Abraham Joy

Control of surface chemistry, substrate stiffness, and cell function in a novel terpolymer methacrylate library

A focused library of methacrylate terpolymers was synthesized to explore the effects of varying surface chemistry and adhesive peptide ligands on cell function. The chemical diversity of methacrylate monomers enabled construction of a library of polymers in which one can systematically vary the chemical composition to achieve a wide range of contact angle, Youngs modulus, and T(g) values. Furthermore, the materials were designed to allow surface immobilization of bioactive peptides. We then examined the effects of these material compositions on protein adsorption and cell attachment, proliferation, and differentiation. We observed that chemical composition of the polymers was an important determinant for NIH 3T3 cell attachment and proliferation, as well as human mesenchymal stem cell differentiation, and correlated directly with the ability of the polymers to adsorb proteins that mediate cell adhesion. Importantly, functionalization of the methacrylate terpolymer library with an adhesive GRGDS peptide normalized cellular responses. RGD-functionalized polymers uniformly exhibited robust attachment, proliferation, and differentiation irrespective of the underlying substrate chemistry. These studies provide a library-based approach to rapidly explore the biological functionality of biomaterials with a wide range of compositions and highlight the importance of cell and protein cell adhesion in predicting their performance.

A library of multifunctional polyesters with "peptide-like" pendant functional groups.

The synthesis and characterization of a library of modular multifunctional polyesters with pendant functional groups are described. The polyesters were synthesized at room temperature by carbodiimide-mediated polymerization of pendant functionalized diols and succinic acid. The pendant groups are designed to mimic the side chains of peptides, and it is shown that the physical properties of the polyesters can be modulated over a wide range by the choice of pendant groups. We also show that the pendant groups can be orthogonally functionalized with ligands such as fluorophores, poly(ethylene glycol) (PEG) or Arg-Gly-Asp (RGD).

Poly(ethylene glycol) as a sensitive regulator of cell survival fate on polymeric biomaterials: the interplay of cell adhesion and pro-oxidant signaling mechanisms

Poly(ethylene glycol) (PEG) is one of the most widely used compounds across a variety of platforms and is increasingly found in medical applications. Polycarbonates containing varying mol% of PEG (Mw 1000) were used to probe the effects of PEG on cell adhesion, proliferation, spreading, and survival. Two contrasting PEG-mediated cell signaling elements affected these cellular behaviors: (i) integrin α5 receptor mediated cellular focal adhesions to the biomaterial surface and (ii) modulation of cellular redox and apoptosis through generation of reactive oxygen species (ROS). At lower PEG1k mol% (5% and 8%) cell attachment and spreading decreased concomitantly due to ROS, whereas at the higher PEG1k mol% studied (10% and 20%) an unusual super-adhesive behavior was observed. At higher PEG1k mol% cells exhibited greatly enhanced spreading, which was confirmed through immunolocalization of integrin α5 receptors and enhanced mRNA expression of the integrin α5 gene. These cellular responses on higher PEG1k mol% co-polymers were sufficient to overcome the ROS-driven effects on caspase activation and cell shrinkage, which dominated at lower PEG1k mol%. These studies elucidate PEG-mediated cellular signaling with the implication that the adhesion and apoptotic activity of PEG-rich materials can be sensitively controlled by anti-oxidant addition. Moreover, this study shows that biomaterials can drive the cell fate in opposing directions through concurrent property changes.

