Coadministration of Oligomeric Hyaluronic Acid-Modified Liposomes

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Coadministration of oligomeric hyaluronic acid modified liposomes with tumor penetrating peptide-iRGD enhances the antitumor efficacy of doxorubicin against melanoma Caifeng Deng, Quan Zhang, Yao Fu, Xun Sun, Tao Gong, and Zhirong Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13738 • Publication Date (Web): 23 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

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ACS Applied Materials & Interfaces

Coadministration of Oligomeric Hyaluronic Acid Modified Liposomes with Tumor Penetrating Peptide-iRGD Enhances the Antitumor Efficacy of Doxorubicin against Melanoma Caifeng Denga, , Quan Zhanga,b,*, Yao Fua, Xun Suna, Tao Gonga,*, Zhirong Zhanga

Authors’ Affiliations:

a Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041,China b School of Pharmacy, Chengdu Medical College, Chengdu 610083, China

* Corresponding author Prof. Tao Gong Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, No. 17, Section 3, RenminSouthRoad, Chengdu, Sichuan 610041, China Tel./fax: + 86 28 8551615. E-mail: [email protected] Dr. Quan Zhang No. 601 Tianhui Road, Rongdu Avenue, School of Pharmacy, Chengdu Medical College, Chengdu, 610083, China. Phone: + 86 28 62308653. E-mail: [email protected]

ABSTRACT

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A safe and efficient tumor targeting strategy based on oligomeric hyaluronic acid (HA) modification and coadministration of tumor penetrating peptide-iRGD was successfully developed. In this study, common liposomes (cLip) were modified by oligomeric HA to obtain HA-Lip. After injection into rats, HA-Lip showed good stealth in blood stream and lower liver distribution compared with cLip. Moreover, our HA-Lip could be internalized into B16F10 cells (CD44-overexpressing tumor cells) through HA-CD44 interaction. After systemic administration to B16F10 melanoma-bearing mice, HA-Lip showed an increased distribution in tumor due to the prolonged blood circulation time and the enhanced penetration and retention effect. When co-administered with iRGD, the tumor penetration of HA-Lip was significantly enhanced, which could promote HA-Lip internalization by tumors cells located at deep tumor regions through receptor-mediated endocytosis. Furthermore, doxorubicin (DOX) loaded HA-Lip co-administering with iRGD showed much better antitumor effect compared to DOX loaded cLip and DOX loaded cLip in combination with iRGD. In systemic toxicity test, DOX loaded HA-Lip could significantly decrease the cardiotoxicity and myelosuppression of DOX. Taken together, our results demonstrated that coadministration of oligomeric HA modified liposomes with iRGD could be a promising treatment strategy for targeted therapy of melanoma. KEYWORDS: Oligomeric hyaluronic acid, iRGD, CD44 receptor, Tumor targeting, Liposomes

1. INTRODUCTION


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Melanoma as the most aggressive type of skin cancer has an increasing incidence rate in human.1-2 Despite surgical and medical advancements, the 5-year survival rate for patients with metastasized melanoma is around 10%.3 Recently, nanomedicines have played an important role in melanoma treatment.4-5 Nanomedicines possess ability to reduce severe side effects of chemotherapeutic drugs and many of them could deliver antitumor drugs to tumors through enhanced permeation and retention (EPR) effect.6-7 Efficient EPR effect-mediated passive targeting enables sufficient antitumor drugs reaching at tumor sites through long blood circulation.8 However, most of passive targeting nanomedicines could be massively internalized by the mononuclear phagocyte system, which resulted in the rapid clearance of nanomedicines in blood.9-10 In addition, the multiple layers of tumor cells and high interstitial fluid pressure greatly hindered nanomedicines from accumulating in tumors.11-12 The short blood circulation time and insufficient drug accumulated in tumor contributed to the limited antitumor efficacy of nanomedcines in melanoma treatment. Therefore, it’s essential to develop a rational drug delivery strategy that could enforce targeting delivery of antitumor drugs to tumor sites and simultaneously facilitate CD44 drugs is highly to penetrate expressed intointumor manytissues. tumors including melanoma, colon cancer and prostate cancer.13-15 This cell surface receptor has been a good drug target candidate based on CD44-hyaluronic acid (HA) interaction. It was reported that all CD44 isoforms have uniform affinity for HA.16-17 HA is a biocompatible, non-toxic and biodegradable glycosaminoglycan polymer.18 Many studies reported that high molecular weight (high Mr) HA modified nanocarriers and high Mr HA conjugates increased the cellular uptake on CD44 expressing tumor cells.19-20 However, CD44 is also expressed in many normal organs including kidney.16 In addition, it was demonstrated that high Mr HA had a rapid clearance in blood due to the quick uptake by sinusoidal endothelial cells in liver.21-22 Further research demonstrated that the clearance of HA by liver endothelial cells is through a Ca21-independent class II endocytic receptor.21, 23 These factors hinder high Mr HA modified nanocarries from exclusively delivering drugs to tumors. Indeed, the affinity of HA oligosaccharides (< 10 kDa) to CD44 is modest.24 But



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the CD44 expression is greatly enriched in melanoma, and the low Mr HA modified nanocarriers would preferentially bind to high expressing CD44 cells rather than to expressing CD44 cells.25 What’s more, our previous study26 demonstrated that oligomer of HA modified liposomes (HA-Lip) decreased drug distribution in liver in comparison with common liposomes. In addition, HA-Lip displayed similar long circulation time compared with PEGylated liposomes (PEG-Lip). Unexpectedly, the repeated administration of PEG-Lip induced complement activation, whereas our HA-Lip did not show such adverse immune response. However, the tumor targeting potency of the HA oligomer has been not investigated in vivo. Here, we speculated the oligomer of HA might serve as a safe and active-targeting ligand and attaching HA oligosaccharides to liposomes would extend their blood circulation time and mediate melanoma cells-targeting. Although the long blood circulation time makes it possible to deliver sufficient antitumor drugs to tumor sites, the drug distribution in melanoma is still limited by the poor tumor penetration of active targeting nanomedicines. Growing studies demonstrated that high interstitial fluid pressure and binding-site barrier greatly hinder active targeting nanomedicines from accumulating in tumor tissues.27-28 Recently, studies have manifested that the tumor penetrating peptide-iRGD could improve tumor penetration of antitumor drugs of various compositions, including small molecules, nanoparticles, and antibodies.29-30 Firstly, iRGD binds the integrins αv over-expressed on the vascular endothelium in tumors, then under the effect of proteolytic cleavage, the CendR motif (RGDK/R) is exposed and binds to neuropilin-1 (NRP-1). The activation of NRP-1 increases tumor vascular and tissue permeability, allowing drugs to extravasate from tumor vessels and penetrate in tumor tissue.29 What’s more, the coadministration of iRGD with nanomedcines was more effective in helping drugs penetrate into tumor tissues compared with the conjugation of iRGD with nanomedcines.30-31 We hypothesized that co-administering iRGD with HA-Lip could increase the vascular extravasation and tumor penetration of HA-Lip so as to effectively deliver drugs to the deep tumor regions. In sum, we pursued an efficient drug delivery strategy of coadministration of


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iRGD with HA oligosaccharides modified liposomes, which could potentiate the antitumor efficacy against melanoma by three steps. Firstly, the blood circulation time of doxorubicin (DOX) loaded liposomes was extended through HA oligosaccharides modification (DOX-HA-Lip) so that DOX-HA-Lip could be effectively delivered to tumor sites by EPR effect. Secondly, the coadministration of iRGD could facilitate DOX-HA-Lip to penetrate into tumor tissues. Finally, DOX-HA-Lip would touch melanoma cells especially for those located at deep tumor regions and then be internalized into tumor cells through receptor-mediated endocytosis.

