Article pubs.acs.org/molecularpharmaceutics
Cyclen Grafted with poly[(Aspartic acid)-co-Lysine]: Preparation, Assembly with Plasmid DNA, and in Vitro Transfection Studies Chunying Ma,†,§ Jin Zhang,†,§ Liwen Guo,† Changguo Du,† Ping Song,† Baojing Zhao,† Ling Li,† Chao Li,*,† and Renzhong Qiao*,†,‡ †
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, 100029 Beijing, P. R. China State Key Laboratory of Natural and Biomimetic Drugs School of Pharmaceutical Sciences, Peking University Health Sciences Center, 100083 Beijing, P. R. China
‡
S Supporting Information *
ABSTRACT: Development of safe and effective gene carriers is the key to the success of gene therapy. Nowadays, it is still required to develop new methods to improve nonviral gene delivery efficiency. Herein, copolymers of poly[(aspartic acid)co-lysine] grafted with cyclen (cyclen-pAL) were designed and evaluated for efficient gene delivery. Two copolymers with different Asp/Lys block ratios were prepared and characterized by NMR and gel permeation chromatography analysis. Agarose gel retardation, circular dichroism, and fluorescent quenching assays showed the strong DNA-binding and protection ability for the title compounds. Atomic force microscopy studies clearly delineated uniform DNA globules with a diameter around 100 nm, induced by cyclen-pAL. By grafting cyclen on Asp, relatively high gene delivery efficiency and low cytotoxicity of the modified copolymers were achieved compared with their parent compounds. The present work might help to develop strategies for design and modification of polypeptide copolymers, which may also be applied to favorable gene expression and delivery. KEYWORDS: cyclen, DNA assembly, drug delivery, gene therapy, polypeptide, synthesis
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be facilitated.10 Polycation polymers, such as polyethylenimine (PEI),11 polyamidoamine dendrimers (PAMAM),12 and poly(2-(dimethylamino)ethyl methacrylate),13 have been widely used to condense DNA and transfer nucleic acid into cells. Poly(L-lysine) (PLL) with great DNA condensation abilities has also been chosen as a gene vector, but it has high cytotoxicity.14 To obtain PLL polymers with excellent cell viability, some grafters, including polyethylene poly(D,L-lacticco-glycolic acid) (PLGA),15 histidine,16 and polyethylene glycol (PEG),17 were usually selected to be conjugated with PLL polymers. Additionally, targeting ligands including galactose18,19 are also good grafters for gene delivery system. These cationic DNA polyplexes have shown the potential ability to mediate gene transfer through electrostatic binding to cell surfaces. For most of current gene carriers, it is the interaction between cationic groups in polymers and negatively charged DNA that promotes DNA condensation or assembly. The negative charge in DNA should be neutralized by polymers so that complex particles can be easily close to
INTRODUCTION Development of biomedical study has driven in-depth research in investigating methods for the introduction of therapeutic molecules such as DNA into cells. However, when entering cytosol of the target cell, naked nucleic acids are susceptible to endogenous nucleases.1 Therefore, it is necessary to achieve DNA protection and assembly when polymers were used as gene delivery vectors. It is an active research field to design and synthesize compounds that can protect DNA temporally or spatially via assembly.2,3 Nowadays, a great deal of effort has been devoted to developing methods for DNA protection such as preparing multilayered polyelectrolyte films,4 developing phosphorylcholine based copolymers,5 or developing aminomodified silica nanoparticles.6 Recently, many researchers have paid attention to developing polycation/DNA complexes that are effective for preventing DNA degradation by enzymes.7,8 Lysine dendrimer as a potential gene delivery strategy has attracted much attention. W. Xue’s group reported that a porphyrin−poly(L-lysine) dendrons (PP−PLLD) with the shape of star can load doxorubicin, suggesting that they can serve as good inhibitor to CNE2 cells.9 Additionally, photoenhanced gene transfection in vitro can be achieved by PP− PLLD-Arg/MMP-9 modified by arginine, and then an obvious reduction of MMP-9 protein expression in HNE-1 cells could © 2015 American Chemical Society
Received: Revised: Accepted: Published: 47
May 21, 2015 October 30, 2015 November 22, 2015 November 22, 2015 DOI: 10.1021/acs.molpharmaceut.5b00396 Mol. Pharmaceutics 2016, 13, 47−54
Article
Molecular Pharmaceutics
