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

A focused library of methacrylate terpolymers was synthesized to explore the effects of varying surface chemistry and adhesive peptide ligands on cell function. The chemical diversity of methacrylate monomers enabled construction of a library of polymers in which one can systematically vary the chemical composition to achieve a wide range of contact angle, Young's modulus, and T(g) values. Furthermore, the materials were designed to allow surface immobilization of bioactive peptides. We then examined the effects of these material compositions on protein adsorption and cell attachment, proliferation, and differentiation. We observed that chemical composition of the polymers was an important determinant for NIH 3T3 cell attachment and proliferation, as well as human mesenchymal stem cell differentiation, and correlated directly with the ability of the polymers to adsorb proteins that mediate cell adhesion. Importantly, functionalization of the methacrylate terpolymer library with an adhesive GRGDS peptide normalized cellular responses. RGD-functionalized polymers uniformly exhibited robust attachment, proliferation, and differentiation irrespective of the underlying substrate chemistry. These studies provide a library-based approach to rapidly explore the biological functionality of biomaterials with a wide range of compositions and highlight the importance of cell and protein cell adhesion in predicting their performance.     

单一分布聚乙二醇的合成及其应用

聚乙二醇(PEG)经过对端基官能团化接枝到药物等生物活性分子上后,可提高药物等的水溶性、抗免疫原性、生物相容性和稳定性并降低毒性。工业级聚乙二醇由于杂质含量高尤其是其中二醇的含量过高、分子量分布过宽,不能直接用于药物等的改性。除利用聚合方法制备窄分布高纯度的聚乙二醇外,采用有机合成方法合成单一聚乙二醇链是有效的手段。对"单一链长(discrete length)"或者"单一分布(mono-disperse)"的聚乙二醇高分子聚合物的近年来主要合成方法的进展进行了综述并简介了其主要应用。     

多肽和蛋白质的聚乙二醇化修饰方法

聚乙二醇是一类具有独特理化性质的大分子聚合物。多肽和蛋白质类药物经聚 乙二醇共价修饰后能明显改善其药代学和药效学性质，如降低免疫原性、增加对蛋 白水解酶的稳定性、增加水溶性及延长体内的半衰期等。蛋白质的聚乙二醇化修饰 研究已取得较好的效果，多肽的聚乙二醇化修饰研究起步较晚。对近年来多肽和蛋 白质的聚乙二醇化修饰方法进行了综述，主要介绍了对多肽和蛋白质的N端、C端及 某些氨基酸侧链进行选择性聚乙二醇化修饰的方法。     

Synthesis and characterization of paramagnetic microparticles through emulsion-templated free radical polymerization

A novel method is described for the preparation of high-magnetization paramagnetic microparticles functionalized with a controlled density of poly(ethylene glycol) (PEG) and carboxyl groups. These microparticles were synthesized using four steps: (1) creation of an oil-in-water emulsion in which hydrophobic iron oxide nanoparticles and a UV-activated initiator were distributed in hexane; (2) formation of uniform microparticles through emulsion homogenization and evaporation of hexane; (3) functionalization of the microparticle with a PEG-functionalized surfactant and acrylic acid; and (4) polymerization of the microparticles. Characterization of the microparticles with electron microscopy and light scattering revealed that they were composed of densely packed iron oxide nanoparticles and that the size of the microparticles may be controlled through the pore size of the membrane used to homogenize the emulsion. The concentration of surfactant and acrylic acid used in the third processing step was found to determine the surface chemistry, iron content, and magnetization of the microparticles. Increasing the PEG surfactant to acrylic acid ratio resulted in higher PEG surface densities, lower iron content, and lower magnetization. The resulting microparticles were readily functionalized with antibodies and showed a low propensity for nonspecific protein adsorption. We believe that these microparticles will be useful for magnetic tweezers measurements and bioanalytical devices that require microparticles with a high magnetization.     