2. EXPERIMENTAL SECTION 2.1 Materials. Doxorubicin hydrochloride was purchased from Huafenglianbo Technology Co., Ltd. (Beijing, China). Sodium hyaluronate (HA) with an average molecular weight of 5.6 kDa was gained from the Shandong Freda Biopharmaceutical Co., Ltd. (Shandong, China). Lipoid E80 (purified ovolecithin) was obtained from Lipoid

Co.,

Ltd

custom-synthesized

(Ludwigshafen, by

GL

Germany).

Biochem

iRGD

(Shanghai)

(CRGDKGPDC)

Ltd.

(Shanghai,

was

China).

1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) was obtained from Corden

Pharma

Switzerland

LLC

(Switzerland).

Cholesterol

and

N-hydroxysuccinimide (NHS) were purchased from Kelong Chemical Company (Chengdu, China). 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide hydrochloride (EDCI) was purchased from Best Reagent Co., Ltd (Chengdu, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

bromide

(MTT)

and

4,6-Diamidino-2-phenylindole (DAPI) were obtained from Sigma-Aldrich Co., LLC. (Saint Louis, USA). Annexvin V-FITC Apoptosis/propidium iodide Dectection kit was purchased from Nanjing Keygen Biotch. CO., Ltd. (Nanjing, China). All the other reagents were of analytical grade and used without further purification. 2.2 Cells and Animals. B16F10 cells and MCF-7 cells were gained from Chinese Academy of Science Cell Bank for Type Culture Collection (Shanghai, China). SD rats (200 ± 20 g) and male C57BL/6 mice (20 ± 2 g) were supported by Chengdu



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Dossy Biological Technology CO., Ltd. (Chengdu, China). All animal experiments were approved by the Laboratory Animal Management Committee of Sichuan University. 2.3 Preparation of DOX-cLip and DOX-HA-Lip. Common liposomes (cLip) was prepared according to the thin-film hydration method.26 Briefly, E80, cholesterol and DPPE (molar ratio, 6:2:1) were dissolved in chloroform-methanol (5:1, v/v). The organic solvent was removed by rotary evaporation at 37 °C and the thin film was hydrated with 123 mM ammonium sulfate at 37 °C for 1 h. The gained multilamellar vesicles was processed by a high pressure homogenizer (Nano DeBBE 45, BBE International, Inc, USA) at an operating pressure of 30000 psi for ten cycles to form blank cLip. DOX was entrapped into cLip by the ammonium sulfate gradient method to gain DOX loaded cLip (DOX-cLip).32 HA modification of DOX-cLip (DOX-HA-Lip) was consistent with our previous study.26 To prepare DOX-HA-Lip, HA was activated by using NHS and EDCI, and pH was adjusted by 100 mM borate buffer to pH 7.8. DOX-cLip was added to incubate with activated HA at room temperature for 12 h. The obtained HA modified liposomes were also purified 26 according to our previousofstudy. 2.4 Characterization Liposomes. The particle size and zeta potential of

DOX-cLip and DOX-HA-Lip were determined by dynamic light scattering (DLS) using Zetasizer Nano ZS90 instrument (Malvern, UK). To determine the encapsulation efficiency (EE %). Liposomes were passed through a Sephadex G75 column to remove un-entrapped DOX, and then the liposomes were diluted with 1% Triton. The DOX in the liposomes (C1) was measured by a fluorescence spectrophotometer (Shimadzu Scientific Instruments, Kyoto, Japan). The excitation wavelength and emission wavelength were at 494 nm and 587 nm, respectively. The same volume of liposomes was diluted with 1% Triton and measured by a fluorescence spectrophotometer to obtain the total amount of DOX (C0). The encapsulation efficiency (EE %) was calculated by the following equation: EE % = C1/C0 * 100 %. The morphology of DOX-cLip and DOX-HA-Lip was observed by transmission electron microscopy (TEM, H-600, Hitachi, Japan). 2.5 In Vitro Drug Release. In vitro drug release was performed by dialysis in saline.


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The molecular weight cut off size of dialysis membrane is 8000-14000 Da. Briefly, mL of DOX-cLip, DOX-HA-Lip and DOX solution were respectively placed into dialysis bag, sealed both ends and immersed in the dissolution medium (50 mL) under 100 rpm constant stirring at 37°C. At the predetermined time intervals, 1 mL of was withdrawn from the release medium and replaced by equal volume of fresh medium. With suitable dilution, the concentrations of DOX in the release medium determined by a fluorescence spectrophotometer. 2.6 Cellular Uptake. 26.1 Qualitative Analysis of Cellular Uptake. B16F10 cells and MCF-7 cells were seeded in glass-bottomed dishes at a density of 1×105 cells / mL, respectively. Cells were allowed to grow for 24 h at 37 °C with 5% CO2. Then, 8 µg/mL DOX equivalents of DOX-cLip and DOX-HA-Lip were added, and cells pre-incubated with 10 mg/mL of HA solution were used for qualitative analysis of competitive inhibition assay. After incubation for 1 h, cells were washed with PBS and fixed with 4% polyoxymethylene for 15 min, followed stained with DAPI for 5 min at a darkness environment. After that, cells were washed thrice with PBS. Finally, the prepared samples were observed using a laser scanning confocal microscope (Olympus Fluoview FV 1000, USA). 2.6.2 Quantitative Analysis of Cellular Uptake. B16F10 cells were seeded in 12-well plates at a density of 1×105 cells per well and allowed to grow for 24 h at 37 °C with 5% CO2. To study the effect of the incubation time on cellular association of different DOX formulations, cells were treated with 8 µg/mL DOX equivalents of DOX-cLip and DOX-HA-Lip for different incubation time of 0.5 h, 1 h, 2 h and 4 h. To investigate the effect of drug concentration on cellular association of different DOX formulations, cells were incubated with different concentrations (4 µg/mL, 8 µg/mL and 16 µg/mL) of DOX formulations, respectively. Then cells were washed thrice with cold PBS (pH = 7.4), followed centrifuged at 2000 rpm for 3min, collected and suspended in PBS for flow cytometry analysis (CytomicsTM FC 500, Beckman Coulter, USA). 2.7 HA Competitive Inhibition Assay. B16F10 and MCF-7 cells were seeded in



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12-well plates at a density of 1×105 cells per well, respectively. Cells were pre-incubated with 10 mg/mL of HA solution for 1 h. Thereafter, 8 µg/mL DOX equivalents of DOX-cLip and DOX-HA-Lip were added for 1 h incubation. The intensity of drug fluorescence was determined by flow cytometry analysis. 2.8 Cell Viability. B16F10 and MCF-7 cells were respectively seeded in 96-well plates at a density of 1×104 cells per well and cultured for 24 h. After that, the culture medium was replaced by different DOX formulations at concentrations of DOX equivalents ranging from 0.078 - 20 µg/mL. Cells were incubated for 48 h and 200 µL of MTT (0.5 mg/mL) was added to each well. After incubation for another 4 h, the MTT was replaced by 100 µL of DMSO with constant shaking at 100 rpm in the darkness. The absorbance at 490 nm was measured by Varioskan flash multimode plate reader (Thermo, NH, USA). Cell viability was calculated according the absorbance values. 2.9 Cell Cycle Arrest and Cell Apoptosis Studies. B16F10 cells were seeded in 6-well plates at a density of 1 × 105 cells per well and cultured for 24 h. For cell cycle arrest study, cell culture medium was removed and cells were incubated with 2 mL of DOX-cLip (50 ng/mL) and DOX-HA-Lip (50 ng/mL) for 24 h. After that, cells were washed thrice with cold PBS, fixed with 75% ethanol (precooled at -20 °C) at 4 °C for 2 h, then after cells were centrifuged at 2000 rpm for 3 min, suspended in PBS, followed incubated with 0.1% Triton X-100 (0.03%) for 45 min, RNase (0.1 mg/mL) for 30 min at 37 °C, and propidium iodide (0.1 mg/mL) at 4 °C for 30 min. For cell apoptosis study, cells were treated with drug free medium, 50 ng/mL DOX equivalents of DOX-cLip and DOX-HA-Lip, incubated for another 24 h. After that, cells were washed thrice with cold PBS, trypsinized, suspended in PBS, stained with an Annexin V-FITC Apoptosis Detection kit according to the manufacturer’s instructions. The prepared cell samples were detected by flow cytometer. 2.10 Safety Evaluation 2.10.1 Bone Marrow Suppression Assay. Myelosupression has been one of the most severe side effects caused by DOX. To assess the bone marrow toxicity of DOX solution, DOX-cLip and DOX-HA-Lip, healthy male SD rats (200 ± 20 g) were