and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Sigma-Aldrich (St. Louis, Mo, USA). All other chemicals were of analytical grade. In the present study, the structure and composition of all title compounds were characterized by NMR spectra obtained with a Bruker AV600 NMR (400 MHz) spectrometer. CD3OD or D2O was used as solvent. A Waters 515−2410 system gel permeation chromatography (GPC) was used to determine the molecular weight (MW) of the copolymer. The detection system includes a 515 high-performance liquid chromatography (HPLC) pump and 996 photodiode array as well as 2410 refractive index detector. Tetrahydrofuran (THF) or water was used as solvent, and the flow rate was 1.0 mL/min. Agarose Gel Electrophoresis Study. Retardation experiments were carried out with plasmid DNA. The 0.3 μL of plasmid DNA solution (0.5 μg/μL) was treated with different concentration polymers, followed by addition of pH 7.4 TrisHCl buffer (100 mM) to get a 10.0 μL final volume. Then polymer/DNA complex solutions were incubated at 37 °C for 12 h, and free DNA without treatment was used as control. After incubation, the samples were mixed with loading buffer (1.0 μL) and then loaded on a 1% (w/v) agarose gel containing 1.0 μg/mL of EB. The electrophoresis assays were carried out in TAE buffer for 60 min at 85 V. Olympus Grab-IT 2.0 Annotating Image Computer System was used to get DNA band photograph in the presence of UV light. The plasmid DNA and cyclen-pAL complex in Tris-HCl buffer solutions were mixed and incubated for 12 h at 37 °C. Then the samples were incubated for another 30 min under the same condition after the addition of DNase I with various conditions. After degradation, DNA samples were loaded on a 1% (w/v) agarose gel for analysis. Fluorescence Quenching. Fluorescence spectra were measured by using a Hitachi model F-4500 spectrofluorimeter. The excitation and emission wavelength were 520 and 620 nm, respectively, and the band bass was 5 nm. Fluorescence quenching experiments were carried out at 25 °C. Ten microliters of CT-DNA (1 mg/mL) and 10 μL of EB (5 mg/mL) were mixed together, and then the mixture was diluted with pH 7.4 Tris-HCl buffer (100 mM) to a final volume of 3 mL. The samples were added in a quartz cuvette. As control, the maximal fluorescence of CT-DNA/EB system was measured. Subsequently, every polymer solution was added to the above system, and a new fluorescence spectrum was obtained after mixing for a constant minute. Circular Dichroism (CD). The CD spectra were obtained by using Jasco-810 spectropolarimeter (sensitivity, 100 mdeg; scan speed, 500 nm/min; wavelength range, 400−220 nm), and a continuous flow of N2 was maintained for the whole measurement process. A quartz cuvette with 1 cm path length cell was used. Free DNA (10 mg/mL) was diluted with pH 7.4 Tris-HCl buffer (100 mM) to a final concentration of 1 mg/ mL, and its CD spectrum was recorded. Then every polymer solution (10 mg/mL) was added into the above DNA system and incubated for 30 min at 37 °C prior to measurement. All the experiments were carried out in triplicate. Zeta-Potential and Particle Size Measurement. Particle size and zeta-potential measurements of polyplex were measured at 25 °C with a Zetasizer Ver.6.20 (Malvern Instruments Ltd. @ Copyright 2008, Malvern, UK). The polymer/DNA complexes at different weight ratios were prepared by the same method mentioned in gel retardation experiments, and then samples were incubated for 30 min at 37
negatively charged plasma membranes in cell. Cationic polymers can interact with DNA by electrostatic interaction, which leads to the compaction of DNA macromolecule and then facilitates DNA penetration into cells due to the size reduction of DNA. Recently, many researchers focus on basic and applied field in 1,4,7,10-tetraazacyclododecane (cyclen) due to its significant properties such as basicity. The cyclen with centralized structure has four N atoms, one or more of which can be replaced by different groups. These unique and various properties of cyclen promote research in designing and synthesizing new cyclen-based compounds, which have valuable applications in biological and chemical field. In the past decade, numerous cyclen-based molecules including chitosan grafted cyclen in our lab,20 uracil-PNA grafted cyclen,21 and cyclengrafted polymers with linear and cross-linked structure,22 have been prepared, and also their interaction with nucleic acid has been well investigated. In the current paper, we report on a kind of water-soluble copolymer, namely poly[(aspartic acid)-co-lysine] grafted with cyclen (cyclen-pAL). The polymers were prepared by thermal polycondensation, and their structures are depicted in Figure 1.
