Statistical versus block fluoropolymers in gene delivery

Echuan Tan , Jia Lv , Jingjing Hu , Wanwan Shen , Hui Wang and Yiyun Cheng *
Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, P. R. China. E-mail: yycheng@mail.ustc.edu.cn

Received 5th June 2018 , Accepted 16th July 2018

First published on 16th July 2018


Fluoropolymers have shown great promise in non-viral gene delivery. The current fluoropolymers developed for gene delivery are synthesized by grafting fluoroalkyls or fluoroaromatics onto cationic polymers. To expand the family of fluoropolymers for the transduction of nucleic acids, new strategies to synthesize fluoropolymers are required. In this study, we synthesized both statistical and block copolymers of poly(2-dimethylaminoethyl methacrylate) (pDMAEMA) and poly(heptafluorobutyl methacrylate) (pHFMA) via reversible addition–fragmentation chain transfer polymerization, and the transfection efficiencies of the fluorocopolymers were evaluated. The statistical fluorocopolymer exhibited dramatically higher performance in gene delivery than the block one, which is attributed to more efficient and sustained DNA uptake by the transfected cells. Moreover, the statistical copolymer of DMAEMA and HFMA showed a fluorine effect in gene delivery, and its efficiency was much superior to non-fluorinated polymers. The results revealed the structure and activity relationships of fluoropolymers consisting of DMAEMA and HFMA, and provided a new insight to guide the design of fluoropolymers for efficient gene delivery.


Introduction

Cationic polymers are widely used as non-viral carriers for the delivery of nucleic acids such as DNA and siRNA.1,2 The polymers usually bind nucleic acids via ionic interactions to form positively charged complexes that can be internalized by cells. To enable efficient cell internalization and endosomal escape, cationic polymers were usually conjugated with hydrophobic fusogenic ligands such as lipids or cholesterols that can fuse with the phospholipid bilayers to facilitate the cell uptake and endosomal escape.3 In addition, the polymers were always incorporated with pH buffering ligands that act as a “proton sponge” to promote the swelling fracture of endolysosomes.2 Since hydrophobic ligands are easily bound with serum proteins, cationic polymers modified with hydrophobic moieties usually showed poor serum resistance,4 and needed to be further engineered with hydrophilic anti-fouling components such as carbohydrates, poly(ethylene glycol) and zwitterions.3,5,6 Considering the co-existence of multiple barriers in gene delivery, polymers should be incorporated with various functional ligands for efficient gene transfection. This involves several challenges, i.e. sophisticated synthesis, difficulty in balancing the hydrophobic and hydrophilic components, and spatial hindrance of multiple components on a single polymer.7,8 As a result, there is an urgent need to seek for a single ligand that can address the multiple barriers existing in gene delivery.

Perfluoroalkanes are both hydrophobic and lipophobic, but prefer to interact with other fluorous ligands via a fluorophilic effect. They are chemically and biologically inert, and were always used as anti-fouling coatings. Incorporation of these ligands endow cationic polymers with excellent self-assembly properties,9 and the yielding polymers possess relatively low critical aggregation concentrations. In this case, fluoropolymers can condense nucleic acids into stable complexes at low positive-to-negative charge ratios.1,10 This property ensures low toxicity of the fluoropolymers in gene delivery. In addition, perfluoroalkanes have a strong tendency to adsorb on cell membranes due to exceedingly low free energy for transferring perfluoroalkanes from water to the interface, and the limited miscibility between perfluoroalkanes and phospholipids minimizes the retention of fluorinated polymers within the membrane.4 These features mean that fluoroalkylated polymers can efficiently penetrate across the bio-membranes with limited membrane disruption. Most importantly, the perfluoroalkane chains are bio-inert and resistant to proteins, thus allowing the fluoroalkylated polymer to achieve high transfection efficiency in the presence of serum.9,11–16 This property is an essential characteristic for in vivo gene delivery. Compared to non-fluorinated amphiphilic polymers, the fluorinated ones exhibited an interesting fluorous effect during the delivery process.4,17,18 As a result, fluorination was reported to be a promising strategy to improve the in vitro and in vivo performance of various cationic polymers in gene delivery.1,9,19–30

Though fluorinated polymers achieved success in cytosolic DNA, RNA and protein delivery, the current materials were mainly synthesized by grafting fluorous ligands such as fluoroalkyls and fluoroaromatics onto cationic polymers.1,3,4,24 The type of fluorinated polymers synthesized by this strategy is limited, and new methods to construct fluoropolymers for efficient gene delivery are required. In addition, the fluorous ligands are randomly distributed on the polymer using the “grafting onto” strategy, and thus it is not available to investigate the effect of fluorous monomer distribution on the efficiency of fluoropolymers in gene delivery, which is an essential parameter for copolymers.31 In this study, we synthesized both statistical and block copolymers of poly(2-dimethylaminoethyl methacrylate) (pDMAEMA) and poly(heptafluorobutyl methacrylate) (pHFMA) via reversible addition–fragmentation chain transfer (RAFT) polymerization (Fig. 1a). pDMAEMA is one of the most investigated cationic polymers for non-viral gene delivery.5,6,32–41 The polymer is positively charged at pH 7.4 (pKa around 7.0), further protonated when entrapped in endolysosomes, and capable of degrading into negatively charged and non-toxic polyacrylic acids, which are beneficial for endosomal escape and intracellular cargo release, respectively.2 For pDMAMEA-mediated gene delivery, the molecular weight of the polymer plays an essential role in transfection efficiency. High molecular weight pDMAEMA was effective, but toxic on the cells, while low molecular weight ones showed poor transfection efficiency. In this case, low molecular weight pDMAEMA was fabricated into branched or star structures,34,42,43 grafted onto/from nanoparticles,44–46 or assembled into nanostructures via supramolecular chemistry.41,47–50 Here, low molecular weight pDMAEMA was incorporated with fluorous monomer HFMA via RAFT polymerization, and the yielding copolymer could assemble into nanostructures due to the excellent assembly behavior of fluoropolymers. The assembled pDMAEMA copolymer was expected to possess both high gene delivery efficiency and minimal toxicity. The effect of the monomer sequence in the constructed fluorocopolymers on transfection efficiency was investigated. The statistical fluorocopolymer exhibited dramatically superior efficiency in gene delivery in comparison with the block one, which is attributed to more efficient and sustained DNA internalization by the statistical fluorocopolymer (Fig. 1b).


