Effect of alkyl chain on cellular uptake and antitumor activity of hydroxycamptothecin nanoparticles based on amphiphilic linear molecules

European Journal of Pharmaceutical Sciences 124 (2018) 266–272

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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Effect of alkyl chain on cellular uptake and antitumor activity of hydroxycamptothecin nanoparticles based on amphiphilic linear molecules ⁎

Yifei Guo, Ting Wang, Shuang Zhao, Hanhong Qiu, Meihua Han, Zhengqi Dong , Xiangtao Wang

T

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Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing 100193, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Linear amphiphilic molecules Alkyl chain effect Antitumor efficacy Biodistribution

Drug-loaded nanoparticles utilizing amphiphilic molecules as nanocarriers were developed broadly for nanoscale drug delivery system. Linear amphiphilic molecule (PEG45C18) based on PEG and alkyl chain was designed and synthesized. To study the influence of alkyl chain on antitumor activity, 10-hydroxycamptothecin (HCPT) was selected as the hydrophobic drug, amphiphilic molecule (PEG45C18) and hydrophilic PEG (PEG45) were applied as nanocarriers to form HCPT-loaded nanoparticles (HCPT/PEG45C18 NPs and HCPT/PEG45 NPs). These two nanoparticles presented high drug-loading content, stability, but different release manner and antitumor efficacy. The HCPT/PEG45C18 NPs existed slower release manner but higher antitumor activity than HCPT/PEG45 NPs, IC50 value was decreased approximately 8.5-fold against 4T1 cells in vitro. Moreover, the antitumor efficacy of HCPT/PEG45C18 NPs on 4T1-bearing mice was promoted significantly, the inhibition rate based on average tumor weight was 1.5-fold higher than HCPT/PEG45 NPs, besides, HCPT/PEG45C18 NPs exhibited better tumor accumulation than HCPT/PEG45 NPs. These results suggested alkyl chain affect the antitumor activity significantly due to nanoparticles decorated with alkyl chains existing higher endocytosis efficacy to cells. According to the enhanced antitumor efficacy, it was suggested that HCPT/PEG45C18 NPs showed the potential application for cancer therapy in clinic, and alkyl chains should be considered for designing biomaterials.

1. Introduction Nanoscale drug delivery systems (NDDSs) are emerging as the potential and effective method for cancer therapy (Bourzac, 2012; Kamaly et al., 2012; Sobot et al., 2016), which could enhance the solubility and anticancer efficacy, reduce the resistance and unfavorable side effects (Farokhzad and Langer, 2006; Patra et al., 2013), on account of the special enhanced permeability and retention (EPR) effects in tumor tissue (Barreto et al., 2011). Hydrophobic drugs have been developed to be entrapped in various nanocarriers (Shi et al., 2010; Devadasu et al., 2013; Park, 2013; Tibbitt et al., 2016), amphiphilic copolymers are generally utilized as nanocarriers to form NDDS based on their selfassembly behavior in aqueous solution (Discher and Kamien, 2004; Zhu et al., 2017). Although these NDDSs present remarkable merits, there still left several problems, including low drug-loading content (almost < 20%) and possible toxicity from nanocarriers, which could induce unsatisfied antitumor efficacy. Therefore, the nanocarriers should be optimized furthermore. When preparing these drug-loaded nanoparticles, the

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component of the nanocarriers should be considered carefully. The appropriate nanocarriers should exhibit several advantages, such as high drug loading capacity, well-controlled release, good cellular uptake efficacy, stimuli-responsive property, and biosafety, which could induce the better bioavailability and antitumor activity (Mura et al., 2013; Daglar et al., 2014). Poly(ethylene glycol) as hydrophilic portion have been widely applied in anticancer drug carriers (Gou et al., 2013; Yue et al., 2013; Tao et al., 2014; Wang et al., 2015; Zhang et al., 2015), owing to their excellent aqueous solubility and biosafety (Joralemon et al., 2010; Nicolas et al., 2013), which have been approved by FDA to be used in clinic (Alconcel et al., 2011). Besides, PEGylated nanoparticles show the stealth property, resulting in nanoparticles could avoid uptake by the mononuclear phagocytes system (Bazile et al., 1995; Langer and Tirrell, 2004). It is reported that the membrane proteins could interact with alkyl chains (Wolfrum et al., 2007; Verma et al., 2013; Ho et al., 2017), inducing alkyl-decorating nanoparticles present higher endocytosis efficacy to mammalian cells, therefore, alkyl chains are utilized broadly in molecular imaging (Feng et al., 2016; Yan et al., 2016;

