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Synthesis of a Cationic Supramolecular Block Copolymer with Covalent and Noncovalent Polymer Blocks for Gene Delivery Wumaier Yasen, Ruijiao Dong, Linzhu Zhou, Jieli Wu, Cheng-Xi Cao, Aliya Aini, and Xinyuan Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15919 • Publication Date (Web): 24 Feb 2017 Downloaded from http://pubs.acs.org on February 25, 2017

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ACS Applied Materials & Interfaces

Synthesis of a Cationic Supramolecular Block Copolymer with Covalent and Noncovalent Polymer Blocks for Gene Delivery Wumaier Yasen,† Ruijiao Dong,*,† Linzhu Zhou,† Jieli Wu,† Chengxi Cao,‡ Aliya Aini# and Xinyuan Zhu*,† †

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix

Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China. ‡

School of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan

Road, Shanghai 200240, P. R. China. #

School of Foreign Languages, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai

200240, China.

ACS Paragon Plus Environment

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ABSTRACT

The design and fabrication of safe and highly efficient nonviral vectors is the key scientific issue for the achievement of clinical gene therapy. Supramolecular cationic polymers have unique structures and specific functions compared to covalent cationic polymers, such as low cytotoxicity, excellent biodegradability and smart environmental responsiveness, thereby showing great application prospect for gene therapy. However, supramolecular gene vectors are facile to be degraded under physiological condition, leading to a significant reduction of gene transfection efficiency. In order to achieve highly efficient gene expression, it is necessary for supramolecular gene vectors being provided with appropriate biostability to overcome various cell obstacles. To this end, a novel cationic supramolecular block copolymer composed of a conventional polymer and a noncovalent polymer was constructed through robust β– cyclodextrin/ferrocene host–guest recognition. The resultant supramolecular block copolymer perfectly combines the advantages of both conventional polymers and supramolecular polymers ranging from structures to functions. This supramolecular copolymer not only has the ability to effectively condense pDNA for enhanced cell uptake, but also release pDNA inside cancer cells triggered by H2O2, which can be utilized as a prospective nonviral delivery vehicle for gene delivery. The block polymer exhibited low cytotoxicity, good biostability, excellent biodegradability and intelligent responsiveness, ascribing to the dynamic/reversible nature of non-covalent linkages. In vitro studies further illustrated that the supramolecular block polymer exhibited greatly improved gene transfection efficiency in cancer cells. This work offers an alternative platform for the exploitation of smart nonviral vehicles for specific cancer gene therapy in future.


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KEYWORDS: supramolecular block copolymer, biostability, redox responsiveness, nonviral vector, gene therapy

INTRODUCTION Supramolecular chemistry, as a relatively young and burgeoning interdiscipline, has recently captured extensive attention in a wide range of areas from material science, nanoscience, information science to life science.1-3 As a perfect bridge between supramolecular chemistry and polymer science, supramolecular polymers based on various non-covalent recognitions not only show unique structures and favorable physicochemical performance, but also have the capability to undergo reversible variations of structure, property, and function under exposure to various external stimuli, making them eminent candidates for versatile applications ranging from scientific research to industrial regions.4-10 These peculiar structures and properties of supramolecular polymers, especially their reversibly dynamic backbone and specific stimuliresponsiveness, have inspired scientists to fabricate versatile robust supramolecular nanoscale vehicles for cancer diagnosis and therapy.9-12 In recent years, in the realm of gene therapy, a rapidly increasing number of supramolecular nonviral vectors have been widely reported, which display comparable transfection efficacy to conventional polymers.13-16 For instance, Yui13 and Li14 reported multifarious supramolecular cationic polyrotaxanes with a necklace-like structure showing fast endosomal escape and improved gene transfer to the cell nucleus. Xu15 reported one kind of supramolecular pseudo-block polymers via host-guest recognition between bioreducible β-cyclodextrin-cored

star

poly(2-dimethyl

amino)ethyl

methacrylate

and

linear

poly(poly(ethylene glycol)ethyl ether methacrylate), which demonstrated high in vitro cellular internalization and in vivo anti-tumor activity. Recently, we developed several cyclodextrin-


