Hypocrellin B‐loaded, folate‐conjugated polymeric micelle for intraperitoneal targeting of ovarian cancer in vitro and in vivo

Photodynamic therapy (PDT) is considered an innovative and attractive modality to treat ovarian cancer. In the present study, a biodegradable polymer poly (ethylene glycol) (PEG)‐poly (lactic acid)(PLA)‐folate (FA‐PEG‐PLA) was prepared in order to synthesize an active‐targeting, water‐soluble and pharmacomodulated photosensitizer nanocarrier. Drug‐loading content, encapsulation efficiency, in vitro and in vivo release were characterized, in which hypocrellin B (HB)/FA‐PEG‐PLA micelles had a high encapsulation efficiency and much slower control release for drugs compared to free drugs (P < .05). To evaluate the targeting ability of the HB/FA‐PEG‐PLA micelles, a cellular uptake study in vitro was carried out, which showed significantly enhanced uptake of HB/FA‐PEG‐PLA micelles in SKOV3 (FR+) compared to A2780 cancer cells (FR−). The enhanced uptake of HB/FA‐PEG‐PLA micelles to cancer cells resulted in a more effective post‐PDT killing of SKOV3 cells compared to plain micelles and free drugs. Binding and uptake of HB/FA‐PEG‐PLA micelles by SKOV3 cells were also observed in vivo after ip injection of folate‐targeted micelles in tumor‐bearing ascitic ovarian cancer animals. Drug levels in ascitic tumor tissues were increased 20‐fold (P < .001), which underscored the effect of a regional therapy approach with folate targeting. Furthermore, the HB‐loaded micelles were mainly distributed in kidney and liver (the main clearance organs) in biodistribution. These results showed that our newly developed PDT photosensitizer HB/FA‐PEG‐PLA micelles have a high drug‐loading capacity, good biocompatibility, controlled drug release, and enhanced targeting and antitumor effect, which is a potential approach to future targeting ovarian cancer therapy.

bambusae growing in the north-western region of Yunnan Province in China. Hypocrellin has advantages of high quantum yields of singlet oxygen, strong photogeneration of anion radicals in deoxygenated media, low dark toxicity, quick clearance from normal tissue and availability in a pure, monomeric form. Therefore, hypocrellin has been widely used in photodynamic therapy as a photosensitizer.
Photodynamic therapy with HB or HB derivatives has been shown to have antitumor activity through induction of tumor cell apoptosis and inhibition of cell viability from several studies. An ideal photosensitizer should have such characteristics as better selectivity for tumor tissue, and higher photocytotoxicity with lower cytotoxicity under dark conditions. However, HB is hydrophobic; hence, it concentrates in the liver and spleen, not the targeted foci. Therefore, there is a need to develop an alternative formulation of HB with good aqueous solubility and high selectivity.
Nano-sized delivery systems such as polymer carriers, 3 liposomes, 4 micelles 5,6 and nanogels 7 have recently been widely used. They have passive targeting characteristics by an enhanced permeation and retention (EPR) effect. The polymeric micelle is one of the most successful drug delivery carriers because of their small size, high drug loading, prolonged blood circulation, and selective tumor accumulation. Therefore, in the present study, we selected a block copolymer of poly (ethylene glycol) (PEG) and poly (lactic acid) (PLA) (PEG-PLA) as the drug delivery system. Furthermore, we selected folic acid as an active targeting molecule to enhance the tumor-targeting effect. Folic acid can conjugate with folate receptor (FR), which is overexpressed in many types of tumors including ovarian, 8 endometrial, 9 breast, 10 renal cell carcinomas, 11 and so forth. Targeting the folate receptor has shown considerable promise in mediating uptake of a variety of drugs, gene therapy products, and radiopharmaceuticals. [12][13][14] Therefore, we have explored a new folic acid-conjugated PEG-PLA micelle delivery system for HB (HB/FA-PEG-PLA micelle). In the present study, the stability and drug release of the HB/FA-PEG-PLA micelles either in vitro or in vivo were studied. Tumor targeting effect and antitumor efficiency of the micelles in vitro were then evaluated. Furthermore, a mouse ovarian ascitic tumor model was established, and a pharmacokinetic study and tissue biodistribution of the HB/FA-PEG-PLA micelles were evaluated in vivo to further confirm their targeting effect. The results of our study showed that the new drug delivery system HB/FA-PEG-PLA micelles had prominently improved biocompatibility, prolonged blood circulation of HB, and had better targeting and antitumor effect against ovarian cancer.  2.2 | Preparation of PEG-PLA and FA-PEG-PLA copolymer PEG-PLA block copolymer was synthesized as described elsewhere. 15 Recrystallized lactide (10 g) was added to a vial in argon gas. Then, THF (20 mL), KHMDS (1.5 mL) (initiator), and epoxyethane (2.4 mL) were added to another vial and stirred continuously for 2 days in an ice bath. Subsequently, the solutions of the 2 vials were mixed and stirred for 2 hours at 25°C. Anhydrous acetic acid (20 mL) was then added to the vial and stirred for 30 minutes at 25°C. Finally, 0.2 mL HCl was added to end the reaction; the PEG-PLA copolymer was well prepared. Folate (1 g) and DMSO (30 mL) were added into thea bottle (50 mL), and then added 0.9 g NHS and, 0.5 g DCC in were added in turn, stirred away from light for 12 hours at 30°C, and then filtereding out of 1,3-dicyclohexylurea (DCU). Added Tthe filtrate and the PEG-PLA (1 g) were added to another bottle (50 mL), and then adjust the pPH was adjusted to 8 with triethylamine, and lucifugal reaction for 24 hours at 30°C was carried out°C, which werewas dialyzed by a dialysis belt (2000 KkDa) in distilled water for 7 days 72 hours to remove the residual organic solvent and unincorporated folic acid. Attachment of the FA peptide and PEG-PLA was achieved by means of the hydroxyl groups of the FA peptides and the N-terminal groups on the surface of nanoparticles combining covalently.

