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There is a constant high demand for blood transfusions worldwide. Donor-derived blood employed as the current clinical standard has several limitations including insufficient availability, short shelf-life, and risk of pathogenic contamination. Thus, the development of artificial red blood cells (RBCs) has become a crucial field of study. Such artificial RBCs encapsulate hemoglobin (Hb), the oxygen-carrying component of RBCs, and aim to mimic the features of the endogenous cells. Currently, mainly microparticles and nanoparticles are used as encapsulated systems in drug delivery research. Polyethylene glycol (PEG)-based hydrogels are the primary choice for material, due to their biocom-patibility. So far, most carriers developed have spherical shapes and are produced from bottom-up approaches that cannot offer the best geometric control. It has been proven that geometry plays a critical role in the vascular behavior of microparticles, thus the need to obtain non-spherical carriers. This project proposed to employ top-down fabrication methods to better overcome this challenge. By aiming to design and fabricate three different PEG-based microgel shapes (rods, stars, and hexagons) designed within the same size range as RBCs. Firstly, Silicon masters with the designed patterns were produced through photolithography and dry etching. Then they were used to produce cyclo olefin polymer (COP) stamps through hot embossing. These stamps were coated with different PEG formulations (with and without Hb) by slot die coating. Next, they were used in UV-assisted punching to form the microgel shapes and mechanically punch through the hydrogel flash layer onto a poly-vinyl alcohol (PVA) substrate. The microgels were harvested by dissolving the PVA. These microgels shapes obtained had the desired aspect ratio of 3:1 retaining good shape features. However, challenges remained in the achievement of individual microgel shapes after harvest. Due to coating thickness limitations that consequently affected the mechanically punching process afterwards. Existe uma constante procura de transfusões sanguíneas. Isto devido ao sangue proveniente de dadores ter inúmeras limitações incluindo disponibilidade insuficiente, baixa longevidade e risco de contaminação pa-togénica. Assim sendo o desenvolvimento de hemácias artificiais tornou-se um crucial ramo de investigação. Estas hemácias artificiais encapsulam a hemoglobina, componente transportador de oxigénio, visando imitar as características das células endógenas. Atualmente maioritariamente micropartículas e nanopartículas são usadas como sistemas de encapsu-lamento em estudos de entrega de fármacos. Sendo os hidrogéis à base de polietilenoglicol (PEG) a maior escolha como material, devido à sua biocompatibilidade. Até agora, a maioria dos portadores desenvolvidos tem formas esféricas e são produzidos a partir de métodos de bottom-up que não conseguem oferecer o melhor controle geométrico. Foi comprovado que a geometria desempenha um papel crítico no comportamento vas-cular das micropartículas, daí a necessidade de obter portadores não esféricos. Este projeto propôs utilizar métodos de fabricação top-down de forma a superar essa limitação. Com o objetivo principal de desenhar e fabricar três formas diferentes de microgéis à base de PEG (hastes, estrelas e hexágonos) projetadas para dimensões semelhantes às hemácias. Primeiramente, foram produzidos templates de silício com os padrões fabricados por fotolitografia e erosão seca. Em seguida, estes foram usados para imprimir moldes de polímero de ciclo olefina (COP) por hot embossing. A estes moldes foram depositadas diferentes formulações de PEG (com e sem Hb) por slot die coating. Consecutivamente, estes foram usados em UV-assisted punching para obter as desejadas formas microgéis com o molde cortando mecanicamente as formas pelo hidrogel para o substrato de álcool polivinílico (PVA). Posteriormente os microgéis foram obtidos através da dissolução do PVA em água. Sendo que estes mantiveram os tamanhos desejados com uma razão de aspeto de 3:1, conservando também as formas. No entanto, os desafios permaneceram na obtenção de mi-crogéis individuais após a dissolução. Devido a limitações de espessura durante a deposição que consequente-mente afetaram o processo de cortamento mecânico posterior.

Single-walled carbon nanotubes (SWCNTs) have attracted great interest regarding drug-delivery applications. However, their application has been limited by some inherent disadvantages. In this study, raw SWCNTs were purified with different oxidizing acids, and the resulting shortened CNTs were conjugated with poly(ethylene glycol) (PEG) and polyethylenimine (PEI). The different nanocarriers, that is, CNTs-COOH (CNTs), CNTs-PEG and CNTs-PEG-PEI, were systematically characterized and evaluated in terms of drug loading, in vitro release, cytotoxicity towards MCF-7 cells and cellular uptake. The results showed that all CNT carriers had a high drug loading capacity. In comparison with CNTs-COOH and CNTs-PEG, CNTs-PEG-PEI showed a more rapid drug release under acidic conditions and a higher antitumor activity. Furthermore, fluorescence detection and flow cytometry (FCM) analysis results indicated that the internalization into cells of CNTs-PEG-PEI was significantly enhanced, thus inducing tumor cell death through apoptosis more efficiently. The above series of benefits of CNTs-PEG-PEI may be attributed to their good dispersibility and comparably higher affinity to tumor cells due to the difunctionalization. In summary, the PEG- and PEI-conjugated CNTs may be used as novel nanocarriers and the findings will contribute to the rational design of multifunctional delivery vehicles for anticancer drugs.

Polymeric-micelle carrier systems have emerged as a novel drug-carrier system and have been actively studied for anticancer drug targeting. In contrast, toxicological and immunological concerns related to not only polymeric-micelle carrier systems, but also other nanocarrier systems, have received little attention owing to researchers’ focus on therapeutic effects. However, in recent clinical contexts, biopharmaceuticals’ effects on immune responses have come to light, requiring that researchers substantively explore the potential negative side effects of nanocarrier systems and of therapeutic proteins in order to develop nanocarrier systems suitable for clinical use. The present review describes current insights into both toxicological and immunological issues regarding polymeric-micelle carrier systems. The review focuses on immunogenicity issues of polymeric-micelle carrier systems possessing poly(ethylene glycol) (PEG). We conclude that PEG-related immunogenicity is deeply related to characteristics of a counterpart block of PEG-conjugates, and we propose future directions for addressing this unresolved issue., Graphical abstract

