In vivo pharmacokinetics, biodistribution and antitumor effect of paclitaxel-loaded micelles based on α-tocopherol succinate-modified chitosan

Abstract

In our previous study, α-tocopherol succinate modified chitosan (CS-TOS) was synthesized and encapsulated paclitaxel (PTX) to form micelles. Preliminary study revealed that the CS-TOS was a potential micellar carrier for PTX. In this study, some further researches were done using Taxol formulation as the control to evaluate the micelle system deeply. In vitro cell experiments demonstrated that the cytotoxic effect of PTX-loaded CS-TOS micelles against MCF-7 cells was comparable with that of Taxol formulation, and the PTX-loaded micelles had excellent cellular uptake ability, which was in a time-dependent manner. The in vivo pharmacokinetic study in rats showed that the micelles prolonged the half-life and increased AUC of PTX than Taxol formulation. From biodistribution study, it was clear that for micelles, the drug concentrations in the liver and spleen were significantly higher than those of Taxol formulation, but much lower in the heart and kidney. Furthermore, the PTX-loaded micelles showed superior antitumor effect, but yielded less toxicity as indicated by the results of antitumor efficacy study and survival study in U14 tumor-bearing mice. These results suggested that CS-TOS micelles could be a potentially useful drug delivery system to improve the performance and safety of PTX.

Keywords:

• α-Tocopherol succinate
• chitosan
• micelle
• paclitaxel
• pharmacokinetics

Introduction

Paclitaxel (PTX) is known as one of the most active antineoplastic agents. It has been used clinically in the treatment of ovarian cancer, metastatic breast cancer, non-small cell lung cancers, and several other malignancies (Spencer & Faulds, 1994; de Bree et al., 2006). However, due to its low water solubility (0.25 μg/ml), the currently widely used preparation of Taxol® is formulated in a vehicle composed of Cremophor EL (polyethoxylated castor oil) and dehydrated alcohol in the ratio of 50:50 (v/v), and the use of Cremophor EL causes serious side effects, such as nephrotoxicity, cardiotoxicity, neurotoxicity and hypersensitivity (Gelderblom et al., 2001), which limited the application of Taxol and caused lots of inconvenience. Furthermore, Cremophor EL was reported to leach diethylhexylphtalate (DHEP) from polyvinyl chloride infusion bags and administration sets (Singla et al., 2002), which was harmful to the patients. Therefore, it is emergent to find a drug delivery system for PTX without Cremophor EL and improve the therapeutic efficacy and safety of the drug.

Over the last decade, polymeric micelles formed by amphiphilic copolymers in aqueous solution have received widespread attention (Liu et al., 2011; Du et al., 2012; Sarisozen et al., 2012). Generally, the micelles possessed a unique core-shell structure, which not only solubilized the poorly soluble drugs in the core but also protected drugs from inactivation. Furthermore, these systems exhibit many advantages, such as passive targeting of tumor via the enhanced permeation and retention (EPR) effect (Danhier et al., 2015), long circulation and easy production, etc (Li et al., 2011; Kwon & Kataoka, 2012). Therefore, the polymeric micelles are considered as promising carriers of water-insoluble anticancer drugs. Currently, several promising candidates are in clinical trials (Lee et al., 2008; Matsumura & Kataoka, 2009).

In our previous study, a novel α-tocopherol succinate-modified chitosan was synthesized to form polymeric micelles, and PTX was encapsulated into the hydrophobic cores of the micelles. The PTX-loaded micelles exhibited small particle size and narrow size distribution, high drug-loading capacity, good compatibility with blood, and comparable cytotoxicity with Taxol (Liang et al., 2012). It can be considered as a potential injectable delivery system for PTX. Thus, this system was further investigated in this paper to assess the in vitro cytotoxicity, cellular uptake, in vivo pharmacokinetics, biodistribution, antitumor activity and mouse survival time with Taxol formulation as reference.

