Tyrosine modified irinotecan-loaded liposomes capable of simultaneously targeting LAT1 and ATB0,+ for efficient tumor therapy

Journal Pre-proof Tyrosine modified irinotecan-loaded liposomes capable of simultaneously targeting LAT1 and ATB0,+ for efficient tumor therapy Zhenjie Wang, Dongxu Chi, Xingchen Wu, Yingli Wang, Xinxin Lin, Zhaochu Xu, Hongzhuo Liu, Jin Sun, Zhonggui He, Yongjun Wang

PII:

S0168-3659(19)30601-7

DOI:

https://doi.org/10.1016/j.jconrel.2019.10.037

Reference:

COREL 9986

To appear in: Received Date:

28 June 2019

Revised Date:

15 October 2019

Accepted Date:

18 October 2019

Please cite this article as: Wang Z, Chi D, Wu X, Wang Y, Lin X, Xu Z, Liu H, Sun J, He Z, Wang Y, Tyrosine modified irinotecan-loaded liposomes capable of simultaneously targeting LAT1 and ATB0,+ for efficient tumor therapy, Journal of Controlled Release (2019), doi: https://doi.org/10.1016/j.jconrel.2019.10.037

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Tyrosine modified irinotecan-loaded liposomes capable of simultaneously targeting LAT1 and ATB0,+ for efficient tumor therapy Zhenjie Wang, Dongxu Chi, Xingchen Wu, Yingli Wang, Xinxin Lin, Zhaochu Xu, Hongzhuo Liu, Jin Sun, Zhonggui He*, Yongjun Wang* Wuya College of Innovation, Shenyang Pharmaceutical University, Wenhua Road, Shenyang, 110016, China

ro of

* Corresponding authors E-mail addresses:

[email protected] (Z. He), [email protected] (Y. Wang)

ur

na

lP

re

-p

Graphical Abstracts

Jo

Abstract

As the demand for nutrients in malignant proliferation of tumors increases, the L-

type amino acid transporter 1(LAT1) and amino acid transporter B0,+ (ATB0,+) of tumor cells are more highly expressed than normal cells which can be used as new targets for active targeting of cancer. However, drug delivery systems often require multi-target design to achieve simultaneous targeting of different receptors or transporters due to the heterogeneity of the tumor. Here we utilized triethylamine-sucrose octasulfate

gradient to actively encapsulate irinotecan into the introliposomal aqueous phase. Targeted ability was achieved through inserting different amino acids modified polyethylene glycol monostearate into the liposomes, and found that glutamateliposomes can be targeted to LAT1, lysine-liposomes can be targeted to ATB0,+, and inspiringly, tyrosine-liposomes can be simultaneously targeted to LAT1 and ATB0,+. The tyrosine-modified liposomes showed the highest cellular uptake in BxPC-3 and MCF-7 cells which were highly expressed both LAT1 and ATB0,+. Moreover, we validated their targeting capabilities and elucidated the transport mechanism of LAT1

ro of

and ATB0,+-mediated endocytosis. The tumor inhibition rate of tyrosine-modified liposomes greatly increased from 39% to 87% compared with commercially available

liposomes loaded CPT-11(Onivyde®). Overall, it showed a good application prospect for efficient tumor therapy and industrial production.

-p

Keywords:

LAT1, ATB0,+, CPT-11, Amino acid transporter, Dual-targeted liposomes, Active

re

drug loading 1. Introduction

lP

Amino acids are the basic unit of protein synthesis in all cells. In addition to this obligatory function, amino acids can also serve as precursors for a variety of

metabolic

na

metabolites and biologically active molecules, including neurotransmitters, hormones, and

epigenetic

regulators[1].

Due

to

their

hydrophilicity

and

physicochemical characteristics, amino acids cannot diffuse through the biofilm

ur

including cell membrane, mitochondrial membrane, lysosomal membrane, etc. They rely on specific transporters to traverse across these biological membranes to provide

Jo

nutrition and participate in cell metabolism. Mammalian cells do not express one, but a variety of amino acid transporters with different substrate selectivity and different tissue expression patterns. All amino acid transporters identified to date at the molecular level in mammalian cells belong to the solute linked carrier (SLC) gene family. The nutritional needs are greatly increased due to the malignant proliferation of cancer cells. Therefore the expression of amino acid transporters in cancer cells is up-regulated compared to normal cells which can be used as a new target for drug therapy[2].

LAT1(SLC7A5) is a Na+/Cl- -independent exchange transporter that transports large neutral amino acids and histidine. Overexpression of LAT1 has been observed in a wide range of tumor cells, such as malignant glioma, prostate, pancreatic and breast cancer[35]. Moreover, LAT1 is used as a target to make prodrugs or carrier to deliver anticancer drugs[6-9]. ATB0,+(SLC6A14) is a Na+/Cl- -dependent unidirectional transporter that has the unique property to transport its substrates even against a concentration gradient from extra to intracellular[10]. ATB0,+ is capable of transporting 18 of 20 natural amino acids, with the exception of glutamate and aspartate. This is the origin of its name: AT

ro of

means amino acid transporter, B means broad substrate, 0 means neutral and + means cationic. ATB0,+ not only transports neutral amino acids but also transports cationic

amino acids. Therefore, ATB0,+ and LAT1 complement each other and coordinate with

each other. When a selective blocker of ATB0,+ (α-methyl-tryptophan) incubated with

-p

cancer cells or the SLC6A14-knockout mice was used, it all induced amino acid

deprivation, inhibited mammalian target of rapamycin (mTOR) pathway and decreased

re

cell proliferation[11, 12]. It suggests that ATB0,+ is an important target for cancer cells and it is also used to increase the effects of radiotherapy and chemotherapy[13-15].

lP

However, tumor cells are heterogeneous, for example, breast cancer is divided into luminal A, luminal B and triple negative breast cancer according to the expression

na

levels of estrogen receptor(ER), progesterone receptor(PR) and human epidermal growth factor receptor 2(HER2)[16]. Pancreatic ductal adenocarcinoma is also divided into different subtypes depending on KRAS, TP53, CDKN2N gene expression[17].

ur

Similarly, the amount of amino acid transporters expressed in the same type of cancer is also different. For example, MCF-7, MDA-MB-231 and T-47D are all human breast

