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1.  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
2.  Progress in MicroRNA Delivery 
MicroRNAs (miRNAs) are non-coding endogenous RNAs that direct post-transcriptional regulation of gene expression by several mechanisms. Activity is primarily through binding to the 3’ untranslated regions (UTRs) of messenger RNAs (mRNA) resulting in degradation and translation repression. Unlike other small-RNAs, miRNAs do not require perfect base pairing, and thus, can regulate a network of broad, yet specific, genes. Although we have only just begun to gain insights into the full range of biologic functions of miRNA, their involvement in the onset and progression of disease has generated significant interest for therapeutic development. Mounting evidence suggests that miRNA-based therapies, either restoring or repressing miRNAs expression and activity, hold great promise. However, despite the early promise and exciting potential, critical hurdles often involving delivery of miRNA-targeting agents remain to be overcome before transition to clinical applications. Limitations that may be overcome by delivery include, but are not limited to, poor in vivo stability, inappropriate biodistribution, disruption and saturation of endogenous RNA machinery, and untoward side effects. Both viral vectors and nonviral delivery systems can be developed to circumvent these challenges. Viral vectors are efficient delivery agents but toxicity and immunogenicity limit their clinical usage. Herein, we review the recent advances in the mechanisms and strategies of nonviral miRNA delivery systems and provide a perspective on the future of miRNA-based therapeutics.
doi:10.1016/j.jconrel.2013.09.015
PMCID: PMC3891846  PMID: 24075926
microRNA; miRNA therapeutics; small RNA delivery; human disease; nonviral delivery
3.  Biodistribution and Bioimaging Studies of Hybrid Paclitaxel Nanocrystals: Lessons Learned of the EPR Effect and Image-Guided Drug Delivery 
Paclitaxel (PTX) nanocrystals (200 nm) were produced by crystallization from solution. Antitumor efficacy and toxicity were examined through a survival study in a human HT-29 colon cancer xenograft murine model. The antitumor activity of the nanocrystal treatments was comparable with that by the conventional solubilization formulation (Taxol®), but yielded less toxicity as indicated by the result of survival study. Tritium-labeled PTX nanocrystals were further produced with a near infrared (NIR) fluorescent dye physically integrated in the crystal lattice. Biodistribution and tumor accumulation of the tritium-labeled PTX nanocrystals were determined immediately after intravenous administration and up to 48 hours by scintillation counting. Whole-body optical imaging of animals was concurrently carried out; fluorescent intensities were also measured from excised tumors and major organs of euthanized animals. It was found that drug accumulation in the tumor was less than 1% of 20 mg/kg intravenous dose. Qualitatively correlation was identified between the biodistribution determined by using tritium-labeled particles and that using optical imaging, but quantitative divergence existed. The divergent results suggest possible ways to improve the design of hybrid nanocrystals for cancer therapy and diagnosis. The study also raises questions of the general role of the enhanced permeability and retention (EPR) effect in tumor targeting and the effectiveness of bioimaging, specifically for hybrid nanocrystals, in tracking drug distribution and pharmacokinetics.
doi:10.1016/j.jconrel.2013.06.039
PMCID: PMC3886194  PMID: 23920039
paclitaxel; nanocrystals; hybrid; theranostics; chemotherapy; EPR; biodistribution
4.  Proteolytically activated anti-bacterial hydrogel microspheres 
Hydrogels are finding increased clinical utility as advances continue to exploit their favorable material properties. Hydrogels can be adapted for many applications, including surface coatings and drug delivery. Anti-infectious surfaces and delivery systems that actively destroy invading organisms are alternative ways to exploit the favorable material properties offered by hydrogels. Sterilization techniques are commonly employed to ensure the materials are non-infectious upon placement, but sterilization is not absolute and infections are still expected. Natural, anti-bacterial proteins have been discovered which have the potential to act as anti-infectious agents; however, the proteins are toxic and need localized release to have therapeutic efficacy without toxicity. In these studies, we explore the use of the glutathione s-transferase (GST) to anchor the bactericidal peptide, melittin, to the surface of poly(ethylene glycol) diacrylate (PEGDA) hydrogel microspheres. We show that therapeutic levels of protein can be anchored to the surface of the microspheres using the GST anchor. We compared the therapeutic efficacy of recombinant melittin released from PEGDA microspheres to melittin. We found that, when released by an activating enzyme, thrombin, recombinant melittin efficiently inhibits growth of the pathogenic bacterium Streptococcus pyogenes as effectively as melittin created by solid phase peptide synthesis. We conclude that a GST protein anchor can be used to immobilize functional protein to PEGDA microspheres and the protein will remain immobilized under physiological conditions until the protein is enzymatically released.
