Enhanced intracellular delivery and improved antitumor efficacy of doxorubicin by sterically stabilized liposomes modified with a synthetic RGD mimetic

Journal of Controlled Release 107 (2005) 262 – 275 www.elsevier.com/locate/jconrel

Enhanced intracellular delivery and improved antitumor efficacy of doxorubicin by sterically stabilized liposomes modified with a synthetic RGD mimetic Xiao-Bing Xiong a, Yue Huang b, Wan-liang Lu a, Xuan Zhang a, Hua Zhang a, Tsuneji Nagai c, Qiang Zhang a,* a School of Pharmaceutical Sciences, Peking University, Beijing, 100083, PR China Department of Vaccine Engineering, Institute of Basic Medical Sciences, 27, Tai-Ping Road, Beijing, 100850, PR China c Department of Pharmaceutics, Hoshi University, Ebara 2-4-41, Shinagawa-ku, Tokyo 142, Japan

b

Received 26 October 2004; accepted 7 March 2005 Available online 25 August 2005

Abstract While sterically stabilized liposomes (SSL) can passively accumulate into tumor tissue due to the effect of enhanced permeability and retention (EPR), the intracellular uptake of the entrapped anticancer drugs by the tumor cells should be a determinant step for their antitumor activities. Therefore, strategies that can enhance the intracellular uptake of SSL into tumor cells could lead to an improved therapeutic efficacy for the drugs. To check this possibility, RGD-mimetic-modified SSL (RGDm-SSL) were constructed aimed to achieve tumor accumulation as well as enhanced intracellular delivery, and were loaded with doxorubicin (DOX), an anticancer drug. Flow cytometry and confocal microscopy reveal that RGDm-SSL facilitated the DOX uptake into the melanoma cells via integrin-mediated endocytosis. DOX-loaded RGDm-SSL (RGDm-SSL-DOX) displayed higher cytotoxicity on melanoma cells than DOX-loaded SSL (SSL-DOX). Tissue distribution and therapeutic experiments were examined in C57BL/6 mice carrying melanoma B16 tumors. RGDm-SSL-DOX displayed similar DOX accumulation in tumor tissue to that of SSL-DOX but showed significantly lower DOX level in blood and remarkably higher DOX level in spleen than SSL-DOX. Administration of RGDm-SSL-DOX at a dose of 5 mg DOX/kg resulted in effective retardation of tumor growth and prolonged survival times compared with SSL-DOX. These results suggest that RGDm-modified SSL may be a promising intracellular targeting carrier for efficient delivery of chemotherapeutic agents into tumor cells. D 2005 Elsevier B.V. All rights reserved. Keywords: Intracellular delivery; RGD mimetic; Doxorubicin; Sterically stabilized liposomes; Biodistribution

* Corresponding author. Tel.: +86 10 82802791; fax: +86 10 82802791. E-mail addresses: [email protected], [email protected] (Q. Zhang). 0168-3659/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2005.03.030

1. Introduction In the last two decades, many studies focused on developing drug delivery systems to achieve con-

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trolled release or enable drug targeting to specific tumor sites. Sterically stabilized liposomes (SSL) were a major breakthrough in prolonging circulation time and achieving improved tumor targeting [1]. It has been demonstrated that SSL can accumulate in tumor tissue due to the effect of enhanced permeability and retention (EPR) [2–4]. However, anticancer drugs accumulation in tumor tissue via SSL seems to be prerequisite but far from sufficient to guarantee a therapeutic improvement. For a variety of chemotherapeutic agents with an intracellular site of action, efficient intracellular uptake by the tumor cells should be the determinant step for their antitumor activity. While introducing PEG enables liposomes accumulate in tumor tissue, it creates a steric barrier that could cause a reduction in liposomes interaction with the target cells [5,6], leading to low uptake of the entrapped drugs via cell endocytosis or membrane fusion. In particular, when the liposomes are composed of rigid lipid and the entrapped drugs cannot be released easily from them, the intracellular delivery by diffusion is limited to a larger extent [7]. Growing solid tumors have capillaries with increased permeability as a result of the disease process (e.g., tumor angiogenesis) [8–10]. Pore diameters in these capillaries can range from 100 to 800 nm. Drugcontaining liposomes that have diameters in the range of approximately 60–150 nm are small enough to extravasate from the blood into the tumor interstitial space through these pores [11]. In contrast, the blood vessels in most normal tissues are nonfenestrated capillaries. These blood vessels are composed of a single layer of endothelial cell with tight junctions. The endothelial barrier may prevent liposomes to traverse intact vessels, and SSL with ~ 120 nm in mean diameter did not accumulate in these normal tissues after i.v. injection [12,13]. The discrepancy of SSL accumulation in tumor tissue and normal tissue encourages us to hypothesize that it is possible to enhance the intracellular delivery of the entrapped drugs accumulated in tumor tissue to obtain an improved therapeutic efficacy. Integrins are a family of cell surface receptors that are responsible for anchoring cells to the extracellular matrix (ECM). These cell surface receptors are universally expressed by tumor cells as well as normal cells. The RGD (arginine–glycine–aspartic acid) se-

