Hepatic-Targeted Gene Delivery Using Cationic Mannan Vehicle

Subscriber access provided by Umea University Library

Article

Hepatic-targeted gene delivery using cationic mannan vehicle Jian-Qing Gao, Gui-Xin Ruan, Tian-Yuan Zhang, Li-Ming Li, Xing-Guo Zhang, Youqing Shen, and Yasuhiko Tabata Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp5000899 • Publication Date (Web): 15 Apr 2014 Downloaded from http://pubs.acs.org on April 23, 2014

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 free 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 accessible to all readers and 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.

Molecular Pharmaceutics 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 24

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

Hepatic-targeted gene delivery using cationic mannan vehicle Gui-Xin Ruana,Tian-Yuan Zhanga, Li-Ming Lia, Xing-Guo Zhangb, You-Qing Shenc, Yasuhiko Tabatad, Jian-Qing Gaoa,*

a

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University,

Hangzhou 310058, P.R.China b

Department of Pharmacy, The First Affiliated Hospital, College of Medicine,

Zhejiang University, P.R.China c

Center for Bionanoengineering and State Key Laboratory of Chemical Engineering,

Zhejiang University, Hangzhou 310027, P.R. China d

Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier

Medical Sciences, Kyoto University, Kyoto, Japan

AUTHOR INFORMATION *Correspondence author: Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, Zhejiang, PR China Tel/Fax: +86-571-88208437 Email: [email protected]

ABSTRACT: The incidence of hepatic diseases continuously increases worldwide and causes significant mortality because of inefficient pharmacotherapy. Gene therapy is a new strategy in the treatment of hepatic diseases, but the disadvantages of insufficient retention in the liver and undesirable side effects hinder its application. In this study, we developed a novel non-viral vehicle targeted to liver. Mannan was cationized with spermine at varying grafted ratios to deliver the gene and was optimized in cytotoxicity and transfection in vitro. Spermine-mannan (SM)-based

ACS Paragon Plus Environment


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

delivery system was proven to be hepatic targeted and was capable of prolonging the gene retention period in the liver. Moreover, SM at N/P of 20 was confirmed to be less interfered by the serum. Optimized SM vehicle has relatively high hepatic transfection with almost no toxicity induction in the liver, which highlighted its potential in the treatment of hepatic diseases. KEYWORDS: hepatic targeting, gene delivery, spermine-mannan, mannose receptor

INTRODUCTION The liver is the largest solid organ in the body and is well known for its prominent function in medication metabolism. The first pass-effect and biological compound uptake in some cases result in the accumulation of xenobiotics in the liver, which contributes to hepatic diseases1. The incidence of hepatic diseases, such as hepatitis, hepatic fibrosis, microbial infection, or hepatic carcinoma, continuously increases worldwide and causes significant mortality because of the inefficient pharmacotherapy for most of these diseases2-4. The main disadvantage of therapy for hepatic diseases arises from the insufficient retention of therapeutic compounds in the liver and the undesirable accumulation in other organs, which tends to induce side effects. Therefore, drug targeting to the liver, particularly to specific cells, represents new therapeutics1. Numerous options of drug targeting to disordered livers have been described, and most studies focus on hepatic-targeted gene delivery1. Nanomedicine using viruses, liposomes, and polymers are extensively applied as the liver-specific vehicles to deliver therapeutic genes after modification5. Moreover, hepatic targeting is achieved via hydrodynamic-based transfection after systemic administration of DNA6, which has been successfully applied to liver disease treatment7, 8. Nevertheless, the occurrence of acute liver damage6 and the lack of cell specificity hinder its application, which is the same problem confronting other delivery systems. Viral delivery systems, such as adenovirus, have already been extensively studied in clinical trials. Adenovirus naturally targets the coxsackie/adenovirus receptor (CAR) expressed in parenchymal cells, but results in the degradation and loss of activity when absorbed


Page 2 of 24

Page 3 of 24

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


by Kupffer cells (KC)9. However, such problems as immunogenicity and non-specific transduction10, 11 still need to be elucidated. Non-viral delivery systems are relatively biocompatible and can conveniently be modified for targeting12-15 compared with viral delivery systems. The receptor-specific targeting systems for hepatic targeting have been investigated to deliver the genes to specific cells in resident hepatocytes1, 16. Ligands, including galactose12, lactose17, mannose18, fructose19, and targeted peptides20, 21 or antibodies22, have been generally studied for modification in carriers to convey drugs, proteins, or genes and to demonstrate the benefit of cell-specific targeting. KC and sinusoidal endothelial cells (SEC) are equipped with the mannose receptors (MRs), which are important for the recognition of foreign yeast that has mannan in the cell wall23. Both cell types are endowed with a high phagocytotic capacity and are part of the reticuloendothelial system (RES) and play an important role in immune-mediated liver diseases such as liver inflammation and fibrosis24. Thus, the design of MR-specific carriers to deliver anti-inflammatory or anti-fibrotic compounds to these cells has great potential for therapy. In this study, we conceptualize a hepatic-targeted vehicle for gene delivery (Figure1). Instead of mannose modification, the vehicle is based on the mannan backbone from yeast, which could specifically target MRs in hepatocytes25. To deliver the genes, mannan is cationized by spermine, a naturally occurring tetramine that exhibits properties superior to those of other amine types26,

27

. The transfection efficiency and

cytotoxicity of spermine-mannan (SM) with different grafted ratios is optimized in vitro. The biodistribution of SM-based delivery system is further investigated, and the interference of the serum is evaluated in vitro. We further assess the liver toxicity induced by SM-based delivery system and applied such system to in vivo hepatic transfection.