doi:10.1016/j.jconrel.2013.06.023
PMCID: PMC3795988  PMID: 23816641
recombinant protein; glutathione s-transferase; glutathione; thrombin; hydrogel; microparticles
5.  Active, soluble recombinant melittin purified by extracting insoluble lysate of Escherichia coli without denaturation 
Biotechnology progress  2013;29(5):1150-1157.
Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U-87 MG glioma cells and inhibits growth of the Gram-positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of approximately 0.5 to 1 mg/L of E. coli culture.
doi:10.1002/btpr.1784
PMCID: PMC3874069  PMID: 23926061
Recombinant protein; fusion protein; protein extraction; surfactant; melittin
6.  Stability of Benzocaine Formulated in Commercial Oral Disintegrating Tablet Platforms 
AAPS PharmSciTech  2013;14(4):1333-1340.
Pharmaceutical excipients contain reactive groups and impurities due to manufacturing processes that can cause decomposition of active drug compounds. The aim of this investigation was to determine if commercially available oral disintegrating tablet (ODT) platforms induce active pharmaceutical ingredient (API) degradation. Benzocaine was selected as the model API due to known degradation through ester and primary amino groups. Benzocaine was either compressed at a constant pressure, 20 kN, or at pressure necessary to produce a set hardness, i.e., where a series of tablets were produced at different compression forces until an average hardness of approximately 100 N was achieved. Tablets were then stored for 6 months under International Conference on Harmonization recommended conditions, 25°C and 60% relative humidity (RH), or under accelerated conditions, 40°C and 75% RH. Benzocaine degradation was monitored by liquid chromatography–mass spectrometry. Regardless of the ODT platform, no degradation of benzocaine was observed in tablets that were kept for 6 months at 25°C and 60% RH. After storage for 30 days under accelerated conditions, benzocaine degradation was observed in a single platform. Qualitative differences in ODT platform behavior were observed in physical appearance of the tablets after storage under different temperature and humidity conditions.
doi:10.1208/s12249-013-0015-5
PMCID: PMC3840786  PMID: 23990120
benzocaine; degradation; oral disintegrating tablet; platform; stability
7.  Characterization of Pore Structure in Biologically Functional Poly(2-Hydroxyethyl Methacrylate) - Poly(Ethylene Glycol) Diacrylate (PHEMA-PEGDA) 
PLoS ONE  2014;9(5):e96709.
A copolymer composed of poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(ethylene glycol) diacrylate (PEGDA) (PHEMA-PEGDA) is structurally versatile. Its structure can be adjusted using the following porogens: water, sucrose, and benzyl alcohol. Using phase separation technique, a variety of surface architectures and pore morphologies were developed by adjusting porogen volume and type. The water and sucrose porogens were effective in creating porous and cytocompatible PHEMA-PEGDA scaffolds. When coated with collagen, the PHEMA-PEGDA scaffolds accommodated cell migration. The PHEMA-PEGDA scaffolds are easy to produce, non-toxic, and mechanically stable enough to resist fracture during routine handling. The PHEMA-PEGDA structures presented in this study may expedite the current research effort to engineer tissue scaffolds that provide both structural stability and biological activity.
doi:10.1371/journal.pone.0096709
PMCID: PMC4016039  PMID: 24816589
8.  Matrix metalloproteases: Underutilized targets for drug delivery 
Journal of drug targeting  2007;15(1):1-20.
