靶向于血小板源性生长因子受体β的干扰素γ脂质体对大鼠肝纤维化治疗作用的研究
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摘要
靶向于血小板源性生长因子受体β的干扰素-γ脂质体对大鼠肝纤维化治疗作用的研究
肝纤维化是慢性肝病重要病理特征,是发展到肝硬化的必经阶段。尽管迄今为止国内外的研究就肝纤维化和早期肝硬化可以逆转,已有共识,并在抑制肝星状细胞(hepatic stellate cell, HSC)活化并促进其凋亡、调节细胞外基质(extracellular matrix, ECM)的合成和降解等方面取得一些成果,对肝纤维化的临床治疗提供了一些药物和方法,但总体疗效仍不理想。目前多认为,限制临床上应用的抗肝纤维化药物效果的原因,可能与药物无法达到靶细胞、或到达靶细胞但无法在其周围形成有效的药物浓度及半衰期短和药物对其他组织细胞的副作用大过增加剂量带来的疗效等有关。
通过靶向载药系统转运药物,可达到靶向、长循环进而增加疗效及减少抗肝纤维化药物的全身副作用的目的。这里主要涉及：①靶向头基的选择；②载体形式的选择；③抗肝纤维化药物的选择。鉴于HSC激活是肝纤维化发展过程中的中心事件,因此,HSC是抗肝纤维化治疗的首选靶细胞。在HSC上,尤其是激活的HSC上特殊表达或过量表达的受体,是最经常被靶向载药系统采用的靶点。血小板源性生长因子(platelet derived growth factor, PDGF)是肝纤维化形成过程中最强的促增殖因子,与转化生长因子β1(transform growth factorβ1, TGF-β1)在肝纤维化的形成中起着重要作用；而且PDGF受体β(PDGF receptorβ, PDGFR-β)己被广泛的证明在激活的HSC上优势过量表达。近年来已有研究证明,环肽C*SRNLIDC* (pPB)能特异性识别PDGFR-β。与其他非多肽、非脂质体的靶向载药系统相比,小分子多肽作为靶向头基具有化学结构明确,高纯度并可被大量制备的优点；脂质体作为载体具有安全、载药谱广的优点。此外,立体稳定脂质体(sterically stabilized liposome, SSL)的聚乙二醇化(polyethylene glycol conjugated, PEGlated)的设计和能够达到纳米粒级别(<100nm)的粒径,能够使它规避网状内皮系统的吞噬,更具有长循环的特点。干扰素-γ(IFN-γ)是目前已经被批准临床应用的抗肝纤维化作用药物,在许多体内外实验中表现出一定的抗肝纤维化作用,但在临床应用中IFN-γ表现出相对低的疗效、相对短的半衰期和一些严重的副作用,如发热、血细胞减少、肝功能损害加重等,严重制约着IFN-γ的临床应用。
本研究中,我们在国内外率先设计了针对PDGFR-β的环肽(pPB)修饰的包封IFN-γ的立体稳定脂质体(pPB-SSL-IFN-γ),并通过体内外的实验研究,从分子、细胞和器官的水平探讨这个主动靶向载药系统能否解决IFN-γ的疗效低、半衰期短、副作用多的临床问题,并对其作用机制进行更深入的探讨,寻求更可能为临床所用的抗肝纤维化的靶向药物治疗途径,为进一步可能的临床研究提供理论依据,以期最终能使病人受益。
本研究的主要内容包括：①环肽和靶向IFN-γ脂质体的制备和表征；②原代大鼠肝星状细胞的分离、培养和鉴定；③环肽和靶向脂质体对肝星状细胞的靶向性验证；④靶向IFN-γ脂质体对肝星状细胞的体外抗肝纤维化作用的研究；⑤靶向IFN-γ脂质体在正常大鼠体内的药代动力学和体内分布的研究,以及靶向IFN-γ脂质体在肝纤维化大鼠的体内分布和肝组织上细胞定位的研究；⑥靶向IFN-γ脂质体对大鼠肝纤维的体内抗肝纤维化作用和改善副作用的研究。
第一部分
环肽和IFN-γ靶向脂质体的制备和表征
目的：人工合成C*SRNLIDC*环肽(pPB),并表征。构建pPB介导的IFN-γ主动靶向脂质体(pPB-SSL-IFN-γ),并表征其特性。
方法：采用固相合成的方法合成pPB,并冻干低温保存,以高效液相分析pPB的纯度,以质谱法检测其分子量。为制备靶向脂质体pPB-SSL-IFN-γ,将胆固醇：蛋磷脂酰胆碱：甲氧基-聚乙二醇2000-二硬酯酰磷脂酰乙醇胺(mPEG2000-DSPE):马来酰亚胺-聚乙二醇3400-二硬酯酰磷脂酰乙醇胺(mal-PEG+3400-DSPE)以2：1：0.1：0.02的摩尔比溶于氯仿,根据水化液不同,采用旋转蒸发-薄膜水化-挤压法制备各种非靶向脂质体。为了制备pPB介导的靶向脂质体,先将pPB与SATP反应,给pPB引入巯基,得到pPB-SH，并质谱验证其分子量,后将pPB-SH与非靶向脂质体反应,得各种靶向脂质体,进一步利用氢谱(1H-NMR)验证pPB-SH与非靶向脂质体膜材料中含有的mal-PEG3400-DSPE的结合。从粒径、包封率、载药量、稳定性等方面表征其理化特性。
结果：合成的pP、pPB-FITC和pPB-SH纯度>95%,质谱检测它们的分子量符合理论值,提示合成正确。进一步通过对比,在mal-PEG3400-DSPE的1H-NMR上清楚显示mal峰,在pPB-PEG3400-DSPE的1H-NMR上消失,提示了pPB与靶向脂质体膜材料mal-PEG3400-DSPE中mal基团通过加成反应相连。制备的pPB-SSL-IFN-γ的平均粒径为83.5nm,多分散指数(PI)为0.067；包封率为32.73±4.61%;载药量为8.99×104IU/μmol磷脂；4℃冷藏情况下,1月后无明显变化,稳定性良好；3月后粒径增大、分布增宽和包封率下降,提示脂质体颗粒有融合、粒径变大和IFN-γ的泄漏。
结论：固相合成法可大量、高纯度合成pPB。成功制备了下一步实验需要的纳米级别的pPB介导的IFN-γ的靶向脂质体。
第二部分
原代大鼠肝星状细胞的分离、培养和鉴定
目的：探讨原代大鼠肝星状细胞的分离、培养方法,并鉴定。为进一步的体外实验提供理想的细胞模型。
方法：采用胶原酶离体灌流、Nycodenz为分离介质的密度梯度离心的方法分离大鼠的肝星状细胞,并培养传代及同步鉴定。
结果：每只大鼠肝星状细胞的得率可达2-5x107/只,细胞存活率>90%。培养过程中出现特征性形态变化,进一步细胞免疫荧光化学鉴定；培养4d,Desmin染色阳性,提示得到的为肝星状细胞；培养10d和传1代后,α-SMA染色阳性,提示为活化表型。
结论：成功获得进一步实验所需的理想细胞模型---原代大鼠活化的肝星状细胞。
第三部分
环肽和靶向脂质体对肝星状细胞的靶向性
目的：探讨pPB环肽和pPB介导的靶向脂质体与原代活化的大鼠肝星状细胞和人肝星状细胞株LX-2体外结合的特性和细胞内定位的情况。
方法：合成异硫氰荧光素(FITC)标记的环肽(pPB-FITC),制备包封FITC的靶向(pPB-SSL-FITC)和非靶向脂质体(SSL-FITC).通过流式细胞仪测定pPB-FITC与原代活化的大鼠肝星状细胞和人LX-2细胞株结合的荧光强度,实施结合-饱和实验和结合-竞争实验,并比较pPB-SSL-FITC和SSL-FITC与原代大鼠肝星状细胞的结合能力。通过荧光显微镜及激光共聚焦显微镜观察pPB-FITC和pPB-SSL-FITC在活化的大鼠肝星状细胞内的定位。
结果：pPB-FITC与原代活化的大鼠肝星状细胞和人LX-2肝星状细胞株的结合具有饱和性和浓度依赖性；未标记的pPB对pPB-FITC与原代活化大鼠肝星状细胞和LX-2细胞株的结合有竞争抑制作用,且呈浓度依赖性。靶向脂质体pPB-SSL-FITC与原代大鼠活化的肝星状细胞结合的能力是非靶向脂质体SSL-FITC的3.01±1.50倍。原代大鼠活化的肝星状细胞能内吞pPB-FITC和pPB-SSL-FITC,绿色荧光信号主要分布于胞浆。
结论：pPB与原代大鼠肝星状细胞和人LX-2肝星状细胞株以受体介导的方式特异结合。靶向脂质体与活化的大鼠肝星状细胞结合的能力明显高于非靶向脂质体,结合后靶向脂质体被肝星状细胞内摄,主要分布在细胞浆,提示pPB-SSL可以作为靶向于HSC的载体来载送相关的药物。
第四部分
IFN-γ靶向脂质体对大鼠肝星状细胞的作用
目的：考察pPB介导的IFN-γ靶向脂质体对活化的大鼠肝星状细胞的体外药效及作用机制。
方法：通过Alamar blue分析法检测IFN-γ脂质体对细胞增殖的影响；通过Hoechst33258和TUNEL的方法检测IFN-γ脂质体对细胞凋亡的影响；通过Western blot分析的方法检测IFN-γ脂质体对细胞α-平滑肌肌动蛋白(α—SMA)表达的影响。
