Novel folated Pluronic/poly(lactic acid) nanoparticles for targeted delivery of paclitaxel

Abstract

The purpose of this study was to explore the in vitro and in vivo targeting behaviors of novel folated Pluronic/poly(lactic acid) block copolymers (FA–Pluronic–PLA) for the anticancer drug, paclitaxel. Both paclitaxel-loaded FA–Pluronic–PLA nanoparticles show in vitro sustained release and in vivo prolonged circulation time. The in vitro actively targeting behavior of paclitaxel-loaded FA–Pluronic–PLA nanoparticles against OVCAR-3 cells (folate receptor positive) was proved by the cytotoxicity tests. The in vivo targeting properties of nanoparticles were also studied in OVCAR-3 ovarian tumor-bearing mice. It was observed that the tumor growth percentage for targeted paclitaxel-loaded FA–F127–PLA nanoparticles is lower than that for non-targeted paclitaxel-loaded PLA–F127–PLA nanoparticles. It was observed from FM images that FA–Pluronic–PLA nanoparticles are mainly localized within the cytoplasm of OVCAR-3 cells.

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Introduction

Paclitaxel is one of the most effective and broad spectrum anticancer drugs used for the treatment of ovarian cancer, breast cancer, lung cancer and other cancers.^1–5 Paclitaxel is a kind of hydrophobic drug and its solubility in water is pretty low. Kolliphor EL is added in the clinical formulation of paclitaxel in order to improve its solubility. However, Kolliphor EL has been found to cause hypersensitivity, nephrotoxicity, neurotoxicity and other side effects.^6,7 In order to improve the solubility of paclitaxel and enhance its antitumor efficacy, many research works have focused on loading paclitaxel into polymeric nanoparticles.^8–10

Amphiphilic block copolymers are able to form nanoparticles composed of hydrophobic core and hydrophilic shell in aqueous solutions. Thus, polymeric nanoparticles have been used widely to load hydrophobic anticancer agents.^11–13 Targeted polymeric nanoparticles by conjugating ligands on the surface can further increase the selectivity and antitumor efficacy of drugs and reduce the systemic toxicity at the same time.^14,15 Folic acid (FA) is a kind of targeting ligands studied by a lot of research groups. FA has a strong specific interaction with folate receptor (FR), which is overexpressed on the cell surface of various carcinomas including ovary, breast, lung, kidney, colon and pancreas.^16–18

Our group has synthesized a novel folate targeted polymeric block copolymer, FA–Pluronic F87–poly(lactic acid) (FA–F87–PLA). The effective targeting behaviors of FA–F87–PLA have been proven by in vitro cytotoxicity tests using paclitaxel as a drug model.¹⁹ In order to study further the effect of different Pluronic block on the targeting properties of FA–Pluronic–PLA nanoparticles, another two folate targeted polymeric block copolymers, FA–F127–PLA and FA–P85–PLA, were also synthesized in this work and their aggregation, in vitro releasing and targeting behaviors were studied. More importantly, the pharmacokinetics and in vivo targeting behaviors of these two copolymers have been explored further so as to understand the efficacy of FA–Pluronic–PLA nanoparticles as targeted drug carriers. Pluronics [poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide), PEO–PPO–PEO] have been used as promising drug carriers as well as multidrug resistance (MDR) reversal agents.^9,20 Compared with PEO block as the hydrophilic block, Pluronic copolymers were proved to be able to enter cells more effectively due to their amphiphilic property.^21,22 Hydrophobic poly(lactic acid) (PLA) is a kind of well-known biodegradable polyesters and widely used in biomedical fields.

Experimental

Materials

Pluronic F127 and P85 were kindly supplied by BASF Corporation. L-Lactide was purchased from Sigma-Aldrich and recrystallized from ethyl acetate. Folic acid (FA), stannous octoate [Sn(Oct)₂] and sodium phosphotungstate were purchased from Sigma-Aldrich and used as received. N,N′-Dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP) was purchased from J&K Chemica. Pyrene was purchased from Acros. Dimethyl sulfoxide (DMSO) and dichloromethane (CH₂Cl₂) was purified by distillation over calcium hydride (CaH₂). Paclitaxel was kindly supplied by Fujian South Pharmaceutical Co., Ltd. Paclitaxel injections was purchased from Sichuan Shenhe Pharmaceutical Co., Ltd. Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Gibco and used as received. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Solarbio and used as received. Tetramethylrhodamine-5-carbonyl azide (TMRCA) was purchased from Invitrogen. All other chemicals were of reagent grade. Human ovarian cancer cells OVCAR-3 were purchased from CICAMS, Beijing. Male Spraguee Dawley (SD) rats and female Balb/c mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. The animal care and handling were performed according to the guidelines issued by WHO and Chinese Academy of Science.

