Carrier-Free, Pure Nanodrug Formed by the Self-Assembly of an

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Carrier-free, pure nanodrug formed by self-assembly of anti-cancer drug for cancer immune therapy Lulu Fan, Bingchen Zhang, Aixiao Xu, Zhichun Shen, Yan Guo, Ruirui Zhao, Huilu Yao, and Jing-Wei Shao Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00444 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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Molecular Pharmaceutics

Carrier-free, pure nanodrug formed by self-assembly of anti-cancer drug for cancer immune therapy Authors: Lulu Fan

a,1

, Bingchen Zhang

a,1

, Aixiao Xu a, Zhichun Shena, Yan Guo a,

Ruirui Zhaoa, HuiluYao b, Jing-Wei Shao a* Affiliations: a

Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of

State Key Laboratory of Photocatalysis on Energy and Environment and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350108, China b

School of Physical Science and Technology , Guangxi University, Guangxi 530004,

China 1 These authors contributed equally to this work. * Corresponding author: Jingwei Shao Address: 2 Xueyuan Road, Sunshine Technology Building, 6FL, Fuzhou University, Fuzhou, Fujian 350116, China. E-mail: [email protected] (J.W. Shao), [email protected] (J.W. Shao) Telephone: + 86-13600802402 No potential conflicts of interests were disclosed.

1

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Ursolic acid (UA) is a food-plant derived natural product which has good anticancer activities and low toxicity. However, the poor water solubility of UA limits its application in clinic. To address this issue, we developed a carrier-free nanodrug by self-assembly of UA. Here, we showed that UA NPs was near-spherical shape with the size of ~150 nm diameter. UA NPs exhibited higher antiproliferative activity, significantly caused apoptosis, decreased the expression of COX-2/VEGFR2/VEGFA and increased the immunostimulatory activity of TNF-α, IL-6 and IFN-β and decrease the activity of STAT-3 in A549 cells in vitro. Furthermore, UA NPs could inhibit tumor growth and has the ability of liver protection in vivo. More importantly, UA NPs could significantly improve the activation of CD4+ T cells, which indicated UA NPs has the potential for immunotherapy. Overall, carrier-free UA nanodrug may be a promising drug for further enhance its anti-cancer efficacy and immune function.

KEY WORDS: Carrier-free nanodrug, Self-assembly, UA NPs, liver protection, immunotherapy.

2


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Background Ursolic acid (UA, 3β-hydroxy-urs-12-en-28-oic acid) is a natural pentacyclic triterpenoid carboxylic that found in many of Chinese traditional herbs and edible plants, including dogwood, self-heal, cranberry and other fruits. UA exhibits a wide range of biologic properties in many of human diseases, such as anti-inflammatory 1-3, anti-angiogenesis 4, anti-diabetic 5, 6， anti-cancer 7-10 , anti-HIV 11-13 and antimalarial activities 14. In recent years, its anticancer activities have been paid close attention 10, 15

. In particular, UA has the advantage of non-toxic to normal cells, which makes it

suitable for cancer metastatic treatment

16, 17

. However, the low tumor-targeting

specificity and poor bioavailability of UA limits its application in clinic. To address this issue, we previously have successfully synthesized a series of UA derivatives UA-5b 15, 18 US597 18 and Asp-UA 19. Among them，US597, and Asp-UA could safe and effective inhibit cancer metastasis both in vitro and in vivo, and being developed by us as potential anti-cancer drug candidates for preventing cancer metastasis. The above UA derivatives are focused on the structure modification to improve UA characteristics, the synthesis procedure is relatively complicated and most of these UA derivatives are lack of water solubility. Others who improve UA characteristics by structure modification obtained similar results

20, 21

. Therefore, it is

essential that develop a new strategy to improve the bioavailability of UA as well as increased its water solubility and anticancer efficacy. Drugs were developed to be encapsulated in a variety of nanocarriers， which in order to improve drugs biocompatibility and recognize cancer tissue, enabling 3



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22

visualization of tumors and delivery of anti-cancer drugs

. Moreover, the

nanocarriers help drugs enhance permeability and retention (EPR) effect [23] and increase water solubility. Although, these drug nanocarriers have above the advantages, there are still some problems, such as low drug loading capacity

