The Development And Characterization Of Heparin-Conjugated Fibrin Hydrogel With Incapsulated Mesenchimal Stem Cells And Growth Factors

Currently, one of the major focuses in regenerative medicine is the development and implementation into practice of composite biomaterials with chondro- and osteoinductive properties, which include human stem cells and growth factors. Heparin-conjugated fibrinogen was obtained using the carbodiimide method, which was further used to create heparin-conjugated fibrin hydrogels (HCFH). As a result of this work, two types of HCFH were obtained: a hydrogel with encapsulated mesenchymal stem cells (MSC) and a hydrogel with TGF-β1 and BMP-4 growth factors. It has been found that synovial MSCs retain viability after encapsulation in HCFH, which indicates that the developed hydrogel is biocompatible and does not have toxic effect to the cells. The results of enzyme-linked immunosorbent assay on the kinetics of BMP-4 and TGF-β1 release from HCFH showed that the developed hydrogel is able to retain BMP-4 and TGF-β1. The kinetics of release from HCFH into phosphate buffer was significantly slower compared to fibrin hydrogel.

Preparation of CD3 Antibody-Conjugated, Graphene Oxide Coated Iron Nitride Magnetic Beads and Its Preliminary Application in T Cell Separation

Immunomagnetic beads (IMBs) for cell sorting are universally used in medical and biological fields. At present, the IMBs on the market are ferrite coated with a silicon shell. Based on a new type of magnetic material, the graphene coated iron nitride magnetic particle (G@FeN-MP), which we previously reported, we prepared a novel IMB, a graphene oxide coated iron nitride immune magnetic bead (GO@FeN-IMBs), and explored its feasibility for cell sorting. First, the surface of the G@FeN-MP was oxidized to produce oxygen-containing groups as carboxyl, etc. by the optimized Hummers’ method, followed by a homogenization procedure to make the particles uniform in size and dispersive. The carboxy groups generated were then condensed and coupled with anti-CD3 antibodies by the carbodiimide method to produce an anti-CD3-GO@FeN-IMB after the coupling efficacy was proved by bovine serum albumin (BSA) and labeled antibodies. Finally, the anti-CD3-GO@FeN-IMBs were incubated with a cell mixture containing human T cells. With the aid of a magnetic stand, the T cells were successfully isolated from the cell mixture. The isolated T cells turned out to be intact and could proliferate with the activation of the IMBs. The results show that the G@FeN-MP can be modified for IMB preparation, and the anti-CD3-GO@FeN-IMBs we prepared can potentially separate T cells.

Trials in developing a nanoscale material for extravascular contrast-enhanced ultrasound targeting hepatocellular carcinoma

Background Medical imaging is an important approach for the diagnosis of hepatocellular carcinoma (HCC), a common life threaten disease, however, the diagnostic efficiency is still not optimal. Developing a novel method to improve diagnosis is necessary. The aim of this project was to formulate a material that can combine with GPC3 of HCC for targeted enhanced ultrasound. Methods A material of sulfur hexafluoride (SF6) filled liposome microbubbles and conjugated with synthesized peptide (LSPMbs) was prepared and assessed in vitro and vivo. Liposome microbubbles were made of DPPC, DPPG, DSPE-PEG2000,and SF6, using thin film method to form shell, followed filling SF6, and conjugating peptide. A carbodiimide method was used for covalent conjugation of peptide to LSMbs. Results The prepared LSPMbs appeared round shaped, with size of 380.9 ± 176.5 nm, and Zeta potential of −51.4 ± 10.4mV. LSPMbs showed high affinity to Huh-7 cells in vitro, presented good enhanced ultrasound effects, did not show cytotoxicity, and did not exhibit targeted fluorescence and enhanced ultrasound in animal xenograft tumors. Conclusion Extravascular contrast-enhanced ultrasound targeted GPC3 on HCC may not be realized, and the reason may be that targeted contrast agents of microbubbles are hard to access and accumulate in the tumor stroma and matrix.

