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1.  Influence of Oligospermines Architecture on Their Suitability for siRNA Delivery 
Biomacromolecules  2014;15(4):1299-1310.
Spermines are naturally abundant polyamines that partially condense nucleic acids and exhibit the proton-sponge effect in an acidic environment. However, spermines show a limited efficiency for transfecting nucleic acids because of their low molecular weight. Therefore, spermines need to be modified to be used as nonviral vectors for nucleic acids. Here, we synthesized linear bisspermine as well as a linear and dendritic tetraspermine with different molecular architectures. These oligospermines were self-assembled into polyplexes with siRNA. The structure–activity relationship of the oligospermines was evaluated in terms of their efficiency for delivering siRNA into a nonsmall cell lung carcinoma cell line. Oligospermines displayed minimal cytotoxicity but efficient siRNA condensation and showed better stability against polyanions than polyethylenimine. The morphology of the polyplexes was strongly affected by the oligospermine architecture. Linear tetraspermine/siRNA polyplexes showed the best gene-silencing efficiency among the oligospermines tested at both the mRNA and protein expression levels, indicating the most favorable structure for siRNA delivery.
doi:10.1021/bm401849d
PMCID: PMC3993926  PMID: 24552396
2.  Hydrophobic Drug-Triggered Self-Assembly of Nanoparticles from Silk-Elastin-Like Protein Polymers for Drug Delivery 
Biomacromolecules  2014;15(3):908-914.
Silk-elastin-like protein polymers (SELPs) combine the mechanical and biological properties of silk and elastin. These properties have led to the development of various SELP-based materials for drug delivery. However, SELPs have rarely been developed into nanoparticles, partially due to the complicated fabrication procedures, nor assessed for potential as an anticancer drug delivery system. We have recently constructed a series of SELPs (SE8Y, S2E8Y, and S4E8Y) with various ratios of silk to elastin blocks and described their capacity to form micellar-like nanoparticles upon thermal triggering. In this study, we demonstrate that doxorubicin, a hydrophobic antitumor drug, can efficiently trigger the self-assembly of SE8Y (SELPs with silk to elastin ratio of 1:8) into uniform micellar-like nanoparticles. The drug can be loaded in the SE8Y nanoparticles with an efficiency around 6.5% (65 ng doxorubicin/μg SE8Y), S2E8Y with 6%, and S4E8Y with 4%, respectively. In vitro studies with HeLa cell lines demonstrate that the protein polymers are not cytotoxic (IC50 > 200 μg/mL), while the doxorubicin-loaded SE8Y nanoparticles showed a 1.8-fold higher cytotoxicity than the free drug. Confocal laser scanning microscopy (CLSM) and flow cytometry indicate significant uptake of the SE8Y nanoparticles by the cells and suggest internalization of the nanoparticles through endocytosis. This study provides an all-aqueous, facile method to prepare nanoscale, drug-loaded SELPs packages with potential for tumor cell treatments.
doi:10.1021/bm4017594
PMCID: PMC3983132  PMID: 24527851
3.  Presentation of BMP-2 Mimicking Peptides in 3D Hydrogels Directs Cell Fate Commitment in Osteoblasts and Mesenchymal Stem Cells 
Biomacromolecules  2014;15(2):445-455.
Many strategies for controlling the fate of transplanted stem cells rely on the concurrent delivery of soluble growth factors that have the potential to produce undesirable secondary effects in surrounding tissue. Such off target effects could be eliminated by locally presenting growth factor peptide mimics from biomaterial scaffolds to control stem cell fate. Peptide mimics of bone morphogenetic protein 2 (BMP-2) were synthesized by solid phase Fmoc-peptide synthesis and covalently bound to alginate hydrogels via either carbodiimide or sulfhydryl-based coupling strategies. Successful peptide conjugation was confirmed by 1H-NMR spectroscopy and quantified by fluorescently labeling the peptides. Peptides derived from the knuckle epitope of BMP-2, presented from both 2D surfaces and 3D alginate hydrogels, were shown to increase alkaline phosphatase activity in clonally derived murine osteoblasts. Furthermore, when presented in 3D hydrogels, these peptides were shown to initiate Smad signaling, upregulate osteopontin production, and increase mineral deposition with clonally derived murine mesenchymal stem cells. These data suggest that these peptide-conjugated hydrogels may be effective alternatives to local BMP-2 release in directly and spatially eliciting osteogenesis from transplanted or host osteoprogenitors in the future.
doi:10.1021/bm401726u
PMCID: PMC3930060  PMID: 24400664
bone morphogenetic protein; peptide mimic; mesenchymal stem cell; osteogenesis
4.  Deconstructing a Plant Macromolecular Assembly: Chemical Architecture, Molecular Flexibility, And Mechanical Performance of Natural and Engineered Potato Suberins 
Biomacromolecules  2014;15(3):799-811.
