海藻酸—磷脂微囊復(fù)合水凝膠的制備、表征及包載siRNA的應(yīng)用研究
發(fā)布時(shí)間：2018-11-13 08:28
【摘要】：癌癥這類(lèi)威脅人類(lèi)生命健康的疾病一直都是醫(yī)學(xué)界的一大難題。目前,化學(xué)療法仍然是常用治療癌癥的手段,而傳統(tǒng)的化療存在毒副作用大、選擇性差、治療效果差等缺陷,因此限制了其應(yīng)用前景和發(fā)展?jié)撃�。為�?人們期望利用納米尺寸顆�；蛩幬镞f送體固有的表面效應(yīng)和小尺寸效應(yīng)增加抗腫瘤化療藥物的生物穩(wěn)定性和生物利用度�；诩{米抗腫瘤化療藥物的生物活性取決于使用的納米材質(zhì)的化學(xué)結(jié)構(gòu)和物理性能的特點(diǎn),通過(guò)優(yōu)化納米藥物載體性能提高藥效,或通過(guò)分子修飾載體以獲得納米藥物遞送體主動(dòng)靶向遞送和定位釋藥的能力�；蛑委熓钱�(dāng)今治療惡性腫瘤、遺傳性疾病及后天疾病較為理想的手段,siRNA能夠降解同源序列的m RNA,特異抑制癌細(xì)胞相關(guān)基因的表達(dá),從而抑制癌細(xì)胞的生長(zhǎng)繁殖、侵襲和擴(kuò)散。但目前可用于將外源性基因?qū)氩∽兲囟ú课坏募夹g(shù)仍然有限,為了進(jìn)一步實(shí)現(xiàn)基因治療的目的,必須首先解決基因傳遞的問(wèn)題。因此,基因治療的關(guān)鍵在于獲得能夠有效包裹基因藥物、攜載其通過(guò)細(xì)胞膜并傳遞進(jìn)入細(xì)胞核使其表達(dá)的載體。脂質(zhì)體因具有無(wú)毒性、靶向性、生物相容性、緩釋性等諸多優(yōu)點(diǎn)備受重視,是目前最具有潛力的藥物、基因、蛋白等的遞送系統(tǒng)之一,但它的應(yīng)用范圍因熱力學(xué)不穩(wěn)定性而受到限制。為此,通過(guò)對(duì)微囊進(jìn)行物理化學(xué)修飾,調(diào)整尺寸大小等各種途徑提高它在體內(nèi)外的穩(wěn)定性,開(kāi)發(fā)出了各種較為穩(wěn)定的脂質(zhì)體,例如:隱形脂質(zhì)體、固體脂質(zhì)納米顆粒等,盡管得到了一些改進(jìn),但其穩(wěn)定性問(wèn)題仍然存在。本文首先是在磷脂微囊膠體中加入含有二價(jià)陽(yáng)離子的電解質(zhì)溶液(氯化錳、氯化鈣、氯化鎂),確保膠體微囊處于較好的穩(wěn)定狀態(tài),然后將穩(wěn)定磷脂微囊體系與海藻酸鈉(Sodium alginate,SA)結(jié)合形成新型水凝膠,克服了磷脂微囊在液體中的熱力學(xué)不穩(wěn)定性,并且利用該材料包載魚(yú)精蛋白-siRNA,并表征其形貌和包封。本研究共分為三章,其主要內(nèi)容如下:第一章概述了基因、藥物治療面臨的困難和存在的缺陷,目前常用的各類(lèi)基因、藥物載體,藥物的主動(dòng)靶向性和被動(dòng)靶向性以及智能響應(yīng)性釋放的手段。第二章采用薄膜分散法合成磷脂微囊,在磷脂微囊膠體中加入含有二價(jià)陽(yáng)離子的電解質(zhì)溶液(氯化錳、氯化鈣、氯化鎂),確保膠體微囊處于較好的穩(wěn)定狀態(tài)。然后將穩(wěn)定的磷脂微囊體系與SA結(jié)合形成復(fù)合水凝膠,對(duì)該材料進(jìn)行了動(dòng)態(tài)光散射和掃描電鏡表征,溶脹率的測(cè)定及毒理分析。第三章利用磷脂微囊包載魚(yú)精蛋白-siRNA,測(cè)定其粒徑和電位,并進(jìn)行穩(wěn)定性考察、透射電鏡表征,然后與SA結(jié)合形成水凝膠。研究結(jié)果如下:1.磷脂微囊中加入電解質(zhì)溶液,測(cè)定其粒徑及Zeta電位值,結(jié)果表明,加入MnCl2、CaCl2溶液,微囊膠體的粒徑?jīng)]有明顯的變化。但加入某些濃度MgCl2電解質(zhì),其粒徑明顯變大。當(dāng)體系中電解質(zhì)溶液的濃度分別為6.00 mmol·L-1 MnCl2、12.50 mmol·L-1CaCl2、27.00 mmol·L-1MgCl2時(shí),其Zeta電位值均可表現(xiàn)出體系具有較好的穩(wěn)定性,Zeta電位值分別為53.11±2.14 mV、54.85±3.65 mV、53.52±1.05 mV。2.將氯化錳、氯化鈣、氯化鎂磷脂微囊膠體分別與SA混合,氯化鎂與SA形成水凝膠極弱,氯化鈣與SA形成水凝膠能力強(qiáng)于氯化錳。對(duì)氯化錳、氯化鈣磷脂微囊與SA形成的水凝膠進(jìn)行掃描電鏡(Scanning electron microscope,SEM)表征、溶脹率測(cè)定。結(jié)果表明,利用PS-Ca2+-SA這種交聯(lián)鍵合方式可以將微囊固定化,且形貌規(guī)整,結(jié)構(gòu)穩(wěn)定,細(xì)胞毒性基本為零,具有良好的細(xì)胞相容性和生物安全性。而氯化錳與SA形成的水凝膠不能將微囊捕獲。3.聚丙烯酰胺凝膠電泳確定魚(yú)精蛋白-siRNA最佳復(fù)合質(zhì)量比為4:1,用磷脂微囊包載魚(yú)精蛋白-siRNA復(fù)合物,測(cè)定其粒徑和Zeta電位分別為124.0 nm、43.42 mV,透射電鏡表征其形貌和包封情況。結(jié)果表明磷脂微囊可以包載魚(yú)精蛋白-siRNA,其粒徑較空白磷脂微囊略微增大,然后將其與SA結(jié)合形成水凝膠。
[Abstract]:Cancer, a disease that threatens the health of human life, has been a major problem in the medical world. At present, chemical therapy is still a common method for treating cancer, and the traditional chemotherapy has the defects of large toxic and side effect, poor selectivity, poor treatment effect and so on, thus limiting the application prospect and the development potential. To this end, it is desirable to increase the biological stability and bioavailability of the anti-tumor chemotherapeutic agent with the inherent surface effects and small size effects of the nano-sized particles or drug delivery body. the biological activity of the drug based on the nano anti-tumor chemotherapy depends on the chemical structure and the physical property of the nano material used, and the drug effect is improved by optimizing the performance of the nano medicine carrier, or by a molecular modification vector to obtain the ability of the nanodrug delivery body to actively target delivery and position release. Gene therapy is an ideal means for the treatment of malignant tumors, genetic diseases and acquired diseases. The siRNA can degrade the m-RNA of the homologous sequence and specifically inhibit the expression of the cancer cell-related genes, thereby inhibiting the growth and reproduction, invasion and diffusion of cancer cells. However, the technique