磷酸膽堿化殼聚糖衍生物的合成、表征及生物學(xué)評(píng)價(jià)
發(fā)布時(shí)間：2018-08-14 11:57
【摘要】：本研究從細(xì)胞膜仿生的角度出發(fā)，設(shè)計(jì)合成一種新的殼聚糖磷�；苌铮粤柞０锋I的方式將細(xì)胞膜結(jié)構(gòu)單元磷酸膽堿基團(tuán)偶聯(lián)到殼聚糖骨架上，研究其合成方法、生物學(xué)性能及其納米化方法。 為了合成磷酸膽堿化殼聚糖衍生物，我們研究比較了三種殼聚糖的均相磷酸膽堿化方法：(A)采用新型高極性六氟異丙醇作反應(yīng)介質(zhì)，基于Antherton-Todd反應(yīng)實(shí)現(xiàn)殼聚糖的直接磷酰化；(B)利用N-鄰苯二甲酰化殼聚糖為中間體，以二氯磷酰膽堿為磷酰化試劑；(C)利用6-O-三苯基甲醚化殼聚糖為中間體，基于Antherton-Todd反應(yīng)實(shí)現(xiàn)磷�；Ｑ芯勘砻骱铣陕肪�(C)適合用來(lái)均相合成磷酸膽堿化殼聚糖衍生物。 應(yīng)用合成路線(C)，，通過(guò)改變投料比，基于Antherton-Todd反應(yīng)合成了三種不同取代度的水溶性磷酸膽堿化殼聚糖衍生物。NMR和FTIR譜圖上對(duì)應(yīng)-N+(CH3)3基團(tuán)吸收峰的出現(xiàn)表明磷酸膽堿基團(tuán)成功偶聯(lián)到殼聚糖骨架的氨基上，根據(jù)1H NMR譜圖的峰強(qiáng)度比計(jì)算出三種殼聚糖衍生物的取代度分別為16%、27%和42%。GPC數(shù)據(jù)顯示，與殼聚糖相比，磷酸膽堿化殼聚糖衍生物的分子量有所降低，分子量分布有所拓寬；XRD、TGA、DSC和水溶性實(shí)驗(yàn)表明，磷酸膽堿化殼聚糖衍生物的結(jié)晶性能和熱穩(wěn)定性均有不同程度地下降，但是其在水中的溶解性能得到了很大地提升，三種取代度的衍生物均可溶于pH=1-12的水溶液中。 細(xì)胞毒性實(shí)驗(yàn)表明，3T3細(xì)胞與磷酸膽堿化殼聚糖衍生物共培養(yǎng)的相對(duì)增殖率在80%-110%之間，細(xì)胞毒性為0級(jí)或1級(jí)，屬于無(wú)細(xì)胞毒性范疇；血液相容性實(shí)驗(yàn)表明，引入磷酸膽堿基團(tuán)可以延緩衍生物的凝血時(shí)間，而且可以有效抑制血小板在其上的黏附與激活；與牛血清白蛋白(BSA)的相互作用表明，磷酸膽堿基團(tuán)的引入可以有效抑制殼聚糖衍生物與BSA之間的相互作用，減小蛋白質(zhì)的構(gòu)象改變，這對(duì)避免激活因蛋白質(zhì)構(gòu)象改變而導(dǎo)致的不良生物反應(yīng)具有重要意義。 磷酸膽堿化殼聚糖衍生物可自組裝形成納米粒子。研究表明，磷酸膽堿化殼聚糖衍生物仍可與三聚磷酸鈉進(jìn)行離子交聯(lián)形成納米粒子，這些納米粒子呈現(xiàn)規(guī)則的球形結(jié)構(gòu)，粒徑在60-120nm之間，Zeta電位介于18-28mV；同時(shí)，磷酸膽堿化殼聚糖衍生物具有兩親性，可在中性水溶液中自組裝成具有疏水核親水殼的納米膠束，由低到高，三種取代度衍生物的臨界膠束濃度分別為0.129mg/mL，0.201mg/mL，0.256mg/mL。所形成的納米膠束粒徑范圍在70-110nm之間，Zeta電位接近于0，介于0-4mV之間。這兩類(lèi)納米粒子有望應(yīng)用于藥物/基因載體。
[Abstract]:In this study, a new phosphorylation derivative of chitosan was designed and synthesized from the point of view of cell membrane bionics. The phosphorylcholine group of cell membrane structure was coupled to the chitosan skeleton by phosphoramide bond, and the synthesis method of the derivative was studied. Biological properties and nanocrystalline methods. In order to synthesize chitosan derivative of choline phosphate, we studied and compared three homogenous choline phosphate methods: (A) used a new high polarity hexafluoroisopropanol as the reaction medium, and realized the direct phosphorylation of chitosan based on Antherton-Todd reaction; (B) used N-phthalic chitosan as intermediate and; (C) as phosphorylation reagent. 6-O- triphenylmethyl ether chitosan was used as intermediate to realize phosphorylation based on Antherton-Todd reaction. The results show that the synthetic route (C) is suitable for homogeneous synthesis of choline phosphate chitosan derivatives. By changing the feed ratio by using the synthetic route (C), Based on Antherton-Todd reaction, three kinds of water-soluble choline phosphate chitosan derivatives were synthesized. NMR and FTIR spectra showed that the corresponding absorption peaks of -N (CH3) _ 3 group were successfully coupled to the amino group of chitosan skeleton. According to the peak intensity ratio of 1H NMR spectrum, the substitution degree of the three chitosan derivatives is 16% and 42%.GPC data show that compared with chitosan, the molecular weight of chitosan derivative of choline phosphate is lower and the molecular weight distribution is wider. DSC and water solubility tests showed that the crystallization and thermal stability of choline phosphate chitosan derivatives decreased in varying degrees, but their solubility in water was greatly improved. All three derivatives of degree of substitution are soluble in the aqueous solution of pH=1-12. The results of cytotoxicity test showed that the relative proliferation rate was between 80% and 110%, and the cytotoxicity was grade 0 or grade 1, which belonged to the category of no cytotoxicity. The introduction of choline phosphate groups can delay the coagulation time of the derivatives and inhibit the adhesion and activation of platelets on them, and the interaction with bovine serum albumin (BSA) (BSA) suggests that, The introduction of choline phosphate group can effectively inhibit the interaction between chitosan derivatives and BSA and reduce the conformation change of protein, which is important for avoiding the activation of adverse biological reactions caused by protein conformation changes. Chitosan derivatives of choline phosphate can be self-assembled to form nanoparticles. The results show that the chitosan derivatives of choline phosphate can still be crosslinked with sodium tripolyphosphate to form nanoparticles. These nanoparticles have regular spherical structure and the potential of Zeta between 60-120nm is 18-28mV, at the same time, The chitosan derivatives of choline phosphate have amphiphilic properties and can self-assemble into nanomicelles with hydrophobic core hydrophilic shells in neutral aqueous solution. The critical micelle concentrations of the three derivatives are 0.129 mg / mL ~ 0.201 mg / mL ~ (-1) ~ 0.256 mg 路m ~ (-1) 路m ~ (-1), respectively, and the critical micelle concentrations of the three derivatives are 0.129 mg / mL ~ (-1) ~ 0.201 mg / mL ~ (-1) ~ 0.256 mg / m ~ (-1). The particle size range of the resulting micelles is close to 0 between 70-110nm and 0-4mV. These two kinds of nanoparticles are expected to be used in drug / gene carriers.