Preparation of well-defined brush-like block copolymers for gene delivery applications under biorelevant reaction conditions

Colloids and Surfaces B: Biointerfaces 169 (2018) 107–117

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Preparation of well-defined brush-like block copolymers for gene delivery applications under biorelevant reaction conditions Joana R. Góis a , Fábio Reis a , Ana M. Almeida b , Patrícia Pereira b , Fani Sousa b , Arménio C. Serra a , Jorge F.J. Coelho a,∗ a b

CEMMPRE, Department of Chemical Engineering, University of Coimbra, Polo II, Rua Sílvio Lima, 3030-790, Coimbra, Portugal CICS-UBI, Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506, Covilhã, Portugal

a r t i c l e

i n f o

Article history: Received 5 February 2018 Received in revised form 10 April 2018 Accepted 1 May 2018 Available online 3 May 2018 Keywords: SARA ATRP Brush-like block copolymers Gene delivery

a b s t r a c t Well-defined oligo(ethylene glycol) methyl ether methacrylate (OEOMA) based block copolymers with cationic segments composed by N,N-(dimethylamino) ethyl methacrylate (DMAEMA) and/or 2(diisopropylamino) ethyl methacrylate (DPA) were developed under biorelevant reaction conditions. These brush-type copolymers were synthesized through supplemental activator and reducing agent (SARA) atom transfer radical polymerization (ATRP) using sodium dithionite as SARA agent. The synthesis was carried out using an eco-friendly solvent mixture, very low copper catalyst concentration, and mild reaction conditions. The structure of the block copolymers was characterized by size exclusion chromatography (SEC) analysis and 1 H nuclear magnetic resonance (NMR) spectroscopy. The pH-dependent protonation of these copolymers enables the efficient complexation with plasmid DNA (pDNA), yielding polyplexes with sizes ranging from 200 up to 700 nm, depending on the molecular weight of the copolymers, composition and concentration used. Agarose gel electrophoresis confirmed the successful pDNA encapsulation. No cytotoxicity effect was observed, even for N/P ratios higher than 50, for human fibroblasts and cervical cancer cell lines cells. The in vitro cellular uptake experiments demonstrated that the pDNA-loaded block copolymers were efficiently delivered into nucleus of cervical cancer cells. The polymerization approach, the unique structure of the block copolymers and the efficient DNA encapsulation presented can open new avenues for development of efficient tailor made gene delivery systems under biorelevant conditions. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Gene therapy is a promising pathway for the treatment of several acquired and inherited genetic diseases, which nowadays still remain impossible to cure. Indeed, gene therapy studies are mostly designed towards cancer research, representing approximately 65% of gene therapy clinical trials worldwide [1]. In particular, tumor suppressor genes, such as p53, are widely explored in gene therapy for cancer treatment [1]. The premise for this approach relies on the fact that cancerous cells lack suitable cell damage regulation, enabling the progression of tumor growth. By inserting into the cells and overexpressing a tumor suppressor gene like p53, it is expected the re-instatement of the mechanisms involved in cancer cell apoptosis [2].

∗ Corresponding author. E-mail address: [email protected] (J.F.J. Coelho). https://doi.org/10.1016/j.colsurfb.2018.05.004 0927-7765/© 2018 Elsevier B.V. All rights reserved.

In the past decades, non-viral vectors based on plasmid DNA (pDNA) have become very popular in gene therapy research, because these type of vectors are able to carry larger DNA information, have a simple and low-cost manufacture as well as very low toxicity [3–5]. However, the delivery of pDNA molecules into eukaryotic cells is currently hampered by several hurdles that need to be overcome such as: molecular instability in the blood stream; lack of cell targeting; insufficient cellular uptake; endosomal degradation; immune system activation; among others [6]. Regarding these issues, important research efforts have been made to improve the safety and the efficiency of the gene delivery strategies. Cationic polymers are undeniably the most studied structures for DNA delivery because their positive charges can complex with the negatively charged phosphate groups of pDNA by electrostatic interactions, yielding the so-called polyplexes [6]. The polymer composition, molecular weight (MW), architecture as well as polymer concentration [7] have a major influence on both cytotoxicity and transfection efficiency of the non-viral gene vectors. In the last decades, the development of reversible deactivation radical poly-

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merization (RDRP) methods have turned possible the synthesis of a wide range of well-defined polymers, with controlled and narrow MW distributions, complex architectures and high chain end functionality [8,9]. Amongst RDRP techniques, atom transfer radical polymerization (ATRP) stands out as a very versatile method, as it can be applied to a wide range of monomers under mild reaction conditions [10]. More recently, in pursuing more eco-friendly and less toxic polymerization systems, several ATRP variations have also been proposed [11–15]. Among those, SARA ATRP is particularly effective to synthesize copolymers under biorelevant reaction conditions [16–19]. Weak cationic polyelectrolytes such as DMAEMA have been reported as promising vectors for gene therapy [20]. The tertiary amine side groups are able to efficiently condense the genetic material into nanoparticles [21]. Polymers based on DMAMEA present a transfection efficiency comparable with the “gold standard”, branched PEI 25 kDa, without inducing toxicity [22]. Different architectures such as linear copolymers [23–25], star-shaped [26], comb-like [27], branched and other structures [28], have been explored using ATRP techniques. Poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) is another tertiary amine methacrylate with a hydrophilic/hydrophobic transition at pH around 6.2 [29,30], turning this polymer very attractive for biomedical applications. Most, of the reported studies using DPA-based copolymers as nonviral gene delivery systems involve the preparation of micellar nanoparticles [31–33] and pH-sensitive polymersomes for DNA encapsulation [34–36]. Often, the use of cationic polymers is associated to several adverse effects that can be mitigated through the functionalization with poly(ethylene glycol) (PEG) [20,37]. This water soluble polymer provides a steric effect to the polyplex with several benefits; the protection of the nanocarrier payload; prevents serum-induced aggregation; shields the nanocarrier from being recognized from the immune system; reduces the polyplex toxicity; and enhances their in vivo stability [38,39]. Recently, the controlled polymerization of PEG based monomers, such as oligo(ethylene glycol) methyl ether methacrylate (OEOMA) proved to be a very efficient tool for polymer PEGylation.[20,40] The architecture and MW of these PEG based monomers influences the size and zeta potential of the PDMAEMA based polyplexes as well as the cellular uptake, transfection efficiency and cell viability [23,41–43]. Stolnik group demonstrated that ‘bottle-brush’ type PDMAEMAb-POEOMA formed compact complexes with phosphorothioate antisense oligonucleotide, with a long term colloidal stability and high cellular uptake, in contrast, to DMAEMA homopolymer and comb-type statistical DMAEMA-co-POEOMA copolymers [42,43]. A similar result was reported by Rudolph and co-workers that synthesized random OEOMA-co-DMAEMA copolymers through normal ATRP and studied the influence of the macromolecular structure of OEOMA block in POEOMA-co-PDMAEMA copolymers on their ability to condense pDNA [23]. The OEOMA segment prevented gene vector aggregation and did not induce cytotoxicity even at higher pDNA concentrations, but a poor cellular transfection was observed. Recently, the conjugation of polycations with different pKa values was proposed as an alternative to enhance the transfection efficiency of POEOMA block copolymers [44,45]. Despite the encouraging results of using these non-linear PEG-based monomers in the development of non-viral pDNA carriers, the available literature reports are still scarce. Moreover, all reported polymers were synthesized using multi step laborious and unattractive synthetic routes, such as the use of toxic organic solvents (e.g. toluene or tetrahydrofuran) and high concentrations of metal complexes (classical ATRP) [46–48]. In this work, we present an efficient SARA ATRP method to afford well-defined block copolymers composed by OEOMA and two distinct stimuli-responsive tertiary amine methacrylate monomers,

