Anti-tumour effects of PIM kinase inhibition on progression and chemoresistance of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a biologically aggressive cancer. Targeted therapy is in need to tackle challenges in the treatment perspective. A growing body of evidence suggests a promising role of pharmacological inhibition of PIM (proviral integration site for Moloney murine leukaemia virus) kinase in some human haematological and solid cancers. Yet to date, the potential application of PIM inhibitors in HCC is still largely unexplored. In the present study we investigated the pre-clinical efficacy of PIM inhibition as a therapeutic approach in HCC. Effects of PIM inhibitors on cell proliferation, migration, invasion, chemosensitivity, and self-renewal were examined in vitro. The effects of PIM inhibitors on tumour growth and chemoresistance in vivo were studied using xenograft mouse models. Potential downstream molecular mechanisms were elucidated by RNA sequencing (RNA-seq) of tumour tissues harvested from animal models. Our findings demonstrate that PIM inhibitors SGI-1776 and PIM447 reduced HCC proliferation, metastatic potential, and self-renewal in vitro. Results from in vivo experiments supported the role of PIM inhibition in suppressing of tumour growth and increasing chemosensitivity of HCC toward cisplatin and doxorubicin, the two commonly used chemotherapeutic agents in trans-arterial chemoembolisation (TACE) for HCC. RNA-seq analysis revealed downregulation of the MAPK/ERK pathway upon PIM inhibition in HCC cells. In addition, LOXL2 and ICAM1 were identified as potential downstream effectors. Taken together, PIM inhibitors demonstrated remarkable anti-tumourigenic effects in HCC in vitro and in vivo. PIM kinase inhibition is a potential approach to be exploited in formulating adjuvant therapy for HCC patients of different disease stages. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Multifunctional Microparticles Incorporating Gold Compound Inhibit Human Lung Cancer Xenograft.

PURPOSE: Gold porphyrin (AuP) is a complex that has been shown to be potent against various tumors. A biocompatible interpenetrating network (IPN) system comprised of polyethyleneglycol diacrylate (PEGdA) and chemically-modified gelatin has been shown to be an effective implantable drug depot to deliver AuP locally. Here we designed IPN microparticles complexed with AuP to facilitate intravenous administration and to diminish systemic toxicity. METHODS: We have synthesized and optimized an IPN microparticle formulation complexed with AuP. Tumor cell cytotoxicity, antitumor activity, and survival rate in lung cancer bearing nude mice were analyzed. RESULTS: IPN microparticles maintained AuP bioactivity against lung cancer cells (NCI-H460). In vivo study showed no observable systemic toxicity in nude mice bearing NCI-H460 xenografts after intravenous injection of 6 mg/kg AuP formulated with IPN microparticles. An anti-tumor activity level comparable to free AuP was maintained. Mice treated with 6 mg/kg AuP in IPN microparticles showed 100% survival rate while the survival rate of mice treated with free AuP was much less. Furthermore, microparticle-formulated AuP significantly reduced the intratumoral microvasculature when compared with the control. CONCLUSION: AuP in IPN microparticles can reduce the systemic toxicity of AuP without compromising its antitumor activity. This work highlighted the potential application of AuP in IPN microparticles for anticancer chemotherapy.

A Multifunctional Hydrogel Delivers Gold Compound and Inhibits Human Lung Cancer Xenograft.

PURPOSE: Interpenetrating network system (IPN), consisting of polyethylene glycol (PEG) -diacrylate (PEGdA) and modified gelatin, is a biocompatible and biodegradable hydrogel and has been studied for the local delivery of bioactive molecules and drugs. Gold(III) porphyrin(AuP) is a stable metal compound in the development for anticancer application when administered systemically. The aim of this work is to develop a novel formulation for AuP based on IPN for local delivery. METHODS: IPN loaded with AuP hydrogel was optimized and synthesized. Drug release kinetics, cytotoxicity against tumor cells, and antitumor activity in lung cancer bearing nude mice were studied. RESULTS: AuP released from the IPN followed a first order kinetics in vitro. The AuP loaded IPN showed higher cytotoxicity against human lung cancer cell lines compared to IPN only. In mice bearing human lung cancer xenograft, AuP loaded IPN inhibited tumor growth and reduced angiogenesis. No sign of systemic toxicity was observed for all treatment groups. CONCLUSION: AuP loaded IPN provides an improved formulation over systemic delivery for tumor inhibition to complement surgical intervention. Graphical Abstract Injectable multifunctional matrix of polyethylene glycol and gelatin derivatives for the delivery of gold porphyrinto inhibit tumor growth.

