Redox-responsive chitosan oligosaccharide-SS-Octadecylamine polymeric carrier for efficient anti-Hepatitis B Virus gene therapy

Accepted Manuscript Title: Redox-responsive Chitosan oligosaccharide-SS-Octadecylamine polymeric carrier for efficient anti-Hepatitis B Virus gene therapy Authors: Jing Miao, Xi-qin Yang, Zhe Gao, Qian Li, Ting-ting Meng, Jia-ying Wu, Hong Yuan, Fu-qiang Hu PII: DOI: Reference:

S0144-8617(19)30189-4 https://doi.org/10.1016/j.carbpol.2019.02.047 CARP 14620

To appear in: Received date: Revised date: Accepted date:

2 October 2018 8 February 2019 14 February 2019

Please cite this article as: Miao J, Yang X-qin, Gao Z, Li Q, Meng T-ting, Wu J-ying, Yuan H, Hu F-qiang, Redox-responsive Chitosan oligosaccharide-SS-Octadecylamine polymeric carrier for efficient anti-Hepatitis B Virus gene therapy, Carbohydrate Polymers (2019), https://doi.org/10.1016/j.carbpol.2019.02.047 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Redox-responsive

Chitosan

oligosaccharide-SS-Octadecylamine

polymeric carrier for efficient anti-Hepatitis B Virus gene therapy

Jing Miaoa,b,1, Xi-qin Yanga,1, Zhe Gaob, Qian Lib, Ting-ting Meng a, Jia-ying Wub,

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Hong Yuana, Fu-qiang Hua,*

College of Pharmaceutical Science, Zhejiang University, Hangzhou 310003, PR

The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou

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b

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China

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These authors contributed equally to this work

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1

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310058, PR China

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*Corresponding author: Tel: 86-571-88208441; Fax: 86-571-88208439; E-mail:

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[email protected]

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Highlights     

Chitosan-SS-Octadecylamine was designed and investigated its properties. CSSO exhibited low cytotoxicity and could bind with DNA. CSSO/DNA could realize subcellular target and released DNA rapidly from endosomes. CSSO/DNA showed a significant increased inhibition ability on HBV secretion. CSSO is a promising redox-responsive gene carrier for efficient anti-HBV therapy.

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Abstract DrzBC and DrzBS (10-23DNAzyme) could block the expression of HBV e- and s- gene respectively. But the application of 10-23DNAzyme was limited owing to the lack of appropriate delivery vehicles. Chitosan oligosaccharide-SS-Octadecylamine

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(CSSO), a redox-responsive nano-sized polymeric carrier, could self-aggregate and bind with DNA by electrostatic interaction at proper mass ratio. Compared with the

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traditional commercial carrier Lipo2000, CSSO exhibited lower cytotoxicity, efficient

cellular uptake by targeting cells, and rapidly DNA released in cytoplasm after

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escaping from endosomes. Including the same DNA concentration, Lipo2000/(DrzBC

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or DrzBS) showed maximum inhibitory rate on HBeAg (47.29±1.86%) and HBsAg

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(33.58±0.72%) secretion after 48h incubation, and then both decreased. In contrast,

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HBeAg secretion inhibition by CSSO/DrzBC and HBsAg secretion inhibition by

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CSSO/DrzBS were up to 73.86±1.77% and 67.80±2.51% at 48h, and further increased to 83.83±2.34% and 76.79±2.18% at 72h, respectively. CSSO is a promising

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redox-responsive polymeric carrier for efficient anti-Hepatitis B Virus gene therapy.

Key words: Chitosan oligosaccharide-SS-Octadecylamine; redox-responsive;

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10–23DNAzyme; anti-Hepatitis B Virus; gene therapy.

1. Introduction Hepatitis B is a viral infectious disease which seriously endangers human’s health. Patients with hepatitis B could develop into liver fibrosis, cirrhosis and even liver

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cancer. At present, the traditional anti-HBV drugs have many problems, such as long course of treatment, large dosage, low efficacy, side effects and drug resistance. Therefore, in order to reduce the side effects and improve the antiviral effect, it is necessary to design new anti-HBV drugs which target the certain steps in the

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replication process of HBV. In recent years, emerging genetic drugs have brought new hope to hepatitis B

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treatment (Liu, Wen, Huang, & Wei, 2013). Among them, deoxyribozyme

10-23DNAzyme has attracted much attention because of its unique advantages in the

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treatment of viral infectious diseases (Kumar, Chaudhury, Kar, & Das, 2009; Sood,

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Gupta, Bano, & Banerjea, 2007; TAKAHASHI, HAMAZAKI, & HABU, 2004).

