Sialic Acid-anchored Micelles: a Hierarchical Targeting Device for

Subscriber access provided by University of Sussex Library

Article

Sialic Acid-anchored Micelles: a Hierarchical Targeting Device for Enhanced Tumor Tissue Accumulation and Cellular Internalization Meng-Lu Zhu, Xiao-Ling Xu, Xiao-Juan Wang, Nan-Nan Zhang, Kong-Jun Lu, Jing Qi, Fei-Yang Jin, Di Liu, and Yong-Zhong Du Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00649 • Publication Date (Web): 15 Aug 2018 Downloaded from http://pubs.acs.org on August 16, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39 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

Molecular Pharmaceutics

Sialic Acid-anchored Micelles: a Hierarchical Targeting Device for Enhanced Tumor Tissue Accumulation and Cellular Internalization Meng-Lu Zhua#, Xiao-Ling Xub#, Xiao-Juan Wangb, Nan-Nan Zhangb, Kong-Jun Lub, Jing Qib, Fei-Yang Jinb, Di Liub, Yong-Zhong Dub* a The Fourth Affiliated Hospital, Zhejiang University, School of Medicine, Yiwu, 322000, China; b Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, 866 Yu-Hang-Tang Road, Hangzhou 310058, China;

#

These authors contributed equally to this work;

*

Corresponding authors: Yong-Zhong Du, Institute of Pharmaceutics, College of

Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China, E-mail: [email protected].

ACS Paragon Plus Environment

Molecular Pharmaceutics 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

Abstract

Targeted Drug Delivery Systems (TDDS) have attracted wide attention with reduced drug side effects and improved anti-tumor efficacy in comparison with traditional preparations. While targeting moiety in existing TDDS principally focused on recognition of receptors on the surface of tumor cells, accumulation into tumor tissue only could be performed by enhanced permeability and retention effect and active transportation into tumor cells. Doxorubicin (DOX)-loaded sialic acid-dextran (Dex)-octadecanoic acid (OA) micelles (SA-Dex-OA/DOX) were designed for targeting hepatocellular carcinoma effectively. The synthesized conjugates could self-aggregate to form micelles with a critical micelle concentration of 27.6 µg·mL-1 and diameter of 54.53±3.23 nm. SA-Dex-OA micelles incorporated with 4.36% DOX loading content could prolong in vitro drug release to 96 h with 80 % of final release. Cellular transportation studies revealed that SA-Dex-OA micelles mediated more efficient DOX delivery into Bel-7402 cells than those without SA modification. In vivo biodistribution test demonstrated that SA-Dex-OA/ICG micelles showed 3.05-fold higher accumulation into Bel-7402 tumors. The recognition of overexpressed E-selectin in inflammatory tumor vascular endothelial cells led to large accumulation of SA-Dex-OA/ICG micelles into tumor tissue, and the E-selectin upregulated on the surface of tumor cells contributed to active cellular transportation into tumor cells. Accordingly, SA-Dex-OA/DOX exhibited prior suppression on Bel-7402 tumor growth to Dex-OA/DOX micelles and free DOX (the tumor inhibition: 79.2 % vs 61.0 % and 51.3%). These results suggest that SA-functionalized


Page 2 of 39

Page 3 of 39 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


micelles with dual targeting properties has high potential for liver cancer therapy. Keywords: Sialic acid, Micelles, E-selectin, Tumor tissue accumulation, Targeting therapy



Page 4 of 39

1 Introduction

Hepatocellular carcinoma (HCC) is the most common malignant tumor, taking the fifth place in the world in terms of tumor occurrence1. Incidence of HCC increases year by year2. Even if remarkable achievements have been obtained in HCC treatment, the HCC related deaths are still high. In our country, about 383,000 people died of HCC each year, accounting for 51% of the global HCC deaths3. The clinical treatment of HCC mainly focusses on conventional surgery, chemotherapeutics, radiotherapy and biotherapeutics. Whereas as one of the major treatment frequently used in HCC therapy, chemotherapeutics is generally limited to its low efficacy induced by fast clearance, insufficient tumor accumulation and severe side effects4, 5. In the past years, nanocarrier-based treatments were expected to reduce side effects of chemotherapeutics and augment their therapeutic index

