Acta Biomaterialia 10 (2014) 1412–1422
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Cyclen-based lipidic oligomers as potential gene delivery vehicles Wen-Jing Yi a, Qin-Fang Zhang a, Ji Zhang a,⇑, Qiang Liu a, Laifeng Ren b, Qian-Ming Chen c, Liandi Guo b, Xiao-Qi Yu a,⇑ a
Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu 610064, People’s Republic of China Laboratory of Genome Stability, Development and Stem Cell Institute, The West China Second Hospital, Sichuan University, Chengdu 610041, People’s Republic of China c State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 7 June 2013 Received in revised form 25 November 2013 Accepted 9 December 2013 Available online 14 December 2013 Keywords: Gene delivery Cyclen Lipopolymer Non-viral vector Structure–activity relationship
a b s t r a c t A series of cyclen-based linear oligomers bearing hydrophobic long chains (lipopolymers Cy-LC, where Cy and LC represent cyclen-based linear backbone and hydrophobic long chain substituents, respectively) were designed and synthesized. The effects of type and degree of substitution (DS) of hydrophobic long chains on the transfection efficiency were systematically studied. The nitrogen atoms with relatively strong basicity on the cyclen ensure their good DNA binding ability, which was confirmed by gel retardation and ethidium bromide exclusion assays. Lipopolyplexes could be formed as nanoparticles with suitable sizes and zeta potentials for gene transfection. In vitro gene delivery experiments revealed that the linoleic acid (LIN) substituted material Cy-LIN has better transfection efficiency than 25 kDa polyethylenimine in the absence or in the presence of serum. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and hemolysis assays showed low cytotoxicity and good biocompatibility of the lipopolyplexes. Fluorescent labeled DNA was used to study the cellular uptake and intracellular distribution of transfected DNA. Flow cytometry results suggested that a long chain is necessary for efficient cellular uptake, and images from confocal laser scanning microscopy showed that after 4 h transfection, most of the fluorescent labeled DNA accumulated in the perinuclear region, which was required for efficient gene expression. Moreover, it was also found that the DS of the hydrophobic moiety can adjust the balance between DNA binding ability and dissociation of polyplexes, significantly affecting the transfection efficiency. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Safe and efficient delivery of genetic material remains the most challenging aspect of human gene therapy [1]. Although viral vectors are considered to have high gene transfer efficiency, their applications have been restricted by security issues such as immunogenicity, insertional mutagenesis, oncogenic effects and toxicity [2]. Non-viral gene carriers have been receiving increasing attention over the past decades because of their low cost, flexibility in chemical design and safety [3–5]. Consequently, numerous nonviral gene delivery vectors have been developed and reported in recent years. These non-viral systems can be divided into liposomes (lipoplexes), polycationic polymers (polyplexes) and organic or inorganic nanoparticles (nanoplexes) [6,7]. However, it should be noted that although non-viral gene delivery vectors such as cationic lipids and polymers have shown potential to overcome the safety problems, low transfection efficiency (TE) limits their use in clinical trials [8].
⇑ Corresponding authors. Fax: +86 28 85415886 (X.-Q. Yu).
The key steps involved in non-viral gene delivery include electrostatic complexation and condensation of DNA molecules into compact particles, uptake of complexes by the target cells and endosome escape and dissociation of the complexes to release DNA to nucleus, which is necessary for expression of the delivered genes [9–11]. Strategies to improve the efficiency and biocompatibility of cationic reagents for gene delivery typically involve grafting functional ligands such as peptides [12], lipids [13,14], sugars [15] or a combination [2] to improve the stability, targeting, uptake and sub-cellular trafficking capabilities of the vectors [16]. In this regard, hydrophobic modifications of cationic regents could facilitate DNA adsorption onto cellular surface by membrane fusion, and as a result, improve the endocytosis and TE [17]. Neamnark et al. reported that substitution of 2 kDa polyethylenimine (PEI) with aliphatic lipids including caprylic, myristic, palmitic, stearic, oleic and linoleic acids led to a TE comparable to 25 kDa PEI [18]. With the palmitic acid modification on poly-L-lysine), the product material gave the TE that was equivalent to adenoviral carrier [19]. Le Gall et al. reported that cationic lipophosphoramide with diunsaturated lipid chains could transfer the target gene into the lung with high efficiency in the in vivo gene delivery [20].
E-mail addresses: [email protected] (J. Zhang), [email protected] (X.-Q. Yu). 1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.12.010
W.-J. Yi et al. / Acta Biomaterialia 10 (2014) 1412–1422
Moreover, hydrophobic modifications of cationic reagents including chitosan [21], poly[2-(dimethylamino)ethyl methacrylate] [22], spermine [23], etc., showed an approximate increase of TE, which is likely due to the balanced protection and release of DNA as well as to their function as membrane-anchoring moieties. Moreover, the alkyl chain length and degree of substitution (DS) may also affect some other properties such as cytotoxicity, biocompatibility and in vivo circulation time [24]. A delicate balance between hydrophilic and hydrophobic components is crucial for the design of new cationic carriers [25]. Optimal chain length and DS should be selected by considering the overall effects of hydrophobic modifications on the gene therapy [26]. However, there are few reports of systematic investigation of the structure–activity relationship of hydrophobic modified non-viral gene delivery vectors. Our group previously reported a series of cyclen-based cationic polymers showing comparable transfection efficiency to 25 kDa PEI with relatively lower cytotoxicity [27–29]. It is of great importance to further investigate the structure–activity relationship of this type of cationic polymer. Herein we studied the effects of the structures and DS of lipidic moiety on the cyclen-based linear oligomer. Results show that the long chain modified complex could transfer target gene into several cell lines with higher TE and lower cytotoxicity compared to 25 kDa PEI, and the increased TE might be attributed to the enhanced cellular uptake and dissociation of the polyplexes inside the cell. We considered that these studies would be particularly important to better understand the relationships between the lipid structure itself (length, amount, saturated vs. unsaturated or polyunsaturated) and the physicochemical properties of the corresponding polyplexes, as well as their transfection activities.