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administered with 5 mg/kg of DOX solution, DOX-cLip and DOX-HA-Lip. Saline was used as control. Blood samples were collected in tubes containing EDTA-2K salt at the day before administration and at day 3 after administration. The amount of white blood cells was counted by MEK-6318K Automated Hematology Analyzer (Nihonkohden, Shinjuku-ku, Japan). 2.10.2 Systematic Toxicity Assay. Healthy male SD rats (200 ± 20 g) were used to study the systematic toxicity of DOX solution, DOX-cLip and DOX-HA-Lip. The rats were fasted for 12 h before administration and were randomly injected with 5 mg/kg dose of DOX solution, DOX-cLip and DOX-HA-Lip every 2 days for 2 times. All rats were sacrificed on day 7, and blood samples, heart, liver, spleen, lung and kidney were collected. The levels of creatine kinase (CK), lactate dehydrogenase (LDH), creatine kinase MB (CK-MB), serum aspartate transaminase (AST), alanine transaminase (ALT), urea nitrogen (BUN), and creatinine (CREA) in serum were detected by Hitachi 7020 automatic biochemical analyzer (Hitachi 7020, Japan). The major organs were stained with hematoxylin & eosin (H&E). 2.11 Pharmacokinetic Studies. Male SD rats weighing 200 ± 20 g were divided into 4 groups (n = 5). Rats were intravenously injected 2 mg/kg of free DOX, DOX-cLip and DOX-HA-Lip with or without 10 mg/kg of iRGD. Blood was withdrawn from the vena ophthalmica at pre-set time intervals and centrifuged at 5000 rpm for 5 min to gain plasma. The obtained plasma was stored at - 40 °C for further analysis. 2.12 In Vivo Distribution and Tumor Penetration Study. 1.5 × 106 B16F10 cells suspended in 200 µl PBS were subcutaneously injected into the right flank of male C57BL/6 mice (20 ± 2 g) to establish melanoma-bearing mice model. Did loaded cLip, HA-Lip, Did loaded cLp in combination with iRGD, HA-Lip in combination with iRGD (150 µg/kg Did, 10 mg/kg iRGD ) were injected into mice via tail vein. After 4 h, mice were sacrificed to collect blood, organs (heat, liver, spleen, lung, and kidney) and tumors. Tissues and tumors were observed by the in vivo imaging system (Quick View 3000, Bio-Real, Austria). The fluorescence intensity of Did in plasma, major organs and tumors was analyzed by semi-quantitative analysis of the ex vivo fluorescent images.



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For tumor penetration study, mice bearing melanoma were treated with DOX solution, DOX-cLip, DOX-cLip in combination with iRGD, DOX-HA-Lip, DOX-HA-Lip in combination with iRGD (DOX dose 5 mg/kg, iRGD dose 10 mg/kg). After 4 h, the obtained tumors were fixed in 4% paraformaldehyde to prepare frozen section (10 µm thickness). The tumor slices were stained with mouse anti-CD31 antibody and DAPI. The prepared tumor sections were observed by confocal microscope (LSM710, Carl Zeiss, Germany). 2.13 Antitumor Effect. C57BL/6 mice (20 ± 2 g) bearing melanoma were established as described above and randomly divided into 6 groups. At day 12, 15 and 18 after B16F10 cells implantation, DOX solution, DOX-cLip, DOX-cLip in combination with iRGD, DOX-HA-Lip, DOX-HA-Lip in combination with iRGD (DOX dose 5 mg/kg, iRGD dose 10 mg/kg) were administered into mice via tail vein. Saline was used as control. Tumor size of each group was determined every other day after first administration. The survival was recorded, presented by Kaplan-Meier plots and analyzed with log-rank test. 2.14 Statistical Analysis. All quantitative parameters in this study are expressed as mean with SD (standard deviations). Statistical analysis was performed by the ANOVA, and survival analysis was presented by Kaplan-Meier plots and compared by the log-rank test using the SPSS software. P value of < 0.05 and < 0.01 are accepted as indicative of statistical differences and significant differences.

3. RESULTS 3.1 Characterization of Liposomes. As shown in Table 1, the particle size of DOX-cLip was 116.4 ± 4.7 nm. After being modified by HA, the particle size of DOX-HA-Lip slightly increased to 128.2 ± 5.9 nm. Average zeta potential of DOX-cLip and DOX-HA-Lip was -2.1 ± 0.9 mV and -7.4 ± 2.2 mV, respectively. DOX encapsulation efficiency of DOX-cLip and DOX-HA-Lip was 94.2 ± 1.8% and 91.6 ± 2.5%. TEM photographs showed that the morphologies of DOX-cLip and DOX-HA-Lip were generally spherical and uniformly dispersed (Figure 1A).


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3.2 In Vitro Drug Release. The profiles of in vitro DOX release are presented in Figure 1B. DOX-cLip and DOX-HA-Lip displayed similar slow and sustained release manners, and less than 50% of total DOX was released from liposomes after a 96 h incubation period. In contrast, in the case of free DOX, complete release was obtained in 8 h. 3.3 Cellular Uptake. To study whether HA modified carriers could actively target tumor cells through HA-CD44 interaction, high CD44-expressing B16F10 murine melanoma cells and low CD44-expressing MCF-7 human breast cancer cells were used to investigate the cellular association of liposomes in vitro. The confocal microscopy images displayed the subcellular localization of DOX-HA-Lip and DOX-cLip in B16F10 cells. As shown in Figure 2, cell nuclei were stained with DAPI. For B16F16 cells, DOX-HA-Lip treated group showed higher intensity of red fluorescence compared with that of DOX-cLip treated group. DOX-HA-Lip was observed mainly located in cell nucleus. For MCF-7 cells, DOX-HA-Lip and DOX-cLip treated cells presented no significant fluorescence signals. However, when B16F10 cells were pre-treated with HA solution, the intensity of fluorescence of DOX-HA-Lip group decreased significantly. On contrary, the pre-incubation of HA had no effect on the uptake of DOX-cLip. What’s more, these differences were disappeared when MCF-7 cells were used. The quantitative analysis of cellular uptake was also carried out by flow cytometry assay. DOX-HA-Lip and DOX-cLip were incubated in CD44 expressing B16F10 cells with different incubation time and at different DOX concentrations, respectively. It was obvious that fluorescence intensity of DOX-HA-Lip group was significant higher than that of DOX-cLip group, which was consistent with the results of confocal microscopy analysis. Besides, with the increase of incubation time and DOX concentration, the fluorescence intensity was greatly elevated for either DOX-HA-Lip group or DOX-cLip group (Figure 3). 3.4 HA Competitive Inhibition Assay. To validate the HA-CD44 interaction involved cellular association of DOX-HA-Lip, the HA competitive inhibition assay was carried out by flow cytometry assay. As depicted in Figure 4A, the cellular uptake