TOF-SIMS analysis of structured surfaces biofunctionalized by a one-step coupling of a spacer-linked GRGDS peptide

Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) was applied to validate GRGDS peptide patterned surfaces. The structuring of the surfaces included several steps: micro contact printing (μCP), chemical etching and aminofunctionalization followed by chemical coupling of spacer-linked GRGDS peptides via an isothiocyanate anchor. TOF-SIMS analysis of characteristic ions and molecular fragments with a lateral resolution of 100 nm allowed proving the change in chemical properties of the surface with each step during the structuring process. We found that the application of polydimethylsiloxane as stamp material resulted in the contamination of the surface with this polymer. TOF-SIMS investigations, however, also showed that during the preparation process the contaminations were removed and do not influence the bio functionality of the surface patterns. The results of the surface analysis carried out with TOF-SIMS were confirmed by complementary cell adhesion experiments with murine fibroblasts. As a result, specific cell adhesion restricted to GRGDS peptide functionalized areas was obvious by the formation of focal adhesion contacts in the fibroblasts. Thus, TOF-SIMS is the method of choice in chemical characterization of surfaces in structuring and functionalization processes, because it offers the opportunity to follow surface contamination during the preparation process and to assess the influence of the contamination on the applicability of the final substrate.     

Synthesis of Porous PEG Microgels Using CaCO3 Microspheres as Hard Templates

Porous poly(ethylene glycol) (PEG) microgels of both 17.6 and 8.3 μm in diameter are synthesized via hard templating with calcium carbonate (CaCO₃) microparticles. The synthesis is performed in three steps: loading of PEG macromonomers into CaCO₃ microparticles, crosslinking via photopolymerization, and removal of the CaCO₃ template under acidic conditions. The resulting porous PEG microgels are inverse replicates of their templates as indicated by light microscopy, cryo‐scanning electron microscopy (cryo‐SEM), and permeability studies. Thus this process allows for the straightforward and highly reproducible synthesis of porous hydrogel particles of two different diameters and porosities that show great potential as carriers for drugs or nanomaterials.     

Novel poly(ethylene glycol) monomers bearing diverse functional groups

Poly(ethylene glycol) (PEG) mono methacrylate ester (MAPEG) has been used, through a variety of reactions, to form several novel monomers, bearing both a polymerizable handle and various functional groups. These new compounds may be conjugated to biomolecules via amine, acid, or thiol moieties or they may form dendrimers via the epoxide. In addition, polymerization of these monomers may result in functionalized nanoparticles and microparticles or coatings, thus altering the acid‐base or electrochemical properties of surfaces and particles. Full synthetic considerations, including interesting intermediates, are reported. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Relationship between interfacial forces measured by colloid-probe atomic force microscopy and protein resistance of poly(ethylene glycol)-grafted poly(L-lysine) adlayers on niobia surfaces

Adsorbed layers of "comb-type" copolymers consisting of PEG chains grafted onto a poly(l-lysine) (PLL) backbone on niobium oxide substrates were studied by colloid-probe AFM in order to characterize the interfacial forces associated with coatings of varying architectures (PEG/PLL ratios and PEG chain lengths) and their relevance to protein resistance. The steric and electrostatic forces measured varied substantially with the architecture of the PLL-g-PEG copolymers. Varying the ionic strength of the buffer solutions enabled discrimination between electrostatic and steric-entropic contributions to the net interfacial force. For high PEG grafting densities the steric component was most prominent, but at low ionic strengths and high grafting densities, a repulsive electrostatic surface force was also observed; its origin was assigned to the niobia charges beneath the copolymer, as insufficient protonated amine groups in the PLL backbone were available for compensation of the oxide surface charges. For lower grafting densities and lower ionic strengths there was a substantial attractive electrostatic contribution arising from interaction of the electrical double layer arising from the protonated amine groups, with that of the silica probe surface (as under low ionic strength conditions, the electrical double layer was thicker than the PEG layer). For these PLL-g-PEG coatings the net interfacial force can thus be a markedly varying superposition of electrostatic and steric-entropic contributions, depending on various factors. The force curves correlate with protein adsorption data, demonstrating the utility of AFM colloid-probe force measurements for quantitative analysis of surface forces and how they determine interfacial interactions with proteins. Such characterization of the net interfacial forces is essential to elucidate the multiple types of interfacial forces relevant to the interactions between PLL-g-PEG coatings and proteins and to advance interpretation of protein adsorption or repellence beyond the oversimplified steric barrier model; in particular, our data demonstrate the importance of an ionic-strength-dependent minimum PEG layer thickness to screen the electrostatic interactions of charged interfaces.     

