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Charge reversible and mitochondria/nucleus dual target lipid hybrid nanoparticles to enhance antitumor activity of doxorubicin Yan-feng Song, Dao-zhou Liu, Ying Cheng, Zeng-hui Teng, Han Cui, Miao Liu, Bang-Le Zhang, Qi-bing Mei, and Si-yuan Zhou Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01109 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018

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Molecular Pharmaceutics

Charge reversible and mitochondria/nucleus dual target lipid hybrid nanoparticles to enhance anti-tumor activity of doxorubicin Yan-feng Song1, 2‡, Dao-zhou Liu 1‡, Ying Cheng1‡, Zeng-hui Teng1, Han Cui1, Miao Liu1, Bang-le Zhang1, Qi-bing Mei3, Si-yuan Zhou1, 3* 1

Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical

University, Xi’an, 710032, China. 2

Department of Pharmacy, Tangtu Hospital, Fourth Military Medical University, Xi’an,

710032, China. 3

Key Laboratory of Gastrointestinal Pharmacology of Chinese Materia Medica of the

State Administration of Traditional Chinese Medicine, Fourth Military Medical University, Xi’an, 710032, China. *Corresponding author; ‡The author contributed equally to this work.

Running title: Charge reversible and mitochondria/nucleus dual target lipid hybrid nanoparticles. Corresponding author: Si-yuan Zhou. The authors declare no conflict of interest. Tel: 86 29 84776783. Fax: +86 29 84779212. E-mail: [email protected] Mail address: Changle West Road 169, Shaanxi province, Xi’an, 710032, China. Source of funding: This work was partly supported by the National Nature Science Foundation (No. 81641185)

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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

Charge reversible and mitochondria/nucleus dual target lipid hybrid nanoparticles to enhance anti-tumor activity of doxorubicin Yan-feng Song1, 2‡, Dao-zhou Liu 1‡, Ying Cheng1‡, Zeng-hui Teng1, Han Cui1, Miao Liu1, Bang-le Zhang1, Qi-bing Mei3, Si-yuan Zhou1, 3*

Graphical abstracts：AA-PEG-hyd-CHOL and TPP-CHOL modified lipid hybrid PLGA nanoparticle (LNPs) deliver DOX to mitochondria and nucleus of cancer cells to improve the therapeutic effect and reduce systemic toxicity of DOX.


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ABSTRACT The experiment aims to increase anti-tumor activity while decreasing the systemic toxicity of doxorubicin (DOX). A charge reversible and mitochondria/nucleus dual target lipid hybrid nanoparticles (LNPs) was prepared. The in vitro experimental results indicated that LNPs released more amount of DOX in acidic environment and delivered more amount of DOX to the mitochondria and nucleus of tumor cells than free DOX did, which resulted in the reduction of mitochondrial membrane potential and the enhancement of cytotoxicity of LNPs on tumor cells. Furthermore, the in vivo experimental results indicated that LNPs delivered more DOX to tumor tissue and significantly prolonged the retention time of DOX in tumor tissue as compared with free DOX, which consequently resulted in the high antitumor activity and low systemic toxicity of LNPs on tumor-bearing nude mice. The above results indicated that charge reversible mitochondria/nucleus dual targeted lipid hybrid nanoparticles greatly enhanced therapeutic efficacy of DOX for treating lung cancer. KEYWORDS: charge

reversible, mitochondria targeting, doxorubicin, lung cancer



1. INTRODUCTION With the rapid worldwide development of science and technology, the cancer treatment has made a great progress. However, cancer is still considered as a leading cause of death in China in recent years. Lung cancer, female breast cancer, stomach cancer, liver cancer, colon-rectum cancer, and esophagus cancer are seen as the most common, which took up about half of all new cancer cases in China in 2013. Lung cancer has become the one with the highest morbidity and mortality rate in China. The budget used in treating cancer in China keeps growing because of the increase of 1

aging population. It is of great significance to set up an effective, safe, and low-priced method to treat lung cancer. Doxorubicin (DOX) is a classical and widely used anti-tumor drug exhibiting wide spectrum, reliable therapeutic effect, and relative low price. However, DOX also shows serious side effect such as cardiac toxicity, bone marrow inhibition and drug resistance, which limit its clinical application.2, 3 Thus, it is imperative to find a proper method to maintain high anti-tumor activity of DOX while decreasing its systemic toxicity in vivo. Many nanocarriers such as liposomes, polymers, solid-lipid nanoparticles and micelles have been applied in the improvement of the therapeutic effect of DOX.4, 5 The action target of DOX is nucleus and mitochondrion of tumor 6

cells. Researchers usually neglect to control the accurate localization of DOX in tumor cells when they design drug delivery system. Theoretically, if DOX can be accurately delivered to the mitochondria and nucleus of tumor cells, its therapeutic effect will be greatly improved. Lipophilic cations, such as triphenylphosphonium (TPP) and dequalinium (DQA), show high specifical affinity with mitochondria and easily accumulate in the matrix of mitochondria due to the high membrane potential across mitochondria inner membrane.7-9 However, TPP modified nanoparticle core usually exhibits positive charge. It can adsorb negatively charged serum protein in blood, and subsequently be uptaken by mononuclear phagocyte system (MPS), which results in a short blood 10

circulation time of positively charged nanoparticles. In addition, after drug loaded nanoparticles are uptaken by the tumor cells through membrane mobile transport, they ACS Paragon Plus Environment

