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生物可降解交聯(lián)納米藥物用于腫瘤高效靶向治療與診斷

發(fā)布時(shí)間:2018-04-30 19:55

  本文選題:生物可降解 + 可逆自交聯(lián)納米藥物; 參考:《蘇州大學(xué)》2016年博士論文


【摘要】:隨著腫瘤發(fā)病率及死亡率急速增長(zhǎng),尋求高效、安全的治療方式刻不容緩。納米藥物由于提高了抗癌藥物溶解性、延長(zhǎng)體內(nèi)循環(huán)時(shí)間、通過(guò)EPR效應(yīng)增強(qiáng)在病灶部位累積量等優(yōu)點(diǎn)而發(fā)展迅速。其中,Doxilò作為首例納米藥物在1995年被FDA批準(zhǔn)主要用于卡波濟(jì)式肉瘤、卵巢癌及多發(fā)性骨髓瘤的治療;Abraxaneò因提高了PTX的溶解度而在2005年上市,用于臨床治療轉(zhuǎn)移性乳腺癌、胰腺癌及非小細(xì)胞肺癌。隨著韓國(guó)三陽(yáng)公司研制的首個(gè)聚合物納米藥物Genexol-PM被批準(zhǔn)用于卵巢癌、肺癌和乳腺癌,研究者們繼續(xù)推動(dòng)包括NK911和NK105在內(nèi)的一系列聚合物納米藥物進(jìn)入臨床試驗(yàn),并取得了階段性成果。納米藥物的初步成功鼓勵(lì)著人們探究更多多功能納米載體用于腫瘤靶向治療,其中,基于脂肪族聚酯、聚碳酸酯和聚氨基酸的聚合物載體由于優(yōu)越的生物相容性、生物可降解性、無(wú)毒的降解產(chǎn)物而受到廣泛青睞。然而,體內(nèi)穩(wěn)定性欠缺、難以在腫瘤部位滲透、腫瘤細(xì)胞內(nèi)吞少、細(xì)胞內(nèi)釋放緩慢是傳統(tǒng)藥物提高治療效率并走向臨床需翻越的四座大山。除此之外,探究特異性強(qiáng)、高效率、無(wú)毒的基因治療及診斷治療一體化多功能納米平臺(tái)也是聚合物納米藥物發(fā)展的兩大趨勢(shì)。論文第一章對(duì)聚合物納米藥物用于腫瘤治療的發(fā)展現(xiàn)狀、面臨的挑戰(zhàn)以及解決方法等作了概述。為此,本論文中我們?cè)O(shè)計(jì)了腫瘤主動(dòng)靶向、細(xì)胞內(nèi)還原敏感、具有自交聯(lián)功能的生物可降解聚合物膠束和囊泡納米藥物平臺(tái)技術(shù),發(fā)現(xiàn)其在不同腫瘤模型中的優(yōu)異表現(xiàn),對(duì)新型抗腫瘤納米藥物的臨床試驗(yàn)起到了積極的推動(dòng)作用。本論文第二章中,我們?cè)O(shè)計(jì)合成了具有自主知識(shí)產(chǎn)權(quán)的新型二硫戊環(huán)三亞甲基碳酸酯(DTC),并基于它制備了一系列腫瘤靶向、可逆自交聯(lián)的生物可降解聚合物膠束及囊泡納米藥物多功能平臺(tái)。基于DTC我們合成了表面偶聯(lián)cRGD的兩親性聚合物聚乙二醇-b-聚二硫戊環(huán)三亞甲基碳酸酯(PEG-b-PDTC),制備了載阿霉素的自交聯(lián)膠束納米藥物,用于主動(dòng)靶向到α_vβ_3整合蛋白過(guò)表達(dá)的新生血管及黑色素瘤細(xì)胞(B16)。該膠束在生理環(huán)境下穩(wěn)定而在模擬細(xì)胞內(nèi)還原環(huán)境下(10 mM GSH),硫硫鍵斷裂解交聯(lián),快速釋放藥物;在小鼠體內(nèi)消除半衰期t1/2,β為6.19小時(shí)(自由dox:0.18小時(shí)),6小時(shí)后腫瘤積累量為6.13%id/g,最大耐受量(mtd100mg/kg)為自由阿霉素的20倍以上,并對(duì)荷b16黑色素瘤小鼠呈現(xiàn)高抗腫瘤活性和低毒副作用,顯示了設(shè)計(jì)的優(yōu)越性。膠束納米藥物的成功鼓舞我們?cè)诘谌卵芯孔越宦?lián)的聚合物囊泡納米藥物。我們?cè)O(shè)計(jì)合成了基于dtc和三亞甲基碳酸酯(tmc)的兩親性聚合物peg-p(tmc-dtc)和crgd-peg-p(tmc-dtc),其疏水鏈可調(diào)控制以適宜囊泡的制備。通過(guò)ph梯度法高效裝載dox·hcl并自交聯(lián)后得到靶向、穩(wěn)定囊泡納米藥物crgd-ps-dox(載藥量為15.4wt.%,120納米)。crgd-ps-dox被α_vβ_3過(guò)表達(dá)的人腦膠質(zhì)瘤細(xì)胞u87mg主動(dòng)靶向攝取,在細(xì)胞內(nèi)和小鼠體內(nèi)有效抑制腫瘤細(xì)胞生長(zhǎng)(半致死率ic50為1.77μg/ml)。其呈現(xiàn)小鼠體內(nèi)循環(huán)時(shí)間長(zhǎng)(t1/2,β為4.49小時(shí))、腫瘤富集多(6.78%id/g)、腫瘤抑制作用強(qiáng)、毒副作用低的特點(diǎn),拓展了以dtc為基礎(chǔ)的自交聯(lián)囊泡用于抗腫瘤藥物的靶向傳遞,為研究該體系在其他腫瘤模型的治療應(yīng)用奠定了基礎(chǔ)。針對(duì)目前全球范圍內(nèi)發(fā)病率及死亡率最高的肺癌,在第四章中基于第三章的自交聯(lián)囊泡,我們分別探究了三種肺癌主動(dòng)靶向的囊泡納米藥物crgd-ps-dox、cngq-ps-dox及cngq-ps-dtx,研究了其在a549肺癌皮下瘤和原位瘤模型的靶向治療效果。三者均對(duì)a549皮下瘤體現(xiàn)了高腫瘤富集量、強(qiáng)腫瘤抑制能力、長(zhǎng)生存期和低毒性。而在荷a549原位瘤裸鼠中的靶向治療效果更佳,尤其是crgd-ps-dox和cngq-ps-dox能有效遏制腫瘤增長(zhǎng)及轉(zhuǎn)移,治療16天后腫瘤體積縮小;對(duì)小鼠主要臟器無(wú)毒副作用;各小鼠在45天實(shí)驗(yàn)期間的生存率為100%,而臨床使用的聚乙二醇化脂質(zhì)體阿霉素里葆多l(xiāng)ipo-dox雖然也能抑制腫瘤增長(zhǎng)但毒副作用大,32天小鼠全部死亡。