image file: c8tb01470a-f1.tif
Fig. 1 Statistical versus block fluorocopolymers consisting of DMAEMA and HFMA in gene delivery. (a) Synthesis of the fluorocopolymers pDMAEMA-st-pHFMA and pDMAEMA-b-pHFMA by RAFT polymerization. (b) Mechanism of pDMAEMA-st-pHFMA and pDMAEMA-b-pHFMA in gene delivery. The statistical fluorocopolymer showed sustained plasmid internalization by the cells and much higher transfection efficiency.

Experimental methods

Materials

1H,1H-Heptafluorobutyl methacrylate (HFMA, 97%) was purchased from Alfa Aesar (Lancaster, England). Cytochalasin D, genistein, methyl-β-cyclodextrin (MβCD), chlorpromazine, LysoTracker Red and Hoechst 33342 were purchased from Sigma-Aldrich (St. Louis, MO, USA). 4-Cyanopentanoic acid dithiobenzoate (CPADB, 97%) was from Macklin (Shanghai, China). Butyl methacrylate (BMA, 99%) and hexyl methacrylate (HMA, 97.5%) were from J&K Scientific (Shanghai, China). 2-(Dimethyl-amino)ethyl methacrylate (DMAEMA, 99%) and 2,2′-azobis(2-methylpropionitrile) (AIBN, 99%) were from Aladdin (Shanghai, China). Tetrahydrofuran (THF), hexane, and chloroform were from Sinopharm (Shanghai, China). Fetal bovine serum (FBS) was from Gemini Bio Products. Lipofectamine 2000 (Lipo 2000) and YOYO-1 were obtained from Invitrogen (Carlsbad, CA). Dulbecco's modified Eagle's medium (DMEM) was obtained from Gibco. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sangon (Shanghai, China). Agarose, GelRed, cell lysis buffer, the luciferase assay kit, and the BCA protein assay kit were from Beyotime (Shanghai, China).

Synthesis of pDMAEMA via RAFT polymerization

DMAEMA (1.0061 g, 6.40 mmol), CPADB (0.0416 g, 0.149 mmol), and the initiator AIBN (0.0073 g, 0.044 mmol) were dissolved in 5 mL THF in a glass vial at a DMAEMA/CPADB/AIBN molar ratio of 43[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]0.3. The bottle was sealed and the reaction mixture was degassed with argon in an ice bath for 5 min. The polymerization was carried out in an oil bath at 60 °C for 24 h and terminated by placing the glass vial in an ice bath for 5 min. The synthesized pDMAEMA was then precipitated in hexane and centrifuged at 6000 rpm for 5 min, and the procedure was repeated three times. The final product was dried in a vacuum oven overnight, yielding a pink solid. The conversion efficiency of DMAEMA and the theoretical molecular weight (Mn,th) were determined by 1H NMR (Varian, 699.804 MHz, CDCl3 as the solvent). The polydispersity index (PDI) of the polymer was measured by gel permeation chromatography (GPC) using THF as the mobile phase.

Synthesis of pDMAEMA-b-pHFMA block and pDMAEMA-st-pHFMA statistical copolymers

pDMAEMA was synthesized as described above and used as a macro-RAFT reagent to synthesize pDMAEMA-b-pHFMA. pDMAEMA (0.2000 g, 0.029 mmol), HFMA (0.0982 g, 0.366 mmol) and AIBN (0.0014 g, 8.52 μmol) were dissolved in 3 mL THF in a glass vial. The block copolymer was synthesized and purified as described above. For pDMAEMA-st-pHFMA, DMAEMA (0.3144 g, 2.000 mmol), HFMA (0.1341 g, 0.500 mmol), CPADB (0.0112 g, 0.040 mmol), and the initiator AIBN (0.0020 g, 0.012 mmol) were dissolved in 5 mL THF in a glass vial. The synthesis and purification procedures were the same as pDMAEMA. The non-fluorinated copolymers pDMAEMA-st-pBMA and pDMAEMA-st-pHMA were synthesized using a similar method. The conversion efficiencies of DMAEMA and the hydrophobic monomer (HFMA, BMA, or HMA) were determined using 1H NMR. The purified copolymers were characterized using 1H NMR. The PDI of the copolymers was determined using GPC.