James G Shamul,1,2,* Sagar R Shah,1–3,* Jayoung Kim,1,2 Paula Schiapparelli,3 Carla A Vazquez-Ramos,3 Ben J Lee,1,2 Kisha K Patel,1,2 Alyssa Shin,1,2 Alfredo Quinones-Hinojosa,3 Jordan J Green1,2,4–6 1Department of Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21231, USA; 2Translational Tissue Engineering Center and Institute for NanoBioTechnology, Johns Hopkins School of Medicine, Baltimore, MD 21231, USA; 3Department of Neurosurgery, Mayo Clinic, Jacksonville, FL 32224, USA; 4Department of Neurosurgery, Johns Hopkins Hospital, Baltimore, MD 21231, USA; 5Department of Oncology, The Sidney Kimmel Comprehensive Cancer, and The Bloomberg∼Kimmel Institute for Cancer Immunotherapy, Johns Hopkins School of Medicine, Baltimore, MD 21231, USA; 6Department of Ophthalmology, Department of Materials Science and Engineering, and Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21231, USA*These authors contributed equally to this workCorrespondence: Alfredo Quinones-Hinojosa; Jordan J Green Email Quinones-Hinojosa.Alfredo@mayo.edu; green@jhu.eduBackground: Nanomedicine can improve traditional therapies by enhancing the controlled release of drugs at targeted tissues in the body. However, there still exists disease- and therapy-specific barriers that limit the efficacy of such treatments. A major challenge in developing effective therapies for one of the most aggressive brain tumors, glioblastoma (GBM), is affecting brain cancer cells while avoiding damage to the surrounding healthy brain parenchyma. Here, we developed poly(ethylene glycol) (PEG)-poly(beta-amino ester) (PBAE) (PEG-PBAE)-based micelles encapsulating verteporfin (VP) to increase tumor-specific targeting.Methods: Biodegradable, pH-sensitive micelles of different shapes were synthesized via nanoprecipitation using two different triblock PEG-PBAE-PEG copolymers varying in their relative hydrophobicity. The anti-tumor efficacy of verteporfin loaded in these anisotropic and spherical micelles was evaluated in vitro using patient-derived primary GBM cells.Results: For anisotropic micelles, uptake efficiency was ∼100% in GBM cells (GBM1A and JHGBM612) while only 46% in normal human astrocytes (NHA) at 15.6 nM VP (p ≤ 0.0001). Cell killing of GBM1A and JHGBM612 vs NHA was 52% and 77% vs 29%, respectively, at 24 hrs post-treatment of 125 nM VP-encapsulated in anisotropic micelles (p ≤ 0.0001), demonstrating the tumor cell-specific selectivity of VP. Moreover, anisotropic micelles showed an approximately fivefold longer half-life in blood circulation than the analogous spherical micelles in a GBM xenograft model in mice. In this model, micelle accumulation to tumors was significantly greater for anisotropic micelle-treated mice compared to spherical micelle-treated mice at both 8 hrs (∼1.8-fold greater, p ≤ 0.001) and 24 hrs (∼2.1-fold greater, p ≤ 0.0001).Conclusion: Overall, this work highlights the promise of a biodegradable anisotropic micelle system to overcome multiple drug delivery challenges and enhance efficacy and safety for the treatment of brain cancer.Keywords: GBM, verteporfin, micelle, anisotropic, poly(ethylene glycol), PEG, poly(beta-amino ester), PBAE

[ES] El estudio del comportamiento durante la inmersión en agua de los materiales compuestos de base biológica es de gran importancia para evaluar su rendimiento en un entorno húmedo como material de embalaje ecológico. En este estudio se prepararon láminas de materiales bionanocompuestos a base de poli(lactida) (PLA), plastificadas con poli(etilenglicol) (PEG) y reforzadas con celulosa nanofibrilada (NFC). Se investigó la presencia de PEG y la influencia de NFC (1, 3, 5% en peso) sobre las propiedades fisicoquímicas tras la inmersión en agua durante diferentes tiempos (7, 15, 30, 60, 90 días) y temperaturas (8, 23, 58, 70 ° C). Se estudió la absorción de agua en términos de saturación, difusividad, solubilidad y permeabilidad. Además, se caracterizaron los cambios de morfología, masa molar y propiedades térmicas mediante microscopía electrónica de barrido, cromatografía de permeación en gel, análisis termogravimétrico y calorimetría diferencial de barrido, respectivamente. A las temperaturas de inmersión más bajas (8 y 23 ° C) se encontró un aumento de masa seguido de un equilibrio estable, mientras que a temperaturas más altas (58 y 70 ° C) las muestras que contenían PEG comenzaron a perder masa linealmente después de la absorción inicial. Tras largos tiempos de inmersión, especialmente a temperaturas altas, las muestras se volvían blancas y frágiles, desintegrándose en pedazos más pequeños. La inmersión en agua promovió una morfología con una superficie rugosa con grietas y poros, así como una disminución de la masa molar y un aumento de la cristalinidad, a causa de la degradación hidrotérmica. En resumen, se determinó el efecto crítico de la temperatura y el tiempo durante la inmersión en agua y la contribución del PEG y la NFC en el rendimiento de estos materiales basados en bioplásticos, resaltando las limitaciones para su uso en un ambiente húmedo a elevadas temperaturas., [EN] The understanding of the short- and long-term behaviour of biobased composites during immersion in water is of great importance for its performance in a wet environment as an eco-friendly packaging material. In this study, bionanocomoposite films based on poly(lactide) (PLA) plasticised with poly(ethylene glycol) (PEG) and reinforced with nanofibrillated cellulose (NFC) were prepared. The presence of PEG and the influence of NFC (1, 3, 5 wt%) on the physio-chemical properties were investigated after immersion in water for different times (7, 15, 30, 60, 90 days) at different temperatures (8, 23, 58, 70 °C). The water absorption behaviour in terms of saturation, diffusivity, solubility, and permeability was assessed. The changes of the morphology, molar mass, and thermal properties of the samples through immersion were characterised with field emission scanning electron microscopy, gel permeation chromatography, thermo gravimetric analysis, and differential scanning calorimetry, respectively. At the lower immersion temperatures (8 and 23 °C) a mass gain followed by a stable equilibrium was found while at higher temperatures (58 and 70 °C) the samples containing PEG started to linearly lose mass after the initial water absorption. After storage in water, especially at higher temperatures, the specimen become white, very fragile and disintegrate into smaller pieces. The immersion in water, dominantly in higher temperatures, also promoted a rough surface with cracks and pores in the morphology as well as a drastic decrease in molar mass. Furthermore, the materials show a reduction of the decomposition and melting temperature and an increase in crystallinity, due to hydrothermal degradation. In summary the critical effect of temperature and time during water immersion and the contribution of PEG and NFC on the performance of the bioplastic materials is assessed and thus the usability and limitations in a wet environment demonstrated.

Made available in DSpace on 2018-12-11T16:50:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-03-01 The partitioning of bacteriocin, a promising alternative to chemical preservatives, in a novel inexpensive and stable aqueous two-phase system (ATPS) comprising poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) was studied. The ATPS was generated by mixing both polymers with Na2SO4 or choline chloride ([Ch]Cl) and a bacteriocin extract from the fermented broth of Lactobacillus plantarum ST16Pa. Bacteriocin showed stability at different pH values (3.0–8.0) and temperatures (50–80 °C), as well as in the presence of ATPS components. Hydrophobic and electrostatic interactions were found to be the major driving forces for bacteriocin partitioning. The peptide partitioned preferentially to the PEG-rich phase (partition coefficient, KBact > 1). However, the highest partition coefficient was achieved in the polymeric-based ATPS using [Ch]Cl as additive, as follows: 8 wt% PEG 10,000 g/mol/8 wt% NaPA 8,000 g/mol/0.5 M [Ch]Cl, resulting in a KBact equal to 32. Moreover, these conditions promoted high selectivity (S = 62.7), since the greater part of total proteins partitioned into the NaPA-rich phase (Kprot = 0.51). In conclusion, ATPS composed of PEG/NaPA using choline-based salts as additive can be considered mainly as a first step for bacteriocin extraction from fermented broth by applying low polymer content and mild conditions. Department of Biochemical and Pharmaceutical Technology School of Pharmaceutical Sciences University of São Paulo – FCF/USP Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences State University of São Paulo – FCFar/UNESP Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences State University of São Paulo – FCFar/UNESP