24

and

almost all nanocarriers have no therapeutic effect by themselves. The nanocarriers are usually inert materials, they are only as the vehicles, but the drug formulation of inert materials may be increased systemic toxicity, which would be imposed an extra burden on the patients. Therefore, it is highly desirable to develop a drug delivery system with high loading drug and without use of inert materials. Self-assemble delivery system has aroused scientists’ attention and it has been well reported in recent years

25-29

. The anticancer drugs could display nanoscale characteristics by

themselves self-assemble without use of nano-carriers, Zhu and Yan et al reported that hydrophilic drugs and hydrophobic drugs conjugated via self-assembly could improve drugs anti-tumor effect

30-32

. Moreover, researchers suggested that the interaction

between the drugs molecular could construct self-delivery systems with improved therapeutic efficiency. For example, Zhu et al reported that the supramolecular nondrugs self-assemble based on non-covalent interactions

33, 34

. Therefore,

self-delivery system is a promising drug system for cancer therapy in the future. In the present study, we reported a simple and green approach to design a carrier-free pure nanodrug by self-assembly of UA molecules.

It was noted that the

UA was nanosized by pure UA self-assemble which was without help of vehicle and water-soluble drugs. UA nanoparticles were synthesized via the interaction between 4


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UA molecular base on electrostatic and hydrophobic interaction. We investigated the antitumor effect of UA NPs on the cell growth, cellular uptake and apoptosis induction in A549 cells in vitro. Additionally, we evaluated whether UA/UA NPs had a potential for the liver prevention in vivo. Moreover, we studied whether UA/UA NPs could suppress tumor growth and improve the T cells population in vivo by the BALB/C nude mice with a human non-small cell lung cancer A549 xenograft.

Experimental section Materials and Methods Ursolic acid (UA, >90% pure) was purchased from Sartorius Scientific Instruments Co., Ltd. (Beijing China). ALT kit and AST kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), methanol was purchased from Sinopharm Chemical Reagent Co.,Ltd, Fluorescein isothiocyanate (FITC) and 4′,6-diamidino-2-phenylindole (DAPI) were procured form Sigma Aldrich and Invitrogen respectively, Trypsin–EDTA and phosphate buffered solution (PBS) were bought from Gibco-BRL (Burlington, ON, Canada). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Life Technologies GmbH (Darmstadt, Germany). The characterizations of UA NPs were performed by Malvern Zetasizer Nano ZS (Malvern Instruments, UK), Transmission Electron Microscope (FEI, USA), Atomic Force Microscopy (Bruker, USA), X-ray Diffractometer (netherlands panalytical, Netherlands), UV-vis spectrophotometer (UV-2700, Shimadzu, Japan) and fluorescence spectrometer (F-7000, Hitachi, Japan), respectively.

All other reagents used in this study were of the highest purity

5



commercially available. Animals were carried out in accordance with the NSFC regulation concerning the care and use of experimental animals and approved by our Animal Care and Use Committee to reduce the suffering and use of animals. All mice were killed with excess amounts of anesthetic. Female nude mice (age 6-8 weeks, body weight 20-25 g) were obtained from the Shanghai Laboratory Animal Center (Shanghai, China), and housed individually in plastic cages at 20-25 ℃, with lighting on from 8:00 AM to 8:00 PM. Throughout the experiments, mice were maintained with free access to pellet food and water. All experimental procedures were approved and carried out in compliance with the related ethical regulations of our university. All efforts were made to minimize the animals suffering and to reduce the number of animals used. Cells and culture A549, HELF and HeLa cells were all obtained from our own laboratory. HeLa cells were cultured in DMEM medium and A549 cells were cultured in RPMI-1640 medium supplemented with fetal bovine serum (FBS, 10 %), penicillin (100 U/mL) and streptomycin (100 µg/mL) in a humidified atmosphere of 5 % CO2 at 37 ℃. Preparation of UA NPs and FITC-UA NPs UA NPs were prepared by a solvent exchanging method. Briefly, 3 mg UA/ethanol solution (1 mL, 6.569 mM) was slowly poured into 10 mL of water at room temperature under vigorous stirring at 1000 rpm. After mixing for 5 min, the sample was ultrasound for 15 min. The methanol in the UA NPs solution was dried by nitrogen, the UA NPs solution was centrifuged for 5 min at 2000 rpm and the 6


