A Facile Multifunctionalized Gene Delivery Platform Based on Î±,Î²

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A facile multi-functionalized gene delivery platform based on #,# cyclodextrin dimers Qi Lei, Hui-Zhen Jia, Wei-Hai Chen, Lei Rong, Si Chen, Guo-Feng Luo, Wen-Xiu Qiu, and Xian-Zheng Zhang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00307 • Publication Date (Web): 05 Oct 2015 Downloaded from http://pubs.acs.org on October 10, 2015

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ACS Biomaterials Science & Engineering

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A facile multi-functionalized gene delivery platform based on

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α,β cyclodextrin dimers

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Qi Lei, Hui-Zhen Jia, Wei-Hai Chen, Lei Rong, Si Chen, Guo-Feng Luo, Wen-Xiu Qiu, Xian-Zheng

4

Zhang*

5

Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry,

6

Wuhan University, Wuhan 430072, PR China

7

ABSTRACT

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In this paper, adamantane (Ad) substituted reduction-sensitive polyethyleneimine (Ad-b-SS-PEI600)

9

was used as cationic polymer gene vector for DNA loading. And then the α,β CD dimers were

10

applied as the bridges between the Ad-b-SS-PEI600/DNA polyplexes and the functional moieties, i.e.

11

targeting moiety and fluorescent probe through effective host-guest interactions. Located in the

12

surface of the assemblies, the post-decorated functional moieties can efficiently exert their own

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function. It was found that gene delivery platforms thus obtained exhibited promising DNA

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compaction capability, rapid stimuli responsiveness, and good biocompatibility, and moreover they

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can mediate efficient gene transfection, targeting and imaging both in vitro and in vivo.

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Keywords: Gene delivery; Post-modification; α,β CD dimer; Host-guest interaction

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1. INTRODUCTION

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Unlike drug carriers that can be sophisticatedly modified both before and after drug loading,

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polymer-based gene vectors often suffer from serious defects in multi-functional decoration.1-3 The

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large-sized plasmid gene would significantly change the topological structures of the polymer-based

*

To whom correspondence should be addressed. Tel. & Fax: 86-27-68754509. E-mail address: [email protected] (X. Z. Zhang).

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carriers through electrostatic interaction4, in which the multi-functional moieties decorated on the

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carrier would be trapped inside the assemblies, and result in poor functional efficiency.5-6 For

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example, the cell targeting moieties should be located on the outer side of the assemblies for better

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efficacy;7-10 the pre-incorporated hydrophilic PEG moieties packed inside the assembly would

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sharply weaken the electrostatic interaction between the cationic carriers and the anionic DNA,

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resulting in loose and unstable complexes.11,12 Therefore, strategies for post-modification of

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carrier/DNA complexes without interrupting their own merits have attracted increasing research

8

interests in recent years. Kataoka et al. reported the pDNA/cationic polymer complexes with anionic

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dendrimer-based photosensitizers (DPc) via mild electrostatic interaction to obtain post-modified

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ternary complexes.13,14 The post-modified DPc on the outer layer can efficiently facilitate

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photochemical internalization (PCI)-mediated gene delivery, and the release of DPc from the carriers

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in endosomes can also avoid photochemical inactivation of DNA. Moreover, Gu and coworkers

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compacted DNA with diselenide-modified oligoethylenimine (OEI-SeSex) to form a cationic

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reduction-sensitive complex.15 And anionic disulfide-conjugated hyaluronic acid derivatives (HA-

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SS-COOH) were further shielded on it to form a ternary complex, which can proceed reduction-

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controlled hierarchical unpacking gene delivery. However, it may be difficult to coordinate multiple

17

functionalities and capabilities in a single carrier via the electrostatic interaction due to their intricate

18

interplay.16-18 Moreover, the electrostatic interaction may be disturbed by ion strength during the

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circulation in blood.19 Thus, an alternative strategy that can coordinate multiple functionalities and

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form stable complexes is in urgent demand for post-modification of carrier/DNA complexes.

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Host-guest interaction, a rapid and efficient reaction under mild condition, is widely applied as a

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better option to electrostatic interaction in post-modification for drug carriers.20-22 Typical

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supramolecular host cyclodextrin (e.g. α-CD and β-CD) is well-known for the high selectivity in

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hydrophobic guest molecules (e.g. benzene and adamantane) to form stable inclusion complexes

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through efficient host-guest interaction.23,24 Because of their promising properties including easy 2 ACS Paragon Plus Environment

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functionalization, non-toxicity and surface hydrophilicity, the cyclodextrins have been thoroughly

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investigated in drug/gene delivery systems.25-27 However, when pre-modified on the carriers, the

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electroneutral CD molecules with large size may impede the drug loading.28 Therefore, the strategy

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that conjugating the guest molecules to the carriers before drug loading, and then decorating the CD

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molecules on the loaded carriers via host-guest interaction seems a preferable alternative

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pathway.29,30 To further simplify the synthesis process and avoid the steric hindrance of macrocyclic

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host molecules, the α,β CD dimer had been reported as a “bridge” in our previous report, which can

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facilitate the effective connection of two separated sophisticated parts under mild conditions.31,32

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Moreover, due to the asymmetry of the α,β CD dimer, the assembly of the different functional

10

components are space-selective and hierarchical.33

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Taking the advantages of α,β CD dimer, herein, we designed a gene delivery platform that can be

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facilely multi-functionalized after DNA loading without interrupting the merits of carrier/DNA

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complexes. As shown in Scheme 1, the amino group of hyperbranched reduction-sensitive

14

polyethyleneimine was partly substituted by adamantane (Ad) to form Ad-b-SS-PEI600, which could

15

be used as cationic polymeric gene vector for DNA loading.34-37 With plenty of Ad groups, cationic

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Ad-b-SS-PEI600 can condense DNA into compact polyplex with some Ad groups randomly hanging

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on the surface. Then the α,β CD dimers were introduced to the Ad-b-SS-PEI600/DNA polyplexes

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through the specific host-guest interactions between Ad and β-CD, obtaining hierarchical ternary

19

complex as a gene delivery platform with many α-CD remaining on the surface. Subsequently, the

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targeting moiety (RGD) and fluorescent probe (RhB) were conveniently decorated on the surface of

21

the assemblies through the efficient host-guest interactions between phenylalanine and α-CD. The

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final multifunctional assemblies can mediate promising in vivo tumor targeting and ex vivo tumor

23

imaging. The method applied in this gene delivery platform can be easily applied in many other gene

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delivery systems, and the functional moiety could be any functional parts needed for the delivery

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system. The novel strategy made delicate post-modification of polymer-based carrier/DNA 3 ACS Paragon Plus Environment


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complexes not only possible but also facile. Our strategy also provided a new perspective for the

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design and synthesis of future gene delivery system.

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2. EXPERIMENTAL SECTION

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2.1. Materials. NucleoBond Xtra Maxi EF plasmid purification was purchased from Macherey-

5

Nagel (Germany). GelRed was bought from Biotium (CA, USA). Dulbecco’s Modified Eagle

6

Medium (DMEM), penicillin-streptomycin, fetal bovine serum (FBS), 3-(4,5-Dimethylthiazol-2-yl)-

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2,5-diphenyltetrazolium bromide (MTT), and phosphate buffered saline (PBS) were purchased from

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Invitrogen Corp. A Micro BCA protein assay kit was purchased from Pierce. Molecular probes

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(Hoechst 33258, YOYO-1 iodide) were purchased from Invitrogen (CA, USA). X-gal staining kit

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was obtained from InvivoGen (USA).