Jo

cancer cells, but the amount of LAT1 and ATB0,+ transporters expression in them is different: MCF-7(LAT1+, ATB0,+ +), MDA-MB-231(LAT1+, ATB0,+ -), T-47D(LAT1-, ATB0,+ +), (+ means high expression and - means low expression)[11, 18]. Therefore, a drug delivery system that targets only a single amino acid transporter is difficult to completely cure cancer in vivo. We designed two dual-targeted nanoformulations that can target both LAT1 and ATB0,+. Glutamate, lysine and tyrosine were used as targeting ligands and covalently

linked to polyethylene glycol stearate(PS). It is then inserted into the phospholipid bilayer of the liposomes to form targeted liposomes. In order to improve the stability of drug loaded liposomes and achieve sustained release effect, we loaded irinotecan(CPT11) into the interior of liposomes by active drug loading method. The dual-targeted liposomes have better anti-tumor effects than Onivyde® and single-targeted liposomes both in vitro and in vivo. 2. Materials and methods 2.1. Materials

ro of

CPT-11 was kindly provided by Jiangsu Hengrui Medicine Co., Ltd (China). Sucrose octasulfate sodium(SOS) was obtained from Hengruikang Reagent Co., Ltd

(Wuhan, China). 1, 2-distearoyl-sn-glycero-3-phosphocholine(DSPC), 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene cholesterol

(Chol)

were

purchased

from

-p

mPEG2000),

glycol)-2000](DSPEA.V.T.(Shanghai)

Pharmaceutical Co.,Ltd. PS, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide(EDCI),

re

4-dimethylaminopyridine (DMAP) and N,N-Diisopropylethylamine(DIEA) were purchased from Aladdin Industrial Corporation(Shanghai, China). Succinic acid was

lP

obtained from Damao Chemical Reagent Factory(Tianjin,China). N-carbobenzyloxyglutamate-α-benzyl ester (Z-Glu-OBzl), N-α-carbobenzyloxy-L-tyrosine(Z-Tyr-OBzl),

na

N-α-carbobenzyloxy-L-lysine (Z-Lys(Z)-OH) were purchased from GL Biochem Ltd. (Shanghai, China). Cation exchange resin-732 and 10% palladium carbon(Pd/C) were obtained from Sinopharm Chemical Reagent Co., Ltd(Shanghai, China). Sepharose CL-

ur

4B, Sephadex G-50, highly efficient RIPA lysate, BCA protein concentration determination kit and 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES)

Jo

were obtained from Solarbio life sciences(Beijing, China). Regenerated cellulose dialysis bag(10 kD, 12 mm) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT) were purchased from Dalian Meilun Biotechnology Co., Ltd(China). Dulbecco's modified eagle medium(DMEM) was purchased from Gibco BRL (Gaithersburg,MD, USA). Fetal bovine serum (FBS) was obtained from Gemini bioproducts(California,

USA).

LAT1

primary

antibody

was

obtained

from

Abcam(Cambridge, UK), ATB0,+ primary antibody and HRP-labeled goat anti-rabbit

secondary antibody were purchased from Invitrogen Thermo(USA). FITC and Alexa Fluor 594 labeled goat anti-rabbit secondary antibody and GAPDH primary antibody were purchased from BIOSS(Beijing, China). PAGE gel fast preparation kit was obtained from EpiZyme(Shanghai, China). Membrane and cytosol protein extraction kit was obtained from Beyotime Biotechnology(Shanghai, China). Annexin V-FITC apoptosis assay kit was purchased from Absin Bioscience Inc. (Shanghai, China). 2.2. Synthesis of amino acid-conjugated PS The carboxyl groups of Z-Glu-OBzl or Z-Lys(Z)-OH are directly esterified with

ro of

the hydroxyl group of PS in the presence of EDCI and DMAP and reacted at 30 °C for 12 h. The hydroxyl group of PS esterified with one carboxyl group of succinic acid and

then the other carboxyl group esterified with phenolic hydroxyl group of Z-Tyr-OBzl in the presence of EDCI, DMAP, DIEA and reacted at 30 °C for 24 h. Then the benzyl

-p

ester protecting groups of amino acid were removed by Pd/C (10%) in H2 at 30 °C for 24 h. The resultant copolymers were purified by silica column chromatography. The

re

structures of the copolymers were confirmed by 1H-NMR(Bruker,AV-400,Switzerland) with d-DMSO and CDCl3 as solvent. PSG means glutamic acid modified PS, PSL

lP

means lysine modified PS and PST means tyrosine modified PS. 2.3. Preparation of triethylamine sucrose octasulfate solution(TEA-SOS)

na

Activated cation exchange resin-732 was put into the exchange column by wet packing method. Then 1 M sucrose octasulfate sodium solution was prepared and slowly passed through the resin for exchange of Na+ and H+. The cationic concentration

ur

of the exchanged solution was detected by Na+ electrode and the Na+ concentration after exchange should be <1% of that before exchange. And then the solution was titrated

Jo

with triethylamine(TEA) until the pH reached 5.5–6.0, thus forming 250 mM TEASOS[19, 20].

2.4. Preparation and characterization of liposomes DSPC 68.1 mg, Chol 22.2 mg, DSPE-mPEG2000 1.2 mg(Onivyde®) or PSG 18

mg(Glutamate-liposomes, G-lipo) or PSL 18 mg(Lysine-liposomes, L-lipo) or PSG 9 mg, PSL 9 mg(Glutamate and Lysine-liposomes, GL-lipo) or PST 18 mg(Tyrosineliposomes, T-lipo) were weighed precisely. Then dissolved them in chloroform and a

thin lipid film was formed by vacuum rotary evaporation. 5 ml TEA-SOS was added and hydrated at 70 °C for 20 min to obtain multilamellar vesicles. And then the multilamellar vesicles were sequentially extruded through 400 nm, 200 nm, 100 nm pore size polycarbonate membrane at 70 °C for 10 times respectively. Subsequently, the liposomes replaced with new external water phase(4.05 mg/ml HEPES and 8.42 mg/ml NaCl) by Sepharose CL-4B column chromatography. The liposomes were diluted in this step and the final concentration of DSPC is 6.81 mg/ml and Chol is 2.22 mg/ml. 4 mg CPT-11 and 1 ml blank liposomes were mixed well by vortex machine

ro of

and coincubated at 70 °C for 1 h. Thereby they became active drug-loaded liposomes via ammonium ion gradient. Liposomal encapsulation efficiency(EE) was determined

by Sephadex G-50 column namely size exclusion chromatography. Then the drugloaded liposomes were destructed by methanol and measured by high performance

-p

liquid chromatography (HPLC, Hitachi, Japan). The column was Ultimate XB-C18,

4.6×150 mm, 5μm(Welch, China ). Column oven temperature was 30 °C and the

re

detection wavelength is 368 nm. The mobile phase was 25 mM NaH2PO4(pH 3.1) and acetonitrile in a ratio of 75:25. The flow rate is 1 ml/min. Liposomal encapsulation

lP

efficiency=the amount of CPT-11 entrapped in liposomes/the amount of totally added CPT-11.

na

The liposomes sizes and zeta potentials were measured by Zetasizer Nano ZS90 (Malvern, UK). All samples were diluted 50-fold in water before analysis and measured at 25 °C. Morphology of liposomes observed by transmission electron microscopy

ur

(TEM, Hitachi HT7700, Japan) at an operating voltage of 80 kV. Different liposomes were placed in 4 °C environment, and their particle sizes were

