High-efficacy subcellular micropatterning of proteins using fibrinogen anchors

Protein micropatterning allows proteins to be precisely deposited onto a substrate of choice and is now routinely used in cell biology and in vitro reconstitution. However, drawbacks of current technology are that micropatterning efficiency can be variable between proteins and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard-to-micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology.     

Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine

NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.     

Surface modification of silicon and gold-patterned silicon surfaces for improved biocompatibility and cell patterning selectivity

Clean silicon and gold-patterned silicon platforms were modified with methoxy-polyethylene glycol (M-PEG silane) via a self-assembly technique, which significantly improved their plasma protein resistance capability and cell patterning selectivity. Fibrinogen and IgG were used as model plasma proteins to study the efficacy of PEG layers in resisting protein adsorption. Selective cell patterning on the gold regions of a gold-patterned silicon substrate and tissue compatibility were studied with macrophage and fibroblast cells. The research also revealed how the presence of gold electrodes on a silicon substrate would influence the cell patterning selectivity. Our experimental results showed that the PEG-modified silicon surfaces had a high resistivity to protein and cell attachment and that the PEG-modified gold-patterned silicon surfaces nearly completely eliminated the protein adsorption and cell attachment on silicon. This study provides a new approach to developing biocompatible surfaces for silicon-based BioMEMS devices, particularly for biosensors where a metal-insulator format must be enforced.     

Bulk and micropatterned conjugation of extracellular matrix proteins to characterized polyacrylamide substrates for cell mechanotransduction assays

Increasing numbers of cell mechanotransduction studies are currently utilizing elastic substrates fabricated from polyacrylamide in the form of thin gels. Their versatility depends on the ability to ensure the appropriate gel stiffness and control the uniformity and geometry of extracellular matrix protein coating of the gel. Beginning with a brief quantitative emphasis on the elastic properties of polyacrylamide gels, we present an inexpensive and highly reproducible method for uniform coating with a wide variety of extracellular matrix proteins. We used a reducing agent, hydrazine hydrate, to modify nonreactive amide groups in polyacrylamide to highly reactive hydrazide groups that can form covalent bonds with aldehyde or ketone groups in oxidized proteins. This simple conjugation method overcomes the limitations of previously used photoactivatable cross-linkers: nonuniform coating due to nonuniformity of irradiation and technically challenging procedures for micropatterning. As demonstrated in our study of cell polarity during constrained migration, this conjugation method is especially effective in gel micropatterning by manual microcontact printing of protein patterns as small as 5 microm and enables numerous studies of constrained cell attachment and migration that were previously unfeasible due to high cost or difficulty in controlling the protein coating.     

Patterning of diverse mammalian cell types in serum free medium with photoablation

Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer‐by‐layer self‐assembly technique and photolithography offer a simple, versatile, and silicon compatible approach that overcomes chemical surface patterning limitations, such as short‐term stability and low‐protein adsorption resistance. In this study, direct photolithographic patterning of two types of multilayers, PAA (poly acrylic acid)/PAAm (poly acryl amide) and PAA/PAH (poly allyl amine hydrochloride), were developed to pattern mammalian neuronal, skeletal, and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a cytophobic surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cell‐selective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108‐15 cells) to a greater extent, while providing little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or in the medium for attachment or differentiation, and could not be patterned using the traditional methods. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cellular,particle and environmental parameters influencing attachment in surface waters: a review

Effective modelling of the fate and transport of water‐borne pathogens is needed to support federally required pollution‐reduction plans, for water quality improvement planning, and to protect public health. Lack of understanding of microbial–particle interactions in water bodies has sometimes led to the assumption that bacteria move in surface waters not associated with suspended mineral and organic particles, despite a growing body of evidence suggesting otherwise. Limited information exists regarding the factors driving interactions between micro‐organisms and particles in surface waters. This review discusses cellular, particle and environmental factors potentially influencing interactions and in‐stream transport. Bacterial attachment in the aquatic environment can be influenced by properties of the cell such as genetic predisposition and physiological state, surface structures such as flagella and fimbriae, the hydrophobicity and electrostatic charge of the cell surface, and the presence of outer‐membrane proteins and extracellular polymeric substances. The mechanisms and degree of attachment are also affected by characteristics of mineral and organic particles including the size, surface area, charge and hydrophobicity. Environmental conditions such as the solution chemistry and temperature are also known to play an important role. Just as the size and surface of chemical particles can be highly variable, bacterial attachment mechanisms are also diverse.     