Corresponding authors. E-mail addresses: ff[email protected] (Y. Guo), [email protected] (Z. Dong), [email protected] (X. Wang).

https://doi.org/10.1016/j.ejps.2018.08.043 Received 29 May 2018; Received in revised form 18 August 2018; Accepted 31 August 2018 Available online 04 September 2018 0928-0987/ © 2018 Published by Elsevier B.V.

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mixed solution was placed into dialysis membrane (MWCO 14000) against deionized water (4 × 1 L) for 4 h, the dialysis medium was changed every hour, then HCPT nanoparticles were obtained as opalescent solution. The actual concentration of HCPT in nanoparticles was measured by UV-HPLC (UltiMate3000, DIONEX) at 384 nm on Thermo C18 column with acetonitrile:water containing 0.1% acetic acid (25:75, v/v) as eluent. The drug-loading content (DLC) was calculated as follows (n = 3).

Chen et al., 2017), gene delivery (Leonard et al., 2004; Ardana et al., 2015; Márquez-Miranda et al., 2016), and drug delivery (Damgé et al., 1988; Doadrio et al., 2006; Xiao et al., 2015). Based on their prominent merits, amphiphilic molecules from alkyl chains decorated with PEG could be possible utilized as potential effective and biocompatible nanocarriers. Combine the good biocompatibility of PEG and alkyl chains, in this study, amphiphilic molecule PEG45C18 from linear PEG (Mn = 2000, PEG45) and octadecylamine (C18H36-NH2) were synthesized and utilized as nanocarriers, 10-hydroxycamptothecin (HCPT) was utilized to prepare HCPT-loaded nanoparticles (HCPT/PEG45C18 NPs) by solvent exchange method. The particle size, morphology, stability, and release profile were studied. The antitumor activity, biodistribution, and systemic toxicity were evaluated at the same time. Besides, it was desirable to illustrate the effect of alkyl chain on antitumor efficacy and biodistribution, the HCPT nanoparticles (HCPT/PEG45) based on hydrophilic linear PEG45 was prepared and their relative properties were researched meanwhile.

DLC = (weight of loaded drug/weight of NPs) × 100% 2.5. Particle diameter The mean diameter, particle size distribution, and zeta potential were detected by dynamic light scattering (DLS) using Zetasizer NanoZS analyzer (Malvern Instruments, UK), which used the integrated 4 mV He-Ne laser (λ = 633 nm) and the backscattering detection (scattering angle θ = 173°) at room temperature. The measurement was performed with the HCPT NPs concentration of 1 mg mL−1 (n = 3).

2. Materials and methods 2.6. Transmission electron microscope 2.1. Materials The morphology of PEG45C18 and PEG45 nanoparticles in aqueous solution were detected by transmission electron microscope (TEM) measurements via negative dyeing method, performing on JEM-1400 (JEOL, Japan) at an accelerating voltage of 80 kV. A drop of samples (100 μg mL−1) was placed into carbon-coated copper grids and air drying at room temperature, then the samples were stained with 2% w/ v uranyl acetate solution for 2 min.

