Peptide PHSCNK as an integrin α5β1 antagonist targets stealth liposomes to integrin-overexpressing melanoma

BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 1152 – 1161

Research Article

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Peptide PHSCNK as an integrin α5β1 antagonist targets stealth liposomes to integrin-overexpressing melanoma Wenbing Dai, PhD, Tingyuan Yang, PhD, Yiguang Wang, PhD, Xueqing Wang, PhD, Jiancheng Wang, PhD, Xuan Zhang, PhD, Qiang Zhang, PhD⁎ State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, People's Republic of China Received 12 May 2011; accepted 16 January 2012

Abstract As an integrin α5β1 antagonist, N-acetyl-proline-histidine-serine-cysteine-asparagine-amide (Ac-PHSCN-NH2) is currently in phase II trials for various cancer therapies. In this study Ac-PHSCNK-NH2 (PHSCNK) was used as a novel homing peptide to prepare ligand-targeted liposomes loaded with doxorubicin (PHSCNK-PL-DOX), with the hypothesis that the therapy target of integrin α5β1 may also serve as a delivery target. The stealth liposomes loaded with doxorubicin (PL-DOX) were used as the control. PHSCNK-PL-DOX demonstrated an enhanced intracellular uptake and a greater cytotoxicity against melanoma B16F10 cells in comparison with PL-DOX. The novel targeted formulation displayed stronger tumor inhibition and prolonged survival time in comparison with controls in C57BL/6 mice bearing B16F10 tumors, and it exhibited less heart toxicity in hematoxylin-eosin (H&E) staining of tissues. Taking the pharmacokinetics and biodistribution results into account, the authors conclude that α5β1 integrin-mediated liposomes might be used as a potential delivery system for targeted tumor therapy. From the Clinical Editor: Lactosyl-norcantharidin TMC nanoparticles were found superior in comparison with Lac-NCTD and Lac-NCTD chitosan nanoparticles from the standpoint of antitumor activity on murine hepatocarcinoma 22 subcutaneous model. © 2012 Elsevier Inc. All rights reserved. Key words: Targeted delivery system; Liposomes; Doxorubicin; PHSCNK; Integrins

Liposomes have been widely studied as delivery system for anticancer agents. Liposomes with diameters of about 100 nm offer advantages in terms of better storage stability in vitro and favorable pharmacokinetic behavior in vivo. 1 However, liposomes usually eliminate rapidly from circulation due to the clearance effect of the reticuloendothelial system (RES). PEGylated liposomes (PL), which can prolong the circulation time of drugs loaded, have shown preferential drug accumulation in tumors by means of the so-called enhanced permeability and retention (EPR) effect. 2,3 However, the increased drug accumulation in tumor tissue is a prerequisite but seems not enough to guarantee a therapeutic improvement. In view of most chemotherapeutic agents with intracellular targets of action, enhanced intracellular drug delivery might be the more determinant step for an antitumor effect. 4

No conflict of interest was reported by the authors of this paper. This work was supported by the National Basic Research Program of China (No.2009CB930300) and National Key program of New Drug innovation (No.2009zx09310-001). ⁎Corresponding author: State Key Laboratory of Natural ad Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, P. R. China. E-mail address: [email protected] (Q. Zhang).

Many effects have been achieved by coupling various targeting moieties (ligands) to the liposomes' surface to increase specificity of interaction of liposomal drugs with targeted cells and therefore enhance the amount of drugs delivered into cells after tumoral accumulation. Monoclonal antibodies or their fragments, peptides, lipoproteins, glycoproteins, growth factors, folate, and so on, which can selectively bind to target antigens or receptors overexpressed on the target cells, have been reported as ligands. 5 Peptide sequences including NGR, 6 APRPG, 7 RGD, 8 and the like have been utilized for tumor and tumor vasculature-targeting carrier. For instance, RGD-modified liposomes, 9-11 nanoparticles (NPs), 12 dendrimers, 13 and micelles, 14 as well as NGR-modified micelles, 15 have been demonstrated to increase the intracellular delivery of chemotherapeutic agents or genes in vitro and in vivo. Integrins are membrane receptors that mediate cell-cell and cell-matrix interactions and play an important role in cell signaling, cell proliferation, apoptosis, invasion, metastasis, gene expression, and tumor angiogenesis. 16-19 Integrin αvβ3 has been relatively well studied. In comparison with αvβ3, α5β1 plays a more significant role in tumor growth and expresses in some tumor cells and angiogenic endothelial cells with even higher selectivity. 20 Tumor cells such as murine melanoma cells B16F10 and human breast cancer cell MDA-MB-231 were

