Shape of Nanoparticles as a Design Parameter to Improve Docetaxel

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The Shape of Nanoparticles as a Design Parameter to Improve Docetaxel Antitumor Efficacy Yifei Guo, Shuang Zhao, Hanhong Qiu, Ting Wang, Yanna Zhao, Meihua Han, Zhengqi Dong, and Xiangtao Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00059 • Publication Date (Web): 09 Feb 2018 Downloaded from http://pubs.acs.org on February 11, 2018

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Bioconjugate Chemistry

The Shape of Nanoparticles as a Design Parameter to Improve Docetaxel Antitumor Efficacy Yifei Guo,a Shuang Zhao,a, b Hanhong Qiu,a Ting Wang,a Yanna Zhao,a, c Meihua Han,a Zhengqi Dong,a, * Xiangtao Wanga,*

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

Research Center on Life Sciences and Environmental Sciences, Harbin University of

Commerce, No. 138, Tongda Street, Daoli District, Harbin 150076, China c

Institute of Biopharmaceutical Research, Liaocheng University, No. 1, Hunan Road,

Liaocheng 252059, China E-mail: [email protected], [email protected]

Abstract It was reported that the shape of nanocarriers played an important role in achieving better therapeutic effect. To optimize the morphology and enhance the antitumor efficacy, in this study based on the amphiphilic PAMAM-b-OEG codendrimer (POD), docetaxel-loaded spherical and flake-like nanoparticles (DTX nanospheres and nanosheets) were prepared via antisolvent precipitation method with the similar particle size, surface charge, stability, and release profiles. The feed weight ratio of DTX/POD and the branched structure of OEG dendron were suggested to influence on the shapes of the self-assembled nanostructures. As expected, DTX nanospheres and nanosheets exhibited strong shape-dependent cellular internalization efficiency and

antitumor

activity.

The

clathrin-mediated

endocytosis

and

macropincytosis-dependent endocytosis were proved to be the main uptake mechanism for DTX nanospheres, while it was clathrin-mediated endocytosis for 1

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DTX nanosheets. More importantly, DTX nanosheets presented obviously superior antitumor efficacy over nanospheres, the tumor inhibition rate was increased 2-fold in vitro and 1.3-fold in vivo. An approximately 2-fold increase in pharmacokinetic parameter (AUC, MRT, and T1/2) and tumor accumulation were observed in DTX nanosheets group. These results suggested the particle shape played a key role in influencing cellular uptake behaviour, pharmacokinetics, biodistribution, and antitumor activity, the shape of drug-loaded nanoparticles should be considered in the design of new generation of nanoscale drug delivery systems for better therapeutic efficacy of anticancer drug. Introduction Owing to the advanced features, including longer blood circulation, better control of drug release kinetics, improved anticancer efficacy, and reduced unfavourable side effects, nanotherapeutics are emerging as the effective method for cancer.1-3 These nanoparticles from self-assembly of amphiphilic polymers could promote antitumor efficacy and reduce side effects due to their high accumulation in tumors through enhanced permeability and retention (EPR) effect and active cellular uptake, have been extensively explored for anticancer drug delivery.4-7 Due to the advanced feature, such as precise control architecture, shape, size, and modifiable surface, dendrimers or dendrons have been developed broadly to prepare drug-loaded nanoparticles, many research groups have done lots of work on dendrimers based nanoparticles.8-12 When preparing these nanocarriers, some parameters including the particle sizes,13, 14

surface modification,15 and drug-loading content,16 are followed to achieve

enhanced antitumor efficacy. Recently, the shape of nanocarriers has been considered to play another important role in influencing cellular uptake, antitumor activity, blood circulation, and biodistribution.17-21 Compared with traditional spherical morphology, several studies have been reported that rod-like or disc-like forms present better cellular uptake efficacy and tumor inhibition rate, and could pass unhindered through the spleen.22-24 For instance, nanodiscs with high aspect-ratio are found to be internalized more efficiently than other shape nanoparticles, the kinetics, efficiency, 2


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and mechanisms of uptake are shape-dependent.24 Besides, based on the polyprodrug amphiphilies

composed

from

PEG

and

polycamptothecin,

four

types

of

self-assembled nanostructures provide prototype models for shape-regulated cellular internalization, trafficking, and drug delivery.25 In our previous study, oligoethylene glycols (OEG) dendron was conjugated with polyamidoamine (PAMAM) to form amphiphilic codendrimers.26,

27

Based on the

amphiphilic codendrimer, honokiol (HK) and 10-hydrocamptothecin (HCPT) were selected as the model drugs to prepare nanoparticles with the spherical and rod-like shape respectively. Through the hydrophobic and electronic interactions, HK and HCPT nanoparticles could be obtained with high drug-loading content and appropriate particle sizes, which showed good biocompatibility and optimized antitumor efficacy. Besides nanospheres and nanorods, the impact of nanosheets on drug delivery systems should be studied furthermore. In this study, based on PAMAM-b-OEG codendrimer POD, DTX as one of the most commonly utilized classes of anticancer agents,28 was selected to prepare drug-loaded nanospheres and nanosheets via ultrasonication method with moderate to good drug-loading content. Then, the physiochemical properties of these nanospheres and nanosheets including the particle size, morphology, self-assembly mechanism, stability, release profiles, cellular uptake, and antitumor efficacy were evaluated. DTX nanospheres and nanosheets exhibited the similar particle sizes, stability, release manners, but nanosheets presented better pharmacokinetics, tumor accumulation, and antitumor efficacy. Results and Discussion Preparation and Characterization of DTX Nanoparticles Based on the codendrimer POD, two shapes of docetaxel (DTX)-loaded nanoparticles (nanospheres and nanosheets) were prepared (Figure 1). DTX and POD were dissolved in DMF, and then injected into deionized water under ultrasonication for 10 min, the resultant white nanospheres and nanosheets were dispersed in deionized 3



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water after dialysis. The drug-loading content (DLC) for nanospheres and nanosheets were approximately 32.6% and 53.2% respectively (Table 1).

