Amphiphilic Hybrid Dendritic-Linear Molecules as Nanocarriers for

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Amphiphilic Hybrid Dendritic-Linear Molecules as Nanocarriers for Shape-Dependent Antitumor Drug Delivery Yifei Guo, Ting Wang, Shuang Zhao, Meihua Han, Zhengqi Dong, Xiangtao Wang, and Yanhong Wang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00190 • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 21, 2018

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Molecular Pharmaceutics

Amphiphilic Hybrid Dendritic-Linear Shape-Dependent Antitumor Drug Delivery

Molecules

as

Nanocarriers

for

Yifei Guo,a Ting Wang,a, b Shuang Zhao,a Meihua Han,a Zhengqi Dong,a, * Xiangtao Wanga, * Yanhong Wang,b, *

a

Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences &

Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing 100193, China b

School of Pharmacy, Heilongjiang University of Chinese Medicine, No. 24, Heping

Road, Xiangfang district, Harbin 150040, China

Abstract Nanoparticles based on hybrid block copolymers had been expected as effective nanocarriers for hydrophobic drug delivery. Herein, the novel dendritic-linear molecules from OEG dendron conjugated with octadecylamine (G2-C18) was designed,

synthesized

and

further

applied

as

nanocarrier

to

prepare

10-hydroxycamptothecin (HCPT) nanoparticles via antisolvent precipitation method. It seemed that the feed weight ratio of HCPT vs. G2-C18 not only affected the drug-loading content of nanoparticles, but also influenced the morphology of HCPT nanoparticles, the morphology of HCPT nanoparticles was changed from nanosphere (NSs) to nanorod (NRs) with increasing the feed weight ratio. Both of HCPT nanoparticles presented good stability and similar drug release profiles, but different anticancer efficacy and cellular uptake mechanism. The cytotoxicity of HCPT NRs was enhanced significant comparing with HCPT NSs, the IC50 value was 2-fold lower than HCPT NSs (p < 0.05). More importantly, HCPT NRs showed apparently higher antitumor activity in vivo, the inhibition rate of HCPT NRs was 1.3-fold higher than HCPT NSs. Based on these results, it suggested that the antitumor activity could be influenced significantly by particle morphology, which should be considered and 1

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optimized during the nanocarriers design. Keywords Dendritic-linear molecules, shape-dependent property, self-assembly mechanism, antitumor efficacy Introduction Nanotherapeutics is emerging as the effective strategy for cancer therapy owing to the higher antitumor efficacy and decreased unfavourable side effects of hydrophobic antitumor agents.1-6 To achieve these purposes, self-assembled nanoarchitectures were often applied as the nanocarriers,7, 8 these drug delivery systems could avoid rapid blood/renal clearance of drugs, and then accomplish the high accumulation of drugs in tumors tissues via the passive targeting through enhanced permeability and retention (EPR) effect.9-11 Nanocarriers can be generally classified into linear amphiphilic copolymers,12 hyperbranched polymers,13, 14 star polymers,22,

23

dendrimers or dendritic polymers,15-21

janus dendritic polymers,24,

copolymers (LDBCs),26,

27

25

and linear-dendritic block

which could be aggregated to form micelles,

polymersomes, nanoparticles, nanogels, nanocapsules, and vesicles.28-31 As an analogue of amphiphilic nanocarriers, LDBCs combine the branched structure of dendrimers with the property of linear block copolymers,32,

33

the

parameter of interfacial curvature of linear-dendritic block copolymers is expected to affect drug delivery efficiency and the self-assembly procedure.34,

35

Traditional

LDBCs as drug carriers reported before are generally conjugated a hydrophilic linear block with a relatively hydrophobic dendritic block,36-39 a lot of drug-loaded nanoparticles are prepared via physical entrapment of hydrophobic drug into the dendritic core.40-42 However, these nanoparticles based on these LDBCs present unsatisfied drug-loading content (almost < 10%) due to the strong steric hindrance of the branched core, which prompt that the composition of the drug carrier should be considered carefully. Besides the drug-loading content, the shape of drug-loaded nanoparticles are expected to act as the key role in blood circulation, biodistribution, and in vivo 2