It has long been a central tenet of biomedical research that coating of nanoparticles with hydrated polymers can improve their performance in biomedical applications. However, the efficacy of the approach in vivo is still debated. In vitro model systems to test the performance of engineered nanoparticles for in vivo applications often use nonrepresentative cell lines and conditions for uptake and toxicity tests. We use our platform of monodisperse iron oxide nanoparticles densely grafted with nitrodopamide-poly(ethylene glycol) (PEG) to probe cell interactions with a set of cell types and culture conditions that are relevant for applications in which nanoparticles are injected into the bloodstream. In the past, these particles have proved to have excellent stability and negligible interaction with proteins and membranes under physiological conditions. We test the influence of flow on the uptake of nanoparticles. We also investigate the transport through endothelial barrier cell layers, as well as the effect that PEG-grafted iron oxide nanoparticles have on cell layers relevant for nanoparticles injected into the bloodstream. Our results show that the dense PEG brush and resulting lack of nonspecific protein and membrane interaction lead to negligible cell uptake, toxicity, and transport across barrier layers. These results contrast with far less well-defined polymer-coated nanoparticles that tend to aggregate and consequently strongly interact with cells, for example, by endocytosis.

International audience; Amphiphilic surfaces are particularly effective at inhibiting the adhesion of microorganisms (bacteria, cells, microalgae, etc.) in liquid media. The aim of this study is to determine the best hydrophilic linker to promote bonding between poly(ethylene glycol) (PEG) as a hydrophilic additive and poly(dimethyl siloxane) (PDMS) as the hydrophobic matrix. Various parameters have been studied (molecular weight, linker type, and polymer end-group), as well as the efficiency of the linking, the capacity of PEG to access to the surface of the film, and overall film homogeneity. According to the results, a PDMS linker paired with a PEG moiety allows for compatibilization of the compounds during cross-linking. This compatibilization seems to provide a good bonding with the matrix and a good surface access to the hydrophilic moiety. Therefore, this structure comprising a linking function attached to the PDMS-PEG copolymer has high potential as a non-releasable additive for amphiphilic coating applications.

Made available in DSpace on 2018-12-11T17:37:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-11-29 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The production of recombinant biopharmaceuticals is highly dependent of a proper choice of the downstream processing stages. Particularly, the purification that must ensure that all the endotoxins (lipopolysaccharides - LPS) are efficiently removed from the final product. The efficient removal of LPS has a direct impact on the manufacturing of therapeutic biopharmaceuticals, since LPS is naturally presented in Gram-negative bacterial expression systems. In order to provide a more simple and faster technique for the purification of green fluorescent protein (GFP) and LPS removal, aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and poly(acrylic acid) (NaPA) and electrolytes were studied. Firstly, the binodal curves of PEG/NaPA + salt systems were established using NaCl, Li2SO4, KI, and KNO3 as additives. It was demonstrated that the formation of ATPS is enhanced following the anion tendency: SO4 2− > Cl− ≫ NO3 − > I−. The stability of GFP in the presence of all different phase forming agents (polymers and salts) was evaluated, and the high biocompatibility of these ATPS demonstrated by the maintenance of GFP fluorescence in all conditions under study. Then, the GFP extraction and LPS removal aptitude of each ATPS was investigated. GFP and LPS were preferentially partitioned into the top (PEG-rich) phase (KGFP > 20), but with a removal of 35% of LPS was attained. Hydrophobic and electrostatic interactions were found to be the major driving forces for GFP partitioning and LPS removal. Moreover, a new effect was found, the presence of high loads of LPS can affect (decrease) the KGFP values (KGFP without LPS > KGFP with 104 EU/mL > KGFP with 106 EU/mL). The ATPS with best GFP extraction performance was selected for the recovery directly from the cell lysates of Escherichia coli. In this experiment, the system composed of 12 wt% PEG 1000 g/mol, 12 wt% NaPA 8000 g/mol, and 0.25 M Li2SO4 lead to a LPS removal (REMLPS) of 13%, KGFP of 20.2, and high selectivity relatively to the total proteins (S = 20), since the majority of contaminants proteins were preferentially partitioned into the bottom (NaPA-rich) phase (purification factor of 4-fold). It is here demonstrated that the ATPS composed of PEG/NaPA + salts as additives can be used as a first step for the recovery of GFP and removal of contaminants (LPS in part, and most contaminant proteins) from cell lysates by applying a system with low polymer content and using mild conditions. Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences São Paulo State University – FCFar/UNESP Ronin Institute Department of Biochemical and Pharmaceutical Technology School of Pharmaceutical Sciences University of São Paulo – FCF/USP Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences São Paulo State University – FCFar/UNESP CAPES: #0366/09-9 FAPESP: 2005/60159-7 FAPESP: 2007/51978-0 FAPESP: 2014/16424-7 FAPESP: 2014/19793-3

Here, the synthesis of two graft copolymers based on ?,?-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) and poly(lactic acid) (PLA), the O-(2-aminoethyl)-O'-galactosyl polyethylene glycol (GAL-PEG-NH2) or the methoxypolyethylene glycol amine (H2N-PEG-OCH3) is described. Starting from the obtained PHEA-PLA-PEG-GAL and PHEA-PLA-PEG copolymers, polymeric nanoparticles were prepared by high pressure homogenization-solvent evaporation method. To demonstrate their biodegradability as a function of the matrix composition, a chemical stability study was carried out until 21 days by incubating systems in two media mimicking physiological compartments (pH 7.4 and pH 5.5). The degradability of both nanosystems was firstly confirmed by the reduction of the pH of the incubation medium. Moreover, the percentage yield of recovered nanoparticles show a gradual reduction while mean size increases as a function of incubation time. Degradation seems to be mainly attributed to the loss of water-soluble portions of PLA, and proceeds with greater speed at pH 5.5, than at pH 7.4 and as a function of matrix composition. KEYWORDS: ?,?-poly-(N-2-hydroxyethyl)-DL-aspartamide (PHEA), poly(lactic acid) (PLA), poly(ethylene glycol) (PEG), graft copolymers, nanoparticles, biodegradability.

Adhesive processes in aqueous media play a crucial role in nature and are important for many technological processes. However, direct quantification of adhesion still requires expensive instrumentation while their sample throughput is rather small. Here we present a fast, and easily applicable method on quantifying adhesion energy in water based on interferometric measurement of polymer microgel contact areas with functionalized glass slides and evaluation via the Johnson–Kendall–Roberts (JKR) model. The advantage of the method is that the microgel matrix can be easily adapted to reconstruct various biological or technological adhesion processes. Here we study the suitability of the new adhesion method with two relevant examples: (1) antibody detection and (2) soil release polymers. The measurement of adhesion energy provides direct insights on the presence of antibodies showing that the method can be generally used for biomolecule detection. As a relevant example of adhesion in technology, the antiadhesive properties of soil release polymers used in today’s laundry products are investigated. Here the measurement of adhesion energy provides direct insights into the relation between polymer composition and soil release activity. Overall, the work shows that polymer hydrogel particles can be used as versatile adhesion sensors to investigate a broad range of adhesion processes in aqueous media.

