http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(5): 570–574 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.853812
ORIGINAL ARTICLE
Transferrin- and folate-modified, double-targeted nanocarriers for gene delivery Fanbo Jing, Dongmei Li, Wen Xu, Yujun Liu, Kai Wang, and Zhongguo Sui
Abstract
Keywords
Context: Surface modification of nanocarriers with specific ligands defines a new biological identity, which assist in targeting and internalization of the nanocarriers to specific cell populations, such as cancers and disease organs. Objective: This study aimed to develop systemically administrable dual ligands modified nanocarriers, which could target the cells through receptor-mediated pathways to increase the nuclear uptake of genetic materials. Materials and methods: In the present work, transferrin (Tf) and folate (Fa) were linked onto polyethylene glycol-phosphatidylethanolamine (PEG-PE) separately to get transferrin-PEG-PE (T-PEG-PE) and folate-PEG-PE (F-PEG-PE) ligands for the surface modification of carriers. The in vivo transfection efficiency of the novel dual ligands modified (D-modified) vectors were evaluated in tumor-bearing animal models. Results: D-Modified solid lipid nanoparticles/enhanced green fluorescence protein plasmid (D-SLN/pEGFP) has a particle size of 226 nm and a gene-loading quantity of 90%. D-SLN/pEGFP displayed over 30% higher transfection efficiency than unmodified SLN/pEGFP and single ligand modified particles in HepG2 cells. Conclusion: It could be concluded that Tf and Fa could function as excellent active targeting ligands to improve the cell-targeting ability of the carriers and the resulting dual ligands modified vectors could be applied as a promising active targeting gene delivery system.
Active targeting system, dual ligands modification, gene therapy, solid lipid nanoparticles
Introduction Nonviral pharmaceutical carriers (Boulaiz et al., 2005), such as liposomes, micelles, polymeric nanoparticles, nanocapsules, solid lipid nanoparticles, niosomes and other vectors, have been widely used for drug/gene delivery because they are less toxic, less immunogenic and easy to be modified (Atkinson & Chalmers, 2010; Fields et al., 2012; Torchilin, 2006; Zhao & Lee, 2004). Much attention has been paid to the use of cationic SLN as gene carriers which may offer a number of technological advantages, including better storage stability in comparison to liposomes, the possibility of steam sterilization and lyophilization, large scale production with qualified production lines and the use of substances that are generally accepted as safe. However, the lack of efficient site-specific delivery systems impeded the prevalent practical realization of nonviral gene therapy (Kim et al., 2010). Surface modification of nanocarriers with specific ligands can assist in targeting and internalization of the nanocarriers to specific cell populations, such as cancers and disease organs (Choi et al., 2006; Yu et al., 2010).
Correspondence: Zhongguo Sui, The Affiliated Hospital of Medical College, Qingdao University, Qingdao, 266003, People’s Republic of China. Tel: +86-532-82911848. E-mail: [email protected]
History Received 7 August 2013 Revised 30 August 2013 Accepted 6 October 2013 Published online 20 November 2013
Poly(ethylene glycol) (PEG) modification of nanocarriers have emerged as common strategies to ensure stealth shielding, long-circulation, and could also provide the nanocarriers active targeting properties by covalent attached with the wide assortment of targeting ligands by amide bonding or disulfide bridge formation, and PEG-containing ligands commonly named PEG-phosphatidylethanolamine (PEG-PE) conjugates were reported by Torchilin’s group (van Vlerken et al., 2007). They demonstrated that PEG-PE conjugates with various PEG lengths and terminal-targeted moieties can provide extremely stable, long-circulating and active-targeting nanocarriers that spontaneously accumulate in specific sites (Lukyanov et al., 2002; Torchilin, 2005, 2007). After that, several ligands-PEG-PE was widely used in nanoparticulate formulations for targeted delivery of drugs/genes (Penate et al., 2011; Reddy et al., 2002; Sawant et al., 2006; Yang et al., 2010). In the previous study, dual ligands modified system which could both target HepG2 cells and KCs in liver was developed, which contain mannose and transferrin as target ligands (Jing et al., 2013). In this research, Tf and Fa were linked onto polyethylene glycol-phosphatidylethanolamine (PEG-PE) separately to get transferrin-PEG-PE (T-PEG-PE) and folate-PEG-PE (F-PEG-PE) ligands for the surface modification of carriers. The in vivo transfection efficiency of the novel dual ligands modified (D-modified) vectors
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Pharmaceutical Biology Downloaded from informahealthcare.com by Western Michigan University on 08/27/14 For personal use only.
Department of Pharmacy, The Affiliated Hospital of Medical College, Qingdao University, Qingdao, China
Double-targeted nanocarriers for gene delivery
DOI: 10.3109/13880209.2013.853812
were evaluated in tumor-bearing animal models. Single ligand modified and unmodified systems were used as controls.
Materials and methods
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Materials Injectable soya lecithin was obtained from Shanghai Taiwei Pharmaceutical Co. Ltd. (Shanghai, China). MaleimidePEG-COOH was purchased from Shanghai Yarebio Co. Ltd. (Shanghai, China). pEGFP-N1 was kindly provided by Shandong University (Shandong, China). Stearic acid, human Tf (iron-free), mannan, dimethyldioctadecylammonium bromide (DDAB), and Cell Counting Kit-8 (CCK-8) were purchased from Sigma-Aldrich Co., Ltd. (St Louis, MO). Quant-iTÔ PicoGreenÕ dsDNA quantitation reagent was obtained from Invitrogen by Life Technologies (Carlsbad, CA). HepG2 cells were obtained from the American type culture collection (USA). All other chemicals were of analytical grade or higher.