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therapeutic efficacy.43-47 As the carriers, amphiphilic block copolymers in aqueous solution have a tendency to self-assemble into aggregates,48, 49 and the morphologies of aggregates are affected by the composition and concentration of block copolymers, temperature, solvent, and so on.50-52 Utilizing amphiphilic copolymers as drug carrier, the drug-loaded nanoparticles also could be prepared with different morphologies, which were related with the structure of the drug carriers and preparation method.53-55 Unfortunately, the drug-loaded nanoparticles always show the spherical morphology based on the reported LDBCs. In this study, dendritic-linear block molecules G2-C18 from oligoethylene glycols dendron (G2) and octadecylamine (C18-NH2) is synthesized as the nanocarrier, and 10-hydroxycamptothecin (HCPT) as one of the most commonly anticancer agents is utilized to construct HCPT-loaded nanoparticles via ultrasonication method. These nanoparticles show similar particle size, stabilitity, drug release profiles, but different morphology (nanospheres and nanorods), which is mainly related to the HCPT-loaded content. Furthermore, it is found that the cellular uptake ratios, anticancer efficacies are dependent with the morphology of HCPT nanoparticles, and nanorods presented better antitumor efficacy. Experimental Section Materials Oligoethylene glycols dendron (G2) pentafluorophenol active ester was prepared following previous papers.56 Hydroxycamptothecin (HCPT) was brought from Ou He Bio-Tech Co., Ltd. (Beijing, China). HCPT injection was brought from Shenghe Pharmaceutical Ltd. (Sichuan, China). Acetonitrile (HPLC grade) were received from Fisher Scientific (Pittsburgh, PA, USA). Dimethylsulfoxide (DMSO) and normal saline were obtained from Sigma-Aldrich Chemicals, Germany. Dialysis membrane (MWCO = 14000 Da) was purchased from Spectrapor. Other reagents and solvents were of analytical grade and used without further purification. Animals and Cell Line The 4T1 cell line was obtained from the Institute of Basic Medical Science, Chinese 3



Academy of Medical Science (Beijing, China) and cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 100 units mL-1 penicicillin G and streptomycin. The culture was performed with 5% CO2 atmosphere at 37 oC. BALB/c mice (20 ± 2 g) and rats (200 ± 20 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All the animals were housed on SPF conditions, 12 h light-darkness cycles, and standard diet of food and water ad libitum. All experimental procedures comply with the Guidelines and Policies for Ethical and Regulatory for Animal Experiments as approved by the Animal Ethics Committee of Peking Union Medical College (Beijing, China). Synthesis of Amphiphilic Compound G2-C18 A solution of oligoethylene glycols dendron (G2) pentafluorophenol active ester (1.0 g, 0.4 μmol) in dichloromethane (20 mL) was added dropwise into a solution of octadecylamine (0.3 g, 0.8 μmol), triethylamine (0.1 g, 1.2 μmol), and N, N-dimethylaminopyridine (DMAP, 10 mg) in dichloromethane (20 mL) at -5 oC with continuously stirring. After stirring for a further 24 h, the mixture was evaporated in vacuo, and the residue was purified by column chromatography with DCM/MeOH (40/1, v/v) to afford colorless G2-C18 (0.9 g, 73%). 1H NMR (300 MHz, CDCl3): δ = 0.80 (t, 3H, CH3), 1.20 (t, 27H, CH3), 1.33 (m, 32H, CH2), 3.31 (t, 2H, CH2), 3.49-3.80 (m, 138H, CH2), 4.18-4.25 (m, 24H, CH2), 4.40 (s, 6H, CH2), 6.61 (s, 6H, CH), 7.12 (s, 2H, CH) ppm. HR-MS: calcd. m/z = 2673.26 [M+H]+; found m/z = 2673.2683 [M+H]+ Preparation of HCPT-Loaded Nanoparticles Spherical and rod-like HCPT-loaded nanoparticles (HCPT NSs and NRs) were obtained via the ultrasonication-dialysis method. Briefly, HCPT (2, 4, and 8 mg) and G2-C18 (4 mg) were dissolved in DMF (1 mL) at 25 oC, which was added into deionized water (5 mL). After ultrasonication for 10 min, the organic phase was dialyzed against deionized water (4 × 1 L) for 4 h with dialysis bag (MWCO 14000). Then, the nanoparticles were obtained and homogenized for 10 times under 1600 bar pressure at 25 oC. The quantitative analysis of HCPT in these nanoparticles (NSs and NRs) was detected by HPLC (UltiMate3000, DIONEX) with a Thermo C18 column 4