1549-9634/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2012.01.003 Please cite this article as: W., Dai, et al, Peptide PHSCNK as an integrin α5β1 antagonist targets stealth liposomes to integrin-overexpressing melanoma. Nanomedicine: NBM 2012;8:1152-1161, doi:10.1016/j.nano.2012.01.003

W. Dai et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 1152–1161

found to be highly expressive of α5β1. Therefore, α5β1 might represent a better tumoral target for drug delivery. ATN-161 (N-acetyl-proline-histidine-serine-cysteine-asparagine-amide, Ac-PHSCN-NH2), as an integrin α5β1 antagonist, is currently in phase II trials for various cancer therapies. 21,22 ATN-161 was found to bind with β subunits of integrin through its cysteine thiol, 23 or interact with tumor cells and tumor endothelial cells that highly express integrin. 24 Hence, the PHSCN sequence might be an ideal targeting moiety for liposomal drug delivery to integrin-overexpressed tumor cells and endothelial cells. In other words, the therapy target of integrin α5β1 may also serve as a delivery target. In this study, N-acetyl-proline-histidine-serine-cysteineasparagine-lysine(amide)-COOH (PHSCNK)-modified and DOX-loaded PEGylated liposomes (PHSCNK-PL-DOX) were prepared and investigated in comparison with DOX-loaded PEGylated liposomes (PL-DOX), with the hypothesis that enhanced uptake of DOX in tumor cells may significantly improve its anticancer effect. The integrin-overexpressing melanoma B16F10 cells were used as the models in vitro and in vivo to provide evidence that PHSCNK-modified PEGylated liposomes will increase intracellular DOX delivery and facilitate tumor-growth inhibition, while decreasing drug toxicity.

Methods Materials Hydrogenated soybean phosphatidylcholine (HSPC) and cholesterol (Chol) were obtained from NOF Corporation (Tokyo, Japan), and the 1, 2-bis (diphenylphosphino) ethane (DPPE) was from Lipoid (Ludwigshafen, Germany). Doxorubicin hydrochloride (DOX) was a gift from Hisun Pharmaceutical Co. (Taizhou, Zhejiang, China). N-acetyl-proline-histidine-serine-cysteine-asparagine-lysine (amide)-COOH (Ac-PHSCNK-NH2, PHSCNK) was supplied by GL Biochem Ltd. (Shanghai, China), and 5, 5′-dithio-bis(2nitrobenzoic acid) (DTNB), Tris base and sulforhodamine B (SRB) were the products of Sigma (St. Louis, Missouri). Polyethylene glycol (PEG) dialdehyde (OHC-PEG-CHO) was provided by State Key Laboratory of Natural and Biomimetic Drugs (Beijing, China). Hoechst 33258 was purchased from Molecular Probes Inc. (Eugene, Oregon). Cell culture media RMPI-1640, penicillin-streptomycin, fetal bovine serum (FBS), Lglutamine and N-(2-hydroxyethyl) piperazine-N′-ethanesulfonic acid (HEPES) buffer were from GIBCO (Langley, Oklahoma). All other chemicals and reagents used were of analytical grade. Murine melanoma cells B16F10 was obstained from the Basic Medical Cell Center, Chinese Academy of Medical Science (Beijing, China) and were maintained in RPMI 1640 medium supplemented with 100 g/mL streptomycin,100 U/mL penicillin, and 10% FBS in a humidified atmosphere containing 5% CO2 at 37°C. Male C57BL/6 mice (6 to 8 weeks old, body weight of 18 to 22 g) were provided by Institute of Materia Medica, Chinese Academy of Medical Sciences (Beijing, China). All care and handling of animals were performed with the approval of Institutional Authority for Laboratory Animal Care.

