表面修饰可生物降解纳米载体的伊马替尼针对脑胶质瘤细胞系的调查：细胞内吸收和细胞毒性的研究

1、Investigation of imatinib loaded surface decorated biodegradablenanocarriers against glioblastoma cell lines: Intracellular uptake andcytotoxicity studies表面修饰可生物降解纳米载体的伊马替尼针对脑胶质瘤细胞系的调查：细胞内吸收和细胞毒性的研究A R S T R A C TOverexpression of P-glycoprotein (P-gp) efflux transporter in glioma cells thwarts the

2、build-up oftherapeutic concentration of drugs usually resulting into poor therapeutic outcome. To surmountaforesaid challenge, Imatinib (IMM) loaded Poly-lactide-co-glycolic acid nanoparticles (IMM-PLGA-NPs)were developed and optimized by Box Behnken Design as a new treatment stratagem in malignantg

3、lioma. Optimized NPs were functionalized with Pluronic® P84, P-gp inhibitor (IMM-PLGA-P84-NPs)which showed size, PDI, zeta potential, drug loading, 182.63士13.56 nm, 0.196士0.021，-15.2士1.49 mV,40.63士2.04 g/mg, respectively. Intracellular uptake study conducted on A172, U251 MG and C6 gliomacells

4、demonstrated significantly high uptake of IMM through NPs when compared with IMM solution(IMM-S), p<0.001. IMM-PLGA-P84-NPs showed better uptake in P-gp expressing cell line (U251 MG andC6) while uncoated NPs showed higher uptake in non-P-gp expressing cell line (A-172). Cytotoxicitystudies demon

5、strated significantly low IC50 for both IMM-PLGA-NPs and IMM-PLGA-P84-NPs whencompared withIC50 of IMM-S. IMM-PLGA-P84-NPs showed a significantly low IC50 against P-gpoverexpressing cell lines when compared with IC50 of IMM-PLGA-NPs. In contrary, IMM-PLGA-NPsshowed lower IC50 against non P-gp expres

6、sing cell line. This study demonstrated the feasibility of targeting surface decorated NPs to multidrug resistant gliomas. However, to address its clinical utilityextensive in vivo studies are required.摘要脑胶质瘤细胞中g-糖蛋白的过度表达会阻碍治疗药物浓度的增加，从而从而影响治疗效果。为了克服上述问题, 给伊马替尼(IMM)加载聚乳酸聚乙二醇酸共聚物纳米粒（Poly-lactide-co-gl

7、ycolic acid nanoparticles）作为修饰（IMM-PLGA-NPs），采用Box-Behnken设计开发和优化设计作为恶性神经胶质瘤新的治疗方案。再利用p-gp抑制剂Pluronic® P84优化NPs功能，其颗粒大小、PDI、电位、载药量分别为182.63士13.56 nm, 0.196士0.021，-15.2士1.49 mV, 40.63士2.04 g/mg。对神经胶质瘤细胞系A172, U251MG和C6进行细胞内吸收研究，结果通过NPs修饰的伊马替尼与伊马替尼溶液（IMM-S）相比，吸收明显增高，p<0.001。其中IMM-PLGA-P84-NPs对

8、p-gp高表达细胞系（U251 MG和C6）表现出较高吸收，而IMM-PLGA-NPs对非p-gp高表达细胞系 (A - 172)表现出较高的吸收。细胞毒性研究中， IMM-PLGA-NPs和IMM-PLGA-P84-NPs的半抑制浓度（IC50）显著低于IMM-S。IMM-PLGA-P84-NPs对p-gp高表达细胞系的IC50较低于IMM-PLGA-NPs。相反,IMM-PLGA-NPs对非p-gp高表达细胞系的IC50较低。本研究证明了表面修饰Nps来针对多药耐药胶质瘤的可行性。然而，为了其在临床上的广泛应用，体内研究是必须的。 1. IntroductionThe advent of

9、rigorous routines of cancer treatment modalities has hardly improved the prognosis of patients in advanced stages of brain gliomas. The overall survival is around a year for patients afflicted with stage III&IV glioma. Poor efficacy of current chemotherapeutic regimens in this diseased state is

