Med Oncol (2015) 32:185 DOI 10.1007/s12032-015-0624-9
ORIGINAL PAPER
Peptide GE11–Polyethylene Glycol–Polyethylenimine for targeted gene delivery in laryngeal cancer Henglei Ren1 • Liang Zhou1 • Min Liu2,3 • Weiyue Lu2,3 • Chunli Gao1,2
Received: 15 April 2015 / Accepted: 21 April 2015 Ó Springer Science+Business Media New York 2015
Abstract The objective of this study was to evaluate the possibility of using GE11–polyethylene glycol– polyethylenimine (GE11-PEG-PEI) for targeted gene delivery to treat epidermal growth factor receptor (EGFR)overexpressing laryngeal cancer. This study described the design, characterization, and in vitro and in vivo study of the nanocarrier GE11-PEG-PEI for gene delivery to treat laryngeal cancer. Analysis of the sizes and zeta potentials indicated that the formation of PEGylated complexes was dependent on the N/P ratio, and these complexes were capable of binding plasmid DNA and condensing DNA into small positively charged nanoparticles. The results also revealed that GE11-PEG-PEI had a weaker effect on cell survival in vitro. Gene transfection was performed on human laryngeal cancer Hep-2 cells in vitro and in vivo. Both the in vitro and in vivo results demonstrated that GE11-PEG-PEI had greater transfection efficiency than mPEG-PEI. Compared with mPEG-PEI/pORF-hTRAIL and saline, GE11-PEG-PEI/pORFh-TRAIL significantly (p \ 0.05) reduced tumor growth in nude mice with laryngeal cancer. Moreover, the GE11-PEG-PEI/pORFhTRAIL-treated groups showed more apoptosis than the mPEG-PEI/pORF-hTRAIL-treated groups. Therefore, our results showed that the peptide GE11 conjugated to PEG-
& Chunli Gao [email protected] 1
Department of Otolaryngology-Head and Neck Surgery, Eye and ENT Hospital, Fudan University, 83 FenYang Road, Shanghai 200031, China
2
Key Laboratory of Smart Drug Delivery, Ministry of Education, Fudan University, Shanghai 201203, China
3
Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, China
PEI delivered significantly more genes to EGFR-overexpressing laryngeal cancer cells in vivo, indicating that GE11-PEG-PEI may be a suitable gene vector for treating EGFR-overexpressing laryngeal cancer. Keywords Laryngeal cancer EGFR Gene delivery Nonviral vector Polyethylenimine
Introduction Nonviral gene delivery systems have been intensively investigated during the last few decades. Compared with viral vectors, nonviral vectors provide several advantages, can be easily synthesized, lack immunogenic potential, and have enhanced biocompatibility [1]. As one of the most successful and efficient nonviral vectors, PEI can condense DNA via electrostatic interactions to form small particles that protect the DNA from degradation and enhance its uptake via endocytosis. The high proton-buffering capacity of PEI facilitates PEI/DNA complexes to escape from endosomes into the cytosol; the complexes are then subsequently transported into the nucleus [2]. Although PEI is inherently toxic because of its highly positive charge, the attachment of PEG reduces the toxicity of PEI. However, PEGylation of PEI interferes with the cellular uptake mechanism, thus leading to low transfection efficiency [3]. Enhanced transfection efficiency could be achieved by modifying with suitable active targeting agents; the hydrophilic PEG spacer presents the targeting moiety on the surface of the complex, and the targeting moiety may behave as a free molecule due to the highly flexible PEG chain, thus leading to active targeted therapy [4]. EGFR has been an important tumor-specific target for many years. Most solid tumors, including lung, liver,
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breast, and bladder cancer, as well as squamous cell carcinoma of the head and neck (SCCHN), express high levels of EGFR, whereas the levels of EGFR are much lower in normal tissues [5]. This makes EGFR a suitable marker for cancer immunotherapy and for actively targeted cancer drug delivery. EGFR-targeted delivery systems could be effective carriers for delivering gene drugs to EGFRpositive cells [6]. Recently, a novel peptide, GE11 (sequence: YHWYGYTPQNVI), was reported as a potent EGFR ligand. GE11 peptide specifically and efficiently binds to EGFR with a much lower mitogenic activity than that of EGF [7, 8]. In a previous study, we reported the use of GE11-modified polyethylene glycol–distearoyl-phosphatidylethanolamine (PEG-DSPE) micelle and demonstrated that our targeted micelle approach results in enhanced intracellular delivery and therefore improved paclitaxel cytotoxicity on the EGFR-overexpressing human laryngeal cancer cell line Hep-2 [9]. For gene delivery systems, GE11-conjugated PEI vectors remain quite efficient for transfecting genes into hepatoma cells and tumor xenografts that highly express EGFR [7]. This peptide may be a suitable active targeting ligand for delivering therapeutic genes to EGFR-overexpressing tumor cells. Therefore, to investigate this further, we constructed and evaluated the potential of a GE11-modified PEG-PEI gene delivery system for treating laryngeal cancer in vitro and in vivo. This study describes the design, characterization, and in vitro and in vivo study of the nanocarrier GE11-PEGPEI for gene delivery to treat laryngeal cancer. The aim of this study was to improve the transfection efficiency both in vitro and in vivo to effectively treat EGFR-overexpressing laryngeal cancer.
Materials and methods Materials High molecular weight PEI (branched, MW: 25,000) was purchased from Sigma-Aldrich (Munich, Germany). Methoxy-poly (ethylene glycol)-maleimide (Mw: 2000) and maleimide-PEG-NHS (Mw: 2000) were obtained from Jenkem Technology (Beijing, China). The EGFR-specific peptide GE11 (YHWYGYTPQNVIGGGGC, MW: 1872) was synthesized by GL Biochem Ltd. (Shanghai, China). The pEGFP–N2 plasmid was purchased from Genechem Co. (Shanghai, China), and the pORF-hTRAIL plasmid was a kind gift from Professor Chen Jiang (School of Pharmacy, Fudan University, China). The TUNEL kit was purchased from Roche Molecular Biochemicals (USA). All other chemicals were of HPLC or reagent grade and were used without further purification.