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were usually restricted in the lysosome, which results in the degradation of drug, and subsequently lowers the activity of drug-loaded nanoparticles. Thus, an ideal drug loaded nanoparticle should be negative charged in blood environment but can be reversed to a positive charged one in acidic lysosome, which is supposed to possess long half time in blood and lysosome escape characteristics.11, 12 Poly(lactic-co-glycolic acid) (PLGA) is widely used in tumor-specific drug delivery system. However, PLGA nanoparticles can be uptaken by MPS and eliminated rapidly from blood circulation, and then accumulate in liver and spleen due to its hydrophobic features. At present, polyethylene glycol (PEG) is usually utilized to hide hydrophobicity and positive charge of PLGA nanoparticle by providing a negative charged and hydrophilic outer layer on its the surface. Besides, the PEG modified PLGA nanoparticles can exhibit active targeting to tumor cells through 13

grafting targeting ligands on its surface.

Sigma receptors are over-expressed in

human tumor cells such as breast tumor, prostate cancer, melanoma, and non-small 14

cell lung carcinoma. Anisamide (AA) exhibited high affinity of to sigma receptor, which has drawn attention for the diagnosis and targeted therapy of tumors including lung cancer.

15

In this study, a pH sensitive anisamide-PEG-hyd-cholesterol (AA-PEG-hyd-CHOL) and a triphenylphosphonium-cholesterol (TPP-CHOL) were used to prepare lipid hybrid PLGA nanoparticle (LNPs). PEG shield the positive charge of TPP modified nanoparticle core to reduce the blood clearance that mediated by binding positive TPP with negative charged blood protein. Once the LNPs enter the lysosome of tumor cells, the positive TPP would be exposed, and TPP modified nanoparticle core subsequently would escape from lysosome and be bound with negative membrane of mitochondria and nucleus, which is supposed to improve the therapeutic effect of DOX while reducing its systemic toxicity. 2. EXPERIMENTAL SECTION 2.1. Materials. PLGA (molar ratio between lactic to glycolic acid is 75:25) was obtained from EVONIK Industries (Essen, Germany). N-hydroxysuccinimide (NHS), cholesterol, (3-carboxypropyl)-triphenylphosphonium bromide (TPP), polyvinyl ACS Paragon Plus Environment


alcohol

(PVA),

dimethylaminopyridine

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(DMAP),

and

1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide (EDC) were purchased from J&K CHEMICA (Beijing, China). H2N-PEG-NH2 (MW2000) was obtained from Yare Biotech Inc (Shanghai, China). Doxorubicin (DOX) was purchased from Hisun Pharmaceutical Co. (Zhejiang, China). 4’,6-diamidino-2-phenylindole (DAPI), MitoTracker green, LysoTracker green and RPMI1640 medium were bought from Invitrogen Technologies (Carlsbad, USA). 16

HepG2 cells (human liver tumor cell line，sigma receptor low-expressed ), A549 cells (human lung adenocarcinoma epithelial cell line, sigma receptor over-expressed 17

18

) and MDA-MB-231 cells (human breast cancer cell, sigma receptor over-expressed )

were purchased from Institute of Biochemistry and Cell Biology, Chinese Academy of Science (Shanghai, China). The doxorubicin resistance of MDA-MB-231 cells (MBA-MD-231/ADR cells) was induced by treating MDA-MB-231 cells with the increasing dosage of DOX for 4 month. The IC50 of DOX to MBA-MD-231/ADR cells was 25 times higher than that to MDA-MB-231 cells. In order to maintain the DOX-resistant property, MBA-MD-231/ADR was cultured with DOX (2 µmol/L) for 24 h once a week. Male nude mice were bought from the Fourth Military Medical University Experimental Animal Center. Animal experimental protocols were approved by the Fourth Military Medical University Animal Care and Use Committee (approval number: 16010). 2.2. Synthesis of pH-sensitive coploymer AA-PEG-hyd-CHOL. The synthetic route of

AA-PEG-hyd-CHOL

is

shown

in

supplementary

Figure

1,

and

19

AA-PEG-4-acetylbenzoic acid was synthesized by using the previous method.