DMAEMA and PDA, to be used as gene delivery vectors. The ability of such block copolymers to condense pDNA is evaluated and the physico-chemical properties of the resultant polyplexes are described. In vitro experiments are also presented to evaluate the cytotoxicity of the polyplexes into two distinct cell lines (human fibroblasts and cervical cancer cells), and their ability to cellular internalization. 2. Experimental section 2.1. Materials Sodium dithionite (Na2 S2 O4 , 85%, ACROS Organics), copper(II) bromide (CuBr2 , 99.9%, Aldrich), ethyl ␣-bromophenyl acetate (EBPA, 97%, Alfa Aesar), isopropanol (IPA, ACS grade, Fisher Scientific), tetrahydrofuran (THF, ACS grade, Fisher Scientific), deuterated chloroform (CDCl3 ) (99.8%, Cambridge Isotope Laboratories), deuterium oxide (D2 O) (99.9%, Aldrich) and sodium hydroxide (NaOH) were used as received. 2-(Diisopropylamino)ethyl methacrylate (DPA, 97%, Scientific Polymer Products Inc.), oligo(ethylene oxide) methyl ether methacrylate (OEOMA, 99%, average molecular weight 475, Aldrich) and N,N-(dimethylamino) ethyl methacrylate (DMAEMA, 98%, Aldrich) were passed over a column of basic alumina to remove the inhibitor prior to use. Tris(pyridin-2-ylmethyl)amine (TPMA) was synthesized as reported in the literature [49]. Hyper Ladder I (Bioline, London, UK) was used as DNA molecular weight marker. GreenSafe Premium and NZY Maxiprep Kit were purchased from NZYTech ® (Lisbon, Portugal). Purified water (Milli-Q , Millipore, resistivity >18 M cm) was obtained by reverse osmosis. 2.2. Methods A KDS Scientific, Legato 101 syringe pump was used for continuous feeding polymerizations. High performance size exclusion chromatography (HPSEC) was performed for POEOMA homopolymer samples, using a Viscotek (ViscotekTDAmax) with a differential viscometer (DV), right-angle laser-light scattering (RALLS, Viscotek), and refractive index (RI) detectors. The column set was composed by a PL 10 ␮m guard column followed by one MIXED-E PLgel column and one MIXED-C PLgel column. Previously filtered THF was used as an eluent at a flow rate of 1.0 mL/min at 30 ◦ C. The samples were filtered through a polytetrafluoroethylene (PTFE) membrane with 0.2 ␮m pore before injection and the system was calibrated with narrow PS standards. The dn/dc of POEOMA in THF at 30 ◦ C was determined as 0.072 (for ␭ = 670 nm) using a Rudolph Research J357 Automatic Refractometer (J357-NDS-670-CC). The Mn SEC and Ð of the synthesized polymers were determined by using a multidetectors calibration (OmniSEC software version: 5.0). For the block copolymers, the SEC analysis was performed using a system equipped with an online degasser, a refractive index (RI) detector and a set of columns: Shodex OHpak SB-G guard column, OHpak SB-802.5HQ and OHpak SB-804HQ columns. The polymers were eluted at a flow rate of 0.5 mL min−1 with 0.1 M Na2 SO4 (aq) −1 wt% acetic acid −0.02% NaN3 at 40 ◦ C. Before injection (50 ␮L) the samples were filtered through a polyester membrane with 0.45 ␮m pore. The system was calibrated with narrow Ð (Mw /Mn ) PEG standards. The number average molecular weight (Mn SEC ) and Ð of the synthesized polymers were determined by conventional calibration using Clarity software version 2.8.2.648. 1 H nuclear magnetic resonance (NMR) spectra of the reaction mixture and pure copolymers samples were recorded on a Bruker Avance III 400 MHz spectrometer, with a 5 mm TXI triple resonance detection probe, in CDCl3 with tetramethylsilane (TMS) as an inter-

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nal standard or D2 O. The conversion of monomers and copolymers composition was determined by integration of characteristic signals using MestReNova software version: 6.0.2-5475. The residual copper content in the block copolymers was evaluated by atomic absorption spectroscopy (AAS) using a Perkin Elmer equipment (model 3300, USA). The block copolymers were dissolved in a 0.1 M HCl solution (5.0 mg/mL) and the pH of the solution was adjusted to 5. At least five measurements were taken for each sample.