Materials and methods

Materials and reagents

α-Tocopherol succinate modified chitosan (CS-TOS) was prepared by us, with the average degree of substitution of about 17%. Paclitaxel (PTX, purity of 99.9%) and docetaxel (DOC, purity >99.5%) were purchased from Tianfeng Bioengineering Technology Co., Ltd., Liaoning, China. Cremophor EL was kindly supplied by BASF Corp., Ludwigshafen, Germany. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), DMSO and fluorescein isothiocyanate (FITC) were obtained from Sigma Chemical Co., St. Louis, MO. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and penicillin–streptomycin mixture were purchased from Gibco BRL, Carlsbad, CA. 0.9% Sodium chloride injection was obtained from Connell Pharmaceutical Co., Ltd, Jilin, China. Heparin sodium for injection (1000 IU/ml) was purchased from Tianjin Biochemistry Reagent Factory, Tianjin, China. All other chemicals and solvents were of analytical grade or higher and used without further purification. Distilled water or Milli-Q water was used in all experiments.

Cell lines and animals

MCF-7 cells (human breast cancer cells, ATCC® HTB-22™) were obtained from American Type Culture Collection (ATCC), Manassas, VA. U14 cells (mouse uterine cervix carcinoma cells) were provided by the Cell Resource Center of Chinese Academy of Medical Sciences, Beijing, China.

Specific pathogen-free (SPF) female Wistar rats (180–220 g, 6–8 weeks old) and female Kunming mice (18–22 g, 5–6 weeks old) were purchased from HFK Bioscience Co., Ltd., Beijing, China. The animals were housed at 21 ± 2 °C with the relative humidity of 50 ± 5% on a 12-hour light/dark cycle and fed with free access to food and water. Prior to experiments, the animals were acclimated for at least one week.

All animal studies were carried out following the protocol approved by the Institutional Animal Care and Use Committee at Shenyang Pharmaceutical University.

Preparation of PTX-loaded CS-TOS micelles

The CS-TOS and PTX-loaded CS-TOS micelles were prepared by the methods as described earlier (Liang et al., 2012). Firstly, the CS-TOS was synthesized by the coupling reaction of carboxyl group of α-tocopherol succinate (TOS) with amino group of chitosan (CS). Then the PTX-loaded CS-TOS micelles were prepared by a probe-type ultrasonic method. Briefly, a CS-TOS solution was prepared by dissolving 10 mg of CS-TOS in 10 ml of distilled water, and stirring overnight at room temperature to ensure complete dispersion. PTX solution was prepared by dissolving PTX in acetone at a concentration of 1 mg/ml. One milliliter of the PTX-acetone solution was added quickly into the aqueous phase under probe-sonication, and the resultant mixture was further ultrasonicated at 400 W for 30 min (JY92-II, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) under cooling conditions. After that, the product was centrifuged at 4000 rpm for 10 min to remove the unloaded PTX. The resultant supernatant was lyophilized to obtain PTX-loaded CS-TOS micelles.

Before in vitro or in vivo study, the micelles were reconstituted and adjusted to required concentration using distilled water or normal saline, then finally sterilized by 0.22 μm filtration, if necessary. The blank micelles were prepared by the same procedure except no PTX was added.

The positive control Taxol formulation was prepared by dissolving PTX into a 50:50 mixture of ethanol and Cremophor EL at a concentration of 6 mg/ml, and stored at 4 °C until use. For injection, the stock solution was diluted with saline to obtain the required concentration.

For fluorescent microscopy observation, the PTX-loaded micelles were labeled with FITC using the technique described by Cho et al. (2007). Firstly, FITC-labeled CS-TOS was synthesized based on the reaction between the isothiocyanate group of FITC and the amino group of CS-TOS. Ten milligrams of FITC was dissolved in 5 ml of ethanol and added dropwise into 1.0 mg/ml of CS-TOS solution. The molar ratio of CS-TOS to FITC was controlled at 2:1. The reaction mixture was stirred for 8 h at room temperature, and then dialyzed against distilled water using dialysis membranes (MWCO: 3.5 kDa, Viskase Companies Inc., Darien, IL) for 24 h to remove the unreacted FITC and ethanol. All procedures were carried out in the dark. Then the PTX was encapsulated as above.