Figure 1. Structure of codendrimer POD, OEG dendron, and the self-assembly procedure of DTX nanoparticles. Table 1. Results of DTX nanospheres and nanosheets Sample

a

DLC a

DLS results b

FALT e

EDS results f

(%)

Dh (nm) c

PDI

ζ (mV) d

(nm)

C

N

O

Powder

-

-

-

-6.3

1.2

76.5

1.7

21.8

Nanospheres

32.6%

247.2

0.05

33.8

12.4

66.4

0.0

33.6

Nanosheets

53.2%

266.2

0.13

37.2

11.0

67.9

0.0

32.1

Measured by HPLC with UV detector.

Hydrodynamic diameter.

d

Zeta potential.

b

Dynamic light scattering, 1 mg mL-1.

c

e

Fixed aqueous layer thickness. f Surface

element analysis. The particle size and zeta potential of DTX nanoshperes and nanosheets in aqueous solution showed no significant difference. The nanospheres presented the hydrodynamic sizes of approximately 247.2 ± 5.0 nm (PDI = 0.05 ± 0.01), 4


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meanwhile, the nanosheets had a mean diameter of approximately 266.2 ± 2.4 nm with the height of 34.5 nm (PDI = 0.13 ± 0.06) (Table 1). The particle size distribution curve and microscope images are shown in Figure 2. To confirm the existing state of DTX, bulk powder, nanospheres, and nanosheets were measured by X-ray powder diffraction (Supporting Information, Figure S1). DTX nanosheets presented the obvious diffraction peaks, while the characteristic diffraction peaks of DTX in nanospheres were disappeared. These results suggested that DTX in these two type nanoparticles presented different existing state, which could be explained by the self-assembled mechanism of DTX nanoparticles.

Figure 2. DLS curves of DTX nanospheres (a), DTX nanosheets (c), and SEM images of DTX nanospheres (b, insert graph: TEM image), DTX nanosheets (d, insert graph: TEM image). Scale bar: 500 nm. Self-assembly Mechanism of DTX Nanoparticles Size and shape of self-assembled nanostructures were critical parameters in biomedical application.29-31 In previous reports the DTX nanoparticles exhibited the 5



spherical morphology in general.32-35 In our study, two shapes of DTX nanoparticles, including nanosheets and nanospheres, were obtained utilizing the fluorescently amphiphilic codendrimer POD as nanocarrier. It was reported that several tunable parameters could influence the shapes of the self-assembled nanostructures, which should be researched briefly. Therefore, several factors were evaluated, including concentration, temperature, injection rate, feed weight ratio, and structure of carrier (Supporting Information, Table S1, Figure S2-S4). These results suggested that the concentration, temperature, and injection rate would not affect the self-assembly morphology of DTX nanoparticles.

Figure 3. SEM image of DTX nanoparticles. POD as carrier with different DTX/POD feed weight ratio of 1/2 (a), 1/1 (b), and 2/1 (c); structure influence at the feed weight ratio of 2/1with linear PEG (d) and OEG dendron (e) as carrier. Scale bar: 1 µm. However, it was found that the DTX/POD feed weight ratio and the structure of branched OEG dendron were the mainly factors in the self-assembly procedure of DTX nanoparticles. For DTX/POD feed weight ratio (Figure 3a-c), it seemed that the DTX nanoparticles showed spherical morphology when the feed weight ratio was 1/2 (Figure 3a), increasing the feed weight ratio to 1/1, DTX nanospheres and nanosheets were observed at the same time (Figure 3b), further increasing the feed weight ratio to 2/1, only nanosheets were formed (Figure 3c). Because POD was self-assembled into spherical aggregates (Supporting information, Figure S5), when DTX was added at 6


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the low feed weight ratio, it could be dispersed well in POD aggregates, and the spherical morphology was maintained, while the particle size was enlarged. Enhanced the feed weight ratio, it was impossible to disperse all DTX in POD aggregates. To overcome this problem, DTX nanocrystals were formed with POD dispersed on the surface, therefore the morphology was changed to nanosheets which could be attributed to flake DTX crystal (Supporting information, Figure S6). Then, the influences of branched OEG dendron on the shapes of DTX nanoparticles were studied (Figure 3d-f), the linear PEG (Mn = 2000) was utilized to prepare drug-loaded nanoparticles for comparison. At the same feed weight ratio (2/1), OEG dendron induced the vast majority of DTX nanosheets and little nanospheres (Figure 3e), which was similar as amphiphilic codendrimer POD (Figure 3c), on the contrary, linear PEG produced the nanospheres (Figure 3d), suggesting the branched structure of OEG dendron influenced on the self-assembly procedure. Different from flexible linear PEG, the rigidity of branched OEG denron was enhanced moderate due to the steric hindrance, DTX was more difficult to be dispersed in OEG dendron at the same feed weight ratio, resulting in the majority of flake-like nanocrystals and a small amount of nanospheres. Furthermore, the rigidity of codendrimer POD was enhanced significantly due to the extremely branched structure, it was almost impossible to disperse DTX in POD aggregates, flake-like nanocrystal was performed. Fixed Aqueous Layer Thickness and Surface Element Analysis The fixed aqueous layer thickness (FALT) of DTX nanospheres and nanosheets were determined by the method introduced by Sadzuka and Shi,36, 37 which was 12.4 and 11.0 nm respective (Table 1). The FALT of DTX nanospheres and nanosheets were almost 10 times thicker than that of DTX bulk powder (1.2 nm), the result suggested that codendrimer POD was distributed on the surface of these nanoparticles and branched OEG chains might form ‘brush’ structures due to the strong steric hindrance, which probably prevented the particles to form aggregation and/or agglomeration and increased the stability of the nanoparticles. The composition and content on the surface of DTX bulk powder, nanospheres, and 7



