http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(8): 1039–1044 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.876655
ORIGINAL ARTICLE
A promising targeted gene delivery system: Folate-modified dexamethasone-conjugated solid lipid nanoparticles Wei Wang1, Fang Zhou2, Linfu Ge2, Ximin Liu2, and Fansheng Kong2 Department of Chinese Medicine Integrated Traditional Chinese Medicine and Western Medicine and 2Department of Hematology, General Hospital of Ji’nan Command, Ji’nan, China
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1
Abstract
Keywords
Context: Non-viral gene delivery could deliver drugs/genes through cellular membranes and nuclear membranes by some modification of materials. Objective: This study develops a kind of vector to target the cells through receptor-mediated pathways. Nuclear localization signal (NLS) was also used to increase the nuclear uptake of genetic materials. Materials and methods: A lipid containing dexamethasone (Dexa) was synthesized as the material of the preparation of solid lipid nanoparticles (SLNs) and folate (Fa)-conjugated PEG-PE (Fa-PEG-PE) ligands were used to modify the SLNs. The in vitro cytotoxicity of the carriers at various concentrations (10, 20, 50, 100, and 200 mg/ml) were evaluated in KB human carcinoma cells (KB cells). In vivo transfection efficiency of the novel modified vectors was evaluated in disseminated peritoneal tumors on mice bearing KB cells. Results: Fa-PEG-PE modified SLNs/enhanced green fluorescence protein plasmid (pEGFP) has a particle size of 258 nm, and the gene loading quantity of the vector was 90%. The in vitro cytotoxicity of Fa-PEG-PE-modified SLNs/pEGFP (Fa-SLNs/pEGFP) was low (cell viabilities were between 80% and 100% compared with controls). Fa-SLNs/pEGFP displayed remarkably higher transfection efficiency (40%) than non-modified SLNs/pEGFP (24%) and the vectors not containing Dexa (30%) in vivo. Conclusion: The results demonstrate that Fa and Dexa could function as excellent active targeting ligands to improve the cell targeting and nuclear targeting ability of the carriers and the resulting vectors could be promising active targeting drug/gene delivery systems.
Active targeted, folate-PEG-PE, gene therapy, nuclear localization
Introduction The success of gene therapy is relying on efficient targeted gene delivery systems. Non-viral gene delivery systems such as polymeric nanoparticles (Fields et al., 2012), liposomes (Kong et al., 2012), and solid lipid nanoparticles (SLNs) (Jiang et al., 2012) have been widely developed in our laboratory for they have advantages such as being less toxic, less immunogenic, and easily modified. One of the barriers for non-viral gene delivery is the cellular membrane (Torchilin, 2006). This could be facilitated through receptor-mediated pathways by surface modification of specific ligands to carriers (Wagner et al., 1998). Folate (Fa) is an attractive ligand because of its low immunogenicity, ease of modification and low cost (Lu & Low, 2002). Fa-targeted gene delivery vectors showed enhanced accumulation in folate receptor expressing tumor model (Morris &
Correspondence: Fansheng Kong, Department of Hematology, General Hospital of Ji’nan Command, PLA, 25 Shifan Road, Ji’nan 250031, China. Tel: +86 053151665200. E-mail: kongfanshengphd@ yahoo.com.cn
History Received 20 August 2013 Revised 18 November 2013 Accepted 14 December 2013 Published online 10 March 2014
Sharma, 2010). Fa-mediated transfection has been shown to facilitate DNA internalization into cells through membrane receptors in vivo. This could improve the internalization of the vectors, hence improving transfection efficiency (Jreyssaty et al., 2012). Another barrier of non-viral gene delivery is the nuclear membrane (Nishikawa & Huang, 2001; Choi et al., 2006). In addition, nuclear localization signal (NLS) has been used to increase the nuclear uptake of genetic materials (Lo et al., 2012; Ma et al., 2009). Glucocorticoid receptor (GR) is a nuclear receptor (Yu et al., 1992). When it binds to the ligand, the receptor–ligand complex translocates from cytoplasm to nucleus (Adcock & Caramori, 2001). It was reported that the glucocorticoid receptor could dilate the nuclear pore up to 60 nm during the translocation process, which is favorable for the translocation of DNA into the nucleus (Shahin et al., 2005). Therefore, the transport of genes into the nucleus may be enhanced by conjugating glucocorticoid to carriers. Dexamethasone (Dexa), an effective synthetic glucocorticoid, could bind to the glucocorticoid receptor after cellular entry, and translocate the complexes into the nucleus (Kim et al., 2010; Rebuffat et al., 2001). The results of our study had also confirmed the observation of other researchers that
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Pharm Biol, 2014; 52(8): 1039–1044
O
NH NH
Maleimide-PEG-COOH
S EDC·HCl, TEA DMSO, Ice bath
Maleimide-PEG-CO-NH-PE
PE
Fa
NH
PEG-CO-NH-PE
S
Fa, RT
O
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Figure 1. General reaction scheme for synthesis of Fa-PEG-PE.
Dexa-conjugated vector/DNA complexes could be efficiently delivered into the nucleus with glucocorticoid receptors, resulting in an increased transgene expression (Wang et al., 2012). PEG-phosphatidylethanolamine (PEG-PE) conjugates with various PEG lengths and terminal-targeted moieties can provide extremely stable, long-circulating, and actively targeted nanocarriers which spontaneously accumulate at specific sites (Lukyanov et al., 2002; Torchilin, 2005). These kinds of ligands were also used previously by our groups for surface modification of vehicles to achieve targeted gene delivery (Jiang et al., 2012; Wang et al., 2012; Wu et al., 2012). In this study, Fa was linked onto PEG-PE to form Fa-PEG-PE as ligands for the surface modification of nanocarriers. In the present study, a Dexa-conjugated lipid was used for the enhancement of nuclear localization of genes. Enhanced green fluorescence protein plasmid (pEGFP) was used as the model gene. After the preparation of gene-loaded cationic SLNs (SLNs/pEGFP), a novel Fa-PEG-PE was synthesized and modified onto the surface of SLNs/pEGFP. The in vivo effects were observed on mice bearing KB human carcinoma cells (KB cells) model. Unmodified SLNs/pEGFP, modified SLNs not containing Dexa (Fa-non-Dexa-SLNs/pEGFP), unmodified non-Dexa-SLNs/pEGFP were applied as controls.