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10-23DNAzyme，the 23rd clone from round 10 during the course of 10 rounds in vitro

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selection (Santoro & Joye, 1997)，is a single strand DNA molecule with the activity of

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phosphatase. 10-23DNAzyme consists of 15 deoxynucleosides to form a catalytic domain with a substrate identification domain consisting of 9 deoxynucleosides (the

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base sequence complements the sequence of the substrate RNA initiating AUG). The

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substrate identification sequence on both sides of the substrate is specific to the RNA substrate, thus 10-23DNAzyme can combine specifically with target mRNA. Meanwhile, the central catalytic domain cleaves mRNA at the purine-pyrimidine

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junctions (A·U sites), and thereby blocks the expression of corresponding mRNA. In our previous research, we designed and synthesized the HBV-specific 10-23 DNAzyme named DrzBC, which can specifically block the expression of HBV e-gene in vitro (Miao et al., 2012), suggesting its potential application in the treatment of

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hepatitis B. However, the application of 10-23DNAzyme was restricted by several problems such as transmembrane ability, stability and intracellular targeting. Thus, an appropriate delivery vehicle is crucial to improve the treatment effect of

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10-23DNAzyme. In fact, there are many kinds of carriers could be chosen, but a selection puzzle still exist. As we known, viral vehicles have high transfection

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characteristics for gene drugs, but there are many safety problems such as immune

response induced by virus protein and potential virus replication in the body (Thomas,

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Ehrhardt, & Kay, 2003). At present, many researchers use cationic liposomes as

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non-viral vehicles for 10-23DNAzyme (Kumar, Chaudhury, Kar, & Das, 2009; Sood,

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Gupta, Bano, & Banerjea, 2007; TAKAHASHI, HAMAZAKI, & HABU, 2004).

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Unfortunately，the high toxicity and the low gene delivery efficiency limited the

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application scope of cationic polymers, especially in vivo. In our previous research (Miao et al., 2012), we synthesized stearic acid grafted chitosan oligosaccharide

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(CSSA), which used as a gene delivery vehicle for DrzBC. However, with amide

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bonds as the linkers, which have a slow degradation rate within the cellular region, the drug release from CSSA relative slowly. Meanwhile, CSSA cannot release rapidly from endosomes. Therefore, in order to achieve the hepatitis B gene therapy by

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10-23DNAzyme, it is urgent to design a novel and advanced carrier by chemical composition improvement. Considering the high variance of glutathione (GSH) level between intracellular and extracellular space (Meister & Anderson, 1983), redox-responsive polymers are

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particularly attractive for intracellular delivery. Those GSH-responsive polymeric carriers usually contain characteristic disulfide linkages in hydrophilic shell, hydrophobic core, or as the cross-linker (Cheng et al., 2011, Cajot et al., 2011, Wang et al., 2013). Before cellular taken-up, the polymeric carriers are stable, once in the

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intracellular high concentration of GSH, the disulfide linkages of polymers are cleaved and the structure of carriers instantly fall apart and rapidly release drug.

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Based on these principles, GSH-responsive polymers is a promising platform for intracellular drug delivery.

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In this study, we have developed a chitosan based glycolipid-like nanocarrier

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(Chitosan oligosaccharide-SS-Octadecylamine, CSSO) with the function of

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endosomal escape and rapid intracellular drug release owing to the structure of

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reducible disulfide bonds ‘–SS-’. Meanwhile, we designed two kinds of

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10-23DNAzyme specific to HBV e-gene ORF A1816UG and s-gene ORF A157UG, namely, DrzBC and DrzBS. By electrostatic interaction，CSSO could bind with

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DrzBC and DrzBS to form CSSO/DrzBC and CSSO/DrzBS complexes. We studied

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the characteristics of CSSO and its complexes. The cytotoxicity, intracellular localization, and in vitro evaluation of anti-HBV efficiency of CSSO/DrzBC and CSSO/DrzBS complexes were further observed in HepG2.2.15 cell lines as model

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HBV-transfected tumor cells.

