Effects of precursor and cross-linking parameters on the properties of dextran-allyl isocyanate-ethylamine/poly(ethylene glycol diacrylate) biodegradable hydrogels and their release of ovalbumin.

In this paper, we studied the effects of molecular weight of poly(ethylene glycol diacrylate) (PEGDA) precursor, the degree of substitution (DS) of both allyl isocyanate (AI) and amine groups in dextran-based precursor (Dex-AE), and photoinitiator concentration on Dex-AE/PEGDA hydrogel formation and its ovalbumin (OVA) release. FT-IR spectra showed chemical bond interaction between amine and urethane groups of the hydrogel carriers with OVA. The increase in PEGDA molecular weight led to a faster OVA release because of a more open gel network structure. The study on the DS of AI in Dex-AE precursor showed that an increase in AI did not result in a prominent gel network structure difference. However, the urethane groups in Dex-AE precursor showed some interactions with OVA and, thus, resulted in a slower release rate. The incorporation of amine group into Dex-AE precursor did not affect the gel network structure, but reduced the OVA release rate, and the level of reduction increased with an increasing amine group substitution into the Dex-AE precursor. This reduction could be attributed to the interaction between the amine groups in the gel carrier and OVA. An increase in the photoinitiator concentration showed no effect on the gel network structure or OVA release.

Cationic poly(VCL-AETA) hydrogels and ovalbumin (OVA) release in vitro.

The objective of this research is to explore the synthesis of a new family of water soluble polycationic copolymeric precursors that could be photo-crosslinked into hydrogels. The in vitro control release of ovalbumin protein (OVA) from this family of hydrogels was also studied to assess the biomedical potential of this new family polycationic hydrogels. A series of novel poly(VCL-AETA) copolymer hydrogels was fabricated in an aqueous medium via photo-induced polymerization and crosslinking of hydrophobic N-vinylcaprolactam (VCL) and hydrophilic [2-(acryloxy)ethyl]trimethylammonium chloride (AETA) monomers over a wide range of VCL to AETA feed molar ratios of 2:1, 1:1, 1:2, 1:5. N,N'-methylene bisacrylamide (MBA) was used as a crosslinker. Ovalbumin (OVA), a model antigen, was preloaded into poly(VCL-AETA) hydrogel precursors and its release profiles in pH 7.4 PBS at 37 degrees C were investigated as a function of VCL to AETA monomer feed ratios over a period of 4 weeks. The in vitro results showed that OVA initial burst and subsequent sustained releases could be controlled by 3 material parameters: the hydrophobic VCL to hydrophilic AETA monomer feed ratios, crosslinking density and hydrogel degradation rate. Thus, the hydrophobic-hydrophilic VCL-AETA hydrogel network for controlled OVA release could offer advantages over organic solvent-based single component polymer system. However, these in vitro OVA release profiles may change in an in vivo environment.

CTLA-4 blockade augments human T lymphocyte-mediated suppression of lung tumor xenografts in SCID mice.

Previous studies by others using transplantable murine tumor models have demonstrated that the administration of antibodies that block CTLA-4 interaction with B7 can provoke the elimination of established tumors, and that the tumor suppression is mediated by T-cells and/or cells expressing NK1.1. Studies from our lab have established in a human/severe combined immunodeficient (SCID) mouse chimeric model that autologous peripheral blood leukocytes (PBL) can suppress the growth of tumor xenografts in a PBL dose-dependent fashion, and that this suppression is dependent upon the patient's T and NK cells. Using this human/mouse chimeric model, we sought to determine whether an antibody blockade of CTLA-4 would enhance the anti-tumor response of a patient's PBL. It was first important to determine whether the tumor suppression observed in the SCID model was dependent upon CD28/B7 co-stimulation. Blockade of B7 with a human CTLA-4-Ig fusion protein completely abrogated the lymphocyte-mediated tumor suppression, confirming in this model that tumor suppression is dependent upon a CD28/B7 co-stimulation. Using two different CTLA-4 specific monoclonal antibodies, we observed that CTLA-4 blockade significantly enhanced the human lymphocyte-mediated tumor suppression in mice co-engrafted with PBL and tumor cells. This enhancement was observed in both an allogeneic setting (in which the PBL were allogeneic with respect to the tumor) and an autologous setting (in which the PBL and tumor were from the same patient). These results sustain the notion that human anti-tumor immune response can be augmented (in vivo) by blocking the interaction between CTLA-4 and B7.

Tissue-specific gene expression of head and neck squamous cell carcinoma in vivo by complementary DNA microarray analysis.

