Chandran R. Sabanayagam

A hydrogel-based tumor model for the evaluation of nanoparticle-based cancer therapeutics

Three-dimensional (3D) tissue-engineered tumor models have the potential to bridge the gap between monolayer cultures and patient-derived xenografts for the testing of nanoparticle (NP)-based cancer therapeutics. In this study, a hydrogel-derived prostate cancer (PCa) model was developed for the in vitro evaluation of doxorubicin (Dox)-loaded polymer NPs (Dox-NPs). The hydrogels were synthesized using chemically modified hyaluronic acid (HA) carrying acrylate groups (HA-AC) or reactive thiols (HA-SH). The crosslinked hydrogel networks exhibited an estimated pore size of 70-100 nm, similar to the spacing of the extracellular matrices (ECM) surrounding tumor tissues. LNCaP PCa cells entrapped in the HA matrices formed distinct tumor-like multicellular aggregates with an average diameter of 50 μm after 7 days of culture. Compared to cells grown on two-dimensional (2D) tissue culture plates, cells from the engineered tumoroids expressed significantly higher levels of multidrug resistance (MDR) proteins, including multidrug resistance protein 1 (MRP1) and lung resistance-related protein (LRP), both at the mRNA and the protein levels. Separately, Dox-NPs with an average diameter of 54 ± 1 nm were prepared from amphiphilic block copolymers based on poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) bearing pendant cyclic ketals. Dox-NPs were able to diffuse through the hydrogel matrices, penetrate into the tumoroid and be internalized by LNCaP PCa cells through caveolae-mediated endocytosis and macropinocytosis pathways. Compared to 2D cultures, LNCaP PCa cells cultured as multicellular aggregates in HA hydrogel were more resistant to Dox and Dox-NPs treatments. Moreover, the NP-based Dox formulation could bypass the drug efflux function of MRP1, thereby partially reversing the resistance to free Dox in 3D cultures. Overall, the engineered tumor model has the potential to provide predictable results on the efficacy of NP-based cancer therapeutics.

Hyaluronic Acid-Based Hydrogels Containing Covalently Integrated Drug Depots: Implication for Controlling Inflammation in Mechanically Stressed Tissues

Synthetic hydrogels containing covalently integrated soft and deformable drug depots capable of releasing therapeutic molecules in response to mechanical forces are attractive candidates for the treatment of degenerated tissues that are normally load bearing. Herein, radically cross-linkable block copolymer micelles (xBCM) assembled from an amphiphilic block copolymer consisting of hydrophilic poly(acrylic acid) (PAA) partially modified with 2-hydroxyethyl acrylate, and hydrophobic poly(n-butyl acryclate) (PnBA) were employed as the drug depots and the microscopic cross-linkers for the preparation of hyaluronic acid (HA)-based, hydrogels. HA hydrogels containing covalently integrated micelles (HAxBCM) were prepared by radical polymerization of glycidyl methacrylate (GMA)-modified HA (HAGMA) in the presence of xBCMs. When micelles prepared from the parent PAA-b-PnBA without any polymerizable double bonds were used, hydrogels containing physically entrapped micelles (HApBCM) were obtained. The addition of xBCMs to a HAGMA precursor solution accelerated the gelation kinetics and altered the hydrogel mechanical properties. The resultant HAxBCM gels exhibit an elastic modulus of 847 ± 43 Pa and a compressive modulus of 9.2 ± 0.7 kPa. Diffusion analysis of Nile Red (NR)-labeled xBCMs employing fluorescence correlation spectroscopy confirmed the covalent immobilization of xBCMs in HA networks. Covalent integration of dexamethasone (DEX)-loaded xBCMs in HA gels significantly reduced the initial burst release and provided sustained release over a prolonged period. Importantly, DEX release from HAxBCM gels was accelerated by intermittently applied external compression in a strain-dependent manner. Culturing macrophages in the presence of DEX-releasing HAxBCM gels significantly reduced cellular production of inflammatory cytokines. Incorporating mechano-responsive modules in synthetic matrices offers a novel strategy to harvest mechanical stress present in the healing wounds to initiate tissue repair.