Figure 1. Structures of cyclen-grafted copolymers with different ratios of aspartic acid and lysine.
The polymers with different Asp/Lys ratios (x:y) were prepared by adjusting Asp/Lys feed ratio and to explore the effects of charges or structures on vector properties. The DNA binding abilities were improved by the introduction of cyclen grafter with protonated amino/imino groups. The parent polymers poly[(aspartic acid)-co-lysine] (pAL) with the corresponding Asp/Lys ratio were tested as credible control compounds. For the purpose of acting as gene delivery carriers, title compounds were evaluated from the following aspects: physiochemical characteristics, morphology, cell viability, and gene delivery abilities. The results indicated that cyclen-pAL can interact with plasmid DNA to form ∼100 nm particles and protect DNA from enzymatic degradation. The further in vitro assays presented low cytotoxicity and favorable transfection activity.
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EXPERIMENTAL METHODS Materials and Methods. L-Aspartic acid (L-Asp) and Llysine (L-Lys) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai) and Beijing Aoboxing Biotech Co., Ltd. (Beijjing), respectively. The polymers were purified by dialysis, and a spectra (MWCO 3500 Da) regenerated cellulose dialysis membrane was used to hold polymer solution. Plasmid DNA (pUC 18) and DNase I were obtained from Takara Biotechnology (Dalian). Calf thymus DNA (CT-DNA) was purchased from Huamei Biotechnology Company. Twenty-five kilodalton PEI (branched, average molecular weight 25 kDa) 48
DOI: 10.1021/acs.molpharmaceut.5b00396 Mol. Pharmaceutics 2016, 13, 47−54
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Molecular Pharmaceutics °C. Prior to measurements, complex solutions were diluted to final 3 mL volume by ultrapure water. Atomic Force Microscopy (AFM). A Digital Instruments multimode NanoScope III was used to obtain AFM image. Before measurement, DNA samples were diluted to a final concentration of 2.5 ng/μL by using AFM buffer. Then the sample was loaded on a freshly cleaved mica plate (1 cm × 1 cm). After 2 min, the surface buffer in the sample was rinsed thoroughly by pure water, and the mica was dried under a stream of nitrogen. All the images were obtained in tapping mode. Cell Viability Study. The tested cells were plated in 96-well plates at the density of 1 × 104 cells/well. Polymers (20 μL) with various concentrations were added to the cells. After 24 h of incubation, fresh complete medium (10 μL) replaced the original medium in each well. Another 4 h incubation was achieved after the addition of MTT (20 μL) solution in PBS (5 mg/mL). Subsequently, the system was incubated for another 30 min after the medium was replaced by DMSO (150 μL). A microplate reader (Beckman DTX 880) was used to determine the optical intensity at the wavelength of 570 nm. The cell viability was obtained by using the following formula: viability (%) = (ODsample/ODcontrol) × 100, where ODsample was got by the addition of title compounds, and ODcontrol was obtained in the absence of compounds. Each value was obtained from five independent experiments. Gene Delivery Assays. In the transfection assays, the reporter plasmid was pEGFPCl. Initially, in the presence of 10% FBS (1 mL), tested cells at the density of 5 × 104 cells/ well were cultured in 24-well plates at 37 °C. CO2 (5%) was maintained in this culture process. When the confluence of the cells was at 80−90%, serum-free medium (200 μL) replaced the culture medium, and cells were incubated for 6 h