Design and synthesis of self-degradable antibacterial polymers by simultaneous chain- and step-growth radical copolymerization.

Self-degradable antimicrobial copolymers bearing cationic side chains and main-chain ester linkages were synthesized using the simultaneous chain- and step-growth radical polymerization of t-butyl acrylate and 3-butenyl 2-chloropropionate, followed by the transformation of t-butyl groups into primary ammonium salts. We prepared a series of copolymers with different structural features in terms of molecular weight, monomer composition, amine functionality, and side chain structures to examine the effect of polymer properties on their antimicrobial and hemolytic activities. The acrylate copolymers containing primary amine side chains displayed moderate antimicrobial activity against E. coli but were relatively hemolytic. The acrylate copolymer with quaternary ammonium groups and the acrylamide copolymers showed low or no antimicrobial and hemolytic activities. An acrylate copolymer with primary amine side chains degraded to lower molecular weight oligomers with lower antimicrobial activity in aqueous solution. This degradation was due to amidation of the ester groups of the polymer chains by the nucleophilic addition of primary amine groups in the side chains resulting in cleavage of the polymer main chain. The degradation mechanism was studied in detail by model reactions between amine compounds and precursor copolymers.

Immobilization of amphiphilic polycations by catechol functionality for antimicrobial coatings.

A new strategy for preparing antimicrobial surfaces by a simple dip-coating procedure is reported. Amphiphilic polycations with different mole ratios of monomers containing dodecyl quaternary ammonium, methoxyethyl, and catechol groups were synthesized by free-radical polymerization. The polymer coatings were prepared by immersing glass slides into a polymer solution and subsequent drying and heating. The quaternary ammonium side chains endow the coatings with potent antibacterial activity, the methoxyethyl side chains enable tuning the hydrophobic/hydrophilic balance, and the catachol groups promote immobilization of the polymers into films. The polymer-coated surfaces displayed bactericidal activity against Escherichia coli and Staphylococcus aureus in a dynamic contact assay and prevented the accumulation of viable E. coli, S. aureus, and Acinetobacter baumannii for up to 96 h. Atomic force microscopy (AFM) images of coating surfaces indicated that the surfaces exhibit virtually the same smoothness for all polymers except the most hydrophobic. The hydrophobic polymer without methoxyethyl side chains showed clear structuring into polymer domains, causing high surface roughness. Sum-frequency generation (SFG) vibrational spectroscopy characterization of the surface structures demonstrated that the dodecyl chains are predominantly localized at the surface-air interface of the coatings. SFG also showed that the phenyl groups of the catechols are oriented on the substrate surface. These results support our hypothesis that the adhesive or cross-linking functionality of catechol groups discourages polymer leaching, allowing the tuning of the amphiphilic balance by incorporating hydrophilic components into the polymer chains to gain potent biocidal activity.

Sequence-regulated vinyl copolymers by metal-catalysed step-growth radical polymerization.

Proteins and nucleic acids are sequence-regulated macromolecules with various properties originating from their perfectly sequenced primary structures. However, the sequence regulation of synthetic polymers, particularly vinyl polymers, has not been achieved and is one of the ultimate goals in polymer chemistry. In this study, we report a strategy to obtain sequence-regulated vinyl copolymers consisting of styrene, acrylate and vinyl chloride units using metal-catalysed step-growth radical polyaddition of designed monomers prepared from common vinyl monomer building blocks. Unprecedented ABCC-sequence-regulated copolymers with perfect vinyl chloride-styrene-acrylate-acrylate sequences were obtained by copper-catalysed step-growth radical polymerization of designed monomers possessing unconjugated C=C and reactive C-Cl bonds. This strategy may open a new route in the study of sequence-regulated synthetic polymers.

Metal-catalyzed simultaneous chain- and step-growth radical polymerization: marriage of vinyl polymers and polyesters.