2.3. Procedures 2.3.1. Typical procedure for the SARA ATRP of POEOMA500 (DP = 50) catalysed by [EBPA]/[Na2 S2 O4 ]/[CuBr2 ]/[TPMA] = 1/0.25/0.01/0.02 in isopropanol/water POEOMA was synthesized by SARA ATRP using Na2 S2 O4 , following procedures adapted from the literature. [50,51] Briefly, 161 ␮L of a stock solution of CuBr2 /TPMA (0.27 mg, 1.20 ␮mol (CuBr2 ), 0.70 mg, 2.40 ␮mol (TPMA)) in water was placed a Schlenk tube reactor that was sealed by using a rubber septa and bubbled with nitrogen. Na2 S2 O4 (6.20 mg, 35.29 ␮mol) and a mixture of OEOMA500 (3.0 g, 6.0 mmol) and EBPA (29.17 mg, 120.0 ␮mol) in IPA (3.061 mL) (previously bubbled with nitrogen for about 15 min) were added to the reactor and frozen in liquid nitrogen. The reaction mixture was deoxygenated by three freeze–pump–thaw cycles and purged with nitrogen. The reactor was placed in an oil bath at 40 ◦ C with stirring (600 rpm) and the reaction proceeded for a pre-stablished time interval. A sample from the reaction mixture was analyzed by 1 H NMR spectroscopy in order to determine the monomer conversion, and by SEC to determine MW and Ð of the polymers. The reaction mixture was dialyzed against distilled water and the pure POEOMA was obtained by freeze drying.

2.3.2. Typical procedure for the synthesis of POEOMA-b-PDMAEMA block copolymers The POEOMA-b-PDMAEMA copolymers were prepared by SARA ATRP using POEOMA-Br as the macromolecular initiator in a solution of IPA/water = 95/5 (v/v). In a typical reaction, 4.58 mL of a POEOMA-Br solution (253 mg, 21.20 ␮mol) (Mn SEC = 11.96 × 103 g mol−1 , Ð= 1.17) in IPA (1.833 mL), DMAEMA (0.50 g, 3.18 mmol) and 50 ␮L of degassed stock solution of Na2 S2 O4 (0.54 mg, 2.64 ␮mol) in water were placed in a Schlenk tube reactor. Next, 100 ␮L of a stock solution of CuBr2 /TPMA (0.24 mg, 1.06 ␮mol (CuBr2 ), 0.50 mg, 1.70 ␮mol (TPMA)) in water was added to the reactor, which was sealed and frozen in liquid nitrogen. The Schlenk tube reactor containing the reaction mixture was deoxygenated with four freeze−vacuum−thaw cycles and purged with nitrogen. An aqueous solution of Na2 S2 O4 (36 mM) in water (previously bubbled with nitrogen for about 15 min) was slowly fed into the reaction mixture using a syringe pump at a feed rate of 120 nL/min. The polymerization proceeded for 12 h at 40 ◦ C. The resultant solution was dialyzed against distilled water and then freeze-dried to yield POEOMA-b-PDMAEMA.

2.3.3. Polymer buffering capacity Potentiometric titration curves of polymers were obtained in ® Milli-Q purified water. Samples of the pure copolymers (10 mg) were dissolved in 0.1 M HCl solution (5 mL) (2.0 mg/mL). The temperature was adjusted to 37 ◦ C using a water bath. The solution was then titrated by adding 100 ␮L aliquots of 0.02 M NaOH under stirring. The pH increase, in the range of 2–11, was monitored as a function of the total added volume of NaOH (VNaOH ). The pH values were measured using a Jenway 3510 pH meter (Stone, Staffs, UK).

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Titration curve of the purified water (without polymer) was used as background control. 2.3.4. Production and pre-purification of plasmid DNA The plasmid pcDNA3-FLAG-p53 was amplified through a cell culture of Escherichia coli (E. coli) DH5␣. Bacterial growth was carried out at 37 ◦ C, under 250 rpm shaking, by using shake flasks containing 0.25 L of Terrific Broth medium (12 g/L tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 0.017 M KH2 PO4 , 0.072 M K2 HPO4 ) supplemented with 30 ␮g/mL of ampicillin. The bacterial growth was suspended at the late log phase (OD600 ± 9). Cells were subsequently recovered by centrifugation and pellets were stored at −20 ◦ C. Plasmid DNA was pre-purified by using the NZYTech Maxiprep kit, according to the manufacturer’s instructions. Briefly, after disrupting the cells membrane and recovering the intracellular bacterial content, the pDNA was bound to the NZYTech anion-exchange resin under appropriate low-salt and pH conditions. Afterwards, the impurities were washed out by ionic strength increase, followed by pDNA elution with high salt concentration. The resulting pDNA preparations were dissolved in 10 mM TrisHCl (pH 8.0) buffer and their concentrations were determined by UV absorbance at 260 nm, before storing the samples at −80 ◦ C. 2.3.5. Preparation of plasmid DNA-copolymer complexes To identify the most suitable concentration for pDNA encapsulation, five different polymer concentrations were used (0.01, 0.05, 0.1, 0.5 and 1.0 mg/mL). For each polymer, a stock solution was prepared in sodium acetate buffer (0.1 M sodium acetate/0.1 M acetic acid, pH 4.5) with a concentration of 1 mg/mL, followed by subsequent dilutions with the same buffer. pDNA stock solution was also prepared in sodium acetate buffer, up to a final concentration of 20 ␮g/mL. To encapsulate the pDNA, a fixed volume of the polymer solution (100 ␮L) of variable concentration was added dropwise to the pDNA solution (400 ␮L), throughout 60 s in a vortex. The pDNA concentration was kept constant (20 ␮g/mL) in all the pDNAcopolymer complexes characterization techniques. Afterwards, all formulated particles were equilibrated at room temperature for 15 min, followed by a 20 min centrifugation at 12,000g, for pDNAcopolymer complexes recovery. 2.3.6. Agarose gel retardation assay Each pDNA-copolymer system was prepared in different concentrations, as previously described. The resulting samples (20 ␮L) were analyzed by horizontal electrophoresis using 1% agarose gels supplemented with Greensafe (0.5 ␮g/mL), for nucleic acid staining. The electrophoresis was carried out in Tris-Acetic Acid (TAE) buffer (40 mM Tris base, 20 mM Acetic acid and 1 mM EDTA, pH 8.0) and run at 150 V for 40 min. Hereafter, the resulting agarose gel was visualized under ultraviolet light. 2.3.7. Encapsulation efficiency To determine the encapsulation efficiency (EE) of the different polymers, the free pDNA concentration in the supernatant resulting from particle centrifugation (15,000 g, 20 min, 4 ◦ C) was quantified. The amount of unbound pDNA was determined by UV absorbance at 260 nm. Three repetitions were performed for each condition. Thus, EE was calculated as follows: EE (%) = [(Total pDNA amount − pDNA supernatant amount)/Total pDNA amount] ∗ 100