Materials and methods
Shandong Province and housed under standard laboratory conditions. Synthesis of Dexa-LHON and Fa-PEG-PE The Dexa-LHON conjugation reaction was performed using the method reported previously (Wang et al., 2012). Briefly, Dexa coupling to LHON was performed with 2 equivalent of Traut’s reagent, and 2 equivalent of dexamethasone mesylate in anhydrous DMSO for 6 h at room temperature (RT). To the reaction mixture, same volume of water was added and it was dialyzed against pure water and filtered through 0.45 mm syringe-type filter to remove insoluble impurities. Then the product was obtained after freezedrying, yielding white crystalline powder. Fa-PEG-PE ligands were synthesized as described in Figure 1. Maleimide-PEG2000-COOH (100 mg) was dissolved with dimethyl sulfoxide (DMSO) and stirred with PE (36 mg) to form a mixture. 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide (EDCHCl) (72 mg) and triethylamine (TEA, 1 equivalent of EDCHCl) were dissolved in DMSO and added drop-wise into the mixture in an ice bath, and stirred for 24 h to produce maleimide-PEG-CO-NH-PE. Fa was then added to the maleimide-PEG2000-COOH solution and it was incubated for 1 h at RT with gentle stirring. The product was dialyzed against Milli-Q water (Billerica, MA) for 18 h to form FaPEG-PE solution. The mixture was centrifuged at 2000g for 10 min at 4 C, and then resuspended in phosphate buffered saline (PBS).
Materials Folic acid, stearic acid, L-a-phosphatidylethanolamine (PE), 2-iminothiolane (Traut’s Reagent), and (3-[4,5-dimehyl2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co., Ltd (St Louis, MO). Injectable soya lecithin was obtained from Shanghai Taiwei Pharmaceutical Co., Ltd (Shanghai, China). Dexamethasone-21-mesylate was purchased from Steraloids Inc. (Newport, RI) Maleimide-PEG2000-COOH was purchased from Shanghai Yare Biotech Inc. (Shanghai, China). pEGFP-N1 was provided by Shandong University (Shandong, China). Quant-iTÔ PicoGreenÕ dsDNA quantitation reagent was obtained from Invitrogen by Life Technologies (Carlsbad, CA). KB cells were obtained from the American type culture collection (Manassas, VA). 6-Lauroxyhexyl ornithinate (LHON) was synthesized according to our previous procedure (Wang et al., 2012). All other chemicals were of analytical grade or higher. Animals BALB/c mice (4–6 weeks old, 20–25 g weight) were purchased from the Medical Animal Test Center of
Preparation of cationic SLNs/pEGFP SLNs were prepared by following the nanoprecipitation method (solvent displacement technique) as described previously (Kong et al., 2011, 2012). Stearic acid (50 mg) and injectable soya lecithin (30 mg) was accurately weighed and dissolved in 10 ml acetone. The organic phase was added drop-wise into the 0.1% Dexa-LHON solution being stirred at 800 rpm at RT. When complete evaporation of the organic solvent had occurred, the redundant stabilizers were separated by ultracentrifugation at 1000g, 4 C for 20 min. The pellet was vortexed and resuspended in Milli-Q water, was washed for three times, filtered through a 0.45 mm membrane, and adjusted to pH 7.0 ± 0.1 with sodium hydroxide. The obtained SLNs suspensions were stored at 2–8 C. Non-DexaSLNs were prepared as the same method using LHON without Dexa. SLNs/pEGFP (non-Dexa-SLNs/pEGFP) complexes were prepared by incubating the SLNs (non-Dexa-SLNs) with pEGFP. Briefly, pEGFP was mixed with SLNs 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 SLNs/pEGFP (non-Dexa-SLNs/pEGFP).
Folate-modified SLNs
DOI: 10.3109/13880209.2013.876655
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Figure 2. Preparation of Fa-PEG-PE modified SLNs/pEGFP.
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Modification of SLNs/pEGFP with Fa-PEG-PE Fa-PEG-PE ligands were dissolved in 5 ml of PBS (pH 7.4). Then the solution was added drop-wise into 20 ml of SLNs/ pEGFP 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 through a membrane with 0.45 mm pore size to obtain Fa-PEG-PE-SLNs/pEGFP (Fa-SLNs/pEGFP) (Figure 2). Optimization of the modification ratio During this procedure, Fa-PEG-PE ligands were continuously coated onto the surface of the SLNs/DNA which would also cause the decrease of zeta potential. To optimize the modification ratio, Fa-PEG-PE ligands dissolved in PBS were designed at different weight ratio to the SLNs (w/w) and the zeta potential of complexes was determined. NonDexa-SLNs/pEGFP was also modified according to the above-mentioned method to form Fa-non-Dexa-SLNs/pEGFP. Characterization of Fa-SLNs/pEGFP Physical-chemical characteristic The mean particle size, polydispersity index (PDI) and zeta potential of SLNs, SLNs/pEGFP, and Fa-SLNs/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 ability PicoGreen-fluorometry assay was used to determine the appropriate modification rate of Fa-PEG-PE ligands and also to quantitate the amount of pEGFP carried by the optimum modified SLNs. Different ratios of Fa-PEG-PE to SLNs/ pEGFP (from 2% to 50%, w/w) were used to prepare Fa-SLNs/pEGFP complexes and the pEGFP was isolated from Fa-SLNs/pEGFP by centrifugation at 800g, 4 C for 10 min. The concentration of pEGFP was determined by fluorescence, comparing with the supernatant from blank SLNs. The amount of pEGFP loaded in the SLNs was calculated according to the linear calibration curve of pEGFP. Gene-loading quantity (%) ¼ (Total amount of pEGFP – The amount of free pEGFP)/Total amount of DNA 100. In vitro release of Fa-SLNs/pEGFP The in vitro release studies of SLNs/pEGFP and Fa-SLNs/ pEGFP were performed in PBS (pH 7.4). Aliquots of complexes were suspended in 1 ml of PBS and vortexed for