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

Figure1. Strategy of liver-targeted SM-based delivery system in vivo

MATERIALS AND METHODS Materials. Mannan from Saccharomyces cerevisiae, 1, 1’-carbonyl-diimidazole (CDI), and spermine were purchased from Sigma-Aldrich (St. Louis, MO, USA). pGL3-control was purchased from Promega (Madison, WI, USA), pEGFP-N1 was purchased from Clontech (Mountain View, CA, USA), and fluorescein isothiocyanate (FITC)-DNA was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), with the sequence of 50 thymine residues and FITC modification. Cells and animals. HepG2 cells were purchased from Cell Bank of the Chinese Academy of Sciences, Shanghai, China and were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Boster, Wuhan, China) containing 10% fetal bovine serum (FBS, v/v; Hyclone, Utah, USA) at 37 °C with 5% CO2. Male C57BL/6 mice, six weeks to eight weeks old, were purchased from Slac Animal Co., Ltd (Shanghai, China). The mice were maintained under SPF conditions, and all experiments were performed under the Care and Use of Animals Committee Guidelines. Synthesis and characterization of SM. Mannan (25 mg) with an average molecular weight of 100 kDa was dissolved in 10 ml of anhydrous DMSO and was


Page 4 of 24

Page 5 of 24

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


stirred to complete dissolution at 60 °C. CDI at 3:1, 6:1, 9:1, and 18:1 molar ratios to the -CH2-OH content of mannan was dissolved in DMSO and gradually added to the dissolved mannan solution to activate the -CH2-OH at 40 °C for 30 min. The activated mannan solution was added dropwise to spermine solution (at a molar ratio of 3:1 to CDI) in 20 ml of DMSO and then allowed to react for 18 h at 37 °C. The mixture was dialyzed for 2d using 8,000 MW to 14,000 MW cut-off membranes in DDW, and the product was collected for lyophilization. Solid SM was prepared for Differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectrophotometric analysis, and elemental analysis and was further prepared in d-H2O for H1-NMR analysis. Preparation and characterization of SM/DNA complex.SM was dissolved in PBS and emerged with pGL3-control or FITC-DNA (100 or 200µg/ml) in an equal volume of PBS at different N/P ratios. The mixture was maintained at room temperature for more than 15 min. The particle size was measured in FBS-free PBS or PBS containing 50% v/v FBS using a Malvern Zetasizer assay (Zetasizer 3000 HSa, Malvern). Transfection evaluation in vitro. HepG2 cells were cultured at a density of 5×104 in 24-well plates for 24h. SM/pGL3-control complexes were prepared with different grafted SMs at varying N/P ratios, and Polyethylenimine (branched 25 kDa PEI, Sigma)/DNA complexes were prepared at an N/P ratio of 1028. The complexes were added to the cells with a plasmid mass of 1 µg/well in serum-free, 10% or 50% v/v FBS containing DMEM for 6h. The medium was replaced by the full medium for another 18h culture. The level of luciferase expressed was quantified by luciferase reporter gene assay kit (Beyotime, Shanghai, China) following manufacturer’s instructions. Relative light unit (RLU) was determined by Luminometer (GloMax Jr Multi-Detection System, Promega). Protein concentration was determined by a BCA protein assay kit (Beyotime, Shanghai, China). Luciferase activity was displayed as RLU/Protein concentration for the final result. MTT assay. HepG2 cells were cultured at a density of 104 in 96-well plates for 24h. SM/pGL3-control complexes were prepared with different grafted SM at varying



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

N/P ratio and added to the cells with a plasmid mass of 0.2 µg/well in serum-free DMEM for 6h. The medium was replaced by the full medium for another 18h culture. Cell viability was determined by MTT assay using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (Sigma, St. Louis, MO, USA) assay. In vivo biodistribution. SM/FITC-DNA complexes were prepared with different grafted ratios of SM at varying N/P ratio, and PEI/DNA complexes were prepared at an N/P ratio of 10 according to a previous study29. The mice were treated with SM/FITC-DNA (20µg/mouse) and PEI/DNA complexes via intravenous injection at a volume of 0.2 ml per mouse. Naked FITC-DNA solution was also injected intravenously with the same amount of FITC-DNA as the control groups. At time points of 1, 6, 24, and 48h after administration, the mice were myocardially perfused by cold saline, and organs including heart, liver, spleen, lungs, and kidneys were harvested and rinsed with saline for observation under MaestroTM In-vivo imaging system (Maestro EX, Cambridge Research & Instrumentation). In vivo toxicity evaluation. SM/pEGFP-N1 complexes were prepared with SM at selected N/P ratios. The mice were treated with SM/pEGFP-N1 (20µg/mouse) complexes via intravenous injection at a volume of 0.2ml per mouse. Naked pEGFP-N1solution and PEI/pEGFP-N1 were also injected intravenously with the same amount of DNA as the control groups. The livers were collected at 24 h after administration for Hemotoxylin-Eosin (HE) staining to facilitate pathological observation. In vivo hepatic transfection evaluation. SM/pEGFP-N1 complexes were prepared with SM at selected N/P ratios. The mice were treated with SM/pEGFP-N1 (20µg/mouse) complex via intravenous injection at a volume of 0.2ml per mouse. Naked pEGFP-N1 solution and PEI/pEGFP-N1 were also injected intravenously with the same amount of DNA as the control groups. The mice were myocardially perfused at 24 h after administration by cold saline, and the livers were harvested and rinsed with saline for observation under MaestroTM In-vivo imaging system. Statistical analysis. All data are presented as mean ± standard deviation. Statistical comparisons between groups were performed using Student’s t-test.


