DOI:
10.1039/C5RA06253E
(Paper)
RSC Adv., 2015,
5, 51592-51599
Design, synthesis and in vivo antitumor efficacy of novel eight-arm-polyethylene glycol–pterostilbene prodrugs
Received
8th April 2015
, Accepted 5th June 2015
First published on 5th June 2015
Abstract
Pterostilbene (PS) is an effective antitumor drug, but its clinical development has been hampered due to its poor solubility and relatively short half-life. To deal with this predicament, an eight-arm-polyethylene glycol–PS (8arm-PEG–PS) prodrug was synthesized by linking PS with eight-arm-polyethylene glycol (8arm-PEG). The obtained 8arm-PEG–PS prodrug possesses high solubility (∼147 fold of free PS) and relatively high drug-binding capacity (∼7.06 wt%). An in vitro cytotoxicity test demonstrated the excellent anticancer activities with a potency similar to that of free PS. In addition, the PS prodrugs significantly improved the therapeutic effect on the inhibition of tumor growth with lower systemic toxicity compared to the native PS. Compared to the group treated by the free PS, the tumor volume of the groups treated by the PS prodrugs decreased dramatically by more than 2 fold or 3 fold in the Lewis lung carcinoma (LLC) tumor-bearing mouse model. On the basis of these results, the 8arm-PEG–PS prodrugs have great promise toward cancer therapy.
1. Introduction
Cancer is a major health problem worldwide, and it is the leading cause of human mortality, exceeded only by cardiovascular disease, that accounts for more than 8 million deaths every year.1 Although there are many methods for the therapy of cancer, chemotherapy has been a mainstay treatment in recent years. Unfortunately, some drawbacks of traditional anti-cancer drugs limit their use in the natural form.2
Pterostilbene (trans-3,5-dimethoxy-4′-hydroxystilbene, PS), a natural, dimethylated analogue of resveratrol that can be extracted from blueberries, is known to have diverse pharmacological activities, including anticancer, anti-inflammation, antioxidant, anti-proliferative and analgesic activities.3 Dietary administration of high doses of PS is not toxic in mice.4 PS has been shown to have potent antitumor activity with low toxicity in various cancer types, including hepatoma,5 breast cancer,6 prostate cancer,7 pancreatic cancer8 and chronic myelogenous leukemia,9 among others. PS also shows better biological activity due to its increased bioavailability, as the substitution of a hydroxy group with a methoxy group.10 In animal studies, PS was shown to have 80% bioavailability compared to 20% of resveratrol making it potentially advantageous as a therapeutic agent.11
Although PS has proved excellent antitumor activity, there are still many limitations that hinder its clinical research, such as the extremely poor water solubility and relatively short half-life. In recent years, extensive studies have been reported to find a way to conquer the problems of PS (e.g. nanoparticles,12,13 liposome,13,14 polymeric micelles,15–18 emulsions,19 and polymeric prodrugs).20,21
Of all above-mentioned methods, the use of polymeric prodrugs have gained considerable attention due to its numerous benefits, such as controlling the release of a drug, altering the drug biodistribution, altering the cellular uptake properties of a drug, taking advantage of the enhanced permeability and retention (EPR) effect, and prolonging the action of a drug.22 Various bioavailable polymers have been selected to produce the polymeric prodrugs to overcome the disadvantages of the conventional drug. Linear polyethylene glycol (PEG) is the most widely used polymeric prodrugs system for the development of drugs because of its high solubility in aqueous solution, simple end-group modification, non-toxic, non-immunogenic, and non-antigenic. However, a traditional linear PEG used in the PS delivery system has only one or two active sites available, which results in the poor loading capacity of small molecule drugs.23,24 In addition, some studies show that the PEG with a comparatively low molecular weight may face the risk of being fast removed by kidney owe to its small size.25–27 Therefore, multiple PEG with more functional groups and appropriate molecular weight (40 kDa) is endowed with the ability to ensure the high drug loading and the greatest drug enhancements.