Preparation and characterization of polymer nanomicelles and nanomicelle/DNA complexes

The polymer nanomicelles for pDMAEMA-b-pHFMA, pDMAEMA-st-pHFMA, pDMAEMA-st-pBMA and pDMAEMA-st-pHMA were prepared using a film dispersion method according to a previous reference.11 Generally, 2 mg of each polymer was dissolved in chloroform, and then the solvent was removed by vacuum rotary evaporation to form a dry film. After that, 20 mL distilled water was added, and the solution was sonicated for 30 min and kept at room temperature for 2 h to allow the formation of nanomicelles. Transmission electron microscopy (TEM) images of the nanomicelles were taken using a Hitachi microscope (HT7700, Hitachi, Japan) operating at an acceleration voltage of 100 kV. The particle size of the nanomicelles was measured using dynamic laser light scattering (DLS, Zetasizer Nano ZS90, Malvern, UK). For the nanomicelle/DNA complexes, polymer nanomicelles (0.1 mg mL−1) were mixed with 0.8 μg of plasmid DNA at different nitrogen-to-phosphorous (N/P) ratios. N here represents the number of tertiary amine groups on pDMAEMA and P represents the number of phosphate groups on plasmid DNA. The mixture of polymer nanomicelles and DNA was vortexed and then incubated at room temperature for 30 min to allow complex formation. The formed complexes were also characterized using TEM and DLS.

Gel electrophoresis assay

The DNA binding ability of the synthesized polymers was evaluated using a gel electrophoresis assay. Generally, the complexes of polymer nanomicelles and DNA were prepared at different N/P ratios as described above. The complexes were mixed with 1 μL DNA loading buffer. The electrophoresis was performed using 1.2% (w/v) agarose gels containing 0.1 μL mL−1 GelRed in a 1 × TAE buffer solution. The samples were run at 80 V for 60 min and the DNA bands in the gel were visualized and imaged under UV illumination using an EC3 Imaging System (UVP Inc.).

In vitro gene transfection experiments

The transfection efficiency of the synthesized polymers was evaluated using plasmid DNA encoding enhanced green fluorescent protein (EGFP) or luciferase. 293T or HeLa cells were cultured in DMEM (10% serum) in 24-well plates at 37 °C. The cells were cultured overnight before gene transfection, and the culture media were replaced with 250 μL DMEM containing nanomicelle/DNA complexes (0.8 μg DNA in each well, N/P ratio ranges from 6[thin space (1/6-em)]:[thin space (1/6-em)]1 to 12[thin space (1/6-em)]:[thin space (1/6-em)]1) in the absence of serum. The cells were replenished with 500 μL DMEM containing 10% serum after 6 h. The transfection efficiency of each polymer was evaluated at 48 h after complex incubation. EGFP expression in the cells was observed using fluorescence microscopy (Olympus, Japan) and quantitatively analyzed using flow cytometry (BD FACSCalibur, San Jose). Transfected luciferase in the cells was measured using a luciferase activity assay kit. Protein mass was determined using a BCA kit. The transfection efficiency of Lipo 2000 was tested according to the manufacturer's protocol and used as a positive control. To evaluate the serum tolerance of the polymer and Lipo 2000, the complexes were diluted in DMEM containing 10%, 20%, 30%, 40% or 50% serum, respectively, before incubation with the cells. The transfection experiments were conducted as described above. Three independent experiments were done for each sample.

Cellular uptake assay

To exclude the influence of EGFP fluorescence on cellular uptake, we used the luciferase plasmid to prepare the polymer nanomicelle/DNA complexes. The luciferase plasmid was labeled with YOYO-1 (0.02 nmol per μg plasmid) for 10 min at room temperature before complexation with the polymer nanomicelles (N/P at 8[thin space (1/6-em)]:[thin space (1/6-em)]1). 293T cells were treated with the complexes for 6, 12, 24 and 48 h, respectively. The detailed procedure was the same as that described in the gene transfection experiments. At each time interval, the cells were washed, digested with trypsin, centrifuged, and re-suspended in PBS buffer. The mean fluorescence intensity and the percent of positive YOYO-1 cells (%) were measured using flow cytometry. The cells incubated with YOYO-1 labeled complexes for 6 h and 12 h were also imaged using confocal laser scanning microscopy (CLSM, Leica TCS SP5) with an argon laser (excitation wavelength: 488 nm). Acidic organelles and nuclei of the transfected cells were stained by LysoTracker Red and Hoechst 33342, respectively. To investigate the endocytosis mechanism for statistical and block fluorocopolymers, the cells were treated with specific endocytosis inhibitors including genistein (an inhibitor of caveolae-dependent endocytosis, 700 μM), MβCD (an inhibitor of lipid raft mediated endocytosis, 10 mM), chlorpromazine (an inhibitor of clathrin-dependent endocytosis, 20 μM), and cytochalasin D (an inhibitor of macropinocytosis-mediated endocytosis, 10 μM) for 1 h before incubation with YOYO-1 labeled complexes. The cells were then washed with PBS buffer after incubation for 24 h and analyzed using flow cytometry to quantitatively determine the internalized complexes.

Cytotoxicity assay

The cytotoxicity of pDMAEMA, pDMAEMA-b-pHFMA, pDMAEMA-st-pHFMA, pDMAEMA-st-pBMA, and pDMAEMA-st-pHMA on 293T was determined using MTT assay as described elsewhere. The cells were cultured in a 96-well plate overnight, and then treated with polymers at a concentration range of 5 to 80 μg mL−1. The cytotoxicity of the nanomicelle/DNA complexes of different polymer concentrations prepared at an N/P ratio of 8[thin space (1/6-em)]:[thin space (1/6-em)]1 was also tested. The procedures were the same as those described in gene transfection experiments. Lipo 2000 was set as a control, and its concentration was equal to that used in gene delivery. Five independent experiments were tested for each sample. All the data in this manuscript were analyzed using Student's t-test, one-tailed.