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the whole was incubated for 4 h at room temperature with gentle stirring. The product was dialyzed against Milli-Q water for 24 h to form T-PEG-PE solution. The mixture was centrifuged at 10 000 g for 30 min at 4 C and then resuspended in PBS (pH 7.4). F-PEG-PE ligands were synthesized as described. Maleimide-PEG2000-COOH (100 mg) was dissolved with dimethyl sulfoxide (DMSO) and stirred with PE (36 mg) as a mixture. 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide (EDCHCl) (72 mg) and triethylamine (TEA, 1 equivalent of EDCHCl) were dissolved in DMSO and added dropwise into the mixture in an ice bath, stirred for 24 h to produce Maleimide-PEG-CO-NH-PE. Fa was then added to the Maleimide-PEG2000-COOH solution and the whole was incubated for 1 h at room temperature with gentle stirring. The product was dialyzed against Milli-Q water for 18 h to form F-PEG-PE solution. The mixture was centrifuged at 2000 g for 10 min at 4 C and then resuspended in PBS (pH 7.4). Preparation of cationic SLN
Animals BALB/c mice (4–6 weeks old, 20 2 g weight) were purchased from the Medical Animal Test Center of Shandong University and housed under standard laboratory conditions. All animal experiments complied with the requirements of the National Act on the Use of Experimental Animals (People’s Republic of China). Synthesis of T-PEG-PE and F-PEG-PE T-PEG-PE ligands were synthesized using the method reported previously (Jing et al., 2013). Maleimide-PEGCOOH (1 g) was dissolved in dimethyl sulfoxide (DMSO) and stirred with PE (0.5 g) as a mixture. 1-[3(Dimethylamino)propyl]-3-ethylcarbodiimide (EDCHCl) (1 g) and triethylamine (TEA, 1 equivalent of EDCHCl) were dissolved in DMSO and added dropwise into the mixture in an ice bath, stirred for 24 h to produce maleimide-PEGCO-NH-PE (PEG-PE). Tf was firstly modified with 1 equivalent of Traut’s reagent to complete thiolation of Tf. The thiolated Tf was then added to the PEG-PE solution and
SLN was prepared following the solvent displacement method (Figure 1) (Endres et al., 2012). Stearic acid (50 mg) and injectable soya lecithin (30 mg) was accurately weighted and dissolved in 10 mL acetone to form the organic phase. This solution was added dropwise into the 0.2% DDAB solution being stirred at 800 rpm at room temperature. When the evaporation of the organic solvent was complete, the redundant stabilizers were separated by ultracentrifugation at 300 g, 4 C for 20 min. The pellet was vortexed and resuspended in Milli-Q water, washed three times, filtered through a 0.45 mm membrane, and adjusted to pH 7.0 0.1 with sodium hydroxide. The obtained SLN suspensions were stored at 2–8 C. Modification of SLN and loading of gene T-PEG-PE ligands were dissolved in 5 mL of PBS (pH 7.4). Then the solution was added dropwise into 20 mL of SLN complexes that was stirred at 600 rpm at RT leading to the immediate modification. The obtained complexes were resuspended in Milli-Q water, washed three times, and filtered
Figure 1. Preparation of dual ligands and single ligand modified SLN/pEGFP.
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through a membrane with 0.80 mm pore size to obtain T-PEGPE modified SLN (T-SLN). The same procedure was done to produce F-PEG-PE modified SLN (F-SLN) (Figure 1). Dual ligands modified SLN/pEGFP complexes (D-SLN/ pEGFP) were obtained by mixing the two kinds of modified vectors together and incubated with pEGFP by vortexing the particles with a 1 mg/mL solution of pEGFP for 20 s. Incubation of the mixture for 20 min at RT facilitated the formation of D-SLN/pEGFP. Single ligand modified SLN/pEGFP complexes (T-SLN/pEGFP and F-SLN/pEGFP) and non-modified SLN/pEGFP complexes was prepared by incubating the single ligand and unmodified SLN with pEGFP (Figure 1). Characterization of D-SLN/pEGFP Size, size distribution and zeta potential The mean particle size, polydispersity index (PDI) and zeta potential of SLN, SLN/pEGFP, T-SLN/pEGFP, F-SLN/ pEGFP and D-SLN/pEGFP were analyzed by photon correlation spectroscopy (PCS) with a Zetasizer 3000 (Malvern Instruments, Malvern, England). The average particle size was expressed as volume mean diameter and the reported value was represented as mean SD (n ¼ 3). Gene loading capacity: PicoGreen-fluorometry assay The pEGFP was isolated from D-SLN/pEGFP by centrifugation at 1000 g, 4 C for 30 min. The concentration of pEGFP was determined by fluorescence, compared with the supernatant from blank SLN. The amount of pEGFP loaded in the SLN was calculated according to the linear calibration curve of pEGFP. Gene-loading quantity (%) ¼ (total amount of pEGFP – Amount of free pEGFP)/total amount of DNA 100. In vitro cytotoxicity evaluation To examine cytotoxicity, HepG2 cells were seeded in 96-well plates at 8 103 cells/well and incubated for 24 h to allow cell attachment (Suzuki et al., 2008). The cells were incubated with SLN/pEGFP and D-SLN/pEGFP complexes at various concentrations (10, 20, 50, 100, and 200 mg/mL) for 48 h at 37 C and 5% CO2 atmosphere, respectively. LipofectamineÔ 2000 (Lip) (200 mg/mL) was used as positive control according to the manufacturer’s procedures. Cells without incubation were used as negative control. Cellular viability was assessed using Cell Counting Kit-8 (CCK-8) according to the manufacturer’s procedures and the absorbance at 450 nm was measured using a microplate reader (Model 680, BIO-RAD, Hercules, CA). Cells without the addition of CCK-8 were used as a blank to calibrate the spectrophotometer to zero absorbance. The relative cell viability (%) was calculated as (Abssample Absblank)/ (Abscontrol Absblank) 100. Animal model preparation Tumor-bearing mice were prepared by inoculating (s.c.) a suspension of HepG2 cells (2 106 cells) into the right armpit of BALB/c mice (Li et al., 2012; Liu et al., 2011). Briefly, the mice were acclimatized at a temperature of 25 2 C and