Pathophysiological molecules in the extracellular environment offer excellent targets that can be exploited for designing drug targeting systems. Matrix metalloproteases (MMPs) are a family of extracellular proteolytic enzymes that are characterized by their overexpression or overactivity in several pathologies. Over the last two decades, the MMP literature reveals heightened interest in the research involving MMP biology, pathology, and targeting. This review describes various strategies that have been designed to utilize MMPs for targeting therapeutic entities. Key factors that need to be considered in the successful design of such systems have been identified based on the analyses of these strategies. Development of targeted drug delivery using MMPs has been steadily pursued; however, drug delivery efforts using these targets need to be intensified and focused to realize the clinical application of the fast developing fundamental MMP research.
doi:10.1080/10611860600968967
PMCID: PMC3782085  PMID: 17365270
drug delivery; drug targeting; matrix metalloprotease (MMP); prodrug; drug activation
9.  Markers Are Shared Between Adipogenic and Osteogenic Differentiated Mesenchymal Stem Cells 
The stem cell differentiation paradigm is based on the progression of cells through generations of daughter cells that eventually become restricted and committed to one lineage resulting in fully differentiated cells. Herein, we report on the differentiation of adult human mesenchymal stem cells (hMSCs) towards adipogenic and osteogenic lineages using established protocols. Lineage specific geneswere evaluated by quantitative real-time PCR relative to two reference genes. The expression of osteoblast-associated genes (alkaline phosphatase, osteopontin, and osteocalcin)was detected in hMSCs that underwent adipogenesis. When normalized, the expression of adipocyte marker genes (adiponectin, fatty acid binding protein P4, and leptin) increasedin a time-dependent manner during adipogenic induction. Adiponectin and leptin were also detected in osteoblast-induced cells. Lipid vacuoles that represent the adipocyte phenotype were only present in the adipogenic induction group. Conforming to the heterogeneous nature of hMSCs and the known plasticity between osteogenic and adipogenic lineages, these data indicatea marker overlap between MSC-derived adipocytes and osteoblasts. Weproposea careful consideration of experimental conditions such as investigated timepoints, selected housekeeping genesand the evidence indicating lack of differentiation into other lineageswhen evaluating hMSC differentiation.
doi:10.5897/JDBTE2013.0065
PMCID: PMC3765027  PMID: 24013643
mesenchymal stem cell; differentiation markers; cell plasticity; differentiation
10.  Hybrid Nanocrystals: University of Kentucky US2006/0280680A1 
This patent application claims an interesting and novel combination of passive accumulation of drug nanocrystals within diseased tissue in combination with active uptake of the nanocrystals by diseased cells. The patent application further claims the hybrid nanocrystals combining imaging or stabilizing molecules as inclusions in the crystal matrix. There is a focus on cancer chemotherapy and imaging, but the initial claims are not disease specific. In this patent evaluation, we examine the novelty and utility of this application and discuss the state of the art in nanocrystal formulations and formulation.