Poly(ε-caprolactone)-based copolymers bearing pendant cyclic ketals and reactive acrylates for the fabrication of photocrosslinked elastomers.

Block copolymers of poly(ethylene glycol) and poly(ε-caprolactone) (PCL) with chemically addressable functional groups were synthesized and characterized. Ring-opening polymerization of ε-caprolactone (CL) and 1,4,8-trioxaspiro-[4,6]-9-undecanone (TSU) using α-methoxy, ω-hydroxyl poly(ethylene glycol) as the initiator afforded a copolymer with cyclic ketals being randomly distributed in the hydrophobic PCL block. At an initiator/catalyst molar ratio of 10/1 and a TSU/CL weight ratio of 1/4, a ketal-carrying copolymer (ECT2-CK) with Mn of 52 kDa and a ketal content of 15 mol.% was obtained. Quantitative side-chain deacetalization revealed the reactive ketones without noticeable polymer degradation. In our study, 10 mol.% of cyclic ketals were deprotected and the ketone-containing copolymer was designated as ECT2-CO. Reaction of ECT2-CO with 2-(2-(aminooxy)acetoxy)-ethyl acrylate gave rise to an acrylated product (ECT2-AC) containing an estimated 3-5 acrylate groups per chain. UV-initiated radical polymerization of ECT2-AC in dichloromethane resulted in a crosslinked network (xECT2-AC). Thermal and morphological analyses employing differential scanning calorimetry and atomic force microscopy operated in PeakForce Tapping mode revealed the semicrystalline nature of the network, which contained stiff crystalline lamellae dispersed in a softer amorphous interstitial. Macroscopic and nanoscale mechanical characterizations showed that ECT2-CK exhibited a significantly lower modulus than PCL of a similar molecular weight. Whereas ECT2-CK undergoes a plastic deformation with a distinct yield point and a cold-drawing region, xECT2-AC exhibits a compliant, elastomeric deformation with a Youngs modulus of 0.5±0.1 MPa at 37°C. When properly processed, the crosslinked network exhibited shape-memory behaviors, with shape fixity and shape recovery values close to 1 and a shape recovery time of less than 4s at 37°C. In vitro studies showed that xECT2-AC films did not induce any cytotoxic effects on the cultured mesenchymal stem cells. The crosslinkable polyester copolymers can be potentially used as tissue engineering scaffolds and minimally invasive medical devices.

Histone-targeted Polyplexes Avoid Endosomal Escape and Enter the Nucleus During Postmitotic Redistribution of ER Membranes.

Nonviral gene delivery is a promising therapeutic approach because of its safety and controllability, yet limited gene transfer efficacy is a common issue. Most nonviral strategies rely upon endosomal escape designs; however, endosomal escape is often uncorrelated with improved gene transfer and membranolytic structures are typically cytotoxic. Previously, we showed that histone-targeted polyplexes trafficked to the nucleus through an alternative route involving caveolae and the Golgi and endoplasmic reticulum (ER), using pathways similar to several pathogens. We hypothesized that the efficacy of these polyplexes was due to an increased utilization of native vesicular trafficking as well as regulation by histone effectors. Accordingly, using confocal microscopy and cellular fractionation, we determined that a key effect of histone-targeting was to route polyplexes away from clathrin-mediated recycling pathways by harnessing endomembrane transfer routes regulated by histone methyltransferases. An unprecedented finding was that polyplexes accumulated in Rab6-labeled Golgi/ER vesicles and ultimately shuttled directly into the nucleus during ER-mediated nuclear envelope reassembly. Specifically, super resolution microscopy and fluorescence correlation spectroscopy unequivocally indicated that the polyplexes remained associated with ER vesicles/membranes until mitosis, when they were redistributed into the nucleus. These novel findings highlight alternative mechanisms to subvert endolysosomal trafficking and harness the ER to enhance gene transfer.