Periderms present in plant barks are essential protective barriers to water diffusion, mechanical breakdown, and pathogenic invasion. They consist of densely packed layers of dead cells with cell walls that are embedded with suberin. Understanding the interplay of molecular structure, dynamics, and biomechanics in these cell wall-associated insoluble amorphous polymeric assemblies presents substantial investigative challenges. We report solid-state NMR coordinated with FT-IR and tensile strength measurements for periderms from native and wound-healing potatoes and from potatoes with genetically modified suberins. The analyses include the intact suberin aromatic–aliphatic polymer and cell-wall polysaccharides, previously reported soluble depolymerized transmethylation products, and undegraded residues including suberan. Wound-healing suberized potato cell walls, which are 2 orders of magnitude more permeable to water than native periderms, display a strikingly enhanced hydrophilic–hydrophobic balance, a degradation-resistant aromatic domain, and flexibility suggestive of an altered supramolecular organization in the periderm. Suppression of ferulate ester formation in suberin and associated wax remodels the periderm with more flexible aliphatic chains and abundant aromatic constituents that can resist transesterification, attenuates cooperative hydroxyfatty acid motions, and produces a mechanically compromised and highly water-permeable periderm.
doi:10.1021/bm401620d
PMCID: PMC3983150  PMID: 24502663
5.  [No title available] 
PMCID: PMC3983133  PMID: 24491174
6.  Influence of Polyelectrolyte Film Stiffness on Bacterial Growth 
Biomacromolecules  2013;14(2):520-528.
Photo-crosslinkable polyelectrolyte films whose nanomechanical properties can be varied under UV light illumination, were prepared from poly(L-lysine) (PLL) and a hyaluronan derivative modified with photoreactive vinylbenzyl groups (HAVB). The adhesion and the growth of two model bacteria, namely Escherichia coli and Lactococcus lactis, were studied on non-crosslinked and crosslinked films to investigate how the film stiffness influences the bacterial behavior. While the Gram positive L. lactis was shown to grow slowly on both films, independently of their rigidity, the Gram negative E. coli exhibited a more rapid growth on non-crosslinked softer films compared to the stiffer ones. Experiments performed on photo-patterned films showing both soft and stiff regions, confirmed a faster development of E. coli colonies on softer regions. Interestingly, this behavior is opposite to the one reported before for mammalian cells. Therefore, the photo-crosslinked (PLL/HAVB) films are interesting coatings for tissue engineering since they promote the growth of mammalian cells while limiting the bacterial colonization.
doi:10.1021/bm301774a
PMCID: PMC4312771  PMID: 23289403
Biofilm; Layer-by-layer assembly; Escherichia coli; Lactococcus lactis; bacterial adhesion; hyaluronic acid
7.  Synthesis of 2-Hydroxypropyl-β-cyclodextrin/Pluronic-Based Polyrotaxanes via Heterogeneous Reaction as Potential Niemann-Pick Type C Therapeutics 
Biomacromolecules  2013;14(12):4189-4197.
Five polyrotaxanes were synthesized by threading 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) onto a variety of α,ω-ditriethylenediamino-N-carbamoyl-poly-(ethylene oxide)-block-poly(propylene oxide)-block-poly-(ethylene oxide) (Pluronic) triblock copolymers using a two-pot strategy under heterogeneous, nonaqueous conditions. The threaded HP-β-CD units were retained on the pseudopolyrotaxane precursors by end-capping the branched diamine termini with sodium 2,4,6-trinitrobenzene sulfonate. Inclusion of the Pluronic copolymers within the HP-β-CD cavities was more favorable in nonpolar solvents, such as diethyl ether and n-hexane, both of which gave better coverage ratios than polar solvents. 1H NMR and MALDI-TOF were used to estimate the average molecular weights of the purified polyrotaxane products. A globular morphology of aggregated polyrotaxanes was observed by tapping-mode AFM imaging of dried samples. Treatment of Niemann-Pick C (NPC) type 2-deficient fibroblasts with the polyrotaxane derivatives produced substantial reductions in sterol accumulation, as seen by diminished filipin staining in these cells, suggesting that Pluronic-based polyrotaxanes may be promising vehicles for delivery of HP-β-CD to cells with abnormal cholesterol accumulation.
doi:10.1021/bm400922a
PMCID: PMC4314287  PMID: 24180231
8.  Toroidal-Spiral Particles for Codelivery of Anti-VEGFR-2 Antibody and Irinotecan: A Potential Implant to Hinder Recurrence of Glioblastoma Multiforme 
Biomacromolecules  2014;15(3):756-762.