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precipitate was removed. Preparation of FITC labeled UA NPs (FITC- UA NPs) is similar to preparation of UA NPs. Briefly, 3 mg UA/ethanol solution (1 mL, 6.569 mM) was poured into 10 mL of water which content 90 µg FITC at room temperature under vigorous stirring at 1000 rpm. After mixing for 5min, the sample was ultrasound for 15 min. The methanol in the UA NPs solution was dried by nitrogen, the UA NPs solution was centrifuged for 5 min at 2000 rpm and the precipitate was removed. Finally, FITC-UA NPs solution was purified by water with a dialysis membrane (MWCO 500) over 24 h and then the dialysate was dried by lyophilization to yield FITC-UA NPs. The UA NPs and FITC-UA NPs were finally formed and further used for characterization. Other formulations with different molar ratios of UA to water were prepared in the same way. The size and surface charge of NPs can be finely modulated by changing some parameters such as ratio of methanol and water, the different concentrations of UA and UA was dissolved in different solvents. Characterization of NPs To determine the particle size and zeta potential of NPs, the Malvern Zetasizer Nano ZS (Malvern Instruments, UK) was performed at 25 ℃. The mean diameter and polydispersity index of the samples were evaluated in order to assess the particle size distribution, (TEM) was performed by a Tecnai G2F20 when a drop of sample was carefully applied to the carbon-coated copper grids and dried in vacuum. atomic force microscopy (AFM, Nano Surface Division, Bruker, USA) was operated in tapping mode and transmission electron microscopy. XRD patterns of UA and UA 7



NPs were measured by Powder X-ray diffraction (X’ pert3 and Empyrean, netherlands panalytical, Netherlands) with Cu Kα radiation. UV data were recorded at room temperature using an UV-vis spectrophotometer (UV-2700, Shimadzu, Japan). The turbidity of the NPs was determined in the form of absorbance at about 210 nm by an UV-vis spectrophotometer. A fluorescence spectrometer (F-7000, Hitachi, Japan) was used to measure the photoluminescence of free FITC and co-assembled nanoparticles FITC-UA NPs in water in a 1.0 cm quartz cuvette. Drug loading and drug release profile Drug loading was calculated similar to previously study 35 which were defined as the weight ratio of the encapsulated drugs to the entire drug-loaded nanomedicines. The methanol in the UA NPs was dried and then the UA NPs solution was centrifuged for 5 min at 2000 rpm. Taking 20 µL of the supernatant was diluted in 180 µL methnol (v/v=1:9), the concentration of functionalized UA NPs was determined by UV-vis measurement at about 210 nm. The drug release profiles were determined by a dialysis method. 1 mL of UA NPs solution was sealed in dialysis bags (Sigma, 1000 MW cutoff), and then immersed in 200 mL phosphate-buffered saline (PBS) solution (pH 7.4 or 5.5) containing 0.1% (v/v) Tween 80 to maintain the sink condition. the beakers were placed into water bath at 37 °C and stirring was maintained at 100 rpm for 24 h. Samples were withdrawn at predetermined time intervals (0.5, 1, 2, 4, 8, 12, 24 h), The drug content of samples was analyzed by HPLC. All the experiments were carried out in triplicate. 8


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HPLC condition A Waters-2695 HPLC system equipped with a 2487 UV detector was used for the analysis of UA concentration. The mobile phase was a mixture of methanol (A) and 0.1% formic acid aqueous solution (B). The injection volume was 20 µL. The flow rate was 1.0 mL/min and the column temperature was ambient. The detection of eluted samples was performed at 210 nm. In vitro antiproliferative activity/viability To determine the effect of UA NPs on in vitro antiproliferative activity, the in vitro cytotoxicity of free UA and UA NPs were tested in two cell lines A549 (human lung adenocarcinoma) and HeLa (human cervical epithelioid carcinoma). Briefly, cells (8×103/well) were cultured on 96-well plates and incubated for 24 h at 37 ℃ with 5 % CO2, free UA and UA NPs diluted in culture medium with 10 % FBS to various concentrations (0, 10, 20, 30, 40 and 50 µM) for 12 h or 24 h at 37 ℃ with 5 % CO2. After the treatment, cells were incubated with the MTT agent in the medium without phenol red and serum for 4 h at 37 ℃. After the medium was removed, 100 µL of DMSO was added to the well for 20 min. The amount of MTT formazan product was determined by detecting the absorbance at 570 nm using an infnite M200 Pro microplate reader (Tecan, Switzerland), triplicate wells were analyzed at each dose. Cellular uptake A549 cells were seeded in 12-well plates with 0.5 mL growth medium and incubated overnight. Then, cells were incubated with free FITC or 10 µM FITC-UA NPs for 4 h at 37 ℃. Subsequently, the cells in each well were washed with PBS for 9