10、attributed to inaccessibility of most of the drugs to tumor due to tissue structure as well as abundance of efflux transporters in blood brain barrier (BBB). Moreover, presence of efflux transporters (majorly P-glycoprotein) on glioma cells further thwarts the build-up of therapeutic concentration o

11、f drugs in the tumor. This necessitates frequent administration which results in severe adverse effects.Opening the BBB to allow feasible access of drugs to cerebrospinal fluid and glioblastoma tissue will thrust the treatment in direction of vastly improved prognosis in glioblasto-ma as well as oth

12、er brain tumors. Nanoparticles are ideal for this purpose since drugs are conveyed across the BBB by nanocarriers without any damage to the BBB. The means adopted by nanoparticles to achieve this end are opening of tight junctions between endothelial cells, transcytosis through the endothelial cell

13、layer, endocytosis by endothelial cells, evading recognition by efflux systems. Moreover, encapsulation of drug into NPs provide protection from metabolizing enzymes and efflux transporters。Matrix material of nanocarriers, their size, surface characteristics, drug load, release profile, physicochemi

14、cal properties of drugs are the critical factors which dictate the efficiency of a nanocarrier for effective delivery of drug across the BBB. Among the various nanocarriers, polymeric nanoparticles have been found to perfectly fit these parameters as they can be easily manipulated while at the same

15、time giving an added advantage of shielding therapeutic agent from adverse systemic effects and RES, enhanced bioavailability and targeting Polymers such as PLGA, Poly(s caprolactone), Polyethylene glycol, Pluronics have beenextensively explored for the purpose. Among these biodegradablepolymers, PL

16、GA systems have been studied at length and found tobe biocompatible, resorbable through natural pathways, haveshown good CNS compatibility, preservation of particle dimensions through extensive degradation levels resulting in controlledrelease of drug, and have drug entrapment efficiency of more tha

17、n80%. Medical andpharmacological applications ofpolylactides have been approved by FDA and WHO.Furthermore, studies have shown that surfacefunctionalization of conventional PLGA nanoparticles by a surfactaut like PVA or Pluronic tends to take their functionality to highernotches.Pluronics though, ha

18、ve outperformed PVAas coating surfactant incomparative efficacy experiments inresistant cancers in terms of cell internalization, intracellulartherapeutic dose build-up, overcoming resistance, and cytotoxicity. This progressive functionality may beattributed to the inherent property of Pluronic bloc

19、k copolymerslike Pluronic® P85, P84 to serve as P-glycoprotein inhibitors viaconsiderable intracellular ATP depletion and alteration of mem-brave fiuidization which modifies structural conformation of theP-glycoprotein receptors. Pluronic based nanoformulationsmerge theproperties of polymeric n

20、anoparticles along with P-gpinhibition,an essential requirement in treatment strategy of advanced stageresistant cancers like glioblastoma。Currently, standard of care and first line agent for the treatmentof glioblastoma after its surgical resection is Temozolamide, TMZ.But given glioblastoma's

21、notoriously consistent resurgence andfatal outcome within 1 year after diagnosis, Imatinib in addition tocurrent treatments might be rewarding.Tyrosinelcinase inhibitor Imatinib has exhibited potential inabating malignant brain tumors like astrocytoma and glioblastomaby inhibition of platelet-derive

22、d growth factor receptor (PDGFR)and tyrosine kinases which facilitate cell growth and proliferationin glioblastoma. Imatinib iscurrently marketed for the indication of chronic myeloid leulcaemia and gastrointestinal stromal tumors。Interestingly, imatinib alone or in combination with hydroxyureaortem

23、ozolamidehave elicited encouragingresponses in glioblastoma。 Furthermore, Dong et. al., have proven theanti-proliferative effectof Imatinib in glioma cells.Imatinib inducessignificant apoptosis and decrease in G2&S phase cells in C6glioma cell line sensitized resistant U87/Pt cells and improves