ChIP and Chips: Introducing the WormPharm for correlative studies employing pharmacology and genome-wide analyses in C. elegans

We present the WormPharm, an automated microfluidic platform that utilizes an axenic medium to culture C. elegans. The WormPharm is capable of sustaining C. elegans for extended periods, while recording worm development and growth with high temporal resolution ranging from seconds to minutes over several days to months. We demonstrate the utility of the device to monitor C. elegans growth in the presence of varying doses of nicotine and alcohol. Furthermore, we show that C. elegans cultured in the WormPharm are amendable for high-throughput genomic assays, i.e. chromatin-immunoprecipitation followed by next generation sequencing, and confirm that nematodes grown in monoxenic and axenic cultures exhibit genetic modifications that correlate with observed phenotypes. The WormPharm is a powerful tool for analyzing the effects of chemical, nutritional and environmental variations on organism level responses in conjunction with genome-wide changes in C. elegans.

Effects of liquid cultivation on gene expression and phenotype of C. elegans

BackgroundLiquid cultures have been commonly used in space, toxicology, and pharmacology studies of Caenorhabditis elegans. However, the knowledge about transcriptomic alterations caused by liquid cultivation remains limited. Moreover, the impact of different genotypes in rapid adaptive responses to environmental changes (e.g., liquid cultivation) is often overlooked. Here, we report the transcriptomic and phenotypic responses of laboratory N2 and the wild-isolate AB1 strains after culturing P0 worms on agar plates, F1 in liquid cultures, and F2 back on agar plates.ResultsSignificant variations were found in the gene expressions between the N2 and AB1 strains in response to liquid cultivation. The results demonstrated that 8–34% of the environmental change-induced transcriptional responses are transmitted to the subsequent generation. By categorizing the gene expressions for genotype, environment, and genotype-environment interactions, we identified that the genotype has a substantial impact on the adaptive responses. Functional analysis of the transcriptome showed correlation with phenotypical changes. For example, the N2 strain exhibited alterations in both phenotype and gene expressions for germline and cuticle in axenic liquid cultivation. We found transcript evidence to approximately 21% of the computationally predicted genes in C. elegans by exposing the worms to environmental changes.ConclusionsThe presented study reveals substantial differences between N2 and AB1 strains for transcriptomic and phenotypical responses to rapid environmental changes. Our data can provide standard controls for future studies for the liquid cultivation of C. elegans and enable the discovery of condition-specific genes.

Microstructured Elastomer-PEG Hydrogels via Kinetic Capture of Aqueous Liquid-Liquid Phase Separation

Abstract Heterogeneous hydrogels with desired matrix complexity are studied for a variety of biomimetic materials. Despite the range of such microstructured materials described, few methods permit independent control over microstructure and microscale mechanics by precisely controlled, single‐step processing methods. Here, a phototriggered crosslinking methodology that traps microstructures in liquid–liquid phase‐separated solutions of a highly elastomeric resilin‐like polypeptide (RLP) and poly(ethylene glycol) (PEG) is reported. RLP‐rich domains of various diameters can be trapped in a PEG continuous phase, with the kinetics of domain maturation dependent on the degree of acrylation. The chemical composition of both hydrogel phases over time is assessed via in situ hyperspectral coherent Raman microscopy, with equilibrium concentrations consistent with the compositions derived from NMR‐measured coexistence curves. Atomic force microscopy reveals that the local mechanical properties of the two phases evolve over time, even as the bulk modulus of the material remains constant, showing that the strategy permits control of mechanical properties on micrometer length scales, of relevance in generating mechanically robust materials for a range of applications. As one example, the successful encapsulation, localization, and survival of primary cells are demonstrated and suggest the potential application of phase‐separated RLP‐PEG hydrogels in regenerative medicine applications.