PEG45, PEG45NHS were purchased from Ruixi Biological Technology Co., Ltd. (Xian, China). Octadecylamine (purity > 97%) was purchased from Sigma-Aldrich Chemicals, Germany. Hydroxycamptothecin (HCPT, purity > 98%) was purchased from Melone pharmaceutical Co., Ltd. (Dalian, China). HCPT injection was obtained from Shenzhen Main Luck Pharmaceuticals Inc. (Shenzhen, China). Dialysis membrane (MWCO 14000) was purchased from SpectraPor (USA). Other reagents and solvents were purchased and used without further purification.

2.7. Scanning electron microscopy The morphology of HCPT NPs were investigated using scanning electron microscopy (SEM, S-4800, Hitachi Limited, Japan). A drop of HCPT NPs solution (100 μg mL−1) was placed on matrix and air-dried. After sputter-coating with Au/Pd for 1 min, samples were observed at 30 mV accelerating potential.

2.2. Cell line and animals The murine breast cancer (4T1 cell line) was purchased from National Infrastructure of Cell Line Resource (Beijing, China) and incubated in RPMI-1640 medium, 10% fetal bovine serum, 100 units mL−1 penicillin G, and streptomycin with 5% CO2 atmosphere at 37 °C (Guo et al., 2018). BALB/c mice (20 ± 2 g) were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and raised according to the normal procedure for 1 week prior to experimentation (Guo et al., 2017). All experimental procedures were performed in line with the Guidelines and Policies for Ethical and Regulatory for Animal Experiments and approved by the Animal Ethics Committee of Peking Union Medical College (Beijing, China).

2.8. Critical aggregation concentration The critical aggregation concentration (CAC) of the compounds PEG45C18 was estimated by pyrene probe method. Pyrene solution in acetone were added to each Eppendorf tube (6.0 × 10−7 mol), then acetone was evaporated. Aqueous sample solutions with different concentration ranging from 1.0 × 10−4 to 2.50 mg mL−1 were added into each tubes. The mixtures were ultrasonicated for 2 min and then stirred at room temperature for 12 h. The spectroscopy measurements were conducted at an excitation wavelength of 334 nm.

2.3. Synthesis of amphiphilic molecules PEG45C18 2.9. Stability study PEG45C18 was synthesized via the active ester route (Guo et al., 2009). Briefly, PEG45NHS active ester (0.50 g, 0.25 μmol), octadecylamine (0.20 g, 0.80 μmol), triethylamine (0.20 g, 2.40 μmol), and N, Ndimethylaminopyridine (DMAP, 10 mg) were added into dichloromethane (20 mL) at −5 °C. After continuous stirring for 24 h and evaporation, the crude product was purified via column chromatography (DCM/MeOH, 20/1), the colorless PEG45C18 was obtained (0.50 g, 75%).

The particle sizes of HCPT NPs after incubating with several media at 37 °C were recorded to estimate the medium stability, including PBS solution, normal saline, glucose solution (5%, wt%), and plasma. At predetermined time intervals, hydrodynamic diameter of nanoparticles was measured by DLS separately (n = 3). 2.10. Release kinetics Dialysis strategy was developed to study the release characteristics of HCPT NPs (Hong et al., 2010). Briefly, sample solutions (HCPT equivalent concentration, 2 mg) were kept in a dialysis tube with 14,000 M weight cutoff, then immersed in PBS solution containing 0.5% SDS (50 mL, pH = 7.4) at 37 °C. At predetermined time intervals, 5 mL external medium was withdrawn, then 5 mL fresh medium was added. The drug release study was performed for 7 days in triplicates

2.4. Preparation of HCPT-loaded nanoparticles Solvent exchange method augmented by ultrasonication was explored to prepare HCPT-loaded nanoparticles (HCPT NPs) (Guo et al., 2017; Guo et al., 2018). Briefly, 8 mg HCPT and 4 mg nanocarriers were dissolved in 1 mL DMF to form organic solution, then injected into 5 mL deionized water under continuous ultrasonication for 10 min. The 267

