Novel factor-loaded polyphosphazene matrices: Potential for driving angiogenesis

Currently employed bone tissue engineered scaffolds often lack the potential for vascularization, which may be enhanced through the incorporation of and regulated release of angiogenic factors. For this reason, the objective here was to fabricate and characterize protein-loaded amino acid ester polyphosphazene (Pphos)-based scaffolds and evaluate the novel sintering method used for protein incorporation, a method which will ultimately allow for the incorporation of proangiogenic agents. To test the hypothesis, Pphos and their composite microspheres with nanocrystalline hydroxyapatite (Pphos-HAp) were fabricated via the emulsion solvent evaporation method. Next, bovine serum albumin (BSA)-containing microsphere matrices were created using a novel solvent–non-solvent approach for protein loading. The resulting protein (BSA) loaded circular porous microsphere based scaffolds were characterized for morphology, porosity, protein structure, protein distribution and subsequent protein release pattern. Scanning electron microscopy revealed porous microsphere scaffolds with a smooth surface and sufficient level of sintering, illustrated by fusion of adjacent microspheres. The porosity measured for the poly(ethyl phenylalanato:glycinato)phosphazene (PNPhGly) and poly(ethyl phenylalanato:glycinato)phosphazene-hydroxyapatite (PNPhGly-HAp) scaffolds were 23 ± 0.11% and 18 ± 4.02%, respectively, and within the range of trabecular bone. Circular dichroism confirmed an intact secondary protein structure for BSA following the solvent sintering method used for loading and confocal microscopy verified that FITC-BSA was successfully entrapped both between adjacent microspheres and within the surface of the microspheres while sintering. For both Pphos and their composite microsphere scaffolds, BSA was released at a steady rate over a 21 day time period, following a zero order release profile. HAp particles in the composite scaffolds served to improve the release profile pattern, underscoring the potential of HAp for growth factor delivery. Moreover, the results of this work suggest that the solvent–non-solvent technique for protein loading is an optimal one that will allow for future development of angiogenic factor-loaded Pphos matrices with the capacity to invoke neovascularization.     

Effective intracellular delivery and Th1 immune response induced by ovalbumin loaded in pH-responsive polyphosphazene polymersomes

A polymersome system for delivering protein antigen ovalbumin (OVA) based on amphiphilic polyphosphazene grafting with N,N-diisopropylethylenediamine (DPA) and poly(ethylene glycol) (PEG) groups (poly[(DPA)_m (PEG)_n phosphazene], PEDP) was designed and constructed. The 200-240?nm-size OVA-loaded polymersomes displayed high stability at physiological pH, slow internalization through clathrin-mediated endocytosis pathway, and then a pH-triggered sustained OVA release in acidic environment, leading to extensive antigen access to cytosol. Prime-boost vaccine kept high antibody titers for 8?weeks and the subcutaneous vaccine of OVA polymersomes biased the immune response towards a type 1?T helper (Th1) response. Animal experiment results showed that the antigen-specific prophylactic vaccination by PEDP polymersomes delivery was much more rapid and efficient in depressing tumor growth and progress when compared with the therapeutic vaccination. These results suggested that PEDP-based polymersomes are very promising in controlled cytosolic delivery of protein antigens, and enhanced Th1 specific immune response.     

PCPP (poly[di(carboxylatophenoxy)-phosphazene]) microparticles co-encapsulating ovalbumin and CpG oligo-deoxynucleotides are potent enhancers of antigen specific Th1 immune responses in mice

We generated poly[di(carboxylatophenoxy)-phosphazene] (PCPP) microparticles encapsulating ovalbumin (OVA) and CpG of 0.5-2.5 μm in diameter with an encapsulation efficiency of approximately 63% and 95% respectively. In mice the microparticles generated high antigen-specific IgG, IgG1 and IgG2a titers with higher IgG2a/IgG1 ratios. Whole body in vivo imaging of mice subcutaneously injected with MPs showed several fold increase of OVA and CpG in draining inguinal lymph nodes compared to soluble formulations. We conclude that PCPP MPs are more effective in enhancing immune responses compared to soluble formulations, due to co-delivery of OVA and CpG resulting in a Th1 type of immune response.     