24、survival in arg kinaseimmunonegative glioblastoma groups. Imatinib is a substrate for the ABC transporter ABCB1(P-glycoprotein, MDRl); this efflux pump mediates substantialelimination of parent drug and given the significant interindividualvariation in the expression of these pumps, this may be a ma

25、jordeterminant of drug resistance development, variable drugexposure, and drug cytotoxicity. Extensiveresearch has led to the observation that the effect of Imatinib inglioblastoma could transcend by carrier assisted BBB contraven-tionand transgression of P glycoprotein transporter's efflux. In

26、light of aforementioned considerations multifunctionalnanoparticle designing is a newer strategy which is being exploitedfor effective drug delivery in glioblastoma. The objective of the present research was to develop anano enabled system carrying a novel drug, Imatinib, that had thepotential to cr

27、oss blood brain barrier and subsequently glioma cellsto achieve therapeutic levels and show cytotoxicity at these sites.We hypothesized a PLGA nanoparticle system surface functionalized by coating with a Pluronic block copolymer. For the selectionof Pluronic polymer the results reported by Batrakova

28、et. al., wereconsidered which propounded that P-gp inhibition and ATPdepletion is the result of optimum number of PO groups andhydrophilic lipophilic balance (HLB) value of a particular Pluronicblock co-polymer。Hence, the premise ofthe current work relied on the exploration of these twodeterminants

29、in P84 which closely resembles P85 (Number ofPO units: P84=43.45, P85=39.66; HLB: P84=14, P85=16)andthusthought to be equally effective.It was hypothesized to use the property of Pluronic whichbreaches BBB as well as resistant glioma cell membrane byswapping diffusion as mode of entry by receptor me

30、diatedendocytosis&transcytosis. After infringement of the resistantcell membrane by Pluronic, the PLGA nanoparticle safely transfersthe drug to its docking site inside the cell and releases the drug at aconsistent rate by virtue of its controlled degradation, thus leadingto build-up of a therape

31、utically significant concentration inside thecell ultimately causing desired cell re-sensitization and cytotoxicity.1 引言缜密的癌症治疗程序几乎没有对晚期脑胶质瘤有所改善。恶性脑胶质瘤III&IV阶段的患者存活期大概为一年。目前，在此治疗阶段过程中，化疗疗效不佳归因于大多数癌症治疗药物由于组织结构（efflux transports）的关系，不能进入blood brain barrier系统。此外，当胶质瘤细胞中表达efflux transports (主要是p-糖基蛋

32、白) 阻碍药物浓度集中在肿瘤组织。因此需要频繁的投药，从而导致不良的效果。开放BBB系统，使药物可以通过脑脊髓液和胶质瘤组织，可以大幅度的提高胶质瘤和其他脑部癌症的预后效果。作为理想的技术手段，经过纳米技术的包装，药物可以在不损伤组织的情况下透过BBB.采用纳米技术的意义在于可以打开内皮细胞间的tight junctions, 通过细胞间的跨膜作用，和内皮细胞的内吞，从而避免受到efflux system的影响。而且，通过纳米技术包裹的药物可以避免代谢酶和efflux transporters带来的药物流失。纳米载体的基质材料，大小，表面特性，载药量，药物释放，药物的物理化学性质是决定纳米载体

33、承载药物通过BBB效率的重要因素。多种纳米载体当中，多聚纳米颗粒被视为最佳选择，因为它们可以被控制对不良影响起到屏蔽作用，提高生物利用度和靶向。多聚物例如PLGA, poly, polyethylene glycol, pluronice都被作为研究对象进行研究。在这些可被生物降解的多聚物当中，PLGA system最为广泛的被大众所为研究对象，并发现，PLGA不存在生物排斥现象，通过自然条件，可被再度吸收，拥有良好的神经系统适应性，可通过维持颗粒的大小，从而达到控制药物释放，对药物的包封率可达到80%以上。FDA和WHO组织已经批准聚乳酸的医学于药理应用。此外，通过表面活性剂（例如PVA或者