2. Materials and Methods 2.1 Materials

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Chitosan oligosaccharide (CSO) with low molecular weight (Mw =18.9 kDa) was obtained by enzymatic degradation of 95% deacetylated chitosan (Mw = 450 kDa, Yuhuan, China). Octadecylamine (ODA) was purchased from Fluka (Milwaukee, WI, USA). The 3, 3′-dithiodipropionic acid (DTPA) was purchased from Tokyo Chemical

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Industry (Tokyo, Japan). NHydroxysuccinimide (NHS) and pyrene were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Fluorescein isothiocyanate (FITC),

were

purchased

from

Sigma

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Methylthiazoletetrazolium (MTT), and 2,4,6-trinitrobenzene sulfonic acid (TNBS) (St.

Louis,

MO,

USA).

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1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from

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Shanghai Medpep Co, Ltd (Shanghai, China). Fetal bovine serum was purchased from

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Sijiqing Biology Engineering Materials Co, Ltd (Zhejiang, China). DrzBC and DrzBS

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were consigned synthesized by Sangon Biotech (Shanghai) C., Ltd. Other chemicals

2.2 Cell culture

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used were of analytical or chromatographic grade.

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HepG2.2.15, hepatitis B virus-transfected human hepatoma cells, were obtained

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from the State Key Lab for Diagnosis and Treatment of Infectious Diseases (Zhejiang University, Hangzhou, China) and cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin, and 380 µg/mL

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geneticin G418 in a humidified atmosphere containing 5% CO2 at 37 ℃. 2.3 Synthesis of CSSO The glycolipid polymer CSSO was synthesized as described in previous study（Hu et al., 2015）. Briefly, ODA was conjugated with 3, 3′-dithiodipropionic acid via amide

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bonding between the amino groups on the ODA and the carboxyl groups on 3, 3′-dithiodipropionic acid. Concretely, 116mg ODA and 90mg DTPA were respectively solved in DMSO solution both at 10.0 mg/mL and mixed, into which 265mg DCC and 16mg DMAP were added. The reaction was performed at 60 °C for 24 h in a nitrogen

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atmosphere and subsequently filtrated to remove the by-products. Then, the mixture was activated by EDC/NHS for 30 min and added dropwise to the chitosan solution.

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The mixture was stirred for 8 h at 60 °C. The resulting mixture was dialyzed against

Deionization water (DI water) for 2 days and centrifuged, and the supernatant was

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lyophilized. The lyophilized product was washed with hot ethanol to remove

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unreacted reagent. Finally, the product was re-dispersed in DI water and lyophilized to

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acquire the CSSO.

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2.4 Preparation of complexes and polyacrylamide gel electrophoresis assay

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Complexes were easily prepared by mixing glycolipid polymers CSSO with DNA (DNA represents DrzBC or DrzBS in this study) followed by vortexing. In brief,

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CSSO was dissolved in DI water for DNA encapsulation. Then DNA solution was

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added to the polymer solution followed by vortex-mixing and incubation for 30 min. A gel retardation assay was performed for the examination of the condensation

ability of polymers to DNA by denaturing 7M urea polyacrylamide gel electrophoresis.

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The complexes (containing 1 μg of DNA), prepared as described above, were equilibrated to 10 μL prior to loading. Electrophoresis was then carried out with a voltage of 100 V for 40 min in TBE buffer solution (1 M Tris, 0.9 M Boric Acid, and 0.01 M EDTA). DNA retardation was visualized by staining with ethidium bromide

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(EB) and a UV lamp. DNase I protection ability was also investigated. Complexes were incubated for 30 min at room temperature, followed by the addition of DNase I. As a control, naked DNA (1 μg) was also treated with DNase I under the same conditions. After 30 min

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incubation, the samples were further treated with EDTA for 10 min to terminate the activation of DNase I, followed by incubation in heparin for 30 min. The released

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DNA from the complexes was assessed as described above. 2.5 Characterization of polymers and complexes

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Glycolipid polymers CSSO was dissolved in D2O of 10.0 mg mL−1, and the

