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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Cyclic Arg-Gly-Asp Peptide-Labeled Liposomes for Targeting Drug Therapy of Hepatic Fibrosis in Rats

Division of Gastroenterology and Hepatology, Department of Internal Medicine, Zhongshan Hospital, Fudan University, Shanghai, China (S.-L.D., J.Wa., J.-Y.W.); Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, China (H.P., W.-Y.L.); and Department of Internal Medicine, Transplant Research Program, University of California, Davis Medical Center, Sacramento, California (J.Wu)

Received March 9, 2007; accepted May 16, 2007.

	Abstract

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Targeting hepatic stellate cells (HSCs) has been challenging due to the lack of specific receptors or motifs on the cells. The aim of the present study was to develop a HSC-specific system for improving drug delivery to HSCs. The affinity of a cyclic peptide containing Arg-Gly-Asp (cRGD) to collagen type VI receptor on HSCs was examined in both in vitro and in vivo experiments. Sterically stable liposomes (SSLs) were modified with this peptide to yield a new carrier, cRGD-SSL. The targeting efficiency of this carrier in delivering interferon (IFN)-1b was investigated in a rat model of liver fibrosis induced by bile duct ligation (BDL). When incubating HSCs or hepatocytes with cyclic RGD peptide, the peptide was bound preferentially to activated HSCs. Biodistribution study showed that the accumulation of cRGD peptide-labeled liposomes in HSCs isolated from BDL rats was 10-fold more than unlabeled SSLs. BDL rats receiving injections of IFN-1b entrapped in cRGD-SSL exhibited significantly reduced extent of liver fibrosis compared with BDL control rats or BDL rats treated with IFN-1b entrapped in SSLs. Thus, cRGD-SSL is an efficient drug carrier, which selectively targets activated HSCs and improves drug therapy for liver fibrosis to a significant extent. This liposomal formulation represents a new means of targeting drug carrier for the treatment of liver fibrosis, and it may have potential clinical applications.

Due to the fact that there are relatively few HSCs in the liver and that there is a lack of specific receptors or motifs on the cell surface, the attempt to target HSCs has been a challenging task. Only a few studies focusing on targeting HSCs have been reported (Beljaars et al., 2002). These include human serum albumin (HSA) modified with mannose 6-phosphate (M6P) (Beljaars et al., 1999) or with a cyclic peptide that recognizes the collagen type VI receptor (Beljaars et al., 2000), and a dominant-negative soluble platelet-derived growth factor- receptor (Borkham-Kamphorst et al., 2004), which inhibits HSC proliferation. However, the carrying capacity and clinical applicability of these studies are questionable. Therefore, there is a tremendous demand to develop approaches that improve drug therapy for the disorder.

Interferon (IFN)- is the most common and effective agent for the treatment of viral hepatitis C (Strader et al., 2004). It has been shown that in IFN--treated patients, along with inhibition of hepatitis C virus replication and improvement of liver injury, there is significant improvement in the inhibition of progression of hepatic fibrosis (Poynard et al., 2002). To investigate whether IFN- itself has any antifibrotic effects, we reported previously that IFN- given by subcutaneous administration was effective in reducing gene expression and deposition of collagen type I and III in a rat model of carbon tetrachloride (CCl₄)-induced liver fibrosis (Zhang et al., 1999). However, IFN- has many adverse effects, such as anemia and flu-like syndromes. Many patients cannot tolerate these side effects, and they discontinue the treatment. We hypothesize that using liposome-mediated targeting delivery of IFN- may improve the therapeutic effects and at the same time reduce its adverse effects. We report here that we have developed a liposomal carrier modulated by the labeling with a cyclic eight-amino acid residue peptide containing Arg-Gly-Asp (RGD) and recognizing collagen type VI receptors, which are up-regulated in activated HSCs of fibrotic livers. Our in vivo experiments demonstrate that the delivery of INF-1b to HSCs with this cyclic RGD peptide-labeled liposomal carrier improved the efficacy of this medication in the treatment of liver fibrosis.