结果：含有相同IFN-γ剂量的靶向IFN-γ脂质体与IFN-γ非靶向脂质体和游离IFN-γ相比,明显抑制活化的肝星状细胞的增殖；Honchst33258和TUNEL染色发现,IFN-γ靶向脂质体明显抑制活化肝星状细胞的凋亡；Western blot分析法证实,IFN-γ靶向脂质体能明显抑制活化肝星状细胞的α-SMA的蛋白表达。
结论：与游离IFN-γ和非靶向IFN-γ脂质体相比,IFN-γ革巴向脂质体能更有效的抑制活化大鼠肝星状细胞的增殖、凋亡和α-SMA的表达。
第五部分
IFN-γ靶向脂质体在大鼠体内的药代动力学、组织分布和细胞定位
目的：探讨IFN-γ靶向脂质体在正常大鼠体内的药代动力学和组织分布的规律,并探讨IFN-γ非靶向脂质体(SSL-IFN-γ)和IFN-γ靶向脂质体(pPB-SSL-IFN-γ)在肝纤维化大鼠体内的动态分布和肝组织上的细胞定位情况。
方法：采用酶联免疫吸附法(ELISA)检测pPB-SSL-IFN-γ和游离IFN-γ静脉注入正常大鼠体内5mi n-24h不同时间点的血浆样本中IFN-γ的浓度,并比较两者的半衰期(t1/2)；进一步通过24h时获得的大鼠的不同组织中IFN-γ的浓度,观察其组织分布情况。制备包含DIR(一种近红外染料)的靶向和非靶向脂质体,采用荧光活体成像仪比较IFN-γ靶向脂质体(DIR-pPB-SSL-IFN-γ)和IFN-γ非靶向脂质体(DIR-SSL-IFN-γ)在正常大鼠和纤维化大鼠体内分布的动态显像的情况。采用双重荧光标记免疫组化染色法,考察靶向和非靶向脂质体携带的IFN-γ在纤维化大鼠肝组织内的细胞定位情况。
结果：药代动力学研究显示：pPB介导的靶向IFN-γ脂质体的半衰期(t1/2)和曲线下面积(AUC)明显长于游离IFN-γ。组织分布研究显示：pPB介导的靶向脂质体运载的IFN-γ在体内主要聚集在肝组织内；而游离IFN-γ在体内主要分布在肝组织和肠；进一步比较在同一种脏器(心、肝、脾、肺、肾、脑和肠)内的分布,pPB介导的靶向脂质体载送的IFN-γ较游离IFN-γ明显增多。活体荧光动态成像显示：①正常大鼠体内：DIR-SSL-IFN-γ和DIR-pPB-SSL-IF-γ在注射后1h,肝脏区域的荧光强度明显增加至最高,后强度逐渐下降直至48h,其它脏器才可看见荧光分布；在成像48h的时间点,结束活体成像,取心、肝、脾、肺、肾和血液组织,立即于体外成像显示：肝组织的荧光强度明显高于其他组织,血液标本的荧光强度甚至接近0,进一步证明了活体成像中肝脏区域的荧光图象确为肝组织中荧光显像,并显示了直至48h,肝组织仍明显的荧光聚集。②纤维化大鼠体内：DIR-pPB-SSL-IFN-γ注入纤维化大鼠体内2h时,肝脏区域的荧光强度已经明显高于DIR-SSL-IFN-γ注入组；至20h时,这种趋势更加明显,甚至在DIR-SSL-IFN-γ处理组的肝脏区域的荧光强度显示出较2h时下降,而DIR-pPB-SSL-IFN-γ处理组大鼠肝脏区域的荧光强度较2h时明显增加。双重荧光标记免疫组织化学染色探测细胞定位的研究显示：DPB-SSL携带的IFN-γ在肝组织中与α-SMA的共同表达率(60.13±9.15%)明显高于SSL携带的IFN-γ(12.11±3.26%)(n=3,p<0.01),且图像显示后者主要定位于肝细胞。
结论：与游离IFN-γ相比,pPB介导的靶向脂质体携带的IFN-γ在体内的药代动力学和组织分布的研究显示了长循环的特性；IFN-γ靶向和IFN-γ非靶向脂质体均显示了明显的肝组织的器官靶向特性,但IFN-γ靶向脂质体在肝纤维化大鼠体内显示出更加明显的肝脏器官靶向。在纤维化肝组织中靶向脂质体携带的IFN-γ主要定位于肝星状细胞；而非靶向脂质体携带的IFN-γ主要定位在肝细胞,验证了靶向脂质体载送的IFN-γ在纤维化大鼠肝组织内的HSC细胞靶向性。
第六部分
IFN-γ靶向脂质体对大鼠肝纤维化的体内抗肝纤维化作用和改善副作用的研究
目的：研究IFN-γ靶向脂质体对大鼠肝纤维化的体内抗肝纤维化作用,并观察其对改善某些副作用的效果。
方法：将40只肝纤维化大鼠,随机分成6组,分别给与(均尾静脉注射,2次/周,持续4周)：①PBS(n=4);②空白脂质体pPB-SSL(n=4)；③IFN-γ(n=8)；④IFN-γ非靶向脂质体(n=8)；⑤IFN-γ靶向脂质体(n=8)；⑥5倍剂量IFN-γ组(n=7),其中①作为模型对照组(model control group)；②作为空白pPB-SSL对照组；③④⑤此3种制剂中游离IFN-γ含量均为2x105IU/m1；⑥中游离的IFN-γ含量为1x10.IU/m1。6只正常大鼠在同等的条件下被饲养,作为正常对照组(normal control group)。最后一次静脉注射后48h,收集大鼠的血液标本,分别检测肝功能(ALT、AST和ALB)、血液分析(WBC、HGB和PLT)和血清肝纤维化指标(HA和IV-C)；收集肝组织标本,分别进行HE、MassOn胶原三色染色、羟脯氨酸含量测定、Ⅰ型胶原的免疫组织化学染色和肝组织的α-SMA蛋白表达的western blOt分析。
结果：与模型对照组、游离IFN-γ组甚至IFN-γ非靶向脂质体组和5倍剂量游离IFN-γ组相比,IFN-γ靶向脂质体明显减轻大鼠的肝纤维化分期,并明显减少肝组织Ⅰ型胶原染色面积、血清IV-C水平和肝组织α-SMA蛋白的表达；与模型对照组和游离IFN-γ低剂量组相比,IFN-γ靶向脂质体明显改善由于肝纤维化造成的HGB和PLT的减少,还可明显降低肝组织羟脯氨酸含量；与模型对照组相比,IFN-γ靶向脂质体亦明显降低肝纤维化大鼠血清ALT和HA水平；与模型对照组和游离IFN-γ高剂量组相比,IFN-γ靶向脂质体亦明显减轻肝纤维化大鼠的肝脏病理炎症HAI分级。同时,IFN-γ靶向脂质体治疗组的血清AST水平较IFN-γ非靶向治疗组明显降低(P<0.05)；而且IFN-γ靶向脂质体治疗组的WBC计数高于游离IFN-γ的低剂量组和高剂量组(P=0.072和P=0.070)。
结论：IFN-γ靶向脂质体显示出更加明显的体内抗肝纤维化作用,而且,IFN-γ靶向脂质体能够明显改善IFN-γ非靶向脂质体非选择性肝细胞摄取造成的AST的升高和避免高剂量IFN-γ造成的肝脏炎症坏死程度加重。另外,IFN-γ靶向脂质体可在一定程度上改善游离IFN-γ造成的血WBC计数的降低。
Effect of Interferon-gamma liposomes targeted to platelet derived growth factor receptorβon hepatic fibrosis in rat
Hepatic fibrosis is a very important pathological feature during the development of chronic hepatic injury. Hepatic fibrosis is also a necessary pathway to liver cirrhosis, which is a very common disease resulting in morbidity and mortality in many countries. Many therapeutic approaches have been examined in experimental and clinical studies of hepatic fibrosis. These approaches include adjusting the balance of extracellular matrix synthesis and degradation, reducing intrahepatic inflammation, and inhibiting activation or promoting apoptosis of hepatic stellate cells (HSC). Through these techniques, the reversibility of fibrosis can be observed. However, no approved anti-fibrotic drug has been clinically applied. The main reason for the lack of clinical use is the inability to specifically target the responsible cells or molecules to increase drug effectiveness and to decrease the side-effects on non-target tissues or cells in vivo.
