Poly(2-propylacrylic acid)/poly(lactic-co-glycolic acid) blend microparticles as a targeted antigen delivery system to direct either CD4+ or CD8+ T cell activation.

Poly(lactic-co-glycolic acid) (PLGA) based microparticles (MPs) are widely investigated for their ability to load a range of molecules with high efficiency, including antigenic proteins, and release them in a controlled manner. Micron-sized PLGA MPs are readily phagocytosed by antigen presenting cells, and localized to endosomes. Due to low pH and digestive enzymes, encapsulated protein cargo is largely degraded and processed in endosomes for MHC-II loading and presentation to CD4+ T cells, with very little antigen delivered into the cytosol, limiting MHC-I antigenic loading and presentation to CD8+ T cells. In this work, PLGA was blended with poly(2-propylacrylic acid) (PPAA), a membrane destabilizing polymer, in order to incorporate an endosomal escape strategy into PLGA MPs as an easily fabricated platform with diverse loading capabilities, as a means to enable antigen presentation to CD8+ T cells. Ovalbumin (OVA)-loaded MPs were fabricated using a water-in-oil double emulsion with a 0% (PLGA only), 3 and 10% PPAA composition. MPs were subsequently determined to have an average diameter of 1 µm, with high loading and a release profile characteristic of PLGA. Bone marrow derived dendritic cells (DCs) were then incubated with MPs in order to evaluate localization, processing, and presentation of ovalbumin. Endosomal escape of OVA was observed only in DC groups treated with PPAA/PLGA blends, which promoted high levels of activation of CD8+ OVA-specific OT-I T cells, compared to DCs treated with OVA-loaded PLGA MPs which were unable activate CD8+ T cells. In contrast, DCs treated with OVA-loaded PLGA MPs promoted OVA-specific OT-II CD4+ T cell activation, whereas PPAA incorporation into the MP blend did not permit CD4+ T cell activation. These studies demonstrate PLGA MP blends containing PPAA are able to provide an endosomal escape strategy for encapsulated protein antigen, enabling the targeted delivery of antigen for tunable presentation and activation of either CD4+ or CD8+ T cells

Galectin-Anchored indoleamine 2,3-dioxygenase tissue-targeted therapeutic enzyme suppresses local inflammation in multiple animal models

Please click Additional Files below to see the full abstract

Water structuring and collagen adsorption at hydrophilic and hydrophobic silicon surfaces

The adsorption of a collagen fragment on both a hydrophobic, hydrogen-terminated and a hydrophilic, natively oxidised Si surface is investigated using all-atom molecular dynamics. While favourable direct protein-surface interactions via localised contact points characterise adhesion to the hydrophilic surface, evenly spread surface/molecule contacts and stabilisation of the helical structure occurs upon adsorption on the hydrophobic surface. In the latter case, we find that adhesion is accompanied by a mutual fit between the hydrophilic/hydrophobic pattern within the protein and the layered water structure at the solid/liquid interface, which may provide an additional driving force to the classic hydrophobic effect

Development of an hydrophobic fluoro-silica surface for studying homotypic cancer cell aggregation–disaggregation as a single dynamic process in vitro

Under normal conditions the detachment of anchorage-dependant cells from their extracellular matrix typically induces programmed cell death which is mediated through a pathway referred to as anoikis. However, a resistance to anoikis in cancer enables the migration of cells from the primary tumour and the establishment of aggressive metastatic disease. Although cancer cell aggregation is known to be an important mechanism within anoikis resistance, research into the underlying mechanisms that govern this process remain problematic as commercially available tissue culture material can only sustain 2D monolayer or 3D aggregate/spheroid cultures. This necessitates the development of a system that can accommodate for cancer cell aggregation–disaggregation as a single dynamic process, without the disruption of passaging cells between alternate substrates. This study describes a procedure for modifying tissue culture polystyrene (TCP) to produce a fluoro-silica (FS) surface which preferentially promotes the deposition of a distinct profile of proteins/factors from serum which mediate the transient aggregation of human breast cancer cell lines. This modified surface therefore provides an experimental platform for better understanding cancer cell aggregation–disaggregation events in vitro, and their influence on the establishment of metastatic disease in patients with cancer

Chimeric protein and nano-construct for tissue-retained enzyme to locally suppress inflammation