2.3.8. Particle size and zeta potential measurements For particle size assessment, the mean particle diameter of the pDNA-copolymer systems and the polydispersity index were deter-

6.40 0.38 0.62

f

e

c

d

a

R3

b

a

POEOMA23 -b-(PDMAEMA18 -co-PDPA20 )

POEOMA26 -b-(PDMAEMA68 -co-PDPA54 )a R2

The copolymers were synthesized via SARA ATRP mediated by Na2 S2 O4 using a pre-synthesized POEOMA-Br macroinitiator. The subscripts indicate the mean degrees of polymerization (DP) of each block determined by 1 H NMR analysis of pure polymer samples. Determined by SEC. Residual copper content determined by AAS (mg Cu/mg Polymer). Molar fraction of the cationic segment calculated from the 1 H NMR spectrum of the purified copolymers. Volume fraction of the cationic segment, calculated from the 1 H NMR spectrum of the purified copolymers.

– 1.57 52.3 22.54

6.39 0.63 0.83 1.29 44.80 28.38

– 1.08 22.3 12.54 PDMAEMA39 -co-PDPA29 R1

96 97 58 25 81 37

34 66 90 110 150 POEOMA16 -b-PDMAEMA26 a POEOMA41 -b-PDMAEMA50 a POEOMA26 -b-PDMAEMA64 a POEOMA26 -b-PDMAEMA91 a POEOMA23 -b-PDMAEMA68 a B2.1 B2.2 B2.3 B2.4 B2.5

40 30 115 85 40 30

12.81 24.00 21.14 23.91 22.34

23.61 37.29 28.70 30.37 25.00

1.14 1.21 1.22 1.29 1.18

1.1 × 10−5 – – – –

2.6 × 10−5

0.53 0.66 0.31 0.44 0.63  (DMAEMA)f 0.34 0.28 0.44 0.52 0.48 (DMAEMA+DPA )f – 0.73 0.82 0.51 0.64 0.80 FDMAEMAexp e 0.62 0.55 0.71 0.78 0.75 F(DMAEMA+DPA)exp e 1 – 2.2 × 10−5 – – – 1.17 1.35 1.22 1.29 1.31 27.36 36.00 25.60 33.05 35.64 55 100 50 217 150 POEOMA16 -b-PDPA43 a POEOMA22 -b-PDPA98 a POEOMA32 -b-PDPA34 a POEOMA50 -b-PDPA90 a POEOMA23 -b-PDPA90 a B1.1 B1.2 B1.3 B1.4 B1.5

90 33 55 61 44

Mn th × 10−3 b Conv. (%)b DP Copolymerb

Table 1 Characterization of the block copolymers.

2.3.11. Cell viability studies To assess the cytotoxic effect of the different copolymers, cell ® viability studies were carried out with Cell Titer 96 AQueous NonRadioactive Cell Proliferation Assay (Promega). For this experiment, human fibroblasts and cervical cancer cells (HeLa) were cultured at 37 ◦ C, in a humidified atmosphere containing 5% CO2 . Three different passages of each cell line were seeded in a 96-well plate, with a density of 1 × 104 per mL. After 24 h, the cell culture medium was replaced by serum-free culture medium. Thereafter, cells were transfected with the pDNA-copolymer systems by using the two concentrations that revealed to be more suitable in the previous characterization studies for each polymer. Cells were incubated for

Mn SEC × 10−3 c

2.3.10. In vitro cellular uptake – cell live imaging To investigate the cellular uptake, pDNA sample was labeled with fluorescein isothiocyanate isomer I (FITC), accordingly to the manufacturer’s instructions. Briefly, FITC was prepared with sterile anhydrous dimethylsulfoxide (DMSO) and added to a reaction mixture containing labeling buffer (0.1 M Sodium Tetraborate, pH 8.5), deionized water and pDNA (20 ␮g/mL), followed by a 4 h incubation-period at room temperature in a shaker oscillating at low speed. Then, the final product was incubated overnight with 3 M NaCl and absolute ice-cold ethanol at −20 ◦ C. Excess FITC was removed by centrifugation at 12,000g for 30 min at 4 ◦ C. After centrifuging, the pellet was washed with 75% ethanol twice, followed by a 5 min centrifugation at 12,000g (4 ◦ C). The resulting FITC-labeled pDNA was encapsulated with 0.1 mg/mL of different polymers [POEOMA50 -b-PDPA90 (B1.4), POEOMA26 b-PDMAEMA91 (B2.4) and POEOMA26 -b-(PDMAEMA68 -co-PDPA54 ) (R2)], as mentioned above. HeLa cells were seeded at a density of 1 × 104 cells/cm2 in ␮-slide 8 well flat bottom imaging plates (Ibidi GmbH, Germany). In the following day, the medium was replaced by fresh serum-free medium and cells were stained with ® Hoechst 33342 nuclear probe for 20 min. Subsequently, the cells were transfected with the complexes prepared with FITC-labeled pDNA in serum-free medium during 4 h and real live transfection was visualized using a Zeiss LSM 710 confocal laser scanning microscope (CLSM; Carl Zeiss SMT Inc., USA) equipped with a planeapocromat 63 × /DIC objective and processed in Zeiss Zen (SP2, 2010) and Imaris software (Bitplane, Switzerland).

14.34 27.68 23.47 45.83 30.52

Ðc

Cures d

FDPAexp e

2.3.9. Stability of plasmid DNA-copolymers complexes All the prepared pDNA-copolymers complexes were ressuspended in PBS buffer, pH 7.4. The enzymatic digestion experiments were performed by incubation of the pDNA-copolymers complexes (100 ␮L) with: 1) DMEM high glucose medium supplemented with 10% FBS (3.9 mL) [52], or DNase I (2 U/␮L, in 5 mM MgCl2 , pH 7.4) [53]. The pDNA-copolymers complexes were incubated in a shaker water bath at 37 ◦ C for distinct periods: 0.5, 1, 2 and 4 h. Then, the pDNA-copolymers complexes were centrifuged at 20,000 g for 15 minutes, to pelletize the complexes, leaving the free pDNA in the supernatant, which was further collected and analyzed by 0.8% agarose gel electrophoresis, to examine the structural integrity of pDNA.