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quence is known to serve as a recognition motif in multiple ligands for several different integrins such as integrin arh3 and a5h1 et al. [14]. Recently, integrinmediated carriers, such as RGD-modified liposomes, nanoparticles, conjugates and so on, have been developed as gene transfer vehicles [15–20]. Potentially, RGD-peptide could be used to enhance SSL binding to tumor cells, from which increased intracellular delivery of the entrapped drugs to these cells can be expected. By coupling an RGD mimetic (RGDm) to the distal end of poly(ethyleneglycol)-coated SSL, we aimed at constructing an enhanced tumor targeting delivery system characterized by both tumor accumulating and enhanced intracellular delivery. To test its targeting effect, doxorubicin (DOX), a widely used anticancer drug, was encapsulated into the RGDmmodified SSL (RGDm-SSL), then DOX uptake and cytotoxicity on melanoma cells were evaluated in vitro. Finally, the tissue distribution and antitumor activity were investigated in C57BL/6 mice bearing B16 tumor.

2. Materials and methods 2.1. Materials Methoxypolyetheleneglycol (Mw 2000)-distearylphosphatidylethanolamine (DSPE-PEG) was purchased from NOF Co. (Tokyo, Japan). DSPE-PEG-BTC [1,2dioleyol-sn-glycero-3-phosphoethanolamine-n-[poly (ethyleneglycol)]-N-benzotriazole carbonate, PEG Mw 3400] was obtained from Shearwater Polymers Inc. (Huntsville, AL, USA), Cholesterol (Chol) and Sephadex G50 from Pharmacia Biotech (Piscataway, NJ, USA), and Soya phosphatidylcholine (SPC) from Lucas Meyer (Hamburg, Germany). Doxorubicin hydrochloride (DOX) was kindly provided as a gift by Haizheng Pharmaceutical Co. (Zhejiang province, China). 4,6-Diamidino-2-phenylindole (DAPI) and 3[4,5-dimethylthiazol-2-yl] -2, 5-diphenltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). All other solvents and reagents were used as chemical grade. The murine B16 and human A375 melanoma cell lines were obtained from the Basic Medical Cell Center, Chinese Academy of Medical Science (CAMS, Beijing, China) and were cultured in DMEM

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medium (Gibco, Paisley, UK) supplemented with 100 U/ml penicillin, 100 Ag/ml streptomycin, and 10% FBS in a humidified atmosphere containing 5% CO2 at 37 8C. Male C57BL/6 mice (6 to 8 weeks old), ranging from 18 to 22 g, were provided by Vital River Laboratory Animal Center (Beijing, China). All care and handling of animals were performed with the approval of Institutional Authority for Laboratory Animal Care. 2.2. Synthesis of RGDm and conjugation of RGDm to DSPE-PEG The RGD mimetic (RGDm) was synthesized according to literature with a modest modification [21]. In brief, 6-amino-hexanoic acid was esterified with ethanol to protect carboxyl in the presence of thionyl dichloride (SOCl2), and then 6-amino-hexanoic acid ethyl ester was coupled to Na-t-butyloxycarbonyl-N u`-nitro arginine [Boc-Arg (NO2)-OH]. The reaction was carried out using the 1,3-dicyclohexycarbodiimide (DCC) and 1-hydroxybenzotriazole in tetrahydrofuran. Removal of the ethyl ester was carried out with sodium hydroxide, pH 9.5. The Boc-protecting group was removed by 50% trifluoroacetic acid in dichloromethane. The nitro protecting group was deducted to amino group under hydrogen atmosphere with 10% Pd. The product was purified by high-performance liquid chromatography (Gilson, France), using a ZORBAX C18 column (2.12 cm  25 cm) with a mobile phase of methanol: H2O (adjusted pH 10.0 with aqueous ammonia). Formation of the compound was confirmed by mass spectroscopy. An activated DSPE-PEG (DSPE-PEG-BTC) was used to conjugate RGDm to DSPE-PEG. This reaction takes place between amines and BTC group, which acts as the linking agent. The RGDm and DSPE-PEGBTC in the molar ratio of 1:2 (RGDm:DSPE-PEGBTC) were dissolved separately in 0.01 M isotonic HEPES buffer, pH 7.5. DSPE-PEG-BTC solution was added in small increments to the RGDm solution at 4 8C with gentle stirring. The reaction was allowed to proceed for 4 h at 4 8C and traced by TLC until the RGDm was approached to complete disappearance. Excess glycine was added to the reaction mixture to consume the remaining BTC moieties. Finally, the reaction mixture was dialyzed extensively against