There is considerable need for new retention strategies of immunomodulatory biologics for localized suppression of inflammation. We developed a chimeric protein as a well as a self-assembled nano-construct incorporating novel approaches for both retention and suppression to induce potent, confined metabolic programming. Immunosuppressive indoleamine 2,3 dioxygenase (IDO), which depletes tryptophan through the kynurenine pathway, was fused to Galectin 3 (Gal3), which binds extracellular glycans and provides tissue anchoring. Using a luciferase-Gal3 fusion reporter, tissue retention was prolonged to ~6 d whereas native luciferase is not retained and undetectable by 24 h. IDO-Gal3 injected subcutaneously controlled local LPS-challenged tissue inflammation. Furthermore, subgingival injection suppressed periodontal disease (PD) in a polymicrobial challenged mouse model. Multiplex analysis of gingival tissue revealed decreased inflammatory (IL-1β, IL-12p70, KC, IP10, MCP1, MIP2) and increased anti-inflammatory (IL-10, TGFβ3) proteins, indicating a shift toward homeostasis. Animals treated with IDO-Gal3 also showed significant decrease in bone loss commonly associated with PD, as determined by µCT analysis

Vitronectin as a micromanager of cell response in material-driven fibronectin nanonetworks

Surface functionalization strategies of synthetic materials for regenerative medicine applications comprise the development of microenvironments that recapitulate the physical and biochemical cues of physiological extracellular matrices. In this context, material-driven fibronectin (FN) nanonetworks obtained from the adsorption of the protein on poly(ethyl acrylate) provide a robust system to control cell behavior, particularly to enhance differentiation. This study aims at augmenting the complexity of these fibrillar matrices by introducing vitronectin, a lower-molecular-weight multifunctional glycoprotein and main adhesive component of serum. A cooperative effect during co-adsorption of the proteins is observed, as the addition of vitronectin leads to increased fibronectin adsorption, improved fibril formation, and enhanced vitronectin exposure. The mobility of the protein at the material interface increases, and this, in turn, facilitates the reorganization of the adsorbed FN by cells. Furthermore, the interplay between interface mobility and engagement of vitronectin receptors controls the level of cell fusion and the degree of cell differentiation. Ultimately, this work reveals that substrate-induced protein interfaces resulting from the cooperative adsorption of fibronectin and vitronectin fine-tune cell behavior, as vitronectin micromanages the local properties of the microenvironment and consequently short-term cell response to the protein interface and higher order cellular functions such as differentiation