Easy and robust micropatterning using fibrinogen anchors

Micropatterning is a process to precisely deposit molecules, typically proteins, onto a substrate of choice with micrometer resolution. Watson et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202009063) describe an innovative yet accessible strategy to enable the reproducible micropatterning of virtually any protein while maintaining its biological activity.

Micropatterning involves depositing molecules, primarily proteins, in a precise pattern on a defined substrate. Since the first applications of micropatterning in biology in the 1970s (1), this technique has become increasingly adopted and led to a variety of important biological discoveries related to the impact and mechanisms of cellular architecture and subcellular cytoskeletal components on cell function (2, 3, 4). The basis of most micropatterning strategies has remained the same for decades: a substrate is coated with a specific, often micron scale, pattern of protein within a background of an anti-fouling agent. This can be achieved in two ways: the substrate is coated with the anti-fouling agent, which is then locally removed in a precisely defined pattern using light projection through a mask, and protein is then deposited in regions free of the anti-fouling agent. Alternatively, protein can be directly printed onto the substrate, for example using an elastomeric stamp, or by photochemical linking via patterned light, and then unpatterned substrate regions are backfilled with the anti-fouling agent. Protein patterns can then be used to control the architecture of whole cells or the organization of subcellular components.While micropatterning is potentially a very useful methodology for a number of applications, widespread adoption of this method was initially limited by the need for access to specialized photolithography equipment to generate either the stamps or to directly pattern the anti-fouling layers. Further, since the masks used to define the stamp or anti-fouling layer patterns are pattern specific, a new mask must be produced for every pattern alteration. Early micropatterning methods also were not ideal for creating patterns containing multiple proteins, as sequential protein stamping or sequential anti-fouling coating removal required tedious and time-consuming stamp or mask alignment to ensure accurate patterning. To overcome these two limitations, Strale and colleagues developed a micropatterning technique called LIMAP (light-induced molecular absorption), in which a UV laser within a microscope is used to photoexcise patterns in the anti-fouling layer without the need for a mask. Further the use of the microscope enables direct visualization of protein patterns and hence easy alignment during multiprotein patterning (5). Despite the increased accessibility achieved by this advance, a number of other challenges still limit the widespread application of LIMAP for micropatterning (Fig. 1). First, pattern robustness remains a challenge, as a proportion of protein attaches outside of the pattern in the anti-fouling region (patterns lacks specificity) and protein distribution on the pattern is not even (pattern lacks homogeneity). Second, maintaining the biological activity of the proteins printed can be a challenge, as LIMAP does not enable control over protein-substrate interactions and the orientation of the deposited protein on the substrate, which can result in inaccessibility of the protein’s biologically active domains. This second limitation in particular dramatically limits the types of protein that can be printed and hence cell types and biological questions that can be explored using the LIMAP technique.Open in a separate windowFigure 1.Overview of fibrinogen anchor micropatterning strategy and advantages over existing patterning techniques.In this issue, Watson and colleagues (6) present a new approach that overcomes these limitations by using fibrinogen anchors, functionalized to bind to any proteins of interest, to enable robust micropatterning of almost any combination of proteins. Specifically, as a first step, LIMAP was used to photopattern fibrinogen molecules conjugated to a specific binding target. In a second step, the protein of interest displaying the corresponding binding partner was then added, under appropriate buffer conditions to maintain protein activity, and bound preferentially to the fibrinogen anchors (Fig. 1). The authors describe generation of a number of conjugates, based on modification of fibrinogen-exposed amines, that enable application of the method to a number of proteins, including fibrinogen–Con A, a lectin that binds to insect cells, fibrinogen-NeutrAvidin to bind to biotinylated targets, fibrinogen-GBP to bind to GFP-tagged proteins (GBP stands for GFP-binding peptide, a nanobody against GFP), and fibrinogen-biotin to bind to streptavidin/NeutrAvidin fusions, as well as to biotinylated targets using a NeutrAvidin sandwich (fibrinogen-biotin::NeutrAvidin::biotinylated protein of interest). Of note, the simplicity of this conjugation process will enable greater adoption of this approach by the biology community.Fibrinogen was selected as the anchor molecule as its properties are such that homogeneous and specific patterning can be routinely achieved using LIMAP. By binding other proteins to the fibrinogen anchor, the authors demonstrate their method can produce micropatterns of the anchor bound with the same high specificity (proteins only pattern where they should) and high homogeneity (protein distribution within the pattern is less variable). Further, the authors take advantage of the library of different fibrinogen conjugates to generate patterns containing three different proteins that bind to three different sequentially patterned fibrinogen conjugates. While the improvements in selectivity and homogeneity achieved by Watson et al. are alone a significant step forward, the most exciting demonstrations in their study is that their method enables maintenance of the biological activity of printed proteins in a number of different contexts (6). For example, the authors pattern proteins to perform a microtubule gliding assay and show that printed kinesin motors maintain their ability to move microtubules with the expected gliding speed and dynamics. In another example, the authors exploit their ability to pattern multiple proteins at the subcellular scale and demonstrate that patterns of EGF can induce relocalization of the corresponding EGFR receptor only to regions of a cell attached to EGF-patterned regions on the substrate.The simplicity of the described approach and compatibility with different proteins make it attractive to explore a broad range of problems in the future. At the multicellular scale, this approach could be applied to understand localized cell–cell interactions during collective phenomena such as organoid patterning and models of early human development (7), cell competition dynamics (8), or collective cell migration (9). In particular, applying sequential patterning to create heterogeneous signaling within a cellular island would be interesting to explore developmental patterning circuits. At the single-cell scale, this approach could be useful for understanding heterogeneity in cell populations, although the patterning throughput achievable using microscope “writing” could limit the number cells that can be assessed for such applications. Integration of this anchoring approach with higher throughput mask-based micropatterning methods could address this, however. Perhaps most excitingly, this approach offers a powerful strategy to broadly control subcellular features in a cell. For example, this opens the possibility to probe which downstream networks are activated upon receptor engagement and to control the spatial locations of signaling components within a cell (10). This clever addition to the micropatterning tool kit significantly expands the types of problems that micropatterning can be used to explore while maintaining the accessibility of the technique to the biological community.     