In this study, the relatively high drug-binding prodrugs, 8arm-PEG–PS, were first synthesized by an esterification reaction between the carboxyl group of 8arm-PEG and the hydroxyl group of PS. The obtained polymeric prodrugs were characterized by 1H-NMR to confirm the successful synthesis. Then, the solubility, drug loading capacity, side effects, in vitro drug release and cytotoxicity were investigated. In addition, in vivo antitumor activities of 8arm-PEG–PS were also assessed on the LLC tumor-bearing mouse model.
2. Experimental
2.1. Materials
Eight arm-polyethylene glycol carboxylic acid (8arm-PEG–COOH, Mw = 20 kDa and 40 kDa) were supplied by JenKem Technology Co., Ltd. (Beijing, China), and were FDA and EU foodgrade materials. Pterostilbene was gained from Chengdu Preferred Biotechnology Co., Ltd (Sichuan, China). 4-Dimethylaminopyridine (DMAP) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) were received from J&K Chemical Reagent Co., Ltd. (Beijing, China). Gibco Dulbecco's Phosphate-Buffered Saline (DPBS), Penicillin and streptomycin, Gibco Dulbecco's Modified Eagle's Medium (DMEM) were all bought from Invitrogen. Fetal bovine serum (FBS) was from HyClone. The Cell Counting Kit-8 (CCK-8) kit was supplied by the Dojindo Laboratories.
Murine LLC cells were supplied by the Peking University Health Science Center (Beijing, China) and were grown in DMEM with 10% FBS, 1% streptomycin–penicillin. The cell line was maintained in an incubator supplied with a 5% CO2/95% air humidified atmosphere at 37 °C.
2.2. Animals and ethics
Female C57BL/6 mice, 6–7 weeks age, were purchased from the National Institute for the Control of Pharmaceutical and Biological Products. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, and approved by the Experimental Animal Ethics Committee in Beijing.
2.3. Synthesis of 8arm-PEG40K–PS and 8arm-PEG20K–PS
The synthesis of 8arm-PEG40K–PS was described here. 8arm-PEG40K–COOH (2.0 g, 0.05 mmol) and PS (0.51 g, 2.0 mmol) were dissolved in 10 mL of dichloromethane (DCM), then EDC (0.38 g, 2.0 mmol) and DMAP (0.48 g, 4.0 mmol) were added at 0 °C, respectively. The solution was stirred at 0 °C for 1 h and then carried out at room temperature for 24 h. The solvent was removed by rotary evaporation and the residue was dissolved in 100 mL of DCM, and the crude product was precipitated by 200 mL ethyl ether. After filtration, the resulting solids were recrystallized with a mixture of N,N-dimethylformamide-dimethylcarbinol (DMF–IPA) (25 mL/100 mL). Then, the solids were filtered, washed twice with 100 mL ethyl ether, and vacuum-dried at 40 °C to obtain 8arm-PEG40K–PS. The synthesis and purification of 8arm-PEG20K–PS were similarly to that of 8arm-PEG40K–PS. The final products were monitored by an Agilent 1200 (Agilent, USA) HPLC instrument, it employs a VYDAC 214TP54 (C18, 300A, 5 μm, 4.6 × 250 mm) with a UV detector at 306 nm, using a gradient of 15–100% of acetonitrile in 0.05% trifluoroacetic acid (TFA) at a flow rate of 1 mL min−1 at room temperature.
2.4. Characterization of 8arm-PEG–PS
The 8arm-PEG20K–PS and 8arm-PEG40K–PS were characterized by proton nuclear magnetic resonance spectroscopy (1H-NMR) to confirm the successful synthesis. The 1H-NMR analyses were recorded on a Varian Mercury Plus 400 MHz spectrometer with deuterated dimethylsulfoxide (DMSO-d6) or deuterium oxide (D2O) as the solvent at room temperature. PS, 8arm-PEG20K–PS and 8arm-PEG40K–PS (5 mg of each) was dissolved in 1 mL of DMSO or D2O, and the products were assessed using an NMR spectrometer.