Jo

measured at 0, 1, 3, 5 and 7 days respectively, to observe their placement stability. And different liposomes were dispersed into PBS 7.4 with 10% FBS, then shake at 100 rpm on a 37 °C water bath shaker. And the particle sizes of different liposomes were measured at 0, 2, 4, 8, 12, 24 h by Zetasizer Nano ZS90 respectively, to observe their stability in buffer and serum 2.5. Drug release in vitro Pipette 0.5 ml of drug-loaded liposomes into 10 kD dialysis bags and tie the ends

of the dialysis bags with cotton ropes. Then the dialysis bags were placed in stoppered conical flasks which contained 20 ml released medium. The released medium is histidine/NH4Cl/glucose(10 mM/ 20 mM/ 250 mM, pH 7.4)[19, 21]. Afterwards, the conical flasks were put in a constant temperature water bath shaker(37 °C, 100 rpm). At designed time intervals(2, 4, 8, 12, 24, 48, 72, 96, 120 h), 2 ml released medium was taken out and replenished with the equal volume of fresh media immediately. The samples were passed through 0.22 μm membrane filters before determined by HPLC. 2.6. Cell culture

ro of

BxPC-3, MCF-7, L-02 and NIH/3T3 cells were obtained from Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and

cultured in DMEM medium with 50 units/mL streptomycin, 100 units/mL penicillin and FBS (10%). Cells were cultured at 37 °C in a humidified incubator with 5% CO2.

-p

2.7. Western blot and immunofluorescence studies of LAT1 and ATB0,+ expression BxPC-3, MCF-7 and NIH/3T3 cells were harvested and lysed in radio assay

buffer

(RIPA)

supplemented

re

immunoprecipitation

with

1

mM

phenylmethylsulfonyl fluoride (PMSF). The concentration of extracted proteins was

lP

determined by BCA protein assay kit. The extracted proteins (20 μg) were subjected to electrophoresis in SDS-PAGE on 10% gels, separated and transferred to Millipore

na

PVDF membranes (0.45 μm). The membranes were blocked with 5% nonfat milk in TBST (10 mM Tris-HCl, 150 mM NaCl and 0.05% Tween-20) for 1 h and incubated with primary antibody to GAPDH, LAT1 and ATB0,+ for 2 h at 25 °C. After being

ur

washed with TBST, the membranes were incubated with goat anti-rabbit IgG-HRP secondary antibody for 1 h at 25 °C. The protein bands were detected by ChemiDOCTM

Jo

XRS+(BIO-RAD, USA) after adding electrochemiluminescence(ECL) solution to the membranes.

BxPC-3, MCF-7 and NIH/3T3 cells were cultured on 12-mm cover glasses for 24

h in cells culture incubator. Then the medium was discarded. The cells were fixed with 4% paraformaldehyde for 20 min and incubated with 0.1% TritonX-100 for 10 min at room temperature. After washing by PBS three times, the cells incubated with primary antibody to LAT1 and ATB0,+ for 3 h at 25 °C. Then the cells which combined with

LAT1 primary antibody were incubated with Alexa Fluor 594 labelled goat anti-rabbit IgG secondary antibody and which combined with ATB0,+ primary antibody were incubated with goat anti-rabbit IgG-FITC secondary antibody for 30 min at 37 °C. The nuclei were labeled with Hoechst 33342 for 10 min. Finally, the cells were observed by confocal laser scanning microscopy (CLSM, Nikon, Japan). 2.8. Cytotoxicity assay BxPC-3, MCF-7, L-02 and NIH/3T3 cells were seeded in 96-well plates at a density of 3×103 cells/well for 12 h. Then the cells were treated with serial dilution of

ro of

Onivyde, G-lipo, L-lipo, GL-lipo, T-lipo at different concentrations. After 48, 72, 96 incubation, 20 μl (5 mg/ml) MTT was added for 4 h more incubation. Then the medium was discarded and 200 μl DMSO was added to dissolve the formazan crystals formed by the living cells. The absorbance was measured at 490 nm using a microplate

-p

reader(Thermo Fisher, USA). 2.9. Cellular uptake in vitro

re

BxPC-3, MCF-7 and NIH/3T3 cells were seeded in 12-well plates at density of 3×105 cells/well for 24 h. Then the cells incubated with Onivyde, G-lipo, L-lipo, GL-

lP

lipo, T-lipo carrying CPT-11 for 12 and 24 h respectively which diluted by cell culture medium and final concentration of CPT-11 is 50 μg/ml. After that, the fluorescence

na

value of cells was analyzed by fluorescence activated cell sorting (FACS) analysis (Becton Dickinson, Germany). Meanwhile, BxPC-3 and MCF-7 were cultured on 12mm cover glasses and incubated with Onivyde, G-lipo, L-lipo, GL-lipo, T-lipo carrying

ur

CPT-11 for 24 h respectively. Then cells were fixed with 4% paraformaldehyde for 10 min and nuclei were labeled with Hoechst 33342 for 10 min at room temperature.

Jo

Finally, the cells were observed by CLSM. For competitive inhibition studies, the cell culture medium extra added 5 mg/ml

L-leucine, 5 mg/ml L-lysine or one of them as special medium. BxPC-3, MCF-7 and NIH/3T3 cells were incubated with different CPT-11(final concentration 50 μg/ml) liposomes for 24 h in normal or special medium respectively. Subsequently, the fluorescence value of cells was measured by FACS analysis. 2.10. Apoptosis assay

MCF-7 cells were seeded in 6-well plates at density of 6×105 cells/well for 12 h. Then the cells incubated with Onivyde, G-lipo, L-lipo, GL-lipo, T-lipo loading CPT11(final concentration at 100 μg/ml) for 48 h. Then the cells were incubated with Annexin V-FITC and PI for another 15 min in dark at room temperature and immediately analyzed by FACS analysis. Cells with no treatment were used as a negative control. 2.11. Mechanistic studies for LAT1 and ATB0,+-mediated endocytosis BxPC-3 cells were cultured on 12-mm cover glasses at density of 3×105 cells/well

ro of

for 24 h. Subsequently, the medium was removed and replenished with fresh Hank's Balanced Salt Solution(HBSS) containing different passive CPT-11 loaded

liposomes(free drug removed through a SephadexG-50 column) for 1 h at 37 °C. After washing by cold PBS three times, the cells incubated with primary antibody to LAT1