Fibronectin fixation on poly(ethyl acrylate)-based copolymer

The aim of this paper is to quantify the adhered fibronectin (FN; by adsorption and/or grafting) and the exposure of its cell adhesive motifs (RGD and FNIII7-10) on poly(ethyl acrylate) (PEA) copolymers whose chemical composition has been designed to increase wettability and to introduce acid functional groups. FN was adsorbed to PEA, poly(ethyl acrylate-co-hydroxyethyl acrylate), poly(ethyl acrylate-co-acrylic acid), and poly(ethyl acrylate-co-methacrylic acid) copolymers, and covalently cross-linked to poly(ethyl acrylate-co-acrylic acid) and poly(ethyl acrylate-co-methacrylic acid) copolymers. Amount of adhered FN and exhibition of RGD and FNIII7-10 fragments involved in cell adhesion were quantified with enzyme-linked immunosorbent assay tests. Even copolymers with a lower content of the hydrophilic component showed a decrease in water contact angle. In addition, FN was successfully fixed on all surfaces, especially on the hydrophobic surfaces. However, it was demonstrated that exposure of its cell adhesion sequences, which is the key factor in cell adhesion and proliferation, was higher for hydrophilic surfaces. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.Contract grant sponsors: Centre for Industrial Technological Development (CDTI) of Ministry of Economy and Competitiveness, Project Customized Eye Care-Oftalmologia personalizada _CEYEC CENIT-Sol 00028336 SFPECEPP and Health Institute Carlos III through the CIBER- BBN (Bioingenieria, Biomateriales y Nanomedicina); CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development FundBriz, N.; Antolinos Turpín, CM.; Alio, J.; Garagorri, N.; Gómez Ribelles, JL.; Gómez-Tejedor, JA. (2013). Fibronectin fixation on poly(ethyl acrylate)-based copolymer. Journal of Biomedical Materials Research Part B: Applied Biomaterials. 101B(6):991-997. https://doi.org/10.1002/jbm.b.32907S991997101B6Roach, P., Eglin, D., Rohde, K., & Perry, C. C. (2007). Modern biomaterials: a review—bulk properties and implications of surface modifications. Journal of Materials Science: Materials in Medicine, 18(7), 1263-1277. doi:10.1007/s10856-006-0064-3Schmidt, D. R., Waldeck, H., & Kao, W. J. (2009). Protein Adsorption to Biomaterials. Biological Interactions on Materials Surfaces, 1-18. doi:10.1007/978-0-387-98161-1_1Ertel, S. I., Ratner, B. D., & Horbett, T. A. (1990). Radiofrequency plasma deposition of oxygen-containing films on polystyrene and poly(ethylene terephthalate) substrates improves endothelial cell growth. Journal of Biomedical Materials Research, 24(12), 1637-1659. doi:10.1002/jbm.820241207Way, T.-D., Hsieh, S.-R., Chang, C.-J., Hung, T.-W., & Chiu, C.-H. (2010). Preparation and characterization of branched polymers as postoperative anti-adhesion barriers. Applied Surface Science, 256(10), 3330-3336. doi:10.1016/j.apsusc.2009.12.029Hsieh, S.-R., Chang, C.-J., Way, T.-D., Kwan, P.-C., & Hung, T.-W. (2009). Preparation and Non-Invasive In-Vivo Imaging of Anti-Adhesion Barriers with Fluorescent Polymeric Marks. Journal of Fluorescence, 19(4), 733-740. doi:10.1007/s10895-009-0469-8Lee, M. H., Ducheyne, P., Lynch, L., Boettiger, D., & Composto, R. J. (2006). Effect of biomaterial surface properties on fibronectin–α5β1 integrin interaction and cellular attachment. Biomaterials, 27(9), 1907-1916. doi:10.1016/j.biomaterials.2005.11.003Keselowsky, B. G., Collard, D. M., & Garcı́a, A. J. (2004). Surface chemistry modulates focal adhesion composition and signaling through changes in integrin binding. Biomaterials, 25(28), 5947-5954. doi:10.1016/j.biomaterials.2004.01.062Tzoneva, R., Faucheux, N., & Groth, T. (2007). Wettability of substrata controls cell–substrate and cell–cell adhesions. Biochimica et Biophysica Acta (BBA) - General Subjects, 1770(11), 1538-1547. doi:10.1016/j.bbagen.2007.07.008Rico, P., Hernández, J. C. R., Moratal, D., Altankov, G., Pradas, M. M., & Salmerón-Sánchez, M. (2009). Substrate-Induced Assembly of Fibronectin into Networks: Influence of Surface Chemistry and Effect on Osteoblast Adhesion. Tissue Engineering Part A, 15(11), 3271-3281. doi:10.1089/ten.tea.2009.0141Gugutkov, D., Altankov, G., Rodríguez Hernández, J. C., Monleón Pradas, M., & Salmerón Sánchez, M. (2010). Fibronectin activity on substrates with controlled OH density. Journal of Biomedical Materials Research Part A, 92A(1), 322-331. doi:10.1002/jbm.a.32374Salmerón-Sánchez, M., Rico, P., Moratal, D., Lee, T. T., Schwarzbauer, J. E., & García, A. J. (2011). Role of material-driven fibronectin fibrillogenesis in cell differentiation. Biomaterials, 32(8), 2099-2105. doi:10.1016/j.biomaterials.2010.11.057Pérez Olmedilla, M., Garcia-Giralt, N., Pradas, M. M., Ruiz, P. B., Gómez Ribelles, J. L., Palou, E. C., & García, J. C. M. (2006). Response of human chondrocytes to a non-uniform distribution of hydrophilic domains on poly (ethyl acrylate-co-hydroxyethyl methacrylate) copolymers. Biomaterials, 27(7), 1003-1012. doi:10.1016/j.biomaterials.2005.07.030Soria, J. M., Martínez Ramos, C., Salmerón Sánchez, M., Benavent, V., Campillo Fernández, A., Gómez Ribelles, J. L., … Barcia, J. A. (2006). Survival and differentiation of embryonic neural explants on different biomaterials. Journal of Biomedical Materials Research Part A, 79A(3), 495-502. doi:10.1002/jbm.a.30803Campillo-Fernandez, A. J., Pastor, S., Abad-Collado, M., Bataille, L., Gomez-Ribelles, J. L., Meseguer-Dueñas, J. M., … Ruiz-Moreno, J. M. (2007). Future Design of a New Keratoprosthesis. Physical and Biological Analysis of Polymeric Substrates for Epithelial Cell Growth. Biomacromolecules, 8(8), 2429-2436. doi:10.1021/bm0703012Campillo-Fernández, A. J., Unger, R. E., Peters, K., Halstenberg, S., Santos, M., Sánchez, M. S., … Kirkpatrick, C. J. (2009). Analysis of the Biological Response of Endothelial and Fibroblast Cells Cultured on Synthetic Scaffolds with Various Hydrophilic/Hydrophobic Ratios: Influence of Fibronectin Adsorption and Conformation. Tissue Engineering Part A, 15(6), 1331-1341. doi:10.1089/ten.tea.2008.0146Cutler, S. (2003). Engineering cell adhesive surfaces that direct integrin α5β1 binding using a recombinant fragment of fibronectin. Biomaterials, 24(10), 1759-1770. doi:10.1016/s0142-9612(02)00570-7Salmerón Sánchez, M., Brı́gido Diego, R., Iannazzo, S. A. ., Gómez Ribelles, J. L., & Monleón Pradas, M. (2004). The structure of poly(ethyl acrylate-co-hydroxyethyl methacrylate) copolymer networks by segmental dynamics studies based on structural relaxation experiments. Polymer, 45(7), 2349-2355. doi:10.1016/j.polymer.2004.01.043Campillo-Fernández, A. J., Salmerón Sánchez, M., Sabater i Serra, R., Meseguer Dueñas, J. M., Monleón Pradas, M., & Gómez Ribelles, J. L. (2008). Water-induced (nano) organization in poly(ethyl acrylate-co-hydroxyethyl acrylate) networks. European Polymer Journal, 44(7), 1996-2004. doi:10.1016/j.eurpolymj.2008.04.032Lan, M. A., Gersbach, C. A., Michael, K. E., Keselowsky, B. G., & García, A. J. (2005). Myoblast proliferation and differentiation on fibronectin-coated self assembled monolayers presenting different surface chemistries. Biomaterials, 26(22), 4523-4531. doi:10.1016/j.biomaterials.2004.11.02