at 37 °C. The polymers/DNA mixtures with various w/w or pH ratios were added in the serum-free medium. After that, complete medium (1 mL) was added in the cells, and a sequential 48 h incubation was carried out. Finally, the medium was removed, and the cells were gently rinsed by PBS, which was in favor of the assessment of the expression of luciferase. The cells were lysed thoroughly by reporter lysis buffer (Promega, 200 μL/well), and then the light emission in a luminometer was detected to characterize the luciferase activity. The BCA protein assay kit was used to investigate the protein content. To ascertain reproducibility, all experiments were assayed in triplicate.
Figure 2. Peaks labeled with “a−g” are protons characteristic to the methylene and methyne of aspartic acid and lysine.
prepared from different Asp/Lys feed ratio. Obviously, the peak intensities at a and b increased with the increase of the Asp/Lys feed ratio, while the peaks at e, f, and g decreased, which indicates that peaks at a and b are attributed to protons in Asp, and peaks at e, f, and g resulted from those in Lys. There was no apparent variation in intensity for peaks at c and d with feed ratio changes of Asp/Lys, indicating that the two were attributed to the protons in the two units. The peaks at c and d showed no apparent variation in intensity with changes in the Asp/Lys feed ratio, suggesting that it derived from protons in both Asp and Lys units. In this work, two copolymers with different Asp/Lys ratios as control, pAL-a (45:10) and pAL-b (10:36), were obtained from the corresponding feed ratio. Subsequently, both control copolymers before hydrolysis were used to prepare the title copolymers cyclen-pAL-a and cyclenpAL-b with the same Asp/Lys ratio, respectively. The degree of substitute cyclen was calculated by following the specific protons in methylene and methyne, giving 31.3% for cyclenpAL-a and 29.3% for cyclen-pAL-b. The MW of the title copolymers was measured by GPC as shown in Figure S2 and Table S1. Interactions of Copolymers with DNA. To explore the interaction between DNA and copolymers, agarose gel electrophoresis experiments at different polymer/DNA weight ratios were carried out. Results in Figure 3 revealed that the
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RESULTS AND DISCUSSION Preparation and Characteristics of Functional Copolymers. The synthetic route of cyclen-pAL was shown in Scheme S1. The polymer of pAL was prepared according to the literature,23 and 1-(4-azyl-butyl)-4,7,10-tris(benzyloxycarbonyl)-1,4,7,10-tetraazacyclodocane (cyclenNH2) was prepared according to the references.24 Cyclen with a flexible linker was introduced to pAL skeleton through the typical hydrolysis reaction of poly(succinimide) (PSI) and NH2-terminal group of cyclen. PSI was synthesized based on relative literature,25 and all the title compounds were prepared by using PSI as a starting material.25 The ungrafted succinimide units were further hydrolyzed by the NaOH solution, and then Cbz group was removed in the presence of Pd/C (10%), giving the title compounds, cyclen-pAL. The NMR data were given in Supporting Information Figure S1. Asp/Lys ratio in polymers was identified by NMR spectra of pAL. Figure 2 shows 1H NMR spectra of p(Asp-co-Lys)
Figure 3. Agarose gel electrophoresis retardation assays of pAL/DNA, cyclen-pAL/DNA complexes at different weight ratios. Lane 1, DNA control (0.015 mg/mL); Lanes 2−6, 0.20, 2.00, 4.00, 8.00, 12.00 mg/ mL; Lane i, DNA control (0.015 mg/mL); Lanes ii−vi, 0.10, 0.20, 0.40, 0.60, 0.80 mg/mL.
