International Journal of Pharmaceutics 492 (2015) 233–243
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Polyamidoamine (PAMAM) dendrimers modified with short oligopeptides for early endosomal escape and enhanced gene delivery Le Thi Thuy, Sudipta Mallick, Joon Sig Choi* Department of Biochemistry, Chungnam National University, Gung-dong 220, Yuseong-gu, Daejeon 305-764, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 March 2015 Received in revised form 15 June 2015 Accepted 7 July 2015 Available online 14 July 2015
Recently, non-viral vectors have become a popular research topic in the field of gene therapy. In this study, we conjugated short oligopeptides to polyamidoamine-generation 4 (PAMAM G4) to achieve higher transfection efficiency. Previous reports have shown that the PAMAM G4-histidine (H)-arginine (R) dendrimer enhances gene delivery by improving cell penetration and internalization mechanisms. Therefore, we synthesized PAMAM G4-H phenylalanine (F) R, PAMAM G4-FHR and PAMAM G4-FR derivatives to determine the best gene carrier with the lowest toxicity. Physicochemical studies were performed to determine mean diameters and surface charge of PAMAM derivatives/pDNA polyplexes. DNA condensation was confirmed using a gel retardation assay. Cytotoxicity and transfection efficiency were analyzed using human cervical carcinoma (HeLa) and human liver carcinoma (HepG2) cells. Similar levels of transfection were achieved in both cell lines by using gold standard transfection reagent PEI 25 kD. Therefore, our results show that these carriers are promising and may help achieve higher transfection with negligible cytotoxicity. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Polyamidoamine dendrimer Oligopeptides Hydrophobic Transfection Polyplex Cytotoxicity
1. Introduction Currently, gene therapy is considered a potential therapy for human diseases such as diabetes and various cancers where the etiological factors include defective genes (Guo and Huang, 2011; Oupický et al., 2000; Zhang et al., 2012). Typical gene therapy procedures involve the introduction of extracellular therapeutic genes that can knock down dysfunctional genes or express proteins. This process has emerged as a multidisciplinary field of research, which emphasizes the physicochemical and pharmaceutical properties of the therapies under development. The development of a superior means of delivering gene products to the site of action would enhance the therapeutic efficacy (Zhang et al., 2012). In particular, nanotechnology has promising applications in the field of gene therapy because it possesses excellent characteristics that satisfy the prerequisites for biomolecule carriers (Mintzer and Simanek, 2008). These characteristics include a prolonged circulation time, negligible immune reactions, enhanced cellular uptake, and early endosomal escape (Mintzer and Simanek, 2008). Therefore, different vectors have been exploited to achieve all the characteristics mentioned above (Kamimura et al., 2011;
* Corresponding author. Tel.: +82 42 821 5489; Fax: +82 42 822 7548. E-mail address: [email protected] (J.S. Choi). http://dx.doi.org/10.1016/j.ijpharm.2015.07.017 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
Nayerossadat et al., 2012). While viral vectors are well known for efficient gene expression, their mutagenicity, and immune reactions are a matter of concern (Kamimura et al., 2011; Nayerossadat et al., 2012; Robbins and Ghivizzani, 1998). Therefore, non-viral vectors have attracted much attention because of their stability, biocompatibility, and surface functionality in vitro and in vivo. However, their transfection efficiency is not as high as that of viral vectors (Akhtar, 2005). Previous studies have proven that cationic polymeric vectors, liposomes, and dendrimers are the best candidates for the complete condensation of negatively charged DNA to nano-sized particles, which they subsequently deliver to cells (Mintzer and Simanek, 2008; Nayerossadat et al., 2012; Zhang et al., 2012). The non-viral vector polyethylenimine (PEI) is considered the gold standard for gene delivery because of its numerous amine groups, which facilitate greater cellular uptake and endosomal escape under mildly acidic conditions (Fu et al., 2012; Kircheis et al., 2001). However, the PEI particle is highly positively charged, which leads to cell membrane disruption and eventual cell death (Tung and Weissleder, 2003). In contrast, the polyamidoamine (PAMAM) dendrimers, which have a uniquely branched structure currently offer a similar level of transfection when modified with cell penetration peptides (CPPs) (Choi et al., 2004; Kim et al., 2009; Kono et al., 2004; Kono et al., 2008; Lee et al., 2011; Majoros et al., 2005; Shcharbin et al., 2009). The previously reported PAMAMgeneration 4 (PAMAM G4) dendrimer conjugated with arginine (R)
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and histidine (H)—R, was previously reported to have excellent transfection efficiency with a negligible cytotoxicity in normal cells (Shi et al., 2013; Zhang et al., 2014). The higher transfection efficiency of this conjugate was attributed to the synergistic effects of the cationic amino acid arginine that enhances cellular uptake
(Futaki, 2005; Luo et al., 2012; Rothbard et al., 2002). In addition, induction of the proton sponge effect by histidine further enhanced this efficiency by overpowering the acidic environment inside the endosomal compartment (Yu et al., 2011).
Fig. 1. Schematic diagram of synthesis of polyamidoamine-generation 4 (PAMAM G4)-histidine-phenylalanine-arginine (HFR), polyamidoamine-generation 4 (PAMAM G4)phenylalanine-histidine-arginine (FHR), polyamidoamine-generation 4 (PAMAM G4)-phenylalanine-arginine (FR).
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Furthermore, CPPs containing hydrophobic amino acid like phenylalanine (F) and leucine, show increased transfection efficiency because of their enhanced interaction with the cell membrane (Choi et al., 2007; Kurisawa et al., 2000; Sang Yoo et al., 2005; Tian et al., 2007; Wang et al., 2009). The endocytosis process is believed to occur when PAMAM G4 is terminally modified with phenylalanine (F) because the hydrophobic environment of the PAMAM G4 facilitates its interaction with the cell membrane fatty acids. In addition, studies on hydrophobic amino acids have shown that F has the highest membrane-disruptive activity in acidic pH induced by osmotic swelling, which leads to the release of endocytosed payloads into the cytoplasm (Castelletto and Hamley, 2009; Liu et al., 2010). However, single amino acid functionalization is not sufficient to surmount the biological barriers. Therefore, in the present study, we modified PAMAM G4 with various combinations of H, R, and F to incorporate the synergistic advantage of the 3 amino acids. We studied the gene transfection efficiency of 3 different oligopeptide sequences including PAMAM G4-HFR, PAMAM G4-FHR, and PAMAM G4-FR in comparison with PAMAM G4 and the conventionally used transfection agent PEI 25 kD. In addition, physicochemical properties and complexation studies, cytotoxicity assay, and transfection assays were performed in HeLa and HepG2 cells. A time-dependent cellular uptake study was performed using confocal microscopy and fluorescenceactivated cell sorting (FACS) analysis to evaluate the effects of altering the positions of the amino acids in the oligopeptides. 