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Cells and animals The human laryngeal cancer Hep-2 cell line was obtained from the Shanghai Institute of Cell Biology. The cells were maintained in RMPI-1640 medium (Gibco, USA) with 10 % fetal bovine serum (Gibco, USA) at 37 °C in a humidified atmosphere containing 5 % CO2. Male BALB/c nude mice, aged 4–6 weeks, were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and kept under SPF conditions. All animal experiments were carried out in accordance with the guidelines evaluated and approved by the ethics committee of Fudan University. The Hep-2 laryngeal squamous cell carcinoma model was established by inoculating 3 9 106 cells (in 100 ll PBS) into the subcutaneous tissue of the right shoulder blades of the mice. Synthesis of mPEG-PEI and GE11-PEG-PEI GE11-PEG-PEI and mPEG-PEI were synthesized as indicated in Fig. 1. To attach the peptide GE11 to maleimidePEG-NHS, the amino group of the peptide was initially coupled to the NHS group of Mal-PEG-NHS to obtain GE11-PEG-maleimide, which was then conjugated with PEI using the maleimide-amino reaction to obtain the final conjugate [10]. Briefly, 24 lmol of the peptide GE11 and 20 lmol of maleimide-PEG-NHS were dissolved in 1 mL of dimethylformamide (DMF), and then, 3 lL of triethylamine (TEA) was added to this mixture in a dropwise manner and stirred for 1 h. The product was further puri¨ KTA explorer 100 system (GE, Fairfield, CT, fied via an A USA) equipped on a Sephadex G-15 column (GE) and was subsequently obtained through lyophilization. The GE11PEG-PEI and mPEG-PEI were synthesized as follows. A fivefold molar ratio of GE11-PEG-maleimide (mPEGmaleimide) was co-dissolved with PEI in PBS (0.2 M, pH 7.4), and the reaction mixture was stirred overnight. After being purified in an Amicon Ultra filter tube (molecular weight cutoff of 10 kDa, Millipore) five times, the resulting products were collected and lyophilized. The products were dissolved in deuterium oxide (D2O) and characterized by 1H-NMR spectroscopy (Varian, Palo Alto, CA, USA). Preparation and characterization of carrier/pDNA complexes Polymer/pDNA complexes with various N/P ratios were prepared via the dropwise addition of equal volumes of varying concentrations of the polymer solution to a pDNA solution dissolved in PBS (pH 7.4) [10]. The complexes were vortexed for 30 s and then incubated for 30 min at room temperature. The sizes and zeta potentials of the
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Fig. 1 Synthesis of GE11-PEG-PEI and mPEG-PEI
carrier/pDNA complexes were detected by a Malvern Zetasizer Nano System (Worcestershire, UK).
(Olympus, Osaka, Japan). For quantitative analysis, cells were washed by PBS and detected by flow cytometry.
Cytotoxicity assay
In vivo antitumor activity
The in vitro cytotoxity of carrier/pDNA complexes were determined by the cell counting kit-8 (CCK8) assay. Hep-2 cells were seeded in 96-well plates in 200 lL of medium at a concentration of 5000 cells per well and incubated overnight to allow for cell attachment. The cells were then incubated with fresh RMPI-1640 medium containing 10 % FBS. Freshly prepared solutions of the PEI/pEGFP-N2, mPEG-PEI/pEGFP-N2, GE11-PEG-PEI/pEGFP-N2 complexes with a variety of different N/P ratios were then added to each well containing 0.8 lg of pDNA. Following an incubation period of 12 h, cell viability was measured using the CCK8 assay, and the plates were then analyzed by a microplate reader (Power Wave XS; Bio-Tek, Winooski, Vermont, USA) at a wavelength of 450 nm. Measurements for each sample were conducted in triplicate.
Once the mean tumor diameter reached 4–6 mm, mice bearing laryngeal tumors were randomly divided into three groups and treated with 200 lL of physiological saline, mPEG-PEI/ pORF-hTRAIL, or GE11-PEG-PEI/pORF-hTRAIL (both containing 40 lg of pDNA with an N/P ratio of 12) via tail vein injection at 10, 12, 14, 16, 18, and 20 days after implantation. The tumor volume was measured three times a week by serial caliper (GuangLu, China) measurement every 2 day, and the volume was recorded as the length 9 (width)2/ 2. The tumor volume measurements were continued until the 26th day, on which the animals were killed. Tumors were excised and fixed with 4 % paraformaldehyde or stored in liquid nitrogen.
Western blot analysis Gene transfection in vitro Transfection studies of mPEG-PEI and GE11-PEG-PEI were performed on Hep-2 cells. Cells were seeded in 48-well plates (20000 cells/well) in 0.5 mL of RMPI-1640 medium with 10 % FBS and incubated overnight. Freshly prepared mPEG-PEI/pEGFP-N2 and GE11-PEG-PEI/ pEGFP-N2 complexes with an N/P ratio of 12 were added into the 48-well plates containing 2 lg of pDNA per well and incubated at 37 °C for 12 h. The medium was exchanged for fresh culture medium and cultured for another 48 h. The cells were observed by fluorescence microscopy
The level of TRAIL expression in Hep-2 laryngeal squamous cell carcinoma xenografts was detected by western blot. Briefly, proteins were prepared from tissue pieces lysed in RIPA buffer. The protein concentrations were measured using the BSA protein reagent. Then, samples were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5 % bovine serum albumin for 2 h, the membranes were incubated overnight at 4 °C with anti-TRAIL antibodies (Abcam). The blots were then incubated with a secondary antibody conjugated to HRP for 2 h at room temperature, and the protein bands
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were visualized by electrochemiluminescence (ECL) and exposed to X-ray film.
modification of the targeting peptide GE11 led only to minor changes in the zeta potential and size properties.
Tumor apoptosis detection
Cytotoxicity assay
We performed TUNEL assays on the subcutaneous tumors to confirm the apoptotic effects induced by the different treatments. The tumor tissues were first fixed with 4 % paraformaldehyde for 48 h and embedded in paraffin. The samples were then sliced into 5-mm-thick sections and processed for TUNEL detection according to the manufacturer’s instructions. The results were captured by fluorescent microscopy (Leica, Germany). To evaluate the TUNEL staining, cells with blue-stained nuclei were considered normal, whereas cells with brown nuclei were considered apoptotic.