nanosheets were investigated by SEM with energy dispersive X-ray analysis (EDS), and the major element percentage is shown in Table 1. The amount of N element in DTX bulk powder was 1.7%, however the value in DTX nanoparticles was 0%; meanwhile, the content of O element in DTX bulk powder was 21.8%, the value in DTX nanoparticles was 32%~34%. These results suggested that the OEG dendron of codendrimer POD was mostly distributed on the surface of DTX nanospheres and nanosheets, because OEG dendrons contained more O atoms than DTX and no N atoms. These data was in good agreement with those results from the fixed aqueous layer thickness measurement. The exposed OEG dendron as the analogue of linear PEG chains, could suppress the interaction between nanoparticles and plasma proteins and prolong their circulation time in vivo.38 Measurement of Stability For the storage stability study, DTX nanospheres and nanosheets were stored at 25 °C. The particle size of these samples was monitored over 14 days by DLS without further ultrasonication (Supporting Information, Figure S7). After 14 days, the particle sizes of these two DTX nanoparticles were approximately 248.7 ± 9.2 and 326.1 ± 11.4 nm. The particle size increased slightly when compared to the initial value at day 0 (p > 0.05), which could be attributed to the Ostwald ripening phenomenon.39 Furthermore, the stabilities of DTX nanospheres and nanosheets in different media were monitored over 24 h by DLS, and the particle size was recorded. After 24 hours, the particle sizes of DTX nanoparticles were maintained in PBS solution (Supporting Information, Figure S8a), plasma (Supporting Information, Figure S8b), normal saline (Supporting Information, Figure S8c), and glucose solution (Supporting Information, Figure S8d); no significant difference (p > 0.05) was obtained when compared with their initial sizes. All these results indicated that DTX nanospheres and nanosheets possessed similar stability, due to the peripheral hydrophilic OEG dendron hindered the aggregation of particles.

In vitro Studies on Release Kinetics 8


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The release of DTX from nanospheres and nanosheets was examined under sink conditions at 37 °C, the release medium was PBS solution containing 0.5% SDS (pH 7.4). A control experiment using DTX solution (DTX dissolved in DMSO) and DTX powder (DTX powder in H2O) were also carried out under similar conditions; the results are shown in Figure 4. For DTX solution, complete diffusion across the dialysis membrane was found to occur within 12 h. For DTX bulk powder, only 25% DTX was released over 6 days. For DTX nanospheres and nanosheets, it was sustained release for 6 days (reaching over 90% cumulative release) and no significant difference was shown between nanosheets and nanospheres. Comparing with the DTX solution, nanospheres and nanosheets presented lower release rate, the plausible explanation was the OEG dendrons might incorporate in the outer layer of DTX nanoparticles (nanosheets and nanospheres) and form a hydrophilic “shell” around the hydrophobic drug which hindered the diffusion of DTX from inner core to outside medium. Compared with DTX powder, the enhanced release rate of DTX from nanoparticles can be attributed to the increased surface area of the nanoscale particles and enhanced solubility, which may further improve the bioavailability of DTX.

Figure 4. Cumulative DTX release from DTX solution, nanospheres, nanosheets, and bulk powder in PBS solution containing 0.5% SDS (pH 7.4) at 37 °C within 6 days (n = 3). Cytotoxicity Assay The hemolytic activity of DTX nanospheres and nanosheets on rat RBC were researched to examine its suitability for intravenous administration (Support 9



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Information, Figure S9). After incubation with the 2% (w/v) RBC suspension at 37 °C for 5 h, the hemolysis rates of DTX nanospheres and nanosheets were below 5% in concentration ranging from 0.01 to 1 mg mL-1 (DTX equivalent concentration), suggesting of no RBC membrane related toxicity and the good blood compatibility.