Page 6 of 24

Page 7 of 24

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


Statistical significance was determined as follows: * p < 0.05 or ** p < 0.01.

RESULTS Synthesis and characterization of SM. The spermine cationization of mannan occurred through the carbamoylation by reactive 6-hydroxyl group in mannose unit by CDI and conjugation with spermine through a terminal amino group (Figure 2A). The grafted ratio of spermine to mannan was controlled based on the addition ratio of CDI at 3:1, 6:1, 9:1,and 18:1 to -CH2-OH, which induced an increase in the grafted spermine in mannan with ratios of 6.75%, 9.64%, 12.03%, and 27.31%, respectively (Table1). The melting temperature of the product was 175.0 °C compared with135 °C for mannan, which indicated successful SM synthesis(Figure 2B). The structure of SM was further confirmed by FTIR analysis and H1-NMR analysis. FTIR (KBr): 1702.35 cm-1 (C=O) and 3377.71 cm-1 (N-H). The appearance of the peak at 1702.35 cm-1 confirmed the presence of the conjugation carbonyl groups of the generated carbamate functionalities after conjugation. The transformation of the cuspidal peak at 3377.71 cm-1 confirmed the presence of the amino group of spermine (Figure 2C). H1-NMR

(d-H2O):

1.5 ppm

to

1.7

ppm

[mannan-CH2OCONH(CH2)3NHCH2CH2CH2CH2NH(CH2)3NH2], 1.7 ppm to1.8 ppm [mannan-CH2OCONHCH2CH2CH2NH(CH2)4NHCH2CH2CH2NH2], 2.6 ppm to 2.9 ppm

[mannan-CH2OCONHCH2CH2CH2NHCH2CH2CH2CH2NHCH2CH2CH2NH2],

3.0 ppm to 3.2 ppm [mannan-CH2OCONH(CH2)3 NH(CH2)4NH(CH2)3 NH2], 3.6 ppm to 4.0 ppm [glycoside hydrogens], and 4.8ppm to 5.0 ppm [anomeric hydrogen] (Figure2D).



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

Page 8 of 24

Table 1 Elemental analysis of SM with varying addition of CDI CDI:[-CH2-OH]

Nitrogen%

Carbon%

Hydrogen%

Grafted%

3:1

4.44

39.47

6.91

6.75

6:1

6.07

41.27

7.23

9.64

9:1

7.20

42.62

7.36

12.03

18:1

11.33

44.47

7.82

27.31

Figure 2. A: Structure of SM, B: DSC analysis, C: FTIR analysis, D: H1-NMR in d-H2O

Cytotoxicity optimization of SM in vitro. To examine the biocompatibility of SM-based delivery system and to select the appropriate grafted ratio and N/P ratio for application, the different grafted ratios of SM incubated with DNA at N/P ratio ranging from 1 to 30 were evaluated in HepG2 cells (Figure 3). No cytotoxicity was found at N/P ratios ranging from 1 to 10 at any grafted ratio. Cytotoxicity was increased at high N/P ratios (N/P ratio of 20 and 30) at all grafted ratios, which indicated that N/P ratio of less than 20 was appropriate for transfection.


Page 9 of 24

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.Cytotoxicity evaluation of SM (grafted ratios of 3:1, 6:1, 9:1, and 18:1) at N/P ratios of 1:1, 3:1, 5:1, 10:1, 20:1, and 30:1 in HepG2 cells (n=6).

Transfection optimization of SM in vitro. Luciferase-report plasmid was used for the transfection evaluation in HepG2 cells to determine the appropriate grafted ratio and N/P ratio with highest transfection efficiency. PEI was chosen as the positive control because it is a classical non-viral vehicle30. The SM-based delivery system achieved superior transfection efficiency when the grafted ratio was 9:1 and 18:1, particularly at N/P ratios of 5 to 20 (Figure 4). The grafted ratio of 3:1 and 6:1 exhibited inferior efficiency, which indicated their inadequacy for transfection. Thus, the grafted ratios of 9:1 and 18:1 were selected to be applied further in vivo.



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 4.Transfection evaluation of PEI at N/P=10 and SM (grafted ratio of 3:1, 6:1, 9:1, and 18:1) at N/P ratios of 1:1, 3:1, 5:1, 7:1, 10:1, and 20:1 in HepG2 cells (n=4).