The mass and molar ratio of drug-to-carrier for the PS prodrugs were detected by a UV-Vis spectrophotometer.28,29 Simply, PS was immersed in 50 mL methanol and filtered and diluted to five kind of different concentrations ranging from 0.4 to 10 μg mL−1. Then the UV absorbance of the various concentrations of PS was measured at 306 nm and drew a standard curve of concentration vs. absorbance. The pretreatment of PS prodrugs were the same as the native PS. The mass or molar of 8arm-PEG–PS (mConjug or nConjug) were diluted in a known concentration, and absorbance at 306 nm and the concentration of PS in the sample was used to obtain the mass and molar of PS (mPS, nPS). The mass and the molar ratio of drug-to-carrier were thus reported as mPS/mConjug and nPS/nConjug.
2.5. Stability study
The stability study of PS prodrugs were described here.30 Briefly, PS prodrugs were dissolved in phosphate buffered saline (PBS) at a concentration of 10 mg mL−1 adjusted to pH 6.1, 7.4, and 8.2 and incubated at 37 °C to encompass the range of pH reported in the literature for the lung fluid.31–33 For HPLC analysis, 1 mL of PBS solution was taken out and the same volume PBS was added to the vials at specified time intervals, high-speed centrifuged to get supernatant, measuring disappearance of the prodrugs by HPLC at 306 nm. The HPLC employs a VYDAC214TP54 (C18, 5 mm, 4.63250 mm) with a UV detector at 306 nm, using a gradient of 60% of acetonitrile in 0.05% TFA at a flow rate of 1 mL min−1 at 37 °C. A stability profile graph was generated by plotting the percentage of the remaining starting material over a time course at different pH values. The percentage was obtained on the basis of the ratio of the peak area of the sample at different time vs. the initial area peak. The release experiments were conducted in triplicate, and the results were expressed as mean ± standard deviation (SD).
2.6. Solubility study
A UV spectrophotometer (VU1601, Shimazu, Japan) was used to examine the solubility of PS and PS prodrugs into deionized water.16 The concentrations of PS, 8arm-PEG20K–PS and 8arm-PEG40K–PS were 1 mg mL−1, which was based on the amount of PS. Then, the transmittance of each sample was measured at 306 nm.
2.7. In vitro cytotoxicity
LLC cell line was kindly provided by the Peking University Health Science Center. The cell line was maintained in DMEM with 10% FBS, 1% streptomycin–penicillin, and subcultured 2–3 times per week at relative cell density of 1:6. All cells were grown at 37 °C, 5% CO2 in a humidified atmosphere.
The in vitro anticancer efficacy of the PS prodrugs against LLC cells were evaluated by a CCK-8 assay.34 The specific experimental process was carried out as follows: approximate 2 × 103 LLC cells per well were seeded in 96-well plates with 180 μL of DMEM medium and incubated overnight at 37 °C before the test. Subsequently, serial dilutions (corresponding PS concentration) of PS, 8arm-PEG20K–PS and 8arm-PEG40K–PS (from 1.0 to 40 μg mL−1) in DMEM were added to the medium of 96-well plates and incubated for 24 h, 48 h and 72 h. At the end of the incubation period, 20 mL of CCK-8 solution was added to all wells of the plate and incubated for another 2 h at 37 °C. The absorbance of each well was measured by using a spectramax M5 at the 450 nm wavelength. The half maximal inhibitory concentration (IC50) of the PS and PS prodrugs were calculated using Origin® 8.6 (OriginLab, Northampton, USA). The experiments were performed in triplicates and data was represented as mean ± SD.
2.8. In vivo antitumor activity assay
The subcutaneous lung cancer model were established by injecting 1 × 106 LLC cells per mouse into the right axillary flank region of female C57BL/6 mice (6–7 weeks) and the mice were randomly divided into four groups. For each study, PS was formulated in a polyvinyl pyrrolidone/PBS (1:99) suspension (polyvinyl pyrrolidone as cosolvent for free PS), and was sterile filtered. The PS prodrugs were prepared in PBS solution, and were sterile filtered. Once the size of the tumors reached 100–150 mm3, the mice were injected intravenously with PS or PS prodrugs (the dose equal to PS) at two day intervals (n = 6, 10 mg kg−1). In the observation phase, tumor volume and body weights data were collected every other day. Tumor size was determined by caliper measurements, with volume calculated as (L × W × W/2), where L is the longest tumor diameter (millimeter) and W is the shortest diameter. For efficacy studies, the percentage of tumor growth inhibition (%TGI) was calculated using the following formula: [(C − T)/C] × 100%, where C is the mean tumor volume of the control group at a specified time and T is the mean tumor volume of the treatment group at the same time. Relative tumor volume (RTV) was calculated at each measurement time point (where RTV was equal to the tumor volume at a given time point divided by the tumor volume prior to initial treatment). For humane reasons, animals were killed and regarded as dead if the implanted tumor volume reached 5000 mm3.