water to remove all impurities and lyophilized. The formation of DSPE-PEG-RGDm was confirmed by reverse-phase high-pressure liquid chromatography and NMR. 2.3. Liposomes preparation Lipid compositions of SPC/Chol/DSPE-PEG (20: 10:2, mol/mol) and SPC/Chol/DSPE-PEG/DSPEPEG-RGDm (20:10:1:1, mol/mol) were used for SSL-DOX and RGDm-SSL-DOX, respectively. Liposomes were prepared by thin lipid film hydration followed by sonication and extrusion as described previously [22,23]. Briefly, lipids of above compositions were dissolved in chloroform, dried into a thin film in a round bottom flask on a rotary evaporator under reduced pressure at 37 8C. The dried lipid film was rehydrated in 123 mM ammonium sulfate (pH 5.4), sonicated with a bath type sonicator, and then sequentially extruded five times through a 0.2-Am pore size polycarbonate filter (Nulcepore, USA). The resulting liposomes were passed through a Sephadex G50 gel-filtration column pre-equilibrated in PBS to exchange the external phase. DOX was remote-loaded via an ammonium sulfate gradient method and the free DOX was removed by gel-filtration. DOX concentrations in the liposomal samples were calculated from absorption at 485 nm following dissolution in 0.1% Triton X-100. DOX loading efficiency obtained by this procedure was consistently found to be greater than 95% at the drug-to-lipid ratio of 1:15 (wt/wt) used in this study. The mean liposomal diameter and particle size distribution were measured by photon correlation spectroscopy (PCS) on a Malvern Zetasizer 3000HS (Malvern instruments, UK). The mean particle diameter was ~ 120 nm for all liposome preparations (polydispersity, 0.30). RGDm was detected by measuring the absorbance at 211 nm. Less than 1% of the added amount of RGDm was detected in free form after conjugation. From this, it was calculated that ~ 1800 RGDm molecules were present on the surface of one liposome, based on the estimation that 80,000 phospholipid molecules form one liposome vesicle of 100 nm [24]. Liposome preparations were stored at 4 8C and used within 2 days of preparation, during which period no significant (b1%) leakage of DOX was found.

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centration of 0.5 mg DOX/ml diluted in the media were placed into a dialysis bag (MW cutoff 12,000– 14,000) sealed at both ends with clips. The liposomesloaded dialysis bag was then placed into a beaker containing 50 ml of the media, and incubated with stirring for 48 h at 37 8C. At various time points, aliquots were withdrawn from the beaker and replaced with equal volume of the media. The DOX concentrations were then measured spectrophotometrically. 2.5. Cellular uptake of DOX by flow cytometry analysis Fig. 1. High-performance liquid chromatography of reaction mixture with different molar ratio of DSPE-PEG-BTC to RGDm: (a) HEPES buffer, (b) RGDm in HEPES buffer, (c) DSPE-PEG-BTC and RGDm with a molar ratio of 1:1 in HEPES buffer and (d) DSPE-PEG-BTC and RGDm with a molar ratio of 2:1 in HEPES buffer.

Melanoma cells A375 or B16 grown as a monolayer were suspended by brief treatment with trypsin and then washed once with fresh culture medium. Aliquots of the melanoma cell suspension were incubated with free DOX, SSL-DOX or RGDm-SSLDOX (containing 20 Ag/ml DOX) diluted in culture medium at 37 8C for 1 h. In competition experiments, excess free RGDm (4 mM) was pre-incubated with the melanoma cells for 15 min, followed by continued co-incubation with liposomal DOX for another 1 h. The cells were then washed three times with cold PBS (pH 7.4) and examined by flow cytometry using a FACScan (Becton Dickinson, San Jose, CA, USA). Cell-associated DOX was excited with an argon laser

2.4. Drug release The in vitro DOX release from liposomes was measured using a dialysis method. DOX-loaded liposomes were passed over a Sephadex G-50 column immediately prior to use to remove any free DOX. The dialysis was conducted in cell culture medium containing 10% fetal bovine serum (FBS) or 50% human plasma in PBS, pH 7.4. Liposomes at a conSSL-DOX in human plasma

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Fig. 2. In vitro release of DOX from liposomes in cell culture media containing 10% FBS or 50% human plasma in PBS. Liposomes at a concentration of 0.5 mg DOX/ml diluted in the media were placed in a dialysis bag (MW cutoff 12000–14000) and incubated at 37 8C in 50 ml of the media. At various time points, aliquots were withdrawn, and the DOX was measured spectrophotometrically as described in Materials and methods (n = 3).