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chemical structures were characterized by 1H NMR on AVANCE DMX 500 NMR

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spectrometer (Burker, Germany). The degrees of amino substitution of polymers were

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determined by the TNBS method (Hu et al., 2012). The critical micelle concentration

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(CMC) of the obtained CSSO in DI water were determined by fluorescence spectroscopy (F-2500, Hitachi Co., Japan) using pyrene as a probe. The particle size

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and zeta potential of the polymers and their corresponding complexes were measured

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by dynamic light scattering (DLS, Zetasizer 3000HS, Malvern Instruments Ltd., U.K.). The morphological examinations were performed by transmission electronic

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microscopy (TEM, STEREOSCAN, LEICA, England). To determine the encapsulation efficiency (EE %) and drug loading (DL %) of

micelles, DNA was labeled with Cy5 to prepare CSSO/Cy5-DrzBC and CSSO/Cy5-DrzBS complexes. After the ultra-centrifugation, the unbound Cy5-DrzBC and Cy5-DrzBS were determined with a fluorescence spectrophotometer (F-2500

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fluorescence spectrophotometer, HITACHI Co, Ltd, Japan). Excitation wavelengths were both at 646nm, emission wavelengths were at 662 nm for DrzBC and 667nm for DrzBS，and slit openings at 5 nm. The EE (%) and DL (%) were calculated by the following equations. (1)

DL%=(Wa-Ws)/ (Wa-Ws+WL)×100%

(2)

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EE%=(Wa-Ws)/Wa×100%

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Where Wa was the amount of total DNA added in system, Ws was the analyzed amount of unbound DNA in supernatant after the ultra-centrifugation, WL was the

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weight of CSSO added in system.

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To measure the GSH-triggered DNA release behavior in mimicking cellular

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environment, Cy5 labeled DNA standard solution with concentration gradient of 0,

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0.5, 1, 2.5, 5.0 ug/mL was prepared. Fluorescence intensity of standard solution was

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determined by fluorescence spectrophotometry (λex = 649 nm, λem = 662 nm for Cy5-DrzBC or 667 nm for Cy5-DrzBS). By linear regression of DNA concentration to

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fluorescence intensity, the standard curve was drawn. Cy5 labeled DNA and CSSO

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complexes were centrifugated for 10 minutes at 13000 rpm, the supernatant was discarded, and then 1 mL Tris hydrochloric acid buffer containing 10 mM GSH (pH 7.2) was added to disperse and precipitate. After incubating in the thermostatic

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oscillator (37°C, 60rpm) for different time, we took samples. The samples were then centrifuged at 13000 rpm for 10 minutes, and 0.6 mL supernatant was removed and measured the fluorescence intensity. The cumulative release of DNA were calculated according to the standard curve.

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2.6 In vitro cytotoxicity Cell cytotoxicity of CSSO, DNA and CSSO/DNA were determined by MTT assay. HepG2.2.15 cells were seeded at a density of 4×103 cells/well in 96-well culture plates and cultured for 24 h under 5% CO2 at 37 °C. Polymers and complexes were

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added into 96-well culture plates. Blank culture medium was treated as a blank control. After 48 h, a 20.0 mL of MTT solution (5.0 mg/mL) was added and further

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incubated for 4 h. Then, the medium was replaced by 200 mL of DMSO to dissolve the purple formazan crystals. The absorbance of each well at 570 nm was measured

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by a microplate reader (Bio-Rad, Model 680, USA). Cell viability was calculated in

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2.7 Intracellular localization

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were plotted from the data of triplicate assays.

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reference to the cells incubated with the culture medium alone. Dose effect curves

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To evaluate DNA intracellular localization in HepG2.2.15, DNA was labeled with Cy5 and glycolipid polymers were labeled with FITC as the methods described before

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(Yan et al., 2013). Then complexes (the DNA concentration was 1μg/mL) were

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prepared as described above. HepG2.2.15 were seeded at a density of 2×10 3 cells/well in 24-well culture plates. After incubation for 12h, cells were treated with prepared complexes for 24h. To label the lysosomes, cells were incubated in prewarmed media

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(37°C) containing Lysotracker blue (LysoTracker blue DND-26, Invitrogen, USA). The corresponding fluorescent images were obtained using confocal laser scanning microscope (CLSM) (Ix81-FV1000, Olympus, Co.). 2.8 In vitro evaluation of anti-HBV efficacy