Synthesis of FA–Pluronic–OH

Folic acid (0.85 g, 1.93 mmol) and DCC (0.06 g, 1.74 mmol) were mixed and dissolved in dried DMSO (50 mL). The reaction mixture was stirred at 25 °C for 24 h. Then Pluronic F127 (20 g, 1.59 mmol) and DMAP (0.20 g, 1.60 mmol) were added, and the reaction mixture was stirred at 25 °C for another 24 h. Following this, the reaction mixture was centrifuged to remove the precipitated byproduct, dicyclohexylurea (DCU). Then the supernatant was dialyzed against DMSO using a dialysis membrane (molecular weight cut-off, MWCO 3500 Da) for 6 h, then dialyzed against water for 48 h, during which the water was renewed every 3 h, and finally freeze-dried to obtain FA–F127–OH copolymer.

FA–P85–OH copolymer was obtained according to the similar procedures as above.

Synthesis of FA–Pluronic–PLA block copolymers

FA–F127–OH (2 g) and LA (2 g) was added at room temperature under argon and was followed by the addition of stannous octoate (about 0.1 wt% of LA). The mixture was degassed by six vacuum-purge cycles and then heated to 150 °C for 6 h. After cooling to room temperature, the reaction mixture was dissolved in CH₂Cl₂ and then precipitated into cold methanol. Following this, the product was dissolved in methylene chloride, and precipitated in cold ethyl ether. The product FA–F127–PLA was then filtered and dried overnight under vacuum. FA–P85–PLA copolymer was synthesized according to the similar procedures as above.

The chemical structures of copolymers were characterized by using nuclear magnetic resonance (¹H NMR) and gel permeation chromatography (GPC). ¹H NMR was measured by a Bruker ACF-400 (400 MHz) Fourier Transform Spectrometer (Bruker BioSpin, Rheinstetten, Germany) with tetramethylsilane (TMS) as the internal standard. The amount of folate groups conjugated to copolymers was determined by ultraviolet spectroscopy (Perkin Elmer UV-Vis Spectrophotometer Lambda 35, USA) at 289 nm. A calibration curve was obtained using DMSO as the solvent. GPC of the copolymers was performed on a Waters 2414 refractive index detector, a Waters 515 binary HPLC pump, and three Waters Styragel Columns (HT2, HT4, and HT5) with THF as an eluent at a flow rate of 0.8 mL min⁻¹ at 40 °C.

The critical micelle concentration (CMC)

The CMC of FA–Pluronic–PLA block copolymers in PBS solutions was determined by fluorescence measurements (HITACHI F2700) using pyrene as a fluorescence probe.^23,24 The pyrene stock solution in acetone (5 μL) was added into test tubes, respectively and then the acetone was evaporated. Following this, the FA–Pluronic–PLA solutions (5 mL) with varying concentrations from 0.001 mg L⁻¹ to 100 mg L⁻¹ were added to each test tube. These test tubes were then sonicated for 2 hours in order to reach equilibrium. The final concentration of pyrene in the solutions was set to be 6 × 10⁻⁷ M.

For the measurement of pyrene excitation spectra, the excitation spectra were scanned from 300 to 360 nm by fixing the emission wavelength at 390 nm. The slit widths for both excitation and emission sides were maintained at 2.5 nm.

Preparation of paclitaxel-loaded FA–Pluronic–PLA nanoparticles

Paclitaxel-loaded FA–Pluronic–PLA nanoparticles were prepared by a dialysis method. Briefly, 3 mg of FA–Pluronic–PLA block copolymer and 0.3 mg of hydrophobic drug paclitaxel were dissolved in 3 mL of DMSO. The resulting organic solution was added drop-wise to 10 g of distilled water under gentle stirring. The excess paclitaxel outside the particles and DMSO were removed by dialysis against distilled water using a dialysis membrane (molecular weight cut-off, MWCO 3500 Da). The water was exchanged at intervals of 1 h.