currently available to introduce an exogenous gene into a specific site of the lesion is still limited, and in order to further achieve the purpose of gene therapy, it is necessary to first address the problem of gene transfer. Therefore, the key to gene therapy is to obtain a vector capable of effectively wrapping a gene drug, carrying it through the cell membrane and transferring it into the nucleus to express it. The liposome is one of the most promising drug, gene, protein and other delivery systems due to many advantages, such as non-toxicity, targeting, biocompatibility, and slow release, but its application range is limited by thermodynamic instability. To this end, through the physical-chemical modification of the micro-capsule, the size and the like are adjusted to improve the stability of the liposome outside the body, and various more stable liposomes are developed, for example, the invisible liposome, the solid lipid nanoparticles, and the like, but its stability problem is still present. The method comprises the following steps of: adding an electrolyte solution (manganese chloride, calcium chloride and magnesium chloride) containing a divalent cation in a phospholipid microcapsule colloid, ensuring that the colloid micro-capsule is in a better stable state, and combining the stable phospholipid microcapsule system with sodium alginate (SA) to form a novel hydrogel, The thermodynamic instability of the phospholipid microcapsule in the liquid is overcome, and the protamine-siRNA is loaded with the material, and the morphology and the encapsulation are characterized. This study is divided into three chapters. The main contents of this study are as follows: The first chapter provides an overview of the difficulties and defects of the gene and drug treatment, the various types of genes commonly used, the drug carrier, the active targeting and the passive targeting of the drugs, and the means of intelligent response release. In the second chapter, the membrane dispersion method is used to synthesize the phospholipid microcapsule, and the electrolyte solution containing the divalent cation (manganese chloride, calcium chloride and magnesium chloride) is added to the phospholipid microcapsule colloid to ensure that the colloid micro-capsule is in a better stable state. and then the stable phospholipid microcapsule system and SA are combined to form a composite hydrogel, and the material is subjected to dynamic light scattering and scanning electron microscopy, and the swelling ratio is determined and the toxicological analysis is carried out. The third chapter uses the phospholipid microcapsule to carry the protamine-siRNA to determine its particle size and potential, and conduct the stability study, the transmission electron microscope characterization, and then combine with SA to form the hydrogel. The results of the study are as follows: 1. The particle size and Zeta potential of the microcapsule were measured by adding the electrolyte solution to the phospholipid microcapsule. The results showed that the particle size of the micro-encapsulated colloid was not changed obviously with the addition of MnCl2 and CaCl2 solution. but some concentration of mgcl2 electrolyte is added, and the particle size of the mgcl2 electrolyte is obviously changed. When the concentration of the electrolyte solution in the system is 6.00 mmol 路 L-1MnCl2, 12.50 mmol 路 L-1CaCl2, and 27.00 mmol 路 L-1MgCl2, the Zeta potential value of the system can show that the system has good stability. The Zeta potential value is 53. 11-2.14mV, 54. 85-3.65 mV, 53. 52-1. 