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(4.60 mm × 250 mm, 5 µm). The HCPT was analyzed using a UV detector at 384 nm, the calibration curve was generated from acetonitrile:water (acetic acid, 0.1%) (25:75, v/v) (y = 1.7874x + 0.1561, R2 = 0.9999). The injection volume was 20 µL and the flow rate was 1.0 mL min-1. The drug-loading content (DLC) was calculated according to the equation as follows. DLC = (weight of loaded drug/weight of drug-loaded NRs) ×100% Particle Size and Zeta Potential Measurements The hydrodynamic diameter, size distribution, and ζ-potential of these HCPT nanoparticles were detected by a dynamic light scattering (DLS) spectrophotometer ( Zetasizer Nano-ZS analyzer, Malvern Instruments, UK), using the detection with scattering angle θ = 173º. Fixed Aqueous Layer Thickness (FALT) Measuremnts After centrifuging at 13000 rpm for 20 min, the HCPT nanoparticles pellets were obtained. Then, the nanoparticles were washed with phosphate buffer solution for three times and dispersed in different NaCl solutions with several concentrations. The FALT (L) was calculated from the zeta potential, according to the linear correlation between ln ζ (zeta potential) and κ (Debye–Huckel parameter): ln ζ = ln A – κL, where A is defined as constant. Transmission Electron Microscope Transmission electronic microscopy (TEM) images were detected at an accelerating voltage of 80 kV (JEM-1400). Before visualization, a drop of HCPT nanoparticle solutions (0.1 mg mL-1) were placed on carbon-coated copper grids and stained with uranyl acetate solution (2%, w/v). Scanning Electron Microscope Scanning electron microscopy (SEM) images were detected at an accelerating potential of 30 mV (SEM-EDS; S-4800). Before visualization, the lyophilized HCPT bulk powders and a drop of HCPT nanoparticle solutions (0.1 mg mL-1) were placed on matrix, then these samples were sputter-coated with a conductive layer of gold-palladium (Au/Pd) for 1 min. Medium Stability Study 5



HCPT NSs and NRs were incubated with different medium at 37 oC, and the hydrodynamic size of the samples was detected at the predetermined time. The experiments were conducted in triplicates. Drug release profile The dialysis method was utilized to estimate the in vitro release manners of HCPT NSs and NRs. After drug loading, the dialysis bag (MWCO 14000) containing HCPT nanoparticle solutions (2 mL) were immersed into 50 mL of PBS solution (pH 7.4) with 5% SDS at 37 °C under sink conditions. At the setting intervals, external solution (5 mL) was withdrawn for analysis and an equal volume of fresh media was added. The quantitative analysis of released HCPT was measured by a UV-HPLC. MTT Assays The cytotoxicity was performed on 4T1 cells by MTT assay in 96-well plates at a density of 1 × 104 cells per well. Before adding the samples, the cells were cultured with RPMI-1640 containing 10% FBS for 2 days. After changing the growth medium to fresh RPMI-1640, HCPT nanoparticles and injection were added and co-cultured for another 48 h, then MTT solution (5 mg mL-1, 20 µL) were added to each well and further incubated for further 4 h. Subsequently, DMSO (200 µL) was added into each well. The absorbance was measured at 570 nm using ELISA plate reader to determine the OD value, from which the cell inhibition rate was calculated: Cell inhibition = (1-ODtreated/ODcontrol) × 100%, where ODtreated was obtained for the cells treated by the nanoparticles and injection, ODcontrol was obtained for the cells treated by the culture medium. Cellular Uptake The 4T1 cells were seeded in a 6-well plate at a density of 1 × 105 cells per well and cultured for 24 h, then the nanoparticles (HCPT-equivalent concentration of 50 µg mL-1) were added and incubated for 4 h. After removing the culture medium, washing three time with PBS, and fixing with 4% of paraformaldehyde solution for 15 min, the cellular uptake images were recorded with Delta Vision Microscopy Imaging Systems. The 4T1 cells were seeded in a 12-well plate at a density of 2 × 105 per well and 6