11

Dimethylformamide (DMF) and diisopropylethylamine (DIEA) were obtained from Shanghai

12

Reagent Chemical Co. (China) and distilled prior to use. Dimethylsulphoxide (DMSO),

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trifluoroacetic acid (TFA), ethanedithiiol (EDT), piperdine, 1-adamantanecarbonyl chloride and

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Rhodamine B (RhB) were provided by Shanghai Reagent Chemical Co. (China) and used directly.

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Cystamine

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methoxycarbonyl (Fmoc)-protected L-amino acids: Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-

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OH, Fmoc-Asp(OtBu)-OH, and 2-chlorotrityl chloride resin (100–200 mesh, loading: 1.08 mmol g-

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1

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hydroxybenzotriazole (HOBt), triisopropylsilane (TIS), and thioanisole were obtained from GL

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Biochem Ltd. (Shanghai, China) and used as received. Hyperbranched 25kDa polyethyleneimine

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(PEI 25k) and hyperbranched 600Da polyethyleneimine (PEI600) were purchased from Sigma-

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Aldrich and used as received. Other reagents were of analytical grade and used as received.

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2.2. Synthesis of RGDFF, RhBF and α,β CD Dimer. The peptide sequences (RGDFF and RhBF)

24

were synthesized manually by applying standard solid-phase methodologies based on classical Fmoc 4

),

bisacrylamide

(CBA) was

bought from

o-benzotriazole-N,N,N’,N’-tetra-methyluronium

HEOWNS (China).

hexafluorophosphate

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N-Fluorenyl-9-

(HBTU),

N-

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chemistry.38 Briefly, in the presence of DIEA/HBTU/HOBt, amino acids or RhB were connected to a

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2-chlorotrityl chloride resin (1.1 mmol g-1) step-by-step. 20% piperidine/DMF (v/v) solution was

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introduced to remove the Fmoc groups in the peptides. The coupling efficacy of each step was

4

monitored by the ninhydrin assay. After the removal of the last N-terminal Fmoc groups, the peptide

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was entirely deprotected and cleaved from resin by using a 15 mL cocktail of TFA/TIS/deionized

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water (95:2.5:2.5 v/v/v) for 100 min at room temperature. The collected solution was concentrated to

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a viscous solution by evaporation in vacuum and then dropped into sufficient cold diethyl ether and

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stored at −20 oC overnight to precipitate the product. The precipitate was centrifuged and dried under

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vacuum for 24 h. And the product was dissolved in distilled water and then lyophilized and stored at

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−20 oC. The purity of the peptide was determined by high-performance liquid chromatography

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(HPLC, Prominence LC−20A, Shimadzu, Japan) with a C18 reversed-phase column by using a

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linear gradient from 95% to 5% of H2O/acetonitrile containing 0.1% trifluoroacetic acid at 1 mL min-

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1

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measured by electrospray ionization mass spectrometry (ESI-MS, LCQ Advantage, Finigan, USA).

for 30 min. The purity of the peptides was at least 90%. The molecular weights of the peptides were

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α,β CD dimer was synthesized according to the literature.32 Briefly, azide-modified β-CD (β-CD-

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N3) and alkyne-modified α-CD (α-CD-C≡CH) were synthesized, respectively. And α,β CD dimer

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was synthesized by typical “click” chemistry. The obtained α,β CD dimer was evaluated by matrix-

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assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis on

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an Axima TOF2 mass spectrometry (Shimadzu, Kyoto, Japan).

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2.3. Synthesis of SS-PEI600 and Ad-b-SS-PEI600. According to the literature39, the reduction-

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sensitive SS-PEI was obtained from the Michael addition between the acrylamide group in CBA and

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the amino group in PEI. In brief, 0.88g of PEI600 was dissolved in 10 mL aqueous methanol, and

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added to a three-necked flask equipped with a condenser under nitrogen protection. And 0.325 g

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CBA was dissolved in 5 mL methanol and added dropwise to the PEI solution via a constant pressure

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funnel. Then the reaction mixture was stirred under nitrogen atmosphere at 45 oC for 36 h. And 5 ACS Paragon Plus Environment


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excess PEI (0.09 g) in 2 mL methanol was added to react with the acrylamide groups for another 12

2

h. Subsequently, the mixture was diluted with deionized water and acidified to pH of 4 with HCl, and

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dialyzed against deionized water in a dialysis membrane filter (molecular weight cutoff (MWCO):

4

3500 Da) for 48 h to remove small molecules. Finally, the solution was lyophilized to obtain the

5

product SS-PEI600. Size-exclusion chromatography and multiangle laser light scattering (SEC-

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MALLS) analysis was actualized to determine the molecular weight distribution of SS-PEI600. A dual

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detector system consisting of a MALLS device (DAWNEOS, Wyatt Technology) and an

8

interferometric refractometer (Optilab DSP, Wyatt Technology) was applied. HAc-NaAc buffer

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solution (0.03 M, pH 2.7) was used as eluent at a flow rate of 0.6 mL min-1. The MALLS detector

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was operated at a laser wavelength of 690.0 nm.

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The amino group of SS-PEI600 was partially substituted with adamantane subsequently. 0.6 g SS-

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PEI600 was dissolved in 20 mL dry dichloromethane in an ice bath, and catalytic amounts of

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trimethylamine was added under stirring. 0.18 g 1-Adamantanecarbonyl chloride was dissolved in 5

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mL dry dichloromethane, and added dropwise to the mixture. The reaction reacted in ice bath for 4 h,

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and then at room temperature for 6 h under constant stirring. The solvent was then evaporated, and

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the residues were dissolved in 0.1 M HCl, and extracted with dichloromethane for three times to

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remove unreacted 1-adamantanecarbonyl chloride. Then the aqueous phase was dropped into

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sufficient cold acetone and stored at −20 oC overnight to precipitate the product. The precipitate was

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filtered and dried under vacuum for 24 h. The substituting degree of adamantane was confirmed by

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1

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2.4. Cell Culture and Amplification of Plasmid DNA. Human cervix adenocarcinoma (HeLa) cells,

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African green monkey SV40-transformed kidney fibroblast (COS7) cells and human brain

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glioblastoma epithelial (U87) cells were incubated in DMEM supplemented with 10% FBS and 1%

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antibiotics (penicillin-streptomycin, 10 000 U mL-1) at 37 oC in a humidified atmosphere containing

25

5% CO2. The luciferase reporter gene (pGL-3) and the β-galactosidase reporter gene (pORF-LacZ)

H-NMR (300 MHz, D2O).


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was transformed in E. coli JM109 and the enhanced green fluorescent gene (pEGFP-C1) was

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transformed in E. coli DH5α. First, the plasmids were amplified in LB media at 37 oC overnight at

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250 rpm, then collected and purified by NucleoBond Xtra Maxi EF plasmid purification. Finally, the

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purified plasmids were dissolved in TE buffer solution at a final concentration of 200 ng µL-1 and

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stored at −20 oC. The quality of each plasmid DNA (pDNA) was tested by agarose gel

6

electrophoresis and ultraviolet (UV) absorbance at 260 and 280 nm.