2. Materials and methods 2.1. Materials Polyamidoamine-generation 4 (PAMAM G4), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N,N-diisopropylethylamine (DIPEA), piperidine, triflouroacetic acid (TFA), triisopropylsilane (TIS), HEPES, agarose and ethidium bromide (EtBr), Tris, EDTA were purchased from Sigma–Aldrich (Seoul, South Korea). N-hydroxybenzotriazole (HOBt), and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-methyluronium (HBTU) and Fmoc-LHis(trt)-OH were purchased from Anaspec (San Jose, CA, USA). Fmoc-L-Arg(pbf)-OH and Fmoc-L-Phe-OH was purchased from Novabiochem (San Diego, CA, USA). EZ-Cytox reagent was purchased from DAEILLAB SERVICE (Seoul, South Korea). Luciferase 1000 assay kit and 5X Reporter Lysis Buffer were purchased from Promega (Madison, WI, USA). The Micro BCA Protein Assay kit was purchased from Pierce (Rockford, IL, USA). The luciferase reporter plasmid DNA (pCN-Luci, 8320 base pairs) was prepared as reported previously (Choi et al., 2004). Picogreen reagent, Alexa flour 488 TFP, Alexa flour 546, Hoechst 33342 was obtained from Invitrogen (Seoul, South Korea). Fetal bovine serum (FBS), 100X Antibiotic-antimycotic agent and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from GIBCO (Gaithersburg, MD, USA). HepG2 and HeLa cell lines were obtained from Korea Cell line Bank. 2.2. Synthesis of PAMAM G4-HFR, PAMAM G4-FHR, PAMAM G4-FR PAMAM G4 (G4) was dried to remove methanol and dissolved in anhydrous DMF-DMSO (v/v 2:1) and the solution was reacted with
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4 equivalent of Fmoc-His(trt)-OH (Fmoc-Phe-OH in case of PAMAM G4-FHR and G4-FR), HOBt, HBTU and 8 equivalent of DIPEA in DMF. The solution was stirred 16–20 h at room temperature (RT). Then product was precipitated and washed in cold ethyl ether for further deprotection of Fmoc group. Deprotection was carried out in 30% piperidine in DMF (v/v) for 2 h at RT and deprotection was confirmed by nynhidrine test. Further amino acid conjugations were achieved as described above. After conjugation reactions, all protecting groups were removed by TFA: TIS: H2O (95: 2.5: 2.5%, v/ v), stirred for 6 h at RT. The final product was collected and purified by cold ether. Then the product was solubilized in water and dialyzed in DW for 24 h. Dialyzed materials were lyophilized and characterized by 1H NMR spectra (400 MHz, D2O). The overall synthesis scheme of PAMAM G4-HFR, PAMAM G4-FHR and PAMAM G4-FR has shown in Fig. 1. 2.3. Size and zeta potential measurement Size and zeta potential (surface charge) of polyplexs prepared at different weight ratios of polymer/pDNA was measured by Particle size Analyzer ELS-Z (Photal, Otuka Electonics, Tokyo, Janpan) and Zetasizer Nano ZS (Malvern Instruments, UK) respectively. All measurements were taken in triplicate and evaluated as z-average (size) and z potential. 2.4. Agarose gel electrophoresis studies Polymers were complexed with pDNA at various weight ratio in HEPES buffer (125 mM HEPES, 150 mM NaCl, pH 7.4). PEI 25 kD (weight ratio, 2:1) and PAMAM G4 (weight ratio, 4:1) were used as control group. PAMAM G4-HFR, PAMAM G4-FHR and PAMAM G4FR at weight ratios 2:1, 4:1, 8:1, 16:1, 20:1 were examined for their DNA condensation ability. Samples were incubated for 1 h and electrophoresed on 0.7% (w/v) agarose gel containing ethidium bromide (EtBr, 0.5 mg/ml of the gel) at 100 V for 20 min, room temperature. 2.5. Picogreen assay Picogreen assay was performed using HEPES buffer and Picogreen reagent (diluted in TE (10 mM Tris, 1 mM EDTA, pH 7.5) buffer). 200 ml picogreen reagents was added to prepared polyplexes and incubated for 2 min. Fluorescence (excitation and emission wavelengths were 480 nm and 520 nm respectively) was measured with a filter fluorometer (QUANTEC, Thermo Scientific). 2.6. Titration The protonatability of the polymers and subsequent acquisition of a positive charge at pH 11–2 range was determined by acid-base titration. Solution A (4 ml 150 mM NaCl and 0.1 ml 1 N NaOH) was titrated with 0.1 N HCl to pH 2 as a negative control. PEI 25 kD and PAMAM G4 were used as positive controls. The polymers were dissolved in solution A at 8 10 8 M concentration, and then titrated with the 0.1 N HCl until pH 2 was attained. The pH values were determined using a pH-meter (pH 211 microprocessor pH meter, HANA Instruments, Seoul, South Korea).
Table 1 Stepwise conjugation yield of amino acids calculated by proton (H1) nuclear magnetic resonance (NMR).
PAMAM G4-HFR PAMAM G4-FHR PAMAM G4-FR
Histidine conjugation yeild (%)
Phenylalanine conjugation yeild (%)
Arginine conjugation yeild (%)
91 99 –
>99 94 98
>99 99 96
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Fig. 2. Proton (H1) nuclear magnetic resonance (NMR) dat of (a) polyamidoamine-generation 4 (PAMAM G4)-histidine-phenylalanine-arginine (HFR), (b) polyamidoaminegeneration 4 (PAMAM G4)-phenylalanine-histidine-arginine (FHR), (c) polyamidoamine-generation 4 (PAMAM G4)-phenylalanine-arginine (FR).
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2.7. Cytotoxicity assay in vitro To check cell viability of polymers, colorimetric WST assay was performed. HeLa and HepG2 cells were cultured in DMEM medium containing 10% (v/v) FBS and 1% (w/v) penicillin/streptomycin at 37 C in a humidified atmosphere of 5% CO2. 12 103cells/well was seeded in a 96 well-plate and incubated in 100 ml media for 24 h. Cells were then treated with 10 ml of sample (PAMAM G4-HFR, PAMAM G4-FHR and PAMAM G4-FR) along with positive controls. After 24 h, 10 ml EZ-Cytox reagent was added to each well (except negative control), incubated 2 h and absorbance was measured at 450 nm wave length. 2.8. Confocal microscopy Alexa Fluor 546 labeled pDNA and Alexa 488 TFP labeled polymer were prepared according to the manufacturer’s protocol. HeLa cells (5 103cells/well) were seeded in confocal dishes (m slide 8 well, ibiTreat, South Korea) and allowed to adhere in 200 ml DMEM media (10% FBS and 1% (w/v) penicillin/streptomycin) at 37 C in a humidified atmosphere of 5% CO2 for 24 h. Polyplexes were prepared at weight ratio 8:1 for PAMAM G4-HFR, PAMAM G4FHR, PAMAM G4-FR, whereas PEI 25 kD (2:1) and PAMAM G4 (4:1) were used. Then cells were incubated with 20 ml of polyplexes for 4 h and 12 h. The culture medium was removed and cells were washed with DPBS twice, the nucleus was stained for 10 min with 100 ml of bisbenzimide (Hoechst 33342). Finally cells were washed and observed using a Zeiss LSM 5 Live confocal laser microscope. 2.9. Flow cytometry Quantitative cellular uptake efficiency was determined by FACS analysis using Alexa Flour 546 labeled pDNA. HeLa cells (2 105cells/ well) were seeded in 2 ml DMEM media (10% FBS and 1% (w/v) penicillin/streptomycin) at 37 C in a humidified atmosphere of 5% CO2 for 24 h. Polyplexes were prepared as described in confocal experiment. After incubating with 200 ml of polyplexes for 4 h and 12 h, cells we harvested using 0.05% trypsin-EDTA and washed by centrifuge with DPBS twice. Then cells were dissolved in DPBS/ absolute EtOH (3:7, v/v) and kept in 20 C for 8 h. Before analysis cells were collected by centrifuge at 7000 rpm for 3 min and DNA fluorescent intensity was measured in cell suspension using a flow cytometer (FACS caliber, Becton Dickinson, USA). 2.10. Transfection experiments HeLa and HepG2 cells were seeded at a density of 12 103cells/ well in 96 well plates and grown in 100 ml of medium containing 10% FBS and 1% (w/v) penicillin/streptomycin at 37 C in a humidified atmosphere of 5% CO2 for 24 h. 10 ml of polyplexs prepared at various weight ratios were injected and incubated at
Fig. 4. PicoGreen assay of polymers with pDNA at various weight ratios.