| Preparation of drug-loaded micelles
The solvent evaporation method was used to load HB into the PEG-PLA diblock copolymer micelles. Briefly, 30 mg diblock copolymer was added to a vial containing 1.3 mL THF and homogenized by sonication for 5 minutes. HB in THF (200 lL; 2 mg/mL) was subsequently added to the vial and mixed uniformly. Then the mixture solutions of HB and PEG-PLA in THF were dropped into 20 mL of purified water maintained under vigorous magnetic stirring, then stirred for 3 hours continuously to form the HB/PEG-PLA micelles, which were dialyzed by a dialysis belt (3500 kDa) overnight to remove the residual organic solvent and unincorporated drugs. HB/ FA-PEG-PLA micelles were also prepared by this method except that the copolymer was substituted with a mixture of FA-PEG-PLA copolymer and PEG-PLA copolymer with weight ratio of 1:9. All the micelles were added to 2.5% F-68 to obtain the solid powder.  To determine the amount of HB encapsulated in the micelles, HPLC was used. Chromatographic separation was achieved using a reverse-phase C18 column (150 9 4.6 mm, 5 lm pore size, Shimadzu VP-ODS, Beijing Shimadzu Medical Equipment Co., Ltd., Beijing, China), the mobile phase solution was acetonitrile/methanoic acid (65:35, v/v); column temperature 25°C; UV detection wavelength: 300 nm; velocity: 0.8 mL/min; quantity: 20 lL. Dried micelles (2 mg) were dissolved in THF (1 mL), then the supernatants were collected after centrifugation (18 800 g, 10 minutes), and dried by N 2 . The dried drugs were redissolved in 1-mL mobile phase solution and analyzed by HPLC. Drug-loading coefficient (DL) and encapsulation ratio (ER) were calculated as follows: DL% ¼ weight of the drug in micelles= weight of the feeding polymer and drug Â 100%; Release study of the micelles was evaluated by the dialysis method.   Average cell viability ¼ ½ðODtreat À ODblankÞ=ðODcontrol À ODblankÞ Â 100%; Percentage of inhibition ¼ ð1 À absorbance of treatment=absorbance of controlÞ Â 100%:

| Pharmacokinetic study in vivo
Calibration curves of HB in serum and tissues were prepared using the HPLC method. Briefly, tumor-bearing mice were anesthetized with diethyl ether, and blood was collected with heparin-treated tubes, which was immediately centrifuged at 18 800 g for 15 minutes to obtain the plasma. Organs such as liver, spleen, kidney, lung, intestine, ovary, and tumor tissues were carefully removed and homogenized with physiological saline to obtain the tissue samples. or those given free HB by tail vein injection (HB concentration 10 mg/kg). After drug injection, rats in every group (3/group) were anesthetized at predetermined time points (10, 20, 30 and 40 minutes, 1, 2, 4, 6, 12, 24, 36, 48 and 60 hours), and blood was collected from the tail vein into heparin-treated tubes. Concentration of HB in every sample was analyzed by HPLC as described earlier.

| Tissue biodistribution study in vivo
For tissue biodistribution studies, SKOV3 tumor-bearing female athymic nude mice were divided into 3 groups (18/group). Each group was given the same ip dose of different micelles or free HB, similar to the pharmacokinetic studies. Three mice from each group were anesthetized at predetermined time points (1, 2, 4, 6, 12 and 24 hours), and blood and tissue samples were collected as described above. Concentrations of HB in blood and tissue samples were determined using a calibration curve by HPLC.

| Statistical analyses
Statistical evaluations of data were carried out by two-tailed Student's t test. Data are expressed as mean AE SD. P < .05 was considered significant.

