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.
drug delivery; drug targeting; matrix metalloprotease (MMP); prodrug; drug activation
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.
mesenchymal stem cell; differentiation markers; cell plasticity; differentiation
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.
cancer; nanocrystal; theranostic; imaging; targeted drug delivery
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.
Matrix metalloproteinase; hydrogel; peptide; enzyme responsive drug delivery; cancer
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.
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.
cancer; chemotherapy; controlled drug delivery; enzyme-triggered drug delivery; matrix metalloproteinase-2; poly(ethylene glycol) diacrylate, hydrogel
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.
Porous; hydrogels; scaffolds; poly(ethylene glycol); mesenchymal stem cells; extracellular matrix; cell proliferation; cell differentiation
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.
Porous; hydrogels; scaffolds; cell encapsulation; tissue engineering; macroporous polymers
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.
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.
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.
Glutathione; PEGDA; Glutathione S-transferase; Batch purification; Recombinant protein
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.
poly(ethylene glycol); poly(ethylene glycol) diacrylate; 2-hydroxy-4’-(2-hydroxyethoxy)-2-metylpropiophenone
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.
integrin; αvβ3; matrix metalloprotease; MMP-2; molecular targets; angiogenesis; HUVEC; RGD peptide
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.
hydrogel; pore; scaffold; poly(ethylene glycol) diacrylate; in vivo; vascularization
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.
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.
Prodrug; cancer; hydrogel; drug targeting; drug activation
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.
nanoparticle, PLGA; chitosan; adsorption isotherm; surface modification
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.
controlled release; cancer; polymer; cisplatin; vaginal delivery; human papilloma virus (HPV)