-p

and ATB0,+ for 3 h at 25 °C. Then the cells were incubated with Alexa Fluor 594 labelled

goat anti-rabbit IgG secondary antibody for 30 min at 37 °C. The nuclei were labelled

re

with Hoechst 33342 for 10 min. Finally, the cells were observed by CLSM. For targeting and endocytotic mechanisms studies, the LAT1 and ATB0,+ proteins

lP

were investigated by western blot. In brief, different blank liposomes that diluted with HBSS incubated with MCF-7 cells for 1 and 4 h respectively. The protein was extracted

na

by membrane and cytosol protein extraction kit and quantified by BCA protein assay kit. The total proteins (20 μg) and cytosol protein (40 μg) and membrane protein (60 μg) were subjected to electrophoresis in SDS-PAGE on 10% gels, separated and

ur

transferred to Millipore PVDF membranes. The membranes were blocked with 5% nonfat milk in TBST for 1 h and incubated with primary antibody to GAPDH, ATB0,+

Jo

for 2 h at 25 °C. After being washed with TBST, the membranes were incubated with goat anti-rabbit IgG-HRP secondary antibody for 1 h at 25 °C. The protein bands were detected by ChemiDOCTM XRS+ after adding ECL solution to the membranes. Subsequently, the antibodies were removed by stripping buffer and blocked with nonfat milk again and incubated with primary antibody to GAPDH, LAT1 overnight at 4 °C. Then PVDF membranes were incubated with goat anti-rabbit IgG-HRP secondary antibody for 1 h at 25 °C and detected by ChemiDOCTM XRS+.

2.12. Animals and tumor model Male Balb/c-nu mice (18-22 g) were purchased from Huafukang Laboratory Animal Technology Co. Ltd (Beijing,China). All animal studies were carried out under Institutional Animal Care and Use Committee-approved protocols of Shenyang Pharmaceutical University. And the mouse tumor model was established by injecting 3×106 BxPC-3 cells in normal saline into the armpit region of nude mice. 2.13. Biodistribution assay The in vivo targeting ability of liposomes into the tumor cells was assessed by

ro of

tumor-bearing BxPC-3 Balb/c-nu mice (n=3). Mice with subcutaneous tumors of approximate 500 mm3 were subjected to tail vein injection of different CPT-11 liposomes at 20 mg/kg. At 6 and 24 h after dosing, mice were sacrificed, and the organs

(heart, liver, spleen, lung and kidney) and tumors were immediately harvested. Then

-p

the organs and tumors were washed in the saline, weighted and homogenized by a tissue homogenizer. Drug concentrations in tissues and tumors were analyzed by fluorescence

re

quantification via microplate reader (λex 368/λem 426 nm). 2.14. In vivo antitumor efficacy and toxicity

lP

The antitumor efficacy was evaluated by using the nude mice armpit tumor model bearing human pancreatic cancer BxPC-3 cells. When the average tumor volume

na

reached 200 mm3, the treatment was initiated at a 5-day interval for 18 days. The mice were randomly divided into six groups (n=7 per group) receiving different CPT-11 liposomes at 10 mg/kg as follows: saline (the control group), Onivyde, G-lipo, L-lipo,

ur

GL-lipo, T-lipo. Tumor volume (V=length×width2/2) and body weight were recorded. At the end of the experiment, mice were sacrificed to prepare organ sections stained by

Jo

hematoxylin and eosin (H&E) for pathological study via optical microscope observation. The obtained tumors were accurately weighed to calculate the tumor burden(Wtumor/Wbody) and tumor inhibition rate [TIR=(Wcontrol tumor-Wsample tumor)/ Wcontrol tumor].

2.15. Statistical analysis All the quantitative data are described using the mean SD (standard deviations), and statistical analysis was performed with Student's t-test and one-way ANOVA.

p<0.05 was considered statistically significant (* p<0.05, ** p<0.01, *** p<0.001.). 3. Results and discussions 3.1. Synthesis of amino acid-modified copolymers Glutamic acid and lysine are directly attached to polyethylene glycol stearate by esterification (PSG and PSL). Tyrosine is linked to polyethylene glycol stearate via a succinic acid linker (PST). The synthetic routes were detailed in Fig.S1S2. Besides, the successful grafting of amino acids to PS backbone was confirmed by 1H-NMR(Fig.S3). All three nuclear magnetic spectra had peaks of PEG(-CH2-) at δ 3.6 ppm and

ro of

octadecyl(-CH2- and –CH3) at δ 1.2-1.4 ppm and δ 0.88 ppm. Meanwhile, the peaks at δ 4.12, 4.17, 4.28 belonged to –COCHN- of PSG, PSL and PST. These results indicated

that the final conjugate of amino acid-modified copolymers were successfully synthesized. The reason for choosing these three amino acids is that glutamic acid is an

-p

acidic amino acid, lysine is a basic amino acid, and tyrosine is a neutral amino acid,

which is more comprehensive and representative. Simultaneously, the substrate of

re

LAT1 requires both α-amino group and α-carboxyl group, and the α-carboxyl group is not necessary for the substrate of ATB0,+, such as basic amino acid and carnitine[14,

lP

22]. Therefore, in order to prepare a ligand that can only be targeted to ATB0,+, lysine was conjugated at α-carboxyl group, and the glutamate and tyrosine were conjugated

na

to PS through their side chains so as to target LAT1. 3.2. Preparation and characterization of liposomes All liposomes prepared by lipid film hydration and made the particle size smaller

ur

and uniform through liposomal extruder. Then the liposomes were loaded with CPT-11 by active drug loading method. The active drug-loading mechanism is shown in Fig.

Jo

1A. In brief, the inner solution of liposomes was 250 mM TEA-SOS and the outer solution was HEPES/NaCl. Then TEA-SOS dissociated to produce [TEAH+] and [SOS-]. It means that the [TEAH+]inner was much higher than [TEAH+]outer, thereby a [TEAH+] gradient was formed. Afterwards the [TEAH+] further dissociation produced protons and TEA. The molecular TEA can freely permeate lipid bilayer, resulting in the acidification of inner liposomal media thus forming a pH gradient and further improving the accumulation and encapsulation of CPT-11. CPT-11 can aggregate with

[SOS-] thus efficient encapsulation inside the liposomes without leaking. Moreover, the encapsulation efficiency was usually higher than 90%, which was the advantage of the active drug-loading method compared to the passive drug-loading method. As shown in Table 1, the drug-loaded liposome has a particle size of about 130 nm and a PDI of about 0.04, which proves that the particle size was very uniform(Fig.1BC and Fig.S4). The zeta potentials were all negative thus conducive to stability in the systemic circulation. Besides, the encapsulation efficiency was above 94% and the drug to lipid ratio was 0.35.

ro of

Fig. 1D showed the results of drug release of different liposomes in vitro. It can be seen from the results that all liposomes have a sustained release effect, because CPT-11 aggregated with TEA-SOS inside the liposomes which is very stable. In contrast, the

release of Onivyde was the slowest which released 19.6% of CPT-11 at 120 h, and the

-p

release of T-lipo was the fastest, releasing 26.5% at 120 h. The reason for this phenomenon may be that the T-lipo modified PEG density is higher than Onivyde,

re

thereby increasing the hydrophilicity of the phospholipid bilayer and increasing the gap between the phospholipids[23, 24]. Therefore, the CPT-11 of T-lipo is more easily

lP

released from the internal aqueous phase than the Onivyde. As seen from Fig. S5, there was no significant change in the particle size of

na

different liposomes within 7 days at 4 °C. It indicates that insertion of 20% amino acid modified polyethylene glycol monostearate(w/w) does not affect the stability of the liposomes. And as shown in Fig. S6, there was no significant change in the particle size

ur

of different liposomes within 12 h in PBS 7.4 with 10% FBS. Although the liposome particle size increased somewhat at 24 h, they were all below 150 nm. It indicates that

Jo

all liposomes are stable in PBS 7.4 with 10% FBS within 24 h.