Characterization and analysis of PTX-loaded CS-TOS micelles

The size as number-weighted hydrodynamic diameter and zeta potential of PTX-loaded CS-TOS micelles were determined in deionized water by the dynamic light scattering (DLS) method using Nicomp 380/ZLS (Nicomp Instruments, Particle Sizing Systems, Santa Barbara, CA).

Chemical quantification of PTX was conducted by a reverse-phase HPLC system with a mobile phase delivery pump (Jasco PU-980 Intelligent HPLC pump, Jasco, Tokyo, Japan), a UV detector (Jasco UV-975 Intelligent UV/Vis detector, Jasco, Tokyo, Japan) at 227 nm, a C18 column (200 mm × 4.6 mm, 5 μm, Dikma Technologies Inc., Beijing, China) used as analytical column, and an ODS C18 column (10 mm × 4.6 mm, 5 µm, Elite, Dalian, China) used as guard column. The mobile phase consisting of acetonitrile and water at the ratio of 60:40 (v/v) was pumped at a flow rate of 1 ml/min. Before sample injection of 20 μl, the column was equilibrated to 30 °C. The encapsulation efficiency (EE%) and the drug loading coefficient (DL%) were calculated as previously described. When biosamples were injected, the mobile phase was composed of acetonitrile and 0.1% phosphoric acid at a ratio of 50:50 (v/v).

Cell experiments

Cell cultures

MCF-7 cells were plated and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin--streptomycin in a humidified incubator containing 5% CO₂ at 37 °C. After 2nd to 4th passage, the cells were processed into particular experiment.

In vitro cytotoxicity study by MTT method

To evaluate the antitumor activity of PTX-loaded CS-TOS micelles preliminarily, the cytotoxicity of micelles against MCF-7 cells was compared with Taxol formulation using the MTT method. Briefly, cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and allowed to grow for 24 h. Then cells were incubated with the PTX-loaded micelles, Taxol formulation, blank micelles or the vehicle of Taxol formulation at a series of PTX concentrations for 24 and 48 h, respectively. The cell viability in each group was determined. Briefly, at predetermined time intervals, the supernatant of each well was removed, and 100 μl of MTT solution (0.5 mg/ml solution in PBS) was added and incubated for additional 4  h at 37 °C. The unreacted MTT was removed by aspiration, and 100 μl of DMSO was added to dissolve the purple formazan crystals. After that, cell viability was evaluated by measuring the absorbance at 570 nm using a Bio-Tek Synergy HT plate reader (Bio-Tek Instruments Inc., Vinooski, VT). PBS-treated cells were taken as the control with 100% viability. Cell viability was calculated as follows:

Cellular uptake study by fluorescent microscopy observation

To investigate the cellular internalization of PTX-loaded micelles by MCF-7 cells, fluorescent microscopy observation was performed using FITC-labeled micelles. The cells were seeded at a density of 1 × 10⁵ cells per well in 6-well plates which included 2 ml of DMEM. After 24 h incubation, the FITC-labeled micelles were added at a PTX concentration of 1.0 μg/ml in each well and allowed for further incubation for 2, 4, 8 and 24 h, respectively. After incubation, the cells were washed with cold PBS 7.4 for three times and fixed with 1 ml of 4% paraformaldehyde for 10 min at 37 °C. Finally, cells were observed under the fluorescence microscope (Olympus IX71, Tokyo, Japan) with FITC-labeled CS as a control.