All polymerization reactions are categorized into two large different families, chain- and step-growth polymerizations, which are typically incompatible. Here, we report the simultaneous chain- and step-growth polymerization via the metal-catalyzed radical copolymerization of conjugated vinyl monomers and designed monomers possessing unconjugated C horizontal lineC and active C-Cl bonds. Especially, almost ideal linear random copolymers containing both vinyl polymer and polyester units in a single polymer chain were formed by the CuCl/1,1,4,7,10,10-hexamethyltriethylenetetramine- or RuCp*Cl(PPh(3))(2)-catalyzed copolymerization of methyl acrylate (MA) for the chain-growth polymerization and 3-butenyl 2-chloropropionate (1) for the step-growth polymerization. In contrast, other transition metal catalysts, such as CuCl with tris[2-(dimethylamino)ethyl]amine or N,N,N',N'',N''-pentamethyldiethylenetriamine and FeCl(2)/PnBu(3), resulted in branched structures via the concomitant chain-growth copolymerization of 1 with MA. The polymerization mechanism was studied in detail by NMR and MALDI-TOF-MS analyses of the polymerizations as well as the model reactions. Furthermore, a series of copolymers changing from random to multiblock polymer structures were obtained by varying the feed ratios of the two monomers. These copolymers can be easily degraded into lower molecular weight oligomers or polymers via methanolysis of the ester-linkages in the main chain using sodium carbonate.

Splicing variations in the ligand-binding domain of ApoER2 results in functional differences in the binding properties to Reelin.

Reelin plays critical roles in brain formation by binding to apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor. Several isoforms and fragments of Reelin are generated by alternative splicing and proteolytic cleavage. In addition, two splice variants of ApoER2 have been recognized, namely, LA1237 and LA12378, that differ in the number of ligand-binding type A (LA) repeats. Here, we quantitatively investigated the affinity between various isoforms/fragments of Reelin and the ApoER2 splice variants. ApoER2-LA1237 bound rather strongly to the Reelin central fragment than to the fragment bearing Reelin repeat 8 (RR8). ApoER2-LA12378 bound comparably to all Reelin fragments without the C-terminal region. These findings suggest that LA8 of ApoER2 and RR8 interfere with the interaction between the Reelin central fragment and ApoER2. Using a monoclonal antibody that only recognizes ApoER2-LA12378, we found that this variant of ApoER2 was expressed in the cerebral cortical wall and in the internal granule cells of the cerebellum during development. Primary-cultured cortical neurons did not express ApoER2-LA12378, and the extent of signal activation by Reelin fragments was well correlated with their affinity for ApoER2-LA1237. Therefore, proteolytic cleavage of Reelin and alternative splicing of ApoER2 may be involved in the fine regulation of Reelin signaling.

Metal-catalyzed radical polyaddition as a novel polymer synthetic route.

A new class of polymerizations was developed via metal-catalyzed C-C bond forming radical polyaddition; the monomers were designed to have a reactive C-Cl bond, which can be activated by the metal catalysts to generate a carbon radical species, along with a C=C double bond, to which the carbon radical generated from another molecule adds to form a C-C backbone polymer with an inactive C-Cl pendant.

Endoglin (CD105) is a target for an oral DNA vaccine against breast cancer.

Endoglin (CD105), a co-receptor in the TGF-beta receptor complex, is over-expressed on proliferating endothelial cells in the breast tumor neovasculature and thus offers an attractive target for anti-angiogenic therapy. Here we report the anti-angiogenic/anti-tumor effects achieved in a prophylactic setting with an oral DNA vaccine encoding murine endoglin, carried by double attenuated Salmonella typhimurium (dam-, AroA-) to a secondary lymphoid organ, i.e., Peyer's patches . We demonstrate that an endoglin vaccine elicited activation of antigen-presenting dendritic cells, coupled with immune responses mediated by CD8+ T cells against endoglin-positive target cells. Moreover, we observed suppression of angiogenesis only in mice administered with the endoglin vaccine as compared to controls. These data suggest that a CD8+ T cell-mediated immune response induced by this vaccine effectively suppressed dissemination of pulmonary metastases of D2F2 breast carcinoma cells presumably by eliminating proliferating endothelial cells in the tumor vasculature. It is anticipated that vaccine strategies such as this may contribute to future therapies for breast cancer.

DNA-based vaccines activate innate and adaptive antitumor immunity by engaging the NKG2D receptor.