Cholesterol hemisuccinate (486.4 mg, 1.0 mmol), DCC (410.4 mg, 2.0 mmol) and DMAP were dissolved in 4 mL dichloromethane and stirred for 6 h. Then Boc-NH-NH2 (132.2 mg, 1.0 mmol) was added into the reaction mixture, and then the reaction mixture was stirred for 12 h at room temperature. The solvent was removed by using a vacuum rotary evaporator. The reaction product was purified by silica gel column. The purified product was dissolved in 2 mL mixture of dichloromethane and ACS Paragon Plus Environment

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trifluoroacetic acid, and the reation mixture was stirred for 2 h at 25 °C. After that, 10 mL water was added into the reaction mixture, and the aqueous phase was set apart by using separating funnel. The pH of aqueous phase was adjusted to 12-13 by using NaOH solution. Then dichloromethane was added into aqueous phase, and the mixture was vigorously shaken. After that, the organic phase was set apart by using separating funnel. Finally, the reaction production (cholesterol succinate hydrazide) was obtained by removing the organic solvent. AA-PEG-4-acetylbenzoic acid (150 mg, 0.03 mmol) and cholesterol succinate hydrazide (15.2 mg, 0.03 mmol) were dissolved into the mixture of 2 mL dichloromethane and 50 µL trifluoroacetic acid. The reaction mixture was stirred for 2 h at 25 °C. After dichloromethane was removed from reaction mixture, the residue was dissolved with water and dialyzed against water for 48 h. Finally, the solution in dialysis bag was lyophilized to get the powder of AA-PEG-hyd-CHOL. 2.3. Synthesis of TPP-CHOL. The synthetic scheme of TPP-CHOL is shown in supplementary Figure 2. Briefly, (3-carboxypropyl)-triphenylphosphonium bromide (429.0 mg, 1.0 mmol), NHS (170.1 mg, 1.5 mmol) and EDC (270.0 mg, 1.5 mmol) were dissolved into 4 mL mixture of dichloromethane and methanol (v/v=1:1), and the reaction mixture was stirred at 25 °C for 3 h. Then 1,6-hexanediamine (116 mg, 1 mmol) and 50 µL triethylamine (TEA) were dropwise added into the above reaction mixture, and the reaction mixture was stirred at 25 °C for 24 h. The reaction product ((3-carboxypropyl)-triphenylphosphonium-hexanediamine)

was

extracted

with

dichloromethane. Finally, the reaction product was purified by silica gel column chromatography. Cholesterol hemisuccinate (105.0 mg, 0.2 mmol), NHS (50.2 mg, 0.4 mmol) and EDC (82.1 mg, 0.4 mmol) were dissolved into dichloromethane (3 mL), and the mixture was

stirred

for

3

h

at

25

°C.

Then

(3-carboxypropyl)-triphenylphosphonium-hexanediamine (116.3 mg, 0.3 mmol) was added, and the reaction mixture was stirred at 25 °C for 24 h. After the organic solvent was removed, the reaction product TPP-CHOL was purified by silica gel column chromatography. ACS Paragon Plus Environment


2.4.

Preparation

and

characterization

of

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DOX-loaded

nanoparticle.

AA-PEG-hyd-CHOL (15 mg) and TPP-CHOL (10 mg) were dissolved into 4 mL dichloromethane (containing 15 mg PLGA) as an oil phase. Meanwhile, 5 mg of DOX was dissolved into 20 mL polyvinyl alcohol solution (3%) as a water phase. Then, the oil phase and water phase were mixed by using ultrasonic wave. The mixture was stirred to completely evaporate the dichloromethane. The nanoparticles (LNPs) were washed for 3 times with deionized water. The naked PLGA nanoparticle (NNPs, without AA-PEG-hyd-CHOL and TPP-CHOL) was prepared by using the same method. The drug loading and encapsulation efficiency of nanoparticle were measured 20

according to the previous parper. The DOX-loaded LNPs (2 mg) were dissolved into 3 mL dimethylsulfoxide (DMSO), and the fluorescence intensity of the sample was detected at 470 nm/597 nm (excitation wavelength/emission wavelength) by using 970 CRT Spectrofluorophotometer (Shanghai, China). The amount of DOX in DOX-loaded LNPs was calculated by using a regression equation of standard curve of DOX in DMSO. The drug loading (DL) and encapsulation efficiency (EE) of DOX-loaded LNPs were calculated as following equation: DL% = (the weight of drug/the total weight)×100% EE% = (measured drug loading/theoretical drug loading)×100% The particle size, polydispersity index, and the surface charge of nanoparticle were determined by using a Beckman Coulter Particle Analyzer (Fullerton, California, USA). The morphology of the nanoparticles was observed via transmission electron microscopy (TEM, JEOL-100CXII, Japan). X-ray photoelectron spectroscopy (XPS) was used to detect the surface element composition of nanoparticle.21 The effect of LNPs on the red cell membrance was investigated by using ultraviolet spectrophotometer. Briefly, the fresh rat blood containing sodium heparin was washed three times with normal saline, and the red blood cells were collected and diluted with isotonic PBS buffer (pH7.4, pH6.5 and pH5.0).