	Materials and Methods

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Chemicals and Reagents. Reagents were obtained from the following sources: egg phosphatidylcholine (EPC), cholesterol (Chol), methoxy-polyethylene glycol) [PEG]₂₀₀₀-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and N-hydroxysulfosuccinimide-polyoxyethylene-maleimide (NHS-PEG-MAL) were from Avanti Polar Lipids (Alabaster, AL); Sephadex G-50 and Sepharose CL-4B were from Pharmacia-LKB (Uppsala, Sweden); Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin, and fetal bovine serum were from Invitrogen (Carlsbad, CA); and cell culture plates were from Corning (New York, NY). All other chemicals were of analytical grade.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Schematic illustration of cRGD-SSLs. The link of cyclic RGD peptide with MAL-PEG-DOPE (A) and its incorporation into the sterically stable liposomes (EPC/Chol/PEG2000-DOPE/MAL-PEG-DOPE) (B). See Materials and Methods for details.

Preparation of MAL-PEG-DOPE. The synthesis of MAL-PEG₂₀₀₀-DOPE was conducted as described previously (Allen et al., 1995). DOPE was added to a NHS-PEG-MAL solution at a ratio of 1:0.2 in chloroform containing triethylamine. The reaction mixture was stirred at 25°C for 6 h. After thin layer chromatography in CHCl₃/CH₃OH/H₂O (65:25:4) showed disappearance of NHS-PEG-MAL with appearance of a more polar material, the solvent was evaporated. Then, 5 ml of acetonitrile was added, and the mixture was kept at 4°C overnight. After centrifugation at 5000 rpm for 10 min, the upper clear solution was collected and evaporated. The product was dried in vacuum overnight, and it was verified by matrix-assisted laser desorption ionization/time of flight mass spectrometric analysis.

Synthesis of Cyclic RGD Peptide and Preparation of SSLs. The sequence of cyclic RGD peptide (Cys-Gly-Arg-Gly-Asp-Ser-Pro-Lys or C*GRGDSPK*) was selected based on its cell adhesion mediated by collagen VI (Beljaars et al., 2000). We changed the sequence of the reported peptide (C*GRGDSPC*) by replacing cysteine with lysine. The cyclic RGD and AGA (C*GAGASPK*) peptides were synthesized at a purity of 95% as determined by solid-phase synthesis, and they were labeled with fluorescein isothiocyanate (FITC). Cyclic RGD peptide was linked via a sulfhydryl group at the cysteine residue to a liposomal formulation, as shown in Fig. 1.

Lipids composed of EPC/Chol/methoxy-PEG₂₀₀₀-DOPE/MAL-PEG-DOPE in a molar ratio of 2:1:0.1:0.02 were dissolved in chloroform, and the solvent was evaporated to form a lipid film under reduced pressure. The lipid mixture was hydrated in an appropriate buffer and extruded through double layers of polycarbonate membranes in 100-nm open mesh 15 times using a mini-extruder (Avanti Polar Lipids) to obtain a homogeneous liposome suspension (Wu et al., 1998a). For loading IFN, IFN-1b was dissolved in phosphate-buffered saline (PBS), and the lipid mixture was hydrated in PBS containing IFN. Resulting liposomes were passed through a Sepharose CL-4B column to remove free IFN-1b. For labeling with cyclic RGD peptide with IFN-loaded liposomes (SSL-IFN), liposome suspension was incubated with cyclic RGD peptide at a molar ratio of 10 to 1 overnight (Blume et al., 1993; Dubey et al., 2004). Unbound cyclic RGD peptide was separated by passing through a Sepharose CL-4B column. For the liposomal formulations used in this study, SSL-IFN refers to liposomes entrapping IFN-1b; RGD-SSL-IFN refers IFN-1b encapsulated in SSLs labeled with cyclic RGD peptide. The diameter of liposomes was determined by dynamic light scattering (Nicomp 380 ZLS; Particle Sizing Systems, Santa Barbara, CA). The entrapping rate of IFN-1b was determined after Sepharose CL-4B filtration to separate the lipid fraction and entrapped IFN-1b and then by using high-performance lipid chromatography and enzyme-linked immunosorbent assay to measure the content of lipids and IFN-1b in the RGD-SSL-IFN-1b, respectively. The entrapping rate was calculated according to the formula entrapping rate = entrapped IFN-1b/total IFN-1b in solution x 100%. The carrying capacity of RGD-SSL was determined according to the equation carrying capacity =W_E/W_LN x 100%, where W_LN refers to total weight of lipids, and W_E refers to the weight of the entrapped substance. The morphology of cyclic RGD peptide-labeled liposomes was examined by a transmission electronic microscope (Hitachi 7000; Hitachi, Tokyo, Japan) after being stained with 2% phosphotungstic acid and dried on carbon-coated grids. The size of liposomes was verified at the same time.

