本文關(guān)鍵詞： 癌癥診斷治療 透明質(zhì)酸 形貌調(diào)控 協(xié)同治療 抗藥性　出處：《浙江大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：構(gòu)建用于癌癥快速診斷和治療的多功能納米粒子是腫瘤治療領(lǐng)域的研究熱點(diǎn)之一。透明質(zhì)酸(HA)是細(xì)胞外基質(zhì)的重要組成成分,具有非常好的生物相容性和體內(nèi)可降解特性。此外,透明質(zhì)酸可與多種癌細(xì)胞表面過(guò)度表達(dá)的CD44受體進(jìn)行特異性結(jié)合,從而增加針對(duì)癌細(xì)胞的靶向作用,所以HA是一種非常好的表面修飾和藥物載體材料。因此,本文選用HA作為主要原料,構(gòu)建用于癌癥診斷治療的多功能納米粒子,研究?jī)?nèi)容主要分為以下兩個(gè)部分:1.多功能納米粒子在癌癥的診斷治療方面引起越來(lái)越多的關(guān)注。除了尺寸和表面化學(xué)特性之外,形貌是影響納米粒子體內(nèi)循環(huán)時(shí)間、生物分布、靶向性以及細(xì)胞內(nèi)吞的重要因素。由于金納米粒子具有無(wú)毒、可以簡(jiǎn)單地大量合成、表面易于修飾、特殊的光學(xué)特性等優(yōu)點(diǎn),能滿足癌癥診斷治療的不同需求。本文首先合成金納米桿(AuNRs)和金納米球(AuNSs)來(lái)構(gòu)建兩種不同形貌的金納米探針,其中AuNRs的長(zhǎng)約50nm,截面直徑約為12mn,AuNSs直徑約為20nm。通過(guò)近紅外光熒光染料尼羅藍(lán)(NB)標(biāo)記的巰基化透明質(zhì)酸(HS-HA-NB)來(lái)包裹兩種不同形貌的金納米粒子,構(gòu)建穩(wěn)定的納米探針AuNR@HS-HA-NB和AuNS@HS-HA-NB。由于透明質(zhì)酸優(yōu)異的生物相容性和體內(nèi)可降解特性,納米探針在不同pH磷酸緩沖液(pH=4-8)和80%濃度的血清中均表現(xiàn)出很好的穩(wěn)定性,同時(shí)乳腺癌(MCF-7)細(xì)胞毒性實(shí)驗(yàn)表明在納米探針表面包裹HS-HA-NB后,毒性顯著降低。對(duì)比在MCF-7細(xì)胞內(nèi)的成像結(jié)果發(fā)現(xiàn),相對(duì)于球形探針,桿形探針可以更加快速地被細(xì)胞內(nèi)吞并產(chǎn)生熒光,這可能是由于其長(zhǎng)軸與細(xì)胞膜表面接觸時(shí)能夠提供更大的接觸面積,因此可以快速有效地被細(xì)胞內(nèi)吞。探針的HA靶向性也通過(guò)競(jìng)爭(zhēng)抑制實(shí)驗(yàn)得到了驗(yàn)證。而且,在近紅外光照后,加入金納米桿探針的實(shí)驗(yàn)組細(xì)胞觀測(cè)到了 MCF-7細(xì)胞的凋亡。以上實(shí)驗(yàn)結(jié)果證明AuNR@HS-HA-NB納米探針能夠?qū)崿F(xiàn)靶向快速準(zhǔn)確成像和光熱治療。2.化療已經(jīng)被廣泛地應(yīng)用于多種類型的癌癥治療當(dāng)中,抗藥性和缺乏癌細(xì)胞的靶向性是導(dǎo)致癌癥化療失敗的主要因素。采用光熱治療產(chǎn)生的過(guò)高熱來(lái)解除癌細(xì)胞的抗藥性受到了廣泛的關(guān)注。本文采用胱胺(Cys)交聯(lián)的HA包裹AuNRs形成HA-Cys@AuNR納米凝膠來(lái)負(fù)載阿霉素(DOX),其載藥量為0.5±0.017%,包封率為51.9±3.5%。并合成了不含有AuNRs的納米凝膠HA-Cys,負(fù)載DOX后其載藥量為0.5±0.014%,包封率為56.8±3.4%。由于Cys上存在二硫鍵,因此在GSH條件下,載藥的HA-Cys@AuNR@DOX及HA-Cys@DOX納米凝膠均能夠有效的響應(yīng)性釋放DOX,4 h藥物釋放量分別為69.9%和72.24%。細(xì)胞實(shí)驗(yàn)表明HA-Cys@AuNR@DOX及HA-Cys@DOX納米凝膠均能對(duì)有效殺傷藥物敏感的MCF-7細(xì)胞,但是對(duì)抗藥性的MCF-7 ADR細(xì)胞卻無(wú)明顯效果。在HA-Cys @AuNR@D OX納米凝膠對(duì)癌細(xì)胞治療的過(guò)程中加入近紅外光照射,AuNRs的光熱作用能有效解除癌細(xì)胞的抗藥性,造成MCF-7ADR凋亡。通過(guò)熒光顯微鏡進(jìn)一步研究發(fā)現(xiàn),增加近紅外光照后,細(xì)胞內(nèi)部的DOX排出過(guò)程被抑制,使細(xì)胞的抗藥性消失,這可能是由于抗藥細(xì)胞在光熱作用產(chǎn)生過(guò)高熱的條件下,細(xì)胞膜通透性改變。在靶向性實(shí)驗(yàn)中,通過(guò)競(jìng)爭(zhēng)抑制實(shí)驗(yàn)驗(yàn)證了納米凝膠對(duì)MCF-7細(xì)胞的靶向性作用。采用HA成功制備了一種針對(duì)抗藥性細(xì)胞的靶向性光熱-藥物協(xié)同治療的多功能納米凝膠。本文采用的基于HA設(shè)計(jì)的納米探針及納米藥物載體,為癌癥診斷治療多功能納米粒子的研究提供了新的思路。
[Abstract]:For the construction of multifunctional nanoparticles rapid diagnosis and treatment of cancer is one of the hot research field of cancer therapy. Hyaluronic acid (HA) is an important component of the extracellular matrix, has very good biocompatibility and in vivo biological degradable properties. In addition, hyaluronic acid can be overexpressed and surface multiple cancer cell CD44 receptor the specificity of binding, thereby increasing in cancer cell targeting, so HA is a very good surface modification and drug carrier material. Therefore, this paper chooses HA as the main raw material, for the construction of multifunctional nanoparticles for cancer diagnosis and treatment, the main content is divided into the following two parts: 1. multifunctional nanoparticles caused more and more attention in the diagnosis and treatment of cancer. In addition to the size and surface chemical properties, morphology is the influence of nanoparticle circulation time in vivo, biodistribution, targeted to Important factors and endocytosis. The gold nanoparticles are nontoxic, simple in large quantities, easy surface modification, special optical properties and other advantages, to meet the different needs of cancer diagnosis and