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based host–guest supramolecular gene vectors that had good biocompatibility and smart responsiveness to various biological stimuli, further achieving favorable gene transfection efficacy in vitro.16-18 However, the existing supramolecular gene vectors constructed from small molecule-based main-chain supramolecular polymers are generally easy to be degraded or depolymerized under variable physiological conditions, further resulting in an evident decrease of in vitro or in vivo gene transfection efficiency.17,18 Therefore, the key issue in this field is how to improve biostability of these supramolecular gene vectors for overcoming cellular and tissue barriers, further achieving highly efficient gene expression. Herein, we report a novel kind of supramolecular gene vectors based on a cationic supramolecular block copolymer (SBC) composed of a conventional polyethylene glycol (PEG) block and a cationic main-chain supramolecular polymer block, which ideally combines these advantages of both covalent polymers and supramolecular polymers ranging from structures to functions, such as low cytotoxicity, good biostability, excellent biodegradability and intelligent responsiveness (Scheme 1).19,20 In this system, the hydrophilic PEG block affords outstanding biocompatibility and biostability.21,22 The cationic supramolecular polymer block endows the supramolecular gene vectors with efficient plasmid DNA (pDNA) binding ability and controlled release of pDNA inside the cells under exposure to specific biological stimuli. As expected, the cationic supramolecular polymer block interacts with pDNA via electrostatic interaction to form DNA polyplexes as inner core, which will be protected by covalent PEG chains in outer shell. The resultant core-shell DNA nanoscale polyplexes not only exhibit greatly enhanced biostability and biocompatibility, but also have the ability to release DNA inside the cells triggered by hydrogen peroxide (H2O2), 23-25 thereby showing superior gene transfer efficacy in cancer cells compared to supramolecular homopolymer (SHP).


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PEG-CD

O

O

NN N

Fe

N H

H N

Fc-PEHA-CD

5

113

Supramolecular polymerization

Supramolecular block copolymer (SBC)

SBC/pDNA polyplex

H2O2

Cytoplasm

Fc+

Nucleus Nucleus

Scheme 1. Schematic illustration of the formation of an unconventional cationic supramolecular block copolymer, and its pDNA binding and H2O2-triggered pDNA release in vitro.

EXPERIMENTAL DETAILS Materials. Poly(ethylene glycol) monomethyl ether (PEG113-OH, Mn = 5.0 kDa, Mw/Mn = 1.06, Aldrich), sodium hydride (NaH) (57-63% in oil, Alfa Aesar), propargyl bromide (80% in toluene, Alfa Aesar), tosyl chloride (TsCl) (99%, Shanghai Sinopharm Chemical Reagent Co. Ltd.), sodium azide (NaN3) (99%, Aldrich), pentaethylenehexamine (PEHA) (95%, Aldrich),


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ferrocenecarboxaldehyde (Fc-CHO) (98%, Aldrich), sodium borohydride (NaBH4) (≥99%, Sigma), 1,1,4,7,7-pentamethyldiethylenetriamine (PMDETA) (98%, Alfa Aesar), 1,7-octadiyne (98%, Shanghai Aladdin Reagent Co. Ltd.), branched polyethyleneimine (PEI) (99%, Mw = 25 kDa, Aldrich), chitosan (CTS) (Mw = 50-190 kDa, Aldrich), lipofectamine 2000 (LIP) (Life technology), glutathione (GSH) (≥98%, Shanghai Aladdin Reagent Co. Ltd.), hydrogen peroxide (H2O2) (30 wt%, Shanghai Sinopharm Chemical Reagent Co. Ltd.), sodium sulfate anhydrous (Na2SO4) (≥99%, Shanghai Sinopharm Chemical Reagent Co. Ltd.), sodium hydrogen carbonate (NaHCO3) (≥99%, Shanghai Sinopharm Chemical Reagent Co. Ltd.), sodium hydroxide (NaOH) (99%, Shanghai Sinopharm Chemical Reagent Co. Ltd.) and hydrochloric acid (HCl) (37%, Shanghai Sinopharm Chemical Reagent Co. Ltd.), were used as received without further purification. β-Cyclodextrin (β-CD) and its derivatives (Shanghai Sinopharm Chemical Reagent Co. Ltd.) were dried for 48 h at 60 oC in vacuum oven prior to use. Copper (I) bromide (CuBr) (98%, Aldrich) was firstly treated with acetic acid under vigorous stirring for several hours, and filtrating, then rinsing with acetic acid, ethanol and diethyl ether in succession until it turned into white, finally stored in vacuo before use. Methanol (MeOH) from Shanghai Sinopharm Chemical Reagent Co. Ltd. was treated with dry molecular sieve and distilled before it could be used. N,NDimethylformamide (DMF) and toluene from Shanghai Sinopharm Chemical Reagent Co. Ltd. were treated with calcium hydride and distilled before use. Ethanol, n-hexane, diethyl ether, acetone and dichloromethane (CH2Cl2) etc. were from Shanghai Sinopharm Chemical Reagent Co. Ltd., and deionized water were used as received. Preparation of Supramolecular Polymers. For supramolecular block copolymer, the constant molar concentration of PEG-CD (1 mM) was mixed with different amounts of FcPEHA-CD building blocks in water at molar ratios from 1:0 to 1:50, followed by a continuous