To improve drug effectiveness and reduce the side-effects on non-target tissues or cells, targeted drug-carrier systems have been employed in recent years that ensure drug delivery for long-term circulation and/or site-specific targeting of specific populations of liver cells, including hepatocytes, liver endothelial cells, Kupffer cells, and HSCs. HSC activation is a central event in hepatic fibrogenesis. Receptors specifically expressed or over-expressed on HSCs, especially on activated HSCs, are selected as the active targeting sites for targeting-drug therapies for hepatic fibrosis. Platelet-derived growth factor (PDGF) is the most prominent mitogen for HSCs during hepatic fibrogenesis, and the PDGF receptor-P (PDGFR-P) is strongly up-regulated on activated HSCs. The cyclic peptide C*SRNLIDC* (pPB) was previously shown to have a specific affinity for PDGFR-β.
In the present study, we utilized pPB to modify sterically-stable liposomes (SSL) to prepare the pPB-SSL carrier. To establish the application, interferon gamma (IFN-y) was selected to be entrapped in pPB-SSL, because IFN-y has anti-fibrotic activities. However, some shortcomings of IFN-y, such as low anti-fibrotic effects, a short blood-circulation half-life, and side effects due to its effects on non-target cells or tissues, have limited its clinic application.
The aim of the present study is to investigate whether the targeted drug-delivery system (pPB-SSL) can resolve these limitations, and to investigate the related action mechanism. The approach of targeting drug-delivered increasing the efficacy of anti-fibrotic drug therapy and reducing side-effects may have the potential clinical value. This study also provide a new option for drug-carriers which could selectively delivery drug to cells over-expressing PDGFR-β.
These works include six parts as described below.
Part One Synthesis and characterization of cyclic peptide and targeted interferon-gamma liposomes
Objective:To synthesize and characterize the cyclic pepetide C*SRNLIDC* (pBP). And to prepare and characterize sterically stable liposomes modified with pPB (pPB-SSL) to deliver interferon-gamma (pPB-SSL-IFN-γ).
Methods:pPB was synthesized using the standard strategy of Boc-protected solid phase peptide synthesis. pPB was analysed and purified by high performance liquid chromatographic (HPLC). The molecular weight of pPB was confirmed by electrospray ionization tandem mass spectrometry (ESI-MS).
pPB-SSL-IFN-γwas prepared by the method of rotary evaporation-thin film hydrated-extruded. The synthesis of pPB-S-mal-PEG34oo-DSPE was confirmed by 'H-NMR spectrometry. To characterize pPB-SSL-IFN-γ, particle diameter and distribution of pPB-SSL-IFN-γwere measured by dynamic light scattering; and the encapsulation efficiency (ee%) of IFN-γwas determined. To investigate the stability of pPB-SSL-IFN-γ, the particle diameter and ee% were compared after pPB-SSL-IFN-γwas prepared and had been stored at 4℃for one month and three month.
Results:HPLC analysis revealed that the purity of prepared pPB exceeded 95%. ESI-MS spectrum showed the molecular weight of pPB was 921.5Da, coinciding with their theoretical values. The conjugation of pPB-SH with mal-PEG34oo-DSPE were indicated by the disappearance of the characteristic resonance of mal (6.7ppm) of pPB-S-mal-PEG3400-DSPE shown in1H-NMR spectrum. The mean particle diameter of pPB-SSL-IFN-y was 83.5nm, and the polydispersity index (PI) was 0.067. The ee% of IFN-y in pPB-SSL (n=3) was 32.73±4.61%. The loading capacity was 8.99x104IU /μM phospholipid. Compared to freshly prepared pPB-SSL-IFN-y, the particle diameter and ee% of pPB-SSL-IFN-y stored at 4℃for one month had no significant difference.
Conclusion:pPB has successfully synthesized. And pPB-SSL-IFN-y was successfully prepared, which showed satisfactory particle size distribution (mostly<100nm), ee% and loading capacity.
Part Two Isolation, culture and identification of primary rat hepatic stellate cells
Objective:To provide activated hepatic stellate cells (HSCs) for the followed in vitro study.
Methods:Primary rat HSCs (pHSCs) was isolated from normal Sprague-Dawley (SD) rats using infusion and combined digestion with pronase E and collagenase followed by Nycodenz density gradient centrifugation. pHSCs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cell viability was detected by Trypan blue dyeing. pHSCs were assessed by auto-fluorescence, immunocytochemical staining of desmin and a-SMA and morphological investigation with microscopy.
Results:About 5X10/ml pHSCs cells were harvested from each rat. The viability of freshly isolated pHSCs was exceeding 90%. Due to the presence of lipid droplets the fresh rat pHSCs showed green-blue spontanenous fluorescence that can be observed at 328nm or 445 nm wavelength by fluorescence microscopy. They stretched out pseudopodia after incubated for 24h to 48h. Atuo-fluorescence and lipid droplets gradually disappeared with time passing. The cells cultured for 10 days to 14 days became stellate or irregular shape and bigger, which represented as activated phenotype. pHSCs were confirmed by positive staining via immunocytochemistry for Desmin, and activated pHSCs were confirmed by positive staining for a-SMA.
Conclusion:Primary rat HSCs were successfully obtained the ideal activated rat HSCs model---cultured for the followed study in vitro.
Part Three Binding and intracellular location of cyclic peptide and cyclic peptide-modified liposomes in hepatic stellate cells
Objective:To investigate the binding and intracellular location of cyclic peptide (pPB) and pPB-modified sterically stable liposomes (pPB-SSL) in hepatic stellate cells (HSCs).
Methods:Activated rat pHSCs were respectively incubate with various concentration of FITC labeling pPB (pPB-FITC,0-150μM) in the darkness. The percentages of the FITC-positive cells at selected cells (%gate) were determined by flow cytometry. For the competitivly experimential study, activated pHSCs were incubated with different concentration (0.035-1411.5μM) of the unlabeled free pPB for 30min before pPB-FITC (36μM) were added. And the total fluosrencence intensity (TFI) was selected as an index analysed. Binding of pPB to LX-2 cells also was measured as describe above. Furthermore the binding of SSL encapsulating FITC (SSL-FITC) or pPB-SSL encapsulating FITC (pPB-SSL-FITC) to HSC were also performed with the folw cytometry.
For investigating the intracellular location, activated rat pHSCs were incubated with free FITC, pPB-FITC (1μM) or pPB-SSL-FITC at 37℃for 1h in darkness. Then the cells were stained with DAPI for 1min. The cells were observed using a fluorescent microscopy or a confocal microscopy.
Results:As pPB-FITC concentration increasing from OμM to 150μM, a increasing percentage of FITC-positive cells (%gate) was shown in the binding study of pPB and pPB-SSL to the activated rat pHSCs or LX-2 cells. And the plateau concentration was observed at the concentration of pPB-FITC>7.5μM in rat pHSCs and>38.5μM in LX-2 cells. The binding-competition experiment showed that the binding of pPB-FITC to pHSCs or LX-2 cells fitted a contro-S curve. The FITC-positive pHSCs rate treated with pPB-SSL-FITC is 3.01±1.50 fold higer than that with SSL-FITC.