This paper describes the evaluation of polymeric nanoparticles (NPs) as a potential carrier for lung administration of fluticasone propionate (FP). The chosen polymeric material to produce NPs was a copolymer based on α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) whose backbone was derivatised with different molecules, such as poly(lactic acid) (PLA) and polyethylenglycol (PEG). The chosen method to produce NPs from PHEA-PLA-PEG2000 was the method based on high-pressure homogenization and subsequent solvent evaporation by adding Pluronic F68 during the process and trehalose before lyophilisation. Obtained colloidal FP-loaded NPs showed a slightly negative surface charge and nanometric dimensions that are maintained after storage for one year at −20 °C and 5 °C. The FP loading was about 2.9 wt % and the drug was slowly released in simulated lung fluid. Moreover, the obtained NPs, containing the drug or not, were biocompatible and did not induce cell necrosis and cell apoptosis on bronchial epithelial cells (16-HBE). Further in vitro testing on cigarette smoke extract (CSE)-stimulated 16-HBE revealed that FP-loaded NPs were able to reduce the survivin expression, while either free FP or empty NPs were not able to significantly reduce this effect.

WOS: 000416432800011, Produced via electrospinning, polyurethane (PU) scaffolds have always attracted the interest of medical applications due of their unique properties such as good adhesion, biocompatibility and excellent mechanical strength. However, the poor hydrophilicity and hemocompatibility of PU presented a problem during PU's application in the manufacturing of biomedical materials. We hypothesized that the incorporation of polyethylene glycol (PEG) and phosphatidylcholine (PC) into electrospinning solution of PU could improve the cell affinity and hemocompatibility. This research focused on fabricating hybrid PU-PEG and PU-PC random/aligned scaffolds through electrospinning technique and comparing their properties as a potential biocompatible scaffold for vascular tissue engineering. PC was doped into a PU solution in order to prepare an electrospun scaffold through the electrospinning technology while crosslinked electrospun PUPEG hybrid scaffolds were fabricated by photoinduced polymerization. The contact angle dramatically decreased from 122.3 +/- 0.8A degrees to 39.1 +/- 0.8A degrees with doping of PC in electrospinning solution while it decreased from 122.3 +/- 0.8A degrees to 41.6 +/- 0.8A degrees with doping of PEG. Furthermore, the mechanical properties of PU scaffolds were altered significantly by the addition of PC. The hemolysis and cytocompatibility assays demonstrated that these composite scaffolds could potentially be used as a smalldiameter vascular graft.

Activation of the thrombotic and complement systems is the main recognition and effector mechanisms in the multiple adverse biological responses triggered when biomaterials or therapeutic cells come into blood contact. We have created a surface which is auto-protective to human innate immunity by combining three fundamentally different strategies, all developed by us previously, which have been shown to induce substantial, but incomplete hemocompatibility when used separately. In summary, we have conjugated a factor H-binding peptide; and an ADP-degrading enzyme; using a PEG linker on both material and cellular surfaces. When exposed to human whole blood, factor H was specifically recruited to the modified surfaces and inhibited complement attack. In addition, activation of platelets and coagulation was efficiently attenuated, by degrading ADP. Thus, by inhibiting thromboinflammation using a multicomponent approach, we have created a hybrid surface with the potential to greatly reduce incompatibility reactions involving biomaterials and transplantation. K.N.E., J.D.L., B.N. and Y.T. equally contributed to this paper as a supervisor.

Model systems have expanded our knowledge of numerous phenomena in Colloid Science, such as the appearance of glasses, order-disorder transitions involving crystals and attraction induced formation of gels. So far, the existing colloidal model systems have been limited mainly to nonaqueous media. Given that water is such an important solvent, an aqueous colloidal model system is called for. Here we present such an aqueous colloidal model system with core-shell particle morphology, where the interior is composed of spherical fluorinated cores and the exterior of a poly(ethylene glycol) (PEG) polymer graft. To synthesize these colloids, we have adopted a semi-batch emulsion polymerization, in which the initiator is slowly fed into the reaction mixture. Using this approach not only can monodisperse, low refractive index and sterically stabilized colloids be produced, but also various lengths of the PEG polymer could be successfully grafted onto the particles. Throughout this thesis, several different instrumental techniques have been used to gain an insight into the collective phenomena of these particles and how particle interactions contribute to the observed phase behavior. Although steric stabilization is very robust way of stabilizing colloidal particles against aggrega- tion, attractions between particles can nevertheless appear, e.g. through the addition of certain salts or addition of a non-solvent. The origin of these attractions is not fully understood. Our results show that colloidal stability increases with decreasing length of the steric stabilizer and that the polymer graft contracts as the solvent quality is worsened. The contraction is accompanied by moderately strong attractions even though the van der Waals force due to core-core interactions is essentially absent. It follows that the attractions are caused by purely polymer-mediated interactions.

The application of stem cells in drug screening and regenerative therapy has led to important advances in basic biology and biomedicine. Such strategies require high cell numbers and the efficient maturation into faithful functional organ or tissue units. An important aspect to gain control over stem cell fate is the recapitulation of their natural instructive extracellular matrix (ECM). In vivo, stem cells reside in microenvironments, termed niches, providing distinct, tissue-specific signals. In a complex interplay, the fate decisions are tightly regulated by various parameters including biophysical and biochemical signals. Biomaterial science interfaced with sophisticated bioengineering techniques has provided powerful tools to recreate stem cell niches in vitro. However, major challenges for the efficient stem cell expansion and differentiation still have not been sufficiently addressed. In this thesis, platforms have been developed to help to reconstruct this dynamic and niche-specific signaling panoply. In one approach, a hydrogel crosslinking scheme based on a Michael-type addition reaction was combined with photosensitive caging molecules. By masking thiol moieties of a poly(ethylene glycol) (PEG) macromer with photolabile groups, the covalent crosslinking with vinylsulfone groups of complementary PEG macromers was spatiotemporally controlled by light illumination. By the generation of precisely controlled stiffness gradients, we were able to spatially direct human mesenchymal stem cell (hMSC) migration. Along the same lines, light-controlled uncaging was applied to tether biomolecules into a hydrogel matrix. PEG macromers were covalently functionalized with a peptide substrate of activated transglutaminase factor XIII (FXIIIa). This allowed for any desired ECM-derived peptide or protein fused to the counter-reactive peptide substrate to be enzymatically tethered into the hydrogel network. Photoprotection of the hydrogel-bound peptide substrate enabled exquisite dynamic control of biomolecule distribution. The patterning platform was exemplified by the guided three-dimensional (3D) invasion of encapsulated hMSCs into the hydrogel matrix. The second part of this work addressed the challenge of dissecting the vast molecular signaling networks that influence stem cell behavior. A droplet microfluidic-based screening platform was developed to study the effect of pivotal niche proteins in unparalleled combinatorial throughput via micron-sized hydrogel beads termed microniches. Randomized compartmentalization of stable inducible mammalian cell lines into microniches translated into myriads of combinations of secreted proteins tethered to the hydrogel matrix. Selected microniches with a desired phenotypic effect were analyzed for the protein combinations by targeted genotypic analysis of the underlying encapsulated stable mammalian lines. Positive instructive effects of microniches could be demonstrated on a model system of engineered HEK cells and are currently under evaluation for mesendodermal differentiation of a human embryonic stem cell (hESC) reporter cell line. The novel light-responsive materials as well as microfluidic-based screening approach presented here strive for a near-physiological niche signaling architecture. These tools can help to fully exploit the stem cell potential and accelerate the translation of stem cell-based applications from the laboratory towards the clinic.