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stirring at ambient temperature for 6 h, and the supramolecular block copolymers with various polymerization degrees will be obtained. In addition, different molar amounts of Fc-PEHA-CD was firstly dissolved in water, followed a subsequent stirring at ambient temperature for 6 h, to produce supramolecular homopolymers with various chain lengths. Characterization. 1H NMR spectra were acquired on a Varian Mercury plus 400 NMR spectrometer (400 MHz) with deuterium oxide (D2O), deuterated dimethyl sulfoxide (DMSO-d6) and deuterated chloroform (CDCl3) as solvents at 293 K. DOSY and 2D-NOESY spectra were acquired on a Bruker Avance III NMR spectrometer (600 MHz) with D2O. The chemical shifts of these deuterated solvents are as follows: D2O (4.79 ppm), DMSO-d6 (2.48 ppm) and CDCl3 (7.26 ppm). The KBr sample holder method was used to record FTIR spectra of these products (e.g., β-CD-N3, PEG-Alkyne and PEG-β-CD) on a Paragon 1000 instrument. In order to remove the residual moisture, the samples were firstly dried for 30 min using infrared lamp before measurement. UPLC & Q-TOF-MS measurement was performed on a Waters-ACQUITYTM UPLC & Q-TOF-MS Premier (Waters Corporation, USA) at ambient temperature with methanol as the solvent. DLS measurement was carried out on a Malvern Zetasizer NanoS apparatus equipped with a 4.0 mW laser (λ = 633 nm). The samples were assayed at a scattering angle of 90o at 25 oC. Before testing, the sample solutions were filtered with some absorbent cotton to eliminate the dust. For the sake of thermal equilibration and chemical equilibration, the sample solution with the addition of certain amount of H2O2 or GSH was put into the cell for around 15 min prior testing. The molecular weight and polydispersity index (PDI) of polymers were determined using gel permeation chromatography/multiangle laser light scattering (GPCMALLS). Tetrahydrofuran (THF) was utilized as the eluent with a flow rate of 1 mL per min at 30 oC. Wyatt Optilab DSP differential refractometer at 690 nm was used to determine the


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refractive index increment (dn/dc). The Malvern Zetasizer NanoS at 25 oC was employed to measure the zeta potential (ζ) of the samples in PBS buffer. The cuvettes were filled with various concentrations of Fc-PEHA-CD solutions in water ranged from 0.5 to 10 mM, and the test was operated in the ζ-model ranging from minimum 10 cycles to maximum 100 cycles. The morphology of Core-Shell DNA polyplex was recorded on an atomic force microscopy (AFM) system in the Dimension 3100 model with a Nanoscope IIIa controller (Veeco, Santa Barbara, CA). 40 µL of SBC/pDNA polyplexes in deionized water (pH 7.4) containing approximate 0.08 µg of pDNA at the N/P ratios of 5 and 20 were dropped onto freshly cleaved mica sheets for 5 min. Subsequently, a piece of filter paper was used to absorb the excess solution, and mica sheets were dried naturally in air for 24 h. The samples were visualized using the tapping mode with setting of 256 × 256 pixels. Agarose Gel Electrophoresis. The PEHA/pDNA, SHP/pDNA and SBC/pDNA polyplexes at different N/P ratios were prepared with the addition of different volumes of PEHA solutions, SHP or SBC solutions to pDNA solutions in PBS buffer, followed by vortexing for 6 s and incubated for ca. 30 min at room temperature. While mixing 5 µL of 0.5 × loading buffer with the polyplex solutions, the 1% agarose gel containing 0.5 µg/mL ethidium bromide was used to analyze the resulting polyplex solution. Gel electrophoresis was performed in 0.5 × Tris-BorateEDTA (TBE) buffer (100 V) for 1 h in a Sub-Cell system (Bio-Rad Laboratories, CA). DNA bands were imaged under a UV lamp by use of a Gel Doc system (Synoptics Ltd., UK). Cell Cultures. COS-7 cells, HeLa cancer cells and MCF-7 breast cancer cells were cultured in DMEM with 4.5 g/L glucose containing 10% fetal bovine serum (FBS), penicillin (100 units/mL) and streptomycin (100 µg/mL) at 37 °C under a 5% CO2 humidified atmosphere. Confluent cells were subcultured every 3 days according to standard procedures.