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Preparation and characterization of liposomes Liposomes were formulated following previously described methods. 25 HSPC/DPPE /Chol/OHC-PEG-CHO (15:5:10:10, mole ratio) were dissolved in chloroform/methanol mixture (10:1, v/v) and dried under reduced pressure at 37°C. The dried lipid film was hydrated in ammonium sulfate solution (123 mM, pH5.4) and then sonicated until transparent blue solution obtained. During this process, a Schiff base product (OHCPEG-DPPE) was formed by means of the reactions between the aldehyde group of excessive OHC-PEG-CHO and the amino group of DPPE. 26 Ammonium sulfate gradient method was used to load DOX into liposomes at a drug-to-lipid ratio of 1:10 (w/w). Free DOX was separated from liposomal DOX by Sephadex G50 gel chromatography. Encapsulation efficiency was calculated from absorption at 485 nm by UV/Visible spectroscopy (TU-1900, Beijing Purkinje General Instrument Co., Ltd., Beijing, China). For coupling of PHSCNK to the liposomal surfaces, PHSCNK solution (PHSCNK: DPPE = 4:10, molar ratio) was added to PL-DOX under gentle stirring at 25°C for 2 hours. In this process, the aldehyde group on the surface of liposomes (OHC-PEG-DPPE) could conjugate to the amino groups of PHSCNK and formed the targeted liposomes (PHSCNK-PL-DOX). 26 The peptide coupling efficiency of PHSCNK-PL-DOX was determined by Ellman test as described above. 27 Liposomes were stored at 4°C and used within 5 days after preparation. The particle size and distribution of the liposomes were evaluated using dynamic light scattering (DLS) with a Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Malvern, United Kingdom). For each measurement, about 1 mL sample was loaded in a disposable sizing cuvette. The liposome surface zeta potential was measured using the same instrument used for the size analysis. For each measurement, the sample was diluted with 0.1 mM phosphate buffer saline (PBS, pH 7.4) at the ratio of 1:10 and performed at 25°C. Each sample was measured at least three times. To visualize the liposomes morphology, liposomes were diluted to 0.5 mg/mL DOX and analyzed by transmission electron microscopy (TEM, Tecnai G2 20ST, FEI, Hillsboro, Oregon) operating at an acceleration voltage of 80 kV after negative staining with uranyl acetate solution (1%, w/v) on a 200-mesh, carbon-coated, copper grid. Drug release in vitro The in vitro release profile of DOX from liposomes was determined at cell culture medium DMEM containing 10% FBS by a dialysis membrane method. Dialysis bags (MW cutoff 12,000∼14,000) were filled with 1 mL of purified liposomes and 1 mL of release medium sealed at both ends with chips and immersed in 50 mL of medium, and then incubated with gentle shaking for 24 hours at 37°C. Next, 0.5 mL aliquots of samples were withdrawn from the medium and replaced with the same volume of fresh release medium at predetermined intervals. The DOX concentrations were quantified by reversed-phase HPLC using the Shimadzu HPLC system of UV-visible detector. The detection wavelength was 233 nm. The separation was conducted on a C18 column (Phenomenex, 4.6 × 250 mm,

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Figure 1. Characterization of liposomes prepared. (A) Illustration of PHSCNK-modified and DOX-loaded PEGylated liposomes. (B) Size distribution of DOX-loaded PEGylated liposomes (PL-DOX) and PHSCNK-modified DOX-loaded PEGylated liposomes (PHSCNK-PL-DOX) by DLS analysis. (C) TEM photomicrograph of PHSCNK-PL-DOX. (D) Cumulative release profiles of DOX from PL-DOX and PHSCNK-PL-DOX in DMEM containing 10% FBS (n = 3).