7

2.5. Preparation of Vector/pDNA Complexes. The Ad-b-SS-PEI600/pDNA binary complexes at

8

various weight ratios (w/w) ranging from 1 to 15 were prepared by adding dropwise 1 µg of pGL-3

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DNA (200 ng µL-1 in TE buffer solution) into appropriate volume of Ad-b-SS-PEI600 solution (1 mg

10

mL-1 in 150 mM NaCl solution), and then the complexes were diluted to a total volume of 100 mL

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with 150 mM NaCl and vortexed for 5 s. The mixtures were incubated at 37 oC for 30 min to form

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the complexes. All the complexes were used immediately after their preparation. The addition of CD

13

dimer or other decorations was in a similar way. CD dimer with proper amount was added to the

14

obtained Ad-b-SS-PEI600/pDNA binary complexes, and vortexed for 5 s, then incubated at 37 oC for

15

15 min to form the ternary complexes. And the multifunctional assemblies were obtained by adding

16

proper amount of RGDFF/RhBF to the Ad-b-SS-PEI600/DNA/CD dimer ternary complexes under

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vortex and incubating at 37 oC for 15 min.

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2.6. Agarose Gel Retardation Assay. The Ad-b-SS-PEI600/DNA complexes at w/w ranging from

19

0.1 to 15 were prepared as mentioned above by adding dropwise 0.1 µg of pGL-3 DNA into

20

appropriate volumes of Ad-b-SS-PEI600 solution. The complexes were then diluted to a constant

21

volume of 8 µL with 150 mM NaCl solution, and incubated at 37 oC for 30 min. Subsequently, the

22

samples were loaded onto the 0.7% (w/v) agarose gel containing GelRed and with Tris-acetate

23

(TAE) running buffer at 80 V for 60 min. DNA was visualized under a UV lamp in the Vilber

24

Lourmat imaging system (France).



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1

To evaluate the ability of DNA release from Ad-b-SS-PEI600/DNA complexes in vitro, reduced

2

glutathione (GSH) was used for simulating the reductive environment of cytoplasm. The obtained

3

Ad-b-SS-PEI600/DNA complexes at w/w ratio of 1:1 were subsequently incubated with equivalent 2

4

µM, 2 mM and 10 mM GSH at 37 oC for 5 min or 30 min, and the samples incubated in 150 mM

5

NaCl solution were used as a control. Then the samples were electrophoresed on the 0.7% (w/v)

6

agarose gel containing GelRed and with Tris-acetate (TAE) running buffer at 80 V for 60 min. After

7

that, DNA was visualized under a UV lamp in the Vilber Lourmat imaging system (France).

8

2.7. Particle Size and Zeta Potential Measurements. The particle size and zeta potential of the

9

complexes were measured with a Nano-ZS ZEN3600 (Malvern, UK) Instruments at 37 oC. Ad-b-SS-

10

PEI600/DNA binary complexes, Ad-b-SS-PEI600/DNA/CD dimer ternary complexes, Ad-b-SS-

11

PEI600/DNA/CD dimer/RGDFF quaternary complexes and Ad-b-SS-PEI600/DNA/CD dimer/RhBF

12

quaternary complexes were prepared as aforementioned, respectively, then diluted to 1 mL volume

13

with deionized water for particle size and zeta potential measurements. To confirm the host-guest

14

interaction between the CD dimer and Ad groups of the Ad-b-SS-PEI600/DNA complexes, SS-

15

PEI600/DNA complexes were prepared and CD dimer was added as aforementioned, and the size and

16

zeta potential variations were explored. Meanwhile, the assemblies using pre-modification methods

17

were also investigated. Ad-b-SS-PEI600 and CD dimer were incubated together for 15 min, and DNA

18

was added to the mixture and incubated for further 30 min (the weight ratio of wAd-b-SS-PEI600/wCD dimer

19

/wDNA was fixed at 10:10:1). Next, the complexes were diluted to 1 mL volume with deionized water

20

for particle size and zeta potential measurements. The particle size distribution of the Ad-b-SS-

21

PEI600/DNA complexes at w/w ratio of 10:1 and Ad-b-SS-PEI600/DNA/CD dimer ternary complexes

22

at w/w/w ratio of 10:1:10 was also measured before and after co-incubation with 10 mM GSH at 37

23

o

24

2.8. BSA Adsorption. The cationic polymer and polyplexes (Ad-b-SS-PEI600/DNA binary

25

complexes at w/w ratio of 10:1, Ad-b-SS-PEI600/DNA/CD dimer ternary complexes at w/w/w ratio 8

C for 30 min or 3 h by a Nano-ZS ZEN3600 (Malvern, UK) Instruments.


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of 10:1:10, Ad-b-SS-PEI600/DNA/CD dimer/RGDFF quaternary complexes at w/w/w/w ratio of

2

10:1:10:3) were prepared respectively, and mixed with bovine serum albumin (BSA) solutions (2

3

mg/ml) of equal volume. Then the mixtures were rapidly shaken at 37 °C for 30 min, and centrifuged

4

at 8000 rpm for 5 min, and the supernatants were collected to detect the BSA concentration using a

5

UV spectrophotometer at 280 nm. The amount of BSA adsorbed [BSA]ad was defined as [BSA]ad =

6

([BSA]t – [BSA]s)/w, [BSA]t was the total amount (mg) of BSA added to the mixture solution

7

initially, and [BSA]s represented the amount (mg) of BSA in the supernatant, w was the total amount

8

(mg) of the cationic polymer and polyplexes in mixture solution.

9

2.9. Cytotoxicity Assay. The cytotoxicity of Ad-b-SS-PEI600 and PEI 25k was estimated in HeLa

10

and COS7 cells by the MTT assay. In brief, HeLa and COS7 cells were seeded in a 96-well plate at a

11

density of 6000 cells per well and incubated in 100 µL DMEM containing 10% FBS for 24 h. Then,

12

these cationic polymers at different concentrations were added to the wells. After 48 h, 20 µL MTT

13

(5 mg mL-1 in PBS buffer solution) was added to each well and further incubated for 4 h. After that,

14

the medium was removed and replaced with 150 µL DMSO. The absorbance of the DMSO solution

15

in the wells at the wavelength of 570 nm was measured by a microplate reader (Model 550, Bio-Rad,

16

USA) to determine cell viability. The relative cell viability was calculated as (OD570sample/OD570control)

17

× 100%, where OD570control was obtained in the absence of vectors and OD570sample was obtained after

18

co-incubation with cationic polymers.

19

The cytotoxicity of PEI 25k/DNA complexes (at w/w ratio of 1.3:1), Ad-b-SS-PEI600/DNA binary

20

complexes and Ad-b-SS-PEI600/DNA/CD dimer ternary complexes was also evaluated in HeLa and

21

COS7 cells by MTT assay. After the cell attached to the plate, 200 µL complete medium containing

22

complexes at different weight ratios was substituted for the medium and the cells were further

23

incubated for 48 h. The amount of DNA was fixed at 0.2 µg per well. Then the cytotoxicity of the

24

complexes was measured using the same protocol as mentioned above.