The intracellular localization of pPB-FITC and pPB-SSL-FITC were found in the cytoplasm of activated rat pHSCs. But when pHSCs were treated with free FITC under the same condition, no available fluorenscence signs were observed.
Conclusion:The binding of pPB to HSCs was in a dose-dependent manner and mediated by receptor on HSCs. The intracellular uptake of pPB-SSL-FITC by HSCs was significant more than that of SSL-FITC.
Part Four Effect of targeted interferon-gamma liposomes on rat hepatic stellate cells
Objective:To investigate the effects of interferon-gamma (IFN-γ) encapsulated in the cyclic peptide (pPB)-modified sterically stable liposomes (pPB-SSL-IFN-y) on primary rat hepatic stellate cells (pHSC).
Methods:The Inhibitory effects of pPB-SSL-IFN-γon cells proliferation were assessed via cell survival assay measured by Alamar blue assay kit and the IC50 values was compared. The effects of pPB-SSL-IFN-γon apoptosis were detected via Honchst33258 and TUNEL staining. The inhibitory effects of pPB-SSL-IFN-γon the expression of a-SMA protein were detected via Western bolt analysis.
Results:The IC50 value was respectively 3.09×105,1.26×105, and 4.27×104 IU/ml in the IFN-γ, SSL-IFN-γ, and pPB-SSL-IFN-γtreatment groups, which indicated the inhibitory effect of pPB-SSL-IFN-γwas respectively 7.24-fold and 2.95-fold higher than that of IFN-γand SSL-IFN-γ. The percentage of apoptosis cells in pPB-SSL-IFN-y treatment group was more than that control group and free IFN-y (2×105IU/ml) treatment group (p<0.05). Compared to the control and free IFN-y (1×104IU/ml) treatment groups, the pPB-SSL-IFN-γtreatment group significantly suppressed a-SMA expression. The marked suppression also was identified in the 1×10°IU/ml IFN-γtreatment group.
Conclusion:pPB-SSL-IFN-γcan inhibit the proliferation of pHSCs, induce apoptosis of pHSCs and suppress the expression of a-SMA protein. These effects of pPB-SSL-IFN-γon pHSCs are superior to free IFN-γ.
Part Five The pharmacokinetics, tissue distribution and cellular location of targeted Interferon-gamma liposomes in normal and fibrotic rats
Objective:To investigate and compare the pharmacokinetics and tissue biodistribution of free interferon-gamma (IFN-γ) and interferon-gamma encapsulated in cyclic peptide (pPB)-modified sterically stable liposomes (pPB-SSL-IFN-γ) in normal and fibrotic rats. And to investigate the intracellular location of IFN-γencapsulated in pPB-SSL and SSL in fibrotic livers.
Methods:IFN-y or pPB-SSL-IFN-γwas injected to normal rats via the tail vein. During 24h period, the blood was collected via intraocular vein at different time point. At 24h point, some tissues were harvested and wet-weighed. The concentration of IFN-y in the blood and 100mg of various tissues were measured by enzyme linked immunosorbent assay (ELISA) kit. Furthermore living-body tracing image analysis of pPB-SSL-DIR and SSL-DIR in normal and fibrotic rats was also performed. After the investigation in normal rats was ended at 48h in vivo, the rats were killed and some major organs were harvested to measured each fluorescent image and intensity in vitro. To identify the intracellular location of IFN-γencapsulated in SSL and pPB-SSLin fibrotic livers, liver sections were performed with immunofluorescent double-staining for IFN-γand a-SMA. The percentage of IFN-γpositive cells in a-SMA positive cells per section was determined.
Results:The pharmacokinetic study determined the half-lives of IFN-γand pPB-SSL-IFN-γto be 0.235±0.022h and 1.146±0.139h, respectively. The study of tissue distribution 24 h after injection showed that IFN-y encapsulated in pPB-SSL accumulated mainly in the liver, while free IFN-γaccumulated in the liver and intestine. Moreover, in the pPB-SSL-IFN-y group, the levels of IFN-y in each 100 mg tissue normalized to the initial injection dose were significantly higher than that of the corresponding tissues in the IFN-γ-injected group (p<0.05). IFN-y tissue distribution was further elucidated through living-body imaging studies with DIR-SSL-IFN-γand DIR-pPB-SSL-IFN-γ. In vivo, fluorescence signals were predominantly found in the liver region following injection, and spread to other tissues 48h after injection. After these rats were sacrificed at 48 h, the fluorescence of some tissue and blood samples was immediately examined in vitro. Fluorescence was observed in the liver and rarely in the blood. Living-body images indicated that DIR-pPB-SSL-IFN-γpredominantly accumulated in the liver of fibrotic rats after 20h. Furthermore, immunofluorescent double-staining for IFN-γand a-SMA showed that IFN-γencapsulated in pPB-SSL (60.13±9.15%) was located in HSCs at a higher level than IFN-γin SSLs (12.11±3.26%)(n=3,p<0.01).
Conclusion:These findings suggest that SSL-IFN-γand pPB-SSL-IFN-γselectively localized to the liver, and IFN-γencapsulated in pPB-SSL had a longer circulation half-life than free IFN-y. Both SSL and pPB-SSL could obtain passive liver-targeted effects in normal and fibrotic rats. But pPB-SSL delivering IFN-y obtain the HSC-specific targeting effects in fibrotic livers.
Part Six Effects of targeted interferon-gamma liposomes on hepatic fibrosis in rats
Objective:To investigate the anti-fibrotic effects and the reducing side-effects of interferon-gamma (IFN-y) encapsulated in cyclic peptide (pPB)-modified modified sterically stable liposomes (pPB-SSL-IFN-y).
Methods:A rat hepatic fibrosis model was induced by thioacetamide (TAA). When obvious hepatic fibrosis was confirmed at 11 w,40 fibrotic rats were randomly divided into six treatment groups and injected intravenously via tail vein with 0.5 ml of IFN-y, SSL-IFN-y and pPB-SSL-IFN-y with the same concentration of IFN-y (2×105 IU/ml), high-dose of IFN-y (1×106 IU/ml), PBS, and pPB-SSL twice a week for 4 w. Six normal rats were maintained under the same conditions. Forty-eight hours after the final injection, blood samples were collected for routine analysis of blood cells and serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), hyaluronic acid (HA) and collagen type IV-C. Some liver specimens were harvested for the histopathological assessment using hematoxylin and eosin staining, Masson staining and immunohistochemical staining for collagen I and the detection of hydroxyproline (Hyp) in livers. Eight microscopic fields were randomly selected per liver tissue section to quantify the positive stain-area of collagen I. Some liver specimens were snap-frozen for western blot analysis ofα-smooth muscle actin (a-SMA) expression.
Results:Hepatic fibrosis was aggravated with prolonged TAA treatment. The Ishak stage score, the level of serum IV-C and the positive-staining area for collagen I were all markedly reduced in the pPB-SSL-IFN-y group compared to low-and high-doses of the free-IFN-y group and SSL-IFN-y group. Furthermore, a-SMA expression in the liver of rats injected with pPB-SSL-IFN-y was lower than that in rats of other groups. In addition, the amounts of blood hemoglobin (HGB), blood platelets (PLT) and Hyp in the fibrotic livers of the pPB-SSL-IFN-y group were significantly higher than those in the control group and could be elevated to the same range as in the normal control group.
To investigate the effects of pPB-SSL-IFN-y on reducing side-effects, serum ALT, AST levels and the amount of total white blood cells (WBC) were detected. The level of serum AST in model control group was significant higher than that of the normal control groups. However, compared to the SSL-IFN-y groups, there was a significant reduction of serum AST levels in the pPB-SSL-IFN-y group, suggesting that the HSC-targeted drug carriers prevented damage caused by general endocytosis of SSLs by hepatocytes. Compared to the model control group, the amounts of WBCs in the low-and high-doses of IFN-y treatment groups were significantly lower, indicating inhibitory effects of IFN-y on leukopoiesis. pPB-SSL-IFN-y was found to increase the amount of WBC. And pPB-SSL-IFN-y could significantly decrease the histological HAI grade of inflammation caused by high-doses free IFN-y.
Conclusion:These data suggest that the anti-fibrotic effects of IFN-y were significantly improved after by pPB-SSL-targeted delivery to the liver. Additionally, IFN-y encapsulation by pPB-SSL improved the damage in liver caused by SSL-IFN-y and the reduction of WBC caused by IFN-y.
