[Show abstract][Hide abstract] ABSTRACT: We present development and use of a 3D synthetic extracellular matrix (ECM) analog with integrin-specific adhesion ligands to characterize the microenvironmental influences in embryonic stem cell (ESC) self-renewal. Transcriptional analysis of 24 integrin subunits followed by confirmation at the translational and functional levels suggested that integrins alpha(5)beta(1), alpha(v)beta(5), alpha(6)beta(1) and alpha(9)beta(1) play important roles in maintenance of stemness in undifferentiated mouse ESCs. Using the well-defined matrix as a tool to activate integrins alpha(5)beta(1) plus alpha(v)beta(5), alpha(6)beta(1) and alpha(9)beta(1), individually and in combination, differential integrin activation was demonstrated to exert exquisite control over ESC fate decisions. Simultaneous ligation of these four integrin heterodimers promoted self-renewal, as evidence by prolonged SSEA-1, Oct4 and Nanog expression, and induced Akt1 kinase signaling along with translational regulation of other stemness-related genes. The biofunctional network we have designed based on this knowledge may be useful as a defined niche for regulating ESC pluripotency through selective cell-matrix interactions, and the method we present may be more generally useful for probing matrix interactions in stem cell self-renewal and differentiation.
[Show abstract][Hide abstract] ABSTRACT: Semi-synthetic, proteolytically degradable polymer hydrogels have proven effective as scaffolds to augment bone and skin regeneration in animals. However, high costs due to expensive peptide building blocks pose a significant hurdle towards broad clinical usage of these materials. Here we demonstrate that tri-amino acid peptides bearing lysine (or arginine), flanked by two cysteine residues for crosslinking, are adequate as minimal plasmin-sensitive peptides in poly(ethylene glycol)-based hydrogels formed via Michael-type addition. Substitution of lysine (or arginine) with serine rendered the matrices insensitive to the action of plasmin. This was demonstrated in vitro by performing gel degradation experiments in the presence of plasmin (0.1 U/mL), and in the in vivo situation of regeneration of critical-sized bone defects. When placed as implants into rat calvaria, gels formed from the minimal plasmin substrates showed clear signs of cell infiltration and gel remodeling that coincided with extensive bone formation.
Journal of Biomedical Materials Research Part A 09/2009; 93(3):870-7. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We designed block copolymer pro-amphiphiles and amphiphiles for providing very long-term release of nitric oxide (NO). A block copolymer of N-acryloylmorpholine (AM, as a hydrophile) and N-acryloyl-2,5-dimethylpiperazine (AZd, as a hydrophilic precursor) was synthesized. The poly(N-acryloyl-2,5-dimethylpiperazine) (PAZd) is water-soluble, but chemical reaction of the secondary amines with NO to form a N-diazeniumdiolate (NONOate) converts the hydrophilic PAZd into a hydrophobic poly(sodium-1-(N-acryloyl-2,5-dimethylpiperazin-1-yl)diazen-1-ium-1,2-diolate) (PAZd.NONOate), driving aggregation. The PAM block guides this process toward micellization, rather than precipitation, yielding ca. 50 nm spherical micelles. The hydrophobic core of the micelle shielded the NONOate from the presence of water, and thus protons, which are required for NO liberation, delaying release to a remarkable 7 d half-life. Release of the NO returned the original soluble polymer. The very small NO-loaded micelles were able to penetrate complex tissue structures, such as the arterial media, opening up a number of tissue targets to NO-based therapy.