treatment. This paper firstly synthesized gold nanorods (AuNRs) and gold nanoparticles (AuNSs) to construct two different morphologies of gold nano probe, which AuNRs is about 50nm, the diameter is about 12mn, the diameter of AuNSs is about 20nm. by near infrared fluorescent dye Nile Blue (NB) thiolated hyaluronic acid (HS-HA-NB) to mark the package of two different morphologies of gold nanoparticles, build stable nano probe AuNR@HS-HA-NB and AuNS@HS-HA-NB. due to excellent hyaluronic acid the in vivo biocompatibility and biodegradable properties, nano probe in different pH phosphate buffer (pH=4-8) and 80% serum concentration showed good stability, and breast cancer (MCF-7) cell toxicity The experiment showed that the coated nano probe after HS-HA-NB toxicity was significantly reduced. Contrast imaging in MCF-7 cells and found that compared to the spherical probe, the rod probe can more quickly by intracellular annexation of fluorescence, which may be due to the long axis of the cell membrane surface contact can provide larger contact area, so you can quickly and efficiently internalized by cells. HA target probe to the competitive inhibition experiment was verified. Moreover, in the near infrared light, the cells in the experimental group with observation of gold nanorods probe to MCF-7 cell apoptosis. These results demonstrated that AuNR@HS-HA-NB nano probe can achieve fast and accurate imaging and targeting photothermal therapy.2. chemotherapy has been widely used in various types of cancer treatment, drug resistance and lack of cancer cell targeting is caused mainly due to the failure of cancer chemotherapy . high heat generated by photothermal therapy to relieve cancer resistance has been widespread concern. This paper used cysteamine (Cys) HA coated AuNRs crosslinked gel to form HA-Cys@AuNR nanoparticles loaded with doxorubicin (DOX), the drug loading was 0.5 + 0.017%, the encapsulation rate was 51.9 + 3.5%. and nano HA-Cys gel not containing AuNRs was synthesized, load DOX after its drug loading was 0.5 + 0.014%, the encapsulation rate was 56.8 + 3.4%. due to the presence of the two disulfide bonds in Cys, so under the condition of GSH, HA-Cys@AuNR@DOX and HA-Cys@DOX nano gel of the drug were able to respond effectively to release DOX, 4 h drug release amount was 69.9% experimental results show that HA-Cys@AuNR@DOX and 72.24%. cells and HA-Cys@DOX nano gel can effectively kill MCF-7 cells in drug sensitive and drug-resistant ADR cells but MCF-7 had no obvious effect. The treatment of cancer cells in HA-Cys @AuNR@D OX nanogel too With near infrared light in the process of resistance AuNRs photothermal effect can effectively relieve cancer cells, resulting in apoptosis of MCF-7ADR. Further study by fluorescent microscope, increase near infrared illumination, the cell's internal DOX discharge process is inhibited, the cell resistance disappeared, this may be due to the excessive heat generated in the resistant cells the photothermal effect under the condition of cell membrane permeability changes. To experiment in the target, the competitive inhibition experiment of nano gel on MCF-7 cell targeting preparation. A needle resistant cell using HA successfully targeted multifunctional nano gel of photothermal therapy. Drug co nano probe and the nano drug carriers based on HA design of this paper provides a new idea for the treatment of multifunctional nanoparticles for cancer diagnosis.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.4;TB383.1

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