肝纤维化是慢性肝病重要病理特征,是发展到肝硬化的必经阶段。尽管迄今为止国内外的研究就肝纤维化和早期肝硬化可以逆转,已有共识,并在抑制肝星状细胞(hepatic stellate cell, HSC)活化并促进其凋亡、调节细胞外基质(extracellular matrix, ECM)的合成和降解等方面取得一些成果,对肝纤维化的临床治疗提供了一些药物和方法,但总体疗效仍不理想。目前多认为,限制临床上应用的抗肝纤维化药物效果的原因,可能与药物无法达到靶细胞、或到达靶细胞但无法在其周围形成有效的药物浓度及半衰期短和药物对其他组织细胞的副作用大过增加剂量带来的疗效等有关。
通过靶向载药系统转运药物,可达到靶向、长循环进而增加疗效及减少抗肝纤维化药物的全身副作用的目的。这里主要涉及：①靶向头基的选择；②载体形式的选择；③抗肝纤维化药物的选择。鉴于HSC激活是肝纤维化发展过程中的中心事件,因此,HSC是抗肝纤维化治疗的首选靶细胞。在HSC上,尤其是激活的HSC上特殊表达或过量表达的受体,是最经常被靶向载药系统采用的靶点。血小板源性生长因子(platelet derived growth factor, PDGF)是肝纤维化形成过程中最强的促增殖因子,与转化生长因子β1(transform growth factorβ1, TGF-β1)在肝纤维化的形成中起着重要作用；而且PDGF受体β(PDGF receptorβ, PDGFR-β)己被广泛的证明在激活的HSC上优势过量表达。近年来已有研究证明,环肽C*SRNLIDC* (pPB)能特异性识别PDGFR-β。与其他非多肽、非脂质体的靶向载药系统相比,小分子多肽作为靶向头基具有化学结构明确,高纯度并可被大量制备的优点；脂质体作为载体具有安全、载药谱广的优点。此外,立体稳定脂质体(sterically stabilized liposome, SSL)的聚乙二醇化(polyethylene glycol conjugated, PEGlated)的设计和能够达到纳米粒级别(<100nm)的粒径,能够使它规避网状内皮系统的吞噬,更具有长循环的特点。干扰素-γ(IFN-γ)是目前已经被批准临床应用的抗肝纤维化作用药物,在许多体内外实验中表现出一定的抗肝纤维化作用,但在临床应用中IFN-γ表现出相对低的疗效、相对短的半衰期和一些严重的副作用,如发热、血细胞减少、肝功能损害加重等,严重制约着IFN-γ的临床应用。
本研究中,我们在国内外率先设计了针对PDGFR-β的环肽(pPB)修饰的包封IFN-γ的立体稳定脂质体(pPB-SSL-IFN-γ),并通过体内外的实验研究,从分子、细胞和器官的水平探讨这个主动靶向载药系统能否解决IFN-γ的疗效低、半衰期短、副作用多的临床问题,并对其作用机制进行更深入的探讨,寻求更可能为临床所用的抗肝纤维化的靶向药物治疗途径,为进一步可能的临床研究提供理论依据,以期最终能使病人受益。
本研究的主要内容包括：①环肽和靶向IFN-γ脂质体的制备和表征；②原代大鼠肝星状细胞的分离、培养和鉴定；③环肽和靶向脂质体对肝星状细胞的靶向性验证；④靶向IFN-γ脂质体对肝星状细胞的体外抗肝纤维化作用的研究；⑤靶向IFN-γ脂质体在正常大鼠体内的药代动力学和体内分布的研究,以及靶向IFN-γ脂质体在肝纤维化大鼠的体内分布和肝组织上细胞定位的研究；⑥靶向IFN-γ脂质体对大鼠肝纤维的体内抗肝纤维化作用和改善副作用的研究。
第一部分
环肽和IFN-γ靶向脂质体的制备和表征
目的：人工合成C*SRNLIDC*环肽(pPB),并表征。构建pPB介导的IFN-γ主动靶向脂质体(pPB-SSL-IFN-γ),并表征其特性。
方法：采用固相合成的方法合成pPB,并冻干低温保存,以高效液相分析pPB的纯度,以质谱法检测其分子量。为制备靶向脂质体pPB-SSL-IFN-γ,将胆固醇：蛋磷脂酰胆碱：甲氧基-聚乙二醇2000-二硬酯酰磷脂酰乙醇胺(mPEG2000-DSPE):马来酰亚胺-聚乙二醇3400-二硬酯酰磷脂酰乙醇胺(mal-PEG+3400-DSPE)以2：1：0.1：0.02的摩尔比溶于氯仿,根据水化液不同,采用旋转蒸发-薄膜水化-挤压法制备各种非靶向脂质体。为了制备pPB介导的靶向脂质体,先将pPB与SATP反应,给pPB引入巯基,得到pPB-SH，并质谱验证其分子量,后将pPB-SH与非靶向脂质体反应,得各种靶向脂质体,进一步利用氢谱(1H-NMR)验证pPB-SH与非靶向脂质体膜材料中含有的mal-PEG3400-DSPE的结合。从粒径、包封率、载药量、稳定性等方面表征其理化特性。
结果：合成的pP、pPB-FITC和pPB-SH纯度>95%,质谱检测它们的分子量符合理论值,提示合成正确。进一步通过对比,在mal-PEG3400-DSPE的1H-NMR上清楚显示mal峰,在pPB-PEG3400-DSPE的1H-NMR上消失,提示了pPB与靶向脂质体膜材料mal-PEG3400-DSPE中mal基团通过加成反应相连。制备的pPB-SSL-IFN-γ的平均粒径为83.5nm,多分散指数(PI)为0.067；包封率为32.73±4.61%;载药量为8.99×104IU/μmol磷脂；4℃冷藏情况下,1月后无明显变化,稳定性良好；3月后粒径增大、分布增宽和包封率下降,提示脂质体颗粒有融合、粒径变大和IFN-γ的泄漏。
结论：固相合成法可大量、高纯度合成pPB。成功制备了下一步实验需要的纳米级别的pPB介导的IFN-γ的靶向脂质体。
第二部分
原代大鼠肝星状细胞的分离、培养和鉴定
目的：探讨原代大鼠肝星状细胞的分离、培养方法,并鉴定。为进一步的体外实验提供理想的细胞模型。
方法：采用胶原酶离体灌流、Nycodenz为分离介质的密度梯度离心的方法分离大鼠的肝星状细胞,并培养传代及同步鉴定。
结果：每只大鼠肝星状细胞的得率可达2-5x107/只,细胞存活率>90%。培养过程中出现特征性形态变化,进一步细胞免疫荧光化学鉴定；培养4d,Desmin染色阳性,提示得到的为肝星状细胞；培养10d和传1代后,α-SMA染色阳性,提示为活化表型。
结论：成功获得进一步实验所需的理想细胞模型---原代大鼠活化的肝星状细胞。
第三部分
环肽和靶向脂质体对肝星状细胞的靶向性
目的：探讨pPB环肽和pPB介导的靶向脂质体与原代活化的大鼠肝星状细胞和人肝星状细胞株LX-2体外结合的特性和细胞内定位的情况。
方法：合成异硫氰荧光素(FITC)标记的环肽(pPB-FITC),制备包封FITC的靶向(pPB-SSL-FITC)和非靶向脂质体(SSL-FITC).通过流式细胞仪测定pPB-FITC与原代活化的大鼠肝星状细胞和人LX-2细胞株结合的荧光强度,实施结合-饱和实验和结合-竞争实验,并比较pPB-SSL-FITC和SSL-FITC与原代大鼠肝星状细胞的结合能力。通过荧光显微镜及激光共聚焦显微镜观察pPB-FITC和pPB-SSL-FITC在活化的大鼠肝星状细胞内的定位。
结果：pPB-FITC与原代活化的大鼠肝星状细胞和人LX-2肝星状细胞株的结合具有饱和性和浓度依赖性；未标记的pPB对pPB-FITC与原代活化大鼠肝星状细胞和LX-2细胞株的结合有竞争抑制作用,且呈浓度依赖性。靶向脂质体pPB-SSL-FITC与原代大鼠活化的肝星状细胞结合的能力是非靶向脂质体SSL-FITC的3.01±1.50倍。原代大鼠活化的肝星状细胞能内吞pPB-FITC和pPB-SSL-FITC,绿色荧光信号主要分布于胞浆。
结论：pPB与原代大鼠肝星状细胞和人LX-2肝星状细胞株以受体介导的方式特异结合。靶向脂质体与活化的大鼠肝星状细胞结合的能力明显高于非靶向脂质体,结合后靶向脂质体被肝星状细胞内摄,主要分布在细胞浆,提示pPB-SSL可以作为靶向于HSC的载体来载送相关的药物。
第四部分
IFN-γ靶向脂质体对大鼠肝星状细胞的作用
目的：考察pPB介导的IFN-γ靶向脂质体对活化的大鼠肝星状细胞的体外药效及作用机制。
方法：通过Alamar blue分析法检测IFN-γ脂质体对细胞增殖的影响；通过Hoechst33258和TUNEL的方法检测IFN-γ脂质体对细胞凋亡的影响；通过Western blot分析的方法检测IFN-γ脂质体对细胞α-平滑肌肌动蛋白(α—SMA)表达的影响。
结果：含有相同IFN-γ剂量的靶向IFN-γ脂质体与IFN-γ非靶向脂质体和游离IFN-γ相比,明显抑制活化的肝星状细胞的增殖；Honchst33258和TUNEL染色发现,IFN-γ靶向脂质体明显抑制活化肝星状细胞的凋亡；Western blot分析法证实,IFN-γ靶向脂质体能明显抑制活化肝星状细胞的α-SMA的蛋白表达。