Results and discussion

The cationic polymers including pDMAEMA, the fluorocopolymers pDMAEMA-b-pHFMA and pDMAEMA-st-pHFMA, and the non-fluorinated copolymers pDMAEMA-st-pBMA and pDMAEMA-st-pHMA were synthesized using RAFT polymerization. The molar ratios of cationic monomer DMAEMA and hydrophobic monomer to the RAFT initiator CPADB were carefully tailored to allow approximately equal numbers of DMAEMA/hydrophobic components in the synthesized copolymers (Fig. 2a). According to the calculated polymerization degrees of DMAEMA and the hydrophobic monomers (HFMA, BMA and HMA) by 1H NMR, the synthesized polymers were termed pDMAEMA40, pDMAEMA42-st-pHFMA12, pDMAEMA42-b-pHFMA13, pDMAEMA42-st-pBMA12, and pDMAEMA44-st-pHMA10, respectively (Fig. 2b and Fig. S1, ESI). NMR spectra in Fig. 2c confirmed a similar ratio of hydrophobic to cationic monomers in the obtained pDMAEMA copolymers. All the synthesized polymers showed relatively narrow molecular weight distributions, and the statistical copolymers showed relatively higher polydispersity indices than the block copolymer pDMAEMA42-b-pHFMA13 and the homopolymer pDMAEMA40.
image file: c8tb01470a-f2.tif
Fig. 2 Synthesis and assembly of pDMAEMA-based polymers. (a) Characterization of the synthesized polymers. *determined using 1H NMR, **determined using GPC. (b) Chemical structures of the five polymers with proton assignments. (c) 1H NMR spectra of the polymers. The number under each peak represents the relative integral area. (d) DLS and TEM images of the nanomicelles for each pDMAEMA copolymer. The scale bar is 200 nm.

The synthesized pDMAEMA copolymers were then fabricated into nanomicelles using a film dispersion method.11 The homopolymer pDMAEMA40 showed poor capability in nanomicelle formation, while all the amphiphilic copolymers were assembled into nanomicelles in the size range of 100–200 nm with a particle distribution index below 0.3 (Fig. 2d and Fig. S2, ESI). The critical micelle concentrations (CMC) of pDMAEMA42-st-pHFMA12 and pDMAEMA42-b-pHFMA13 were determined using a light scattering method.51 As shown in Fig. S3 (ESI), both fluoropolymers showed a similar and low CMC value around 4 μg mL−1. The hydrodynamic sizes of fluorocopolymer nanomicelles were relatively larger than those of non-fluorinated materials, probably due to the low cohesive energy densities and weak van der Waals interactions between the fluoroalkyl chains relative to hydrogenated lipids.52 TEM images of the prepared nanomicelles inserted in Fig. 2d also confirmed the assembly of pDMAEMA copolymers into nanoparticles. The sizes of nanomicelles observed in TEM are much smaller than those determined using DLS, which is probably due to the collapse of polymer nanomicelles in dry states.

The polymer nanomicelles were then incubated with plasmid DNA at different N/P ratios to prepare complexes. The hydrodynamic size of pDMAEMA42-b-pHFMA13 complexes was relatively smaller than those of other complexes, especially at low N/P ratios of 4[thin space (1/6-em)]:[thin space (1/6-em)]1, 6[thin space (1/6-em)]:[thin space (1/6-em)]1 and 8[thin space (1/6-em)]:[thin space (1/6-em)]1 (Fig. 3a). This is probably due to the relatively concentrated positive charges on the block copolymer compared to the statistical one. The consecutive fluorous segments in the block copolymer may improve the charge density on the pDMAEMA copolymer and endow the polymer with higher DNA binding capacity.53 All the polymers formed positively charged complexes with DNA at N/P ratios of 4[thin space (1/6-em)]:[thin space (1/6-em)]1 to 12[thin space (1/6-em)]:[thin space (1/6-em)]1 (Fig. 3b), and successful condensation of plasmid DNA into small-sized nanoparticles was further confirmed using TEM (Fig. 3c). The gel electrophoresis assay in Fig. S4 (ESI) also confirmed that pDMAEMA42-b-pHFMA13 had better DNA binding capability than the other polymers. Though all the polymer nanomicelles condensed the plasmid DNA into small nanoparticles at the investigated N/P ratios in DLS studies, they showed weak association with DNA in the formed complexes as the bound DNA was separated from the complexes in the gel electrophoresis assay. This is probably due to the low charge density of the pDMAEMA-based polymers, and this phenomenon is also observed for other fluoropolymers with low positive charge densities.9


image file: c8tb01470a-f3.tif
Fig. 3 Characterization of the polymer nanomicelle/DNA complexes. (a) Size and (b) zeta potential of the complexes prepared at N/P ratios of 4[thin space (1/6-em)]:[thin space (1/6-em)]1, 6[thin space (1/6-em)]:[thin space (1/6-em)]1, 8[thin space (1/6-em)]:[thin space (1/6-em)]1, 10[thin space (1/6-em)]:[thin space (1/6-em)]1, and 12[thin space (1/6-em)]:[thin space (1/6-em)]1, respectively. (c) TEM images of the complexes prepared at an N/P ratio of 8[thin space (1/6-em)]:[thin space (1/6-em)]1. The scale bar is 200 nm.