three times, after that, cells were fixed for 10 min by paraformal, washed with PBS for three times. Then, cells were stained with 200 µL Hoechst 33342 for 15 min, washed with PBS for three times. At last, the cells were measured using a Leica confocal microscope (SP-8, Germany). Cellular accumulation of UA NPs A549 cells were seeded in 24-well plates at a density of 5×104 cells/well/ml and incubated overnight. The cells were treated with 30 µM UA NPs or UA for 0.5, 1, and 2 h. cells were washed with PBS twice and then lysed with PBS containing 1% Triton X-100 (300 µl/well). Cell lysates were lyophilized and then extracted with methanol (1 ml/sample) for 4 h. Methanol extracts were centrifuged at 5000 rpm for 10 min at 4 °C and the supernatants were subjected to HPLC analysis. Cell apoptosis The effects of free UA and UA NPs on cell apoptosis were evaluated by flow cytometry (FCM). A549 cells seeded on 6-well plates (105 cells per well) were incubated with free UA and UA NPs, Untreated cells were used as control. After the treatment, the cells were collected by centrifugation, washed twice with PBS, carefully trypsinized to avoid mechanical damage of the membrane and the mixture solution of containing Annexin V-FITC and PI was added to the samples to stain the cells for 15 min in the dark according to the instructions. Then, the cell populations at early and late apoptotic stages in A549 cells were measured by flow cytometry (BD Bioscience, FACS AriaIII). Western blot 10


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A549 cells were treated with UA (20 µM) and UA NPs (10 and 20 µM) for 24 h. Briefly, cell lysates were collected using immunoprecipitation (RIPA) lysis buffer. Samples with equivalent amounts of proteins using BCA protein assay kit were denatured in sample buffer at 100 ℃ for 5 min. And the samples were prepared resolved on 10 % gel SDS-PAGE. Proteins were electrophoretically transferred to a polyvinyldene difluoride (PVDF) membrane for 2 h. After the transfer, the membranes were blocked by blocking solution. The blots were washed three times with TBST, and they were incubated with specifc primary antibodies diluted 1:1000 in TBST solution overnight at 4 ℃. Subsequently, the blots were washed three times with TBST, followed by incubation with a secondary antibody conjugated with horseradish peroxidase diluted 1:5000 in TBST solution for 2 h at 37 ℃. The membranes were washed with TBST and TBS. Chemiluminescent signals were generated using a Super Signal West Pico Chemiluminescent Substrate kit (Pierce), and detected by using the ChemiDoc XRS system (Bio-Rad). The target proteins expression was quantifed by use of Image Lab analysis software (Bio-Rad). Real-time PCR assay Gene expression of TNF-α, STAT-3, IL-6 and IFN-β was analyzed through real-time RT-PCR method. Master mixture of the following reaction was prepared as follows: 5 µL master mix SYBER Green, 0.4 µL forward primer, 0.4 µL reverse primer and 2.2 µL RNase-free water. Then 2 µL cDNA was added to 18 µL of master mixture and the mixture was placed in real-time RT-PCR. The real-time RT-PCR run procedure was as follows: denaturation program (94 ℃ for 10 min), amplification, 11



and quantification program repeated 45 times (94 ℃ for 15 s, 55 ℃ for 15 s, and 72 ℃ for 15 s). In vivo antitumor growth assay To establish the A549 cancer xenograft growth model, A549 cells were harvested, washed and re-suspended with appropriate concentrations in PBS to a final density of 5×106 cells /200 µL, then injected into the oxter of BALB/C nude mice by the hypodermic inoculation to form the tumor tissue in the oxter. Nude mice were randomly divided into three groups (n = 6 for each group), and then they were given with PBS, free UA and UA NPs (at the dose of 8 mg/kg) by tail vein injection for 21 days, respectively. Mice weight and tumor volume were measured every two days for 11 times. Nude mice were sacrificed at the 21st days and tumor weight was performed. The level of transminase in mice serum BALB/C nude mice about 8 weeks old weighing about 20–25 g were housed for 3 day to acclimatize. The mice were established the A549 cancer xenograft growth model and the mice were randomly divided into 6 groups each comprised of six mice. The first group, control group, was only injected PBS in caudal vein. The second group, CCl4 group, 20 % of CCl4 dissolved in olive oil was injected in abdominal cavity at the dose of 0.4 mL/kg.