polymers pAL-a and pAL-b do not retard the migration of DNA until 12 mg/mL. However, cyclen-pAL-a based complexes could retard plasmid DNA completely at 0.8 mg/ mL (lane vi), while cyclen-pAL-b/DNA complexes displayed full retardation at 0.4 mg/mL (lane iv). These results indicated that near-neutral complexes can be formed via electrostatic interaction between positive amine/imino groups of cyclen49
DOI: 10.1021/acs.molpharmaceut.5b00396 Mol. Pharmaceutics 2016, 13, 47−54
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Figure 4. Fluorescence quenching of CT-DNA/EB system after adding (A) cyclen-pAL-a and (B) cyclen-pAL-b polymers. (C) Linear fitting according the relative intensity (F0/F) in different concentrations of the four polymers.
Figure 5. CD spectra of (A) DNA/control compounds (black dashed, DNA only; green, DNA/pAL-a; orange, DNA/pAL-b; blue, DNA/cyclen) and (B) DNA/cyclen-pAL complexes (black dashed, DNA only; red, DNA/cyclen-pAL-a; black, DNA/cyclen-pAL-b).
bound to DNA. The fluorescence quenching results were also in accordance with the classical Stern−Volmer equation: F0/F = 1 + Ksv[Q], where Ksv represents the binding constant (also namely quenching constant), which reflects the binding ability of the polymer to DNA, and Q is the mole concentration (mol/ L) of the polymer.27 As shown in Figure 4, panel C for linear fitting, the Ksv values of the polymer originated from pAL-a, pAL-b, cyclen-pAL-a, and cyclen-pAL-b were obtained as 0.24 × 104 M−1 (R2 = 0.9306), 0.67 × 104 M−1 (R2 = 0.9684), 1.62 × 104 M−1 (R2 = 0.9850), 2.19 × 104 M−1 (R2 = 0.9770), respectively, showing the order of the EB-displacement ability of cyclen-pAL-b > cyclen-pAL-a > pAL-b > pAL-a. Both types of cyclen-pAL showed stronger DNA binding ability than pAL copolymers, which were mainly derived from the introduction of cyclen with positive charge. The configuration of DNA before and after polymers added was investigated by CD spectrum (Figure 5). Free B-type DNA shows a typical CD spectrum. The positive band around 245 nm is due to right-handed helicity, and the negative band around 275 nm arises from base stacking (black dashed line). As depicted in Figure 5, panel A, no CD signal change was observed after addition of pAL or cyclen to the DNA solution, which indicates that ligands can not lead to changes of DNA configuration. Under the same conditions, however, after addition of cyclen-pAL to DNA solution, detectable changes in the CD spectra were displayed in Figure 5, panel B. From free DNA (black dashed line) to the DNA-polycationic polymer (red and black solid line), the intensity of the major negative CD signal at 245 nm increased, while the intensity of the positive band at 275 nm decreased, suggesting that a secondary structural change of DNA appeared due to its condensation.28,29 Nevertheless, there is no CD spectral red- or blue-shift of DNA after addition, indicating that the B-
pAL and negatively charged phosphate groups of DNA. Meanwhile, cyclen-pAL-b has more lysine segments than cyclen-pAL-a, so the more amine groups retarded plasmid DNA completely at low weight ratio. Lysine and cyclen of cyclen-pAL contributed more positive charges to bind to DNA and interacted electrostatically with DNA and formed complexes. DNA assembly is necessary for preventing DNA degradation by nuclease via gene delivery process. It is well accepted that cationic polymers can assemble with DNA to form complexes, which can effectively protect DNA from degradation by nucleases. Agarose gel electrophoresis was used to investigate the protection effect of polyplex against DNase I degradation. We chose cyclen-pAL-b copolymer as a representative sample to illustrate the effect of protection of cyclen-pAL/DNA polyplex against biological enzymes, and the result was shown in Figure S3. Supercoiled DNA as control was digested after 30 min (lanes 4 and 5); however, no degradation of DNA was observed (lanes 6 and 7) when cyclen-pAL-b was added to the solution. Consistently, DNA was fully bound with cyclen-pALb at 0.4 mg/mL in agarose gel