Cervical Cancer Gene Therapy by Gene Loaded PEG-PLA Nanomedicine

BACKGROUND AND AIMS
Advances in the treatment of cervical cancer over the last decade have predominantly involved the development of genes directed at molecular targets. Gene therapy is recognized to be a novel method for the treatment of cervical cancer. Genes can be administered into target cells via nanocarriers. This study aimed to develop systemically administrable nano-vectors. Floate (Fa) containing gene loaded nanoparticles (NPs) could target HeLa human cervical cancer cells through combination with receptors on the cells to increase the nuclear uptake of genetic materials.


METHODS
Fa was linked onto Poly (ethylene glycol)-b-poly (D, L-lactide) (PEG-PLA) to form Fa-PEG-PLA, and the resulting material was used to load plasmids of enhanced green fluorescence protein (pEGFP) to obtain gene loaded nanoparticles (Fa-NPs/DNA). Physical-chemical characteristics, in vitro release and cytotoxicity of Fa-NPs/DNA were evaluated. The in vitro transfection efficiency of Fa-NPs/ DNA was evaluated in HeLa cells and human umbilical vein endothelial cells (HUVEC). PEG-PLA without Fa was used to load pEGFP from NPs/DNA as a control.


RESULTS
Fa-NPs/DNA has a particle size of 183 nm and a gene loading quantity of 92%. After 72h of transfection, Fa-NPs/DNA displayed over 20% higher transfection efficiency than NPs/DNA and 40% higher than naked DNA in HeLa cells. However, in HUVECs, no significant difference appeared between Fa-NPs/DNA and NPs/DNA.


CONCLUSIONS
Fa-PEG-PLA NPs could function as excellent materials for gene loading. This nano-approach could be used as tumor cell targeted medicine for the treatment of cervical cancer.


Introduction
Cervical cancer is one of the most common gynecological malignancies; it is a significant cause of morbidity and mortality among women worldwide, and its prevalence has increased during the last decades, especially in Asia countries (Zhou et al., 2012;Luu et al., 2013;Antic et al., 2014;Filipi et al., 2014;Karadag et al., 2014;Tungsrithong et al., 2014).Due to non-existent or inadequate screening, disease is normally detected at late stage.Improved treatment options for this type of malignancy are highly needed (Takai et al., 2011).
Gene therapy is recognized to be a novel method for the treatment of various disorders.Gene therapy strategies involve gene manipulation on broad biological processes responsible for the spreading of diseases.Cancer, monogenic diseases, vascular and infectious diseases are the main targets of gene therapy (Podolska et al., 2012).Recent technological advances in high-throughput genomics and the application of integrative approaches have greatly accelerated gene discovery, facilitating the identification of molecular targets (Narayan and Murty, 2010).New expression systems, viral and bacterial vectors for gene delivery, were widely used for cervical cancer
As an alternative to the viral vectors, a variety of non-viral vectors have been developed for gene delivery (Vachani et al., 2011).Several general strategies have been developed to achieve this end, including liposomes (Chen et al., 2010;Buyens et al., 2011), polymers (Elzoghby 2013;Lee et al., 2014), and molecular conjugates (Elfinger et al., 2007;Geiger et al., 2010).As a research focus in recent years, polyethylene glycol-polylactic acid (PEG-PLA) block copolymer and its end-group derivative nanoparticles can enhance the drug loading of hydrophobic drugs, reduce the burst effect, avoid being engulfed by phagocytes, increase the circulation time of drugs in blood, and improve bioavailability (Xiao et al, 2010).There are also some reports on using PEG-PLA nanocarriers in vivo, showed its excellent property and safety for drug/gene delivery (Schädlich et al, 2011;Xia et al, 2012;Shi et al, 2013).
By adding end groups onto the PEG-PLA block copolymers, various nanocarriers may be prepared in order to increase the therapeutic effect of genes, such as long-circulating nanoparticles, immunonanoparticles, thermosensitive nanoparticles, and pH-sensitive nanoparticles (Xiao et al, 2010).Due to their core shell structure, the surface of PEG-PLA block copolymer nanoparticles is often modifed by folic acid, peptide, lectin, albumin, and so on (Gao et al., 2006;Yu et al., 2010;Wang et al., 2013).Compared with PEG-PLA block copolymer nanoparticles with passive targeting, these modifed nanoparticles can actively target special locations for enhancing gene transfection efficiency and decreasing cytotoxicity.
In the present study, Fa was linked onto PEG-PLA to form Fa-PEG-PLA, the resulting material was used to load pEGFP to get Fa-NPs/DNA.The physical-chemical characteristics, in vitro release and cytotoxicity of Fa-NPs/ DNA were evaluated.The in vitro transfection efficiency of Fa-NPs/DNA was evaluated on HeLa cells and HUVEC.PEG-PLA without Fa was used to load pEGFP to from NPs/DNA, applied as control.