Folate Receptor–Targeted Polymeric Micellar Nanocarriers for Delivery of Orlistat as a Repurposed Drug against Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a recalcitrant malignancy with no available targeted therapy. Off-target effects and poor bioavailability of the FDA-approved antiobesity drug orlistat hinder its clinical translation as a repurposed new drug against TNBC. Here, we demonstrate a newly engineered drug formulation for packaging orlistat tailored to TNBC treatment. We synthesized TNBC-specific folate receptor–targeted micellar nanoparticles (NP) carrying orlistat, which improved the solubility (70–80 μg/mL) of this water-insoluble drug. The targeted NPs also improved the delivery and bioavailability of orlistat to MDA-MB-231 cells in culture and to tumor xenografts in a nude mouse model. We prepared HEA–EHA copolymer micellar NPs by copolymerization of 2-hydroxyethylacrylate (HEA) and 2-ethylhexylacrylate (EHA), and functionalized them with folic acid and an imaging dye. Fluorescence-activated cell sorting (FACS) analysis of TNBC cells indicated a dose-dependent increase in apoptotic populations in cells treated with free orlistat, orlistat NPs, and folate-receptor–targeted Fol-HEA-EHA-orlistat NPs in which Fol-HEA-EHA-orlistat NPs showed significantly higher cytotoxicity than free orlistat. In vitro analysis data demonstrated significant apoptosis at nanomolar concentrations in cells activated through caspase-3 and PARP inhibition. In vivo analysis demonstrated significant antitumor effects in living mice after targeted treatment of tumors, and confirmed by fluorescence imaging. Moreover, folate receptor–targeted Fol-DyLight747-orlistat NP–treated mice exhibited significantly higher reduction in tumor volume compared to control group. Taken together, these results indicate that orlistat packaged in HEA-b-EHA micellar NPs is a highly promising new drug formulation for TNBC therapy. Mol Cancer Ther; 15(2); 221–31. ©2015 AACR.

Thermoresponsive dual emission nanosensor based on quantum dots and dye labeled poly(N-isopropylacrylamide)

Dual end-functionalized telechelic poly(N-isopropylacrylamide) (PNIPAM) was synthesized using reversible addition–fragmentation chain-transfer (RAFT) polymerization. One end was coupled to a fluorescent dye and the other end was covalently coupled to CdSe/ZnS quantum dots (QDs) through carbodiimide chemistry. The hybrid nanoparticle shows ratiometric changes in fluorescence emission upon temperature cycling between 25 °C and 45 °C.

Nontoxic Cationic Coumarin Polyester Coatings Prevent Pseudomonas aeruginosa Biofilm Formation

The rapid increase in bacterial infections and antimicrobial resistance is a growing public health concern. Infections arising from bacterial contamination of surgical tools, medical implants, catheters, and hospital surfaces can potentially be addressed by antimicrobial polymeric coatings. The challenge in developing such polymers for in vivo use is the ability to achieve high antimicrobial efficacy while at the same time being nontoxic to human cells. Although several classes of antimicrobial polymers have been developed, many of them cannot be used in the clinical setting due to their nonselective toxicity toward bacteria and mammalian cells. Here, we demonstrate that coumarin polyesters with cationic pendant groups are very effective against Gram negative Pseudomonas aeruginosa. Coumarin polyesters with pendant cationic amine groups were coated onto glass coverslips and tested for their antimicrobial activity against P. aeruginosa colonization of the surface. The results demonstrate that the cationic coumarin polyester kills the surface attached bacterial cells preventing biofilm formation but does not show any hemolytic activity or discernible toxicity toward mammalian cells. The antimicrobial polyesters described in this work have several advantages desired in antimicrobial coatings such as high antimicrobial activity, low toxicity toward mammalian cells, visualization and ease of synthesis and fabrication, all of which are necessary for translation to the clinic.

Photoresponsive polyesters by incorporation of alkoxyphenacyl or coumarin chromophores along the backbone

The synthesis and photochemical characterization of two classes of photoresponsive polyesters are described. These polyesters contain either alkoxyphenacyl or coumarin chromophores embedded along the polymer chain. The alkoxyphenacyl polyesters undergo efficient photoinduced chain scission upon irradiation at 300 nm in solution or as a nanoparticle suspension. At 254 nm the coumarin polyesters undergo polymer chain scission. Irradiation of the coumarin polyesters in solution at 350 nm results in both chain crosslinking and chain scission behavior, while irradiation of the coumarin polyesters as nanoparticles results in chain crosslinking. The properties of the alkoxyphenacyl and coumarin polyesters are influenced by the choice of diacid as seen from their thermal behavior. The use of glutamic acid enabled surface or bulk functionalization of the photoresponsive polymers. In addition, controlled release of Nile Red from coumarin polyester nanoparticles is demonstrated by modulation of the wavelength and intensity of irradiation.