Convenient and Efficient Syntheses of Peptide Nucleic Acid Purine Monomers

Currently, peptide nucleic acids (PNAs) play an important role as therapeutic agents, molecular tools for diagnosis and detection of genetic diseases as well as in biosensor probes. This research aims to optimize the synthesis of aeg- and γ-(S)-Me PNA monomers based on L-Ala, intended for oligomerization according to the Boc protocol. The monomers were obtained through the condensation of the corresponding pseudopeptides with carboxymethyl purine nucleic bases. During the work, the optimization of benzyloxycarbonyl- N6-adenine-9-yl-acetic acid and benzyloxycarbonyl-N2-guanine-9-ylacetic acid was carried out. The synthesis of benzyloxycarbonyl-N6-adenine-9-yl-acetic acid was conducted in three stages based on adenine with an overall yield of 22%. At the same time, the conditions for effective recrystallization of the mixture after alkylation of benzyloxycarbonyl-N6-adenine with ethyl bromoacetic acid ether have been developed to isolate the desired N9-regioisomer. Also, the optimization of a known method for producing benzyloxycarbonyl-N2-guanine-9-ylacetic acid from 2-amino-6-chloropurine was carried out. The total yield of the five-stage scheme was 55%. Condensation of aeg- and γ-(S)-Me pseudopeptides with benzyloxycarbonyl-N6-adenine-9-yl-acetic acid and benzyloxycarbonyl-N2-guanine-9-yl-acetic acid was performed by the standard carbodiimide method, DCC/HOBt in DMF followed by the removal of C-terminal methyl protective group by alkaline hydrolysis. The structure of the new compounds obtained was confirmed by spectral analysis methods. This work provides simple and optimized methods for obtaining protected carboxymethyl purine bases and increasing the efficiency of the synthesis and synthesized purine PNA monomers in an acceptable yield.

SYNTHESIS AND PROPERTIES OF NEOGLYCOLIPIDS BASED ON 2-AMINO-2-HYDROXYMETHYLPROPANE-1,3-DIOL

The use of TRIS derivatives as the kernels of branching fragments is a modern method of preparing carbohydrate-containing dendrimer-like amphiphiles. A scheme of the synthesis of derivatives of trivalent neoglycolipids with terminal residues of D-mannose and a branching component based on 2-amino-2-hydroxymethylpropene-1,3-diol (TRIS) differing in the degree of saturation of the hydrocarbon chains is developed. The preparation of the hydrophilic part of the target molecules was carried out with the use of the reaction of 1,3-dipolar cycloaddition followed by conjugation with the hydrophobic component according to the carbodiimide method with the addition of HOBt as a catalyst. Approaches to the formation of target designs - liposomes based on phosphatidylcholine and synthesized neoglycolipids - and their physical and chemical properties, such as the size of particles and stability are investigated. The activity of the obtained compounds in the composition of liposomes loaded with the antibiotic meropenem with respect to Escherichia coli strain is carried out. An opportunity of changing the profile of the action of a liposome sample containing neoglycolipids by choosing various methods of its preparation that is promising for further research in this direction is revealed.

Prepared Polyclonal Antibody of the Ciprofloxacin

To detect Ciprofloxacin (CPFX) residues in food stuffs of animal origin rapidly by immune method, preparation of high quality polyclonal antibody against CPFX was the most critical step of project. We produced two kinds of antigens for CPFX, using carrier protein bovine serum albumin (BSA) and ovalbumin (OVA) by a modified carbodiimide method, and then the hapten-protein conjugates were characterized by ultraviolet spectrophotometry in detecting qualitatively comparable hapten density, before being used for the immunization and detection purposes. The production of polyclonal antibodies (PcAbs) were sought following the generation of appropriate CPFX-BSA conjugate. One PcAb against Ciprofloxacin (CPFX) was produced and the titer polyclonal antibody could reach 6.25×105. In the optimized ELISA, the PcAb showed 50% inhibition at 10.93 ng/mL for CPFX in buffer.

Determination of Sudan I by High Performance Liquid Chromatography with Immuno-Affinity Column Clean-Up

To prepare an immuno-affinity column (IAC) for Sudan I, Sepharose 4FF was activated by epichlorohydrin, and the density of epoxy is 80 μmol/g. Then it was coupled with 6-aminocaproic acid. The Sudan I immunoaffinity column was prepared by carbodiimide method in which modified Sepharose 4FF was used as carrier to couple with monoclonal antibody against Sudan I. A reversed-phase high performance liquid chromatographic method was used to determine the samples elution after the IAC cleaning up. Sudan I was determined by HPLC with a retention time of 5.82 min (flow rate of 1 ml/min). The column capacity is 1582.8 ng Sudan I when using 0.3 g Sepharose 4FF. Average recoveries of Sudan I from red chili powder spiked at levels of 0.25-3 mg/kg range from 44.52% to 77.40%. The coefficient of variation is 4.6% to 8.3%. The IAC method was found to be highly effective, sensitive and convenient for isolating the target analyte from sample.