2. Materials and methods 2.1. Materials and general measurements Anhydrous ethanol, dichloromethane and epichlorohydrin were dried and purified under nitrogen by using standard methods and were distilled immediately before use. 1,7-Bis (tert-butyloxycarbonyl)-1,4,7,10-tetraazacyclodocane was prepared according to the literature [30]. High molecular weight PEI (branched, average molecular weight 25 kDa: 25 kDa PEI), and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Sigma–Aldrich (St Louis, MO, USA). The plasmids used in the study were pGL-3 (Promega, Madison, WI, USA) coding for luciferase DNA and pEGFP-N1 (Clontech, Palo Alto, CA, USA) coding for EGFP DNA. Cy™5 was obtained from Molecular Probe (Mirus, Madison, WI, USA). Dulbecco’s modified Eagle’s medium (DMEM), 1640 medium and fetal bovine serum (FBS) were purchased from Invitrogen Corp. MicroBCA protein assay kit was obtained from Pierce (Rockford, IL, USA). Luciferase assay kit was purchased from Promega (Madison, WI, USA). Endotoxin free plasmid purification kit was purchased from TIANGEN (Beijing, China). 293T human embryonic kidney cell lines, A549 lung cancer cell lines and U2OS human osteosarcoma cancer cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. All other reagents used in the synthesis, if not specified, were obtained from Sigma–Aldrich and used without further purification. Characterization and structural confirmation of intermediates and products were performed by proton nuclear magnetic resonance (1H NMR) (Varian INOVA-400 spectrometer) and electrospray ionization time-of-flight (ESI-TOF, Waters Q-TOF Premier) mass spectrometry. The molecular weights of the oligomers were determined by a Waters 717 plus autosampler (Waters Corp., Milford, MA, USA) using tetrahydrofuran (THF) as eluent at a flow
1413
rate of 1.0 ml min1. Two Waters Styragel columns (HR3 and HR4) were used in series, and the detector (Waters 2414 refractive index detector) and columns were maintained at 45 °C throughout the runs. Linear polystyrene standards were used for calibration. 100 ll of each sample prepared at 2 mg ml1 was injected, and each sample was given 40 min to elute off of the column. Fluorescence spectra were measured by a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer. 2.2. Synthesis of lipid-substituted compound and polymers 2.2.1. Synthesis of lipid-diethanolamine compound (1) Aliphatic acid (caprylic, lauric, palmitic, stearic, oleic and linoleic acid, 0.04 mol) was dissolved in anhydrous dichloromethane (CH2Cl2, 50 ml). The solution was stirred at 0 °C for 10 min followed by the addition of isobutyl chloroformate (5.46 g, 0.04 mol) and N-methylmorpholine (4.05 g, 0.04 mol). After addition of diethanolamine (5.05 g, 0.048 mol), the solution was stirred at 0 °C for 30 min and at 25 °C for 6 h. The solution was washed with sat. NaHCO3 (2 50 ml), and brine (2 50 ml). The organic layer was dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 1, whose detailed analytical data are listed in the Supplementary data. 2.2.2. Synthesis of epoxy-lipid-diethanolamine cross-linkers (2) Compounds 2 were prepared according to the literature [31]. In a typical procedure, a mixture of epichlorhydrin (9.34 ml, 0.12 mol), lipid-diethanolamine 1 (0.02 mol), sodium hydroxide pellets (4.8 g, 0.12 mol), water (1 ml, 0.056 mol) and tetrabutylammonium chloride (0.3224 g, 0.001 mol) was vigorously stirred for 2 h at 40 °C. The solid produced in the reaction was filtered off and washed with dichloromethane. The combined organic layer was dried with anhydrous magnesium sulfate. The solvent and excess epichlorhydrin were distilled off to give oily products. The residue was purified by silica gel column chromatography to give the product 2, whose detailed analytical data are listed in the Supplementary data. 2.2.3. Synthesis of hydrophobically modified polymer (Cy-LC) 1,7-Bis(tert-butyloxycarbonyl)-1,4,7,10-tetraazacyclodocane (223.5 mg, 0.6 mmol) was dissolved in 0.5 ml of C2H5OH, then compound 2 (0.6 mmol) was added to the solution. Under the protection of N2, the reaction mixture was stirred at 80 °C for 80 h. The reaction mixture was stirred overnight after adding 50 ml saturated MeOH–HCl solution at room temperature and purifying by dialysis (MWCO 3.5 kDa) against pure water for 3 days. The retentate was lyophilized to give the product. Using 1H NMR (400 MHz, D2O), the integrals of the characteristic proton peaks of the substituted fatty acids (d 0.8 ppm, –CH3 and d 1.3 ppm, –CH2) and of polymer backbone (d 2.8–4.0 ppm) were obtained. 2.2.4. Synthesis of hydrophobically modified polymer with different DS (Cy-LIN0.53) Cyclen-based linear oligomer (LCPA) (0.036 mmol) was prepared according to the literature [28], and was dissolved in CH2Cl2. The solution was stirred at 0 °C for 10 min followed by the addition of various amounts of linoleic acid (fatty acid:LCPA ratios = 0.5, 1, 2 and 3), HOBt (N-hydroxybenzotriazole, 0.036 mmol) and EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.036 mmol). After addition of DIEA (N,N-diisopropylethylamine, 0.036 mmol), the solution was stirred at 25 °C for 6 h. The solvent was removed under reduced pressure and added to 30 ml saturated MeOH–HCl solution at room temperature and purified by dialysis (molecular weight cut-off (MWCO) 1000 kDa) against pure water for 3 days. The retentate was lyophilized to give the product
1414
W.-J. Yi et al. / Acta Biomaterialia 10 (2014) 1412–1422
Cy-LIN0.53. Using 1H NMR (400 MHz, D2O), the integrals of the characteristic proton peaks of the substituted fatty acids (d 0.86 ppm, –CH3; d 1.27 ppm, –CH2 and d 5.28 ppm, –CH@CH) and of polymer backbone (d 2.5–4.1 ppm) were obtained.
was added to each well. After 4 h, unreacted dye was removed by aspiration. The formazan crystals were dissolved in 150 ll dimethyl sulfoxide per well and measured spectrophotometrically in an ELISA plate reader (model 550, BioRad) at a wavelength of 570 nm. The cell survival was expressed as follows: cell viability = (ODtreated/ODcontrol) 100%.
2.3. Gel retardation assay Plasmid pEGFP-N1 (BD Biosciences Clontech) and pGL3-control (Promega) were amplified in Escherichia coli JM109 and extracted with Endo-Free Plasmid Maxi Kit (E.Z.N.A.™, Omega). Polymers/ DNA complexes were freshly prepared at various w/w ratios. 5 ll complex solution containing 0.2 lg plasmid pGL3-control was incubated for 30 min at room temperature and run on 1% (w/v) agarose gel at 80 V for 40 min. Gel was stained with ethidium bromide (EB) and the mobility of pDNA bands were analyzed using a Molecular Imager ChemiDoc XRS + (BIO-RAD, USA). 2.4. EB displacement assay The ability of polymers to condense DNA was studied using EB exclusion assays [14]. Fluorescence spectra were measured at room temperature in air by a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer and corrected for the system response. EB (2.5 ll, 1 mg ml1) was put into a quartz cuvette containing 2.5 ml of 10 mM Hepes solution (pH = 7.4). After shaking, the fluorescence intensity of EB was measured. Then CT DNA (10 ll, 1 mg ml1) was added to the solution and mixed symmetrically, and the measured fluorescence intensity is the result of the interaction between DNA and EB. Subsequently, the solutions of polymer (1 mg ml1, 2 ll for each addition) were added to the above solution for further measurement. All the samples were excited at 520 nm and the emission was measured at 600 nm. 2.5. Measurement of particle size and zeta potential The complexes of polymers/DNA at various w/w ratios ranging from 2 to 16 were prepared as described above. 1 lg pGL3-control and the corresponding amount of polymers were respectively dissolved in 100 ll PBS buffer (pH = 7.4), and mixed. After 30 min incubation at room temperature, polymers/DNA complexes were diluted with 900 ll H2O, and the zeta potentials and the hydrodynamic diameters were measured using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd, UK) at 25 °C. 2.6. Transmission electron microscopy (TEM) TEM images were obtained on a JEM-100CX (JEOL) transmission electron microscope at an acceleration voltage of 100 kV. The TEM samples were prepared by dipping a copper grid with Formvar film into the freshly prepared nanoparticles solution (1.0 lg ml1). A few minutes after the deposition, the aqueous solution was blotted away with a strip of filter paper and then the samples were dried for 2 min at room temperature. The samples were stained with phosphotungstic acid (ATP) aqueous solution and dried in air. 2.7. Cytotoxicity of the complexes evaluation Cytotoxicity of polymer/DNA and PEI/DNA complexes were evaluated by MTT assay. 1.5 104 cells per well 293T, U2Os and A549 cells were respectively seeded in 96-well plates. After 24 h culture, the lipopolyplexes (vector-DNA complexes) or free vectors were added to cells at different chosen w/w ratios and concentrations. For complexes, the final pGL-3 concentration is 0.2 lg per well in a total volume of 100 ll for another 24 h. After that, the medium was replaced again with 200 ll of fresh medium, and 20 ll of sterile filtered MTT (5 mg ml1) stock solution in PBS