22, 23

The red blood cell suspension

were incubated with LNPs (or NNPs) at 37 °C for 4 h. Then the supernatant was isolated by centrifugation, and its absorbance at 414 nm was determined by using ACS Paragon Plus Environment

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ultraviolet spectrophotometer. The water and isotonic PBS buffer were respectively used as a positive control and a negative control. The in vitro DOX release from DOX-loaded LNPs was investigated by dialysis method.

24

DOX-loaded LNPs (10 mg) was dispersed into 5 mL PBS with different

pH. Then the LNPs suspension solution was removed into a dialysis bag (molecular weight cut off 5000 Da) to dialyze against 30 mL PBS with the same pH as what was in dialysis bag. At previous determined time points, 0.5 mL release medium out of dialysis bag was taken out, and the same volume of fresh PBS was supplemented to the release medium. The released DOX was determined by using fluorescence spectroscopy at 470 nm/597 nm (excitation wavelength/emission wavelength). 2.5. Cytotoxicity assay. A549 cells, HepG2 cells, MDA-MB-231 cells and MDA-MB-231/ADR cells were cultured in 96-well plate (5×103 cells per well) for 24 h. Then, the cells were incubated with fresh culture medium containing free DOX, LNPs or NNPs for 48 h. MTT solution (20 µL, 5 mg/mL) was added into each wells, and the cells kept being incubated for another 4 h. Next, the culture medium was discarded, and 200 µL DMSO was added into the well. Finally, the absorbance of liquid in each well was measured at 490 nm by using a Bio-Rad Microplate Reader (Bio-Rad Laboratories, Richmond, California, USA). Besides, the effect of LNPs on the cleaved caspase3 activity in A549 cell was evaluated by using the cleaved 20

caspase3 activity assay kit (Beyotime Institute of Biotechnology, Jiangsu, China) . The effect of DOX-loaded LNPs on the mitochondrial membrane potential of A549 cells (or MDA-MB-231/ADR cells) was determined according to the previous reported method.

20

5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide

(JC-1) was used as a specific dye of mitochondrial membrane potential. When bonded with normal mitochondria, JC-1 exhibits red fluorescence; but when bonded with damaged mitochondria, JC-1 exhibits green fluorescence. A549 cells (or MDA-MB-231/ADR cells) were incubated with DOX or LNPs (equivalent DOX concentration was 2 µmol/L) for 4 h. Then the cell was incubated with JC-1 solution (2 mL, 2 µmol/L) for 20 min. Finally, the red fluorescence intensity and green fluorescence intensity were detected by fluorescent spectrophotometer, and the ACS Paragon Plus Environment


change of mitochondrial membrane potential was calculated by the ratio between red fluorescence intensity and green fluorescence intensity. 2.6. Cellular uptake of LNPs. A549 cells or HepG2 cells were planted into coverglass-containing 24-well plates (0.5×106 cells/well) and were incubated at 37 °C for 24 h. The culture medium was replaced with new medium containing DOX or LNPs (2 µmol/L) for 0.5 h or 2 h. After the removal of the medium, cells were fixed with 4% paraformaldehyde. Then, the cells were incubated with Triton X-100 solution (0.1%, w/w) for DAPI staining (100 µg/mL) for 15 min. Cover glass was placed onto glass microscope slide, and then the cellular uptake of LNPs was observed by using laser confocal scan microscopy (CLSM, Leica, Wetzler, Germany). Besides, in order to identify the role of AA incorporated into the particles, the exogenous AA was added into the culture medium 30 min before A549 cells were cultured with LNPs. The following methods were derived in the same as what was described above. 2.7. The subcellular distibution of DOX delivered by LNPs. A549 cells were planted into coverglass-containing 24-well plates (0.5×106 cells/well) and were cultured for 24 h. After the removal of culture medium, the cells were cultured with new medium containing DOX or LNPs (2 µmol/L) for 30 min or 4 h. Cells were incubated with LysoTracker green (600 nmol) or MitoTracker green (100 nmol) for 30 min. After the removal of the culture medium, the cells were fixed with 4% paraformaldehyde. Lastly, the cells were incubated with Triton X-100 solution (0.1%, w/w) for DAPI staining (100 µg/mL). The subcellular distibution of DOX was observed by CLSM. 2.8. In vivo anti-tumor activity of LNPs. A549 cells were subcutaneously implanted in the rear left flank of nude mice (1×107 cells/0.1 mL/animal). The tumor-bearing nude mice were divided into four groups when the tumor volume reached about 50 mm3. DOX (10 µmol/kg) and LNPs (the equivalent DOX dose was 2 µmol/kg and 10 µmol/kg) were injected into tumor-bearing nude mice by tail vein every 7 days (from day 1 to 14), respectively. Body weight and tumor volume were observed every four days. The long (L) and short (W) diameter of the tumor were measured to calculate ACS Paragon Plus Environment

