Self-Assembled Pectin-Conjugated Eight-Arm Polyethylene Glycol

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Self-Assembled Pectin-Conjugated Eight-Arm Polyethylene Glycol− Dihydroartemisinin Nanoparticles for Anticancer Combination Therapy Yanxue Liu, Qi Qi, Xiaomin Li, Jing Liu, Luying Wang, Jing He, and Jiandu Lei* Beijing Key Laboratory of Lignocellulosic Chemistry, and MOE Engineering Research Center of Forestry Biomass Materials and Bioenergy, Beijing Forestry University, No. 35 Tsinghua East Road, Haidian District, Beijing, 100083, China ABSTRACT: Most individual anticancer drugs have poor water-solubilities and difficulties in controlling drug release, which significantly limit their clinical applications for cancer treatment. In order to solve these problems, this study reports a novel synthesis method for encapsulating hydroxycamptothecin (HCPT) into pectin−(eight-arm polyethylene glycol)− dihydroartemisinin nanoparticle (PPD) conjugates to form self-assembled PPDH nanoparticles (PPDH NPs). The prepared PPDH NPs contained hydrophilic carriers of pectin and eight-arm polyethylene glycol and the hydrophobic anticancer drugs of dihydroartemisinin and hydroxycamptothecin, for stabilizing particle size, improving water solubility, and achieving drug controlled release. The results indicated that the obtained nanoparticles possessed appropriate size (∼84 nm), drug-loaded efficiency (∼9.13 wt % DHA), encapsulation efficiency (∼12.03 wt % HCPT), and good stability and were pH-dependent. The time-dependent cytotoxicity tests of the PPDH NPs showed that only 4% 4T1 cell and 2% MCF-1 cell survived after 72 h. The PPDH NPs exhibited a higher cytotoxicity, longer blood retention time of free drug (8.0-fold DHA, 7.4-fold HCPT), and more effective cellular uptake. 4T1 tumor-bearing mice treated with the nanoparticles also showed a considerable survival advantage (90.6%) in comparison to those with the free DHA (15.5%) as well as the free HCPT (14.1%). Moreover, it is clearly an elaborate certification that PPDH NPs could reduce the risk of hypersensitivity reactions substantially. Therefore, PPDH NPs have a promising potential for anticancer combination therapy. KEYWORDS: petctin, Eight-arm polyethylene glycol, Dihydroartemisinin, 10-Hydroxycamptothecin, Self-assembled, Nanoparticle

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INTRODUCTION Great progress in the history of medicine has taken place in oncology during the last two to three decades, and these advances have improved survival rates and even cured some certain tumors during recent years.1 Nevertheless, cancer remains the second leading cause of death globally, and its overall incidence continues to rise.2 According to the World Health Organization as of May 30, 2017, cancer caused 7.9 million deaths in 2007 which accounted for 13% of all deaths and 8.2 million deaths worldwide in 2012. The annual number of new cases is projected to rise from 14.1 million in 2012 to 21.6 million in 2030. Around 75% of cancer deaths occur in low- and middle-income countries, where the number of cancer cases is rising most rapidly.3,4 Therefore, more challenges are faced for cancer treatment including relatively safe, combination, and targeted therapies.2,5 At present, a variety of drug carriers’ nanoparticles have been developed to improve the safety of drugs. Of all these drug-carriers’ systems, nanoparticle carriers are applied to ameliorate the bioavailability of drugs. Generally, the existence of abnormal tissue in the tumor could cause extensive leakage of macromolecules,6−10 which contributes to longer retention of macromolecules from the interstitial space of the tumor than normal vessels, namely the enhanced permeability and retention (EPR) effect.11−14 Polymeric nanoparticles ( 1 shows an antagonistic effect. Cellular Uptake Study. Cellular uptake and distribution of HCPT in the PPD NPs and PPDH NPs were observed by confocal laser scanning microscopy (CLSM, TCS SP5, Leica). After 4T1 cells achieved about 80% confluency, the cells were trypsinized to cultivated onto culture slides (BD Falcon, Bedford, MA) with a density of 1.0 × 105 cell mL−1. A 1 mL portion of cell suspension was separated into 4 cm2 confocal Petri dishes at 37 °C overnight. After 24 h of incubation, a certain concentration of HCPT (IC50) and embedded HCPT (IC50) was added along the inner wall, and incubated at 37 °C for 4 h. The liquid of the drug was removed, the cells were washed four times repeatedly, and then the cells were fixed with 4% formaldehyde solution for 15 min before formaldehyde was aspirated. A 1 mL portion of 0.5 μg mL−1 DAPI solution was added for 5 min and, then, sucked out. The cells were washed many times with PBS until removing excess stains. Nanoparticles were detected by the absorbance of HCPT at 488 nm. 8100