Mean diameters and size distributions of paclitaxel-loaded FA–Pluronic–PLA nanoparticles were determined by the dynamic light scattering (DLS) method on a Zeta Plus instrument (Brookhaven Instruments Corporation, NY, USA) at 25 °C and at a scattering angle of 90 °C. The morphology of nanoparticles was characterized by transmission electron microscope (TEM, HITACHI H-600, Hitachi Corp., Osaka, Japan) at an acceleration voltage of 75 kV. Sodium phosphotungstate (2 wt%) was used as the negative stain.

The amount of paclitaxel loaded in FA–Pluronic–PLA nanoparticles was determined by high performance liquid chromatography (HPLC). An Agilent HPLC Series 1100 equipped with a UV-Vis detector set at 227 nm, connected with a Zorbax Eclipse XDB-C18 column, acetonitrile/PBS aqueous solution (70/30 v/v) as the mobile phase, and a flow rate of 1 mL min⁻¹ was used.

The loading efficiency (LE) and loading capacity (LC) of paclitaxel into FA–Pluronic–PLA nanoparticles were calculated by the following equations:

In vitro drug release

The in vitro drug release behaviors of paclitaxel-loaded FA–Pluronic–PLA nanoparticles were studied by a dialysis bag diffusion technique.^5,25

The paclitaxel-loaded FA–Pluronic–PLA nanoparticles solution (10 mL) was placed into a dialysis bag (MWCO 3500 Da) and dialyzed against phosphate buffered saline (PBS, pH 7.4, 0.01 M) aqueous solution (100 mL) under continuous mechanical shaking in water bath at 37 °C. At predetermined time intervals, 5 mL of released paclitaxel solution was withdrawn and replaced with an equal volume of fresh medium in order to maintain a constant volume. The drug content in the samples was determined by HPLC using the same method as that for determining the drug encapsulation.

In vivo pharmacokinetic studies

Ten male Spraguee Dawley (SD) rats (250 ± 20 g) were randomly assigned to two groups for pharmacokinetic studies. Group 1 and 2 were injected intravenously through the tail vein with free paclitaxel and paclitaxel-loaded FA–Pluronic–PLA nanoparticle at an equivalent dose of 0.2 mg kg⁻¹ paclitaxel vs. the body weight, respectively. At predetermined time, blood samples were taken from the tail vein and centrifuged at 4500 g for 10 min. 200 μL of plasma samples were mixed with 1 mL ethyl acetate, followed by vortexing for 2 min and centrifugation for 15 min at 14 000 rpm. Then the organic layers were taken out and dried under nitrogen gas stream at 40 °C. The residue was redissolved in acetonitrile/PBS aqueous solution (70/30 v/v) and measured by HPLC system as above.

In vitro cytotoxicity studies

OVCAR-3 (folate receptor positive) cells were cultured in DMEM supplemented with 2.0 mmol L⁻¹ glutamine, 10% fetal bovine serum (FBS), 100 μg mL⁻¹ streptomycin sulfate and 100 U mL⁻¹ penicillin at 37 °C in humidified 5% CO₂.

The cytotoxicity assays through a MTT method were used to investigate the tumor cell inhibitions of paclitaxel-loaded nanoparticles, and the cytotoxicity of blank FA–Pluronic–PLA and PLA–Pluronic–PLA nanoparticles.

OVCAR-3 cells were seeded into 96-well plates at the density of 1 × 10⁴ cells per well for 8 hours to allow cell attachment. The attached cells were then incubated with targeted paclitaxel-loaded FA–Pluronic–PLA nanoparticle, non-targeted paclitaxel-loaded PLA–Pluronic–PLA nanoparticle and free paclitaxel injections at 0.02, 0.05, 0.1 and 0.2 mg L⁻¹ equivalent paclitaxel concentrations for 48 h. Following this, the medium was discarded and the cells were washed by PBS aqueous solution. MTT solution (10 μL, 5 mg mL⁻¹) was then added to each well and incubated for 4 h. After that, all media was removed and 150 μL dimethylsulfoxide (DMSO) was added into each well of the plate and the plate was shaked for 10 min. The absorbance of the transformed MTT solution was immediately measured at 492 nm by a microplate reader (Thermo Labsystems MK3). The cell viability was calculated as a percentage compared to a control that had not been treated with the nanoparticles, using the following equation:

where A(test) and A(control) are the absorbance of surviving cells treated with nanoparticles and untreated cells, respectively.

