Introduction
Renal cell carcinoma (RCC) is the third most common urological cancer with an incidence of approximately 5-10 per 100,000 and comprises 2-3% of all malignancies [
1]. The majority (~80%) type is defined as clear cell RCC (CCRCC) which has bad prognosis and does not sensitive to radiotherapy and chemotherapy [
2,
3]. Therefore, a novel therapy strategy against CCRCC needs to be developed. In the past decade, gene therapy was studied world-widely and demonstrated as a novel method to treat many cancers [
4]. Thus, the use of gene therapy may be a new way to treat CCRCC.
In the field of cancer gene therapy, it is well known that the success of therapy is greatly dependent on the gene delivery vectors which ensure the gene to reach target cells. Recent years, cationic polymers, which can interact with negatively charged DNA through electrostatic interaction to form nanocomplexes, are widely attempted to use as gene delivery systems. The advantages associated with this kind of vectors include that they can protect DNA from nuclease digestion, and thus enhance the gene expression within target cells, they hold low immunogenic response and can also be modified selectively, and so on [
5‐
7]. Therefore, various cationic polymers, such as poly(L-lysine) [
8] and polyethyleneimine (PEI) [
9], have been synthesized and used as a gene delivery vehicle.
Among the total non-viral gene vectors, PEI, with high transfection efficiency, has bright prospects in application [
9,
10]. However, PEI is not fit for keeping on gene expression [
10,
11] due to its serious cytotoxicity. Actually, the transfection efficiency and cytotoxicity are almost antagonistic. PEI with a low molecular weight (MW), including 800-Da, 2000-Da MW or less, displays a low cytotoxicity and transfection efficiency. On the contrary, PEI with a high 25-kD MW shows higher transfection efficiency and cytotoxicity [
12,
13]. In order to balance the transfection efficiency and toxicity, investigators attempt to make some modifications against PEI properties. Up to now, PEI has been modified with chloroquine, polyethylene glycol (PEG), folic acid (FA), heparin and so on [
4]. Furthermore, several PEI-based delivery vehicles have been used to carry DNA for gene therapy [
9,
14,
15]. For example, a liner PEI-cholesterol conjugation was encapsulated with interleukin-12 to treat RCC mice intravenously, which was demonstrated to be effective for treatment of RCC-induced pulmonary metastases [
15]. In addition, folate-target gene therapy vectors have been found to promote much higher levels of tumor-specific gene expression than nontargeted vectors [
16].
In this study, we compared cytotoxicity and the transfection efficiency of the three PEI-derived materials, including poly(ε-caprolactone)-pluronic-poly(ε-caprolactone)-grafted-PEI (PCFC-g-PEI), FA-PCFC-isophorone diidocyanate-PEI (FA-PEAs) and heparin-PEI (HPEI) in vitro and in gene therapy carrying with Von Hippel-Lindau (VHL) on mice RCC model. These new attempts provide potential methods to treat CCRCC by VHL gene therapy, which may have a bright prospect in future.
Methods
Cell Lines
The human CCRCC cell line OS-RC-2 and mouse macrophage cell line Ana-1 were purchased from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Human umbilical vein endothelial cell line (HUVEC) was ordered from ATCC. All of them were maintained in RPMI-1640 media supplemented with 10% fetal bovine serum which contained 100 units/ml of penicillin and 100 units/ml of streptomycin. All cells were routinely maintained at 37°C in humidified air containing 5% CO2.
Reagents
Dimethyl sulfoxide (DMSO) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were ordered from Sigma Company, USA. The PCFC-g-PEI, FA-PEAs and HPEI, all were synthesized. PCFC-g-PEI was obtained by Michael addition reaction with glycidyl methacrylate-PCFC-glycidyl methacrylate (GMA-PCFC-GMA) and the 25-kD PEI [
4]. PCFC was synthesized by ring-opening polymerization of ε-caprolactone initiated by pluronic 105 (poly (ethylene glycol)-poly (propylene glycol)-poly (ethylene glycol), PEG-PPG-PEG, MW = 1900 ) [
17]. The cationic HPEI nanogel was conjugated by 2-kD PEI and heparin. Heparin is a biodegradable negative polysaccharide with many carboxylic groups, and PEI is a cationic polymer with many primary amine groups in its molecular structure. Thus, in presence of EDC/NHS, the reaction between heparin and PEI occurs [
18]. PEAs (PCFC-IPDI-PEI) were synthesized by 2-kD PEI and PCFC copolymers using isophorone diidocyanate (IPDI) as a cross-linker [
19]. The folic acid-coupled PEAs were prepared by the reaction of the activated folate ester with the amine group on the PEAs.