Cytotoxicity is an important factor to be considered when selecting polymeric gene vectors. The cytotoxicity of a variety of different PEI-derivative/pEGFP-N2 complexes was evaluated in Hep-2 cells by CCK-8 assays. As shown in Fig. 2, in all of the complexes at the N/P ratios tested in the current study, the PEI group appeared to be the most cytotoxic, whereas the cytotoxicities of mPEG-PEI/ pEGFP-N2 and GE11-PEG-PEI/pEGFP-N2 were significantly lower, with cell viabilities greater than 80 %. Our results may indicate that PEGylation led to a reduction in the positive charge, thereby reducing the cytotoxicity. However, there was no obvious difference between mPEGPEI/pEGFP-N2 and GE11-PEG-PEI/pEGFP-N2. Based on the cytotoxicity levels, we selected an N/P ratio of 12 for further examination of the PEGylated polymers.
Results Polymer synthesis and characterization The PEI derivatives were synthesized according to the above method and characterized by 1H-NMR. The substituted degrees of PEG and PEI were calculated via the characteristic peaks of 3.6 and 2.6 ppm, respectively. The degrees of substitution and molecular weights of the resulting polymers are given in Table 1. The degree of substitution of PEG was 4.3 for mPEG-PEI and 4.4 for GE11-PEG-PEI, respectively, which were close to the theoretical value of 5.0.
Table 2 Size and zeta potential of the polymer/pEGFP-N2 complexes with different N/P ratios Sample
N/P
PEI
Characterization of carrier/pDNA complexes To investigate the abilities of the PEI derivatives to bind and condense DNA, the particle sizes and zeta potentials of the polymers/pEGFP-N2 complexes are measured and shown in Table 2. In all of the complexes, the sizes increased as the N/P ratios decreased, whereas the zeta potentials sharply decreased. All of the polymers had positive zeta potentials over the N/P ratios tested, and PEGylation could reduce the sizes and zeta potentials of the carrier/ pDNA complexes at the same N/P ratio. In addition,
Table 1 Molecular composition of mPEG-PEI GE11-PEG-PEI
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Materials
mPEG-PEI
GE11-PEG-PEI
Size (nm)
Zeta potential (mV)
4
1504.8 ± 169.34
18.5 ± 0.76
8
1003.4 ± 98.72
23.4 ± 0.74
12
679.5 ± 23.24
29.2 ± 0.98
16
357.8 ± 20.4
37.2 ± 0.48
4
359.2 ± 15.56
1.8 ± 0.77
8
260.4 ± 6.38
2.9 ± 0.84
12
229.5 ± 1.90
5.9 ± 0.49
16
257.8 ± 3.74
7.9 ± 0.55
4
504.3 ± 20.44
2.3 ± 0.74
8 12
308.8 ± 4.13 248.4 ± 6.54
5.1 ± 0.42 6.8 ± 0.39
16
298.8 ± 4.58
8.7 ± 0.46
Data points represent the mean ± SD (n = 3)
SDa of PEG
Mw (kDa) b
Theoretical
Calculated
4.3
35.0
33.6
4.4
42.5
37.4
Theoretical
Calculated
mPEG-PEI
5.0
GE11-PEG-PEI
5.0
a
SD stands for substituted degree
b
Calculated according to the 1H-NMR (hydrogen 1-nuclear magnetic resonance) data
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Gene transfection in vitro
In vivo antitumor activity
The in vitro gene transfection efficiency of mPEG-PEI/ pEGFP-N2 and GE11-PEG-PEI/pEGFP-N2 was investigated, the results are shown in Fig. 3. Due to modification of the targeting peptide GE11, the gene transfection efficiency was significantly increased from 32.68 to 46.60 %. It is reasonable to assume that the modification of GE11 facilitated the increase in the mPEG-PEI transfection efficiency in vitro.
To study the in vivo antitumor activity of mPEG-PEI/pORFhTRAIL and GE11-PEG-PEI/pORF-hTRAIL, we investigated tumor volumes and growth progression in a murine model of Hep-2 laryngeal squamous cell carcinoma. The inhibitory effect of different treatments against nude mice with Hep-2 cell xenograft tumors is shown in Fig. 4a. Compared with mPEG-PEI/pORF-hTRAIL and saline, GE11-PEG-PEI/pORFh-TRAIL significantly (p \ 0.05) reduced tumor growth in the nude mice with laryngeal cancer. The body weights of each group were not significantly different (data not shown). Western blot analysis The expression of TRAIL protein in Hep-2 cell xenograft tumors was also investigated. The western blot results demonstrated that the GE11-PEG-PEI/pORFh-TRAIL complexes had higher transfection efficiency in the tumors compared with the mPEG-PEI/pORF-hTRAIL complexes (Fig. 4b). This was consistent with the results of tumor growth inhibition. Tumor apoptosis detection
Fig. 2 Cytotoxicity induced by PEI/pEGFP-N2, mPEG-PEI/pEGFPN2, GE11-PEG-PEI/pEGFP-N2 in Hep-2 cells. The relative cell viability values were compared with the untreated cells. Data are expressed as the mean ± SD (n = 3)
The TUNEL assay has been increasingly applied to investigate cell apoptosis. As shown in Fig. 4c, after all of the treatments, tumor cells of the saline- and mPEG-PEI/ pORF-hTRAIL-treated groups were slightly apoptotic,
Fig. 3 Fluorescent images of the gene expression of a mPEG–PEI/pEGFP-N2 and b GE11–PEG–PEI/pEGFP-N2 in Hep-2 cells at an N/P ratio of 12. c Quantitative evaluation of the EGFP expression by flow cytometry. Data are expressed as the mean ± SD (n = 3)
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Fig. 4 In vivo anti-tumor effect of different treatments. a The change in tumor volume as a function of time after different in Hep-2 laryngeal squamous cell carcinoma-bearing nude mice. The results are expressed as mean ± SD (n = 6). b Western blot analysis of TRAIL expression in Hep-2 laryngeal squamous cell carcinoma xenografts. GAPDH was used as a loading control. *p \ 0.05 versus
saline group and p [ 0.05 versus mPEG-PEI; **p \ 0.05 versus saline group and p \ 0.05 versus mPEG-PEI; ***p \ 0.01 versus saline group and p \ 0.05 versus mPEG-PEI. c Immunohistochemical staining of TUNEL in Hep-2 laryngeal squamous cell carcinoma xenografts treated with different schedules in nude mice. a Saline, b mPEG-PEI/pORF-hTRAIL c GE11-PEG-PEI/pORF-hTRAIL
whereas modification with GE11 could further improve the induction of apoptosis.