Heterogeneous toroidal-spiral particles (TSPs) were generated by polymer droplet sedimentation, interaction, and cross-linking. TSPs provide a platform for encapsulation and release of multiple compounds of different sizes and physicochemical properties. As a model system, we demonstrate the encapsulation and independently controlled release of an anti-VEGFR-2 antibody and irinotecan for the treatment of glioblastoma multiforme. The anti-VEGFR-2 antibody was released from the TS channels and its binding to HUVECs was confirmed by confocal microscopy and flow cytometry, suggesting active antibody encapsulation and release. Irinotecan, a small molecule drug, was released from the dense polymer matrix of poly(ethylene glycol) diacrylate (MW ∼ 700 g/mol; PEGDA 700). Released irinotecan inhibited the proliferation of U251 malignant glioma cells. Since the therapeutic compounds are released through different pathways, specifically diffusion through the polymer matrix versus TS channels, the release rate can be controlled independently through the design of the structure and material of particle components.
doi:10.1021/bm401550r
PMCID: PMC3983134  PMID: 24460101
9.  Enzymatic Surface Erosion of High Tensile Strength Polycarbonates Based on Natural Phenols 
Biomacromolecules  2014;15(3):830-836.
Surface erosion has been recognized as a valuable design tool for resorbable biomaterials within the context of drug delivery devices, surface coatings, and when precise control of strength retention is critical. Here we report on high tensile strength, aromatic–aliphatic polycarbonates based on natural phenols, tyrosol (Ty) and homovanillyl alcohol (Hva), that exhibit enzymatic surface erosion by lipase. The Young’s moduli of the polymers for dry and fully hydrated samples are 1.0 to 1.2 GPa and 0.8 to 1.2 GPa, respectively. Typical characteristics of enzymatic surface erosion were confirmed for poly(tyrosol carbonate) films with concomitant mass-loss and thickness-loss at linear rates of 0.14 ± 0.01 mg cm–2 d–1 and 3.0 ± 0.8 μm d–1, respectively. The molecular weight and the mechanical properties of the residual films remained constant. Changing the ratio of Ty and Hva provided control over the glass transition temperature (Tg) and the enzymatic surface erosion: increasing the Hva content in the polymers resulted in higher Tg and lower enzymatic erosion rate. Polymers with more than 50 mol % Hva were stable at 37 °C in enzyme solution. Analysis on thin films using quartz crystal microbalance with dissipation (QCM-D) demonstrated that the onset temperature of the enzymatic erosion was approximately 20 °C lower than the wet Tg for all tested polymers. This new finding demonstrates that relatively high tensile strength polycarbonates can undergo enzymatic surface erosion. Moreover, it also sheds light on the connection between Tg and enzymatic degradation and explains why few of the high strength polymers follow an enzyme-meditated degradation pathway.
doi:10.1021/bm4016539
PMCID: PMC3983148  PMID: 24432806
10.  [No title available] 
PMCID: PMC4295786  PMID: 24164501
11.  High Antimicrobial Effectiveness with Low Hemolytic and Cytotoxic Activity for PEG/Quaternary Copolyoxetanes 
Biomacromolecules  2014;15(2):456-467.