DOI: 10.1021/acssuschemeng.7b01715 ACS Sustainable Chem. Eng. 2017, 5, 8097−8107

Research Article

ACS Sustainable Chemistry & Engineering

Figure 3. 1H NMR spectra of DHA in CDCl3, pectin in D2O, 8arm-PEG-DHA in D2O, and pectin-8arm-PEG-DHA in D2O.

%TGI = [(C − T )/C ] × 100%

(4)

Table 1. Particle Size and Drug-Loading Efficiency of Different Nanoparticles

where C is the mean tumor volume of the control group and T is the mean tumor volume of the treatment group. Detection of Allergic Reaction. Determination of allergic reaction is particularly important to protect the current chemotherapeutical drugs from toxic side-effects. Five groups (control, DHA, HCPT, PPD NPs, and PPDH NPs) of tumor-bearing mice (n = 6) were used for allergy testing studies. Mice were administered with five samples via tail intravenous injection every 2 days (DHA 7 mg kg−1; HCPT 6 mg kg−1; PPD NPs and PPDH NPs with equal DHA concentration). After 10 days, five different groups of mice blood were collected and centrifuged, and serum samples were analyzed by the procedure of Mouse IgE ELISA. Statistical Analysis. All experiments in this work were repeated three times, and data statistics was performed by variance analysis (ANOVA) by the usage of GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). All graphical data is reported as mean ± standard deviation (SD). Significance levels were set at * p < 0.05.

compound

size (nm)

DLE of DHA (wt %)

DLE of HCPT (wt %)

PDD NPs PDDH NPs

83.17 ± 5.66 91.33 ± 7.25

8.58 ± 0.55 7.33 ± 0.39

11.41 ± 0.62

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RESULTS AND DISCUSSION Preparation of DHA Conjugates and Control of Drug Loading. 8arm-PEG10K-DHA and Pet-8arm-PEG10K-DHA

Figure 5. Particle size distribution of PPDH NPs with different amounts of DHA weight percent, (a) 20, (b) 40, (c) 60, and (d) 70%, and a certain amount of HCPT.

conjugates were synthesized by the scheme of Figure 2. Figure 3 shows the 1H NMR spectra of pectin, DHA, 8arm-PEG10KDHA, and Pet-8arm-PEG10K-DHA,44 where the signals at δ 0.8−2.9 are all attributed to the most characteristic peak protons of DHA (CDCl3) and those at δ 3.2−4.0 are the characteristic peak of pectin (D2O). Peaks at δ 3.50−3.85 (4nH, −(CH2CH2O)n−)) and δ 4.20 (2H, −CH2OC(O)O−) are attributed to the methylene protons of PEG.45,46 The pectin

Figure 4. Optimization of PPDH NPs formulation. (a) DHA was fed into the reactions at different ratios. The pectin-8arm-PEG-DHA conjugates were characterized for the mass fraction of DHA. (b) PPD conjugates formed nanoparticles when they contained a defined range of DHA contents. Conjugates with compositions outside this range formed particles, or precipitated. 8101

DOI: 10.1021/acssuschemeng.7b01715 ACS Sustainable Chem. Eng. 2017, 5, 8097−8107

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ACS Sustainable Chemistry & Engineering Table 2. In Vitro Cytotoxicity Analysisa compound DHA HCPT PPD NPs PPDH NPs a

4T1 5.97 0.28 2.03 0.05

(0.46155) (0.02977) (0.17220) (0.00173)

MCF-1 7.04 0.37 3.48 0.07

(0.5189) (0.03831) (0.31914) (0.00265)

IC50, μg mL−1.