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HepG2.2.15 cells were seeded in 24-well plate at a density of 3×104 cells/well and incubated at 37℃ in a humid atmosphere with 5% CO2. After 24 h incubation, cells were supplemented with OPTI-MEMI after washing with serum-free culture medium. CSSO, DrzBC, DrzBS, Lipo2000/DrzBC, Lipo2000/DrzBS, CSSO/DrzBC and

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CSSO/DrzBS were added at a final DNA concentrations of 1μmol/L. After incubation for a certain period of time, the cell supernates was replaced with complete medium

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and cells were continually cultured for different periods of time. Enzyme-linked immunosorbent assay(ELISA) Kits (AXSYM System, Abbott, Wiesbaden, Germany)

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were used to determine the content of HBsAg and HBeAg in supernate according to

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3. Results and Discussion

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the manufacturer’s instructions.

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3.1 Synthesis and characterization of polymers Glycolipid polymer CSSO was constructed as shown in Fig. 1A. Firstly, 3,

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3′-dithiodipropionic acid was used as the coupling agent and modified one side of the

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carboxyl groups with octadecylamine at a molar ratio of 1:1. Then the remaining carboxyl group was reacted with the free amino groups of the chitosan by an

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amine-reactive coupling to produce the target CSSO.

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Fig. 1. Synthesis of CSSO. (A) Synthetic scheme of CSSO. (B) 1H NMR spectra,

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from top to bottom: CSO, DTPA, ODA, and CSSO. (C) 1H NMR spectra, from top to

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bottom: DTPA, ODA, and Intermediate A. (D) FT-IR spectra, from top to bottom: CSO, DTPA, ODA, and CSSO.

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The chemical structure of CSSO was analyzed by 1H NMR as shown in Fig.

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1B. The peaks of CSSO at about 1.2ppm were attributed to -CH2- of ODA, indicating CSSO was synthesized. As shown in Table 1, the degree of amino substitution (SD%) of the chitosan chain on CSSO was measured as 6.79±0.15% (molar ratio). The FT-IR

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spectra (Fig. 1D) confirmed the successful synthesis of CSSO. In the spectra of DTPA, the peak 1667.16 cm-1 represented the -C=O carbonyl stretching vibration peak of DTPA. During the preparation of CSSO, the amidation reaction between DTPA and CSO took place. As a result, the stretching vibration absorption of –C=O on DTPA

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shifted to lower wave number, which came into the range of the N-H bending vibration absorption peaks of -NH2 (~1640 cm-1) as shown in the spectra of CSSO. Table 1. Characterizations of CSSO and CSSO/DNA complexes. Zeta potential

Diameter

PDI

CMC

SD (%)

(mV)

(nm)

CSSO

21.10±0.50

123.00±10.98

0.29±0.02

65.84±1.11

6.79±0.15

CSSO/DrzBC

17.90±1.19

214.75±3.43

0.19±0.04

-

-

CSSO/DrzBS

17.43±1.03

230.70±6.16

0.23±0.02

-

EE (%)

DL (%)

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(µg/mL) -

-

96.48±0.27

1.582±0.004

96.45±0.33

1.581±0.005

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Micelles

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The PDI, CMC, SD, EE, and DL values represent the polydispersity index, critical micelle

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concentration, degree of amino substitution, drug encapsulation efficiency and drug loading,

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respectively. Data represent the mean ± standard deviation (n = 3).

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The synthesized glycolipid polymers CSSO could self-assemble easily into

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nano-scaled micelles. Fig. 2A showed the variation of the I1/I3 ratio against the

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logarithmic concentration (Log C) of CSSO. The inflection point corresponds to the critical micelle concentration (CMC) value of CSSO was 65.84±1.11 µg/mL.

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The average size of CSSO was determined as 123.00±10.98 nm, the zeta potential

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of CSSO was determined as 21.10±0.50 mV (Table 1). The spherical morphologies of CSSO micelles was revealed by TEM image (Fig. 2B). The encapsulation efficiency (EE%) of CSSO/Cy5-DrzBC and CSSO/Cy5-DrzBS

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complexes were both around 96%, which inhibited excellent DNA encapsulation ability of CSSO. However, the drug loading (DL %) of the complexes were just around 1.58%, owing to the preparation mass ratio was up to 60:1 (chosen by the following examination results).