Electrophoretic analysis of substrate-attached proteins from normal and virus-transformed cells.

The proteins which have been left tightly bound to the tissue culture substrate after ethylenebis (oxyethyl-enenitrilo) tetraacetic acid (EGTA)-mediated removal of normal, virus-transformed, and revertant mouse cells and which have been implicated in the substrate adhesion process have been analyzed by slab sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three size classes of hyaluronate proteoglycans were resolved in the 5% well gel; approximately half of the protein in the substrate-attached material coelectrophoresed with these polysaccharides-so-called glycosaminoglycan-associated protein(GAP). A portion of the GAP was shown to be highly heterogeneous and displaced from the polysaccharide by preincubation with calf histone before electrophoresis. The relative proportions of the proteoglycans varied in material deposited during a variety of cellular attachment and growth conditions. The remainder of the cellular protein in substrate-attached material was resolved as several major and distinct protein bands in 8 or 20% separating gels (a limited number of distinct serum proteins have also been identified as substrate bound). Protein C0 (molecular weight 220 000) was a prominent component in the material from a variety of normal and virus-transformed cells and resembled the so-called LETS or CSP glycoprotein in several respects; protein Ca was myosin-like in several respects; protein C2 was shown to be actin; and protein C1 (molecular weight 56 000) does not appear to be tubulin. Histones were also present in most preparations of substrate-attached material, particularly at high levels in transformed cell meterial, and may result from EGTA-mediated leakiness of the cell and subsequent binding to the negatively charged polysaccharide. These substrate-attached proteins were (a) prominent in substrate-attached material from many cell types in characteristic relative proportions, (b) deposited by EGTA-subcultured cells during the first hour of attachment to fresh substrate, (c) deposited by cells growing on plastic or glass substrates (three additional) components were also prominent in glass-attached material), and (d) deposited during long-term growth on or initial attachment to substrates coated wit 3T3 substrate-attached material. Pulse-chase analyses with radioactive leucine indicated that these proteins exhibit different turn-over behaviors. These results are discussed with regard to the possible involvement of these substrate-attached proteins in the substrate adhesion process, with particular interest in the interaction of cytoskeletal microfilaments with other surface membrane components and with regard to alteration of substrate adhesion by virus transformation.     

Nanoparticle-assisted micropatterning of active proteins on solid substrate

Micropatterning of proteins on silica substrate was achieved using a new method. Proteins were first immobilized onto silica nanoparticles which were then dispensed into arrayed microwells on silicon. Atomic force microscopy (AFM), fluorescence microscopy and Fourier transform infrared (FTIR) spectroscopy were used to characterize the samples. The results showed that, compared to a planar surface, curved surfaces of nanoparticles provide more space for attaching proteins and thus increases the intensity of fluorescence signal. Furthermore, after attaching to silica nanoparticles, bovine serum albumin (BSA) maintains its major structure and the cytokine IFN-gamma maintains its ability to bind to its antibody. Use of this method can be extended to micropatterning of other biomolecules, such as DNA and enzymes.     