After successful preparation of complexes for the polymers, we first evaluated the transfection efficiencies of the five polymers on 293T cells using the EGFP reporter gene. As shown in Fig. 4a and Fig. S5–S10 (ESI), the homopolymer pDMAEMA40 showed extremely low transfection efficiency in the delivery of EGFP plasmids into 293T cells. The incorporation of hydrophobic monomers such as BMA and HMA into pDMAEMA improved its transfection efficiency, which is in accordance with previous reports on the construction of amphiphilic pDMAEMA for gene delivery.2,35,41,54 Interestingly, the statistical fluorocopolymer pDMAEMA42-st-pHFMA12 showed significantly improved EGFP expressions in the transfected cells compared to the non-fluorinated copolymers, while the block polymer pDMAEMA42-b-pHFMA13 showed poor transfection efficiency at all the tested N/P ratios. The result suggested that the monomer sequence on fluorocopolymers consisting of DMAEMA and HFMA plays a critical role in gene delivery. This situation is very similar to PCL-b-P(GMA-TEPA)-st-P(OEGMA), a block-statistical copolymer, which is much more efficient than the triblock copolymers with a similar chemical composition.31 The cells transfected by pDMAEMA42-st-pHFMA12 showed compatible fluorescence intensity with those by Lipo 2000, a commercial transfection reagent (Fig. 4b). It is reported that fluorous ligands on polymers enhance the serum-resistance of polymers in gene delivery,1,12,15 and we therefore tested the efficiencies of pDMAEMA42-st-pHFMA12 (N/P ratio of 8[thin space (1/6-em)]:[thin space (1/6-em)]1) and Lipo 2000 in the presence of 10–50% serum. As shown in Fig. 4c, both the statistical fluorocopolymer and Lipo 2000 showed decreased EGFP expressions with increasing serum concentration; however, the fluorocopolymer showed much better serum tolerance than Lipo 2000, especially in the range of 20–50% serum. This result confirmed the beneficial effect of fluorous ligands to improve the serum tolerance of cationic polymers.10


image file: c8tb01470a-f4.tif
Fig. 4 EGFP transfection efficiency of the polymers on 293T cells for 48 h. (a) Fluorescence microscopy images of the cells treated with the polymer nanomicelle/EGFP complexes at an N/P ratio of 8[thin space (1/6-em)]:[thin space (1/6-em)]1 for 48 h. Lipo 2000 was used as a positive control at its optimal dose. The scale bar is 200 μm. (b) Mean fluorescence intensity and EGFP positive cells of the transfected cells. The N/P ratios for polymers are 6[thin space (1/6-em)]:[thin space (1/6-em)]1, 8[thin space (1/6-em)]:[thin space (1/6-em)]1, 10[thin space (1/6-em)]:[thin space (1/6-em)]1, and 12[thin space (1/6-em)]:[thin space (1/6-em)]1, respectively. (c) Transfection of pDMAEMA42-st-pHFMA12 (N/P = 8[thin space (1/6-em)]:[thin space (1/6-em)]1) and Lipo 2000 in the presence of 10–50% serum. The EGFP positive cells by pDMAEMA42-st-pHFMA12 and Lipo 2000 at 0% FBS were normalized to 100%. The data were analyzed using Student's t-test, one-tailed. *p < 0.05 and **p < 0.01, respectively.

We further tested the efficiency of pDMAEMA copolymers on 293T cells using the luciferase reporter gene. As shown in Fig. 5a, pDMAEMA42-st-pHFMA12 was again the most efficient polymer among the five polymers. The luciferase expression in 293T cells delivered by pDMAEMA42-st-pHFMA12 was two or three orders of magnitude higher than those transfected with the block copolymer pDMAEMA42-b-pHFMA13. Similarly, pDMAEMA42-st-pHFMA12 was one or two orders of magnitude more efficient than the non-fluorinated copolymers pDMAEMA42-st-pBMA12 and pDMAEMA44-st-pHMA10, which confirmed the fluorous effect in the cytosolic delivery of biomacromolecules.1,4 In addition, pDMAEMA42-st-pHFMA12 showed high efficiency when transfecting luciferase DNA on HeLa cells (Fig. 5b). The cytotoxicity of pDMAEMA and the copolymers was also tested on 293T cells. The polymer concentration was equal to those of the complexes (N/P = 8[thin space (1/6-em)]:[thin space (1/6-em)]1) in the gene transfection experiments. It was reported that the contribution of a CF2 group to hydrophobicity was about 1.5 times that of a CH2 group,52,55 and thus the hydrophobicity of HFMA could be roughly equivalent to that of HMA. As shown in Fig. 5c and Fig. S11 (ESI), both pDMAEMA42-st-pHFMA12 and pDMAEMA42-b-pHFMA13 were less toxic than the non-fluorinated control pDMAEMA44-st-pHMA10 with equivalent hydrophobicity at different polymer concentrations. This phenomenon was also observed for PEI-based fluoroamphiphiles, which exhibited much lower cytotoxicity than hydrogenated amphiphiles due to decreased polymer absorption and retention on cell membranes.4 In addition, both fluorocopolymers showed lower toxicity than pDMAEMA42-st-pBMA12, pDMAEMA44-st-pHMA10 and Lipo 2000 in the presence of plasmid DNA (N/P ratio of 8[thin space (1/6-em)]:[thin space (1/6-em)]1). It is worth noting that the pDMAEMA42-st-pHFMA12/DNA complex was slightly more toxic than the block fluorocopolymer complex. This is due to the high transfection efficiency of the pDMAEMA42-st-pHFMA12/DNA complex, and overexpression of plasmid DNA in the cells may generate toxicity during gene transfection. These results confirmed that the incorporation of fluorous monomers into pDMAEMA does not cause additional toxicity compared to non-fluorinated pDMAEMA copolymers.


image file: c8tb01470a-f5.tif
Fig. 5 Luciferase gene transfection efficiency of the polymers on 293T (a) and HeLa (b) cells for 48 h. Cytotoxicity of polymers on 293T cells without plasmid (c) or with luciferase plasmid (d) for 48 h. The polymer concentration ranges from 5 μg mL−1 to 80 μg mL−1. The N/P ratio of the polymer/plasmid complexes was fixed at 8[thin space (1/6-em)]:[thin space (1/6-em)]1.