formation of ester bonds in 8arm-PEG5K-DHA failed to find due to the insoluble of DHA. The relative weight percent of 8arm-PEG10K-DHA and Pet8arm-PEG10K-DHA conjugates was obtained by UV. Pet-8armPEG10K-DHA conjugate was prepared with a range of 8armPEG10K-DHA feeds (10, 20, 30, 40, 50, 60, 80, and 90 wt %). As showed in Figure 4a and Table 1, the weight percent for two kinds of complexes increased with increasing DHA% concentrations. The drug-loaded rate was 9.13% in the Pet8arm-PEG10K-DHA conjugate. In addition, derivatives were prepared across the 5, 10, 20, 30, 40, 50, 60, 80, and 90 wt % feed, the corresponding particle sizes were tested in Figure 4b. The particle sizes range from 136 to 84 nm, which means a better choice of 50−80% for DHA weight percent feeds; the larger nanoparticles easily clear the RES, and the smaller nanoobjects prolong the retention time in the bloodstream.47,48 In particular, nanoparticle diameters less than 100 nm have been revealed to be an ideal size for cancer therapy, due to the favorable biodistribution, clearance, and accumulation behavior.49−51 Stability of PPDH Nanoparticles. In our study, HCPT as a kind of water-insoluble drug was encapsulated into the PPDH nanoparticle and formed the core in preparation of selfassemble (Table 1). Distribution of particle size for PPDH nanoparticles was evaluated by self-assembled with different amounts of DHA wt % (20, 40, 60, 70%) and a certain amount of HCPT. As is shown in Figure 5a−d, PPDH nanoparticles had a good dispersibility and homogeneity. When nanoparticles were loaded with HCPT, the particle sizes of PPDH NP were increased due to the insertion of the hydrophobic drug into the nanoparticles and the increased drug loading of DHA. The variety of particle size of the nanoparticles was observed in an interval of 2 days (Figure 6a). A surprising discovery was that the stability of the PPDH nanoparticles showed a smooth trend that was better than PPD nanoparticles, which indicated an increased hydrophobic performance of HCPT. Hemolysis Study. In order to research the influence of PPD NPs and PPDH NPs for drug delivery in blood circulation and avoid allergic reactions due to injection, we need to consider and determine the blood transmission of the drug. Erythrocytes were incubated with nanoparticles by two concentrations of 1 and 0.1 mg mL−1 at 37 °C for 1 h. Hemolysis was determined by detecting the amount of hemoglobin released in the supernatant at 541 nm. 1% Triton X-100 was chosen as a positive control, which could induce full hemoglobin release. As seen from Figure 6b, the drug with concentrations of 1 and 0.1 mg mL−1 showed a similar hemoglobin release to blank values ( PPD NPs > HCPT > PPDH NPs (Table 2). The cytotoxicity of PPD NPs was more than 2.94 and 2.02 times free DHA in 4T1 and MCF-1 cells, and the cytotoxicity of PPDH NPs was more than 119.40 and 100.57 times DHA in 4T1 and MCF-1 cells, respectively. Compared to the free HCPT, PPDH NPs were more than 5.61 and 5.29 times HCPT in 4T1 and MCF-1 cells. It is clear to reveal that PPD NPs and PPDH NPs were more effective than the free drug in vitro. Moreover, the increased PPDH NPs toxicity was different between 4T1 and MCF-1 cells which may be due to the chemosensitivity of each cell line and drugs’ slow release. In addition, polymers internalization and strong release in lysosomes may further enhance drugs’ efficacies. In the PPDH NPs, IC50 values in 4T1 and MCF-1 were 0.05 and 0.07 μg mL−1, and the calculated combination indices (CIs) of DHA and HCPT in the PPDH NPs was 0.04, which suggested that PPDH NPs had achieved the significant synergistic effect by codelivery of DHA and HCPT. Cellular Uptake of PPDH NPs. To provide evidence of cell compatibility and evaluate the drug delivery efficiency of nanoparticles, 4T1 cells were incubated with HCPT and PPDH nanoparticles for 4 h. In order to compare the cellular uptake, the concentrations of free HCPT and HCPT in PPDH NPs were the same as IC50, respectively. The fluorescence of HCPT (green) and DAPI (blue) can be visualized by confocal microscopy. PPDH NPs (green fluorescence) was more

Figure 10. Blood circulation curves and half-time of PPDH NPs compared with free DHA (a), and PPDH NPs compared with free HCPT (b). Error bars were based on six mice per group at each time point.