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R318.08
本文編號(hào)：2182786
[Abstract]:In this study, a new phosphorylation derivative of chitosan was designed and synthesized from the point of view of cell membrane bionics. The phosphorylcholine group of cell membrane structure was coupled to the chitosan skeleton by phosphoramide bond, and the synthesis method of the derivative was studied. Biological properties and nanocrystalline methods. In order to synthesize chitosan derivative of choline phosphate, we studied and compared three homogenous choline phosphate methods: (A) used a new high polarity hexafluoroisopropanol as the reaction medium, and realized the direct phosphorylation of chitosan based on Antherton-Todd reaction; (B) used N-phthalic chitosan as intermediate and; (C) as phosphorylation reagent. 6-O- triphenylmethyl ether chitosan was used as intermediate to realize phosphorylation based on Antherton-Todd reaction. The results show that the synthetic route (C) is suitable for homogeneous synthesis of choline phosphate chitosan derivatives. By changing the feed ratio by using the synthetic route (C), Based on Antherton-Todd reaction, three kinds of water-soluble choline phosphate chitosan derivatives were synthesized. NMR and FTIR spectra showed that the corresponding absorption peaks of -N (CH3) _ 3 group were successfully coupled to the amino group of chitosan skeleton. According to the peak intensity ratio of 1H NMR spectrum, the substitution degree of the three chitosan derivatives is 16% and 42%.GPC data show that compared with chitosan, the molecular weight of chitosan derivative of choline phosphate is lower and the molecular weight distribution is wider. DSC and water solubility tests showed that the crystallization and thermal stability of choline phosphate chitosan derivatives decreased in varying degrees, but their solubility in water was greatly improved. All three derivatives of degree of substitution are soluble in the aqueous solution of pH=1-12. The results of cytotoxicity test showed that the relative proliferation rate was between 80% and 110%, and the cytotoxicity was grade 0 or grade 1, which belonged to the category of no cytotoxicity. The introduction of choline phosphate groups can delay the coagulation time of the derivatives and inhibit the adhesion and activation of platelets on them, and the interaction with bovine serum albumin (BSA) (BSA) suggests that, The introduction of choline phosphate group can effectively inhibit the interaction between chitosan derivatives and BSA and reduce the conformation change of protein, which is important for avoiding the activation of adverse biological reactions caused by protein conformation changes. Chitosan derivatives of choline phosphate can be self-assembled to form nanoparticles. The results show that the chitosan derivatives of choline phosphate can still be crosslinked with sodium tripolyphosphate to form nanoparticles. These nanoparticles have regular spherical structure and the potential of Zeta between 60-120nm is 18-28mV, at the same time, The chitosan derivatives of choline phosphate have amphiphilic properties and can self-assemble into nanomicelles with hydrophobic core hydrophilic shells in neutral aqueous solution. The critical micelle concentrations of the three derivatives are 0.129 mg / mL ~ 0.201 mg / mL ~ (-1) ~ 0.256 mg 路m ~ (-1) 路m ~ (-1), respectively, and the critical micelle concentrations of the three derivatives are 0.129 mg / mL ~ (-1) ~ 0.201 mg / mL ~ (-1) ~ 0.256 mg / m ~ (-1). The particle size range of the resulting micelles is close to 0 between 70-110nm and 0-4mV. These two kinds of nanoparticles are expected to be used in drug / gene carriers.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R318.08
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