The alkyl chain length of quaternary ammonium/PEG copolyoxetanes has been varied to discern effects on solution antimicrobial efficacy, hemolytic activity and cytotoxicity. Monomers 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx) and 3-((2-(2-methoxyethoxy)ethoxy)methyl)-3-methyloxetane (ME2Ox) were used to prepare precursor P[(BBOx)(ME2Ox)-50:50–4 kDa] copolyoxetane via cationic ring opening polymerization. The 1:1 copolymer composition and Mn (4 kDa) were confirmed by 1H NMR spectroscopy. After C–Br substitution by a series of tertiary amines, ionic liquid Cx-50 copolyoxetanes were obtained, where 50 is the mole percent of quaternary repeat units and “x” is quaternary alkyl chain length (2, 6, 8, 10, 12, 14, or 16 carbons). Modulated differential scanning calorimetry (MDSC) studies showed Tgs between −40 and −60 °C and melting endotherms for C14–50 and C16–50. Minimum inhibitory concentrations (MIC) were determined for Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. A systematic dependence of MIC on alkyl chain length was found. The most effective antimicrobials were in the C6–50 to C12–50 range. C8–50 had better overall performance with MICs of 4 μg/mL, E. coli; 2 μg/mL, S. aureus; and 24 μg/mL, P. aeruginosa. At 5 × MIC, C8–50 effected >99% kill in 1 h against S. aureus, E. coli, and P. aeruginosa challenges of 108 cfu/mL; log reductions (1 h) were 7, 3, and 5, respectively. To provide additional insight into polycation interactions with bacterial membranes, a geometric model based on the dimensions of E. coli is described that provides an estimate of the maximum number of polycations that can chemisorb. Chain dimensions were estimated for polycation C8–50 with a molecular weight of 5 kDa. Considering the approximations for polycation chemisorption (PCC), it is surprising that a calculation based on geometric considerations gives a C8–50 concentration within a factor of 2 of the MIC, 4.0 (±1.2) μg/mL for E. coli. Cx-50 copolyoxetane cytotoxicity was low for human red blood cells, human dermal fibroblasts (HDF), and human foreskin fibroblasts (HFF). Selectivities for bacterial kill over cell lysis were among the highest ever reported for polycations indicating good prospects for biocompatibility.
doi:10.1021/bm401794p
PMCID: PMC3998775  PMID: 24422429
12.  Antigen delivery by lipid-enveloped PLGA microparticle vaccines mediated by in situ vesicle shedding 
Biomacromolecules  2014;15(7):2475-2481.
Lipid-coated poly(lactide-co-glycolide) microparticles (LCMPs) consist of a solid polymer core wrapped by a surface lipid bilayer. Previous studies demonstrated that immunization with LCMPs surface-decorated with nanograms of antigen elicit potent humoral immune responses in mice. However, the mechanism of action for these vaccines remained unclear, as LCMPs are too large to drain efficiently to lymph nodes from the vaccination site. Here we characterized the stability of the lipid envelope of LCMPs and discovered that in the presence of serum, the lipid coating of the particles spontaneously delaminates, shedding antigen-displaying vesicles. Lipid delamination generated 180 nm liposomes in a temperature- and lipid/serum-dependent manner. Vesicle shedding was restricted by inclusion of high-TM lipids or cholesterol in the LCMP coating. Administration of LCMPs bearing stabilized lipid envelopes generated weaker antibody responses than shedding-competent LCMPs, suggesting that in situ release of antigen-loaded vesicles plays a key role in the remarkable potency of LCMPs as vaccine adjuvants.
doi:10.1021/bm500337r
PMCID: PMC4115588  PMID: 24894061
Vaccine; Adjuvant; Microparticle; Liposomes; Lipid membrane
13.  Nanofibers Support Oligodendrocyte Precursor Cell Growth and Function as a Neuron-Free Model for Myelination Study 
Biomacromolecules  2013;15(1):319-326.
Nanofiber-based scaffolds may simultaneously provide immediate contact guidance for neural regeneration and act as a vehicle for therapeutic cell delivery to enhance axonal myelination. Additionally, nanofibers can serve as a neuron-free model to study myelination of oligodendrocytes. In this study, we fabricated nanofibers using a polycaprolactone and gelatin co-polymer. The ratio of the gelatin component in the fibers was confirmed by energy dispersive x-ray spectroscopy. The addition of gelatin to the polycaprolactone (PCL) for nanofiber fabrication decreased the contact angle of the electrospun fibers. We showed that both polycaprolactone nanofibers as well as polycaprolactone and gelatin co-polymer nanofibers can support oligodendrocyte precursor cell (OPC) growth and differentiation. OPCs maintained their phenotype and viability on nanofibers and were induced to differentiate into oligodendrocytes. The differentiated oligodendrocytes extend their processes along the nanofibers and ensheathed the nanofibers. Oligodendrocytes formed significantly more myelinated segments on the PCL and gelatin co3polymer nanofibers than those on PCL nanofibers alone.
doi:10.1021/bm401558c
PMCID: PMC3909489  PMID: 24304204
Oligodendrocyte precursor cells; nanofibers; myelination; differentiation
14.  Structural Proteins from Whelk Egg Capsule with Long Range Elasticity Associated with a Solid-state Phase Transition 
Biomacromolecules  2014;15(1):30-42.