由此可見(jiàn),可逆交聯(lián)囊泡無(wú)論裝載親水或疏水藥物均不改變表面性質(zhì),能有效抑制肺癌皮下和原位肺癌增長(zhǎng),向臨床應(yīng)用前進(jìn)了一大步。這些令人振奮的實(shí)驗(yàn)結(jié)果加之囊泡本身和脂質(zhì)體類似的結(jié)構(gòu)特性,促使我們?cè)诘谖逭聦⒃撃遗菁{米藥物和里葆多l(xiāng)ipo-dox在治療惡性卵巢癌方面來(lái)進(jìn)行頭對(duì)頭的比較,因后者是卵巢癌臨床治療的一線用藥。我們制備了多肽ge11表面修飾的交聯(lián)囊泡(ge11-ps-dox),其中g(shù)e11對(duì)卵巢癌細(xì)胞表面過(guò)表達(dá)的egfr受體特異性靶向的。ge11-ps-dox最大耐受劑量為160mg/kg,是lipo-dox至少8倍,大大提高了治療窗。更令人振奮的是,ge11-ps-dox在60mg/kg單劑量靜脈給藥治療荷skov-3卵巢瘤小鼠時(shí),可有效遏制腫瘤增長(zhǎng),減小毒副作用,延長(zhǎng)小鼠生存期。故ge11-ps-dox為替代lipo-dox治療卵巢癌提供了可能性。針對(duì)納米藥物應(yīng)用中腫瘤滲透性低這一難題,我們?cè)诘诹略O(shè)計(jì)將具有強(qiáng)腫瘤滲透功能、同時(shí)能靶向腫瘤新生血管的多肽irgd偶聯(lián)到囊泡表面得到irgd-ps-dox,系統(tǒng)研究了其中irgd表面密度對(duì)血液循環(huán)、腫瘤富集、滲透及治療的影響。irgd-ps-dox實(shí)現(xiàn)了它在血管豐富、α_vβ_3整合蛋白過(guò)表達(dá)的b16腫瘤模型中高效的滲透性及抗腫瘤活性。體內(nèi)外實(shí)驗(yàn)表明,含50%irgd的irgd50-ps-dox有最長(zhǎng)的循環(huán)時(shí)間(4.19小時(shí))、最高的腫瘤富集量(4.89%id/g)、最佳的滲透及腫瘤抑制效率、最長(zhǎng)的生存期,體現(xiàn)了高腫瘤滲透性囊泡對(duì)提高納米藥物體內(nèi)療效的重要性。生物大分子藥物如核酸和蛋白質(zhì)具有小分子藥物無(wú)法比擬的優(yōu)點(diǎn),包括特異性強(qiáng)和高效無(wú)毒等,尤其是小干擾rna(sirna)具有直接的抗腫瘤作用。第七章中我們拓展了囊泡納米藥物技術(shù),設(shè)計(jì)了囊泡內(nèi)層為短鏈帶正電荷的支化聚乙烯亞胺pei的囊泡,其pei內(nèi)殼可高效復(fù)合sirna(sirna-cngq/rccps)并助其更好逃逸內(nèi)涵體,實(shí)現(xiàn)了sirna的高效體內(nèi)遞送。該囊泡sirna在體內(nèi)循環(huán)的半衰期為1.78小時(shí),在荷a549原位肺癌裸鼠肺中富集量為4.02%id/g,復(fù)合治療基因siplk1后的囊泡可有效抑制腫瘤增長(zhǎng)且小鼠中值為54.0天,遠(yuǎn)高于其他對(duì)照組。該囊泡納米藥物為低毒、高效的生物療法,尤其是基因療法靶向治療腫瘤提供了技術(shù)平臺(tái)。癌癥死亡率攀高的重要原因之一就是癌癥缺乏安全有效精準(zhǔn)的早期診斷,發(fā)現(xiàn)時(shí)已是中晚期且已轉(zhuǎn)移,給癌癥的治療帶來(lái)巨大挑戰(zhàn)。包括ct在內(nèi)的非侵入性影像學(xué)診斷方法在臨床上較常使用,但是需要使用大量的造影劑。在第八章中我們制備了基于生物可降解、含碘聚碳酸酯的囊泡納米造影劑,用于腫瘤的診斷及診療。首先我們?cè)O(shè)計(jì)合成了具有知識(shí)產(chǎn)權(quán)的含雙碘的碳酸酯單體2,2'-碘代甲基-三亞甲基碳酸酯(ic),通過(guò)可控開(kāi)環(huán)聚合得到含碘量高達(dá)60.4wt.%的兩親性聚合物peg-pic和peg-p(dtc-ic)。制備的peg-pic囊泡ips在相同碘量下在體內(nèi)外的造影效果均優(yōu)于小分子造影劑碘海醇,其體內(nèi)t1/2,β為3.73小時(shí),尾靜脈注射入荷u87mg皮下瘤小鼠中發(fā)現(xiàn)腫瘤處ct值增加了43.2hu,而在荷a549原位瘤小鼠中ct值增加量為121.3hu,為腫瘤的早期診斷奠定了基礎(chǔ)。接著我們探究了表面偶聯(lián)crgd、可逆自交聯(lián)囊泡顯影劑納米藥物crgd-rcips-dox在荷b16皮下瘤小鼠的診斷與治療。其呈現(xiàn)優(yōu)異的體內(nèi)外穩(wěn)定性,t1/2,β長(zhǎng)達(dá)6.84小時(shí);尾靜脈注射在荷b16皮下瘤小鼠體內(nèi)4小時(shí)后,發(fā)現(xiàn)腫瘤的特異性ct成像,腫瘤的ct增長(zhǎng)量為碘海醇的16.4倍。此外,DOX在腫瘤積累量達(dá)6.68%ID/g,佐證了其特異性CT成像和高抗腫瘤活性且小毒副作用。cRGD-RCIPs-Dox集特異性CT成像診斷、主動(dòng)靶向、高效裝載抗癌藥物、細(xì)胞內(nèi)觸發(fā)藥物釋放、高效抑瘤等優(yōu)點(diǎn)于一身,在腫瘤的早期診斷及靶向治療上前景廣闊。第九章對(duì)本論文工作進(jìn)行了全面總結(jié),并展望了今后可開(kāi)展的工作。