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Fig. 2. Characterizations of CSSO. (A) Variation of intensity ratio (I1/I3) vs concentration of CSSO (▲). (B)The transmission electron microscopy (TEM) images

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of CSSO.

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3.2 Polyacrylamide gel electrophoresis assay

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The binding ability between the DNA and polymers was assessed using a

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denaturing 7M urea polyacrylamide gel electrophoresis assay. Considering the similar physical properties of DrzBC and DrzBS, we chose DrzBC as representative to

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investigate the proper mass ratio of CSSO/DNA complexes. As shown in Fig. 3A,

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when the mass ratio of CSSO/DrzBC complexes was 20:1 (critical value), DrzBC started to be retarded in the sample hole. As a critical value pot, there was just a little

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difference in comparison to naked DrzBC, but suggested that polymers were able to compact DNA when the mass ratio was 20:1 or above 20:1. Therefore, we investigated the ratio of 60:1 and 100:1, and finally chose the mass ratio of 60:1 to

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prepare CSSO/DNA complexes for the following studies. It is noteworthy that in the lane 2 (Lipo2000/DrzBC), either in the sample hole or the electrophoretic band, there were no stained DNA observed. The reason for this phenomenon might be the powerful ability of Lipo2000 to compact DNA, leading to the difficulty in staining DNA by EB. However, too powerful compact ability would made the DNA release to 14

be difficult. Complexes at the mass ratio of 60:1 were prepared and the protection effect of polymers to DrzBC against DNase I was displayed in Fig. 3B. Naked pDNA was completely digested (i.e., no bands were observed). By contrast, clear pDNA

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indicating polymers were able to protect DrzBC from enzymolysis.

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migration bands for CSSO/DrzBC was noted via the substitution of heparin,

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Fig. 3. Gel retardation analyses and DNase I protection assay. (A) Gel retardation analyses of the complexes. Lane 1 is naked DrzBC; lane 2 is Lipo/DrzBC; lanes 3-5

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are CSSO/DrzBC complexes prepared at mass ratios of 20, 60, 100, respectively. (B)

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DNase I protection assay. Lane 1 is naked DrzBC untreated with DNase I; lane 2 is naked DrzBC treated with DNase I; lanes 3 is CSSO/DrzBC untreated with DNase I;

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lane 4 is CSSO/DrzBC treated with DNase I. CSSO/DrzBC were prepared at a mass ratio of 60:1.

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3.3 Preparation and characterization of complexes Complexes were easily prepared by mixing polymers with DrzBC or DrzBS at the

mass ratio of 60:1. The characterizations of CSSO/DrzBC and CSSO/DrzBS were exhibited in Table 1. The zeta potentials of complexes were all positive, and the average diameter were 210-240 nm which were larger than CSSO polymers owing to 15

DNA adherence on the surface of CSSO by electrostatic interaction. There was no obvious difference between CSSO/DrzBC and CSSO/DrzBS in zeta potential or average diameter because of the similarity in physical properties between DrzBC and DrzBS. In addition, all the polymers in Table 1 shown low polydispersity index (PDI

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< 1). Herein, PDI measured by dynamic light scattering (DLS) indicates the size distribution by scattering intensity. Therefore, the PDI value is smaller, the particle

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size is more uniform. Fig. 4A showed the TEM images of CSSO/DrzBC and CSSO/DrzBS, which verified that the micelles had a spherical and homogeneous

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morphology with a narrow size distribution. The average diameters of complexes in

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of complexes after vacuum extraction.

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DI water were larger than that shown in TEM images because of the severe shrinkage

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Glutathione (GSH), an abundant reducing agent in living cells, has an intracellular

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concentration of about 1-10 mM and 2-20 µM in blood and extracellular matrix. With the structure of reducible disulfide bonds ‘–SS-’, the glycolipid-like nanocarrier

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CSSO is redox-responsive. As shown in Fig.4B and C, in 10 mM GSH, the release of

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DNA from CSSO showed a significant increase compared with that in 0 mM GSH.