The robust, proteinaceous egg capsules of marine prosobranch gastropods (genus Busycotypus) exhibit unique biomechanical properties such as high elastic strain recovery and elastic energy dissipation capability. Capsule material possesses long-range extensibility that is fully recoverable and is the result of a secondary structure phase transition from α-helix to extended β-sheet rather than of entropic (rubber) elasticity. We report here the characterization of the precursor proteins that make up this material. Three different proteins have been purified and analyzed, and complete protein sequences deduced from messenger ribonucleic acid (mRNA) transcripts. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectra indicate that the proteins are strongly α-helical in solution and primary sequence analysis suggests that these proteins have a propensity to form coiled-coils. This is in agreement with previous wide-angle x-ray scattering (WAXS) and solid-state Raman spectroscopic analysis of mature egg capsules.
doi:10.1021/bm401598z
PMCID: PMC3956310  PMID: 24350603
Egg capsule; elasticity; coiled-coil; α-β transition; shape memory; self-healing
15.  Electrically Conductive Chitosan/Carbon Scaffolds for Cardiac Tissue Engineering 
Biomacromolecules  2014;15(2):635-643.
In this work, carbon nanofibers were used as doping material to develop a highly conductive chitosan-based composite. Scaffolds based on chitosan only and chitosan/carbon composites were prepared by precipitation. Carbon nanofibers were homogeneously dispersed throughout the chitosan matrix, and the composite scaffold was highly porous with fully interconnected pores. Chitosan/carbon scaffolds had an elastic modulus of 28.1 ± 3.3 KPa, similar to that measured for rat myocardium, and excellent electrical properties, with a conductivity of 0.25 ± 0.09 S/m. The scaffolds were seeded with neonatal rat heart cells and cultured for up to 14 days, without electrical stimulation. After 14 days of culture, the scaffold pores throughout the construct volume were filled with cells. The metabolic activity of cells in chitosan/carbon constructs was significantly higher as compared to cells in chitosan scaffolds. The incorporation of carbon nanofibers also led to increased expression of cardiac-specific genes involved in muscle contraction and electrical coupling. This study demonstrates that the incorporation of carbon nanofibers into porous chitosan scaffolds improved the properties of cardiac tissue constructs, presumably through enhanced transmission of electrical signals between the cells.
doi:10.1021/bm401679q
PMCID: PMC3983145  PMID: 24417502
16.  Interfacial Protein-Protein Associations 
Biomacromolecules  2013;15(1):66-74.
While traditional models of protein adsorption focus primarily on direct protein-surface interactions, recent findings suggest that protein-protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein-protein associations of bovine serum albumin on poly(ethylene glycol) modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for longer times. The appearance of three distinct RET states suggested a spatially heterogeneous surface – with areas of high protein density (i.e. strongly-interacting clusters) coexisting with mobile monomers. Distinct association states exhibited characteristic behavior, i.e. partial-RET (monomer-monomer) associations were shorter-lived than complete-RET (protein-cluster) associations. While the fractional surface area covered by regions with high protein density (i.e. clusters) increased with increasing concentration, the distribution of contact times between monomers and clusters was independent of solution concentration, suggesting that associations were a local phenomenon, and independent of the global surface coverage.
doi:10.1021/bm401302v
PMCID: PMC3895929  PMID: 24274729
Protein adsorption; protein-protein interactions; single-molecule; total internal reflection fluorescence microscopy (TIRFM); solid-liquid interface; bovine serum albumin
17.  Effect of Pendant Group on pDNA Delivery by Cationic-β-Cyclodextrin:Alkyl-PVA-PEG Pendant Polymer Complexes 
Biomacromolecules  2013;15(1):12-19.
