Effect Of Interferon-gamma Liposomes Targeted To Platelet Derived Growth Factor Receptor ? On Hepatic Fibrosis In Rat

Effect of Interferon-gamma liposomes targeted to platelet derived growth factor receptor?on hepatic fibrosis in ratHepatic 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-?.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-?.These works include six parts as described below.Part One Synthesis and characterization of cyclic peptide and targeted interferon-gamma liposomesObjective: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-?).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-?was 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-?, particle diameter and distribution of pPB-SSL-IFN-?were measured by dynamic light scattering; and the encapsulation efficiency (ee%) of IFN-?was determined. To investigate the stability of pPB-SSL-IFN-?, the particle diameter and ee% were compared after pPB-SSL-IFN-?was 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 /?M 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 cellsObjective: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,0-150?M) 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 (0.035-1411.5?M) of the unlabeled free pPB for 30min before pPB-FITC (36?M) 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 (1?M) 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 O?M to 150?M, 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>7.5?M in rat pHSCs and>38.5?M 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 cellsObjective:To investigate the effects of interferon-gamma (IFN-?) 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-?on 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-?on apoptosis were detected via Honchst33258 and TUNEL staining. The inhibitory effects of pPB-SSL-IFN-?on 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-?, SSL-IFN-?, and pPB-SSL-IFN-?treatment groups, which indicated the inhibitory effect of pPB-SSL-IFN-?was respectively 7.24-fold and 2.95-fold higher than that of IFN-?and SSL-IFN-?. 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-?treatment group significantly suppressed a-SMA expression. The marked suppression also was identified in the 1×10°IU/ml IFN-?treatment group.Conclusion:pPB-SSL-IFN-?can inhibit the proliferation of pHSCs, induce apoptosis of pHSCs and suppress the expression of a-SMA protein. These effects of pPB-SSL-IFN-?on pHSCs are superior to free IFN-?.Part Five The pharmacokinetics, tissue distribution and cellular location of targeted Interferon-gamma liposomes in normal and fibrotic ratsObjective:To investigate and compare the pharmacokinetics and tissue biodistribution of free interferon-gamma (IFN-?) and interferon-gamma encapsulated in cyclic peptide (pPB)-modified sterically stable liposomes (pPB-SSL-IFN-?) in normal and fibrotic rats. And to investigate the intracellular location of IFN-?encapsulated in pPB-SSL and SSL in fibrotic livers.Methods:IFN-y or pPB-SSL-IFN-?was 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-?encapsulated in SSL and pPB-SSLin fibrotic livers, liver sections were performed with immunofluorescent double-staining for IFN-?and a-SMA. The percentage of IFN-?positive cells in a-SMA positive cells per section was determined.Results:The pharmacokinetic study determined the half-lives of IFN-?and pPB-SSL-IFN-?to 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-?accumulated 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-?-injected group (p<0.05). IFN-y tissue distribution was further elucidated through living-body imaging studies with DIR-SSL-IFN-?and DIR-pPB-SSL-IFN-?. 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-?predominantly accumulated in the liver of fibrotic rats after 20h. Furthermore, immunofluorescent double-staining for IFN-?and a-SMA showed that IFN-?encapsulated in pPB-SSL (60.13±9.15%) was located in HSCs at a higher level than IFN-?in SSLs (12.11±3.26%)(n=3,p<0.01).Conclusion:These findings suggest that SSL-IFN-?and pPB-SSL-IFN-?selectively localized to the liver, and IFN-?encapsulated 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 of?-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.