doi:10.1517/13543776.2012.665877
PMCID: PMC3742739  PMID: 22364361
cancer; nanocrystal; theranostic; imaging; targeted drug delivery
11.  Improving Matrix Metalloproteinase-2 Specific Response of a Hydrogel System using Electrophoresis 
Matrix metalloproteinases (MMPs) overexpression plays a critical role in cancer invasion and metastasis. We utilized this key feature of tumor microenvironment to develop a disease-stimuli triggered drug delivery system. Poly(acrylic acid) hydrogels were synthesized by UV polymerization and pendant MMP-2 sensitive peptides (Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys) conjugated throughout using EDC/sulfo-NHS chemistry. There were significantly more peptides released in the presence of MMP-2 compared with the control groups. The released peptide fragments were analyzed by HPLC and MALDI-MS and confirmed to be the expected fragments. In order to avoid nonspecific release of nonconjugated (i.e. unreacted) peptides, a novel method of electrophoretic washing was developed disrupting the strong electrostatic interactions between the peptides and the pendant groups of the hydrogel. After electrophoresis, the nonspecific peptide release in the absence of MMP-2 was minimized. This newly developed purification system significantly improved the control of release to be in response of the magnitude of the stimuli, i.e. MMP. Specifically, peptides were released proportionally to the concentration of MMP-2 present. Now that many of the design parameters have been examined, anticancer drugs will be conjugated to the MMP sensitive peptide linkers with the goal of implantation in a tumor void releasing anticancer reagent in response to elevated level of MMPs.
doi:10.1016/j.ijpharm.2012.03.012
PMCID: PMC3327771  PMID: 22440150
Matrix metalloproteinase; hydrogel; peptide; enzyme responsive drug delivery; cancer
12.  Decellularized Human Cornea for Reconstructing the Corneal Epithelium and Anterior Stroma 
In this project, we strived to develop a decellularized human cornea to use as a scaffold for reconstructing the corneal epithelium and anterior stroma. Human cadaver corneas were decellularized by five different methods, including detergent- and nondetergent-based approaches. The success of each method on the removal of cells from the cornea was investigated. The structural integrity of decellularized corneas was compared with the native cornea by electron microscopy. The integrity of the basement membrane of the epithelium was analyzed by histology and by the expression of collagen type IV, laminin, and fibronectin. Finally, the ability of the decellularized corneas to support the growth of human corneal epithelial cells and fibroblasts was assessed in vitro. Corneas processed using Triton X-100, liquid nitrogen, and poly(ethylene glycol) resulted in incomplete removal of cellular material. Corneas processed with the use of sodium dodecyl sulfate (SDS) or with sodium chloride (NaCl) plus nucleases successfully removed all cellular material; however, only the NaCl plus nuclease treatment kept the epithelial basement membrane completely intact. Corneas processed with NaCl plus nuclease supported both fibroblast and epithelial cell growth in vitro, while corneas treated with SDS supported the growth of only fibroblasts and not epithelial cells. Decellularized human corneas provide a scaffold that can support the growth of corneal epithelial cells and stromal fibroblasts. This approach may be useful for reconstructing the anterior cornea and limbus using autologous cells.
doi:10.1089/ten.tec.2011.0072
PMCID: PMC3338110  PMID: 22082039
13.  Effects of Molecular Weight and Loading on Matrix Metalloproteinase-2 Mediated Release from Poly(Ethylene Glycol) Diacrylate Hydrogels 
The AAPS Journal  2012;14(3):482-490.
Herein, we report on continued efforts to understand an implantable poly(ethylene glycol) diacrylate (PEGDA) hydrogel drug delivery system that responds to extracellular enzymes, in particular matrix metalloproteinase-2 (MMP-2) to provide controlled drug delivery. By attaching peptide as pendant groups on the hydrogel backbone, drug release occurs at an accelerated rate in the presence of active protease. We investigated MMP-2 entry and optimized parameters of the drug delivery system. Mesh size for different PEGDA molecular weight macromers was measured with PEGDA 3,400 hydrogels having a mesh size smaller than the dimensions of MMP-2 and PEGDA 10,000 and PEGDA 20,000 hydrogels having mesh sizes larger than MMP-2. Purified MMP-2 increased release of peptide fragment compared to buffer at several loading concentrations. Cell-stimulated release was demonstrated using U-87 MG cells embedded in collagen. GM6001, an MMP inhibitor, diminished release and altered the identity of the released peptide fragment. The increase in ratio of release from PEGDA 10,000 and PEGDA 20,000 hydrogels compared to PEGDA 3,400 hydrogels suggests MMP-2 enters the hydrogel. PEGDA molecular weight of 10,000 and 15 % (w/V) were the optimal conditions for release and handling. The use of protease-triggered drug delivery has great advantage particularly with the control of protease penetration as a parameter for controlling rate of release.