Pharm Biol, 2014; 52(5): 570–574
a relative humidity of 70 5% under natural light/dark conditions for one week before dosing. Then the mice were injected subcutaneously in the right armpit with HepG2 cells suspended in PBS. Tumors were permitted to reach 8–10 mm in diameter before initiation of the studies. In vivo transfection studies In vivo transfection activity of D-SLN/pEGFP and other vectors was evaluated against HepG2 solid tumors in mice. Five groups of tumor-bearing mice, six per group, were used. The mice were injected intravenously with naked DNA, SLN/ pEGFP, T-SLN/pEGFP, F-SLN/pEGFP and D-SLN/pEGFP. The mice were sacrificed at 48 h or 72 h after injection and the tumor tissue samples and liver were taken out. The tumor tissues were homogenized by pressing the samples through a 30 mm cell mesh with the plunger of a 10 mL syringe; erythrocyte lysis buffer was added during homogenization to lyse the red blood cells. The homogenates were washed three times with PBS containing 0.5% bovine serum albumin and then filtered. The cells were finally obtained after centrifugation (4 C, 100 g, 5 min) and were seeded into 24-well plates in 1 mL of Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum (FBS). Flow cytometry was applied to quantitate the amount of cells that have been successfully transfected. The cells were washed with 1 mL of PBS (200 g, 4 C for 5 min) and were detached with trypsin/EDTA. The supernatant was discarded and resuspended with 300 mL of PBS and added into the flow cytometry to quantitate the amount of HepG2 cells, which have been successfully transfected. Statistical analysis All studies were repeated three times and all measurements were carried out in triplicate. Results were reported as means SD (SD ¼ standard deviation). Statistical significance was analyzed using the Student’s t-test. Differences between experimental groups were considered significant when the p value was less than 0.05 (p50.05).
Results and discussion Characterization of D-SLN/pEGFP Mean particle size, polydispersity index (PDI) and zeta potential of SLN, SLN/pEGFP, T-SLN/pEGFP, F-SLN/ pEGFP and D-SLN/pEGFP as well as gene-loading capacity are characterized and summarized in Table 1. During the modification procedure, T-PEG-PE ligands and F-PEG-PE were coated onto the SLN surface separately to form two kinds of single ligand modified nanocarriers first (T-SLN and F-SLN), and then they were mixed carefully and incubated with pEGFP to form a uniform dual ligand gene loaded system (D-SLN/pEGFP). The mean particle size of D-SLN/pEGFP is around 238 nm (Table 1), which is slightly different from T-SLN/pEGFP (215 nm) and F-SLN/pEGFP (221 nm). D-SLN/pEGFP has a zeta potential of þ 22 mV, and it is also in accordance with the single ligand system (T-SLN/pEGFP þ 22 mV, F-SLN/ pEGFP þ 20 mV). The gene-loading efficiency of D-SLN/ pEGFP was 90%, which marked no significant difference
Double-targeted nanocarriers for gene delivery
DOI: 10.3109/13880209.2013.853812
Table 1. Particle size, zeta potential and gene-loading quantity of different vectors.
95.3 2.8 115.8 2.9 129.6 3.1 215.4 4.2 221.6 3.7 238.4 4.8
Samples SLN T-SLN F-SLN T-SLN/pEGFP F-SLN/pEGFP D-SLN/pEGFP
Polydispersity index (PDI)
Zeta potential (mV)
Gene-loading quantity (%)
0.10 0.03 0.15 0.01 0.13 0.02 0.18 0.03 0.21 0.04 0.19 0.02
þ41.8 2.8 þ32.1 2.3 þ33.8 3.1 þ21.8 1.9 þ20.4 2.2 þ22.6 1.9
0 0 0 91 90 90
SLN/pEGFP T-SLN/pEGFP F-SLN/pEGFP
100
D-SLN/pEGFP Cell viabilities (%)
Mean particle size (nm)
80 60 40 20 0
10
F-SLN/pEGFP
100
20
50 100 200 Concentration (µg/ml)
Lipo
Figure 3. In vivo transfection of vectors in HepG2 solid tumors bearing mice.
D-SLN/pEGFP Cell viabilities (%)
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SLN/pEGFP T-SLN/pEGFP
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80 60 40 20 0
10
20
50 100 200 Concentration (µg/ml)
Lipo
Figure 2. In vitro cytotoxicity evaluation of vectors in HepG2 cells.
from T-SLN/pEGFP (91%) and F-SLN/pEGFP (90%). These results could demonstrate the uniformity and stability of the dual ligands system. In vitro cytotoxicity evaluation In vitro cytotoxicity of SLN/pEGFP, T-SLN/pEGFP, F-SLN/ pEGFP and D-SLN/pEGFP were evaluated by CCK-8 in HepG2 cells (Figure 2) at different concentrations. The cell viabilities of the vectors over the studied concentration range (10200 mg/mL) were between 80% and 100% compared with controls. D-SLN/pEGFP showed no higher cytotoxicity than other vectors at all concentrations. These results demonstrate the safety of the modified vectors for cell targeted delivery.