Biodistribution investigation. Most drugs tend to accumulate in the liver because of the first-pass effect and existence of RES, but are degraded and eliminated rapidly. Gene delivery systems can specifically target and prolong the retention period in the liver, which are important for gene delivery. In this study, we investigated the biodistribution of SM/FITC-DNA at 1h after intravenous injection in mice (Figures 5A and 5B). Naked FITC-DNA was extensively absorbed by the liver after elimination from plasma, but was eliminated and excreted rapidly as fluorescence appeared in the kidneys. PEI delivered FITC-DNA to the liver (Figure 5A) and exhibited increasing retention in the liver compared with naked FITC-DNA (Figure5B). As regards the SM-based delivery system, 9:1 grafted SM at N/P ratios of 2, 5, 10, 20, and 18:1 grafted SM at an N/P ratio of 2 exhibited an increasing retention in the liver. However, 9:1 grafted SM at N/P ratio of 40 and 18:1 grafted SM at N/P ratio of 5 illustrated the FITC-DNA distribution in the lungs (Figure5A), which remained to be elucidated further. The size of the particle is one of the physicochemical properties of the vehicle gene complex that determines the targeted delivery after systemic administration. The particle size of PEI and most of the SM/DNA complexes (Figure 5C) maintained the size of ~100 nm except for 18:1 SM at N/P ratio of 2 (236nm). The combined results of biodistribution (Figure 5) and transfection evaluation (Figure4), which is 9:1 SM at N/P ratios of 5, 10, and 20were capable of both hepatic targeting and transfection and would be elucidated in further studies.


Page 10 of 24

Page 11 of 24

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 5.A: Biodistribution of naked FITC-DNA, PEI/FITC-DNA at N/P=10 and SM/FITC-DNA (9:1 grafted at N/P ratios of 2, 5, 10, 20, 40, and 18:1 grafted at N/P ratios of 2 and 5) at 1h after intravenous injection in C57BL/6. B: Total signal statistic of FITC-DNA delivered by none, PEI, and SM in the liver. C: Particle size measurements.

Prolonged retention in the liver. Further investigation continued at time points of 6, 24, and 48h after intravenous injection to evaluate the retention of DNA in the liver (Figure 6). Naked FITC-DNA was eliminated and excreted rapidly from the liver as less fluorescence was detected in the liver and kidney at 6, 24, and 48h after injection. By contrast, the fluorescence intensity of PEI/FITC-DNA remained high in the liver at 6 h and was eliminated and excreted gradually. The distribution of PEI/FITC-DNA at 6, 24, and 48h in the lungs was also remarkable. The optimized SM/FITC-DNA complexes prolonged the retention of FITC-DNA in the liver at 24h after injection (Figure 6) compared with PEI, without the distribution of other organs.



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 6. Biodistribution of SM/FITC-DNA at N/P ratio of 5, 10, and 20 at 6, 24, and 48h after intravenous injection in C57BL/6.

Less interference by serum. Plasma constitutes more than 50% of the blood fluid. The plasma, except for the blood cells, contains dissipated proteins and other essential compounds. These negatively charged macromolecules in the serum are among the major limitations of positively charged vehicle complexes because of potential adverse interaction31,

32

. In this study, the existence of 50% v/v serum

significantly affected PEI/DNA complex in terms of particle size (Figure 7A), which increased from 119nm to 797nm. By contrast, SM/DNA at N/P ratios of 5 and 10 increased from ~100nm to ~200nmand to 407nm at an N/P ratio of 20. As regards the interference in the transfection in vitro, PEI- and SM-based delivery systems exhibited decreased transfection efficiency in the presence of 10% and 50% v/v serum. SM at N/P ratios of 5 and 10 was inferior in transfection in the presence of 10% v/v serum compared with PEI. For that with 50% v/v serum, SM at an N/P ratio of 5 exhibited a decrease in transfection, and SM at an N/P ratio of 10 similarly exhibited a decrease in ratio compared with PEI. However, SM at an N/P ratio of 20 had a similar and smaller decrease in ratio with 10% and 50% v/v serum in contrast to PEI (Figure 7B).


Page 12 of 24

Page 13 of 24

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.A: Particle size detection in the presence of 50 % v/v serum and, B: Transfection evaluation in the presence of 10% and 50 % v/v serum in HepG2 cells (n=4).

Acute toxicity evaluation. An efficient vehicle should be sufficient in gene delivery and biocompatibility. As regards in vivo toxicity, SM at varying N/P ratios exhibited no histological change in HE staining slices compared with the untreated blank and DNA groups (Figure 8), which indicated that the SM-based delivery system is not harmful to the liver despite prolonged retention. PEI at an N/P ratio of 10 also exhibited no obvious pathological change in the slice (Figure 8B) at the dose.

Figure 8.Acute hepatic toxicity evaluation (24h) of A: Blank, B: PEI, C: Naked DNA,



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

D: SM at N/P=5, E: SM at N/P=10, F: SM at N/P ratio=20.