electrophoresis experiments (Figure 3). The enzyme-degradation experiments demonstrated that polymer cyclen-pAL can effectively protect DNA from enzymatic degradation. For further study, the DNA binding ability of the polymer was investigated by fluorescent spectroscopy in the presence of ethidium bromide (EB) on the basis of related literature,26 and the result was shown in Figures 4 and S4. The quenching phenomenon was not obvious after addition of the polymer pAL (shown in Figure S4, Supporting Information), and appreciable decrease in the emission intensity was observed after addition of the cyclen-pAL to DNA/EB system, suggesting that cyclen-pAL partially replaced EB, which 50
DOI: 10.1021/acs.molpharmaceut.5b00396 Mol. Pharmaceutics 2016, 13, 47−54
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Figure 6. (A) Zeta-potential and (B) particle size of pAL/DNA and cyclen-pAL/DNA complexes at various weight ratios.
shown in Figure 7, panel A, the free DNA without any treatment exists as a little loose nicked and supercoiled forms.
conformation of DNA does not change after assembly by cyclen-pAL copolymer.30 Physiochemical Properties and Morphology of Copolymers Micelles. The surface charges on the polymer/ DNA complexes can be characterized by zeta potential. A positively charged surface is in favor of electrostatic interaction between polyplexes and anionic cell surfaces and then allows cellular uptake.31 The zeta-potential values of cyclen-pAL/ DNA polyplex increased along with weight ratios increase, as indicated in Figure 6, panel A. The zeta-potential of naked DNA without treatment is −34.3 mV. When the weight ratio of cyclen-pAL-a/DNA was above 26.7, the copolymers could completely occupy DNA surface to form positive charge complexes, suggesting that at this w/w ratio, almost full DNA assembly was achieved. Similarly, at the 13.3 weight ratio, cyclen-pAL-b/DNA polyplex also showed positive zetapotential. Over the testing range of weight ratio, the polyexes cyclen-pAL-b/DNA presented higher zeta-potential than cyclen-pAL-a/DNA at the same weight ratio. However, both complexes of pAL/DNA possessed ∼0 mV zeta-potential until the weight ratio at 120. These observations were consistent with the data in Figure 3, indicating that cyclen-pAL can assemble with DNA to form complexes and carry extra positive charges to target cell membrane. An appropriate particle size of polymer/DNA nanoparticles is important for gene carriers since it would allow the endocytosis and subsequently facilitate gene transfer. Figure 6, panel B showed the particle sizes of cyclen-pAL-a/DNA and cyclen-pAL-b/DNA complexes. It was shown that both cyclenpAL could not fully assemble with DNA at the weight ratio lower than 15; instead, they gathered into big and loose clumps,32 so the particle sizes were relatively bigger than 400 nm. At the weight ratio of around 20−50, DNA was efficiently compacted into small nanoparticles with the diameter around 100 nm in the presence of copolymers. In a similar study, Cai et al. reported a triblock copolymers PEG−PLL-PLLeu as biodegradable micelles.33 It spontaneously formed selfassembled micelles with sizes ranging from in the range of 43.6−89.2 nm, which was closely related to PLLeu segments and the length of PLL. In contrast, Caruso et al. constructed a large diblock copolymers PEG−PLLs with size of ∼1.5 μm, which can deliver a siRNA targeting the antiapoptotic factor in prostate cancer cells.34 These results provided a favorable size and surface charges for further exploring the transfection studies as potential gene vectors. The morphological characteristics of DNA before and after mixing with cyclen-pAL-b were further studied by AFM. As
Figure 7. AFM images of (A) plasmid DNA and (B) cyclen-pAL-b/ DNA polyplex at weight ratio of 20. The inset shows the profile delineated by the blue line in the main panel. (C) Surface plot delineated by the dashed square in the main panel. (D) Distribution of the frequencies of height and diameter of cyclen-pAL-b/DNA polyplex. The scale of images is 2.5 μm × 2.5 μm.