Cell-based tissue engineering strategies have shown tremendous promise for the repair of bone mass deficiencies, but the efficient and appropriate induction of stem cells down osteogenic pathways remains a significant roadblock to the effective implementation of cell-based therapies. When grown in culture, human Mesenchymal Stromal/Stem Cells (hMSCs) remain multipotent, requiring specific exogenous signals to induce osteogenic differentiation. hMSCs used in transplantations, therefore, must be presented with local signals, often provided by the host's own tissues, to be directed down bone-related lineages. This process is relatively inefficient and remains difficult to control. In an effort to enhance osteogenesis, hMSCs were transfected with specific miRNA mimics and inhibitors that had originally identified for their ability to increase Alkaline Phosphatase (ALP) activity. Transfection with miRNA reagents had the effect of sensitizing hMSCs to soluble osteogenic factors, resulting in a rapid and robust induction of bone-related markers, including ALP activity and calcium deposition. Synthetic 3D tissue constructs prepared with miRNA-transfected hMSCs demonstrated similar responses to soluble osteogenic signals, suggesting that controlling miRNA activity in hMSCs can be an effective tool for enhancing the induction of osteogenesis for tissue engineering purposes. Copyright © 2011 John Wiley & Sons, Ltd.

Zwitterionic polymers and poly(ethylene glycol) (PEG) have been reported as promising nonfouling materials, and strong surface hydration has been proposed as a significant contributor to the nonfouling mechanism. Better understanding of the similarity and difference between these two types of materials in terms of hydration and protein interaction will benefit the design of new and effective nonfouling materials. In this study, sum frequency generation (SFG) vibrational spectroscopy was applied for in situ and real-time assessment of the surface hydration of the sulfobetaine methacrylate (SBMA) and oligo(ethylene glycol) methacrylate (OEGMA) polymer brushes, denoted as pSBMA and pOEGMA, in contact with proteins. Whereas a majority of strongly hydrogen-bonded water was observed at both pSBMA and pOEGMA surfaces, upon contact with proteins, the surface hydration of pSBMA remained unaffected, but the water ordering at the pOEGMA surface was disturbed. The effects of free sulfobetaine, free PEG chains with two different molecular weights, and PEG coated gold nanoparticles on the surface hydration of proteins were investigated. The results indicated that free sulfobetaine could strengthen the protein hydration layer, but free PEG chains greatly disrupt the protein hydration layer and likely directly interact with the protein molecules. In contrast to free PEG, the PEG chains anchored on the nanoparticles behave similarly to the pOEGMA surface and could induce strong hydrogen bonding of the water molecules at the protein surfaces. Refereed/Peer-reviewed

International audience; This chapter focuses on olefin metathesis transformations in green(er) organic solvents, including supercritical carbon dioxide (scCO2) and organic carbonates, in particular dimethyl carbonate (DMC) and poly(ethylene glycol) (PEG). It reviews the domain of a solvent very often quoted as the greenest or best solvent in green chemistry textbooks, namely, no solvent or solventless conditions. DCM is often used in ruthenium-catalyzed metathesis reactions as it is in general a good catalyst solvent. Recently, ring closing metathesis (RCM) of diethyl diallylmalonate (DEDAM) and cross metathesis of allylbenzene with cis-1,4-diacetoxy-2-butene were studied, both under solvent-free conditions and in methyl decanoate, a renewable and environmentally benign solvent derived from fatty acid derivatives. More recently, the synthesis of nitrogen containing heterocycles by ruthenium catalyzed RCM using low catalyst loading was studied by Grubbs.

International audience; α,ω-Di(glycerol carbonate) telechelic poly(propylene glycol) (PPG), poly(ethylene glycol) (PEG), poly(ester ether) (PEE), and poly(butadiene) (PBD) have been synthesized through chemical modification of the corresponding α,ω-dihydroxy telechelic polymers (PPG-OH2, PEG-OH2, PEE-OH2 and PBD-OH2, respectively). Tosylation of the polymer diols with 4-tosylmethyl-1,3-dioxolan-2-one (GC-OTs) afforded, in high yields, the desired PPG, PEG, PEE and PBD end-capped at both termini with a five-membered ring cyclic glycerol carbonate (4-hydroxymethyl-1,3-dioxolan-2-one, GC). The GC-functionalization of the polymers at both chain-ends has been confirmed by NMR (1H, 13C, 1D and 2D) and FTIR spectroscopies. Using PPG-GC2 to demonstrate the concept, the corresponding polyhydroxyurethanes (PHUs/non-isocyanate polyurethanes (NIPUs)) have been subsequently prepared following a non-isocyanate method upon ring-opening catalyst-free polyaddition of the PPG-GC2 with JEFFAMINEs (Mn = 230-2000 g mol−1). The effect of various additives introduced during the polyaddition reaction has been studied at different temperatures. In particular, addition of LiBr (5 mol%) to the reaction medium was found to slightly promote the cyclocarbonate/amine reaction. The polymerization process was supported by FTIR and SEC analyses.

Size exclusion chromatography coupled with light scattering (SEC/MALS), dynamic light scattering (DLS), steady-state and time-resolved fluorescence, as well as molecular dynamics (MD) simulations are used to study the behavior of several poly(ethylene glycol) (PEG)/α-cyclodextrins (αCDs) polyrotaxanes (PRs) in solution. The number of CD units in any of the PRs studied is always smaller than that required to saturate the PEG chains. These PRs seem to aggregate in dimethyl sulfoxide (DMSO) solution. The presence of hairpins in the non-saturated PRs contributes to diminishing their expected large dimensions. Intra- and inter­molecular interactions and forces responsible for hairpins and aggregation are investigated. This work was supported by the University of Alcalá (GC2011–002 and UAH2011/EXP-036) and Nanomod (MAT2010–17091) from the Spanish Ministerio de Ciencia e Innovación. The authors acknowledge the assistance of M. L. Heijnen with the preparation of the manuscript.