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Fig. 4. Characterizations of CSSO/DNA complexes. (A) The transmission electron microscopy (TEM) images of CSSO/DrzBC and CSSO/DrzBS. (B) DrzBC release behavior from CSSO/DrzBC with 10 mM GSH. (C) DrzBS behavior from

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CSSO/DrzBS release with 10 mM GSH. 3.4 In vitro cytotoxicity A MTT evaluation of the in vitro cytotoxicity of polymers and their corresponding complexes against HepG2.2.15 cells was carried out. As shown in Fig. 5A, when the

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concentration of CSSO polymers was 200μg/mL, which was higher than the in vitro administration dosage, the cell viabilities of HepG2.2.15 treated with CSSO was still above 80%, implying CSSO had relative low cytotoxicity. The in vitro cytotoxicity of complexes was displayed in Fig. 5B and C. All the

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complexes exhibited a dose-dependent cytotoxicity. When the DNA concentration was 2μg/mL, which was higher than the in vitro administration dosage, the cell viabilities

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of HepG2.2.15 treated with CSSO/DNA complexes were all above 85%, suggesting no obvious cytotoxicity. Overall, both CSSO polymers and CSSO/DNA complexes

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had negligible effect on HepG2.2.15 cells viability.

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Fig. 5. In vitro cytotoxicity of CSSO (A), CSSO/DrzBC (B) and CSSO/DrzBS (C) against HepG2.2.15 cells after incubation for 48 h. (n=6). 3.5 Intracellular trafficking Encapsulation of DNA within nanoparticles is an attractive alternative method for gene delivery. In order to promote high efficiency of gene delivery, DNA must escape 18

from the endosome before degrading within the late endosome and lysosome (Morachis et al., 2012). In this study, redox-responsive polymer CSSO was synthesized and hoped to deliver DNA effectively. To investigate whether CSSO/DNA(Considering the similarity in physical properties between DrzBC and

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DrzBS, we only choose DrzBC to as one of them for experiment) could escape from the endosome to achieve efficient DNA delivery after cellular uptake, the intracellular

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distribution of complexes in HepG2.2.15 cells was observed by CLSM (Fig. 6). The

FITC-labeled polymers and Cy5-labeled DrzBC were used to prepare complexes by

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the vortex, and lysosomes were stained with Lysotracker blue. The colocalization of

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Cy5-labeled DrzBC (red) with lysosomes (blue) produced purple fluorescence in the

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merged images. The confocal images revealed that at 24 h after incubation, red dots

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were mainly observed in the cytoplasm, indicating that CSSO/DrzBC complexes were

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able to achieve lysosome escape and hinting the ability to achieve gene therapy.

Fig. 6. The intracellular localization of DrzBC in HepG2.2.15 cells. DrzBC was labeled by Cy5 (red points), CSSO was labeled by FITC (green points), and endosomes were labeled by Lyso-Tracker blue (blue points). White arrow indicated

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the endosomal escape of CSSO/DrzBC. 3.6 In vitro anti-HBV efficacy Virus particles are present in large quantities in blood during HBV infection, which consist of a membrane composed of envelope and nucleocapsid proteins

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containing circular DNA molecule. The envelope protein carries a hepatitis B surface antigen (HBsAg) while the capsid contains the hepatitis B core antigen (HBcAg) and

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hepatitis B e antigen (HBeAg). The in vitro anti-HBV efficacy can be investigated by measuring the HBsAg and HBeAg levels in HepG2.2.15 cell culture (Xiang et al.,

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2010). To determine the inhibitory effects of complexes on HBV antigen secretion,

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HepG2.2.15 cells were treated with different DNA delivery systems in the same DNA

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concentration (1µmol/L). The result displayed in Table 2 showed that no significant

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differences were found in DrzBC solution group compared to the untreated group.