Folate‐Conjugated Micelles and Their Folate‐Receptor‐Mediated Endocytosis

A folate‐conjugated copolymer PEG‐PLA‐PLL/folate was synthesized and mixed with pure PEG‐PLA‐PLL and a fluorescent model drug mFITC to prepare folate‐conjugated micelles. The distribution of micelles was studied on cancer‐cell‐bearing mice via frozen slicing. The r e sults show that mFITC is successfully encapsulated into folate(+) and folate(?)micelles; PEG‐PLA‐PLL micelles the latter can be internalized by both HeLa and CHO cells without selectivity due to their cationic surface charges, while folate(+)micelles exhibit more preferential endocytosis by HeLa cells than by CHO cells. The folate(?)micelles showed retention in both organs and tumors. The folate(+)micelles are a promising active targeting drug delivery system for FR over‐expressing cells and they accumulate in tumor beds.

     

Organic functionalization of luminescent oxide nanoparticles toward their application as biological probes

Luminescent inorganic nanoparticles are now widely studied for their applications as biological probes for in vitro or in vivo experiments. The functionalization of the particles is a key step toward these applications, since it determines the control of the coupling between the particles and the biological species of interest. This paper is devoted to the case of rare earth doped oxide nanoparticles and their functionalization through their surface encapsulation with a functional polysiloxane shell. The first step of the process is the adsorption of silicate ions that will act as a primary layer for the further surface polymerization of the silane, either aminopropyltriethoxysilane (APTES) or glycidoxypropyltrimethoxysilane (GPTMS). The amino- or epoxy- functions born by the silane allow the versatile coupling of the particles with bio-organic species following the chemistry that is commonly used in biochips. Special attention is paid to the careful characterization of each step of the functionalization process, especially concerning the average number of organic functions that are available for the final coupling of the particles with proteins. The surface density of amino or epoxy functions was found to be 0.4 and 1.9 functions per square nanometer for GPTMS and APTES silanized particles, respectively. An example of application of the amino-functionalized particles is given for the coupling with alpha-bungarotoxins. The average number (up to 8) and the distribution of the number of proteins per particle are given, showing the potentialities of the functionalization process for the labeling of biological species.     

Protein microparticles with controlled stability prepared via layer-by-layer adsorption of biopolyelectrolytes

This review covers the experimental data on the preparation and characterization of protein microparticles with controlled stability that are formed by layer-by-layer adsorption of oppositely charged macromolecules. Variants of using proteins as adsorbed polyelectrolyes, methods of incorporating proteins into matrixes (aggregates and microspheres) for further deposition of biopolyelectrolytes, and immobilization of proteins in preformed multilayered polyelectrolyte particles due to a change in the permeability of their shells are considered. Special attention is given to biocompatible and biodegradable microparticles characterized by depot functions, that is, the ability to reliably protect biologically active compounds from aggregative media of the body and to quantitatively release protein preparations (hormones, enzymes, and peptides) into solution when a certain acidity of solution is attained. This feature is especially important for designing peroral means of protein delivery.     

Carboxyl Group (CO2H) Functionalized Coordination Polymer Nanoparticles as Efficient Platforms for Drug Delivery

Functionalization of nanoparticles can significantly influence their properties and potential applications. Although researchers can now functionalize metal, metal oxide, and organic polymer nanoparticles with a high degree of precision, controlled surface functionalization of nanoscale coordination polymer particles (CPPs) has remained a significant challenge. The lack of methodology is perhaps one of the greatest roadblocks to the advancement of CPPs into high added‐value drug delivery applications. Here, we report having achieved this goal through a stepwise formation and functionalization protocol. We fabricated robust nanoparticles with enhanced thermal and colloidal stabilities by incorporation of carboxyl groups and these surface carboxyl groups could be subsequently functionalized through well‐known peptide coupling reactions. The set of chemistries that we employed as proof‐of‐concept enabled a plethora of new functional improvements for the application of CPPs as drug delivery carriers, including enhanced colloidal stabilities and the incorporation of additional functional groups such as polyethylene glycol (PEG) or fluorescent dyes that enabled tracking of their cellular uptake. Finally, we ascertained the cytotoxicity of the new CPP nanoparticles loaded with camptothecin to human breast adenocarcinoma (MCF‐7). Efflux measurements show that the encapsulation of camptothecin enhances the potency of the drug 6.5‐fold and increases the drug retention within the cell.     