结论：与游离IFN-γ和非靶向IFN-γ脂质体相比,IFN-γ革巴向脂质体能更有效的抑制活化大鼠肝星状细胞的增殖、凋亡和α-SMA的表达。
第五部分
IFN-γ靶向脂质体在大鼠体内的药代动力学、组织分布和细胞定位
目的：探讨IFN-γ靶向脂质体在正常大鼠体内的药代动力学和组织分布的规律,并探讨IFN-γ非靶向脂质体(SSL-IFN-γ)和IFN-γ靶向脂质体(pPB-SSL-IFN-γ)在肝纤维化大鼠体内的动态分布和肝组织上的细胞定位情况。
方法：采用酶联免疫吸附法(ELISA)检测pPB-SSL-IFN-γ和游离IFN-γ静脉注入正常大鼠体内5mi n-24h不同时间点的血浆样本中IFN-γ的浓度,并比较两者的半衰期(t1/2)；进一步通过24h时获得的大鼠的不同组织中IFN-γ的浓度,观察其组织分布情况。制备包含DIR(一种近红外染料)的靶向和非靶向脂质体,采用荧光活体成像仪比较IFN-γ靶向脂质体(DIR-pPB-SSL-IFN-γ)和IFN-γ非靶向脂质体(DIR-SSL-IFN-γ)在正常大鼠和纤维化大鼠体内分布的动态显像的情况。采用双重荧光标记免疫组化染色法,考察靶向和非靶向脂质体携带的IFN-γ在纤维化大鼠肝组织内的细胞定位情况。
结果：药代动力学研究显示：pPB介导的靶向IFN-γ脂质体的半衰期(t1/2)和曲线下面积(AUC)明显长于游离IFN-γ。组织分布研究显示：pPB介导的靶向脂质体运载的IFN-γ在体内主要聚集在肝组织内；而游离IFN-γ在体内主要分布在肝组织和肠；进一步比较在同一种脏器(心、肝、脾、肺、肾、脑和肠)内的分布,pPB介导的靶向脂质体载送的IFN-γ较游离IFN-γ明显增多。活体荧光动态成像显示：①正常大鼠体内：DIR-SSL-IFN-γ和DIR-pPB-SSL-IF-γ在注射后1h,肝脏区域的荧光强度明显增加至最高,后强度逐渐下降直至48h,其它脏器才可看见荧光分布；在成像48h的时间点,结束活体成像,取心、肝、脾、肺、肾和血液组织,立即于体外成像显示：肝组织的荧光强度明显高于其他组织,血液标本的荧光强度甚至接近0,进一步证明了活体成像中肝脏区域的荧光图象确为肝组织中荧光显像,并显示了直至48h,肝组织仍明显的荧光聚集。②纤维化大鼠体内：DIR-pPB-SSL-IFN-γ注入纤维化大鼠体内2h时,肝脏区域的荧光强度已经明显高于DIR-SSL-IFN-γ注入组；至20h时,这种趋势更加明显,甚至在DIR-SSL-IFN-γ处理组的肝脏区域的荧光强度显示出较2h时下降,而DIR-pPB-SSL-IFN-γ处理组大鼠肝脏区域的荧光强度较2h时明显增加。双重荧光标记免疫组织化学染色探测细胞定位的研究显示：DPB-SSL携带的IFN-γ在肝组织中与α-SMA的共同表达率(60.13±9.15%)明显高于SSL携带的IFN-γ(12.11±3.26%)(n=3,p<0.01),且图像显示后者主要定位于肝细胞。
结论：与游离IFN-γ相比,pPB介导的靶向脂质体携带的IFN-γ在体内的药代动力学和组织分布的研究显示了长循环的特性；IFN-γ靶向和IFN-γ非靶向脂质体均显示了明显的肝组织的器官靶向特性,但IFN-γ靶向脂质体在肝纤维化大鼠体内显示出更加明显的肝脏器官靶向。在纤维化肝组织中靶向脂质体携带的IFN-γ主要定位于肝星状细胞；而非靶向脂质体携带的IFN-γ主要定位在肝细胞,验证了靶向脂质体载送的IFN-γ在纤维化大鼠肝组织内的HSC细胞靶向性。
第六部分
IFN-γ靶向脂质体对大鼠肝纤维化的体内抗肝纤维化作用和改善副作用的研究
目的：研究IFN-γ靶向脂质体对大鼠肝纤维化的体内抗肝纤维化作用,并观察其对改善某些副作用的效果。
方法：将40只肝纤维化大鼠,随机分成6组,分别给与(均尾静脉注射,2次/周,持续4周)：①PBS(n=4);②空白脂质体pPB-SSL(n=4)；③IFN-γ(n=8)；④IFN-γ非靶向脂质体(n=8)；⑤IFN-γ靶向脂质体(n=8)；⑥5倍剂量IFN-γ组(n=7),其中①作为模型对照组(model control group)；②作为空白pPB-SSL对照组；③④⑤此3种制剂中游离IFN-γ含量均为2x105IU/m1；⑥中游离的IFN-γ含量为1x10.IU/m1。6只正常大鼠在同等的条件下被饲养,作为正常对照组(normal control group)。最后一次静脉注射后48h,收集大鼠的血液标本,分别检测肝功能(ALT、AST和ALB)、血液分析(WBC、HGB和PLT)和血清肝纤维化指标(HA和IV-C)；收集肝组织标本,分别进行HE、MassOn胶原三色染色、羟脯氨酸含量测定、Ⅰ型胶原的免疫组织化学染色和肝组织的α-SMA蛋白表达的western blOt分析。
结果：与模型对照组、游离IFN-γ组甚至IFN-γ非靶向脂质体组和5倍剂量游离IFN-γ组相比,IFN-γ靶向脂质体明显减轻大鼠的肝纤维化分期,并明显减少肝组织Ⅰ型胶原染色面积、血清IV-C水平和肝组织α-SMA蛋白的表达；与模型对照组和游离IFN-γ低剂量组相比,IFN-γ靶向脂质体明显改善由于肝纤维化造成的HGB和PLT的减少,还可明显降低肝组织羟脯氨酸含量；与模型对照组相比,IFN-γ靶向脂质体亦明显降低肝纤维化大鼠血清ALT和HA水平；与模型对照组和游离IFN-γ高剂量组相比,IFN-γ靶向脂质体亦明显减轻肝纤维化大鼠的肝脏病理炎症HAI分级。同时,IFN-γ靶向脂质体治疗组的血清AST水平较IFN-γ非靶向治疗组明显降低(P<0.05)；而且IFN-γ靶向脂质体治疗组的WBC计数高于游离IFN-γ的低剂量组和高剂量组(P=0.072和P=0.070)。
结论：IFN-γ靶向脂质体显示出更加明显的体内抗肝纤维化作用,而且,IFN-γ靶向脂质体能够明显改善IFN-γ非靶向脂质体非选择性肝细胞摄取造成的AST的升高和避免高剂量IFN-γ造成的肝脏炎症坏死程度加重。另外,IFN-γ靶向脂质体可在一定程度上改善游离IFN-γ造成的血WBC计数的降低。
Effect of Interferon-gamma liposomes targeted to platelet derived growth factor receptorβon hepatic fibrosis in rat
Hepatic fibrosis is a very important pathological feature during the development of chronic hepatic injury. Hepatic fibrosis is also a necessary pathway to liver cirrhosis, which is a very common disease resulting in morbidity and mortality in many countries. Many therapeutic approaches have been examined in experimental and clinical studies of hepatic fibrosis. These approaches include adjusting the balance of extracellular matrix synthesis and degradation, reducing intrahepatic inflammation, and inhibiting activation or promoting apoptosis of hepatic stellate cells (HSC). Through these techniques, the reversibility of fibrosis can be observed. However, no approved anti-fibrotic drug has been clinically applied. The main reason for the lack of clinical use is the inability to specifically target the responsible cells or molecules to increase drug effectiveness and to decrease the side-effects on non-target tissues or cells in vivo.