The cellular uptake, endocytosis mechanism and intracellular localization of the statistical fluorocopolymer pDMAEMA42-st-pHFMA12 were further investigated to explain its superior transfection efficiency to other polymers, especially the block fluorocopolymer pDMAEMA42-b-pHFMA13 with approximately equal chemical compositions. For the cellular uptake assay, luciferase plasmid was labeled with YOYO-1 before complexation with the polymers (N/P ratio fixed at 8[thin space (1/6-em)]:[thin space (1/6-em)]1). As shown in Fig. 6a, the internalized plasmid delivered by pDMAEMA42-st-pHFMA12 increase directly in proportion to the incubation time up to 48 h and was much higher than those delivered by other polymers at 48 h, while the cellular uptake of the pDMAEMA42-b-pHFMA13/DNA complexes is almost saturated after incubation for 12 h. It is known that the intracellular released plasmids will be degraded by nuclease abundant in the cytosol, and thus sustained DNA internalization is beneficial for efficient gene expression. This result may explain why the statistical fluorocopolymer showed much higher transfection efficiency than the block one, and suggests the beneficial effect of the statistical fluorocopolymer during endocytosis. We further investigated the specific endocytosis pathways of pDMAEMA42-st-pHFMA12 and pDMAEMA42-b-pHFMA13 using specific endocytosis inhibitors. As shown in Fig. 6b, both complexes were internalized by 293T cells via a macropinocytosis-mediated endocytosis rather than caveolae- and clathrin-dependent endocytosis. This endocytosis pathway is in accordance with our previous report on fluorinated dendrimers,1 and suggests that the monomer sequence on the fluorocopolymer does not influence its internalization pathway. We further investigated the localization of the pDMAEMA42-st-pHFMA12/DNA complex in the transfected cells. As shown in Fig. 6c and Fig. S12 (ESI), the plasmid DNA delivered by pDMAEMA42-st-pHFMA12 and pDMAEMA42-b-pHFMA13 nanomicelles was not co-localized with the acidic organelles stained with LysoTracker Red at 12 h, and more released plasmid DNA was observed in the cytosol with increasing incubation time, suggesting the successful escape of the complex from endosomes. This could be explained by the pH buffering capability of pDMAEMA as well as the role of fluorous ligands in facilitated endosomal escape.1,4 Taken together, the statistical fluorocopolymer showed more sustained DNA cellular uptake and much higher transfection efficiency compared to the block one.


image file: c8tb01470a-f6.tif
Fig. 6 Cellular uptake of the polymer nanomicelle/DNA complexes by 293T cells. (a) Quantitative determination of the cell uptake of YOYO-1 labeled complexes at 6, 12, 24, and 48 h, respectively. (b) Endocytic pathways of pDMAEMA42-st-pHFMA12 and pDMAEMA42-b-pHFMA13 complexes on 293T cells. Cytochalasin D, genistein, MβCD and chlorpromazine were used as specific endocytosis inhibitors. The YOYO-1 labeled complexes internalized by 293T cells were quantitatively determined using flow cytometry at 24 h. The data are analyzed using Student's t-test, ***p < 0.001. (c) Confocal images of 293T cells treated with the YOYO-1 labeled pDMAEMA42-st-pHFMA12 complex for 6 h and 12 h, respectively. The scale bar is 20 μm.

Conclusions

In summary, several fluorocopolymers were constructed using RAFT polymerization of DMAEMA and the fluorous monomer. The effect of the monomer sequence in the fluorocopolymers on gene transfection efficiency was investigated. The statistical copolymer pDMAEMA-st-pHFMA showed much higher efficiency than the block copolymer with a similar chemical component. Moreover, the statistical fluorocopolymer was much more efficient than non-fluorinated copolymers with a similar carbon number or hydrophobicity, which is attributed to the fluorous effect of fluoropolymers in the cytosolic delivery of biomacromolecules. The statistical fluorocopolymer showed compatible transfection efficiency with Lipo 2000, but better serum tolerance and lower cytotoxicity. The reason why the statistical fluorocopolymer showed more efficient and sustained DNA cell internalization than the block polymer needs further exploration. The results in this study are helpful for the development of fluoropolymers for efficient gene delivery.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We greatly appreciate the grants from the NSFC (21725402 and 21474030) and the SMSTC (17XD1401600) for this work.