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cultured for 48 h. After incubated with different inhibitors for 1 h, the HCPT nanoparticles (HCPT-equivalent concentration of 50 µg mL-1) were added for additional 4 h co-culturing. Then, removing the culture medium, washing with PBS three times, adding 0.2 mL of RIPA lysis buffer and 1 mL of ethyl acetate, and centrifuging at 10000 rpm for 15 min, the supernatant was collected and reconstituted in 200 µL methanol. The quantitative analysis was carried out on fluorescence-HPLC system (excitation/emission wavelengths 375/435 nm) using C18 column, and the calibration curves was generated from acetonitrile/water containing 0.1% acetic acid (30/70, v/v). A flow rate was 1.0 mL min-1, and the sample injection volume was 20 µL. Cellular uptake ratio = (HCPT concentration in 4T1 cells/50 µg mL-1) ×100% In vivo Antitumor Efficacy BALB/c mice were injected 0.2 mL cell suspension (5 × 106 4T1 cells) in the right armpit to induce 4T1 tumor. After the tumor volume exceeding 100 mm3, the mice were randomly divided into 4 groups (10 mice per group), which were administrated separately by normal saline (control group), HCPT injection (positive group, 3 mg Kg-1), HCPT nanoparticles (NSs and NRs groups, 3 mg Kg-1) via intravenous (i.v.) injected with 200 µL every 2 days for 6 times. On the 12th day, the mice were sacrificed, then, tumors and main organs were excised. The inhibitory rate (IR) of the tumor was calculated as follows. IR = (1 – tumor weight of treated group/tumor weight of the control group) × 100%. Biodistribution in 4T1-tumor Bearing Mice After accurately weighed, the organs and tumors were homogenized with 0.9% NaCl solution. The HCPT concentration in main tissues and tumors were measured by fluorescence-HPLC system (excitation/emission wavelengths 375/435 nm) using C18 column. Calibration curves were established respectively for the tumor and organs; all of the correlation coefficients were more than 0.99. Histological Analysis After fixed in 10% neutral formalin, the fixed tissue was embedded in paraffin, 7



sectioned, and stained by hematoxylin and eosin (H&E). Statistical Analysis Data were presented as the mean values ± standard deviation (> 3 independent experiments. Statistical evaluation was according to one-way analysis of variance (ANOVA) (SPSS 19.0, USA), P < 0.05 was considered as significant. Results and Discussion Synthesis and Characterization of Hybrid Compound G2-C18 According to our previous papers, dendritic-linear hybrid compound G2-C18 was synthesized from OEG dendron active ester (G2) and octadecylamine (C18-NH2). After purification by column chromatography, G2-C18 was obtained with a yield of 73%. The 1H NMR spectrum identified the successful synthesis of G2-C18, the signals at 0.8 and 1.3 ppm were attributed to the methyl and methylene protons of octadecylamine (Figure 1). Benefiting from hydrophilic OEG dendron and hydrophobic octadecylamine, amphiphilic G2-C18 was dispersed in deionized water and self-assembled into nanoaggregates (Figure 4a), the mean hydrodynamic diameter was approximately 172.4 ± 5.8 nm and PDI was 0.13 ± 0.02 (Table 1).

Figure 1. 1H NMR spectrum of G2-C18.

8


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Table 1. Results of HCPT nanospheres and nanorods Sample

a

DLC a

DLS results b

FALT e

(%)

Dh (nm) c

PDI

ζ (mV) d

(nm)

G2-C18

0

172.4 ± 5.8

0.13 ± 0.02

n.d.f

n.d.

Nanospheres

26.3 ± 2.1

337.4 ± 6.4

0.21 ± 0.01

18.8 ± 0.3

12.8

Nanorods

61.8 ± 3.7

241.4 ± 4.6

0.11 ± 0.03

19.0 ± 0.3

10.5

Drug-loading content measured by UV-HPLC.

mL-1.

c

Hydrodynamic diameter.

d

Zeta potential.

b

Dynamic light scattering, 1 mg

e

Fixed aqueous layer thickness.

f

Not detected.