[Abstract]:With the rapid growth of tumor incidence and mortality, it is urgent to seek efficient and safe treatment. Nanodrugs have developed rapidly because of improving the solubility of anticancer drugs, prolonging the circulation time in the body, and increasing the accumulation of the lesions by the EPR effect. In 1995, Doxil was approved by FDA as the first drug. Mainly used for the treatment of kapoji's sarcoma, ovarian cancer and multiple myeloma; Abraxane was listed in 2005 for improving the solubility of PTX, for clinical treatment of metastatic breast, pancreatic and non-small cell lung cancer. The first polymeric nanomoto drug Genexol-PM was approved for ovarian cancer and lung cancer with the development of South Korea's San Yang company. And breast cancer, the researchers continue to promote a series of polymer nanometers, including NK911 and NK105, to enter clinical trials and achieve stage results. The initial success of nanomedicine encourages people to explore more multifunctional nanoscale for tumor targeting therapy based on aliphatic polyester, polycarbonate and polyamino acids. Polymer carriers are widely favored because of their superior biocompatibility, biodegradability and non-toxic degradation products. However, the lack of stability in the body is difficult to infiltrate in the tumor sites, the tumor cells are swallowed up, and the intracellular release is slow to be the four mountains that traditional drugs increase the therapeutic efficiency and move towards the clinical need. In addition, It is also the two trend of the development of polymer nanomaterials to explore the specific, high efficiency, non-toxic gene therapy and diagnosis and treatment integrated multi-functional nano platform. Chapter 1 of the thesis is a summary of the current development of polymer nanometers for cancer treatment, the challenges faced and the solution method. In this paper, we designed the paper. The tumor active target, the cell reduction sensitivity, the biodegradable polymer micelles and the vesicular nano drug platform technology, which have the self intersection function, found their excellent performance in different tumor models, and played an active role in the clinical trials of new anti-tumor nanoscale drugs. In the second chapter of this paper, we designed and synthesized A new type of two sulfur amyl ring Sanya methyl carbonate (DTC) with independent intellectual property rights was used to prepare a series of tumor targeting, reversible self crosslinked biodegradable polymer micelles and vesicular nano drug multifunction platforms. Based on DTC, we synthesized the two Pro polymer polyethylene glycol two sulphate ring three on the surface of the coupling of cRGD. Methylene carbonate (PEG-b-PDTC), prepared a self crosslinked micellar nano drug carrying doxorubicin, used to actively target the overexpressed alpha _v beta _3 integrin's neovascularization and melanoma cells (B16). The