The cytotoxicity of blank FA–Pluronic–PLA and PLA–Pluronic–PLA nanoparticles was explored using the same procedure as above. The tested concentrations of the blank FA–Pluronic–PLA and PLA–Pluronic–PLA nanoparticles are from 20 to 400 mg L⁻¹.

Study on anti-cancer targeting efficacy in OVCAR-3 ovarian tumor-bearing mice

OVCAR-3 tumor model was established to study the in vivo targeting efficacy of FA–Pluronic–PLA nanoparticles. Female Balb/c nude mice (6 weeks, 18 ± 2 g) were injected subcutaneously in the right flank with 0.1 mL of cell suspension containing 6 × 10⁶ cells per mouse OVCAR-3 cells. When the tumor volumes of mice reach 50–150 mm³, the mice were divided into four groups (n = 8).

Three groups were administered intraperitoneally with paclitaxel-loaded FA–Pluronic–PLA nanoparticles, paclitaxel-loaded PLA–Pluronic–PLA nanoparticles, and paclitaxel injections at the same 0.6 mg kg⁻¹ dose of paclitaxel, respectively. One group was injected intraperitoneally with 0.9% saline as a control. The injections were given at day 0, 7 and 21 for four groups. The change of tumor for all nude mice was monitored closely. The tumor volume was calculated using the following formula: (length) × (width)²/2.

FA–Pluronic–PLA block copolymers chemically conjugated with TMRCA and their intracellular fate by fluorescence microscopy (FM)

A fluorescence probe TMRCA was chemically attached to the hydroxyl end groups of PLA blocks in FA–Pluroinc–PLA block copolymers according to the published procedures.^23,24,26,27 FA–Pluronic–PLA (30 mg) and TMRCA (1.2 mg) dissolved in dried toluene were reacted at 80 °C for 5 hours under argon. After that, the unreacted TMRCA was removed by dialysis against DMF for 5 days and then the DMF was removed by dialysis against distilled water for 3 days. The final FA–Pluronic–PLA–TMRCA aqueous solutions were lyophilized.

FM was also performed to study the intracellular fate of FA–Pluronic–PLA nanoparticles. FA–Pluronic–PLA–TMRCA nanoparticles (0.2 mg mL⁻¹) were incubated in OVCAR-3 cells for 6 h. Following this, cells were stained with nucleus-selective dye, Hoechst 33342 (5 μg mL⁻¹) for 0.5 h.

Fluorescent images were imaged with an Olympus IX71 inverted fluorescence microscope using a 40× objective and an Olympus DP71 digital colour camera.

Statistical studies

Values from different groups were compared with the control groups by the Students t-test. A difference was considered to be statistically significant when the P value was less than 0.05. The results are expressed as mean ± standard deviation (S.D.).

Results and discussion

Synthesis and characterization of FA–Pluronic–PLA block copolymers

In order to develop targeted drug delivery systems, folic acid has been attached to one end of Pluronic/PLA block copolymers to obtain FA–Pluronic–PLA block copolymers. The aggregation behavior, drug loading, releasing, in vitro and in vivo targeting behaviors, and intracellular localization of FA–Pluronic–PLA block copolymers were investigated in this study.

FA–Pluronic–PLA block copolymers were synthesized by two steps. Firstly, folic acid was grafted to one OH end of Pluronic block copolymers to obtain FA–Pluronic–OH. Secondly, FA–Pluronic–PLA block copolymers were obtained by ring opening polymerization of the monomer L-lactide using FA–Pluronic–OH as the initiator and stannous octoate [Sn(Oct)₂] as the catalyst (Scheme 1). The possible byproduct FA–Pluronic–FA generated in the first step can be removed by methanol in the second step.

Scheme 1 The synthesis scheme of FA–Pluronic–PLA and FA–Pluronic–PLA–TMRCA copolymers.

