Ying Chau

Incorporation of a matrix metalloproteinase-sensitive substrate into self-assembling peptides - a model for biofunctional scaffolds.

Controlling and guiding cell behavior requires scaffolding materials capable of programming the three-dimensional (3-D) extracellular environment. In this study, we devised a new self-assembling peptide template for synthesizing nanofibrous hydrogels containing cell-responsive ligands. In particular, the insertion of a matrix metalloproteinase-2 (MMP-2) labile hexapeptide into the self-assembling building blocks of arginine-alanine-aspartate-alanine (RADA) was investigated. A series of peptides, varied by the position of the MMP-2 hexapeptide substrate and the length of RADA blocks, were prepared by parallel synthesis. Their self-assembling capabilities were characterized and compared by circular dichroism spectroscopy and dynamical mechanical analysis. Among all the different insertion patterns, the sequence comprising a centrically positioned MMP-2 substrate was flanked with three RADA units on each side self-assembled into a hydrogel matrix, with mechanical properties and nanofiber morphology comparable to the native material built with (RADA)(4) alone. Exposure of the new gel to MMP-2 resulted in peptide cleavage, as confirmed by mass spectroscopy, and a decrease in surface hardness, as detected by nanoindentor, indicating that the enzyme mediated degradation was localized to the gel surface. The new design can be used for introducing biological functions into self-assembling peptides to create scaffolding materials with potential applications in areas such as tissue engineering and regenerative medicine.

Antitumor efficacy of a novel polymer-peptide-drug conjugate in human tumor xenograft models.

We have designed a new dextran–peptide–methotrexate conjugate to achieve tumor‐targeted delivery of chemotherapeutics. The dextran carrier was selected to allow passive targeting and enhanced permeation and retention (EPR). The peptide linker has also been optimized to allow drug release in the presence of matrix‐metalloproteinase‐2 (MMP‐2) and matrix‐metalloproteinase‐9 (MMP‐9), 2 important tumor‐associated enzymes. The new conjugate was assessed for its in vivo antitumor efficacy and systemic side effects. It was compared with free methotrexate (MTX) and a similar conjugate, differing by an MMP‐insensitive linker, at equivalent intraperitoneal dosages. The MMP‐sensitive conjugate demonstrated tolerable in vivo side effects and effective inhibition of in vivo tumor growth by 83% in each of the 2 separate tumor models that overexpress MMP (HT‐1080 and U‐87). The antiproliferative effect of the drug contributed to the inhibition of tumor growth. In contrast, free MTX resulted in no significant tumor reduction in the same models. Neither free MTX nor the conjugate caused any tumor inhibition in the mice bearing RT‐112, a slower growing model that does not overexpress MMP. MMP‐insensitive conjugates, though able to inhibit tumor growth, caused toxicity in the small intestine and bone marrow.

Size-dependent internalisation of folate-decorated nanoparticles via the pathways of clathrin and caveolae-mediated endocytosis in ARPE-19 cells

We aim to quantify the effect of size and degree of folate loading of folate‐decorated polymeric nanoparticles (NPs) on the kinetics of cellular uptake and the selection of endocytic pathways in retinal pigment epithelium (RPE) cells.

Synthesis of Linear Polyether Polyol Derivatives As New Materials for Bioconjugation

Linear polyether polyol (PEP) consisting of glycidol as repeating units is a flexible hydrophilic aliphatic polymer. The polyether main chain is similar to the widely used, biocompatible polymer poly(ethylene glycol) (PEG). While linear PEG has one or two terminal hydroxyl group(s), linear PEP distinguishes itself by the large number of pendant hydroxyl groups along the polyether main chain. We propose that this property of PEP represents a major advantage over PEG, namely, by providing multiple anchorage points and increasing the possibility for introducing different functional groups. As a first step to establishing PEP as a bioconjugation material, we modified the pendant hydroxyl groups on PEP and prepared a series of mono- and heterobifunctional derivatives with the potential to join various drug entities and biomolecules. The synthesis methods and the results of characterization are reported here.