of 4.5, 7.4, and 8.0 can simulate biological fluids to investigate release kinetics of DHA and HCPT by HPLC analysis in vitro. As expected, DHA at pH = 7.4 or 8.0 was very slowly released without the phenomenon of burst release in PPDH nanoparticle (Figure 7a and b). However, PPDH nanoparticle at pH = 4.5 released DHA faster than that at pH = 7.4 or 8.0, which attributed to the swelled effect of pectin at a certain pH value.52 Therefore, the release of DHA were obviously pH-dependent, presenting a similar increment tendency to release time. Esterase was added to promote HCPT release because of the hydrolysis of PPDH nanoparticle (Figure 7b). The PPDH nanoparticle could be as a prodrug for DHA and HCPT intracellular release. The pH-responsive DHA and HCPT release data are shown in Figure 7a and Table 1. Cytotoxicity Evaluation of PPDH Nanoparticle in Vitro. 4T1 is a 6-thioglycine resistant cell line that has been screened from 410.4 tumor strain without mutagenesis and is an animal model of human breast cancer in stage VI. The growth and metastasis of 4T1 cells in BALB/c mice is very similar to that in breast cancer. The kinetics of 4T1-induced tumors are similar in both postoperative and nonsurgical conditions, which can be used as a postoperative and nonsurgical model. In contrast to other breast tumor models, tiny metastatic cell masses (less than one) can also be detected in many distal organs due to the 4T1 anti-6-thioglycine properties. Thus, 4T1 is a representative cell line in breast cancer. In vitro cytotoxicity should be considered to guarantee the effectiveness of the nanoparticles before entry into the 8103

DOI: 10.1021/acssuschemeng.7b01715 ACS Sustainable Chem. Eng. 2017, 5, 8097−8107

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Figure 11. In vivo antitumor activity of free DHA, free HCPT, and nanoparticles in the subcutaneous mouse model of 4T1. (a) Tumor volumes of mice during treatment with different groups. (b) Survival of mice in different treatments. (c) Tumor photographs from each treatment group excised on day 20.

Table 3. 4T1 Xenograft Model (q2d × 5): Efficacy Comparison compound control DHA HCPT PPD NPs PPDH NPs a b

mean TV ± SDa (mm3) 5587 ± 1624 3234 ± 1074 3599 ± 1214 774 ± 210 330 ± 188

RTVa

TGIa (%)

curesb (%)

± ± ± ± ±

0 38.7 33.0 90.3 95.0

0 15.5 14.1 75.9 90.6

42.0 27.1 27.9 8.0 4.0

13.7 13.1 12.3 1.1 1.0

concentration in the cytoplasm region. The results also demonstrated that PPDH NPs increased green fluorescence which were densely located around the cytoplasm region near the cell membrane and primarily located on the surface through visualization. Pharmacokinetics Experiment. The pharmacokinetics study was undertaken by intravenous administrations of HCPT, DHA, PPD NPs, and PPDH NPs injection to 4T1bearing mice. The results displayed in Figure 10, showed that HCPT and DHA concentrations in plasma gradually declined with time for PPD NPs and PPDH NPs injection by intravenous administrations. Obviously, HCPT and DHA of PPDH NPs are retained at a higher concentration in the plasma up to 40 and 70 h, respectively. Free DHA and free HCPT in the blood circulation after intravenous administration of injection was disappeared rapidly with the plasma concentration below 9%. However, PPDH NPs exhibited prolonged clearance with the DHA and HCPT levels of 19.0%, 18.1% ID

Mean tumor volume (TV), RTV, and TGI date were taken at day 23. Percent cures were taken at day 28.

effectively delivered than free HCPT which was almost no attachment to the cell surface in 4T1 cells (Figure 9). HCPTloaded nanoparticle has been known for taking up through an endocytic pathway of cells, thereby escaping from the effect of P-glycoprotein. HCPT in nanoparticles could maintains a high

Figure 12. (a) Animal weights recorded once per week and expressed over the 20-day observation. (b) IgE levels of mice treated with free DHA, free HCPT, and nanoparticles for 30 min. Data as mean ± SD; n = 6. (c) White blood cell (WBC) changes during four administrations in normal mice with free DHA, free HCPT, and nanoparticles. The blood sample was collected from mice on day 2 after the last dosage treatment. 8104