doi:10.1208/s12248-012-9356-3
PMCID: PMC3385811  PMID: 22535508
cancer; chemotherapy; controlled drug delivery; enzyme-triggered drug delivery; matrix metalloproteinase-2; poly(ethylene glycol) diacrylate, hydrogel
14.  Stem Cell Derived Extracellular Matrix Enables Survival and Multi Lineage Differentiation within Superporous Hydrogels 
Biomacromolecules  2012;13(4):963-973.
Hydrophilic poly(ethylene glycol) diacrylate (PEGDA) hydrogel surfaces resist protein adsorption and are generally thought to be unsuitable for anchorage dependent cells to adhere. Intriguingly, our previous findings revealed that PEGDA superporous hydrogel scaffolds (SPHs) allow anchorage of bone marrow derived human mesenchymal stem cells (hMSCs) and support their long term survival. Therefore, we hypothesized that the physicochemical characteristics of the scaffold impart properties that could foster cellular responses. We examined if hMSCs alter their microenvironment to allow cell attachment by synthesizing their own extracellular matrix (ECM) proteins. Immunofluorescence staining revealed extensive expression of collagen type I, collagen type IV, laminin and fibronectin within hMSC-seeded SPHs by the end of the third week. Whether cultured in serum-free or serum-supplemented medium, hMSC ECM protein gene expression patterns exhibited no substantial changes. The presence of serum proteins is required for initial anchorage of hMSCs within the SPHs but not for the hMSC survival after 24 hours. In contrast to 2D expansion on tissue culture plastic (TCP), hMSCs cultured within SPHs proliferate similarly in the presence or absence of serum. To test whether hMSCs retain their undifferentiated state within the SPHs, cell-seeded constructs were cultured for 3 weeks in stem cell maintenance medium and the expression of hMSC-specific cell surface markers were evaluated by flow cytometry. CD105, CD90, CD73 and CD44 were present to a similar extent in the SPH and in 2D monolayer culture. We further demonstrated multi lineage potential of hMSCs grown in the PEGDA SPHs whereby differentiation into osteoblasts, chondrocytes and adipocytes could be induced. The present study demonstrates the potential of hMSCs to alter the “blank” PEGDA environment to a milieu conducive to cell growth and multi-lineage differentiation by secreting adhesive ECM proteins within the porous network of the SPH scaffolds.
doi:10.1021/bm300332w
PMCID: PMC3322260  PMID: 22404228
Porous; hydrogels; scaffolds; poly(ethylene glycol); mesenchymal stem cells; extracellular matrix; cell proliferation; cell differentiation
15.  Critical Factors Affecting Cell Encapsulation in Superporous Hydrogels 
We recently showed that superporous hydrogel scaffolds promote long-term stem cell viability and cell driven mineralization when cells were seeded within the pores of pre-fabricated SPH scaffolds. The possibility of cell encapsulation within SPH hydrogel matrix during its fabrication was further explored in this study. The impact of each chemical component used in the SPH fabrication and each step of the fabrication process on cell viability was systematically examined. Ammonium persulfate, an initiator, and sodium bicarbonate, the gas-generating compound, were the two components having significant toxicity toward encapsulated cells at the concentrations necessary for SPH fabrication. Cell survival rates were 55.7±19.3% and 88.8±9.4% after 10 minute exposure to ammonium persulfate and sodium bicarbonate solutions, respectively. In addition, solution pH change via the addition of sodium bicarbonate had significant toxicity toward encapsulated cells with cell survival of only 50.3±2.5%. Despite toxicity of chemical components and the SPH fabrication method, cells still exhibited significant overall survival rates within SPHs of 81.2±6.8 and 67.0±0.9%, respectively, 48 and 72 hours after encapsulation. This method of cell encapsulation holds promise for use in vitro an in vivo as a scaffold material for both hydrogel matrix encapsulation and cell seeding within the pores.
