Versatile Supermolecular Inclusion Complex Based on HostâGuest

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Versatile supermolecular inclusion complex based on host-guest interaction for targeted gene delivery Yunxia Sun, Jingyi Zhu, Wen-Xiu Qiu, Qi Lei, Si Chen, and Xian-Zheng Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14963 • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 21, 2017

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ACS Applied Materials & Interfaces

Versatile supermolecular inclusion complex based on host-guest interaction for targeted gene delivery Yun-Xia Sun, Jing-Yi Zhu, Wen-Xiu Qiu, Qi Lei, Si Chen, Xian-Zheng Zhang* Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China.

ACS Paragon Plus Environment

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ABSTRACT: A facile and targeted gene delivery system was prepared by conjugating βcyclodextrin modified polyethylenimine (PEI-CD) and adamantyl peptide (AdGRGDS) based on host-guest interaction. With the rational design between PEI-CD and AdGRGDS, PEICD/AdGRGDS gene delivery system showed excellent DNA binding capability, and exhibited good ability to compact DNA into uniform spherical nanoparticles. In vitro luciferase assay showed that gene expression transfected by PEI-CD/AdGRGDS was stronger than that by PEICD in HeLa cells, whereas, gene expression transfected by PEI-CD/AdGRGDS and PEI-CD was similar to each other in COS7 cells. Internalization of complexes was qualitatively studied using confocal laser scanning microscope (CLSM) and quantitatively analyzed by flow cytometry respectively, and targeting specificity was also evaluated by CLSM. Results of CLSM and flow cytometry indicated that PEI-CD/AdGRGDS had good targeting specificity to tumor cells with integrin αvβ3 over-expression. To further evaluate the targeting specificity and transfection efficiency in vivo, a rat model with murine hepatic carcinoma cell line H22 was used. PEICD/AdGRGDS showed stronger gene expression efficiency than PEI-CD via in vivo transfection of pORF-LacZ and pGL-3 plasmids after subcutaneous injection. Interestingly, PEICD/AdGRGDS also showed high targeting specificity and transfection distribution to tumor xenograft after tail-vein injection. In vitro and in vivo assays highlighted importance of GRGDS targeting specificity to tumor cells with integrin αvβ3 over-expression, and demonstrated that PEI-CD/AdGRGDS gene delivery system would have great potential for targeted tumor therapy.

KEYWORDS: gene delivery system, host-guest interaction, transfection efficiency, targeting specificity, integrin receptor


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INTRODUCTION Gene therapy as a potential strategy for cancer treatment requires an efficient gene delivery system to deliver nucleic acid molecules into targeted cells.1-5 Among a variety of gene delivery systems, polycations as the promising gene vectors have attracted increasing attention due to easy

to

synthesize,

flexible

structural

modification,

and

low

immunogenicity.6-8

Polyethylenimine (PEI) as the gold standard of polycations has been widely used for gene delivery because of its efficient gene expression efficiency and the effect of proton sponge.9,10 Transfection efficiency mediated by polycation gene vector, including PEI, is usually perfect in vitro, but the result in vivo study is commonly unsatisfactory. To some extent, the inefficient transfection is due to net positive charge on polycations. Injecting into in vitro serum condition or in vivo circumstance, the polycations would aggregate with serum molecules due to electrostatic interaction between polycations with positive charge and serum molecules with negative charge, and then aggregation would be cleared by reticuloendothelial system (RES).11 In order to prolong the systematic circulation time of polycation and avoid the clearance by RES in vivo, many strategies have been adopted, such as PEGylation,12-19 and the introduction of natural polysaccharide including dextrans,20 chitosan,21 as well as cyclodextrins (CDs).22 Polysaccharides are biocompatible, biodegradable, and suitable for functional delivery systems. Moreover, with respect to CDs, besides the characteristic of polysaccharides, CDs have distinct advantage due to the special molecular structure, which have the hydrophilic exterior surface and hydrophobic interior cavity.23,24 Owing to this special structure, CDs are commonly used as the host molecule in the hydrophobic cavity to bind a variety of guest molecules, such as nucleic acid, contrast agents, and drugs, to construct supramolecular structures in aqueous solution based on host-guest interaction.25,26 Host-guest interaction systems are widely used in delivery of drug,