Materials
NH2-PEG-PLA was purchased from Ji'nan Daigang Biology Co. Ltd. (Ji'nan, Shandong, China).pEGFP was obtained from Zhejiang University (Hangzhou, Zhejiang, China).Folic acid, and Cell Counting Kit-8 (CCK-8) were purchased from Sigma-Aldrich Co., Ltd.(St Louis, MO, USA).Quant-iT™ PicoGreen® dsDNA quantitation reagent was obtained from Invitrogen by Life Technologies (Carlsbad, CA, USA).HeLa cells were obtained from the American type culture collection (Manassas, VA, USA).Other reagents were commercially available and were used as received.

Synthesis of Fa-PEG-PLA
Fa-PEG-PLA was synthesized using the following modified method (Tsai et al., 2010;Zhang et al., 2010;Wang et al., 2013): Fa (1 g) was dissolved in hydrous DMSO (30 mL).Then Fa, DCC, DMAP were added to the NH2-PEG-PLA containing DMSO solution to react with each other at 50°C water bath in dark for 36 h to get Fa-CO-NH-PEG-PLA (Fa-PEG-PLA).The raw materials molar ratio of Fa, DCC and DMAP to the initial of NH2-PEG-PLA was 1:1.2:1.2:1.The finished product Fa-PEG-PLA was obtained after purifying by dialysis against water and freeze drying.

Preparation and characterization of Fa-NPs/DNA
Fa-NPs/DNA was prepared by solvent evaporationinduced interfacial self-assembly method (Wang et al., 2013) (Figure 1).Briefly, Fa-PEG-PLA was dissolved in 1 mL of acetone, the solution was added dropwise into 20 mL pEGFP containing distilled water within 20 min under 400rpm of stirring at room temperature.Then the mixture was maintained with continuous stirring for 2 h, allowing the polymer to self-assemble to form gene loaded NPs while the organic solvent was evaporating.The NPs were separated from the remaining organic solvent through high speed centrifugation (100 g, 4°C, 10 min), and then washed with distilled water for 3 times and freeze dried.The blank Fa-NPs were prepared using the same method without the experience of pEGFP.Unmodified NPs/DNA was prepared as the same method using PEG-PLA without Fa ligands.
The mean diameter and zeta potential of the fresh made Fa-NPs and Fa-NPs/DNA were determined by photon correlation spectroscopy (PCS) with a Zetasizer 3000 (Malvern Instruments, Malvern, UK).The average particle size was expressed as volume mean diameter.The Gene loading capacity of the vectors was characterized by PicoGreen-fluorometry assay.Briefly, pEGFP was isolated from Fa-NPs/DNA and NPs/DNA by centrifugation at 1000 g, 4°C for 30 min.The concentration of pEGFP was determined by fluorescence, comparing with the supernatant from blank Fa-NPs.The amount of pEGFP loaded in the carriers was then calculated.

Cell culture and cytotoxicity test
For cytotoxicity studies, HeLa cells were seeded in 24 well plates at 3×104 cells/mL and cultured for 24 h allow cell attachment.The cells were incubated with Fa-NPs, NPs/DNA, and Fa-NPs/DNA various concentrations (25, 50, 100 μg/ml) for 48 h at 37°C and 5% CO 2 atmosphere, respectively.Cells without incubation were used as control.After incubation for 48 h, the culture medium was removed.Cellular viability was assessed using Cell Counting Kit-8 (CCK-8) according to the manufacturer's procedures and the absorbance at 450 nm was measured using a microplate reader (Bio-Rad 680, USA).Cells without the addition of CCK-8 were used as a blank control.

In vitro gene delivery efficiency
The HeLa cells were seeded into 24-well plates at a density of 3×104 cells/well in 1 mL of DMEM with 10% FBS, 24 h prior to transfection.When the cells were at about 80% confluence, the media were replaced with 300 µL of serum-free media containing Fa-NPs/DNA and NPs/ DNA complexes at 37°C.Naked DNA was used as control.After incubation for 4 h at 37°C in a 5% CO 2 incubator, the original incubation medium was replaced with 1 mL of complete medium and then cells were incubated sequentially until 72 h post transfection (Yu et al., 2010).HUVEC were applied as the some method as HeLa cells mentioned above.
Flow cytometry was applied to quantitate the amount of cells that have been successfully transfected.The cells were washed with 1 mL of PBS (200 g, 4°C for 5 min) and were detached with trypsin/EDTA.The supernatant was discarded and resuspended with 300 μL of PBS and added into the flow cytometry to quantitate the successfully transfected HeLa cells and HUVEC.