Table 1. The size, PDI, zeta potential, encapsulation efficiency(EE), drug to lipid ratio(D/L) of different liposomes.

Onivyde

Size(nm)

PDI

Zeta potential(mV)

EE(%)

D/L

126.5±1.4

0.042±0.027

-8.21±0.29

98.58

0.42

G-lipo

139.7±1.4

0.058±0.028

-5.93±0.39

94.22

0.35

L-lipo

128.2±1.5

0.025±0.010

-2.37±0.12

94.24

0.35

GL-lipo

127.0±1.8

0.013±0.007

-4.10±0.07

96.14

0.35

T-lipo

110.9±0.7

0.042±0.018

-9.20±0.52

99.65

0.35

3.3. Western blot and immunofluorescence studies of LAT1 and ATB0,+ expression Western blot using antibody specific for LAT1 and ATB0,+ yielded proteins with molecular weight 55 kD and 72 kD (Fig. S7). Immunofluorescence of the two

ro of

transporters was studied by Alexa Fluor 594 labelled LAT1 that is red color and FITC labelled ATB0,+ which is green color (Fig. S8S9). Both results showed high expression of LAT1 and ATB0,+ in BxPC-3 and MCF-7 cells while low expression in NIH/3T3

control cells in the subsequent experiments. 3.4. Cytotoxicity assay

-p

cells. Therefore we used BxPC-3 and MCF-7 as positive cells and NIH/3T3 as negative

re

The in vitro antitumor activity of different liposomes was investigated in BxPC-3, MCF-7 and NIH/3T3 cells by the MTT assay and the IC50 values were also calculated.

lP

As shown in Table 2, the cytotoxicity of T-lipo and GL-lipo (dual targeted liposomes) were stronger than G-lipo and L-lipo (single targeted liposomes) and stronger than

na

Onivyde (untargeted liposome) in BxPC-3 and MCF-7 cells (LAT1 and ATB0,+ high expression). However, the difference in cytotoxicity of different liposomes was not apparent in NIH/3T3 cells (LAT1 and ATB0,+ low expression) which means the

ur

increased liposome cytotoxicity associated with LAT1 and ATB0,+ transporters. Furthermore, we examined the cytotoxicity of different liposomes to human normal

Jo

hepatocytes L-02. Compared to Onivyde, dual-targeted liposomes T-lipo did not significantly increase toxicity to human normal cells (228.1 vs 190.3 μg/ml in 48 h, 98.74 vs 131.2 μg/ml in 72 h, 21.06 vs 25.52 μg/ml in 96 h). This indicated that T-lipo increased the cytotoxicity against cancer cells with high expression of LAT1 and ATB0,+ whereas without significantly increasing the toxicity to human normal cells.

f oo

Table 2. IC50 value of different liposomes in BxPC-3, MCF-7, NIH/3T3 and L-02 cells. MCF-7 IC50(μg/ml)

pr

BxPC-3 IC50(μg/ml)

NIH/3T3 IC50(μg/ml)

L-02 IC50(μg/ml)

72 h

96 h

48 h

72 h

96 h

48 h

72 h

96 h

48 h

72 h

96 h

Onivyde

253.2

40.66

16.86

481.3

184.9

82.32

203.8

77.86

45.56

228.1

98.74

21.06

G-lipo

145.0***

29.99**

8.428***

137.0***

69.06**

31.76**

193.4

47.31

50.14

133.8

71.50

26.16

L-lipo

220.1

33.99

8.574***

451.3

108.5*

29.91**

143.8

40.78

64.95

194.0

48.51

22.68

GL-lipo

129.0***

22.89***

5.057***

96.15***

38.98***

26.13***

169.9

38.74

47.48

116.5

71.06

16.99

T-lipo

76.99***

17.26***

3.054***

61.06***

38.47***

15.00***

320.9

57.12

63.21

190.3

131.2

25.52

na l

Pr

e-

48 h

Jo ur

*Statistical difference analysis compared with Onivyde group (* p<0.05, ** p<0.01, *** p<0.001.).

3.5. Cellular uptake in vitro Cell uptake efficiency represents tumor targeting efficiency. The higher tumor targeting efficiency means the more cellular uptake. As shown in Fig. 2A, cell uptake of liposomes is time dependent that the cumulative concentration of intracellular drugs for 24 h was greater than 12 h in all three cell lines via FACS analysis. The dual-targeted liposome(GL-lipo, T-lipo) uptake efficiency was higher than the single-targeted formulation(G-lipo, L-lipo) at 12 h or 24 h, while the single-targeted liposomes were

ro of

higher than the commercially available formulation(Onivyde) in BxPC-3 and MCF-7 cells. For example, dual-targeted liposomes were 2.3-fold more efficient than Onivyde uptake in BxPC-3 cells at 12 hours, while single-targeted liposomes were up to 1.6

times more efficient than Onivyde and dual-targeted liposomes were 1.4-fold more

-p

efficient than single-targeted one. However, no similar differences in cellular uptake

were observed in NIH/3T3 cells and even T-lipo uptake efficiency was lower than

re

Onivyde. This means that the increased uptake efficiency of T-lipo in BxPC-3 and MCF-7 cells was associated with LAT1 and ATB0,+ transporters. The blue channel was

lP

the cell nucleus of Hoechst 33342 staining, and the green channel was CPT-11 fluorescence in Fig. 2B. The stronger green fluorescence represented the more CPT-11

na

taken up by the cells. Similar experimental results were shown in BxPC-3 and MCF-7 cells that was dual-targeted liposomes uptake the most, followed by single-targeted liposomes, with a minimum of Onivyde. It was consistent with the results of FACS.

ur

As shown in Fig. 2C, after adding leucine as a competitive inhibitor of LAT1, the uptake of BxPC-3 cells in group G-lipo decreased to 54% compared to control group,