Binding of Cyclic RGD Peptide to HSCs. FITC-conjugated cyclic RGD or AGA peptides were incubated at concentrations of 20 to 1000 nM with quiescent or activated HSCs (1 x 10⁶ cells/well) at either 4°C for 1 h or 37°C for 1 to 4 h. In competition experiments, the cells were preincubated with non-FITC-conjugated cyclic RGD peptide (20–100,000 nM) at 4 or 37°C for 30 min and then with FITC-conjugated cyclic RGD peptide (200 nM). After the incubation, the cells were washed three times with ice-cold PBS, pH 7.4. The FITC-positive cells were counted by flow cytometry, and the data are expressed as percentage of counted cells.

In separate experiments, cells were plated at 2 x 10⁶ cells/well in 24-well plates, and they were incubated with FITC-conjugated cyclic RGD or AGA peptides at 200 nM for 1 h. After washing three times with PBS and staining by 4,6-diamidino-2-phenylindole (DAPI), the cells were visualized with a TCS-SP2 laser-scanning confocal microscope (Leica Microsystems, Wetzlar, Germany), using an ultraviolet laser with emission at 488 and 372 nm for scanning. Cells were optically sectioned, and digital images were acquired. All instrumental parameters pertaining to fluorescence detection, and image analyses were held constant to allow sample comparison.

Inhibition of HSC Proliferation by IFN. In vitro inhibition of HSC proliferation by free IFN or IFN in various liposomal formulations was determined using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) proliferation assay. In brief, HSCs at 8 x 10⁴/ml were seeded in 96-well plates, and then they were incubated with free IFN (IFN-1b), SSL-IFN, or RGD-SSL-IFN. After the cells were incubated for 2, 24, or 48 h under these treatments, medium was replaced with 50 µl of MTT at l0.5 mg/ml in DMEM, and the mixture was incubated at 37°C for another 4 h. Acid-isopropanol was added into each well (0.1 ml of 40 mM HCl in isopropanol) and mixed thoroughly until all crystals were dissolved. The plates were read immediately in a Titerk Multiskan Plus MK II plate reader (Flow Laboratories, Mississauga, ON, Canada) using test (570-nm) and reference (650-nm) wavelengths. The IC₅₀ (the concentration of an inhibitor needed to inhibit ligand binding by 50%) was calculated by nonlinear regression using GraphPad Prism software (GraphPad Software Inc., San Diego, CA).

Animal Model of Liver Fibrosis Induced by Bile Duct Ligation. Male Wistar rats (250–300 g) were obtained from the Fudan University Animal Care Center (Shanghai, China). The protocol of animal experiments was approved by the Institutional Ethical Committee of Animal Experimentation, and the experiments were performed strictly according to governmental and international guidelines on animal experimentation. Liver fibrosis was induced in rats by ligation of the common bile duct as reported previously (Zhan et al., 2006). The bile duct-ligated (BDL) rats were randomly divided into four groups: BDL control, BDL + IFN, SSL-IFN, or RGD-SSL-IFN. One day after completing the common bile duct ligation procedure, rats were injected with IFN (5 x 10⁴ U/rat) in various formulations every other day via the tail vein. After 4 weeks of the treatment, rats were sacrificed, and blood samples were collected for the determination of serum levels of alanine aminotransferase (ALT) and total bilirubin (TB) with routine methods in a clinical laboratory of the hospital. Liver specimens were collected for formalin fixation and for snap-freezing. The frozen tissue was used for the assay for hydroxyproline content and RNA extraction. Liver total RNA was extracted by TRIzol reagent (Invitrogen), and it was quantitated spectrophotometrically. cDNA was generated by reserve transcriptase reaction, and liver procollagen type I/III gene expression was semiquantitated by reserve transcriptase-polymerase chain reaction (RT-PCR), using glyceraldehyde phosphate dehydrogenase (GAPDH) as a house-keeping gene control. The primer pairs used are shown in Table 1. The agarose gel images of the polymerase chain reaction products after 28 cycles of amplification were densitometrically analyzed, and the average density ratios of the genes of interest over GAPDH were used to reflect the relative gene expression levels (Jiang et al., 2004). Liver total hydroxyproline content was determined spectrophotometrically as reported previously (Zhu et al., 1999).