doi:10.1088/1748-6041/7/2/024108
PMCID: PMC3358450  PMID: 22455976
Porous; hydrogels; scaffolds; cell encapsulation; tissue engineering; macroporous polymers
16.  In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins 
BMC Biotechnology  2012;12:63.
Background
Many branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST)-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins.
Results
Glutathione was conjugated to low molecular weight poly(ethylene glycol) diacrylate (PEGDA) via thiol-ene “click” chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH) homogenates, we were able to purify a glutathione S-transferase (GST) green fluorescent protein (GFP) fusion protein (GST-GFP) from the soluble fraction of E. coli lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads.
Conclusions
GSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.
doi:10.1186/1472-6750-12-63
PMCID: PMC3463477  PMID: 22989306
Glutathione; PEGDA; Glutathione S-transferase; Batch purification; Recombinant protein
17.  Fabrication of Poly(ethylene glycol) Hydrogel Structures for Pharmaceutical Applications using Electron beam and Optical Lithography 
Soft-polymer based microparticles are currently being applied in many biomedical applications, ranging from bioimaging and bioassays to drug delivery carriers. As one class of soft-polymers, hydrogels are materials, which can be used for delivering drug cargoes and can be fabricated in controlled sizes. Among the various hydrogel-forming polymers, poly(ethylene glycol) (PEG) based hydrogel systems are widely used due to their negligible toxicity and limited immunogenic recognition. Physical and chemical properties of particles (i.e., particle size, shape, surface charge, and hydrophobicity) are known to play an important role in cell-particle recognition and response. To understand the role of physicochemical properties of PEG-based hydrogel structures on cells, it is important to have geometrically precise and uniform hydrogel structures. To fabricate geometrically uniform structures, we have employed electron beam lithography (EBL) and ultra-violet optical lithography (UVL) using PEG or PEG diacrylate polymers. These hydrogel structures have been characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), optical microscopy, and attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) confirming control of chemistry, size, and shape.
doi:10.1116/1.3517716
PMCID: PMC3060791  PMID: 21423572
poly(ethylene glycol); poly(ethylene glycol) diacrylate; 2-hydroxy-4’-(2-hydroxyethoxy)-2-metylpropiophenone
18.  In vitro evaluation of functional interaction of integrin αvβ3 and matrix metalloprotease-2 
Molecular pharmaceutics  2009;6(6):1856-1867.
Integrin αvβ3 and matrix metalloprotease (MMP)-2 are two established molecular targets of angiogenesis. Basic understanding of various forms of functional interaction of integrin αvβ3 and active MMP-2 may be used to develop therapeutic approaches. Based upon the idea that integrins are present on the surface of invasive cells and MMP-2 may be localized to this and other cell-surface receptors, we investigated the hypothesis that integrin binding will alter cleavage of MMP-2 substrates. To investigate this hypothesis, integrin-binding and MMP-2 cleavable motifs were combined in a single peptide, MMP-RGD, designed with fluorescent probes for monitoring peptide cleavage. MMP-RGD was bound to integrin αvβ3 with equal affinity compared to the integrin-binding motif and was cleaved with equal specificity by active MMP-2. MMP-RGD bound to human umbilical vein endothelial cells (HUVECs). MMP-2 from HUVECs cleaved MMP-RGD but the cleavage was not altered due to integrin binding. Our results indicate that integrin αvβ3 and active MMP-2 may not be as functionally collaborative for substrate cleavage as expected based on the current knowledge of their cell surface co-localization.