Jo

while in group GL-lipo and T-lipo both decreased to approximately 76%. However, cellular uptake in group Onivyde and group L-lipo was almost unaffected by leucine addition. It indicated that the increase in cellular uptake in groups G-lipo, GL-lipo and T-lipo was dependent on LAT1, whereas GL-lipo and T-lipo groups were dual-targeted, blocking LAT1, ATB0,+ could still increase uptake, so GL-lipo and T-lipo do not decrease as much as G-lipo. Similarly, after the addition of lysine to inhibit the ATB0,+ transporter, the cell uptake in group L-lipo, GL-lipo and T-lipo reduced, and the uptake

of Onivyde and G-lipo remained unchanged. When leucine and lysine were simultaneously added as inhibitors, cell uptake was decreased in all groups except the Onivyde. And the cell uptake of GL-lipo and T-lipo was reduced to about 54% compared to control group. It was close to the level of cellular uptake of Onivyde which was no targeting effect. It suggested again that the increased cellular uptake of different liposomes is associated with the ability to target both LAT1 and ATB0,+ transporters. 3.6. Apoptosis assay As shown in Fig. 3, after 48 hours of incubation with different liposomes, the

ro of

Onivyde group was 23.6% apoptotic, G-lipo group was 42.4% apoptotic, L-lipo group was 37.5% apoptotic, GL-lipo group was 58.9% apoptotic, T-lipo group was 65.0%

apoptotic, compared with the control group(8.2%). This suggests that dual-targeted

liposomes can significantly promote cell apoptosis compared to single-targeted

-p

liposomes and Onivyde.

3.7. Mechanistic studies for LAT1 and ATB0,+-mediated endocytosis

re

To investigate the colocalization of different liposomes and amino acid transporters in cells, we used red fluorescent secondary antibody (Alexa Fluor 594) to

lP

label the LAT1 and ATB0,+ transporters, and the liposomes entrapped CPT-11 that emitted green fluorescence. After incubating liposomes at 37 °C with the BxPC-3 cells

na

for 1 h in HBSS, the cells were observed on CLSM. As shown in Fig. 4A, the yellow part after merging was the portion where the liposomes and the transporters were colocalized. The most of G-lipo, GL-lipo and T-lipo were targeted to LAT1.

ur

Simultaneously, the majority of L-lipo, GL-lipo and T-lipo were targeted to ATB0,+. However, Onivyde had almost no targeting effect on both amino acid transporters and

Jo

G-lipo cannot be targeted to the ATB0,+ transporter meanwhile L-lipo cannot be targeted to the LAT1. It suggested that the increase in cellular uptake of different liposomes was due to the modification of different amino acid targets and thus can be targeted to LAT1 and ATB0,+ transporters. In order to study the intracellular transport mechanism of different liposomes after combining with amino acid transporters, the membrane and cytosol protein extraction kit was used. After incubation of different liposomes with MCF-7 cells for 1 h and 4 h, their membrane proteins and cytoplasmic proteins were

extracted and then characterized by Western blotting technique. As shown in Fig. 4B, in the case where the total protein was unchanged, the content of LAT1 protein in the cell membrane was decreased, and the content in the cytoplasm was increased after 1 h incubation with G-lipo, GL-lipo and T-lipo(circled with a red frame in Fig. 4B). Simultaneously, the content of ATB0,+ protein in the cell membrane was decreased, and the content in the cytoplasm was increased after 1 h incubation with L-lipo, GL-lipo and T-lipo(circled with a red frame in Fig. 4B). However, LAT1 and ATB0,+ proteins in the cell membrane and cytoplasm returned to normal, with no significant differences

ro of

between the different groups after 4 h incubation with liposomes. Therefore, we speculated that the possible mechanism of liposomes endocytosis was shown in Fig. 5. First the LAT1 and ATB0,+ recognized the amino acid ligands and combined with them then form endocytic vesicles by pinocytosis. Second the vesicles encapsulating

-p

liposomes and transporters complexes separated from the cell membrane with the help

of dynamin. The pH reduction of the early endosomes usually drove the dissociation of

re

the receptor from the ligand and transported the receptor back to the cell membrane[25, 26]. Third, the liposomes/amino acid transports complex was dissociated and sorted in

lP

early endosomes. Finally, LAT1 and ATB0,+ recycled back to the cell membrane, while the liposomes enter the lysosomes[7, 27]. And the other possible mechanism is that the

na

endocytosed transporters are degraded while new transporters are synthesized to achieve dynamic balance. This requires further experimental verification. 3.8. Biodistribution assay

ur

The accumulative concentrations of CPT-11 in the heart, liver, spleen, lung, kidney and tumors were quantified 6 h and 24 h after the tail vein liposomal injection of nude

Jo

mice measured by a microplate reader. As shown in Fig. 6AB, different liposomes groups all had the highest accumulative concentration in the liver and spleen. Because when liposomes enter the systemic circulation, the most of them were swallowed by the liver and spleen. More notable were the accumulative concentrations of CPT-11 loaded in different liposomes in the tumors. Compared to Onivyde, the accumulation of single-targeted liposomes(G-lipo and L-lipo) at the tumor site increased by an average of 1.3 times and 1.6 times at 6 h and 24 h, respectively. Even more interesting

is that the tumor accumulation of dual-targeted liposomes increased by an average of 3 and 2.4 times at 6 h and 24 h respectively. What is impressive is that the accumulation of T-lipo had actually increased by 3.6 times compared to Onivyde at 6 h after tail vein injection. It manifested that dual-targeted liposomes especially T-lipo can significantly increase tumor targeting efficiency in vivo compared to Onivyde. 3.9. In vivo antitumor efficacy and toxicity After verifying the targeting ability of different liposomes in vivo, the antitumor efficacy and systemic toxicity were evaluated by a pharmacodynamics study. The tumor

ro of

volume and body weight of BxPC-3 tumor-bearing nude mice were measured after intravenous administration of normal saline and different CPT-11 liposomes at a dose

of 10 mg CPT-11/kg every 5 days. Dosing was initiated when the average tumor volume is about 200 mm3. As shown in Fig. 7A, Onivyde showed a poor tumor suppressing

-p

effect that tumor volume was 784±155 mm3 (mean ± SD, n=7) compared to saline group (1259±264 mm3) after 18-day treatment. In contrast, single-targeted liposomes

re

exhibit better antitumor effects (G-lipo 456±69 mm3, L-lipo 517±58 mm3) than Onivyde. Impressively, dual-targeted liposomes showed the best tumor suppression and

lP

the tumor barely grew (GL-lipo 222±36 mm3, T-lipo 192±51 mm3). It can be seen from Fig. 7B that all liposomes have no significant systemic toxicity, because the body

na

weight of the nude mice is relatively stable during these 18 days. In Fig. 7CD, the tumor burden(%) and TIR(%) of the different administration groups were also calculated, respectively. Dual-targeted T-lipo showed the lowest tumor burden(0.67%) and highest

ur

TIR(87%) compared to Onivyde(3.13%, 39%). The histological analysis of tissue sections was also performed to determine the