37 C for 24 h. Old medium was removed and cells were washed with DPBS and lysed for 30 min at room temperature using 50 ml of Reporter lysis buffer (Promega). The protein content was measured by Micro BCA assay reagent kit (Pierce, Rockford, IL). Absorption was measured at 570 nm by a microplate reader (VERSAmax, Molecular Devices, Sunnyvale, CA, USA) and compared to a standard curve calibrated with BSA samples of known concentration. Results are expressed as RLU per milligram of cell protein lysate (RLU/mg protein). 2.11. Statistical analysis The statistical analysis was performed using the unpaired Student’s t-test (GraphPad Prism 5 for Window). Results are given as the mean standard deviation (SD). Differences between groups were considered statistically significant at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). 3. Results and discussion 3.1. Synthesis of PAMAM G4-FHR, PAMAM G4-HFR, and PAMAM G4-FR Cationic nano-carriers are suitable candidates for gene delivery because of their ability to condense negatively charged DNA, siRNA, and miRNA (Cloninger, 2002; Dufès et al., 2005). However, the condensation process insufficient to achieve optimum gene expression. The cellular uptake, early endosomal escape, and nuclear co-localization are physiologically related factors that also need to be addressed. Numerous attempts to develop specialized systems to overcome all the potential limitations mentioned above have been previously reported (Chen et al., 2009). In this study, PAMAM G4 was a suitable dendrimer vector because of its terminal primary amines that enable it to condense DNA, as well as its secondary and tertiary amines that facilitate early endosomal escape. The complete procedure for the synthesis of the short
Fig. 3. Agarose gel retardation data of polyplxes (a) pDNA (lane 1), PEI 25 kD (lane 2), polyamidoamine-generation 4 (PAMAM G4, lane 3), polyamidoamine-generation 4 (PAMAM G4)-histidine-phenylalanine-arginine (HFR); (b) polyamidoamine-generation 4 (PAMAM G4)-phenylalanine-histidine-arginine (FHR); (c) polyamidoaminegeneration 4 (PAMAM G4)-phenylalanine-arginine (FR). Each number is the weight ratio of polymer/pDNA.
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Table 2 Number of positive charges present per polymer and per 1.0 mg of polymer.
MW(Da) No. of (+)/ploymer No. of (+)/1.0mg
PEI 25 kD
PAMAM G4
PAMAM G4-HFR
PAMAM G4-FHR
PAMAM G4-FR
25000 581 1.40 1016
14242.22 64 2.71 1015
41987.19 122 1.75 1015
41665.54 122 1.76 1015
33070.88 123 2.24 1015
Fig. 5. (a) Average diameter and (b) zeta potential values of polyamidoamine (PAMAM) derivatives/pDNA polyplexes. Data are expressed as mean standard deviation (n = 3).
oligopeptides, which were subsequently conjugated to PAMAM G4, is illustrated in Fig. 1. We successfully synthesized the PAMAM G4HFR, PAMAM G4-FHR, and PAMAM G4-FR amino acid conjugated dendrimers with sufficiently high conjugated yields that were calculated from the proton (1H) nuclear magnetic resonance (NMR) spectral data as shown in Table 1. The NMR spectra for the polymer conjugated oligopeptides are illustrated in Fig. 2. PAMAM G4-HFR. d 2.699 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 2.443 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 3.813 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 3.312 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit and NCH2CH2N of PAMAM G4 unit), 4.637 ( COCH(CH2 (CCHNCHNH))NH of histidine unit), 2.86 ( COCH (CH2(CCHNCHNH))NH of histidine unit), 7.236 ( COCH (CH2(CCHNCHNH))NH of histidine unit), 7.827 ( COCH (CH2(CCHNCHNH))NH of histidine unit), 4.4 ( COCH (CH2C6H5)NH of phenylalanine unit), 3.211 ( COCH(CH2C6H5) NH of phenylalanine unit), 7.321 ( COCH(CH2C6H5)NH of phenylalanine unit), 3.331 ( COCH(NH2)CH2CH2CH2NHC(NH) NH2 of arginine unit), 1.7 ( COCH(NH2)CH2CH2CH2NHC(NH) NH2 of arginine unit), 1.472 ( COCH(NH2)CH2CH2CH2NHC(NH) NH2 of arginine unit), 2.929 ( COCH(NH2)CH2CH2CH2NHC(NH) NH2 of arginine unit). PAMAM G4-FHR. d 2.484 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 2.381 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 3.813 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 2.6 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit and NCH2CH2N of PAMAM G4 unit), 4.4 ( COCH(CH2C6H5) NH of phenylalanine unit), 2.926 ( COCH(CH2C6H5)NH of phenylalanine unit), 7.2 ( COCH(CH2C6H5)NH of phenylalanine unit), 4.4 ( COCH(CH2(CCHNCHNH))NH of histidine unit), 2.926 ( COCH(CH2(CCHNCHNH))NH of histidine unit), 7.191 ( COCH(CH2(CCHNCHNH))NH of histidine unit), 7.78 ( COCH (CH2(CCHNCHNH))NH of histidine unit), 3.265 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit), 1.714 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit), 1.449 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit), 2.6 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit).
PAMAM G4-FR. d 2.63 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 2.329 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 3.686 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 3.234 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit and NCH2CH2N of PAMAM G4 unit), 4.4 ( COCH(CH2C6H5)NH of phenylalanine unit), 2.751 ( COCH (CH2C6H5)NH of phenylalanine unit), 7.2 ( COCH(CH2C6H5) NH of phenylalanine unit), 3.064 ( COCH(NH2) CH2CH2CH2NH2C(NH)NH2 of arginine unit), 1.666 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit), 1.436 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit), 2.593 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit). 3.2. Agarose gel retardation and PicoGreen assay To evaluate the formation of the polymer/DNA complexes, agarose gel electrophoresis of the polyplexes was performed at different weight ratios (Fig. 3). In Fig. 3a, lanes 1, 2, and 3 were
Fig. 6. Acid-base titration profiles of PEI 25 kD, polyamidoamine-generation 4 (PAMAM G4), polyamidoamine-generation 4 (PAMAM G4)-histidine-phenylalanine-arginine (HFR), polyamidoamine-generation 4 (PAMAM G4)-phenylalaninehistidine-arginine (FHR), polyamidoamine-generation 4 (PAMAM G4)-phenylalanine-arginine (FR).
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were positively charged, which is essential for efficient cellular uptake whereas PAMAM G4-FR had the highest positive charge compared to the other derivatives. 3.4. Titration The mechanism by which the polyplexes escape the endosomal/lysosomal compartments following internalization has not been elucidated. The hypothetical proton sponge effect is responsible for releasing the internalized payload into the cytoplasm (Varkouhi et al., 2011). The protonatability of the PAMAM G4-HFR, PAMAM G4-FHR, and PAMAM G4-FR in acidic pH is shown in Fig. 6. The PEI 25 kD showed the strongest buffering capacity as reported previously, and this is attributable to its functional amine groups, which have pK values of around 8.5– 9.5 and tend to be protonated at the physiological pH. The PAMAM G4 derivatives showed a higher buffering capacity than that of PAMAM G4 at a pH range of 3.5–6. However, the PAMAM G4-HFR and PAMAM G4-FHR appeared to have a similar buffering capacity owing to the protonation of their imidazole rings at the acidic pH. 3.5. Cytotoxicity
Fig. 7. Cell viability of polymers at different concentrations measured in (a) HeLa and (b) HepG2 cells.