PEG-PLA micelles in vitro
Cell viability rate of the PEG-PLA micelles without drug loading was very high, and they could reach 67.2% AE 2.2% at a concentration as high as 100 lg/mL. Therefore, the unloaded micelles themselves had no toxicity to cells and were very safe for drug delivery. Table 3 shows that HB/FA-PEG-PLA micelles had higher cytotoxicities toward SKOV3 cells than that of plain HB micelles and free HB solution at all concentrations (P < .05) after photodynamic treatment. However, there was no statistically significant difference between plain HB micelles and free HB solution at most doses (P > .05). At the dose of 50 lg/mL in particular, the inhibition rate of SKOV3 cells with HB/FA-PEG-PLA micelles was 69.5% AE 4.3%, which was 2-fold higher than that of plain micellar HB (32.5% AE 9.6%) and free HB (30.3% AE 5.6%) solution. The results indicated that FA-PEG-PLA micelles can enhance cytotoxicity of HB in SKOV3 cells on account of folate receptor-mediated internalization of micelles and amplification of the drug effect. Table 4 shows the dark toxicity of SKOV3 cells for 3 treatment groups, which indicated that there were no significant differences between the 3 groups at most doses (P > .05).

| Biodistribution study in vivo
Distributions of the micelles in major organs after ip injection were studied. As shown in Figures 5 and 6, peak time of both micelles In most tissues, accumulations of folate-conjugated micelles were much higher than those of plain micelles and free drugs, as shown in Figure 7. Especially in kidney, HB/FA-PEG-PLA micelles were found to have much greater access to kidney, compared to the other 2 drug formulations. This observation suggests that kidney may be the major clearance organ for micelle systems and the bioactivity accounts for the higher concentration there. In contrast, free HB in liver was 413.3 AE 1.8 lg/g at peak, which was much higher compared to the micelles. The lower uptake of the micelles in the liver may be attributed to PEGylation of polymer, which can decrease the uptake of nanoparticles by the reticuloendothelial system in the liver.

| DISCUSSION
Photodynamic therapy is a novel and promising therapy for advanced-stage ovarian cancer which is uncontrolled by routine treatment. The first key process in PDT therapy is the accumulation of photosensitizers in tumor tissues. To gain good aqueous solubility and high selectivity for the tumor, in the present study, we developed a novel active targeted system, FA-PEG-PLA copolymer micelles, to deliver HB.
An ideal targeted anticancer drug delivery system should have characteristics such as avoiding uptake by the reticuloendothelial system (RES), EPR, and active tumor-targeting. [16][17][18] As a novel drug delivery system, FA-PEG-PLA copolymer has several advantages.
First, the copolymer PEG-PLA micelles have the advantages of small size, high drug loading, and good biocompatibility. Second, PEG chain shell have an effective protein resistant property, which have the characteristic of steric repulsion effect, thus PEG could circumvent the uptake of RES. Also, the folate receptor is highly overexpressed in a number of human tumors including ovarian, 19 breast, lung, brain, and renal cell cancers. [20][21][22] Third, we selected folic acid as the targeting ligand, which is cheap, non-immunogenic, retains high binding affinity, and is stable in storage and in the circulation. 23 In this study, HB/FA-PEG-PLA micelles showed a prolonged time for HB in the blood circulation, and better targeting and antitumor effect against ovarian cancer.
The drug release study in vitro showed that both micelles were released slower than the free HB group. Meanwhile, the pharmacokinetic and biodistribution study in vivo showed similar results. The elimination phase (t 1/2,b ) of HB/FA-PEG-PLA and HB/PEG-PLA was 13.5 AE 1.1 hours and 13.6 AE 2.3 hours, respectively; whereas t 1/2,b of free HB was only 7.4 AE 1.2 hours (P < .05). Furthermore, in tumor-bearing mice, free HB rapidly reached its peak at the 2nd hour in every organ; however, the peak times of both micelles were postponed by at least 2 hours. As previously reported, the release mechanism of the PEG-PLA copolymer micelles is mainly diffusioncontrolled release. 24 In our study, HB was dispersed in PLA polymers, which, inside the PEG membrane, was required to diffuse to the membrane surface first and then release successfully. Therefore, the newly developed HB/FA-PEG-PLA micelles showed a well-controlled slow release for HB.   In the present study, we used ip injection instead of iv injection.

CONFLI CTS OF INTEREST
Authors declare no conflicts of interest for this article. F I G U R E 7 Distributions of micelles in blood, tumor tissues, and major organs at 2 and 6 h after drug delivery (n = 3). A, Drug distribution in different tissues at 2 h after drug delivery; B, the drug distribution in different tissues at 6 h after drug delivery; C, the drug distribution of three HB micelles in different tissues at 2 h after drug delivery; D, the drug distribution of three HB micelles in different tissues at 2 h after drug delivery. In each organ, hypocrellin B (HB) concentration was significantly higher in the order of FA-PEG-PLA, PEG-PLA, and free HB, except in the liver and lung. Kidney may be the major clearance organ. Accumulation of HB/FA-PEG-PLA micelles in tumors nearly reached the peak after 2 h, whereas it took at least 6 h to reach the peak in other peritoneal organs. *P < .05, **P < .01, ***P < .001 vs HB. #P < .05, ##P < .01, ###P < .001 vs HB/PEG-PLA-M. HB/FA-PEG-PLA, hypocrellin B poly (ethylene glycol) (PEG)-poly (lactic acid)(PLA)-folate