54 77 70 45 58

(DPA) f

pKa

mined by dynamic light scattering (DLS) using a Zetasizer Nano ZS particle analyzer (Malvern Instruments, Worcestershire, UK), equipped with a He-Ne laser. Particle diameters of the freshly prepared samples were measured at 25 ◦ C, with 173◦ scattering angle in fully automatic mode. On the other hand, the surface charges (zeta potential) of the prepared samples were determined in disposable capillary cells and computed by using Henry’s [F(Ka) 1.5], and Smoluchowsky models, using the equipment previously described, at 25 ◦ C. For both, the average values of size and zeta potential measurements were calculated with the data obtained from three repetitions ± SD.

5.92 5.83 5.99 5.94 5.89 pKa 6.32 6.31 6.43 6.22 6.51 pKa 6.77

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Sample

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111

Fig. 1. Chemical structure of the different block copolymers synthesized by SARA ATRP: POEOMA-b-PDMAEMA, POEOMA-b-PDPA and POEOMA-b-(PDMAEMA-co-PDPA).

48 h, followed by medium exchange. A mixture of MTS/phenazine metasulfate (PMS) was added to each well, and cells were subsequently incubated for 2 h at 37 ◦ C in a humidified atmosphere containing 5% CO2 , protected from light. Finally, the formazan produced by MTS metabolisation was analyzed by absorbance measurements performed in a microplate reader at 490 nm. Nontransfected cells incubated with absolute ethanol were used as a positive control for cytotoxicity levels. All experiments were repeated at least three times. 3. Results and discussion 3.1. Block copolymer synthesis and characterization The control of radical polymerization processes has opened unprecedented possibilities for developing tailor-made (co)polymers with pre-determined composition, MW and architecture that can be used as non-viral gene delivery systems [54–59]. During the last years, the atom transfer radical polymerization (ATRP) has evolved towards the development of new variations that enable the use of mild reaction conditions, non-toxic solvents and very low concentrations of metal catalyst [10]. Amongst the different ATRP variations, the SARA ATRP in the presence of inorganic sulfites, particularly sodium dithionite (Na2 S2 O4 ), proved to be a powerful route for different monomer families, allowing the straightforward synthesis of well-defined block copolymers [19,50,51,60]. Its convenient reaction features such as: the tiny catalyst concentrations required; the high monomer conversions obtained; the high chain-end fidelity; and the possibility to synthesize block copolymers with narrow MW distributions turns the SARA ATRP method very suitable for the preparation of (co)polymers for biomedical applications. In this work, copolymers composed by OEOMA and either DPA or DMAEMA, were synthesized by ATRP using Na2 S2 O4 as SARA agent in a mixture of isopropanol (IPA) and water. The structures of the block copolymers are shown in Fig. 1. The first segment, POEOMA, was obtained with different MWs and narrow MW distributions (Ð ≤ 1.2) and was used as macroinitiator for the polymerization of the second blocks through chain extension experiments. This strategy allowed the preparation of a library of well-defined block copolymers with distinct compositions and MWs (Fig. 1). The results revealed that the use of TPMA as ligand allowed a significant reduction of the copper concentrations required to achieve a controlled polymerization (up to 10 times less), when compared to other results using Me6 TREN [50]. Notably, when the comparison is done with other ATRP methods reported in the literature for the synthesis of similar block copolymers, our system uses 100 times less the ratio of the initiator to metal complex, and consequently fully transparent reaction solutions are obtained instead of common dark brown [46,47]. This issue is particular important in the synthesis of (co)polymers for biomedical field, since it reduces substantially the metal contamination in the final polymers and

Fig. 2. SEC chromatographs of the POEOMA before (left line) and after the chain extension with DPA to obtain POEOMA-b-PDPA (right line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

simplifies the purification procedures to ensure the safety of these materials. The success of the block copolymer synthesis was confirmed by a clear shift of the SEC traces of the POEOMA macroinitiator towards high MWs, as shown in Fig. 2 and through 1 H NMR analysis (Supporting Information (SI), Figs. S1–S4). The chemical structure of the copolymers was confirmed by 1 H NMR spectroscopy (SI, Figs. S1–S5) and are in agreement with the characteristic proton signals of POEOMA [61], PDPA [50] and PDMAEMA [62]. Despite the identification of the signals from the protons of the initiator fragment in the 1 H NMR spectrum of the POEOMA-Br macroinitiator (SI, Fig. S1) and in the block copolymers, the poor signal to noise ratio hampered the accurate calculation of Mn NMR . Therefore, the molecular weight (MW) of the copolymers was only determined by SEC analysis, by using a multidetector calibration system (Table 1). The value of Mn SEC from the macroinitiator POEOMA-Br was used as reference for the calculation of the length of OEOMA segment, since all the samples exhibited a very narrow MW distribution (Ð < 1.2). For the block copolymers, the molar ratios of monomers present in copolymers were calculated from the 1 H NMR spectra of the purified block copolymers (SI, Figs. S2–S5). The ratio of OEOMA to DPA was determined by comparing the signal integrals of the three protons of the characteristic methyl group from OEOMA monomer units at 3.38 ppm (SI, Fig. S3, j) and the two protons of DPA units at 3.02 ppm (SI, Fig. S3, x), while for POEOMA-b-PDMAEMA copolymers, the signal at 4.08 ppm (SI, Fig. S2, y, f), that correspond to the two protons from both OEOMA and DMAEMA units, was the selected signal. The molar fraction of the cationic monomers in the copolymer was varied from 0.5 up to 1 as shown in Table 1. The volume fraction (˚) of the cationic segments was determined from the ratio [DPDPA MDPA /(DPOEOMA MOEOMA + DPDPA MDPA )], with MDPA

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Fig. 3. Patterns of electrophoretic mobility of plasmid DNA (pDNA) in the presence of block copolymers at different polymer concentrations (0.01; 0.05; 0.1; 0.5 and 1.0 mg/mL). pDNA lane represents the mobility of free pDNA as a control.

and MOEOMA the molecular mass of DPA and OEOMA monomers respectively. The atomic absorption spectroscopy (AAS) was used to determine the residual copper contamination of the block copolymers synthesized (Table 1). The results reveal a very low remaining amount of copper, in the range of 1.1 × 10−5 –2.6 × 10−5 mg Cu/mg polymer, depending on the copolymer composition, which evidences the safety of the copolymers synthesized in respect to copper contamination. In contrast to the traditional ATRP methods, the SARA ATRP system presented here allows the reactions to be carried out with tiny amounts of metal catalyst [50] resulting in a neglected contamination of the final (co)polymer and simplifying the purification procedures.