Notes and references

  1. M. Wang, H. Liu, L. Li and Y. Cheng, Nat. Commun., 2014, 5, 3053 CrossRef PubMed.
  2. N. P. Truong, W. Gu, I. Prasadam, Z. Jia, R. Crawford, Y. Xiao and M. J. Monteiro, Nat. Commun., 2013, 4, 1902 CrossRef PubMed.
  3. J. Yang, Q. Zhang, H. Chang and Y. Cheng, Chem. Rev., 2015, 115, 5274–5300 CrossRef PubMed.
  4. Z. Zhang, W. Shen, J. Ling, Y. Yan, J. Hu and Y. Cheng, Nat. Commun., 2018, 9, 1377 CrossRef PubMed.
  5. F. Dai and W. Liu, Biomaterials, 2011, 32, 628–638 CrossRef PubMed.
  6. Y. Li, B. Xu, T. Bai and W. Liu, Biomaterials, 2015, 55, 12–23 CrossRef PubMed.
  7. F. Wang, Y. Wang, H. Wang, N. Shao, Y. Chen and Y. Cheng, Biomaterials, 2014, 35, 9187–9198 CrossRef PubMed.
  8. F. Wang, L. Deng, J. Hu and Y. Cheng, Bioconjugate Chem., 2016, 27, 638–646 CrossRef PubMed.
  9. H. Wang, Y. Wang, Y. Wang, J. Hu, T. Li, H. Liu, Q. Zhang and Y. Cheng, Angew. Chem., Int. Ed., 2015, 54, 11647–11651 CrossRef PubMed.
  10. Y. Cheng, Acta Polym. Sin., 2017, 1234–1245 Search PubMed.
  11. L. H. Wang, D. C. Wu, H. X. Xu and Y. Z. You, Angew. Chem., Int. Ed., 2016, 55, 755–759 CrossRef PubMed.
  12. L. Li, L. Song, X. Liu, X. Yang, X. Li, T. He, N. Wang, S. Yang, C. Yu, T. Yin, Y. Wen, Z. He, X. Wei, W. Su, Q. Wu, S. Yao, C. Gong and Y. Wei, ACS Nano, 2017, 11, 95–111 CrossRef PubMed.
  13. L. Li, L. Song, X. Yang, X. Li, Y. Wu, T. He, N. Wang, S. Yang, Y. Zeng, L. Yang, Q. Wu, Y. Wei and C. Gong, Biomaterials, 2016, 111, 124–137 CrossRef PubMed.
  14. X. Cai, R. Jin, J. Wang, D. Yue, Q. Jiang, Y. Wu and Z. Gu, ACS Appl. Mater. Interfaces, 2016, 8, 5821–5832 CrossRef PubMed.
  15. G. Chen, K. Wang, Y. Wang, P. Wu, M. Sun and D. Oupický, Adv. Healthcare Mater., 2018, 7, 1700978 CrossRef PubMed.
  16. G. Chen, K. Wang, Q. Hu, L. Ding, F. Yu, Z. Zhou, Y. Zhou, J. Li, M. Sun and D. Oupický, ACS Appl. Mater. Interfaces, 2017, 9, 4457–4466 CrossRef PubMed.
  17. X.-Q. Yu, Y.-P. Xiao, J. Zhang, Y.-H. Liu, Z. Huang, B. Wang and Y.-M. Zhang, J. Mater. Chem. B, 2017, 5, 8542–8553 RSC.
  18. M. E. Johnson, J. Shon, B. M. Guan, J. P. Patterson, N. J. Oldenhuis, A. C. Eldredge, N. C. Gianneschi and Z. Guan, Bioconjugate Chem., 2016, 27, 1784–1788 CrossRef PubMed.
  19. B. He, Y. Wang, N. Shao, H. Chang and Y. Cheng, Acta Biomater., 2015, 22, 111–119 CrossRef PubMed.
  20. H. Wang, J. Hu, X. Cai, J. Xiao and Y. Cheng, Polym. Chem., 2016, 7, 2319–2322 RSC.
  21. H. Liu, H. Chang, J. Lv, C. Jiang, Z. Li, F. Wang, H. Wang, M. Wang, C. Liu, X. Wang, N. Shao, B. He, W. Shen, Q. Zhang and Y. Cheng, Sci. Rep., 2016, 6, 25069 CrossRef PubMed.
  22. D. Gao, M. Xu, Z. Cao, J. Gao, Y. Chen, Y. Li, Z. Yang, X. Xie, Q. Jiang, W. Wang and J. Liu, ACS Appl. Mater. Interfaces, 2015, 7, 13524–13537 CrossRef PubMed.
  23. H. Liu, Y. Wang, M. Wang, J. Xiao and Y. Cheng, Biomaterials, 2014, 35, 5407–5413 CrossRef PubMed.
  24. M. Wang and Y. Cheng, Biomaterials, 2014, 35, 6603–6613 CrossRef PubMed.
  25. J. Kretzmann, D. Ho, C. W. Evans, J. Plani-Lam, B. García-Bloj, E. Mohamed, M. O’Mara, E. Ford, D. Tan, R. Lister, P. Blancafort, M. Norret and S. Iyer, Chem. Sci., 2017, 8, 2923–2930 RSC.
  26. J. Lv, H. Chang, Y. Wang, M. Wang, J. Xiao, Q. Zhang and Y. Cheng, J. Mater. Chem. B, 2015, 3, 642–650 RSC.
  27. Y. Wang, M. Wang, H. Chen, H. Liu, Q. Zhang and Y. Cheng, J. Mater. Chem. B, 2016, 4, 1354–1360 RSC.
  28. W. Shen, H. Wang, Y. Ling-hu, J. Lv, H. Chang and Y. Cheng, J. Mater. Chem. B, 2016, 4, 6468–6474 RSC.
  29. H. Chang, H. Wang, N. Shao, M. Wang, X. Wang and Y. Cheng, Bioconjugate Chem., 2014, 25, 342–350 CrossRef PubMed.