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images were shown in Fig. 2. The particle size of PEG45C18 was 208 nm and the distribution curve showed single peak with polydispersity index (PDI) of 0.29 ± 0.03 (Fig. 2a), the nanoaggregates exhibited spherical morphology (Fig. 2b). The critical aggregation concentration of PEG45C18 was 0.2 mg mL−1 detected by pyrene probe method. These results suggested amphiphilic structure could induce copolymer to form uniform micelles.

and quantified by a UV-HPLC (n = 3). 2.11. In vitro cytotoxicity assay To evaluate the cytotoxicity effect of HCPT NPs, the cell viability of 4T1 cells was detected using MTT assay (Zhou et al., 2013; Zhou et al., 2016). Cells were seeded in 96-well plates (~10,000 cells per well), after incubating with cultural medium for 48 h, the medium was changed to fresh RPMI-1640. Subsequently, samples at varying concentrations (HCPT equivalent concentration) were added. After culturing for 48 h, MTT solution (20 μL, 5 mg mL−1) was added to each well and cultured for another 4 h. The medium was removed, followed by adding 200 μL DMSO and pipetting up and down for several times, the formazan in each well was dissolved and the OD value was recorded. Cell inhibition = (1 − ODtreated/ODcontrol) × 100%, where ODtreated and ODcontrol was absorbance treated by the HCPT samples and the culture medium respectively (n = 5).

3.2. Preparation and characterization HCPT nanoparticles According to our previous papers, hydroxycamptothecin (HCPT)loaded nanoparticles (HCPT/PEG45C18 NPs) were prepared based on amphiphilic PEG45C18. To investigate the effect of alkyl chain, the HCPT-loaded nanoparticles (HCPT/PEG45 NPs) was prepared under the same conditions based on hydrophilic PEG with the molar mass of 2000 (PEG45) (Fig. 1c). HCPT and PEG45C18 or PEG45 were dissolved in DMF, then the organic phased was dropped into MiliQ water, the HCPT/PEG45C18 NPs and HCPT/PEG45 NPs (HCPT NPs) were obtained after dialysis against deionized water. For HCPT/PEG45C18 NPs with the drug-loading content (DLC) of approximately 48.8%, the hydrodynamic diameter (Dh) and zeta potential in aqueous solution were 255.7 ± 4.9 nm (polydispersity index, PDI = 0.14 ± 0.01) and 0.15 ± 0.03 mV (Fig. 3a), meanwhile, for HCPT/PEG45 NPs, they exhibited the similar particle diameter of 240.1 ± 7.1 nm (PDI = 0.09 ± 0.01) and zeta potential of 0.17 ± 0.02 mV (Fig. 3c), but the lower DLC of 34.1%. The SEM images showed that the morphology of the HCPT NPs were the mixture of nanorods and seldom nanospheres (Fig. 3b, d).

2.12. In vivo antitumor efficacy The 4T1 mice model was generated by subcutaneous injection of 5 × 106 4T1 cells in 0.2 mL PBS into the right armpit of BALB/c mice (Yang et al., 2014; Yu et al., 2015). After inoculation for 1 week, the tumor volume exceeded 100 mm3, the mice were randomly separately to 4 groups (n = 10), which were treated with normal saline (control group), HCPT injection (positive group), HCPT/PEG45C18 NPs and HCPT/PEG45 NPs (test groups) via intravenous (i.v.) administration. The treated dose was HCPT equivalent concentration of 3 mg Kg−1, and the volume was 0.2 mL. The mice were further injected every 2 days for 6 times, meanwhile, the body weight and tumor volume were recorded. Tumors and main organs were excised and weighed, tumor volume and inhibition rate (IR) were calculated according to previous report (Guo et al., 2017).

3.3. Measurement of stability

The actual HCPT concentration in main organs and tumors were quantified by HPLC with fluorescence detector (excitation/emission wavelengths 375/435 nm). The heart, liver, spleen, lung, kidney, tumor were collected, washed, accurately weighed, and homogenized with normal saline solution (0.9% NaCl), and then analysed by HPLC.