微图形化技术及其在生物医学研究中的应用

结合生物物理学与生物化学的微细加工技术已可以获得与生物大分子相近的特征尺寸,推动了微图形化技术在药物筛选与新药开发、组织工程、疾病诊断等领域的应用．综述了微图形化技术在生物医学领域的发展,讨论了光刻、软光刻、模板辅助构图、扫描探针加工、喷墨构图、激光诱导图形化等方法,分析了各种方法的优势、局限性与适用范围,指出分辨力与精度、图形化规模、实验加工条件等是选择不同图形化方法的主要依据．而基于生物物理学和生物化学等对纳米尺度的处理过程进行定量分析、进一步提高其生物兼容性及材料适应性、发展适合图形化芯片的体内微环境模拟技术等是微图形化技术进一步发展的方向．     

Effect of magnetic nanoparticles Fe3O4 on viability, attachment, and spreading of isolated fetuses and newborn rats

This study has shown that the toxic effect of nonmodified Fe₃O₄ nanoparticles in vitro depends on the metabolic and morphological conditions of cells from rat fetuses and newborns. In the process of cultivation, cells with magnetic nanoparticles bind to their surface and penetrate the intracellular space. The sorption of nanoparticles on the cell surface hinders their attachment to the substrate and absorption by spread cells can prevent their proliferation. Magnetic nanoparticles are well sorbed by the upper layer of cell aggregates, whereas cells of the inner layer remain intact. As a result, the cell aggregates acquire the property of responding to a constant magnetic field. These aggregates could potentially be used in cell transplantation for directed cell delivery.     

In vitro studies with mammalian cell lines and gum arabic-coated magnetic nanoparticles

Iron oxide magnetic nanoparticles (MNPs) were synthesized by the chemical co-precipitation method and coated with gum arabic (GA) by physical adsorption and covalent attachment. Cultures of mammalian cell lines (HEK293, CHO and TE671) were grown in the presence of uncoated and GA-coated MNPs. Cellular growth was followed by optical microscopy in order to assess the proportion of cells with particles, alterations in cellular density and the presence of debris. The in vitro assays demonstrated that cells from different origins are affected differently by the presence of the nanoparticles. Also, the methods followed for GA coating of MNPs endow distinct surface characteristics that probably underlie the observed differences when in contact with the cells. In general, the nanoparticles to which the GA was adsorbed had a smaller ability to attach to the cells' surface and to compromise the viability of the cultures.     

Regulation of the ubiquitin-mediated proteolytic pathway: role of the substrate alpha-NH2 group and of transfer RNA

Degradation of intracellular proteins via the ubiquitin pathway involves several steps. In the initial event, ubiquitin becomes covalently linked to the protein substrate in an ATP-requiring reaction. Following ubiquitin conjugation, the protein moiety of the adduct is selectively degraded with the release of free and reusable ubiquitin. Ubiquitin modification of a variety of protein targets in the cell plays a role in basic cellular functions. Modification of core nucleosomal histones is probably involved in regulation of gene expression at the level of chromatin structure. Ubiquitin attachment to cell surface proteins may play roles in processes of cell-cell interaction and adhesion, and conjugation of ubiquitin to other yet to be identified protein(s) could be involved in the progression of cells through the cell cycle. Despite the considerable progress that has been made in the elucidation of the mode of action and cellular roles of the ubiquitin pathway, many major problems remain unsolved. A problem f central importance is the specificity in the ubiquitin ligation system. Why are certain proteins conjugated and committed for degradation, whereas other proteins are not? A free α-NH₂ group is an important feature of the protein structure recognized by the ubiquitin conjugation system, and tRNA is required for the conjugation of ubiquitin to selective proteo-lytic substrates and for their subsequent degradation. These findings can shed light on some of the features of a substrate that render it susceptile to ubiquitin-mediated degradation.     