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MTT Analysis. COS-7 cells were seeded into 96-well plates with a seeding density of 5000 cells/well in 200 µL media. After 24 h, the culture media was then removed and replaced with 200 µL of media containing 50 µL of PEI solutions or SBC solutions with a series of concentrations, such as 0.001, 0.005, 0.01, 0.05, 0.1,0.25, 0.5 and 1.0 mg/mL. The cells were continuously cultured for 48 h, and then 20 µL of 5 mg/mL MTT assays stock solution in PBS buffer was added to 96-well plates. After 4 h incubation, the unreacted dye-containing media was fully removed. The resulting blue formazan crystals were dissolved in 200 µL DMSO, and the absorbance was determined in a Perkin-Elmer 1420 Multi-label counter at 490 nm. In Vitro Transfection Assay. With regard to luciferase expression studies, three different cells including COS-7 cells, HeLa cancer cells and MCF-7 cancer cells, were seeded at a density of 104 cells/well in 96-well plates and incubated for 16-24 h until 60-70% confluent at 37 °C and 5% CO2. The media was immediately removed prior to transfection. And these cells were rinsed and replaced with fresh and prewarmed DMEM with or without 10% FBS. Polyplexes (Note: Polyplexes at various N/P ratios were prepared with the addition of different volumes of SHP or SBC solutions to pDNA solutions in PBS buffer, followed by vortexing for 6 s and incubated for 30 min at room temperature.) were added to each well, and these cells were incubated at 37 °C for 4 h. Then the media was replaced with fresh DMEM supplemented with 10% FBS, 10% FBS/0.05 mM H2O2 or 10% FBS/0.10 mM H2O2, and then incubated for an extra 48 h. We conducted the luciferase assay in accordance with manufacturer’s protocol (Promega, Madison, WI). Relative light units (RLUs) were determined with GloMaxTM 96 microplate luminometer (Promega). The resulting RLUs were normalized with regard to protein concentration in the cell extract that was measured by the BCA protein assay kit (Beyotime, China). RESULTS AND DISCUSSION


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Synthesis of PEG-CD and Fc-PEHA-CD. In order to acquire the linear SBC, a βcyclodextrin (β-CD)-monosubstituted polyethylene glycol (PEG-CD) and a cationic βCD/ferrocene (Fc)-terminated pentaethylenehexamine (Fc-PEHA-CD) were synthesized according to the synthesis route shown in Scheme 2. PEG–CD was fabricated via click chemistry between mono-azido-substituted-β-CD and mono-alkynyl-terminated PEG with copper (I) bromide (CuBr) as catalyst and 1,1,4,7,7-pentamethyldiethylenetriamine (PMDETA) as ligand.26,27 Fc-PEHA-CD was synthesized through a nucleophilic substitution reaction between mono-tolylsulfonyl-β-CD and pentaethylenehexamine (PEHA) followed by a Schiff-base condensation with ferrocenecarboxaldehyde.17,28 The successful synthesis of PEG-CD and FcPEHA-CD was clearly confirmed by 1H NMR, 13C NMR, FTIR, GPC and UPLC & Q-TOF-MS (Figures S1-S10). The association constant of β-CD host with Fc guest was determined to be 2.95×104 M-1 by using UV-Vis spectroscopy.17 (a) O

OH 113

NaH, toluene, 60 oC, 18 h

Br

O

O

113

CuBr, PMDETA, DMF, 70 oC, 48 h O

OH

7

TsCl, NaOH, H 2O, r.t, 30 min

O

N N N

113

NaN 3, H 2O, o OTs 80 C, 12 h

N3

O

(b)