micelles are stable under the physiological environment and in the simulated cell reduction environment (10 mM GSH), sulfur sulfur bond cleavage and crosslinking, and the rapid release of drugs In mice, the half-life t1/2, beta 6.19 hours (free dox:0.18 hours), 6 hours after the tumor accumulation was 6.13%id/g, the maximum tolerance (mtd100mg/kg) was more than 20 times that of free adriamycin, and showed high anti-tumor activity and low toxicity against B16 melanoma mice. It showed the superiority of the design. We have inspired us to study self crosslinked polymer vesicles in the third chapter. We designed and synthesized two amphiphilic polymer peg-p (tmc-dtc) and crgd-peg-p (tmc-dtc) based on DTC and Sanya methyl carbonate (TMC). The hydrophobic chain is adjustable for the preparation of the vesicles. The DOX. HCl and self crosslinking are efficiently loaded by the pH gradient method. Targeting, stable vesicle nanoscale drug crgd-ps-dox (15.4wt.%, 120 nm).Crgd-ps-dox is active targeting of human glioma cells that are overexpressed by alpha _v beta _3, and effectively inhibits the growth of tumor cells in the cells and in mice (the semi lethal rate IC50 is 1.77 mu g/ml). It presents a long cycle time in mice (t1/2, beta 4.49 small). When the tumor is enriched and enriched (6.78%id/g), the tumor suppresses and the side effects are low, the DTC based self crosslinking vesicle is used to target the target delivery of antitumor drugs. It lays the foundation for the study of the system in the treatment of other tumor models. In the four chapter, based on the third chapter, we explored three kinds of lung cancer active targeted vesicle nanometers, crgd-ps-dox, cngq-ps-dox and cngq-ps-dtx, and studied the target therapy effect in A549 lung cancer subcutaneous tumor and in situ tumor model. All of the three were high tumor accumulation, strong tumor suppressor ability and long tumor suppressor ability in A549 subcutaneous tumor. The survival time and low toxicity. But in the nude mice bearing A549 tumor in situ, the target therapy was better, especially the crgd-ps-dox and cngq-ps-dox could effectively inhibit the tumor growth and metastasis. The tumor volume reduced after 16 days of treatment and the nontoxic side effect on the main organs of the mice; the survival rate of each mouse during the 45 day experiment was 100%, and the clinical use of polyb two. The alcohol liposomes, adriamycin lipo-dox, can also inhibit the growth of the tumor but the toxic and side effects are large, and all the mice died in the 32 day. Thus, the reversible cross-linked vesicles, regardless of the hydrophilic or