Isomeric Folate-Conjugated Polymeric Micelles Bind to Folate Receptors and Display Anticancer Effects

Folate receptors (FRs), which are GPI-anchored membrane protein involved in folate endocytosis, are often over-expressed on tumor cells including malignancies of the ovary, brain, kidney, breast, myeloid cells and lungs (Elnakat and Ratnam, 2004; Chen et al., 2013). Although a few normal tissues have also been found expresses folate receptors, these receptors located on the apical membrane of polarized epithelia are not accessible to blood-borne folate-derived conjugates. With high affinity towards the FRs, as a targeting ligand, folic acid has various advantages, such as low molecular weight and stability. In order to construct a folic acid-modified drug delivery system, folic acid is often covalently conjugated with PEG through its αor γ-carboxyl group to connect with drug delivery system. Nevertheless, the binding ability for αand γ-folate regioisomers to FRs was disputed. Wang and colleagues showed that the cell surface FRs recognizes folate derivatives at the γbut not α-carboxylate (Wang et al., 1996; Wang et al., 1997). In contrast, it was recently reported that both αand γ-folate derivatives are able to bind to folate receptor-positive cells at virtually identical levels (Leamon et al., 1999; Bettio et al., 2006; Zong et al., 2012).


Introduction
Folate receptors (FRs), which are GPI-anchored membrane protein involved in folate endocytosis, are often over-expressed on tumor cells including malignancies of the ovary, brain, kidney, breast, myeloid cells and lungs (Elnakat and Ratnam, 2004;Chen et al., 2013).Although a few normal tissues have also been found expresses folate receptors, these receptors located on the apical membrane of polarized epithelia are not accessible to blood-borne folate-derived conjugates.With high affinity towards the FRs, as a targeting ligand, folic acid has various advantages, such as low molecular weight and stability.In order to construct a folic acid-modified drug delivery system, folic acid is often covalently conjugated with PEG through its α-or γ-carboxyl group to connect with drug delivery system.Nevertheless, the binding ability for αand γ-folate regioisomers to FRs was disputed.Wang and colleagues showed that the cell surface FRs recognizes folate derivatives at the γ-but not α-carboxylate (Wang et al., 1996;Wang et al., 1997).In contrast, it was recently reported that both α-and γ-folate derivatives are able to bind to folate receptor-positive cells at virtually identical levels (Leamon et al., 1999;Bettio et al., 2006;Zong et al., 2012).
Qing Dong 1& , Zuo-Xu Xie 1& , Cao Xie 1 , Wei-Yue Lu 1 , Qian Zhang 2 , Xue Li 1 , Min Liu 1 * Doxorubicin (DOX) is widely used antitumor drug in the treatment of many types of cancers; however, it exhibits serious cardiac toxicity (Ashrafi and Roshan, 2012).In order to reduce side effect of antitumor agents, various drug delivery systems have been developed, such as polymer conjugates, polymeric micelles and liposomes (Torchilin, 2004;Chang et al., 2009;Khemapech et al., 2013;Xu et al., 2013).As an important member of drug delivery systems, polymeric micelles have attracted significant attention in tumor-targeted drug delivery systems recently.Polymeric micelles formed from amphiphilic copolymers are found to have high stability, controllable drug release profiles, long systemic circulation time and increased accumulation in tumor tissues via EPR effects (Torchilin, 2007;Wang et al., 2010).Amphiphilic copolymers containing hydrophobic block and hydrophilic block play a critically essential role in the form of polymeric micelles.Even though there are many options for the hydrophobic block, for the hydrophilic segment, polyethylene glycol (PEG) is the most frequently used one.Micelles coated with PEG have the ability to escape from the reticuloendothelial system and circulate in the blood for long periods of time (Hawley et al., 1995;VertutDoi et al., 1996;Gabizon et al., 2004).As a hydrophobic segment, distearoylphosphatidylethanol-amine (DSPE) represents Qing Dong et al good biocompatibility.When this hydrophobic DSPE conjugates to PEG, PEG-DSPE micelles are formed, which have suitable nanoscale (-20 nm), high stability, and low toxicity (Lukyanov et al., 2002;Torchilin, 2002).
Due to the study on the difference in the ability to bind to FRs between α-and γ-folate regioisomers, furthermore, the affinity of the two folate regioisomers for FRs was controversial, it cannot be assumed that the necessity of preparing the pure γ-folate regioisomers.In order to figure out the perplexity of the folate regioisomers, we planned to apply DOX as model drug and polymeric micelles as drug delivery system to study the folate derivatives.We adopted solid phase peptide synthesis to synthesize two isomeric copolymers, α-and γ-Fol-PEG-DSPE.Then we prepared both α-and γ-Fol-PEG-DSPE-DOX micelles to investigate their antitumor efficacy in vitro and in vivo.