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plasmid DNA, and siRNA.27-29 One of the typical host-guest supramolecular gene delivery systems is based on noncovalent interaction between adamantane (Ad) and β-CD.30-32 The coating of CD-Ad on gene vector not only reduces the polycationic cytotoxicity but also improves the gene transfection efficiency under in vitro serum condition.23 Although introduction of electroneutral CDs would prolong the systematic circulation time of gene vector, the in vivo application would also be severely restricted due to the nonspecific interaction between the vector and anionic molecules in blood.33 In order to improve the targeting property of gene vector, targeting ligands were widely introduced into the gene delivery system.9,22,34,35 Cancer cell surface can over-express many kinds of biomarkers or receptors. Integrin αvβ3, as one of typical targeting receptors, plays an important role in cell adhesion and mediating the tumor growth, invasion, and metastasis.36,37 The targeting ligand of arginine-glycine-aspartate (RGD) synthetic peptide shows high ability to bind integrin αvβ3, and polymer-RGD conjugates are being used for targeted gene delivery and cancer therapy.38,39 Here, a host-guest chemistry was used to construct gene delivery system containing RGD ligand for safe and efficient targeting gene transfection. The 5 kDa branched PEI (PEI 5 kDa) was selected as a scaffold polycation and mono-tosylated β-cyclodextrin (TsO-β-CD) was introduced as the host component. The targeting peptide GRGDS was conjugated onto adamantanecarboxylic acid (AdCOOH) as the guest component. The targeting ability of PEICD/AdGRGDS was evaluated via in vitro gene transfection and cellular internalization. To further evaluate targeting specificity and transfection distribution in tumor and normal orangs, the gene vector was injected into mice via tail-vein. As shown in Scheme 1, after tail-vein injection of PEI-CD/AdGRGDS/DNA complexes, the targeting GRGDS moiety would improve cellular internalization of complexes via receptor-mediated endocytosis. After internalization, the


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effect of proton sponge of PEI would induce the quick endosomal escape of complexes to cytoplasm, and plasmid DNA would enter nucleus efficiently. In vitro and in vivo studies validated that PEI-CD/AdGRGDS had high transfection efficiency and good tumor targeting specificity.

Scheme 1. Schematic illustration of targeted gene delivery system PEI-CD/AdGRGDS for gene therapy. The PEI-CD/AdGRGDS can targetedly deliver plasmid DNA into the tumor site via the ligand-receptor specific endocytosis. EXPERIMENTAL SECTION Materials and Chemicals. PEI 5 kDa (PEI 5000, PEI 5k), β-cyclodextrin (β-CD), and 1Adamantanecarboxylic acid (AdCOOH) were obtained from Sigma-Aldrich and used directly. 2-chlorotrityl chloride resin (100-200 mesh, loading: 1.32 mmol/g), N-fluorenyl-9methoxycarbonyl (FMOC) protected L-amino acids (FMOC-Gly-OH, FMOC-Ser(tBu)-OH, FMOC-Arg(Pbf)-OH,

FMOC-Asp-OH),

o-benzotriazol-N,N,N’,N’

tetramethyluroniumhexafluorophosphate (HBTU) were purchased from GL Biochem Ltd.


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(Shanghai, China) and used directly. Diisopropylethylamine (DIEA) was provided by Shanghai Reagent Chemical Co. (China) and used after distillation. N-hydroxybenzotriazole (HOBt), triisopropylsilane (TIS), piperidine, trifluoroacetic acid (TFA), N,N-Dimethylformamide (DMF), p-toluenesulfonyl chloride (p-TsCl), dichloromethane (DCM), and ammonium chloride were purchased from Shanghai Reagent Chemical Co. (China) and used directly. Haematoxylin and eosin (H&E), 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside substrate (X-gal), YOYO-1, and Hoechst 33258 were obtained from Invitrogen Corp (USA). Dulbecco’s Modified Eagle’s Medium