30 s. The tubes were then placed in a 37 C shaking water bath (100 rpm). Separate tubes were used for different data points. At predetermined time intervals, the suspensions were centrifuged (1000g, 30 min) and the amount of DNA released in the supernatant was analyzed by PicoGreen assay mentioned above. Background readings were obtained using the supernatants from the blank SLNs. In vitro cytotoxicity evaluation To examine the cytotoxicity, KB cells were seeded in 48-well plates at 1 104 cells/well and incubated for 24 h to allow cell attachment. The cells were incubated with SLNs/pEGFP and Fa-SLNs/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 (Lipo, Carlsbad, CA 200 mg/ml) was used as a positive control according to manufacturer’s procedures. Cells without incubation were used as a negative control. Cellular viability was assessed using Cell Counting Kit-8 (CCK-8, Beijing, China) 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 CCK8 were used as a blank to calibrate the spectrophotometer to zero absorbance. The relative cell viability (%) was calculated as (Abssample Absblank)/(Abscontrol Absblank) 100. In vivo transfection studies Tumor-bearing mice were prepared by inoculating (s.c.), a suspension of KB cells (1 106 cells) into the right armpit of BALB/c mice (Liu et al., 2011; Li et al., 2012). Briefly, the mice were acclimatized at a temperature of 25 ± 2 C and 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 KB cells suspended in PBS. Tumors were permitted to reach 8–10 mm in diameter before initiation of the studies. In vivo anticancer activity of Fa-SLNs/pEGFP was evaluated against KB solid tumors in mice. Five groups of tumor-bearing mice (eight per group) were selected. The mice were injected intravenously with naked DNA, SLNs/pEGFP, non-Dexa-SLNs/pEGFP, Fa-non-Dexa-SLNs/pEGFP, and Fa-SLNs/pEGFP. The mice were sacrificed at 48 h or 72 h after injection and the tumor tissue samples were excised. 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
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after centrifugation (4 C, 100g, 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). The fluorescent cells were observed using an inversion fluorescence microscope, at which time pictures were taken for the record. After that, the cells were washed with 1 ml of PBS (100 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 KB cells that have been successfully transfected.
All studies were repeated three times and all measurements were carried out in triplicate. The results were reported as means ± SD (SD ¼ standard deviation). Statistical significance was analyzed using Student’s t-test. Differences between experimental groups were considered significant when the p-value was less than 0.05 (p50.05).
Results Optimization of the modification ratio Fa-PEG-PE ligands were continuously coated onto the surface of the SLNs/DNA. This procedure would mask the cationic surface charge SLNs/DNA complexes and cause the decrease of zeta potential. The best ratio of the ligands to the carriers was optimized by measuring the change of zeta potential. As illustrated in Figure 3, the optimized ratio of the Fa-PEG-PE to SLNs/DNA was 25%. This ratio was determined and used for the further experiments. Characterization of Fa-SLNs/pEGFP Size, zeta potential and gene-loading capacity, mean particle size, polydispersity index (PDI), and zeta potential of SLNs, SLNs/pEGFP, and Fa-SLNs/pEGFP were characterized and are summarized in Table 1.
The in vitro release profiles of Fa-SLNs/pEGFP and unmodified SLNs/pEGFP are illustrated in Figure 4. Both the Fa-SLNs/pEGFP and unmodified SLNs/pEGFP reached over 80% gene release at the time point of 72 h. In vitro cytotoxicity evaluation In vitro cytotoxicity of Fa-SLNs/pEGFP and SLNs/pEGFP was evaluated by CCK-8 in KB cells at different concentrations. The cell viabilities of the vectors over the studied concentration range (10 200 mg/ml) were between 80% and 100% compared with controls (Figure 5). In vivo gene delivery In vivo anticancer activity of Fa-SLNs/pEGFP was evaluated against KB solid tumors in mice. The in vivo transfection efficiency of Fa-SLNs/pEGFP, naked DNA, unmodified SLNs/pEGFP, non-Dexa-SLNs/pEGFP, Fa-non-Dexa-SLNs/ pEGFP were observed and extrapolated in Figure 6. Fa-SLNs/ pEGFP had higher transfection efficiency at different time 100 Fa-SLNs/pEGFP 90
SLNs/pEGFP
80 70 60 50 40 30 20 10
35
0 Zeta potential (mV)
0
30
12
24
36
48
60
72
84
Time (h) 25
Figure 4. The in vitro release profiles of Fa-SLNs/pEGFP and SLNs/ pEGFP.
20 15 0%
5%
10% 15% 20% 25% 30% 35% 40% 45% 50% 55%
Figure 3. Optimization of the modification ratios of Fa-PEG-PE to SLNs/DNA.
Table 1. Particle size, zeta potential and gene loading quantity of different vectors.
Characteristics Samples
Mean particle Polydispersity size (nm) index (PDI)
SLNs 97.6 ± 2.9 SLNs/pEGFP 138.6 ± 3.2 Fa-SLNs/pEGFP 203.4 ± 4.7
0.11 ± 0.04 0.16 ± 0.03 0.15 ± 0.02
SLNs/pEGFP
100
Ratios of Fa-PEG-PE to SLNs/pEGFP
Zeta potential (mV)
Gene loading quantity (%)
+39.3 ± 2.6 +28.4 ± 2.2 +16.3 ± 1.8
0 90.5 89.9
Cell viabilities (%)
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Statistical analysis
In vitro release of Fa-SLNs/pEGFP
Accumulated pEGFP release (%)
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Fa-SLNs/pEGFP
80 60 40 20 0 10
20
50
100
200
Lipo
Concentrations (µg/ml)
Figure 5. In vitro cytotoxicity evaluation of Fa-SLNs/pEGFP and SLNs/ pEGFP.
Folate-modified SLNs
DOI: 10.3109/13880209.2013.876655
Naked DNA Non-Dexa-SLNs/pEGFP SLNs/pEGFP In vivo gene expression (%)
50 40
Fa-non-Dexa-SLNs/pEGFP Fa-SLNs/pEGFP
30 20 10 0 24
48
72
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Time (h)
Figure 6. Quantitation of in vivo transfection efficiencies of Fa-SLNs/ pEGFP and other vectors at 24, 48, and 72 h post-transfection.
intervals compared to other vectors. Flow cytometry was applied to future quantitate the amount of cells which have been successfully transfected. As shown in Figure 6, Fa-SLNs/pEGFP displayed remarkably higher transfection efficiency than SLNs/pEGFP and others (p50.05).