Analysis of the sizes and zeta potentials indicated that the formation of PEGylated complexes was dependent on the N/P ratio; these complexes were capable of binding plasmid DNA and condensing DNA into small positive nanoparticles. At the same N/P ratio, the PEI/pDNA complexes showed larger sizes and higher zeta potentials compared with PEGylated polymer/pDNA complexes, this was consistent with other studies indicating that PEGylation could reduce aggregation and decrease particle sizes [14]. To ensure the safe utilization of the cationic polymer PEG-PEI, its cytotoxicity was detected using CCK8 assays (Fig. 2). The results indicated that PEGylation of the PEI complexes led to a reduction in their cytotoxicities, thereby increasing cell viability via the shielding effect of PEG [15, 16]. The results also revealed that GE11-PEG-PEI had a weaker effect on the cell survival rate in vitro. Thus, GE11PEG-PEI may serve as a low-toxicity reagent to transfer DNA into tumor cells in vivo. Based on cytotoxicity levels and the sizes and zeta potentials, we selected an N/P ratio of 12 for our remaining experiments. In addition to reducing cytotoxicity, a key issue for the systemic delivery of therapeutic genes in clinical applications is the improvement of the gene transfection efficiency. In vitro qualitative EGFP expression assays and quantitative assays revealed a significant increase in the transfection efficiency of GE11-PEG-PEI/pEGFP-N2 compared with that of the mPEG-PEI/pEGFP-N2 polymer in Hep-2 cells (Fig. 3). Receptor-mediated active internalization of the particles is triggered by binding of the vehicle surface decoration of the GE11 peptides to EGFR, which is highly expressed on the surfaces of head and neck cancer cells [17]. This is consistent with a previous report showing that the peptide GE11 may be able to mediate the
Discussion The availability of a safe and efficient delivery vehicle is an important requirement for gene therapy. Because of safety concerns with host immune responses, ease of production, and the potential for repeat administration, nonviral vectors continue to be indicated as attractive alternatives to viral vectors in a number of studies [11, 12]. However, the gene transfection efficiency is the key issue of using nonviral vectors [13]. When administered through the blood circulation, most DNA-loaded colloidal particles are rapidly engulfed by residential macrophages in the spleen and by Kupffer cells in the liver. In addition, a major requirement for DNA delivery systems is the efficient transport of DNA through the cell membrane. Therefore, we designed GE11-PEG-PEI for gene delivery to treat laryngeal cancer, we supposed that PEI could condense plasmid DNA (pDNA) into small particles, that the PEG modification could reduce the toxicity of PEI, and that the GE11 conjugation could bind to EGFR on cancer cell membranes to deliver more genes to EGFR-overexpressing laryngeal cancer cells. In the current study, the EGFR-specific peptide GE11 was conjugated with branched PEI via heterobifunctional PEG to give the GE11–PEG–PEI polymer for targeted gene delivery to treat laryngeal cancer; formation of the resulting polymer was confirmed by 1H-NMR analysis. The existence of the PEG spacer ensured that the GE11 residue was extended on the surface of the complexes and was beneficial in maintaining the binding affinity of GE11.
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target-specific delivery of reporter genes to EGFR-overexpressing tumor cells in vitro [7]. We also evaluated the efficacy and potential of using this EGFR-targeted PEG-PEI nanocarrier in mice with laryngeal cancer. We chose tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a therapeutic gene. After intravenous administration of the carrier/pDNA complexes, GE11 modification resulted in enhanced growth inhibition of xenograft tumors (Fig. 4a). We further investigated the in vivo transfection efficiency of TRAIL protein expression in Hep-2 cell xenograft tumors. The western blot results demonstrated that GE11-PEG-PEI had better transfection efficiency than that of mPEG-PEI (Fig. 4b). Moreover, the GE11-PEG-PEI/pORF-hTRAILtreated group showed more cell apoptosis than the mPEGPEI/pORF-hTRAIL-treated group (Fig. 4c). Therefore, our results indicated that GE11 conjugated to PEG-PEI delivered significantly more genes to EGFR-overexpressing laryngeal cancer cells in vivo. Tumor-targeted anticancer drugs and gene nanoparticles have become significant cancer treatment strategies because they can be used to specifically target tumor cells and improve drug delivery, respectively [18]. It has been established that the enhanced permeation and retention (EPR) effect facilitates the accumulation of chemotherapeutics or therapeutic genes in the tumor area [19, 20]. However, the active targeting device provides the selective delivery of drugs or genes to targeted cells mainly via specific ligand recognition by receptors as well as specific cellular uptake via receptor-mediated endocytosis [21]. In combination with the results of gene transfection in vitro and in vivo, the GE11 modification was believed to be the main reason for the high transfection efficiency in gene delivery using PEG-PEI. This was consistent with our previous study showing that the GE11PEG-DSPE micelle could deliver more paclitaxel to Hep-2 cells [9]. Future intensive studies should be performed to explore the potential of co-delivering GE11-modified chemotherapeutic agents and genes for laryngeal cancer therapy. In conclusion, we systematically evaluated the targeting properties of the gene carrier GE11-PEG-PEI to treat EGFR-overexpressing laryngeal cancer. Compared with mPEG-PEI, GE11-PEG-PEI had higher tumor-targeting and gene transfection efficiency in vitro and in vivo. Based on this study, we concluded that GE11-PEG-PEI may be a suitable gene vector for treating EGFR-overexpressing laryngeal cancer. Acknowledgments This study was financially supported by National Natural Science Foundation of China (No. 81001200) and School of Pharmacy, Fudan University & The Open Project Program of Key Lab of Smart Drug Delivery (Fudan University), Ministry of Education, China.