Synthesis of α-Fol-Cys and γ-Fol-Cys
Two Isomeric Fol-Cys conjugates were synthesized through the solid phase peptide synthesis method.The commercially available 2-chlorotritylchloride resin was coupled to Fomc-Cys (Trt)-OH.Resin (1.17 g) was swelled with dichlormethane (DCM) and washed with N, N-Dimethylformamide (DMF) three times, and the resin-bound Fomc-Cys was successively treated with 25% piperidine in DMF.Next, keeping all the operations in the dark, 0.7 g PyBop and 0.18 g HOBt were added into 20 ml of DMF, dissolving 0.6 mg of folic acid, and stirred for 5min, and added the solution into the resin mixture to react for 24 hours.Then, added trifluoroacetic acid (TFA) to the reaction solution and placed it in an ice bath for 1.5 hours.After that, the filtrate was collected by vacuum filtration under pressure.Then cold anhydrous diethyl ether was added into the filtrate to obtain a precipitate, which represented crude Fol-Cys.In order to obtain pure α-Fol-Cys and γ-Fol-Cys, the crude Fol-Cys was purified by preparative HPLC (Waters, 600 E, USA) using a Waters System PrepTM C18 reversed phase column (7 μm, 19×30 mm, Eluent A, water with 0.1% TFA; Eluent B, acetonitrile with 0.1% TFA; gradient, 5B to 25% B over 30 minutes; flow rate, 10 mL/min; UV @280 nm).

Synthesis of α-and γ-Fol-PEG-DSPE
Mal-PEG-DSPE was dissolved in a solution of DMF.Then excessive α-Fol-Cys was added into the solution and stirred at room temperature in the dark.The reaction was monitored by HPLC analysis (Agilent 1100 series, USA).Excess α-Fol-Cys was removed by preparative HPLC (Sepax C4 10×250 mm 5 μm, Eluent A, water with 0.1% TFA; Eluent B, acetonitrile with 0.1% TFA; gradient, 70 B to 90% B over 30 minutes; flow rate, 3 mL/min; UV @ 220 nm), and the purity of the α-Fol-PEG-DSPE obtained was confirmed by NMR analysis.γ-Fol-PEG-DSPE was prepared and purified using the same methodologies as described above.

Preparationand Characterization of Micelles
Micelles were successfully prepared using the film formation method.Briefly, 0.6 mg Doxorubicin hydrochloride was stirred with 2 molar ratio of triethylamine in methanol to remove the hydrochlorate salt and then mixed with 5 mg MPEG-DSPE and α-or γ-Fol-PEG-DSPE/MPEG-DSPE (molar ratio = 100:1) dissolved in chloroform and dried in a rotary evaporation to form a thin film at 37℃, which was re-suspended in 10 mM HEPES-buffered saline (pH 7.4) at room temperature.To remove un-incorporated doxorubicin, eluting with 10 mM HEPES-buffered saline, the micelles were passed through a gel column filled with Sephadex G-50.The solution was determined by high-performance liquid chromatography (HPLC) show that the final concentration of doxorubicin in micelles was 1 mg/mL Micelle size was measured by dynamic light scattering (DLS) using NICOMP 380 ZLS Zeta-potential/Particle System (Malvern Zetasizer Nano Series, Britain).The morphological examination of micelles was performed using a transmission electron microscope (TEM, Joel JEM-1230, Japan).Fluorescent dye DiR and Rodamine B loaded micelles were prepared by dissolving fluorescein in acetonitrile and the later steps were the same as the preparation of DOX-loaded micelles.