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tumor volume by using the formulation LW2/2. At the end of the treatment, the tumor-bearing mice were executed euthanasia, and tumor tissue was stripped and weighted. The organs and tumor tissue was stained with H&E to observe histopathological changes. 2.9. Biodistribution of DOX. Tumor-bearing nude mice were executed euthanasia at 12 h or 24 h after they had been treated with free DOX (10 µmol/kg) or LNPs (the equivalent DOX dose was 10 µmol/kg). The main organs and tumor tissue were collected, and they were imaged by the IVIS™ Imaging System (Xenogen Imaging Technologies, Alameda, CA, USA). After that, the frozen tumor tissues and organs was cut into 5 µm thick section and stained with DAPI. Finally, the distribution of DOX in sections was detected by fluorescence microscopy. 2.10. Statistical analysis Statistical significance was evaluated by using one-way ANOVA analysis of SPSS software. 3. RESULTS 3.1. Characterization of the copolymer. The 1HNMR spectrum of AA-PEG-hyd-CHOL is shown in supplementary Figure 3. The AA in AA-PEG-hyd-CHOL was comfirmed by the peak a, b, c, d, f (δ=2.38 ppm, δ=8.2 ppm, δ=7.74 ppm, δ=6.19 ppm, δ=1.46 ppm). The PEG in the copolymer was comfirmed by peak g (δ=3.6 ppm). The CHOL in the copolymer was comfirmed by the peak h (δ=5.35 ppm). The infrared spectrum of AA-PEG-hyd-CHOL is shown in supplementary Figure 4. The characteristic absorbance peaks for PEG were at 1113/cm (C-O-C) and 774/cm (C-H). The absorption peak at 1661/cm was attributed to C＝N vibration. The 1HNMR spectrum of TPP-CHOL is shown in supplementary Figure 5. The structure of TPP-CHOL was comfirmed by the signal at δ=8.403 ppm, δ=7.78 ppm, δ=6.28 ppm, δ=5.38 ppm, δ=4.64 ppm, δ=3.71 ppm, δ=3.48 ppm, δ=3.24 ppm, δ=2.79 ppm, δ=2.63 ppm, δ=2.33 ppm, δ=1.98 ppm and δ=0.68-1.5 ppm. The infrared spectrum of TPP-CHOL is shown in supplementary Figure 6. The characteristic absorbance peak at 1732/cm was attribute to C=O (ester bond) vibration, and peak at ACS Paragon Plus Environment


1655/cm was due to C=O (amide bond) vibration. 3.2. Characterization of LNPs. According to the particle size and drug loading, the formulation of nanoparticle was optimized by using different mass ratio of PLGA, AA-PEG-hyd-CHOL and TPP-CHOL, and the results are shown in supplementary Table 1. The LNPs exhibited smaller particle size and higher drug loading when the mass ratio of PLGA/AA-PEG-hyd-CHOL/TPP-CHOL was 3:2:3. As exhibited in Figure 1A-B, the LNPs showed relatively spherical shape. The drug loading and the average particlesize were 5.4% and 180 nm, respectively. Figure 1C-D showed the XPS spectrum of NNPs and LNPs. The results indicated that the chemical composition weight percentages of nitrogen element and phosphorus element on the surface of NNPs were 0.0 and 0.0, respectively. However, the chemical composition weight percentages of nitrogen element and phosphorus element on the surface of LNPs were 2.67% and 1.49%, respectively. This result implied that the AA and TPP were on the surface of LNPs. As shown in Figure 2A-B, the particle size of LNPs was stable in 9 days in pH7.4 medium, and it became big in pH5.0 medium. The zeta potential of LNPs was -25.3 mV in pH7.4 medium, and it became +5.8 mV in pH 5.0 medium. Besides, there was no significant hemolysis effect of LNPs on red blood cell in pH7.4 medium. However, LNPs exhibited strong hemolysis effects on red blood cell (Figure 2C) in pH5.0 medium. The in vitro release of DOX from the nanoparticles in different pH (5.0, 6.5 and 7.4) medium is shown in Figure 2D-E. The results showed that 90%, 40% and 30% of loaded DOX were released from LNPs within 96 h in pH5.0, pH6.5 and pH7.4 release medium, respectively. Meanwhile, 23%, 33% and 35% of loaded DOX were released from NNPs within 96 h in pH5.0, pH6.5 and pH7.4 release medium, respectively. The above data indicated that the release of DOX from LNPs exhibited pH-dependent manner. 3.3. Cytotoxicity of LNPs. As shown in Figure 3A-F, the blank nanoparticle exhibited no significant cytotoxicity on A549 cells and HepG2 cells when its concentration ranged from 0.5 to 5 mg/mL. LNPs exhibited marked cytotoxicity on ACS Paragon Plus Environment