Jo

microscopic changes in six important organs harvested from tumor-bearing nude mice. After staining with H&E(Fig. 8A), there was no significant difference in the microstructure of heart, liver, spleen, lung and kidney between the different liposomeadministered mice compared to normal mice. It proved again that the prepared liposomes had no systemic toxicity. In sections of tumors, T-lipo generated more necrosis areas associated with inflammatory cells and connective tissue hyperplasia compared to other groups. To monitor whether different liposomes affect liver and

kidney function, we measured four indicators of ALT, AST, CR and BUN. As shown in Fig. 8B, these four indicators did not differ significantly in the five liposomes groups compared to normal group. It means that there was no damage to the liver and kidney after liposome-administration. In general, this suggested that tyrosine-modified dual targeted liposomes significantly improve tumor targeting efficiency and cellular uptake efficiency, with the best anti-tumor effect compared to other groups and without systemic toxicity. 4. Conclusion

ro of

In this study, we successfully synthesized PS with the conjugation of glutamate, lysine and tyrosine to modify the surfaces of CPT-11 loaded liposomes for targeting

LAT1 and ATB0,+ simultaneously. Amino acids as small molecule ligands were readily modified to the surface of liposomes and were stable in the systemic circulation. The

-p

uniform particle size of all liposomes loaded with CPT-11 by active drug loading method was about 130 nm and encapsulation efficiency is over 94%. Dual-targeted

re

liposomes have superior cytotoxicity, apoptosis and higher cellular uptake via LAT1 and ATB0,+-mediated endocytosis compared to Onivyde and single-targeted liposomes

lP

in vitro experiments. Uptake mechanism study indicated that LAT1 and ATB0,+ were recycled in cell membranes and cytoplasm. Moreover, dual-targeted liposomes showed

na

better tumor accumulation and preferable anti-tumor effects than Onivyde and singletargeted liposomes in vivo. It showed a good application prospect for overcoming tumor

ur

heterogeneity and industrial production.

Acknowledgements

Jo

This work was financially supported from the National Nature Science Foundation

of China (U1608283) and the Career Development Program for Young and Middleaged Teachers in Shenyang Pharmaceutical University.

Appendix A. Supplementary data Supplementary data to this article can be found online

[1] M.O.F. Sikder, S. Yang, V. Ganapathy, Y.D. Bhutia, The Na(+)/Cl(-)-Coupled, Broad-Specific, Amino Acid Transporter SLC6A14 (ATB(0,+)): Emerging Roles in Multiple Diseases and Therapeutic Potential for Treatment and Diagnosis, AAPS J 20(1) (2017) 12. [2] L. Lin, S.W. Yee, R.B. Kim, K.M. Giacomini, SLC transporters as therapeutic targets: emerging opportunities, Nat Rev Drug Discov 14(8) (2015) 543-60. [3] R. Yan, X. Zhao, J. Lei, Q. Zhou, Structure of the human LAT1-4F2hc heteromeric amino acid transporter complex, Nature 568(7750) (2019) 127-130. [4] Y. Zhao, L. Wang, J. Pan, The role of L-type amino acid transporter 1 in human tumors, Intractable Rare Dis Res 4(4) (2015) 165-9. [5] B. Altan, K. Kaira, A. Watanabe, N. Kubo, P. Bao, G. Dolgormaa, E.O. Bilguun, K. Araki, Y. Kanai, T. Yokobori, T. Oyama, M. Nishiyama, H. Kuwano, K. Shirabe, Relationship between LAT1 expression and resistance to chemotherapy in pancreatic ductal adenocarcinoma, Cancer Chemother Pharmacol 81(1) (2018) 141-153.

ro of

[6] E. Puris, M. Gynther, J. Huttunen, A. Petsalo, K.M. Huttunen, L-type amino acid transporter 1 utilizing prodrugs: How to achieve effective brain delivery and low systemic exposure of drugs, J Control Release 261 (2017) 93-104.

[7] L. Li, X. Di, M. Wu, Z. Sun, L. Zhong, Y. Wang, Q. Fu, Q. Kan, J. Sun, Z. He, Targeting tumor highlyexpressed LAT1 transporter with amino acid-modified nanoparticles: Toward a novel active targeting

-p

strategy in breast cancer therapy, Nanomedicine: Nanotechnology, Biology and Medicine 13(3) (2017) 987-998.

[8] L. Li, X. Di, S. Zhang, Q. Kan, H. Liu, T. Lu, Y. Wang, Q. Fu, J. Sun, Z. He, Large amino acid transporter Biointerfaces 141 (2016) 260-267.

re

1 mediated glutamate modified docetaxel-loaded liposomes for glioma targeting, Colloids Surf B [9] S. An, X. Lu, W. Zhao, T. Sun, Y. Zhang, Y. Lu, C. Jiang, Amino Acid Metabolism Abnormity and

lP

Microenvironment Variation Mediated Targeting and Controlled Glioma Chemotherapy, Small 12(40) (2016) 5633-5645.

[10] A. Muller, A. Chiotellis, C. Keller, S.M. Ametamey, R. Schibli, L. Mu, S.D. Kramer, Imaging tumour ATB0,+ transport activity by PET with the cationic amino acid O-2((2-[18F]fluoroethyl)methyl-

na

amino)ethyltyrosine, Mol Imaging Biol 16(3) (2014) 412-20. [11] S. Karunakaran, S. Ramachandran, V. Coothankandaswamy, S. Elangovan, E. Babu, S. PeriyasamyThandavan, A. Gurav, J.P. Gnanaprakasam, N. Singh, P.V. Schoenlein, P.D. Prasad, M. Thangaraju, V.

ur

Ganapathy, SLC6A14 (ATB0,+) protein, a highly concentrative and broad specific amino acid transporter, is a novel and effective drug target for treatment of estrogen receptor-positive breast cancer, J Biol Chem 286(36) (2011) 31830-8.

Jo

[12] E. Babu, Yangzom D. Bhutia, S. Ramachandran, Jaya P. Gnanaprakasam, Puttur D. Prasad, M. Thangaraju, V. Ganapathy, Deletion of the amino acid transporter Slc6a14 suppresses tumour growth in spontaneous mouse models of breast cancer, Biochemical Journal 469(1) (2015) 17-23. [13] P. Wongthai, K. Hagiwara, Y. Miyoshi, P. Wiriyasermkul, L. Wei, R. Ohgaki, I. Kato, K. Hamase, S. Nagamori, Y. Kanai, Boronophenylalanine, a boron delivery agent for boron neutron capture therapy, is transported by ATB0,+, LAT1 and LAT2, Cancer Sci 106(3) (2015) 279-86. [14] Q. Luo, P. Gong, M. Sun, L. Kou, V. Ganapathy, Y. Jing, Z. He, J. Sun, Transporter occluded-state conformation-induced endocytosis: Amino acid transporter ATB0,+-mediated tumor targeting of liposomes for docetaxel delivery for hepatocarcinoma therapy, Journal of Controlled Release 243 (2016) 370-380.