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The authors declare no conflict of interest.
Ethical standard All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
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ORIGINAL PAPER
Peptide GE11–Polyethylene Glycol–Polyethylenimine for targeted gene delivery in laryngeal cancer Henglei Ren1 • Liang Zhou1 • Min Liu2,3 • Weiyue Lu2,3 • Chunli Gao1,2
Received: 15 April 2015 / Accepted: 21 April 2015 Ó Springer Science+Business Media New York 2015
Abstract The objective of this study was to evaluate the possibility of using GE11–polyethylene glycol– polyethylenimine (GE11-PEG-PEI) for targeted gene delivery to treat epidermal growth factor receptor (EGFR)overexpressing laryngeal cancer. This study described the design, characterization, and in vitro and in vivo study of the nanocarrier GE11-PEG-PEI for gene delivery to treat laryngeal cancer. Analysis of the sizes and zeta potentials indicated that the formation of PEGylated complexes was dependent on the N/P ratio, and these complexes were capable of binding plasmid DNA and condensing DNA into small positively charged nanoparticles. The results also revealed that GE11-PEG-PEI had a weaker effect on cell survival in vitro. Gene transfection was performed on human laryngeal cancer Hep-2 cells in vitro and in vivo. Both the in vitro and in vivo results demonstrated that GE11-PEG-PEI had greater transfection efficiency than mPEG-PEI. Compared with mPEG-PEI/pORF-hTRAIL and saline, GE11-PEG-PEI/pORFh-TRAIL significantly (p \ 0.05) reduced tumor growth in nude mice with laryngeal cancer. Moreover, the GE11-PEG-PEI/pORFhTRAIL-treated groups showed more apoptosis than the mPEG-PEI/pORF-hTRAIL-treated groups. Therefore, our results showed that the peptide GE11 conjugated to PEG-
& Chunli Gao [email protected] 1
Department of Otolaryngology-Head and Neck Surgery, Eye and ENT Hospital, Fudan University, 83 FenYang Road, Shanghai 200031, China
2
Key Laboratory of Smart Drug Delivery, Ministry of Education, Fudan University, Shanghai 201203, China
3
Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, China
PEI delivered significantly more genes to EGFR-overexpressing laryngeal cancer cells in vivo, indicating that GE11-PEG-PEI may be a suitable gene vector for treating EGFR-overexpressing laryngeal cancer. Keywords Laryngeal cancer EGFR Gene delivery Nonviral vector Polyethylenimine
Introduction Nonviral gene delivery systems have been intensively investigated during the last few decades. Compared with viral vectors, nonviral vectors provide several advantages, can be easily synthesized, lack immunogenic potential, and have enhanced biocompatibility [1]. As one of the most successful and efficient nonviral vectors, PEI can condense DNA via electrostatic interactions to form small particles that protect the DNA from degradation and enhance its uptake via endocytosis. The high proton-buffering capacity of PEI facilitates PEI/DNA complexes to escape from endosomes into the cytosol; the complexes are then subsequently transported into the nucleus [2]. Although PEI is inherently toxic because of its highly positive charge, the attachment of PEG reduces the toxicity of PEI. However, PEGylation of PEI interferes with the cellular uptake mechanism, thus leading to low transfection efficiency [3]. Enhanced transfection efficiency could be achieved by modifying with suitable active targeting agents; the hydrophilic PEG spacer presents the targeting moiety on the surface of the complex, and the targeting moiety may behave as a free molecule due to the highly flexible PEG chain, thus leading to active targeted therapy [4]. EGFR has been an important tumor-specific target for many years. Most solid tumors, including lung, liver,
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breast, and bladder cancer, as well as squamous cell carcinoma of the head and neck (SCCHN), express high levels of EGFR, whereas the levels of EGFR are much lower in normal tissues [5]. This makes EGFR a suitable marker for cancer immunotherapy and for actively targeted cancer drug delivery. EGFR-targeted delivery systems could be effective carriers for delivering gene drugs to EGFRpositive cells [6]. Recently, a novel peptide, GE11 (sequence: YHWYGYTPQNVI), was reported as a potent EGFR ligand. GE11 peptide specifically and efficiently binds to EGFR with a much lower mitogenic activity than that of EGF [7, 8]. In a previous study, we reported the use of GE11-modified polyethylene glycol–distearoyl-phosphatidylethanolamine (PEG-DSPE) micelle and demonstrated that our targeted micelle approach results in enhanced intracellular delivery and therefore improved paclitaxel cytotoxicity on the EGFR-overexpressing human laryngeal cancer cell line Hep-2 [9]. For gene delivery systems, GE11-conjugated PEI vectors remain quite efficient for transfecting genes into hepatoma cells and tumor xenografts that highly express EGFR [7]. This peptide may be a suitable active targeting ligand for delivering therapeutic genes to EGFR-overexpressing tumor cells. Therefore, to investigate this further, we constructed and evaluated the potential of a GE11-modified PEG-PEI gene delivery system for treating laryngeal cancer in vitro and in vivo. This study describes the design, characterization, and in vitro and in vivo study of the nanocarrier GE11-PEGPEI for gene delivery to treat laryngeal cancer. The aim of this study was to improve the transfection efficiency both in vitro and in vivo to effectively treat EGFR-overexpressing laryngeal cancer.
Materials and methods Materials High molecular weight PEI (branched, MW: 25,000) was purchased from Sigma-Aldrich (Munich, Germany). Methoxy-poly (ethylene glycol)-maleimide (Mw: 2000) and maleimide-PEG-NHS (Mw: 2000) were obtained from Jenkem Technology (Beijing, China). The EGFR-specific peptide GE11 (YHWYGYTPQNVIGGGGC, MW: 1872) was synthesized by GL Biochem Ltd. (Shanghai, China). The pEGFP–N2 plasmid was purchased from Genechem Co. (Shanghai, China), and the pORF-hTRAIL plasmid was a kind gift from Professor Chen Jiang (School of Pharmacy, Fudan University, China). The TUNEL kit was purchased from Roche Molecular Biochemicals (USA). All other chemicals were of HPLC or reagent grade and were used without further purification.