6-10

. Polymeric

micelles, as one of the potent nanocarriers, have gained widespread attention11-13. They were fabricated with hydrophobic cores and hydrophilic shells. The inner core offers space and capacity to encapsulate hydrophobic drugs, while the outer shell enables micelles remain stable in the blood circulation14, 15. Many benefits of micelles had spread out before us, such as the accumulation into tumors via the passive enhanced permeability and retention (EPR) effect and the prolonged circulation time in case of phagocytosis of the reticuloendothelial system. Thanks to some specific receptors upregulated on the surface of tumor cells, ligand-functionalized micelles could be further prepared in terms of active targeting delivery16, 17. Folate receptor is one of the specific proteins up-regulated on cell


Page 5 of 39 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


membrane of tumor cells. As a ligand, folic acid was frequently used to augment targeting therapy18. Based on the synergistic effects between enhanced accumulation and controlled release, Tanshinone IIA-loaded micelles showed tumor growth inhibition and prolonged survival time in a mouse HCC-xenograft model19. However, recognition of receptors on tumor cells by micelles only succeeded in dealing with cellular uptake, while the tumor tissue accumulation is still limited with EPR effect. Therefore, to achieve desirable effectiveness, a hierarchical targeting device for improved tumor tissue accumulation and active cellular uptake is indispensable. Sialic acid (SA), found at the terminal of glycoproteins and glycolipids on the surface of cells, is a family of 9-carbon carboxylated monosaccharides. It is a component of sialyl Lewisx antigen which participates in E-selectin binding20. E-selectin is an adhesion molecule that is specifically expressed on the surface of inflammatory vascular endothelial cells21 (VECs) and tumor cells22-24. Consequently, TDDS depending on E-selectin was on track to meet the goal for hierarchical targeting therapy. Firstly, upregulated E-selectins on tumor VECs could promote distribution of micelles into tumor tissues. Once arrived, overexpression of E-selectin on the surface of tumor cells could further recognize micelles and realize an active transportation into tumor cells. Several TDDSs have been reported to anchor SA onto the surface of nanocarrier to improve their distribution at lesion by interaction with E-selectin. The E-selectin-mediated targeting therapy had produced a marked effect on ameliorating inflammatory response25, suppressing phagocytosis of immunocyte26, together with elevating quality of life in acute kidney injury murine model27. Above



Page 6 of 39

all, E-selectin-mediated hierarchical targeting nanocarrier for both tumor tissue and tumor cell accumulation hasn’t been investigated. In this regard, a novel DOX-loaded Sialic acid-Dextran-Octadecanoic Acid (SA-Dex-OA/DOX) conjugate was designed to improve tumor tissue accumulation and tumor cells transportation effectively. SA-Dex-OA conjugates were synthesized through esterification reaction, and SA-Dex-OA/DOX micelles were then prepared by solvent diffusion method. The chemical structure, critical micelle concentration (CMC), size, morphology, drug-loading efficiency and drug release kinetics of SA-Dex-OA/DOX micelles were then investigated. Moreover, in vitro anti-tumor activity, biodistribution and therapeutic potential of SA-Dex-OA/DOX micelles were further studied, taking Dex-OA/DOX micelles and free DOX as control.

2 Materials and methods

2.1 Materials

Dex

(Mw=10

kDa)

was

obtained

from

Bio

Basic

(USA).

4-Dimethylaminopyridine (DMAP) and N, N′-dicyclohexylcarbodiimide (DCC) were obtained from Shanghai Medped (PRC). HCl·DOX was gifted from Hisun Pharm (PRC). Thiazolyl blue tetrazolium bromide (MTT), fluorescein-5-lsothiocyanalte (FITC) and indocyanine green (ICG) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies (Caspase-3, CD62E) were purchased from Abcam. Cell death detection kit (TUNEL Apoptosis Assay Kit) was purchased from Roche (Nutley, NJ, USA). Trypsin and Dulbecco’s Modified Eagle Medium (DMEM) were


Page 7 of 39 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


obtained from Gibco (USA). Fetal bovine serum (FBS) was purchased from Sijiqing Biologic (PRC). The human hepatoma cells (Bel-7402) and human normal cells (LO2) were got from the Second Affiliated Hospital, Zhejiang University School of Medicine. BALB/C+ nu/F1 nude mices (aged 4-6 weeks, 18-20g) were approved from the Zhejiang Medical Animal Centre for in vivo experiments. All the mice received humane care and all animal research experiments were performed on the basis of the National Institutes of Health (NIH, USA) guidelines and Laboratory Animal Ethics Committee of Zhejiang University.