(DMEM),

penicillin-streptomycin,

0.25

%

Trypsin-EDTA

solution,

3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and fetal bovine serum (FBS) were obtained from Lonza Corp (USA). All other reagents and solvents were of analytical grade and used directly. Synthesis of Mono-6-(p-Tolylsulfonyl)-β-Cyclodextrin (TsO-β-CD). TsO-β-CD was synthesized according to previous established procedure.40,41 10 g of β-CD was dissolved in 240 mL of deionized water at room temperature, and 2.6 g of p-toluenesulfonyl chloride was slowly added into the β-CD aqueous solution under magnetic stirring to make sure that substitution only occurred at C6 position. After mixture solution stirring for 2 h, 40 mL of NaOH solution (2.5 M) was added, and suspension was obtained and filtered. Then 12.0 g of ammonium chloride was added to filtrate for adjusting the pH value to 8.0, and filtrate was further cooled at 4 oC for overnight. After that, precipitation was obtained via vacuum filtration and further washed three times by acetone. In order to further remove the unreacted β-CD and p-TsCl, the precipitation was recrystallized three times at 60 oC. The final product of TsO-β-CD was obtained after drying at 50 oC for 2 days under vacuum.


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Synthesis of PEI-CD. PEI-CD was prepared according to previous report.42 Briefly, 2.5 g of PEI 5k was dissolved in 20 mL of DMSO, and then 10 mL of DMSO solution of TsO-β-CD (1.3 g) was added to PEI 5k solution under N2 atmosphere at 90 oC for 2 h. The mixture solution was further stirred under N2 atmosphere at 90 oC for 3 days. After that solution in dialysis tube (MWCO 2000) was dialyzed in deionized water for 2 days, and the light yellow powder was obtained after freeze drying for 3 days. 26,43 Synthesis of AdGRGDS. Peptide was manually synthesized according to previous methods using standard Fmoc solid phase peptide synthesis (SPPS).44-47 In brief, 2-chlorotrityl chloride resin was soaked in DMF for 30 min, and then 2 equiv. of (relative to the substitution degree of resin) first Fmoc-protected amino acid and 4 equiv. of DIEA in DMF was loaded on resin for 2 h. After that, Fmoc groups was deprotected by 20 % piperidine-DMF (v/v), and remaining amino acids were loaded in turn by adding 2 equiv. of Fmoc-protected amino acid, 4 equiv. of HBTU, HOBt, and DIEA for 3 h. The coupling efficacy was monitored via the ninhydrin assay. The AdCOOH was loaded on resin as the last amino acid. After completion of peptide synthesis, resin was washed by DMF and DCM, and then dried under vacuum for overnight. For cleaving the expected peptides from resin, dried resin was dispersed in a cleavage cocktail containing TFA (95%), TIS (2.5%) and H2O (2.5%) at room temperature for 2 h. The filtrate was collected and concentrated by rotary evaporation. The concentrated solution was precipitated in cold ether and dried under vacuum overnight, and then the product was further dissolved in deionized water and lyophilized. The molecular weight of AdGRGDS was confirmed by electrospray ionization mass spectrometry (ESI-MS). Preparation of PEI-CD/AdGRGDS Polymer Inclusion Complex. 0.315 g of PEI-CD and 0.063 g of AdGRGDS were dissolved in 15 mL of deionized water and stirred at room