The successful synthesis and chemical structure of FA–Pluronic–PLA block copolymers were confirmed and determined by NMR and GPC techniques. Fig. 1A shows the ¹H-NMR spectrum of FA–P85–PLA block copolymer in CDCl₃. The appearance of the small peak at δ 4.2–4.4 ppm belongs to methylene protons of PLA–CO–OCH̲₂–CH₂–O–PEO– segment, indicating the successful synthesis of FA–P85–PLA block copolymer. The weight fraction of PLA block in FA–P85–PLA copolymer was calculated to be 56.5% from the peak intensity ratio of methyl protons of PLA (O–CH(CH̲₃)–CO–: δ = 1.60 ppm) and methyl protons of Pluronic (–OCH₂–CH(CH̲₃)–: δ = 1.13 ppm). The molecular weight of FA–P85–PLA copolymer was then calculated to be 17 200. A single and narrow peak was observed from the GPC trace of FA–P85–PLA copolymer as shown in Fig. 1B and the index of molecular weight distribution (M̄_w/M̄_n) was determined to be 1.11, indicating that the synthesized FA–P85–PLA block copolymer is pure and narrowly distributed. FA–F127–PLA copolymer was characterized similarly and the relevant data were shown in Table 1.

Fig. 1 (A) ¹H NMR spectra of FA–P85–PLA block copolymer (CDCl₃). (B) The GPC trace of FA–P85–PLA copolymer. (C) UV-Vis spectra of FA–Pluronic–PLA copolymers and folic acid in DMSO.

Table 1 The characterizations and drug loading of FA–Pluronic–PLA block copolymers and nanoparticles

Samples	Copolymers				Drug loading of nanoparticles
	M̄n	M̄w/M̄n	wt% (PLA)	Mol% (FA)	Size (nm)	LE	LC
FA–F127–PLA	27 000	1.45	51.7	12.6	258.8	43.5%	4.8%
FA–P85–PLA	17 200	1.11	56.5	9.5	185.3	42.1%	4.2%

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The molar content of folate groups on FA–Pluronic–PLA copolymer was measured by UV-Vis spectrum.^18,28 It can be seen from Fig. 1C that FA–Pluronic–PLA copolymers show the similar characteristic peaks at 285 and 360 nm as folic acid does, indicating that folate groups have been grafted successfully on the end of FA–Pluronic–PLA block copolymers. The molar content of folate groups on FA–F127–PLA and FA–P85–PLA copolymers was determined to be 12.6% and 9.5%, respectively. Too many folate groups on the surface of nanoparticles could affect the stability and targeting efficacy of nanoparticles.^29,30 Therefore, the content of folate groups on both FA–Pluronic–PLA copolymers might be a suitable one for their application in targeted drug delivery systems.

Characterization of blank and paclitaxel-loaded FA–Pluronic–PLA nanoparticles

Amphiphilic block copolymer is able to self-assemble into nanoparticles composed of inner hydrophobic core and outside hydrophilic shell in aqueous solution. The CMC values are used to characterize the thermodynamic stability of polymeric nanoparticles.^23,31

A fluorescence spectroscopy measurement using pyrene as a probe was carried out in order to determine the CMC values of FA–Pluronic–PLA block copolymers. When pyrene was transferred from the aqueous phase to the hydrophobic environment provided by the polymer nanoparticles, a redshift can be detected for the excitation spectra of pyrene. The peak intensity ratio (I₃₃₇/I₃₃₄) in the excitation spectra of pyrene was plotted against the concentration of FA–Pluronic–PLA copolymers (see Fig. 2A). The CMC values of FA–F127–PLA and FA–P85–PLA nanoparticles were determined to be 1.4 and 1.9 mg L⁻¹ at room temperature. The low CMC values of FA–Pluronic–PLA copolymers indicate the good stability of both nanoparticles and a great resistance to dissociation due to a larger volume of blood in the body, which thus augurs well for their potential applications in drug delivery systems.

Fig. 2 (A) The concentration dependencies of peak intensity ratio (I₃₃₇/I₃₃₄) in the excitation spectra of pyrene for FA–Pluronic–PLA block copolymer aqueous solutions. (B) Size distributions of FA–Pluronic–PLA nanoparticles. (C) and (D) are TEM pictures of FA–F127–PLA and FA–P85–PLA nanoparticles, respectively.