3.2. Plasmid DNA-copolymer complexes – polyplex formation The tertiary amine groups of the DPA (or the DMAEMA) monomer units are responsible for the pH-dependent protonation of the block copolymers. For solutions with pH below the pKa of the copolymers, the tertiary amine becomes protonated allowing the water solubilization of the block copolymers. The potentiometric titration curves of the block copolymers in water, at 37 ◦ C, were obtained, by plotting the solution pH against the volume of added NaOH (VNaOH ). All the pH titration curves shown a plateau region associated with the polymer buffer effect. The degree of protonation (␣) was traced from the potentiometric titration curves (SI, Fig. S5) and the pKa values were determined for each sample (Table 1). As expected, the buffer capacity was more evidenced in the case of PDPA block copolymers, and their pKa values are slightly lower than the values for PDMAEMA polymers.[63] Furthermore, the close values of pKa obtained regardless the second segment of the POEOMA block copolymers, PDMAEMA or PDPA, suggest that the length of the cationic segment has no major effect on the final value of the copolymer pKa .

As evidenced by the pH titration curves, the solution pH strongly influences the degree of ionization of the polymers. Therefore, the pH of the encapsulation solution will influence the cationic charge density of the copolymers and thus affect the extent of complexation with anionic pDNA. [64] The correlation between the non-protonated species (A− ) and protonated species from (AH) is  given  the Henderson-Hasselback equation, pH = pKa + [A− ] log10 [AH]+[A− ] and allows the calculation of the percentage of protonation of each block copolymer in the encapsulation assay. Therefore, at the pH of the encapsulation assay (pH = 4.5), all the block copolymer samples were fully protonated (≥96% protonation). DMAEMA is a well-known polymer in gene delivery. Due to the lower pKa value of DPA, these copolymers are mostly used in the development of pH-responsive micellar vehicles for the delivery of hydrophobic drugs [30,65,66]. The literature available concerning DPA block copolymers for gene delivery applications is very scarce and it is mainly related with the development of pH-responsive nanocapsules or vesicles able to encapsulate siRNA into the internal aqueous phase [34,36,67] or micelleplexes for the co-delivery of siRNA and hydrophobic drugs [32,33]. In an endeavor to understand the potential of PDPA based block copolymers as non-viral vectors, the plasmid pcDNA-3-FLAG-p53 was condensed with the synthesized block copolymers into polyplexes. The pDNA-copolymer complexes were formed at different copolymer concentrations in sodium acetate buffer at pH = 4.5. The concentration of genetic material was kept constant throughout the assays (20 ␮g/mL). The value of the ratio between the copolymer nitrogen residues and nucleic acid phosphate groups (N/P ratio) was determined assuming that all the amine groups of the pH-responsive monomers were fully protonated (protonation ≥ 96%) at the pH of the assay (Table 2). The ability of copolymers to bind and condense the genetic material was confirmed by agarose gel electrophoresis. The images

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Table 2 Characterization of particle size and charge distribution for the different pDNA-copolymer complexes. Sample

Structure

[Polymer]a

N/P

Size (nm)

Zeta (mV)

EE (%)

B1.1

POEOMA16 -b-PDPA43

B1.2

POEOMA22 -b-PDPA98

B1.3

POEOMA32 -b-PDPA34

B1.4

POEOMA50 -b-PDPA90

0.1 0.5 0.1 0.5 0.5 1 0.5 1

10 50 12 58 24 48 32 65

662.83 ± 37.50 626.43 ± 100.37 291.77 ± 19.91 231.23 ± 2.27 216.60 ± 15.36 370.13 ± 17.44 518.83 ± 18.78 299.10 ± 23.35

33.00 ± 0.89 31.82 ± 2.60 30.95 ± 0.99 32.54 ± 2.29 25.79 ± 0.48 25.70 ± 2.64 31.63 ± 1.57 22.05 ± 2.03

87.10 ± 2.38 80.80 ± 1.68 90.03 ± 2.54 60.80 ± 3.52 61.61 ± 2.67 55.71 ± 2.95 64.29 ± 3.26 58.50 ± 4.62

B2.1

POEOMA16 -b-PDMAEMA26

B2.2

POEOMA41 -b-PDMAEMA50

B2.3

POEOMA26 -b-PDMAEMA64

B2.4

POEOMA26 -b-PDMAEMA91

0.1 0.5 0.1 0.5 0.1 0.5 0.1 0.5

7 34 7 34 10 50 14 71

491.47 ± 32.90 318.03 ± 2.90 – 484.53 ± 38.71 331.13 ± 31.42 288.77 ± 6.75 568.10 ± 30.12 370.90 ± 10.42

−14.26 ± 0.70 31.59 ± 1.63 28.20 ± 1.15 38.60 ± 0.96 24.33 ± 1.12 33.15 ± 1.65 29.81 ± 0.93 30.21 ± 0.90

55.69 ± 1.35 69.60 ± 1.61 87.85 ± 2.64 70.69 ± 1.07 84.44 ± 3.71 61.18 ± 2.32 80.85 ± 5.53 70.47 ± 5.70

R1

PDMAEMA39 -co-PDPA29

R2 R3

POEOMA23 -b-(PDMAEMA18 -co-PDPA20 ) POEOMA26 -b-(PDMAEMA68 -co-PDPA54 )

0.1 0.5 0.5 0.5 1

18 90 28 72 143

673.25 ± 138.38 413.03 ± 23.39 310.27 ± 3.46 282.40 ± 7.75 279.03 ± 1.96

40.8 ± 2.83 39.24 ± 0.91 33.18 ± 2.16 33.35 ± 3.41 26.10 ± 1.86

95.63 ± 0.55 87.89 ± 1.88 77.10 ± 5.88 66.18 ± 5.64 54.74 ± 3.36

a

[Polymer] in mg/mL.