Figure 2. DLS curves of HCPT NSs (a), HCPT NRs (c), and TEM images of HCPT NSs (b), HCPT NRs (d). HCPT-Loaded Nanoparticles Based on the amphiphilic compound G2-C18, hydroxycamptothecin (HCPT)-loaded nanospheres (NSs) and nanorods (NRs) were prepared. The solution of HCPT and G2-C18 in DMF was injected into distilled water. After ultrasonication for 10 min, the nanospheres and nanorods were obtained via dialysis method with the 9



drug-loading content (DLC) of approximately 26.3% and 61.8% respectively (Table 1). The crystalline structure of HCPT was maintained during the preparing procedure, which was confirmed by XRD (Supporting Information, Figure S1). The hydrodynamic sizes of NSs was approximately 337.4 ± 6.4 nm (PDI = 0.21 ± 0.01), meanwhile, the NRs was approximately 241.4 ± 4.6 nm (PDI = 0.11 ± 0.03) (Table 1), Figure 2a and 2c were the particle size distribution curves. The surface charges of these two nanoparticles were approximately 18.8 ± 0.3 and 19.0 ± 0.3 mV respectively (Table 1). Investigating by TEM and SEM observation, the HCPT nanoparticles showed the spherical and rod-like morphology with well dispersed as individual particles (Figure 2b, 2d). HCPT NRs presented similar morphology with bulk HCPT powder but smaller particle sizes (Supporting Information, Figure S2). Based on TEM measurement, the diameter of nanospheres was approximately 200 nm, the length and width of nanorods were approximately 330 and 50 nm with the aspect ratio of 6.6. The stability of nanoparticles in medium and plasma was critical to their application in drug delivery via intravenous injection.57 Both of these HCPT nanoparticles possessed the good stability in glucose solution (5%) and plasma for 6 h, which was attributed to the branched OEG dendron hindered the aggregation of nanoparticles (Supporting Information, Figure S3). Self-Assembly Mechanism of HCPT Nanoparticles The fixed aqueous layer thickness (FALT) for HCPT NRs was 10.5 nm, thinner than 12.8 nm for HCPT NSs (Table 1). Moreover, surface element analysis of these two nanoparticles determined by EDS analysis (Figure 3), HCPT NRs showed higher C element content. These results implied that these two HCPT nanoparticles presented different structural properties, and HCPT NRs expressed more hydrophobic property comparing with NSs.

10


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Figure 3. The major element percentage on the surface of HCPT NSs (a) and HCPT NRs (b). It is reported that the simple modulation of the weight ratio between nanocarriers and bioactive drug molecules could construct specific nanostructures and morphologies.52 The similar phenomenon was found here, HCPT/G2-C18 feed weight ratio affected the self-assembly morphology of HCPT nanoparticles. When the feed weight ratio was low (1/2), the HCPT nanoparticles presented spherical morphology (Figure 4b). Increasing the amount of HCPT (feed weight ratio of 1/1), the mixture of nanospheres and nanorods was shown (Figure 4c). Further increasing the amount of HCPT (2/1), nanospheres disappeared completely, only nanorods were formed (Figure 4d). These could be explained by the critical phase behavior, it was reported that the hydrophilic/hydrophobic volume fraction (f) could affect the self-assembly behavior of block copolymers. With increasing the hydrophobic volume fraction, the block copolymers could be self-assembled into disordered structures, spheres, cylinders, gyroid, and lamellae.35 Based on the unique structure, hybrid molecules G2-C18 presented low hydrophobic fraction,

which could be

self-assembled into disordered structure in aqueous solution (Figure 4a). When the hydrophobic HCPT was added, the hydrophobic fraction of the whole drug-loaded nanoparticles was increased, and the self-assembly behavior was induced to form spherical structure (Figure 4b). Increasing the drug-loaded content furthermore, the hydrophobic fraction of nanoparticles was enhanced enough to form cylinders (Figure 4d). 11