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temperature for 12 h.26 After that solution in dialysis tube (MWCO 2000) was dialyzed in deionized water for 2 days, and the white powder was obtained by lyophilisation after 2 days.43 Characterization of Polymers. The chemical structure of PEI-CD was characterized by 1H nuclear magnetic resonance (1H NMR) spectra on a Varian Unity 300 MHz spectrometer, and D2O as the solvent. The host-guest interaction of PEI-CD/AdGRGDS was characterized by twodimensional nuclear overhauser effect (NOE) spectroscopy nuclear magnetic resonance (2DNOESY NMR) spectra on a Bruker Avance III HD 400 MHz NMR spectrometer. The molecular weight of AdGRGDS was detected by ESI-MS systems, and molecular weight of PEI-CD as well as PEI-CD/AdGRGDS was measured by gel permeation chromatography (GPC) with a Waters-2690D HPLC equipped with Ultrahydrogel 120 and 250 columns. HAc–NaAc buffer solution (0.1 M, pH 2.8) was used as eluent for PEI-CD and PEI-CD/AdGRGDS at a flow rate of 1.0 mL/min. Preparation of Polymer/DNA Complexes. To prepare PEI-CD/AdGRGDS/DNA and PEICD/DNA complexes at different N/P ratios, the different volume of PEI-CD/AdGRGDS or PEICD solution was added to 1 µg of DNA (200 ng/µL in 40 mM Trise HCl buffer solution), and then mixture solution was diluted to 100 µL by PBS solution and vortexed for 30 s. After that, mixture solution was incubated at 37 oC for 30 min. All complexes were prepared freshly before characterization. Characterization of Polymer/DNA Complexes. The DNA binding capability of PEICD/AdGRGDS and PEI-CD was evaluated by agarose gel retardation assay. Briefly, the PEICD/AdGRGDS/DNA and PEI-CD/DNA complexes at various N/P ratios (from 0.5 to10) were loaded onto the 0.7 % (w/v) agarose gel containing GelRedTM, and conducted in tris-acetate


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(TAE) running buffer at 80 V for 60 min. After that, DNA was observed using a Vilber Lourmat imaging system equipped with a UV lamp (Paris, France). To further evaluate the DNA encapsulation amount by PEI-CD/AdGRGDS and PEI-CD, the concentration of non-encapsulated DNA was measured by a PerkinElmer Lambda 35 UV spectrophotometer at 260 nm. After incubating of PEI-CD/AdGRGDS/DNA and PEI-CD/DNA complexes (10 µg DNA) at N/P ratios of 1, 10, and 20 at 37 oC for 30 min, various complexes were centrifuged at 12,000 rpm for 30 min, and then supernatant was measured and amount of non-encapsulated DNA was measured according to DNA concentration standard operation curve. The DNA encapsulation efficiency was measured according to equation: encapsulation

efficiency (%)= The diameter and zeta potential of PEI-CD/AdGRGDS/DNA complexes at various N/P ratios (from 2 to 30) were carried out by Nano-ZSZEN3600 (Malvern Instruments) at 25 oC. After complexes were prepared and incubated for 30 min, complexes were diluted to 1 mL volume with deionized water before measurement. The stability of PEI-CD/AdGRGDS/DNA complexes at N/P ratio of 10 in PBS solution at different incubation time was measured by Nano-ZSZEN3600 (Malvern Instruments) at 25 oC. The morphology and distribution of complex at N/P ratio of 10 were observed by transmission electron microscopy (TEM, JEOL-2100) at an acceleration voltage of 200 kV. Cell Culture and Amplification of Plasmid DNA. COS7 cells (African green monkey SV40-transformed kidney fibroblast) and HeLa cells (human cervix carcinoma) in high glucose DMEM media with 10% FBS and 1% antibiotics (penicillin-streptomycin, 10 000 U mL-1) were incubated in incubator containing 5% CO2 at 37 oC.