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Cells and animals The human laryngeal cancer Hep-2 cell line was obtained from the Shanghai Institute of Cell Biology. The cells were maintained in RMPI-1640 medium (Gibco, USA) with 10 % fetal bovine serum (Gibco, USA) at 37 °C in a humidified atmosphere containing 5 % CO2. Male BALB/c nude mice, aged 4–6 weeks, were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and kept under SPF conditions. All animal experiments were carried out in accordance with the guidelines evaluated and approved by the ethics committee of Fudan University. The Hep-2 laryngeal squamous cell carcinoma model was established by inoculating 3 9 106 cells (in 100 ll PBS) into the subcutaneous tissue of the right shoulder blades of the mice. Synthesis of mPEG-PEI and GE11-PEG-PEI GE11-PEG-PEI and mPEG-PEI were synthesized as indicated in Fig. 1. To attach the peptide GE11 to maleimidePEG-NHS, the amino group of the peptide was initially coupled to the NHS group of Mal-PEG-NHS to obtain GE11-PEG-maleimide, which was then conjugated with PEI using the maleimide-amino reaction to obtain the final conjugate [10]. Briefly, 24 lmol of the peptide GE11 and 20 lmol of maleimide-PEG-NHS were dissolved in 1 mL of dimethylformamide (DMF), and then, 3 lL of triethylamine (TEA) was added to this mixture in a dropwise manner and stirred for 1 h. The product was further puri¨ KTA explorer 100 system (GE, Fairfield, CT, fied via an A USA) equipped on a Sephadex G-15 column (GE) and was subsequently obtained through lyophilization. The GE11PEG-PEI and mPEG-PEI were synthesized as follows. A fivefold molar ratio of GE11-PEG-maleimide (mPEGmaleimide) was co-dissolved with PEI in PBS (0.2 M, pH 7.4), and the reaction mixture was stirred overnight. After being purified in an Amicon Ultra filter tube (molecular weight cutoff of 10 kDa, Millipore) five times, the resulting products were collected and lyophilized. The products were dissolved in deuterium oxide (D2O) and characterized by 1H-NMR spectroscopy (Varian, Palo Alto, CA, USA). Preparation and characterization of carrier/pDNA complexes Polymer/pDNA complexes with various N/P ratios were prepared via the dropwise addition of equal volumes of varying concentrations of the polymer solution to a pDNA solution dissolved in PBS (pH 7.4) [10]. The complexes were vortexed for 30 s and then incubated for 30 min at room temperature. The sizes and zeta potentials of the
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Fig. 1 Synthesis of GE11-PEG-PEI and mPEG-PEI
carrier/pDNA complexes were detected by a Malvern Zetasizer Nano System (Worcestershire, UK).
(Olympus, Osaka, Japan). For quantitative analysis, cells were washed by PBS and detected by flow cytometry.
Cytotoxicity assay
In vivo antitumor activity
The in vitro cytotoxity of carrier/pDNA complexes were determined by the cell counting kit-8 (CCK8) assay. Hep-2 cells were seeded in 96-well plates in 200 lL of medium at a concentration of 5000 cells per well and incubated overnight to allow for cell attachment. The cells were then incubated with fresh RMPI-1640 medium containing 10 % FBS. Freshly prepared solutions of the PEI/pEGFP-N2, mPEG-PEI/pEGFP-N2, GE11-PEG-PEI/pEGFP-N2 complexes with a variety of different N/P ratios were then added to each well containing 0.8 lg of pDNA. Following an incubation period of 12 h, cell viability was measured using the CCK8 assay, and the plates were then analyzed by a microplate reader (Power Wave XS; Bio-Tek, Winooski, Vermont, USA) at a wavelength of 450 nm. Measurements for each sample were conducted in triplicate.
Once the mean tumor diameter reached 4–6 mm, mice bearing laryngeal tumors were randomly divided into three groups and treated with 200 lL of physiological saline, mPEG-PEI/ pORF-hTRAIL, or GE11-PEG-PEI/pORF-hTRAIL (both containing 40 lg of pDNA with an N/P ratio of 12) via tail vein injection at 10, 12, 14, 16, 18, and 20 days after implantation. The tumor volume was measured three times a week by serial caliper (GuangLu, China) measurement every 2 day, and the volume was recorded as the length 9 (width)2/ 2. The tumor volume measurements were continued until the 26th day, on which the animals were killed. Tumors were excised and fixed with 4 % paraformaldehyde or stored in liquid nitrogen.
Western blot analysis Gene transfection in vitro Transfection studies of mPEG-PEI and GE11-PEG-PEI were performed on Hep-2 cells. Cells were seeded in 48-well plates (20000 cells/well) in 0.5 mL of RMPI-1640 medium with 10 % FBS and incubated overnight. Freshly prepared mPEG-PEI/pEGFP-N2 and GE11-PEG-PEI/ pEGFP-N2 complexes with an N/P ratio of 12 were added into the 48-well plates containing 2 lg of pDNA per well and incubated at 37 °C for 12 h. The medium was exchanged for fresh culture medium and cultured for another 48 h. The cells were observed by fluorescence microscopy
The level of TRAIL expression in Hep-2 laryngeal squamous cell carcinoma xenografts was detected by western blot. Briefly, proteins were prepared from tissue pieces lysed in RIPA buffer. The protein concentrations were measured using the BSA protein reagent. Then, samples were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5 % bovine serum albumin for 2 h, the membranes were incubated overnight at 4 °C with anti-TRAIL antibodies (Abcam). The blots were then incubated with a secondary antibody conjugated to HRP for 2 h at room temperature, and the protein bands
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were visualized by electrochemiluminescence (ECL) and exposed to X-ray film.
modification of the targeting peptide GE11 led only to minor changes in the zeta potential and size properties.
Tumor apoptosis detection
Cytotoxicity assay
We performed TUNEL assays on the subcutaneous tumors to confirm the apoptotic effects induced by the different treatments. The tumor tissues were first fixed with 4 % paraformaldehyde for 48 h and embedded in paraffin. The samples were then sliced into 5-mm-thick sections and processed for TUNEL detection according to the manufacturer’s instructions. The results were captured by fluorescent microscopy (Leica, Germany). To evaluate the TUNEL staining, cells with blue-stained nuclei were considered normal, whereas cells with brown nuclei were considered apoptotic.