A coacervate-forming biodegradable polyester with elevated LCST based on bis-(2-methoxyethyl)amine

Recently, we reported a new class of biodegradable, thermoresponsive polyesters (TR-PEs) inspired by polyacrylamides and elastin-like peptides (ELPs). The polyesters exhibit tunable cloud point temperatures (Tcp) and thermoresponsive coacervation in aqueous solution as shown via UV-vis spectroscopy, 1H NMR, and DLS. However, the Tcp of all TR-PEs remained low (<15 °C), and higher thermoresponsivity would be beneficial for many applications. This study examines the synthesis, polymerization, and analysis of a new monomer bearing a more hydrophilic pendant group, bis-2-methoxyethylamine (bMoEtA). The resulting TR-PE, TR-bMoEtAPE, displays a threefold increase in Tcp (ca. 50 °C) that is affected by solution (DI water vs. phosphate buffered saline), concentration (1–40 mg mL−1) molecular weight (20–130 kDa), and cosolutes (Hofmeister salts and urea). The Tcp and Tg of random TR-bMoEtAPE copolymers can be tuned via comonomer feed. Variable temperature 1H NMR indicated a cooperative coacervation mechanism above Tcp, further reinforced by DLS measurements. As evidenced by UV-vis and SEC analysis, TR-bMoEtAPE underwent rapid degradation over a period of 7 days in DI water and PBS. Finally, cytotoxicity studies suggested that TR-bMoEtAPE is non-cytotoxic even at high concentrations (ca. 1000 μg mL−1). The increased Tcp and tunability suggests TR-bMoEtAPE as a potential candidate for future functionalized TR-PE therapeutic-delivery systems.

Micropatterned Coumarin Polyester Thin Films Direct Neurite Orientation

Guidance and migration of cells in the nervous system is imperative for proper development, maturation, and regeneration. In the peripheral nervous system (PNS), it is challenging for axons to bridge critical-sized injury defects to achieve repair and the central nervous system (CNS) has a very limited ability to regenerate after injury because of its innate injury response. The photoreactivity of the coumarin polyester used in this study enables efficient micropatterning using a custom digital micromirror device (DMD) and has been previously shown to be biodegradable, making these thin films ideal for cell guidance substrates with potential for future in vivo applications. With DMD, we fabricated coumarin polyester thin films into 10×20 μm and 15×50 μm micropatterns with depths ranging from 15 to 20 nm to enhance nervous system cell alignment. Adult primary neurons, oligodendrocytes, and astrocytes were isolated from rat brain tissue and seeded onto the polymer surfaces. After 24 h, cell type and neurite alignment were analyzed using phase contrast and fluorescence imaging. There was a significant difference (p<0.0001) in cell process distribution for both emergence angle (from the body of the cell) and orientation angle (at the tip of the growth cone) confirming alignment on patterned surfaces compared to control substrates (unpatterned polymer and glass surfaces). The expected frequency distribution for parallel alignment (≤15°) is 14% and the two micropatterned groups ranged from 42 to 49% alignment for emergence and orientation angle measurements, where the control groups range from 12 to 22% for parallel alignment. Despite depths being 15 to 20 nm, cell processes could sense these topographical changes and preferred to align to certain features of the micropatterns like the plateau/channel interface. As a result this initial study in utilizing these new DMD micropatterned coumarin polyester thin films has proven beneficial as an axon guidance platform for future nervous system regenerative strategies.

Biocompatibility and In Vivo Tolerability of a New Class of Photoresponsive Alkoxylphenacyl-Based Polycarbonates

Potential toxicities of chromophoric or polymeric units of most photoresponsive delivery systems have impacted clinical relevance. Herein, we evaluated the biocompatibility and tolerability of alkoxylphenacyl-based polycarbonates (APPs) as a new class of photoresponsive polymers. The polymers were applied as homopolymer or copolymers of polyethylene glycol (10%, w/w) or polycaprolactone (10%, w/w). APP polymers were comparable to poly(lactic-co-glycolic acid) (PLGA) based on cytotoxicity, macrophage activation, and blood compatibility. Data from biodistribution studies in BALB/c mice showed preferential accumulation in kidney and liver. Meanwhile, potential application of APP polymers as immediate or sustained (implants) drug delivery systems indicated that liver and kidney functions were not distorted. Also, plasma levels of tumor necrosis factor-alpha and interleukin-6 were comparable to PLGA-treated mice (p > 0.05). A histological analysis of liver and kidney sections showed no detectable damage for APP polymers. The overall data strongly supported potential consideration of APP polymers as photoresponsive delivery systems especially as implantable or tissue-mimicking photopatterned biomaterials.