hydrophobic drugs, do not change the surface properties, can effectively inhibit the growth of lung cancer subcutaneous and in situ lung cancer, and make a big step forward to clinical application. The exhilarating experimental results, coupled with the structural characteristics of the vesicle itself and liposomes, prompted us to compare the vesicle nanoscale with the Li Pao lipo-dox in the treatment of malignant ovarian cancer in the fifth chapter, because the latter is the first line of clinical treatment for ovarian cancer. We have prepared the cross-linked sac of the peptide ge11 surface modification. Ge11-ps-dox, in which the maximum tolerance dose of ge11 to the EGFR receptor specific targeting of the ovarian cancer cell surface is 160mg/kg, at least 8 times as much as lipo-dox, which greatly improves the treatment window. It is more exciting that ge11-ps-dox can effectively contain the tumor when the single dose of ge11-ps-dox is administered intravenously to the mice with SKOV-3 ovarian tumor. Increase, reduce toxic side effects and prolong the survival time of mice. Therefore, ge11-ps-dox provides a possibility to replace lipo-dox in the treatment of ovarian cancer. In the sixth chapter, we design a peptide irgd that has strong tumor permeability and can target the neovascularization of tumor to the surface of the vesicle. Irgd-ps-dox, a systematic study of the effects of irgd surface density on blood circulation, tumor enrichment, infiltration and treatment,.Irgd-ps-dox realized its high permeability and anti-tumor activity in the vascular rich, _v beta _3 integrin's overexpressed B16 tumor model. In vivo and in vivo, it was shown that irgd50-ps-dox containing 50%irgd had the longest cycle time. Between 4.19 hours, the highest concentration of tumor (4.89%id/g), the best osmosis and tumor inhibition efficiency, the longest life period, reflects the importance of high tumor osmotic vesicles to improve the efficacy of nanoscale drugs. Biological macromolecules such as nucleic acids and proteins have advantages unmatched by small molecular drugs, including specific and high efficiency. Non-toxic, especially small interference RNA (siRNA) has direct anti-tumor effect. In the seventh chapter, we expanded the vesicle nano drug technology, designed the vesicle of the vesicle with a short chain and positive charge of the branched polyethyleneimine Pei, and its Pei inner shell can efficiently compound siRNA (sirna-cngq /rccps) and help it to escape the endosomes better and realize siRNA The half-life of the vesicle siRNA in the body is 1.78 hours, and the concentration of the lung in nude mice bearing