34、pluronic tends）可以增强传统的PLGA纳米颗粒的表面效应。作为包裹表面活性剂，就细胞内在化，细胞内药物浓度增进，克服抗药性和药物毒性等方面而言，Pluronics胜于PVA。这种功能上的优势可能归功于pluronic固有的可以阻断类似Pluronic® P85的共多聚物，作为磷酸化-糖基蛋白抑制剂， P84抑制细胞内ATP, 并且调节细胞膜的流动性，从而达到调节磷酸化-糖基蛋白受体的结构变化。以pluronic为基础的纳米规划将多聚纳米颗粒融入到抑制糖基蛋白磷酸化的过程当中，例如成为恶性胶质瘤治疗过程当中必不可少的所需条件。目前，手术切除胶质瘤后首选标准药物是temoz

35、olamide（TMZ）但是众所周知的是，被判定为胶质瘤后的一年里，会出现致命的影响，目前，治疗过程中，添加imatinib也被大众所期待。-氨基对羟苯丙酸激酶抑制剂imatinib 已经被证实可以通过对血小板衍生的生长因子受体（PDGFR）和-氨基对羟苯丙酸激酶引起的细胞生长与增殖的抑制，从而降低恶性脑肿瘤，例如星形细胞瘤和恶性胶质瘤。当前，imatinib被当作治疗慢性粒细胞白血病和胃肠间质瘤进行贩卖。有趣的是，单独使用imatinib或者与hydroxyure或temozolamide同时使用可在胶质瘤治疗当中起到正向的效果。此外， dong et.al,. 已经在胶质瘤细胞中证实了im

36、atinib具有抑制细胞增殖的效果，而且imatinib可以引起细胞凋亡并且减少C6胶质瘤细胞的G2/S phase cells, 激活抵抗U87/Pt cells, 并且提高无ARG激酶的胶质瘤存活率。Imatinib是ABCB1的作用底物，流出泵（ABC transporter）可调节大量的母体药物并且象征着个体之间transporter表达的差异，这种现象可能成为耐药性发展，可变的药物暴露和药物毒性的决定因素。大量的研究结果表明，imatinib在胶质瘤中的影响为可以超越对BBB抵触和磷酸化糖基蛋白transporter的外流。根据上述的内容，治疗胶质瘤过程当中，利用多功能纳米颗粒的设计

37、来改善药物的传输效率将成为一个新的挑战项目。本研究项目的是开发可以承载新药-imatinib的纳米系统，来达到可以穿过BBB并且达到有效的治疗水平。我们假设pluronic阻断的共聚物包裹可激活PLGA纳米系统表面活性。为了筛选pluronic聚合物，Batrakova et. al提出最适宜的精确pluronic阻断共聚物的亲水亲油平衡值和最适宜的PO数量导致提出糖基蛋白磷酸化和ATP耗减。因此，当前工作的前取决于P84，P85中两个被认为几乎相近的因素（PO的数量和亲水亲油平衡值）。目前，使用pluronic被认为是通过受体介导的内在化作用和跨膜作用来透过BBB和胶质瘤细胞膜。Pluron

38、ic透过细胞膜之后， PLGA可以安全的传送药物到细胞内的docking site, 并且控制药物的释放速度，减少药物降解，使药物可以达到有效的治疗浓度，最终可达到期望的细胞脱敏作用和细胞毒性。2. Materials and methodsImatinibmesylate was given ex gratia by Fresenius Kabi Pvt. Ltd,Gurgaon, Haryana, India. PLGA 50:50 (Resomer 503H) was giftedby Evonik Degussa, India, Pvt. Ltd, Mumbai, India. Plu

39、ronic® P84was also obtained ex gratia from BASF Corporation, NJ 07932, USA.Polyvinyl alcohol (PVA, MW-25,000), Trypsin-EDTA, penicillin,streptomycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tet-razolium bromide (MTT) were purchased from Sigma-Aldrich, Inc.(St. Louis, M0, USA). Dulbecco

40、9;s Modified Eagle Medium (DMEM)and Fetal bovine serum (FBS) were procured from Invitrogen(Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO, HPLC grade)Dichloromethane (DCM, AR grade), acetonitrile (ACN, HPLC grade),methanol (HPLC grade), 0-phosphoric acid (HPLC Grade), sodiumdihydrogen phosphate dihydr

