韓智豪,呂麗偉,顧月清
(1.中國(guó)藥科大學(xué) 生物醫(yī)學(xué)工程教研室,江蘇 南京 210009)
近紅外光響應(yīng)的光熱敏感型脂質(zhì)體載藥系統(tǒng)在腫瘤靶向治療和緩控藥物釋放中的應(yīng)用
韓智豪,呂麗偉,顧月清1
(1.中國(guó)藥科大學(xué) 生物醫(yī)學(xué)工程教研室,江蘇 南京 210009)
藥物遞送系統(tǒng)如能通過(guò)光熱觸控將包裹的藥物釋放,同時(shí)進(jìn)行癌癥成像和治療,這將是一個(gè)新興的探索焦點(diǎn)。我們合成了一種新型多功能的納米脂質(zhì)體藥物遞送系統(tǒng),即所述的光熱敏感脂質(zhì)體(PTSL),它的水溶性層摻雜了阿霉素作為治療劑,同時(shí)它的疏水層包裹了cypate作為診斷劑。我們測(cè)試了PTSL的物理性質(zhì),穩(wěn)定性以及脂質(zhì)體的裝載性能,合成的納米復(fù)合材料具有針對(duì)性,穩(wěn)定性和精準(zhǔn)性,具有載藥量9±1.5%和包封率82.7±2.1%。其Tm值落在43.1℃,提高脂質(zhì)體的Tm溫度達(dá)到熱敏脂質(zhì)體的熱敏溫度,脂質(zhì)體崩解同時(shí)包裹的化療藥物釋放。這表明該P(yáng)TSL可用于腫瘤的診斷和靶向治療。
Cypate;光熱敏感脂質(zhì)體;EPR效應(yīng)觸發(fā)釋放;光熱轉(zhuǎn)換;光化學(xué)內(nèi)化;光熱治療
截至到目前,臨床上的化療藥物總是低水溶性,缺乏穩(wěn)定性,代謝快速和非選擇性的分布,這可能將導(dǎo)致高器官毒性和劑量限制性的副作用[1,2]。納米脂質(zhì)體遞送系統(tǒng)正引起研究者的關(guān)注,它作為一種替代品可以克服現(xiàn)有的抗癌藥的局限性[3-6]。 我們?cè)O(shè)計(jì)并合成了一種新型多官能的納米脂質(zhì)體藥物遞送系統(tǒng),即所述的光熱敏感脂質(zhì)體(PTSL)。多柔比星(DOX)被包裹在親水層和cypate被摻雜在熱敏感脂質(zhì)體(TSL)的疏水層,以得到DOX@PTSL復(fù)合物。這里的cypate可實(shí)現(xiàn)多種功能[7-13],包括近紅外成像,光熱轉(zhuǎn)化和誘導(dǎo)產(chǎn)生單線態(tài)氧。PTSL,粒子直徑為100-200nm,一旦靜脈注射到體內(nèi),則通過(guò)EPR效應(yīng)聚集在腫瘤部位。當(dāng)暴露在780nm近紅外光下[14],cypate可以用于輔助成像,將激光能量轉(zhuǎn)化為熱能,并產(chǎn)生單線態(tài)氧。綜合以上各種因素,DOX將被釋放到細(xì)胞質(zhì)中,然后進(jìn)入細(xì)胞核[15],發(fā)揮它們的藥理作用。
圖1 (A)-(C)的流體動(dòng)力學(xué)直徑和PTSL,DOX @ PTSL和DOX @ PTSL納米粒子的TEM照片(D)的熒光光譜(Ex=780 nm)和cypate和PTSL的紫外吸收光譜。內(nèi)部:cypate和PTSL的近紅外圖像780納米激光輻射下(E)DOX的紫外吸收光譜,CYP以及DOX @ PTSL和DOX @ PTSL作了妥善處理1%TX100Fig. 1. (A)-(C) The hydrodynamic diameter and TEM images of PTSL, DOX@PTSL and DOX@PTSL nanoparticles. (D) The fluorescence spectrum (Ex=780 nm) and UV absorbance spectrum of cypate and PTSL. Insert: the near infrared image of cypate and PTSL under 780 nm laser irradiation. (E) The UV absorbance spectrum of DOX, CYP as well as DOX@PTSL and DOX@PTSL dealed with 1% TX100.
圖2 (A)游離DOX和DOX @ PTSL的CLSM圖片與U87細(xì)胞系的定位性能研究培養(yǎng)后DOX @PTSL與U87細(xì)胞系加780近紅外激光照射培養(yǎng),DOX @ PTSL的動(dòng)力學(xué)孵育后U87細(xì)胞系和近紅外激光(1.5W)5分鐘,在不同的孵育時(shí)間點(diǎn),20分鐘(B)和40分鐘(C)照射兩個(gè)不同信道的熒光強(qiáng)度的半定量的結(jié)果,紅色為阿霉素信號(hào),綠色為溶酶體信號(hào)(D)在U87腦膠質(zhì)瘤細(xì)胞中的細(xì)胞毒性試驗(yàn)Fig. 2. (A) The CLSM pictures of free DOX and DOX@PTSL after incubated with U87 cell lines for the locating properties study and that of DOX@PTSL incubated with U87 cell lines plus 780 NIR laser irradiation. Dynamics of DOX@PTSL after incubated with U87 cell lines and irradiated with NIR laser (1.5W) for 5 minutes, at different incubation time points, the semi-quantitative results 20 minutes (B) and 40 minutes (C)for flurescence intensity of two different channels. In vitro cytotoxicity studies of five kinds of treatments performed on U87 cell lines(D) by MTT assays.