5 μm) with a flow rate of 1 mL/minute, mobile phase consisting of 55% methanol, 40% acetonitrile, and 5% acetic acid. Confocal laser microscopy analysis For visualizing the DOX internalization and distribution in cells, B16F10 cells were cultured on sterile 35-mm glass-bottom dishes and grew overnight. Then cells were incubated with liposomal DOX or free DOX at the same concentration of 20 μg DOX/mL for 2 hours and washed three times with cold PBS (pH 7.4), fixed with formaldehyde and stained with Hoechst 33258 for cell nuclei. The samples were analyzed by a Leica SP2 confocal microscope (Heidelberg, Germany). The excitation/emission wavelengths of Hoechst 33258 and DOX were 352/460 nm and 480/540 nm, respectively. Flow cytometry analysis Flow cytometry was used to determine total cell-associated DOX, including that bound to the cell surface and that delivered into cells. B16F10 cells were seeded in a 6-well plate and attached for 24 hours. Then the completed medium was replaced with DOX-loaded liposomes diluted with basel culture medium at a

final concentration of 10 μg DOX/mL and incubated for different time at 37°C. The cells were then washed three times with cold PBS (pH 7.4) and trypsinized; then cell-associated DOX was evaluated using flow cytometry (FACS Vantage TM, BD Bioscience, Franklin Lakes, New Jersey, USA) at an excitation wavelength of 488 nm and emission wavelength of 560 nm. In vitro cytotoxicity test In vitro cytotoxicity of DOX-loaded liposomes was evaluated by SRB assay. B16F10 cells suspension were transferred into 96-well microplates at a density of 2500 cells/well and allowed to attach to the microplates overnight. The attached cells were exposed to various liposomal DOX at a series of concentrations for 48 hours. Then, the cells were fixed by adding cold 10% trichloroacetic acid (TCA). The fixed cells were washed five times with water and dried. Fixed cells were stained with 0.4% SRB solution at room temperature (25°±5°C) for 10 minutes and then quickly rinsed four times with 1% acetic acid to remove unbound SRB dye and dried. Aliquots of 100 μL Tris base (pH10.5) was added later to dissolve the protein-bound dye. Finally, the ODs (optical densities) were measured on a

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Figure 2. (A) Celluar uptake kinetics of PL-DOX or PHSCNK-PL-DOX by B16F10 cells. Cells' monolayer was incubated with DOX liposomes at a final concentration of 10 μg DOX/mL diluted in culture medium at 37°C for various times. Cells were washed, trypsinized, and evaluated using flow cytometry. (B) Cell inhibition ratio of PL-DOX, PHSCNK-PL-DOX and free DOX against B16F10 cells at various concentrations. Data are illustrated as the mean ± SD. (n = 3).

Figure 3. Confocal microscopy images of B16F10 cells incubated with PL-DOX (A), PHSCNK-PL-DOX (B) and free DOX (C) at a DOX concentration of 40 μg/mL at 37°C for 2 h. Blue, fluorescence of Hoechst 33258. Red, fluorescence of DOX. Co-localization (lower right) of Hoechst 33258 and DOX are also presented (the overlapped fluorescence of blue and red).

microplate reader at 540 nm. SPSS12.0 was used to calculate 50% cell-killing concentration (IC50). Results are shown from three independent experiments (n = 3). Pharmacokinetics and tissue distribution of DOX in tumor-bearing mice of DOX The B16F10 melanoma (1×10 6 cells) were carefully inoculated into the right flank of male C57BL/6 mice. Tumors were left to grow to an estimated size of 1 cm 3, and then the animals were randomly divided into three groups (3 to 5 mice per group). Free or liposomal DOX was injected intravenously via the tail vein at a dose of 6 mg DOX/kg bodyweight. At a predetermined time, whole blood was collected from the retroorbital sinus after ether anesthesia, and plasma were separated by centrifugation at 10,000 g for 20 minutes. Mice were immediately sacrificed by cervical dislocation, and their heart, liver, spleen, lung, kidney, and tumor were excised immediately. The organs and tumors were carefully washed with distilled water, weighed, and homogenized with distilled water. Homog-