Discussion The aim of this study was to develop a kind of vector which could overcome the main barriers of non-viral gene delivery including cellular membrane and nuclear membrane. It is acknowledged that efficient gene delivery rely on multifunctional gene vectors that possess long circulation time, cellular or tissue targeting, and nuclear targeting (Li et al., 2012). To overcome the barriers of cellular membrane and nuclear membrane and achieve efficient gene therapy, new synthetic Fa-containing ligand (Fa-PEG-PE) was applied as a modifier, which was coated on to the nanocarrier’s surface after the preparation of gene-loaded cationic SLNs (SLNs/pEGFP). Fa is an essential B vitamin, which plays a pivotal role in cell survival by participating in the biosynthesis of nucleic and amino acids. Folate is internalized into the cells via a lowaffinity reduced folate carrier protein or via high-affinity folate receptors. The best studied of these receptors is folate receptor (FaR), a cell surface glycosyl phosphatidylinositolanchored glycoprotein that can internalize bound folates and folate-conjugated 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 KB cells. A series of PEG containing ligands, commonly named PEG-phosphatidylethanolamine (PEG-PE) conjugates, were reported and used for the modification of various vehicles to achieve active-targeting nanocarriers that spontaneously accumulate in specific sites. Thus, PEG-PE was used at the anchor. Fa-PEG-PE was applied as the target ligand of the system. During the modification procedure, Fa-PEG-PE ligands were extensively coated onto the SLNs/pEGFP carriers due to the electric charge and lipophilic interaction, which covered their original surface charge and caused the decrease in zeta potential. The optimization of ligand-to-carrier ratio was carried out by measuring the zeta potential. As shown in
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Figure 3 and Table 1, the optimum ratio was obtained at 25% (Fa-PEG-PE to SLNs/DNA, w/w) and the optimum Fa-SLNs/ pEGFP had a size of 203.4 nm and zeta potential of +16.3 mV. After the optimization of the modification procedure, the Fa-PEG-PE to SLNs/DNA ratio was determined as 25% and used throughout the further experiments. PicoGreen-fluorometry assay was applied to determine the binding ability and in vitro release of Fa-SLNs/pEGFP and SLNs/pEGFP. The gene-loading efficiency of Fa-SLNs/pEGFP was 90.5%, which marked no significant difference from SLNs/pEGFP (89.9%) (Table 1). The results demonstrated that the binding of Fa-PEG-PE ligand did not detach the pEGFP from the complexes. The in vitro release profile (Figure 4) of Fa-SLNs/ pEGFP showed slightly slower release than SLNs/pEGFP during the first 12 h. This could be explained if the coating of ligand hindered the release of DNA initially. After that, FaPEG-PE might detach from the vectors, allowing the release of DNA more freely. At the end of the release study, the total amount of DNA delivered from the two kinds of vehicles was nearly the same (over 80%). In vitro cytotoxicity analysis was carried out in KB cells. The cell viabilities of Fa-SLNs/pEGFP and SLNs/pEGFP over the studied concentration range were between 80% and 100% compared with controls (Figure 5). Fa-SLNs/pEGFP showed no higher cytotoxicity than SLNs/pEGFP at all concentrations. The gene delivery ability of Fa-SLNs/pEGFP could be tested in animal models. In vivo gene delivery and expression studies were evaluated against KB tumor-bearing mice. After intravenous injection, the rats were euthanized and the Kupffer cells were isolated at 24, 48 and 72 h, and were analyzed. Fa-SLNs/pEGFP displayed remarkably higher transfection efficiency than the unmodified SLNs/pEGFP and other vectors without Dexa at all time points, as shown in Figure 6. This may be explained by the receptor-mediated active targeting mechanism. The Fa-SLNs/pEGFP was more likely to bind to KB cells via the FaR on the cells and deliver DNA more easily into the cells. Fa-non-Dexa-SLNs/pEGFP (or non-Dexa-SLNs/pEGFP), had higher transfection efficiency at all time points compared to Fa-SLNs/pEGFP (or SLNs/pEGFP) with Dexa-conjugated lipid carrier. This could be the evidence of the presence of Dexa that facilitates the binding of the vectors to the glucocorticoid receptor after cellular entry, and translocates the complexes into the nucleus, resulting in an increased gene expression. These observations strongly support the active targeting ability of Fa-PEG-PE-modified SLNs/pEGFP, and the resulting vectors would be useful for in vivo gene delivery.
Conclusions This study showed that a modified ligand (Fa-PEG-PE) glucocorticoid-mediated targeting can significantly improve the transfection efficiency of the vector, and successfully enhance the gene expression in cancer cells. It could also demonstrate that, in SLNs formulations Fa-SLNs/pEGFP had remarkably higher transfection efficiency both in vitro and in vivo than unmodified SLNs/pEGFP and non-Dexa. The conclusion is that Fa and Dexa could function as an excellent
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active targeting ligand to improve the cell targeting and nuclear targeting ability of the carriers, and the resulting nanomedicine could be a promising active targeting drug/ gene delivery system.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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Lo WL, Chien Y, Chiou GY, et al. (2012). Nuclear localization signalenhanced RNA interference of EZH2 and Oct4 in the eradication of head and neck squamous cell carcinoma-derived cancer stem cells. Biomaterials 33:3693–709. Lu Y, Low PS. (2002). Folate-mediated delivery of macromolecular anticancer therapeutic agents. Adv Drug Deliv Rev 54:675–93. Lukyanov AN, Gao Z, Mazzola L, Torchilin VP. (2002). Polyethylene glycol-dia-cyllipid micelles demonstrate increased accumulation in subcutaneous tumors in mice. Pharm Res 19:1424–9. Ma K, Hu MX, Qi Y, et al. (2009). PAMAM-triamcinolone acetonide conjugate as a nucleus-targeting gene carrier for enhanced transfer activity. Biomaterials 30:6109–18. Morris VB, Sharma CP. (2010). Folate mediated histidine derivative of quaternised chitosan as a gene delivery vector. Int J Pharm 389: 176–85. Nishikawa M, Huang L. (2001). Nonviral vectors in the new millennium: Delivery barriers in gene transfer. Hum Gene Ther 12:861–70. 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. Rebuffat A, Bernasconi A, Ceppi M, et al. (2001). Selective enhancement of gene transfer by steroid-mediated gene delivery. Nat Biotechnol 19:1155–61. Shahin V, Albermann L, Schillers H, et al. (2005). Steroids dilate nuclear pores imaged with atomic force microscopy. J Cell Physiol 202: 591–601. 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. Wagner E, Ogris M, Zauner W. (1998). Polylysine-based transfection systems utilizing receptor-mediated delivery. Adv Drug Deliv Rev 30: 97–113. Wang W, Zhou F, Ge L, et al. (2012). Transferrin-PEG-PE modified dexamethasone conjugated cationic lipid carrier mediated gene delivery system for tumor-targeted transfection. Int J Nanomedicine 7:2513–22. Wu G, Zhou F, Ge L, et al (2012). Novel mannan-PEG-PE Modified bioadhesive PLGA nanoparticles for targeted gene delivery. J Nanomaterials 2012:981670. Yu VC, Naar AM, Rosenfeld MG. (1992). Transcriptional regulation by the nuclear receptor superfamily. Curr Opin Biotechnol 3:597–602.