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of DOX-HA-Lip on high CD44-expressing B16F10 cells was significantly higher compared with that of DOX-cLip (P < 0.01). What’s more, the pre-incubation of HA solution for 1 h significantly decreased the cellular uptake of DOX-HA-Lip (P < 0.01). While the above trends weren’t observed on low CD44-expressing cells, the DOX-HA-Lip and DOX-cLip treated MCF-7 cells displayed similar fluorescence intensity with or without the pre-treatment of HA solution (Figure 4B). These results were also consistent with that of confocal microscopy analysis. 3.5 Cell Viability. Dose-response curves of DOX cytotoxicity against B16F10 and MCF-7 cells were performed by MTT assay and shown in Figure 4C. Obviously, DOX-HA-Lip presented higher cytotoxicity against B16F10 cells compared with DOX-cLip. The IC50 were 0.55 ± 0.23 µg/mL for DOX-HA-Lip and 1.67 ± 0.52 µg/mL for DOX-cLip (Table 2 ). However, for MCF-7 cells, DOX-HA-Lip and DOX-cLip displayed no significant difference in the anti-proliferation activity and the value of IC50 (Figure 4D and Table 2 ). 3.6 Cell Cycle Arrest and Cell Apoptosis. The cell cycle of B16F10 cells treated with different DOX formulations was examined by flow cytometry assay. As shown in Figure 5D, to compare with control group, accumulation of cells in G2/M phase after incubation with DOX-cLip and DOX-HA-Lip was increased to 31.6% and 41.8%, respectively. Apparently, DOX-HA-Lip arrested more cells in G2/M phase than DOX-cLip treated group (P < 0.05). Cell apoptosis induced by DOX preparations was performed on B16F10 cells by using Annexin V-FITC Apoptosis Detection Kit. As shown in Figure 5(A, B, C), the percentage of cell apoptosis treated with DOX-cLip was 9.6%. While those treated with DOX-HA-Lip was significantly increased to 20.9%. DOX-HA-Lip had the advantage in causing B16F10 cells apoptosis over DOX-cLip. 3.7 Safety Evaluation 3.7.1 Bone Marrow Suppression Assay. Overcome the systematic side effects of chemotherapeutic drugs is an important consideration for drug delivery system.33 The myelosuppression is one of serious toxicities induced by DOX treatment. The white blood cell counts in serum has been considered as useful index for assessing


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myelosuppression.34 The normal value of WBC counts for healthy rats is 5-25 (× 109/L). As shown in Figure 6, mean WBC counts of healthy rats treated with DOX solution decreased from 10.05 × 109/L to 3.26 × 109/L 3 days after administration. Obviously, the mean WBC value of DOX solution treated group was less than normal range (P < 0.01). DOX-cLip and DOX-HA-Lip also induced decrease of WBC counts to 5.56 × 109/L and 6.41 × 109/L, respectively. Besides, the mean WBC value of DOX-HA-Lip treated group was within normal range and significantly higher than that of DOX-cLip group (P < 0.05). 3.7.2 Systematic Toxicity Assay. In order to explore the general toxicity of different DOX formulations, we investigated the blood biochemistry parameters, major organs histopathology. The detected biochemistry parameters were showed in Table 3. Serum ALT, AST, and ALP levels are helpful for the evaluation of liver function.35 No significant difference of serum ALT, AST levels was found among all the treated groups except that the AST levels increased in DOX solution treated rats compared with control group (P < 0.01). When comparing the renal function, DOX solution treated rats displayed a significant increase in serum levels of BUN-a useful maker for the evaluation of renal function,36 compared to the saline group (P < 0.05). While the serum BUN levels of DOX-cLip and DOX-HA-Lip treated rats showed no difference compared with that of saline group. The serum levels of CK, CK-MB, and LDH are useful markers for assessing cardiotoxicity.37-38 When compared with saline group, rats treated with DOX solution displayed a significant increase in CK and CK-MB levels (P < 0.01) and DOX-cLip induced slight rise of CK and CK-MB (P < 0.05). In contrast, DOX-HA-Lip showed no significant effects on serum levels of CK, CK-MB, and LDH. To further evaluate the in vivo safety of our drug delivery platform, we examined the toxicity of major organs induced by DOX, DOX-cLip and DOX-HA-Lip through pathological tissue section technique. Compared with saline group, DOX solution caused obvious damage to heart, liver and spleen (Figure 7). The myocardial fiber breakage, cell necrosis and inflammation were obvious in heart samples from DOX solution treated rats. The results showed dilatation of blood sinus and atrophy of liver



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cells in liver samples of DOX solution group. Cell necrosis was also obvious in spleen tissue samples when rats exposed to free DOX. Whereas, rats treated by DOX-cLip showed light pathology in liver tissue, heart, and spleen tissue. What’s more, rats receiving DOX-HA-Lip displayed no damages to heart, liver and spleen. Histological examination of lung and kidney did not show any gross pathology in any of the treatment groups. 3.8 Pharmacokinetic Studies. In order to investigate whether HA modification could improve the long circulating capacity of cLip, we examined the in vivo pharmacokinetic behavior of DOX-HA-Lip. DOX showed a rapid elimination from the circulation system and displayed a dramatically high clearance rate of 146.36 ± 28.63 mL/min/kg, DOX-cLip and DOX-HA-Lip significantly decreased the clearance rate of DOX to 0.40 ± 0.13 mL/min/kg (P < 0.05) and 0.24 ± 0.04 mL/min/kg (P < 0.05), respectively. Besides, the area under the curve (AUC0-t) were 24.33 ± 2.84 µg/mL*min for DOX and 3785.67 ± 1505.91 µg/mL*min for DOX-cLip, while DOX-HA-Lip had the largest AUC0-t of 6794.24 ± 1092.84 µg/mL*min. What’s more, DOX-cLip and DOX-HA-Lip showed higher values of MRT0-t and t1/2z compared with that of DOX (P < 0.05), and DOX-HA-Lip presented the highest MRT0-t and t1/2z among all the groups. Those results indicated that the modification of HA could extend the blood circulation of DOX loaded liposomes. To study the influence of iRGD on the blood circulation of liposomes, we co-administered iRGD with DOX-HA-Lip and DOX-cLip. The pharmacokinetic profiles of DOX-HA-Lip + iRGD and the DOX-cLip + iRGD were both closed to that of DOX-HA-Lip and DOX-cLip (Figure 8 and Table 4). 3.9 In Vivo Distribution and Tumor Penetration Study. The in vivo distribution was examined by ex vivo imaging analysis. HA-Lip did not exhibit specific accumulation in liver and spleen compared with cLip. Besides, the fluorescence signals of HA-Lip detected in blood were much stronger than that of cLip, and HA-Lip displayed higher tumor distribution compared with cLip (P < 0.05). When co-administering with iRGD, the tumor distributions of cLip and HA-Lip were both significantly increased. In addition, HA-Lip + iRGD displayed the highest tumor distribution among all the