We have previously shown that cationic-β-CD:R-poly(vinyl alcohol)-poly(ethylene glycol) pendant polymer host:guest complexes are safe and efficient vehicles for nucleic acid delivery, where R = benzylidene-linked adamantyl or cholesteryl esters. Herein, we report the synthesis and biological performance of a family of PVA-PEG pendant polymers whose pendant groups have a wide range of different affinities for the β-CD cavity. Cytotoxicity studies revealed that all of the cationic-β-CD:pendant polymer host:guest complexes have 100 – 1000-fold lower toxicity than bPEI, with pDNA transfection efficiencies that are comparable to branched polyethylenimine (bPEI) and Lipofectamine 2000 (L2K). Complexes formed with pDNA at N/P ratios greater than 5 produced particles with diameters in the 100 – 170 nm range and ζ-potentials of 15 – 35 mV. Gel shift and heparin challenge experiments showed that the complexes are most stable at N/P ≥ 10, with adamantyl- and noradamantyl-modified complexes displaying the best resistance toward heparin-induced decomplexation. Disassembly rates of fluoresceinated-pDNA:CD+:R-PVA-PEG-rhodamine complexes within HeLa cells showed a modest dependence on host:guest binding constant, with adamantyl-, noradamantyl-, and dodecyl-based complexes showing the highest FRET efficiency 9 h after cellular exposure. These findings suggest that the host:guest binding constant has a significant impact on the colloidal stability in the presence of serum, cellular uptake efficiency, endosomal disassembly, and transfection performance of cationic-β-CD:R-poly(vinyl alcohol)-poly(ethylene glycol) pendant polymer complexes.
doi:10.1021/bm401096v
PMCID: PMC3899244  PMID: 24295406
Gene delivery; Cyclodextrins; Pendant Polymers; Transfection; pDNA
18.  Synthesis, Physicochemical Characterization, and Cytocompatibility of Bioresorbable, Dual-Gelling Injectable Hydrogels 
Biomacromolecules  2013;15(1):132-142.
Injectable, dual-gelling hydrogels were successfully developed through the combination of physical thermogellation at 37°C and favorable amine:epoxy chemical crosslinking. Poly(N-isopropylacrylamide)-based thermogelling macromers with a hydrolyzable lactone ring and epoxy pendant groups, and a biodegradable diamine-functionalized polyamidoamine crosslinker were synthesized, characterized, and combined to produce non-syneresing and bioresorbable hydrogels. Differential scanning calorimetry and oscillatory rheometry demonstrated the rapid and dual-gelling nature of the hydrogel formation. The post-gelation dimensional stability, swelling, and mechanical behavior of the hydrogel system were shown to be easily tuned at the synthesis and formulation stages. The leachable products were found to be cytocompatible at all conditions, while the degradation products demonstrated a dose- and time-dependent response due to solution osmolality. Preliminary encapsulation studies showed MSC viability could be maintained for 7 days. The results suggest that injectable, thermally and chemically crosslinkable hydrogels are promising alternatives to prefabricated biomaterials for tissue engineering applications, particularly for cell delivery.
doi:10.1021/bm401413c
PMCID: PMC3916782  PMID: 24320599
injectable hydrogel; poly(N-isopropylacrylamide); tissue engineering; thermogelling
19.  Injectable MMP-sensitive alginate hydrogels as hMSC delivery systems 
Biomacromolecules  2013;15(1):380-390.
Hydrogels with the potential to provide minimally invasive cell delivery represent a powerful tool for tissue-regeneration therapies. In this context, entrapped cells should be able to escape the matrix becoming more available to actively participate in the healing process. Here, we analyzed the performance of proteolytically-degradable alginate hydrogels as vehicles for human mesenchymal stem cells (hMSC) transplantation. Alginate was modified with the matrix metalloproteinase (MMP)-sensitive peptide Pro-Val-Gly-Leu-Iso-Gly (PVGLIG), which did not promote dendritic cell maturation in vitro, neither free nor conjugated to alginate chains, indicating low immunogenicity. hMSC were entrapped within MMP-sensitive and MMP-insensitive alginate hydrogels, both containing cell-adhesion RGD peptides. Softer (2 wt% alginate) and stiffer (4 wt% alginate) matrices were tested. When embedded in a Matrigel™ layer, hMSC-laden MMP-sensitive alginate hydrogels promoted more extensive outward cell migration and invasion into the tissue mimic. In vivo, after 4 weeks of subcutaneous implantation in a xenograft mouse model, hMSC-laden MMP-sensitive alginate hydrogels showed higher degradation and host tissue invasion than their MMP-insensitive equivalents. In both cases, softer matrices degraded faster than stiffer ones. The transplanted hMSC were able to produce their own collagenous extracellular matrix, and were located not only inside the hydrogels, but also outside, integrated in the host tissue. In summary, injectable MMP-sensitive alginate hydrogels can act as localized depots of cells, and confer protection to transplanted cells while facilitating tissue regeneration.
doi:10.1021/bm4016495
PMCID: PMC3918418  PMID: 24345197
Injectable biomaterials; hydrogels; alginate; protease-sensitive; MMPs; Cell delivery
20.  Microfluidics-Assisted Fabrication of Gelatin-Silica Core-Shell Microgels for Injectable Tissue Constructs 
Biomacromolecules  2014;15(1):283-290.