Figure 5. Cytotoxicities of DTX nanospheres and nanosheets toward 4T1 cells after incubation for 48 h (n = 5). The cytotoxicity of DTX nanospheres and nanosheets against murine 4T1 breast cancer cell line (4T1 cells) was studied using an MTT assay, with the concentration ranging from 0.01 to 500 µg mL-1 (DTX equivalent concentration) (Figure 5). The IC50 values for free DTX, nanospheres, and nanosheets were 7.84, 2.73, and 1.46 µg mL-1 (DTX equivalent concentration) respectively after incubation for 48 h. Compared with free DTX, both DTX nanospheres and nanosheets exerted a higher cytotoxicity effect against 4T1 cells at the same dose (p < 0.001), which suggested much more DTX was transferred into the tumor cells and proved the activity of DTX nanoparticles were greater than that of the free drug. The enhanced cytotoxicity of nanoparticles could be attributed to the facilitated endocytotic transport, relative to passive diffusion of free DTX through the cell membrane. This was probably due to the mechanism of endocytosis that might effectively circumvent the multi-drug resistance (MDR) effect that occurs for free drug. In the previous study of DTX-loaded nanoparticles, the similar enhanced uptake was observed.40,

41

Importantly, nanosheets presented higher antitumor efficacy in vitro than nanospheres, the IC50 was decreased 2-fold (p < 0.05), suggesting the particle shape also played a 10


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key role in specificity of internalization.42, 43 Cellular Uptake The cell internalization of DTX nanospheres and nanosheets were evaluated in comparison in 4T1 cell lines using fluorescent microcopy imaging system, meanwhile DTX solution was used as control (Figure 6). To qualitatively investigate the cellular uptake of the DTX nanoparticles, 4T1 cells were incubated with the DTX solution, nanospheres, and nanosheets at the equivalent cy5.5 concentration for 3 h. The DTX solution showed weak fluorescence signals in 4T1 cells, on the contrary, nanospheres and nanosheets exhibited greater fluorescence signals (Figure 6). Calculated from the fluorescence intensity (189.7 ± 10.1 vs. 934.6 ± 16.0, 1399.7 ± 17.4, DTX solutions vs. nanospheres and nanosheets), the uptake efficacy of DTX nanoparticles was enhanced significantly (p < 0.001), indicating DTX nanoparticles were taken up better by 4T1 cells (Supporting Information, Figure S10). It was possible that nanoparticles could be preferentially internalized via endocytosis pathway, while free DTX was transported into cells by passive diffusion, which was in good agreement with the cytotoxicity results. Furthermore, nanosheets presented higher endocytosis efficacy than nanospheres, the intracellular fluorescence intensity was increased by 50% (Supporting Information, Figure S10), which could be explained by nanosheets could be accumulated in cellular membrane and internalized more efficiently compared with nanospheres.24 The interaction of particles with cells is known to be affected by particle size and shape. To verify the endocytosis pathway taking part in the internalization process furthermore, 4T1 cells was incubated with various endocytosis inhibitors of caveolae-mediated

endocytosis,

clathrin-mediated

endocytosis,

and

macropincytosis-dependent endocytosis (Figure 6). After incubating for 3 h, nanosheets and nanospheres exhibited different results. For nanospheres, the methyl-β-cyclodextrin (MβCD)-blocking caveolae-mediated endocytosis pathway and the cytochalasin D (CCD)-blocking macropincytosis-dependent endocytosis pathway resulted in significant decrease in the intracellular fluorescence intensity, 11



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approximately 50-60% decrease. Meanwhile, as the inhibitor of clathrin-mediated endocytosis, the sucrose caused no decrease in the intracellular fluorescence intensity. These

results

suggested

that

clathrin-mediated

endocytosis

and

macropincytosis-dependent endocytosis were the main uptake mechanism which contributed to the effective uptake of nanospheres. For nanosheets, only the MβCD resulted in significant decrease in the intracellular fluorescence intensity, approximately 65% decrease, CCD and sucrose didn’t affect the cellular uptake, suggesting clathrin-mediated endocytosis was the main uptake mechanism of nanosheets. These results suggested the shape-dependent cellular uptake efficacy.

Figure 6. Representative fluorescent microscopy images of DTX solution, nanospheres, and nanosheets incubated with different endocytosis inhibitor: methyl-β-cyclodextrin (MβCD), cytochalasin D (CCD), and sucrose. Red: cy5.5. Besides, the DTX concentration in 4T1 cells was analyzed by HPLC with UV detector to verify the cellular uptake of two nanoparticles furthermore (50 µg mL-1, DTX equivalent concentration, Supporing Information, Figure S11). After incubated for 3 h, the cellular uptake rate of DTX were 12.0%, 17.8%, and 22.4% for DTX solution, nanoparticles, and nanosheets respectively, which was calculated based on the actually determined DTX concentration from cell lysis buffer. Similar as the 12


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results of fluorescent images, the cellular uptake of nanospheres and nanosheets were promoted significantly compared to DTX solution (p < 0.01). Besides, nanosheets presented higher uptake ratio than nanospheres (p < 0.05). After incubated with endocytosis inhibitors, the cellular uptake rates of these two particles were decreased. It seemed that the methyl-β-cyclodextrin (MβCD)-blocking caveolae-mediated endocytosis

pathway

and

the

cytochalasin

D

(CCD)-blocking

macropincytosis-dependent endocytosis pathway were the main uptake mechanism for nanospheres (p < 0.05); on the contrary, the cellular uptake rate of nanosheets only influenced by MβCD (p < 0.05), suggesting the caveolae-mediated endocytosis pathway was the main uptake mechanism for nanosheets, which was in accordance with the results of fluorescent microscopy images.