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A549 cells, MDA-MB-231 cells, MDA-MB-231/ADR cells and HepG2 cells in dose-dependent manner. When equvalent DOX concentration was 10 µmol/L, the cell viability was about 74%, 65% and 43% in NNPs, free DOX and LNPs treated A549 cells, respectively. This result indicated that LNPs showed higher cytotoxicity on A549 cells as compared with NNPs and free DOX. The cytotoxicity of LNPs on MDA-MB-231 cells, MDA-MB-231/ADR cells and HepG2 cells showed the same tendency as what was exhibited on A549 cells. Those results implied that LNPs not only showed great cytotoxicity to wild type tumor cells but also exhibited strong cytotoxicity to DOX resistant tumor cells. Besides, the effect of LNPs on the cleaved caspase3 level in tumor cells is shown in Figure 3G, and the results indicated that LNPs increased the cleaved caspase3 level on A549 cells with the increase of dosage. When equivalent DOX concentration was 10 µmol/L, the caspase3 level was about 1.3 fold, 1.1 fold and 2.3 fold in NNPs, free DOX and LNPs treated A549 cells, respectively. This result indicated that LNPs manifested greater effects on the cleaved caspase3 level on A549 cells as compared with NNPs and free DOX. Furthermore, the effect of LNPs on the mitochondrial membrane potential of tumor cells is shown in Figure 3H, and the results indicated that LNPs reduced the mitochondrial membrane potential in A549 cells and MDA-MB-231/ADR cells as compared with free DOX. The above data also implied that LNPs exerted cytotoxicity on tumor cells through increasing the cleaved caspase3 level and decreasing the mitochondrial membrane potential of the cells. 3.4. Cellular uptake of LNPs. Figure 4 showed the uptake of LNPs in A549 cells. The DOX red fluorescence intensity in A549 cells increased in time-dependent manner after the treatments with LNPs and DOX. Compared with free DOX, LNPs delivered more amount of DOX into A549 cells. At the same time, exogenous AA obviously decreased the accumulation of DOX in A549 cells. Furthermore, HepG2 cell is a sigma receptor-negative cell line. The cellular uptake of nanoparticle in HepG2 cell is shown in supplementary Figure 7. The result showed that the accumulation of DOX in HepG2 cell was lower than that in A549 cell after the treatment of LNP. The above results indicated that LNPs was uptaken by A549 cells ACS Paragon Plus Environment


through sigma receptor mediated endocytosis. 3.5. Subcellular distribution of DOX delivered by LNPs. The dynamic distribution of DOX in the lysosome is shown in Figure 5. When A549 cells were incubated with LNPs for 0.5 h, the mean fluorescence intensity (MFI) of DOX in the lysosome and in the nucleus was about 60 and 18, respectively. When the incubation time prolonged to 4 h, the MFI of DOX in the lysosome and in the nucleus was about 12 and 90, respectively. This result implied that LNPs could destroy the lysosome membrane, and subsequently escaped from the lysosome. The above data were consistent with the results of hemolysis experiment. After A549 cells were incubated with free DOX and LNPs, the dynamic distribution of DOX in mitochondria and nucleus is shown in Figure 6. When A549 cells were incubated with LNPs for 0.5 h, the MFI of DOX in the mitochondria and in the nucleus was about 27 and 48, respectively. When the incubation time was prolonged to 4 h, the MFI of DOX in the mitochondria and in the nucleus was about 43 and 100, respectively. Compared with free DOX, more DOX was released in mitochondria when the cells were treated with LNPs. This indicated that LNPs exhibited mitochondria targeting characteristic, which resulted in the greater cytotoxicity of LNPs on A549 cells than that of free DOX. The dynamic distribution of DOX in nucleus and mitochondria of MDA-MB-231/ADR cells is shown in supplementary Figure 8. The results showed the same tendency as what was observed in A549 cells. 3.6. Anti-tumor activity of LNPs in vivo. The anti-tumor activity of LNPs in vivo is shown in Figure 7A-D. At the end of experiment, the tumor volume was about 680 mm3, 270 mm3, 150 mm3 and 50 mm3 in normal saline, 10 µmol/kg free DOX, 2 µmol/kg LNPs (equivalent DOX dosage) and 10 µmol/kg LNPs (the equivalent DOX dosage) treated groups, respectively. Meanwhile, the tumor weight was about 0.79 g, 0.56 g, 0.31 g and 0.19 g in normal saline, 10 µmol/kg free DOX, 2 µmol/kg LNPs (equivalent DOX dosage) and 10 µmol/kg LNPs (the equivalent DOX dosage) treated groups at the end of the experiment, respectively. The above data indicated that free DOX and LNPs delayed the growth of tumor in dose-dependent manner. Compared with free DOX, LNPs ACS Paragon Plus Environment