loaded with DNA only, PEI 25 kD, and PAMAM G4 respectively. The PAMAM G4-HFR, PAMAM G4-FHR, and PAMAM G4-FR polymer/ pDNA complexes are shown in Fig. 3a–c. Complete retardation of all the PAMAM G4 derivatives was observed at a weight ratio of 16. This effect was attributable to the presence of the neutral F in all the oligopeptide sequences, which decreased the net positive charge required for pDNA condensation (Liu et al., 2010). Therefore, higher weight ratios appear necessary to form complete and stable polyplexes. For a more precise analysis, a PicoGreen assay was performed, and Fig. 4 shows an increase in the inhibition of the fluorescence with increasing weight ratios of the polymer/pDNA. In addition, the gel retardation data shows the complete condensation of DNA at 8 and 16 weight ratios. Moreover, PAMAM G4-HFR, PAMAM G4-FHR, and PAMAM G4-FR showed higher condensation than PAMAM G4 owing to an increase in the number of positive charges (Table 2). Based on this result, the weight ratio 8 was selected for use in further experiments. 3.3. Size and zeta potential measurement The particle sizes of the polyplexes were determined using dynamic light scattering. The nano-sized particles were formed at a weight ratio of 8 for the PAMAM derivatives and control groups. The polyplex sizes were 291.9 and 263.67 nm for the PEI 25 kD and PAMAM G4 controls, respectively (Fig. 5a) while the PAMAM G4HFR, PAMAM G4-FHR, and PAMAM G4-FR polyplexes were smaller than the controls were. This result is attributable to the ability of the hydrophobic L-phenylalanines to create stable and compact polyplexes in the aqueous milieu. The condensation of the pDNA to nano-sized particles is certainly a beneficial feature to achieve an enhanced permeability and retention (EPR) effect. Surface charges of the polyplexes were also determined along with control groups. As shown in Fig. 5b, all polyplexes including the control groups
During the synthesis and development of biocompatible gene carriers, evaluation of their cytotoxicity is essential to ensure the safety of the final product. The toxicity of cationic polymers has been reported to be associated with their interaction with the cell membrane (Choi et al., 2004). The toxicity analysis of the oligopeptide conjugated PAMAM G4 derivatives was performed using an EZ-Cytox cell viability assay kit. The HepG2 and HeLa cells were treated with polymers for 24 h, and the absorbance was measured to evaluate the percentage cell viability (Fig. 7). As expected, the PEI 25 kD showed the highest level of toxicity in both the HeLa and HepG2 cells because of its cationic property. The PAMAM G4-FR was the derivative that showed toxicity at a high concentration of 200 mg/ml. This was an expected observation because PAMAM G4-FR/pDNA complexes exhibited the highest positive charge compared to the other derivatives (Fig. 5b). Interestingly, the addition of a histidine group remarkably lowered the cytotoxicity. This result is consistent with previous reports that the PAMAM G4-R conjugate is more toxic than the PAMAM G4-HR (Yu et al., 2011). Based on the cytotoxicity data it was presumed that the histidines grafted between PAMAM G4 and other amino acids may act as a neutralizing layer minimizing the toxicity. Additional experiments are required to elucidate the effects of histidine on the cytotoxicity. However, all the PAMAM derivatives showed a lower toxicity than the PEI 25 kD did in both cell lines at all the concentrations tested. 3.6. Cellular uptake by confocal microscopy and FACS analysis Endocytosis is composed of two steps including endocytic uptake followed by the endosomal escape. This process is known to be the most common entry mechanism for nonviral gene carriers. The PAMAM G4-oligopeptides of this study should also follow the similar pathway but with enhanced efficacy. This is because the hydrophobic environment around the PAMAM G4 induced by phenylalanines facilitates its interaction with cell membrane fatty acids (Varkouhi et al., 2011). Furthermore, studies on hydrophobic amino acids have shown that F has the highest membrane-disruptive activity in acidic pH induced by osmotic swelling, which releases the endocytosed payloads into the cytoplasm (Santos et al., 2010; Sunshine et al., 2011; Yoksan and Akashi, 2009). Confocal microscopy was performed to observe the biological responses and cellular uptake of the PAMAM G4 derivatives and the control group. The labeled polymers (green) were complex with the labeled pDNA (red) at a weight ratio of 8 and
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Fig. 8. Confocal laser scanning microscopy image of HeLa cells incubated with polyplexes for (a) 4 and (b) 12 h. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
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Fig. 9. Flow cytometry analysis of HeLa cells incubated with polyplexes for 4 and 12 h.
transfected into HeLa cells. The cellular distribution of the polyplexes was then analyzed after incubation for 4 and 12 h (Fig. 8). PAMAM G4 derivatives were efficiently taken up by cells after a 4 h incubation (Fig. 8a), whereas larger aggregations of the PEI 25 kD polyplexes were found in the cytoplasm of cells. A previous report has proven that PEI 25 kD/pDNA complexes tend to aggregate in physiological conditions owing to charged serum protein and difference in ionic strength (Goula et al., 1998). However, PAMAM G4 appeared to exhibit the lowest cellular uptake, which corresponds to lower transfection efficiency. Interestingly, the cellular internalization was efficient for the PAMAM G4 derivatives after 12 h incubation (Fig. 8b). Further quantitative cellular uptake was confirmed using flow cytometry (Fig. 9) after the cells were incubated with polyplexes for 4 and 12 h. Fluorescence intensity was more than 10-fold higher in the control PEI 25 kD sample than it was in the PAMAM derivatives, suggesting a greater cellular internalization of polyplexes. However, in the case of the PAMAM derivatives, the cellular uptake was higher than that of PAMAM G4. This may be due to the synergistic effect of the combination of phenylalanines and histidines, which facilitated cellular uptake, and a consequently higher payload were released into the cytoplasm.
3.7. Transfection The luciferase reporter gene was used to evaluate the transfection efficiency of the PAMAM G4 derivatives and the control groups. The luciferase enzyme catalyzes the luciferin reaction in the presence of adenosine triphosphate (ATP), thereby producing visible light. The luciferase expression is quantifiable by measuring the light intensity. As shown in Fig. 10, all the PAMAM G4 derivatives exhibited a comparable gene expression to that of the PEI 25 kD because of their enhanced cellular uptake and buffering capacity in the HeLa and HepG2 cells. As previously proposed with the cellular uptake results, higher cellular internalization of the PAMAM G4 derivatives probably led to higher transfection efficiency and negligible toxicity than that of the PAMAM G4. This result is consistent with a previous report showing that the combination of hydrophobic and hydrophilic groups improves transfection efficiency (Wang et al., 2014; Zhi et al., 2010). Therefore, the combination of arginine, histidine, and phenylalanine to achieve similar levels of gene expression can be a novel strategy for the production of safe and effective gene carriers.
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Fig. 10. Transfection efficiency of polymers in (a) HeLa and (b) HepG2 cells.
4. Conclusions Acknowledgments In this study, PAMAM G4 derivatives conjugated to H, R, and F were synthesized with a conjugation yield of >90%. A complete DNA condensation was achieved, and we obtained nano-sized polyplexes, which were further analyzed in vitro. The suggested acid-base titration profile improved the buffering capacity of the histidine residues, thereby synergistically inducing early endosomal escape. The higher cellular internalization was confirmed by confocal microscopy and is probably attributable to a greater interaction between the hydrophobic F residue and the cell membrane. Moreover, the PAMAM G4 derivatives exhibited a lower toxicity than that of the PEI 25 kD, which makes them safer non-viral gene carriers with a similar level of transfection efficiency. In summary, the PAMAM G4 derivatives are highly promising candidates for gene delivery because of their low toxicity, improved buffering capacity, and higher transfection efficiency.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2064737). References Akhtar, S., 2005. Non-viral cancer gene therapy: beyond delivery. Gene Ther. 13, 739–740. Castelletto, V., Hamley, I.W., 2009. Self assembly of a model amphiphilic phenylalanine peptide/polyethylene glycol block copolymer in aqueous solution. Biophys. Chem. 141, 169–174. Chen, R., Khormaee, S., Eccleston, M.E., Slater, N.K.H., 2009. The role of hydrophobic amino acid grafts in the enhancement of membrane-disruptive activity of pHresponsive pseudo-peptides. Biomaterials 30, 1954–1961. Choi, J.S., Nam, K., Park, J.-y., Kim, J.-B., Lee, J.-K., Park, J.-s., 2004. Enhanced transfection efficiency of PAMAM dendrimer by surface modification with Larginine. J. Control. Release 99, 445–456.