Figure 4. SEM images of G2-C18 (a) and HCPT nanoparticles with HCPT/G2-C18 feed weight ratio of 1/2 (b), 1/1 (c), 2/1 (d). Scale bar: 500 nm. To confirm the influence of hydrophobic fraction of drug-loaded nanoparticles on the morphology furthermore, a series of the OEG dendron derivatives, including OEG dendron and PAMAM-co-OEG codendrimers (PGD) were utilized as nanomaterials to prepare HCPT nanoparticles at the same conditions.58 OEG dendron and PGD presented similar sphere-like morphology without hydrophobic HCPT (Supporting Information, Figure S4a and S4c), after added hydrophobic HCPT, the hydrophobic fraction of these nanoparticles were enhanced, inducing the morphologies were changed from spheres to nanorods (Supporting Information, Figure S4b and S4d), which was similar as G2-C18. This phenomenon indicated the drug-loading content could affect the hydrophobic fraction, further result in the different self-assembly behavior. Drug release profile The HCPT release manners from NSs and NRs were studied in the PBS solution (pH 7.4, containing 5% SDS) at 37 °C, HCPT injection was utilized as control (Supporting Information, Figure S5). For injection, completely diffusion of HCPT from injection 12


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was shown within 4 h, because HCPT was the carboxylate salt form in injection. While, HCPT NSs and NRs presented the similar release properties, both of them could sustained released over 144 h. The different release manners between HCPT nanoparticles and injection could be attributed to the structure of nanoparticles, in which the hydrophilic OEG dendron hindered the diffusion of HCPT due to its steric hindrance. The similar release property of NSs and NRs could be explained by these two shape nanoparticles was formed by the same HCPT nanocrystals, which was verified by XRD.

Figure 5. Cytotoxicities of HCPT NSs and NRs toward 4T1 cells after incubation for 48 h (n = 5). In vitro antitumor efficacy The cytotoxicity of HCPT NSs and NRs against 4T1 cells was studied via MTT assay with the concentration ranging from 0.1 to 50 µg mL-1 (HCPT equivalent concentration, Figure 5). Amphiphilic hybrid compound G2-C18 showed no significant cytotoxicity towards 4T1 cells during the whole test concentration. After incubated 48 h against 4T1 cells, the significantly enhanced cytotoxicities were observed for these two HCPT nanoparticles, the IC50 value for free HCPT, NSs, and NRs was 1.56, 0.54, and 0.23 µg mL-1 respectively. At the same dose, NSs and NRs exerted higher cytotoxicity effect than HCPT injection (p < 0.001), which confirmed the anticancer activity of HCPT nanoparticles were better than free drug. According to previous reports, this phenomenon could be explained by the facilitated endocytotic transport of nanoparticles, while, free HCPT across the cell membrane via the passive diffusion. Besides, the higher antitumor efficacy in vitro was observed from NRs, the 13



IC50 was 2-fold lower than NSs (p < 0.05), suggesting the morphology of nanoparticles could affect the ratio of internalization. Cellular Uptake Fluorescent microcopy imaging system was utilized to detect the cellular uptake of HCPT nanoparticles against 4T1 cell lines at the equivalent HCPT concentration, and HCPT injection were characterized as control under the similar condition. After co-cultured 4 h and repeated washing, the cells treated by HCPT injection emitted weak HCPT fluorescence signals (Figure 6a, Injection), however, strong fluorescence signals was captured from the cells treated by HCPT NSs and NRs (Figure 6a, Nanospheres and Nanorods), indicating the higher internalization ratio of HCPT nanoparticles against cancer cells. These results demonstrated that nanoparticles employed the different mechanism to enter the cancer cells, according to the previous papers, nanoparticles could be preferentially internalized via endocytosis pathway, while free drug across the cell membrane via the passive diffusion. Moreover, NRs exerted stronger fluorescence intensity than NSs, which was attributed to the more effective adhesion with cell membrane and different endocytic mechanisms. HCPT NRs presented higher hydrophobic property and hence induced more effective adhesion to the cell membrane, furthermore, enhanced the endocytosis opportunity.59 During endocytosis procedure, it seemed that the symmetry of curvature energy landscape and the angle of entry could be affected by the nanoparticles shape,60 nanorods exhibited unique wrapping modes owing to their aspect ratio.61, 62 To identify the endocytosis mechanism, the HCPT nanoparticles were incubated with 4T1 cells and several endocytosis inhibitors. After co-cultured 4 h and washed several times, the qualification analysis of HCPT in 4T1 cells was measured by fluorescence-HPLC system (Figure 6b). Based on the HCPT concentration determined from cell lysis buffer, the cellular uptake ratio was calculated as 5.8%, 8.3%, and 16.9% for HCPT injection, NSs, and NRs. Consistent with the fluorescent images, the cellular uptakes of two HCPT nanoparticles were enhanced significantly comparing with HCPT injection (p < 0.05). After co-cultured with endocytosis inhibitors, the cellular uptake rate of NSs and NRs presented different results, 14