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exhibited greater antitumor activity in vivo. Body weight indicates the systemic toxicity of drug delivery system. Free DOX significantly decreased body weight of tumor-bearing nude mice while LNPs did not cause significant loss of body weight. This result indicated that LNPs showed less systemic toxicity in tumor-bearing nude mice. Furthermore, the histological structure of tumor tissues and major organs are shown in Figure 8. After LNPs was administered to tumor-bearing nude mice, there were no histological changes in heart, liver, spleen, lung, kidney, or brain tissue as compared with normal saline treated tumor-bearing mice. In normal saline treated tumor-bearing nude mice, tumor tissue exhibited many living tumor cells. However, in LNPs treated tumor-bearing nude mice, tumor tissue exhibited less viable tumor cells and more serious necrosis. In addition, the significant histological structure changes, such as cardiomyocyte hypertrophy and neutrophils infiltration, could be found in cardiac tissue from DOX treated tumor-bearing nude mice. These results indicated that LNPs reduced the systemic toxicity of DOX but enhanced the toxicity effect on tumor tissue. 3.7. Biodistribution of DOX. Figure 7E showed the distribution of DOX in tumor-bearing nude mice. After LNPs was injected via tail vein, DOX red fluorescence mainly distributed in tumor tissue of tumor-bearing mice. However, DOX red fluorescence distributed through the whole body after free DOX was administered via tail vein injection. Besides, a large amount of red DOX fluorescence could be still found in tumor tissue at 24 h after LNPs was administered to tumor-bearing nude mice. However, there was a little of DOX red fluorescence in tumor tissue at 24 h after the free DOX was administered to tumor-bearing nude mice. This indicated that LNPs markedly prolonged the retention time of DOX in tumor tissue. The distribution of DOX in the tumor tissue and organs section was investigated by using fluorescence microscope, and the results are shown in supplementary Figure 9. After LNPs was administered to tumor-bearing nude mice, a large amount of DOX red fluorescence could be found in the section of tumor tissue and little amount of red ACS Paragon Plus Environment


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DOX fluorescence was found in the section of normal organs. However, more amount of red DOX fluorescence was found in the normal organs section and little amount of red DOX fluorescence was found in the tumor tissue section after free DOX was administered to tumor-bearing mice. 4. DISCUSSION TPP was widely used to modify drug carrier to deliver anti-tumor drug to mitochondria.

This

kind

of

mitochondria targeting

TPP-poly(ethyleneglycol)-phosphatidylethanolamine liposomes (load paclitaxel) PEI-TPP-DOX

drug

carrier

(TPP-PEG-PE)

includes modified

25, 26

, TPP-stearyl modified liposome (load DOX)27,

nanoparticle28,

and

PLGA-b-PEG-TPP

nanoparticles

(load

lonidamine)29. Because TPP is a lipophilic cation, the above mitochondria targeting drug carriers exhibited positive charge, and it is easy to adsorb serum protein in blood, which may result in a short blood circulation time30. Besides, the negatively charged nanoparticles show low capacity of lysosome escape, but they hardly adsorb serum protein and show a rather longer blood circulation31,

32

. pH-triggered charge

conversion has been proved as an efficient strategy to solve this dilemma

33, 34

. Based

on citraconic amides, a charge-conversional polymer was prepared. This kind of polymer was stable under neutral conditions. In acidic conditions, the negatively charged carboxylate functionalities transformed into positively charged primary amines34. Thereafter, many charge-conversional drug carriers such as PEG-b-PCL copolymer micelles35, the PVA nanogels36, hybrid polymer micelles37, PEGylated polymeric micelles38 and PEG-polypeptide polyionic complex nanoparticles[11] have been reported to enhance the lysosome escape of drug carrier. In this study, we designed a new mitochondria/nucleus dual targeting and charge-conversional nanoparticle. In order to ensure that the nanoparticle can work in

vivo, a pH-sensitive amphiphilic material AA-PEG-hyd-CHOL and mitochondria targeting amphiphilic material TPP-CHOL were used to modify the PLGA nanoparticle to form lipid hybrid PLGA nanoparticle (LNPs). LNPs exhibited double shell structures. In blood circulation, the outer shell shielded the positive charged inner core, increased the hydrophilic of PLGA nanoparticle and decreased the ACS Paragon Plus Environment