of gels obtained using different concentrations of the different families of block copolymers synthesized are shown in Fig. 3 (the results from the agarose gel electrophoresis for the remaining block copolymers are presented in SI, Fig. S6). Naked pDNA was used as a control. As revealed by the agarose gel electrophoresis assay, all the copolymers investigated were able to form complexes with DNA from concentrations above 0.05 mg/mL (that corresponds to a NP ratio ≥ 5). Another pivotal parameter for gene delivery systems is their ability to complex as much genetic material as possible without inducing cytotoxicity. Therefore, the encapsulation efficiency (EE) of the different block copolymers was determined for different polymer concentrations (SI, Tables S1 and S2). All the copolymer samples were able to complex a significant content of genetic material (up to 84%, 90% and 95% in the case of POEOMA26 -b-PDMEMA64 , POEOMA22 -b-PDPA98 and POEOMA23 b-(PDMAEMA18 -co-PDPA20 ), respectively). However, no linear correlation was observed between polymer concentration and encapsulation efficiency. The EE results suggest that the ability to complex pDNA is related with an optimal polymer concentration (0.05–0.1 mg/mL) associated with an N/P ratio from 5 to 10 (Fig. 4). These results are in accordance with similar pDNA-copolymer complexes reported in the literature [41,44,68,69].

3.3. Size and zeta potential measurements The properties of the obtained pDNA-copolymer complexes were evaluated by DLS and zeta potential measurements (Table 2). A uniform and unimodal size distribution was observed for all the pDNA-copolymer complexes analyzed. The majority of the pDNA-copolymer complexes present a narrow size distribution with particle sizes from 216 nm up to 673 nm, depending on copolymer composition, molecular weight, length of the cationic segment and copolymer concentration. The DLS results suggest that high copolymer/DNA ratios are associated with smaller and more compact complexes, in accordance with other reports in the literature [20,42]. The hydrodynamic diameter of the obtained polyplexes is in the required size range for passive targeting tumor-targeted drug delivery (100–600 nm) [70]. Therefore, it is expected that these particles would be able to penetrate into the leaky vasculature at the tumor site and accumulate in the tumor microenviron-

Fig. 4. Plasmid DNA encapsulation efficiencies (%) of the studied polymers at different N/P ratios in sodium acetate buffer at pH 4.5 (the lines are guides for the eye).

ment. From all the polymers synthesized, the copolymers with controlled structure POEOMA26 -b-PDMAEMA64 , POEOMA32 -bPDPA34 , POEOMA22 -b-PDPA98 , POEOMA23 -b-(PDPA20 -co-PDPA18 ), POEOMA26 -b-(PDPA55 -co-PDPA68 ) are the most promising vectors taking into account the particle size. The reduced diameter facilitates the diffusion over the tumor vasculature and consequently enhances the access to the extravascular tumor tissue [71]. On the contrary, the large particle sizes observed for random PDMAEMA39 -co-PDPA29 copolymers are mostly associated with particle agglomeration due to the lack of any steric stabilization mechanism [37]. The surface charge of the nanocarrier is another important characteristic for gene delivery systems as it influences not only the interactions between the polyplex and cell membrane, but also the colloidal stability. The zeta potential results summarized in Table 2 demonstrate that the pDNA-copolymer complexes exhibit a strong positive charge on their surface, around 30 mV, for all copolymers tested which confirms the stability of the polyplex. It is important to notice that the pDNA concentration was kept constant throughout the assays. Therefore, the use of higher polymer concentrations

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Fig. 5. Agarose gel electrophoresis of: A) pDNA-copolymers complexes in cell culture medium supplemented with 10% FBS (Lane 1 represent DNA marker; Lanes 2 and 3 represent naked pDNA and naked pDNA after incubation in serum-containing medium, respectively), and B) pDNA-copolymers complexes incubated with DNase I. Incubations were carried out at 37 ◦ C for 0.5, 1, 2, and 4 h.

increases the N/P ratio and consequently increases the cationic surface charge density of the resultant nanocarriers. No correlation was observed between the surface charge of the polyplex and the MW of the cationic segment. 3.4. Stability of pDNA-copolymers complexes The ability of the pDNA-copolymers complexes to protect the condensed pDNA from serum nucleases mediated degradation was confirmed by incubation of the complexes in serum. The rationale behind this approach is that nanoparticles must

be able to resist to the capacity of serum nucleases to rapidly degrade naked pDNA. As shown in Fig. 5A, pDNA degradation was visualized by the appearance of pDNA bands in the lanes of the POEOMA26 -b-PDMAEMA91 (B2.4) complexes. On the other hand, when complexed with the copolymers [POEOMA50 -b-PDPA90 (B1.4) and POEOMA23 -b-(PDMAEMA18 -co-PDPA20 ) (R2)], there are no signs of pDNA degradation, even after incubation with 10% FBS for 4 h at 37 ◦ C. These observations contrast with those from the supercoiled isoform of the free pDNA, which was rapidly degraded after 0.5 h of incubation at 37 ◦ C. In general, the results showed that POEOMA50 -b-PDPA90 and POEOMA23 -b-(PDMAEMA18 -co-PDPA20 )

Fig. 6. Live cell imaging 3D and orthogonal view of HeLa cells transfected with different FITC-labeled pDNA-copolymer complexes. The cell nuclei are represented in blue staining and FITC-labeled pDNA-polymer complexes are represented in green staining. Cells were transfected with 0.1 mg/mL of POEOMA50 -b-PDPA90 (B1.4) (A), POEOMA26 b-PDMAEMA91 (B2.4) (B) and POEOMA26 -b-(PDMAEMA68 -co-PDPA54 ) (R2) (C). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 7. Effect of the polymer concentration and composition of the pDNA-polymer complexes on cell viability in human fibroblasts (a, b) and cervical cancer (HeLa) cell lines (c,d).

