Enhancement of Gene Transfer to Cervical Cancer Cells Using Transferrin- Conjugated Liposome

ABSTRACT

Transferrin-conjugated cationic liposome (T_f -DDAB liposome) was developed as a targeted gene delivery system by using heterobifunctional cross-linking agent, N-succimidyl-3-(2-pyridyldithio)propionate (SPDP), and gradient metrizamide ultracentrifugation method. Physico-chemical properties of T_f -liposome were determined by scanning/transmission electron microscopy (SEM/TEM) and dynamic laser-light scattering method (DLS) with a mean diameter of 584±15 nm. Gel retardation assay was performed using various DDAB:DNA ratios, and proved that the 6:1 weight ratio formulation gave the most neutralized complex. In vitro transfection was done in human cervical cancer cells, HeLa, and the transfection efficiency of T_f -liposome was found to be fivefold higher than that of unconjugated (plain) DDAB liposome and twofold higher than that of Lipofectin?. In conclusion, a target-oriented T_f -DDAB liposome was made successfully and proved to be very efficient in DNA delivery into the cervical cancer cells in culture.     

免疫活性蛋白的开发及其在乳制品中的应用

母乳化奶粉目前的研究重点是模拟母乳中具有生物免疫活性的功能因子,其中最重要的有免疫球蛋白（Immunoglobulin)、转铁蛋白（Transferrin)及溶菌酶（Lysozyme).美国及日本的公司已开发了含免疫球蛋白或乳铁蛋白等活性物质的婴儿配方奶粉,我国刚开始这方面的工作。鸡蛋被认为是丰富和优良的高生物活性免疫球蛋白、转铁蛋白及溶菌酶的资源。但由于分离的难度较大,除溶菌外还没有工业化生产的报道,从而影响了它的广泛应用。浙江长兴艾格生物制品有限公司在国家“八五”重点科技（攻关）项目－《鸡蛋中免疫球蛋白的分离提取》的基础上,通过对中试工艺的进一步改良,生产出了含优质高生产活性的蛋黄免疫球蛋白和转铁蛋白的免疫复合产品。为开发生物免疫活性的婴儿配方奶粉奠定了良好的基础。     

转铁蛋白放射免疫分析法的建立及其初步应用

现已建立的转铁蛋白放射免疫分析法具有较高的灵敏度与特异性，结果稳定。标准曲线及标本的定量测量值重复性好；血清标本的批内ＣＶ＝１．８０％（ｎ＝１４），批间ＣＶ＝９．９４％（ｎ＝１０）；回收率高、中、低浓度平均为１０２．９１％；可测范围为７．８－５００μｇ／ｌ。所检测的血清、尿和唾液的正常值分别为３．０３±０．６６ｇ／ｌ（Ｘ±Ｓ）；１９８±９８μｇ／ｌ；５５７．８９±２０６．３２μｇ／ｌ。肾功能异常者     

Stepwise functionalization of SiNx surfaces for covalent immobilization of antibodies

A stepwise functionalization of silicon nitride surfaces is followed by X-ray photoelectron spectroscopy (XPS). The first step involves a silanization reaction leading to the formation of a silane film with a thickness estimated by XPS of one or two molecular layers. A monoprotected homobifunctionalized linker is then used to avoid the formation of bridge structures on the surface. The linker reacts quantitatively with the amino groups of the surface as outlined by the absence of residual unreacted CNH₂/CNH₃⁺ groups in XPS analyses. Deprotection of the ester groups of the immobilized linker and subsequent reaction with N-hydroxysuccinimid lead to N-hydroxysuccinimid activated surfaces able to react with biological species. These surfaces were then incubated with anti-transferrin antibodies. As seen by XPS and atomic force microscopy analyses, the concentration and incubation conditions of antibodies are important to obtain a compact layer of antibodies on the surface. All chemical steps of the procedure are compatible with microelectronic process on silicon. Moreover, antibodies introduced under native conditions at physiological pH, in the last step of the immobilization process, recognized specifically antigens, as shown by fluorescence competitive assay.     