The stabilities of HCPT/PEG45C18 NPs and HCPT/PEG45 NPs in several media were measured over 6 h, the particle size was utilized to evaluate the stability of nanoparticles. After incubating 6 h in 5% glucose solution and plasma, the particle sizes of both of HCPT NPs didn't show significant change (p > 0.05) comparing with their initial sizes (Fig. 4). These results revealed the good stability of HCPT NPs, this phenomenon could be attributed to the effect of peripheral hydrophilic PEG chains, which hindered the aggregation of particles. The good stability indicated HCPT NPs met the stability requirements of animal experiments in vivo.

2.14. Statistical analysis

3.4. Controllable release profiles of HCPT NPs

All experiments were conducted at least in triplicate (> 3 independent experiment). Data were presented as the mean values ± SD. Comparison between groups was estimated by one-way analysis of variance (ANOVA) (SPSS 19.0, USA), p < 0.05 indicated statistical significance.

The release manners of HCPT NPs were evaluated in PBS solution containing 0.5% SDS, as the control, HCPT injection was also performed under similar conditions (Fig. 5). For injection, it was found that HCPT released completely within 4 h due to the carboxylate HCPT in injection was water soluble. Meanwhile, for HCPT/PEG45C18 NPs, HCPT was sustained release for 7 days, the release of HCPT was approximately 35% within the initial 1 day, and the survival was sustained release in the following 6 days. It seemed that the structure of HCPT nanoparticles affected the release procedure, the PEG chains might extend in the surface of nanoparticles and form the hydrophilic shell, which hampered the HCPT release from nanoparticles. Besides, comparing with HCPT/PEG45C18 NPs, HCPT/PEG45 NPs showed the similar release in the initial 1 day, but faster release in the following 6 days. This phenomenon could be explained by the effect of alkyl chain, HCPT released slowly due to the hydrophobic interaction between alkyl chain and HCPT.

2.13. Biodistribution in vivo

3. Results and discussion 3.1. Synthesis of amphiphilic molecules PEG45C18 Amphiphilic molecules PEG45C18 was synthesized via the active ester method from PEG45NHS and octadecylamine (Fig. 1a), and the purified PEG45C18 was obtained (yield of 75%). The characteristic peaks of octadecylamine was shown in the 1H NMR spectrum of PEG45C18, the peaks at 0.8 and 1.2 ppm were assigned to signals of the methyl and methylene protons in octadecylamine, the resonance peak of PEG was found at 3.2–3.8 ppm (Fig. 1b). Besides, the molar mass of PEG45C18 was 2270.92 detected by Maldi-tof. These results implied the successful conjugation of PEG45C18. The self-assembly behaviors of PEG45C18 in aqueous solution were detected, which was dissolved well in aqueous solution with the concentration of 1 mg mL−1, the particle size distribution curves and TEM

3.5. In vitro cytotoxicity Murine breast cancer (4T1) cells were incubated with HCPT NPs in varied concentration (HCPT equivalent concentration) to explore the cytotoxicity of HCPT NPs in vitro with MTT assay (Fig. 6). The IC50 268

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Fig. 1. Synthetic procedure for amphiphilic molecules and HCPT NRs: a) synthetic procedure of PEG45C18, b) 1H spectrum of PEG45C18, c) HCPT NPs preparing procedure.

Fig. 2. The particle size distribution curves (a) and TEM images (b) of PEG45C18.

value for HCPT injection were 9.55 μg mL−1, while, the IC50 values for HCPT/PEG45 NPs and HCPT/PEG45C18 NPs were 8.36 and 0.98 μg mL−1 respectively. Comparing with the HCPT injection, the IC50 values of both nanoparticles were decreased, indicating higher cytotoxicity against 4T1 cells after incubating 48 h (p < 0.05), and suggesting more HCPT nanoparticles was uptake by 4T1 cells. The enhanced antitumor efficacy of nanoparticles could be explained by the facilitated endocytosis transport, while, free HCPT crossed the cell membrane via passive diffusion. In previous study, the similar enhanced cytotoxicity were observed (Zhou et al., 2013; Guo et al., 2017). Moreover, HCPT/PEG45C18 NPs showed higher cytotoxicity than HCPT/PEG45 NPs, the IC50 value was decreased approximately 8.5-fold, suggesting HCPT/PEG45C18 NPs exhibited higher endocytosis efficacy to mammalian cells due to the better interactions with cellular membrane (Janas et al., 2011; Meyer et al., 2014; Selvaraj et al., 2015; Yadav et al., 2017).