To improve drug effectiveness and reduce the side-effects on non-target tissues or cells, targeted drug-carrier systems have been employed in recent years that ensure drug delivery for long-term circulation and/or site-specific targeting of specific populations of liver cells, including hepatocytes, liver endothelial cells, Kupffer cells, and HSCs. HSC activation is a central event in hepatic fibrogenesis. Receptors specifically expressed or over-expressed on HSCs, especially on activated HSCs, are selected as the active targeting sites for targeting-drug therapies for hepatic fibrosis. Platelet-derived growth factor (PDGF) is the most prominent mitogen for HSCs during hepatic fibrogenesis, and the PDGF receptor-P (PDGFR-P) is strongly up-regulated on activated HSCs. The cyclic peptide C*SRNLIDC* (pPB) was previously shown to have a specific affinity for PDGFR-β.
In the present study, we utilized pPB to modify sterically-stable liposomes (SSL) to prepare the pPB-SSL carrier. To establish the application, interferon gamma (IFN-y) was selected to be entrapped in pPB-SSL, because IFN-y has anti-fibrotic activities. However, some shortcomings of IFN-y, such as low anti-fibrotic effects, a short blood-circulation half-life, and side effects due to its effects on non-target cells or tissues, have limited its clinic application.
The aim of the present study is to investigate whether the targeted drug-delivery system (pPB-SSL) can resolve these limitations, and to investigate the related action mechanism. The approach of targeting drug-delivered increasing the efficacy of anti-fibrotic drug therapy and reducing side-effects may have the potential clinical value. This study also provide a new option for drug-carriers which could selectively delivery drug to cells over-expressing PDGFR-β.
These works include six parts as described below.
Part One Synthesis and characterization of cyclic peptide and targeted interferon-gamma liposomes
Objective:To synthesize and characterize the cyclic pepetide C*SRNLIDC* (pBP). And to prepare and characterize sterically stable liposomes modified with pPB (pPB-SSL) to deliver interferon-gamma (pPB-SSL-IFN-γ).
Methods:pPB was synthesized using the standard strategy of Boc-protected solid phase peptide synthesis. pPB was analysed and purified by high performance liquid chromatographic (HPLC). The molecular weight of pPB was confirmed by electrospray ionization tandem mass spectrometry (ESI-MS).
pPB-SSL-IFN-γwas prepared by the method of rotary evaporation-thin film hydrated-extruded. The synthesis of pPB-S-mal-PEG34oo-DSPE was confirmed by 'H-NMR spectrometry. To characterize pPB-SSL-IFN-γ, particle diameter and distribution of pPB-SSL-IFN-γwere measured by dynamic light scattering; and the encapsulation efficiency (ee%) of IFN-γwas determined. To investigate the stability of pPB-SSL-IFN-γ, the particle diameter and ee% were compared after pPB-SSL-IFN-γwas prepared and had been stored at 4℃for one month and three month.
Results:HPLC analysis revealed that the purity of prepared pPB exceeded 95%. ESI-MS spectrum showed the molecular weight of pPB was 921.5Da, coinciding with their theoretical values. The conjugation of pPB-SH with mal-PEG34oo-DSPE were indicated by the disappearance of the characteristic resonance of mal (6.7ppm) of pPB-S-mal-PEG3400-DSPE shown in1H-NMR spectrum. The mean particle diameter of pPB-SSL-IFN-y was 83.5nm, and the polydispersity index (PI) was 0.067. The ee% of IFN-y in pPB-SSL (n=3) was 32.73±4.61%. The loading capacity was 8.99x104IU /μM phospholipid. Compared to freshly prepared pPB-SSL-IFN-y, the particle diameter and ee% of pPB-SSL-IFN-y stored at 4℃for one month had no significant difference.
Conclusion:pPB has successfully synthesized. And pPB-SSL-IFN-y was successfully prepared, which showed satisfactory particle size distribution (mostly<100nm), ee% and loading capacity.
Part Two Isolation, culture and identification of primary rat hepatic stellate cells
Objective:To provide activated hepatic stellate cells (HSCs) for the followed in vitro study.
Methods:Primary rat HSCs (pHSCs) was isolated from normal Sprague-Dawley (SD) rats using infusion and combined digestion with pronase E and collagenase followed by Nycodenz density gradient centrifugation. pHSCs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cell viability was detected by Trypan blue dyeing. pHSCs were assessed by auto-fluorescence, immunocytochemical staining of desmin and a-SMA and morphological investigation with microscopy.
Results:About 5X10/ml pHSCs cells were harvested from each rat. The viability of freshly isolated pHSCs was exceeding 90%. Due to the presence of lipid droplets the fresh rat pHSCs showed green-blue spontanenous fluorescence that can be observed at 328nm or 445 nm wavelength by fluorescence microscopy. They stretched out pseudopodia after incubated for 24h to 48h. Atuo-fluorescence and lipid droplets gradually disappeared with time passing. The cells cultured for 10 days to 14 days became stellate or irregular shape and bigger, which represented as activated phenotype. pHSCs were confirmed by positive staining via immunocytochemistry for Desmin, and activated pHSCs were confirmed by positive staining for a-SMA.
Conclusion:Primary rat HSCs were successfully obtained the ideal activated rat HSCs model---cultured for the followed study in vitro.
Part Three Binding and intracellular location of cyclic peptide and cyclic peptide-modified liposomes in hepatic stellate cells
Objective:To investigate the binding and intracellular location of cyclic peptide (pPB) and pPB-modified sterically stable liposomes (pPB-SSL) in hepatic stellate cells (HSCs).
Methods:Activated rat pHSCs were respectively incubate with various concentration of FITC labeling pPB (pPB-FITC,0-150μM) in the darkness. The percentages of the FITC-positive cells at selected cells (%gate) were determined by flow cytometry. For the competitivly experimential study, activated pHSCs were incubated with different concentration (0.035-1411.5μM) of the unlabeled free pPB for 30min before pPB-FITC (36μM) were added. And the total fluosrencence intensity (TFI) was selected as an index analysed. Binding of pPB to LX-2 cells also was measured as describe above. Furthermore the binding of SSL encapsulating FITC (SSL-FITC) or pPB-SSL encapsulating FITC (pPB-SSL-FITC) to HSC were also performed with the folw cytometry.
For investigating the intracellular location, activated rat pHSCs were incubated with free FITC, pPB-FITC (1μM) or pPB-SSL-FITC at 37℃for 1h in darkness. Then the cells were stained with DAPI for 1min. The cells were observed using a fluorescent microscopy or a confocal microscopy.
Results:As pPB-FITC concentration increasing from OμM to 150μM, a increasing percentage of FITC-positive cells (%gate) was shown in the binding study of pPB and pPB-SSL to the activated rat pHSCs or LX-2 cells. And the plateau concentration was observed at the concentration of pPB-FITC>7.5μM in rat pHSCs and>38.5μM in LX-2 cells. The binding-competition experiment showed that the binding of pPB-FITC to pHSCs or LX-2 cells fitted a contro-S curve. The FITC-positive pHSCs rate treated with pPB-SSL-FITC is 3.01±1.50 fold higer than that with SSL-FITC.