The interaction of NKG2D, a stimulatory receptor expressed on natural killer (NK) cells and activated CD8(+) T cells, and its ligands mediates stimulatory and costimulatory signals to these cells. Here, we demonstrate that DNA-based vaccines, encoding syngeneic or allogeneic NKG2D ligands together with tumor antigens such as survivin or carcinoembryonic antigen, markedly activate both innate and adaptive antitumor immunity. Such vaccines result in highly effective, NK- and CD8(+) T cell-mediated protection against either breast or colon carcinoma cells in prophylactic and therapeutic settings. Notably, this protection was irrespective of the NKG2D ligand expression level of the tumor cells. Hence, this strategy has the potential to lead to widely applicable and possibly clinically useful DNA-based cancer vaccines.

T cell-mediated suppression of angiogenesis results in tumor protective immunity.

Antiangiogenic intervention is known to inhibit tumor growth and dissemination by attacking the tumor's vascular supply. Here, we report that this was achieved for the first time using an oral DNA minigene vaccine against murine vascular endothelial growth factor receptor 2 (FLK-1), a self-antigen overexpressed on proliferating endothelial cells in the tumor vasculature. Moreover, we identified the first H-2Db-restricted epitope, FLK400 (VILT-NPISM), specifically recognized by cytotoxic T lymphocytes (CTLs). Such CTLs were capable of killing FLK-1+ endothelial cells, resulting in suppression of angiogenesis and long-lived tumor protection. The specificity of this immune response was indicated because the DNA vaccine encoding the entire FLK-1 gene also induced a FLK400-specific CTL response. This minigene vaccine strategy provides a more flexible alternative to whole-gene vaccination and facilitates in-depth mechanism studies to tailor DNA vaccines for optimal T-cell activation and tumor protection.

A DNA vaccine targeting Fos-related antigen 1 enhanced by IL-18 induces long-lived T-cell memory against tumor recurrence.

A novel vaccination strategy induced specific CD8(+) T cell-mediated immunity that eradicated spontaneous and experimental pulmonary cancer metastases in syngeneic mice and was also effective in a therapeutic setting of established breast cancer metastases. This was achieved by targeting transcription factor Fos-related antigen 1(Fra-1), overexpressed by many tumor cells, with an ubiquitinated DNA vaccine against Fra-1, coexpressing secretory IL-18. Insight into the immunologic mechanisms involved was provided by adoptive transfer of T lymphocytes from successfully immunized BALB/c mice to syngeneic severe combined immunodeficient (SCID) mice. Specifically, long-lived T memory cells were maintained dormant in nonlymphoid tissues by IL-18 in the absence of tumor antigen. Importantly, a second tumor cell challenge of these SCID mice restored both, robust tumor-specific cytotoxicity and long-lived T-cell memory, capable of eradicating established pulmonary cancer metastases, suggesting that this vaccine could be effective against tumor recurrence.

A DNA vaccine targeting survivin combines apoptosis with suppression of angiogenesis in lung tumor eradication.

A novel strategy achieved the eradication of lung tumor metastases by joint suppression of angiogenesis in the tumor neovasculature and induction of tumor cell apoptosis. This was accomplished by CTLs induced by a DNA vaccine encoding secretory chemokine CCL21 and the inhibitor of apoptosis protein survivin, overexpressed by both proliferating endothelial cells in the tumor vasculature and tumor cells. Oral delivery of this DNA vaccine by doubly attenuated Salmonella typhimurium (dam(-) and AroA(-)) to such secondary lymphoid organs as Peyer's patches in the small intestine, elicited marked activation of antigen-presenting dendritic cells, and an effective CD8(+)T cell immune response against the survivin self-antigen. This resulted in eradication or suppression of pulmonary metastases of non-small cell lung carcinoma in both prophylactic and therapeutic settings in C57BL/6J mice. Moreover, the suppression of angiogenesis induced by the vaccine did not impair wound healing or fertility of treated mice. It is anticipated that such novel DNA vaccines will aid in the rational design of future strategies for the prevention and treatment of cancer.

A novel transgenic mouse model for immunological evaluation of carcinoembryonic antigen-based DNA minigene vaccines.