doi:10.1021/mp900152t
PMCID: PMC2790922  PMID: 19799453
integrin; αvβ3; matrix metalloprotease; MMP-2; molecular targets; angiogenesis; HUVEC; RGD peptide
19.  Initial Evaluation of Vascular Ingrowth into Superporous Hydrogels 
There is a need of new materials and architectures for tissue engineering and regenerative medicine. Based upon our recent results developing novel scaffold architecture, we hypothesized that this new architecture would foster vascularization, a particular need for tissue engineering. We report on the potential of superporous hydrogel (SPH) scaffolds for in vivo cellular infiltration and vascularization. Poly(ethylene glycol) diacrylate (PEGDA) SPH scaffolds were implanted in the dorsum of severe combined immunodeficient (SCID) mice and harvested after four weeks of in vivo implantation. The SPHs were visibly red and vascularized as apparent when compared to the non-porous hydrogel controls which were macroscopically avascular. Host cell infiltration was observed throughout the SPHs. Blood cells and vascular structures, confirmed through staining for CD 34 and smooth muscle alpha actin, were observed throughout the scaffolds. This novel soft material may be utilized for cell transplantation, tissue engineering, and in combination with cell therapies. The neovasularization and limited fibritic response suggest that the architecture may be conducive to cell survival and rapid vessel development.
doi:10.1002/term.183
PMCID: PMC2766576  PMID: 19526620
hydrogel; pore; scaffold; poly(ethylene glycol) diacrylate; in vivo; vascularization
20.  In Vitro Evaluation of Macroporous Hydrogels to Facilitate Stem Cell Infiltration, Growth, and Mineralization 
Tissue Engineering. Part A  2009;15(7):1695-1707.
Hydrogels have gained acceptance as biomaterials in a wide range of applications, including pharmaceutical formulations, drug delivery, and tissue sealants. However, exploiting the potential of hydrogels as scaffolds for cell transplantation, tissue engineering, and regenerative medicine still remains a challenge due to, in part, scaffold design limitations. Here, we describe a highly interconnected, macroporous poly(ethylene glycol) diacrylate hydrogel scaffold, with pores ranging from 100 to 600 μm. The scaffold exhibits rapid cell uptake and cell seeding without the need of any external force or device with high incorporation efficiency. When human mesenchymal stem cells are seeded within the porous scaffolds, the scaffolds were found to promote long-term stem cell viability, and on exposure to osteogenic medium, elicit an mineralization response as evaluated by an increased alkaline phosphatase activity (per cell) and calcium and phosphate content within the constructs. The atomic composition of the mineralized matrix was further determined by energy dispersive spectroscopy and found to be similar to calcium-deficient hydroxyapatite, the amorphous biological precursor of bone. The macroporous design of the hydrogel appears advantageous over similar porous hydrogel scaffolds with respect to ease of synthesis, ease of stem cell seeding, and its ability to support long-term stem cell survival and possible differentiation.
doi:10.1089/ten.tea.2008.0238
PMCID: PMC2810413  PMID: 19119921
21.  Matrix Metalloprotease Selective Peptide Substrates Cleavage within Hydrogel Matrices for Cancer Chemotherapy Activation 
Peptides  2008;29(11):1965-1973.
To utilize biologic mechanisms to elicit controlled release in response to disease, protease-sensitive devices have been created. Hydrogels were created with pendant peptide-drug complexes. For the matrix metalloproteases (MMPs) examined, a length of six amino acids greatly improved the specificity of the peptide (kcat/Km ~ 2.4±0.1×104 M−1s−1) over shorter sequences (kcat/Km ~ 4.4±0.2×102 M−1s−1). The peptides did not exhibit anti-proliferative effects upon cancer cells, and peptide-platinum complexes showed similar anti-proliferative effects upon the cancer cells compared to the free platinum drugs. Once the peptide-drug complex was incorporated into the hydrogels, the release was dependent upon the presence of MMP in the solution with approximately 35% of platinum released from hydrogels in the presence of MMP and only 10% without MMP in the week examined. The released drug exhibited the expected anti-proliferative activity over several days of incubation. The MMP selective drug delivery holds much potential for treatment of cancer and other diseases.