After incubation with 3 μg/mL cyclen-pAL-b for 30 min at 25 °C in Tris-HCl buffer (pH 7.4), the original supercoiled and nicked DNA turned into uniform globules with a diameter around 100 nm, which is much more compacted than the original DNA (Figure 7B). The AFM images presented a dramatic change with the addition of the copolymer, indicating that DNA was enclosed by the copolymer or assembled with the positively charged copolymer. Histograms showed the detailed diameter and height distributions for cyclen-pAL-b/ DNA complex in Figure 7, panel D. The cyclen-pAL-a/DNA complexes show similar AFM results, as indicated in Figure S5. Uniform particle size (narrow particle size distribution) of copolymer/DNA complex provided a good physical condition for further gene delivery. The strong interaction between 51
DOI: 10.1021/acs.molpharmaceut.5b00396 Mol. Pharmaceutics 2016, 13, 47−54
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Figure 8. (A) Cytotoxicity of cyclen-pAL and p(SI)L at various concentrations against HepG2 (solid) and Hela (hollow) cell lines. (B) the structure of p(SI)L as control copolymers.
Figure 9. Expression of pEGFP-Cl mediated by cyclen-pAL/DNA and pAL/DNA polyplexes at various w/w ratios for (A) HepG2 and (B) Hela cell.
viable after exposure to p(SI)L-a and p(SI)L-b with 60 μg/mL for 24 h, respectively. Interestingly, increased cell viability for hybrid polymers can be obtained through breaking cationic groups into short segments,37 which was also consistent with our previous results.20 Gene Transfection in Vitro. To examine whether the introduction of cyclen has an effect on transfection efficiency, we carried out transfection experiments on cyclen-pAL and pAL, respectively. HepG2 and Hela cells were selected as tested cells, and pEGFP-Cl was used as reporter gene. Under the same w/w ratio, both cyclen-pAL-a and cyclen-pAL-b displayed higher transfection efficiency than pAL copolymers (Figure 9A,B). The increased transfection efficiency of cyclen-pAL was attributed to the introduction of cyclen with the high amine content. Compared with that for PAL, the interaction with DNA increased for cyclen-pAL due to the introduction of cyclen and the cell uptake ability increased accordingly. The results also suggested that the presence of “ungrafted” carboxyl moieties in Asp could partially neutralize the positive charges in Lys, and reduce cell-penetrating ability. Therefore, cyclen grafted in Asp may facilitate DNA delivery and further be favor for cellular uptake. The copolymers-mediated enhanced green fluorescent protein expression in HepG2 and Hela cells was obtained by an inverted fluorescent microscope, and the fluorescent microscope images of transfected HepG2 cells were shown in Figure 10 (the data for Hela were not shown). On the basis of
copolymer and DNA arose from amino/imino both in Lys and cyclen, resulting in efficient compaction of DNA into small nanoparticles. Cell Viabilities of Copolymers Micelles. The cell viabilities of pAL and cyclen-pAL copolymers against HepG2 and Hela cell line were evaluated by a standard MTT test. PEI (average molecular weight 25 kDa) and lipofectamin 2000 were used as control. As shown in Figure 8, panel A, cytotoxicity increased with the increase of polymer concentration. Reduced cytotoxicity was observed for both cyclen-pAL copolymers compared with that for their respective skeleton compound. Additionally, at the concentration of 20 μg/mL, the cell viabilities ranged from 86−94% after 24 h of exposure to cyclen-pAL-a and cyclen-pAL-b. Cell viabilities of both cyclenpAL-a and cyclen-pAL-b were around 85% even at the concentration of 60 μg/mL. It is widely accepted that the reduced cell