Zirconium-containing nanoscale coordination polymers for positron emission tomography and fluorescence-guided cargo delivery to triple-negative breast tumors.

Nanoscale coordination polymer (NCP) is a class of hybrid materials formed by self-assembly of metal ions and organic ligands through coordination. The applications of NCP in biomedicine are quite extensive due to the diversity choice of metal ions and organic ligands. Here we designed Zr-P1 NCP based on Zr4+ selected as metal ion nodes and tetrakis(4-carboxyphenyl) ethylene as bridging ligands. Zr-P1 NCP was modified with functionalized pyrene derived polyethylene glycol (Py-PAA-PEG-Mal) on the surface and further conjugated with cRGD for active targeting of integrin αvß3 overexpressed in triple-negative breast cancer. Doxorubicin was loaded on Zr-P1 NCP with encapsulation efficiency up to 22 % for the treatment of triple negative breast cancer. 89Zr-P1 NCP can be used for in vivo tumor imaging due to the fluorescence properties resulting from the enhanced aggregation-induced Emission (AIE) behavior of P1 ligands and its positron emission tomography (PET) capability. Cellular evaluation indicated that the functionalized Zr-P1@PEG-RGD presented a good function for tumor cell targeting imaging and doxorubicin could be targeted to triple negative breast cancer when it was loaded onto Zr-P1@PEG-RGD, which corroborated with the in vivo results. In summary, 89Zr-P1@PEG-RGD can serve as a biocompatible nanoplatform for fluorescence and PET image-guided cargo delivery. STATEMENT OF SIGNIFICANCE: Nanoscale coordination polymer (NCP) is a class of hybrid materials formed by self-assembly of metal ions and organic ligands through coordination. The diversity of available metals and ligand structures upon NCP synthesis plays an advantage in establishing multimodal imaging platforms. Here we designed 89Zr-P1@PEG-RGD NCP based on Zr4+ selected as metal ion nodes and tetrakis(4-carboxyphenyl) ethylene as bridging ligands. 89Zr-P1@PEG-RGD nanomaterials have positron emission tomography (PET) capability due to the incorporation of zirconium-89, which can be used for in vivo tumor imaging with high sensitivity. The chemotherapeutic drug DOX was loaded on Zr-P1 NCP for the treatment of triple-negative breast cancer, and dual modality imaging can provide visual guidance for drug delivery.

Biomimetic-Functionalized, Tannic Acid-Templated Mesoporous Silica as a New Support for Immobilization of NHase.

Tannic acid-templated mesoporous silica (TAMS) was synthesized using a simple nonsurfactant template method and dopamine-functionalized TAMS (Dop-TAMS), which was prepared via a biomimetic coating, was developed as a new support for immobilization of NHase (NHase@Dop-TAMS). The Dop-TAMS was thoroughly characterized by the transmission electron microscopy (TEM), scanning electron microscopy (SEM), Brunauer-Emmett-Teller (BET), and Fourier transform infrared (FT-IR) and the results showed that the Dop-TAMS possessed sufficiently large pore size and volume for the accommodation of NHase. Studying the thermal stability, storage, shaking stability, and pH stability of the free and immobilized NHase indicated that the catalytic properties of NHase@Dop-TAMS were significantly enhanced. Moreover, the NHase@Dop-TAMS exhibited good reusability. All the results demonstrated that Dop-TAMS could be used as an excellent matrix for the immobilization of NHase.

Chitosan-mediated formation of biomimetic silica nanoparticles: an effective method for manganese peroxidase immobilization and stabilization.