2.8. Gene transfection efficiency assay in vitro 2.8.1. Gene delivery studies: expression of the Luc gene A549, U2Os and 293T cells were seeded at a density of 7 104 cells per well in a 24-well plate in DMEM (U2Os and 293T cells) or RPMI-1640 medium (A549 cells) containing 10% FBS and grown to reach 70–80% confluence prior to transfection. For transfection in the absence of serum, the medium of each well was exchanged for fresh serum-free medium. For transfection in the presence of serum, the medium of each well was not changed at this time. Subsequently, the cells were treated with polyplexes (containing 1 lg of pGL3-control or pEGFP-N1) at different (w/w) weight ratios and PEI/DNA at weight ratio of 1.4 (N/P = 10) for 4 h at 37 °C. The medium was then completely refreshed with the completed culture media. After an additional 24 h incubation, the medium was removed and the cells were washed with 500 ll 1 PBS (pH 7.4) twice, lysed with 100 ll 1 lysis reporter buffer (Promega) and centrifuged at 12,000g for 3 min at 4 °C. Luciferase activity in 20 ll supernatant was evaluated with a luciferase assay system (Promega). The gene transfection efficiency of each sample was represented by firefly luciferase expression and calculated as relative light units per milligram of total protein (RLU/mg protein). Protein concentrations in cell lysates were determined using BCA Protein Assay Kit (Pierce) with bovine serum albumin (BSA) as standard. 2.8.2. Gene delivery studies: expression of the EGFP gene Enhanced green fluorescent protein expression studies were carried out as mentioned above for the Luc gene expression. 24 h after transfection, cells were observed with an inverted fluorescence microscope (Nikon Eclipse TE 2000E) equipped with a cold Nikon camera. Digital image recording and image analysis were performed with the NIS Elements Advanced Research (version 2.31) software. GFP expressions were quantitatively measured using flow cytometry. The cell suspensions were evaluated using Aria 2 flow cytometry and 10,000 cells were evaluated in each experiment. Data acquisition and analysis were performed using Aria 2 flow cytometry (Becton, Dickinson and Company). GFP fluorescence from the expression of the plasmid DNA was measured in the FL1 channel using the 488 nm blue laser. Untransfected cells were used to set the background. 2.9. Cellular uptake of plasmid DNA The cellular uptake of the polymer/fluorescein labeled-DNA complexes was analyzed by flow cytometry. The Label IT Cy5 Labeling Kit was used to label pDNA with Cy5 according to the manufacturer’s protocol. Briefly, A549 cells were seeded onto 12well plates (1.6 105 cells per well) and allowed to attach and grow for 24 h. For transfection in the absence of serum, the medium was exchanged with serum-free medium. As for transfection in the presence of serum, the medium was exchanged with serum-containing medium. Cells were incubated with Cy5 labeled DNA nanoparticles (2 lg DNA per well, optimal N/P ratio of each sample) in media for 4 h at 37 °C. Subsequently, the cells were washed with 1 PBS and harvested with 0.25% trypsin/ethylenediaminetetraacetic acid and resuspended in 1 PBS. Mean fluorescence intensity was analyzed using FACSCalibur flow
W.-J. Yi et al. / Acta Biomaterialia 10 (2014) 1412–1422
cytometer (Becton Dickinson and Company). Cy5-labeled plasmid DNA uptake was measured in the FL4 channel using the red diode laser (633 nm). Data from 10,000 events were gated using forward and side scatter parameters to exclude cell debris. The flow cytometer was calibrated for each run to obtain a background level of 1% for control samples (i.e., untreated cells). 2.10. Confocal laser scanning microscopy (CLSM) analysis A549 cells were seeded at a density of 6 104 cells per well onto the 24-well plate with a sterile cover glass (8 mm 8 mm) in each well and incubated for 24 h. For transfection in the absence of serum, the medium was exchange with serum-free medium. Complexes of polymers and Cy5-labeled pGL3 at a given concentration were added to each well. After 4 h, the cells on the cover glass were washed three times with PBS buffer and fixed with 4% paraformaldehyde (dissolved with PBS buffer) for 10 min; nuclear staining was done with 40 ,6-diamidino-2-phenylindole (DAPI). The CLSM observation was performed using an Olympus FV1000 – IX81 (Leica, Germany) with a 40 objective at excitation wavelengths of 405 nm for DAPI (blue), 633 nm for Cy5 (red), respectively. 2.11. Hemolytic activity test The hemolytic activity was measured based on the reported method [32]. A total of 200 ll of diluted rabbit blood was added to different polyplexes and the volume was adjusted to 1 ml with sterile PBS (pH = 7.4). 200 ll of rabbit blood mixed with 800 ll of the PBS served as the positive control, and 200 ll of rabbit blood mixed with 800 ll of the PBS containing an excess of ammonium chloride to cause complete hemolysis was used as the negative control. After the vials were incubated at 37 °C for 90 min, the solutions were centrifuged at 2000 rpm for 10 min. A total of 200 ll of the supernatant was collected and seeded in each well of a 96-well plate. The absorbance at a wavelength of 543 nm was recorded on a Synergy HT Multi-Mode Microplate Reader (BioTek, USA). The percentage hemolysis (PH%) (mean% ± SD, n = 3) was calculated using the following formula: PH% = (As Ap)/(An Ap) %, where As, An and Ap represent the absorbance of sample, negative and positive controls, respectively.
3. Results and discussion 3.1. Synthesis of the hydrophobic long chain modified oligomers Cy-LC Scheme 1 summarizes the general synthetic methods for the hydrophobic-modified oligomers, which were named according to the abbreviations of the hydrophobic groups. Diol glycidyl ethers 2 with various long chains were first prepared to act as the bridge molecule. Subsequent ring-opening polymerization took place between 1,7-diprotected cyclen and 2. The final deprotection by HCl and dialysis gave the comb-like products Cy-LC. The dialysis yields of the oligomers were not greatly different, suggesting that similar polymerization degrees were achieved for the six products. These new compounds were obtained as hydrochlorides and characterized by 1H NMR (400 MHz) using D2O as solvent. Since the molecular weights of Cy-LC could not be regularly measured by gel permeation chromatography (GPC), the indirect method was used by employing Boc-substituted substrates, whose molecular weights were measured to be in the range of 2599–3603 (polydispersity index: 1.1–1.2, Table S.1 of Supplementary data) by GPC using THF as mobile phase, indicating an average of 4–5 repeating
1415
units in one molecule. All of the target oligomers were found to be well soluble in pure water. 3.2. Interaction with plasmid DNA The ability of polymers to bind DNA to form polyplex is a prerequisite for efficient DNA transfection. Gel electrophoresis has been widely used to reveal the interaction between different polymers and DNA. As reported in previous work, hydrophobic moieties could facilitate a cooperative binding of polymer with DNA [33]. To identify the appropriate vector/DNA weight ratio required for efficient condensation, a gel mobility assay was carried out. As shown in Fig. 1A, full retardation of plasmid DNA was observed from the w/w ratio of 4. For compound Cy-LIN, the ratio for full retardation was even lower. These results show that all the oligomers can effectively bind DNA at a relatively low dosage. On the other hand, the binding abilities of these vectors were further evaluated through EB exclusion assay. The fluorescence intensity of EB is significantly increased by nucleic acid intercalation. When a cationic agent binds to the plasmid, some of the intercalated EB is substituted, resulting in measurable reduction of the fluorescence intensity. Fig. 1B shows that the addition of Cy-LC to DNA pretreated with EB caused considerable decrease of fluorescence intensity, which was almost completely quenched at the w/w ratio of 2. Generally, the oligomer with the shorter aliphatic chain showed the better fluorescent quenching ability. This might be due to its weaker ability to screen the positive charge on the cyclen moiety which was necessary for DNA condensation. Having the proper size and shape of polymer/DNA complexes is essential for efficient gene delivery. The effective diameters of the various alkyl-modified polyplexes were measured by dynamic light scattering (DLS). As shown in Fig. 2A, polyplex particles were formed with the sizes in the range of 100–250 nm at different w/w ratios. For oligomers with longer hydrophobic chain (Cy-STE, CyOLE and Cy-LIN), the 18-C length substituent might facilitate the formation of micelle-like structure, which would condense DNA more compactly. Consequently, the average sizes of the polyplexes formed from these three vectors were distinctly smaller (95%) than PEI, indicating that the hydrophobic chain has a significant positive effect on the endocytosis by membrane fusion. CLSM was also used to study the intracellular distribution of Cy5-labeled DNA transfected by Cy-LINx, and Fig. 10C shows the typical results involving Cy-LIN2. It was shown that after 4 h transfection, most of the fluorescent labeled DNA was accumulated in the perinuclear region, which was necessary for efficient gene expression.
hydrophobic moiety plays an important role in the gene delivery, and it may affect the balance between DNA binding ability and dissociation of polyplexes. Good TE together with excellent biocompatibility mean that this type of lipopolymer is promising as a non-viral gene delivery vector. Subsequent studies will focus on further development of the lipopolymers, such as the introduction of biodegradable or cell-targeting moieties.
Acknowledgements This work was financially supported by the National Program on Key Basic Research Project of China (973 Program, 2012CB720603 and 2013CB328900), the National Science Foundation of China (No. 21232005) and State Key Lab of Oral Diseases (Sichuan University) (SKLODSCUKF2012-02). J. Z. thanks the Program for New Century Excellent Talents in University (NCET11-0354). We also thank Analytical & Testing Center of Sichuan University for structural analysis of the compounds.
4. Conclusions Appendix A. Figures with essential colour discrimination In this report, LCPA was modified with hydrophobic long chains through two different methods. One is the polymerization between diBoc-protected cyclen and long chain contained bridge, and the other is direct acylation of LCPA. The former method helps us to study the effect of different hydrophobic chains, and the latter method gives a way to investigate the effect of the DS on gene delivery. Compared to LCPA, the hydrophobic modified materials gave much higher TE, which was also higher than 25 kDa PEI. The structure–activity relationship of the lipopolymers was studied, and the long chain group (especially linoleic acid substituent) was found to be crucial for efficient cellular uptake and nuclear orientation. Furthermore, it was also found that the DS of
Certain figures in this article, particularly Figs. 1–10 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2013. 12.010.
Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2013. 12.010.