H2N OTs

H

H N H 5

75 oC, 8 h

Fe N H

Fe

NH2 5

i) MeOH, 70 oC, 24 h; ii) NaBH 4, 0 oC, r.t., 12 h

N H

H N 5

Scheme 2. Synthesis of (a) PEG-CD and (b) Fc-PEHA-CD. Formation of Supramolecular Block Copolymer. The supramolecular polymerization behavior of Fc-PEHA-CD and PEG-CD/Fc-PEHA-CD in aqueous medium was investigated by


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concentration-dependent 1H NMR, 2D NOESY NMR and 2D DOSY NMR spectroscopy. The 2D NOESY spectra for both Fc-PEHA-CD and PEG-CD/Fc-PEHA-CD systems (Figure 1a and Figure S11) demonstrates the intermolecular correlations between the internal H3, H5 protons of β-CD host and the protons of Fc guest, indicating the inclusion complexation between β-CD and Fc in water. 1H NMR titration experiments further offered a vital insight into the concentrationdependent polymerization behavior of Fc-PEHA-CD and PEG-CD/Fc-PEHA-CD in water. With raising the solution concentration up to 50 mM, the proton signals of Fc groups thereupon become wide, demonstrating the formation of high-molecular-weight supramolecular polymers (Figure 1b and Figure S12).29,30 In Figure 1c, the critical polymerization concentration (CPC), above which concentration-driven supramolecular polymerization will predominantly occur, was found to be ca. 7.5 mM for SHP system (Note: SHP refers to a non-covalent homopolymer composed only of Fc-PEHA-CD monomer.). With the introduction of a certain amount of PEGCD, no obvious CPC can be detected for SBC system. The diffusion coefficient of SBC is slightly lower than that of SHP at the same concentration, verifying the resulting SBC having higher molecular weight. Also, the surface charge density of both supramolecular polymer systems was determined through zeta potential measurement, and the SBC has relatively lower zeta potential than that of homopolymer system at the equivalent concentration due to the partial shielding effect by neutral, hydrophilic PEG shell (Figure S13). Furthermore, the molecular weight of SBC at 10 mM was calculated to be 32 kDa with degree of polymerization (DP) of 17 according to the equation of DP = (Ka[Conc.])1/2 (Table S1).31,32 We try to determine the molecular weight of SBC at 10 mM using GPC (Figure S14), but unfortunately no valid value was acquired. It can be mainly attributed to the fact that the high-molecular-weight supramolecular polymer formed via dynamic non-covalent interaction gradually depolymerized


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during the process of continuous elution in GPC column. In view of these results, we can generally conclude that a linear SBC has been successfully acquired at high solution concentrations. * H3,H5 Ha-c

(a)

m

Supramolecular polymerization

(b)

Supramolecular block copolymer

(c)

Ha-c

H3,H5

*

1:2

3.0

1:5

3.5

1:10

4.0

1:20

4.5

Diffusion coefficient (m2/s)

1:0 2.5

Ha-c

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1:30 1:40

5.0 1:50 5.5

5.0 4.5 4.0

3.5 3.0

5.5 2.5 (ppm)

5.5

5.0

1.5×10-9

Without SHP PEG WithPEG SBC

1.0×10-9

5.0×10-10

CPC = 7.5 mM 0.0 0

4.5

4.0

3.5

3.0

2.5

(ppm)

10

20

30

40

50

60

Concentration (mM)

Figure 1. (a) Partial 2D NOESY NMR spectrum of the SBC solution containing 1 mM PEG-CD and 20 mM Fc-PEHA-CD in D2O at 30 oC. (b) 1H NMR spectrum of the SBC solution with the molar ratios of PEG-CD/Fc-PEHA-CD from 1:0 to 1:50 in D2O. (c) The diffusion coefficient of SBC and SHP in D2O as a function of molar concentration, and the plot of diffusion coefficient of SHP in water versus molar concentration. Redox Responsiveness of Supramolecular Block Copolymer. The resultant SBC displays the redox-triggered reversible polymerization/depolymerization behavior upon alternative addition of H2O2 and glutathione (GSH). 1H NMR spectroscopy was utilized to evaluate the redox response of this copolymer. In Figure 2a, the proton signal peaks of Fc-PEHA-CD completely disappear after the addition of H2O2 into the initial polymer solution owing to the electron paramagnetic resonance triggered by the oxidized Fc (Fc+) unit. Hereupon, the exclusion of the water-soluble Fc+ unit from the hydrophobic inner cavity of β-CD would lead to the depolymerization of SBC.33 With the addition of GSH, the proton peaks of Fc-PEHA-CD