The anticancer drug paclitaxel was then loaded into FA–Pluronic–PLA nanoparticles. The size distributions of paclitaxel-loaded FA–Pluronic–PLA nanoparticles were shown in Fig. 2B and their mean diameters were summarized in Table 1. The morphologies of both paclitaxel-loaded FA–Pluronic–PLA nanoparticles were observed to be spherical core–shell micelles by TEM (see Fig. 2C and D). The sample nanoparticles are stained by sodium phosphotungstate, which is a negative stain. That is to say the dark part stained by sodium phosphotungstate is the hydrophilic part. It is shown in Fig. 2C and D that the dark part is hydrophilic shell and the light part is hydrophobic core. Thus it was proved that FA–Pluronic–PLA nanoparticles are spherical core–shell micelles. The loading efficiency (LE) and loading capacity (LC) of paclitaxel-loaded FA–Pluronic–PLA nanoparticles were calculated and summarized in Table 1 according to the HPLC results, respectively. The LE and LC of both paclitaxel-loaded FA–Pluronic–PLA nanoparticles have similar values and those of paclitaxel-loaded FA–F127–PLA nanoparticles are a bit larger.

In vitro release studies of paclitaxel-loaded FA–Pluronic–PLA nanoparticles

Fig. 3A shows the cumulative release profiles of paclitaxel-loaded FA–Pluronic–PLA nanoparticles in PBS solution at 37 °C. It was obvious that the release behaviors of paclitaxel-loaded FA–Pluronic–PLA nanoparticles showed a biphasic release pattern. An initial burst release (47% for FA–F127–PLA and 51.7% for FA–P85–PLA) in the first 4 hours was observed and the drug release profile reaches a plateau (about 85% for FA–F127–PLA and 72% for FA–P85–PLA) about 33 hours later. Similar results were published by other research work.^32–34 The biphasic release profile could be related to the location of paclitaxel in the FA–P85–PLA nanoparticles. The initial fast release might be because part of paclitaxel was located at the edges of hydrophobic PLA core. The rest part of paclitaxel located inside the hydrophobic core resulted in the following sustained release.³⁵ PLA is a kind of biodegradable polyesters. Thus the release kinetics of paclitaxel from FA–Pluronic–PLA nanoparticles could be affected by both diffusion and degradation of polymers. Based on other research groups' and our results, the release mechanism for the initial fast release of paclitaxel from nanoparticles could be mainly dominated by the diffusion control one due to the relatively slow degradation rate of PLA block.^36,37

Fig. 3 (A) The release behavior of PTX-loaded FA–Pluronic–PLA nanoparticles in PBS (pH 7.4, 0.01 M) solutions at 37 °C. (B) Plasma concentration–time curves of free PTX injections and PTX-loaded FA–Pluronic–PLA nanoparticles after i.v. administration to SD rats at the same 0.2 mg kg⁻¹ PTX dose (n = 5).

Compared with FA–P85–PLA, the relative higher cumulative release amount of paclitaxel from FA–F127–PLA could be due to its a bit higher loading efficiency. Compared with the cumulative release percentage of paclitaxel from FA–F87–PLA nanoparticles (about 60%) we synthesized previously,¹⁹ those of both FA–F127–PLA and FA–P85–PLA nanoparticles are higher. These could be due to the relative higher loading efficiency of paclitaxel in the latter two nanoparticles.

pharmacokinetics of paclitaxel-loaded FA–Pluronic–PLA nanoparticles

Fig. 3B shows the plasma paclitaxel concentration vs. time profiles of paclitaxel injections and paclitaxel-loaded FA–Pluronic–PLA nanoparticles aqueous solution. For all the three samples, it is clear that the plasma paclitaxel concentration decreases with increasing the circulation time. However, the plasma paclitaxel concentration for both paclitaxel-loaded FA–Pluronic–PLA nanoparticles decreased much more slowly than that for paclitaxel injections. It was reported that the maximum tolerable level and minimum effective level for paclitaxel are 8540 and 43 ng mL⁻¹, respectively.³⁸ For paclitaxel-loaded FA–P85–PLA nanoparticles, the plasma paclitaxel concentration decreased below 43 ng mL⁻¹ after about 108 hours later. The time is even longer for paclitaxel-loaded FA–F127–PLA nanoparticles. Nevertheless, the plasma paclitaxel concentration of paclitaxel injections decreased below 43 ng mL⁻¹ after just about 32 hours later. These results indicate that the circulation period keeping the effective plasma paclitaxel concentration for both paclitaxel-loaded FA–Pluronic–PLA nanoparticles is over three times longer than that for free paclitaxel injections. The longer in vivo circulation time of paclitaxel-loaded FA–Pluronic–PLA nanoparticles is consistent with their in vitro sustained release behavior discussed above. Other paclitaxel-loaded nanoparticles also showed similar prolonged circulation time in vivo.^6,38,39

In vitro cytotoxicity studies of paclitaxel-loaded nanoparticles

In order to study the targeting properties of paclitaxel-loaded FA–Pluronic–PLA nanoparticles, MTT assays using Human ovarian cancer cells OVCAR-3 (folate receptor positive) were performed.