Liver Distribution of RGD-SSL or SSL in BDL Rats, and HSC and Hepatocyte Distribution in BDL Rats. Both SSL and RGD-SSL formulations were labeled by ^99mTc by the reaction between of ^99mTcO₄ and the lipid diethylenetriaminepentaacetic-1,2-DOPE (Avanti Polar Lipids) in SSLs in the presence of SnCl₂ (Ahkong and Tilcock, 1992), and unlabeled isotope was separated by Sephadex G-50. ^99mTc-labeled liposomes were injected via tail vein in BDL rats. Four hours after injection, hepatocytes and HSCs were isolated from the same rats as described above (Wu et al., 1996; Zhu et al., 1999). The radioactivity in each cell type was determined by a liquid scintillation counter (LS 6500; Beckman Coulter, Inc., Fullerton, CA) for the evaluation of the cell type-selective distribution based on cell number. In a separate experiment, ^99mTc-labeled SSL or RGD-SSL was injected via tail vein in BDL rats. Whole-body scanning was conducted with single photon emission tomography (Philips-IRIX; Skannerudvikling Corp., Veenendaal, The Netherlands) under anesthesia of ketamine 15 min after the injection. The radioactivity in each organ was estimated by calculating the radioactivity of the image area of individual organ, expressed by a ratio of radioactivity in the organ area over that of the whole body.

Statistical Analysis. All data are shown in mean values ± S.D. Data were analyzed by analysis of variance test plus multiple comparisons between two given groups with Newman-Keuls test. p < 0.05 was considered statistically significant.

	Results

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Selective Binding of Cyclic RGD Peptide to HSCs. To determine the affinity of the cyclic RGD peptide with HSCs, quiescent HSCs, activated HSCs, hepatocytes, and Cos-7 cells were incubated with FITC-conjugated cyclic RGD or AGA peptides. The cells with cellular internalization of FITC-conjugated peptides were considered as positive binding. As shown in Fig. 2, activated HSCs incubated with cyclic RGD peptide at 37°C had a much higher FITC-positive rate as determined by flow cytometry compared with either quiescent HSCs or hepatocytes, whereas cyclic AGA peptide did not display any affinity to all cell types examined. This suggests that activated HSCs selectively bound to the cyclic RGD peptide. The FITC-positive HSC number was significantly increased when the incubation temperature was elevated from nonpermissive (4°C) to permissive temperature (37°C) (Fig. 3, A and B), suggesting that the endocytosis of cyclic RGD peptide was boosted with the elevation in temperature. Moreover, the endocytosis of the peptide seemed to be concentration-saturated, because a plateau was observed when the peptide reached approximately 500 nM (Fig. 3A). Meanwhile, when HSCs were preincubated with the cyclic RGD peptide at 37°C, the subsequent cell binding of FITC-conjugated cyclic RGD peptide was inhibited in a concentration-dependent manner (Fig. 3C), but the inhibitory concentration of non-FITC conjugated cyclic RGD peptide was higher than we expected.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Binding of cyclic RGD peptide to activated HSCs. The binding of cyclic RGD peptide at 200 nM to quiescent and activated rat HSCs was determined at 37°C for 4 h. Cyclic AGA peptide was used as a control. Both cyclic peptides were conjugated with FITC. The FITC-positive cells were counted by flow cytometry, and the data are expressed as percentage of counted cells. **, p < 0.01 compared with quiescent HSCs (n = 5).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Binding of cyclic RGD peptide to HSCs in a dose- and temperature-dependent manner. A, binding of activated HSCs with FITC-conjugated cyclic RGD peptide in different concentrations at 4 or 37°C for 4 h. FITC-conjugated cyclic AGA peptide was used as a control. FITC-positive cells were counted by flow cytometry after the incubation, and the data are expressed as percentage of counted cells. B, binding of activated HSCs with cyclic RGD peptide at different time points at 4 or 37°C. The levels of RGD peptide binding to HSCs peaked at 4 h, and they were sustained until 8 h. C, competitive binding of unlabeled cyclic RGD peptide with HSCs at 37°C for 4 h. HSCs were preincubated with increasing concentration of unconjugated cyclic RGD peptide at 37°C, and the binding of FITC-conjugated cyclic RGD peptide to HSCs was determined in the presence of non-FITC-conjugated cyclic RGD peptide. n = 5 in all groups of each panel.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Affinity of FITC-conjugated cyclic RGD peptide with HSCs. Micrographs are the representatives of FITC-conjugated cyclic RGD peptide under a fluoroscope (A and B) and laser-scanning microscope (C). A, activated HSCs with positive FITC fluorescent signal in the cytoplasm. B, HSCs were cocultured with hepatocytes to simulate in vivo HSC activation. FITC-conjugated cyclic RGD peptide fluorescent signals were localized exclusively within activated HSCs, but not in hepatocytes. Activated HSCs are morphologically stellate-like, with many projections. Their cytoplasm was stained in green, and the nucleus was stained in blue by counterstaining with DAPI. Hepatocytes are relatively smaller in this image, and they are only positive for DAPI staining in nuclei. C, activated HSCs. HSCs with internalized FITC-conjugated cyclic RGD peptide showed green fluorescence within the cytoplasm with DAPI counterstaining in the nucleus.