2.9. Evaluation of hypersensitivity
To evaluate allergic reaction, four groups of tumor bearing mice with weight of 26–28 g (n = 6) was injected with physiological saline, PS, 8arm-PEG20K–PS, and 8arm-PEG40K–PS every two days at a PS dose of 10 mg kg−1, respectively. After being injected for 10 days, the orbit blood of mice in different groups was collected and centrifuged. Serum samples were analyzed according to the procedure of mouse IgE ELISA using a hematology analyzer (MEK-7222K, Nihon Kohden Celltac E). To further evaluate the hematological toxicity of different PS formulations, 200 μL of blood of each mouse was collected to test the white blood cell (WBC) number after final administration by a blood cell analyzer (MEK-7222K, Japan).
2.10. Statistical analysis
All data in this study were reported as the mean ± SD unless otherwise illustrated. The statistical analysis was measured using ANOVA. A p < 0.05 was considered to be statistically significant for evaluating the differences between the treatment groups.
3. Results and discussion
3.1. Synthesis of 8arm-PEG40K–PS and 8arm-PEG20K–PS
A novel polymeric prodrug was synthesized to overcome the low solubility of PS against deionized water and to enhance its drug-binding capacity. Linear PEG is the most widely used nonionic polymer because of its high aqueous solubility in the field of polymer-based drug delivery,27,35 but it is limited by its low drug-binding capacity. Here, 8arm-PEG was selected due to its high drug-binding capacity compared with linear PEG. The prodrug was synthesized by a coupling reaction between the carboxyl group of 8arm-PEG–COOH and the hydroxyl group of PS (Fig. 1A and B).
|
| Fig. 1 Synthesis of 8arm-PEG–PS. | |
The chemical constructions of PS prodrugs were measured by 1H-NMR. Samples were dissolved in DMSO-d6 or D2O for analysis by 1H-NMR (Bruker DRX-600 Avance III spectrometer). Fig. 2 shows the 1H-NMR spectra of PS, 8arm-PEG20K–PS and 8arm-PEG40K–PS, where the signals at 6.22–7.82 attributed to the most characteristic peaks protons of PS, 3.50–3.67 (4nH, –(CH2CH2O)n–) and 3.88 (2H, –CH2OC(O)O–) attributed to the methylene protons of PEG. Due to the formation of ester bond, a proton peak δ 6.70 (1H, s) of PS moved to a′ δ 7.29 (1H, s), b proton peak δ 7.38 (1H, d) of PS moved to b′ δ 7.82 (1H, d).
|
| Fig. 2 1H-NMR spectra of PS (B), 8arm-PEG20K–PS (C) and 8arm-PEG40K–PS (D). They were solubilized in DMSO-d6 and D2O, respectively. | |
3.2. Drug-to-carrier ratios
The absorbance spectra of PS and 8arm-PEG–PS were measured with a UV-Vis spectrophotometer.36,37 The UV absorbance of free PS in methanol was determined at 306 nm for five kind of different concentrations ranging from 0.4 to 10 μg mL−1. From the standard plot of concentration vs. absorbance, the linear equation for PS was calculated (Fig. 3). The 8arm-PEG20K–PS and 8arm-PEG40K–PS were respectively diluted into 1.0 mg mL−1 and 3.0 mg mL−1, and then their UV absorbance at 306 nm was measured after pretreating solutions. Using this value, and employing the linear equation obtained from above, the concentration of PS in the sample was measured. Thus, the percentage of PS in the prodrugs was obtained using this value divided by the 8arm-PEG–PS concentration (Table 1). The mean mass ratio of drug-to-carrier for 8arm-PEG20K–PS, 8arm-PEG40K–PS was 7.06 ± 0.24 and 4.16 ± 0.20 and their molar ratio was 5.94 ± 0.21 and 6.78 ± 0.22, respectively. The mass/molar ratio indicates that approximately 1.22 ± 0.04 or 2.06 ± 0.09 functional groups of 8arm-PEG remained unconjugated.