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2.10. Transfection Assays. The in vitro transfection efficiency of complexes was evaluated in HeLa

2

and COS7 cells. Ad-b-SS-PEI600/DNA complexes were prepared at various weight ratios ranging

3

from 1 to 15 (w/w). PEI 25k at w/w ratio of 1.3 was served as positive control. The cells were seeded

4

in the 24-well plate at a density of 6 × 104 cells per well and cultured with 1 mL DMEM containing

5

10% FBS at 37 oC until reaching about 80% confluence. Then the cells were cultured with

6

complexes in DMEM containing 10% FBS for 4 h at 37 oC. After that, the medium was replaced

7

with fresh complete medium and the cells were further cultured for 24 h. For luciferase assay, the

8

medium was discarded and cells were washed with 200 µL PBS, then the cells lysates were collected

9

by treating with 200 µL reporter lysis buffer (Pierce). The relative lightunits (RLUs) were measured

10

with a chemiluminometer (Lumat LB9507, EG&G Berthold, Germany). The total cellular protein

11

was measured by the BCA protein assay kit (Pierce) and luciferase activity was defined as RLU per

12

mg protein. For green fluorescent protein assay, transfections mediated by Ad-b-SS-PEI600/pEGFP-

13

C1 complexes were further evaluated in HeLa cells. The complexes were prepared at the respective

14

optimal w/w ratios determined from the luciferase assay. The cells expressing GFPs were directly

15

observed by an inverted microscope (IX 70, Olympus, Japan). The images were obtained at the

16

magnification of 100×. And the transfection efficiency of Ad-b-SS-PEI600/DNA/CD dimer ternary

17

complexes also evaluated by adding proper amount of CD dimer. The luciferase assay and green

18

fluorescent protein assay were repeated according to the protocols as aforementioned.

19

Furthermore, the free ligand competition transfection assays for Ad-b-SS-PEI600/DNA/CD

20

dimer/RGDFF quaternary complexes were conducted to evaluate the function of the targeting

21

moiety. The HeLa cells were incubated with 100mM RGDFF for 2 hours, and washed with PBS for

22

three times. Then the cells were cultured with the quaternary complexes in DMEM containing 10%

23

FBS for 4 h at 37 oC. After that, the medium was replaced with fresh complete medium and the cells

24

were further cultured for 24 h. And RGDFF was added freely to the Ad-b-SS-PEI600/DNA/CD dimer

25

ternary complexes (the weight ratio of wAd-b-SS-PEI600/wDNA/wCD dimer/wRGDFF was fixed at 10:1:10:3) as a 10 ACS Paragon Plus Environment

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control. Briefly, the ternary complexes were prepared, and diluted with complete medium, then

2

added proper amount of RGDFF. The cells were cultured with the obtained complexes for 4 h at 37

3

o

4

cultured for 24 h. The subsequent measurements of luciferase were according to the protocols as

5

aforementioned.

C. After that, the medium was replaced with fresh complete medium and the cells were further

6

The in vivo transfection efficiency of complexes was evaluated in BALB/c mice models with H22

7

tumor xenograft on the left forelimb armpit. According to the in vitro transfection efficiency,

8

complexes at optimizing weight ratios were injected subcutaneously, and the dosage of β-

9

galactosidase reporter gene pORF-LacZ was fixed at 7.5 µg. 48 h after injection, the mice were

10

sacrificed, and the tumors were collected for further analysis. According the protocol of X-gal

11

staining kit, the tumor tissues were fixed and stained overnight, and imaged by a digital camera. To

12

evaluate the biocompatibility of the complexes, the tumors were fixed in 4% paraformaldehyde and

13

embedded with paraffin for histology analysis. The paraffin sections of 7 µm thickness were

14

mounted on glass slide and finally stained with Hematoxylin/eosin for histological analysis, and then

15

imaged by light microscopy.

16

2.11. Gene Delivery Assays. To improve the tumor targeting property of the gene delivery platform,

17

targeting moiety RGD was further introduced to the Ad-b-SS-PEI600/DNA/CD dimer ternary

18

complexes via the host-guest interaction between the surface α-CD and phenylalanine on the RGDFF

19

peptide sequence. In order to assess the tumor-targeting ability, Ad-b-SS-PEI600/DNA/CD

20

dimer/RGDFF quaternary complexes at w/w/w/w ratio of 10:1:10:3 were prepared and the

21

complexes without RGDFF at the same ratio was used as control. Free ligand competition

22

endocytosis assays were further conducted to investigate the internalization process. And RGDFF

23

was added freely to the Ad-b-SS-PEI600/DNA/CD dimer ternary complexes to confirm the function

24

of the host-guest interaction.



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Page 12 of 38

1

The YOYO-1 labeled pGL-3 plasmid delivered to cytoplasm by complexes was observed in U87,

2

COS7 and HeLa cells by confocal laser scanning microscopy (CLSM). After co-incubation for 2 h,

3

the medium was removed and the cells were washed three times with PBS. Then the nucleus was

4

stained with Hoechst 33342 for 15 min at 37 oC; after that the cells were further washed with PBS

5

for three times and incubated with 1 mL complete medium for observation. The fluorescence was

6

visualized on a confocal laser scanning microscope (CLSM, C1-Si, Nikon, Japan).

7

The tumor-targeting cellular uptake was also quantitatively estimated by flow cytometry. The

8

U87, COS7 and HeLa cells were seeded in 6-well plates at a density of 2 × 105 cells/well and

9

incubated with 2 mL complete medium overnight. Ad-b-SS-PEI600/DNA/CD dimer/RGDFF

10

quaternary complexes and Ad-b-SS-PEI600/DNA/CD dimer ternary complexes were prepared

11

respectively, and the DNA applied was labeled by YOYO-1. Then the complexes were diluted to 2

12

mL with complete medium, and added to the plates. After 2 h incubation, the complexes were

13

removed and the cells were washed with PBS for three times. All the cells were trypsinized and

14

collected by centrifugation, and subsequently washed with PBS for three times. Finally, the

15

suspended cells were filtrated and examined with flow cytometry (BD FACSAria III, USA). The

16

instrument was calibrated with non-transfected cells (negative control) to identify viable cells, and

17

the transfected cells were determined from a fluorescence scan performed with 1 × 104 cells using

18

the FL1-H channel.

19

To monitor the gene delivery process in HeLa cells, Ad-b-SS-PEI600/DNA/CD dimer/RhBF

20

quaternary complexes at w/w/w/w ratio of 10:1:10:3 were prepared, the DNA applied was labeled by

21

YOYO-1. After co-incubation for 4 h, the complexes were removed and the cells were washed with

22

PBS for three times. Then the nucleus was stained with Hoechst 33342 for 15 min at 37 oC; after that

23

the cells were further washed with PBS for three times and incubated with 1 mL complete medium

24

for observation. The fluorescence was visualized on a confocal laser scanning microscope (CLSM,

25

C1-Si, Nikon, Japan). 12 ACS Paragon Plus Environment

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1

The in vivo distribution assays were evaluated in BALB/c mice models with H22 tumor xenograft

2

on the right flank. When the tumor volume reached 300 mm3, the mice were intravenously injected

3

with Ad-b-SS-PEI600/DNA/CD dimer/RhBF+RGDFF quinary complexes (at w/w/w/w+w ratio of

4

10:1:10:1.5+1.5) and Ad-b-SS-PEI600/DNA/CD dimer/RhBF quaternary complexes (at w/w/w/w

5

ratio of 10:1:10:1.5), and the dosage of DNA was fixed at 7.5 µg per mouse. After 12 h, the mice

6

were sacrificed, and the main organs (heart, liver, spleen, lung and kidney) and the tumor tissue were

7

dissected and washed with PBS, and subsequently imaged by In Vivo Imaging System (Maestro 2,

8

Cambridge Research &Instrumentation, Inc., USA) at excitation wavelength of 550 and emission

9

wavelengths of 580 nm.

