understanding of the metallicity-dependence of functional group interactions with nanotubes is still lacking; this lack is compounded by the absence of methods to directly measure these interactions. Herein, a novel technology platform based on a recently developed atomic force microscopy (AFM) mode is reported which directly quantifies the adhesion forces between a chosen functional group and individual nanotubes of known metallicity, permitting comparisons between different metallicity. These results unambiguously show that this technology platform is able to discriminate the subtle adhesion force differences of a chosen functional group with pure metallic as opposed to pure semiconducting nanotubes. This new method provides a route towards rapid advances in understanding of non-covalent interactions of large libraries of compounds with nanotubes of varying metallicity and diameter; presenting a superior tool to assist the discovery of more effective metallicity-based SWNT separation agents.

Biomaterials patterned with discontinuous microwalls for vascular smooth muscle cell culture: biodegradable small diameter vascular grafts and stable cell culture substrates

Abstract The medial layer of small diameter blood vessels contains circumferentially aligned vascular smooth muscle cells (vSMC) that possess contractile phenotype. In tissue-engineered constructs, these cellular characteristics are usually achieved by seeding planar scaffolds with vSMC, rolling the cell-laden scaffold into a tubular structure, and maturing the construct in a pulsatile bioreactor, a lengthy process that can take up to two months. During the maturation phase, the cells circumferentially orient, their contractile protein expression increases, and they obtain a contractile phenotype. Generating cell culture platforms that enable the rapid production of directionally oriented vSMC with increased contractile protein expression would be a major step forward for blood vessel tissue engineering and would greatly facilitate the in vitro study of vSMC biology. Previously, we developed a micropatterned cell culture surface that promotes orientation and contractile protein expression of vSMC. Herein, we explore two potential applications of this technology. First, we fabricate tubular and biodegradable scaffolds that possess the micropatterning on their exterior surface. When vSMC are seeded on these scaffolds, they initially proliferate in order to fill the microchannels and as confluence is reached the cells align in the direction of the micropatterning resulting in a biodegradable scaffold that is inhabited by circumferentially aligned vSMC within a week. Second, we illustrate that we can generate biostable cell culture surfaces that allow the in vitro study of the cells in a more contractile state. Specifically, we explore contractile protein expression of cells cultured on the micropatterned surfaces with the addition of soluble transforming growth factor beta one (TGFβ1).