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(488 nm) and fluorescence was detected at 560 nm. Files were collected of 20,000 gated events and analyzed with the FACStation software program. 2.6. Intracellular uptake and distribution of DOX by confocal microscopy analysis A confocal fluorescent microscopy was used to compare the intracellular uptake of DOX (excitation/ emission: 480/540 nm) and investigate their cellular distribution. Melanoma cells A375 or B16 were grown on coverslips to 50% confluence and incubated with RGDm-SSL-DOX, SSL-DOX or free DOX (containing 6 Ag/ml DOX) diluted in culture medium at 37 8C for 1 h. The cells were then washed three times with PBS, fixed in 95% ethanol, and then treated with DAPI (excitation/emission: 345/661 nm) for 15 min for nuclei staining. Fluorescent images of cells were analyzed using a TCS SP2 confocal microscope (Leica, Heidelberg, Germany).

6 mice (6–8 weeks), and tumor was allowed to grow for 15 days, at which time the tumor volume reached ~ 1 cm3. The mice in groups of 4–6 animals were given free DOX, SSL-DOX and RGDm-SSL-DOX at a single injection of 5 mg DOX/kg via tail vein. At indicated times after injection, blood samples (0.5 ml) were collected from retro-orbital sinus. The mice were then sacrificed by cervical dislocation, and the liver, heart, lungs, kidneys, spleen and tumor were immediately excised. These tissues were then lightly blotted to remove any excess blood, stored at 20 8C until assay. The tissues as well as 0.2 ml serum

2.7. In vitro cytotoxicity assay A comparison of in vitro cytotoxicity of various DOX formulations was performed on A375 and B16 cells with an in vitro proliferation using MTT method [25]. Briefly, 5000 cells were plated in 96-well plates and incubated for 2 days to allow the cells to attach. Thus, the cells were exposed to serial concentrations of free DOX, SSL-DOX, RGDm-SSLDOX or free DOX plus RGDm-SSL at 37 8C for 8 h, followed by washing with PBS and replacing with fresh media. Next, the A375 and B16 cells were additionally incubated at 37 8C for 16 h and 40 h, respectively. At the end of incubation time, 20 Al of MTT solution was added and incubated for another 3 h at 37 8C, and then the media were replaced with 200 Al DMSO. The absorbance was read on a Sunrise Absorbance Microplate Reader (TECAN, Austria) at dual wavelengths of 570 nm and 650 nm. The data reported represent the means of triplicate measurement; the standard errors of the mean were less than 15%. 2.8. Tissue distribution of DOX in tumor-bearing mice Tumor-bearing mice were prepared by inoculating B16 cells (1  106) in the right flank of male C57BL/

Fig. 3. Flow cytometric measurement of doxorubicin uptake by melanoma A375 (A) and B16 (B) cells after incubated with SSLDOX, RGDm-SSL-DOX or free DOX (containing 20 Ag DOX/ml) at 37 8C for 1 h. Untreated cells served as negative control while free doxorubicin solution was used as positive control. DOX uptake by melanoma cells was determined based on DOX fluorescence by flow cytometry, as described in Materials and methods.

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were homogenized and extracted with chloroform/ methanol (4:1, v/v), the extracts were then subjected to HPLC assay according to the method of Shinozawa et al. [26]. The HPLC system consisted of an Agilent 3400 series high-pressure pump, an Agilent FLD fluores-

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cence detector (EX, 470 nm; EM, 585 nm) and a Kromasil-C18 column. The mobile phase was methanol/water/acetic acid (50:45:5, v/v) and a flow rate of 1.0 ml/min was used. Measurements were made using the ratio of the peak area of DOX to that of an internal standard (daunomycin).

Fig. 4. Confocal images of melanoma cells B16 (1, 2) and A375 (3, 4) incubated with SSL-DOX (A), RGDm-SSL-DOX (B) and free DOX (C) for 1 h at 37 8C. All the three DOX formulations have DOX concentrations of 6.0 Ag/ml. Cells were fixed in 95% ethanol and treated with DAPI (blue) for nuclei staining. Red—fluorescence of DOX. Blue—fluorescence of DAPI. Co-localization of DAPI and DOX are also represented (the merged fluorescence of blue and red).