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treated groups (Figure 9). To illustrate the tumor-targeting and tumor-penetrating ability of different formulations,

the

tumor

penetration

study

was

performed

by

using

immunohistochemical technology. As shown in Figure 10, tumor blood vessel was stained with anti-CD31 antibody. It’s obvious that the accumulation of DOX-HA-Lip group was much greater than that of DOX-cLip and comparable to that of DOX-cLip + iRGD. Furthermore, the intratumoral drug for DOX-cLip group was mainly observed around the tumor blood vessels with a little extravasation. However, the drug fluorescence distributions of DOX-cLip + iRGD and DOX-HA-Lip + iRGD group were significantly far way from the tumor vessels and much wider. Besides, DOX-HA-Lip + iRGD group showed the highest drug fluorescence and best accumulation among all the treated groups. 3.10 Antitumor Effect. The antitumor effect was evaluated in male C57BL/6 mice bearing melanoma. As shown in Figure 11A, DOX solution and DOX-cLip displayed undesirable antitumor efficiency. DOX-HA-Lip had better inhibitory effect against tumor growth than free DOX and DOX-cLip. The coadministration of iRGD greatly enhanced the antitumor efficacy of DOX-cLip and DOX-HA-Lip. What’s more, DOX-HA-Lip + iRGD group showed the highest inhibition rate of tumor growth, with 68.23% reduction of tumor volume compared with saline group. The median survival of male C57BL/6 mice bearing melanoma administrated with DOX-HA-Lip + iRGD (34.0 days) was significantly longer than those of mice treated with saline (24.0 days, P < 0.01), DOX (24.0 days, P < 0.01), DOX-cLip (26.0 days, p < 0.01), DOX-HA-Lip (28.0 days, p < 0.05) and DOX-cLip + iRGD (30.0 days, P < 0.05) (Figure 11B and Table 5)

4. DISCUSSION Melanoma, originated from the malignant transformation of melanocytes, is notorious for its low survival rate and its incidence has increased most rapidly among human cancers in the last decades.39 As one of indispensible therapies, chemotherapy



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has played an crucial role in controlling the development of melanoma. However, the antitumor efficacy of chemotherapeutic agents is limited in melanoma treatment due to their short blood circulation time and poor penetration into the melanoma parenchyma. Understanding of this fact highlights the necessary of developing more safe and rational drug delivery strategies. Liposomes have been extensively used as drug carriers for they can be readily modified and generally exhibit excellent biocompatibility and good compatibility towards kinds of therapeutic agents, including small molecule drugs, proteins and even RNA.40-42 Since the prolonged circulation of drug carriers after systemic administration in vivo is an important determinant to deliver sufficient antitumor drugs to tumor sites by EPR effect,43-44 numerous hydrophilic polymers have been used to modify liposomes to form hydrophilic shells, such as poly(ethylene glycol) (PEG)45-46, poly(N-vinyl-2-pyrrolidone) (PVP)47-48, dextran49-50, and heparin51-52. Of these polymers, PEG has been extensively investigated for tumor-targetable liposomes, because it shows good biocompatibility and the PEG surface enables liposomes to escape from the uptake by the reticuloendothelial system of liver.53-54 Recently, many investigations showed that PEGylated liposomes would trigger “Accelerated Blood Clearance” effect (ABC effect) and complement activation.55-56 Furthermore, this passive targeting strategy in the effective diagnosis or therapy of tumors is partially limited by its low internalization ability into the target cells.57-58 To overcome this limitation, those carriers have been modified with targeting moieties, including antibodies59-60 and various ligands61-63 to develop them capable of actively targeting cancer cells. However, it is important to notice that targeting moieties on carriers do not necessarily insure successful delivery of the payloads. For example, antibodies could suffer from their potential immunogenicity and depressed binding affinity with their receptors after chemical conjugation.64 Folate, as another example, has been extensively used as a targeting moiety for its receptor is over expressed on various cancer cells.65-66 However, a significant portion of folate modified carriers in vivo were also found in kidney, brain and liver, owing to the high levels of the folate receptor on those organs.67


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Fortunately, our group has demonstrated that oligomeric HA modified liposomes showed similar prolonged blood circulation compared with PEGylated liposomes. Furthermore, our HA modified liposomes would not induce ABC effect and complement activation.26 In addition, it was reported that the high and low molecular weight forms of HA have distinct effects on CD44 clustering. Although the high Mr HA has higher affinity to CD44 compared with the low Mr HA,24, 68 a significant portion of the carriers modified with high Mr HA was also prone to distribute in the liver site due to their cellular uptake by liver sinusoidal endothelial cells expressing another HA receptor (Ca21-independent class II endocytic receptor).23,

25

Taken

together, we proposed that the low Mr HA fragments modification would overcome the uptake of liposomes by sinusoidal endothelial cells of liver to prolong their blood circulation and the developed liposomes could exclusively target melanoma tumors. To optimize the accumulation and penetration of nano-carriers in tumors, particle size of carriers was considered as an important factor. It was demonstrated that the nanoparticles with particle size ranging from 5 to 250 nm displayed higher transport efficiency.69 In our study, we reported that the average particle size of DOX-cLip was 116.4 ± 4.7 nm, and the modification of HA oligosaccharides slightly increased the particle size of liposomes to 128.2 ± 5.9 nm (Table 1). Therefore, the DOX-cLip and DOX-HA-Lip both presented the desired particle sizes. We also investigated whether the drug loading capacity of the liposomes was affected by the modification of HA, and comparable encapsulation efficiency was obtained for DOX-HA-Lip compared with that of DOX-cLip (Table 1). What’s more, DOX-cLip and DOX-HA-Lip presented similar sustained drug release manners, which suggested the modification of HA also exhibited no impact on the drug release behavior of liposomes (Figure 1B). Interestingly, we found that DOX-HA-Lip could significantly increase drug fluorescence signal in B16F10 cells compared with DOX-Lip. While on low CD44-expressing MCF-7 human breast cancer cells, DOX-HA-Lip and DOX-cLip displayed similar drug fluorescence signal (Figure 2). The quantitative analysis of cellular uptake presented the same above trend (Figure 3 and Figure 4). In addition,



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the pretreatment of HA inhibited the cellular uptake of DOX-HA-Lip by B16F10 melanoma cells, while the equal treatment resulted no impact on the cellular uptake of DOX-cLip (Figure 4A). We didn’t observe such effect of HA pretreatment on the cellular uptake of DOX-HA-Lip by MCF-7 cells (Figure 4B). These results were consistent with that of literature reported by Eliaz et al.,25 in which low Mr HA fragments facilitates the recognition of nanocarriers by high CD44-expressing cells rather than by low CD44-expressing cells, and suggested that the cellular association of DOX-HA-Lip on B16F10 cells was based on HA-CD44 interaction. Besides, the enhanced tumor cell apoptosis and increased tumor cell cytotoxicity of DOX-HA-Lip compared with DOX-cLip (Figure 4C and Figure 5A-C) also illustrated that HA could improve the internalization of liposomes into B16F10 cells. We also conducted safety evaluation to assess the in vivo safety of our developed drug delivery system. DOX could induce severe systematic toxicities, especially the bone marrow toxicity and cardiac toxicity of DOX could have negative impact on the living quality of patients.70 In our study, DOX caused obvious myelosupression by inducing the decreasing of WBC (Figure 6). Besides, obvious organ injuries were observed in DOX treated group. By contrast, both DOX-cLip and DOX-HA-Lip reduced the toxicity of DOX to bone marrow and displayed negligible injuries and damages to major organs (Figure 7). What’s more, DOX-HA-Lip treated rats showed the lowest side effects induced by DOX. The results of blood biochemistry parameters were consistent with the above trends. Those results confirmed that DOX-HA-Lip exhibited the ability of reducing the side effects of DOX. The side effects of DOX decreased significantly by DOX-HA-Lip can be explained by the in vivo distribution behaviors. The DOX levels in heart, liver and spleen were significantly reduced when mice were injected with DOX-HA-Lip (Figure 9). In addition to the reduced unexpected toxicity, we observed that DOX-HA-Lip showed longer t1/2, lower clearance rate and higher AUC0-t than DOX-Lip (Figure 8 and Table 4). Those results indicated that the modification of HA could extend the blood circulation of DOX loaded liposomes. Furthermore, the liver distribution of cLip was obviously higher than that of HA-Lip (Figure 9), which suggested that the