ORIGINAL ARTICLE
A promising targeted gene delivery system: Folate-modified dexamethasone-conjugated solid lipid nanoparticles Wei Wang1, Fang Zhou2, Linfu Ge2, Ximin Liu2, and Fansheng Kong2 Department of Chinese Medicine Integrated Traditional Chinese Medicine and Western Medicine and 2Department of Hematology, General Hospital of Ji’nan Command, Ji’nan, China
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1
Abstract
Keywords
Context: Non-viral gene delivery could deliver drugs/genes through cellular membranes and nuclear membranes by some modification of materials. Objective: This study develops a kind of vector to target the cells through receptor-mediated pathways. Nuclear localization signal (NLS) was also used to increase the nuclear uptake of genetic materials. Materials and methods: A lipid containing dexamethasone (Dexa) was synthesized as the material of the preparation of solid lipid nanoparticles (SLNs) and folate (Fa)-conjugated PEG-PE (Fa-PEG-PE) ligands were used to modify the SLNs. The in vitro cytotoxicity of the carriers at various concentrations (10, 20, 50, 100, and 200 mg/ml) were evaluated in KB human carcinoma cells (KB cells). In vivo transfection efficiency of the novel modified vectors was evaluated in disseminated peritoneal tumors on mice bearing KB cells. Results: Fa-PEG-PE modified SLNs/enhanced green fluorescence protein plasmid (pEGFP) has a particle size of 258 nm, and the gene loading quantity of the vector was 90%. The in vitro cytotoxicity of Fa-PEG-PE-modified SLNs/pEGFP (Fa-SLNs/pEGFP) was low (cell viabilities were between 80% and 100% compared with controls). Fa-SLNs/pEGFP displayed remarkably higher transfection efficiency (40%) than non-modified SLNs/pEGFP (24%) and the vectors not containing Dexa (30%) in vivo. Conclusion: The results demonstrate that Fa and Dexa could function as excellent active targeting ligands to improve the cell targeting and nuclear targeting ability of the carriers and the resulting vectors could be promising active targeting drug/gene delivery systems.
Active targeted, folate-PEG-PE, gene therapy, nuclear localization
Introduction The success of gene therapy is relying on efficient targeted gene delivery systems. Non-viral gene delivery systems such as polymeric nanoparticles (Fields et al., 2012), liposomes (Kong et al., 2012), and solid lipid nanoparticles (SLNs) (Jiang et al., 2012) have been widely developed in our laboratory for they have advantages such as being less toxic, less immunogenic, and easily modified. One of the barriers for non-viral gene delivery is the cellular membrane (Torchilin, 2006). This could be facilitated through receptor-mediated pathways by surface modification of specific ligands to carriers (Wagner et al., 1998). Folate (Fa) is an attractive ligand because of its low immunogenicity, ease of modification and low cost (Lu & Low, 2002). Fa-targeted gene delivery vectors showed enhanced accumulation in folate receptor expressing tumor model (Morris &
Correspondence: Fansheng Kong, Department of Hematology, General Hospital of Ji’nan Command, PLA, 25 Shifan Road, Ji’nan 250031, China. Tel: +86 053151665200. E-mail: kongfanshengphd@ yahoo.com.cn
History Received 20 August 2013 Revised 18 November 2013 Accepted 14 December 2013 Published online 10 March 2014
Sharma, 2010). Fa-mediated transfection has been shown to facilitate DNA internalization into cells through membrane receptors in vivo. This could improve the internalization of the vectors, hence improving transfection efficiency (Jreyssaty et al., 2012). Another barrier of non-viral gene delivery is the nuclear membrane (Nishikawa & Huang, 2001; Choi et al., 2006). In addition, nuclear localization signal (NLS) has been used to increase the nuclear uptake of genetic materials (Lo et al., 2012; Ma et al., 2009). Glucocorticoid receptor (GR) is a nuclear receptor (Yu et al., 1992). When it binds to the ligand, the receptor–ligand complex translocates from cytoplasm to nucleus (Adcock & Caramori, 2001). It was reported that the glucocorticoid receptor could dilate the nuclear pore up to 60 nm during the translocation process, which is favorable for the translocation of DNA into the nucleus (Shahin et al., 2005). Therefore, the transport of genes into the nucleus may be enhanced by conjugating glucocorticoid to carriers. Dexamethasone (Dexa), an effective synthetic glucocorticoid, could bind to the glucocorticoid receptor after cellular entry, and translocate the complexes into the nucleus (Kim et al., 2010; Rebuffat et al., 2001). The results of our study had also confirmed the observation of other researchers that
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O
NH NH
Maleimide-PEG-COOH
S EDC·HCl, TEA DMSO, Ice bath
Maleimide-PEG-CO-NH-PE
PE
Fa
NH
PEG-CO-NH-PE
S
Fa, RT
O
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Figure 1. General reaction scheme for synthesis of Fa-PEG-PE.
