Enhanced anticancer efficacy and tumor targeting through folate-PEG modified nanoliposome loaded with 5-fluorouracil

Cancer targeted therapies have attracted considerable attention over the past year. Recently, 5-fluouracil (5-FU), which has high toxicity to normal cells and short half-life associated with rapid metabolism, is one of the most commonly used therapies in the treatment of cancer. In this study the folic acid-conjugated pegylated nanoliposomes were synthesized and then loaded into them with 5-FU to improve the anti-tumor efficacy. The average size of liposomes (LPs) was about 52.7 nm which was identified by TEM. In the liposome uptake studies, the level uptake of folate-conjugated liposomes has increased compared to non-conjugated LPs according to LPs concentration, incubation time and presence of concentration of free folic acid (FA). The MTT assay and apoptotic test were carried out in HCT116 and MCF-7 cells for 24 or 48 h. The results revealed that the folate-PEG modified 5-Fu loaded nanoliposomes had strong cytotoxicity to cancer cell compared to pure 5-FU or PEG modified 5-FU loaded liposomes in a concentration- and time-dependent manner, and mainly enhanced the cancer cell death through folate-mediated endocytosis. Hence, the folate-PEG modified nanoliposome is a potential targeted drug-delivery system for the treatment of FR-positive cancers.

liposome, respectively [9,10]. Due to these characteristics, liposomes has received FDA approval and are widely applied as drug carriers or diagnostic agents in clinic [11,12].
In order to achieve tumor-targeted drug delivery, folic acid (FA) was commonly employed as a targeting ligand to modify nanocarriers [13][14][15][16][17][18][19]. FA plays a critical role in DNA and RNA synthesis, one-carbon metabolism and cellular growth. Cellular uptake of folate is mediated by cell membrane folate receptor (FR) that have a low expression level in normal cell, but is overexpressing within the tumor cells [20,21].
Here, we aimed at development of 5-FU loading with FA modified nanoliposomes for active targeting to tumor tissues. Then, the liposome targeting ability and cytotoxicity were investigated by evaluating the cellular uptake, MTT assay, and apoptosis test in HCT116 and MCF-7 cells.
Human breast adenocarcinoma cell line (MCF-7) and human colon cancer cell line (HCT116) were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Cell Biology of Shanghai Institute, Shanghai, China). Roswell park memorial institute (RPMI) 1640 media, Dulbecco's modified eagle medium (DMEM) 1% penicillin/streptomycin were purchased from Gibco-Invitrogen (Carlsbad, CA). Fetal calf serum was purchased from Hyclone Laboratories Inc., Australia.

Preparation of folate-modified PEG-liposomes.
Liposomes were prepared by thin film hydration technique followed by high-pressure homogenization, previously described by Mourtas et al [22] and Chen et al [23]. In brief, the lipid mixture composed of CHEMS/NHS/TEA/PEG (PEG-CHEM or folate-PEG-CHEM, synthesized as reported by Wu et al [24]) were dissolved in a chloroform/methanol (2:1 v/v) mixture in a pear-shaped flask. The lipids were subsequently dried by rotary evaporation until a thin film was formed. Residual organic solvents were further dried under vacuum at room temperature overnight. The lipid film was hydrated with the appropriate volume of phosphate-buffered saline (PBS) buffer (pH 7.4), which contained 5-fluouracil by sonication in a water bath. The drug was passively loaded. Non-encapsulated drug was removed by centrifugation at 16 000 rpm for 30 min. FR-targeted liposome rhodamine B (F-RhB/PEG/LPs) and 5-FU-loaded folate-PEG modified nanoliposomes (F-5-FU/PEG/LPs) were prepared by the same method as previously reported [23,24]. These liposome suspensions were passed through high-pressured homogenizer (HPH ATS Engineering, Canada) at 600 bar homogenization cycles. All the samples were lyophilized for long stability and frozen at −20 °C for 12 h before being lyophilized for 48 h. The freeze-dried powders were rehydrated with double distilled water when required for further experiments.

Dynamic lights scattering and zeta potential
measurements. The mean particle sizes, polydispersity index (PDI), and zeta potentials were measured by dynamic light scattering (DLS) using the Delsa ™ Nano C Particle Analyzer (Beckman Coulter, USA) at a fixed angle of 165 °C and 25 °C. The mean diameter was calculated from the volume distribution curves given by the particle analyser.

Encapsulation efficiency and drug loading in
liposomes. The concentration of 5-FU incorporated into liposome vesicles was monitored by centrifugation method. The samples were centrifuged at 16 000 rpm for 30 min at 4 °C. The ultrafiltrate contained the non-entrapped 5-FU was diluted in PBS and determined using a UV-Vis spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan) at 265 nm. The drug content (DL) was calculated using the standard calibration curve and the percent of the drug encapsulation (EE) was calculated using the following formulas: where W T is the total amount of 5-FU added to the system, W UL is the amount of the non-entrapped drug, W L is the total weight of liposomes.  (FBS) (Hyclone Laboratories Inc., Australia) and 1% penicillin/streptomycin. The cells are incubated in a humidified atmosphere of 5% CO 2 and 95% air at 37 °C.