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The green fluorescent protein reporter gene (pEGFP), luciferase reporter gene (pGL-3), and pORF-LacZ were amplified and purified according to our previous reports.43,48 Plasmid DNA was diluted to 200 ng/µL by Trise HCl buffer solution and stored at -20oC. Cytotoxicity of PEI-CD/AdGRGDS/DNA Complexes. In vitro cytotoxicity of complexes was examined in COS7 and HeLa cells by MTT assay. Cells in 100 µL of DMEM with 10% FBS were seeded in a 96-well plate at a density of 6000 cells/well and incubated for 24 h. After that, complexes at different N/P ratios was added to each well and incubated for 48 h. And then, 20 µL of MTT was added to each well and incubated for 4 h. Thereafter, 150 µL of DMSO was added to replace the medium. The optical density (OD) was measured by a microplate reader (BIO-RAD, Model 550, USA) at 570 nm. The cell viability was calculated as: Cell viability = (ODsamples-ODDMSO/ODcontrolODDMSO)*100. In Vitro Gene Transfection. The gene transfection efficiency was evaluated in HeLa and COS7 cells. Before transfection, the cells were seeded into a 24-well plate at a density of 6 ×104 cell/well and cultured in DMEM with 10% FBS for 24 h. After that, 1 mL of complexes in DMEM with 10% FBS at various N/P ratios were added to 24-well plate and cultured for 4h, and then medium was replaced by 1 mL of fresh DMEM with 10% FBS and cultured for 44 h. For qualitative study (green fluorescence protein assay), the pEGPF plasmid was used as reported gene, and transfected cells mediated by PEI-CD/AdGRGDS/pEGPF as well as PEI-CD/pEGPF complexes at an optimal N/P ratio of 10 were directly observed using inverted fluorescence microscope (Leica). For quantitative study (green fluorescence protein assay and luciferase assay), the pEGPF and pGL-3 plasmid was respectively used as reported gene. The fluorescence intensity of transfected cells mediated by PEI-CD/AdGRGDS/pEGPF as well as PEI-CD/pEGPF


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complexes at an optimal N/P ratio of 10 was quantified by flow cytometry. And PEICD/AdGRGDS/pGL-3 as well as PEI-CD/pGL-3 at N/P ratios ranging from 5 to 40 was evaluated. After removing of complexes, cells were washed for 3 times by PBS and further lysed by adding 200 µL of reporter lysis buffer. The relative light unit (RLU) was measured using a chemiluminometer (Lumat LB9507, EG&G Berthold, Germany), and total cellular protein was measured using a BCA protein assay kit (Pierce). The luciferase expression activity was defined as RLU/mg protein. Confocal Laser Scanning Microscopy. For evaluating the capability of polycation transfer plasmid DNA to cells, cellular uptake experiments were investigated by a CLSM (Nikon C1-Si, TE2000, Japan). Cells were seeded on cover-glass slides at an original density of 1×105 cells and cultured for 24 h. Before preparing of complexes, the DNA was labelled with 2.5 µL of green fluorescent dye YOYO-1 (10 mmol/L) at 37 oC for 15 min. Complexes were removed after incubating for 4 h, and cells were washed three times by PBS. Then nucleus was stained with 20 µL of Hoechst 33258 (2 µg/µL) at 37 oC for 15 min. Cells were incubated with 200 µL of fresh DMEM after further washing with PBS for three times. To further evaluate targeting specificity of PEI-CD/AdGRGDS/DNA complexes to integrin expression positive cells, excess free GRGDS was pre-incubated with HeLa and COS7 cells for 30 min to block αvβ3 on surface of HeLa cell membrane, and then PEI-CD/AdGRGDS/DNA complex at N/P ratio of 10 was added and incubated for 4 h. Green ﬂuorescence was collected by CLSM using a 405 nm diode for Hoechst 33258, and blue fluorescence was collected using a 488 nm argon laser for YOYO-1. Flow Cytometry Quantitative Analysis. Flow cytometry was quantitatively investigated the cellular uptake and transfection efficiency. Cells were seeded in a 6-well plate at a density of 1×106 cell/well. After YOYO-1 stained complexes at N/P ratio of 10 incubating for 4 h, DMEM