In vivo Antitumor Efficacy The toxicity of codendrimer POD in vivo was investigated first, normal mice were administrated by POD in normal saline solution with the concentration ranging from 0.1 to 20 mg kg-1 via intravenous administration. Even the concentration was elevated to 20 mg kg-1, no weight loss (Supporting Information, Figure S12), no signs of distress (unresponsive, labored breathing, discharge), and no death were shown, indicating the good biosafety of POD. Then, the in vivo antitumor activity of DTX nanoparticles were investigated using BALB/c mice bearing 4T1 breast tumor model, while the saline, POD, and DTX solution were used as control. Tumor-bearing mice were randomly divided into five groups (n = 10): saline (blank control), DTX solution (positive control, 10 mg Kg-1), POD, nanospheres and nanosheets (test groups, 10 mg Kg-1, DTX equivalent concentration), which were administrated every two days for 6 times. The tumor volume was monitored every two days for 12 days. As shown in Figure 7a, the time-related tumor volume increase was observed, compared to saline control group, codendrimer POD didn’t show any antitumor activity, DTX solution group only showed moderate antitumor efficacy. In contrast, both nanospheres and nanosheets groups expressed enhanced antitumor activity than DTX solution group (p < 0.01). Besides, tumor volume of the mice administrated with DTX nanosheets 13



showed the slower growth than those treated with DTX nanospheres (p < 0.05), suggesting the better antitumor efficacy of nanosheets. The tumor inhibition rates calculated based on the average weight of the tumors, further indicating the promoted tumor inhibition activity of nanosheets. The tumor weights were 1.23 ± 0.20, 1.18 ± 0.18, 0.80 ± 0.07, 0.59 ± 0.12, and 0.38 ± 0.05 g for saline, POD, DTX solution, nanospheres, and nanosheets respectively, with the corresponding tumor inhibition rate of 4%, 34%, 52%, and 69% for POD, DTX solution, nanospheres, and nanosheets respectively (Figure 7b). The tumor inhibition rate of nanosheets was 2-fold higher than DTX solution (69% vs. 34%), and 1.3-fold as nanospheres (69% vs. 52%), suggesting the DTX nanoparticles presented advanced antitumor activity, and the shape of nanoparticles showed a significant effect on the antitumor efficacy. Either from tumor growth curves or the final weight of tumor tissue, the DTX nanosheets exhibited good antitumor efficacy, which was in good agreement with the results from the in vitro antitumor study.

Figure 7. Comparison of the in vivo tumor growth inhibition of nanospheres and nanosheets versus DTX solution in BALB/c mice bearing 4T1 breast tumor model: tumor volume change curves (a), tumor inhibition rate (b). For each animal, six consecutive doses were given (marked by arrows). ** p < 0.01 vs. DTX solution, # p < 0.05 vs. DTX nanospheres. Pharmacokinetic and Biodistribution For pharmacokinetic analysis and biodistribution, fluorescence probe DiR was 14


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utilized

to

label

nanospheres

and

nanosheets.

Rats

were

administrated

DiR/nanoparticles at DTX equivalent concentration of 20 mg Kg-1 and DiR equivalent concentration of 0.5 mg Kg-1. Consistent with previously reported pharmacokinetic profiles of DTX nanoparticles,44-46 in vivo pharmacokinetic studies showed that both nanospheres and nanosheets exhibited longer circulation times (Supporting Information, Figure S13), which could be explained by the OEG dendron as an analogue of PEG presented stealth properties.

47, 48

However, nanospheres and

nanosheets showed different parameters (Table 2). Compared with nanospheres, the area-under-the-curve (AUC), the mean residence time (MRT), the half-time (t1/2), and Cmax of nanosheets were promoted significantly, which was 2.7-fold (2723.4 vs. 1011.1), 1.1-fold (10.2 vs. 9.3), 1.3-fold (17.7 vs. 13.6), and 1.3-fold (172.7 vs. 136.1) respectively. These results suggested that DTX in nanosheets was greater exposure to the tumor, resulting in the promoted bioavailability of DTX nanosheets. Table 2. Mean estimates of pharmacokinetic parameters for DTX nanospheres and nanosheets in rats. (n = 3) Samples

AUC0-∞

MRT

T1/2

Cmax

Tmax

(µg h mL-1)

(h)

(h)

(µg mL-1)

(h)

Nanospheres

1011.1

9.3

13.6

136.1

0.5

Nanosheets

2723.4

10.2

17.7

172.7

2.0

Figure 8. Ex-vivo fluorescence images (b), and biodistribution of DTX in main tissues (c). 1, tumor; 2, heart; 3, liver; 4, spleen; 5, lung; 6, kidney. 15


*

p < 0.05 vs.