10

3. RESULTS AND DISCUSSION

11

3.1. Chemical Characterization. Based on classical Fmoc chemistry, the peptides were successfully

12

synthesized. As shown in Figure S1, RGDFF: molecular weight, calculated 641 (M + H)+, found

13

641.3; calculated 663 (M + Na)+, found 663.3. RhBF: molecular weight, calculated 590 (M − H)−,

14

found 590.2. And the synthesis of α,β CD dimer was also monitored by mass spectrometry. As

15

shown in Figure S2, α,β CD dimer: molecular weight, calculated 2289 (M + Na)+, found 2289.

16

The molecular weight (Mw) of the obtained SS-PEI600 was 8800 Da by SEC-MALLS analysis and

17

the polydispersity index (Mw/Mn, PDI) was about 1.35. And the successful synthesis of Ad-b-SS-

18

PEI600 was confirmed by 1H-NMR (300 MHz, D2O) (Figure S3). The peaks between δ 2.5-3.2 ppm

19

in 1H NMR spectra were contributed to -NHCH2CH2- of PEI. And the obvious proton peaks of

20

adamantane displayed between δ 1.5-2.0 ppm, implying that the adamantane groups were

21

successfully grafted on the SS-PEI600. And the substituted degree of adamantane in Ad-b-SS-PEI600

22

was calculated to be 25. The high substituted degree is important for the subsequent decoration of

23

CD dimers.



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Page 14 of 38

1

3.2. DNA Compacting Capability. The ability of cationic polymers to condense DNA into stable

2

polyplexes is a prerequisite for applying as gene carriers.40 The obtained electrostatic positive

3

polyplexes can protect the DNA from enzymatic degradation and facilitate cellular internalization.41

4

The DNA binding capability of Ad-b-SS-PEI600 was first evaluated by agarose gel electrophoresis.

5

As shown in Figure. 1A, with increasing amount of Ad-b-SS-PEI600, the mobility of DNA was

6

retarded due to the decrease of dissociative DNA. Ad-b-SS-PEI600 was efficient in hindering DNA

7

migration at a low w/w ratio of 1:1, demonstrating the promising DNA binding capability of the

8

cationic polymer.

9

Meanwhile, the unpacking capability under reductive conditions was also monitored by agarose

10

gel electrophoresis. When incubated with glutathione (GSH), the disulfide bonds of Ad-b-SS-PEI600

11

would be rapidly cleaved, then the DNA binding capability would be sharply weakened, and thus the

12

compacted DNA would be released.42,43 As shown in Figure 1B, incubation in NaCl solution or low

13

concentration of GSH (2 µM, mimicking the extracellular GSH level) for 30 min did not affect the

14

DNA compaction at the w/w ratio of 1:1. While incubated with high concentration of GSH (2-10

15

mM, mimicking the intracellular GSH level), bright bands of dissociative DNA were visible at the

16

same w/w ratio, indicating the immediately DNA unpacking and release in cytoplasm with a high

17

GSH concentration.

18

To further evaluate the Ad-b-SS-PEI600/DNA polyplexes, the particle size and zeta potential were

19

also investigated. As shown in Figure 1C, the sizes of the polyplexes decreased before w/w ratio of

20

10:1, and then slightly increased, which revealed the aggregation tendency at high w/w ratios. And

21

the sizes of the polyplexes are between 250 and 350 nm, which can be internalized via nonspecific or

22

caveolae-mediated endocytosis.44 The positive charge of polyplexes is important for nonspecific

23

cellular uptake.45 The zeta potential was also measured at respective w/w ratios. As shown in Figure

24

1D, the zeta potential of Ad-b-SS-PEI600/DNA increased gradually, and reached a plateau at w/w


Page 15 of 38

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1

ratio of 10:1, and the value of the binary complexes is about 17 mV. The electropositive property

2

would facilitate the cellular internalization of the obtained binary polyplexes.

3

The addition of hydrophilic α,β CD dimers may alter the assembling structure and surface

4

properties of the polyplexes. To confirm these changes, the particle sizes and zeta potential were also

5

evaluated after incorporating different amount of α,β CD dimers to the Ad-b-SS-PEI600/DNA binary

6

complexes at w/w ratio of 10:1. The feasibility of CD dimer assemble to the complexes was first

7

calculated. According to the NMR results, the Ad substituted degree was high in the Ad-b-SS-PEI600,

8

about 25 Ad groups (Mr = 163 Da) in an Ad-b-SS-PEI600 chain (Mr = 8800 Da), and at the operation

9

w/w ratio of 10/1, the Ad-b-SS-PEI600/DNA binary complexes would process about 0.020 e.q.

10

(=10/(8800+163×25)×25) Ad groups, which can interact with CD dimer (Mr = 2267 Da) to form Ad-

11

b-SS-PEI600/DNA/CD dimer ternary at w/w/w ratio as high as 10/1/45. The distribution of Ad groups

12

in the Ad-b-SS-PEI600/DNA binary complexes may be random, and some of them may locate inside.

13

Thus, the weight ratio of Ad-b-SS-PEI600/DNA/CD dimer ternary was set at w/w/w ratio 10/1/5 to

14

10/1/20. As shown in Figure 1E, the sizes of the complexes decreased after the α,β CD dimer

15

addition, and reached a plateau at Ad-b-SS-PEI600/DNA/CD dimer weight ratio of 1:10:10. The sizes

16

of the obtained ternary complexes are about 230 nm. The CD dimer addition may further compress

17

the DNA via host-guest interaction between the CD dimers and the Ad groups of the binary

18

complexes. And the zeta potential histograms (Figure 1F) also demonstrated the incorporation of CD

19

dimer would change the surface charge of the complexes. With increasing addition amount of CD

20

dimer, the zeta potential distinctly dropped, and the values of zeta potential were close to zero when

21

the added weight ratio reached 10. It is deduced that the introduction of CD dimer with large size

22

shields the electropositive amino group of the Ad-b-SS-PEI600/DNA binary complexes, and the

23

abundant hydroxyl groups of CD dimer make the surface of the ternary complexes approximately

24

electroneutral. Then the SS-PEI600/DNA complexes were also incubated with CD dimer to confirm

25

the host-guest interaction between CD and Ad groups in Ad-b-SS-PEI600/DNA/CD dimer ternary 15 ACS Paragon Plus Environment


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1

complexes. As shown in Figure S4, the CD dimer addition didn’t change the size and zeta potential

2

of the SS-PEI600/DNA complexes. Without guest molecule Ad groups on the surface of complexes,

3

the added CD dimer failed to alter the assembling structure and surface properties of the SS-

4

PEI600/DNA complexes. Meanwhile, as for the Ad-b-SS-PEI600/DNA binary complexes, the added

5

CD dimer can assemble on the complexes through host-guest interaction with the Ad groups

6

displayed on the surface, and alter the surface property of the complexes. Simultaneously, the

7

complexes obtained by pre-modification methods were also prepared, the size and zeta potential

8

were about 1700 nm and -4 mV (Figure S4), respectively, demonstrating the opposite formulation

9

failed to compact DNA. In consideration of the complex stability and cellular uptake, the ternary

10

complexes of Ad-b-SS-PEI600/DNA/CD dimer at weight ratio of 1:10:10 were applied in further

11

investigations. Then the size and zeta potential of the complexes decorated with functional moieties

12

were measured, as shown in Figure S4, the size of the obtained quaternary complexes is similar to

13

that of the unmodified ternary complexes, and the zeta potential decreased slightly.