Use of Natural Monomer in the Synthesis of Nano- and Microparticles of Polyurethane by Suspension-Polyaddition Technique

Summary: Polyurethane nano- and microparticles were synthesized by suspension-polyaddition technique, using aqueous polymerization medium. Castor oil, a vegetable triglyceride possessing hydroxyl groups was used as natural polyol and methylene diphenyl diisocyanate (MDI) as isocyanate. The levofloxacin, an antibacterial drug was used as model drug to measure the particles encapsulation efficiency. The effect of the addition of a second polyol, the poly(ethylene glycol) (PEG), and the stirring rate on the mean diameter and morphology of particles was also investigated. The poly(ethylene glycol) has an important effect in the reduction of particles size and their porosity. On the other hand, the poly(ethylene glycol) reduced the yield of encapsulation from 70% for the formulation without PEG to 20% for formulations with PEG. FTIR analysis confirmed the polyurethane formation. Dynamic light scattering study, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to determine the nanoparticles size and shape. Spectrofluorimetric analysis was used to detect the levofloxacin.     

Solid-Phase Peptide Modification via Deaminative Photochemical Csp3-Csp3 Bond Formation Using Katritzky Salts

Introduction of unnatural amino acids can significantly improve the binding affinity and stability of peptides. Commercial availability of such amino acids is limited, and their synthesis is a long and tedious process. We here describe a method that allows the functionalization of peptides directly on solid-support by converting lysine residues to Katritzky salts, and subjecting them to a photochemical Giese reaction under mild reaction conditions. The method avoids the need for amino acid synthesis and instead offers a late-stage modification route for rapid peptide diversification. While numerous modification approaches at the lysine amine have been described, this work provides the first example of deaminative functionalization of peptides at lysine. The two-step protocol is compatible with various substrates, lysine analogues, resins, and all proteinogenic amino acids. Finally, by leveraging solid-phase modification, this protocol facilitates the functionalization of longer peptides as was demonstrated using biologically relevant peptides of up to 15 amino acids.     

Single component and selective competitive protein adsorption in a patchy polymer brush: opposition between steric repulsions and electrostatic attractions

This work explores the use of "patchy" polymer brushes to control protein adsorption rates on engineered surfaces and to bind targeted species from protein mixtures with high selectivity but without invoking molecular recognition. The brushes of interest contain embedded cationic "patches" composed of isolated adsorbed poly(l-lysine) coils (PLL) that are about 10 nm in diameter and are randomly arranged on a silica substrate. Around these patches is a protein-resistant poly(ethylene glycol) (PEG) brush that is formed from the adsorption of a PLL-g-PEG graft copolymer on the remaining silica surface. Electrostatic attractions between individual cationic patches and the negative regions of approaching proteins may be energetically insufficient to bind proteins. Furthermore, protein-patch attractions are reduced by steric repulsions between proteins and the PEG brush. We show that protein adsorption, gauged by ultimate short-term coverages and by the initial protein adsorption rate, exhibits an adhesion threshold: pure PEG brushes of the architectures employed here and brushes containing sparse loadings of PLL patches do not adsorb protein. Above a critical PLL patch loading or threshold, protein adsorption proceeds, often dramatically. The PLL patch thresholds are specific to the protein of interest, allowing surfaces to be engineered to adhesively discriminate different proteins within a mixture. The separation achieved is remarkably sharp: one protein adsorbs, but the second is completely rejected from the interface. The surfaces in this study, by virtue of their well-controlled and well-characterized patchy nature, distinguish themselves from multicomponent brushes or brushes used to end-tether peptide sequences and nucleotides.     

Peptide-mediated nanoengineering of inorganic particle surfaces: a general route toward surface functionalization via peptide adhesion domains

The peptide-mediated functionalization of inorganic particle surfaces is demonstrated on gadolinium oxide (GdO) particles, revealing specific means to functionalize nano- or microparticles. Phage display screening is exploited to select 12mer peptides, which exhibit sequence-specific adhesion onto surfaces of GdO particles. These peptide adhesion domains are exploited to effectively decorate GdO particles with fluorescently labeled poly(ethylene oxide) (PEO), proving to result in a stable surface modification as shown by significant reduction of protein adsorption by 80%, compared to nonfunctionalized particles. Peptide adhesion and stability of the noncovalent coating are investigated by adsorption/elution experiments and Langmuir isotherms. Fluorescence microscopy, contact angle, and energy dispersive X-ray (EDX) measurements confirmed the sequence specificity of the interactions by comparing adhesion sequences with scrambled peptide sequences. Noncovalent, but specific modification of inorganic particle surfaces represents a generic strategy to modulate functionality and function of nano- or microparticle surfaces.     