1422
W.-J. Yi et al. / Acta Biomaterialia 10 (2014) 1412–1422
References [1] Santos JL, Oliveira H, Pandita D, Rodrigues J, Pêgo AP, Granja PL, et al. Functionalization of poly (amidoamine) dendrimers with hydrophobic chains for improved gene delivery in mesenchymal stem cells. J Control Release 2010;144:55–64. [2] Mintzer MA, Simanek EE. Nonviral vectors for gene delivery. Chem Rev 2009;109:259–302. [3] Russ V, Günther M, Halama A, Ogris M, Wagner E. Oligoethylenimine-grafted polypropylenimine dendrimers as degradable and biocompatible synthetic vectors for gene delivery. J Control Release 2008;132:131–40. [4] Merkel OM, Beyerle A, Librizzi D, Pfestroff A, Behr TM, Sproat B, et al. Nonviral siRNA delivery to the lung: investigation of PEG-PEI polyplexes and their in vivo performance. Mol Pharmacol 2009;6:1246–60. [5] Kogure K, Moriguchi R, Sasaki K, Ueno M, Futaki S, Harashima H. Development of a non-viral multifunctional envelope-type nano device by a novel lipid film hydration method. J Control Release 2004;98:317–23. [6] Ravi Kumar MN, Sameti M, Mohapatra SS, Kong X, Lockey RF, Bakowsky U, et al. Cationic silica nanoparticles as gene carriers: synthesis, characterization and transfection efficiency in vitro and in vivo. J Nanosci Nanotechnol 2004;4:876–81. [7] Kumar MN, Mohapatra SS, Kong X, Jena PK, Bakowsky U, Lehr CM. Cationic poly (lactide-co-glycolide) nanoparticles as efficient in vivo gene transfection agents. J Nanosci Nanotechnol 2004;4:990–4. [8] Forrest ML, Meister GE, Koerber JT, Pack DW. Partial acetylation of polyethylenimine enhances in vitro gene delivery. Pharm Res 2004;21:365–71. [9] Ogris M, Steinlein P, Kursa M, Mechtler K, Kircheis R, Wagner E. The size of DNA/transferrin–PEI complexes is an important factor for gene expression in cultured cells. Gene Ther 1998;5:1425–33. [10] Neu M, Fischer D, Kissel T. Recent advances in rational gene transfer vector design based on poly (ethylene imine) and its derivatives. J Gene Med 2005;7:992–1009. [11] Nimesh S, Aggarwal A, Kumar P, Singh Y, Gupta KC, Chandra R. Influence of acyl chain length on transfection mediated by acylated PEI nanoparticles. Int J Pharm 2007;337:265–74. [12] Nie Y, Günther M, Gu Z, Wagner E. Pyridylhydrazone-based PEGylation for pHreversible lipopolyplex shielding. Biomaterials 2011;32:858–69. [13] Gusachenko Simonova O, Kravchuk Y, Konevets D, Silnikov V, Vlassov VV, Zenkova MA. Transfection efficiency of 25-kDa PEI-cholesterol conjugates with different levels of modification. J Biomater Sci Polym Ed 2009;20:1091–110. [14] Dehshahri A, Oskuee RK, Shier WT, Hatefi A, Ramezani M. Gene transfer efficiency of high primary amine content, hydrophobic, alkyl-oligoamine derivatives of polyethylenimine. Biomaterials 2009;30:4187–94. [15] Wakebayashi D, Nishiyama N, Yamasaki Y, Itaka K, Kanayama N, Harada A, et al. Lactose-conjugated polyion complex micelles incorporating plasmid DNA as a targetable gene vector system: their preparation and gene transfecting efficiency against cultured HepG2 cells. J Control Release 2004;95:653–64. [16] Incani V, Lavasanifar A, Uludagˇ H. Lipid and hydrophobic modification of cationic carriers on route to superior gene vectors. Soft Matter 2010;6:2124–38. [17] Liu WG, Zhang X, Sun SJ, Sun GJ, Yao KD, Liang DC, et al. N-alkylated chitosan as a potential nonviral vector for gene transfection. Bioconjug Chem 2003;14:782–9. [18] Neamnark A, Suwantong O, Bahadur KCR, Hsu CYM, Supaphol P, Uludag˘ H. Aliphatic lipid substitution on 2 kDa polyethylenimine improves plasmid delivery and transgene expression. Mol Pharm 2009;6:1798–815.
[19] Incani V, Tunis E, Clements BA, Olson C, Kucharski C, Lavasanifar A, et al. Palmitic acid substitution on cationic polymers for effective delivery of plasmid DNA to bone marrow stromal cells. J Biomed Mater Res A 2007;81:493–504. [20] Le Gall T, Loizeau D, Picquet E, Carmoy N, Yaouanc JJ, Burel-Deschamps L, et al. A novel cationic lipophosphoramide with diunsaturated lipid chains: synthesis, physicochemical properties, and transfection activities. J Med Chem 2010;25(53):1496–508. [21] Zhang X, Ercelen S, Tikhonov VE, Karaeva SZ, Slita AV, Zarubaev VV, et al. Alkylated chitosans of low molecular weight as non-viral transfection vectors for gene therapy. Russ J Gen Chem 2008;78:1093–102. [22] Jeong JH, Kim SW, Park TG. Molecular design of functional polymers for gene therapy. Prog Polym Sci 2007;32:1239–74. [23] Ahmed OA, Pourzand C, Blagbrough IS. Varying the unsaturation in N4, N9dioctadecanoyl spermines: nonviral lipopolyamine vectors for more efficient plasmid DNA formulation. Pharm Res 2006;23:31–40. [24] Liu ZH, Zhang ZY, Zhou CR, Jiao YP. Hydrophobic modifications of cationic polymers for gene delivery. Prog Polym Sci 2010;35:1144–62. [25] Wang B, He C, Tang C, Yin C. Effects of hydrophobic and hydrophilic modifications on gene delivery of amphiphilic chitosan based nanocarriers. Biomaterials 2011;32:4630–8. [26] Doody AM, Korley JN, Dang KP, Zawaneh PN, Putnam D. Characterizing the structure/function parameter space of hydrocarbon-conjugated branched polyethylenimine for DNA delivery in vitro. J Control Release 2006;116:227–37. [27] Yi WJ, Feng ZH, Zhang QF, Zhang J, Li LD, Zhu W, et al. Diol glycidyl etherbridged cyclens: preparation and their applications in gene delivery. Org Biomol Chem 2011;9:2413–21. [28] Xiang YZ, Feng ZH, Zhang J, Liao YL, Yu CJ, Yi WJ, et al. Linear cyclen-based polyamine as a novel and efficient reagent in gene delivery. Org Biomol Chem 2010;8:640–7. [29] Li S, Wang Y, Wang S, Zhang J, Wu SF, Wang BL, et al. Biodegradable cyclenbased linear and cross-linked polymers as non-viral gene vectors. Bioorg Med Chem 2012;20:1380–7. [30] De León-Rodríguez LM, Kovacs Z, Esqueda-Oliva AC, Miranda-Olvera AD. Highly regioselective N-trans symmetrical diprotection of cyclen. Tetrahedron Lett 2006;47:6937–40. [31] Peng W, Liu PY, Jiang N, Lin HH, Zhang GL, Liu Y, et al. Dinuclear macrocyclic polyamine zinc(II) complexes linked with flexible spacers: Synthesis, characterization, and DNA cleavage. Bioorg Chem 2005;33:374–85. [32] Yang J, Liu Y, Wang H, Liu L, Wang W, Wang C, et al. The biocompatibility of fatty acid modified dextran-agmatine bioconjugate gene delivery vector. Biomaterials 2012;33:604–13. [33] Kuhn PS, Levin Y, Barbosa MC. Charge inversion in DNA-amphiphile complexes: possible application to gene therapy. Phys A 1999;274:8–18. [34] Wang HF, Luo XH, Liu CW, Feng J, Zhang XZ, Zhuo RX. A smart micellar system with an amine-containing polycarbonate shell. Acta Biomater 2012;8:589–98. [35] Hsu CY, Hendzel M, Uludagˇ H. Improved transfection efficiency of an aliphatic lipid substituted 2 kDa polyethylenimine is attributed to enhanced nuclear association and uptake in rat bone marrow stromal cell. J Gene Med 2011;13:46–59. [36] Hsu CY, Uludag˘ H. Cellular uptake pathways of lipid-modified cationic polymers in gene delivery to primary cells. Biomaterials 2012;33:7834–48. [37] van der Aa LJ, Vader P, Storm G, Schiffelers RM, Engbersen JF. Optimization of poly(amido amine)s as vectors for siRNA delivery. J Control Release 2011;150:177–86.