TfR on their surface (Maruyama, 2011). So Tf containing ligand (T-PEG-PE) was used as the first target moiety, which could bind to the TfR on the HepG2 cells. Fa is an essential B vitamin. It plays a pivotal role in cell survival by participating in the biosynthesis of nucleic and amino acids. Fa is internalized into the cells via a low-affinity reduced Fa carrier protein or via high-affinity Fa receptors (FaR). FaR is a cell surface glycosyl phosphatidylinositolanchored glycoprotein that can internalize bound Fa and Faconjugated compounds via receptor-mediated endocytosis (Paulos et al., 2004). So Fa was used as the target moiety which could bind to the FaR on the cells. In this study, these two kinds of vectors were applied as ligands for in vivo gene delivery. The gene delivery ability of D-SLN/pEGFP and others were tested in tumor-bearing animal models – HepG2 tumors bearing mice. After intravenous injection, the rats were euthanized at 24, 48 and 72 h, and at each time point HepG2 cells were isolated and analyzed. As shown in Figure 3, D-SLN/pEGFP achieved remarkably higher transfection efficiency at different time intervals when compared with T-SLN/pEGFP other vectors (p50.05). These observations strongly support the active targeting ability of Tf and Fa modified SLN/pEGFP in tumor cells and the resulting vectors would be very useful for in vivo gene delivery.
In vivo gene delivery
Conclusion
The in vivo transfection efficiency of SLN/pEGFP, T-SLN/ pEGFP, F-SLN/pEGFP and D-SLN/pEGFP, were evaluated against HepG2 solid tumors in mice. In HepG2 cells (Figure 3), D-SLN/pEGFP achieved higher transfection efficiency (42.8%) at different time intervals compared to other vectors (p50.05). HepG2 is a human liver carcinoma cell line that is widely used for the study of polarized human hepatocytes (Pinti et al., 2003). Tf is an iron-binding glycoprotein. When Tf loaded with iron encounters Tf receptor (TfR) on the surface of cell, they bind and are consequently transported into the cell (Bellocq et al., 2003; Singh, 1999). Tf is especially useful in targeting to cancer cells, as many cancer cells overexpress
This study supports the view that dual ligands (Tf and Fa) mediated targeting can successfully enhance gene expression in liver cancer cells. The results could also demonstrate that having the modification ligands in SLN formulations can significantly improve the transfection efficiency of the carriers in target cells. D-SLN/pEGFP had remarkably higher transfection efficiency in vivo which illustrate the double-targeted capability of the vectors. It could be concluded that Tf and Fa could function as an excellent active targeting ligand to improve the cell-targeting ability of the carriers, and the resulting dual ligands nanomedicine could be used as a promising double-targeted gene delivery system.
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Declaration of interest The work was supported by the Natural Science Foundation of Shandong Province (ZR2011HQ032, ZR2011HM030). The authors do not have any conflict of interest with the content of the manuscript.
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References Atkinson H, Chalmers R. (2010). Delivering the goods: Viral and nonviral gene therapy systems and the inherent limits on cargo DNA and internal sequences. Genetica 138:485–98. Bellocq NC, Pun SH, Jensen GS, Davis ME. (2003). Transferrincontaining, cyclodextrin polymer-based particles for tumor-targeted gene delivery. Bioconjug Chem 14:1122–32. Boulaiz H, Marchal JA, Prados J, et al. (2005). Non-viral and viral vectors for gene therapy. Cell Mol Biol (Noisy-le-grand) 51:3–22. Choi JS, Ko KS, Park JS, et al. (2006). Dexamethasone conjugated poly(amidoamine) dendrimer as a gene carrier for efficient nuclear translocation. Int J Pharm 320:171–8. Endres T, Zheng M, Beck-Broichsitter M, et al. (2012). Optimising the self-assembly of siRNA loaded PEG-PCL-lPEI nano-carriers employing different preparation techniques. J Control Release 160:583–91. Fields RJ, Cheng CJ, Quijano E, et al. (2012). Surface modified poly(b amino ester)-containing nanoparticles for plasmid DNA delivery. J Control Release 3659:00708–10. Jing F, Li J, Liu D, et al. (2013). Dual ligands modified double targeted nano-system for liver targeted gene delivery. Pharm Biol 51:643–9. Kim SK, Park KM, Singha K, et al. (2010). Galactosylated cucurbiturilinclusion polyplex for hepatocyte-targeted gene delivery. Chem Commun (Camb) 46:692–4. Li P, Liu D, Miao L, et al. (2012). A pH-sensitive multifunctional gene carrier assembled via layer-by-layer technique for efficient gene delivery. Int J Nanomed 7:925–39. Liu C, Yu W, Chen Z, et al. (2011). Enhanced gene transfection efficiency in CD13-positive vascular endothelial cells with targeted poly(lactic acid)-poly(ethylene glycol) nanoparticles through caveolaemediated endocytosis. J Control Release 151:162–75. Lukyanov AN, Gao Z, Mazzola L, Torchilin VP. (2002). Polyethylene glycoldiacyllipid micelles demonstrate increased acculumation in subcutaneous tumors in mice. Pharm Res 19:1424–9.