14

The size distribution of the binary and ternary complexes in saline or in 10 mM GSH revealed the

15

dissociation of the assemblies under reductive conditions (Figure 1G). The binary and ternary

16

complexes in 10 mM GSH both swelled at first 30 min, and then dissociated to form small fragments

17

and large aggregates at 3 h. The size of the ternary complexes was smaller than that of the binary

18

complexes in GSH at 30 min, which was ascribed to the incorporation of the CD dimer. The CD

19

dimer with large steric hindrance may delay the interaction between GSH and disulfide bonds in Ad-

20

b-SS-PEI600. But after treated with GSH for 3 h, the swollen ternary complexes also dissociated,

21

indicating the degradation of carriers and the gene release.15

22

In summary, the cationic polymer exhibited optimizing DNA compaction capability under normal

23

physiological conditions, and the incorporation of CD dimer would further compress the Ad-b-SS-

24

PEI600/DNA binary complexes. Meanwhile, the disulfide bonds in the cationic polymer would be

25

readily cleaved by GSH, and thus the DNA would be successfully unpacked in the cytoplasm. 16 ACS Paragon Plus Environment

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1

However, when applied in vivo, the serum proteins would non-specifically bind to cationic/DNA

2

polyplexes via electrostatic attraction and led aggregation or sedimentation of the polyplexes. To

3

investigate this phenomenon, herein, the bovine serum albumin (BSA) was used as a model protein

4

to adsorp the cationic polymer and polyplexes. As shown in Figure 1H, Ad-b-SS-PEI600 presented

5

weakest protein adsorption inhibition, and Ad-b-SS-PEI600/DNA binary complexes also exhibited

6

high level protein adsorption, which is attributed to the abundant electropositive amino groups on the

7

surface. When incorporated the CD dimer to the Ad-b-SS-PEI600/DNA binary complexes, the protein

8

adsorption sharply decreased, and so did the complexes that further decorated with the targeting

9

module RGDFF. This decline of protein adsorption was mainly ascribed to the reduction of zeta

10

potential after introducing CD dimer. The electroneutral CD dimer with large size would readily

11

shield the amino groups on the surface of the binary complexes, and moreover the abundant hydroxyl

12

groups would prevent electrostatic attraction of serum proteins.46 Therefore, our gene delivery

13

platform is provided with robust serum-tolerability apart from excellent DNA compaction capability

14

and rapid stimuli responsiveness.

15

3.3. Cell Cytotoxicity. The inevitable cytotoxicity of the gene delivery platform seriously hinders

16

their further application, which heavily relies on the chemical structure of the platform.47 To evaluate

17

the cytotoxicity of the cationic polymer and the gene delivery platform, MTT assays were

18

investigated in cancerous HeLa cells and normal COS7 cells. As shown in Figure 2A, the cationic

19

polymer Ad-b-SS-PEI600 exhibited low cytotoxicity for both HeLa and COS7 cell lines, while the

20

cell viability profiles showed a sudden drop at a low PEI 25k concentration in both cell lines. The

21

outstanding biocompatibility of Ad-b-SS-PEI600 was attributed to the incorporation of reduction-

22

sensitive disulfide bond in the cationic polymer, which can be cleaved and allow rapid degradation

23

into small non-toxic PEI600.5 But the PEI 25k without reduction-sensitive linkages may initiate

24

cumulative cytotoxicity. Meanwhile, in Ad-b-SS-PEI600, the cationic amino groups of hyperbranched

25

reduction-sensitive polyethyleneimine were partly substituted by electroneutral adamantane, which 17 ACS Paragon Plus Environment


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Page 18 of 38

1

may reduce the positive charge density of the PEI, and thus alleviate the destruction of negative cell

2

membrane during electrostatic interaction-mediated internalization. The cytotoxicity of the obtained

3

complexes at various weight ratios was also evaluated (Figure 2B). All the complexes displayed

4

excellent biocompatibility, and the cell viabilities were higher than 95%. This is ascribed to the

5

charge neutralization between cationic polymer and electronegative DNA. It is worth mentioning

6

that the incorporation of CD dimer into the Ad-b-SS-PEI600/DNA binary complexes had not altered

7

the cytotoxicity. With reduction-sensitive linkages and lower positive charge density, the complexes

8

exhibited insignificant cytotoxicity to both cancerous and normal cell lines. And the PEI 25k/DNA

9

complexes exhibited higher cytotoxicity in both cell lines (the cell viability is about 85%), which

10

may impede their further application.

11

3.4. In Vitro Gene Transfection. High-performance gene transfection is the foremost feature of

12

gene delivery platform. To evaluate the transfection efficiency of our gene delivery platform,

13

luciferase plasmid DNA pGL-3 and enhanced green fluorescent protein plasmid DNA pEGFP-C1

14

were applied as model reporter genes. As shown in Figure A, the trend of pGL-3 transfection

15

efficiency in the HeLa and COS7 cell lines are similar, although the in vitro transfection capabilities

16

are cell line-dependent. At the w/w ratio of 1:1, the transfection level was seriously limited due to the

17

suboptimal DNA compaction and ineffective cellular internalization at low weight ratio. And when

18

the w/w ratio reached 10, the transfection efficiency of Ad-b-SS-PEI600/DNA binary complexes was

19

higher than that of the “golden standard” PEI 25k/DNA at w/w ratio of 1.3 in both cell lines. And the

20

incorporated CD dimer had slightly weaken the transfection efficiency (Figure 3B), which may be

21

ascribed to the decrease of zeta potential, and the slight abatement of cellular uptake. The more

22

intuitive characterization of gene transfection was implemented by the cellular expression of green

23

fluorescent protein when treated with the pEGFP-C1 complexes. As shown in Figure 3C and D, the

24

green fluorescent spots can be clearly observed in cells. It meant that the pEGFP-C1 had been

25

transported into HeLa cells, and GFP was successfully expressed. The protein expression level 18 ACS Paragon Plus Environment

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1

mediated by Ad-b-SS-PEI600/DNA binary complexes was slightly higher than that of Ad-b-SS-

2

PEI600/DNA/CD dimer ternary complexes, which was in accordance with the results of pGL-3

3

transfection. All the aforementioned quantitative and qualitative results have demonstrated the

4

promising gene transfection capability of our gene delivery platforms. Though with relatively low

5

transfection efficiency, the Ad-b-SS-PEI600/DNA/CD dimer ternary complexes are still satisfactory

6

as gene carriers in vitro, and can be further decorated at will.