Size and zeta potential of Fa-NPs/DNA
Mean diameter, polydispersity index (PDI), and Zeta potential of Fa-NPs, NPs/DNA, Fa-NPs/DNA, F-SLN/ pEGFP, and D-SLN/pEGFP as well as gene loading capacity were characterized and summarized in Table 1.The volume mean diameter of Fa-NPs and gene loaded Fa-NPs/DNA is around 97 and183 nm respectively.Both Fa-NPs and Fa-NPs/DNA demonstrated narrow particle size distribution (below 0.1), narrow size-range of NPs DOI:http://dx.doi.org/10.7314/APJCP.2014.15.12.4915Cervical Cancer Gene Therapy by Gene Loaded PEG-PLA Nanomedicine acts as a crucial factor in determining the transport and retention of NPs in tissues showing enhanced permeability and retention (EPR) effect (Parveen et al., 2012).Fa-NPs/ DNA has a zeta potential of -33 mV, it is in the stable range of electric charge of the nanoparticles.These results could demonstrate the uniformity and stability of the dual ligands system.

In vitro cytotoxicity test
Figure 2 showed the in vitro cytotoxicity of NPs/DNA and Fa-NPs/DNA evaluated by CCK-8 in HeLa cells at different concentrations.The cell viabilities of the vectors over the studied concentration range (25~100 μg/ml) were over 80% compared with controls.Fa-NPs/DNA showed no obvious cytotoxicity at all concentrations.
Cytotoxicity was a major hurdle for clinical feasibility of gene carriers (Wang et al., 2012;Yang et al., 2013).The nanocarriers showed no higher cytotoxicity at all concentrations.This could facilitate its usage as gene delivery vectors without unexpected damage to the cells.

In vitro gene delivery effect
The in vitro gene delivery effect of NPs/DNA and Fa-NPs/DNA were evaluated against HeLa cells (Figure 3) and HUVEC (Figure 4).Fa-NPs/DNA achieved displayed over 20% higher transfection efficiency than NPs/DNA and 40% higher than naked DNA in HeLa cells at 72h (P < 0.01).At the same incubation time (24, 48, and 72 h), the uptake of the Fa-NPs/DNA for HeLa cells was significantly higher than of NPs/DNA.To further determine the effect of Fa receptor mediated targeted delivery, one kind of common cell line, HUVEC were used for the transfection.The appearance in HUVEC showed no significant difference between Fa-NPs/DNA and NPs/ DNA.The studies of cervical cancer gene diagnosis and therapy in Asia countries have been carried out by several researches and gained excellent results (Kahla et al, 2014;Lin et al, 2014;Ma et al, 2014;Zhao et al, 2014).The results in this study suggesting much higher affinity and selecting capability of the Fa containing gene loaded NPs to cervical cancer cells.It is known that the cellular uptake of Fa in physiological environments is mainly controlled by receptor-mediated endocytosis (Wang et al., 2013).The more Fa receptor (FR) in cell membrane allows more uptake of Fa into cells.HeLa cell line is one of the types of tumor cells over-express Fa receptors compared to normal cells.So in this section, Fa in the surface of Fa-NPs/DNA would target to HeLa cells surfaces through receptor-ligands interactions and facilitate the entry of the particles to the cells by means of endocytosis.In fact, EPR effect due to the leaky vasculature of tumor tissues is another effective way to target NPs to tumor tissues rather than normal tissues.Thus, it may be expected that both receptor-mediated endocytosis and particle size-dependent EPR effects would lead to the much stronger selecting capability of Fa-NPs/DNA to cervical cancer parts than to normal tissues and cells of the body.
In conclusion: In this study, a novel Fa containing lipid Fa-PEG-PLA was used as a polymeric carrier for tumor targeting gene delivery.The results from the CCK-8 assay revealed that the obtained Fa-NPs/DNA is non-toxic to tumor cells HeLa.Transfection study against cervical cells demonstrated that the existence of folate on the surface of the carriers significantly increased the selecting capability of the NPs to the tumor cells, suggesting the receptor-mediated endocytosis pathway of Fa-NPs/DNA.Therefore, it can be concluded that the resulting gene loaded polymeric nanoparticles could be used as a safe and effective nanomedicine for the treatment of cervical cancer.
Figure 1.General Scheme for the Preparation of Fa-NPs/DNA