viability of copolymers derives from cationic groups due to their interactions with negatively charged cell components and proteins or the plasma membrane.35,36 For pAL, carboxyl in Asp will partially neutralize positive charges in Lys and reduce cytotoxicity of copolymer. Thus, we chose p(SI)L with a neutral succinimide moiety (Figure 8B) as control compound to compare cytotoxicity of copolymers after grafting cyclen. The result showed that the cytotoxicity of both p(SI)L copolymers was higher than that of cyclen-pAL at the same concentration, giving 49.9 and 46.9% of HepG2 cells that were 52
DOI: 10.1021/acs.molpharmaceut.5b00396 Mol. Pharmaceutics 2016, 13, 47−54
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Molecular Pharmaceutics Author Contributions §
C.M. and J.Z. contributed equally to this work. C.M., J.Z., L.G., and C.D. performed experiments and calculations. P.S., B.Z., and L.L. analyzed the data. C.L. wrote the paper. R.Q. contributed reagents and analytical tools. Notes
The authors declare no competing financial interest.
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optimal results of luciferase assays, the w/w ratios of 40 were used. As depicted in Figure 10, the strongest fluorescence intensities were observed for both cyclen-pAL among the tested copolymers, while the transfected cells were less than those transfected by lipofectamine 2000. Copolymers containing more Lys moieties (x < y) gave better result than those containing more Asp (x > y) either for cyclen-pAL or pAL.
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CONCLUSIONS In summary, a new polypeptide copolymer p(Asp-co-Lys) (pAL) with different block ratios was designed and synthesized. Cyclen moiety was further grafted on Asp to modulate physicochemical properties. The signal changes in fluorescence quenching assays and CD spectra indicated suitable interaction between DNA and the modified copolymers including p[Asp(cyclen)-co-Lys] (cyclen-pAL). The morphological study was performed by AFM, showing extremely uniform particles with a diameter of 100 nm. Compared with their parent polymers, the grafting of cyclen does not lead to an increase of cell viability, whereas it benefits the improvement of transfection efficiency. It can be concluded that the introduction of small molecule cyclen could be used as an effective approach to design effective gene vectors by improving the physicochemical properties. ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00396. Synthetic route of p[SI-co-Lys], p[Asp-co-Lys], and p[Asp(cyclen)-co-Lys]; 1H and 13C NMR of the new compound and its intermediates; GPC and the GPC results of cyclen-pAL; protection assay of cyclen-pAL/ DNA by DNase I; fluorescence quenching of CT-DNA/ EB system by addition of pAL; AFM images of cyclenpAL-a/DNA polyplex (PDF)
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ACKNOWLEDGMENTS
Support of this research by the National Nature Science Foundation of China is gratefully acknowledged (Nos. 21372024, 21202005, 21572018, and 21232005). Supported by the Fundamental Research Funds for the Central Universities (No. YS 1407).
Figure 10. Fluorescent microscope images of HepG2 cells transfected by (A) control, (B) pAL-a, (C) pAL-b, (D) cyclen-pAL-a, (E) cyclenpAL-b, and (F) lipofectamine 2000 based w/w ratio of 40. The cells were transfected for 24 h and then photographed by fluorescence microscopy.
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AUTHOR INFORMATION
Corresponding Authors
*Phone: +86 10 64413899. E-mail:
[email protected]. *E-mail:
[email protected]. 53
DOI: 10.1021/acs.molpharmaceut.5b00396 Mol. Pharmaceutics 2016, 13, 47−54
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