[15] Q. Luo, B. Yang, W. Tao, J. Li, L. Kou, H. Lian, X. Che, Z. He, J. Sun, ATB0,+ transporter-mediated targeting delivery to human lung cancer cells via aspartate-modified docetaxel-loading stealth liposomes, Biomaterials Science 5(2) (2017) 295-304. [16] A.G. Waks, E.P. Winer, Breast Cancer Treatment: A Review, JAMA 321(3) (2019) 288-300. [17] J. Cros, J. Raffenne, A. Couvelard, N. Pote, Tumor Heterogeneity in Pancreatic Adenocarcinoma, Pathobiology 85(1-2) (2018) 64-71. [18] S. Karunakaran, N.S. Umapathy, M. Thangaraju, T. Hatanaka, S. Itagaki, D.H. Munn, P.D. Prasad, V. Ganapathy, Interaction of tryptophan derivatives with SLC6A14 (ATB0,+) reveals the potential of the transporter as a drug target for cancer chemotherapy, Biochem J 414(3) (2008) 343-55. [19] W. Yang, Z. Yang, J. Fu, M. Guo, B. Sun, W. Wei, D. Liu, H. Liu, The influence of trapping agents on the antitumor efficacy of irinotecan liposomes: head-to-head comparison of ammonium sulfate, sulfobutylether-beta-cyclodextrin and sucrose octasulfate, Biomater Sci 7(1) (2018) 419-428. [20] J. Liu, D. Chi, S. Pan, L. Zhao, X. Wang, D. Wang, Y. Wang, Effective co-encapsulation of doxorubicin octasulfate as drug trapping agent, Int J Pharm 557 (2019) 264-272.

ro of

and irinotecan for synergistic therapy using liposomes prepared with triethylammonium sucrose

[21] J. Cui, C. Li, W. Guo, Y. Li, C. Wang, L. Zhang, L. Zhang, Y. Hao, Y. Wang, Direct comparison of two pegylated liposomal doxorubicin formulations: is AUC predictive for toxicity and efficacy?, J Control Release 118(2) (2007) 204-15.

-p

[22] L.F. Kou, Q. Yao, S. Sivaprakasam, Q.H. Luo, Y.H. Sun, Q. Fu, Z.G. He, J. Sun, V. Ganapathy, Dual targeting of L-carnitine-conjugated nanoparticles to OCTN2 and ATB(0,+) to deliver chemotherapeutic agents for colon cancer therapy, Drug Delivery 24(1) (2017) 1338-1349.

re

[23] D. Molnar, J. Linders, C. Mayer, R. Schubert, Insertion stability of poly(ethylene glycol)-cholesterylbased lipid anchors in liposome membranes, Eur J Pharm Biopharm 103 (2016) 51-61. [24] Y.H. Hongwei Ren, Jianming Liang, Zhekang Cheng, Meng Zhang, Ying Zhu, Chao Hong, Jing Qin,

lP

Xinchun Xu, and Jianxin Wang, Role of liposomes size, surface charge and PEGylation on rheumatoid arthritis targeting therapy, ACS Applied Materials & Interfaces Just Accepted Manuscript (2019). [25] B.D. Grant, J.G. Donaldson, Pathways and mechanisms of endocytic recycling, Nat Rev Mol Cell Biol 10(9) (2009) 597-608.

na

[26] S. Xu, B.Z. Olenyuk, C.T. Okamoto, S.F. Hamm-Alvarez, Targeting receptor-mediated endocytotic pathways with nanoparticles: Rationale and advances, Advanced Drug Delivery Reviews 65(1) (2013) 121-138.

ur

[27] Y.Q. Du, C.T. Tian, M.L. Wang, D. Huang, W. Wei, Y. Liu, L. Li, B.J. Sun, L.F. Kou, Q.M. Kan, K.X. Liu, C. Luo, J. Sun, Z.G. He, Dipeptide-modified nanoparticles to facilitate oral docetaxel delivery: new insights

Jo

into PepT1-mediated targeting strategy, Drug Delivery 25(1) (2018) 1403-1413.

ro of -p re lP

Jo

ur

na

Fig.1 A: Schematic diagram of active CPT-11 loading mechanism of liposomes. The TEM images of CPT-11 loaded Onivyde(B) and T-lipo(C) respectively, scale bar: 200 nm. D: Drug release profiles of different liposomes in vitro(n=3).

ro of -p re lP na ur Jo

Fig.2 A: Cellular uptake of the different liposomes in BxPC-3, MCF-7 and NIH/3T3 cells at 12 h and 24 h via FACS analysis(n=3). B: Cellular uptake of the different liposomes in BxPC-3 and MCF-7 cells at 24 h through CLSM analysis, scale bar: 10 μm. The blue channel was Hoechst 33342-stained nuclei and green channel was CPT11 fluorescence. C: Cellular uptake of different liposomes after the addition of leucine, lysine, leucine and lysine competitively inhibited LAT1 and ATB0,+ transporters in BxPC-3 cells at 24 h (n=3).

ro of -p

Jo

ur

na

lP

re

Fig.3 The apoptosis of MCF-7 cells after incubating with different liposomes for 48 h.

ro of -p re lP na

Jo

ur

Fig.4 A: Co-localization pictures of different liposomes and amino acid transporters at 1 h in BxPC-3 cells via immunofluorescence assay. The blue channel was Hoechst 33342-stained nuclei, green channel was CPT-11 fluorescence and red channel was LAT1 or ATB0,+ fluorescence, the part enclosed by the dotted line are highmagnification images, scale bar: 10 μm. B: Endocytic transport mechanism after liposomes targeting to LAT1 or ATB0,+ transporter via western blotting analysis in MCF-7 cells after incubating with different liposomes for 1 h and 4 h.

ro of -p re

lP

Fig.5 One possible schematic diagram of dual-targeted liposome endocytosis and amino

Jo

ur

na

acid transporters recycling mechanism.

Fig.6 The accumulative concentration of CPT-11 in the heart, liver, spleen, lung and kidney after 6 h(A), 24 h(B) of injection of different liposomes in the tail vein. The accumulation of CPT-11 in tumor(C), n=3.

ro of

Jo

ur

na

lP

re

-p

Fig.7 The antitumor efficacy in vivo. The tumor volume (A), body weight (B), tumor burden(C), and TIR(D) of BxPC-3 tumor-bearing Balb/c-nu mice(n=7). The arrow indicates the day of dosing via tail vein injection.

ro of -p re lP na

Jo

ur

Fig.8 Histopathological section of the hearts, livers, spleens, lungs and tumors of nude mice after different liposomes administration for 18 days(A), scale bar: 100 μm. Liver(ALT, AST) and kidney(CR, BUN) functional parameters at day 18 after nude mice sacrificed(B).