Pharmacokinetic study in rats

For pharmacokinetic study, 12 healthy adult female Wistar rats weighing 200 ± 20 g were randomly assigned to two groups. Before the experiment, rats were fasted overnight with free access to water. Either of the groups received an intravenous (IV) injection of PTX-loaded CS-TOS micelles or Taxol formulation with equivalent dose of 10 mg/kg (PTX versus the body weight) through the tail vein, respectively. Both formulations were diluted with normal saline to a final concentration of 1 mg/ml just before use. Blood samples (about 300 µl) were taken via retro-orbital venous plexus puncture and collected in heparinized cryotubes before drug administration and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 hours after dosing. Then the collected blood samples were immediately centrifuged at 4000 rpm for 10 min at 4 °C, and the plasma was obtained and stored at −70 °C until analysis.

Biodistribution study in mice

For biodistribution study, 60 female Kunming mice weighing 18–22 g were randomly assigned to two groups and dosed with PTX-loaded micelles and Taxol formulation at an equivalent dose of 10 mg/kg (PTX versus the body weight) via the tail vein, respectively. For each group, 30 mice were allocated into 10 time points accordingly (n = 3): 0.05, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h. Before administration, all mice were fasted overnight with free access to water. At predetermined time points, the mice were sacrificed by cervical dislocation after collecting blood from the lateral canthus. The heart, liver, spleen, lung and kidney were excised and thoroughly washed with normal saline, then blotted dry with paper towel and weighed. Immediately after blood collection, the blood was centrifuged at 4000 rpm for 10 min at 4 °C to obtain plasma. The plasma and tissues were labeled and stored at −70 °C until the PTX concentrations analysis.

Pharmacokinetic data analysis

Concentrations of PTX in plasma or tissues were determined using the HPLC method. For determination, the weighed tissue samples were homogenized with a two-fold weight of normal saline. Subsequently, to 100 µl of plasma or tissue homogenate, 100 µl of methanol, 200 µl of internal standard (IS) working solution (DOC in acetonitrile, 2 μg/ml), and 250 mg of anhydrous sodium sulfate were added. Methanol and acetonitrile were used for both protein precipitation and PTX extraction. The sample mixture was vortexed for 5 min and put into the refrigerator at 4 °C for 30 min, then centrifuged at 10 000 rpm for 10 min at 4 °C. After that, aliquots of 20 µl of the clear supernatant were injected into the HPLC system. The ratio of the peak area of PTX to that of IS was calculated to estimate the PTX concentration using the calibration curve accordingly.

Furthermore, the pharmacokinetic parameters of PTX were obtained using a non-compartmental model with WinNonlin Professional version 5.2 software (Pharsight Corporation, Mountain View, CA). The data were the area under the plasma concentration–time curve from time 0 to the last sampling time (AUC_0–24), elimination half-life (t_{1/2_Lambda_z}), apparent volume of distribution (Vss) and the total clearance (CL).

In vivo tumor growth-inhibition study

Establishment of tumor model

To further compare the behavior of PTX-loaded micelles with Taxol formulation, the in vivo evaluation was performed using female mice bearing U14 tumors. For the tumor xenograft establishing, U14 cells of the third passage in vivo were harvested, and suspended in sterile normal saline at a concentration of 1.0 × 10⁷ cells/ml, then kept in ice. For the inoculation, 0.2 ml of the above cell suspension was inoculated beneath the armpit of the right anterior limb of each mouse via subcutaneous injection. Three or four days later, when tumors became palpable, the tumor model was established.