Development of cisplatin-loaded hydrogels for trans-portal vein chemoembolization in an orthotopic liver cancer mouse model.

Transarterial chemoembolization is a standard treatment for intermediate-stage hepatocellular carcinoma (HCC). This study evaluated the anti-tumor effect of the semi-interpenetrating network (IPN) hydrogel as a novel embolic material for trans-portal vein chemoembolization (TPVE) in vivo. A nude mice orthotopic HCC model was established, followed by TPVE using IPN hydrogel loaded with or without cisplatin. Portal vein blockade was visualized by MRI and the development of tumor was monitored by IVIS Spectrum Imaging. Tumor proliferation and angiogenesis were evaluated by Ki67 and CD34 staining respectively. Intra-tumor caspase 3, Akt, ERK1/2, and VEGF activation were detected by Western Blot. 18 F-FMISO uptake was evaluated by microPET-MRI scanning. IPN hydrogel first embolized the left branch of portal vein within 24 hours and further integrated into the intra-tumor vessels during 2 weeks after the treatment. Mice treated with cisplatin-loaded hydrogels exhibited a significant decrease in tumor growth, along with lower plasma AFP levels as compared to hydrogel-treated and untreated tumor-bearing mice. By Ki67 and CD34 staining, the TPVE with IPN hydrogel suppressed tumor proliferation and angiogenesis. In addition, increased tumor apoptosis shown by up-regulation of caspase 3 with decreased expressions of tumor cell survival indicators Akt and ERK1/2 were observed in the treatment groups. Consistent with the decreased expression of VEGF after TPVE, hypoxia level in the tumor was also reduced as indicated by 18 F-FMISO uptake level. IPN hydrogel-based TPVE significantly suppressed the tumor development by regulating intra-tumor angiogenesis and cell survival in an orthotopic HCC mouse model, suggesting a viable embolic agent for transarterial chemoembolization.

Drug release kinetics and transport mechanisms from semi-interpenetrating networks of gelatin and poly(ethylene glycol) diacrylate.

PURPOSE: To elucidate the key parameters affecting solute transport from semi-interpenetrating networks (sIPNs) comprised of poly(ethylene glycol) diacrylate (PEGdA) and gelatin that are partially crosslinked, water-swellable and biodegradable. Effects of material compositions, solute size, solubility, and loading density have been investigated. MATERIALS AND METHODS: sIPNs of following gelatin/PEGdA weight-to-weight ratios were prepared: 10:15, 10:20, 10:30, 15:15, 20:15. Five model solutes of different physicochemical properties were selected, i.e. silver sulfadiazine (AgSD), bupivacaine hydrochloride (Bup), sulfadiazine sodium (NaSD), keratinocyte growth factor (KGF), and bovine serum albumin conjugated with fluorescein isothiocyanate (BSA-FITC). Release studies were performed and the results were analyzed using three hydrogel based common theories (free volume, hydrodynamic and obstruction). RESULTS: The release kinetics of model solutes was influenced by each factor under investigation. Specifically, the initial release rates and intra-gel diffusivity decreased with increasing PEGdA content or increasing solute molecular weight. However, the initial release rate and intra-gel diffusivity increased with increasing gelatin content or increasing solute water solubility, which contradicted with the classical hydrogel based solute transport theories, i.e. increasing polymer volume leads to decreased solute diffusivity within the gel. CONCLUSION: This analysis provides structure-functional information of the sIPN as a potential therapeutic delivery matrix.

Extended interaction of beta1 integrin subunit-deficient cells (GD25) with surfaces modified with fibronectin-derived peptides: Culture optimization, adhesion and cytokine panel studies.