Evaluation of cytotoxicity of three PEI-derived materials
The cytotoxicity of the three modified PEI-derived materials were determined by MTT assay. According to the reported methods [
20], the renal cancer cell line, phagocytic cells and endothelial cells were used to detect the toxicity of three modified PEI-derived materials
in vitro.
The OS-RC-2 cells were seeded in 96-well plates at a density of 5 × 10
3 cells/well in 0.1 ml growth medium and incubated overnight, then a series of concentrations of each PEI-derived material (FA-PEAs, PCFC-g-PEI and HPEI), solved in 0.1 ml fresh RPMI-1640 medium, were respectively added into each well to incubate for another 24 h. Untreated cells were used as a control. Then, 20 μl of 5 mg/ml MTT solution was added to each well for incubation 4 h. Finally, the MTT was removed, and 200 μl DMSO was added to dissolve the MTT-formazan crystals. The absorbance was measured at 490 nm by an ELISA microplate reader (Bio-Rad). Besides that, the toxicity on Ana-1 and HUVEC cells were evaluated with the same method. The cell viability (%) was calculated according to the following formula. All data were presented as the mean ± SD (Standard Deviation).
VHL- expressing plasmid
The VHL gene was cloned into the mammalian expression vector pVITRO2-neo-mcs (Invitrogen, San Diego, CA), which can be stably transfected in mammalian cells so that the genes of interest are expressed at high levels. Moreover, it can also allow the ubiquitous and constitutive co-expression of two genes of interest. Therefore, it has usually been used as an expression vector in gene therapy [
21]. The recombinant plasmid pVITRO2-VHL was validated by DNA sequencing.
Transfection in vitro
In order to evaluate the transfection efficiency of the three PEI derivatives
in vitro, 2 μg of GFP (green fluorescent protein) plasmids, was respectively encapsulated with FA-PEAs, PCFC-g-PEI and HPEI at different ratios to transfect into OS-RC-2 cells to detect GFP expression profiling. For FA-PEAs:GFP complexes, the weight ratio of FA-PEAs
versu s GFP plasmids (pGFP) was 10:1, 20:1, 30:1 and 40:1 to optimize the transfection activity. Similarly, the HPEI: GFP complex, with a gradient weight ratio of 10:1, 15:1, 20:1 and 25:1, was utilized to transfect OS-RC-2 cells. As a control, based on our previous studies, 2-kD PEI was used to transfer GFP into OS-RC-2 cells at 5:1 weight ratio of PEI (2-kD) versus pGFP [
18].
Furthermore, the transfection effects between PCFC-g-PEI and 25-kD PEI were also compared, and the N/P ratio of PCFC-g-PEI versus GFP was used from 5:1, 7:1, to 10:1. Correspondingly, the weight ratio between PCFC-g-PEI and GFP plasmids was 0.7:1, 1:1 and 1.3:1. This relation was acquired by the formula: N/P ratio = 7.53 × weight ratio of PEI/DNA [
10]. Where N is the number of polymer nitrogen atoms and P is the number of DNA phosphorus atoms. As controls, same quantity (1:1) of 25-kD PEI and GFP was used to detect transfection activity based on our previous studies [
18].
OS-RC-2 cells (1 × 105 cells/well) were seeded on 6-well plates to detect transfection activity. Before transfection, the medium 1640 in each well was replaced with 0.8 ml of fresh serum-free medium, then added a different ratio of PEI: pGFP complex to mix in 0.2 ml serum-free medium for incubation 4 h. Then the complete medium 1640 was added, and the plate was maintained at 37°C for 24 h to observe green fluorescence expression under Fluorescence Inverted Microscope (IX71, OLYMPUS).
The transfection efficiency was determined based on cell percent with GFP expressing. The number of GFP-expressing cells versus the total cell quantity in the microscope was defined as the transfection efficiency. Cell counting was performed randomly in microscopic observation scope under 10 × magnification with 3 repeats. All data were presented as the mean ± standard deviation (SD).
Measurement of particle size and zeta potential of FA-PEAs:DNA complexes
The particle size and zeta potential of the free PEI derivatives and FA-PEAs:pVHL complexes were measured by Malvern Zetasizer 3000HS (Malvern, UK) at 25°C. Different concentration, including 1, 2, 5, 10 and 15 mg/ml of PCFC-g-PEI or FA-PEAs was separately resolved in water to test. The FA-PEAs:pVHL complexes, ranging from 5:1 to 30:1 weight ratios, were prepared by adding FA-PEAs to suitable volume of VHL plasmids to incubate at room temperature for 30 min. Then the complexes were diluted by phosphate buffered saline (PBS) buffer to 1 mL for measurement. Meanwhile, the particle size and zeta potential of PEI (2-kD):pVHL complexes, at a series of ratios from 5 to 30, were measured with the same protocol. All results were measured three times.