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Polyamidoamine (PAMAM) dendrimers modified with short oligopeptides for early endosomal escape and enhanced gene delivery Le Thi Thuy, Sudipta Mallick, Joon Sig Choi* Department of Biochemistry, Chungnam National University, Gung-dong 220, Yuseong-gu, Daejeon 305-764, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 March 2015 Received in revised form 15 June 2015 Accepted 7 July 2015 Available online 14 July 2015
Recently, non-viral vectors have become a popular research topic in the field of gene therapy. In this study, we conjugated short oligopeptides to polyamidoamine-generation 4 (PAMAM G4) to achieve higher transfection efficiency. Previous reports have shown that the PAMAM G4-histidine (H)-arginine (R) dendrimer enhances gene delivery by improving cell penetration and internalization mechanisms. Therefore, we synthesized PAMAM G4-H phenylalanine (F) R, PAMAM G4-FHR and PAMAM G4-FR derivatives to determine the best gene carrier with the lowest toxicity. Physicochemical studies were performed to determine mean diameters and surface charge of PAMAM derivatives/pDNA polyplexes. DNA condensation was confirmed using a gel retardation assay. Cytotoxicity and transfection efficiency were analyzed using human cervical carcinoma (HeLa) and human liver carcinoma (HepG2) cells. Similar levels of transfection were achieved in both cell lines by using gold standard transfection reagent PEI 25 kD. Therefore, our results show that these carriers are promising and may help achieve higher transfection with negligible cytotoxicity. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Polyamidoamine dendrimer Oligopeptides Hydrophobic Transfection Polyplex Cytotoxicity
1. Introduction Currently, gene therapy is considered a potential therapy for human diseases such as diabetes and various cancers where the etiological factors include defective genes (Guo and Huang, 2011; Oupický et al., 2000; Zhang et al., 2012). Typical gene therapy procedures involve the introduction of extracellular therapeutic genes that can knock down dysfunctional genes or express proteins. This process has emerged as a multidisciplinary field of research, which emphasizes the physicochemical and pharmaceutical properties of the therapies under development. The development of a superior means of delivering gene products to the site of action would enhance the therapeutic efficacy (Zhang et al., 2012). In particular, nanotechnology has promising applications in the field of gene therapy because it possesses excellent characteristics that satisfy the prerequisites for biomolecule carriers (Mintzer and Simanek, 2008). These characteristics include a prolonged circulation time, negligible immune reactions, enhanced cellular uptake, and early endosomal escape (Mintzer and Simanek, 2008). Therefore, different vectors have been exploited to achieve all the characteristics mentioned above (Kamimura et al., 2011;
* Corresponding author. Tel.: +82 42 821 5489; Fax: +82 42 822 7548. E-mail address: [email protected] (J.S. Choi). http://dx.doi.org/10.1016/j.ijpharm.2015.07.017 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
Nayerossadat et al., 2012). While viral vectors are well known for efficient gene expression, their mutagenicity, and immune reactions are a matter of concern (Kamimura et al., 2011; Nayerossadat et al., 2012; Robbins and Ghivizzani, 1998). Therefore, non-viral vectors have attracted much attention because of their stability, biocompatibility, and surface functionality in vitro and in vivo. However, their transfection efficiency is not as high as that of viral vectors (Akhtar, 2005). Previous studies have proven that cationic polymeric vectors, liposomes, and dendrimers are the best candidates for the complete condensation of negatively charged DNA to nano-sized particles, which they subsequently deliver to cells (Mintzer and Simanek, 2008; Nayerossadat et al., 2012; Zhang et al., 2012). The non-viral vector polyethylenimine (PEI) is considered the gold standard for gene delivery because of its numerous amine groups, which facilitate greater cellular uptake and endosomal escape under mildly acidic conditions (Fu et al., 2012; Kircheis et al., 2001). However, the PEI particle is highly positively charged, which leads to cell membrane disruption and eventual cell death (Tung and Weissleder, 2003). In contrast, the polyamidoamine (PAMAM) dendrimers, which have a uniquely branched structure currently offer a similar level of transfection when modified with cell penetration peptides (CPPs) (Choi et al., 2004; Kim et al., 2009; Kono et al., 2004; Kono et al., 2008; Lee et al., 2011; Majoros et al., 2005; Shcharbin et al., 2009). The previously reported PAMAMgeneration 4 (PAMAM G4) dendrimer conjugated with arginine (R)
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and histidine (H)—R, was previously reported to have excellent transfection efficiency with a negligible cytotoxicity in normal cells (Shi et al., 2013; Zhang et al., 2014). The higher transfection efficiency of this conjugate was attributed to the synergistic effects of the cationic amino acid arginine that enhances cellular uptake
(Futaki, 2005; Luo et al., 2012; Rothbard et al., 2002). In addition, induction of the proton sponge effect by histidine further enhanced this efficiency by overpowering the acidic environment inside the endosomal compartment (Yu et al., 2011).
Fig. 1. Schematic diagram of synthesis of polyamidoamine-generation 4 (PAMAM G4)-histidine-phenylalanine-arginine (HFR), polyamidoamine-generation 4 (PAMAM G4)phenylalanine-histidine-arginine (FHR), polyamidoamine-generation 4 (PAMAM G4)-phenylalanine-arginine (FR).