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Lipo2000/DrzBC increased within 48h and with maximum inhibitory rate of 47.29±1.86% at 48h, and then decreased rapidly which may be due to higher

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cytotoxicity of Lipo2000. In contrast, CSSO/DrzBC significantly inhibited HBeAg

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secretion, which was up to 73.86±1.77% at 48h, and surprisingly up to 83.83±2.34% after 72h incubation. Similar results were obtained for HBsAg secretion assay. As shown in Table 3, compared to the untreated group, no significant decrease of HBsAg

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expression levels were detected in DrzBS solution group. Lipo2000/DrzBS increased within 48h and with maximum inhibitory rate of 33.58±0.72% at 48h, and then also decreased rapidly. CSSO/DrzBS were significantly reduced HBsAg expression levels, and the HBsAg secretion inhibition of CSSO/DrzBS was 67.80±2.51% at 48h, and

20

further increased to 76.79±2.18% after 72h incubation, which was slightly lower than the HBeAg secretion inhibition of CSSO/DrzBC. From Table 2 and Table 3, we discovered that CSSO also exhibited inhibition activity on HBeAg and HBsAg secretion to some extent, which could be a synergistic

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action on anti-Hepatitis B Virus. As reported in our previous research (Li et al., 2010), stearic acid-g-chitosan oligosaccharide (CSO-SA) micelles with glycolipid-like

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structure possessed antiviral activity, and further deduced that with saccharide

segment might inhibit the HBV replication. Removal of the CSSO antiviral factor, the

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inhibitory effects originated purely from DrzBC or DrzBS were still remarkable when

DrzBC

CSSO/DrzBC

3.84±0.22%

35.12±0.77%

68.30±2.79%

18.89±0.71%

4.21±0.25%

47.29±1.86%

73.86±1.77%

21.65±0.79%

4.89±0.19%

23.98±0.81%

83.83±2.34%

DrzBS

Lipo2000/DrzBS

CSSO/DrzBS

12.58±0.26%

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72

Lipo2000/DrzBC

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24 48

CSSO

ED

Time post treatment (h)

M

Table 2. The inhibitory rate of HBeAg secretion.

A

in efficient anti-HBV gene therapy.

N

they were delivered by CSSO, which revealed the potential of CSSO/DNA complexes

Data represent the mean ± standard deviation (n = 3).

A

Table 3. The inhibitory rate of HBsAg secretion.

Time post treatment (h)

CSSO

24

10.29±0.21%

2.25±0.29%

19.35±0.31%

55.84±1.03%

48

15.25±0.35%

4.56±0.41%

33.58±0.72%

67.80±2.51%

21

72

19.24±0.68%

3.95±0.15%

22.76±0.67%

76.79±2.18%

Data represent the mean ± standard deviation (n = 3).

4. Conclusion

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In conclusion, we have developed a chitosan based glycolipid-like polymers

Chitosan oligosaccharide-SS-Octadecylamine (CSSO) which selectively responded to

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intracellular environment by reducible disulfide bonds ‘–SS-’. CSSO could

self-aggregate above 65.84±1.11µg/mL in aqueous medium to form nano-size

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micelles (123.00±10.98nm). As a polymeric carrier with low cytotoxicity, CSSO

N

could bind with our designed gene drug (10-23DNAzyme) by electrostatic interaction

A

with proper mass ratio of 60:1, which could efficiently protect the condensed DNA

M

from enzymatic degradation by DNase I. Further, CSSO showed the function of

ED

endosomal escape and rapid intracellular DNA release in response to the high reducing milieus, thus delivery the DNA into the gene therapeutic target in the

PT

subcellular level. Besides, CSSO exhibited inhibition activity on HBsAg and HBeAg

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secretion to some extent, which could be a synergistic action on anti-Hepatitis B Virus. In consequence, compared with the traditional commercial carrier Lipo2000, the

A

complexes of CSSO/DrzBC and CSSO/DrzBS showed a significant increased inhibition ability on HBeAg and HBsAg secretion respectively. Given these encouraging results, CSSO is a promising redox-responsive polymeric carrier for efficient anti-Hepatitis B Virus gene therapy.

Acknowledgment 22

We are grateful for financial support of the Zhejiang provincial natural science foundation-Qing Shan

Hu

Science ﹠ Technology City Union

Foundation

(LQY19H300001), the National Nature Science Foundation of China under contract 81402862, the hospital pharmacy special research project of Zhejiang Pharmaceutical Association (2018ZYY02), the Zhejiang provincial natural science foundation (LQ15H280005), the Zhejiang Traditional Chinese Medicine Science and Technology

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Program (2016ZA116) and the Zhejiang Medical Technology Program (2017178266).

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