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reappear, which means that the Fc+ unit is turned into its reduced state. The reversible polymerization occurs accordingly when the reduced Fc unit goes back to the inner cavum of βCD. The dynamic light scattering (DLS) was further carried out to detect the size change of supramolecular copolymer at 10 mM upon alternative addition of H2O2 and GSH. In Figure 2b, the hydrodynamic diameter of SBC significantly reduces from approximately 120 nm to 70 nm with the addition of H2O2, while the subsequent additive GSH leads to an invertible size raise to ca. 180 nm, meaning the H2O2-induced depolymerization and invertible formation of SBC. Moreover, the alternating addition of H2O2 and GSH further triggers the reversible polymerization and depolymerization behavior of this copolymer, which are mainly attributed to the reversible association and disassociation between β-CD and Fc along the supramolecular copolymer backbone.

Figure 2. (a) 1H NMR spectra of the SBC in D2O at 10 mM before and after addition of H2O2 and GSH. (b) Periodic changes in the hydrodynamic diameter of a SBC upon alternate addition of H2O2 and GSH.


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pDNA Condensation Behavior. The surface charge property of SBC was evaluated using zeta potential experiment. In Figure S13, with a raise of the concentration, the zeta potentials for both SBC and SHP systems nonlinearly reduced owing to the fact that the entanglement and aggregation of the supramolecular polymer chains at high concentrations lead to the partial embedment of surface charges. In comparison to SHP, the SBC shows lower zeta potential at identical concentration owing to the charge shielding effect by neutral, hydrophilic PEG segment. Because of their cationic nature, both SBC and SHP are expected to condense pDNA effectively via electrostatic interactions. Agarose gel electrophoresis was carried out to evaluate the pDNA binding ability of SBC and SHP. In Figure 3a, at a nitrogen-to-phosphorus ratio (N/P ratio) of below 5, the SBC only partially retarded the migration of pDNA in agarose gel, whereas the SBC could condense pDNA fully at an N/P ratio of above 10. In addition, a similar phenomenon was also clearly observed for SHP system (Figure S15). However, the cationic PEHA small molecule is unable to condense pDNA even if the N/P ratio reaches up to 50 (Figure S15a). Atomic force microscopy (AFM) was further utilized to visualize the size and morphology of the SBC/pDNA complexes. At an N/P ratio of 5, pDNA was partially condensed by SBC to generate a typical morphological trait with few large-sized particles (ca. 250 nm) and lots of supercoiled naked pDNA in Figure 3b. In sharp contrast, Figure 3c presents the tight nano-sized particles with an average size of 100 nm without naked pDNA at an N/P ratio of 20, demonstrating the intact condensation of pDNA by SBC. Combining the results of agarose gel electrophoresis and AFM, it can therefore be concluded that this block copolymer has great potential to efficiently bind pDNA into nanoscale polyplexes appropriate for in vitro or in vivo gene delivery.


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Figure 3. (a) Agarose gel electrophoresis retardation of pDNA by a cationic SBC at the N/P ratios of 1, 3, 5, 10, 20, 30 and 50. AFM images of SBC/pDNA complexes at the N/P ratios of (b) 5 and (c) 20. Biostability. Next, it is critical to determine the biostability of these supramolecular polymers under physiological environments to allow their use in vitro or in vivo. The real-time dynamic light scattering (DLS) measurement was employed to monitor the size variation of SBC and SHP under various physiological environments. In Figure 4, while being incubated in both PBS buffer (pH 7.4) and serum at 37 °C, the SBC nearly maintains a constant hydrodynamic diameter of 100-120 nm, further exhibiting outstanding physiological stability in both PBS buffer (pH 7.4) and serum. In sharp contrast, the size of SHP remarkably raises up to several micrometers along with precipitation occurring. As shown in the inset of Figure 4a, the SBC in PBS buffer (pH 7.4) appears to be homogenous, while the SHP promptly precipitates in PBS buffer. These results


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further demonstrate that the SBC has much better physiological stability compared to the SHP, benefiting from excellent water-solubility and improved biostability of hydrophilic PEG block. In addition, the SBC will possibly depolymerize under low concentration, however, the resulting SBC/pDNA polyplexes having unique core-shell nanostructure, which was surrounded by a large number of hydrophilic PEG chains in the outer layer, showing greatly improved biostability at low concentration conditions. Meanwhile, the AFM image of SBC/pDNA polyplexes (N/P ratio = 20) at a concentration of 0.1 mg/mL further provides direct evidence for its superior stability at low concentration (Figure 3c). Therefore, the SBC has great potential for in vivo gene delivery.