OBJECTIVES: To identify distinct gene expression profiles of human head and neck squamous cell carcinomas (HNSCCAs) using complementary DNA (cDNA) microarray analysis and to create a preliminary, comprehensive database of HNSCCA gene expression. PATIENTS AND METHODS: Nine patients with histologically confirmed HNSCCAs, staged according to the American Joint Committee on Cancer, were enrolled. The HNSCCA tumor tissue and normal mucosal tissue were harvested at the time of surgery. A cDNA library was constructed from the paired fresh-frozen human surgical specimens of HNSCCAs and nonmalignant epithelial tissues. Biotinylated RNA was transcribed from the cDNA library and hybridized to high-density microarrays containing approximately 12 000 human genes. Altered gene expression of HNSCCAs was identified by comparison to corresponding normal mucosal tissues after a bayesian statistical analysis of variance. Results were analyzed using the gene database of the National Institutes of Health. Hierarchical clustering of the genomic data sets was determined by similarity metrics based on Pearson correlation. RESULTS: Hierarchical clustering analysis revealed that the gene expression profiles obtained from the nonselected panel of 12 000 genes could distinguish the tumors from nonmalignant tissues. Gene expression changes were reproducibly observed in 227 genes representing previously identified chemokines, tumor suppressors, differentiation markers, matrix molecules, membrane receptors, and transcription factors that correlated with neoplasia, including 46 previously uncharacterized genes. Moreover, significant expression of the collagen type XI alpha1 gene and a novel gene was reproducibly observed in all 9 tumors, whereas these genes were virtually undetectable in their corresponding, adjacent nonmalignant tissues. CONCLUSIONS: Complementary DNA microarray analysis of human HNSCCAs has produced a preliminary, comprehensive database of tumor-specific gene expression profiles and provided important insights into modeling gene expression changes implicated in carcinogenesis. A large-scale analysis of gene expression carries the future potential of identifying sensitive molecular markers for early tumor detection, prognosis, and novel targets for interceptive therapeutics.

Biodegradable polymer-mediated intratumoral delivery of cisplatin for treatment of human head and neck squamous cell carcinoma in a chimeric mouse model.

BACKGROUND: The effectiveness of chemotherapeutic agents is proportional to the dose of the agents at their targets; however, the dose is limited by systemic toxicity. Attempts have been made to improve therapeutic effectiveness by increasing maximum tolerated dose (MTD) of chemotherapeutic agents using various local and regional drug delivery systems. Herein we report the use of an injectable biodegradable polymer to deliver cisplatin for intratumoral treatment of human head and neck squamous cell carcinoma (HNSCC) in a chimeric mouse model. The objectives of this research project were (1) to determine the release kinetics of cisplatin from the polymer delivery system, (2) to identify the MTD of polymer-delivered cisplatin, and (3) to evaluate its therapeutic efficacy. METHODS: To determine the in vivo release kinetics, cisplatin-loaded polymer was injected subcutaneously into rats. Implants were removed and analyzed for remaining cisplatin by a high-performance liquid chromatography technique. Sera from these rats were assayed for platinum by atomic absorption spectrophotometry. For MTD determination, SCID mice were engrafted subcutaneously with fresh biopsy specimens of HNSCC. Various doses of free or polymer-loaded cisplatin were injected intratumorally. MTD was estimated based on the threshold at which all mice survived. The antitumor efficacy of free and polymer-loaded cisplatin at their respective MTD was assayed on the same chimeric mouse model. RESULTS: The polymer delivery system released 80% of the loaded cisplatin in vivo over a 7-day period. The polymer-delivered cisplatin exhibited higher MTD (36 mg/kg) than free cisplatin (18 mg/kg) and had a statistically significant tumor suppression effect compared with free cisplatin when used at their respective MTD. CONCLUSIONS: The polymer delivery system can sustain cisplatin release for a period of 7 days. It can increase MTD and potentially enhance the antitumor efficacy of cisplatin against human head and neck cancers.

Human CD4+ effector T cells mediate indirect interleukin-12- and interferon-gamma-dependent suppression of autologous HLA-negative lung tumor xenografts in severe combined immunodeficient mice.

A human/severe combined immunodeficient mouse chimeric model was used to demonstrate that peripheral blood leukocytes (PBLs) from a patient with lung cancer completely suppress the growth of an autologous tumor in a PBL dose-dependent fashion repeatedly and over a 4-year period. Suppression of the patient's tumor required CD4+ T cells, CD56+ natural killer cells, and CD14+ monocytes/macrophages, but was completely independent of CD8+ T cells. The CD4+ effector cells promoted tumor killing indirectly because direct tumor recognition and killing are precluded by the absence of MHC class I and II molecules on the tumor cells. Tumor suppression was found to require both human interleukin-12 (IL-12) and IFN-gamma, which were produced and released by the patient's monocytes and T cells, respectively. These results establish that human CD4+ T cells present in the peripheral blood of a patient with lung cancer are able to orchestrate cytokine-dependent killing of an autologous MHC-negative tumor indirectly and without codependence on CD8+ T cells. We conclude that human tumor suppression is achieved in vivo even in the absence of MHC molecules on tumor cells. This tumor suppression is mediated indirectly by cytokines produced by the patient's PBLs that ultimately initiate tumor killing via several, presently incompletely defined mechanisms.