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Maruyama K. (2011). Intracellular targeting delivery of liposomal drugs to solid tumors based on EPR effects. Adv Drug Deliv Rev 63: 161–9. Paulos CM, Reddy JA, Leamon CP, et al. (2004). Ligand binding and kinetics of folate receptor recycling in vivo: Impact on receptormediated drug delivery. Mol Pharmacol 66:1406–14. Penate Medina O, Haikola M, Tahtinen M, et al. (2011). Liposomal tumor targeting in drug delivery utilizing MMP-2- and MMP-9binding ligands. J Drug Deliv 2011:160515. Pinti M, Troiano L, Nasi M, et al. (2003). Hepatoma HepG2 cells as a model for in vitro studies on mitochondrial toxicity of antiviral drugs: Which correlation with the patient? J Biol Regul Homeost Agents 17: 166–71. Reddy JA, Abburi C, Hofland H, et al. (2002). Folate-targeted, cationic liposome-mediated gene transfer into disseminated peritoneal tumors. Gene Ther 9:1542–50. Sawant RM, Hurley JP, Salmaso S, et al. (2006). ‘‘SMART’’ drug delivery systems: Double-targeted pH-responsive pharmaceutical nanocarriers. Bioconjug Chem 17:943–9. Singh M. (1999). Transferrin as a targeting ligand for liposomes and anticancer drugs. Curr Pharm Des 5:443–51. Suzuki R, Takizawa T, Kuwata Y, et al. (2008). Effective anti-tumor activity of oxaliplatin encapsulated in transferrin-PEG-liposome. Int J Pharm 346:143–50. Torchilin VP. (2005). Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 4:145–60. Torchilin VP. (2006). Multifunctional nanocarriers. Adv Drug Deliv Rev 58:1532–55. Torchilin VP. (2007) Micellar nanocarriers: Pharmaceutical perspectives. Pharm Res 24:1–16. van Vlerken LE, Vyas TK, Amiji MM. (2007). Poly(ethylene glycol)modified nanocarriers for tumor-targeted and intracellular delivery. Pharm Res 24:1405–14. Yang L, Wang L, Su XQ, et al. (2010) Suppression of ovarian cancer growth via systemic administration with liposomeencapsulated adenovirus-encoding endostatin. Cancer Gene Ther 17: 49–57. Yu W, Liu C, Liu Y, et al. (2010). Mannan-modified solid lipid nanoparticles for targeted gene delivery to alveolar macrophages. Pharm Res 27:1584–96. Zhao XB, Lee RJ. (2004) Tumor-selective targeted delivery of genes and antisense oligodeoxyribonucleotides via the folate receptor. Adv Drug Deliv Rev 56:1193–204.
ORIGINAL ARTICLE
Transferrin- and folate-modified, double-targeted nanocarriers for gene delivery Fanbo Jing, Dongmei Li, Wen Xu, Yujun Liu, Kai Wang, and Zhongguo Sui
Abstract
Keywords
Context: Surface modification of nanocarriers with specific ligands defines a new biological identity, which assist in targeting and internalization of the nanocarriers to specific cell populations, such as cancers and disease organs. Objective: This study aimed to develop systemically administrable dual ligands modified nanocarriers, which could target the cells through receptor-mediated pathways to increase the nuclear uptake of genetic materials. Materials and methods: In the present work, transferrin (Tf) and folate (Fa) were linked onto polyethylene glycol-phosphatidylethanolamine (PEG-PE) separately to get transferrin-PEG-PE (T-PEG-PE) and folate-PEG-PE (F-PEG-PE) ligands for the surface modification of carriers. The in vivo transfection efficiency of the novel dual ligands modified (D-modified) vectors were evaluated in tumor-bearing animal models. Results: D-Modified solid lipid nanoparticles/enhanced green fluorescence protein plasmid (D-SLN/pEGFP) has a particle size of 226 nm and a gene-loading quantity of 90%. D-SLN/pEGFP displayed over 30% higher transfection efficiency than unmodified SLN/pEGFP and single ligand modified particles in HepG2 cells. Conclusion: It could be concluded that Tf and Fa could function as excellent active targeting ligands to improve the cell-targeting ability of the carriers and the resulting dual ligands modified vectors could be applied as a promising active targeting gene delivery system.
Active targeting system, dual ligands modification, gene therapy, solid lipid nanoparticles
Introduction Nonviral pharmaceutical carriers (Boulaiz et al., 2005), such as liposomes, micelles, polymeric nanoparticles, nanocapsules, solid lipid nanoparticles, niosomes and other vectors, have been widely used for drug/gene delivery because they are less toxic, less immunogenic and easy to be modified (Atkinson & Chalmers, 2010; Fields et al., 2012; Torchilin, 2006; Zhao & Lee, 2004). Much attention has been paid to the use of cationic SLN as gene carriers which may offer a number of technological advantages, including better storage stability in comparison to liposomes, the possibility of steam sterilization and lyophilization, large scale production with qualified production lines and the use of substances that are generally accepted as safe. However, the lack of efficient site-specific delivery systems impeded the prevalent practical realization of nonviral gene therapy (Kim et al., 2010). Surface modification of nanocarriers with specific ligands can assist in targeting and internalization of the nanocarriers to specific cell populations, such as cancers and disease organs (Choi et al., 2006; Yu et al., 2010).