Preparation and characterization of 5-FU liposomes
The PEGylated liposome and folate-modified liposome was prepared. Figure 1 shows the high-resolution transmission electron micrographs of the folate modified PEGylated liposomes. The 5-FU liposomes were found to have a spherical shape with one or two layer of lipid molecules. The mean diameter of 5-FU liposomes formulation was about 52.7 nm. The PDI is 0.181. The drug encapsulation efficiency and drug loading of FA/PEG/LP are 16.14% and 1.189%, respectively. Nano-size of drug-loaded liposomes have been prepared and modified with folate ligand to obtain tumor targeting.

Liposomes uptake by tumor cells in vitro
In order to understand the uptake mechanism of liposomes into cells via folate-mediated endocytosis, the cellular uptake study was evaluated in MCF-7 at different conditions. As To represent the role of cell surface FRs in liposome binding, we were further performed free folate competition study. As showed in figure 4, the high folate concentration (200 µg ml -1 ) significantly reduced the F-Rh-loaded liposome uptake in MCF-7 cells compared with low folate concentration (0, 1 and 10 µg ml -1 ). But, there is no effect in reducing Rh/PEG/LP uptake.  These experimental results indicated the folate-modified liposomes had a marked increase in cellular uptake compared to non-modified liposomes, and it was dependent on liposome concentration and incubated times. Furthermore, the uptake of folate-modified liposomes could be inhibited by the competition of high concentration of free FA. So, it suggested that the FA-modified liposome might be endocytosed via the FR in MCF-7 cell surface. In this study, we did not observed the saturation of the uptake capacity of the cells because of cell treatment with low concentration of liposomes.

Cytotoxicity study by MTT methods
Phospholipids are cell membrane component, so liposome made from lipid mixture (CHEMS/NHS/TEA/PEG) is wellknown non-cytotoxicity [25]. As show in figure 5, there was no significant difference in cell viability over the liposome concentration range of 0 to 4 mg ml -1 for 24 h or 48 h in MCF-7 cells (figure 5(a)) and HCT116 cells ( figure 5(b)). In addition, in the cellular uptake study, tumor cells have been showed significantly increased uptake to the folate-modified liposome. Therefore, any side effects in drug delivery process can be associated with the encapsulated drug alone and FA/ PEG/LP, on the other hand, only acts as vehicles for drug. It suggests that FA/PEG/LP is a safe drug delivery system for biomedical applications.
The in vitro antitumor activity of 5-FU-loaded liposomes and pure drugs were determined by MTT assay. Two cancer cells, MCF-7 and HCT116, were treated with pure 5-FU

Apoptotic analysis
Tumor cell general underwent apoptosis in response to anticancer agents. To elucidate drug-loaded liposomes induced apoptosis in MCF-7 cells, cell nuclei was stained by Hoechst 33342 after different treatments. As shown in figure 7, the control and B-LPs groups seem to stain cytoplasm (figures 7(A) and (B)). But the cells treated with pure 5-FU or 5-FU-loaed liposomes exhibited chromatin condensation and nuclear fragmentation (figures 7(C)-(F)). Apoptotic features of 5-FU/ PEG/LP and F-5-FU/PEG/LP treated groups notably higher than pure 5-FU or 5-FU/LP treated groups. It is indicated that the 5-FU loaded liposome had an enhanced apoptosis effect in cancer cells. The enhanced uptake of the folate-modified liposomes via FR-mediated endocytosis is one of reason that folate-modified liposomes had a higher effect than pure 5-FU or other liposome formulations. This result again demonstrated cytotoxicity or apoptosis effect of drug-loaded liposomes only attributed to the effect of the 5-FU release from it.

Conclusion
Some biocompatible polymers such as poly(lactide)-tocopheryl polyethylene glycol, chitosan and PEG, have been widely used for the tumor-targeted delivery and site-specifically release of of anticancer drugs (paclitaxel, curcumin and doxorubicin) [26][27][28]. In recent years, the use of liposomes to increase the therapeutic index of chemotheraputic drugs and to offers drug targeting and controlled release has emerged as a promising strategy against cancer [8,29,30]. PEGylated phospholipids are commonly used to protect the liposome surface from penetration and disintegration by plasma proteins or to avoid monocyte/macrophage clearance [31,32]. In this case, liposomes can passively accumulate in tumor tissues due to the enhanced permeability and retention (EPR) effect. Here, 5-FU loaded folate-PEG modified nanoliposomes were prepared and evaluated for their in vitro antitumor activity in HCT116 and MCF-7 cells. The average size of this FR-targeting 5-FU-delivery system was about 52.7 nm, which can allow them preferentially accumulate at tumor sites [27,33]. Additionally, the liposome with FA modification can provide active targeting capability to FR-positive cancer. The MTT assay showed that the blank liposomes had no significant toxic effect on the growth of two tumor cell lines (HCT116 and MCF-7). Further, the testing of cytotoxicity and apoptosis of 5-FU-loaded liposomes indicated that folate-modified liposomes effectively enhanced anticancer activity of drug through folate-mediated endocytosis. The F-5-FU/PEG/ LP was found to have significantly greater cancer-inhibitory effect than without folate conjugation or pure 5-FU. Therefore, the results of this study illustrated this drug delivery system could be beneficial in the cancer treatment by targeting 5-FU to the tumor cells and reducing its toxicities. Moreover, further studies are in progress to improve drug loading of this system.