DOI: 10.1021/acssuschemeng.7b01715 ACS Sustainable Chem. Eng. 2017, 5, 8097−8107

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ACS Sustainable Chemistry & Engineering g−1 at 24 h after administration. PPD NPs and PPDH NPs could prolong the blood circulation half-time of DHA from 1.0 to 5.8 and 8.0 h, respectively, which were far longer (5.8- and 8.0-fold compared with free DHA) than values of DHA. PPDH nanoparticles could prolong the blood circulation half-time of HCPT from 0.5 to 3.7 h. In Vivo Anticancer Activity Studies of PPDH NPs. For humane care, animals were killed after the tumor volume reached 5000 mm3 or over the time of experiment (>6 weeks). The drug delivery efficacies of nanoparticles were considered for equivalent doses of 10 mg kg−1 DHA and 10 mg kg−1 HCPT, respectively. In Figure 11, 4T1 tumor-bearing mice were utilized to test the in vivo anticancer activity of PPDH NPs. After 40 days of treatment, a significant difference in the mice tumor volumes of the experimental groups could be seen in Figure 11a. 4T1 tumor-bearing mice treated with the nanoparticles showed a considerable survival advantage in comparison with the free DHA and free HCPT. Antitumor capacity of DHA, HCPT, and nanoparticles was PPDH NPs > PPD NPs > free DHA and free HCPT. The survival rates and TGI of PPDH NPs were 90.6% (28 days) and 95.0% (23 days), while DHA was 15.5% (28 days) and 38.7% (23 days), and HCPT was 14.1% (28 days) and 33.0% (23 days) (Figures 11a−c, and Table 3). It suggested that the tumor volumes in the PPDH NPs-treated group were much smaller than those treated with DHA and HCPT injection. These findings coincided with the foregoing results of in vitro evaluations. Throughout the experiment, the mice’s average body weights were no obvious changes in all treated mice (Figure 12a), which suggested the nanoparticle as a drug was safe at such a dose. Generally, the increased tumor volume has an effect on the weight of mice. However, although the tumor volume changes, the effect on body weight is not significant due to the tumor volume of mice is much smaller than that of mice. Therefore, the prepared PPDH NPs is significantly inhibition of the tumor with small changes in body weight.54,55 Evaluation of the Side Effects. Parameter IgE levels (DHA, HCPT, PPD NPs, and PPDH NPs) were selected for rapid evaluation of type I hypersensitivity reactions. From Figures 12b, c, we can see that 4T1 tumor-bearing mice treated with DHA and HCPT displayed a higher IgE level than the control group, and no significant change in the PPD NPs and PPDH NPs groups. This may be attributed to the bad water solubility of DHA and HCPT; meanwhile, it is clearly an elaborate certification that nanoparticles could decrease the hazard of hypersensitivity reactions substantially. The mice blood was collected to test the WBC count after treatment with different formulations, which is often used as an indicator of hematologic toxicity. No discernible decreases in the WBC number of the mice treated with the PPD NPs and PPDH NPs groups were observed, indicating that the nanoparticles designed in this study could avoid severe hematologic toxicity.

In vitro assays demonstrated that the dual-functionalized nanoparticles exhibited enhanced cellular uptake, strong cell apoptosis-induction and cell-viability inhibition ability in 4T1 cells and MCF-1 cells. Confocal microscopy analysis showed that nanoparticles could actively capture 4T1 cells. In addition, drug delivery systems of PPDH nanoparticles remarkably inhibited tumor growth by eliminating bulk tumor cells in a 4T1 orthotopic tumor murine model and realized a satisfactory effect. In this study, we developed an alternative technology of Pet-multiarm-PEG conjugate, which can also be applied to other insoluble anticancer drugs, but the development of other functional groups on this conjugate remains to be resolved.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +8610-62337251. Tel.: +8610-62337251. ORCID

Jiandu Lei: 0000-0002-1432-0588 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (No. 21576029; 21406013) and the Sichuan Science and Technology Department (No. 2017JY0139).

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CONCLUSIONS Our study highly indicates that combination therapy for eliminating bulk tumor cells could achieve a synergistic antitumor effect and may be an attractive strategy for breast cancer treatment. A satisfactory size (∼84 nm) was obtained from amphiphilic PPDH nanoparticles platform to codelivery insoluble anticancer drug DHA and HCPT for favorable biodistribution and clearance/accumulation behavior. Biodegradable polymer nanoparticles showed 9.13 wt % drug-loaded rate of DHA and 12.03 wt % encapsulation efficiency of HCPT. 8105

DOI: 10.1021/acssuschemeng.7b01715 ACS Sustainable Chem. Eng. 2017, 5, 8097−8107

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DOI: 10.1021/acssuschemeng.7b01715 ACS Sustainable Chem. Eng. 2017, 5, 8097−8107

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DOI: 10.1021/acssuschemeng.7b01715 ACS Sustainable Chem. Eng. 2017, 5, 8097−8107