doi:10.1016/j.peptides.2008.06.021
PMCID: PMC2592099  PMID: 18652863
Prodrug; cancer; hydrogel; drug targeting; drug activation
22.  Understanding the adsorption mechanism of chitosan onto poly(lactide-co-glycolide) particles 
Polyelectrolyte-coated nanoparticles or microparticles interact with bioactive molecules (peptides, proteins or nucleic acids) and have been proposed as delivery systems for these molecules. However, the mechanism of adsorption of polyelectrolyte onto particles remains unsolved. In this study, cationic poly(lactide-co-glycolide) (PLGA) nanoparticles were fabricated by adsorption of various concentrations of a biodegradable polysaccharide, chitosan (0–2.4 g/L), using oil-in-water emulsion and solvent evaporation techniques. The particle diameter, zeta-potential, and chitosan adsorption of chitosan coated PLGA nanoparticles confirmed the increase of polyelectrolyte adsorption. Five adsorption isotherm models (Langmuir, Freundlich, Halsey, Henderson and Smith) were applied to the experimental data in order to better understand the mechanism of adsorption. Both particle diameter and chitosan adsorption increased with chitosan concentration during adsorption. A good correlation was obtained between PLGA-chitosan nanoparticle size and adsorbed chitosan on the surface, suggesting the increased particle size was primarily due to the increased chitosan adsorption. The zeta-potential of chitosan-coated PLGA nanoparticles was positive and increased with chitosan adsorbed until a maximum value (+55 mV) was reached at approximately 0.4–0.6 g/L; PLGA nanoparticles had a negative zeta-potential (−20 mV) prior to chitosan adsorption. Chitosan adsorption on PLGA nanoparticles followed a multilayer adsorption behavior, although the Langmuir monolayer equation held at low concentrations of chitosan. The underlying reasons for adsorption of chitosan on PLGA nanoparticles were thought to be the cationic nature of chitosan, high surface energy and microporous non-uniform surface of PLGA nanoparticles.
doi:10.1016/j.ejpb.2008.06.008
PMCID: PMC2612535  PMID: 18602994
nanoparticle, PLGA; chitosan; adsorption isotherm; surface modification
23.  Cervical cancer treatment with a locally insertable controlled release delivery system 
Local delivery of cancer chemotherapeutics enables sustained drug levels at the site of action thereby reducing systemic side effects. A novel insertable polymeric drug delivery system for cervical cancer treatment is presented. Cisplatin, the first line of therapy employed for cervical cancers, was incorporated in a poly(ethylene-co-vinyl acetate) (EVAc) device that is similar to those currently used for vaginal contraceptive delivery. Cisplatin crystals were uniformly dispersed in the polymeric system without undergoing significant dissolution in the polymer matrix. Cisplatin dissolution from the devices was biphasic, consistent with a matrix-type controlled-release system with an initial rapid release phase followed by a slower, linear release phase. Depending on the drug loading in the polymeric devices, the near-linear release phase varied in rate according both empirical, linear curve-fitting (0.38±0.15 μg/day to 46.9±10.0 μg/day) and diffusion analysis based upon diffusion through a porous structure (Dapp from 1.3±0.5×10−9 cm2/s to 5.8±0.3×10−12 cm2/s). The devices were tested for in vitro activity and found to be effective against both HPV positive and HPV negative cervical cancer cell lines. Preliminary studies indicate that this delivery system would be a good candidate for investigation as a choice of treatment in cervical cancers.
doi:10.1016/j.jconrel.2006.08.014
PMCID: PMC1769315  PMID: 17034891
controlled release; cancer; polymer; cisplatin; vaginal delivery; human papilloma virus (HPV)

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