The modification of biomaterials with extracellular matrix-mimicking factors is a common technique used to influence the cellular response through integrin-mediated signaling. The inherent limitations of antibody-inhibition studies necessitate the use of complementary methods to block integrin function to confirm cell-surface interaction. In this study, we employed a beta1 integrin-deficient cell line, GD25, to investigate the role of beta1 subunit in cell adhesion and subsequent cytokine (granulocyte macrophage colony stimulating factor; interleukin (IL)-1alpha; IL-1beta; IL-6; monocyte chemoattractant protein-1; regulated upon activation, normal T-cell expressed, and secreted; tumor necrosis factor-alpha) release kinetics in the presence of tissue culture polystyrene (TCPS) and semi-interpenetrating polymer networks (sIPN) modified with fibronectin (FN)-mimic peptides (RGD, PHSRN). Culture conditions (i.e. seeding density, medium, serum supplementation) were optimized for long-term observation. Differences in cell adhesion, cell viability and cytokine release behavior were dependent on the presence of the beta1 integrin subunit, FN, sIPN cast method and peptide identity. By comparing two complementary techniques for assaying integrin function, we observed both similarities (i.e. decreased adhesion to FN-absorbed TCPS and increased IL-1beta release at 96h) and differences (i.e. no difference in adhesion or IL-1beta release in the presence of different sIPN surfaces) when the function of the beta1 subunit was blocked in cell adhesion and signaling in the presence of biomaterials.

The interrelated role of fibronectin and interleukin-1 in biomaterial-modulated macrophage function.

Macrophages play a critical role in mediating the host response to biomaterials, perhaps most notably by guiding the host inflammatory response through the release of inflammatory molecules such as the cytokine interleukin-1 (IL-1). The extent of the macrophage response following interaction with the biomaterial surface contributes greatly to device efficacy, yet the molecular mechanisms of this interaction are still unclear. The extracellular matrix (ECM) protein fibronectin (FN) is recognized by macrophages and frequently used in biomaterial modification to elicit greater cellular adhesion and tissue integration. Macrophage interaction with FN and other ECM molecules on the biomaterial surface has been shown to induce a variety of inflammatory responses, thus both FN and IL-1 can be utilized as model molecules to better understand the mechanisms of material-mediated macrophage responses. This literature review presents a comprehensive survey of past and current research on the interrelated role of IL-1, FN, and FN-derivatives in determining biomaterial-modulated macrophage function.

Macrophage matrix metalloproteinase-2/-9 gene and protein expression following adhesion to ECM-derived multifunctional matrices via integrin complexation.

Macrophages are commonly observed at the biomaterial-tissue interface and interact with the extracellular matrix (ECM) mainly by integrin receptors to play a critical role in ECM turnover by secreting matrix metalloproteinases (MMPs). To investigate beta1 and beta3 containing integrin-mediated adhesion and subsequent MMP-2/-9 protein and gene expression in human blood-derived monocytes, biofunctional peptides immobilized onto flexible polyethylene glycol (PEG) arms were grafted onto a gelatin-based interpenetrating network (IPN). Adherent monocyte density was dramatically greater in the presence of RGD immobilized onto flexible PEG arms of the gelatin-based IPN. Pretreatment of monocytes with either anti-integrin beta1 or beta3 led to a significant decrease in adherent cell density on RGD-PEG-grafted IPNs. MMP-2 and MMP-9 protein and MMP-9 mRNA expression increased in the presence of IPNs initially, independent of ligand identity. Anti-integrin beta1 or beta3 antibody pretreatment of monocytes led to a general decrease in MMP-2/-9 protein expression. These results demonstrate the importance of beta1 and beta3 containing integrins in mediating monocyte adhesion onto RGD immobilized onto flexible PEG arms of the IPN. The results also reveal that MMP-2/-9 protein and gene expression is influenced by the presence of gelatin and not the ligands immobilized on the PEG arms of the IPN.

Monocytic U937 adhesion, tumor necrosis factor-alpha and interleukin-1 beta expression in response to gelatin-based networks grafted with arginine-glycine-aspartic acid and proline-histidine-serine-arginine-asparagine oligopeptides.