2.2 Synthesis of SA-Dex-OA conjugates

There were two steps about synthesis of SA-Dex-OA: (1) The carboxyl group of OA was esterified with the hydroxyl group of Dex. (2) By acylation reaction for synthesis of SA-Dex-OA. Briefly, OA was activated with DCC and DMAP in anhydrous dimethyl sulfoxide (DMSO) in a round-bottom flask and left to stir for 2 h at 60℃ under nitrogen atmosphere (OA:DCC:DMAP=1:3:0.3, mol:mol, OA=2.635g). Then Dex (Dex: OA: DCC: DMAP=500:263.5:573.2:33.8, mg:mg, Dex=5g) was then added and reacted at 60℃ for 48 h under stirring and protection of nitrogen. After the reaction, the final products were further purified via dialysis through a dialysis bag (MWCO: 3.5 kDa) against deionized water for 48 h. The dialyzed solution was then centrifuged at 5000 rpm to remove water-insoluble by-products. After lyophilization, the crude products were purified by ethanol to remove unreacted OA. Dex-OA



(3.015g, 39% yield) was finally obtained. For synthesis of SA-Dex-OA, dropwise 4-toluene sulfonyl chloride was transferred to 5 mL of SA dissolved DMSO (anhydrous), followed by stirring with nitrogen protection at room temperature for 4 h (SA:4-toluene sulfonyl chloride: DMAP=1:1.2:5, mol: mol). 1,6-diaminohexane was then added and reacted at 100℃ for 48 h under nitrogen atmosphere (SA:1,6-Diaminohexane=1:1, mol: mol). Dex-OA, DMAP and 4-toluene sulfonyl chloride in 10 mL of DMSO were reacted under magnetic stirring with nitrogen protection at room temperature for 4 h (Dex-OA: DMAP: 4-toluene sulfonyl chloride=1:150:36, mol: mol). Then SA-Hex was added to the reaction system and reacted at 100℃ for 48 h under nitrogen atmosphere. The mixture was transferred to a dialysis bag (MWCO 3.5 kDa) when the reaction was over and dialyzed against deionized water for 48 h with intermittent replacement of water. The SA-Dex-OA powder was obtained by centrifugation and lyophilization of the dialyzed solution.

2.3 Preparation of blank and DOX-loaded SA-Dex-OA micelles

SA-Dex-OA/DOX micelles were prepared through solvent diffusion method28. Firstly, HCl·DOX was reacted with double-mole triethylamine in DMSO solution for 24 h in order to harvest DOX base29 and added dropwise to the polymer solution. The preparation of Dex-OA/DOX micelles was the same as above.


Page 8 of 39

Page 9 of 39 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


2.4 Characterization of SA-Dex-OA/DOX micelles

The structure confirmation of SA-Dex-OA/DOX was conducted via proton nuclear magnetic resonance (1H NMR) spectrophotometer. The CMC values of Dex-OA and SA-Dex-OA micelles were evaluated by fluorescent spectroscopy. Pyrene dissolved in acetone (1.2×10-3 mg·mL-1) was used as fluorescent probe according to previous references30. The acetone was evaporated when the oven temperature was set at 50℃. 5 mL of polymer solution (3.0×10-3 to 1.0 mg·mL-1) were then added. Fluorescence spectra of the polymer/pyrene solutions were collected with F-2500 Hitachi Japan luminescence spectrometer. The excitation wavelength was set at 337 nm. And the emission intensity ratio of peak at 374 nm and 385 nm was recorded to calculate CMC. The size distribution and morphologies of micelles were monitored using zetasizer (3000HS, Malvern Co.,UK) and transmission electron microscopy (TEM, JEM-1230; JEOL,Japan). Each sample was deposited onto a copper grid and stained with 2% (w/v) phosphotungstic acid for viewing.