for 6 times (Fig. 7). The tumor volume of all four groups were timerelative increased, the tumor sizes were increased by 17.8-fold, 11.2fold, 8.8-fold, and 5.9-fold for control, positive, and two test groups correspondingly (Fig. 7a). Compared to control group, positive group exhibited moderate antitumor activities, on the contrary, HCPT NPs produced higher antitumor activity than HCPT injection group (p < 0.01). Furthermore, it was found that HCPT/PEG45C18 NPs group existed better antitumor efficacy (p < 0.05, vs. HCPT/PEG45 NPs). Then, the tumor inhibition rate further indicated the good tumor inhibition activity of nanoparticles (Fig. 7b). The average weight of the tumors were 0.91, 0.57, 0.49, and 0.32 g for blank, positive, and test groups, the relative tumor inhibition rates were 37.4%, 45.6%, and 64.8% for positive, HCPT/PEG45 NPs, and HCPT/PEG45C18 NPs groups respectively. The promoted tumor inhibition rate of HCPT nanoparticles could be attributed to longer blood circulation escaping from RES system, higher accumulation in tumor tissue via EPR effect, and higher cell endocytosis efficacy. It was confirmed that the alkyl chain could affect the antitumor activity, due to nanoparticles decorated with alkyl chains may affect cellular uptake (Wender et al., 2000; Moody et al., 2002; Raffa et al., 2010).

3.6. In vivo antitumor effect After tumor volume exceeded 100 mm3, mice were divided into four groups (n = 10), including saline group (control group), HCPT injection group (positive group), HCPT/PEG45C18 NPs and HCPT/PEG45 NPs group (test groups). 4T1 tumor-bearing mice were administrated with these formulations at the concentration of HCPT equivalent 3 mg Kg−1, and the tumor volume and body weight were monitored every two days

3.7. Biodistribution and toxicity evaluation The quantitative analyses of HCPT in tumor and major organs, including heart, liver, spleen, lung, and kidney, were carried out by HPLC 269

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Fig. 3. Particle size distribution curves of HCPT/PEG45C18 NPs (a) and HCPT/PEG45 NPs (c) in aqueous solutions, and SEM image of HCPT/PEG45C18 NPs (b) and HCPT/PEG45 NPs (d). Scale bar: 200 nm.

Fig. 4. Particle size of the HCPT NPs in 5% glucose solution (a) and plasma (b) at 37 °C.

Fig. 5. Cumulative HCPT release procedure in PBS solution at 37 °C (n = 3).

Fig. 6. Cytotoxicities of HCPT NPs towards 4T1 cells after 48 h incubation (n = 5).

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Fig. 7. In vivo antitumor efficacy: tumor volume change curves (a) and tumor inhibition rate (b). Six consecutive doses were given (marked by arrows) (n = 10). p < 0.01 vs. HCPT injection, # p < 0.05 vs. HCPT/PEG45 NPs.

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HARE-mediated endocytosis, therefore the majority of nanoparticles were accumulated, disintegrated, and cleared by the hepatobiliary system (Ganesh et al., 2013). The accumulation of nanoparticles in kidney might be attributed to the renal excretion, which was an elimination pathway through renal system (Choi et al., 2007; Choi et al., 2010). These results were consistent with other published papers (Parveen and Sahoo, 2011; Xiao et al., 2011; Liang et al., 2017). Due to the high accumulation in liver and kidney, the hepatic/renal function markers, including alanine amino transferanse (ALT), aspartate amino transferase (AST), blood urea nitrogen (BUN), and creatinine (CRE), were detected. HCPT NPs showed no significant difference comparing with the other two groups (p > 0.05) (Table 1), including saline group and injection group. These results revealed that HCPT/ PEG45C18 NPs presented no obvious hepatic or renal toxicity, although showing high accumulation in liver and kidney. 4. Conclusions