In vivo antitumor efficacy study

To confirm the antitumor potential of PTX-loaded micelles in vivo, the antitumor efficacy was evaluated. Briefly, 60 tumor-bearing mice were labeled, weighed and randomly divided into five groups (n = 12): a negative control group (normal saline), two positive control groups (Taxol formulation, PTX equivalent dose of 10 and 20 mg/kg) and two PTX-loaded micelles groups (PTX equivalent dose of 10 and 20 mg/kg). The mice were given intravenous injection via the tail vein once every third day for four times. Furthermore, their weights were monitored throughout the study as weight loss is an indicator for toxicity. The day that mice received treatment was set as day 1. On day 13, after weighed, all the mice were sacrificed by cervical vertebra dislocation and the tumor mass was harvested, photographed and weighted. Tumor growth inhibition rate (TIR) which was used to assess the therapeutic efficacy against tumor was calculated by the following equation. The body weight change was also plotted for each group. where, W_m is the mean tumor weight of PTX-loaded micelles or Taxol formulation group and W_n is the mean tumor weight of the normal saline group.

Mouse survival time evaluation

For antitumor drugs, the time of subjects survived after the treatment over a period of time was an important criterion to assess the efficacy and safety of the drug. To further study the PTX-loaded micelles deeply, the survival time of each group of mice was determined.

Briefly, a total of 36 mice bearing U14 tumor were randomized into three groups with 12 animals each. To two groups, PTX-loaded micelles and Taxol formulation were injected at a dose of 10 mg/kg via tail vein on days 1, 4, 7 and 10, respectively. For the control group, equivalent volume of normal saline was administered correspondingly. The tumor-bearing mice of each group were allowed unrestricted access to food and water until died. The Kaplan–Meier curves were generated and the comparison of overall survival across treatment groups was performed by the log-rank test. The mean survival time of each group was estimated. Furthermore, the survival time was used to calculate the life extension value as follows: where, T_m is the mean survival time of the PTX-loaded micelles group or the Taxol formulation group, and T_n is the mean survival time of normal saline group.

Statistical analysis

All data were expressed as the mean ± standard deviation (SD) unless otherwise noted. Comparisons between two groups were performed using Student’s t-test. The differences were considered to be significant at p < 0.05.

Results and discussion

Characterization of PTX-loaded micelles

The PTX-loaded CS-TOS micelles were successfully prepared by the probe-type ultrasonic method. As a result from the HPLC determination, the drug-loading content and encapsulation efficiency of the PTX-loaded micelles was 7.4 ± 0.8 and 68.2 ± 5.3%, respectively. The prepared micelles exhibited a narrow particle size distribution with an average diameter of 81.4 ± 10.5 nm and polydispersity index of 0.287. The zeta potential was measured as + 26.3 ± 2.3 mV. For micelles are known to have many advantages in delivering anticancer drugs, further studies were performed to know this delivery system deeply.

In vitro cytotoxicity study

The in vitro cytotoxicity of PTX-loaded micelles, Taxol formulation, blank micelles and Cremophor EL was assessed by the MTT method using MCF-7 cell line. The assay was based on the ability of active mitochondrial dehydrogenase to convert dissolved MTT to water-insoluble purple formazan crystals.

According to Figure 1, it was clear that drug-free micelles did not exhibit obvious cytotoxicity to the cells during the test period, while Cremophor EL displayed significant cytotoxicity, which was well consistent with previous reports (Wei et al., 2009). Furthermore, the PTX-loaded micelles and Taxol formulation significantly decreased the viability of MCF-7 cells with increasing drug concentration and incubation time. Over the concentration range studied, the cytotoxicity of PTX-loaded micelles was comparable to Taxol formulation, which indicated that the PTX-loaded micelles maintained anticancer activity. For different incubation time, the cell viability at 48 h were much lower than those at 24 h, confirming that sufficient exposure time is essential for the drug to effectively kill tumor cells. This may be explained that for longer incubation periods, a larger number of cells enter the G2 and M cell cycle phases during which PTX is more active (Zhang et al., 2012).

Figure 1. In vitro cytotoxicity of PTX-loaded micelles, Taxol formulation, Cremophor EL and blank micelles against MCF-7 cells after 24 h (A) and 48 h (B) incubation (mean ± SD, n = 3).