A facile pathway to prepare enzymatically degradable microcapsules with tunable capsule shell properties

Dextran sulfate (DS)/poly-l-lysine (PLL) microcapsules are fabricated by an in situ coacervation method using DS-doped CaCO₃ microparticles as templates. Twinned superstructures or spherical CaCO₃ microparticles are produced depending on DS concentration in the starting solution. DS/PLL microcapsules with ellipsoidal or spherical outline are obtained after removal of templates in disodium ethylene diamine tetraacetate dehydrate (EDTA) without PLL. Their shell thickness and negative surface charges increase with the DS weight percentage in the templates. The surface potential of DS/PLL microcapsules, fabricated by core removal in an EDTA/PLL solution, can be easily tuned by altering PLL concentration in template removal solution. DS/PLL microcapsules fabricated by template removal in solution with or without PLL are both degraded by α-chymotrypsin, and different degradation profiles are observed because of shell thickness differences. DS/PLL may be used as transport vehicles for various compounds regardless of their charge sign in biomedical fields.     

Synthesis of magnetite nanoparticles for bio- and nanotechnology: genetic engineering and biomimetics of bacterial magnetosomes

Magnetotactic bacteria (MTB) have the ability to navigate along the Earth's magnetic field. This so-called magnetotaxis is a result of the presence of magnetosomes, organelles which comprise nanometer-sized intracellular crystals of magnetite (Fe(3)O(4)) enveloped by a membrane. Because of their unique characteristics, magnetosomes have a high potential for nano- and biotechnological applications, which require a specifically designed particle surface. The functionalization of magnetosomes is possible either by chemical modification of purified particles or by genetic engineering of magnetosome membrane proteins. The second approach is potentially superior to chemical approaches as a large variety of biological functions such as protein tags, fluorophores, and enzymes may be directly incorporated in a site-specific manner during magnetosome biomineralization. An alternative to the bacterial production of magnetosomes are biomimetic approaches, which aim to mimic the bacterial biomineralization pathway in vitro. In MTB a number of magnetosome proteins with putative functions in the biomineralization of the nanoparticles have been identified by genetic and biochemical approaches. The initial results obtained by several groups indicate that some of these proteins have an impact on nanomagnetite properties in vitro. In this article the key features of magnetosomes are discussed, an overview of their potential applications are given, and different strategies are proposed for the functionalization of magnetosome particles and for the biomimetism of their biomineralization pathway.     

Reversible Diels–Alder Reactions with a Fluorescent Dye on the Surface of Magnetite Nanoparticles

Diels–Alder reactions on the surface of nanoparticles allow a thermoreversible functionalization of the nanosized building blocks. We report the synthesis of well-defined magnetite nanoparticles by thermal decomposition reaction and their functionalization with maleimide groups. Attachment of these dienophiles was realized by the synthesis of organophosphonate coupling agents and a partial ligand exchange of the original carboxylic acid groups. The functionalized iron oxide particles allow a covalent surface attachment of a furfuryl-functionalized rhodamine B dye by a Diels–Alder reaction at 60 °C. The resulting particles showed the typical fluorescence of rhodamine B. The dye can be cleaved off the particle surface by a retro-Diels–Alder reaction. The study showed that organic functions can be thermoreversibly attached onto inorganic nanoparticles.     

Differences in electrophoretic behavior between linear and branched PEG‐conjugated proteins

The objective of this study was to characterize the differences in electrophoretic behavior between linear and branched PEG‐conjugated proteins. Human growth hormone and alpha‐lactalbumin modified by linear or branched PEGs with molecular weight of 10 kDa were analyzed by SEC, MALDI‐TOF MS, SDS‐PAGE, and microchip CGE (MCGE). Chromatographic and mass spectrometric differences between the linear and branched PEG‐proteins on SEC and MALDI‐TOF MS were small, but their electrophoretic behaviors on SDS‐PAGE and MCGE were significantly different. In particular, MCGE showed significant differences in the peak width and the migration times of linear and branched PEG‐proteins, in which the branched PEG‐proteins exhibited a narrower peak and longer migration time than the linear PEG‐proteins. This phenomenon may explain the longer circulation half‐life for the branched PEG‐proteins observed in previously reported in vivo studies. Consequently, this study indicates that MCGE may be a valuable tool for differentiating linear and branched PEG‐proteins.