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Cyclen-based lipidic oligomers as potential gene delivery vehicles Wen-Jing Yi a, Qin-Fang Zhang a, Ji Zhang a,⇑, Qiang Liu a, Laifeng Ren b, Qian-Ming Chen c, Liandi Guo b, Xiao-Qi Yu a,⇑ a
Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu 610064, People’s Republic of China Laboratory of Genome Stability, Development and Stem Cell Institute, The West China Second Hospital, Sichuan University, Chengdu 610041, People’s Republic of China c State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 7 June 2013 Received in revised form 25 November 2013 Accepted 9 December 2013 Available online 14 December 2013 Keywords: Gene delivery Cyclen Lipopolymer Non-viral vector Structure–activity relationship
a b s t r a c t A series of cyclen-based linear oligomers bearing hydrophobic long chains (lipopolymers Cy-LC, where Cy and LC represent cyclen-based linear backbone and hydrophobic long chain substituents, respectively) were designed and synthesized. The effects of type and degree of substitution (DS) of hydrophobic long chains on the transfection efficiency were systematically studied. The nitrogen atoms with relatively strong basicity on the cyclen ensure their good DNA binding ability, which was confirmed by gel retardation and ethidium bromide exclusion assays. Lipopolyplexes could be formed as nanoparticles with suitable sizes and zeta potentials for gene transfection. In vitro gene delivery experiments revealed that the linoleic acid (LIN) substituted material Cy-LIN has better transfection efficiency than 25 kDa polyethylenimine in the absence or in the presence of serum. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and hemolysis assays showed low cytotoxicity and good biocompatibility of the lipopolyplexes. Fluorescent labeled DNA was used to study the cellular uptake and intracellular distribution of transfected DNA. Flow cytometry results suggested that a long chain is necessary for efficient cellular uptake, and images from confocal laser scanning microscopy showed that after 4 h transfection, most of the fluorescent labeled DNA accumulated in the perinuclear region, which was required for efficient gene expression. Moreover, it was also found that the DS of the hydrophobic moiety can adjust the balance between DNA binding ability and dissociation of polyplexes, significantly affecting the transfection efficiency. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Safe and efficient delivery of genetic material remains the most challenging aspect of human gene therapy [1]. Although viral vectors are considered to have high gene transfer efficiency, their applications have been restricted by security issues such as immunogenicity, insertional mutagenesis, oncogenic effects and toxicity [2]. Non-viral gene carriers have been receiving increasing attention over the past decades because of their low cost, flexibility in chemical design and safety [3–5]. Consequently, numerous nonviral gene delivery vectors have been developed and reported in recent years. These non-viral systems can be divided into liposomes (lipoplexes), polycationic polymers (polyplexes) and organic or inorganic nanoparticles (nanoplexes) [6,7]. However, it should be noted that although non-viral gene delivery vectors such as cationic lipids and polymers have shown potential to overcome the safety problems, low transfection efficiency (TE) limits their use in clinical trials [8].
⇑ Corresponding authors. Fax: +86 28 85415886 (X.-Q. Yu).
The key steps involved in non-viral gene delivery include electrostatic complexation and condensation of DNA molecules into compact particles, uptake of complexes by the target cells and endosome escape and dissociation of the complexes to release DNA to nucleus, which is necessary for expression of the delivered genes [9–11]. Strategies to improve the efficiency and biocompatibility of cationic reagents for gene delivery typically involve grafting functional ligands such as peptides [12], lipids [13,14], sugars [15] or a combination [2] to improve the stability, targeting, uptake and sub-cellular trafficking capabilities of the vectors [16]. In this regard, hydrophobic modifications of cationic regents could facilitate DNA adsorption onto cellular surface by membrane fusion, and as a result, improve the endocytosis and TE [17]. Neamnark et al. reported that substitution of 2 kDa polyethylenimine (PEI) with aliphatic lipids including caprylic, myristic, palmitic, stearic, oleic and linoleic acids led to a TE comparable to 25 kDa PEI [18]. With the palmitic acid modification on poly-L-lysine), the product material gave the TE that was equivalent to adenoviral carrier [19]. Le Gall et al. reported that cationic lipophosphoramide with diunsaturated lipid chains could transfer the target gene into the lung with high efficiency in the in vivo gene delivery [20].
E-mail addresses: [email protected] (J. Zhang), [email protected] (X.-Q. Yu). 1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.12.010
W.-J. Yi et al. / Acta Biomaterialia 10 (2014) 1412–1422
Moreover, hydrophobic modifications of cationic reagents including chitosan [21], poly[2-(dimethylamino)ethyl methacrylate] [22], spermine [23], etc., showed an approximate increase of TE, which is likely due to the balanced protection and release of DNA as well as to their function as membrane-anchoring moieties. Moreover, the alkyl chain length and degree of substitution (DS) may also affect some other properties such as cytotoxicity, biocompatibility and in vivo circulation time [24]. A delicate balance between hydrophilic and hydrophobic components is crucial for the design of new cationic carriers [25]. Optimal chain length and DS should be selected by considering the overall effects of hydrophobic modifications on the gene therapy [26]. However, there are few reports of systematic investigation of the structure–activity relationship of hydrophobic modified non-viral gene delivery vectors. Our group previously reported a series of cyclen-based cationic polymers showing comparable transfection efficiency to 25 kDa PEI with relatively lower cytotoxicity [27–29]. It is of great importance to further investigate the structure–activity relationship of this type of cationic polymer. Herein we studied the effects of the structures and DS of lipidic moiety on the cyclen-based linear oligomer. Results show that the long chain modified complex could transfer target gene into several cell lines with higher TE and lower cytotoxicity compared to 25 kDa PEI, and the increased TE might be attributed to the enhanced cellular uptake and dissociation of the polyplexes inside the cell. We considered that these studies would be particularly important to better understand the relationships between the lipid structure itself (length, amount, saturated vs. unsaturated or polyunsaturated) and the physicochemical properties of the corresponding polyplexes, as well as their transfection activities.
2. Materials and methods 2.1. Materials and general measurements Anhydrous ethanol, dichloromethane and epichlorohydrin were dried and purified under nitrogen by using standard methods and were distilled immediately before use. 1,7-Bis (tert-butyloxycarbonyl)-1,4,7,10-tetraazacyclodocane was prepared according to the literature [30]. High molecular weight PEI (branched, average molecular weight 25 kDa: 25 kDa PEI), and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Sigma–Aldrich (St Louis, MO, USA). The plasmids used in the study were pGL-3 (Promega, Madison, WI, USA) coding for luciferase DNA and pEGFP-N1 (Clontech, Palo Alto, CA, USA) coding for EGFP DNA. Cy™5 was obtained from Molecular Probe (Mirus, Madison, WI, USA). Dulbecco’s modified Eagle’s medium (DMEM), 1640 medium and fetal bovine serum (FBS) were purchased from Invitrogen Corp. MicroBCA protein assay kit was obtained from Pierce (Rockford, IL, USA). Luciferase assay kit was purchased from Promega (Madison, WI, USA). Endotoxin free plasmid purification kit was purchased from TIANGEN (Beijing, China). 293T human embryonic kidney cell lines, A549 lung cancer cell lines and U2OS human osteosarcoma cancer cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. All other reagents used in the synthesis, if not specified, were obtained from Sigma–Aldrich and used without further purification. Characterization and structural confirmation of intermediates and products were performed by proton nuclear magnetic resonance (1H NMR) (Varian INOVA-400 spectrometer) and electrospray ionization time-of-flight (ESI-TOF, Waters Q-TOF Premier) mass spectrometry. The molecular weights of the oligomers were determined by a Waters 717 plus autosampler (Waters Corp., Milford, MA, USA) using tetrahydrofuran (THF) as eluent at a flow
1413