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RGDm was consumed almost completely. The disappearance of N-benzotriazole identified by NMR (data not shown) indicates that there was almost no existence of DSPE-PEG-BTC or impurities (small molecules such as free RGDm or N-benzotriazole) in the product. The resulting product was then used for preparing liposomes without further purification. 3.2. In vitro release of DOX from liposomes Fig. 5. Confocal images of melanoma cells A375 treated with SSLDOX (A) and RGDm-SSL- DOX (B) for 1 h at 37 8C. Both DOX formulations have DOX concentrations of 6.0 Ag/ml. Cells were fixed in 95% ethanol and treated with DAPI for nuclei staining. Red—fluorescence of DOX. Blue—fluorescence of DAPI.

2.9. In vivo therapeutic efficacy in mice bearing B16 tumor The therapeutic efficacies were compared in C57BL/6 mice inoculated B16 cells (1  106) subcutaneously in the right flank of the mice. Treatment were given at 24 h post-inoculation and consisted of saline control, free DOX, SSL-DOX and RGDm-SSL-DOX. The mice were dosed at 5 mg DOX/kg according to the mean weight of each treatment group. Mice were then checked for survival every day, and tumor size was measured daily with a caliper in two dimensions. Individual tumor volumes (V) were calculated by the formula: V = [length (width)2] / 2. 2.10. Statistics Values are presented as mean F the standard deviation (S.D.). Statistical significance of differences was tested using the 2-tailed Student’s t-test, and differences in survival curves were evaluated by the log-rank test. A p-value below 0.05 was considered significant.

3. Results 3.1. The conjugation of RGDm to DSPE-PEG Chromatograms of the reaction mixture with different ratios of DSPE-PEG-BTC to RGDm were shown in Fig. 1. Under the conditions of reaction (4 h at 4 8C, pH 7.5, gentle stirring and 2:1 molar ratio),

The results of in vitro DOX release experiments are presented in Fig. 2. SSL-DOX and RGDm-SSL-DOX showed very similar DOX leakage in culture medium or in human plasma within 48 h of incubation. There were no pronounced differences in DOX release from them at every time point evaluated. These DOX encapsulated liposomes showed minimal DOX leakage in culture medium within 12 h of incubation, and more than 95% of the encapsulated DOX was still retained in liposomes after 12 h of incubation at 37 8C. When the Leakage experiments were conducted in human plasma, DOX leakage from liposomes showed a marked increase compared to that in culture medium. The leakage of DOX from SSL-DOX and RGDm-SSL-DOX was approached to 30% after 24 h of incubation in human plasma. 3.3. Cellular uptake of DOX by flow cytometry Flow cytometry was used to quantify the total DOX uptake by melanoma cells A375 and B16 for different Table 1 Cytotoxicity of various DOX formulations against melanoma cells A375 and B16 in vitro (IC50, AM) Treatment

IC50, B16

IC50, A375

RGDm-SSL-DOX SSL-DOX Free DOX RGDm-SSL + Free DOX IC50 ratio, SSL-DOX/RGDm-SSL-DOX

1.40 F 0.27 2.70 F 0.80 0.84 F 0.06 0.92 F 0.13 1.9

0.62 F 0.03 0.91 F 0.04 0.46 F 0.02 0.52 F 0.08 1.5

B16 or A375 cells (5000/well) were incubated with various test samples for 8 h at 37 8C, and then cells were washed and placed in wells containing fresh media. The incubation continued up to a total incubation time of 48 h for B16 and 24 h for A375, respectively. Then MTT solution was added to each well and the plates were further incubated for 3 h at 37 8C. The colored formazan product was dissolved using 200 Al of DMSO and the plates were read on a Sunrise Absorbance Microplate Reader.

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B16 (Fig. 4-1, -2) and A375 (Fig. 4-3, -4) cells visualized after 1 h of incubation with SSL-DOX (Fig. 4A), RGDm-SSL-DOX (Fig. 4B) or free DOX (Fig. 4C). After B16 and A375 cells were treated with various DOX formulations, the majority of visible fluorescence was mainly in the nuclear compartment. DOX fluorescence in cells treated with SSL-DOX was discernible, but less intense, while DOX fluorescence treated with RGDm-SSL-DOX was evident and intense, indicating an increased uptake of DOX by these cells. The most intense fluorescence was observed in the cells treated with free DOX. In addition, the melanoma cells treated with RGDm-SSL-DOX also showed speckled DOX fluorescence in cytoplasm, as

DOX formulations. As shown in Fig. 3, the cellular DOX level for RGDm-SSL-DOX in the melanoma cells was about twice that for SSL-DOX. Competition with excess free RGDm was able to inhibit the uptake of DOX for RGDm-SSL-DOX by the cells, indicating that the observed uptake could be partly mediated by the RGDm receptor. Free DOX formulations displayed the highest cellular uptake with 1 h of incubation. 3.4. Intracellular uptake and distribution of DOX by confocal microscopy analysis Cellular uptake and distribution of DOX were also analyzed by laser confocal scanning. Fig. 4 illustrates

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Fig. 6. Drug levels in various tissues after i.v. injection of free DOX ( ), SSL-DOX (D) and RGDm-SSL-DOX (n) into tumor-bearing mice at a dose of 5 mg DOX/kg. Results are given as mean F S.D. (n = 5–6).