Cellular uptake study by fluorescence microscopy

To investigate the cellular uptake of PTX-loaded CS-TOS micelles, the FITC-labeled micelles and FITC-labeled CS nanoparticles were incubated with MCF-7cells for 2, 4, 8 and 24 h, respectively. Fluorescence images of cell-associated FITC are shown in Figure 2, with green fluorescence imaging utilized to visualize FITC. As a control, there was no fluorescence inside MCF-7 cells after the cells were incubated with FITC-labeled CS nanoparticles over the 24 h. For FITC-labeled micelles, it can be observed that after 2 h incubation, the FITC fluorescence was found to be aggregated in the MCF-7 cells along the membranes. After 4 h, the obvious fluorescence was observed, and the fluorescence intensity was enhanced with the incubation time within 8 h. This implied that more micelles entered into the cells, which was in accordance with the results reported previously (Zhou et al., 2010). At 24 h, the fluorescence was weak, and the outline of MCF-7 cells was not visible, which implied the apoptosis of cells by micelles.

Figure 2. Fluorescence images of MCF-7 cells after incubation with FITC-labeled PTX-loaded CS-TOS micelles for (A) 2 h, (B) 4 h, (C) 8 h, (D) 24 h, and (E) FITC-labeled CS for 8 h.

It was reported that nanoparticles were generally internalized into cells via fluid phase endocytosis, receptor-mediated endocytosis or phagocytosis (Hu et al., 2009; Xu et al., 2013; Banerjee et al., 2014). The possible mechanism for this uptake may be that the cell membrane is negative-charged, and the micelles with positive surface charge density are affinity for cell endocytosis (Miller et al., 1998). Furthermore, the CS-TOS was amphiphilic. It can dissolve in both polar and non-polar solvents and have high solubility in cell membranes. Therefore, the cellular entry of micelles is greatly enhanced. However, since the entry and subsequent trafficking of nanocarriers depend on many factors, such as the particle size, shape, material composition, surface chemistry and charge as well as cell type, etc (Sahay et al., 2010; Verma & Stellacci, 2010), the exact mechanism of cellular uptake might be far more complicated than our current understanding and further studies are clearly needed.

HPLC method validation for biosamples determination

Under the conditions for biosamples determination, PTX and IS (DOC) were eluted at the desirable retention times of 17.4 and 13.5 min, respectively, and they were well separated from endogenous interfering components in rat or mice tissues and plasma (data not shown). Linear calibration curves were obtained in the concentration range of 0.1–20.0 μg/ml with corresponding correlation coefficients (r) of >0.9950. The intra- and inter-day precision and accuracy values for the detection of PTX conformed to the acceptable criteria. Therefore, this analytical method is valid in terms of specificity, linearity, precision and accuracy.

Pharmacokinetic studies in rats

To investigate the in vivo application of PTX-loaded CS-TOS micelles, the pharmacokinetic behavior of micelles and Taxol formulation was compared after IV administration to rats at the same PTX dose of 10 mg/kg. The mean plasma concentration–time profiles of PTX for both formulations and the relevant pharmacokinetic parameters are shown in Figure 3 and Table 1, respectively.

Figure 3. Plasma concentration-time curves of PTX after intravenous administration of PTX-loaded micelles and Taxol formulation to rats at the dose of 10 mg/kg (mean ± SD, n = 6). *p < 0.05, compared with Taxol formulation.

Table 1. Pharmacokinetic parameters of PTX in rats after intravenous administration of PTX-loaded micelles and Taxol formulation at the dose of 10 mg/kg (mean ± SD, n = 6).

	Taxol formulation	PTX-loaded micelles
t1/2_Lambda_z (h)	3.72 ± 0.45	8.13 ± 1.39*
AUC0–24 (µg·h/ml)	8.08 ± 2.33	10.03 ± 1.86
Vss (L)	1.14 ± 0.39	2.35 ± 0.75*
CL (L/h)	0.25 ± 0.06	0.18 ± 0.03

*p < 0.05 compared with Taxol formulation.