|
| Fig. 3 Absorbance spectrum of PS and 8arm-PEG–PS prodrugs in UV-Vis buffer. Linear regression fit of PS standards to calculate the concentration for PS (n = 3 tests, 4 scans per test, R2 = 0.999). | |
Table 1 Drug carrier ratio, solubility and hydrolysis half-lives of PS prodrugs
Compound |
Drug carrier ratio |
Hydrolysis t1/2a (h) |
Mass (%) |
Molar |
Solubility (%T) |
pH 8.2 |
pH 7.4 |
pH 6.1 |
Based on the release of PS. |
PS |
|
|
0.65 ± 0.02 |
|
|
|
8arm-PEG40K–PS |
4.16 ± 0.20 |
6.78 ± 0.22 |
91.20 ± 2.85 |
19.5 ± 0.82 |
38.4 ± 1.98 |
157.8 ± 5.96 |
8arm-PEG20K–PS |
7.06 ± 0.24 |
5.94 ± 0.21 |
95.70 ± 2.96 |
24.5 ± 1.06 |
47.6 ± 2.14 |
189.5 ± 6.74 |
3.3. Stability study
To ensure the hydrolysis of the ester bonds would occur, the in vitro drug release of PS prodrugs were tested in PBS (pH 6.1, 7.4 and 8.2) at 37 °C.38 Hydrolysis studies demonstrated that the hydrolysis rate is strongly dependent on pH. As shown in Fig. 4A, the drug initial burst release of 8arm-PEG40K–PS was up to 73% and that of 8arm-PEG20K–PS was 66% at the first 40 h (pH 8.2). After the relative rapid release of PS in the early stage, the release rate slowed down greatly during the following long period, maintaining a slow and steady release of PS. By the end of 90 h, the cumulative release of PS was 94.24% and 89.35% for 8arm-PEG40K–PS and 8arm-PEG20K–PS, respectively. In addition, the hydrolysis rate of PS prodrugs at pH 7.4 and 6.1 were relatively stable compare to that of pH 8.2. The final release of 8arm-PEG40K–PS and 8arm-PEG20K–PS were 81.46% and 75.21% at pH 7.4 and that of pH 6.1 were 35.32% and 30.91%, respectively (Fig. 4B and C). The hydrolysis curves appeared in Fig. 4, and the half-lives for these curves are given in Table 1. Thus, we concluded that the two prodrugs were much more stable at pH 6.1 than at pH 7.4 or 8.2, and the stability trend was the same as the trend of hydrolysis rates for the two prodrugs (8arm-PEG20K–PS < 8arm-PEG40K–PS). The hydrolysis half-lives of the 8arm-PEG40K–PS increased 4-fold and 8-fold at pH 6.1 over pH 7.4 and pH 8.2 values, respectively.
|
| Fig. 4 Stability of 8arm-PEG–PS prodrugs in PBS at 37 °C. Experiments were done at pH 8.2 (A), at pH 7.4 (B), and at pH 6.1 (C) for 8arm-PEG20K–PS and 8arm-PEG40K–PS. Data was acquired by HPLC analysis. | |
3.4. Solubility study
Generally, PS has very poor solubility in aqueous solutions. Therefore, the use of PS may cause side effects due to the excipient, such as Cremophor EL.15,39,40 In this study, the polymeric prodrugs were prepared to overcome the poor solubility of PS in aqueous solutions, which resulted in the significant increase in the solubility of PS (Fig. 5). The concentration of each sample (PS, 8arm-PEG20K–PS and 8arm-PEG40K–PS) was based on the amount of PS (final PS concentration: 1 mg mL−1). The transmittances of PS, 8arm-PEG40K–PS and 8arm-PEG20K–PS were 0.65%, 91.20%, and 95.70% at 306 nm, respectively (Table 1). The results showed that the obtained polymeric prodrugs greatly improved the solubility compared with free PS.