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Table 2 AUC values in various tissues after i.v. injection of free DOX, SSL-DOX and RGDm-SSL-DOX into the tumor-bearing mice at a dose of 5 mg DOX/kg Formulation

Free DOX SSL-DOX RGDm-SSL-DOX

Tissue AUCa (hd Ag/g) Bloodb

Liver

Spleen

Kidney

Heart

Lung

Tumor

4.8 329.0*** 165.1*,***

111.9 161.3*** 182.5***

91.5 151.2*** 218.2*,***

94.0 112.2*** 121.8***

76.3 82.4 79.4

59.9 81.2** 70.4**

27.4 67.4*** 54.3***

a

AUC values are calculated for 1–48 h. AUC value in blood is given as h Ag/ml. * P b 0.01, vs. SSL-DOX. ** P b 0.05, vs. free DOX. *** P b 0.01, vs. free DOX. b

3.5. Cytotoxic activity The cytotoxicity of RGDm-SSL-DOX, SSL-DOX, and free DOX to melanoma cells was also compared. DOX concentrations leading to 50% cell-killing (IC50) were determined from concentration-dependent cell viability curves. As shown in Table 1, the IC50 of RGDm-SSL-DOX was 1.40 AM and 0.62 AM for B16 and A375 cells, respectively, which was 1.9- and 1.5fold lower than that of SSL-DOX, but higher than that of free DOX. The cytotoxicity of free DOX plus RGDm-SSL to B16 or A375 cells was comparable to that of free DOX alone, suggesting that RGDmSSL showed no cytotoxicity against cultured B16 or A375 cells. It was also observed that IC50 value for A375 cells was lower than that for B16 cells despite that A375 cells had a shorter incubation time, indicating that A375 cells were more sensitive to DOX than B16 cells. 3.6. Tissue distribution of DOX in mice bearing melanoma B16 tumors The DOX levels in blood, liver, spleen, kidneys, heart and lungs after i.v. injection of SSL-DOX,

RGDm-SSL-DOX or free DOX at a dose of 5 mg DOX/kg are presented in Fig. 6, and the area under the concentration–time curves (AUC) in these tissues is listed in Table 2. As anticipated, RGDm-SSLDOX and SSL-DOX displayed a much greater systemic circulation time than free DOX, which showed rapid clearance kinetics, and the calculated AUC of SSL-DOX and RGDm-SSL-DOX in blood was 68.5and 34.4-fold higher than that of free DOX, respectively. In addition, the liposomal DOX also produced significantly increased AUC in liver ( P b 0.01), spleen ( P b 0.01), kidneys ( P b 0.01) and lungs ( P b 0.05). However, similar AUC was observed in heart for the three DOX formulations. It is noteworthy that the mice treated with RGDm-SSL-DOX demonstrated remarkably lower DOX levels ( P b 0.05) and AUC ( P b 0.01) in blood, but showed significantly higher levels and AUC in spleen ( P b 0.01) compared to those treated with SSL-DOX. 3

ug DOX/g Tumor

shown in Fig. 5. It should be noted that the contrast of the confocal images has been adjusted to facilitate comparison of the DOX fluorescence in melanoma cells treated with different DOX formulations. Intensity quantification of unprocessed, raw image data confirmed a statistically significant increase in DOX uptake of approximately twofold due to the effect of RGDm ligand.

2.5 Tumor

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Fig. 7. Drug levels in tumor after i.v. injection of free DOX ( ), SSL-DOX (D) and RGDm-SSL-DOX (n) into tumor-bearing mice at a dose of 5 mg DOX/kg. Results are given as mean F S.D. S.D. (n = 5–6).

X.-B. Xiong et al. / Journal of Controlled Release 107 (2005) 262–275 Saline Free DOX SSL-DOX RGDm-SSL-DOX

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Table 3 Therapeutic efficacy of various formulations of DOX in C57BL/6 mice inoculated with B16 cells

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Days following inoculation Fig. 8. Tumor growth inhibition by RGDm-SSL-DOX, SSL-DOX or free DOX. C57BL/6 mice with B16 tumor xenografts were given a single i.v. injection of 5 mg/kg DOX at 24 h post-inoculation. Tumor size was measured for each animal daily. Results are given as means F S.D. (n = 6–11).