In this study we synthesized gelatin-based, tissue-engineering, interpenetrating network (IPN) scaffolds immobilized with fibronectin (FN)-derived peptides to assess monocyte-biomaterial interaction. Human promonocytic U937 cells were seeded onto peptide-grafted IPN or tissue-culture polystyrene plate (TCPS) pre-adsorbed with FN or FN-derived peptides. The presence of RGD influenced U937 density on IPN. Interleukin-1 beta (IL-1beta) messenger ribonucleic acid (mRNA) expression in adherent U937 on treated TCPS was slightly upregulated at 4 h. Tumor necrosis factor alpha (TNF-alpha) and IL-1beta mRNA expression in adherent U937 on all IPNs was generally downregulated at 4 h. This downregulation of IL-1beta mRNA apparently varied in IPNs grafted with different ligand and was still present at 24 h. TNF-alpha and IL-1beta proteins released from U937 on treated TCPS were comparable with the control at 24 h, but TNF-alpha and IL-1beta protein expression in U937 on IPNs was lower at 24 h than on the TCPS control. The results indicate that the tissue-engineering substrate and the bioactive peptides modulate the initial U937 adhesion and the subsequent inflammatory cytokine gene and protein expression.

Monocyte activation in response to polyethylene glycol hydrogels grafted with RGD and PHSRN separated by interpositional spacers of various lengths.

Polyethylene glycol (PEG) is often cited as a "stealth" polymer, capable of resisting both protein adsorption and cell adhesion. By extension, PEG would then be expected to limit the host response. Monocyte-derived macrophages play an integral role in inflammation, and thus their response to a material can potentially dictate the overall host response to a biomaterial. In the present study, monocyte responses following interaction with a photopolymerized PEG hydrogel were compared with those from standard tissue culture polystyrene (TCPS). Additionally, the effect of the spacing between RGD and PHSRN, the corresponding synergy sequence on fibronectin (FN), was evaluated using peptides with differing spacer lengths grafted to the PEG hydrogel. Monocyte adherent density on the PEG-only hydrogel was comparable with that of TCPS; however, the secretion of the proinflammatory molecules interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF) increased dramatically following monocyte interaction with PEG-only hydrogels as compared with TCPS. The matrix metalloproteinase-2 (MMP-2) concentration was similar for all surfaces, while both the matrix metalloproteinase-9 (MMP-9) and FN concentrations were above the range of the assay for all substrates. Cell density was higher on the PHSRNG(13)RGD grafted substrate as compared with PHSRNG(6)RGD, but neither sequence increased cell density versus RGD alone. Although protein concentration did sometimes vary with different peptides, this variation was minimal in comparison with the surface effects between TCPS and the PEG-only hydrogel. This study explores the roles of PEG and FN-derived peptides on monocyte activation.

Interpenetrating polymer networks containing gelatin modified with PEGylated RGD and soluble KGF: synthesis, characterization, and application in in vivo critical dermal wound.

The purpose of this study was to evaluate the biocompatibility and the efficacy in wound healing of a gelatin-based interpenetrating polymer network (IPN) containing poly(ethylene glycol) (PEG)-ylated RGD and soluble KGF-1 (RGD-IPN+KGF). IPNs were applied to full-thickness wounds on a rat model. Wound healing was assessed through histological grading of the host response and percent area contraction at 2 days, 1 week, 2 weeks, and 3 weeks. A control IPN containing unmodified gelatin (unmod-IPN) and a conventional clinical bandage were applied to similar wounds and also evaluated. During the first week of healing, the unmod-IPN and conventional dressing wound showed a greater amount of contraction than that of RGD-IPN+KGF. However, by 3 weeks the extent of wound contraction was comparable between treatments. The RGD-IPN+KGF treated wound demonstrated lower macrophage and fibroblast densities at 3 weeks as compared to unmod-IPN treated wounds. RGD-IPN+KGF acted as a tissue scaffold while preventing the entry of foreign bodies, advantages not seen with the conventional dressing. The extent of cellularity and extracellular matrix organization was higher for wounds healed with RGD-IPN+KGF than those healed with unmod-IPN. These results indicate that both soluble and immobilized bioactive factors can be incorporated into our IPN platform to enhance the rate and the quality of dermal wound healing.

Effect of surface-adsorbed proteins and phosphorylation inhibitor AG18 on intracellular protein expression in adherent macrophages.