Dexa-conjugated vector/DNA complexes could be efficiently delivered into the nucleus with glucocorticoid receptors, resulting in an increased transgene expression (Wang et al., 2012). PEG-phosphatidylethanolamine (PEG-PE) conjugates with various PEG lengths and terminal-targeted moieties can provide extremely stable, long-circulating, and actively targeted nanocarriers which spontaneously accumulate at specific sites (Lukyanov et al., 2002; Torchilin, 2005). These kinds of ligands were also used previously by our groups for surface modification of vehicles to achieve targeted gene delivery (Jiang et al., 2012; Wang et al., 2012; Wu et al., 2012). In this study, Fa was linked onto PEG-PE to form Fa-PEG-PE as ligands for the surface modification of nanocarriers. In the present study, a Dexa-conjugated lipid was used for the enhancement of nuclear localization of genes. Enhanced green fluorescence protein plasmid (pEGFP) was used as the model gene. After the preparation of gene-loaded cationic SLNs (SLNs/pEGFP), a novel Fa-PEG-PE was synthesized and modified onto the surface of SLNs/pEGFP. The in vivo effects were observed on mice bearing KB human carcinoma cells (KB cells) model. Unmodified SLNs/pEGFP, modified SLNs not containing Dexa (Fa-non-Dexa-SLNs/pEGFP), unmodified non-Dexa-SLNs/pEGFP were applied as controls.
Materials and methods
Shandong Province and housed under standard laboratory conditions. Synthesis of Dexa-LHON and Fa-PEG-PE The Dexa-LHON conjugation reaction was performed using the method reported previously (Wang et al., 2012). Briefly, Dexa coupling to LHON was performed with 2 equivalent of Traut’s reagent, and 2 equivalent of dexamethasone mesylate in anhydrous DMSO for 6 h at room temperature (RT). To the reaction mixture, same volume of water was added and it was dialyzed against pure water and filtered through 0.45 mm syringe-type filter to remove insoluble impurities. Then the product was obtained after freezedrying, yielding white crystalline powder. Fa-PEG-PE ligands were synthesized as described in Figure 1. Maleimide-PEG2000-COOH (100 mg) was dissolved with dimethyl sulfoxide (DMSO) and stirred with PE (36 mg) to form a mixture. 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide (EDCHCl) (72 mg) and triethylamine (TEA, 1 equivalent of EDCHCl) were dissolved in DMSO and added drop-wise into the mixture in an ice bath, and stirred for 24 h to produce maleimide-PEG-CO-NH-PE. Fa was then added to the maleimide-PEG2000-COOH solution and it was incubated for 1 h at RT with gentle stirring. The product was dialyzed against Milli-Q water (Billerica, MA) for 18 h to form FaPEG-PE solution. The mixture was centrifuged at 2000g for 10 min at 4 C, and then resuspended in phosphate buffered saline (PBS).
Materials Folic acid, stearic acid, L-a-phosphatidylethanolamine (PE), 2-iminothiolane (Traut’s Reagent), and (3-[4,5-dimehyl2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co., Ltd (St Louis, MO). Injectable soya lecithin was obtained from Shanghai Taiwei Pharmaceutical Co., Ltd (Shanghai, China). Dexamethasone-21-mesylate was purchased from Steraloids Inc. (Newport, RI) Maleimide-PEG2000-COOH was purchased from Shanghai Yare Biotech Inc. (Shanghai, China). pEGFP-N1 was provided by Shandong University (Shandong, China). Quant-iTÔ PicoGreenÕ dsDNA quantitation reagent was obtained from Invitrogen by Life Technologies (Carlsbad, CA). KB cells were obtained from the American type culture collection (Manassas, VA). 6-Lauroxyhexyl ornithinate (LHON) was synthesized according to our previous procedure (Wang et al., 2012). All other chemicals were of analytical grade or higher. Animals BALB/c mice (4–6 weeks old, 20–25 g weight) were purchased from the Medical Animal Test Center of
Preparation of cationic SLNs/pEGFP SLNs were prepared by following the nanoprecipitation method (solvent displacement technique) as described previously (Kong et al., 2011, 2012). Stearic acid (50 mg) and injectable soya lecithin (30 mg) was accurately weighed and dissolved in 10 ml acetone. The organic phase was added drop-wise into the 0.1% Dexa-LHON solution being stirred at 800 rpm at RT. When complete evaporation of the organic solvent had occurred, the redundant stabilizers were separated by ultracentrifugation at 1000g, 4 C for 20 min. The pellet was vortexed and resuspended in Milli-Q water, was washed for three times, filtered through a 0.45 mm membrane, and adjusted to pH 7.0 ± 0.1 with sodium hydroxide. The obtained SLNs suspensions were stored at 2–8 C. Non-DexaSLNs were prepared as the same method using LHON without Dexa. SLNs/pEGFP (non-Dexa-SLNs/pEGFP) complexes were prepared by incubating the SLNs (non-Dexa-SLNs) with pEGFP. Briefly, pEGFP was mixed with SLNs 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 SLNs/pEGFP (non-Dexa-SLNs/pEGFP).
Folate-modified SLNs
DOI: 10.3109/13880209.2013.876655
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Figure 2. Preparation of Fa-PEG-PE modified SLNs/pEGFP.