In vivo transfection evaluation. We further applied the optimized SM-based delivery system in liver transfection using EGFP-report plasmids. Branched 25kDa PEI at an N/P ratio of 10 exhibited sufficient transfection in the liver29 and was selected as the positive control. No expression of fluorescence was detected in SM N/P ratio=5 group, as well as in the naked-DNA group (Figure 9). The PEI-treated group displayed relatively high fluorescence intensity. SM at N/P=10 displayed similar transfection as the PEI group, and SM at N/P=20 displayed the highest transfection efficiency among the SM-treated groups, which a value even higher than that of the PEI group.

Figure 9. In vivo hepatic transfection observation at 24h after intravenous injection of naked EGFP plasmid, PEI, or SM (N/P=5, 10, 20)/plasmid complexes.

DISCUSSION The strategies for drug or gene delivery to specific cells identified for each liver disease served as the focus of various studies in recent years. Parenchymal cells represent more than 75% of the total resident hepatocytes, and the hepatic uptake of the compound without cell-type identification mostly occurs in parenchymal cells after RES phagocytosis. The asialoglycoprotein receptor (ASGPR) has been used as the specific receptor for drug delivery12,

14, 33

in the majority of the cases in

parenchymal cells targeting because it is abundantly expressed in the cells34. However,


Page 14 of 24

Page 15 of 24

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


in several diseases, parenchymal cells are not the most relevant cells in disease occurrence and regulation. KC and SEC particularly in hepatic hepatitis and fibrosis24 have important function in acute or chronic inflammation in those diseases and are very important targets for therapy. These cells are endowed with high phagocytotic capacity and are parts of RES. Specific receptor-mediated uptake in these cells is administered by negatively-charged delivery vehicles via scavenger receptors35 and mannose/fucose- modified delivery system via MRs13, 18. Moreover, hepatic stellate cells (HSC) serve a pivotal function in cirrhosis, which is the end stage of many liver diseases. Previous researches successfully used the mannose6-phosphate residue36, Vitamine A15, or RGD peptide21 to modify the delivery system to selectively target HSC. In this study, we developed a non-viral gene delivery vehicle theoretically targeted to hepatocyte-high expressing MRs. The vehicle was based in mannan backbone and cationized by spermine, and was optimized in vitro for invivo gene delivery. Naked DNA was reported to be rapidly eliminated from the plasma and absorbed by the liver after the intravenous injection. This process is preferentially proceeded by non-parenchymal cells that were eliminated and showed no gene expression in the liver37. The degradation of DNA by nuclease is another limitation that contributes to inefficient transfection. Condensing DNA with cationized polymers enables protection from degradation in vivo. Branched PEI 25kDa, for instance, has been reported to exhibit an increase in residence period in the liver38. However, relatively high accumulation was also observed in the lungs, which indicate specific transfection problems in the liver using simple cationic polymers. Previous study speculated that distribution of PEI/DNA to the lungs is related to the rapid crossing of pulmonary epithelial barriers39, 40 with enlarged particle size in the plasma. In our study, we found out that grafted ratio and N/P ratio affect the in vivo distribution of SM/DNA complexes, however, similar particle size was detected. Different grafted ratio at varying N/P ratio tend to form complexes with different physicochemical property, thus it interacted with negatively-charged compounds in different serum differently, which leads to non-identical transfection efficiency in the tumor cell line.



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

We also confirmed that optimized-grafted SM with certain N/P ratios prolong liver compared with naked DNA and PEI/DNA complexes, which was probably because of the specific targeting to SEC and KC in hepatocytes. In most cases however, these cells in RES are unwanted targeting cells. Viruses41 or liposomes42 opsonized by complements43 in plasma resulted in unwanted uptake in RES and degradation in the lysosomes of KC. In spite of this limitation, the drugs of genes targeted to SEC and KC are important in anti-inflammatory or anti-fibrotic treatment in acute or chronic hepatic inflammatory diseases. SM-based delivery system was absorbed by the macrophage in a mannose-receptor specific pathway and exhibited promoted transfection efficiency in vitro, which was elucidated in our previous study (Data unpublished). Although PEI and SM complexes displayed no pathological change in this study at 1d after injection, several researches reported that PEI induce multiple responses, such as apoptosis44, liver necrosis, activation of lung endothelium adhesion of aggregated platelets, and shock45 after systemic injection of elevated doses. Branched PEI complexes were also reported to induce pro-inflammatory cytokine producing46. PEI exhibited superior transfection efficiency than SM in the tumor cell line in vitro, but was inferior when applied in vivo. SM at N/P=20 exhibited better transfection efficiency than PEI in vivo because of the capacity against the serum interference. In this study, we successfully developed a cationized mannan vehicle theoretically targeted to hepatocyte that high expressing MRs. SM vehicles were optimized in the tumor cell line in cytotoxicity and transfection. SM was further proved to be hepatic targeted at optimized N/P ratios, and was able to prolong the retention period of DNA in the liver as compared with naked DNA and PEI. Moreover, SM at N/P ratio of 20 displayed less interference by the serum, which was further confirmed to have relatively high hepatic transfection with almost no toxicity induction in the liver. The results emphasized the potential of SM in hepatic diseases particularly inflammatory disease therapy.