It was observed from Fig. 4A and B that the blank FA–Pluronic–PLA and PLA–Pluronic–PLA nanoparticles showed no obvious cytotoxicity at the concentrations from 20 to 400 mg L⁻¹. PLA homopolymer is a kind of well-known biocompatible and biodegradable polyester. Pluronic F127 and P85 are also approved by FDA to be used in medical fields. The above results indicate that FA–Pluronic–PLA and PLA–Pluronic–PLA block polymers also show good biocompatibility.

Fig. 4 (A) and (B) are cytotoxicity of FA–Pluronic–PLA and PLA–Pluronic–PLA nanoparticles over OVCAR-3 cells, respectively. Each point represents the mean ± S.D. of five cultures. (C) and (D) are cytotoxicity of OVCAR-3 cells treated with PTX-loaded FA–Pluronic–PLA nanoparticle, PTX-loaded PLA–Pluronic–PLA nanoparticle and free PTX injections depending on the PTX concentrations, respectively. The incubation time was 48 h. Each point represents the mean ± SD of five cultures. *P < 0.05, **P < 0.01 vs. the PTX-loaded PLA–Pluronic–PLA nanoparticle group.

The cell viability of paclitaxel-loaded FA–Pluronic–PLA nanoparticle, paclitaxel-loaded PLA–Pluronic–PLA nanoparticle and free paclitaxel injections at equivalent paclitaxel concentrations (0.02–0.2 mg L⁻¹) was shown in Fig. 4C and D, respectively. It was obvious that the cytotoxicity of paclitaxel-loaded nanoparticles and free paclitaxel enhanced with increasing the drug dose. Furthermore, it was observed that the cell viability of all paclitaxel-loaded nanoparticles was lower than that of free paclitaxel injections, which means that the anticancer efficacy of paclitaxel can be enhanced through being loaded inside nanoparticles. The low anti-tumor efficacy of paclitaxel injections could be due to the P-glycoprotein (P-gp) efflux pump, which is responsible for cellular multidrug resistance (MDR) to a wide variety of anticancer drugs.⁷ Biocompatible polymeric nanoparticles found to be able to overcome the efflux of P-gp substrates, including loading these drugs in nanoparticles to enable entry into cells by endocytosis and thus bypassing P-gp.^40,41 FA–Pluronic–PLA and PLA–Pluronic–PLA nanoparticles have been proved to show good biocompatibility. Therefore, the higher anticancer efficacy of paclitaxel-loaded FA–Pluronic–PLA and PLA–Pluronic–PLA nanoparticles could be because paclitaxel loaded in nanoparticles was easier to enter cells by endocytosis mechanism than free paclitaxel was.

Furthermore, compared with non-targeted PLA–Pluronic–PLA nanoparticle, paclitaxel loaded in both FA–Pluronic–PLA nanoparticle showed better cell suppression, proving the targeting efficacy of FA–Pluronic–PLA nanoparticle. This could be related to the specific targeting interaction between folate groups on the surface of FA–Pluronic–PLA nanoparticles and folate receptor on the surface of OVCAR-3 cells, thus enhancing the uptake of FA–Pluronic–PLA nanoparticle by folate receptor-mediated endocytosis. It was also noticed that the cell viability of paclitaxel in FA–F127–PLA was slightly lower than that in FA–P85–PLA and FA–F87–PLA.¹⁹ Based on the above in vivo and in vitro results, FA–F127–PLA could be a better candidate for targeted delivery carriers among these three nanoparticles.