Specific uptake of folate-decorated triamcinolone-encapsulating nanoparticles by retinal pigment epithelium cells enhances and prolongs antiangiogenic activity

We are proposing folate-decorated polymeric nanoparticles as carriers of poorly soluble drug molecules for intracellular and prolonged delivery to retinal pigment epithelium (RPE) cells. RPE is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). In this study, folate-functionalized poly(ethylene glycol)-b-polycaprolactone (folate-PEG-b-PCL) were synthesized for assembling into nanoparticles of ~130nm. These nanoparticles were internalized into ARPE-19 (human RPE cell line) via receptor-mediated endocytosis, and the cellular uptake was significantly higher than particles without folate modification. Triamcinolone acetonide (TA) was efficiently encapsulated (>97%) into the folate-decorated nanoparticles and was slowly released over a period of 4 weeks at pH 5.5 and 8 weeks at pH 7.4. The enhanced uptake and controlled release resulted in prolonged anti-angiogenic gene expression of RPE cells. In cell culture, the down-regulation of vascular endothelial growth factor (VEGF) and up-regulation of pigment epithelium derived factor (PEDF) lasted for at least 3 weeks. Unlike benzyl alcohol, the surfactant found in commercial formulation, folate-modified nanoparticles were non-toxic. Furthermore, TA became less cytotoxic by being encapsulated in the nanoparticles. Our findings suggest that folate-PEG-PCL nanoparticles are promising drug carriers for RPE targeting.

One-Step “Click” Method for Generating Vinyl Sulfone Groups on Hydroxyl-Containing Water-Soluble Polymers

■ INTRODUCTION “Click chemistry” is a concept for chemical synthesis proposed by Sharpless et al. in 2001. It requires the reactions to be selective in reactivity while highly efficient. Importantly, the reaction conditions are mild and the procedure is simple. This idea has since received overwhelming responses and has been extended to other disciplines of chemistry. One class of click reactions that involves Michael addition between an electrophilic double bond and a thiol, termed “thiol-Michael addition”, has attracted much attention in the field of bioconjugates and in situ gelation. This is because some of its reactions can proceed efficiently at physiological pH and temperature in aqueous conditions, which is required to preserve the labile biomolecules or cells. At physiological pH, thiols are partially deprotonated and become thiol anions (S−, Michael acceptors), which can readily react with certain electron-deficient double bonds (Michael acceptor). Among the electron-deficient double bonds, the vinyl sulfone group (VS) is a popular choice because it is effective under mild aqueous conditions. Various VS-modified polymers, including dextran-VS, HA-VS, and PEG-VS have been successfully synthesized and shown to be biocompatible and useful for forming bioconjugates and in situ hydrogels with a thiol-containing counterpart. However, most of the methods for producing VS-modified polymers require organic solvents and often involve multiple and lengthy steps. These synthesis schemes are also specific to a particular polymer, which may not be easily adapted to a new material. We appreciate the ability of VS to participate in “click chemistry” toward thiol, and aimed to take a “click chemistry” approach to generate VS groups in biopolymers. We envisioned that the reaction for generating VS should be simple, efficient, and capable of a controllable degree of modification. To maximize the yield and minimize the potential hazard when applying to living systems, the reaction should also take place in aqueous environment. Here, we present a one-step reaction method to prepare vinyl sulfone (VS)-functionalized polymers by a simple “click” reaction that fulfils all the proposed requirements. This synthesis method is applicable to all hydroxyl-containing water-soluble polymers in general, including hyaluronic acid (HA), polyethylene glycol (PEG), dextran, alginate and polyvinyl alcohol (PVA). We further show that the VSfunctionalized polymers can participate in a subsequent thiolMichael “click” reaction with a thiol counterpart. This novelty is made possible by our insights of a reaction for the preparation of cross-linked hyaluronic acid (HA) hydrogel, in which the polymer chains were cross-linked by a small molecule, divinyl sulfone (DVS), via the hydroxyl groups of HA. Our understanding of the reaction mechanism between DVS and hydroxyl allows us to modify the reaction conditions, essentially by increasing DVS to OH molar ratio and optimizing reaction parameters, including pH and time. The result is that DVS is not used as a cross-linker, but a reagent to modify polymers by a simple “click” reaction. Despite that the changes in the reaction condition seem subtle, the consequences are significant. This simple “click” method enables the polymers to become “clickable” subsequently under physiological conditions. The modified polymers, instead of forming precross-linked hydrogels, generate “clickable” precursors that are suitable for a wide range of biomedical use.