|
| Fig. 5 Solubility photography of PS, 8arm-PEG20K–PS, and 8arm-PEG40K–PS against aqueous solution and transmittance was measured using UV. | |
3.5. In vitro cytotoxicity
Considering the growing safety concerns about the in vivo application of the prodrugs and evaluating whether the PS prodrugs were efficient carriers for anticancer drug delivery, the cytotoxicity of PS, 8arm-PEG and PS prodrugs were assessed using a CCK-8 assay. The LLC cells were treated with PS, 8arm-PEG and PS prodrugs at concentrations ranging from 1.0 to 40 μg mL−1 for 24 h, 48 h or 72 h, in which the concentration of 8arm-PEG and PS prodrugs were match to the PS. As shown in Fig. 6, the free PS exhibited high anti-cancer activity when the concentration up to 40 μg mL−1 and the in vitro anti-cancer efficiencies of both PS and PS prodrugs were time-dependent. Treatment of LLC cells with free PS, 8arm-PEG20K–PS and 8arm-PEG40K–PS, the viability of cells were 36.56%, 59.32% and 55.06% when the incubation time was 24 h, but there was 32.83%, 46.96% and 43.36% after 48 h, and it was only 29.26%, 34.03%, 30.09% when time up to 72 h. But the viability of cells treated by 8arm-PEG20K and 8arm-PEG40K were not found decreased obviously.
|
| Fig. 6 CCK-8 assay of PS, 8arm-PEG and 8arm-PEG–PS prodrugs with different concentration in LLC cell lines (A). Cell viability of LLC cells treated with 40 μg mL−1 of PS, 8arm-PEG and 8arm-PEG–PS (equivalent to free PS) was measured by CCK-8 assay (B). | |
To compare the potency of prodrugs, the concentrations of drug that killed 50% of the cells (IC50) were estimated from survival curves, including obtained from replicate experiments the curves shown in Fig. 6A. The IC50 of PS, 8arm-PEG40K–PS and 8arm-PEG20K–PS were 5.31, 6.69 and 7.92 μg mL−1, respectively (Table 2). Two prodrugs were sensitive to cells with the trend for the IC50 values for the samples remaining the same (8arm-PEG20K–PS > 8arm-PEG40K–PS > PS). These values are virtually equivalent to that for the free drug, indicating that PS is being released into the medium. 8arm-PEG20K–PS was slightly greater than that of 8arm-PEG40K–PS, which was related to that the slower release of 8arm-PEG20K–PS. Also, the IC50 values of prodrugs correlated with the hydrolytic stabilities of the compounds in PBS, indicating that more prodrugs were needed to kill an equivalent fraction of cells, because the prodrugs released PS over time whereas incubation with free drug resulted in a bolus dose. In vitro experiments of the tumor cell culture do not capture the advantages of PS prodrugs compared to free PS, such as improved pharmacokinetics, and hence may underestimate the efficacy of PS prodrugs.
Table 2 In vitro cytotoxicity of PS prodrugs (IC50, μg mL−1)
Compound |
LLC |
PS |
5.31 ± 0.135 |
8arm-PEG40K–PS |
6.69 ± 0.152 |
8arm-PEG20K–PS |
7.92 ± 0.216 |
3.6. In vivo anticancer activity assay
Inspired by the above results, we moved on to explore the antitumor efficacy of different PS formulations. The tumor growth rate was examined after an intravascular injection (i.v. injection) of PBS, PS and polymeric drugs into the LLC tumor-bearing mice. It is vital to noted that the concentration of PS prodrugs were match to the PS. During the treatment period, although all the groups showed tumor inhibition compared with the PBS-treated group, the tumor growth rates of mice treated with PS was faster than PS prodrugs (Fig. 7D). Moreover, for the two PS prodrugs, 8arm-PEG40K–PS treatment exhibited higher tumor inhibition than 8arm-PEG20K–PS (Fig. 7A). As shown in Fig. 7C, the groups treated with PS and different PS prodrugs showed varied levels of survival time and they were ranked as 8arm-PEG40K–PS > 8arm-PEG20K–PS > PS, which was consistent with the results of tumor growth inhibition. The treatment with 8arm-PEG40K–PS and 8arm-PEG20K–PS resulted in 84.2%, 74.4% TGI (day 20) and 83.3%, 50.0% survival of animals (day 26), respectively. In contrast, the free PS treatment resulted in 35.3% TGI (day 20) and 16.7% survival of animals (day 26) (Fig. 7A and C, Table 3). Importantly, in line with the literature, no signs of systemic toxicity were observed by monitoring general behavior, appetite and mice body weight (Fig. 7B).