enates (150 μL) or plasma (50 μL) was extracted at -20°C for 24 hours by adding first 200 μL of 10% Triton-X100, vortexing for 1 minute, and then adding acidified isopropanol (0.75M HCl) to make a final volume of 1 mL. The samples were vortexed for 3 minutes and centrifuged at 10,000 g for 20 minutes. The supernatants were stored at -20°C until measured. The DOX concentration in the homogenized tissue was measured by fluorescence spectrophotometer. The excitation and emission wavelengths were 490 nm and 570 nm, respectively. To correct for background fluorescence of tissue and plasma, standards curves were obtained with standard DOX solution of blank tissue or plasma of mice that had not received drug. 28 The plasma or tissue concentration data were fitted by NonCompartment Analysis (NCA) using Win NonLin, to calculate the area under the curve (AUC) and plasma clearance (Cl). In vivo antitumor activity Mice were implanted with B16F10 melanoma as described above. After 24 hours of tumor inoculation (day 1), normal saline

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-3.349 ± 0.448 mV (n = 3) for these two systems, respectively. The encapsulation efficiency of DOX in both liposome preparations was greater than 95%. The coupling efficiency of PHSCNK to PL-DOX was evaluated to be 75.6% by determining the SH of the peptide in the separated liposomes. A nearly spherical structure of PHSCNK-PL-DOX could be observed, and the thickness of lipid bilayer could also be determined (15–20 nm) in TEM (Figure 1, C). The diameters determined by TEM were similar with that by DLS. Figure 1, D shows the cumulative percent of DOX released from the two kinds of liposomes. Less than 6% of drug released within 24 hours for both groups and two liposome systems showed similar release kinetics in the medium tested. Flow cytometry analysis Figure 4. Plasma DOX concentration-time profiles of free DOX ( × ), PL-DOX (◇) and PHSCNK-PL-DOX (□), following IV administration at a dose of 6 mg/kg in C57BL/6 mice (n = 3∼5).

(NS), free DOX or liposomal DOX was injected intravenously via tail vein at a dose of 1.25 mg DOX/kg bodyweight for the first time. The drugs were then given in five other doses (on day 4, 6, 8, 11, and 13). Tumor size was measured every day with a caliper in two dimensions. The tumor volume was calculated using the formula, Tumor volume(mm 3) =( length × width 2)/2. Animal survival was monitored daily for survival analysis. C57BL/6 mice were sacrificed by cervical dislocation and the tumor was removed and weighed on day 15 for tumor growthinhibition analysis.

The changes of DOX fluorescence after incubation of B16F10 cells with liposomal DOX at various times are shown in Figure 2, A. It was clear that the fluorescence intensity of PHSCNK-PL-DOX was higher than that of PL-DOX at all test times, and concretely it was about 1.73-fold, 1.4-fold, 1.4-fold, and 1.24-fold higher than that of PL-DOX in 0.5 hours, 1 hour, 2 hours, and 3 hours, respectively. During the test period, the uptake of each liposome system seems to be a linear kinetics as the function of time without saturation. Confocal microscopy analysis

The bodyweight loss of the mice was recorded during the antitumor activity in vivo. For pathological analysis, hearts were removed when the treatments were finished as described in experiments of antitumor activity and fixed in 10% formalin and embedded in paraffin. The paraffin-embedded tissues were cut into 5-μm-thick sections, stained with H&E, and examined microscopically.

As shown in Figure 2, after incubation of B16F10 cells with different DOX formulations at 37°C for 2 hours, the strongest fluorescence was observed in the cells treated with free DOX (Figure 3, C). It was found that the fluorescence of DOX in PHSCNK-PL-DOX group was significantly stronger than that in PL-DOX group (Figure 3, B and Figure 3, A). In addition, there was some difference in the fluorescence distribution of two kinds of liposomes. The fluorescence of intracellular DOX almost all distributed in the nuclear compartment after incubation with PL-DOX, which was similar to that of free DOX, whereas red fluorescence was also observed in the cytoplasm of cells treated with PHSCNK-PL-DOX (as shown by green arrow), in spite of the majority of fluorescence in the nucleus.