rate of 1.0 ml min1. Two Waters Styragel columns (HR3 and HR4) were used in series, and the detector (Waters 2414 refractive index detector) and columns were maintained at 45 °C throughout the runs. Linear polystyrene standards were used for calibration. 100 ll of each sample prepared at 2 mg ml1 was injected, and each sample was given 40 min to elute off of the column. Fluorescence spectra were measured by a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer. 2.2. Synthesis of lipid-substituted compound and polymers 2.2.1. Synthesis of lipid-diethanolamine compound (1) Aliphatic acid (caprylic, lauric, palmitic, stearic, oleic and linoleic acid, 0.04 mol) was dissolved in anhydrous dichloromethane (CH2Cl2, 50 ml). The solution was stirred at 0 °C for 10 min followed by the addition of isobutyl chloroformate (5.46 g, 0.04 mol) and N-methylmorpholine (4.05 g, 0.04 mol). After addition of diethanolamine (5.05 g, 0.048 mol), the solution was stirred at 0 °C for 30 min and at 25 °C for 6 h. The solution was washed with sat. NaHCO3 (2 50 ml), and brine (2 50 ml). The organic layer was dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 1, whose detailed analytical data are listed in the Supplementary data. 2.2.2. Synthesis of epoxy-lipid-diethanolamine cross-linkers (2) Compounds 2 were prepared according to the literature [31]. In a typical procedure, a mixture of epichlorhydrin (9.34 ml, 0.12 mol), lipid-diethanolamine 1 (0.02 mol), sodium hydroxide pellets (4.8 g, 0.12 mol), water (1 ml, 0.056 mol) and tetrabutylammonium chloride (0.3224 g, 0.001 mol) was vigorously stirred for 2 h at 40 °C. The solid produced in the reaction was filtered off and washed with dichloromethane. The combined organic layer was dried with anhydrous magnesium sulfate. The solvent and excess epichlorhydrin were distilled off to give oily products. The residue was purified by silica gel column chromatography to give the product 2, whose detailed analytical data are listed in the Supplementary data. 2.2.3. Synthesis of hydrophobically modified polymer (Cy-LC) 1,7-Bis(tert-butyloxycarbonyl)-1,4,7,10-tetraazacyclodocane (223.5 mg, 0.6 mmol) was dissolved in 0.5 ml of C2H5OH, then compound 2 (0.6 mmol) was added to the solution. Under the protection of N2, the reaction mixture was stirred at 80 °C for 80 h. The reaction mixture was stirred overnight after adding 50 ml saturated MeOH–HCl solution at room temperature and purifying by dialysis (MWCO 3.5 kDa) against pure water for 3 days. The retentate was lyophilized to give the product. Using 1H NMR (400 MHz, D2O), the integrals of the characteristic proton peaks of the substituted fatty acids (d 0.8 ppm, –CH3 and d 1.3 ppm, –CH2) and of polymer backbone (d 2.8–4.0 ppm) were obtained. 2.2.4. Synthesis of hydrophobically modified polymer with different DS (Cy-LIN0.53) Cyclen-based linear oligomer (LCPA) (0.036 mmol) was prepared according to the literature [28], and was dissolved in CH2Cl2. The solution was stirred at 0 °C for 10 min followed by the addition of various amounts of linoleic acid (fatty acid:LCPA ratios = 0.5, 1, 2 and 3), HOBt (N-hydroxybenzotriazole, 0.036 mmol) and EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.036 mmol). After addition of DIEA (N,N-diisopropylethylamine, 0.036 mmol), the solution was stirred at 25 °C for 6 h. The solvent was removed under reduced pressure and added to 30 ml saturated MeOH–HCl solution at room temperature and purified by dialysis (molecular weight cut-off (MWCO) 1000 kDa) against pure water for 3 days. The retentate was lyophilized to give the product
1414
W.-J. Yi et al. / Acta Biomaterialia 10 (2014) 1412–1422
Cy-LIN0.53. Using 1H NMR (400 MHz, D2O), the integrals of the characteristic proton peaks of the substituted fatty acids (d 0.86 ppm, –CH3; d 1.27 ppm, –CH2 and d 5.28 ppm, –CH@CH) and of polymer backbone (d 2.5–4.1 ppm) were obtained.
was added to each well. After 4 h, unreacted dye was removed by aspiration. The formazan crystals were dissolved in 150 ll dimethyl sulfoxide per well and measured spectrophotometrically in an ELISA plate reader (model 550, BioRad) at a wavelength of 570 nm. The cell survival was expressed as follows: cell viability = (ODtreated/ODcontrol) 100%.
2.3. Gel retardation assay Plasmid pEGFP-N1 (BD Biosciences Clontech) and pGL3-control (Promega) were amplified in Escherichia coli JM109 and extracted with Endo-Free Plasmid Maxi Kit (E.Z.N.A.™, Omega). Polymers/ DNA complexes were freshly prepared at various w/w ratios. 5 ll complex solution containing 0.2 lg plasmid pGL3-control was incubated for 30 min at room temperature and run on 1% (w/v) agarose gel at 80 V for 40 min. Gel was stained with ethidium bromide (EB) and the mobility of pDNA bands were analyzed using a Molecular Imager ChemiDoc XRS + (BIO-RAD, USA). 2.4. EB displacement assay The ability of polymers to condense DNA was studied using EB exclusion assays [14]. Fluorescence spectra were measured at room temperature in air by a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer and corrected for the system response. EB (2.5 ll, 1 mg ml1) was put into a quartz cuvette containing 2.5 ml of 10 mM Hepes solution (pH = 7.4). After shaking, the fluorescence intensity of EB was measured. Then CT DNA (10 ll, 1 mg ml1) was added to the solution and mixed symmetrically, and the measured fluorescence intensity is the result of the interaction between DNA and EB. Subsequently, the solutions of polymer (1 mg ml1, 2 ll for each addition) were added to the above solution for further measurement. All the samples were excited at 520 nm and the emission was measured at 600 nm. 2.5. Measurement of particle size and zeta potential The complexes of polymers/DNA at various w/w ratios ranging from 2 to 16 were prepared as described above. 1 lg pGL3-control and the corresponding amount of polymers were respectively dissolved in 100 ll PBS buffer (pH = 7.4), and mixed. After 30 min incubation at room temperature, polymers/DNA complexes were diluted with 900 ll H2O, and the zeta potentials and the hydrodynamic diameters were measured using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd, UK) at 25 °C. 2.6. Transmission electron microscopy (TEM) TEM images were obtained on a JEM-100CX (JEOL) transmission electron microscope at an acceleration voltage of 100 kV. The TEM samples were prepared by dipping a copper grid with Formvar film into the freshly prepared nanoparticles solution (1.0 lg ml1). A few minutes after the deposition, the aqueous solution was blotted away with a strip of filter paper and then the samples were dried for 2 min at room temperature. The samples were stained with phosphotungstic acid (ATP) aqueous solution and dried in air. 2.7. Cytotoxicity of the complexes evaluation Cytotoxicity of polymer/DNA and PEI/DNA complexes were evaluated by MTT assay. 1.5 104 cells per well 293T, U2Os and A549 cells were respectively seeded in 96-well plates. After 24 h culture, the lipopolyplexes (vector-DNA complexes) or free vectors were added to cells at different chosen w/w ratios and concentrations. For complexes, the final pGL-3 concentration is 0.2 lg per well in a total volume of 100 ll for another 24 h. After that, the medium was replaced again with 200 ll of fresh medium, and 20 ll of sterile filtered MTT (5 mg ml1) stock solution in PBS
2.8. Gene transfection efficiency assay in vitro 2.8.1. Gene delivery studies: expression of the Luc gene A549, U2Os and 293T cells were seeded at a density of 7 104 cells per well in a 24-well plate in DMEM (U2Os and 293T cells) or RPMI-1640 medium (A549 cells) containing 10% FBS and grown to reach 70–80% confluence prior to transfection. For transfection in the absence of serum, the medium of each well was exchanged for fresh serum-free medium. For transfection in the presence of serum, the medium of each well was not changed at this time. Subsequently, the cells were treated with polyplexes (containing 1 lg of pGL3-control or pEGFP-N1) at different (w/w) weight ratios and PEI/DNA at weight ratio of 1.4 (N/P = 10) for 4 h at 37 °C. The medium was then completely refreshed with the completed culture media. After an additional 24 h incubation, the medium was removed and the cells were washed with 500 ll 1 PBS (pH 7.4) twice, lysed with 100 ll 1 lysis reporter buffer (Promega) and centrifuged at 12,000g for 3 min at 4 °C. Luciferase activity in 20 ll supernatant was evaluated with a luciferase assay system (Promega). The gene transfection efficiency of each sample was represented by firefly luciferase expression and calculated as relative light units per milligram of total protein (RLU/mg protein). Protein concentrations in cell lysates were determined using BCA Protein Assay Kit (Pierce) with bovine serum albumin (BSA) as standard. 2.8.2. Gene delivery studies: expression of the EGFP gene Enhanced green fluorescent protein expression studies were carried out as mentioned above for the Luc gene expression. 24 h after transfection, cells were observed with an inverted fluorescence microscope (Nikon Eclipse TE 2000E) equipped with a cold Nikon camera. Digital image recording and image analysis were performed with the NIS Elements Advanced Research (version 2.31) software. GFP expressions were quantitatively measured using flow cytometry. The cell suspensions were evaluated using Aria 2 flow cytometry and 10,000 cells were evaluated in each experiment. Data acquisition and analysis were performed using Aria 2 flow cytometry (Becton, Dickinson and Company). GFP fluorescence from the expression of the plasmid DNA was measured in the FL1 channel using the 488 nm blue laser. Untransfected cells were used to set the background. 2.9. Cellular uptake of plasmid DNA The cellular uptake of the polymer/fluorescein labeled-DNA complexes was analyzed by flow cytometry. The Label IT Cy5 Labeling Kit was used to label pDNA with Cy5 according to the manufacturer’s protocol. Briefly, A549 cells were seeded onto 12well plates (1.6 105 cells per well) and allowed to attach and grow for 24 h. For transfection in the absence of serum, the medium was exchanged with serum-free medium. As for transfection in the presence of serum, the medium was exchanged with serum-containing medium. Cells were incubated with Cy5 labeled DNA nanoparticles (2 lg DNA per well, optimal N/P ratio of each sample) in media for 4 h at 37 °C. Subsequently, the cells were washed with 1 PBS and harvested with 0.25% trypsin/ethylenediaminetetraacetic acid and resuspended in 1 PBS. Mean fluorescence intensity was analyzed using FACSCalibur flow