05mV. 2, respectively. the colloid of the manganese chloride, the calcium chloride and the magnesium chloride phospholipid is respectively mixed with the SA, and the magnesium chloride and the SA form the hydrogel very weak; and the calcium chloride and the SA form the hydrogel with the capability of being stronger than that of the manganese chloride. Scanning electron microscopy (SEM) was used to characterize the water-gel formed by the micro-capsule of manganese chloride, calcium chloride and the SA, and the swelling rate was determined. The results show that the micro-capsule can be immobilized by using PS-Ca2 +-SA, and the morphology is regular, the structure is stable, the cytotoxicity is basically zero, and it has good cell compatibility and biological safety. and the hydrogel which is formed by manganese chloride and the sa cannot capture the microcapsules. The optimal composite mass ratio of protamine-siRNA was 4: 1, and the particle size and Zeta potential were 124.0nm, 432.42mV, respectively. The results showed that the phospholipid microcapsules can be encapsulated with protamine-siRNA, whose particle size is slightly larger than that of the blank phospholipid microcapsule, and then it is combined with SA to form a hydrogel.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：O648.17
本文編號(hào)：2328559
[Abstract]:Cancer, a disease that threatens the health of human life, has been a major problem in the medical world. At present, chemical therapy is still a common method for treating cancer, and the traditional chemotherapy has the defects of large toxic and side effect, poor selectivity, poor treatment effect and so on, thus limiting the application prospect and the development potential. To this end, it is desirable to increase the biological stability and bioavailability of the anti-tumor chemotherapeutic agent with the inherent surface effects and small size effects of the nano-sized particles or drug delivery body. the biological activity of the drug based on the nano anti-tumor chemotherapy depends on the chemical structure and the physical property of the nano material used, and the drug effect is improved by optimizing the performance of the nano medicine carrier, or by a molecular modification vector to obtain the ability of the nanodrug delivery body to actively target delivery and position release. Gene therapy is an ideal means for the treatment of malignant tumors, genetic diseases and acquired diseases. The siRNA can degrade the m-RNA of the homologous sequence and specifically inhibit the expression of the cancer cell-related genes, thereby inhibiting the growth and reproduction, invasion and diffusion of cancer cells. However, the technique currently available to introduce an exogenous gene into a specific site of the lesion is still limited, and in order to further achieve the purpose of gene therapy, it is necessary to first address the problem of gene transfer. Therefore, the key to gene therapy is to obtain a vector capable of effectively wrapping a gene drug, carrying it through the cell membrane and transferring it into the nucleus to express it. The liposome is one of the most promising drug, gene, protein and other delivery systems due to many advantages, such as non-toxicity, targeting, biocompatibility, and slow release, but its application range is limited by thermodynamic instability. To this end, through the physical-chemical modification of the micro-capsule, the size and the like are adjusted to improve the stability of the liposome outside the body, and various more stable liposomes are developed, for example, the invisible liposome, the solid lipid nanoparticles, and the like, but its stability problem is still present. The method comprises the following steps of: adding an electrolyte solution (manganese chloride, calcium chloride and magnesium chloride) containing a divalent cation in a phospholipid microcapsule