2.5 Encapsulation efficiency and drug loading capacity

SA-Dex-OA/DOX micelles were diluted in DMSO to dissociate the micelles, and the DOX amount was then detected using a fluorescence spectrophotometry (Ex= 505 nm, Em=565 nm, slit=5.0 nm). The drug encapsulation efficiency (EE%) and drug loading (DL%) were calculated according to the following equations, respectively:



×100 %, ×100 %.

2.6 In vitro drug release behavior

The DOX release from Dex-OA/DOX and SA-Dex-OA/DOX micelles at pH 7.4 were studied using dialysis method. Briefly, 1 mL of DOX-loaded micelles were transferred into a dialysis membrane (MWCO: 3.5 kDa) and the whole dialysis bag was put into a tube filled with 25 mL of phosphate-buffered saline (PBS) solution at pH 7.4. The experiment was performed in an incubation shaker at 37°C at 70 rpm. At predetermined time intervals (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h), all the medium was picked up and replaced with new PBS. DOX content was detected using fluorescence spectrophotometer.

2.7 Cell culture

Bel-7402 and LO2 cells were cultured in DMEM medium at 37℃ in an atmosphere containing 5% CO2. The media consisted of 10% FBS and penicillin/streptomycin (100 U mL-1, 100 U mL-1). Cells were digested regularly using trypsin/EDTA.

2.8 In vitro cytotoxicity assay

Cell viability assay was measured via MTT assay30 as reported before. Bel-7402 and LO2 cells were chosen as model cells for in vitro cytotoxicity assay. Each well in 96-well plate was filled with 1×104 cells and they were incubated 24 h to allow cell


Page 10 of 39

Page 11 of 39 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


attachment. Cells were then exposed to HCl·DOX, Dex-OA-loaded micelles and blank micelles with serial concentrations (including 2, 5, 10, 20, 50, 100, 200, 500 and 1000 µg·mL-1) for 48 h. After that, 20 µL of MTT solution (5mg·mL-1) in PBS was added to incubate for another 4 h at 37℃. The absorbance at 570 nm of each well was detected by a microplate reader (Bio-Rad, Model 680, USA). Cell viability was calculated as below: ×100% Bel-7402 cells were incubated for 24 h with an equivalent concentration of DOX (1µg·mL-1) in different preparations (HCl·DOX, Dex-OA and SA-Dex-OA), then cells were stained using Calcein-AM cell viability assay kit (Fluorometric, Invitrogen, USA). The cellular fluorescence was captured using fluorescence microscopy.

2.9 Cellular uptake

For cellular uptake, each well in 24-well plate was filled with 3×104 cells (Bel-7402 or LO2 cells) and they were incubated 24 h to allow cell attachment. Cells were then exposed to ODA-FITC-loaded micelles and incubated for another 1 h, 3 h, 6 h, and 12 h. The cellular fluorescence was captured using fluorescence microscope. For the competitive experiment, Bel-7402 cells were incubated firstly with free SA (1.0, 2.0, 4.0, 6.0 and 8.0 mg·mL-1) for 1 h and then incubated with ODA-FITC-loaded SA-Dex-OA micelles. Afterwards, nuclei were stained with 20 uL of Hoechst 33342 (10-2 mg·mL-1) for 30 min. The cellular fluorescence was examined under fluorescence microscope. For the quantitative analysis, cellular fluorescence



was measured by flow cytometry (FC 500 MCL; Beckman Coulter, USA).

2.10 Immunofluorescence

Each well in 6-well plate was filled with 1×105 cells (Bel-7402 or LO2 cells). After 24 h, cells were exposed to SA-Dex-OA/DOX and Dex-OA/DOX micelles for 3 h. Then Bel-7402 and LO2 cells were fixed with 4% paraformaldehyde for 15 min. 5% bovine serum albumin in PBS were used to block nonspecific sites for 1 h. Next, cells were simultaneously labeled with a mouse monoclonal CD62E antibody (10 µg·mL-1) overnight at 4℃. Afterwards, cells were incubated with secondary antibody (1:200 dilution) for 2 h at room temperature in a dark chamber. Nuclei was stained with DAPI and cells were analyzed using confocal laser scanning microscopy.