  30. M. Wang and Y. Cheng, Acta Biomater., 2016, 46, 204–210 CrossRef PubMed.
  31. H. Wei, L. R. Volpatti, D. L. Sellers, D. O. Maris, I. W. Andrews, A. S. Hemphill, L. W. Chan, D. S. H. Chu, P. J. Horner and S. H. Pun, Angew. Chem., Int. Ed., 2013, 52, 5377–5381 CrossRef PubMed.
  32. S. Han, Q. Cheng, Y. Wu, J. Zhou, X. Long, T. Wei, Y. Huang, S. Zheng, J. Zhang, L. Deng, X. Wang, X. J. Liang, H. Cao, Z. Liang and A. Dong, Biomaterials, 2015, 48, 45–55 CrossRef PubMed.
  33. C. Zhu, M. Zheng, F. Meng, F. M. Mickler, N. Ruthardt, X. Zhu and Z. Zhong, Biomacromolecules, 2012, 13, 769–778 CrossRef PubMed.
  34. Y. Cheng, H. Wei, J. K. Y. Tan, D. J. Peeler, D. O. Maris, D. L. Sellers, P. J. Horner and S. H. Pun, Small, 2016, 12, 2750–2758 CrossRef PubMed.
  35. Y. Cheng, D. L. Sellers, J. K. Y. Tan, D. J. Peeler, P. J. Horner and S. H. Pun, Biomaterials, 2017, 127, 89–96 CrossRef PubMed.
  36. P. Yan, R. Wang, N. Zhao, H. Zhao, D. F. Chen and F. J. Xu, Nanoscale, 2015, 7, 5281–5291 RSC.
  37. X. Yang, N. Zhao and F. J. Xu, Nanoscale, 2014, 6, 6141 RSC.
  38. J. J. Nie, W. Zhao, H. Hu, B. Yu and F. J. Xu, ACS Appl. Mater. Interfaces, 2016, 8, 8376–8385 CrossRef PubMed.
  39. X. B. Dou, Y. Hu, N. N. Zhao and F. J. Xu, Biomaterials, 2014, 35, 3015–3026 CrossRef PubMed.
  40. X. Yue, Y. Qiao, N. Qiao, S. Guo, J. Xing, L. Deng, J. Xu and A. Dong, Biomacromolecules, 2010, 11, 2306–2312 CrossRef PubMed.
  41. S. Guo, Y. Huang, W. Zhang, W. Wang, T. Wei, D. Lin, J. Xing, L. Deng, Q. Du, Z. Liang, X. J. Liang and A. Dong, Biomaterials, 2011, 32, 4283–4292 CrossRef PubMed.
  42. T. Zhao, H. Zhang, B. Newland, A. Aied, D. Zhou and W. Wang, Angew. Chem., Int. Ed., 2014, 53, 6095–6100 CrossRef PubMed.
  43. X. Qian, L. Long, Z. Shi, C. Liu, M. Qiu, J. Sheng, P. Pu, X. Yuan, Y. Ren and C. Kang, Biomaterials, 2014, 35, 2322–2335 CrossRef PubMed.
  44. M. Krishnamoorthy, D. Li, A. S. Sharili, T. Gulin-Sarfraz, J. M. Rosenholm and J. E. Gautrot, Biomacromolecules, 2017, 18, 4121–4132 CrossRef PubMed.
  45. A. P. Majewski, U. Stahlschmidt, V. Jéroîme, R. Freitag, A. H. E. Müller and H. Schmalz, Biomacromolecules, 2013, 14, 3081–3090 CrossRef PubMed.
  46. J. Pan, Y. Yuan, H. Wang, F. Liu, X. Xiong, H. Chen and L. Yuan, ACS Appl. Mater. Interfaces, 2016, 8, 15138–15144 CrossRef PubMed.
  47. D. Lin, Y. Huang, Q. Jiang, W. Zhang, X. Yue, S. Guo, P. Xiao, Q. Du, J. Xing, L. Deng, Z. Liang and A. Dong, Biomaterials, 2011, 32, 8730–8742 CrossRef PubMed.
  48. M. Omedes Pujol, D. J. L. Coleman, C. D. Allen, O. Heidenreich and D. A. Fulton, J. Controlled Release, 2013, 172, 939–945 CrossRef PubMed.
  49. D. J. Gary, H. Lee, R. Sharma, J. S. Lee, Y. Kim, Z. Y. Cui, D. Jia, V. D. Bowman, P. R. Chipman, L. Wan, Y. Zou, G. Mao, K. Park, B. S. Herbert, S. F. Konieczny and Y. Y. Won, ACS Nano, 2011, 5, 3493–3505 CrossRef PubMed.
  50. H. Yu, Y. Zou, Y. Wang, X. Huang, G. Huang, B. D. Sumer, D. A. Boothman and J. Gao, ACS Nano, 2011, 5, 9246–9255 CrossRef PubMed.
  51. Ö. Topel, B. A. Çakir, L. Budama and N. Hoda, J. Mol. Liq., 2013, 177, 40–43 CrossRef.
  52. M. P. Krafft, Adv. Drug Delivery Rev., 2001, 47, 209–228 CrossRef PubMed.
  53. Z. Gu, J. Cheng, M. Zhang, J. He and P. Ni, Chin. J. Polym. Sci., 2017, 35, 1061–1072 CrossRef.
  54. Z. Zha, J. Li and Z. Ge, ACS Macro Lett., 2015, 4, 1123–1127 CrossRef.
  55. M. C. Z. Kasuya, S. Nakano, R. Katayama and K. Hatanaka, J. Fluorine Chem., 2011, 132, 202–206 CrossRef.

Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c8tb01470a

This journal is © The Royal Society of Chemistry 2018