A lack of relevant animal models has hampered preclinical screening and critical evaluation of the efficacy of human vaccines in vivo. Carcinoembryonic antigen-A2Kb (CEA-A2Kb) double transgenic mice provide a biologically relevant model for preclinical screening and critical evaluation of human CEA vaccine efficacy in vivo, particularly because such animals are peripherally tolerant of CEA. We established the utility of this model by demonstrating that an oral DNA minigene vaccine induces effective HLA-A2-restricted, CEA-specific antitumor CTL responses. This finding is supported by three lines of evidence: (a). an effective HLA-A2-restricted, CEA(691)-specific CTL response; (b). specific in vitro killing of CEA-A2Kb transduced MC-38 colon carcinoma cells; and (c). protective immunity induced in vaccinated mice against challenges of these tumor cells. Importantly, peripheral T cell tolerance against CEA in CEA-A2Kb double transgenic mice was broken by the CEA(691) (IMIGVLVGV) minigene vaccine. In conclusion, CEA-A2Kb double transgenic mice were demonstrated to be good candidates for in vivo testing of human CEA-based vaccines. This result suggests a potential for these vaccines in future human vaccine development. The feasibility of using nonmutated self-antigens as targets for therapeutic vaccinations was indicated, provided that such antigens are presented in an immunogenic context; that is, as a DNA minigene in a bacterial carrier system.

Transcription factor Fos-related antigen 1 is an effective target for a breast cancer vaccine.

Protection against breast cancer was achieved with a DNA vaccine against murine transcription factor Fos-related antigen 1, which is overexpressed in aggressively proliferating D2F2 murine breast carcinoma. Growth of primary s.c. tumor and dissemination of pulmonary metastases was markedly suppressed by this oral DNA vaccine, carried by attenuated Salmonella typhimurium, encoding murine Fos-related antigen 1, fused with mutant polyubiquitin, and cotransformed with secretory murine IL-18. The life span of 60% of vaccinated mice was tripled in the absence of detectable tumor growth after lethal tumor cell challenge. Immunological mechanisms involved activation of T, natural killer, and dendritic cells, as indicated by up-regulation of their activation markers and costimulatory molecules. Markedly increased specific target cell lysis was mediated by both MHC class I-restricted CD8+ T cells and natural killer cells isolated from splenocytes of vaccinated mice, including a significant release of proinflammatory cytokines IFN-gamma and IL-2. Importantly, fluorescence analysis of fibroblast growth factor 2 and tumor cell-induced vessel growth in Matrigel plugs demonstrated marked suppression of angiogenesis only in vaccinated animals. Taken together, this multifunctional DNA vaccine proved effective in protecting against growth and metastases of breast cancer by combining the action of immune effector cells with suppression of tumor angiogenesis.

Protective effects of glutathione on 5-fluorouracil-induced myelosuppression in mice.

The protective effects of glutathione (GSH) administration on myelosuppression induced by 5-fluorouracil (5-FU) were investigated in female BALB/c mice. Animals were allocated to four groups (16 mice/group). GSH was given orally at a dose of 800 mg/kg to groups 3 and 4 for 21 consecutive days (day 0 to day 20). 5-FU was repeatedly administered at a dose of 40 mg/kg to groups 2 and 3 for 1 week (day 7 to day 13) by gavage. Group 3 served as a combined treatment group and group 1 as a non-treated control group. The total observation period was 3 weeks. Body weight was measured once a week. A decrease in body weight due to 5-FU treatment was observed in groups 2 and 3 on day 14. Although the body weight in group 2 had not increased by 1-week after cessation of 5-FU treatment, the value in group 3 markedly recovered. Hematology, total nucleated myelocyte count and histopathology of bone marrow were carried out on day 14 and day 21. In groups 2 and 3, these examinations showed thrombocytopenia, leukopenia, reticulocytopenia and myelosuppression on day 14. However, platelets and bone marrow were less affected in group 3 than in group 2. On day 21, the thrombocytopenia in groups 2 and 3 was resolved. The myelosuppression, leukopenia and reticulocytopenia resolved in group 3, but not in group 2. Although simple microcytic anemia occurred delayed on day 21, it was less severe in group 3 than in group 2. Therefore, GSH may have preventive effects against 5-FU-induced hematopoietic toxicity, and accelerate recovery after cessation of 5-FU treatment.