Enhanced immune responses and protection by vaccination with respiratory syncytial virus fusion protein formulated with CpG oligodeoxynucleotide and innate defense regulator peptide in polyphosphazene microparticles

Although respiratory syncytial virus (RSV) is the leading cause of serious respiratory tract disease in children, to date no RSV vaccine is available. To produce an effective subunit vaccine, a truncated secreted version of the F protein (ΔF) was expressed in mammalian cells, purified and shown to form trimers. The ΔF protein was then formulated with a CpG oligodeoxynucleotide (ODN) and an innate defense regulator (IDR) peptide in polyphosphazene microparticles (ΔF-MP). Mice immunized either intramuscularly (IM) or intranasally (IN) with ΔF-MP developed significantly higher levels of virus-neutralizing antibodies in the sera and lungs, as well as higher numbers of IFN-γ secreting cells than mice immunized with the ΔF protein alone. In contrast, the IM delivered ΔF induced high production of IL-5 while the IN delivered ΔF did not elicit a measurable immune response. After RSV challenge, essentially no virus and no evidence of immunopathology were detected in mice immunized with ΔF-MP regardless of the route of delivery. While the mice immunized IM with ΔF alone also showed reduced virus replication, they developed enhanced levels of pulmonary IgE, IL-4, IL-5, IL-13 and eotaxin, as well as eosinophilia after challenge. The level of protection induced by the ΔF-MP formulation was equivalent after IM and IN delivery. The efficacy and safety of the ΔF-MP formulation was confirmed in cotton rats, which also developed enhanced immune responses and were fully protected from RSV challenge after vaccination with ΔF-MP. In conclusion, formulation of recombinant ΔF with CpG ODN and IDR peptide in polyphosphazene microparticles should be considered for further evaluation as a safe and effective vaccine against RSV.     

Immunization with PCEP microparticles containing pertussis toxoid, CpG ODN and a synthetic innate defense regulator peptide induces protective immunity against pertussis

We investigated the efficacy of a novel microparticle (MP) based vaccine formulation consisting of pertussis toxoid (PTd), polyphosphazene (PCEP), CpG ODN 10101 and synthetic cationic innate defense regulator peptide 1002 (IDR) against Bordetella pertussis in mice. We studied whether encapsulation of these IDR-CpG ODN complexes into polyphosphazene-based microparticles further enhanced their immunomodulatory activity compared to soluble formulations containing PCEP (SOL), or without PCEP (AQ). In vitro stimulation of murine macrophages showed MP induced significantly higher levels of pro-inflammatory cytokines. When assessed in a B. pertussis infection challenge model, a single immunization with MP formulation led to significantly lower bacterial loads compared to other formulations and non-vaccinated animals. ELISPOT of splenocytes showed that MP group mice had significantly higher number of antigen-specific IL-17 secreting cells. The cytokine profile in lung homogenates of MP group mice after challenge showed significantly higher amounts of MCP-1, TNF-α, IFN-γ, IL-12 and IL-17 and significantly lowered IL-10 levels suggesting a strong Th1 shift. Protection was observed against challenge infection with B. pertussis. On the other hand protective immune responses elicited in Quadracel^® immunized mice were Th2 skewed. Hence, we conclude that formulation of PTd, PCEP, CpG ODN and IDR into MP generates a protective immune response in mice against pertussis emphasizing the potential of MP as a delivery vehicle for the potential development of single-shot vaccines.     

聚膦腈共混膜在动物体内的降解和组织相容性

研究了聚磷腈／降酯或聚膦腈共混膜（PGP／PLGA或PGP／PSTP）在小鼠体内的降解行为和组织相容性。初步结果表明，共混物的降解速率随共混组成的变化而变化，PGP／PLGA（70：30质量比）降解55d重量损失68．4％，PGP/PSTP(70:30质量比）12d即降解88．5％；PGP／PLGA的降解机制包括水解和酶解，PGP／PSTP主要是水解；PGP／PLGA的组织相容性较PGP／PSTP更好，而且在PGP／PSTP体系中PGP含量的增加有利于共混物组织相容性的改善。这些结果提示该共混物在医药领域具有潜在的应用前景。     

The novel adjuvant combination of CpG ODN,indolicidin and polyphosphazene induces potent antibody- and cell-mediated immune responses in mice

The need to enhance the immunogenicity of purified subunit antigens and modulate resulting immune responses has prompted the development of new adjuvants. Here, the ability of CpG oligodeoxynucleotides (ODN), a bovine host defence peptide indolicidin, and polyphosphazene to synergistically combine and enhance innate and adaptive immune responses was examined in mice. In vitro, the adjuvant combination of CpG ODN, indolicidin and polyphosphazene (CpG/indol/PP) enhanced the secretion of TNF-α, IL-12p40, and IL-6 by bone marrow-derived DCs (BMDCs) when compared to the individual components. When co-formulated with ovalbumin (OVA), CpG/indol/PP formed antigen-adjuvant complexes, and enhanced antibody and cell-mediated responses in mice, via both MHC I and II pathways, promoting a more balanced antibody-mediated and type 1-biased cell-mediated immune response. Furthermore, substitution of the proline residues of indolicidin with arginine increased the synergistic adjuvant effect of the peptide, and induced significantly higher IgG1 and IgG2a titers and IFN-γ secretion, as well as increased uptake by antigen presenting cells. These results clearly demonstrate that the use of a combination of CpG ODN, indolicidin, and polyphosphazene as adjuvant can significantly enhance an antigen-specific immune response.     