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indicating they internalized via different endocytosis pathway. For HCPT NSs, when cytochalasin

D

(CCD)

was

used

as

endocytosis

inhibitor

of

the

macropincytosis-dependent pathway, the cellular uptake ratio was significant decreased (p < 0.05), indicating that the effective uptake mechanism of NSs was macropincytosis-dependent endocytosis. For HCPT NRs, as the inhibitor of clathrin-mediated endocytosis and macropincytosis-dependent endocytosis, sucrose and CCD caused dramatic decrease in the cellular uptake rate (p < 0.05), suggesting that the cellular uptake mechanism of nanorods were clathrin-mediated endocytosis and macropincytosis-dependent endocytosis, which was in accordance with previous reports.63 From these results, it seemed that the morphology of nanoparticles could affect the cellular uptake procedure.

Figure 6. Representative fluorescent microscopy images (a) and cellular uptake ratio (b) of HCPT injection, nanospheres, and nanorods. Blue: HCPT. Endocytosis inhibitor: sucrose, methyl-β-cyclodextrin (CD), and cytochalasin D (CCD). Cellular uptake rate was calculated based on the actually determined HCPT concentration from cell lysis buffer. * p < 0.05, ** p < 0.01 vs. injection group,

★

p < 0.05 vs. NSs group, # p < 0.05

vs. NRs group. In vivo Antitumor Efficacy BALB/c mice bearing 4T1 breast tumor model was utilized to evaluate the in vivo antitumor activity of HCPT nanoparticles, in which the normal saline and HCPT injection was performed as control. Tumor-bearing mice were randomly divided into four groups (n = 10): saline, HCPT injection (control group), NSs and NRs (test 15



Page 16 of 26

groups), the equivalent concentration of HCPT was 3 mg Kg-1. These mice were administrated every two days for 6 times and the tumor volume was monitored every two days for 12 days. The tumor volume of all four groups showed time-dependent increase, which enhanced 9.0-fold, 5.3-fold, 4.1-fod, and 2.7-fold comparing with the initial values respectively (Figure 7a). HCPT injection group showed moderate antitumor activity, however, both NSs and NRs exerted higher antitumor efficacy than HCPT injection group (p < 0.01). More important, NRs existed optimizing antitumor efficacy comparing with NSs, the tumor volume change of the mice administrated with HCPT NRs was smaller than those treated with HCPT NSs (p < 0.05). The average weight of the tumors was utilized to calculate the tumor inhibition rates, the HCPT injection group resulted in the inhibition rate of 47.6%, meanwhile, the HCPT NSs group was 60.6% and NRs group was 78.5% (Figure 7b). The inhibition rate of NRs was 1.6-fold as HCPT injection (78.5% vs. 47.6%, p < 0.01), and 1.3-fold as HCPT NSs (78.5% vs. 60.6%, p < 0.05), prompting HCPT nanoparticles showed better antitumor efficacy which was related with the morphology of nanoparticles. Both from tumor volume growth curves and inhibition rate calculated from the average weight of tumor tissue, the HCPT NRs presented better antitumor efficacy, which was consistent with the results of the MTT assay.

Figure 7. Tumor volume change curves (a) and tumor inhibition rate (b) for BALB/c mice bearing 4T1 breast tumor model. For each animal, six consecutive doses were given (marked by arrows). ** p < 0.01 vs. injection group,

★

p < 0.05 vs. NSs group.