The third group, free UA group, was only injected 8

mg/kg UA in caudal vein. The fourth group, UA NPs group, was only injected 8 mg/kg UA NPs in caudal vein. The fifth group, UA+CCl4 group, was injected 8 mg/kg UA in caudal vein, and 2 h later, was injected 20 % CCl4 in abdominal cavity at the 12


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dose of 0.4 mL/kg. The sixth group was injected 8 mg/kg UA NPs in caudal vein, and 2 h later, was injected 20 % CCl4 in abdominal cavity at the dose of 0.4 mL/kg. After 24 h, the mice were sacrificed and the blood was collected. The blood was centrifuged for 15 min at 1500 g. The ALT and AST kit were used for quantitation of transaminase in mice serum in diﬀerent groups. Flow cytometric analysis of the relative abundance of tumour-infiltrating T-cell populations. The peripheral blood of the nude mice was harvested at the 21th. The buffer (PBS+2% FBS) added to the blood and centrifugated for 10 min at 1000 rpm. Then, red blood cells were lysed using ACK Lysis Buffer (Life Technologies) and the suspended at a density 1 ×106/mL. After, the cells were stained with surface anti-mouse CD8 and CD4 for 30 min on ice. The cells were collected by centrifugation, washed twice with buffer. Finally, the CD8+ and CD4+ T cells were measured by flow cytometry (BD Bioscience, FACS AriaIII). Tumors deposition of UA NPs BALB/C nude mice were established the A549 cancer xenograft growth model and the mice were randomly divided into 2 groups each comprised of six mice. The mice were sacrificed after intravenous injection of 20 mg/kg free UA or UA NPs for 4 h, respectively, and then tumor was excised and weighed. Tumors extracts were prepared by adding 2 mL of ethyl acetate to 1 g of homogenize tumors. The mixtures were vortexed for 5 min followed by centrifugation at 3,500 × g for 15 min. The organic phases were removed and volatilized. The residue was reconstituted in 100 µL 13



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of methanol. The supernatant of 20 µL was injected into HPLC system for analysis. Statistical analysis All data were presented as mean ± S.D of three determinations. Statistical analysis was evaluated using Student’s t-test and one-way analysis of variance. Multiple comparisons of the means were performed by the least significantly difference (LSD) test. Differences were considered statistically significant at P < 0.05. Data variation analysis was made by using SPSS statistical software (version 16.0).

Results Preparation and characterization of UA NPs and FITC-UA NPs Among the modes of fabricating drug delivery carriers, self-assembly plays an important role in the fields of both nanobiotechnology and biomedical

36-38

. In this

study, The UA NPs were prepared and optimized depend on a simple and green approach by solvent exchanging method. It is known to that UA is the hydrophobic drug with poor solubility in water. We used different solvents (DMSO, CH2Cl2, CHCl3, EtOH and MeOH) with a ratio of 1:10 to dissolve UA, and then UA solution was slowly poured into the water with vigorous string. It was found that used EtOH dissolved UA could obtain the better size and PDI which mean EtOH was a good solvents for UA (Table1). We found that UA could formed into the UA NPs with different proportion gradients of 1:1 to 1:10, but the ratio 1:10 with 163.3 nm and 0.162 PDI was better than others (Table2). At the same time, we found that UA was finely nanosized with different concentration gradients of 75, 150, 300, 600, 800 and 1200 µM, there was the smaller size and PDI in the concentration of 600 µM (Table3). 14


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Hence, it was indicated that the best size of self-assemble NPs could be adjusted by solvents, the ratio of EtOH/water and concentrations. In our study, it was interesting that UA nanosized by itself, it was hypothesized that UA NPs formed into nanoparticles by hydrophobic interactions and hydrogen bond interactions between UA molecules (Fig.1). We also used DS (Discovery Studio) 4.0 to analysis of UA NPs self-assemble. As shown in Fig. S1, the UA self-assembly into UA NPs were mainly via hydrophobic interactions (in purple) and hydrogen bond interactions (in red). With the optimal condition, we found that the size of UA NPs and FITC-UA NPs were approximately 150 nm (Fig.2A). It was indicated that the small molecular of FITC not change the particle size of NPs. Of course, UA NPs and FITC-UA NPs were relatively small, therefore these particles administrated into the blood, it will render a good enhanced permeability and retention (EPR) effect. In addition, the surface charge of UA NPs was about -7 mV (Fig.2B) and the surface charge of FITC-UA NPs was about -23 mV (Fig.2B). The surface charge of FITC-UA NPs was higher than UA NPs, it may be FITC negatively charged. The previously study indicated that negative charged nanoparticles had ability to display a slow or reduced opsonization via reticular endothelial system (RES), which promoted the blood circulation time

39-41

.