41、ate, potassium dihydrogenphosphate and sodium hydroxide were purchased from SD finechemical, Mumbai, India. Water was obtained from Milli-qwaterpurification system (Millipore, MA,USA). Cell lines were obtainedfrom National Centre for Cell Science, Pune, India.2. 材料与方法甲磺酸伊马替尼由印度哈里亚纳邦古尔加翁费森尤斯卡比私营有限公司特

42、惠提供。PLGA 50:50(Resomer 503H)由印度孟买赢创德固赛印度分公司提供。Pluronic® P84也由美国巴斯夫公司（新泽西州07932）特惠提供。聚乙烯醇（PVA，MW-25 000），胰蛋白酶-EDTA，青霉素，链霉素，和 3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐（MTT）购自美国西格玛奥德里奇（Sigma Aldrich）公司（圣路易斯，M0，美国）。细胞培养基（DMEM）和胎牛血清（FBS）购自美国加利福尼亚州卡尔斯巴德英杰公司。二甲基亚砜（DMSO，HPLC级）二氯甲烷（DCM，AR级），乙腈（ACN，HPLC级），甲醇（HPL

43、C级），0-磷酸（HPLC级），磷酸二氢钠二水合物，磷酸二氢钾和氢氧化钠购自印度孟买SD精细化工。水是用美国Milli-Q水净化系统（Millipore, MA, USA）纯化而得。细胞系来自印度浦那国立细胞科学中心。2.1. Statistical optimization of Imatinib Loaded PLGA NPs by Box Behnken designImatinibmesylate (IMM) loaded poly-lactide-co-glycolic acidnanoparticles (IMM-PLGA-NPs) were prepared by earlier

44、 reportedmethod with some modification (Tariq et al., 2015). Briefly, drugwas dissolved in internal aqueous phase and PLGA (100 mg) wasdissolved in 2 ml of dichloromethane. Drug solution was emulsi-fled in polymeric solution under sonication over an ice bath for 60s(25 W, 40% duty cycles, Sonopuls,

45、Bandelin, Germany). Formed primary emulsion (w/o) was added into external aqueous phase (4, 8,12 ml as per design) under sonication over an ice bath at 25% amplitude and resultant dispersion was subjected to solvent evaporation under mild magnetic stirring (400 rpm) at room temperature followed by c

46、entrifugation at 18,000 rpm for 20 min. The obtained pellet was washed, dispersed and finally lyophilized for 24h at -50 and 0.015millibar pressure (Lab Conco., LPYH, Lock 6, USA freeze dryer). The effect of different independent variables on dependent variables (nanoparticles size and drug loading)

47、 was studied for the optimization of IMM-PLGA-NPs. The studied independent variables were sonication time, stabilizer's concentration, phase volume ratio and volume of internal aqueous phase. Said variables were optimized statistically by Box-Behnken design using software (Design Expert® So

48、ftware Version 9.0, Stat-Ease Inc, and Minneapolis). A detailed study protocol is shown in Table 1. 2.1 以Box-Behnken设计加载PLGA NPs (聚乳酸聚乙二醇酸共聚物纳米粒)的伊马替尼的统计学优化甲磺酸伊马替尼(IMM)加载PLGA NPs (IMM-PLGA-NPs)是通过先前报道的方法稍加修改制备而成(Tariq et al .,2015)。简单说，将药物溶解于内部水相中，并将PLGA100mg溶解于2ml二氯甲烷中。药物溶液在超声波下在聚合物溶液中冰浴60s被乳化。形成的乳

49、液（W / O）添加到外部水相中（25%超声波下，冰浴），再将合成分散在室温下温和磁力搅拌（400转）进行溶剂蒸发，随后以18000rpm进行离心20分钟。将所得到的沉淀经洗涤，分散，最后在-50和0.015millibar压力冻干24小时。为对IMM-PLGA-NPs进行优化，研究了不同的自变量对因变量的影响(纳米粒子大小和药物加载)。所研究的独立变量是超声处理时间，稳定剂浓度，相体积比和内部水相的体积。变量采用Box-Behnken设计法统计优化，实用软件（设计Expert®软件版本9.0，Stat-Ease 公司, 明尼阿波利斯）。详细研究记录示于表1。表1 独立变量及其依赖性