Ultrasound-enhanced intrascleral delivery of protein

We aim to investigate ultrasound on enhancing protein penetration into the sclera, a non-invasive method to overcome the first barrier in taking the transscleral route for delivering therapeutics. Rabbit eyes were immersed in a fluorescein isothiocyanate conjugated bovine serum albumin solution. The distances of protein penetration, with and without ultrasound (30s continuous wave, 1MHz, 0.05W/cm(2)) applied on the sclera, and at different immersion time intervals (0, 5, 15, 30 and 60min), were measured by examining the cryo-sectioned tissues under fluorescence microscope (≥60 measurements from 3 eyes for each condition). Retina was examined for structural damage by histology. It was found that ultrasound enhances the intrascleral penetration of protein, increasing the diffusivity by 1.6-folds while causing no damage to the retinal tissues. This physical modulation of the sclera is temporary, as evident by the restoration of the diffusional resistance at 15min after ultrasound treatment. The negligible effect of ultrasound-induced convection and the minimal temperature rise (<0.5°C), together with cavitation detected by acoustic emission and a decreased penetration distance at higher ultrasound frequency (30s continuous wave, 3MHz, 0.05W/cm(2)), suggest that cavitation is a possible mechanism for increasing the permeability of the sclera for diffusive transport.

Neural differentiation directed by self-assembling peptide scaffolds presenting laminin-derived epitopes

We prepared biofunctionalized matrices for cell growth using (RADA)(3)IKVAV(RADA)(3) ((Arg-Ala-Asp-Ala)(3)-Ile-Lys-Val-Ala-Val-(Arg-Ala-Asp-Ala)(3)) and (RADA)(4)IKVAV ((Arg-Ala-Asp-Ala)(4)-Ile-Lys-Val-Ala-Val), self-assembling peptides with a laminin-derived sequence inserted between and attached terminally to the repeats of RADA, respectively. The material-cell interactions were investigated with PC12, a cell line commonly used as a model for studying neural differentiation. The behavior of PC12 and especially the neural differentiation was guided by the presence of IKVAV. Furthermore, the cell-material interactions were dependent on the culture dimensionality and the position of IKVAV in the self-assembling peptide template. In the two-dimensional (2-D) culture, matrices containing IKVAV stimulated significantly longer neurite outgrowths from PC12 cells than did (RADA)(4). More pronounced effect was observed in (RADA)(3)IKVAV(RADA)(3) than in (RADA)(4)IKVAV. In the three-dimensional (3-D) culture, neurite outgrowth was not observed in the biofunctionalized matrices. Instead, cells displayed higher proliferation rate and survived longer culture time than in the 2-D culture, with such enhancement being most significant in (RADA)(3)IKVAV(RADA)(3.) Despite the lack of differentiation phenotype, the cells grown in 3-D biofunctionalized matrices were primed for differentiation, as evident by enhanced neurite outgrowth, increased neurite networking, and up-regulated expression of differentiation markers upon their reintroduction to the 2-D culture condition on petri dish. With the ease of incorporating biofunctional epitopes, and the flexibility to support either 2-D or 3-D culture, self-assembling peptides provide versatile scaffolds to study the multiple facets of biomaterial-cell interactions.

Effectiveness of Muscimol‐containing Microparticles against Pilocarpine‐induced Focal Seizures

Summary:  Purpose: To investigate the efficacy of in situ lipid–protein–sugar particles (LPSPs) in mitigating the epileptogenic and histologic effects of intrahippocampal pilocarpine in rats.

Synthesis, characterization, and in vivo evaluation of poly(ethylene oxide-co-glycidol)-platinate conjugate.

Poly(ethylene oxide-co-glycidol) (poly(EO-co-Gly)), a member of polyether polyol (PEP), resembles polyethylene glycol (PEG) in the polymer backbone but distinguishes itself by having multiple pendent groups along the main chain. We showed that this new bioconjugation material is biocompatible by its lack of toxicity on fibroblast cell growth, inactivity in hemolysis, and the absence of side effects after injection in mice. The usefulness of poly(EO-co-Gly) as a polymeric drug carrier was demonstrated via the preparation and characterization of a new anticancer polymer-drug conjugate, poly(EO-co-Gly)-platinate. The drug loading was 9.1-12.6% (cisplatin/conjugate w/w), at least four times higher than a PEG conjugate of similar molecular weight. The aqueous solubility of cisplatin was increased by around 10 folds after conjugation. Platinum complexes were released from the conjugate in a sustained manner over 2 days. The release of active drugs was confirmed by the antitumor activity of poly(EO-co-Gly)-platinate in vitro against HONE-1 (human nasopharyngeal carcinoma) and MCF-7 (human breast cancer), albeit at a potency lower than free cisplatin. Poly(EO-co-Gly)-platinate improved the therapeutic index of cisplatin in vivo. The conjugate had a similar antitumor activity as free cisplatin in nude mice bearing HONE-1 xenografts, and achieved 52% inhibition of tumor growth at the conclusion of the study. While free cisplatin injection caused a severe loss in body weight (>20%), poly(EO-co-Gly)-platinate resulted in mild side effects. These findings support that poly(EO-co-Gly) is a suitable drug carrier.