Fig. 8. Biodistribution of HCPT in 4T1-bearing mice (n = 10) determined by HPLC. ⁎⁎ p < 0.01 vs. HCPT injection, ⁎ p < 0.05 vs. HCPT injection, ## p < 0.01 vs. HCPT/PEG45 NPs.

Based on the linear amphiphilic molecules PEG45C18, HCPT-loaded nanoparticles (HCPT/PEG45C18 NPs) was prepared via solvent exchange method augment ultrasonication. Meanwhile, HCPT/PEG45 NPs was prepared under the same conditions utilizing linear PEG45 as the nanocarrier to verify the effect of alkyl chain on antitumor activity. The drug-loading content of HCPT/PEG45C18 NPs and HCPT/PEG45 NPs was 48.8% and 34.1% correspondingly. Both HCPT NPs existed good stability in 5% glucose solution and plasma, the accumulative release could sustain to 5–7 days, besides, HCPT NPs presented better antitumor activity comparing with HCPT injection due to the higher cellular uptake efficacy and passive targeting. Owing to the effect of alkyl chain, HCPT/PEG45C18 NPs presented the slower release kinetics and better antitumor activity comparing with HCPT/PEG45 NPs, the cytotoxicity of HCPT/PEG45C18 NPs was promoted significantly and the IC50 value was decreased approximately 8.5-fold, meanwhile, the tumor inhibition rate was enhanced 1.5-fold. The accumulation of HCPT NPs in tumor tissue was increased significantly, especially for HCPT/ PEG45C18 NPs. Although the biodistribution of HCPT NPs in liver and kidney was high, no obvious hepatic/renal toxicity was determined. These results revealed the alkyl chain affected the antitumor activity significantly due to nanoparticles decorated with alkyl chains existing higher endocytosis efficacy to cells, and could be considered carefully when designed the nanocarriers in the future.

Table 1 Plasma biochemical levels of 4T1 bearing BALB/c mice. Sample

ALT (IU L−1)

AST (IU L−1)

CRE (μmol L−1)

BUN (mmol L−1)

Saline Injection HCPT/PEG45C18 NPs

9.02 11.69 10.61

8.76 12.39 10.22

15.93 10.54 19.88

5.09 4.12 4.94

with fluorescence detector (Fig. 8). HCPT NPs existed better tumor accumulation than injection, the biodistribution were enhanced significantly (p < 0.01 for HCPT/PEG45C18 NPs, and p < 0.05 for HCPT/PEG45 NPs), due to the passive targeting of nanoparticles via EPR effects. Moreover, HCPT/PEG45C18 NPs showed the better tumor accumulation than HCPT/PEG45 NPs, because the alkyl chains may affect cellular uptake. Beside tumor tissue, HCPT nanoparticles were also mainly detected with high concentration in liver and kidney, on account of the phagocytosis effect of reticuloendothelial system (RES) (Sharifi et al., 2012). It was well known that nanoparticles could be easily cleared by the reticuloendothelial system (RES) or the mononuclear phagocytic system, which comprises phagocytic cells located in the liver, spleen, and other reticular connective tissues, this system played an important role to clear the foreign particles in blood circulation and tissues (Choi et al., 2011; Yu and Zheng, 2015). These foreign particles might be internalized by the non-specific adsorption or the

Acknowledgments This work is financially supported by CAMS Innovation Fund for Medical Sciences (CIFMS, no. 2017-I2M-1-013), CAMS Innovation Fund 271

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for Medical Sciences (CIFMS, no. 2016-I2M-1-012), National Natural Science Foundation of China (no. 81573622), and National Natural Science Foundation of China (no. U1401223).

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