colloid, ensuring that the colloid micro-capsule is in a better stable state, and combining the stable phospholipid microcapsule system with sodium alginate (SA) to form a novel hydrogel, The thermodynamic instability of the phospholipid microcapsule in the liquid is overcome, and the protamine-siRNA is loaded with the material, and the morphology and the encapsulation are characterized. This study is divided into three chapters. The main contents of this study are as follows: The first chapter provides an overview of the difficulties and defects of the gene and drug treatment, the various types of genes commonly used, the drug carrier, the active targeting and the passive targeting of the drugs, and the means of intelligent response release. In the second chapter, the membrane dispersion method is used to synthesize the phospholipid microcapsule, and the electrolyte solution containing the divalent cation (manganese chloride, calcium chloride and magnesium chloride) is added to the phospholipid microcapsule colloid to ensure that the colloid micro-capsule is in a better stable state. and then the stable phospholipid microcapsule system and SA are combined to form a composite hydrogel, and the material is subjected to dynamic light scattering and scanning electron microscopy, and the swelling ratio is determined and the toxicological analysis is carried out. The third chapter uses the phospholipid microcapsule to carry the protamine-siRNA to determine its particle size and potential, and conduct the stability study, the transmission electron microscope characterization, and then combine with SA to form the hydrogel. The results of the study are as follows: 1. The particle size and Zeta potential of the microcapsule were measured by adding the electrolyte solution to the phospholipid microcapsule. The results showed that the particle size of the micro-encapsulated colloid was not changed obviously with the addition of MnCl2 and CaCl2 solution. but some concentration of mgcl2 electrolyte is added, and the particle size of the mgcl2 electrolyte is obviously changed. When the concentration of the electrolyte solution in the system is 6.00 mmol 路 L-1MnCl2, 12.50 mmol 路 L-1CaCl2, and 27.00 mmol 路 L-1MgCl2, the Zeta potential value of the system can show that the system has good stability. The Zeta potential value is 53. 11-2.14mV, 54. 85-3.65 mV, 53. 52-1. 05mV. 2, respectively. the colloid of the manganese chloride, the calcium chloride and the magnesium chloride phospholipid is respectively mixed with the SA, and the magnesium chloride and the SA form the hydrogel very weak; and the calcium chloride and the SA form the hydrogel with the capability of being stronger than that of the manganese chloride. Scanning electron microscopy (SEM) was used to characterize the water-gel formed by the micro-capsule of manganese chloride, calcium chloride and the SA, and the swelling rate was determined. The results show that the micro-capsule can be immobilized by using PS-Ca2 +-SA, and the morphology is regular, the structure is stable, the cytotoxicity is basically zero, and it has good cell compatibility and biological safety. and the hydrogel which is formed by manganese chloride and the sa cannot capture the microcapsules. The optimal composite mass ratio of protamine-siRNA was 4: 1, and the particle size and Zeta potential were 124.0nm, 432.42mV, respectively. The results showed that the phospholipid microcapsules can be encapsulated with protamine-siRNA, whose particle size is slightly larger than that of the blank phospholipid microcapsule, and then it is combined with SA to form a hydrogel.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：O648.17
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