7

Then the transfection of the final functional complexes were further investigated. The tumor

8

targeting moiety was incubated with the ternary complexes to form the functional quaternary

9

complexes, and the transfection efficiency of the obtained Ad-b-SS-PEI600/DNA/CD dimer/RGDFF

10

complexes was much higher than that of the Ad-b-SS-PEI600/DNA/CD dimer complexes in αvβ3

11

integrin-positive HeLa cells (Figure S5). And the RGDFF ligands were added freely to the ternary

12

complexes, which had no effect on the transfection process. And the efficiency was almost

13

unchanged. Meanwhile, in the competition assay, free RGDFF were incubated with HeLa cells to

14

block the receptors, and then the Ad-b-SS-PEI600/DNA/CD dimer/RGDFF complexes were added to

15

transfect the cells. The transfection efficiency distinctly decreased, which may be mainly ascribed to

16

the inhibition of receptor-mediated uptake. And according to the Figure S4, the zeta potential of the

17

Ad-b-SS-PEI600/DNA/CD dimer/RGDFF complexes was negative, which may result in electrostatic

18

repulsion by cell membrane. Without efficient access into cells, the transfection efficiency of the

19

quaternary complexes is limited. Based on the above results, it can be deduced that the RGDFF

20

moieties located on the complexes via efficient host-guest interactions can exert their tumor-targeting

21

property, and enhance the subsequent gene transfection.

22

3.5. In Vivo Gene Transfection. Encouraged by above results, in vivo gene transfection of model

23

reporter gene pORF-lacz was carried out in BALB/c mice with H22 cells xenograft model. As

24

expected from the in vitro study, the tumor sections with blue color indicated high β-galactosidase 19 ACS Paragon Plus Environment


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Page 20 of 38

1

expression for PEI 25k/DNA complexes, Ad-b-SS-PEI600/DNA binary complexes and Ad-b-SS-

2

PEI600/DNA/CD dimer ternary complexes (Figure 4A). And the tumor section treated with naked

3

DNA was only partly light blue, implying the insufficient gene transfection. To validate the

4

biocompatibility of our gene delivery platforms, H&E staining assays were carried out on the excised

5

tumor tissues. The tumors incubated with naked DNA, Ad-b-SS-PEI600/DNA binary complexes and

6

Ad-b-SS-PEI600/DNA/CD dimer ternary complexes were composed of abundant and compact tumor

7

cells (Figure 4B, D and E), indicating the good biocompatibility of them. While the tumor incubated

8

with PEI 25k/DNA complexes showed partial nuclei absence (Figure 4C), implying the inevitable

9

tissue toxicity of PEI 25k. Overall, the Ad-b-SS-PEI600/DNA/CD dimer ternary complexes can

10

effectively transfect tumor cells both in vitro and in vivo, possessing good biocompatibility.

11

3.6. In Vitro Gene Delivery. Next, targeting moiety RGD was further introduced to the Ad-b-SS-

12

PEI600/DNA/CD dimer ternary complexes via the host-guest interaction between the surface α-CD

13

and phenylalanine on the RGDFF peptide sequence. The obtained quaternary complexes was co-

14

incubated with αvβ3 integrin-positive U87 and HeLa cells and αvβ3 integrin-negative COS7 cells. As

15

shown in Figure 5A-F, the green fluorescence spots of YOYO-1 labeled DNA were increased in U87

16

and HeLa cells after adding the targeting moiety RGD to the complexes, while the green

17

fluorescence in COS7 cells remained at the low strength. And the quantitative data of cellular uptake

18

by flow cytometry also revealed similar tendency in the three cell lines (Figure S7). These

19

differences were ascribed to the incorporation of RGD moiety, which can be recognized by αvβ3

20

integrin overexpressed on most tumor cells, and internalized via receptor-mediated endocytosis.

21

With low zeta potential, the Ad-b-SS-PEI600/DNA/CD dimer ternary complexes were not good at cell

22

entry in the three investigated cell lines. To further confirm the function of the targeting moieties, the

23

RGDFF ligands were added freely to the ternary complexes, which had no effect on cellular uptake.

24

As shown in Figure S6B, the fluorescence intensity in cells was same to that in cells treated with the

25

Ad-b-SS-PEI600/DNA/CD dimer ternary complexes (Figure S6A). And as shown in Figure S6D, the 20 ACS Paragon Plus Environment

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1

internalization of the Ad-b-SS-PEI600/DNA/CD dimer/RGDFF complexes was significantly

2

restrained after the αvβ3 integrin of HeLa cells blocked by free RGD. The results of the control

3

assays both demonstrated that the RGDFF ligands located on the complexes played important roles

4

in receptor-mediated internalization of the tumor-targeting complexes. When incorporated with

5

targeting moiety to the ternary complexes, the targeted cells would be prone to uptake the further

6

modified complexes. Other targeting moiety such as folate and galactose, can be also facilely

7

introduce to our gene delivery platforms to implement more specific cellular internalization and gene

8

transfection.

9

To real-time observe the gene delivery platforms, fluorescent probe (RhB) was also incorporated

10

into the Ad-b-SS-PEI600/DNA/CD dimer ternary complexes via similar method. As shown in Figure

11

6, the green fluorescence of YOYO-1 labeled DNA was in coincidence with the red fluorescence of

12

probe RhB in cytoplasm, which meant the DNA was compacted by the carriers. The fluorescent

13

signals of the white line in Figure 6C revealed the good overlap of the green and red fluorescence,

14

demonstrating the successful fluorescence labeling of the gene delivery platform using this

15

convenient method. And the green fluorescence was also found in the nuclei, which is not

16

overlapped with the red fluorescence, indicating the DNA unpacking and nuclei entry. Other

17

functional fluorescent probe can be also conveniently modified on the gene delivery platform at will.

18

3.7. In Vivo Gene Delivery. With good biocompatibility and facile multi-functionalization, targeting

19

moiety and fluorescent probe are both introduced to the Ad-b-SS-PEI600/DNA/CD dimer ternary

20

complexes for real-time monitor and tumor-targeted in vivo gene delivery. The targeting moiety

21

RGD and fluorescent probe RhB are conjugated to the obtained complexes via aforementioned host-

22

guest interactions, and the final gene delivery platform was applied in BALB/c mice with H22 tumor

23

xenograft via intravenous injection. Limited by the fluorescent emission wavelength of RhB, the in

24

vivo fluorescence distribution are often interfered by the blood and skin. Therefore, mice were 21 ACS Paragon Plus Environment


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Page 22 of 38

1

sacrificed at the 12th h after injection of the final gene delivery platform. The excised tissues of

2

heart, liver, spleen, lung, kidney and tumor were collected and imaged. As shown in Figure 7, the ex

3

vivo fluorescence mainly distributed in liver, kidney and tumor, and the intensity in liver was

4

strongest. It was attributed to the nanoparticle retention effect of liver and subsequent the

5

nanoparticle excretion effect of kidney. Thanks to the impaired lymphatic drainage of tumor, the

6

nano-size final gene delivery platforms can be also accumulated in tumor via enhanced permeability

7

and retention (EPR) effects. Compared with the platform without targeting moiety, the platform

8

decorated with RGD exhibited better tumor accumulation, and meanwhile, the fluorescence intensity

9

in liver and kidney was relatively weaker, which demonstrated the enhanced tumor targeting

10

property of RGD. Overall, the post-modified functional moieties on the gene delivery platform

11

exerted excellent activity and efficacy.