Figure 4. Size variation of supramolecular block copolymer and supramolecular homopolymer at 10 mM in PBS buffer (pH 7.4) (a) and serum (b) at 37 oC monitored by DLS. The insets are photographs of the supramolecular homopolymer (SHP, left) and supramolecular block copolymer (SBC, right) in PBS buffer (pH 7.4) at ambient temperature. Cell Viability. Furthermore, the cell cytotoxicity of the resulting supramolecular polymers was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.34 In Figure 5, compared to commercially available branched PEI (25 kDa) and


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lipofectamine 2000, both SBC and SHP demonstrate much lower cytotoxicity after 48 h incubation in COS-7 cells. In particular, when the sample concentration is higher than 0.05 mg/mL, the cell viability of PEI and lipofectamine 2000 is less than 20%, whereas that of both SBC and SHP is more than 40% (Figure 5). When the polymer concentration reaches up to 1.0 mg/mL, the SBC shows much higher cell viability than that of SHP mainly owing to the charge screening effect by hydrophilic and biocompatible PEG chains of the SBC.

Figure 5. Cell viability assay of supramolecular block copolymer (SBC), supramolecular homopolymer (SHP), lipofectamine 2000 (LIP) and branched PEI (PEI, 25 kDa) with sample concentrations ranging from 0.001 to 1.0 mg/mL were incubated in COS-7 cells for 48 h. Black bars represent the mean values (n = 5). In Vitro Gene Delivery. The in vitro transfection efficiency for these two supramolecular polymers was examined using luciferase assay. Both the SBC/pDNA and SHP/pDNA polyplexes


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at the N/P ratios of 10, 20, 30, 50 and 80 were incubated in COS-7 cells, HeLa cells and MCF-7 cells for 48 h. The corresponding polymer dose of SBC at the above N/P ratios is 0.019, 0.038, 0.057, 0.095 and 0.155 mg/mL, respectively. It is worth noting that the cell viability of SBC with the N/P ratio up to 80 is still above 50% (Figure 5). In Figure 6b, both the SBC and SHP present favorable gene transfer efficacy in COS-7 cells (ca. 6 ×106), which is lower than that of branched PEI (25 kDa) (ca. 1 × 108), but comparable to that of lipofectamine 2000 (ca. 7 ×106), even evidently higher than that of chitosan (1×106) in COS-7 cells. A slight reduction in the transfection efficacy was also observed with increasing the N/P ratio from 10 to 80 resulting from their higher cytotoxicity at a higher N/P ratio. Figure 6a and Figure S15c indicate the introduction of H2O2 triggers pDNA release from the complexes induced by H2O2-induced depolymerization of supramolecular polymers. Thereby, the addition of a certain amount of H2O2 leads to a reduction of transfection efficacy of both SBC and SHP to some degree in COS-7 cells partially resulting from the dissociation of the polyplexes at the early stage of gene transfer. We also noticed that the addition of H2O2 leads to a reduction of transfection efficacy of PEI in COS7 cells possibly owing to the increasing cytotoxicity towards COS-7 cells induced by external H2O2. Compared with SHP, the reduction tendency of transfection efficiency of SBC in COS-7 cells induced by H2O2 has been effectively suppressed because of its better biostability of SBC/pDNA polyplex. The gene expression for these two supramolecular polymers in cancer cells was further evaluated. In Figure 6c-d, both SBC and SHP shows slightly lower transfection efficacy in HeLa cells and MCF-7 cells compared to branched PEI (25 kDa) ascribed to the intracellular disintegration of polyplexes caused by excess H2O2 in cancer cells.35 However, the gene transfection efficiency of SBC is comparable to that of lipofectamine 2000, and even evidently higher than that of chitosan in HeLa cells and MCF-7 cells.