W.-J. Yi et al. / Acta Biomaterialia 10 (2014) 1412–1422
cytometer (Becton Dickinson and Company). Cy5-labeled plasmid DNA uptake was measured in the FL4 channel using the red diode laser (633 nm). Data from 10,000 events were gated using forward and side scatter parameters to exclude cell debris. The flow cytometer was calibrated for each run to obtain a background level of 1% for control samples (i.e., untreated cells). 2.10. Confocal laser scanning microscopy (CLSM) analysis A549 cells were seeded at a density of 6 104 cells per well onto the 24-well plate with a sterile cover glass (8 mm 8 mm) in each well and incubated for 24 h. For transfection in the absence of serum, the medium was exchange with serum-free medium. Complexes of polymers and Cy5-labeled pGL3 at a given concentration were added to each well. After 4 h, the cells on the cover glass were washed three times with PBS buffer and fixed with 4% paraformaldehyde (dissolved with PBS buffer) for 10 min; nuclear staining was done with 40 ,6-diamidino-2-phenylindole (DAPI). The CLSM observation was performed using an Olympus FV1000 – IX81 (Leica, Germany) with a 40 objective at excitation wavelengths of 405 nm for DAPI (blue), 633 nm for Cy5 (red), respectively. 2.11. Hemolytic activity test The hemolytic activity was measured based on the reported method [32]. A total of 200 ll of diluted rabbit blood was added to different polyplexes and the volume was adjusted to 1 ml with sterile PBS (pH = 7.4). 200 ll of rabbit blood mixed with 800 ll of the PBS served as the positive control, and 200 ll of rabbit blood mixed with 800 ll of the PBS containing an excess of ammonium chloride to cause complete hemolysis was used as the negative control. After the vials were incubated at 37 °C for 90 min, the solutions were centrifuged at 2000 rpm for 10 min. A total of 200 ll of the supernatant was collected and seeded in each well of a 96-well plate. The absorbance at a wavelength of 543 nm was recorded on a Synergy HT Multi-Mode Microplate Reader (BioTek, USA). The percentage hemolysis (PH%) (mean% ± SD, n = 3) was calculated using the following formula: PH% = (As Ap)/(An Ap) %, where As, An and Ap represent the absorbance of sample, negative and positive controls, respectively.
3. Results and discussion 3.1. Synthesis of the hydrophobic long chain modified oligomers Cy-LC Scheme 1 summarizes the general synthetic methods for the hydrophobic-modified oligomers, which were named according to the abbreviations of the hydrophobic groups. Diol glycidyl ethers 2 with various long chains were first prepared to act as the bridge molecule. Subsequent ring-opening polymerization took place between 1,7-diprotected cyclen and 2. The final deprotection by HCl and dialysis gave the comb-like products Cy-LC. The dialysis yields of the oligomers were not greatly different, suggesting that similar polymerization degrees were achieved for the six products. These new compounds were obtained as hydrochlorides and characterized by 1H NMR (400 MHz) using D2O as solvent. Since the molecular weights of Cy-LC could not be regularly measured by gel permeation chromatography (GPC), the indirect method was used by employing Boc-substituted substrates, whose molecular weights were measured to be in the range of 2599–3603 (polydispersity index: 1.1–1.2, Table S.1 of Supplementary data) by GPC using THF as mobile phase, indicating an average of 4–5 repeating
1415
units in one molecule. All of the target oligomers were found to be well soluble in pure water. 3.2. Interaction with plasmid DNA The ability of polymers to bind DNA to form polyplex is a prerequisite for efficient DNA transfection. Gel electrophoresis has been widely used to reveal the interaction between different polymers and DNA. As reported in previous work, hydrophobic moieties could facilitate a cooperative binding of polymer with DNA [33]. To identify the appropriate vector/DNA weight ratio required for efficient condensation, a gel mobility assay was carried out. As shown in Fig. 1A, full retardation of plasmid DNA was observed from the w/w ratio of 4. For compound Cy-LIN, the ratio for full retardation was even lower. These results show that all the oligomers can effectively bind DNA at a relatively low dosage. On the other hand, the binding abilities of these vectors were further evaluated through EB exclusion assay. The fluorescence intensity of EB is significantly increased by nucleic acid intercalation. When a cationic agent binds to the plasmid, some of the intercalated EB is substituted, resulting in measurable reduction of the fluorescence intensity. Fig. 1B shows that the addition of Cy-LC to DNA pretreated with EB caused considerable decrease of fluorescence intensity, which was almost completely quenched at the w/w ratio of 2. Generally, the oligomer with the shorter aliphatic chain showed the better fluorescent quenching ability. This might be due to its weaker ability to screen the positive charge on the cyclen moiety which was necessary for DNA condensation. Having the proper size and shape of polymer/DNA complexes is essential for efficient gene delivery. The effective diameters of the various alkyl-modified polyplexes were measured by dynamic light scattering (DLS). As shown in Fig. 2A, polyplex particles were formed with the sizes in the range of 100–250 nm at different w/w ratios. For oligomers with longer hydrophobic chain (Cy-STE, CyOLE and Cy-LIN), the 18-C length substituent might facilitate the formation of micelle-like structure, which would condense DNA more compactly. Consequently, the average sizes of the polyplexes formed from these three vectors were distinctly smaller (95%) than PEI, indicating that the hydrophobic chain has a significant positive effect on the endocytosis by membrane fusion. CLSM was also used to study the intracellular distribution of Cy5-labeled DNA transfected by Cy-LINx, and Fig. 10C shows the typical results involving Cy-LIN2. It was shown that after 4 h transfection, most of the fluorescent labeled DNA was accumulated in the perinuclear region, which was necessary for efficient gene expression.
hydrophobic moiety plays an important role in the gene delivery, and it may affect the balance between DNA binding ability and dissociation of polyplexes. Good TE together with excellent biocompatibility mean that this type of lipopolymer is promising as a non-viral gene delivery vector. Subsequent studies will focus on further development of the lipopolymers, such as the introduction of biodegradable or cell-targeting moieties.
Acknowledgements This work was financially supported by the National Program on Key Basic Research Project of China (973 Program, 2012CB720603 and 2013CB328900), the National Science Foundation of China (No. 21232005) and State Key Lab of Oral Diseases (Sichuan University) (SKLODSCUKF2012-02). J. Z. thanks the Program for New Century Excellent Talents in University (NCET11-0354). We also thank Analytical & Testing Center of Sichuan University for structural analysis of the compounds.
4. Conclusions Appendix A. Figures with essential colour discrimination In this report, LCPA was modified with hydrophobic long chains through two different methods. One is the polymerization between diBoc-protected cyclen and long chain contained bridge, and the other is direct acylation of LCPA. The former method helps us to study the effect of different hydrophobic chains, and the latter method gives a way to investigate the effect of the DS on gene delivery. Compared to LCPA, the hydrophobic modified materials gave much higher TE, which was also higher than 25 kDa PEI. The structure–activity relationship of the lipopolymers was studied, and the long chain group (especially linoleic acid substituent) was found to be crucial for efficient cellular uptake and nuclear orientation. Furthermore, it was also found that the DS of
Certain figures in this article, particularly Figs. 1–10 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2013. 12.010.
Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2013. 12.010.