Journal of the American Chemical Society 09/2009; 131(40):14413-8. · 10.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We present a versatile scheme to rationally modulate the hydrolysis rate of ester bonds in hydrophilic polymer networks via adjacent charged amino acids. As soluble model systems, two cysteine-bearing oligopeptides containing either positively charged arginine (GRCRGGRCRG, termed R-linker) or negatively charged aspartic acid (GDCDGGDCDG, termed D-linker) were linked to monomethoxy PEG-acrylate via Michael-type addition, and the hydrolysis rate of the conjugates was monitored using HPLC. A ca. 6-fold difference in hydrolysis kinetics of the conjugates was determined, positively charged arginine leading to an increased hydrolysis rate (t1/2 of 6.56 days vs. 36.1 days for the R- and D-linker containing conjugates, respectively). As a first step towards utilizing this concept to create tunable matrices for drug delivery and tissue engineering, the above peptides were crosslinked into hybrid hydrogels (R-gels and D-gels) by mixing with 4-arm PEG-acrylate at variable stoichiometric ratios. The physicochemical gel properties were characterized and gel degradation kinetics were quantified by monitoring the gel weight change over time at pH 7.4 and 37 °C. Differences in ester hydrolysis rates of individual chains translated into a ca. 12-fold difference in hydrogel degradation rate (R-gels: t1/2 = 7.53 days, D-gels: t1/2 = 86.6 days). Finally, the gel release kinetics of covalently linked bovine serum albumin (BSA) was also shown to be highly dependent on the charge of adjacent amino acids (R-gels: t1/2 = 3.32 days, D-gels: t1/2 = 32.1 days). With the availability of 20 natural amino acids as building blocks to modulate the chemical environment in close proximity of labile esters, we expect this work will provide a generalizable framework for the engineering of hybrid polymer-co-peptide gels with tunable and predictive degradation and drug release properties.
[Show abstract][Hide abstract] ABSTRACT: We present new polymeric amphiphiles derived from N-acryloyl derivatives of the cyclic secondary amines: morpholine, piperidine, and azocane polymerized by reversible addition -fragmentation transfer (RAFT) polymerization. Both homopolymerization and block copolymerization of N-acryloylmorpholine (AM), N-acryloylpiperidine (AP), and N-acryloylazocane (AH) were carried out. The block copolymeric amphiphiles, poly[(N-acryloylmorpholine)-block-(N-acryloylpiperidine)] (PAM-PAP) and poly[(N-acryloylmorpholine)-block-(N-acryloylazocane)] (PAM-PAH) were investigated, PAM being a hydrophile, and PAP and PAH being hydrophobes. Moreover, to compare PAM as a hydrophilic block with poly(ethylene glycol) (PEG), poly[(ethylene glycol)block-(N-acryloylpiperidine)] (PEG-PAP) was also formed. In all cases, the degree of polymerization was well-controlled and polymers were obtained in monomodal distributions. The macroamphiphile aggregates in water were reproducibly well-formed by dialysis with a size range between 10 and 70 nm as characterized by dynamic light scattering (DLS). The morphology of the aggregates was examined by transmission electron microscopy (TEM). All aggregates formed from PAM-PAP and PAM-PAH series, up to 0.76 and 0.85 hydrophobic weight fraction, respectively, revealed spherical micelles, whereas coexistence of spherical micelles and/or polymersomes was observed from PEG-PAP at a hydrophobic weight fraction of 0.91. From study of copolymer segregation behavior, PEG-PAP and PAM-PAH span the weak segregation region (WSR) as well as the strong segregation region (SSR), whereas PAM-PAP is positioned in the WSR, owing to the greater hydrophobicity of PAH than PAP. PAM yielded similar aggregation results to PEG when copolymerized with hydrophobic blocks. As a model drug, everolimus was loaded in PAM(0.15)-PAH(0.85) micelles. After loading the drug, the micelle hydrodynamic diameter was slightly increased from 43 +/- 0.1 to 52 +/- 1.8 nm. Everolimus was encapsulated with 60 +/- 7.8% of efficiency and was released over 3 wk in PBS (pH 7.4, 10 mM) at 37 degrees C.