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stealthiness of HA-Lip in the bloodstream was attributed to the less RES uptake in liver. HA could guarantee the long blood circulation of nano-carriers so as to promote them reach to tumor sites by EPR effect. And HA modification also facilitated the internalization of carriers into cancer cells in vitro. But those conditions would not necessarily insure efficient drug penetration in tumor tissues. When HA modified liposomes were injected into animals, those liposomes mainly stayed around the blood vessels owing to the high interstitial fluid pressure in tumors and binding-site barrier.27-28 Therefore, those HA modified liposomes could not effectively touch cancer cells especially for those located at deep tumor regions. In order to increase the tumor extravasation and tumor penetration of our developed drug delivery system, we co-administered iRGD with DOX-HA-Lip. From the results of in vivo distribution study, we observed that the coadministration of iRGD significantly increased drug distribution in tumors, and HA-Lip in combination with iRGD displayed the highest fluorescence tumor distribution (Figure 9). Furthermore, the tumor penetration study displayed that the drug fluorescence of co-administering iRGD with DOX formulations could be found at tumor parenchyma that far away from tumor vessels (Figure 10). In sum, DOX-HA-Lip displayed extended blood circulation time and lower unexpected toxicity. Besides, DOX-HA-Lip could preferentially bind to high CD44-expressing B10F10 cells and increase the toxicity to B10F10 cells. When co-administered with iRGD, the tumor accumulation and penetration of DOX-HA-Lip was greatly enhanced. Accordingly, mice treated by iRGD in combination with DOX-HA-Lip displayed highest tumor inhibition rate and longest whole survival among all the treated groups (Figure 11).

CONCLUSION we developed a safe and rational drug delivery strategy of co-administering iRGD with DOX-HA-Lip for melanoma treatment. We proved that oligomeric HA fragments had higher affinity to high CD44-expressing B10F10 cells than to low



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CD44-expressing cells, which reduced DOX-HA-Lip distribution in normal organs including liver and kidney. We also demonstrated that HA oligomer modification extended the blood circulation time of DOX loaded liposomes, and the coadministration of iRGD with DOX-HA-Lip dramatically increased the DOX distribution in tumors. Our drug delivery strategy satisfied the notion that exclusively targeting delivering drugs to tumor cells.

ACKNOWLEDGMENTS This work was supported by the financial support from the National Natural Science Foundation of China (No. 81673359), the National Basic Research Program of China (973 Program, 2013CB932504) and the National Natural Science Foundation of China (No. 81603045).

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected].

Notes The authors declare no competing financial interest.

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47. Torchilin, V. P.; Shtilman, M. I.; Trubetskoy, V. S.; Whiteman, K.; Milstein, A. M., Amphiphilic Vinyl Polymers Effectively Prolong Liposome Circulation Time In Vivo. Biochimica et Biophysica Acta (BBA) - Biomembranes 1994, 1195 (1), 181-184. 48. Ishihara, T.; Maeda, T.; Sakamoto, H.; Takasaki, N.; Shigyo, M.; Ishida, T.; Kiwada, H.; Mizushima, Y.; Mizushima, T., Evasion of the Accelerated Blood Clearance Phenomenon by Coating of Nanoparticles with Various Hydrophilic Polymers. Biomacromolecules 2010, 11 (10), 2700-2706. 49. Ning, S.; Huang, Q.; Sun, X.; Li, C.; Zhang, Y.; Li, J.; Liu, Y. N., Carboxymethyl Dextran-Coated Liposomes: Toward a Robust Drug Delivery Platform. Soft Matter 2011, 7 (19), 9394-9401. 50. Torchilin, V. P., How Do Polymers Prolong Circulation Time of Liposomes? J. Liposome Res. 1996, 6 (1), 99-116. 51. Su, L. C.; Chen, Y. H.; Chen, M. C., Dual Drug-Eluting Stents Coated with Multilayers of Hydrophobic Heparin and Sirolimus. ACS Appl. Mater. Interfaces 2013, 5 (24), 12944-12953. 52. Sahli, A.; Cansell, M.; Tapon-Bretaudière, J.; Letourneur, D.; Jozefonvicz, J.; Fischer, A. M., The Stability of Heparin-Coated Liposomes in Plasma and Their Effect on its Coagulation. Colloids Surf. B Biointerfaces 1998, 10 (4), 205-215. 53. Wang, Y. Y.; Lu, L. X.; Shi, J. C.; Wang, H. F.; Xiao, Z. D.; Huang, N. P., Introducing RGD Peptides on PHBV Films through PEG-Containing Cross-Linkers to Improve the Biocompatibility. Biomacromolecules 2011, 12 (3), 551-559. 54. Lasic, D. D.; Martin, F. J.; Gabizon, A.; Huang, S. K.; Papahadjopoulos, D., Sterically Stabilized Liposomes: A Hypothesis on the Molecular Origin of the Extended Circulation Times. Biochimica et Biophysica Acta (BBA) - Biomembranes 1991, 1070 (1), 187-192. 55. Ishida, T.; Harada, M.; Wang, X. Y.; Ichihara, M.; Irimura, K.; Kiwada, H., Accelerated Blood Clearance of Pegylated Liposomes Following Preceding Liposome Injection: Effects of Lipid Dose and PEG Surface-Density and Chain Length of the First-Dose Liposomes. J. Control. Release 2005, 105 (3), 305-317. 56. Szebeni, J.; Muggia, F.; Gabizon, A.; Barenholz, Y., Activation of Complement by Liposomes and Other Lipid Excipient-Based Therapeutic Products: Prediction and Prevention. Adv. Drug Delivery Rev. 2011, 63 (12), 1020-1030.



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57. Byrne, J. D.; Betancourt, T.; Brannon-Peppas, L., Active Targeting Schemes for Nanoparticle Systems in Cancer Therapeutics. Adv. Drug Delivery Rev. 2008, 60 (15), 1615-1626. 58. Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R., Nanocarriers as an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2 (12), 751-760. 59. Xiang, Y.; Kiseleva, R.; Reukov, V.; Mulligan, J.; Atkinson, C.; Schlosser, R.; Vertegel, A., Relationship between Targeting Efficacy of Liposomes and the Dosage of Targeting Antibody Using Surface Plasmon Resonance. Langmuir 2015, 31 (44), 12177-12186. 60. ElBayoumi, T. A.; Torchilin, V. P., Tumor-Specific Anti-Nucleosome Antibody Improves Therapeutic Efficacy of Doxorubicin-Loaded Long-Circulating Liposomes against Primary and Metastatic Tumor in Mice. Mol. Pharm. 2009, 6 (1), 246-254. 61. Agrawal, A. K.; Harde, H.; Thanki, K.; Jain, S., Improved Stability and Antidiabetic Potential of Insulin Containing Folic Acid Functionalized Polymer Stabilized Multilayered Liposomes Following Oral Administration. Biomacromolecules 2014, 15 (1), 350-360. 62. Han, Q.; Wang, W.; Jia, X.; Qian, Y.; Li, Q.; Wang, Z.; Zhang, W.; Yang, S.; Jia, Y.; Hu, Z., Switchable Liposomes: Targeting-Peptide-Functionalized and pH-Triggered Cytoplasmic Delivery. ACS Appl. Mater. Interfaces 2016, 8 (29), 18658-18663. 63. Miyajima, Y.; Nakamura, H.; Kuwata, Y.; Lee, J. D.; Masunaga, S.; Ono, K.; Maruyama, K., Transferrin-Loaded Nido-Carborane Liposomes: Tumor-Targeting Boron Delivery System for Neutron Capture Therapy. Bioconjug. Chem. 2006, 17 (5), 1314-1320. 64. Torchilin, V. P., Recent Advances with Liposomes as Pharmaceutical Carriers. Nat. Rev. Drug Discovery 2005, 4 (2), 145-160. 65. Yang, S. J.; Lin, F. H.; Tsai, K. C.; Wei, M. F.; Tsai, H. M.; Wong, J. M.; Shieh, M. J., Folic Acid-Conjugated Chitosan Nanoparticles Enhanced Protoporphyrin IX Accumulation in Colorectal Cancer Cells. Bioconjug. Chem. 2010, 21 (4), 679-689. 66. Chen, Y.; Cao, W.; Zhou, J.; Pidhatika, B.; Xiong, B.; Huang, L.; Tian, Q.; Shu, Y.; Wen, W.; Hsing, I. M.; Wu, H., Poly(L-Lysine)-Graft-Folic Acid-Coupled Poly(2-Methyl-2-Oxazoline) (PLL-G-PMOXA-C-FA):