Statistical analysis

In vitro cytotoxicity test

The statistical analysis of the samples was performed by a Student's t-test or ANOVA test. Differences were considered significant when the P value was less than 0.05, though P value less than 0.01 was considered highly significant. Survival data were analyzed using the method of Kaplan and Meier.

The IC50 of DOX was determined from dose-dependent cellinhibition curves, as shown in Figure 2, B, and the value for PL-DOX, PHSCNK-PL-DOX, and free DOX were 2.21 μM, 1.76 μM and 0.57 μM against B16F10 cells, respectively. In comparison with PL-DOX, the enhanced cytotoxicity of PHSCNK-PL-DOX indicated that modification with PHSCNK was effective in promoting the internalization of liposomal DOX to the tumor cells (P b 0.01), which was consistent with the increased uptake of PHSCNK-PL-DOX by B16F10 cells as shown in Figure 3 and Figure 2, A.

Toxicity study

Results Preparation and characterization of liposomes PHSCNK was coupled to PL-DOX via imine bond between the aldehyde of PEG terminus on the surface of PL-DOX and amino groups of PHSCNK. The mean diameters for PL-DOX and PHSCNK-PL-DOX were 95.4 ± 2.5 nm and 96.0 ± 3.1 nm (Figure 1, B), respectively, with a polydispersity index (PDI) less than 0.2, and the zeta potential was -2.119 ± 0.971 mV and

The pharmacokinetics and tissue distribution The profiles of the DOX concentration in the plasma after intravenous injection of the free DOX, PL-DOX, or PHSCNKPL-DOX at a dose of 6 mg/kg are illustrated in Figure 4. It was seen that free DOX was eliminated very rapidly from circulation,

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Figure 5. Tissue distribution of DOX after IV administration of free DOX ( × ), PL-DOX (◇) or PHSCNK-PL-DOX (□) at a dose of 6 mg/kg in C57BL/6 mice (n = 3∼5).

and it was detectable only at 0.5 hours. In contrast, both of the liposomal DOX groups showed a much longer circulation time than the free drug. Notably, PHSCNK-PL-DOX (Cl, 25.0 mL/h/ kg) had a quicker clearance in comparison with that of PL-DOX (Cl, 13.6 mL/h/kg). The AUC of PHSCNK-PL-DOX and PLDOX were 233.0 and 439.4 h⁎μg/mL, respectively. The DOX distribution in the tumor, heart, liver, spleen, kidney, and lung after i.v.IV injection of different formulations are depicted in Figure 5, and the AUC values are listed in Table 1. As anticipated, the concentrations of liposomal DOX in the tumor, as well as in the liver and spleen, were significantly higher than those of free DOX (P b 0.01). However, the DOX distribution in the heart in both liposomal DOX groups was

significantly lower than that in the free DOX group. Although there were some differences between the two liposome formulations in some tissues, there was no significance in terms of AUC values between the two groups. Antitumor efficacy Tumor growth-inhibition curves based on mean tumor volume are presented in Figure 6, A. The tumor growthinhibition effects of DOX formulations were significantly superior to those of the NS group (P b 0.01). PHSCNK-PLDOX displayed stronger tumor inhibition than PL-DOX (P b 0.05) did, whereas the latter was stronger than that of free DOX

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Table 1 AUC0-48h values in plasma and various tissues after IV injection of free DOX, PL-DOX, or PHSCNK-PL-DOX into tumor-bearing mice at a dose of 6 mg DOX/ kg (n = 5) Formulation

Free DOX PL-DOX PHSCNK-PL-DOX

AUC0-48h (h ⋅ μg/g) Plasma

Heart

Liver

Spleen

Lung

Kidney

Tumor

/ 439.4 233.0 ‡

267.1 227 189.8*

385.3 775.9 † 965.6 †

412.8 1568.3 † 1889.6 †

270.6 290.7 274.5

760.6 720.5 633.9

63.4 91.4 † 79.3 †

/ : data not available. * P b 0.05. † P b 0.01 vs. free-DOX group. ‡ P b 0.05 vs. PL-DOX group.