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Furthermore, CPPs containing hydrophobic amino acid like phenylalanine (F) and leucine, show increased transfection efficiency because of their enhanced interaction with the cell membrane (Choi et al., 2007; Kurisawa et al., 2000; Sang Yoo et al., 2005; Tian et al., 2007; Wang et al., 2009). The endocytosis process is believed to occur when PAMAM G4 is terminally modified with phenylalanine (F) because the hydrophobic environment of the PAMAM G4 facilitates its interaction with the cell membrane fatty acids. In addition, studies on hydrophobic amino acids have shown that F has the highest membrane-disruptive activity in acidic pH induced by osmotic swelling, which leads to the release of endocytosed payloads into the cytoplasm (Castelletto and Hamley, 2009; Liu et al., 2010). However, single amino acid functionalization is not sufficient to surmount the biological barriers. Therefore, in the present study, we modified PAMAM G4 with various combinations of H, R, and F to incorporate the synergistic advantage of the 3 amino acids. We studied the gene transfection efficiency of 3 different oligopeptide sequences including PAMAM G4-HFR, PAMAM G4-FHR, and PAMAM G4-FR in comparison with PAMAM G4 and the conventionally used transfection agent PEI 25 kD. In addition, physicochemical properties and complexation studies, cytotoxicity assay, and transfection assays were performed in HeLa and HepG2 cells. A time-dependent cellular uptake study was performed using confocal microscopy and fluorescenceactivated cell sorting (FACS) analysis to evaluate the effects of altering the positions of the amino acids in the oligopeptides. 2. Materials and methods 2.1. Materials Polyamidoamine-generation 4 (PAMAM G4), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N,N-diisopropylethylamine (DIPEA), piperidine, triflouroacetic acid (TFA), triisopropylsilane (TIS), HEPES, agarose and ethidium bromide (EtBr), Tris, EDTA were purchased from Sigma–Aldrich (Seoul, South Korea). N-hydroxybenzotriazole (HOBt), and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-methyluronium (HBTU) and Fmoc-LHis(trt)-OH were purchased from Anaspec (San Jose, CA, USA). Fmoc-L-Arg(pbf)-OH and Fmoc-L-Phe-OH was purchased from Novabiochem (San Diego, CA, USA). EZ-Cytox reagent was purchased from DAEILLAB SERVICE (Seoul, South Korea). Luciferase 1000 assay kit and 5X Reporter Lysis Buffer were purchased from Promega (Madison, WI, USA). The Micro BCA Protein Assay kit was purchased from Pierce (Rockford, IL, USA). The luciferase reporter plasmid DNA (pCN-Luci, 8320 base pairs) was prepared as reported previously (Choi et al., 2004). Picogreen reagent, Alexa flour 488 TFP, Alexa flour 546, Hoechst 33342 was obtained from Invitrogen (Seoul, South Korea). Fetal bovine serum (FBS), 100X Antibiotic-antimycotic agent and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from GIBCO (Gaithersburg, MD, USA). HepG2 and HeLa cell lines were obtained from Korea Cell line Bank. 2.2. Synthesis of PAMAM G4-HFR, PAMAM G4-FHR, PAMAM G4-FR PAMAM G4 (G4) was dried to remove methanol and dissolved in anhydrous DMF-DMSO (v/v 2:1) and the solution was reacted with
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4 equivalent of Fmoc-His(trt)-OH (Fmoc-Phe-OH in case of PAMAM G4-FHR and G4-FR), HOBt, HBTU and 8 equivalent of DIPEA in DMF. The solution was stirred 16–20 h at room temperature (RT). Then product was precipitated and washed in cold ethyl ether for further deprotection of Fmoc group. Deprotection was carried out in 30% piperidine in DMF (v/v) for 2 h at RT and deprotection was confirmed by nynhidrine test. Further amino acid conjugations were achieved as described above. After conjugation reactions, all protecting groups were removed by TFA: TIS: H2O (95: 2.5: 2.5%, v/ v), stirred for 6 h at RT. The final product was collected and purified by cold ether. Then the product was solubilized in water and dialyzed in DW for 24 h. Dialyzed materials were lyophilized and characterized by 1H NMR spectra (400 MHz, D2O). The overall synthesis scheme of PAMAM G4-HFR, PAMAM G4-FHR and PAMAM G4-FR has shown in Fig. 1. 2.3. Size and zeta potential measurement Size and zeta potential (surface charge) of polyplexs prepared at different weight ratios of polymer/pDNA was measured by Particle size Analyzer ELS-Z (Photal, Otuka Electonics, Tokyo, Janpan) and Zetasizer Nano ZS (Malvern Instruments, UK) respectively. All measurements were taken in triplicate and evaluated as z-average (size) and z potential. 2.4. Agarose gel electrophoresis studies Polymers were complexed with pDNA at various weight ratio in HEPES buffer (125 mM HEPES, 150 mM NaCl, pH 7.4). PEI 25 kD (weight ratio, 2:1) and PAMAM G4 (weight ratio, 4:1) were used as control group. PAMAM G4-HFR, PAMAM G4-FHR and PAMAM G4FR at weight ratios 2:1, 4:1, 8:1, 16:1, 20:1 were examined for their DNA condensation ability. Samples were incubated for 1 h and electrophoresed on 0.7% (w/v) agarose gel containing ethidium bromide (EtBr, 0.5 mg/ml of the gel) at 100 V for 20 min, room temperature. 2.5. Picogreen assay Picogreen assay was performed using HEPES buffer and Picogreen reagent (diluted in TE (10 mM Tris, 1 mM EDTA, pH 7.5) buffer). 200 ml picogreen reagents was added to prepared polyplexes and incubated for 2 min. Fluorescence (excitation and emission wavelengths were 480 nm and 520 nm respectively) was measured with a filter fluorometer (QUANTEC, Thermo Scientific). 2.6. Titration The protonatability of the polymers and subsequent acquisition of a positive charge at pH 11–2 range was determined by acid-base titration. Solution A (4 ml 150 mM NaCl and 0.1 ml 1 N NaOH) was titrated with 0.1 N HCl to pH 2 as a negative control. PEI 25 kD and PAMAM G4 were used as positive controls. The polymers were dissolved in solution A at 8 10 8 M concentration, and then titrated with the 0.1 N HCl until pH 2 was attained. The pH values were determined using a pH-meter (pH 211 microprocessor pH meter, HANA Instruments, Seoul, South Korea).
Table 1 Stepwise conjugation yield of amino acids calculated by proton (H1) nuclear magnetic resonance (NMR).
PAMAM G4-HFR PAMAM G4-FHR PAMAM G4-FR
Histidine conjugation yeild (%)
Phenylalanine conjugation yeild (%)
Arginine conjugation yeild (%)
91 99 –
>99 94 98
>99 99 96
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Fig. 2. Proton (H1) nuclear magnetic resonance (NMR) dat of (a) polyamidoamine-generation 4 (PAMAM G4)-histidine-phenylalanine-arginine (HFR), (b) polyamidoaminegeneration 4 (PAMAM G4)-phenylalanine-histidine-arginine (FHR), (c) polyamidoamine-generation 4 (PAMAM G4)-phenylalanine-arginine (FR).
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2.7. Cytotoxicity assay in vitro To check cell viability of polymers, colorimetric WST assay was performed. HeLa and HepG2 cells were cultured in DMEM medium containing 10% (v/v) FBS and 1% (w/v) penicillin/streptomycin at 37 C in a humidified atmosphere of 5% CO2. 12 103cells/well was seeded in a 96 well-plate and incubated in 100 ml media for 24 h. Cells were then treated with 10 ml of sample (PAMAM G4-HFR, PAMAM G4-FHR and PAMAM G4-FR) along with positive controls. After 24 h, 10 ml EZ-Cytox reagent was added to each well (except negative control), incubated 2 h and absorbance was measured at 450 nm wave length. 2.8. Confocal microscopy Alexa Fluor 546 labeled pDNA and Alexa 488 TFP labeled polymer were prepared according to the manufacturer’s protocol. HeLa cells (5 103cells/well) were seeded in confocal dishes (m slide 8 well, ibiTreat, South Korea) and allowed to adhere in 200 ml DMEM media (10% FBS and 1% (w/v) penicillin/streptomycin) at 37 C in a humidified atmosphere of 5% CO2 for 24 h. Polyplexes were prepared at weight ratio 8:1 for PAMAM G4-HFR, PAMAM G4FHR, PAMAM G4-FR, whereas PEI 25 kD (2:1) and PAMAM G4 (4:1) were used. Then cells were incubated with 20 ml of polyplexes for 4 h and 12 h. The culture medium was removed and cells were washed with DPBS twice, the nucleus was stained for 10 min with 100 ml of bisbenzimide (Hoechst 33342). Finally cells were washed and observed using a Zeiss LSM 5 Live confocal laser microscope. 2.9. Flow cytometry Quantitative cellular uptake efficiency was determined by FACS analysis using Alexa Flour 546 labeled pDNA. HeLa cells (2 105cells/ well) were seeded in 2 ml DMEM media (10% FBS and 1% (w/v) penicillin/streptomycin) at 37 C in a humidified atmosphere of 5% CO2 for 24 h. Polyplexes were prepared as described in confocal experiment. After incubating with 200 ml of polyplexes for 4 h and 12 h, cells we harvested using 0.05% trypsin-EDTA and washed by centrifuge with DPBS twice. Then cells were dissolved in DPBS/ absolute EtOH (3:7, v/v) and kept in 20 C for 8 h. Before analysis cells were collected by centrifuge at 7000 rpm for 3 min and DNA fluorescent intensity was measured in cell suspension using a flow cytometer (FACS caliber, Becton Dickinson, USA). 2.10. Transfection experiments HeLa and HepG2 cells were seeded at a density of 12 103cells/ well in 96 well plates and grown in 100 ml of medium containing 10% FBS and 1% (w/v) penicillin/streptomycin at 37 C in a humidified atmosphere of 5% CO2 for 24 h. 10 ml of polyplexs prepared at various weight ratios were injected and incubated at
Fig. 4. PicoGreen assay of polymers with pDNA at various weight ratios.