Addition of ionized terminal groups to PEG graft layers may cause additional interfacial forces to modulate the net interfacial interactions between PEG graft layers and proteins. In this study we investigated the effect of terminal sulfonate groups, characterizing PEG-aldehyde (PEG-CHO) and sulfonated PEG (PEG-SO3) graft layers by XPS and colloid probe AFM interaction force measurements as a function of ionic strength, in order to determine surface forces relevant to protein resistance and models of bio-interfacial interaction of such graft coatings. On the PEG-CHO surface the measured interaction force does not alter with ionic strength, typical of a repulsive steric barrier coating. An analogous repulsive interaction force of steric origin was also observed on the PEG-SO3 graft coating; however, the net interaction force changed with ionic strength. Interaction forces were modelled by steric and electrical double layer interaction theories, with fitting to a scaling theory model enabling determination of the spacing and stretching of the grafted chains. Albumin, fibrinogen, and lysozyme did not adsorb on the PEG-CHO coating, whereas the PEG graft with terminal sulfonate groups showed substantial adsorption of albumin but not fibrinogen or lysozyme from 0.15 M salt solutions. Under lower ionic strength conditions albumin adsorption was again minimized as a result of the increased electrical double-layer interaction observed with the PEG-SO3 modified surface. This unique and unexpected adsorption behaviour of albumin provides an alternative explanation to the "negative cilia" model used by others to rationalize observed thromboresistance on PEG-sulfonate coatings. Refereed/Peer-reviewed

The role of a protein-repelling coating is to limit the interaction between a device and its physiological environment. Plasma-polymerized-PEG (pp-PEG) surfaces are of great interest since they are known to avoid protein adsorption. and cell attachment. However, in all the studies previously published in the literature, the PEG coatings have been prepared using low pressure processes. In this thesis, we synthesize biocompatible pp-PEG coatings using atmospheric pressure plasma. Two original methods are developed to obtain these pp-PEG films. 1. Atmospheric pressure plasma liquid deposition (APPLD) consists in the injection of the precursor, tetra(ethylene glycol)dimethylether (tetraglyme), by means of a liquid spray, directly in the post-discharge of an atmospheric argon plasma torch. 2. In atmospheric pressure plasma-enhanced chemical vapor deposition (APPECVD), tetraglyme vapors are brought in the post-discharge trough a heating sprinkler. The chemical composition, as well as the non-fouling properties of the APPLD and APPECVD films, are compared to those of PEG coatings synthesized by conventional low pressure plasma processes.In the first part of the study, the effect of the power on the chemical composition of the films has been investigated by infrared reflection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS) and secondary ions mass spectroscopy (SIMS). The surface analysis reveals that for the APPECVD samples, the fragmentation of the precursor increases as the power of the treatment is increased. In other terms, the lower the plasma power is, the higher the “PEG character” of the resulting films is. Indeed, the C-O component (286.5 eV) of the XPS C 1s peak is decreasing while the hydrocarbon component (285 eV) is increasing as the power of the plasma is increased. The same conclusion can be drawn from the signature ToF-SIMS peaks (m/z = 45 (CH3&61485;O&61485;CH2+ and +CH2CH2&61485;OH), 59 (CH3&61485;O&61485;CH2&61485;CH2+), 103 (CH3&61485;(O&61485;CH2&61485;CH2)2+)) that are decreasing in the case of high power treatments. Accordingly, IRRAS measurements show that the C-O stretching band is decreasing for high power plasma deposition. This is in agreement with the observations made from the analysis of the LP PECVD coatings and from the literature.The films deposited by the APPLD process do not show the same behavior. Indeed, whatever the power injected into the discharge is, we are able to achieve films with a relatively high PEG character (&61566;83 %).The second part of this study is dedicated to the evaluation of the non-fouling properties of the coatings by exposing them to proteins (bovine serum albumin and human fibrinogen) and cells (mouse fibroblasts (L929 and MEF)) and controlling the adsorption with XPS (proteins) and SEM (cells).For the APPECVD samples, a low plasma power (30 W) leads to an important reduction of protein adsorption and cell adhesion (over 85%). However, higher-powered treatments tend to reduce the non-fouling ability of the surfaces (around 50% of reduction for a 80 W deposition). The same order of magnitude (over 90% reduction of the adsorption) is obtained for the APPLD surfaces, whatever is the power of the treatment. Those results show an important difference between the two processes in terms of power of the plasma treatment, and a strong relationship between the surface chemistry and the adsorption behavior: the more the PEG character is preserved, the more protein-repellent and cell-repellent is the surface. / Le rôle d’une couche empêchant l’adsorption de protéines est de limiter les interactions entre un implant et le milieu physiologique auquel il est exposé. Les films de poly(éthylène glycol) polymérisés par plasma (pp-PEG) sont d’intérêt majeur car ils sont connus pour empêcher l’adsorption de protéines ainsi que l’attachement cellulaire. Cependant, dans toutes les études publiées précédemment, les couches de type PEG ont été réalisées sous vide.Dans cette thèse de doctorat, nous synthétisons des couches de type pp-PEG biocompatibles par plasmas à pression atmosphérique. A cette fin, deux méthodes originales ont été développées. 1. La première méthode consiste en l’injection du précurseur, le tetra(éthylène glycol) diméthyl éther (tetraglyme), en phase liquide, en nébulisant ce dernier au moyen d’un spray, directement dans la post-décharge d’une torche à plasma atmosphérique fonctionnant à l’argon. En anglais, nous appelons ce procédé « Atmospheric pressure plasma liquid deposition (APPLD) ». 2. Dans la deuxième méthode, appelée en anglais « Atmospheric pressure plasma-enhanced chemical vapor deposition (APPECVD)», le tetraglyme est amené en phase vapeur dans la post-décharge, au moyen d’un diffuseur chauffant. La composition chimique des dépôts de type APPLD et APPECVD, ainsi que leurs propriétés d’anti-adsorption sont évaluées, et comparées aux dépôts pp-PEG obtenus par les méthodes à basse pression conventionnelles.Dans la première partie de cette étude, nous nous focalisons sur la composition chimique des films déposés, et plus particulièrement sur l’influence de la puissance injectée dans le plasma sur cette composition chimique. A cette fin, nous avons fait appel à des techniques d’analyse telles que la spectroscopie de réflexion-absorption infrarouge (IRRAS), la spectroscopie des photoélectrons X (XPS) et la spectrométrie de masse des ions secondaires (SIMS). Il en ressort que les films de type APPECVD perdent progressivement leur « caractère PEG » à mesure que la puissance de la décharge plasma est élevée. Cela serait dû à une plus grande fragmentation du précurseur dans la post-décharge d’un plasma plus énergétique. Cette tendance est cohérente avec ce que nous avons observé pour les dépôts à basse pression ainsi que dans la littérature.Dans le cas des films de type APPLD, un tel comportement n’a pas été mis en évidence :quelle que soit la puissance dissipée dans le plasma, les films présentent un « caractère PEG » relativement élevé.La deuxième partie de cette thèse est dédiée à l’évaluation des propriétés d’anti-adsorption des films synthétisés, en les exposant à des protéines (albumine de sérum bovin et fibrinogène humain) et des cellules (fibroblastes de souris, L929 et MEF). L’adsorption de protéines est contrôlée par XPS tandis que l’attachement cellulaire est contrôlé par imagerie SEM.Pour les échantillons de type APPECVD, un dépôt à faible puissance (30 W) mène à une importante réduction de l’adsorption de protéines et de cellules (> 85%) tandis qu’à de plus hautes puissances (80 W), l’anti-adsorption est sensiblement diminuée (50% de réduction). Dans le cas des dépôts de type APPLD, quelle que soit la puissance du plasma, une forte diminution de l’adsorption de protéines et de cellules est observée (> 90 %).Ces résultats montrent une différence majeure entre les deux procédés quant à l’influence de la puissance du plasma ainsi qu’une forte relation entre la composition chimique de la surface synthétisée et son pouvoir d’anti-adsorption :plus le « caractère PEG » du dépôt est conservé, plus la surface empêchera l’interaction avec les protéines et les cellules., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Hybrid systems formed from polymers and transition metals have now their physical and chemical properties extensively investigated for use in electronic devices. In this work, Titanium Dioxide (TiO2) from the precursor of titanium tetrabutoxide and the composite system Poly(Ethylene Glycol)-Titanium Dioxide (TiO2-PEG) were synthesized by sol-gel method. The PEG as acquired and TiO2 and composites powders were analyzed by X-Ray Diffraction (XRD), Spectroscopy in the Infrared region with Fourier transform (IRFT), Thermogravimetric Analysis (TGA), Scanning Electron Microscopy (SEM) and Electrochemical Impedance Spectroscopy (EIS). In the XRD analysis were observed in the TiO2 crystal faces of one of its polymorphs - anatase phase, crystal planes in Poly (Ethylene Glycol) with considerable intensity and in the composite systems the mixture of crystal faces of their precursors isolated and reduction of crystallinity. The TG / DTG suggested increasing the thermal instability of PEG in the composite powders as TiO2 is incorporated into the system. Spectral analysis presented in the infrared overlapping bands for the polymer and metal oxide, reducing the intensity of symmetric stretching of ligand groups in the main chain polymer and angular deformations; were observed using SEM micrographs of the morphological changes suffered by composite systems with the variation of the oxide concentration. Analyses by impedance spectroscopy indicated that the increased conductivity in composite occurs in line with the addition of the metal oxide concentration in the composite system Sistemas híbridos formados a partir de polímeros e metais de transição atualmente têm suas propriedades físicas e químicas extensivamente investigadas para aplicação em dispositivos eletrônicos. Neste trabalho, o Dióxido de Titânio (TiO2) a partir do precursor Tetrabutóxido de Titânio e o sistema compósito Poli (Etileno Glicol)-Dióxido de Titânio (PEG-TiO2) foram sintetizados pelo método sol-gel. O PEG como adquirido e os pós de TiO2 e compósitos obtidos foram analisados por Difração de Raios X (DRX), Espectroscopia de Absorção na região do Infravermelho com Transformada de Fourier (EIV-TF), Análise Termogravimétrica (TG), Microscopia Eletrônica de Varredura (MEV) e Espectroscopia de Impedância Eletroquímica (EIE). Nas análises por DRX foram observadas no TiO2, faces cristalinas de um de seus polimorfos fase anatásio, planos cristalinos no PEG com intensidade substancial e nos sistemas compósitos a mistura de faces cristalinas dos seus precursores na forma isolada, bem como, redução da cristalinidade. As curvas TG/DTG sugeriram o aumento da instabilidade térmica do PEG nos pós compósitos à medida que TiO2 é incorporado ao sistema. A análise espectral na região do infravermelho apresentou sobreposição de bandas relativas ao polímero e ao óxido metálico, redução da intensidade de estiramentos simétricos dos grupos ligantes da cadeia principal polimérica e deformações angulares; foram observadas através das micrografias de MEV modificações morfológicas sofridas pelos sistemas compósitos com a variação da concentração do óxido. As análises por impedância eletroquímica indicaram que o aumento da condutividade nos compósitos ocorre em consonância com a adição da concentração do óxido metálico no sistema compósito