A549 in situ lung cancer is 4.02%id/g. The vesicles after the compound therapy gene siplk1 can effectively inhibit the growth of the tumor and the median value of the mice is 54 days, far higher than that of the other control groups. The vesicle nanoscale drug is low toxic and efficient biotherapy, In particular, gene therapy provides a technical platform for cancer treatment. One of the important reasons for the rise of cancer mortality is the lack of effective and accurate early diagnosis of cancer. It has been found in the middle and late stages and has been transferred to a great challenge for the treatment of cancer. The noninvasive imaging diagnosis, including CT, is more often in clinical practice. Use, but need to use a large number of contrast agents. In the eighth chapter, we have prepared a biodegradable, iodine containing polycarbonate vesicle nano contrast agent for the diagnosis and diagnosis of cancer. First, we designed and synthesized the iodized carbonate monomer 2,2'- iodide methyl Sanya methyl carbonate (IC) with intellectual property. The two Pro polymer peg-pic and peg-p (dtc-ic) with iodine content up to 60.4wt.% were obtained by the controlled open loop polymerization. The effect of peg-pic vesicle IPS in the same iodine amount in vivo and in vivo was better than that of small molecule contrast agent iodiol, and the t1/2, beta was 3.73 hours in its body, and the CT value increased in the tumor at the tail vein injected into the tumor of U87MG subcutaneous tumor in mice. 43.2hu, in A549 in situ tumor bearing mice, the increase of CT value was 121.3hu, which laid the foundation for the early diagnosis of the tumor. Then we explored the diagnosis and treatment of the surface coupled crgd, the reversible self crosslinking vesicle nano drug crgd-rcips-dox in the subcutaneous tumor bearing mice of B16, which showed excellent stability, t1/2, and beta as long as 6.84 small. 4 hours after the injection of the tail vein in the mice bearing B16 subcutaneous tumor, the tumor specific CT imaging was found, and the tumor's CT growth was 16.4 times as much as iodiol. In addition, the accumulation of DOX in the tumor was 6.68%ID/g, which confirmed the specific CT imaging and high antitumor activity and the small toxic side effect.CRGD-RCIPs-Dox set specific CT imaging diagnosis, active targeting The ninth chapters make a comprehensive summary of the work of this paper and look forward to the work that can be carried out in the future.

【學(xué)位授予單位】:蘇州大學(xué)
【學(xué)位級(jí)別】:博士
【學(xué)位授予年份】:2016
【分類號(hào)】:TQ460.1


本文編號(hào):1825973

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