Page 16 of 24

Page 17 of 24

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


ACKNOWLEDGEMENT The project was supported by National Basic Research Program of China (No.2014CB931901),

International Joint Program(No. 81011140077) supported by

both NSFC, China and JSPS, Japan, and Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents.

REFERENCE 1.

Poelstra, K.; Prakash, J.; Beljaars, L.

Drug targeting to the diseased liver. J

Control Release 2012, 161, (2), 188-97. 2.

Wieckowska, A.; McCullough, A. J.; Feldstein, A. E.

Noninvasive diagnosis

and monitoring of nonalcoholic steatohepatitis: present and future. Hepatology 2007, 46, (2), 582-9. 3.

Friedman, S. L.; Bansal, M. B.

Reversal of hepatic fibrosis -- fact or fantasy?

Hepatology 2006, 43, (2 Suppl 1), S82-8. 4.

Nordenstedt, H.; White, D. L.; El-Serag, H. B.

The changing pattern of

epidemiology in hepatocellular carcinoma. Dig Liver Dis 2010, 42 Suppl 3, S206-14. 5.

Reddy, L. H.; Couvreur, P.

Nanotechnology for therapy and imaging of liver

diseases. J Hepatol 2011, 55, (6), 1461-6. 6.

Liu, F.; Song, Y.; Liu, D.

Hydrodynamics-based transfection in animals by

systemic administration of plasmid DNA. Gene Ther 1999, 6, (7), 1258-66. 7.

Jiang, J.; Yamato, E.; Miyazaki, J.

Intravenous delivery of naked plasmid DNA

for in vivo cytokine expression. Biochem Biophys Res Commun 2001, 289, (5), 1088-92. 8.

Yang, J.; Chen, S.; Huang, L.; Michalopoulos, G. K.; Liu, Y.

Sustained

expression of naked plasmid DNA encoding hepatocyte growth factor in mice promotes liver and overall body growth. Hepatology 2001, 33, (4), 848-59. 9.

Haisma, H. J.; Bellu, A. R.

Pharmacological interventions for improving

adenovirus usage in gene therapy. Mol Pharm 2011, 8, (1), 50-5. 10. Descamps,

D.;

Benihoud,

K.

Two

key

challenges


for

effective


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

adenovirus-mediated

liver

gene

therapy:

innate

Page 18 of 24

immune

responses

and

hepatocyte-specific transduction. Curr Gene Ther 2009, 9, (2), 115-27. 11. Yao, X. L.; Yoshioka, Y.; Ruan, G. X.; Chen, Y. Z.; Mizuguchi, H.; Mukai, Y.; Okada, N.; Gao, J. Q.; Nakagawa, S.

Optimization and internalization mechanisms

of PEGylated adenovirus vector with targeting peptide for cancer gene therapy. Biomacromolecules 2012, 13, (8), 2402-9. 12. Zhang, X. Q.; Wang, X. L.; Zhang, P. C.; Liu, Z. L.; Zhuo, R. X.; Mao, H. Q.; Leong, K. W.

Galactosylated ternary DNA/polyphosphoramidate nanoparticles

mediate high gene transfection efficiency in hepatocytes. J Control Release 2005, 102, (3), 749-63. 13. Takei, Y.; Maruyama, A.; Ikejima, K.; Enomoto, N.; Yamashina, S.; Lemasters, J. J.; Sato, N.

Genetic manipulation of sinusoidal endothelial cells. J Gastroenterol

Hepatol 2007, 22 Suppl 1, S68-72. 14. Nie, C.; Liu, C.; Chen, G.; Dai, J.; Li, H.; Shuai, X. psiRNA

delivery

mediated

by

Hepatocyte-targeted

galactosylated

poly(ethylene

glycol)-graft-polyethylenimine in vitro. J Biomater Appl 2011, 26, (3), 255-75. 15. Sato, Y.; Murase, K.; Kato, J.; Kobune, M.; Sato, T.; Kawano, Y.; Takimoto, R.; Takada, K.; Miyanishi, K.; Matsunaga, T.; Takayama, T.; Niitsu, Y.

Resolution of

liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. Nat Biotechnol 2008, 26, (4), 431-442. 16. Hashida, M.; Nishikawa, M.; Yamashita, F.; Takakura, Y.

Cell-specific delivery

of genes with glycosylated carriers. Adv Drug Deliv Rev 2001, 52, (3), 187-96. 17. Singh, M.; Ariatti, M. Targeted gene delivery into HepG2 cells using complexes containing DNA, cationized asialoorosomucoid and activated cationic liposomes. J Control Release 2003, 92, (3), 383-94. 18. Higuchi, Y.; Kawakami, S.; Oka, M.; Yabe, Y.; Yamashita, F.; Hashida, M. Intravenous administration of mannosylated cationic liposome/NFkappaB decoy complexes effectively prevent LPS-induced cytokine production in a murine liver failure model. FEBS Lett 2006, 580, (15), 3706-14. 19. Higuchi, Y.; Nishikawa, M.; Kawakami, S.; Yamashita, F.; Hashida, M.


Uptake

Page 19 of 24

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


characteristics of mannosylated and fucosylated bovine serum albumin in primary cultured rat sinusoidal endothelial cells and Kupffer cells. Int J Pharm 2004, 287, (1-2), 147-54. 20. Du, S. L.; Pan, H.; Lu, W. Y.; Wang, J.; Wu, J.; Wang, J. Y.