Macrophages are believed to play an important role in the host inflammatory response to implanted biomaterials. However, the mechanism of macrophage adhesion to protein-adsorbed substrates and the subsequent activation and inflammation is unresolved. Previously the effect of various surface-adsorbed proteins and increasing concentrations of phosphorylation inhibitor AG18 on intracellular protein expression levels in adherent human monocytic cell line U937 was identified using SDS-PAGE and densitometry. The protein ligands and AG18 concentrations up or down regulated the expression of a set of proteins ranging from approximately 200 to approximately 23 kDa. In the present work, HPLC coupled tandem mass spectroscopy (LC/MS) was used to identify proteins in these bands. We hypothesized that key proteins in macrophage adhesion and activation could be identified by observing protein expression resulting from various surface-adsorbed ligands and AG18 concentrations. Increasing concentrations of AG18 down or up regulate protein expression in adherent U937 on PBS-adsorbed TCPS at approximately 52, approximately 42 and approximately 23 kDa. AG18 concentrations had no effect on cells on albumin (Alb)-adsorbed surfaces but regulated different protein expression in adherent U937 on fibronectin (FN)-adsorbed TCPS at 40 and 80 microm AG18. Both Alb and FN regulate distinct sets of proteins in adherent cells as surface-adsorbed ligands. Based on the data from LC/MS, both surface associated ligand and increasing concentrations of AG18 modulate shifts in intracellular signaling.

Keratinocyte-fibroblast paracrine interaction: the effects of substrate and culture condition.

Interactions between epidermal-dermal cells via soluble factors provide important signals in regulating the reepithelialization of wounded skin. For example, keratinocytes regulate the expression of keratinocyte growth factor (KGF) in fibroblasts through the release of interleukin-1beta (IL-1beta). In this study, a previously developed polyethyleneglycol-based interpenetrating network (IPN) system was utilized as a platform for the delivery of keratinocyte-active factors. The effect of substrate chemistry, culture condition, and the delivery of exogenous keratinocyte-active factors on the keratinocyte behavior and the keratinocyte-fibroblast paracrine relationship was delineated. Adherent keratinocyte density on TCPS and glutaraldehyde-fixed gelatin hydrogels but not on IPN was significantly increased with culture time in the presence of growth supplements independent of the released KGF from the gelatin hydrogel and IPN. In the presence of fibroblasts, adherent keratinocyte density on gelatin hydrogels was higher than that without fibroblasts. This phenomenon was not observed on IPN and polycarbonate membrane. In summary, the delivered exogenous huKGF (i.e., released from a biomaterial matrix) operates in tandem with fibroblasts in regulating keratinocyte activation (i.e., IL-lbeta release and adhesion) in a surface-dependent manner. Immunoassay analysis of cell culture keratinocyte-fibroblast paracrine relationship as characterized by IL-1beta and KGF could not be established in the presence of IPNs, 0.1% glutaraldehyde-fixed gelatin hydrogels, and polycarbonate membranes.

Intracellular protein phosphorylation in adherent U937 monocytes mediated by various culture conditions and fibronectin-derived surface ligands.

Macrophages play a central role in the normal healing process after tissue injury and the host response to foreign objects such as biomaterials. The process leading to macrophage adhesion and activation on protein-adsorbed substrates is complex and unresolved. While the use of primary cells offers clinical relevancy, macrophage cell lines offer unique advantages such as availability and relatively homogeneous phenotype as models to probe the molecular mechanism of cell-surface interaction. Our goal was to better characterize the effect of the culture condition and surface-associated ligands on the extent of U937 adhesion. Tyrosine phosphorylation of intracellular proteins was surveyed as a basis to seek a greater understanding of the molecular mechanism involved in mediating U937 adhesion on various ligand-adsorbed surfaces. U937 viability and adhesion on tissue culture polystyrene (TCPS) increased with (i) increasing serum level, (ii) decreasing tyrosine phosphorylation inhibitor AG18 concentration, or (iii) increasing culture time. The adsorption of various adhesion proteins such as fibronectin and peptide ligands (i.e., RGD, PHSRN) on TCPS did not significantly increase the adherent density of U937 when compared with albumin and PBS ligand controls. However, ligand identity and the presence of phorbol myristate acetate dramatically affected the extent (i.e., increase or decrease) and the identity (i.e., molecular weight) of phosphotyrosine proteins in adherent U937 in a time-dependent manner. The extent and identity of phosphotyrosine proteins did not exhibit a clear AG18 dose dependency, rather the level of tyrosine phosphorylation for a distinct group of proteins was either increased or decreased for a given AG18 concentration.