RCC model and VHL-gene therapy mediated by PEI system
The following animal experiments were in compliance with all regulatory guidelines and were approved by the Institutional Animal Care and Use Committee of Sichuan University. Six to eight week-old female BALB/c nude mice were purchased from the West China Experimental Animal Center of Sichuan University (Sichuan, China). Mice were permitted one week to acclimate to their environment before studies. The CCRCC model was established in BALB/c nude mice, inoculated with 5 × 106 OS-RC-2 cells/each mouse in the right flank. Primary tumors usually became palpable on the inoculation day 9-10 and with an average 3-mm size.
On the inoculation day 11, the tumor-bearing mice were randomly assigned into 3 groups, including VHL-treated, pVITRO2 and PBS group, and each group contained 6 mice. The nanomaterial:DNA polyplexes were composed of 100 μg FA-PEAs or 100 μg HPEI solved in 0.1 ml PBS and 5 μg pVITRO2-VHL plasmids, and each mouse in the VHL-treated group was injected polyplexes by tail vein for 10 times at 2-days intervals. The mice in the pVITRO2 and PBS groups were separately injected 0.1 ml solution containing 5 μg pVITRO2:100 μg FA-PEAs (or 5 μg pVITRO2:100 μg HPEI), and 0.1 ml PBS. While, different from the FA-PEA and HPEI system, 5 μg PCFC-g-PEI:5 μg plasmids were used to transfer gene into a mouse based on the optimal transfection activity in vitro.
Tumor size was measured with calipers before every treatment, and tumor volumes were calculated according to the formula: width2 × length × 0.52. After treatment for 10 times, all mice were sacrificed and tumor tissues were collected. One part of tissues was stored -20°C, and the other tissues were fixed in 4% formaldehyde solution for immunohistochemistry staining.
Semiquantitative RT-PCR
Total RNA from tumor tissues was isolated using Trizol reagent (Invitrogen) to take as templates to amplify each target cDNA fragment, which was synthesized by using the cDNA Synthesis Kit (#K1622, Fermentas, USA ). The primers of VHL and β-actin for RT-PCR were designed as following. The forward primer for VHL was 5'- TCA CCT TTG GCT CTT CAG AGA TGC A -3' (25bp), and the reverse primer was 5'- GTC TTT CTG CAC ATT TGG GTG GTC T -3' (25bp). The amplified VHL fragment was 250bp in length. The designed primers for β-actin were 5'-CGG GAA ATC GTG CGT GAC-3'(18 bp, forward) and 5'-TGG AAG GTG GAC AGC GAG G-3' (19bp, reverse), and the length of the amplified cDNA was 434 bp.
PCR was performed as follows: first cycle at 95°C for 2 min, and then 30 cycles at 94°C for 45 s, 54°C for 1 min, 72°C for 1 min and a final extension cycle of 72°C for 5 min. The house-keeping gene β-actin was taken as a loading control. HEK293T cells were used as a positive control in VHL expression.
Immunohistochemistry
The IHC analysis was performed mainly according to our previous protocols [
22,
23]. Tumor tissue sections with 4 μm thickness were cut from formalin-fixed and paraffin-embedded tissues for immunohistochemistry (IHC). The endogenous peroxidase was blocked with 3% H
2O
2, and the antigen retrieval was carried out in citrate buffer (pH6.0). The VHL expression level in tumor tissues was detected by indirect immunohistochemical staining using the labeled streptavidin-biotin method. The anti-VHL mouse monoclonal antibodies (Abcam, ab11191) were used as the primary antibodies, and the second antibody was a biotinylated anti-mouse IgG. The antigen-antibody complex was then visualized with horseradish peroxidase-streptavidin reagents and 3, 3'-diaminobenzidine solution and counterstained with hematoxylin.
Data statistical analysis
The SPSS program (version 15.0, SPSS Inc., USA) was used for statistical analysis. Comparisons between two groups were performed by Student's t test, and comparisons among multiple groups were performed by One-way ANOVA. The difference was considered significant if p < 0.05.
Discussion
Recently, several PEI-modified nanomaterials, as nonviral delivery vectors, have already been extensively used to carry DNA for gene therapy. For example, the intraperitoneal injection of DNA:PEI complexes is a promising delivery method to transduce genes into disseminated cancer nodules that induced by pancreatic tumor in the peritoneal cavity [
9]. Moreover, one PEI derivative, which was removed the N-acyl moieties from commercial linear 25-kD PEI, enhanced its DNA delivery efficiency 21 times
in vitro, as well as 10,000 times in mice with a concomitant 1,500-fold enhancement in lung specificity [
24]. Another water-soluble polymer, heparin-conjugated PEI exhibited significantly higher in target gene expression than 25-kD PEI [
25].