Microfabrication technology provides a highly versatile platform for engineering hydrogels used in biomedical applications with high-resolution control and injectability. Herein, we present a strategy of microfluidics-assisted fabrication photocrosslinkable gelatin microgels, coupled with providing protective silica hydrogel layer on the microgel surface to ultimately generate gelatin-silica core-shell microgels for applications as in vitro cell culture platform and injectable tissue constructs. A microfluidic device having flow-focusing channel geometry was utilized to generate droplets containing methacrylated gelatin (GelMA), followed by a photocrosslinking step to synthesize GelMA microgels. The size of the microgels could easily be controlled by varying the ratio of flow rates of aqueous and oil phases. Then, the GelMA microgels were used as in vitro cell culture platform to grow cardiac side population cells on the microgel surface. The cells readily adhered on the microgel surface and proliferated over time while maintaining high viability (~90%). The cells on the microgels were also able to migrate to their surrounding area. In addition, the microgels eventually degraded over time. These results demonstrate that cell-seeded GelMA microgels have a great potential as injectable tissue constructs. Furthermore, we demonstrated that coating the cells on GelMA microgels with biocompatible and biodegradable silica hydrogels via sol-gel method provided significant protection against oxidative stress which is often encountered during and after injection into host tissues, and detrimental to the cells. Overall, the microfluidic approach to generate cell-adhesive microgel core, coupled with silica hydrogels as a protective shell, will be highly useful as a cell culture platform to generate a wide range of injectable tissue constructs.
doi:10.1021/bm401533y
PMCID: PMC3922064  PMID: 24344625
21.  Gelation chemistries for the encapsulation of nanoparticles in composite gel microparticles for lung imaging and drug delivery 
Biomacromolecules  2013;15(1):252-261.
The formation of 10 to 40 μm Composite Gel MicroParticles (CGMPs) comprising ~100 nm drug containing nanoparticles (NPs) in a poly(ethylene glycol)(PEG) gel matrix is described. The CGMP particles enable targeting to the lung by filtration from the venous circulation. UV radical polymerization and Michael addition polymerization reactions are compared as approaches to form the PEG matrix. A fluorescent dye in the solid core of the NP was used to investigate the effect of reaction chemistry on the integrity of encapsulated species. When formed via UV radical polymerization, the fluorescence signal from the NPs indicated degradation of the encapsulated species by radical attack. The degradation decreased fluorescence by 90% over 15 minutes of UV exposure. When formed via Michael addition polymerization, the fluorescence was maintained. Emulsion processing using controlled shear stress enabled control of droplet size with narrow polydispersities. To allow for emulsion processing, the gelation rate was delayed by adjusting the solution pH. At a pH= 5.4 the gelation occurred at 3.5 hours. The modulus of the gels was tuned over the range of 5 to 50 kPa by changing the polymer concentration between 20 and 70 vol %. NPs aggregation during polymerization, driven by depletion forces, was controlled by the reaction kinetics. The ester bonds in the gel network enabled CGMP degradation. The gel modulus decreased by 50% over 27 days, followed by complete gel degradation after 55 days. This permits ultimate clearance of the CGMPs from the lungs. The demonstration of uniform delivery of 15.8 ± 2.6 μm CGMPs to the lungs of mice, with no deposition in other organs, is shown, and indicates the ability to target therapeutics to the lung while avoiding off-target toxic exposure.
doi:10.1021/bm4015232
PMCID: PMC3981107  PMID: 24410445
microgel particle; venous filtration pathway; drug delivery; lung targeting; hydrogel; nanoparticle
22.  The Microstructure and Bulk Rheology of Human Cervicovaginal Mucus are Remarkably Resistant to Changes in pH 
Biomacromolecules  2013;14(12):4429-4435.