The intracellular localization of pPB-FITC and pPB-SSL-FITC were found in the cytoplasm of activated rat pHSCs. But when pHSCs were treated with free FITC under the same condition, no available fluorenscence signs were observed.
Conclusion:The binding of pPB to HSCs was in a dose-dependent manner and mediated by receptor on HSCs. The intracellular uptake of pPB-SSL-FITC by HSCs was significant more than that of SSL-FITC.
Part Four Effect of targeted interferon-gamma liposomes on rat hepatic stellate cells
Objective:To investigate the effects of interferon-gamma (IFN-γ) encapsulated in the cyclic peptide (pPB)-modified sterically stable liposomes (pPB-SSL-IFN-y) on primary rat hepatic stellate cells (pHSC).
Methods:The Inhibitory effects of pPB-SSL-IFN-γon cells proliferation were assessed via cell survival assay measured by Alamar blue assay kit and the IC50 values was compared. The effects of pPB-SSL-IFN-γon apoptosis were detected via Honchst33258 and TUNEL staining. The inhibitory effects of pPB-SSL-IFN-γon the expression of a-SMA protein were detected via Western bolt analysis.
Results:The IC50 value was respectively 3.09×105,1.26×105, and 4.27×104 IU/ml in the IFN-γ, SSL-IFN-γ, and pPB-SSL-IFN-γtreatment groups, which indicated the inhibitory effect of pPB-SSL-IFN-γwas respectively 7.24-fold and 2.95-fold higher than that of IFN-γand SSL-IFN-γ. The percentage of apoptosis cells in pPB-SSL-IFN-y treatment group was more than that control group and free IFN-y (2×105IU/ml) treatment group (p<0.05). Compared to the control and free IFN-y (1×104IU/ml) treatment groups, the pPB-SSL-IFN-γtreatment group significantly suppressed a-SMA expression. The marked suppression also was identified in the 1×10°IU/ml IFN-γtreatment group.
Conclusion:pPB-SSL-IFN-γcan inhibit the proliferation of pHSCs, induce apoptosis of pHSCs and suppress the expression of a-SMA protein. These effects of pPB-SSL-IFN-γon pHSCs are superior to free IFN-γ.
Part Five The pharmacokinetics, tissue distribution and cellular location of targeted Interferon-gamma liposomes in normal and fibrotic rats
Objective:To investigate and compare the pharmacokinetics and tissue biodistribution of free interferon-gamma (IFN-γ) and interferon-gamma encapsulated in cyclic peptide (pPB)-modified sterically stable liposomes (pPB-SSL-IFN-γ) in normal and fibrotic rats. And to investigate the intracellular location of IFN-γencapsulated in pPB-SSL and SSL in fibrotic livers.
Methods:IFN-y or pPB-SSL-IFN-γwas injected to normal rats via the tail vein. During 24h period, the blood was collected via intraocular vein at different time point. At 24h point, some tissues were harvested and wet-weighed. The concentration of IFN-y in the blood and 100mg of various tissues were measured by enzyme linked immunosorbent assay (ELISA) kit. Furthermore living-body tracing image analysis of pPB-SSL-DIR and SSL-DIR in normal and fibrotic rats was also performed. After the investigation in normal rats was ended at 48h in vivo, the rats were killed and some major organs were harvested to measured each fluorescent image and intensity in vitro. To identify the intracellular location of IFN-γencapsulated in SSL and pPB-SSLin fibrotic livers, liver sections were performed with immunofluorescent double-staining for IFN-γand a-SMA. The percentage of IFN-γpositive cells in a-SMA positive cells per section was determined.
Results:The pharmacokinetic study determined the half-lives of IFN-γand pPB-SSL-IFN-γto be 0.235±0.022h and 1.146±0.139h, respectively. The study of tissue distribution 24 h after injection showed that IFN-y encapsulated in pPB-SSL accumulated mainly in the liver, while free IFN-γaccumulated in the liver and intestine. Moreover, in the pPB-SSL-IFN-y group, the levels of IFN-y in each 100 mg tissue normalized to the initial injection dose were significantly higher than that of the corresponding tissues in the IFN-γ-injected group (p<0.05). IFN-y tissue distribution was further elucidated through living-body imaging studies with DIR-SSL-IFN-γand DIR-pPB-SSL-IFN-γ. In vivo, fluorescence signals were predominantly found in the liver region following injection, and spread to other tissues 48h after injection. After these rats were sacrificed at 48 h, the fluorescence of some tissue and blood samples was immediately examined in vitro. Fluorescence was observed in the liver and rarely in the blood. Living-body images indicated that DIR-pPB-SSL-IFN-γpredominantly accumulated in the liver of fibrotic rats after 20h. Furthermore, immunofluorescent double-staining for IFN-γand a-SMA showed that IFN-γencapsulated in pPB-SSL (60.13±9.15%) was located in HSCs at a higher level than IFN-γin SSLs (12.11±3.26%)(n=3,p<0.01).
Conclusion:These findings suggest that SSL-IFN-γand pPB-SSL-IFN-γselectively localized to the liver, and IFN-γencapsulated in pPB-SSL had a longer circulation half-life than free IFN-y. Both SSL and pPB-SSL could obtain passive liver-targeted effects in normal and fibrotic rats. But pPB-SSL delivering IFN-y obtain the HSC-specific targeting effects in fibrotic livers.
Part Six Effects of targeted interferon-gamma liposomes on hepatic fibrosis in rats
Objective:To investigate the anti-fibrotic effects and the reducing side-effects of interferon-gamma (IFN-y) encapsulated in cyclic peptide (pPB)-modified modified sterically stable liposomes (pPB-SSL-IFN-y).
Methods:A rat hepatic fibrosis model was induced by thioacetamide (TAA). When obvious hepatic fibrosis was confirmed at 11 w,40 fibrotic rats were randomly divided into six treatment groups and injected intravenously via tail vein with 0.5 ml of IFN-y, SSL-IFN-y and pPB-SSL-IFN-y with the same concentration of IFN-y (2×105 IU/ml), high-dose of IFN-y (1×106 IU/ml), PBS, and pPB-SSL twice a week for 4 w. Six normal rats were maintained under the same conditions. Forty-eight hours after the final injection, blood samples were collected for routine analysis of blood cells and serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), hyaluronic acid (HA) and collagen type IV-C. Some liver specimens were harvested for the histopathological assessment using hematoxylin and eosin staining, Masson staining and immunohistochemical staining for collagen I and the detection of hydroxyproline (Hyp) in livers. Eight microscopic fields were randomly selected per liver tissue section to quantify the positive stain-area of collagen I. Some liver specimens were snap-frozen for western blot analysis ofα-smooth muscle actin (a-SMA) expression.
Results:Hepatic fibrosis was aggravated with prolonged TAA treatment. The Ishak stage score, the level of serum IV-C and the positive-staining area for collagen I were all markedly reduced in the pPB-SSL-IFN-y group compared to low-and high-doses of the free-IFN-y group and SSL-IFN-y group. Furthermore, a-SMA expression in the liver of rats injected with pPB-SSL-IFN-y was lower than that in rats of other groups. In addition, the amounts of blood hemoglobin (HGB), blood platelets (PLT) and Hyp in the fibrotic livers of the pPB-SSL-IFN-y group were significantly higher than those in the control group and could be elevated to the same range as in the normal control group.
To investigate the effects of pPB-SSL-IFN-y on reducing side-effects, serum ALT, AST levels and the amount of total white blood cells (WBC) were detected. The level of serum AST in model control group was significant higher than that of the normal control groups. However, compared to the SSL-IFN-y groups, there was a significant reduction of serum AST levels in the pPB-SSL-IFN-y group, suggesting that the HSC-targeted drug carriers prevented damage caused by general endocytosis of SSLs by hepatocytes. Compared to the model control group, the amounts of WBCs in the low-and high-doses of IFN-y treatment groups were significantly lower, indicating inhibitory effects of IFN-y on leukopoiesis. pPB-SSL-IFN-y was found to increase the amount of WBC. And pPB-SSL-IFN-y could significantly decrease the histological HAI grade of inflammation caused by high-doses free IFN-y.
Conclusion:These data suggest that the anti-fibrotic effects of IFN-y were significantly improved after by pPB-SSL-targeted delivery to the liver. Additionally, IFN-y encapsulation by pPB-SSL improved the damage in liver caused by SSL-IFN-y and the reduction of WBC caused by IFN-y.
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