Cytotoxicity is an important factor to be considered when selecting polymeric gene vectors. The cytotoxicity of a variety of different PEI-derivative/pEGFP-N2 complexes was evaluated in Hep-2 cells by CCK-8 assays. As shown in Fig. 2, in all of the complexes at the N/P ratios tested in the current study, the PEI group appeared to be the most cytotoxic, whereas the cytotoxicities of mPEG-PEI/ pEGFP-N2 and GE11-PEG-PEI/pEGFP-N2 were significantly lower, with cell viabilities greater than 80 %. Our results may indicate that PEGylation led to a reduction in the positive charge, thereby reducing the cytotoxicity. However, there was no obvious difference between mPEGPEI/pEGFP-N2 and GE11-PEG-PEI/pEGFP-N2. Based on the cytotoxicity levels, we selected an N/P ratio of 12 for further examination of the PEGylated polymers.
Results Polymer synthesis and characterization The PEI derivatives were synthesized according to the above method and characterized by 1H-NMR. The substituted degrees of PEG and PEI were calculated via the characteristic peaks of 3.6 and 2.6 ppm, respectively. The degrees of substitution and molecular weights of the resulting polymers are given in Table 1. The degree of substitution of PEG was 4.3 for mPEG-PEI and 4.4 for GE11-PEG-PEI, respectively, which were close to the theoretical value of 5.0.
Table 2 Size and zeta potential of the polymer/pEGFP-N2 complexes with different N/P ratios Sample
N/P
PEI
Characterization of carrier/pDNA complexes To investigate the abilities of the PEI derivatives to bind and condense DNA, the particle sizes and zeta potentials of the polymers/pEGFP-N2 complexes are measured and shown in Table 2. In all of the complexes, the sizes increased as the N/P ratios decreased, whereas the zeta potentials sharply decreased. All of the polymers had positive zeta potentials over the N/P ratios tested, and PEGylation could reduce the sizes and zeta potentials of the carrier/ pDNA complexes at the same N/P ratio. In addition,
Table 1 Molecular composition of mPEG-PEI GE11-PEG-PEI
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Materials
mPEG-PEI
GE11-PEG-PEI
Size (nm)
Zeta potential (mV)
4
1504.8 ± 169.34
18.5 ± 0.76
8
1003.4 ± 98.72
23.4 ± 0.74
12
679.5 ± 23.24
29.2 ± 0.98
16
357.8 ± 20.4
37.2 ± 0.48
4
359.2 ± 15.56
1.8 ± 0.77
8
260.4 ± 6.38
2.9 ± 0.84
12
229.5 ± 1.90
5.9 ± 0.49
16
257.8 ± 3.74
7.9 ± 0.55
4
504.3 ± 20.44
2.3 ± 0.74
8 12
308.8 ± 4.13 248.4 ± 6.54
5.1 ± 0.42 6.8 ± 0.39
16
298.8 ± 4.58
8.7 ± 0.46
Data points represent the mean ± SD (n = 3)
SDa of PEG
Mw (kDa) b
Theoretical
Calculated
4.3
35.0
33.6
4.4
42.5
37.4
Theoretical
Calculated
mPEG-PEI
5.0
GE11-PEG-PEI
5.0
a
SD stands for substituted degree
b
Calculated according to the 1H-NMR (hydrogen 1-nuclear magnetic resonance) data
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Gene transfection in vitro
In vivo antitumor activity
The in vitro gene transfection efficiency of mPEG-PEI/ pEGFP-N2 and GE11-PEG-PEI/pEGFP-N2 was investigated, the results are shown in Fig. 3. Due to modification of the targeting peptide GE11, the gene transfection efficiency was significantly increased from 32.68 to 46.60 %. It is reasonable to assume that the modification of GE11 facilitated the increase in the mPEG-PEI transfection efficiency in vitro.
To study the in vivo antitumor activity of mPEG-PEI/pORFhTRAIL and GE11-PEG-PEI/pORF-hTRAIL, we investigated tumor volumes and growth progression in a murine model of Hep-2 laryngeal squamous cell carcinoma. The inhibitory effect of different treatments against nude mice with Hep-2 cell xenograft tumors is shown in Fig. 4a. Compared with mPEG-PEI/pORF-hTRAIL and saline, GE11-PEG-PEI/pORFh-TRAIL significantly (p \ 0.05) reduced tumor growth in the nude mice with laryngeal cancer. The body weights of each group were not significantly different (data not shown). Western blot analysis The expression of TRAIL protein in Hep-2 cell xenograft tumors was also investigated. The western blot results demonstrated that the GE11-PEG-PEI/pORFh-TRAIL complexes had higher transfection efficiency in the tumors compared with the mPEG-PEI/pORF-hTRAIL complexes (Fig. 4b). This was consistent with the results of tumor growth inhibition. Tumor apoptosis detection
Fig. 2 Cytotoxicity induced by PEI/pEGFP-N2, mPEG-PEI/pEGFPN2, GE11-PEG-PEI/pEGFP-N2 in Hep-2 cells. The relative cell viability values were compared with the untreated cells. Data are expressed as the mean ± SD (n = 3)
The TUNEL assay has been increasingly applied to investigate cell apoptosis. As shown in Fig. 4c, after all of the treatments, tumor cells of the saline- and mPEG-PEI/ pORF-hTRAIL-treated groups were slightly apoptotic,
Fig. 3 Fluorescent images of the gene expression of a mPEG–PEI/pEGFP-N2 and b GE11–PEG–PEI/pEGFP-N2 in Hep-2 cells at an N/P ratio of 12. c Quantitative evaluation of the EGFP expression by flow cytometry. Data are expressed as the mean ± SD (n = 3)
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Fig. 4 In vivo anti-tumor effect of different treatments. a The change in tumor volume as a function of time after different in Hep-2 laryngeal squamous cell carcinoma-bearing nude mice. The results are expressed as mean ± SD (n = 6). b Western blot analysis of TRAIL expression in Hep-2 laryngeal squamous cell carcinoma xenografts. GAPDH was used as a loading control. *p \ 0.05 versus
saline group and p [ 0.05 versus mPEG-PEI; **p \ 0.05 versus saline group and p \ 0.05 versus mPEG-PEI; ***p \ 0.01 versus saline group and p \ 0.05 versus mPEG-PEI. c Immunohistochemical staining of TUNEL in Hep-2 laryngeal squamous cell carcinoma xenografts treated with different schedules in nude mice. a Saline, b mPEG-PEI/pORF-hTRAIL c GE11-PEG-PEI/pORF-hTRAIL
whereas modification with GE11 could further improve the induction of apoptosis.