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Modification of SLNs/pEGFP with Fa-PEG-PE Fa-PEG-PE ligands were dissolved in 5 ml of PBS (pH 7.4). Then the solution was added drop-wise into 20 ml of SLNs/ pEGFP 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 through a membrane with 0.45 mm pore size to obtain Fa-PEG-PE-SLNs/pEGFP (Fa-SLNs/pEGFP) (Figure 2). Optimization of the modification ratio During this procedure, Fa-PEG-PE ligands were continuously coated onto the surface of the SLNs/DNA which would also cause the decrease of zeta potential. To optimize the modification ratio, Fa-PEG-PE ligands dissolved in PBS were designed at different weight ratio to the SLNs (w/w) and the zeta potential of complexes was determined. NonDexa-SLNs/pEGFP was also modified according to the above-mentioned method to form Fa-non-Dexa-SLNs/pEGFP. Characterization of Fa-SLNs/pEGFP Physical-chemical characteristic The mean particle size, polydispersity index (PDI) and zeta potential of SLNs, SLNs/pEGFP, and Fa-SLNs/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 ability PicoGreen-fluorometry assay was used to determine the appropriate modification rate of Fa-PEG-PE ligands and also to quantitate the amount of pEGFP carried by the optimum modified SLNs. Different ratios of Fa-PEG-PE to SLNs/ pEGFP (from 2% to 50%, w/w) were used to prepare Fa-SLNs/pEGFP complexes and the pEGFP was isolated from Fa-SLNs/pEGFP by centrifugation at 800g, 4 C for 10 min. The concentration of pEGFP was determined by fluorescence, comparing with the supernatant from blank SLNs. The amount of pEGFP loaded in the SLNs was calculated according to the linear calibration curve of pEGFP. Gene-loading quantity (%) ¼ (Total amount of pEGFP – The amount of free pEGFP)/Total amount of DNA 100. In vitro release of Fa-SLNs/pEGFP The in vitro release studies of SLNs/pEGFP and Fa-SLNs/ pEGFP were performed in PBS (pH 7.4). Aliquots of complexes were suspended in 1 ml of PBS and vortexed for
30 s. The tubes were then placed in a 37 C shaking water bath (100 rpm). Separate tubes were used for different data points. At predetermined time intervals, the suspensions were centrifuged (1000g, 30 min) and the amount of DNA released in the supernatant was analyzed by PicoGreen assay mentioned above. Background readings were obtained using the supernatants from the blank SLNs. In vitro cytotoxicity evaluation To examine the cytotoxicity, KB cells were seeded in 48-well plates at 1 104 cells/well and incubated for 24 h to allow cell attachment. The cells were incubated with SLNs/pEGFP and Fa-SLNs/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 (Lipo, Carlsbad, CA 200 mg/ml) was used as a positive control according to manufacturer’s procedures. Cells without incubation were used as a negative control. Cellular viability was assessed using Cell Counting Kit-8 (CCK-8, Beijing, China) 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 CCK8 were used as a blank to calibrate the spectrophotometer to zero absorbance. The relative cell viability (%) was calculated as (Abssample Absblank)/(Abscontrol Absblank) 100. In vivo transfection studies Tumor-bearing mice were prepared by inoculating (s.c.), a suspension of KB cells (1 106 cells) into the right armpit of BALB/c mice (Liu et al., 2011; Li et al., 2012). Briefly, the mice were acclimatized at a temperature of 25 ± 2 C and 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 KB cells suspended in PBS. Tumors were permitted to reach 8–10 mm in diameter before initiation of the studies. In vivo anticancer activity of Fa-SLNs/pEGFP was evaluated against KB solid tumors in mice. Five groups of tumor-bearing mice (eight per group) were selected. The mice were injected intravenously with naked DNA, SLNs/pEGFP, non-Dexa-SLNs/pEGFP, Fa-non-Dexa-SLNs/pEGFP, and Fa-SLNs/pEGFP. The mice were sacrificed at 48 h or 72 h after injection and the tumor tissue samples were excised. 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
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after centrifugation (4 C, 100g, 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). The fluorescent cells were observed using an inversion fluorescence microscope, at which time pictures were taken for the record. After that, the cells were washed with 1 ml of PBS (100 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 KB cells that have been successfully transfected.
All studies were repeated three times and all measurements were carried out in triplicate. The results were reported as means ± SD (SD ¼ standard deviation). Statistical significance was analyzed using Student’s t-test. Differences between experimental groups were considered significant when the p-value was less than 0.05 (p50.05).
Results Optimization of the modification ratio Fa-PEG-PE ligands were continuously coated onto the surface of the SLNs/DNA. This procedure would mask the cationic surface charge SLNs/DNA complexes and cause the decrease of zeta potential. The best ratio of the ligands to the carriers was optimized by measuring the change of zeta potential. As illustrated in Figure 3, the optimized ratio of the Fa-PEG-PE to SLNs/DNA was 25%. This ratio was determined and used for the further experiments. Characterization of Fa-SLNs/pEGFP Size, zeta potential and gene-loading capacity, mean particle size, polydispersity index (PDI), and zeta potential of SLNs, SLNs/pEGFP, and Fa-SLNs/pEGFP were characterized and are summarized in Table 1.
The in vitro release profiles of Fa-SLNs/pEGFP and unmodified SLNs/pEGFP are illustrated in Figure 4. Both the Fa-SLNs/pEGFP and unmodified SLNs/pEGFP reached over 80% gene release at the time point of 72 h. In vitro cytotoxicity evaluation In vitro cytotoxicity of Fa-SLNs/pEGFP and SLNs/pEGFP was evaluated by CCK-8 in KB cells at different concentrations. The cell viabilities of the vectors over the studied concentration range (10 200 mg/ml) were between 80% and 100% compared with controls (Figure 5). In vivo gene delivery In vivo anticancer activity of Fa-SLNs/pEGFP was evaluated against KB solid tumors in mice. The in vivo transfection efficiency of Fa-SLNs/pEGFP, naked DNA, unmodified SLNs/pEGFP, non-Dexa-SLNs/pEGFP, Fa-non-Dexa-SLNs/ pEGFP were observed and extrapolated in Figure 6. Fa-SLNs/ pEGFP had higher transfection efficiency at different time 100 Fa-SLNs/pEGFP 90
SLNs/pEGFP
80 70 60 50 40 30 20 10
35
0 Zeta potential (mV)
0
30
12
24
36
48
60
72
84
Time (h) 25
Figure 4. The in vitro release profiles of Fa-SLNs/pEGFP and SLNs/ pEGFP.
20 15 0%
5%
10% 15% 20% 25% 30% 35% 40% 45% 50% 55%
Figure 3. Optimization of the modification ratios of Fa-PEG-PE to SLNs/DNA.
Table 1. Particle size, zeta potential and gene loading quantity of different vectors.
Characteristics Samples
Mean particle Polydispersity size (nm) index (PDI)
SLNs 97.6 ± 2.9 SLNs/pEGFP 138.6 ± 3.2 Fa-SLNs/pEGFP 203.4 ± 4.7
0.11 ± 0.04 0.16 ± 0.03 0.15 ± 0.02
SLNs/pEGFP
100
Ratios of Fa-PEG-PE to SLNs/pEGFP
Zeta potential (mV)
Gene loading quantity (%)
+39.3 ± 2.6 +28.4 ± 2.2 +16.3 ± 1.8
0 90.5 89.9
Cell viabilities (%)
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Statistical analysis
In vitro release of Fa-SLNs/pEGFP
Accumulated pEGFP release (%)
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80 60 40 20 0 10
20
50
100
200
Lipo
Concentrations (µg/ml)
Figure 5. In vitro cytotoxicity evaluation of Fa-SLNs/pEGFP and SLNs/ pEGFP.