2.11 Biodistribution

To investigate the biodistribution, female nude mice bearing Bel-7402 tumors were treated with ICG-tetrabutylammonium iodide loaded Dex-OA micelles or SA-Dex-OA micelles (2 mg·kg-1). The anesthetized mice were imaged at the pre-setting time (6, 12, 24 and 48 h) after different treatment using IVIS® Spectrum system (Cambridge Research & Instrumentation, Inc., Woburn, MA, USA). At 48 h, the mice were sacrificed and representative organs were collected and imaged to observe the fluorescence signals. Tumors were then transferred into liquid nitrogen and stained with anti-CD62E antibody. Fluorescence signals in dissected organs were determined using IVIS® Spectrum system. To analyze the DOX concentration in


Page 12 of 39

Page 13 of 39 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


tumors, representative organs of mice treated with DOX-loaded SPD micelles (5.0 mg/kg) were harvested at predetermined time (6, 12, 24 and 48 h) and imaged by the IVIS® Spectrum system.

2.12 In vivo therapeutic efficacy

Antitumor effect of SA-Dex-OA/DOX micelles was determined using Bel-7402 tumor bearing nude mice as model. Bel-7402 cells were inoculated subcutaneously in the athymic nude mice. After tumor volume reached ~100 mm3, mice were randomly grouped into four groups (n=6), and were intravenously administered with saline, Adriamycin, Dex-OA/DOX and SA-Dex-OA/DOX micelles at an equivalent DOX dose of 2mg/kg every day for a total of 24 days. The body weight and tumor volume were recorded every 3 days thereafter. Tumor volumes were calculated as below:

. L stands for the longest diameter, while W means the shortest one (mm). The

inhibition

of

tumor

growth

(%)

was

calculated

as

below:

. Wc and Wt denote the average tumor volume for the control group and treatment group, respectively. Tumors were fixed in 4% buffered formalin and embedded in paraffin blocks for histological analysis

by

hematoxylin

and

eosin

(H&E)

staining.

For

immunohistochemical staining, Ki67 antibody was applied to tumor sections. Besides, apoptotic cells in tumors were detected using TUNEL apoptosis kit. All the images were captured using fluorescence microscopy.



2.13 Western blot

Protein from Tumor tissues and Bel-7402 cells was extracted in cell-lysis buffer (RIPA). The concentration of protein was then detected by bicinchoninic acid assay (Beyotime Biotechnology, Shanghai, China). An equivalent protein was fractionated by 10% SDS-PAGE, and transferred onto PVDF membranes. 5% milk dissolved in TBST was used to block nonspecific sites on membranes for 90 min at room temperature. Then primary antibodies were used to incubate with membrane overnight at 4℃. Afterwards, appropriate secondary antibodies were used to incubate with membranes for 1 h at room temperature. Finally, proteins on the membrane were analyzed using enhanced chemiluminescence kit (Bio-Rad) and quantified by densitometry using Image Lab 3.0 software. GAPDH was used as control protein31, 32. 2.14 Adverse effects After 24 days, whole blood samples of all the mice were collected and used to detect cell counts including white blood cells (WBC) and platelets (PLT). Serums were separated after centrifuged at 2000 rpm for 10 min. They were used to assess alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in livers. Whole blood and serum samples were detected by an automated Beckman Analyzer (Beckman Instruments GmbH, Munich, Germany). Major organs were fixed in 4% buffered formalin and embedded in paraffin blocks for histological analysis by H&E staining. Hearts were dissected and homogenized in a high-speed blender. After centrifugation, the supernatants were picked up to test the Ca2+-ATPase activity in hearts using Ca2+-ATPase kit.


Page 14 of 39

Page 15 of 39 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


2.14 Statistical analysis

The results were displayed as mean ± SD, followed by statistical analysis by SPSS statistical package (version 14.0). The difference between two groups was compared using Student’s t-test, while multigroup comparisons were measured using two-way ANOVA followed by Bonferroni post hoc tests. A significant difference was set at **p

Sialic Acid-anchored Micelles: a Hierarchical Targeting Device for

Recommend Documents