Analysis of the sizes and zeta potentials indicated that the formation of PEGylated complexes was dependent on the N/P ratio; these complexes were capable of binding plasmid DNA and condensing DNA into small positive nanoparticles. At the same N/P ratio, the PEI/pDNA complexes showed larger sizes and higher zeta potentials compared with PEGylated polymer/pDNA complexes, this was consistent with other studies indicating that PEGylation could reduce aggregation and decrease particle sizes [14]. To ensure the safe utilization of the cationic polymer PEG-PEI, its cytotoxicity was detected using CCK8 assays (Fig. 2). The results indicated that PEGylation of the PEI complexes led to a reduction in their cytotoxicities, thereby increasing cell viability via the shielding effect of PEG [15, 16]. The results also revealed that GE11-PEG-PEI had a weaker effect on the cell survival rate in vitro. Thus, GE11PEG-PEI may serve as a low-toxicity reagent to transfer DNA into tumor cells in vivo. Based on cytotoxicity levels and the sizes and zeta potentials, we selected an N/P ratio of 12 for our remaining experiments. In addition to reducing cytotoxicity, a key issue for the systemic delivery of therapeutic genes in clinical applications is the improvement of the gene transfection efficiency. In vitro qualitative EGFP expression assays and quantitative assays revealed a significant increase in the transfection efficiency of GE11-PEG-PEI/pEGFP-N2 compared with that of the mPEG-PEI/pEGFP-N2 polymer in Hep-2 cells (Fig. 3). Receptor-mediated active internalization of the particles is triggered by binding of the vehicle surface decoration of the GE11 peptides to EGFR, which is highly expressed on the surfaces of head and neck cancer cells [17]. This is consistent with a previous report showing that the peptide GE11 may be able to mediate the
Discussion The availability of a safe and efficient delivery vehicle is an important requirement for gene therapy. Because of safety concerns with host immune responses, ease of production, and the potential for repeat administration, nonviral vectors continue to be indicated as attractive alternatives to viral vectors in a number of studies [11, 12]. However, the gene transfection efficiency is the key issue of using nonviral vectors [13]. When administered through the blood circulation, most DNA-loaded colloidal particles are rapidly engulfed by residential macrophages in the spleen and by Kupffer cells in the liver. In addition, a major requirement for DNA delivery systems is the efficient transport of DNA through the cell membrane. Therefore, we designed GE11-PEG-PEI for gene delivery to treat laryngeal cancer, we supposed that PEI could condense plasmid DNA (pDNA) into small particles, that the PEG modification could reduce the toxicity of PEI, and that the GE11 conjugation could bind to EGFR on cancer cell membranes to deliver more genes to EGFR-overexpressing laryngeal cancer cells. In the current study, the EGFR-specific peptide GE11 was conjugated with branched PEI via heterobifunctional PEG to give the GE11–PEG–PEI polymer for targeted gene delivery to treat laryngeal cancer; formation of the resulting polymer was confirmed by 1H-NMR analysis. The existence of the PEG spacer ensured that the GE11 residue was extended on the surface of the complexes and was beneficial in maintaining the binding affinity of GE11.
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target-specific delivery of reporter genes to EGFR-overexpressing tumor cells in vitro [7]. We also evaluated the efficacy and potential of using this EGFR-targeted PEG-PEI nanocarrier in mice with laryngeal cancer. We chose tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a therapeutic gene. After intravenous administration of the carrier/pDNA complexes, GE11 modification resulted in enhanced growth inhibition of xenograft tumors (Fig. 4a). We further investigated the in vivo transfection efficiency of TRAIL protein expression in Hep-2 cell xenograft tumors. The western blot results demonstrated that GE11-PEG-PEI had better transfection efficiency than that of mPEG-PEI (Fig. 4b). Moreover, the GE11-PEG-PEI/pORF-hTRAILtreated group showed more cell apoptosis than the mPEGPEI/pORF-hTRAIL-treated group (Fig. 4c). Therefore, our results indicated that GE11 conjugated to PEG-PEI delivered significantly more genes to EGFR-overexpressing laryngeal cancer cells in vivo. Tumor-targeted anticancer drugs and gene nanoparticles have become significant cancer treatment strategies because they can be used to specifically target tumor cells and improve drug delivery, respectively [18]. It has been established that the enhanced permeation and retention (EPR) effect facilitates the accumulation of chemotherapeutics or therapeutic genes in the tumor area [19, 20]. However, the active targeting device provides the selective delivery of drugs or genes to targeted cells mainly via specific ligand recognition by receptors as well as specific cellular uptake via receptor-mediated endocytosis [21]. In combination with the results of gene transfection in vitro and in vivo, the GE11 modification was believed to be the main reason for the high transfection efficiency in gene delivery using PEG-PEI. This was consistent with our previous study showing that the GE11PEG-DSPE micelle could deliver more paclitaxel to Hep-2 cells [9]. Future intensive studies should be performed to explore the potential of co-delivering GE11-modified chemotherapeutic agents and genes for laryngeal cancer therapy. In conclusion, we systematically evaluated the targeting properties of the gene carrier GE11-PEG-PEI to treat EGFR-overexpressing laryngeal cancer. Compared with mPEG-PEI, GE11-PEG-PEI had higher tumor-targeting and gene transfection efficiency in vitro and in vivo. Based on this study, we concluded that GE11-PEG-PEI may be a suitable gene vector for treating EGFR-overexpressing laryngeal cancer. Acknowledgments This study was financially supported by National Natural Science Foundation of China (No. 81001200) and School of Pharmacy, Fudan University & The Open Project Program of Key Lab of Smart Drug Delivery (Fudan University), Ministry of Education, China.
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The authors declare no conflict of interest.
Ethical standard All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
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