nanospheres. Figure 8a showed the tumor and tissues accumulation of fluorescent DiR after the mice were treated with DiR/nanospheres and DiR/nanosheets. Nanoparticles frequently accumulate in the mononuclear phagocyte system, including liver and spleen, no significant differences were obtained between nanospheres and nanosheets. While, DTX nanospheres showed weak fluorescence intensity in tumor, on the contrary, nanosheets exhibited greater fluorescence signals, indicating nanosheets possessed greater tumor accumulation. And then, the concentration of DTX in the tumor and major organs (heart, live, spleen, lung, and kidney) was examined (Figure 8b). Both nanoparticles were mainly distributed in the tumor tissue, liver, and spleen. Moreover, the drug concentration of DTX nanosheets in tumor was increased by 2.1-fold (p < 0.05). These results prompted DTX nanosheets could generate high antitumor efficacy, consistent with antitumor efficacy. Conclusion In this study, codendrimer (POD) was utilized to prepare docetaxel spherical and flake-like nanoparticles (nanospheres and nanosheets) via antisolvent precipitation method. Followed by homogenization, these nanospheres and nanosheets were obtained with the similar hydrodynamic size and surface charge. It was found that the feed weight ratio of DTX/POD and the branched structure of OEG dendron influenced the self-assembled shape of drug-loaded nanoparticles. Due to the amphiphilic codendrimer POD possibly dispersed on the surface of these nanoparticles, DTX nanospheres and nanosheets showed good stability and sustained release for 6 days in vitro and no significant difference was observed. Both of these nanoparticles exhibited advanced antitumor efficacy in vitro and in vivo, besides, they showed strong shape dependent cellular internalization efficiency and in vivo antitumor efficacy. The clathrin-mediated endocytosis and macropincytosis-dependent endocytosis were proved to be the main uptake mechanism of nanospheres, while, it was clathrin-mediated endocytosis for nanosheets. More importantly, DTX nanosheets presented obviously superior antitumor efficacy over nanospheres, the IC50 was 16


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decreased 2-fold against 4T1 cells in vitro and the tumor inhibition rate was enhanced 1.3-fold in vivo. Enhancement in pharmacokinetics, including AUC, MRT, and T1/2 were obtained in nanosheets, which also showed 2.1-fold increase in tumor accumulation. These results suggested the particle shape played a key role in influencing cellular uptake behaviour, pharmacokinetics profiles, biodistribution, and antitumor activity, the shape optimization should be considered in the design of new drug nanocarriers to enhance the therapeutic efficacy of cancer therapy. Materials and Methods Materials Codendrimer (POD) was synthesized according to pervious papers.26 Docetaxel (DTX, purity >98%) was purchased from Beijing Ou He Bio-Tech Co., Ltd. (Beijing, China). Dialysis membrane of molecular weight cut-off of 14000 Da was obtained from Spectrapor. Normal saline solution was purchased from Sigma Chemical Co. (America). Other reagents and solvents were purchased of analytical grade and obtained from commercial company. Animals and Cell Line The murine breast cancer (4T1) cell line was purchased from the Institute of Basic Medical Science, Chinese 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 at 37 oC in a humidified 5% CO2 atmosphere. BALB/c mice (20 ± 2 g) and rats were purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All the animals were acclimatized in a laminar flow room at controlled temperature of 25 ± 2 oC, relative humidity of 50-60% and 12 h light-dark cycles with standard diet ad libitum for 1 week prior to experimentation. 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). 17



Preparation of Docetaxel-loaded Nanoparticles Docetaxel-loaded POD nanoparticles (DTX nanosheets and nanospheres) were prepared by the antisolvent precipitation method. Briefly, DTX, fluorescent probe (cy5.5 and DiR), and carries (POD, OEG dendron, PEG) were dissolved in DMF (1 mL) at room temperature, and then the DMF solution was quickly injected into deionized water (5 mL) under continuous ultrasonication for 10 min. The mixed solution was transferred into the dialysis bag (MWCO 14000) and dialyzed against deionized water (4 × 1 L) for 4 h to remove DMF and the free drug. Then, the nanoparticles were homogenized with 5 cycles at 25 oC under 2000 bar pressure using a JN-3000 PLUS homogenizer (JNBIO Inc. China). To quantify the drug-loading content (DLC), the drug in the nanoparticles was collected by lyophilized, dissolved in methanol, and analyzed by HPLC (UltiMate3000, DIONEX) using a UV detector operated at 230 nm. The quantitative analysis was carried out on a Thermo C18 (4.60 mm × 250 mm, 5 µm) and compared to a calibration curve generated from acetonitrile:acetic acid solution (0.1%) (35:65, v/v) (y = 0.44x + 0.02, R2 = 0.9999). A flow rate was 1.0 mL min-1, and the sample injection volume was 20 µL. The DLC was calculated as follows, the experiments were conducted in triplicates, and the data were shown as the mean values plus standard deviation (± SD). DLC = (weight of loaded drug/weight of drug-loaded NPs) ×100% Particle Size and Zeta Potential Measurements The particle size, size distribution, and zeta potential of these DTX nanoparticles were determined by the dynamic light scattering (DLS) analysis using a Zetasizer Nano-ZS analyzer (Malvern Instruments, UK) with an integrated 4 mV He-Ne laser, λ = 633 nm, which used the backscattering detection (scattering angle θ = 173º) at room temperature. Experiments were performed in triplicates, and the data were shown as the mean values plus standard deviation (± SD). Transmission Electron Microscope. Transmission electronic microscopy (TEM) measurements were performed on a 18


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JEM-1400, operating at an acceleration voltage of 80 kV. A drop of DTX nanoparticles (0.2 mg mL-1) was placed on carbon-coated copper grid. After 2 min, the grid was drained by filter paper to remove the aqueous solution, air-drying at room temperature, and then dyeing with uranyl acetate solution (2%, w/v). Scanning Electron Microscope and Surface Element Analysis The morphology and surface chemical composition assay of DTX bulk powders and two nanoparticles were investigated by scanning electron microscopy (SEM) with Energy Dispersive Spectrometer (SEM-EDS; S-4800, Hitachi Limited., Tokyo, Japan). DTX bulk powders were lyophilized directly, a drop of DTX nanoparticles solution (0.2 mg mL-1) was placed on matrix and air-dried, then these samples were sputter-coated with a conductive layer of gold-palladium (Au/Pd) for 1 min. An accelerating potential of 30 mV was used for the observation and analysis. Measurement of the Fixed Aqueous Layer Thickness (FALT) The DTX nanoparticles were centrifuged at 25 °C for 20 min at 13000 rpm and the pellet was washed with a phosphate buffer solution. Then the pellet was resuspended in NaCl solutions with different ion concentrations. The zeta potential was measured and the calculation of FALT (L) was based on the linear correlation between ln ζ (zeta potential) and κ (Debye–Huckel parameter): ln ζ = ln A − κL. Where A is regarded as constant and κ is Debye–Huckel parameter, expressed as κ =