From Figure 3, it was obvious that both profiles showed the typical biphasic pattern which can be characterized as a rapid distribution phase, followed by an elimination phase. Generally, the concentrations of PTX delivered by micelles were lower than those of Taxol formulation in the distribution phase and a little higher in the elimination phase. After 12 h, the drug concentrations in Taxol formulation group were below the HPLC quantification limit, while for the micelles group, they were still detectable till 24 h. Moreover, PTX-loaded micelles changed the PTX pharmacokinetic parameters in comparison with Taxol formulation. The area under the curve (AUC_0–24) for micelles was 10.03 µg·h/ml, which was more than 20% higher than that of Taxol formulation. There was also a significant increase in half-life (t_1/2) of the PTX-loaded micelles. These may be attributable to an almost 30% reduction in the CL of PTX in the micelles, which imply a longer retention of the drug in blood circulation. In addition, the mean volume of distribution at steady state (Vss) of micelles was significantly larger than that of Taxol formulation (2.35 L versus 1.14 L).

The slower plasma elimination rate and longer systemic circulation time of PTX-loaded micelles was inspiring. For sufficient retention time, it is essential for the drug to effectively kill tumor cells; the PTX-loaded micelles with long retention time were supposed to have improved therapeutic efficacy of PTX.

Tissue distribution in mice

The biodistribution of PTX delivered by micelles and Taxol formulation was investigated in mice. Figure 4 revealed the biodistribution data of both formulations. After intravenous injection, PTX was largely distributed to the body organs. For micelles, the drug concentrations in the liver and spleen were significantly higher than those of Taxol formulation. This suggested that the micelles were sequestered by the macrophage phagocytic system (MPS) within few minutes of intravenous administration. The liver may temporarily act as a depot site of the drug and release the drug back into the systemic circulation, which could prolong the retention time of PTX (Hollis et al., 2013). Specially, for cancer therapy, it is important to reduce drug accumulation in the key organs, such as the heart and kidney, to avoid or minimize systemic side effects. The biodistribution results demonstrated that drug accumulation in the heart following micelles administration was significantly less than that achieved with Taxol formulation. It can be deduced that the micelles may decrease the cardiac toxicity and nephrotoxicity.

Figure 4. Biodistribution of PTX in the heart, liver, spleen, lungs and kidneys in mice after intravenous injection of (A) PTX-loaded micelles and (B) Taxol formulation at 10 mg/kg (mean ± SD, n = 3).

In vivo antitumor efficacy and treatment toxicity study

To evaluate the antitumor activity, PTX-loaded micelles and Taxol formulation were injected intravenously each third day for four times into the mice bearing U14 tumors with doses 10 and 20 mg/kg, respectively. As shown in Figure 5, the tumors in mice treated with both formulations were obviously smaller in comparison with the saline control group. At the end of this experiment, the group treated with PTX-loaded micelles showed comparable antitumor activity with Taxol formulation group at a dose of 10 mg/kg, but much higher at 20 mg/kg, with tumor growth inhibition of 54.7 versus 50.3% (10 mg/kg) and 74.9 versus 67.3% (20 mg/kg), respectively. This may be explained as follows: for the micellar formulation, drug was released in a slower and sustained manner. At 20 mg/kg, the micellar delivery system can maintain a relatively high drug concentration for a long time. So the PTX-loaded micelles exhibited superior antitumor efficacy than Taxol formulation.

Figure 5. Photographs of tumors from each treatment group excised after IV treatment of PTX-loaded micelles and Taxol formulation on U14 tumor-bearing mice.