Inhibition of HSC Proliferation by IFN in Various Liposomal Formulations. Inhibitory effects of free IFN, empty RGD-SSL, SSL-IFN, RGD-SSL-IFN, or IFN plus SSL on HSC proliferation were evaluated by incubating with these agents for various times. As shown in Table 2, the inhibitory effect of RGD-SSL-IFN was approximately 20-fold higher than SSL-IFN (p < 0.001) at 2 and 24 h, and it remained higher (9-fold) even after 48 h of the incubation (p < 0.001), when the release and uptake of IFN from the unlabeled liposomes would be expected to be high. The RGD-labeled formulation exerted the inhibition, and it reached its maximum effect within 2 to 24 h. The inhibitory effect of RGD-SSL-IFN at 3 h of the incubation was 3-fold higher than that seen in SSL-IFN at 48 h (p < 0.01). At all time points, SSL-IFN was significantly less effective than free IFN in the inhibition of HSC proliferation (p < 0.001). A high IC₅₀ (1000 µM) for RGD-SSL implied that neither cyclic RGD peptide, lipids, nor the combination of the two resulted in any inhibition on HSC proliferation. Moreover, IC₅₀ for free IFN is similar to free IFN plus SSL (p > 0.05), which suggests that SSLs did not affect the inhibition of IFN on HSC proliferation in vitro.

View this table: [in this window] [in a new window]   TABLE 2 Inhibitory effect of interferon-1b in different formulations on HSC proliferation in vitro The proliferation of HSCs was determined with an MTT test in a 96-well plate (n = 5 in each group). Data are expressed as IC50 (U/ml of IFN, unless otherwise stated), and they were extrapolated from dose-response curves.

Morphology and Liver Distribution of Cyclic RGD-Labeled Liposomes. Morphology of cyclic RGD-labeled liposomes was examined by a transmission electron microscope. As shown in Fig. 5A, the liposomes are unilamellar, round, and regular in size. The average size of liposomes determined by laser-based light scattering was 101 ± 17.7 nm, which was further verified by transmission electron microscopic examination. The entrapping rate of IFN-1b in RGD-SSL was 40.2%, and the entrapping capacity was 1 x 10⁴U IFN/µmol lipid as determined by measuring the phosphorus content. After labeling with ^99mTc, both SSL and RGD-SSL were injected intravenously in BDL rats, and the total radioactivity in the liver 15 min after the injection was higher in animals receiving RGD-SSL than those receiving SSL injection (Fig. 5B). There was no significant difference in liposomal radioactivity accumulation between these two formulations in other organs. In separate experiments, ^99mTc-labeled SSL or RGD-SSL was injected intravenously in rats 4 weeks after BDL. Four hours after the injection, hepatocytes and HSCs were isolated, and the radioactivity in hepatocytes and HSCs was determined. As shown in Fig. 5C, it is evident that unlabeled SSLs were largely distributed in hepatocytes, whereas the distribution of cyclic RGD peptide-labeled SSLs was markedly increased more than 10-fold in in vivo-activated HSCs, and it dropped nearly 14-fold in hepatocytes. These findings demonstrated that cyclic RGD peptide-labeled SSLs had a preferential liver uptake, and they were largely distributed in activated HSCs rather than hepatocytes in vivo.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Microscopic morphology and in vivo distribution of RGD-SSL. A, RGD-SSLs were stained with phosphotungstic acid, and then they were examined under a transmission electronic microscope. The scale bar indicates the liposome size (30,000x). B, radioactivity in each organ was determined 15 min after intravenous injection of 99mTc-RGD-SSL or 99mTc-SSL. The percentage of the radioactivity in each organ was calculated based on the total whole body radioactivity (n = 6) after correction with time. C, distribution of cyclic RGD peptide-labeled SSLs or unlabeled SSLs in HSCs or hepatocytes from BDL rats. Radioactivity of HSCs or hepatocytes 4 h after intravenous injection of either 99mTc-SSL or 99mTc-RGD-SSL in BDL rats was determined, and the data are expressed as percentage of radioactivity (dpm) in cells/total liver radioactivity after correction with time. **, p < 0.01 compared with unlabeled SSL. n = 3.