In Vitro Antitumor Efficacy of Micelles
KB cells (4×10 3 cells/ml) were seeded onto a 96-well plate in 200 μl of RPMI 1640 medium containing 10% FBS.After 24 hours, the medium was replaced with folatefree RPMI 1640 medium.DOX-loaded micelles (DOX concentration 3.45 μmol/L, equivalently) in folate-free RPMI1640 medium were added to the 96-well plate for 2 hours or 4 hours at 37°C.The cells were subsequently incubated in fresh growth media for a total of 48 hours DOI:http://dx.doi.org/10.7314/APJCP.2014.15.17.7363 Isomeric Folate-Conjugated Polymeric Micelles Bind to Folate Receptors and Display Anticancer Effects before cell viability was assessed in each group.The viability of cells was measured using the tetrazolium dye method (MTT assay).

Uptake of macrophages
Flow cytometry was used to compare the intracellular uptake of Rhodamine B-loaded micelles by Macrophages cell.RAW 264.7 cells were seeded in 6-well plates (each well 105 cells) and grown for 1 day in DMEM medium containing 10% FBS.The cells were incubated for 30 minutes at 37°C with free Rhodamine B or Rhodamine B-loaded micelles.After the medium was removed, RAW 264.7 cells was washed with PBS, detached by 0.25% Trypsin-EDTA, fixed by 1% formaldehyde in PBS, resuspended in 1 ml of PBS, and immediately analyzed by flow cytometry.

In vivo optical imaging of micelle biodistribution
In vivo, biodistribution tests were carried out with DiR-loaded micelles using optical imaging.KB cells were transplanted into the shoulder of male nude mice by subcutaneous injection of 1×10 6 cells suspended in cell culture media.When the size of the tumor had reached a size of approximately 300 mm 3 , DiR loaded MPEG-DSPE, α-and γ-Fol-PEG-DSPE micelles were intravenously injected into KB tumor-bearing nude mice through the tail vein.2h and 8h after injection, the mice were transferred to a Maestro In-Vivo Imaging System for observation.

In vivo antitumor activity
The same manner as described above was used to establish the KB tumor-bearing nude mice model.The mice were kept on a folate-deficient diet exclusively throughout the study.Two weeks after the tumor transplanted, the mice were randomly assigned to five groups (n=6) and were treated five times at 2-day intervals with free DOX, MPEG-DSPE-DOX micelles, α-and γ-Fol-PEG-DSPE-DOX micelles (2 mg/kg per dose), and 200 μl of physiological saline (blank control) via tail vein injection.Tumor size was determined using serial calipers (GuangLu ® , China) and measurements were made every two days to calculate the volumes using the formula length× (width) 2/2.The inhibition rate of tumor growth (IR%): = (Vc-Vt)/Vc×100%, where Vc and Vt are the mean tumor size of the blank control and treated mice respectively.Vt/Wt stands for the relationship between tumor size and mice weight, where Wt is the mice weight.

Histopathological Analysis
After the study of in vivo antitumor activity, all of the surviving nude mice were sacrificed and dissected.The hearts of mice were fixed in 10% neutral buffered formalin and embedded in paraffin to make tissue slice.All the groups of mice' heart tissue slice were stained with haematosylin and eosin.Then the stained sections were observed under microscope and photographed.

Statistical analysis
Differences of the vivo antitumor effect between test groups were assessed using an Unpaired Student's t-Test using the significance level of p<0.05.

Characterization of Micelles
The particle size and polydispersity index (PDI) of DOX-loaded micelles were 20 nm ±5 nm and 0.1 respectively.The morphology of the micelles was investigated by TEM (Figure 1).It could be seen that the Qing Dong et al self-assembled micelles were well dispersed as individual nanoparticles with regular spherical shape and no drug crystal was visible, which confirmed that the micellization process did occur.We stored the micelles solution at 4°C for 4 months and then made it passed through a gel column (Sephadex G-50).That no drug dissociation was determined proved that DOX-loaded micelles which diluted in HEPES-buffered saline (pH 7.4) are sufficiently stable at 4°C in 4 months.The encapsulation and drug loading efficiency of DOX incorporated into micelles were 85.2% ± 0.6% and 10% ± 1.2%, respectively.The α-and γ-Fol-PEG-DSPE-DOX micelles showed no difference in size, encapsulation efficiency and drug loading efficiency when compared with MPEG-DSPE-DOX micelles.