Our work here, for the first time, reported the use of chitosan-mediated biomimetic silica nanoparticles in enzyme immobilization. In order to make clear the relationship among silicification process, silica nanoparticle structure and immobilized enzyme activity, a mechanism of chitosan-mediated silicification using sodium silicate as the silica source was primarily evaluated. Chitosan was demonstrated effectively to promote the silicification not only in accelerating the aggregation rate of sodium silicate, but also in templating the formation of silica nanoparticles. Although the whole biomimetic silicification process contained polycondensation-aggregation-precipitation three stages, the elemental unit in precipitated silica was confirmed to be nanoparticles with 100 nm diameter regardless of the chitosan and silicate concentration used. Furthermore, the effect of enzyme on silicification process was also investigated. The introducing of manganese peroxidase (MnP) to silica precursor solution had no obvious effect on the silicification rate and nanoparticle morphology. The residual activity and embedding rate of immobilized MnP were 64.2% and 36.4% respectively under the optimum conditions. In addition, compared to native MnP, the MnP embedded in chitosan/silica nanoparticles exhibited improved stability against organic solvent and ultrasonic wave. After ultrasonic treatment for 20 min, 77% of the initial activity was remained due to the protective effect of chitosan/silica nanoparticles, while native MnP lost almost all of its original activity.

Biosilica-coated kappa-carrageenan microspheres for yeast alcohol dehydrogenase encapsulation.

In this study, a novel polysaccharide/inorganic hybrid biocomposite was prepared through biomineralization-inspired process for efficient immobilization of yeast alcohol dehydrogenase (YADH). YADH-encapsulated kappa-carrageenan microspheres (KCM) were first coated with chitosan, which was used to guide and catalyze the biomimetic formation of silica, and then coated with silica derived from tetraethoxysiliane (TEOS). Scanning electron microscopy (SEM) equipped with a energy dispersive X-ray spectrometer (EDX) was employed to investigate the composition and thickness of silica film. Compared with KCM, the silica-coated kappa-carrageenan microspheres (SKCM) containing YADH exhibited improved anti-swelling and catalytic properties. Enzyme leakage of YADH in KCM was detected to be 63.9% after 1 h, while the enzyme leakage in SKCM was as low as 18.2%. The KCM adsorbed water promptly and after 1 h maximum water uptake can be as high as 1576 wt%, while the water uptake of SKCM was only 143 wt% even after 48 h. The optimum pH and temperature for encapsulated YADH were pH 6.5 and 25 degrees C, which were just the same as those for free YADH, but the encapsulated YADH exhibited broader pH and temperature ranges for high activity. Furthermore, the relative activity of YADH in KCM declined almost to zero after 8 recycles, while the relative activity of YADH in SKCM still maintained more than 50%. The significantly increased recycling stability of YADH in SKCM may be attributed to the effective inhibition of enzyme leakage by the compact biosilica layer.

Expression and self-assembly of HCV structural proteins into virus-like particles and their immunogenicity.

BACKGROUND: The synthesis of virus-like particles (VLPs) provides an important tool to determine the structural requirements for viral particle assembly and virus-host interactions. Our purpose was to express simultaneously all three structural proteins of hepatitis C virus (HCV) in insect cells to investigate the proteins assembly into VLPs and the immunogenicity of these particles. METHODS: HCV gene sequences encoding the structural proteins C, E1, and E2 were amplified with PCR, and recombinant baculoviruses were constructed using recombinant DNA techniques. The expression of HCV structural proteins in insect cells was analyzed by immunofluorescence and SDS-PAGE. The interaction of expressed structural proteins was investigated by immunoprecipitation and immunoblotting. The VLPs in the insect cells were visualized by electron microscopy (EM). VLPs were then purified by sucrose gradient centrifugation and used to immunize BALB/c mice. Antibodies against HCV were tested for in mouse serum samples by an ELISA assay. RESULTS: The recombinant baculoviruses reBV/C and reBV/E1-E2 were constructed successfully. Insect cells co-infected with reBV/C and reBV/E1-E2 expressed HCV C, E1, and E2 proteins with the expected molecular weights of 20kD, 35kD, and 66kD, respectively. The results of immunoprecipitation and immunoblotting assays revealed the coimmunoprecipitation of C, E1, and E2 proteins, indicating association of the three structural proteins. Electron microscopy of insect cells co-infected with reBV/C and reBV/E1-E2 demonstrated spherical particles (40 to 60 nm in diameter) similar to the HCV virions from serum samples or hepatic tissue samples of HCV infected humans. The VLPs were partially purified. Antibodies to HCV were detectable in the serum of mice immunized with VLPs. CONCLUSION: HCV structural proteins simultaneously expressed in insect cells can interact with each other and assemble into HCV-like particles, which are shown to be immunogenic in mice.