圖3 (A)用激光照射或不不同的藥劑注射后荷瘤小鼠的紅外圖像(B)腫瘤解剖圖片,15天以來(lái)的治療情況(C)用激光照射或不不同的藥劑注射后荷瘤小鼠的腫瘤組織切片的顯微HE染色照片F(xiàn)ig.3. (A) The infrared images of tumor-bearing mice after injected with different agents with laser irradiated or not. (B) the dissected tumor pictures, 15 days since treatment. (C) the microscopic pictures of tumor histological section(HE)
首先,我們用薄膜水化和浴超聲法合成了DSPEPEG2000的不同比例的TSL,然后將脂質(zhì)體4℃儲(chǔ)存三周,研究其穩(wěn)定性和分散性。為了研究藥物裝載能力和穩(wěn)定性,通過(guò)在4℃進(jìn)行三周和在37℃進(jìn)行60分鐘HBS透析。當(dāng)確認(rèn)DSPE-PEG2000的最佳比例,DOX和cypate分別摻入親水和疏水層以得到DOX@ PTSL。DOX@ PTSL的基本物理性質(zhì)進(jìn)行了研究,包括粒徑,TEM電鏡成像,熒光光譜,Tm值,載藥量,體內(nèi)紅外熱成像和包封率。在細(xì)胞實(shí)驗(yàn)中,我們研究了細(xì)胞毒性,細(xì)胞定位和細(xì)胞內(nèi)cypate介導(dǎo)的溶酶體逃逸。(圖2)生物分布,光熱成像,治療效果和功能脂質(zhì)體的組織病理學(xué)研究是在荷瘤小鼠中進(jìn)行。
首先,5%的DSPE-PEG2000被確認(rèn)為合成的最佳比例,因其良好的穩(wěn)定性以及高包裹效率。所有的表征結(jié)果顯示DOX@ PTSL是結(jié)構(gòu)完整性,水和粒徑約160納米(圖1),并且能夠產(chǎn)生較好的EPR腫瘤富集效果。通過(guò)體外溫度依賴的釋放研究確認(rèn)Tm值為43.1℃。另一方面,無(wú)論是體外光熱轉(zhuǎn)化研究和體內(nèi)光熱成像,都表明摻入DOX@ PTSL的cypate可以在1.5W近紅外激光照射5分鐘下到達(dá)TSL的Tm溫度。在體內(nèi)動(dòng)力學(xué)及生物分布研究表明,注入體內(nèi)后1.5小時(shí)后,脂質(zhì)體開(kāi)始在腫瘤部位積聚并呈現(xiàn)明顯的EPR效應(yīng)。細(xì)胞研究表明DOX@ PTSL進(jìn)行激光光照,在U87細(xì)胞系呈現(xiàn)出良好的治療效果。40μmol/ L的DOX@ PTSL可以達(dá)到60%致死率。高腫瘤抑制活性在荷瘤小鼠上也得以證實(shí)。(圖3)
總之,本文建立了多功能光熱敏感脂質(zhì)體載藥系統(tǒng)。包裹了阿霉素的納米復(fù)合材料具有各種良好的物理化學(xué)性質(zhì),穩(wěn)定性和生物活性??偠灾?,DOX@PTSL這里展現(xiàn)了吸引人的功能,在癌癥治療中表現(xiàn)了較廣闊的前景。
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NIR-responsive Thermosensitive Nanoliposomes for Targeted, Prolonged and Controlled-release of Drugs
Han Zhihao, Lv Liwei, Gu Yueqing1
(1.School of Engineering, China Pharmaceutical University, Nanjing, Jiangsu 210009, China)
It is an emerging focus to explore a drug delivery system displayed a photothermal release of entrapped drug for simultaneous cancer imaging and therapy. We synthesized a novel multifunctional nano liposome drug delivery system, the photothermal sensitive liposome(PTSL), with doxorubicin wrapped in the hydropholic layer as the therapeutical agents and cypate dopped in the hydrophobic layer as the diagnostic agents. The physical properties, stabilities and loading properties of the liposomes were tested, the nano composites we synthesized were targeted, stable and delicate, with a drug loading (DL) of 9±1.5% and encapsulation efficiency (EE) of 82.7±2.1%. The Tm value falls on 43.1℃, and it can raise the temperature of liposome to the Tm of thermal sensitive liposome, the liposome cracks and the wrapped chemotherapy drugs release. It indicates that the PTSL is a kind of promising drug delivery system for tumor diagnosis and targeting therapy.
Cypate; photothermal sensitive liposome; ERP effect; Triggered release; Photothermal conversion
R730.5
A
10. 11967/ 2017150111
R730.5
ADOI:10. 11967/ 2017150111
973國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(2015CB755504),國(guó)家自然科學(xué)基金項(xiàng)目資助(81220108012)。
顧月清(1963—),女,江蘇靖江,博士生導(dǎo)師, 研究方向?yàn)榧{米靶向藥物,郵箱:guyueqingsubmission@hotmail.com.