View larger version (171K): [in this window] [in a new window]   Fig. 6. Representative images of liver histology treated with IFN in different liposomal formulations in BDL rats. Liver fibrosis was induced by BDL. Liver sections were stained with hematoxylin and eosin. BDL rats were randomly divided into four groups: BDL controls, BDL + IFN, SSL-IFN, or RGD-SSL-IFN. One day after the BDL procedure, rats were injected with IFN, SSL-IFN, or RGD-SSL-IFN via tail vein in every other day for 4 weeks. A, BDL control. B, BDL+ IFN. C, BDL + SSL-IFN. D, BDL + RGD-SSL-IFN (all 100x). Brown dots in A and B are the accumulated bile in microbile ducts. Hepatocellular damage is obvious, thick fibrotic septa are seen, and there were infiltrated inflammatory cells in the portal triads in A and B. However, there was less hepatocellular injury, inflammatory infiltration, and thinner fibrotic septa infiltration in both C and D compared with A and B, and further improvements in liver injury and fibrosis in D are evident compared with C.

View larger version (42K): [in this window] [in a new window]   Fig. 7. Serum ALT, total TB levels, and liver hydroxyproline content. A, serum ALT and TB levels were determined after the animals were sacrificed by a routine method. B, liver content of hydroxyproline was determined spectrophotometrically. *, p < 0.05 compared with sham-operated group. , p < 0.05 compared with BDL controls. , p < 0.05 compared with BDL + SSL-IFN. n = 5 in all groups in both panels except the sham-operated group (n = 7).

The mRNA levels of procollagen type I/III in BDL (Fig. 8) were significantly higher than in sham-operated controls. Consistent with histology, intravenous administration of SSL-IFN significantly reduced the procollagen type I/III mRNA levels in these BDL rats. The amount of liver procollagen type I/III mRNA in RGD-SSL-IFN-treated animals was much lower than in BDL controls and in the SSL-IFN-treated group, and it almost reached the levels in the sham-operated group. Thus, RGD-SSL-IFN seemed to be the most effective in suppressing the development of liver fibrosis by inhibiting procollagen type I/III mRNA expression compared with IFN encapsulated in SSL without cyclic RGD peptide labeling.

View larger version (43K): [in this window] [in a new window]   Fig. 8. Procollagen type I/III mRNA levels in BDL rats treated with IFN in different formulations. A, levels of type I/III procollagen mRNA was determined by semiquantitative RT-PCR using GAPDH as a house-keeping gene control. B, polymerase chain reaction product agarose gel images were analyzed by densitometry. *, p < 0.05 compared with sham-operated group. , p < 0.05 compared with BDL controls. , p < 0.05 compared with BDL + SSL-IFN. n = 5 in all groups in both panels except the sham-operated group (n = 7).

	Discussion

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The peptide sequence we used to label the liposome formulation was used previously to label HSA (Beljaars et al., 2000). We modified the sequence by replacing cysteine with lysine, and the modified peptide was easily conjugated to the liposomal formulation via a sulfhydryl group in the cysteine residue. The modified cyclic RGD peptide tends to form a more stable cyclic peptide than the original peptide, which was cyclized by forming an instable disulfo bond (-S–S-) between two neighboring cysteine residues. In contrast, our new cyclic peptide forms a cycle with a peptide bond (-NH–CO-) between the lysine and cysteine residues. The peptide bond is much more stable than the disulfo bond due to less possibility to be oxygenized. Although HSA can be used to deliver agents that react with it, the list of agents that react with HSA, but without changes in their chemical and pharmacological features, is very short. Thus, developing drug carriers that possess much more delivering capacity and that are clinically applicable is a critical step toward selective drug delivery for the treatment of liver fibrosis. Based on our experience in targeting drug and gene delivery in the treatment of liver injury (Wu et al., 1998a, 2004; Liu et al., 2003) and in the establishment of liposomal formulations (Pan et al., 2006), we established a new formulation of sterically stable liposomes, which are approximately 100 nm in diameter and which contain PEG as a spacer for the cyclic peptide linkage. The entrapping rate for IFN-1b was very high, and the liposome size is in the range of the fenestrae of SECs for easily crossing through the fenestrae to reach the Disse space. The cyclic RGD peptide-labeled liposomes are long-circulating, which is a crucial property for in vivo drug delivery (Lee and Low, 1994). Moreover, cyclic RGD peptide labeling significantly enhanced the biodistribution of the liposomes in the fibrotic liver of BDL rats, resulted in a more than 10-fold increase of the liposomal accumulation in activated HSCs, and improved the antifibrotic efficacy of IFN-1b in treated animals. Thus, we have successfully established a new formulation of sterically stable liposomes that are clinically applicable and readily conjugated by molecules, such as peptides, for tissue- or cell type-specific recognition or delivery.