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(a) (a) pDNA

N/P ratio N/P ratio 10

30

50

(b)

10/H2O2 30/H 2O2 50/H2O2

N/P ratio 20

108

10 30

10 50

10 80

107 106 105 104 103 PEI LIP CTS SBC SHP +H2O2 (mM) [0 0.05 0.1] [0 0.05 0.1]

(c)

(d)

107 N/P ratio 20

10 30

10 50

10 80

106 105 104 103

Luciferase Expression (RLU/mg protein)


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N/P ratio 20

106

10 30

10 50

10 80

105 104 103 102

PEI

LIP

CTS

SBC

SHP

PEI

LIP

CTS

SBC

SHP

Figure 6. (a) Agarose gel electrophoresis retardation of pDNA by SBC at the N/P ratios of 10, 30 and 50 before and after addition of H2O2. Luciferase expression of SBC or SHP/pDNA polyplexes at the N/P ratios of 10, 20, 30, 50 and 80 (with 0, 0.05 and 0.1 mM of H2O2), and branched PEI, lipofectamine 2000 (LIP) or chitosan (CTS)/pDNA polyplexes at a N/P ratio of 10 after 48 h in (b) COS-7 cells, (c) HeLa cells and (d) MCF-7 cells. In order to better illustrate the advantages of the SBC for in vivo gene delivery, we further carried out the gene delivery experiments of SBC in COS-7 cells, Hela cells and MCF-7 with the addition of 10% fetal bovine serum (FBS) into DMEM (Figure S16). As shown in Figure S16, the addition of serum leads to a slight reduction of the transfection efficacy of SBC in COS-7 cells (Figure S16a), whereas almost no measureable variation in transfection efficiency of the SBC can be observed in both HeLa cells and MCF-7 cells using 10% FBS-containing media


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(Figure S16b-c). On the other hand, the transfection efficiency of SBC is a little lower than that of PEI and lipofectamine 2000, but significantly higher than that of chitosan in these three cell lines in serum-containing media (Figure S16). The result indicates that the SBC presents remarkably improved gene transfer efficacy in various cell lines in the presence of serum. Furthermore, the SBC having better physiological stability and biocompatibility compared to the SHP under various physiological environments (i.e., PBS buffer or serum) facilitates the transport and delivery of pDNA across cellular barriers (Figure 4), further achieving greatly improved gene transfection efficacy in cancer cells (Figure 6c-d and Figure S16b-c). Therefore, it can be concluded that the cationic SBC will be an alternative to exploit highly efficient genedelivery vehicles for gene therapy.

CONCLUSION In conclusion, a novel cationic supramolecular block copolymer, by the combination of excellent biostability and biocompatibility of traditional polymer with exclusive biodegradability and smart responsiveness of supramolecular polymers, has been successfully developed. The resultant supramolecular block polymer not only has the capability to condense pDNA into nanosized particles for enhanced cell uptake, but also release pDNA inside the cancer cells in response to H2O2. Because of its unique structure and fascinating properties, this supramolecular block copolymer shows greatly improved gene transfection efficiency in cancer cells compared to the supramolecular homopolymer. This work aims to offer an alternative platform for the development of smart nonviral vehicles for specific cancer gene therapy in future. We expect to spark new ideas and inspire persistent endeavors in this emerging field.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:********. Synthesis details of PEG-CD and FC-PEHA-CD; 1H NMR, 13C NMR, GPC, FTIR and UPLC & Q-TOF-MS results for the materials and Supplemented Figures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] ORCID ID Ruijiao Dong: 0000-0002-3091-7712 Xinyuan Zhu: 0000-0002-2891-837X Author Contributions X. Z. and R. D. conceived and supervised the project. W. Y. and R. D. designed and carried out the detailed experiments, participated in result analysis and wrote the whole paper. W. Y.


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prepared the materials and carried out the spectroscopic measurements. L. Z. and J. W. assisted with gene delivery experiments in vitro and DOSY assay. C. C. and A. A. contributed to the article writing. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was granted by the National Basic Research Program of China (2015CB931801), National Natural Science Foundation of China (51690151, 21504054), and Postdoctoral Science Foundation of China (2015M580321).

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Table of Contents (TOC)


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Synthesis of a Cationic Supramolecular Block ... - ACS Publications

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