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was removed, and cells were washed with PBS. And then, cells were trypsinized by 0.25% trypsin and collected in centrifuge tubes after centrifugation at 1000 rpm for 3 min. The collected cells were resuspended in PBS and filtrated. The filtrate was tested by a Beckman Flow Cytometer (Epics XL) equipped with Flowjo 7.6 software. Tumor Model and in Vivo Gene Transfection. The animal experiments were agreed with ethics of institutional and guidelines for care and use of research animals from Wuhan University. H22 cells are suspended growth and have excellent tumor formation rate, and have been widely used in mouse-based tumor models, hence, the murine hepatic carcinoma cell line H22 tumor xenograft model was established in this work. Female BALB/c nude mice (5-weeks old, ≈ 22.0 g) were obtained from Wuhan University Animal Biosafty Level III Lab (Wuhan, China). The tumors were established by injecting 100 µL of H22 cell suspensions in PBS (1 × 107 cells) into the armpits of BALB/c nude mice. Upon tumor size reaching to 200 mm3, the in vivo gene transfection experiments were conducted for 2 days. 100 µL of PEI-CD/AdGRGDS/DNA and PEI-CD/DNA complexes at N/P ratio of 10 in PBS was subcutaneously injected into tumors. After 2 days injection of complexes, the mice were sacrificed and tumor xenografts were resected. After washing with cold PBS for three times, the tumor xenografts injected with PEI-CD/AdGRGDS/pORF-LacZ and PEI-CD/pORF-LacZ complexes (containing 10 µg of pORF-LacZ) were stained overnight according to X-gal staining kit (InvivoGen, USA). And then, images of stained tumor xenografts were obtained photographically. After that, tumor xenografts were fixed in 4% formaldehyde for 1 day, and fixed tumor xenografts were embedded into paraffin and cut into 5 µm of thick sections. The tumor xenografts sections were placed on polylysinecoated slides and stained by haematoxylin


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and eosin (H&E) for histological examination. The stained slides were observed by fluorescent microscope (BX60, Olympus, Japan) at 200 × magnifications. Tumor xenografts injected with PEI-CD/AdGRGDS/pGL-3 and PEI-CD/pGL-3 complexes (containing 10 µg of pGL-3) were dissolved in cell lysis buffer and homogenized by an IKAUltra-Turrax homogenizer. The tissue lysates supernatant was collected via centrifugation (12,000 g/min) at 4 oC for the quantification of luciferase activity and protein content. The transfection efficiency of lysates was expressed as RLU/mg protein. To demonstrate the targeting specificity of PEI-CD/AdGRGDS, the in vivo transfection efficiency and transfection distribution was further evaluated via tail-vein injection into H22 tumor xenograft model. After 2 days tail-vein injection of 100 µL of PEI-CD/AdGRGDS/pORFLacZ and PEI-CD/pORF-LacZ complexes into mice, the mice were sacrificed and tumor xenografts as well as normal organs including heart, liver, spleen, lung, and kidney were collected for X-gal staining and eosin staining for observing the pORF-LacZ expression. The Xgal staining and eosin staining procedure was similar to that in subcutaneous injection experiment. Statistical Analysis. Statistical analysis was conducted by the Student’s t test, and a p value < 0.05 was considered to be statistically significant among different groups.

RESULTS AND DISCUSSION

Synthesis and Characterization of PEI-CD/AdGRGDS. In order to construct an efficient targeted polycation gene delivery system for potential gene therapy, the AdCOOH modified targeting molecule GRGDS (AdGRGDS) was introduced onto the CD modified branching PEI (PEI-CD) via host-guest interaction. Polymers of PEI-CD and AdGRGDS were synthesized


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respectively, and then the PEI-CD/AdGRGDS supramolecular inclusion complex was obtained simply through the modular self-assembly between the appropriate amount of PEI-CD and AdGRGDS in an aqueous solution. The preparation of PEI-CD, and PEI-CD/AdGRGDS inclusion complex were illustrated in Scheme 2. PEI-CD/AdGRGDS was used as the targeted gene delivery system. The AdGRGDS was manually synthesized through standard FMOC SPPS technique. The molecular weight of AdGRGDS was measured by ESI-MS (LCQ Advantage, Finigan, USA). Molecular mass of AdGRGDS was calculated 652.3, and m/z of ESI-MS was found 653.3 ([M+H]+) and 1305.4 ([2M+H]+). The spectrum of ESI-MS was listed in Figure S1.


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Scheme 2. (A) Preparation of PEI-CD and (B) construction of PEI-CD/AdGRGDS inclusion complex via host-guest interaction.