CpG oligonucleotide,host defense peptide and polyphosphazene act synergistically,inducing long-lasting,balanced immune responses in cattle

Vaccines consisting of subunit or protein antigens are less immunogenic than traditional vaccines, and therefore require formulation with an adjuvant. Conventional adjuvants, however, often cause undesirable injection site reactions and Th2-biased immune responses. Therefore, novel vaccine adjuvants which can safely enhance and selectively bias the resulting immune response are required. Here the adjuvant combination of CpG ODN, indolicidin and polyphosphazene (CpG + indol + PP) was evaluated for its ability to enhance and modulate the immune response when formulated with the antigen hen egg lysozyme (HEL). Cattle immunized with HEL co-adjuvanted with CpG + indol + PP developed higher antigen-specific humoral responses, and long-lasting cell-mediated immune responses, as evidenced by elevated levels of IFN-γ secretion by re-stimulated PBMCs, that were superior even to EMULSIGEN^®, an oil-in-water based adjuvant that was used as positive control. Physical characterization of the vaccines indicated that formulation of HEL with CpG + indol + PP resulted in the formation of antigen-adjuvant complexes, which may have contributed to their enhanced immunogenicity. Furthermore, the addition of polyphosphazene to CpG ODN and indolicidin dose-dependently enhanced the secretion of the cytokines IFN-α, TNF-α and IFN-γ in vitro, indicating that polyphosphazene can also synergize with CpG ODN and indolicidin to stimulate innate immune responses.     

Biocompatibility and Recanalization Characteristics of Hydrogel Microspheres with Polyzene-F as Polymer Coating

The objective of this study was to evaluate inflammatory response and recanalization after embolization with a new spherical embolic agent based on a core and shell design with a hydrogel core of polymethylmethacrylate (PMMA) and a Polyzene-F nanoscale coating in a porcine kidney model. Thirty-six minipigs were enrolled for superselective renal embolization. Polyzene-F-coated PMMA particles and uncoated PMMA particles with a diameter of 300-600 mum were used. Either 4 or 12 weeks post-embolization, arteriography of the embolized kidneys was performed and then compared with pre- and immediate post-embolization arteriograms using a specific recanalization score to determine the extent of recanalization. Using a microscopic inflammation score (Banff classification), the embolized organs were examined for local inflammatory effects which occurred in response to the embolic agent. In Polyzene-F-coated particles, the Banff classification showed an average inflammation score of 0.26 +/- 0.58 at 4 weeks and of 0.08 +/- 0.28 at 12 weeks. In uncoated particles, the Banff score measured 0.37 +/- 0.6 at 4 weeks, which was higher, but without a statistically significant difference. According to the recanalization score used in this study, mild angiographic recanalization was evident in all groups, without statistically significant differences (3.0 +/- 0.71 in coated particles, 3.09 +/- 0.81 in uncoated particles; p = 0.74). We conclude that both uncoated hydrogel particles and Polyzene-F-coated embolic agents triggered virtually no inflammatory response and effectively occluded target arteries. This study demonstrates good biocompatibility of the new embolic material. As in other spherical embolic agents, recanalization can occur to some degree.     

添加纳米聚磷腈对聚氨酯材料微生物黏附性能的影响

目的:观察纯聚氨酯材料(PU)、含1%和5%纳米聚磷腈的聚氨酯材料(PZS-1%、PZS-5%)表面白念菌和变形链球菌黏附情况。方法:测定3种材料(PU、PZS-1%、PZS-5%)表面粗糙度,并将其分别于白念菌和变链球菌菌液中进行黏附实验,分析材料表面微生物的黏附情况。采用SAS8.02软件包对数据进行统计学分析。结果:PU、PZS-1%和PZS-5%材料的表面粗糙度由大到小依次为PU>PZS-1%>PZS-5%(P<0.001);白念菌和变链球菌黏附实验中,黏附微生物量从多到少依次为PU>PZS-1%>PZS-5%(P<0.001)。结论:添加不同比例的纳米聚磷腈,对聚氨酯材料微生物黏附性能有一定影响。