The in vivo targeting efficacy of paclitaxel-loaded nanoparticles

In order to further study the in vivo targeting properties of FA–Pluronic–PLA nanoparticles, paclitaxel-loaded FA–F127–PLA nanoparticles, paclitaxel-loaded PLA–F127–PLA nanoparticles and paclitaxel injections were injected intraperitoneally into OVCAR-3 bearing nude mice. The change of tumor growth for all groups was observed for 28 days and the average tumor growth percentage was shown in Fig. 5. It was indicated that the average tumor growth percentage is less for both paclitaxel-loaded nanoparticles than for saline control group. Compared with non-targeted paclitaxel-loaded PLA–F127–PLA nanoparticles, targeted paclitaxel-loaded FA–F127–PLA nanoparticles showed even better tumor suppression ability. These results mean that FA–Pluronic–PLA nanoparticles could be a promising candidate in targeted drug delivery systems.

Fig. 5 Change in tumor volume of OVCAR-3 implanted in Balb/c nude mice over a period of 28 days. Each point represents the mean ± SD of eight mice.

The cellular uptake and intracellular distribution of FA–Pluronic–PLA nanoparticles

FM technique was used to study the cellular uptake and intracellular distribution of FA–Pluronic–PLA nanoparticles. Fluorescence probes were loaded physically in the nanoparticles for most published research work.^25,42 The location of nanoparticles may not be really indicated by the fluorescence dyes due to the possible leakage of dyes from nanoparticles. Therefore, in this study, a fluorescence dye TMRCA was grafted chemically to the OH end group of hydrophobic PLA block of FA–Pluronic–PLA copolymers to obtain FA–Pluronic–PLA–TMRCA copolymers (see Scheme 1). The acyl azide group of TMRCA was rearranged into isocyanate and then the hydroxyl end group in the PLA block reacted the isocyanate group to form a urethane.^26,27,43

The fluorescence images of OVCAR-3 cells after incubation with FA–Pluronic–PLA–TMRCA nanoparticles and nucleus-selective dye were shown in Fig. 6. The red fluorescence in OVCAR-3 cells shown in Fig. 6A and D corresponds to FA–F127–PLA–TMRCA and FA–P85–PLA–TMRCA nanoparticles, respectively. The blue fluorescence in OVCAR-3 cells shown in Fig. 6B and E corresponds to the nucleus of OVCAR-3 cells. Fig. 6C and F show the position of OVCAR-3 cells. Compared with these pictures, it is clear that both FA–Pluronic–PLA–TMRCA nanoparticles are mainly localized in the cytoplasm of OVCAR-3 cells. Similar results were obtained for other polymeric nanoparticles.^19,42,44

Fig. 6 Internalization and localization of FA–Pluronic–PLA–TMRCA nanoparticles in the cytoplasmic compartment of OVCAR-3 cells. Fluorescent images for cells incubated with FA–F127–PLA–TMRCA (A) and FA–P85–PLA–TMRCA (D) nanoparticles, and subsequently stained with nucleus-selective dye Hoechst 33342 (B and E), (C and F) are the corresponding phase-contrast photograph. Scale bar, 30 μm.

Conclusions

In this paper, two FA–Pluronic–PLA block copolymers were prepared for their potential application in targeted drug delivery systems. FA–Pluronic–PLA copolymers were successfully synthesized and characterized. The molar content of folate groups on the end of both FA–Pluronic–PLA copolymers was determined to be 12.6% and 9.5%, respectively. Based on published results, about 10% of folate groups on the surface of nanoparticles might be a suitable one for their application in targeted drug delivery systems. The effect of various folate contents on the targeting properties of FA–Pluronic–PLA nanoparticles will be studied in future. FA–Pluronic–PLA nanoparticles in aqueous solutions were examined to be stable and small size. Paclitaxel-loaded FA–Pluronic–PLA nanoparticles showed sustained release, prolonged circulation time, in vitro and in vivo effective targeting property over ovarian cancer cells and cytoplasmic localization. These results indicate that both FA–Pluronic–PLA nanoparticles could be promising targeting drug carriers of anticancer drugs.

Acknowledgements

The authors would like to acknowledge the financial support from the National Natural Science Foundation of China (No. 21264009 and 31360376), the Cultivation Project for Academic and Technical Leaders of Major Disciplines of Jiangxi Province (20153BCB22009), the Scientific and Technological Landing Project of Higher Education of Jiangxi Province (KJLD13071), and the research project for undergraduates of JSTNU (20140802023).

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