[Show abstract][Hide abstract] ABSTRACT: In this paper, the positive effect of a gold layer on cell viability is demonstrated by examining the results given by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfop henyl)-2H-tetrazolium (MTS) assay and two-color cell fluorescence viability (TCCV) assay. These cytotoxicity tests were performed with human cervical adenocarcinoma cells (HeLa cell line) and transformed African green monkey kidney fibroblast cells (Cos-7 cell line). To fabricate the nanostructures as drug vehicles, first, poly(l,l-lactide-co-ethylene glycol) (PLLA-PEG) and poly(N-isopropylacrylamide-co-D,D-lactide) (PNIPAAm-PDLA) were synthesized, and then two kinds of thermosensitive nanospheres comprising "shell-in-shell" structures without a gold layer (PLLA-PEG@PNIPAAm-PDLA) and with a gold layer (Au@PLLA-PEG@PNIPAAm-PDLA) were constructed by a modified double-emulsion method (MDEM). Both of them displayed a unique thermosensitive character exhibiting the lower critical solubility temperature (LCST) at 36.7 degrees C which was confirmed by UV-vis spectroscopy and differential scanning calorimetry (DSC). The release profiles of entrapped bovine serum albumin (BSA) were monitored at 22 and 37 degrees C, respectively, to reveal the thermal dependence on the release rate. In cell viability tests, both PLLA-PEG@PNIPAAm-PDLA and Au@PLLA-PEG@PNIPAAm-PDLA showed excellent cell viability, and furthermore, Au@PLLA-PEG@PNIPAAm-PDLA, particularly at high doses, exhibited more enhanced cell viability than PLLA-PEG@PNIPAAm-PDLA. This effect is mainly attributed to the gold layer which binds the protein molecules first and consequently facilitates transmembrane uptake of essential nutrients in the cell media, resulting in favorable cell proliferation.
[Show abstract][Hide abstract] ABSTRACT: A method to construct synchronous delivery systems via direct self-assembly of Au nanoparticles on the poly[(N-isopropylacrylamide-r-acrylamide)-b-L-lactic acid] (PNAL) nanospheres has been presented in this paper. To achieve amphiphilic diblock terpolymer, hydrophobic poly (L-lactic acid) (PLLA) block was added to poly(N-isopropylacrylamide- r-acrylamide) (PNA) block via Michel-type addition reaction. Lower critical solubility temperature (LCST) was modulated at 35.6 degrees C which is close to the body temperature, but higher than poly(N-isopropylacrylamide) (PNIPAAm) homopolymer by controlling the ratio between isopropylacrylamide (IPAAm) monomers and acrylamide (AAm) monomers. Using this amphiphilic diblock terpolymer, PNAL nanospheres were fabricated by emulsion/evaporation technique followed by direct self-assembly of Au nanoparticles on the PNAL nanospheres due to the high affinity of amino groups donated from PNA block. The 'core' site of Au@PNAL nanospheres can load various lyphophilic drugs. Moreover, Au nanoparticles in the 'shell' domain of PNAL nanospheres give optimal environment to conjugate various biomolecules. Therefore, it is expected that Au@PNAL hybrid nanospheres can be utilized in synchronous delivery of both biomolecules in the 'shell' domain and various therapeutic drugs in the 'core' domain.
Journal of Materials Science Materials in Medicine 01/2005; 15(12):1291-5. · 2.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effect of the surface coating on the magnetic properties of superparamagnetic iron oxide nanoparticles (SPION) with 8 nm in size has been studied. Four different biocompatible coating layers are considered: poly L,L-lactic acid (PLLA), poly -caprolactone (PCL), bovine serum albumin (BSA) and gold. The presence of coating layer on the surface of SPION is confirmed by FT-IR spectroscopy. Mössbauer spectroscopy and magnetic susceptibility measurements show that for uncoated SPION and Au@SPION the superparamagnetic fraction is retained. The formation of clusters in the case of BSA@SPION and chain-like structure for PCL@SPION and PLLA@SPION increase the inter-particle interactions resulting in hyperfine magnetic structure observed in the Mössbauer spectra at ambient temperature.