Figure 6. Antitumor activity and heart toxicity of different treatment groups on B16F10-bearing C57BL/6 mice. (A) Tumor growth curves. Results are means ± SD (n = 6). (B) Survival of B16F10-bearing C57BL/6 mice. (C) Tumors excised at the end of the tests. (D) H&E staining of heart tissue after different treatments. Mice were given six IV injections at a dose of 1.25 mg DOX/kg, at 1 day, 4 days, 6 days, 8 days, 11 days, and 13 days after tumor inoculation.

(P b 0.01). The average weight of excised tumors in various treated groups (data not shown) at the end of the test also confirmed the highest efficiency of PHSCNK-PL-DOX. The survival of mice bearing B16F10 tumors in response to the different treatments is indicated in a Kaplan-Meier plot in Figure 6, B. The mean survival time (MST) and the percent of increased life span (ILS) for each group are summarized in Table 2. The three DOX formulations were significantly more effective in prolonging mouse survival (P b 0.01) than saline. PHSCNK-PL-DOX showed significantly increased

MST in comparison with to PL-DOX or free DOX (P b 0.05). However, no significant difference was observed between PL-DOX and free DOX. Tumors excised at the end of the tests are shown in Figure 6, C, and again, the highest efficacy was observed in PHSCNK-PL-DOX group. Toxicity study The group treated with each of liposomal DOX systems did not cause significant bodyweight loss in the mice, whereas the

W. Dai et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 1152–1161 Table 2 Survival time of B16F10-bearing C57BL/6 mice after IV injection of NS, free DOX, PL-DOX, or PHSCNK-PL-DOX Formulation

MST (days ± S.D.)

ILS (%)

NS Free DOX PL-DOX PHSCNK-PL-DOX

19.5 ± 0.4 32.0 ± 0.6* 31.9 ± 1* 35.9 ± 1.3 †

— 68.4 63.2 84.2

Mice were given six IV injections at a dose of 1.25 mg DOX/kg, at 1 day, 4 days, 6 days, 8 days, 11 days, and 13 days after tumor inoculation. NS: normal saline. * P b 0.01 vs. NS. † P b 0.05 vs. PL-DOX.

group treated with free DOX showed some loss of the bodyweight (data not shown). Additionally, the results of heart staining with H&E revealed the reduced cardiac toxicity of PHSCNK-PL-DOX in comparison with both free DOX and PL-DOX (Figure 6, D).

Discussion Because ATN-161 cannot directly covalently attach to the surface of liposomes, we selected the ATN-161 derivative Ac-PHSCNK-NH2 to modify liposomes. It was reported previously that the moderate derivation from the C terminal of ATN-161 did not affect its binding properties to either related cells or isolated integrins α5β1. 24 PHSCNK-PL-DOX was investigated in comparison with PL-DOX in vitro and in vivo in this study to achieve the proofof-principle for the hypothesis that PHSCNK-modified liposomes may actively deliver the DOX into the tumor cells after accumulation in tumor tissue by the EPR effect to realize the improved therapeutic indices. As can be seen from Figure 1, B, the size distributions of PHSCNK-PL-DOX and PL-DOX were slightly different. On one hand, the preparation process of PHSCNK-PL-DOX, stirring the solution containing PL-DOX and PHSCNK solution at 25°C for 2 hours, may have some effect on the size distribution of the modified liposomes. On the other hand, peptide modification may also affect the size distribution profile of liposomes somewhat. Moreover, PHSCNK-PL-DOX and PL-DOX showed very close negative zeta potential, which is similar to that found in a previous study. 29-31 Due to the very low zeta potential, its effect on the properties of liposomes in vitro and in vivo seemed not very much. It was found that the in vitro release of DOX was very similar between PL-DOX and PHSCNK-PL-DOX in culture medium. The reasons may include the same lipid compositions used in the preparation of liposomes and no substantial change in the structure of liposomes after the conjugation of peptide to PL-DOX. In fact, similar results were reported previously. 10,32 The integrin-overexpressing melanoma B16F10 cells were used first as the models in vitro and in vivo for the evaluation of PHSCNK-PL-DOX. It was demonstrated in this study that tests in all cell levels conducted by flow cytometry analysis, confocal laser microscopy analysis, or SRB assay, achieved the same