Uptake of macrophages
In order to evaluate the influences of PEG-DSPE micelles on macrophage phagocytosis, RAW 264.7 cells were incubated with Rhodamine B-loaded micelles for 30min, and the uptake was monitored by flow cytometry.For the free Rhodamine B, 36.6 percent of macrophages phagocytized the Rhodamine B, nevertheless, when Rhodamine B was encapsulated in micelles, the fluorescent uptake of macrophages decreased significantly.The percentages of RAW 264.7 cells phagocytizing the Rhodamine B are 4.40%, 1.51% and 1.80%, which were incubated with MPEG-DSPE-RhB micelles, α-Fol-PEG-DSPE-RhB micelles and γ-Fol-PEG-DSPE-RhB micelles respectively.As expected, the PEG-DSPE micelles could inhibit the uptake of Rbodamine B by RAW 264.7 cells, in comparison with free Rhodamine B, which was inhibited up to 94.2% (Figure 2b).These results demonstrated that no matter the α-Fol-PEG-DSPE-RhB micelles or the γ-Fol-PEG-DSPE-RhB could avoid macrophage phagocytosis to prolong the circulation time.

In Vivo Optical Imaging of Micelle Biodistribution
After DiR loaded micelles were injected, through near infrared spectroscopy, we observed the distribution of micelles in KB tumor-bearing nude mice.The fluorescence of micelles was found to accumulate mainly in tumors at 2h and 8h in vivo (Figure 3).The tumors could be delineated from the surrounding normal tissue as early as 2h after injection.Compared with MPEG-DSPE-DOX micelles, theα-and γ-Fol-PEG-DSPE micelles showed a stronger intensity at all tested time points..2014.15.17.7363 Isomeric Folate-Conjugated Polymeric Micelles Bind to Folate Receptors and Display Anticancer Effects In Vivo Antitumor Activity In the KB tumor-bearing mice model, free DOX and DOX-loaded micelles exhibited the different in vivo antitumor efficiency.The tumor volume growth (Figure 4a) and the changes in the body weight of mice (Figure 4b) were examined over a 26-dayperiod.It can be seen that the tumor growth in mice treated withα-or γ-Fol-PEG-DSPE-DOX micelles was suppressed more strongly than those treated with DOX and MPEG-DSPE-DOX micelles and was considered as significant (p<0.05).After 26 days, the IRs (%) were 50.4%, 42%, 69.6% and 74.8% for free DOX, MPEG-DSPE-DOX micelles, α-and γ-Fol-PEG-DSPE-DOX micelles respectively.Indeed, the α-and γ-folate-modified micelles suppressed the tumor growth more than that of MPEG-DSPE-DOX micelles at this stage.Moreover, no significant difference in the size of tumors was observed between α-and γ-Fol-PEG-DSPE-DOX micelle treated animals.
Although free DOX and all kinds of DOX-loaded micelles possessed antitumor activity, a sharp decrease in body weight during the treatment was observed in the free DOX group.The results suggested serious toxicity levels of free DOX at the given dose compared with DOX-loaded micelles.Additionally, empty micelles showed no signs of toxicity (data not shown).The ratio of tumor volume to mice weight was calculated and shows that the Vt/Wt of free DOX and MPEG-DSPE-DOX micelles was higher than α-and γ-Fol-PEG-DSPE-DOX micelles (Figure 4c).That means theα-and γ-Fol-PEG-DSPE-DOX micelles have a better antitumor effect and lower toxicity as compared to free DOX and MPEG-DSPE-DOX micelles.

Histopathological Analysis
In order to observe the doxorubicin-induced cardiac toxicity in mice, the hearts of mice were prepared for histological analysis.The control group showed regular myofiber, maintained sarcotubular structure and abundant blood vessel among myocardium.Whereas myocardial fibers necrosis and myocardial damage were observed in the group treated with free doxorubicin.There is little evidence of morphological damage were seen in the three groups: MPEG-DSPE-DOX micelles group, α-Fol-PEG-DSPE-DOX micelles group and γ-Fol-PEG-DSPE-DOX micelles group (Figure 5).The comparisons between groups demonstrate that polymeric micelles encapsulating doxorubicin could lower the cardiac toxicity of doxorubicin.

Figure 3 .Figure 5 .
Figure 3. Optical Imaging Result for Tumor Bearing Nude Mice taken using DiR loading Micelles in at Times Periods (a: 2h; b: 8h).Tumors are highlighted by the white arrows (from left to right, MPEG-DSPE, α-and γ-Fol-PEG-DSPE micelles in order)