Macrophage adhesion on gelatin-based interpenetrating networks grafted with PEGylated RGD.

Human blood-derived macrophage adhesion on interpenetrating networks (IPNs) composed of PEGylated RGD-modified gelatin and poly(ethylene glycol) diacrylate was studied. The interaction between biomaterial immobilized with biofunctional peptides such as RGD and macrophages is central in the design of tissue-engineering scaffolds. PEGylated RGD-modified gelatin was synthesized via several steps involving PEG derivations and characterized by high-performance liquid chromatography, mass spectroscopy, gel permeation chromatography, and the trinitrobenzenesulfonic acid method. IPNs containing modified or unmodified gelatin were cultured with human macrophages and monitored at 2, 24, 96, and 168 h. At each time point, IPNs containing gelatin modified with PEGylated RGD showed a comparable adherent macrophage density as tissue culture polystyrene and a significantly higher cell density than other IPN formulations containing unmodified gelatin or gelatin modified with PEGylated triglycine. Although surface-immobilized RGD can serve to mediate the adhesion of different cell types on the biomaterial surface, the interaction of RGD with immune/inflammatory cells such as macrophages should also be considered when assessing the potential host response of tissue-engineering scaffolds.

Human macrophage adhesion on fibronectin: the role of substratum and intracellular signalling kinases.

Fibronectin and Arg-Gly-Asp (RGD)- and/or Pro-His-Ser-Arg-Asn (PHSRN)-containing oligopeptides were immobilized onto physicochemically distinct substrata: polyethyleneglycol-based networks or tissue culture polystyrene (TCPS). The role of selected signalling kinases in the adhesion of human primary blood-derived macrophages on these modified substrata was investigated. We demonstrated that the protein tyrosine kinase (PTK) or protein serine/threonine kinase (PSK) dependency and the PTK-PSK cross-talk compensation for macrophage adhesion varied dynamically with the substratum modification and the culture time. The inhibition of MAPK kinase (MAPKK) decreased macrophage adhesion on TCPS, whereas the inhibition of phosphoinositide-3 kinase (PI3 kinase) decreased macrophage adhesion on networks at 24 h. The PI3 kinase-protein kinase C (PKC)-MAPK cascade was involved in macrophage adhesion on fibronectin-preadsorbed TCPS or networks but not on fibronectin-grafted networks. This fibronectin-mediated adhesion signalling involved both RGD and PHSRN sequences in a form of G(3)RGDG(6)PHSRNG on TCPS but not on networks. Furthermore, G(3)RGDG(6)PHSRNG grafted onto networks evoked unique signalling in macrophage adhesion from that preadsorbed onto networks. Thus, macrophage adhesion and the role of selected signalling kinases were modulated by the substratum and the ligand conjugation method.

Mesenchymal Stromal/Stem Cell and Minocycline-Loaded Hydrogels Inhibit the Growth of Staphylococcus aureus that Evades Immunomodulation of Blood-Derived Leukocytes.

Mesenchymal stromal/stem cells (MSCs) have demonstrated favorable wound healing properties in addition to their differentiation capacity. MSCs encapsulated in biomaterials such as gelatin and polyethylene glycol (PEG) composite hydrogels have displayed an immunophenotype change that leads to the release of cytokines and growth factors to accelerate wound healing. However, therapeutic potential of implanted MSC-loaded hydrogels may be limited by non-specific protein adsorption that facilitates adhesion of bacterial pathogens such as planktonic Staphylococcus aureus (SA) to the surface with subsequent biofilm formation resistant to immune cell recognition and antibiotic activity. In this study, we demonstrate that blood-derived primary leukocytes and bone marrow-derived MSCs cannot inhibit colony-forming abilities of planktonic or biofilm-associated SA. However, we show that hydrogels loaded with MSCs and minocycline significantly inhibit colony-forming abilities of planktonic SA while maintaining MSC viability and multipotency. Our results suggest that minocycline and MSC-loaded hydrogels may decrease the bioburden of SA at implant sites in wounds, and may improve the wound healing capabilities of MSC-loaded hydrogels.

Intracellular protein tyrosine phosphorylation of adherent human macrophages on adsorbed fibronectin.