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non-specific uptake of PLGA nanoparticle by MPS. Once LNPs entered the lysosome of tumor cells, positive TPP would be exposed, and subsequently escaped from lysosome and binded with negative membrane of mitochondria and nucleus. In a word, LNPs combined the merit of charge-conversion and mitochondria/nucleus dual targeting while overcoming the disadvantage of traditional TPP mediated mitochondria targeting drug carrier. A biomaterial was considered as non-hemolytic when its hemolysis rate was less than 0-5%, and was considered as hemolytic when its hemolysis rate was larger than 5% 39. The experimental results indicated that the hemolytic rate of LNPs was less than 4% in pH7.4 and pH6.5 medium, but it became 20% in pH5.0 medium. The above data indicated that the LNPs was considered as a non-hemolytic biomaterial in blood environment, and was considered as a hemolytic biomaterial in pH5.0 environment. This also implied that LNPs was safe in blood circulation. Because the pH value in lysosome was about 4-5,40 thus LNPs could damage the lysosome membrane, which could enhance the escape of DOX-loaded PLGA core from lysosome, consequently increasing the anti-tumor activity of LNPs. Therefore, the cytotoxicity of LNPs on A549 cells was stronger as compared with the same dose of NNPs. The action target of DOX includes nucleus and mitochondria of tumor cells. Thus, it is important for DOX-loaded nanoparticle to escape efficiently from lysosomes. The zeta potential of LNPs was respective -25 mV and -18 mV in pH7.4 and pH6.5 medium, and it became +5.8 mV in pH 5.0 medium. The surface charge of LNPs became positive in acidic buffer due to the breakage of the hydrazone bond in AA-PEG-hyd-CHOL, which subsequently sheded the negative charged outer shell and exposing the positive charged TPP modified core. This indicated that LNPs was not sensitive to the extracellular microenvironment of tumor, and it was response to the lysosome microenvironment of tumor cells. In addition, after the exposure of the positive charged nanoparticle core, the interaction between the nanoparticle core and the membrane of lysosome was enhanced, subsequently resulted in the escapement of nanoparticle core from lysosome. Consequently, more amount of DOX was delivered to mitochondria and nucleus by electrostatic interaction.41-43 ACS Paragon Plus Environment


When a large amount of DOX is accumulated in the mitochondria, it can cause the lipid peroxidation of mitochondria and dysfunction of mitochondria such as the decreased mitochondrial membrane potential and the increased permeability of the mitochondrial membrane. The experimental results indicated that compared with free DOX, LNPs significantly decreased the mitochondrial membrane potential, which could result in the increase of permeability of the mitochondrial membrane and the release of cytochrome c from the mitochondria membrane.44, 45 After cytochrome c was released from the mitochondria, it could activate the caspase9 and lead to the activation of the caspase3, which was responsible for the apoptosis and cell death.46, 47 In addition, when a large amount of DOX is accumulated in the nucleus, the replication of nucleus DNA will be completely blocked, which is also responsible for the apoptosis and cell death.48 The experimental results indicated that compared with free DOX, LNPs markedly increased the cleaved caspase3 level in tumor cells, which led to the greater cytotoxicity on tumor cells. The above data also implied when DOX was simultaneously delivered to mitochondria and nucleus, the cytotoxicity of DOX greatly enhanced through the synergistic effect of two different DOX action targets in tumor cells. Compared with free DOX, LNPs significantly inhibited the growth of tumor in dose-dependent manner due to the following reasons. (1) LNPs specifically accumulated in tumor tissue through enhanced permeability and retention effect. (2) LNPs increased the retention time of DOX in tumor tissue. (3) The active targeting of LNPs to the AA receptor increased the accumulation of DOX in AA receptor over-expressed A549 cells. (4) The charge reversible and mitochondria/nucleus dual target LNPs enhanced the accumulation of DOX in mitochondria and nucleus of tumor cell. 5.CONCLUSION Charge reversible and mitochondria/nucleus dual target LNPs not only accumulated in tumor tissue but also simultaniously delivered DOX to mitochondria and nucleus of tumor cells. Consequently, LNPs greatly enhanced the antitumor activity of DOX in

vivo while reducing the systemic toxicity of DOX. LNPs exhibited great potential in ACS Paragon Plus Environment

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lung cancer treatment. Acknowledgments This research was partly supported by the National Nature Science Foundation (No. 81641185). Supporting Information Synthetic scheme of AA-PEG-hyd-CHOL and TPP-CHOL; the 1HNMR spectrum of AA-PEG-hyd-CHOL and TPP-CHOL; the infrared spectrum of AA-PEG-hyd-CHOL and TPP-CHOL; the cellular uptake of LNPs on HepG2 cells and A549 cells; the dynamic distribution of DOX in mitochondria and nucleus of MDA-MB-231/ADR cell; the distribution of DOX red fluorescence in section of main organs and tumor tissues;

characterization

of

LNPs

formed

by

different

ratio

of

PLGA/AA-PEG-hyd-CHOL/TPP-CHOL.

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Figure legends ACS Paragon Plus Environment


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Figure 1. Characteristics of nanoparticles. Panel A is TEM image of LNPs. Panel B is particle size distribution of LNPs detected by DLS. Panel C and panel D are the XPS spectrum of NNPs and LNPs, respectively. Figure 2. The in vitro properties of LNPs. The stability of LNPs in deferent pH medium (panel A). The effect of the medium pH on the zeta potential of LNPs (panel B). The hemolysis rate of LNPs in different pH medium (panel C). The release profiles of DOX from NNPs (panel D) and LNPs (panel E) in different pH medium. Data are presented as mean±SD, n=3. **p

nucleus dual target lipid hybrid

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