37 C for 24 h. Old medium was removed and cells were washed with DPBS and lysed for 30 min at room temperature using 50 ml of Reporter lysis buffer (Promega). The protein content was measured by Micro BCA assay reagent kit (Pierce, Rockford, IL). Absorption was measured at 570 nm by a microplate reader (VERSAmax, Molecular Devices, Sunnyvale, CA, USA) and compared to a standard curve calibrated with BSA samples of known concentration. Results are expressed as RLU per milligram of cell protein lysate (RLU/mg protein). 2.11. Statistical analysis The statistical analysis was performed using the unpaired Student’s t-test (GraphPad Prism 5 for Window). Results are given as the mean standard deviation (SD). Differences between groups were considered statistically significant at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). 3. Results and discussion 3.1. Synthesis of PAMAM G4-FHR, PAMAM G4-HFR, and PAMAM G4-FR Cationic nano-carriers are suitable candidates for gene delivery because of their ability to condense negatively charged DNA, siRNA, and miRNA (Cloninger, 2002; Dufès et al., 2005). However, the condensation process insufficient to achieve optimum gene expression. The cellular uptake, early endosomal escape, and nuclear co-localization are physiologically related factors that also need to be addressed. Numerous attempts to develop specialized systems to overcome all the potential limitations mentioned above have been previously reported (Chen et al., 2009). In this study, PAMAM G4 was a suitable dendrimer vector because of its terminal primary amines that enable it to condense DNA, as well as its secondary and tertiary amines that facilitate early endosomal escape. The complete procedure for the synthesis of the short
Fig. 3. Agarose gel retardation data of polyplxes (a) pDNA (lane 1), PEI 25 kD (lane 2), polyamidoamine-generation 4 (PAMAM G4, lane 3), polyamidoamine-generation 4 (PAMAM G4)-histidine-phenylalanine-arginine (HFR); (b) polyamidoamine-generation 4 (PAMAM G4)-phenylalanine-histidine-arginine (FHR); (c) polyamidoaminegeneration 4 (PAMAM G4)-phenylalanine-arginine (FR). Each number is the weight ratio of polymer/pDNA.
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Table 2 Number of positive charges present per polymer and per 1.0 mg of polymer.
MW(Da) No. of (+)/ploymer No. of (+)/1.0mg
PEI 25 kD
PAMAM G4
PAMAM G4-HFR
PAMAM G4-FHR
PAMAM G4-FR
25000 581 1.40 1016
14242.22 64 2.71 1015
41987.19 122 1.75 1015
41665.54 122 1.76 1015
33070.88 123 2.24 1015
Fig. 5. (a) Average diameter and (b) zeta potential values of polyamidoamine (PAMAM) derivatives/pDNA polyplexes. Data are expressed as mean standard deviation (n = 3).
oligopeptides, which were subsequently conjugated to PAMAM G4, is illustrated in Fig. 1. We successfully synthesized the PAMAM G4HFR, PAMAM G4-FHR, and PAMAM G4-FR amino acid conjugated dendrimers with sufficiently high conjugated yields that were calculated from the proton (1H) nuclear magnetic resonance (NMR) spectral data as shown in Table 1. The NMR spectra for the polymer conjugated oligopeptides are illustrated in Fig. 2. PAMAM G4-HFR. d 2.699 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 2.443 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 3.813 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 3.312 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit and NCH2CH2N of PAMAM G4 unit), 4.637 ( COCH(CH2 (CCHNCHNH))NH of histidine unit), 2.86 ( COCH (CH2(CCHNCHNH))NH of histidine unit), 7.236 ( COCH (CH2(CCHNCHNH))NH of histidine unit), 7.827 ( COCH (CH2(CCHNCHNH))NH of histidine unit), 4.4 ( COCH (CH2C6H5)NH of phenylalanine unit), 3.211 ( COCH(CH2C6H5) NH of phenylalanine unit), 7.321 ( COCH(CH2C6H5)NH of phenylalanine unit), 3.331 ( COCH(NH2)CH2CH2CH2NHC(NH) NH2 of arginine unit), 1.7 ( COCH(NH2)CH2CH2CH2NHC(NH) NH2 of arginine unit), 1.472 ( COCH(NH2)CH2CH2CH2NHC(NH) NH2 of arginine unit), 2.929 ( COCH(NH2)CH2CH2CH2NHC(NH) NH2 of arginine unit). PAMAM G4-FHR. d 2.484 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 2.381 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 3.813 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 2.6 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit and NCH2CH2N of PAMAM G4 unit), 4.4 ( COCH(CH2C6H5) NH of phenylalanine unit), 2.926 ( COCH(CH2C6H5)NH of phenylalanine unit), 7.2 ( COCH(CH2C6H5)NH of phenylalanine unit), 4.4 ( COCH(CH2(CCHNCHNH))NH of histidine unit), 2.926 ( COCH(CH2(CCHNCHNH))NH of histidine unit), 7.191 ( COCH(CH2(CCHNCHNH))NH of histidine unit), 7.78 ( COCH (CH2(CCHNCHNH))NH of histidine unit), 3.265 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit), 1.714 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit), 1.449 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit), 2.6 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit).
PAMAM G4-FR. d 2.63 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 2.329 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 3.686 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit), 3.234 ( NHCH2CH2CONHCH2CH2NH of PAMAM G4 unit and NCH2CH2N of PAMAM G4 unit), 4.4 ( COCH(CH2C6H5)NH of phenylalanine unit), 2.751 ( COCH (CH2C6H5)NH of phenylalanine unit), 7.2 ( COCH(CH2C6H5) NH of phenylalanine unit), 3.064 ( COCH(NH2) CH2CH2CH2NH2C(NH)NH2 of arginine unit), 1.666 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit), 1.436 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit), 2.593 ( COCH(NH2) CH2CH2CH2NHC(NH)NH2 of arginine unit). 3.2. Agarose gel retardation and PicoGreen assay To evaluate the formation of the polymer/DNA complexes, agarose gel electrophoresis of the polyplexes was performed at different weight ratios (Fig. 3). In Fig. 3a, lanes 1, 2, and 3 were
Fig. 6. Acid-base titration profiles of PEI 25 kD, polyamidoamine-generation 4 (PAMAM G4), polyamidoamine-generation 4 (PAMAM G4)-histidine-phenylalanine-arginine (HFR), polyamidoamine-generation 4 (PAMAM G4)-phenylalaninehistidine-arginine (FHR), polyamidoamine-generation 4 (PAMAM G4)-phenylalanine-arginine (FR).