On the other hand, UA NPs and FITC-UA NPs displayed spherical shapes with monodispersed morphology, as shown in the TEM and AFM (Fig.2C and 2D), indicating that a good spherical stability depends on stronger hydrophobic interactions and hydrogen bond interactions. XRD patterns of UA showed high intensity peaks at the diffraction angles 2θ: 5.5, 8.38, 10.98, 14.9, 16.8, 22.01 and 27.12, suggesting the 15



crystalline pattern of the drug (Fig.2E). XRD of UA NPs was exhibited only broad bands, characterizing amorphous state of UA NPs (Fig.2F). When the UA molecules self-assemble into UA NPs, the particles solutions of UA NPs and FITC-UA NPs were displayed blue color and yellow color (Fig.3A), respectively. Tyndall effect can occur when the solution of UA NPs and FITC-UA NPs were irradiated with a laser, it was showed that the UA molecules had been formed

into NPs (Fig.3B). Where UA molecules interacted with each other and

formed into NPs at nanoscale

42, 43

. UV spectra showed that the assembled UA NPs

and FITC-UA NPs had the typical absorbance peaks from UA (∼210 nm). We found that the UA NPs and FITC-UA NPs were happened red-shifted absorption (Fig.3C), indicates that the pentacyclic triterpene was able to interaction each other by hydrophobic interactions. In addition, the intensity of fluorescence emission peaks of FITC inside the NPs were dramatically decreased compared to monomeric FITC (Fig. 2D), indicating excitonic migration between stacked FITC molecules 44. Furthermore, when sodium dodecyl sulfate (SDS, 0.2% w/v) was added to the NPs aqueous solution, the UV- vis absorption of UA NPs was decreased (Fig.3E), it is an indicator for changes in the assembled architecture of UA NPs, were observed may due to the hydrophobic interactions by SDS

44, 45

. The results showed that hydrophobic

interactions may be the main driving forces for formation of the NPs. Drug loading efficiency and release from UA NPs To investigate stability of self-assemble NPs, we measured the size and zeta in different time points. As shown in Fig.4A and Fig.4B, it is found that there was no 16


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significant changes in both size (Fig.4A) and zeta potential (Fig.4B) of NPs during storage for up to 7 days in water. It is displayed that the self-assemble NPs maintain stability possibly due to the hydrophobic interactions and electrostatic force. The outcomes indicated that self-assemble NPs had a great stability in water which improved the water solubility of UA. It has been reported that tumor in a slightly acidic environment

46

. Thus, we

investigated the release profiles of assembly UA NPs and free UA in vitro under different pH conditions. In the Fig.4C, It is no significant difference in the character for free UA under the environment of pH=7.4 and pH=5.5, free UA released fastly from dialysis bag. Free UA was releasing about 94.4% and 91.8% in the pH=7.4 and pH=5.5 after 24 h, respectively. In contrast, the NPs release slower than free UA under the same conditions. Approximately 62.7 % of UA release from the NPs in acid solution (pH=5.5) during 24 h, under the PBS solution of pH = 7.4, the NPs release of UA gets a much lower than pH=5.5, which indicated that UA NPs could increase the amount of UA reaching to the tumor site.

According to Fig.4D, the concentration

was calculated as follows: (0.158 + 0.09379)⁄0.006412 = 39.2685 μM . The concentration (v/v=1:9) of UA NPs was 39.2685 × 10 = 392.69 μM. The volume of suspensions is 10 mL, thus the weight of encapsulated drugs is W = C × V × M = 392.69 μM × 10 × 10 L × 456.68 g⁄mol = 1.793337 × 10 g. The weight of the entire drug-loaded nanomedicines is the total weight of drugs. Therefore, the drugs loading is about 60 % (1.793337 × 10 ⁄3 × 10 × 100 = 60%). Effect of UA NPs on cell viability 17