Correspondence: Zhongguo Sui, The Affiliated Hospital of Medical College, Qingdao University, Qingdao, 266003, People’s Republic of China. Tel: +86-532-82911848. E-mail: [email protected]
History Received 7 August 2013 Revised 30 August 2013 Accepted 6 October 2013 Published online 20 November 2013
Poly(ethylene glycol) (PEG) modification of nanocarriers have emerged as common strategies to ensure stealth shielding, long-circulation, and could also provide the nanocarriers active targeting properties by covalent attached with the wide assortment of targeting ligands by amide bonding or disulfide bridge formation, and PEG-containing ligands commonly named PEG-phosphatidylethanolamine (PEG-PE) conjugates were reported by Torchilin’s group (van Vlerken et al., 2007). They demonstrated that PEG-PE conjugates with various PEG lengths and terminal-targeted moieties can provide extremely stable, long-circulating and active-targeting nanocarriers that spontaneously accumulate in specific sites (Lukyanov et al., 2002; Torchilin, 2005, 2007). After that, several ligands-PEG-PE was widely used in nanoparticulate formulations for targeted delivery of drugs/genes (Penate et al., 2011; Reddy et al., 2002; Sawant et al., 2006; Yang et al., 2010). In the previous study, dual ligands modified system which could both target HepG2 cells and KCs in liver was developed, which contain mannose and transferrin as target ligands (Jing et al., 2013). In this research, Tf and Fa were linked onto polyethylene glycol-phosphatidylethanolamine (PEG-PE) separately to get transferrin-PEG-PE (T-PEG-PE) and folate-PEG-PE (F-PEG-PE) ligands for the surface modification of carriers. The in vivo transfection efficiency of the novel dual ligands modified (D-modified) vectors
20 14
Pharmaceutical Biology Downloaded from informahealthcare.com by Western Michigan University on 08/27/14 For personal use only.
Department of Pharmacy, The Affiliated Hospital of Medical College, Qingdao University, Qingdao, China
Double-targeted nanocarriers for gene delivery
DOI: 10.3109/13880209.2013.853812
were evaluated in tumor-bearing animal models. Single ligand modified and unmodified systems were used as controls.
Materials and methods
Pharmaceutical Biology Downloaded from informahealthcare.com by Western Michigan University on 08/27/14 For personal use only.
Materials Injectable soya lecithin was obtained from Shanghai Taiwei Pharmaceutical Co. Ltd. (Shanghai, China). MaleimidePEG-COOH was purchased from Shanghai Yarebio Co. Ltd. (Shanghai, China). pEGFP-N1 was kindly provided by Shandong University (Shandong, China). Stearic acid, human Tf (iron-free), mannan, dimethyldioctadecylammonium bromide (DDAB), and Cell Counting Kit-8 (CCK-8) were purchased from Sigma-Aldrich Co., Ltd. (St Louis, MO). Quant-iTÔ PicoGreenÕ dsDNA quantitation reagent was obtained from Invitrogen by Life Technologies (Carlsbad, CA). HepG2 cells were obtained from the American type culture collection (USA). All other chemicals were of analytical grade or higher.
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the whole was incubated for 4 h at room temperature with gentle stirring. The product was dialyzed against Milli-Q water for 24 h to form T-PEG-PE solution. The mixture was centrifuged at 10 000 g for 30 min at 4 C and then resuspended in PBS (pH 7.4). F-PEG-PE ligands were synthesized as described. Maleimide-PEG2000-COOH (100 mg) was dissolved with dimethyl sulfoxide (DMSO) and stirred with PE (36 mg) as a mixture. 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide (EDCHCl) (72 mg) and triethylamine (TEA, 1 equivalent of EDCHCl) were dissolved in DMSO and added dropwise into the mixture in an ice bath, stirred for 24 h to produce Maleimide-PEG-CO-NH-PE. Fa was then added to the Maleimide-PEG2000-COOH solution and the whole was incubated for 1 h at room temperature with gentle stirring. The product was dialyzed against Milli-Q water for 18 h to form F-PEG-PE solution. The mixture was centrifuged at 2000 g for 10 min at 4 C and then resuspended in PBS (pH 7.4). Preparation of cationic SLN
Animals BALB/c mice (4–6 weeks old, 20 2 g weight) were purchased from the Medical Animal Test Center of Shandong University and housed under standard laboratory conditions. All animal experiments complied with the requirements of the National Act on the Use of Experimental Animals (People’s Republic of China). Synthesis of T-PEG-PE and F-PEG-PE T-PEG-PE ligands were synthesized using the method reported previously (Jing et al., 2013). Maleimide-PEGCOOH (1 g) was dissolved in dimethyl sulfoxide (DMSO) and stirred with PE (0.5 g) as a mixture. 1-[3(Dimethylamino)propyl]-3-ethylcarbodiimide (EDCHCl) (1 g) and triethylamine (TEA, 1 equivalent of EDCHCl) were dissolved in DMSO and added dropwise into the mixture in an ice bath, stirred for 24 h to produce maleimide-PEGCO-NH-PE (PEG-PE). Tf was firstly modified with 1 equivalent of Traut’s reagent to complete thiolation of Tf. The thiolated Tf was then added to the PEG-PE solution and
SLN was prepared following the solvent displacement method (Figure 1) (Endres et al., 2012). Stearic acid (50 mg) and injectable soya lecithin (30 mg) was accurately weighted and dissolved in 10 mL acetone to form the organic phase. This solution was added dropwise into the 0.2% DDAB solution being stirred at 800 rpm at room temperature. When the evaporation of the organic solvent was complete, the redundant stabilizers were separated by ultracentrifugation at 300 g, 4 C for 20 min. The pellet was vortexed and resuspended in Milli-Q water, washed three times, filtered through a 0.45 mm membrane, and adjusted to pH 7.0 0.1 with sodium hydroxide. The obtained SLN suspensions were stored at 2–8 C. Modification of SLN and loading of gene T-PEG-PE ligands were dissolved in 5 mL of PBS (pH 7.4). Then the solution was added dropwise into 20 mL of SLN complexes that was stirred at 600 rpm at RT leading to the immediate modification. The obtained complexes were resuspended in Milli-Q water, washed three times, and filtered
Figure 1. Preparation of dual ligands and single ligand modified SLN/pEGFP.