Fig. 7 shows the DOX levels in B16 tumor after administration of various DOX formulations at a dose of 5 mg DOX/kg. The tumor concentration for free DOX at 1 h after injection was 0.5 Ag/g and did not increase thereafter. RGDm-SSL-DOX and SSL-DOX produced increased accumulation of DOX in the tumors compared to free DOX, and resulted in a tumor concentration of 1.5 Ag/g at 24 h after injection. The AUC value of tumor (Table 2) for RGDm-SSL-DOX or SSLDOX was ~ 2-fold higher than that of free DOX. RGDm-SSL-DOX did not show significant improvement in tumor accumulation compared to SSL-DOX. 3.7. Evaluation of antitumor activity Various treatments (free DOX, SSL-DOX, RGDmSSL-DOX, at a dose of 5 mg DOX /kg and saline)

Formulation

MST F S.D. (days) Median (days) ILS (%)

Saline control Free DOX SSL-DOX RGDm-SSL-DOX

30.9 F 3.5 33.9 F 4.5 34.6 F 4.3 38.7 F 4.1

31 32 34 39

– 9.7 12.0 25.2

C57BL/6 mice received B16 cells (1  106 ) subcutaneously 24 h before a single treatment with 5 mg/kg DOX. MST denotes mean survival time and ILS denotes increased life span (n = 11).

were intravenously injected via tail vein at 24 h postinoculation. The tumor growth rate in terms of mean tumor size (cm3) is presented in Fig. 8. All the DOX formulations were effective in preventing tumor growth compared to saline. Treatment with RGDmSSL-DOX displayed stronger tumor inhibition than treatment with SSL-DOX. However, no significant difference was observed between treatments with SSL-DOX and free DOX. Survival of mice carrying B16 tumors in response to the above treatments was also determined. The results are represented in a Kaplan–Meier plot as indicated in Fig. 9. The mean survival time (MST) and the percent of increased life spans (ILS) for each treatment group are presented in Table 3. The three DOX formulations were significantly more effective in prolonging mouse survival ( P b 0.05) than saline. The mice treated with RGDm-SSL-DOX showed significantly increased MST compared to those that received SSL-DOX or free DOX ( P b 0.05). However, no significant difference was observed between SSLDOX and free DOX.

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40 20 0

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Days following inoculation Fig. 9. Effects of various DOX formulations on the survival of C57BL/6 mice inoculated with B16 cells. The mice were treated with saline, free DOX, SSL-DOX or RGDm-SSL-DOX via a single i.v. injection at a dose of 5 mg/kg DOX (n = 11).

DOX is one of the most widely used broad spectrum anticancer agents and it has been in clinical use against a wide range of human cancers for decades. Nevertheless, a number of issues critical to the therapeutic success and safety of the drug, such as cardiotoxicity, drug resistance, and specificity remain to be improved. In addition, DOX exerts antitumor activity after intercalating with the double-stranded helix DNA in nuclei [27]. Thus, intracellular delivery of DOX into tumor cells will be essential to its antitumor activity.

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It is well established that RGD sequence is present in many extracellular matrix components such as fibronectin and vitronectin and can bind to integrins. Integrins are used for natural targets by a variety of bacteria and viruses to integrate them with their host cells for receptor-mediated cell entry [28,29]. It is suggested that RGD-modified liposomes increased the gene transfection depending on their increased cellular uptake [30]. In addition, RGDm as a model ligand for integrin has several potential advantages, including (a) low immunogenicity due to its low molecular weight (Mw 287), (b) chemical and functional stability, and (c) ease of synthesis and being economically acceptable for the further research and development. As expected, the RGDm-modified liposomes were able to enhance the uptake of entrapped DOX into the melanoma cells in vitro compared to non-modified ones (Figs. 3 and 4). Free DOX enters the cells by diffusion, leading to significantly higher drug levels than found with the liposomal DOX, so one possibility is that the higher levels of DOX in the tumor cells after incubated with RGDm-SSL-DOX were caused by the augmented leakage of DOX due to the conjugation of RGDm to SSL. However, the DOX release in cell culture medium for RGDm-SSL-DOX was very similar to that for SSL-DOX and it could be concluded that conjugation of RGDm to SSL did not increase the DOX leakage. The weak DOX fluorescence in the cells for SSL-DOX (Fig. 4A) also indicates that DOX uptake by diffusion for RGDmSSL-DOX was very limited. Drugs entrapped in liposomes could enter tumor cells by diffusion, membrane fusion or endocytosis, depending on the liposomes and cell characteristics [31]. Liposomes with the composition in this study hardly fuse with the cell membranes, so DOX uptake by membrane fusion would be limited to a large extent. In addition, it is suggested that liposomes without an internalizing ligand have rare chance to be endocytosed by tumor cells [11,32,33]. Our observation that melanoma cells treated with SSL-DOX did not show any DOX fluorescence in cytoplasm (Fig. 5A) also confirmed that endocytosis was not involved in the DOX uptake. Therefore, the DOX uptake for SSL-DOX was mainly by the mechanism of diffusion. In fact, SSL-DOX displayed some DOX leakage (~ 2%) when incubated with cell culture medium for