12

4. CONCLUSION

13

To overcome the defects of traditional polymer-based gene delivery systems in multi-

14

functionalization, we constructed a sophisticated gene delivery platform decorated with α,β CD

15

dimer, which can be applied as a substantial bridge between the polyplexes and the functional

16

moieties. Moreover, the hydrophilic CD dimer with rich hydroxyl groups can significantly improve

17

the stability of the polyplexes in serum, without disturbing the pre-loaded DNA. Tumor targeting

18

moiety and fluorescence probe were facilely introduced to the gene delivery platforms, and

19

efficiently exerted their own functions both in vitro and in vivo. This gene delivery platform was a

20

variable formulation, which can be modified with versatile moieties for more specific and further

21

applications, such as drug and gene synergistic therapy and real-time tracking of in vivo gene

22

delivery. The novel strategy made delicate post-modification of polymer-based carrier/DNA

23

complexes not only possible but also facile, and simultaneously, provided a new perspective for the

24

design and synthesis of future gene delivery system. 22 ACS Paragon Plus Environment

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ASSOCIATED CONTENT

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Supporting Information. The ESI-MS profile of the peptide RGDFF and RhBF; the MALDI-TOF

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mass spectrum of α,β CD dimer; 1H-NMR spectra of Ad-b-SS-PEI600; size and zeta potential of

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different complexes; in vitro gene transfection mediated by different complexes; CLSM images of

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cellular internalization; and the flow cytometry analysis of cellular uptake. This material is available

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free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

8

Corresponding Author

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* E-mail address: [email protected] (X.Z. Zhang).

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Author Contributions

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The manuscript was written through contributions of all authors. All authors have given approval to

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the final version of the manuscript.

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENT

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This work was supported by the National Natural Science Foundation of China (51125014,

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51233003 and 51273165) and the Natural Science Foundation of Hubei Province of China

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(2013CFA003).

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REFERENCES

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Scheme 1 Schematic illustration of the facilely multi-functionalized gene delivery platform. (I) Ad-b-SS-PEI600 compacts DNA into polyplexes via electrostatic interaction. (II) The α,β CD dimers are introduced to the Ad-b-SS-PEI600/DNA polyplexes through the host-guest interactions between Ad and β-CD, obtaining ternary complex with many α-CD assembled on the surface. (III) The targeting moiety (RGD) and fluorescent probe (RhB) are decorated on the surface of the assemblies through the host-guest interactions between phenylalanine (Phe or F) and α-CD, obtaining the final multifunctional assemblies. (IV) The targeting moiety RGD on the surface of the assemblies can be recognized by the αvβ3 integrin on tumor cell membrane. (V) The final multifunctional platforms can be internalized via αvβ3 integrin-mediated endocytosis. (VI) After endosomal escape, the assemblies would be rapidly disassembled under the reduction of GSH in cytoplasm. (VII) The released DNA are transferred into nuclei for gene expression.


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Figure 1. DNA compacting capability of the gene delivery platform. Agarose gel electrophoresis retardation assay of (A) Ad-b-SS-PEI600/DNA polyplexes at different w/w ratios, and (B) Ad-bSS-PEI600/DNA polyplexes at w/w of 1 treated with different concentration of GSH (2 µM, 2 mM and 10 mM)for different time periods (Left: 5 min; Right: 30 min). Particle size of (C) Adb-SS-PEI600/DNA polyplexes at different w/w ratios, and (E) the Ad-b-SS-PEI600/DNA polyplexes at w/w ratio of 10 added with different amounts of α,β CD dimer. Zeta potential of (D) Ad-b-SS-PEI600/DNA polyplexes at different w/w ratios, and (F) the Ad-b-SS-PEI600/DNA polyplexes at w/w ratio of 10 added with different amounts of α,β CD dimer. (G) The hydrodynamic size distribution of the Ad-b-SS-PEI600/DNA binary complexes (BC) and Ad-b-


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SS-PEI600/DNA/CD dimer ternary complexes (TC) in saline or in 10 mM GSH (for 30 min and 3 h) by intensity. (H) BSA adsorption of the cationic polymer Ad-b-SS-PEI600, Ad-b-SSPEI600/DNA binary complexes, Ad-b-SS-PEI600/DNA/CD dimer ternary complexes and Ad-bSS-PEI600/DNA/CD dimer/RGDFF quaternary complexes. Data are shown as mean ± S.D. (n = 3).


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Figure 2. (A) Cell viability of Ad-b-SS-PEI600 and PEI 25k in HeLa cells and COS7 cells. (B) Cell viability of Ad-b-SS-PEI600/DNA binary complexes (at w/w ratio of 1:1, 2.5:1, 5:1, 10:1 and 15:1) and Ad-b-SS-PEI600/DNA/CD dimer ternary complexes (at w/w/w ratio of 10:1:5, 10:1:10, 10:1:15 and 10:1:20) in HeLa cells and COS7 cells, naked DNA and PEI 25k/DNA complexes (at w/w ratio of 1.3:1) are applied as control. Data are shown as mean ± S.D. (n = 3).


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Figure 3. In vitro gene transfection. Luciferase expression mediated by (A) Ad-b-SSPEI600/DNA binary complexes and (B) Ad-b-SS-PEI600/DNA/CD dimer ternary complexes at different weight ratios. Data are shown as mean ± S.D. (n = 3). Enhanced green fluorescent protein expression mediated by (C) Ad-b-SS-PEI600/DNA binary complexes at w/w ratio of 10:1 and (D) Ad-b-SS-PEI600/DNA/CD dimer at w/w/w ratio of 10:1:10.


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Figure 4. In vivo gene transfection of β-galactosidase reporter gene (pORF-LacZ). (A) Photograph of the tumors dissected from mice 48 h post-injection with X-gal staining. H&E staining of the tumor sections treated with different polyplexes (B) Naked DNA, (C) PEI 25k/DNA complexes, (D) Ad-b-SS-PEI600/DNA binary complexes and (E) Ad-b-SSPEI600/DNA/CD dimer ternary complexes.


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Figure 5. CLSM images of cellular internalization at 2 h treated with (A, C and E) Ad-b-SSPEI600/DNA/CD dimer ternary complexes and (B, D and F) Ad-b-SS-PEI600/DNA/CD dimer/RGDFF quaternary complexes in (A and B) U87 cells, (C and D) COS7 cells, and (E and F) HeLa cells. (A1-F1) Green fluorescence of pGL-3 labeled with YOYO-1. (A2-F2) Blue fluorescence of nuclei stained with Hoechst. (A3-F3) Merge images of green and blue fluorescence fields. (A4-F4) Merge images of bright and fluorescent fields. The scale bar is 20 µm.


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Figure 6. CLSM images of cellular internalization at 4 h treated with Ad-b-SS-PEI600/DNA/CD dimer/RhBF quaternary complexes in HeLa cells. (A) Green fluorescence of pGL-3 labeled with YOYO-1. (B) Red fluorescence of RhB probe. (C) Merge images of green and red fluorescence fields, and the orange fluorescence was the overlap of pGL-3 and RhB. (D) Blue fluorescence of nuclei stained with Hoechst. (E) Merge images of bright and fluorescent fields. (F) Fluorescence signals based on the white line in C, green signal of pGL-3 and red signal of RhB. The scale bar is 20 µm.


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Figure 7. Optical and fluorescent images of excised organs and tumor tissues intravenously treated with the final multifunctional gene delivery platforms with or without tumor targeting moiety RGDFF at 12 h post-injection.


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The Table of Contents Graphic


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A Facile Multifunctionalized Gene Delivery Platform Based on Î±,Î²

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