|
| Fig. 7 Antitumor efficacies of PS and different PS prodrugs in the subcutaneous mouse model of LLC. Tumor volumes of mice during treatment with PS and different PS prodrugs (A). Body weight after treatment (B). Survival of mice in different treatment groups (C). Tumor images of different groups after treatment on day 20 (D). | |
Table 3 LLC xenograft model (10 mg kg−1) efficacy comparison
Compound |
Mean TV ± SDa (mm3) |
RTVa |
TGIa (%) |
Curesb (%) |
Mean tumor volume (TV), RTV, and %TGI data were taken on day 20 (by day 20, a significant percentage of control animals were euthanized due to excess tumor burden). %cures were taken on day 26. |
Control |
4850 ± 1400 |
38.5 ± 17.2 |
0 |
0 |
PS |
3137 ± 1290 |
24.9 ± 11.2 |
35.3 ± 7.8 |
16.7 |
8arm-PEG20K–PS |
1240 ± 402 |
10.0 ± 3.2 |
74.4 ± 18.2 |
50.0 |
8arm-PEG40K–PS |
808 ± 295 |
6.4 ± 3.1 |
84.2 ± 20.2 |
83.3 |
3.7. Evaluation of hypersensitivity
Although PS prodrugs had shown significant therapeutic effects in vivo, the side effect level was still unclear. During the early development of the drugs, type I hypersensitivity is the most common type of the hypersensitivity reaction.41 Some of the natural anticancer drugs, such as paclitaxel, docetaxel, and teniposide cyclosporine, were usually associated with a high incidence of type I hypersensitivity reaction. It has been demonstrated that IgE antibodies play an important role in mediating type I hypersensitivity responses. We thus selected IgE levels as the parameter for rapid evaluation of type I hypersensitivity reactions. The blood IgE levels of mice in different groups (PS, 8arm-PEG20K–PS, and 8arm-PEG40K–PS) are shown in Fig. 8A. The IgE level of mice treated with PS was significantly higher than that of the PBS group, which might be ascribed to the bad water solubility. As expected, no significant change of IgE level was observed in the 8arm-PEG20K–PS or 8arm-PEG40K–PS groups, which suggested that the use of PS prodrugs could reduce the risk of hypersensitivity reactions substantially. The blood of mice was also collected to analyze the blood cell counts, which are often used as the indicator of hematotoxicity. The total WBC count of mice treated with free PS showed a little decrease over the normal group. No discernible decrease in the WBC number of the mice treated with PS prodrugs were observed (Fig. 8B), which indicated that the PS prodrugs designed in this study could avoid severe hematotoxicity.
|
| Fig. 8 Subacute toxicities of different groups were reflected by IgE levels (A) and the WBC change of mice (B). | |
4. Conclusions
In this study, we have designed and successfully synthesized 8arm-PEG–PS prodrugs using 8arm-PEG linkers that enable significantly increased drug binding capacity, water solubility, and a significantly enhanced therapeutic index in lung xenograft models. The hydrolysis studies demonstrated that the PS molecule could be released from 8arm-PEG–PS prodrugs and the hydrolysis rate is strongly dependent on pH. In addition, the WBC count in the mice, as an indication of hematopoietic toxicity, was not affected after i.v. PS-treatment. Together, these results indicate that PS prodrugs have the potential to slow the outgrowth of tumors from lung carcinomas, thereby prolonging life, without inducing systemic adverse effects. Compared with 8arm-PEG20K–PEG, 8arm-PEG40K–PEG retained the biological activity well in vitro and showed higher tumor inhibition capacity.
Acknowledgements
This work was supported by the China State Forestry Administration 948 Project (no. 2014-4-35), Beijing Natural Science Foundation of China (Grant no. 2142024), and the National Nature Science Foundation of China (no. 20976179).
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