Biomedical applications of electrostatic layer-by-layer nano-assembly of polymers,enzymes, and nanoparticles

The introduction of electrostatic layer-by-layer (LbL) self-assembly has shown broad biomedical applications in thin film coating, micropatterning, nanobioreactors, artificial cells, and drug delivery systems. Multiple assembly polyelectrolytes and proteins are based on electrostatic interaction between oppositely charged layers. The film architecture is precisely designed and can be controlled to 1-nm precision with a range from 5 to 1000 nm. Thin films can be deposited on any surface including many widely used biomaterials. Microencapsulation of micro/nanotemplates with multilayers enabled cell surface modification, controlled drug release, hollow shell formation, and nanobioreactors. Both in vitro and in vivo studies indicate potential applications in biology, pharmaceutics, medicine, and other biomedical areas.     

Receptor tyrosine kinases.

A major process through which environmental information is transmitted into cells is via activation of protein tyrosine kinases. Receptor tyrosine kinases contain extracellular ligand recognition, single membrane spanning, and cytoplasmic protein tyrosine kinase domains. The cytoplasmic kinase core is flanked by regulatory segments, which in some family members are also inserted into the core kinase domain. Ligand binding initiates receptor signaling from the cell surface. Activated receptors autophosphorylate to remove alternate substrate/inhibitory constraints and to provide loci for assembly of proteins that contain SRC homology regions. Information is transmitted and diffused by tyrosine phosphorylation of the assembled proteins and of cellular substrates that include protein kinases with specificity for serine/threonine residues. Signaling, which is strictly ligand-dependent, is attenuated by down-regulation of receptors and by feed-back inhibitory loops that involve receptor phosphorylation by cellular kinases. The tyrosine kinase receptors are essential for normal growth, development, and reparative processes. Mutations that remove normal regulatory constraints on the approximately 290 amino acid kinase core of these large proteins result in constitutive function and cell transformation.     

Embryonic neural retinal cell response to extracellular matrix proteins: developmental changes and effects of the cell substratum attachment antibody (CSAT)

Cell attachment and neurite outgrowth by embryonic neural retinal cells were measured in separate quantitative assays to define differences in substrate preference and to demonstrate developmentally regulated changes in cellular response to different extracellular matrix glycoproteins. Cells attached to laminin, fibronectin, and collagen IV in a concentration-dependent fashion, though fibronectin was less effective for attachment than the other two substrates. Neurite outgrowth was much more extensive on laminin than on fibronectin or collagen IV. These results suggest that different substrates have distinct effects on neuronal differentiation. Neural retinal cell attachment and neurite outgrowth were inhibited on all three substrates by two antibodies, cell substratum attachment antibody (CSAT) and JG22, which recognize a cell surface glycoprotein complex required for cell interactions with several extracellular matrix constituents. In addition, retinal cells grew neurites on substrates coated with the CSAT antibodies. These results suggest that cell surface molecules recognized by this antibody are directly involved in cell attachment and neurite extension. Neural retinal cells from embryos of different ages varied in their capacity to interact with extracellular matrix substrates. Cells of all ages, embryonic day 6 (E6) to E12, attached to collagen IV and CSAT antibody substrates. In contrast, cell attachment to laminin and fibronectin diminished with increasing embryonic age. Age-dependent differences were found in the profile of proteins precipitated by the CSAT antibody, raising the possibility that modifications of these proteins are responsible for the dramatic changes in substrate preference of retinal cells between E6 and E12.     

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.     

Cellular uptake of Poly-(D,L-lactide-co-glycolide) (PLGA) nanoparticles synthesized through solvent emulsion evaporation and nanoprecipitation method

Poly-(D,L-lactide-co-glycolide) (PLGA) nanoparticles have been widely studied for drug delivery. The aim of this study is to determine how cellular uptake of these nanoparticles is influenced by different surface properties, incubation time, particle concentration and cell types. Spherical coumarin-6 loaded PLGA nanoparticles with a size of about 100 nm were synthesized through solvent emulsion evaporation and nanoprecipitation methods. In vitro cellular uptake efficiency was determined using human bronchial epithelial cells (BEAS-2B) and murine monocyte-derived macrophage (RAW264.7) cells. PLGA nanoparticles were incubated with these cells in a concentration range of 10-300 μg/ml for different time periods. The results show that cellular uptake decreased for nanoparticles surface coated with PVA surfactant and was especially limited for severely aggregated particles. At higher particle concentration, the total amount of particles taken up by cells increased while the uptake efficiency decreased. In addition, cells could take up more particles with longer incubation time, although the uptake rate decreased gradually with time. Finally, RAW264.7 cells show increased uptake compared to BEAS-2B cells. The information drawn from this study would provide important clues on how nanomaterials interact with cells and how these interactions can influence biocompatibility or toxicity.