The objective of this work was to create three-dimensional (3D) hydrogel matrices with defined mechanical properties, as well as tunable degradability for use in applications involving protein delivery and cell encapsulation. Thus, we report the synthesis and characterization of a novel hydrolytically degradable poly(ethylene glycol) (PEG) hydrogel composed of PEG vinyl sulfone (PEG-VS) cross-linked with PEG-diester-dithiol. Unlike previously reported degradable PEGbased hydrogels, these materials are homogeneous in structure, fully hydrophilic and have highly specific cross-linking chemistry. We characterized hydrogel degradation and associated trends in mechanical properties, i.e., storage modulus (G′), swelling ratio (QM), and mesh size (ξ). Degradation time and the monitored mechanical properties of the hydrogel correlated with crosslinker molecular weight, cross-linker functionality, and total polymer density; these properties changed predictably as degradation proceeded (G′ decreased, whereas QM and ξ increased) until the gels reached complete degradation. Balb/3T3 fibroblast adhesion and proliferation within the 3D hydrogel matrices were also verified. In sum, these unique properties indicate that the reported degradable PEG hydrogels are well poised for specific applications in protein and cell delivery to repair soft tissue.

Throughout the study of polymeric Langmuir monolayers at the air/water (A/W) interface, the Wilhelmy plate and Langmuir-Blodgett (LB) techniques along with Brewster angle microscopy (BAM) have been identified as key methods for acquiring structural, thermodynamic, rheological and morphological information. These techniques along with surface light scattering (SLS), a method for probing a monolayer's dynamic dilational rheological properties, will be used to characterize homopolymers, poly(ethylene oxide) (PEO) and poly(ethylene glycol) (PEG), and a new class of novel polymeric surfactants, telechelic (POSS-PEG-POSS) and hemi-telechelic (POSS-PEG) polyhedral oligomeric silsesquioxane (POSS) derivatives of PEG. PEO with number average molar mass, Mn > ~ 18 kg·mol-1 form stable spread Langmuir films at the A/W interface, while oligomeric PEG have ï -A isotherms that deviate from high molar mass PEO. Nonetheless, SLS reveals that the dynamic dilational viscoelastic properties of any Mn PEG(PEO) only depend on ï and not Mn. Likewise, POSS-PEG-POSS telechelics exhibit molar mass dependent ï -A isotherms, where low ï regimes (ï < 1 mN·m-1) have PEG-like behavior, but high ï regimes were dominated by POSS-POSS interactions. SLS studies reveal that the dynamic dilational moduli of POSS-PEG-POSS are greater than either PEO or an analogous POSS compound, trisilanolcyclohexyl-POSS. The ability to control rheological properties and the hydrophilic-lipophilic balance even allows one POSS-PEG-POSS (PEG Mn = 1 kg·mol-1) to form Y-type LB-multilayer films. For POSS-PEG systems, comparisons at comparable POSS:PEG ratios reveal short PEG chains (PEG Mn ~ 0.5 kg·mol-1) yield similar viscoelastic properties as POSS-PEG-POSS (PEG Mn ~ 1 kg·mol-1), while longer PEG chains (PEG Mn ~ 2 kg·mol-1) yield lower modulus films than comparable POSS-PEG-POSS. These differences are attributed to brush-like PEG conformations in short POSS-PEG versus mushroom-like PEG conformations in long POSS-PEG at the A/W interface. These results provide insight for designing PEG-based amphiphilic nanoparticles with controlled interfacial rheology. Ph. D.