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Tables Table 1. Characteristics of DOX-cLip and DOX-HA-Lip. Data are represented as mean ± SD (n = 3). Particle size (nm)

PDI

Zeta potential

Encapsulation

(mV)

efficiency (%)



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DOX-cLip

116.4 ± 4.7

0.152 ± 0.037

-2.1 ± 0.9

94.2 ± 1.8

DOX-HA-Lip

128.2 ± 5.9

0.206 ± 0.053

-7.4 ± 2.2

91.6 ± 2.5

Table 2. IC50 values of DOX-cLip and DOX-HA-Lip on B16F10 and MCF-7 cells. Data represent mean ± SD (n = 3). IC50 (µg/mL) DOX-cLip

DOX-HA-Lip

B16F10

1.67 ± 0.47

0. 55 ± 0.23**

MCF-7

4.74 ± 0.35

4.24 ± 0.52

** P < 0.01 versus DOX-cLip group.

Table 3. The blood biochemistry parameters of rats determined 48 h after the second administration with DOX formulations. Saline

DOX solution

DOX-cLip

DOX-HA-Lip

ALT (U/L)

39.21 ± 7.55

42.02 ± 6.21

38.71 ± 8.01

36.05 ± 5.84

AST (U/L)

218.44 ± 46.32

443.25 ± 31.64**

228.09 ± 48.48

234.01 ± 28.90

BUN (mmol)

5.83 ± 1.49

14.55 ± 4.92*

6.83 ± 2.84

6.04 ± 2.36

CREA (µmol)

32.73 ± 3.71

36.01 ± 2.65

33.70 ± 2.01

28.46 ± 4.09

LDH (U/L)

1850.32 ± 477.11

2254.93 ± 512.76

2093.05 ± 373.44

1942.99 ± 287.44

CK-MB

1032.00

2142.77**

1493.32*

1178.09

(U/L)

± 285.42

± 435.88

± 302.49

± 412.67

CK

820.40

2507.53**

1301.11*

763.00

(U/L)

± 237.99

± 444.28

± 282.95

± 397.84

* P < 0.05, ** P < 0.01 versus saline group. Table 4. Pharmacokinetic parameters of DOX solution, DOX-cLip, DOX-cLip

in

combination with iRGD, DOX-HA-Lip and DOX-HA-Lip in combination with iRGD after intravenous injection in rats (mean ± SD, n = 5). Parameters

DOX solution

DOX-cLip

DOX-cLip

DOX-HA-Lip

(iRGD)


DOX-HA-Lip (iRGD)

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AUC0-t

24.33

3785.67a

3201.43

6794.24ab

5260.76

(µg/mL*min)

± 2.84

± 1505.91

± 910.57

± 1092.84

± 1388.91

t1/2z (min)

75.94

344.64a

292.96

505.51ab

440.94

± 13.09

± 81.28

± 61.19

± 104.32

± 78.09

21.55

322.98a

278.71

448.33ab

365.45c

± 6.83

± 49.06

± 34.42

± 54.29

± 63.21

CLz

146.36

0.40a

0.51b

0.24ab

0.33c

(mL/min/kg)

± 28.63

± 0.13

± 0.08

± 0.04

± 0.07

MRT0-t (min)

a

P < 0.05 versus DOX solution. b P < 0.05 versus DOX-cLip. c P < 0.05 versus DOX-HA-Lip.

Table 5. Effect of DOX formulations on survival time of the xenografted melanoma B16F10-bearing C57BL/6 mice. Survival time (days) Groups

Compare with

Means

Medians

DOX

DOX-cLip +

(SD)

(SD)

Saline

solution

DOX-cLip

iRGD

DOX-HA-Lip

Saline

23.2 (0.5)

24.0 (0.4)

--

--

--

--

--

DOX solution

24.6 (0.8)

24.0 (0.7)

N.S.

--

--

--

--

DOX-cLip

27.0 (0.8)

26.0 (0.8)

**

*

-

--

--

DOX-cLip +

30.0 (1.4)

28.0 (1.3)

**

**

*

--

--

DOX-HA-Lip

29.6 (1.5)

30.0 (1.5)

**

**

*

N.S.

--

DOX-HA-Lip +

35.0 (1.4)

34.0 (1.5)

**

**

**

*

*

iRGD

iRGD

N.S. P > 0.05, * P < 0.05, ** P < 0.01 of log-rank analysis.

Figure Legends

Figure 1. (A) Transmission electron micrographs of DOX-cLip and DOX-HA-Lip. (B) DOX release profiles from DOX-cLip and DOX-HA-Lip in saline at 37 °C. Scale bar represents 100 nm.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Confocal images (200 ×) of cellular uptake on B16F10 and MCF-7 cells after being treated with DOX-cLip and DOX-HA-Lip for 1 h in the absence or presence of 10 mg/mL HA. Cell nuclei were stained with DAPI (blue) and DOX fluorescence was displayed in red.

Figure 3. (A) Quantitative cellular association of DOX-cLip and DOX-HA-Lip on B16F10 cells after incubation for 0.5 h, 1 h, 2 h and 4 h at the DOX concentration of 8µg/mL. (B) Quantitative cellular association of DOX-cLip and DOX-HA-Lip on B16F10 cells after 1 h incubation with different concentrations (4 µg/mL, 8 µg/mL and 16 µg/mL) of DOX formulations. * P < 0.05, ** P < 0.01. Data represent the mean ± SD (n = 3).

Figure 4. HA competitive inhibition assay and cell viability assay. Quantitative cellular association of DOX-cLip and DOX-HA-Lip on B16F10 cells (A) and MCF-7 cells (B) in the absence or presence of 10 mg/mL HA. Cytotoxicity of DOX-cLip and DOX-HA-Lip on B16F10 cells (C) and MCF-7 cells (D) after incubation for 48 h. * P < 0.05, ** P < 0.01. Data represent the mean ± SD (n = 3).

Figure 5. (A-C) The percentages of apoptotic and necrotic cells induced by blank culture medium (A) and culture medium containing DOX-cLip (B) or DOX-HA-Lip (C) were determined by flow cytometer. (D) Relative percentages of cells at each cell phase after treatment with different DOX formulations for 24 h, and cells treated by blank culture medium were used as control. * P < 0.05, ** P < 0.01. Data represent the mean ± SD (n = 3).