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were positively charged, which is essential for efficient cellular uptake whereas PAMAM G4-FR had the highest positive charge compared to the other derivatives. 3.4. Titration The mechanism by which the polyplexes escape the endosomal/lysosomal compartments following internalization has not been elucidated. The hypothetical proton sponge effect is responsible for releasing the internalized payload into the cytoplasm (Varkouhi et al., 2011). The protonatability of the PAMAM G4-HFR, PAMAM G4-FHR, and PAMAM G4-FR in acidic pH is shown in Fig. 6. The PEI 25 kD showed the strongest buffering capacity as reported previously, and this is attributable to its functional amine groups, which have pK values of around 8.5– 9.5 and tend to be protonated at the physiological pH. The PAMAM G4 derivatives showed a higher buffering capacity than that of PAMAM G4 at a pH range of 3.5–6. However, the PAMAM G4-HFR and PAMAM G4-FHR appeared to have a similar buffering capacity owing to the protonation of their imidazole rings at the acidic pH. 3.5. Cytotoxicity
Fig. 7. Cell viability of polymers at different concentrations measured in (a) HeLa and (b) HepG2 cells.
loaded with DNA only, PEI 25 kD, and PAMAM G4 respectively. The PAMAM G4-HFR, PAMAM G4-FHR, and PAMAM G4-FR polymer/ pDNA complexes are shown in Fig. 3a–c. Complete retardation of all the PAMAM G4 derivatives was observed at a weight ratio of 16. This effect was attributable to the presence of the neutral F in all the oligopeptide sequences, which decreased the net positive charge required for pDNA condensation (Liu et al., 2010). Therefore, higher weight ratios appear necessary to form complete and stable polyplexes. For a more precise analysis, a PicoGreen assay was performed, and Fig. 4 shows an increase in the inhibition of the fluorescence with increasing weight ratios of the polymer/pDNA. In addition, the gel retardation data shows the complete condensation of DNA at 8 and 16 weight ratios. Moreover, PAMAM G4-HFR, PAMAM G4-FHR, and PAMAM G4-FR showed higher condensation than PAMAM G4 owing to an increase in the number of positive charges (Table 2). Based on this result, the weight ratio 8 was selected for use in further experiments. 3.3. Size and zeta potential measurement The particle sizes of the polyplexes were determined using dynamic light scattering. The nano-sized particles were formed at a weight ratio of 8 for the PAMAM derivatives and control groups. The polyplex sizes were 291.9 and 263.67 nm for the PEI 25 kD and PAMAM G4 controls, respectively (Fig. 5a) while the PAMAM G4HFR, PAMAM G4-FHR, and PAMAM G4-FR polyplexes were smaller than the controls were. This result is attributable to the ability of the hydrophobic L-phenylalanines to create stable and compact polyplexes in the aqueous milieu. The condensation of the pDNA to nano-sized particles is certainly a beneficial feature to achieve an enhanced permeability and retention (EPR) effect. Surface charges of the polyplexes were also determined along with control groups. As shown in Fig. 5b, all polyplexes including the control groups
During the synthesis and development of biocompatible gene carriers, evaluation of their cytotoxicity is essential to ensure the safety of the final product. The toxicity of cationic polymers has been reported to be associated with their interaction with the cell membrane (Choi et al., 2004). The toxicity analysis of the oligopeptide conjugated PAMAM G4 derivatives was performed using an EZ-Cytox cell viability assay kit. The HepG2 and HeLa cells were treated with polymers for 24 h, and the absorbance was measured to evaluate the percentage cell viability (Fig. 7). As expected, the PEI 25 kD showed the highest level of toxicity in both the HeLa and HepG2 cells because of its cationic property. The PAMAM G4-FR was the derivative that showed toxicity at a high concentration of 200 mg/ml. This was an expected observation because PAMAM G4-FR/pDNA complexes exhibited the highest positive charge compared to the other derivatives (Fig. 5b). Interestingly, the addition of a histidine group remarkably lowered the cytotoxicity. This result is consistent with previous reports that the PAMAM G4-R conjugate is more toxic than the PAMAM G4-HR (Yu et al., 2011). Based on the cytotoxicity data it was presumed that the histidines grafted between PAMAM G4 and other amino acids may act as a neutralizing layer minimizing the toxicity. Additional experiments are required to elucidate the effects of histidine on the cytotoxicity. However, all the PAMAM derivatives showed a lower toxicity than the PEI 25 kD did in both cell lines at all the concentrations tested. 3.6. Cellular uptake by confocal microscopy and FACS analysis Endocytosis is composed of two steps including endocytic uptake followed by the endosomal escape. This process is known to be the most common entry mechanism for nonviral gene carriers. The PAMAM G4-oligopeptides of this study should also follow the similar pathway but with enhanced efficacy. This is because the hydrophobic environment around the PAMAM G4 induced by phenylalanines facilitates its interaction with cell membrane fatty acids (Varkouhi et al., 2011). Furthermore, studies on hydrophobic amino acids have shown that F has the highest membrane-disruptive activity in acidic pH induced by osmotic swelling, which releases the endocytosed payloads into the cytoplasm (Santos et al., 2010; Sunshine et al., 2011; Yoksan and Akashi, 2009). Confocal microscopy was performed to observe the biological responses and cellular uptake of the PAMAM G4 derivatives and the control group. The labeled polymers (green) were complex with the labeled pDNA (red) at a weight ratio of 8 and
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Fig. 8. Confocal laser scanning microscopy image of HeLa cells incubated with polyplexes for (a) 4 and (b) 12 h. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
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Fig. 9. Flow cytometry analysis of HeLa cells incubated with polyplexes for 4 and 12 h.
transfected into HeLa cells. The cellular distribution of the polyplexes was then analyzed after incubation for 4 and 12 h (Fig. 8). PAMAM G4 derivatives were efficiently taken up by cells after a 4 h incubation (Fig. 8a), whereas larger aggregations of the PEI 25 kD polyplexes were found in the cytoplasm of cells. A previous report has proven that PEI 25 kD/pDNA complexes tend to aggregate in physiological conditions owing to charged serum protein and difference in ionic strength (Goula et al., 1998). However, PAMAM G4 appeared to exhibit the lowest cellular uptake, which corresponds to lower transfection efficiency. Interestingly, the cellular internalization was efficient for the PAMAM G4 derivatives after 12 h incubation (Fig. 8b). Further quantitative cellular uptake was confirmed using flow cytometry (Fig. 9) after the cells were incubated with polyplexes for 4 and 12 h. Fluorescence intensity was more than 10-fold higher in the control PEI 25 kD sample than it was in the PAMAM derivatives, suggesting a greater cellular internalization of polyplexes. However, in the case of the PAMAM derivatives, the cellular uptake was higher than that of PAMAM G4. This may be due to the synergistic effect of the combination of phenylalanines and histidines, which facilitated cellular uptake, and a consequently higher payload were released into the cytoplasm.
3.7. Transfection The luciferase reporter gene was used to evaluate the transfection efficiency of the PAMAM G4 derivatives and the control groups. The luciferase enzyme catalyzes the luciferin reaction in the presence of adenosine triphosphate (ATP), thereby producing visible light. The luciferase expression is quantifiable by measuring the light intensity. As shown in Fig. 10, all the PAMAM G4 derivatives exhibited a comparable gene expression to that of the PEI 25 kD because of their enhanced cellular uptake and buffering capacity in the HeLa and HepG2 cells. As previously proposed with the cellular uptake results, higher cellular internalization of the PAMAM G4 derivatives probably led to higher transfection efficiency and negligible toxicity than that of the PAMAM G4. This result is consistent with a previous report showing that the combination of hydrophobic and hydrophilic groups improves transfection efficiency (Wang et al., 2014; Zhi et al., 2010). Therefore, the combination of arginine, histidine, and phenylalanine to achieve similar levels of gene expression can be a novel strategy for the production of safe and effective gene carriers.
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Fig. 10. Transfection efficiency of polymers in (a) HeLa and (b) HepG2 cells.
4. Conclusions Acknowledgments In this study, PAMAM G4 derivatives conjugated to H, R, and F were synthesized with a conjugation yield of >90%. A complete DNA condensation was achieved, and we obtained nano-sized polyplexes, which were further analyzed in vitro. The suggested acid-base titration profile improved the buffering capacity of the histidine residues, thereby synergistically inducing early endosomal escape. The higher cellular internalization was confirmed by confocal microscopy and is probably attributable to a greater interaction between the hydrophobic F residue and the cell membrane. Moreover, the PAMAM G4 derivatives exhibited a lower toxicity than that of the PEI 25 kD, which makes them safer non-viral gene carriers with a similar level of transfection efficiency. In summary, the PAMAM G4 derivatives are highly promising candidates for gene delivery because of their low toxicity, improved buffering capacity, and higher transfection efficiency.
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