complexes are able to upkeep encapsulation of pDNA, whilst protecting it from nucleases present in serum-supplemented culture medium (Fig. 5A), suggesting that they are suitable delivery vehicles for in vitro gene delivery applications. Moreover, to ensure that the prepared copolymers can protect the encapsulated pDNA, the pDNA-copolymers complexes were also incubated with a specific nuclease (DNase I) that possesses high ability to induce DNA cleavage. In the case of POEOMA26 -bPDMAEMA91 (B2.4) and POEOMA50 -b-PDPA90 (B1.4) complexes, pDNA degradation was observed for all incubation times studied (Fig. 5B). However, it should be noted that pDNA encapsulated in the complexes produced with POEOMA23 -b-(PDMAEMA18 -coPDPA20 ) (R2) did not show any signs of degradation by DNase I (Fig. 5B). Therefore, the structure of pDNA seems to be complexed more loosely in POEOMA26 -b-PDMAEMA91 (B2.4) and POEOMA50 b-PDPA90 (B1.4) complexes and hence DNase partially degrades the pDNA, whereas POEOMA23 -b-(PDMAEMA18 -co-PDPA20 ) (R2) provide a complete and more prolonged protection (Fig. 5B). 3.5. Cellular uptake of pDNA-copolymers complexes Many factors are known to interfere with the cell entry of foreign complexes. As a matter of fact, cell membranes may exhibit different interactions with nanoparticles, since they can differ in membrane fluidity, type of receptors, receptor density, and recycling rate of receptors [72]. Taking this into account, it is imperative to assess if the system is able to successfully cross the cell membrane and reach the cell nucleus so that the pDNA can be correctly expressed. For this purpose, live cell imaging was per-

formed to follow the FITC-labeled pDNA entry into the nuclei of HeLa cells. In Fig. 6 is perceived the 3D and orthogonal view of fluorescence images obtained for HeLa cells transfection with POEOMA50 -b-PDPA90 (B1.4) (Fig. 6A), POEOMA26 -b-PDMAEMA91 (B2.4) (Fig. 6B) and POEOMA26 -b-(PDMAEMA68 -co-PDPA54 ) (R2) (Fig. 6C) copolymers, at 0.1 mg/mL complexed with FITC-labeled pDNA. Each sample was selected to represent the different copolymer families. As shown in all images, the FITC-labeled pDNA (green) was able to enter into the cell and reach the nucleus core (blue). Overall, this data suggests that all copolymer families studied in this work have the ability to cross the cell membrane and deliver the pDNA to the nucleus, for consequent pDNA expression. 3.6. Cell viability As already mentioned, the use of cationic polymers for gene delivery is often associated with high levels of cell toxicity. To evaluate the viability of the developed block copolymers as gene transfer agents, the cytotoxicity of the polyplexes was investigated on both human fibroblasts and cervical cancer cells (HeLa) (Fig. 7). No significant cytotoxicity was observed for all the copolymers tested in both cell lines. The results reveal high cellular viability for both human fibroblasts and HeLa cell lines, regardless the polymer composition, degree of cationic groups or molecular weight and cytotoxicity. Moreover, it should be highlighted the high levels of cellular viability observed even for high copolymer concentrations (i.e. 1000 and 500 ␮g/mL, that corresponds to N/P ratios of 10 and 50, respectively). Also, contrary to what might be expected, the absence of POEOMA segment in the polyplex formed with

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the copolymer PDMAEMA39 -co-PDPA29 does not contributed to an increase in cell toxicity, which may indicate a very effective pDNA complexation capacity, especially for higher copolymer concentrations (0.5 mg/mL, N/P ratio of 90). The reduced levels of cell toxicity observed might also be associated with the conditions adopted for the synthesis of the block copolymers that ensured minor copper contamination. 4. Conclusions In the present work, well defined brush-type copolymers of PDMAEMA, PDPA and PDMAEMA-co-PDPMA were successfully synthesized by SARA ATRP mediated by Na2 S2 O4 , under biorelevant reaction conditions. These block copolymers were evaluated as non-viral gene vectors. Complexation of these copolymers with pDNA was achieved, resulting in high encapsulation efficiencies of pDNA. In general, suitable sizes and surface charge densities were found for all pDNA-copolymer complexes. Furthermore, it was shown that higher copolymer concentrations were associated with formation of smaller complexes, simultaneously presenting higher cationic surface charge density. In vitro studies indicate that all pDNA-copolymer complexes are able to successfully enter the eukaryotic cells without loss of cell viability, suggesting that these nanocarriers are not cytotoxicity. Taking all these data together, it can be inferred that the newly synthetized copolymers are worthy of exploring for the delivery of genetic material. Conflicts of interest There are no conflicts to declare. Acknowledgements Joana R. Góis acknowledges MATIS – Materiais e Tecnologias Industriais Sustentáveis (CENTRO-01-0145-FEDER-000014). Jorge F.J. Coelho acknowledges FCT-MCTES for financial support of the projects PTDC/CTMPOL/6138/2014 and CMUP-ERI/TIC/0021/2014. The research from CICS-UBI was supported by FEDER funds through the POCI – COMPETE 2020 – Operational Programme Competitiveness and Internationalization in Axis I − Strengthening research, technological development and innovation (Project POCI-01-0145FEDER-007491) and National Funds by FCT – Foundation for Science and Technology (Project UID/Multi/00709/2013). A.M. Almeida acknowledges doctoral fellowship (SFRH/BD/102284/2014). The 1 H NMR data was collected at the UC-NMR facility which is supported in part by FEDER – European Regional Development Fund through the COMPETE Programme (Operational Programme for competitiveness) and by National Funds through FCT − Fundac¸ão para a Ciência e a Tecnologia (Portuguese Foundation for Science and Technology) through grants REEQ/481/QUI/2006, RECI/QEQ-QFI/0168/2012, CENTRO-07-CT62-FEDER-002012, and Rede Nacional de Ressonância Magnética Nuclear (RNRMN). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.colsurfb.2018.05. 004. References [1] S.L. Ginn, I.E. Alexander, M.L. Edelstein, M.R. Abedi, J. Wixon, Gene therapy clinical trials worldwide to 2012–an update, J. Gene Med. 15 (2013) 65–77. [2] Y. Li, B. Li, C.J. Li, L.J. Li, Key points of basic theories and clinical practice in rAd-p53 (Gendicine) gene therapy for solid malignant tumors, Expert Opin. Biol. Ther. 15 (2015) 437–454.

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