The effects of UA NPs on cell viability were tested in three different cancer cell lines A549, HeLa and normal cells HELF. As shown in Fig.5A-D, free UA and UA NPs could inhibit A549 and HeLa cells growth in a dose- and time- dependent manner. In addition, the IC50 of HeLa induced by free UA and UA NPs at 12 h and 24 h were relatively higher than A549 cells, indicating that A549 cells were more sensitive to the UA NPs and UA. Moreover, we found that UA and UA NPs treatments at the same concentration, UA NPs were more effectively suppressed the cancer cells growth no matter what A549 cells or HeLa cells. Especially, UA NPs could significantly inhibit A549 cells proliferation at the low concentration 10 µM. However, UA slightly inhibited A549 cells growth the concentration of 10 µM. It was suggested that UA NPs was better to inhibit tumor cells growth, which contributed to UA NPs improved UA water solubility. Furthermore, to determine the cytotoxicity of UA and UA NPs on normal human cells, we treated UA and UA NPs to the normal lung cells HELF by MTT assay. UA and UA NPs inhibited HELF cells viability at a much higher concentration with an IC50 value 48.12 µM and 39.74 µM at 24 h, respectively. It was showed that the UA/UA NPs has the low toxicity to normal cells comparison to tumor cells (Fig.5E-F). Cellular uptake of FITC-UA NPs To visualize the cellular uptake performance of free FITC and FITC labeled UA NPs were prepared and studied by confocal microscopy. As shown in Fig.S2, Hoechst 33342 channel (blue), FITC channel (green), in the free FITC group, the nuclei of A549 cells showed a weak green after staining with Hoechst 33342, and the cells 18


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nuclei are complete without damage. When the cells were treated with FITC-UA NPs, strong green fluorescent signals originating from FITC were detected around the A549 cells. It is showed that UA NPs are able to permeate by passive diffusion efficiently. Effect of UA NPs on cell apoptosis The effect of free UA and UA NPs on cell apoptosis in A549 cells were evaluated by flow cytometry. We exposed A549 cells to 20 µM free UA and UA NPs for 12 h, respectively. Then cells were stained with the AnnexinV-FITC/PI double staining kit for analysis by flow cytometry. The induced cell apoptosis was characterized by counting the cell populations at early and late apoptotic stages. As shown in Fig.6A and 6B, when cells were treated with 20 µM free UA, the induced apoptosis percentages by free UA was 23.23 % in A549cells. The UA NPs possessed higher efficacy of inducing A549 cells apoptosis compared with that of free UA, which the apoptotic cell percentage was 32.65 %. It was showed that UA NPs improve UA cell apoptosis. Effect of UA NPs on cell accumulation In older to determine the cell accumulation of free UA and UA NPs in A549 cells, we evaluated the intracellular accumulation of UA by HPLC. At the same concentration, the cells were treated with UA NPs or UA at different time 0.5, 1 and 2 h. As shown in Fig.6C, UA NPs were significantly increased UA accumulation in A549 cells. After 2 h, the cell accumulation of UA NPs was ~2.4-fold higher than the UA, it is indicated that UA NPs could increase the UA uptake. Effect of UA NPs on the COX-2/VEGFA pathway 19



COX-2/VEGFA pathway plays an important role in the control of tumor cells growth, survival, progression, and apoptosis. Therefore, in order to understand the mechanism of apoptosis induced by UA/UA NPs, we further tested the effect of UA/UA NPs on the expression of COX-2, VEGFA and VEGFR2 in A549 cell lines. As shown in Fig.6D, UA/UA NPs down-regulated the expression of COX-2, VEGFA and VEGFR2 in A549 cells compared with their control groups. Moreover, when UA NPs was treated, the effect was prominent compared with UA. Furthermore, UA NPs significantly decreased the expression of three proteins in a concentration- dependent manner compares to control group. The results indicated that UA NPs could significantly induce apoptosis of A549 cells through COX/VEGFA/VEGFR2 signaling pathway. Effect of UA NPs on cytokine and signal transducer and activator of transcription The extracellular immune stimulatory efficacy of mRNA was evaluated by measuring the release of cytokines TNF-α, IL-6, STAT-3 and IFN-β. A549 cells were pretreated with PBS, UA (30 µM) and UANPs (30 µM) for 24 h, the expression of TNF-α, IL-6, STAT-3 and IFN-β were measured by PCR method. As shown in Fig.7A-B, UA NPs significantly increased the mRNA expression of TNF-α (P

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