Fibronectin (FN) was pre-adsorbed onto physicochemically distinct substrates: polyethyleneglycol-based networks or tissue culture polystyrene (TCPS). The role of these substrates in modulating FN-mediated intracellular protein tyrosine phosphorylation and cell adhesion was analyzed with human primary blood derived macrophages. Although macrophage adhesion on both FN-pre-adsorbed TCPS and networks was similarly dependent on protein tyrosine kinase (PTK) and protein serine/threonine kinase (PSK), the compensation between PTK and PSK, and the involvement of signaling molecules (such as protein kinase C (PKC) isoforms) were distinct between the substrates. The pattern and the extent of tyrosine phosphorylation of several proteins (i.e. approximately 70, approximately 44, approximately 30kDa) were differentially regulated by PKCs. FN-derived peptides were employed to probe this material-dependency in macrophage adhesion and tyrosine phosphorylation. The PHSRN domain in the peptide sequence was predominant in mediating this substrate-dependent FN signaling event. We conclude that the tyrosine phosphorylation and the cross talk between PTK and PSK are modulated by FN and the substrate onto which the protein is adsorbed.

Synthesis and physicochemical analysis of gelatin-based hydrogels for drug carrier matrices.

This study examined the interrelated effect of environmental pH, gelatin backbone modification and crosslinking modality on hydrogel morphology, surface hydrophilicity, in vitro swelling/degradation kinetics, in vitro drug release kinetics and in vivo degradation, inflammatory response and drug release activity. The percent glutaraldehyde fixation had a greater impact on the morphology of the dehydrated hydrogels than gelatin modification. Any decrease in percent glutaraldehyde fixation and/or modification of gelatin with polyethylene glycol dialdehyde (PEG-dial) and/or ethylenediaminetetraacetic dianhydride (EDTAD) increased hydrogel surface hydrophilicity. Swelling/degradation studies showed that modification of gelatin with PEG-dial generally increased the time to reach the maximum swelling weight ratio (T(max)) and the time to failure by hydrolysis (T(fail)), but had little effect on the maximum swelling weight ratio (R(max)) and the weight ratio at failure (R(fail)). Modification of gelatin with EDTAD generally had no effect on T(max) and T(fail), but increased R(max) and R(fail). Modification of gelatin with PEG-dial and EDTAD increased R(max), but had no effect on T(max), R(fail), or T(fail). Decreasing percent glutaraldehyde fixation generally increased R(max) and R(fail) but decreased T(max) and T(fail). Decreasing environmental pH from 7.4 to 4.5 had no effect on any swelling/degradation properties. In vitro drug release studies showed that modification of gelatin with PEG-dial and/or EDTAD generally decreased the maximum mass ratio of drug released (D(max)) and the time to reach D(max) (T(dmax)). Percent glutaraldehyde fixation did not significantly affect D(max) or T(dmax) (except for EDTAD-modified gelatin hydrogels). In vivo studies showed that gelatin-based hydrogels elicited comparable levels of acute and chronic inflammatory response as that of the empty cage control by 21 d.

Cell interaction with protein-loaded interpenetrating networks containing modified gelatin and poly(ethylene glycol) diacrylate.

The effects of modification to an interpenetrating network (IPN) system composed of gelatin and poly(ethylene glycol) diacrylate were characterized by protein release kinetics, fibroblast adhesion, and in vivo host response. The maximum cumulative percent of parvalbumin released from various IPN formulations ranged from 17.6+/-3.2% to 56.9+/-35.4% over 2-96h. Despite modifying gelatin with ethylenediaminetetraacetic dianhydride and/or monomethoxy poly(ethylene glycol) monoacetate ester or increasing the gelatin content, the largest amount of parvalbumin released occurred within 24h, prior to material bulk degradation. Over the time period evaluated, little (i.e. <1%) of the basic fibroblast growth factor (bFGF) loaded into the IPNs evaluated was released, independent of modifications made to the IPN formulation. Fibroblast adhesion to IPNs with or without loaded bFGF was quantified. The adherent fibroblast density on the IPNs was significantly lower than that of TCPS controls at all times independent of the IPN formulation tested and bFGF loading. Select IPN formulations elicited a comparable level of acute and chronic inflammatory response in vivo when compared with the gelatin and poly(ethylene glycol) diacrylate starting materials. IPNs provide a minimal cell-active surface that could be employed as delivery matrices and for further bioconjugation to mediate specific cellular response.