PEI was modified with mono-tosylated β-cyclodextrin (mono-TsO-β-CD) to obtain the PEICD. Mono-substituted β-CD was used to prevent the intermolecular crosslinking of PEI. On the other hand, it was beneficial to form inclusion complex between the minimal modifying host CD cavity and guest molecules.42 To prevent excess of CD conjugation onto the surface of each same PEI molecular, TsO-β-CD solution was added dropwise to PEI solution under magnetic stirring. The structure of PEI-CD was confirmed by 1H NMR and spectrum was shown in Figure S2. The multiple peaks of δ 2.3-2.8 ppm attributed to the methylene protons of PEI, and the typical proton signal of δ 4.9 ppm belonged to the H1 of β-CD, indicating that the β-CD has been integrated into PEI. The degree of substitution (DS) of CD on per PEI molecule was obtained through the integral peak areas ratio between H1 protons belonging to CD and methylene protons in PEI, and it was 2.0. For evaluating the host-guest interaction between PEI-CD and AdGRGDS, the 2D-NOESY NMR was characterized, and the spectrum was listed in Figure S3. The 2D-NOESY cross-peaks from correlation between the protons of β-CD (H3, 5, 6) and adamantly protons (Hb, c, d) demonstrated the conformational information on the PEI-CD/AdGRGDS complex model. DNA Binding Ability and Encapsulation Efficiency. The capability of polycation to bind DNA via electrostatic interaction is a prerequisite for polycation as gene vector.49 The binding capability of PEI-CD and PEI-CD/AdGRGDS for DNA was demonstrated via agarose gel retardation assay, and result of PEI/DNA was shown as control. As presented in Figure 1, free DNA in PEI/DNA complexes (Figure 1A) was completely retarded at an N/P ratio of 1, while free DNA in PEI-CD/DNA (Figure 1B) and PEI-


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CD/AdGRGDS/DNA complexes (Figure 1C) was bound at an N/P ratio of 2. Moreover, at an N/P ratio of 1, mobility of free DNA in PEI-CD/DNA complexes was reduced than that in PEI-CD/AdGRGDS/DNA complexes. These results indicated that, as comparing with PEI, PEI-CD and PEI-CD/AdGRGDS were inclined to condense DNA at low N/P ratios, but PEI-CD/AdGRGDS had slight weaker DNA binding capability as compared to PEI-CD, which was ascribed to the decreased density of amines after interaction with AdGRGDS.

Figure 1. Agarose gel electrophoresis retardation assay of PEI/DNA (A), PEICD/DNA (B), and PEI-CD/AdGRGDS/DNA complexes (C) at N/P ratios ranging from 0.5 to 10.

The DNA encapsulation efficiency was evaluated by spectrophotometer to measure the nonencapsulated DNA concentration at 260 nm, and the results were listed in Figure S4. With increasing of N/P ratios, DNA encapsulation efficiency increased correspondingly, and increasing trend was consistent with that measured by agarose gel retardation assay. Biophysical Characterization of PEI-CD/AdGRGDS/DNA Complexes. The proper particle size, zeta potential, and morphology of complexes are vital for cellular uptake and efficient gene transfection.30,50,51 As shown in Figure 2A, with increasing of N/P ratios, particle size of PEI-


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CD/DNA and PEI-CD/AdGRGDS/DNA decreased at an average diameter varying from 380 to 200 nm. Complexes with diameter around 200 nm would be effective for endocytosis and gene delivery.52 Furthermore, it was found that diameter of PEI-CD/AdGRGDS/DNA complexes was obviously smaller than that of PEI-CD/DNA complexes at the same N/P ratios. For evaluating the stability of PEI-CD/AdGRGDS/DNA in physiological solution, the particle size of PEI-CD/AdGRGDS/DNA at N/P ratio of 10 in PBS with different incubation times was measured. The particle size of PEI-CD/AdGRDS/DNA increased with increasing of incubation time in PBS condition (Figure S5), which was ascribed to abundant positive charge on PEI and low substituted degree of CD.