A novel process for production of hepatitis A virus in Vero cells grown on microcarriers in bioreactor.

AIM: To develop a novel process for production of HAV in Vero cells grown on microcarriers in a bioreactor. METHODS: Vero cells infected with HAV strain W were seeded at an initial density of 1 x 10(5) cells/mL into a 7-L bioreactor containing Cytodex-I microcarriers. During the stage of cell proliferation, the following conditions were applied: pH 7.2 +/- 0.2, temperature 37 +/- 0.2 degrees, dissolved oxygen 40% of air saturation and agitation rate 40 r/min. After the stage of virus culture started, the culture conditions were altered to pH 7.2 +/- 0.2, temperature 35 +/- 0.2 degrees, dissolved oxygen 25% of air saturation, agitation rate 50 r/min and perfusion of fresh medium at a flux of 20 mL/h. During the course of fermentation, cell density, HAV antigen titre, glucose, lactate and ammonia levels were monitored. A control experiment using conventional static culture was conducted in the T150 flask. RESULTS: After a 28-d cultivation, cell density increased to 14.0 x 10(6) cells/mL in the bioreactor, 5.6 x 10(9) viable cells and 4,000 mL virus suspension with a titre of 1:64 were harvested. The viral antigen output per cell unit in the bioreactor was 3-fold higher than that in the T150 flask. Meanwhile the metabolic mode of Vero cells did not change after the infection with HAV strain W. CONCLUSION: The process for production of HAV in Vero cells grown on microcarriers in a bioreactor is a novel, efficient and practical way to obtain virus antigen for vaccine purpose. This approach produces more cells and HAV antigen than the conventional static culture. With further improvement, it is possible to be used for the production of hepatitis A vaccine.

No requirement of HCV 5'NCR for HCV-like particles assembly in insect cells.

AIM: To express all three HCV structural proteins in the presence or absence of HCV 5'NCR to investigate the requirement of 5'NCR for the assembly of HCV-like particles in insect cells. METHODS: HCV structural protein encoding sequences CE1E2 and 5'NCR-CE1E2 were amplified with PCR. Recombinant baculovirus were constructed with recombinant DNA techniques. HCV structural proteins expressed in insect cells were analyzed by immunofluorescence and SDS-PAGE. Immunoprecipitation experiment of insect cell lysates with anti-E2 monoclonal antibody (MAb) was carried out and the immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotting with anti-C, anti-E2 MAbs and HCV positive serum. The virus-like particles in insect cells were visualized by electron microscopy (EM). The HCV-like particles were purified by sucrose gradient centrifugation and identified by EM and immune aggregation EM. RESULTS: The recombinant baculovirus reBV/CE1E2 containing HCV C, E1, E2 genes and reBV/CS containing the same structural protein genes plus 5'NCR were constructed. The insect cells infected with either reBV/CE1E2 or reBV/CS expressed HCV C, E1 and E2 proteins with a molecular weight of 20 kD, 35 kD and 66 kD respectively. The results of immunoprecipitation and the immunoblotting revealed the coimmunoprecipitation of C, E1, and E2 proteins, indicating the interaction of HCV structural proteins expressed in insect cells. Electron microscopy of insect cells infected with reBV/CE1E2 or reBV/CS demonstrated spherical particles (40 to 60 nm in diameter) similar to the HCV virions from sera or hepatic tissues of HCV infected humans. The HCV-like particles were partially purified by sucrose gradient centrifugation, and the purified VLPs showed immuno-reactivity with anti-HCV antibodies. CONCLUSION: HCV 5'NCR is not required for the assembly of HCV-like particles in insect cells, HCV core and envelope proteins are sufficient for viral particle formation.