Enhancement of gene transfer to cervical cancer cells using transferrin-conjugated liposome

Transferrin-conjugated cationic liposome (T_f -DDAB liposome) was developed as a targeted gene delivery system by using heterobifunctional cross-linking agent, N-succimidyl-3-(2-pyridyldithio)propionate (SPDP), and gradient metrizamide ultracentrifugation method. Physico-chemical properties of T_f -liposome were determined by scanning/transmission electron microscopy (SEM/TEM) and dynamic laser-light scattering method (DLS) with a mean diameter of 584±15 nm. Gel retardation assay was performed using various DDAB:DNA ratios, and proved that the 6:1 weight ratio formulation gave the most neutralized complex. In vitro transfection was done in human cervical cancer cells, HeLa, and the transfection efficiency of T_f -liposome was found to be fivefold higher than that of unconjugated (plain) DDAB liposome and twofold higher than that of Lipofectin™. In conclusion, a target-oriented T_f -DDAB liposome was made successfully and proved to be very efficient in DNA delivery into the cervical cancer cells in culture.     

Enhanced oral absorption of salmon calcitonin-encapsulated PLGA nanoparticles by adding organic substances

Two organic compounds with potential absorption enhancing effects, bile acids and transferrin, were examined by the gastro-intestinal (GI) absorption of therapeutic salmon calcitonin (sCT) as encapsulated by poly(lactide-co-glycolide) (PLGA) for the treatment of osteoporosis. The sCT-loaded PLGA nanocapsules were prepared by O/W emulsification approach. Either additive of a designated content was mixed with sCT dissolved in methanol. For bile acids, their content (0–7.5 mg to sCT 6 mg) was observed to have a substantial effect both on the emulsification process and the encapsulation efficiency. When 1.5 mg of bile acids was added, sCT-loaded PLGA nanocapsules of about 700 nm in diameter and with a fairly high encapsulation efficiency greater than 35% were produced. Accordingly, this formulation gave the most significant hypocalcemic effect in an in vivo experiment with SD rats. On the other hand, a too high bile acids loading resulted in a poor encapsulation efficiency of less than 7%. Two principal roles of bile acids were proposed: emulsifying agent and absorption enhancer. Transferrin, a human glycoprotein of 80 kDa molecular weight, turned out to have potential as absorption enhancer as well.     

Cu-MOF nanosheets modified nickel foam: A versatile platform for highly sensitive electrochemical detection of transferrin in simulated human blood serum

Transferrin (TRF) is a type of glycoprotein present in blood plasma that plays a vital role in facilitating the absorption, storage, and movement of iron(III) ions within the circulatory system of multiple vertebrates. Herein, we report one-pot solvothermal synthesis of Copper Metal-Organic Framework nanosheets modified nickel foam (Cu-MOF/NF) for electrochemical detection of transferrin (TRF) in simulated human blood serum. The crystal structure of the Cu-MOF nanosheets was determined through X-ray diffraction (XRD), while Raman spectroscopy and Fourier-transform infrared spectroscopy (FTIR) analysis were employed to investigate the vibrational modes and the presence of surface OH groups, respectively. The nanosheet morphology of the Cu-MOF was observed and characterized using scanning electron microscopy (SEM) micrographs.The as-fabricated Cu-MOF/NF exhibits a sensitivity of 1.36 mA/x (x = mg.mL⁻¹cm⁻²) for a linear range of concentrations from 1 mg mL⁻¹ – 100 mg mL⁻¹ with a low limit of detection (LOD) of 0.81 mg mL⁻¹. The sensor exhibited good selectivity for TRF, even in the presence of interfering ions such as bovine serum albumin, ascorbic acid, uric acid, and urea, with concentrations 100-fold higher than TRF. The porous nickel foam with high conductivity, offers numerous active sites and pathways for ion transport, as well as pathways for electron transport during electrochemical catalytic reactions. This performance of the sensor is attributed to Cu ²⁺/Cu⁺ redox couple at the octahedral sites of the Cu-MOF facilitating the electro-oxidation of TRF and charge-carrier delocalization during the catalytic reaction. The as-fabricated sensor is successful in the determination of trace level TRF concentration the human blood serum with excellent recovery percentages of 108.33% This proves Cu-MOF/NF as a promising platform for various electrochemical analytical applications.