Cyclic Arg-Gly-Asp

peptide-labeled liposomes for targeting drug therapy of hepatic fibrosis in rats. J Pharmacol Exp Ther 2007, 322, (2), 560-8. 21. Li, F.; Sun, J. Y.; Wang, J. Y.; Du, S. L.; Lu, W. Y.; Liu, M.; Xie, C.; Shi, J. Y. Effect of hepatocyte growth factor encapsulated in targeted liposomes on liver cirrhosis. J Control Release 2008, 131, (1), 77-82. 22. Elrick, L. J.; Leel, V.; Blaylock, M. G.; Duncan, L.; Drever, M. R.; Strachan, G.; Charlton, K. A.; Koruth, M.; Porter, A. J.; Wright, M. C.

Generation of a

monoclonal human single chain antibody fragment to hepatic stellate cells--a potential mechanism for targeting liver anti-fibrotic therapeutics. J Hepatol 2005, 42, (6), 888-96. 23. Okawa, Y.; Abe, K.; Watanabe, T.; Sasaki, H.; Suzuki, M.

Production of

interleukin-1 activity of Kupffer cells from mice treated with the acidic mannan fraction of baker's yeast. Biol Pharm Bull 2002, 25, (11), 1506-8. 24. Szabo, G.; Mandrekar, P.; Dolganiuc, A.

Innate immune response and hepatic

inflammation. Semin Liver Dis 2007, 27, (4), 339-50. 25. Tanaka, T.; Fujishima, Y.; Hanano, S.; Kaneo, Y.

Intracellular disposition of

polysaccharides in rat liver parenchymal and nonparenchymal cells. Int J Pharm 2004, 286, (1-2), 9-17. 26. Chen, Y. Z.; Ruan, G. X.; Yao, X. L.; Li, L. M.; Hu, Y.; Tabata, Y.; Gao, J. Q. Co-transfection gene delivery of dendritic cells induced effective lymph node targeting and anti-tumor vaccination. Pharm Res 2013, 30, (6), 1502-12. 27. Jo, J.; Nagane, K.; Yamamoto, M.; Tabata, Y.

Effect of amine type on the

expression of plasmid DNA by cationized dextran. J Biomater Sci Polym Ed 2010, 21, (2), 225-36. 28. Zhao, Q. Q.; Chen, J. L.; Lv, T. F.; He, C. X.; Tang, G. P.; Liang, W. Q.; Tabata, Y.; Gao, J. Q.

N/P Ratio Significantly Influences the Transfection Efficiency and



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

Cytotoxicity

of

a

Page 20 of 24

Polyethylenimine/Chitosan/DNA Complex.

Biological

&

Pharmaceutical Bulletin 2009, 32, (4), 706-710. 29. Jeong, G. J.; Byun, H. M.; Kim, J. M.; Yoon, H.; Choi, H. G.; Kim, W. K.; Kim, S. J.; Oh, Y. K.

Biodistribution and tissue expression kinetics of plasmid DNA

complexed with polyethylenimines of different molecular weight and structure. Journal of Controlled Release 2007, 118, (1), 118-125. 30. Lungwitz, U.; Breunig, M.; Blunk, T.; Gopferich, A.

Polyethylenimine-based

non-viral gene delivery systems. Eur J Pharm Biopharm 2005, 60, (2), 247-66. 31. Plank, C.; Mechtler, K.; Szoka, F. C., Jr.; Wagner, E.

Activation of the

complement system by synthetic DNA complexes: a potential barrier for intravenous gene delivery. Hum Gene Ther 1996, 7, (12), 1437-46. 32. He, C. X.; Li, N.; Hu, Y. L.; Zhu, X. M.; Li, H. J.; Han, M.; Miao, P. H.; Hu, Z. J.; Wang, G.; Liang, W. Q.; Tabata, Y.; Gao, J. Q.

Effective gene delivery to

mesenchymal stem cells based on the reverse transfection and three-dimensional cell culture system. Pharm Res 2011, 28, (7), 1577-90. 33. Singh, M.; Ariatti, M. Targeted gene delivery into HepG2 cells using complexes containing DNA, cationized asialoorosomucoid and activated cationic liposomes. Journal of Controlled Release 2003, 92, (3), 383-394. 34. Rensen, P. C.; Sliedregt, L. A.; Ferns, M.; Kieviet, E.; van Rossenberg, S. M.; van Leeuwen, S. H.; van Berkel, T. J.; Biessen, E. A.

Determination of the upper size

limit for uptake and processing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo. J Biol Chem 2001, 276, (40), 37577-84. 35. Rensen, P. C.; Gras, J. C.; Lindfors, E. K.; van Dijk, K. W.; Jukema, J. W.; van Berkel, T. J.; Biessen, E. A.

Selective targeting of liposomes to macrophages using a

ligand with high affinity for the macrophage scavenger receptor class A. Curr Drug Discov Technol 2006, 3, (2), 135-44. 36. Zhu, L.; Mahato, R. I. Targeted delivery of siRNA to hepatocytes and hepatic stellate cells by bioconjugation. Bioconjug Chem 2010, 21, (11), 2119-27. 37. Mahato, R. I.; Kawabata, K.; Takakura, Y.; Hashida, M.