50、反应调查水平2.2. Surface modification of IMM-PLGA-NPs by coating with Pluronic®P84 (IMM-PLGA-P84-NPs) 以普朗尼克®P84表面修饰IMM-PLGA-NPs (IMM-PLGA-P84-NPs) Surface modification of IMM-PLGA-NPs was carried out according to previously reported method with slight change (Shah et al., 2009). Equal volumes of

51、 aqueous particles suspension(0.2% w/v) and aqueous solution of Pluronic® P84 (1% w/v) were mixed together and incubated overnight under mild magnetic stirring (200 rpm) followed by ultracentrifugation at 18,000 rpm for 30 min. The obtained pellet was washed, dispersed and lyophilized for 24h a

52、t -50 and 0.015 millibar pressure (Lab Conco., LPYH, Lock 6, USA freeze dryer).2.3. Particle size and zeta potential 粒子大小和电位The particle size, size distribution (polydispersity index, PDI) and zeta potential analyses of IMM-PLGA-NPs were performed using the dynamic light scattering (DLS) method with

53、 a computerized inspection system (Malvern Zetasizer, Nano-ZS, Malvern, UK) and analyzed by 'DTS nano' software. For analysis, 1 mg of NPs were dispersed in 2 mL of Milliqwater and subjected to size and zeta potential measurement. Similar procedure was adopted for IMM-PLGA-P84-NPs.2.4. Drug

54、content or drug Loading 药物含量或载药量载药率药物包封总量Drug loading of IMM-PLGA-NPs and IMM-PLGA-P84-NPs was determined according to previously reported method (Kalaria et al., 2009). Two mg each of lyophilized IMM-PLGA-NPs and IMM-PLGA-P84-NPs were dissolved in 2 mL of dimethyl sulfoxide (HPLC grade) followed by

55、 filtration through 0.2 m syringe filter and analyzed by HPLC (Agilent) coupled with a UV detector. The separation was achieved at 30 with flow rate of 1 mL/min using C18 analytical column (Develosil ODS-HG-S, Nomula chemical 50 mm) and detection was made at 267 nm. Mobile phase consisting of aqueou

56、s buffer phase pH 8 (55% v/v) and organic phase (45% v/v) comprising of acetonitrile and methanol (6:4 ratio) was used (Negi et al., 2013a). Drug loading was calculated by using following formula;制剂重量2.5. Shape and surface morphology 形状和表面形态 Surface morphological and size analyses of IMM-PLGA-NPs an

57、d IMM-PLGA-P84-NPs were performed by using transmittance electron microscope (TEM). Particles were suspended in Milli Q water and adsorbed on copper grid having 300 meshes followed by treatment with l%uranyl acetate for negative staining and later dried. Dried samples were then analyzed in transmiss

58、ion electron microscope (TOPCON002B, Tokyo, Japan), at an accelerating voltage of 200 kV.2.6. Physical state characterization and release studies of IMM after formulating it into NPs IMM制成NPs后的物理状态描述和释放的研究2.6.1. Differential scanning calorimetric (DSC) analysis 差示扫描量热（DSC）分析 DSC analysis was carried

59、 out by Perkin-Elmer Pyris 1 DSC instrument, equipped with Intra-cooler 2 P cooling accessory. Accurately weighed (5 mg); IMM, PLGA, physical mixture of IMM and PLGA, placebo PLGA-NPs, and IMM-PLGA-NPs were separately sealed into a standard aluminium pans. DSC scanning was performed from 30 to 300 keeping heating rates of 10/min with a nitrogen purge of 10 mL/min.2.6.2. X-ray diffractometric (XRD) analysis X射线衍射（XRD）分析XRD analysis of IMM, PLGA, physical mixture of IMM&PLGA, placebo PLGA-NPs and IMM-PLGA-N