C /0.3 for univalent

salts where C is the molarity of electrolytes. The slope L gives the position of the slipping plane or thickness of the ﬁxed aqueous layer in nm units. The experiments were conducted in triplicates. The data were shown as the mean values plus standard deviation (± SD). X-ray Diffraction Analysis X-ray diffraction analysis was performed using graphite filtered CuKα radiation ( λ = 1.54 Å) at 40 KV and 100 mA with a scanning rate of 8 degree per minute (2θ from 3 ° to 80 °) with an X-ray diffractometer (Rigaku D/Max-2500; Rigaku Denki Co., Tokyo, 19



Japan) at room temperature. Stability Study For the storage stability study, DTX nanoparticles were stored at 25 °C over 14 days separately. The particle size and polydispersity index (PDI) of the samples were measured at 0, 1, 3, 7, and 14 days. The experiments were conducted in triplicates, and the data were shown as the mean values plus standard deviation (± SD). For the solution stability study, DTX nanoparticles were mixed with PBS (pH 7.4), glucose solution (5%, wt-%), normal saline, and plasma at 37 oC, and the particle size of the samples was measured after at 0, 1, 3, 6, 9, and 24 h. The experiments were conducted in triplicates, and the data were shown as the mean values plus standard deviation (± SD).

In vitro Studies on Release Kinetics In vitro release characteristics of DTX nanoparticles were studied by the dialysis method. Briefly, DTX nanoparticles solution (2 mL) was placed in a dialysis bag (MWCO 14000), then immersed in 50 mL of PBS solution containing 0.5% SDS (pH 7.4) at 37 °C with continuous magnetic stirring at 100 rpm under sink conditions. A control experiment using DTX solution (dissolved in DMSO) and DTX bulk powder were also carried out under similar conditions. At predetermined time intervals, 5 mL external solution was withdrawn for analysis and an equal volume of fresh media was replenished. The drug release study was performed for 192 h and all release experiments were performed in triplicates. The amount of DTX released was quantified by a UV-HPLC, the data were shown as the mean values plus standard deviation (± SD). Hemolytic Effect The rat red blood cell (RBC, 2% w/v) solution was prepared and centrifuged at 5000 rpm for 5 min. The plasma supernatant was removed, and the erythrocytes were resuspended in normal saline solution. The DTX nanoparticles were incubated with 20


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the 2% (w/v) RBC suspension at 37 °C for 5 h with different concentrations. Then the RBCs were removed by centrifugation, 150 µL of the supernatant was pipetted into a 96-microwell plate, and the absorbance was measured at 540 nm using a microplate reader (Versamax Tunable Microplate Reader). The results were expressed as percentage hemolysis with the assumption that deionized water causes 100% hemolysis and normal saline solution 0% hemolysis. The experiments were conducted in triplicate, the data were shown as the mean values plus standard deviation (± SD). MTT Assays 4T1 cells were seeded in RPMI-1640 medium supplemented with 10% fetal calf serum, 100 units mL-1 penicicillin G, and streptomycin at 37 oC with 5% CO2 in 96-well plates at a density of 1 × 104 cells per well. After incubation for 48 h, the growth medium was replaced with fresh RPMI-1640. Then, DTX nanoparticles and free DTX solution were added into the wells. After incubation for 48 h, 20 µL MTT solution (5 mg mL-1) were added to each well and incubation was continued for another 4 h. The medium was removed and 200 µL DMSO was added into each well to dissolve the formazan by pipetting up and down for several times. The absorbance of solution in each well was measured using ELISA plate reader at 570 nm to determine the OD value. The cell inhibition rate was calculated as follows. Cell inhibition = (1-ODtreated/ODcontrol) × 100%, where ODtreated was obtained for the cells treated by the nanoparticles, ODcontrol was obtained for the cells treated by the culture medium, and the other culture conditions were the same. Each experiment was done in quintuplicate. The data were shown as the mean values plus standard deviation (± SD). The half maximal inhibitory concentration (IC50 value) was calculated according to the MTT results.