Targeting HSCs has been much more difficult than hepatocytes and Kupffer cells due to no specific motifs or receptors existing on the cell surface, relatively fewer cell numbers, and residence of HSCs in the Disse space side of SECs (Beljaars et al., 2002; Wu et al., 2002). Ideally, targeting therapeutics should have a high degree of site preference, i.e., they should be specifically delivered to their target site(s) to achieve a high level of therapeutic efficacy and a low level of adverse effects, because many potential antifibrotic agents have a wide range of effects on many other organs or cell types (Wu and Zern, 2000; Yen et al., 2006). One means that has been used to increase the selectivity of antifibrotic agents is to be encapsulated in liposomes, which have been approved by the Food and Drug Administration for drug delivery in chemotherapy, such as doxorubicin. Further improvements in the targeting effects of antifibrotic drugs might be achieved by coupling ligands selective for the target cells to the liposome surface. However, a previously confirmed targeting approach, incorporation of M6P-HSA-modified human albumin into liposomes, did not show any improvement in anti-inflammatory and antifibrotic effects of bioactive lipid dilinoleoylphosphatidylcholine (DLPC) in a BDL model of rat hepatic fibrosis, and the inflammatory responses were even worse in animals receiving M6P-HSA-liposomes containing DLPC than in those receiving DLPC-liposomes without M6P-HSA incorporation (Adrian et al., 2007). We are not clear why the conflicting results were obtained. One possible explanation is the different model systems that were used. In that work, the liposomes with DLPC were administered only once; the BDL rats were evaluated 1 day after the therapeutics. Our animals were injected every other day for 4 weeks, thus providing a more optimal antifibrotic regimen.

Small molecules, e.g., peptides, carbohydrates, or antibody fragments, may ultimately be useful targeting ligands. Small peptides have the advantage of being chemically defined, and they are also able to be manufactured in large quantities and at high purity without biological contaminants. They can be selected for specific targets with "one-bead one-compound" combinatorial libraries (Aina et al., 2005). The cyclic peptide C*GRGDSPC* has been shown to specifically inhibit the attachment of collagen type VI to cells (Marcelino and McDevitt, 1995). We linked the cyclic RGD peptide to the distal end of PEG, and we obtained a stable drug delivery system, SSLs. The in vitro experiment with rat HSCs showed that IFN-1b entrapped in the RGD-labeled liposomes exerted much greater effects in inhibiting HSC proliferation than that in nonlabeled formulation. The mechanism underlying IFN--induced inhibition of HSC proliferation is associated with its effects on promoting an apoptotic process of activated HSCs (Saile et al., 2003). IFN- has been found to promote gene expression of antioxidant enzymes, such as copper/zinc superoxide dismutase and glutathione peroxidase in hepatocytes and HSCs, to enhance antioxidative defense capability, and in turn, to improve hepatocellular function and inhibit HSC activation (Lu et al., 2002). The biodistribution of cyclic RGD peptide-labeled liposomes showed the preferential liver uptake and much increased HSC accumulation in comparison with unlabeled liposomes.