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through a membrane with 0.80 mm pore size to obtain T-PEGPE modified SLN (T-SLN). The same procedure was done to produce F-PEG-PE modified SLN (F-SLN) (Figure 1). Dual ligands modified SLN/pEGFP complexes (D-SLN/ pEGFP) were obtained by mixing the two kinds of modified vectors together and incubated with pEGFP by vortexing the particles with a 1 mg/mL solution of pEGFP for 20 s. Incubation of the mixture for 20 min at RT facilitated the formation of D-SLN/pEGFP. Single ligand modified SLN/pEGFP complexes (T-SLN/pEGFP and F-SLN/pEGFP) and non-modified SLN/pEGFP complexes was prepared by incubating the single ligand and unmodified SLN with pEGFP (Figure 1). Characterization of D-SLN/pEGFP Size, size distribution and zeta potential The mean particle size, polydispersity index (PDI) and zeta potential of SLN, SLN/pEGFP, T-SLN/pEGFP, F-SLN/ pEGFP and D-SLN/pEGFP were analyzed by photon correlation spectroscopy (PCS) with a Zetasizer 3000 (Malvern Instruments, Malvern, England). The average particle size was expressed as volume mean diameter and the reported value was represented as mean SD (n ¼ 3). Gene loading capacity: PicoGreen-fluorometry assay The pEGFP was isolated from D-SLN/pEGFP by centrifugation at 1000 g, 4 C for 30 min. The concentration of pEGFP was determined by fluorescence, compared with the supernatant from blank SLN. The amount of pEGFP loaded in the SLN was calculated according to the linear calibration curve of pEGFP. Gene-loading quantity (%) ¼ (total amount of pEGFP – Amount of free pEGFP)/total amount of DNA 100. In vitro cytotoxicity evaluation To examine cytotoxicity, HepG2 cells were seeded in 96-well plates at 8 103 cells/well and incubated for 24 h to allow cell attachment (Suzuki et al., 2008). The cells were incubated with SLN/pEGFP and D-SLN/pEGFP complexes at various concentrations (10, 20, 50, 100, and 200 mg/mL) for 48 h at 37 C and 5% CO2 atmosphere, respectively. LipofectamineÔ 2000 (Lip) (200 mg/mL) was used as positive control according to the manufacturer’s procedures. Cells without incubation were used as negative control. Cellular viability was assessed using Cell Counting Kit-8 (CCK-8) according to the manufacturer’s procedures and the absorbance at 450 nm was measured using a microplate reader (Model 680, BIO-RAD, Hercules, CA). Cells without the addition of CCK-8 were used as a blank to calibrate the spectrophotometer to zero absorbance. The relative cell viability (%) was calculated as (Abssample Absblank)/ (Abscontrol Absblank) 100. Animal model preparation Tumor-bearing mice were prepared by inoculating (s.c.) a suspension of HepG2 cells (2 106 cells) into the right armpit of BALB/c mice (Li et al., 2012; Liu et al., 2011). Briefly, the mice were acclimatized at a temperature of 25 2 C and
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a relative humidity of 70 5% under natural light/dark conditions for one week before dosing. Then the mice were injected subcutaneously in the right armpit with HepG2 cells suspended in PBS. Tumors were permitted to reach 8–10 mm in diameter before initiation of the studies. In vivo transfection studies In vivo transfection activity of D-SLN/pEGFP and other vectors was evaluated against HepG2 solid tumors in mice. Five groups of tumor-bearing mice, six per group, were used. The mice were injected intravenously with naked DNA, SLN/ pEGFP, T-SLN/pEGFP, F-SLN/pEGFP and D-SLN/pEGFP. The mice were sacrificed at 48 h or 72 h after injection and the tumor tissue samples and liver were taken out. The tumor tissues were homogenized by pressing the samples through a 30 mm cell mesh with the plunger of a 10 mL syringe; erythrocyte lysis buffer was added during homogenization to lyse the red blood cells. The homogenates were washed three times with PBS containing 0.5% bovine serum albumin and then filtered. The cells were finally obtained after centrifugation (4 C, 100 g, 5 min) and were seeded into 24-well plates in 1 mL of Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum (FBS). Flow cytometry was applied to quantitate the amount of cells that have been successfully transfected. The cells were washed with 1 mL of PBS (200 g, 4 C for 5 min) and were detached with trypsin/EDTA. The supernatant was discarded and resuspended with 300 mL of PBS and added into the flow cytometry to quantitate the amount of HepG2 cells, which have been successfully transfected. Statistical analysis All studies were repeated three times and all measurements were carried out in triplicate. Results were reported as means SD (SD ¼ standard deviation). Statistical significance was analyzed using the Student’s t-test. Differences between experimental groups were considered significant when the p value was less than 0.05 (p50.05).