Figure 2. Particle size (A) and zeta potential (B) of PEI-CD/DNA and PEI-CD/AdGRGDS/DNA complexes at N/P ratios ranging from 2 to 30. TEM images of PEI-CD/DNA (C) and PEICD/AdGRGDS/DNA (D) complexes at N/P ratio of 10.

The morphology of complexes at an N/P ratio of 10 observed by TEM was shown in Figure 2C&D. TEM investigation of PEI-CD/AdGRGDS/DNA and PEI-CD/DNA complexes showed


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well dispersed and analogous to spherical nanoparticle with mean diameters of 50 nm and 80 nm, respectively. The diameter of complexes evaluated by TEM was reduced compared with that measured by DLS. This discrepancy could be ascribed to fact that DLS measures the hydrodynamic diameter in solution, whereas, TEM observes shrinkage of nanoparticle after complexes air-dried onto the TEM grids.43 The surface charge is an important parameter for stability of complexes, cellular uptake and transfection efficiency of complexes.43,53 In this study, zeta potential was measured by DLS. As illustrated in Figure 2B, the surface charge of complexes improved with increasing of N/P ratios ranging from 2 to 30, but above N/P ratio of 10, surface charge had no obvious change, indicating that the excess of polycation had no obvious effect on zeta potential after complexes became stable. Moreover, as compared with the surface charge of PEI-CD/DNA complexes at each N/P ratio, the surface charge of PEI-CD/AdGRGDS/DNA was slightly lower, which was ascribed to the decrease of density of amine with the introduction of AdGRGDS. In Vitro Cytotoxicity of Complexes. In vitro cytotoxicity of complexes is crucial for its further application in vivo or in clinical.43,54 As shown in Figure 3, cell viability of PEICD/AdGRGDS/DNA and PEI-CD/DNA complexes was dose-dependent and decreased gradually with increasing of N/P ratios. PEI-CD/AdGRGDS/DNA complexes showed lower cytotoxicity as comparing with PEI-CD/DNA and PEI-CD/DNA complexes at N/P ratios ranging from 5 to 35, which could be attributed to different surface charge on complexes. The cell viability of both complexes was over 80% in HeLa and COS7 cell lines. The results demonstrated that the complexes were adaptable for gene delivery and also for in vivo application.


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Figure 3. Cell viability of PEI/DNA, PEI-CD/DNA, and PEI-CD/AdGRGDS/DNA complexes at N/P ratios ranging from 5 to 35 in COS7 (A) and HeLa cells (B). In vitro gene transfection. Serum resistance is one of important parameters for polymeric gene vector used as in vivo or clinical gene therapy.6,43,54 In order to assess the serum stability of complexes, all of in vitro assays was investigated under condition in presence of 10% FBS. The transfection efficiency of complexes was evaluated by green fluorescence protein assay and luciferase assay, respectively. Luciferase gene expression mediated by PEI-CD/AdGRGDS and PEI-CD was evaluated in HeLa and COS7 cells at N/P ratios ranging from 5 to 40. As illustrated in Figure 4, trend of luciferase gene expression in both cell lines was similar, i.e., luciferase gene expression increased firstly and then decreased slightly, and luciferase gene expression at N/P ratio of 10 was higher than that at other N/P ratios. Moreover, in HeLa cells, luciferase gene expression of PEI-CD/AdGRGDS/DNA complexes was higher than that of PEI-CD /DNA complexes at each N/P ratio, and luciferase gene expression of PEI-CD/AdGRGDS/DNA at N/P ratio of 10 was higher than that of 25 kDa PEI/DNA at N/P ratio of 10. In COS7 cells, luciferase gene expression of PEI-CD/AdGRGDS/DNA and PEI-CD/DNA complexes was similar to each other at each N/P ratio, and luciferase gene expression of PEI-CD/AdGRGDS/DNA at N/P ratio of 10 was comparable to that of 25 kDa PEI/DNA at N/P ratio of 10.


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Figure 4. Luciferase gene expression of PEI-CD/DNA and PEI-CD/AdGRGDS/DNA complexes at N/P ratios ranging from 5 to 40 under condition of 10% serum in COS7 (A) and HeLa cells (B). *p

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