In vitro Cellular Uptake Efficiency The 4T1 cells (1 × 105 per cell) were seeded in a 6-well plate and cultured at 37 °C for 24 h in a humidified atmosphere with 5% CO2, then the cy5.5 labelled agents (1 µg mL-1) were added into the 6-well plate. After 3 h of additional incubation at 37 °C, the 21



medium was removed and the cells were washed with PBS three times, fixed with 4% of paraformaldehyde PBS solution for 15 min. The cellular uptake images were recorded with Delta Vision Microscopy Imaging Systems, and all the images and average fluorescence intensity were recorded under the same condition. The 4T1 cells (5 × 105 per well) were seeded in a 12-well plate and cultured at 37 oC for 48 h in a humidified atmosphere with 5% CO2, then the DTX nanoparticles or solution (DTX equivalent concentration of 50 µg mL-1) were added into the 12-well plate respectively. After additional incubation for 3 h at 37 oC, the medium was removed and the cells were washed with PBS for three times. Then the cells were trypsinized and recollected, and 0.2 mL of RIPA lysis buffer was added to release the intracellular DTX. Furthermore, 1 mL of ethyl acetate was added to precipitate the protein. After vortexing for 1 min, the mixture was centrifuged at 10000 rpm for 15 min. The supernatant was collected, evaporated to dryness under nitrogen at 40 °C, reconstituted in 200 µL methanol, and filtered through a 0.22 µm filter before HPLC analysis. 20 µL of the supernatant was injected into a HPLC system (UltiMate3000, DIONEX) using a UV detector. All experiments were performed in triplicate. Cellular uptake ratio = (DTX concentration in 4T1 cells/50 µg mL-1) ×100%

In vivo Antitumor Efficacy on 4T1-tumor Bearing Mice In vivo antitumor activity was evaluated using 4T1-tumor bearing mice models. Briefly, BALB/c mice (5-6 weeks, 18-22g) were induced for 4T1 tumor by subcutaneously injection of 0.2 mL cell suspension (5 × 106 4T1 cells) in the right armpit. When the tumor exceeded 100 mm3 (10 days after implantation), the 4T1-tumor bearing mice were randomly divided into 5 groups (10 mice per group). Mice were treated with normal saline (control group), POD, DTX injection at a concentration of 10 mg Kg-1 (positive group), DTX nanosheets and nanospheres at concentrations of 10 mg Kg-1 (test groups) in the volume of 0.2 mL via intravenous (i.v.) administration every 2 days for 6 times. During the administration process, the relative body weight of the mice was monitored as an index of systemic toxicity. Tumor volume was measured daily with a caliper and calculated as: tumor volume 22


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(mm3) = 0.5 × L × W2, where L and W represent the largest diameter and the smallest diameter, respectively. Mice were sacrificed on the 12th day, tumors and main organs were excised, and weighed. The inhibitory rate (IR) of the tumor was calculated as follows, and the data were shown as the mean values plus standard deviation (± SD). IR = (1 - tumor weight of treated group/tumor weight of the control group) × 100%. Pharmacokinetic Studies in Rats The pharmacokinetic behaviors of nanospheres and nanosheets were evaluated by the determination of the DiR content in rat plasma, 10 SD rats (200-220 g) were used and randomly divided into two groups (n = 5). Rats were treated with DiR labelled DTX nanosheets and nanospheres in the volume of 1.0 mL via intravenous (i.v.) administration at DTX equivalent concentration of 20 mg Kg-1 (DiR equivalent concentration of 0.5 mg Kg-1). The blood samples (0.5 mL) were collected at 0.08, 0.17, 0.33, 0.5, 1, 2, 4, 12, and 24 h after administration. All blood samples were immediately centrifuged at 3000 rpm for 20 min to obtain plasma. Plasma was stored at -20 °C. Furthermore, 1 mL of ethyl acetate was added to 100 µL plasma to precipitate the protein. After vortexing for 5 min, the mixture was centrifuged at 10000 rpm for 10 min. The supernatant was collected, evaporated to dryness under nitrogen at 40 °C, reconstituted in 100 µL acetonitrile. After centrifuged at 10000 rpm for 10 min, 20 µL of the supernatant was injected into a HPLC system (UltiMate3000, DIONEX) using a fluorescence detector (excitation/emission wavelengths 748/780 nm). The DiR quantitative analysis was carried out on a Thermo C18 (4.60 mm × 250 mm, 5 µm) and compared to the calibration curves generated from acetonitrile with a flow rate of 1.0 mL min-1. PK parameters were assessed with Phoenix WinNonlin. Biodistribution in Tumor-bearing Mice BALB/c mice (5-6 weeks, 18-22 g) were induced for 4T1 tumor by subcutaneously injection of 0.2 mL cell suspension (5 × 106 4T1 cells) in the right armpit. When tumor exceeded 100 mm3 (10 days after implantation), mice were randomly divided into 2 groups (10 mice per group). Mice were administrated with DTX nanospheres 23



and nanosheets at a concentation of 10 mg Kg-1 in the final volume of 0.2 mL via the tail vein. All mice were sacrificed after administration at 24 h, the heart, liver, spleen, lung, kidney, tumor were collected, washed, accurately weighed and homogenized with normal saline solution. The DiR concentration in the tissues was analyzed by HPLC (UltiMate3000, DIONEX) using a fluorescence detector. Calibration curves were established respectively for the tumor and organs; all of the correlation coefficients were more than 0.99. Statistical Analysis One-way analysis of variance (ANOVA) (SPSS 19.0, USA) was utilized for the statistical evaluation. P < 0.05 was considered as significant and P < 0.001 was considered as highly significant. 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 is available free of charge on the ACS Publications website.

Author Information Corresponding Author: Zhengqi Dong, E-mail: [email protected] Xiangtao Wang, E-mail: [email protected] Notes The authors declare no competing financial interest.

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TOC graphic

DTX nanoparticles exhibited strong shape-dependent cellular internalization efficiency and antitumor activity, which was influenced by feed weight ratio of DTX/POD and the branched structure of OEG dendron.

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Shape of Nanoparticles as a Design Parameter to Improve Docetaxel

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