1 h, which provided the source of free DOX for intracellular-directed diffusion. Because the lipid composition and the release behavior for RGDmSSL-DOX were similar to that for SSL-DOX, the mechanism of diffusion should be equally involved in the DOX uptake for RGDm-SSL-DOX. Further, besides displaying intense DOX fluorescence in nuclei, melanoma cells treated with RGDm-SSL-DOX also showed speckled DOX fluorescence in cytoplasm as evidenced by Fig. 5B, indicating that endocytosis was involved in the DOX uptake and mainly responsible for the improved intracellular uptake of DOX for RGDm-SSL-DOX compared to SSL-DOX. The nuclei treated with RGDm-SSL-DOX showed stronger DOX fluorescence than that with SSL-DOX within 1 h of incubation, indicating that process of endocytosis is rapid, and DOX was quickly released from the endocytotic vesicles and concentrated in the nuclei. It is noteworthy that since the DOX fluorescence inside these liposomes was greatly quenched as suggested by Lee and Low [34], the cytoplasm may contain significantly more RGDm-SSL-DOX than indicated by their relative fluorescence intensity. Several studies also suggested that the integrin-mediated endocytosis was responsible for the enhanced intracellular delivery [17,30]. The combined effect of passive accumulating and enhanced intracellular delivery should be responsible for the improved therapeutic efficacy of RGDm-SSLDOX over SSL-DOX or free DOX. RGDm-SSLDOX and SSL-DOX can passively accumulate into the tumor tissues by the effect of EPR. Park et al. demonstrated that following intravenous administration, the DOX-entrapped liposomes predominantly accumulated in the interstitial fluid of extracellular and perivascular space of the tumor [35]. Liposomes diffusion into the interstitial fluid of the tumor was heavily dependent on the liposomal AUC in the blood stream. Therefore, higher concentrations of liposomal DOX should be expected in the interstitial fluid for SSL-DOX than that for RGDm-SSL-DOX. However, as mentioned above, SSL-DOX cannot directly enter the tumor cells, so the extracellular release in the interstitial fluid becomes the determinant step for the intracellular delivery of DOX by tumor cells. The released DOX from SSL-DOX will follow the same pathway as for free drugs in terms of cellular drug uptake, metabolism and efflux. In the dynamic in vivo

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environment, the accumulated SSL-DOX or the released DOX in the interstitial fluid, if not be arrested timely by the tumor cells, will redistribute away from the tumor cells. As a result, DOX that actually delivered into the tumor cells by SSL-DOX would be not different from that by free DOX, leading to a similar therapeutic efficiency for them. In contrast, RGDmSSL-DOX can efficiently deliver the drugs into the tumor cells by the receptor-mediated endocytosis and lead to a high concentration of DOX in the tumor cells, despite that the liposomal levels in the interstitial fluid for RGDm-SSL-DOX could be much lower than that for SSL-DOX. The observation that RGDmSSL-DOX and SSL-DOX showed an apparent similar tumor accumulation also supported this inference. Similar observations have been found in other studies where various ligands are employed [36–39], so whether it means a similar targeting mechanism was involved needs to be further investigated. The increased DOX uptake by spleen for RGDmSSL-DOX can explain the rapid clearance of DOX from the circulation as compared to SSL-DOX. It was suggested that mononuclear cells abundant in the spleen seemed to be responsible for the increased splenic uptake of RGD-modified liposomes, since these phagocytic cells showed integrin avh3 expression [40]. Our observations of tissue distribution were in good agreement with those of Schiffelers and coworkers [36] and previous studies suggested that RGD-peptide could bind to both tumor and tumorendothelial cells [41], so vasculature-targeting associated with integrin avh3 may partly be involved in the improved therapeutic efficacy for RGDm-SSL-DOX. The adverse effect is a major concern for the RGDm-SSL as a potential drug delivery system, because integrins that recognize RGDm could also be found in some normal cells. Thus, the intracellular DOX uptake by normal cells and the cytotoxicity on these cells might be enhanced by RGDm-SSL. However, no significant signs of toxicity were observed with the mice treated with different DOX formulations when the weight loss was used as the indicator for toxicity measurement (data not shown). Further, the over-expression of integrin avh3 or avh5 on tumor endothelium or tumor cells could strength the targeting ability of the RGDm-modified SSL. Meanwhile, we found that RGDm-SSL-DOX did not increase DOX accumulation in liver, lungs or kidney and,

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