Biodistribution in 4T1 Tumor-bearing Mice The biodistribution of HCPT nanoparticles in tumor tissue and main organs (heart, 16


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liver, spleen, lung, and kidney) were determined by HPLC with fluorescence detector, the results are shown in Figure 8. Comparing with HCPT injection, the concentration of HCPT NSs and NRs in tumor tissue was enhanced 2.8 and 3.5-fold separately, moreover, the significant difference was shown between NSs and NRs (p < 0.05), the HCPT NRs presented higher tumor accumulation. These results consisted with the antitumor efficacy, which could be explained by the nanoparticles could be accumulated in tumor better via EPR effects and the morphology of nanoparticles may affect the uptake ratio by cancer cell. Besides, HCPT NSs and NRs were mainly distributed in reticuloendothelial system (RES) organs such as liver and kidney. Due to this high accumulation in RES system, the relative toxicity should be evaluated.

Figure 8. Biodistribution of HCPT in main tissues (n = 10). Toxicity Evaluation To evaluate the in vivo toxicity of HCPT nanoparticles, a histological analysis of organs was performed (Supporting Information, Figure S6). These images didn’t show any significant difference. To estimate safety of HCPT NSs and NRs furthermore, the liver and kidney function markers including alanine amino transferase (ALT), aspartate amino transferase (AST), blood urea nitrogen (BUN), and creatinine (CRE) were measured (Supporting Information, Table S1). Comparing with control saline group, HCPT injection and NSs groups presented significant differences in ALT, AST, and BUN (p < 0.05), on the contrary, HCPT NRs showed no significant difference (p > 0.05), suggesting no obvious hepatic or renal toxicity of HCPT NRs and indicating the good biosafety. Conclusion 17



Page 18 of 26

Dendritic-linear hybrid compound G2-C18 was synthesized from OEG dendron and octadecylamine, which could be dissolved in deionized water and self-assembled into nanoaggregates with the particle size of approximately 172.4 ± 5.8 nm. Then, HCPT nanospheres (NSs) and nanorods (NRs) were prepared via dialysis method augmenting ultrasonication, utilizing G2-C18 as the drug carrier. It seemed that the feed weight ratio of HCPT vs. G2-C18 not only affected the drug-loading content of HCPT nanoparticles, but also influenced the self-assembled morphology of drug-loaded nanoparticles. With increasing the feed weight ratio, the morphology of HCPT nanoparticles was changed from nanosphere to nanorod. HCPT nanoparticles presented good stability and similar release profiles, but different anticancer efficacy and cellular uptake mechanism. The cytotoxicity of HCPT NRs was enhanced significant comparing with HCPT NSs, the IC50 value was decreased 2-fold (p < 0.05), which could be explained by the different uptake mechanism. More importantly, HCPT NRs showed obviously better antitumor efficacy in vivo, the inhibition rate enhanced 1.3-fold as that of HCPT NSs, according to higher tumor accumulation of HCPT NRs. These results proved the particle morphology influenced antitumor activity significantly, which should be considered in the design of nanocarriers. Acknowledgments This work is financially supported by CAMS Innovation Fund for Medical Sciences (CIFMS, no. 2017-I2M-1-013), CAMS Innovation Fund 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). Supporting Information Supporting information is available free of charge

via the internet at

http://pubs.acs.org. XRD patterns of dried HCPT powder, NSs, and NRs, SEM image of HCPT crystals, particle sizes of HCPT nanoparticles in glucose solution and plasma, TEM and SEM images of different nanocarriers and HCPT nanoparticles respectively, cumulative 18


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release curves, histological analysis, liver/kidney functional markers. Author Information Corresponding Author: Yifei Guo, E-mail: [email protected] Xiangtao Wang, E-mail: [email protected] Notes The authors declare no competing financial interest.

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TOC graphic

HCPT nanoparticles exhibited strong shape-dependent cellular internalization efficiency and antitumor activity, which was influenced by feed weight ratio of HCPT/G2-C18.

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Amphiphilic Hybrid Dendritic-Linear Molecules as Nanocarriers for

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