Due to its intrinsic transfection properties, PEI has been used to provide the backbone of PEI-derived vector formulations. Therefore, PEI-based vector improvements are needed with regard to the efficiency and specificity of the gene transfer. In our studies, we mainly investigated the cytotoxicity and the transfection efficiency of the three PEI derivatives (PCFC-g-PEI, FA-PEAs and HPEI)
in vitro and on a RCC model
in vivo. The toxicity of three PEI derivatives was relatively lower than their corresponding PEI precursor on OS-RC-2 tumor cells and on other cell types, including Ana-1 and HUVEC cells. The partial reasons are probably due to the decrease in charge of complexes with decrease in primary amine amount [
4]. Meanwhile, the toxicity is often associated with materials uptake by cells. HPEI has a proton-buffering effect [
25] and HPEI:pVHL has higher blood compatibility and a lower cytotoxicity than PEI (25kD):pVHL.
On the other hand, with an optimized weight ratio, the transfection efficiency of PCFC-g-PEI is also a little increased than 25-kD PEI. An important part of polyplex transfection activity depends on the polyplex physico-chemical characteristics [
26]. Because the PCFC complex contains a pluronic 105, in which the pluronic block copolymer could enhance polycation-mediated gene transfer
in vitro[
4,
27]. Moreover, the particle size of the PEI:DNA complex was also important for its uptake by cells. For efficient endocytosis and gene transfer, the complex must be small (below 200 nm) and compact [
19]. The particle size and zeta potential of the PCFC-g-PEI:DNA and HPEI:DNA complexes had been detected in our previous reports [
4,
18]. The size of PCFC-g-PEI:DNA complexes was relatively stable around 200 nm [
4]. Therefore, when the copolymer bind to DNA, the complexes size will be condensed obviously and may be uptaken easily by cells through endocytosis. In the case of HPEI/DNA complexes, along with an increase in HPEI:DNA weight ratio, the particle size was decreased [
18], which was also supported by the FA-PEAs:pVHL polyplexes.
Similarly, the transfection efficiency of FA-PEAs and HPEI was increased and the cytotoxicity was decreased compared to 2-kD PEI, especially the
in vivo effects of VHL gene therapy mediated by FA-PEAs polymer were better than other PEI-polyplexes. Except to the same chemical structure, pluronic block copolymer, such as PCFC-g-PEI, the poly(ε-caprolactone) (PCL) segments in FA-PEAs can increase the circulating half-life [
28], which is contributed to enhance gene transfer efficiency
in vitro. Furthermore, the cross-linker IPDI stabilizes the polyplexes and prolongs circulation times at the same time [
19]. In addition, the relative smaller particle size and larger zeta potential of FA-PEAs:pVHL complexes were also helpful for the nonspecific cell interaction with them. Meanwhile, FA can interact with folate receptor (FR) which is usually overexpressed in cancer cells [
29]. These resulted in its better transfection efficiency
in vitro and
in vivo. Furthermore, both FA and heparin could be degraded easily by enzyme in cells, and the PEI amino group, which is harmful to cells, is reduced too. All these factors might contribute to low cytotoxicity of FA-PEAs and HPEI.
In order to validate the potential transport potent of the three novel modified PEI
in vivo, we used VHL gene, a tumor suppressor gene that is usually inactivation or absence in RCC [
30], to treat the RCC model on nude mice. The mean tumor volume in FA-PEAs:VHL-treated mice was decreased about 30% compared to the control group. The FR exhibits limited expression on healthy cells, but overexpression in many types of tumors, such as ovarian, colorectal and renal cell carcinomas [
26,
31]. Therefore, FA-PEAs:pVHL complexes could bind to FR that locates in cell surface with nanomolar affinity. The specific interactions between polyplexes and cell surface are targeted
via a specific ligand-receptor incorporating mechanism, which is very important for
in vivo targeting gene therapy. And the FA-PEAs:VHL complexes might be released into cytosol through endocytosis [
32]. This is may be the most important reason that FA-PEAs:VHL exhibits an obvious therapeutic efficacy.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
XZ performed the experiments and wrote the paper draft; LS conceived, instructed the experiments and revised the paper; SG and XX performed experiments and analyzed data; ZX and ZP collected and validated tissue samples; WH and GQ prepared PEI-derived material; QZ guided the PEI synthesis; WY provided experimental devices and gave suggestions on this project. All authors read and approved the final manuscript.