On the other hand, as important indicators for the toxicity of formulation, animal behavior and bodyweight change after drug administration were also studied. It was noted that the mice in Taxol formulation groups huddled up, became restless, or screamed post-injection, and in 20 mg/kg group, two mice developed sudden convulsion and then died, whereas no perceptible change in activity was observed after administration of PTX-loaded micelles. The behavioral difference may be related to the use of Cremophor EL and ethanol in Taxol formulation, which was reported, could cause serious side effects, such as cardiotoxicity (Gelderblom et al., 2001).

Furthermore, the body weight curve in Figure 6 demonstrated that there was no severe body weight loss observed with the exception of Taxol formulation at the dose of 20 mg/kg. In that group, a significant body weight loss was observed, which may also due to the side effect of Cremophor EL. It is well known that the key drawback of Taxol formulation is its toxic side effects associated with Cremophor EL, thus, the low toxicity of PTX-loaded micelles is of great value, and this also can endow the formulation with a high dosage tolerance.

Figure 6. Body weight changes after IV treatment of PTX-loaded micelles and Taxol formulation on U14 tumor-bearing mice (mean ± SD, n = 12).

In a word, the PTX-loaded micelles exhibited comparable or higher antitumor activity, but offered reduced toxic effects in comparison with Taxol formulation at the same dosage. This was in accordance with the observations in cytotoxicity study. There are several explanations for that. First, the micelles prolonged the circulation time of PTX, so that tumor cells can be exposed to the drug for a longer time to enhance the therapeutic effect. Secondly, the PTX-loaded micelles could solubilize the drug in the core without any cosolvent addition, which can minimize the adverse effects of the formulation. Therefore, PTX-loaded micelles were considered to be much more effective and safer than Taxol formulation.

Mouse survival time

As the PTX-loaded CS-TOS micelles showed improvement than Taxol formulation, the micelles were further evaluated by the survival time of U14 tumor-bearing mice. The Kaplan–Meier survival curves are plotted in Figure 7. It was obvious that the survival time of PTX-loaded micelles and Taxol formulation treated tumor-bearing mice was significantly prolonged, compared with the control. The mean survival time of the control, Taxol formulation and PTX-loaded micelles was 19.2, 21.2 and 24.8 days, respectively. It was worth noting that, the micelles group showed much higher life extension value than Taxol formulation, with the ILS being 29.1 versus 10.4%. This was consistent with the former results. Thus, the above results indicated that the PTX-loaded micelles could be a promising carrier for PTX delivery.

Figure 7. Kaplan–Meier survival curves of U14 tumor-bearing mice after IV treatment of PTX-loaded micelles and Taxol formulation (n = 12).

Conclusions

In conclusion, PTX-loaded CS-TOS micelles were successfully prepared by the probe-type ultrasonic method with the average diameter of 81.4 nm. The in vitro cytotoxicity study demonstrated that the cytotoxic effect of PTX-loaded micelles was comparable with that of Taxol formulation. Cellular uptake study revealed that the PTX-loaded micelles had excellent cellular uptake ability which was in a time-dependent manner. In vivo pharmacokinetic results indicated that the PTX-loaded micelles had longer systemic circulation time and slower elimination rate than those of Taxol formulation. From biodistribution study, it was clear that for micelles, the drug concentrations in the liver and spleen were significantly higher than those of Taxol formulation, but much lower in the heart and kidney. Furthermore, in vivo antitumor study and survival study showed that the PTX-loaded polymeric micelles could significantly inhibit U14 tumor growth and reduce the toxicity of the formulation. Therefore, the PTX-loaded CS-TOS micelles might be a potentially effective and safety drug delivery system for PTX.

Declaration of interest

The authors report no declarations of interest.

The National Natural Science Foundation of China (No. 51403057), the General Financial Grant from the China Postdoctoral Science Foundation (No. 2015M570305), the Heilongjiang Returned Overseas Fund (No. LC2013C24), the Heilongjiang Postdoctoral Fund (No. LBH-Z14179), and the Doctoral Scientific Research Startup Foundation of Harbin Normal University (XKB201304) are gratefully acknowledged for financial support.