1422
W.-J. Yi et al. / Acta Biomaterialia 10 (2014) 1412–1422
References [1] Santos JL, Oliveira H, Pandita D, Rodrigues J, Pêgo AP, Granja PL, et al. Functionalization of poly (amidoamine) dendrimers with hydrophobic chains for improved gene delivery in mesenchymal stem cells. J Control Release 2010;144:55–64. [2] Mintzer MA, Simanek EE. Nonviral vectors for gene delivery. Chem Rev 2009;109:259–302. [3] Russ V, Günther M, Halama A, Ogris M, Wagner E. Oligoethylenimine-grafted polypropylenimine dendrimers as degradable and biocompatible synthetic vectors for gene delivery. J Control Release 2008;132:131–40. [4] Merkel OM, Beyerle A, Librizzi D, Pfestroff A, Behr TM, Sproat B, et al. Nonviral siRNA delivery to the lung: investigation of PEG-PEI polyplexes and their in vivo performance. Mol Pharmacol 2009;6:1246–60. [5] Kogure K, Moriguchi R, Sasaki K, Ueno M, Futaki S, Harashima H. Development of a non-viral multifunctional envelope-type nano device by a novel lipid film hydration method. J Control Release 2004;98:317–23. [6] Ravi Kumar MN, Sameti M, Mohapatra SS, Kong X, Lockey RF, Bakowsky U, et al. Cationic silica nanoparticles as gene carriers: synthesis, characterization and transfection efficiency in vitro and in vivo. J Nanosci Nanotechnol 2004;4:876–81. [7] Kumar MN, Mohapatra SS, Kong X, Jena PK, Bakowsky U, Lehr CM. Cationic poly (lactide-co-glycolide) nanoparticles as efficient in vivo gene transfection agents. J Nanosci Nanotechnol 2004;4:990–4. [8] Forrest ML, Meister GE, Koerber JT, Pack DW. Partial acetylation of polyethylenimine enhances in vitro gene delivery. Pharm Res 2004;21:365–71. [9] Ogris M, Steinlein P, Kursa M, Mechtler K, Kircheis R, Wagner E. The size of DNA/transferrin–PEI complexes is an important factor for gene expression in cultured cells. Gene Ther 1998;5:1425–33. [10] Neu M, Fischer D, Kissel T. Recent advances in rational gene transfer vector design based on poly (ethylene imine) and its derivatives. J Gene Med 2005;7:992–1009. [11] Nimesh S, Aggarwal A, Kumar P, Singh Y, Gupta KC, Chandra R. Influence of acyl chain length on transfection mediated by acylated PEI nanoparticles. Int J Pharm 2007;337:265–74. [12] Nie Y, Günther M, Gu Z, Wagner E. Pyridylhydrazone-based PEGylation for pHreversible lipopolyplex shielding. Biomaterials 2011;32:858–69. [13] Gusachenko Simonova O, Kravchuk Y, Konevets D, Silnikov V, Vlassov VV, Zenkova MA. Transfection efficiency of 25-kDa PEI-cholesterol conjugates with different levels of modification. J Biomater Sci Polym Ed 2009;20:1091–110. [14] Dehshahri A, Oskuee RK, Shier WT, Hatefi A, Ramezani M. Gene transfer efficiency of high primary amine content, hydrophobic, alkyl-oligoamine derivatives of polyethylenimine. Biomaterials 2009;30:4187–94. [15] Wakebayashi D, Nishiyama N, Yamasaki Y, Itaka K, Kanayama N, Harada A, et al. Lactose-conjugated polyion complex micelles incorporating plasmid DNA as a targetable gene vector system: their preparation and gene transfecting efficiency against cultured HepG2 cells. J Control Release 2004;95:653–64. [16] Incani V, Lavasanifar A, Uludagˇ H. Lipid and hydrophobic modification of cationic carriers on route to superior gene vectors. Soft Matter 2010;6:2124–38. [17] Liu WG, Zhang X, Sun SJ, Sun GJ, Yao KD, Liang DC, et al. N-alkylated chitosan as a potential nonviral vector for gene transfection. Bioconjug Chem 2003;14:782–9. [18] Neamnark A, Suwantong O, Bahadur KCR, Hsu CYM, Supaphol P, Uludag˘ H. Aliphatic lipid substitution on 2 kDa polyethylenimine improves plasmid delivery and transgene expression. Mol Pharm 2009;6:1798–815.
[19] Incani V, Tunis E, Clements BA, Olson C, Kucharski C, Lavasanifar A, et al. Palmitic acid substitution on cationic polymers for effective delivery of plasmid DNA to bone marrow stromal cells. J Biomed Mater Res A 2007;81:493–504. [20] Le Gall T, Loizeau D, Picquet E, Carmoy N, Yaouanc JJ, Burel-Deschamps L, et al. A novel cationic lipophosphoramide with diunsaturated lipid chains: synthesis, physicochemical properties, and transfection activities. J Med Chem 2010;25(53):1496–508. [21] Zhang X, Ercelen S, Tikhonov VE, Karaeva SZ, Slita AV, Zarubaev VV, et al. Alkylated chitosans of low molecular weight as non-viral transfection vectors for gene therapy. Russ J Gen Chem 2008;78:1093–102. [22] Jeong JH, Kim SW, Park TG. Molecular design of functional polymers for gene therapy. Prog Polym Sci 2007;32:1239–74. [23] Ahmed OA, Pourzand C, Blagbrough IS. Varying the unsaturation in N4, N9dioctadecanoyl spermines: nonviral lipopolyamine vectors for more efficient plasmid DNA formulation. Pharm Res 2006;23:31–40. [24] Liu ZH, Zhang ZY, Zhou CR, Jiao YP. Hydrophobic modifications of cationic polymers for gene delivery. Prog Polym Sci 2010;35:1144–62. [25] Wang B, He C, Tang C, Yin C. Effects of hydrophobic and hydrophilic modifications on gene delivery of amphiphilic chitosan based nanocarriers. Biomaterials 2011;32:4630–8. [26] Doody AM, Korley JN, Dang KP, Zawaneh PN, Putnam D. Characterizing the structure/function parameter space of hydrocarbon-conjugated branched polyethylenimine for DNA delivery in vitro. J Control Release 2006;116:227–37. [27] Yi WJ, Feng ZH, Zhang QF, Zhang J, Li LD, Zhu W, et al. Diol glycidyl etherbridged cyclens: preparation and their applications in gene delivery. Org Biomol Chem 2011;9:2413–21. [28] Xiang YZ, Feng ZH, Zhang J, Liao YL, Yu CJ, Yi WJ, et al. Linear cyclen-based polyamine as a novel and efficient reagent in gene delivery. Org Biomol Chem 2010;8:640–7. [29] Li S, Wang Y, Wang S, Zhang J, Wu SF, Wang BL, et al. Biodegradable cyclenbased linear and cross-linked polymers as non-viral gene vectors. Bioorg Med Chem 2012;20:1380–7. [30] De León-Rodríguez LM, Kovacs Z, Esqueda-Oliva AC, Miranda-Olvera AD. Highly regioselective N-trans symmetrical diprotection of cyclen. Tetrahedron Lett 2006;47:6937–40. [31] Peng W, Liu PY, Jiang N, Lin HH, Zhang GL, Liu Y, et al. Dinuclear macrocyclic polyamine zinc(II) complexes linked with flexible spacers: Synthesis, characterization, and DNA cleavage. Bioorg Chem 2005;33:374–85. [32] Yang J, Liu Y, Wang H, Liu L, Wang W, Wang C, et al. The biocompatibility of fatty acid modified dextran-agmatine bioconjugate gene delivery vector. Biomaterials 2012;33:604–13. [33] Kuhn PS, Levin Y, Barbosa MC. Charge inversion in DNA-amphiphile complexes: possible application to gene therapy. Phys A 1999;274:8–18. [34] Wang HF, Luo XH, Liu CW, Feng J, Zhang XZ, Zhuo RX. A smart micellar system with an amine-containing polycarbonate shell. Acta Biomater 2012;8:589–98. [35] Hsu CY, Hendzel M, Uludagˇ H. Improved transfection efficiency of an aliphatic lipid substituted 2 kDa polyethylenimine is attributed to enhanced nuclear association and uptake in rat bone marrow stromal cell. J Gene Med 2011;13:46–59. [36] Hsu CY, Uludag˘ H. Cellular uptake pathways of lipid-modified cationic polymers in gene delivery to primary cells. Biomaterials 2012;33:7834–48. [37] van der Aa LJ, Vader P, Storm G, Schiffelers RM, Engbersen JF. Optimization of poly(amido amine)s as vectors for siRNA delivery. J Control Release 2011;150:177–86.