The findings in the present study demonstrate that cyclic RGD peptide-labeled liposomes are specifically recognized by activated HSCs. IFN exerts better pharmacological actions, such as inhibiting their proliferation, reducing ECM synthesis, and suppressing fibrogenic transforming growth factor- release when it was delivered with targeting liposomes. The recognition of cyclic RGD peptide-labeled liposomes by activated HSCs is critical for its preferential localization in HSCs, because liposomes without cell-specific labeling after being taken up by the liver are normally cleaned up by Kupffer cells (Wu et al., 1998a,b). The intravenous injection of IFN in various formulations did not cause any elevation of serum ALT levels, but it displayed significant benefits in improving the extent of liver injury and fibrosis as evidenced by reduced serum ALT and total bilirubin levels, decreased liver hydroxyproline content, lessened connective tissue deposition in liver histology, and reduced mRNA levels of procollagen type I and III genes in the groups of BDL + SSL-IFN and BDL + RGD-SSL-IFN, and further reduction in the BDL + RGD-SSL-IFN group. Although the reduction in the BDL + RGD-SSL-IFN group was not as robust as we expected, it is statistically significant, and it proves the usefulness of the targeting strategy. This may be due to the fact that liposomes and entrapped IFN- are taken up by cells via endocytosis, and endocytosed IFN- may undergo lysosomal or endosomal degradation. Antifibrotic agents that can be encapsulated in liposomes for site-selected delivery have a large spectrum (Wu and Zern, 2000). Many of these agents are not degradable in endosomes or lysosomes, and they are expected to exhibit a much better improvement. Taken together, it seemed that IFN encapsulated in RGD-SSL further improved its antifibrotic activity, which involves the inhibition of procollagen type I gene expression at both steady-state and activated levels of HSCs (Inagaki et al., 2003), as well as promotion of an apoptotic process of activated HSCs (Saile et al., 2003), which is an important step toward cessation or reversal of the fibrogenic process (Canbay et al., 2004).

In conclusion, the present study demonstrates that cyclic RGD peptide-labeled liposomes are selectively taken up by activated HSCs via receptor-mediated endocytosis and that interferon-1b encapsulated in the sterically stable liposome formulation displayed better suppression on HSC proliferation in vitro. The cyclic RGD peptide labeling markedly increased the liposomal accumulation in activated HSCs in vivo, and it improved the efficiency of IFN to a significant extent in blocking the fibrogenesis in a rat model of bile duct ligation. Thus, the cyclic RGD peptide-labeled sterically stable liposomes represent a new means of targeting drug carrier for the treatment of liver fibrosis, and they may have potential clinical applications.

	Acknowledgements

 
The synthesis of cyclic peptide was kindly conducted by Dr. Wuyuan Lu (Division of Basic Science, Institute of Human Virology, University of Maryland Biotechnology Institute, Baltimore, MD).

	Footnotes

 
The study was supported in part by Grant 30270595 from the National Nature Science Foundation, People's Republic of China (to J.-Y.W.). J.Wu was supported by the National Institute of Diabetes, Digestive, and Kidney Diseases Grant DK069939.

Part of this study was presented at the Annual Meeting of the American Gastroenterology Association; 2005 May 14–19; Chicago, IL, and it was published in an abstract form in Gastroenterology 1284:A15, 2005.

Both Drs. Ji-Yao Wang and Jian Wu share the equal authorship. Dr. Jian Wu's contact information: Department of Internal Medicine, Transplant Research Program, UC Davis Medical Center, 4635 2nd Ave., Suite 1001, Sacramento, CA 95817. E-mail: jdwu{at}ucdavis.edu.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.122481.

ABBREVIATIONS: ECM, extracellular matrix; SEC, sinusoidal endothelial cell; HSC, hepatic stellate cell; HSA, human serum albumin; M6P, mannose-6-phosphate; IFN, interferon; RGD, Arg-Gly-Asp; EPC, egg phosphatidylcholine; Chol, cholesterol; PEG, polyethylene glycol; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; NHS, N-hydroxysulfosuccinimide; MAL, maleimide; SSL, sterically stable liposome; DMEM, Dulbecco's modified Eagle's medium; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; AGA, Arg-Gly-Arg; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; DAPI, 4,6-diamidino-2-phenylindole; ^99mTc, Technetium-99m; BDL, bile duct ligation/ligated; ALT, alanine aminotransferase; TB, total bilirubin; RT-PCR, reverse transcriptase-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DLPC, dilinoleoylphosphatidylcholine; cRGD, cyclic Arg-Gly-Asp.

Address correspondence to: Dr. Ji-Yao Wang, Department of Internal Medicine, Division of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, 180 Fenglin Rd., Shanghai 200032, China. E-mail: jiyao_wang{at}hotmail.com

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