Results and discussion Characterization of D-SLN/pEGFP Mean particle size, polydispersity index (PDI) and zeta potential of SLN, SLN/pEGFP, T-SLN/pEGFP, F-SLN/ pEGFP and D-SLN/pEGFP as well as gene-loading capacity are characterized and summarized in Table 1. During the modification procedure, T-PEG-PE ligands and F-PEG-PE were coated onto the SLN surface separately to form two kinds of single ligand modified nanocarriers first (T-SLN and F-SLN), and then they were mixed carefully and incubated with pEGFP to form a uniform dual ligand gene loaded system (D-SLN/pEGFP). The mean particle size of D-SLN/pEGFP is around 238 nm (Table 1), which is slightly different from T-SLN/pEGFP (215 nm) and F-SLN/pEGFP (221 nm). D-SLN/pEGFP has a zeta potential of þ 22 mV, and it is also in accordance with the single ligand system (T-SLN/pEGFP þ 22 mV, F-SLN/ pEGFP þ 20 mV). The gene-loading efficiency of D-SLN/ pEGFP was 90%, which marked no significant difference
Double-targeted nanocarriers for gene delivery
DOI: 10.3109/13880209.2013.853812
Table 1. Particle size, zeta potential and gene-loading quantity of different vectors.
95.3 2.8 115.8 2.9 129.6 3.1 215.4 4.2 221.6 3.7 238.4 4.8
Samples SLN T-SLN F-SLN T-SLN/pEGFP F-SLN/pEGFP D-SLN/pEGFP
Polydispersity index (PDI)
Zeta potential (mV)
Gene-loading quantity (%)
0.10 0.03 0.15 0.01 0.13 0.02 0.18 0.03 0.21 0.04 0.19 0.02
þ41.8 2.8 þ32.1 2.3 þ33.8 3.1 þ21.8 1.9 þ20.4 2.2 þ22.6 1.9
0 0 0 91 90 90
SLN/pEGFP T-SLN/pEGFP F-SLN/pEGFP
100
D-SLN/pEGFP Cell viabilities (%)
Mean particle size (nm)
80 60 40 20 0
10
F-SLN/pEGFP
100
20
50 100 200 Concentration (µg/ml)
Lipo
Figure 3. In vivo transfection of vectors in HepG2 solid tumors bearing mice.
D-SLN/pEGFP Cell viabilities (%)
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SLN/pEGFP T-SLN/pEGFP
573
80 60 40 20 0
10
20
50 100 200 Concentration (µg/ml)
Lipo
Figure 2. In vitro cytotoxicity evaluation of vectors in HepG2 cells.
from T-SLN/pEGFP (91%) and F-SLN/pEGFP (90%). These results could demonstrate the uniformity and stability of the dual ligands system. In vitro cytotoxicity evaluation In vitro cytotoxicity of SLN/pEGFP, T-SLN/pEGFP, F-SLN/ pEGFP and D-SLN/pEGFP were evaluated by CCK-8 in HepG2 cells (Figure 2) at different concentrations. The cell viabilities of the vectors over the studied concentration range (10200 mg/mL) were between 80% and 100% compared with controls. D-SLN/pEGFP showed no higher cytotoxicity than other vectors at all concentrations. These results demonstrate the safety of the modified vectors for cell targeted delivery.
TfR on their surface (Maruyama, 2011). So Tf containing ligand (T-PEG-PE) was used as the first target moiety, which could bind to the TfR on the HepG2 cells. Fa is an essential B vitamin. It plays a pivotal role in cell survival by participating in the biosynthesis of nucleic and amino acids. Fa is internalized into the cells via a low-affinity reduced Fa carrier protein or via high-affinity Fa receptors (FaR). FaR is a cell surface glycosyl phosphatidylinositolanchored glycoprotein that can internalize bound Fa and Faconjugated compounds via receptor-mediated endocytosis (Paulos et al., 2004). So Fa was used as the target moiety which could bind to the FaR on the cells. In this study, these two kinds of vectors were applied as ligands for in vivo gene delivery. The gene delivery ability of D-SLN/pEGFP and others were tested in tumor-bearing animal models – HepG2 tumors bearing mice. After intravenous injection, the rats were euthanized at 24, 48 and 72 h, and at each time point HepG2 cells were isolated and analyzed. As shown in Figure 3, D-SLN/pEGFP achieved remarkably higher transfection efficiency at different time intervals when compared with T-SLN/pEGFP other vectors (p50.05). These observations strongly support the active targeting ability of Tf and Fa modified SLN/pEGFP in tumor cells and the resulting vectors would be very useful for in vivo gene delivery.
In vivo gene delivery
Conclusion
The in vivo transfection efficiency of SLN/pEGFP, T-SLN/ pEGFP, F-SLN/pEGFP and D-SLN/pEGFP, were evaluated against HepG2 solid tumors in mice. In HepG2 cells (Figure 3), D-SLN/pEGFP achieved higher transfection efficiency (42.8%) at different time intervals compared to other vectors (p50.05). HepG2 is a human liver carcinoma cell line that is widely used for the study of polarized human hepatocytes (Pinti et al., 2003). Tf is an iron-binding glycoprotein. When Tf loaded with iron encounters Tf receptor (TfR) on the surface of cell, they bind and are consequently transported into the cell (Bellocq et al., 2003; Singh, 1999). Tf is especially useful in targeting to cancer cells, as many cancer cells overexpress
This study supports the view that dual ligands (Tf and Fa) mediated targeting can successfully enhance gene expression in liver cancer cells. The results could also demonstrate that having the modification ligands in SLN formulations can significantly improve the transfection efficiency of the carriers in target cells. D-SLN/pEGFP had remarkably higher transfection efficiency in vivo which illustrate the double-targeted capability of the vectors. It could be concluded that Tf and Fa could function as an excellent active targeting ligand to improve the cell-targeting ability of the carriers, and the resulting dual ligands nanomedicine could be used as a promising double-targeted gene delivery system.
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Declaration of interest The work was supported by the Natural Science Foundation of Shandong Province (ZR2011HQ032, ZR2011HM030). The authors do not have any conflict of interest with the content of the manuscript.
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