The protective barrier, lubricant and clearance functions of mucus are intimately coupled to its microstructure and bulk rheology. Mucus gels consist of a network of mucin biopolymers along with lipids, salts and other proteins, and exhibit similar biochemical and physical properties across diverse mucosal surfaces. Nevertheless, mucus is exposed to a broad range of pH throughout the human body. Protein functions are typically sensitive to small changes in pH, and prior investigations using reconstituted, purified mucin gels suggested mucus transitions from a low viscosity liquid at neutral pH to a highly viscoelastic solid at low pH. We sought to determine whether those observations hold for fresh, minimally-perturbed human mucus ex vivo, by using different-sized muco-inert nanoparticles to probe microstructure, and cone-and-plate rheometry to measure bulk rheology. We demonstrate that both the microstructure and bulk rheology of fresh, undiluted and minimally perturbed cervicovaginal mucus exhibit relatively minor changes from pH 1–2 to 8–9, in marked contrast with the pH sensitivity of purified mucin gels. Our work also suggests additional components in mucus secretions, typically eliminated during mucin purification and reconstitution, may play an important role in maintaining the protective properties of mucus.
doi:10.1021/bm401356q
PMCID: PMC3918948  PMID: 24266646
biophysics; glycoprotein; microscopy; mucins; multiple particle tracking; nanotechnology
23.  Sensitization of Transforming Growth Factor-β Signaling by Multiple Peptides Patterned on DNA Nanostructures 
Biomacromolecules  2013;14(12):4157-4160.
We report sensitization of a cellular signaling pathway by addition of functionalized DNA nanostructures. Signaling by transforming growth factor β (TGFβ) has been shown to be dependent on receptor clustering. By patterning a DNA nanostructure with closely spaced peptides that bind to TGFβ we observe increased sensitivity of NMuMG cells to TGFβ ligand. This is evidenced by translocation of secondary messenger proteins to the nucleus and stimulation of an inducible luciferase reporter at lower concentrations of TGFβ ligand. We believe this represents an important initial step towards realization of DNA as a self assembling and biologically compatible material for use in tissue engineering and drug delivery.
doi:10.1021/bm4011722
PMCID: PMC4219643  PMID: 24206086
DNA; Nanostructure; TGFβ; Nanotechnology
24.  Synthetically Simple, Highly Resilient Hydrogels 
Biomacromolecules  2012;13(3):584-588.
Highly resilient synthetic hydrogels were synthesized by using the efficient thiol-norbornene chemistry to cross-link hydrophilic poly(ethylene glycol) (PEG) and hydrophobic polydimethylsiloxane (PDMS) polymer chains. The swelling and mechanical properties of the hydrogels were well-controlled by the relative amounts of PEG and PDMS. In addition, the mechanical energy storage efficiency (resilience) was more than 97% at strains up to 300%. This is comparable with one of the most resilient materials known: natural resilin, an elastic protein found in many insects, such as in the tendons of fleas and the wings of dragonflies. The high resilience of these hydrogels can be attributed to the well-defined network structure provided by the versatile chemistry, low cross-link density, and lack of secondary structure in the polymer chains.
doi:10.1021/bm300015s
PMCID: PMC4251582  PMID: 22372639
Hydrogels; Resilience; Thiol-Ene; Resilin
25.  Photocrosslinked PLA-PEO-PLA Hydrogels from Self-Assembled Physical Networks: Mechanical Properties and Influence of Assumed Constitutive Relationships 
Biomacromolecules  2008;9(10):2784-2791.
Poly(lactide) – block – poly(ethylene oxide) – block – poly(lactide) [PLA-PEO-PLA] triblock copolymers are known to form physical hydrogels in water, due to the polymer's amphiphilicity. Their mechanical properties, biocompatibility, and biodegradability have made them attractive for use as soft tissue scaffolds. However, the network junction points are not covalently crosslinked and in a highly aqueous environment these hydrogels adsorb more water, transform from gel to sol, and lose the designed mechanical properties. In this report, a hydrogel was formed by using a novel two step approach. In the first step end-functionalized PLA-PEOPLA triblock was self-assembled into a physical hydrogel through hydrophobic micelle network junctions, and then, in the second step, this self-assembled physical network structure was locked into place by photocrosslinking the terminal acrylate groups. In contrast to physical hydrogels, the photocrosslinked gels remained intact in phosphate buffered solution at body temperature. The swelling, degradation, and mechanical properties were characterized and demonstrated extended degradation time (~ 65 days), exponential decrease in modulus with degradation time, and tunable shear modulus (1.6 – 133 kPa) by varying concentration. We also discuss the various constitutive relationships (Hookean, Neo-Hookean, and Mooney-Rivlin) that can be used to describe the stress-strain behavior of these hydrogels. The chosen model and assumptions used for data fitting influences the obtained modulus values by as much as a factor of 3.5, demonstrating the importance of clearly stating one's data fitting parameters so that accurate comparisons can be made within the literature.
doi:10.1021/bm800557r
PMCID: PMC4251597  PMID: 18817440

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