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Shann S Yu1,2, Cheryl M Lau1, Susan N Thomas3, W Gray Jerome4, David J Maron5, James H Dickerson2,6, Jeffrey A Hubbell3, Todd D Giorgio1,2,7,81Department of Biomedical Engineering, Vanderbilt University, Nashville, 2Vanderbilt Institute of Nanoscale Science and Engineering, Nashville, TN, USA; 3Institute of Bioengineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland, 4Department of Pathology, Vanderbilt University Medical Center, Nashville, 5Vanderbilt Heart and Vascular Institute, Nashville, 6Department of Physics and Astronomy, Vanderbilt University, Nashville, 7Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, 8Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USAAbstract: The assessment of macrophage response to nanoparticles is a central component in the evaluation of new nanoparticle designs for future in vivo application. This work investigates which feature, nanoparticle size or charge, is more predictive of non-specific uptake of nanoparticles by macrophages. This was investigated by synthesizing a library of polymer-coated iron oxide micelles, spanning a range of 30–100 nm in diameter and -23 mV to +9 mV, and measuring internalization into macrophages in vitro. Nanoparticle size and charge both contributed towards non-specific uptake, but within the ranges investigated, size appears to be a more dominant predictor of uptake. Based on these results, a protease-responsive nanoparticle was synthesized, displaying a matrix metalloproteinase-9 (MMP-9)-cleavable polymeric corona. These nanoparticles are able to respond to MMP-9 activity through the shedding of 10–20 nm of hydrodynamic diameter. This MMP-9-triggered decrease in nanoparticle size also led to up to a six-fold decrease in nanoparticle internalization by macrophages and is observable by T2-weighted magnetic resonance imaging. These findings guide the design of imaging or therapeutic nanoparticles for in vivo targeting of macrophage activity in pathologic states.Keywords: macrophage targeting, poly(ethylene glycol) (PEG), poly(propylene sulfide) (PPS), iron oxides, opsonization

Hao Yan1,2, Weimin Jiang1,2, Yinxing Zhang1,2, Ying Liu1,2, Bin Wang1,2, Li Yang1,2, Lihong Deng1,2, Gurinder K Singh1,2, Jun Pan1,21Bioengineering College, Chongqing University, 2Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, Chongqing, People's Republic of ChinaAbstract: Targeted drug delivery requires novel biodegradable, specific binding systems with longer circulation time. The aim of this study was to prepare biotinylated poly(lactic acid) (PLA) nanoparticles (NPs) which can meet regular requirements as well conjugate more biotins in the polymer to provide better binding with streptavidin. A biotin-graft-PLA was synthesized based on previously published biodegradable poly(ethylene glycol) (PEG)-graft-PLA, with one polymer molecule containing three PEG molecules. Newly synthesized biotin-graft-PLA had three biotins per polymer molecule, higher than the previous biotinylated PLA (≤1 biotin per polymer molecule). A PEG with a much lower molecular weight (MW ~1900) than the previous biotinylated PLA (PEG MW ≥3800), and thus more biocompatible, was used which supplied good nonspecific protein-resistant property compatible to PEG-graft-PLA, suggesting its possible longer stay in the bloodstream. Biotin-graft-PLA specifically bound to streptavidin and self-assembled into NPs, during which naproxen, a model small molecule (MW 230 Da) and hydrophobic drug, was encapsulated (encapsulation efficiency 51.88%). The naproxen-loaded NPs with particle size and zeta potential of 175 nm and —27.35 mV realized controlled release within 170 hours, comparable to previous studies. The biotin-graft-PLA NPs adhered approximately two-fold more on streptavidin film and on biotin film via a streptavidin arm both in static and dynamic conditions compared with PEG-graft-PLA NPs, the proven nonspecific protein-resistant NPs. The specific binding of biotin-graft-PLA NPs with streptavidin and with biotin using streptavidin arm, as well as its entrapment and controlled release for naproxen, suggest potential applications in targeted drug delivery.Keywords: targeted drug delivery, bioactive, biodegradable, poly(ethylene glycol) (PEG), controlled release, naproxen

Biomolecular signaling is of utmost importance in governing many biological processes such as morphogenesis during tissue development where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion and in the context of a soft and hydrated microenvironment. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro- scale is far from ideal due to issues related to medium evaporation, limited space for cell growth, shear stress and a lack of cell-instructive microenvironments that might adversely impact cell-fate. As a result, microfluidic cell culture systems are not yet suitable to unravel complex multicellular phenomena. This may explain why they are not yet widely used in biology laboratories. Consequently, the overall goal of this thesis was to overcome this technology gap by developing next generation microfluidics through a combination of microfabrication and smart biomaterials. A special emphasis was placed on decoupling biomolecule presentation at micro-scale from macro-scale cell culture in order to enable long-term stem cell culture within user-friendly multiwell plate formats. First, a novel microfluidic concept was invented to rapidly immobilize linear protein gradients on the surface of poly(ethylene glycol) (PEG)-based hydrogels whose biophysical properties are reminiscent of natural extracellular matrices. This method allows efficient capture of steady-state gradients of tagged proteins (e.g. using Fc or biotin tags) in just a few minutes on engineered hydrogels that display the corresponding auxiliary proteins (e.g. ProteinA or NeutrAvidin). The selectivity and orthogonality of the chosen binding schemes enables the formation of parallel and orthogonal overlapping gradients of multiple proteins, which is impossible using existing platforms. After patterning, the microfluidic chip can be readily removed from the gel for cell culture applications. Using quantitative single-cell time-lapse microscopy, this platform was validated here by probing the effect of fibronectin concentration on the directionality and speed of cell migration. Next, the resolution, flexibility and throughput of the protein patterning on hydrogels was substantially enhanced by the introduction of hydrodynamic flow focusing, that is, the generation of user-defined patterns by spatially controlled step- wise deposition of biomolecules. To this end, a microfluidic device was conceived that enabled the generation of arrays of parallel and crossed overlapping gradients. Application of the platform to generate gradients of immobilized leukemia inhibitory factor (LIF) showed an influence of the LIF concentration on ESC self-renewal. This platform should be useful to systematically probe the effect of biomolecule dose, singly or in combinations, on stem cell behavior in vitro; however, it lacks the ability to dynamically vary the presentation of biomolecules that might be crucial to control stem cell fate towards establishing functional in vitro tissue models. To address this limitation, in the last part of the thesis work a hydrogel-based microfluidic chip was developed that decouples macro-scale cell culture on the gel surface from the precise spatiotemporal biomolecule delivery at the micro-scale, i.e. through the gel layer. The hydrogel chip, used here as a simple insert that is compatible with conventional multiwell plate formats, was optimized to support long- term ESC maintenance in both adherent format and as uniformly sized ESC aggregates termed embryoid bodies. The platform was successfully used for the spatially controlled neuronal commitment of ESC via delivery of gradients of the morphogen retinoic acid. Taken together, in this thesis several innovative microengineered cell culture platforms are presented which enable, perhaps for the first time, the long-term culture and manipulation of stem cell fate at the micro-scale. These systems should be useful to systematically probe in vitro the effect of biomolecule dose and delivery dynamics on stem cell behavior, ultimately facilitating the development of complex in vitro tissue models.