Anti-thrombogenicity by Layer-by-Layer

Anti-thrombogenic films with high durability were fabricated in a wet process. Anti-thrombogenicity was achieved with polyelectrolyte multilayer thin film prepared from poly(vinyl alcohol)-poly(acrylic acid)(PVA-PAA) blends, deposited in alternate layers with poly(allylamine hydrochloride) (PAH). Film durability, assessed by abrasion resistance and water resistance, was enhanced by forming crosslinks via amide bonds induced by heat treatment of the film. The film was found to be resistant to protein adsorption, as measured by the amount of fibrinogen adsorbed from an aqueous solution. Anti-thrombogenic efficacy was assessed in ex vivo experiments by the ability of stainless steel mesh, coated with the polyelectrolyte and inserted into a pig blood vessel, to inhibit thrombus formation. Mesh coated with the polyelectrolyte did not reduce blood flow over a period of 15 minutes, whereas with uncoated mesh blood flow stopped within 6 minutes because of blood vessel blockage by thrombus formation

Hot embossing for fabrication of a microfluidic 3D cell culture

Clinically relevant studies of cell function in vitro require a physiologically-representative microenvironment possessing aspects such as a 3D extracellular matrix (ECM) and controlled biochemical and biophysical parameters. A polydimethylsiloxane (PDMS) microfluidic system with a 3D collagen gel has previously served for analysis of factors inducing different responses of cells in a 3D microenvironment under controlled biochemical and biophysical parameters. In the present study, applying the known commercially-viable manufacturing methods to a cyclic olefin copolymer (COC) material resulted in a microfluidic device with enhanced 3D gel capabilities, controlled surface properties, and improved potential to serve high-volume applications. Hot embossing and roller lamination molded and sealed the microfluidic device. A combination of oxygen plasma and thermal treatments enhanced the sealing, ensured proper placement of the 3D gel, and created controlled and stable surface properties within the device. Culture of cells in the new device indicated no adverse effects of the COC material or processing as compared to previous PDMS devices. The results demonstrate a methodology to transition microfludic devices for 3D cell culture from scientific research to high-volume applications with broad clinical impact.National Cancer Institute (U.S.) (award R21CA140096)Charles Stark Draper Laboratory (IR&D Grant