Figure 6. (A) WBC cell count of saline, DOX solution, DOX-cLip and DOX-HA-Lip treated rats before and 3 days after intravenous injection. (B) WBC inhibition ratio. Each value represents the mean ± SD (n = 10). * P < 0.01 versus DOX solution group; & P < 0.05 versus DOX-cLip group.

Figure 7. Hematoxylin and eosin (H&E) staining of the major organs (heart, liver, spleen, lung, and kidney) collected from rats that were intravenously injected with 5 mg/kg DOX equivalents of free DOX, DOX-cLip and DOX-HA-Lip for two times. Organs sections were magnified by 40


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times and 200 times (black boxes) (n = 5).

Figure 8. Pharmacokinetics behaviors of DOX-cLip and DOX-HA-Lip in rats after intravenous injection of different DOX formulations with or without iRGD at an equivalent dose of 2 mg/kg of DOX and 10 mg/kg of iRGD (n = 5).

Figure 9. (A) Ex vivo imaging of liposomes in main organs of B16F10 melanoma-bearing C57BL/6 mice at 4 h post-injection with Did labeled cLip, cLip in combination with iRGD, HA-Lip and HA-Lip in combination with iRGD (Did dose 150 µg/kg, iRGD dose 10 mg/kg). (B) The statistical graphs of the fluorescence intensity of blood, organs and tumors based on the semi-quantitative analysis of the ex vivo fluorescence images of mice attained at 4 h after i.v. administration of Did labeled liposomes. * P < 0.05, ** P < 0.01. Data represent mean ± SD (n = 3).

Figure 10. Distribution of DOX solution, DOX-cLip, DOX-cLip in combination with iRGD, DOX-HA-Lip, DOX-HA-Lip in combination with iRGD in B10F10 tumors of melanoma-bearing C57BL/6 mice 4 h after i.v. administration (DOX dose 5 mg/kg, iRGD dose 10 mg/kg). Tumors sections were stained with anti-CD31 (red) for blood vessels and DAPI (blue) for nuclei. The green fluorescence represents DOX. Bars represent 100 µm.

Figure 11. Antitumor effect. (A) Average tumor volumes of mice bearing B16F10 melanoma tumors after treated with DOX solution, DOX-cLip, DOX-cLip in combination with iRGD, DOX-HA-Lip, DOX-HA-Lip in combination with iRGD (DOX dose 5 mg/kg, iRGD dose 10 mg/kg) at day 12, 15 and 18 post implantation. The saline group was used as control. Data represent the mean ± SD (n = 10). (B) Kaplan-Meier survival curves of different formulations treated B16F10 tumor-bearing mice. Data represent mean ± SD (n = 12).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. (A) Transmission electron micrographs of DOX-cLip and DOX-HA-Lip. (B) DOX release profiles from DOX-cLip and DOX-HA-Lip in saline at 37 °C. Scale bar represents 100 nm. 349x379mm (72 x 72 DPI)


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Figure 2. Confocal images (200 ×) of cellular uptake on B16F10 and MCF-7 cells after being treated with DOX-cLip and DOX-HA-Lip for 1 h in the absence or presence of 10 mg/mL HA. Cell nuclei were stained with DAPI (blue) and DOX fluorescence was displayed in red. 660x408mm (72 x 72 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. (A) Quantitative cellular association of DOX-cLip and DOX-HA-Lip on B16F10 cells after incubation for 0.5 h, 1 h, 2 h and 4 h at the DOX concentration of 8µg/mL. (B) Quantitative cellular association of DOXcLip and DOX-HA-Lip on B16F10 cells after 1 h incubation with different concentrations (4 µg/mL, 8 µg/mL and 16 µg/mL) of DOX formulations. * P < 0.05, ** P < 0.01. Data represent the mean ± SD (n = 3). 2291x874mm (72 x 72 DPI)


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Figure 4. HA competitive inhibition assay and cell viability assay. Quantitative cellular association of DOXcLip and DOX-HA-Lip on B16F10 cells (A) and MCF-7 cells (B) in the absence or presence of 10 mg/mL HA. Cytotoxicity of DOX-cLip and DOX-HA-Lip on B16F10 cells (C) and MCF-7 cells (D) after incubation for 48 h. * P < 0.05, ** P < 0.01. Data represent the mean ± SD (n = 3). 2374x1833mm (72 x 72 DPI)



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Figure 5. (A-C) The percentages of apoptotic and necrotic cells induced by blank culture medium (A) and culture medium containing DOX-cLip (B) or DOX-HA-Lip (C) were determined by flow cytometer. (D) Relative percentages of cells at each cell phase after treatment with different DOX formulations for 24 h, and cells treated by blank culture medium were used as control. * P < 0.05, ** P < 0.01. Data represent the mean ± SD (n = 3). 458x333mm (72 x 72 DPI)


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Figure 6. (A) WBC cell count of saline, DOX solution, DOX-cLip and DOX-HA-Lip treated rats before and 3 days after intravenous injection. (B) WBC inhibition ratio. Each value represents the mean ± SD (n = 10). * P < 0.01 versus DOX solution group; & P < 0.05 versus DOX-cLip group. 2333x874mm (72 x 72 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7. Hematoxylin and eosin (H&E) staining of the major organs (heart, liver, spleen, lung, and kidney) collected from rats that were intravenously injected with 5 mg/kg DOX equivalents of free DOX, DOX-cLip and DOX-HA-Lip for two times. Organs sections were magnified by 40 times and 200 times (black boxes) (n = 5). 705x396mm (72 x 72 DPI)


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Figure 8. Pharmacokinetics behaviors of DOX-cLip and DOX-HA-Lip in rats after intravenous injection of different DOX formulations with or without iRGD at an equivalent dose of 2 mg/kg of DOX and 10 mg/kg of iRGD (n = 5). 1237x874mm (72 x 72 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 9. (A) Ex vivo imaging of liposomes in main organs of B16F10 melanoma-bearing C57BL/6 mice at 4 h post-injection with Did labeled cLip, cLip in combination with iRGD, HA-Lip and HA-Lip in combination with iRGD (Did dose 150 µg/kg, iRGD dose 10 mg/kg). (B) The statistical graphs of the fluorescence intensity of blood, organs and tumors based on the semi-quantitative analysis of the ex vivo fluorescence images of mice attained at 4 h after i.v. administration of Did labeled liposomes. * P < 0.05, ** P < 0.01. Data represent mean ± SD (n = 3). 635x589mm (72 x 72 DPI)


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Figure 10. Distribution of DOX solution, DOX-cLip, DOX-cLip in combination with iRGD, DOX-HA-Lip, DOXHA-Lip in combination with iRGD in B10F10 tumors of melanoma- bearing C57BL/6 mice 4 h after i.v. administration (DOX dose 5 mg/kg, iRGD dose 10 mg/kg). Tumors sections were stained with anti-CD31 (red) for blood vessels and DAPI (blue) for nuclei. The green fluorescence represents DOX. Bars represent 100 µm. 559x423mm (72 x 72 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 11. Antitumor effect. (A) Average tumor volumes of mice bearing B16F10 melanoma tumors after treated with DOX solution, DOX-cLip, DOX-cLip in combination with iRGD, DOX-HA-Lip, DOX-HA-Lip in combination with iRGD (DOX dose 5 mg/kg, iRGD dose 10 mg/kg) at day 12, 15 and 18 post implantation. The saline group was used as control. Data represent the mean ± SD (n = 10). (B) Kaplan-Meier survival curves of different formulations treated B16F10 tumor-bearing mice. Data represent mean ± SD (n = 12). 1249x1666mm (72 x 72 DPI)


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Graphical abstract 655x423mm (72 x 72 DPI)


Coadministration of Oligomeric Hyaluronic Acid-Modified Liposomes

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