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result, namely, that PHSCNK modification was effective in terms of improving the internalization of liposomal DOX into B16F10 and increasing its cytotoxicity. These observations were also in good accord with those of HUVEC and MDA-MB-231 cells in our previous studies. 25 In this paper, the in vivo behavior of PHSCNK-PL-DOX has been evaluated for the first time. In comparison with free DOX, both liposome formulations significantly prolonged the circulation time of DOX, whereas the DOX plasma concentrations in the PHSCNK-PL-DOX group were significantly lower than those of PL-DOX, which indicated that the long circulation characteristic of PEGylated liposomes partially remained but was somewhat affected by the modification of PHSCNK at the terminal of the PEG chain. Ligand-targeted liposomes leading to a faster clearance were also previously reported. 33-35 As mentioned in the introduction, PEGylated liposomes can prolong the circulation time of drug-loaded liposomes, resulting in preferential drug accumulation in the tumors by so-called EPR effect. 2,3 This explains the data in Table 1 that drug levels in tumors were higher in liposome groups. As we know, the opsonization of plasma on liposomes can lead to a rapid capture by the cells in the RES, such as liver and spleen. 36,37 PEG modification of liposomes can decrease the drug distribution in RES by partially preventing the opsonization. However, in comparison with the fast elimination of free drug in circulation, it seems that DOX in liposomes still have more opportunity to accumulate in RES. That is why the drug level in liver and spleen was higher in liposome groups than the level in the free drug group. On the other hand, the higher DOX level in the heart of the free-DOX group may be closely related to the inherent cardiac affinity of DOX. Many previous studies 38,39 had already shown that encapsulation inside the liposomes was able to decrease the serious cardiac toxicity of DOX, as did our study. In addition, the drug accumulation in the heart also depends on the drug concentration in the blood circulation. As mentioned above, PHSCNK-PL-DOX group showed a faster clearance and lower blood concentration than found with PL-DOX, which may lead to decreased heart distribution and less cardiotoxicity than found in the control liposome group. The result of heart staining with H&E supports this hypothesis. It was noticed that less than 6% of DOX was released within 24 hours in a release medium containing FBS, as shown in Figure 1, D, whereas in liposomal DOX groups, the drug levels in plasma and most tissues changed little after 24 hours, as seen in Figures 4 and 5. Therefore. it can be concluded that the drug levels in plasma and most tissues within 24 hours in liposomal DOX groups basically represent the levels of liposomal DOX. The results from tumor growth inhibition, the average weight of excised tumors at the end of the test, as well as the survival of B16F10-bearing C57BL/6 mice, all demonstrated that the antitumor activity of PHSCNK-PL-DOX was significantly increased in comparison with those of PL-DOX, which agrees with these cell experiments, but not with the findings of pharmacokinetics and biodistribution studies. It is understandable for these facts because the antitumor efficacy may directly and closely relate to enhanced intracellular DOX delivery. On the contrary, higher tissue level cannot necessarily guarantee more intracellular drug delivery. 10 Although the ligand

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modification has some impact on the long circulation of liposomes, it is still beneficial to the improvement of therapy. It was proved in this study that PHSCNK-PL-DOX effectively transported drug into the tumor cells, inhibited tumor growth in vivo, prolonged the survival time of tumor-bearing mice, and decreased drug toxicity. The present studies suggest that PHSCNK-PL-DOX is a potential drug delivery system for some integrin-overexpressing tumors, and ligand-targeted liposomes present exciting opportunities for cancer therapy. It is also noteworthy that the improved antitumor efficacy of PHSCNK-PL-DOX may be a combination of the long circulation effect of PEGylated liposomes, the active targeting effect of ligand to tumor cells, and the antitumor drug DOX itself.

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