In vivo disposition

characteristics of plasmid DNA complexed with cationic liposomes. J Drug Target


Page 21 of 24

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


1995, 3, (2), 149-57. 38. Jeong, G. J.; Byun, H. M.; Kim, J. M.; Yoon, H.; Choi, H. G.; Kim, W. K.; Kim, S. J.; Oh, Y. K.

Biodistribution and tissue expression kinetics of plasmid DNA

complexed with polyethylenimines of different molecular weight and structure. J Control Release 2007, 118, (1), 118-25. 39. Rudolph, C.; Schillinger, U.; Ortiz, A.; Plank, C.; Golas, M. M.; Sander, B.; Stark, H.; Rosenecker, J.

Aerosolized nanogram quantities of plasmid DNA mediate highly

efficient gene delivery to mouse airway epithelium. Mol Ther 2005, 12, (3), 493-501. 40. Goula, D.; Becker, N.; Lemkine, G. F.; Normandie, P.; Rodrigues, J.; Mantero, S.; Levi, G.; Demeneix, B. A.

Rapid crossing of the pulmonary endothelial barrier by

polyethylenimine/DNA complexes. Gene Ther 2000, 7, (6), 499-504. 41. Smith, J. S.; Xu, Z.; Byrnes, A. P.

A quantitative assay for measuring clearance

of adenovirus vectors by Kupffer cells. J Virol Methods 2008, 147, (1), 54-60. 42. Liu, D.; Liu, F.; Song, Y. K.

Recognition and clearance of liposomes containing

phosphatidylserine are mediated by serum opsonin. Biochim Biophys Acta 1995, 1235, (1), 140-6. 43. Yan, X.; Morselt, H. W.; Scherphof, G. L.; Poelstra, K.; Kamps, J. A.

The role

of beta2-glycoprotein I in liposome-hepatocyte interaction. Biochim Biophys Acta 2004, 1667, (2), 208-14. 44. Regnstrom, K.; Ragnarsson, E. G.; Koping-Hoggard, M.; Torstensson, E.; Nyblom, H.; Artursson, P.

PEI - a potent, but not harmless, mucosal

immuno-stimulator of mixed T-helper cell response and FasL-mediated cell death in mice. Gene Ther 2003, 10, (18), 1575-83. 45. Chollet, P.; Favrot, M. C.; Hurbin, A.; Coll, J. L.

Side-effects of a systemic

injection of linear polyethylenimine-DNA complexes. J Gene Med 2002, 4, (1), 84-91. 46. Kawakami, S.; Ito, Y.; Charoensit, P.; Yamashita, F.; Hashida, M. proinflammatory

cytokine

production

induced

by

linear

Evaluation of and

branched

polyethylenimine/plasmid DNA complexes in mice. J Pharmacol Exp Ther 2006, 317, (3), 1382-90.



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 CAPATIONS Figure 1. Strategy of liver-targeted SM-based delivery system in vivo

Figure 2. A: Structure of SM, B: DSC analysis, C: FTIR analysis, D: H1-NMR in d-H2O

Figure 3.Cytotoxicity evaluation of SM (grafted ratios of 3:1, 6:1, 9:1, and 18:1) at N/P ratios of 1:1, 3:1, 5:1, 10:1, 20:1, and 30:1 in HepG2 cells (n=6).

Figure 4.Transfection evaluation of PEI at N/P=10 and SM (grafted ratio of 3:1, 6:1, 9:1, and 18:1) at N/P ratios of 1:1, 3:1, 5:1, 7:1, 10:1, and 20:1 in HepG2 cells (n=4).

Figure 5.A: Biodistribution of naked FITC-DNA, PEI/FITC-DNA at N/P=10 and SM/FITC-DNA (9:1 grafted at N/P ratios of 2, 5, 10, 20, 40, and 18:1 grafted at N/P ratios of 2 and 5) at 1h after intravenous injection in C57BL/6. B: Total signal statistic of FITC-DNA delivered by none, PEI, and SM in the liver. C: Particle size measurements.

Figure 6. Biodistribution of SM/FITC-DNA at N/P ratio of 5, 10, and 20 at 6, 24, and 48h after intravenous injection in C57BL/6.

Figure 7.A: Particle size detection in the presence of 50 % v/v serum and, B: Transfection evaluation in the presence of 10% and 50 % v/v serum in HepG2 cells (n=4).

Figure 8.Acute hepatic toxicity evaluation (24h) of A: Blank, B: PEI, C:Naked DNA, D:SM at N/P=5, E: SM at N/P=10, F:SM at N/P ratio=20.


Page 22 of 24

Page 23 of 24

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. In vivo hepatic transfection observation at 24h after intravenous injection of naked EGFP plasmid, PEI, or SM (N/P=5, 10, 20)/plasmid complexes.



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

203x135mm (300 x 300 DPI)


Page 24 of 24

Hepatic-Targeted Gene Delivery Using Cationic Mannan Vehicle

Recommend Documents