Folate-modified SLNs
DOI: 10.3109/13880209.2013.876655
Naked DNA Non-Dexa-SLNs/pEGFP SLNs/pEGFP In vivo gene expression (%)
50 40
Fa-non-Dexa-SLNs/pEGFP Fa-SLNs/pEGFP
30 20 10 0 24
48
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Time (h)
Figure 6. Quantitation of in vivo transfection efficiencies of Fa-SLNs/ pEGFP and other vectors at 24, 48, and 72 h post-transfection.
intervals compared to other vectors. Flow cytometry was applied to future quantitate the amount of cells which have been successfully transfected. As shown in Figure 6, Fa-SLNs/pEGFP displayed remarkably higher transfection efficiency than SLNs/pEGFP and others (p50.05).
Discussion The aim of this study was to develop a kind of vector which could overcome the main barriers of non-viral gene delivery including cellular membrane and nuclear membrane. It is acknowledged that efficient gene delivery rely on multifunctional gene vectors that possess long circulation time, cellular or tissue targeting, and nuclear targeting (Li et al., 2012). To overcome the barriers of cellular membrane and nuclear membrane and achieve efficient gene therapy, new synthetic Fa-containing ligand (Fa-PEG-PE) was applied as a modifier, which was coated on to the nanocarrier’s surface after the preparation of gene-loaded cationic SLNs (SLNs/pEGFP). Fa is an essential B vitamin, which plays a pivotal role in cell survival by participating in the biosynthesis of nucleic and amino acids. Folate is internalized into the cells via a lowaffinity reduced folate carrier protein or via high-affinity folate receptors. The best studied of these receptors is folate receptor (FaR), a cell surface glycosyl phosphatidylinositolanchored glycoprotein that can internalize bound folates and folate-conjugated 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 KB cells. A series of PEG containing ligands, commonly named PEG-phosphatidylethanolamine (PEG-PE) conjugates, were reported and used for the modification of various vehicles to achieve active-targeting nanocarriers that spontaneously accumulate in specific sites. Thus, PEG-PE was used at the anchor. Fa-PEG-PE was applied as the target ligand of the system. During the modification procedure, Fa-PEG-PE ligands were extensively coated onto the SLNs/pEGFP carriers due to the electric charge and lipophilic interaction, which covered their original surface charge and caused the decrease in zeta potential. The optimization of ligand-to-carrier ratio was carried out by measuring the zeta potential. As shown in
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Figure 3 and Table 1, the optimum ratio was obtained at 25% (Fa-PEG-PE to SLNs/DNA, w/w) and the optimum Fa-SLNs/ pEGFP had a size of 203.4 nm and zeta potential of +16.3 mV. After the optimization of the modification procedure, the Fa-PEG-PE to SLNs/DNA ratio was determined as 25% and used throughout the further experiments. PicoGreen-fluorometry assay was applied to determine the binding ability and in vitro release of Fa-SLNs/pEGFP and SLNs/pEGFP. The gene-loading efficiency of Fa-SLNs/pEGFP was 90.5%, which marked no significant difference from SLNs/pEGFP (89.9%) (Table 1). The results demonstrated that the binding of Fa-PEG-PE ligand did not detach the pEGFP from the complexes. The in vitro release profile (Figure 4) of Fa-SLNs/ pEGFP showed slightly slower release than SLNs/pEGFP during the first 12 h. This could be explained if the coating of ligand hindered the release of DNA initially. After that, FaPEG-PE might detach from the vectors, allowing the release of DNA more freely. At the end of the release study, the total amount of DNA delivered from the two kinds of vehicles was nearly the same (over 80%). In vitro cytotoxicity analysis was carried out in KB cells. The cell viabilities of Fa-SLNs/pEGFP and SLNs/pEGFP over the studied concentration range were between 80% and 100% compared with controls (Figure 5). Fa-SLNs/pEGFP showed no higher cytotoxicity than SLNs/pEGFP at all concentrations. The gene delivery ability of Fa-SLNs/pEGFP could be tested in animal models. In vivo gene delivery and expression studies were evaluated against KB tumor-bearing mice. After intravenous injection, the rats were euthanized and the Kupffer cells were isolated at 24, 48 and 72 h, and were analyzed. Fa-SLNs/pEGFP displayed remarkably higher transfection efficiency than the unmodified SLNs/pEGFP and other vectors without Dexa at all time points, as shown in Figure 6. This may be explained by the receptor-mediated active targeting mechanism. The Fa-SLNs/pEGFP was more likely to bind to KB cells via the FaR on the cells and deliver DNA more easily into the cells. Fa-non-Dexa-SLNs/pEGFP (or non-Dexa-SLNs/pEGFP), had higher transfection efficiency at all time points compared to Fa-SLNs/pEGFP (or SLNs/pEGFP) with Dexa-conjugated lipid carrier. This could be the evidence of the presence of Dexa that facilitates the binding of the vectors to the glucocorticoid receptor after cellular entry, and translocates the complexes into the nucleus, resulting in an increased gene expression. These observations strongly support the active targeting ability of Fa-PEG-PE-modified SLNs/pEGFP, and the resulting vectors would be useful for in vivo gene delivery.
Conclusions This study showed that a modified ligand (Fa-PEG-PE) glucocorticoid-mediated targeting can significantly improve the transfection efficiency of the vector, and successfully enhance the gene expression in cancer cells. It could also demonstrate that, in SLNs formulations Fa-SLNs/pEGFP had remarkably higher transfection efficiency both in vitro and in vivo than unmodified SLNs/pEGFP and non-Dexa. The conclusion is that Fa and Dexa could function as an excellent
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active targeting ligand to improve the cell targeting and nuclear targeting ability of the carriers, and the resulting nanomedicine could be a promising active targeting drug/ gene delivery system.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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