pH-Triggered Surface Charge Reversed Nanoparticle with Active

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pH-triggered surface charge reversed nanoparticle with active targeting to enhance the antitumor activity of doxorubicin Jiang-bo Du, Ying Cheng, Zeng-hui Teng, Meng-lei Huan, Miao Liu, Han Cui, Bang-le Zhang, and Si-yuan Zhou Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00158 • Publication Date (Web): 21 Mar 2016 Downloaded from http://pubs.acs.org on March 22, 2016

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Molecular Pharmaceutics

pH-triggered surface charge reversed nanoparticle with active targeting to enhance the antitumor activity of doxorubicin Jiang-bo Du#, Ying Cheng#, Zeng-hui Teng#, Meng-lei Huan, Miao Liu, Han Cui, Bang-le Zhang, Si-yuan Zhou* Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi’an, 710032, China.

Running title: Charge reversed PLGA nanoparticle.

*Corresponding author: Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical University, Changle West Road 169, Xi’an, Shaanxi, 710032, China. Tel: +86-29-84776783; Fax: +86-29-84779212; E-mail: [email protected] (S.Zhou) #

These author contributed equally to this work.

ACS Paragon Plus Environment


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Abstract PLGA nanoparticles are widely used in tumor targeting drug delivery system. However, the naked PLGA nanoparticles (NNPs) not only have low drug loading but also can be rapidly removed from blood circulation by the immune system. The aim of this study was to prepare pH-triggered surface charge reversed lipid hybrid PLGA nanoparticles (LNPs) to enhance drug loading and drug delivery efficiency. CHO-Arg-His-OMe and FA-PEG-DSPE were synthesized to modify PLGA nanoparticles to prepare LNPs. The drug loading and encapsulation rate of LNPs were greatly improved as compared with NNPs. In pH7.4 medium, doxorubicin (DOX)-loaded LNPs showed negatively charged and released DOX slowly. In pH5.0 medium, DOX-loaded LNPs exhibited positively charged and released DOX quickly. DOX-loaded LNPs delivered more amount of DOX to the nucleus of KB cells and MBA-MD-231/ADR cells than what free DOX did. In addition, DOX-loaded LNPs significantly inhibited the proliferation of KB cells and MBA-MD-231/ADR cells. Compared with free DOX, the same dose of the DOX-loaded LNPs delivered more DOX to tumor tissue. Thus, DOX-loaded LNPs significantly inhibited the growth of tumor in tumor-bearing nude mice and obviously reduced the systemic toxicity of DOX. In conclusion, pH-triggered surface charge reversed DOX-loaded LNPs significantly enhanced the antitumor activity of DOX in vitro and in vivo. DOX-loaded LNPs had great potential in tumor targeted chemotherapy. Keywords: PLGA; doxorubicin; lipid hybrid nanoparticles; folate;


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Introduction Many chemotherapeutic drugs used for tumor treatment are far from perfect with undesirable severe side effects and development of drug resistance. Over the past few decades, in order to overcome those problems, various drug delivery systems were designed to improve therapeutic efficacy, reduce toxicity and lower the frequency of drug administration[1,2]. PLGA (poly-lactic-co-glycolic acid) is the most commonly used drug carrier

[3,4]

. PLGA can be metabolized to lactic acid and glycolic acid that

are endogenous and degraded via the Krebs cycle[5]. However, as a drug carrier system, naked PLGA nanoparticles (NNPs) have the following deficiencies: (1) the drug loading efficiency of NNPs was lower [6]; (2) NNPs can be easily eliminated from blood circulation by the mononuclear macrophage system (MPS) due to its hydrophobic property, which resulted in the reduction of accumulation at targeting site; (3) it is difficult for NNPs to escape from the endosomes or lysosomes. NNPs are easily locked in lysosomes, or directly pumped out of tumor cells, which lead to the decrease of delivery efficiency and efficacy of the drugs[7]. In order to overcome these deficiencies, surface modification of NNPs has drawn much more attention. The coating of poly(ethylene glycol) (PEG) to nanoparticle surface can prolong circulation time of NNPs by increasing the hydrophilicity and inhibiting nonspecific protein adsorption, opsonization, and subsequent clearance. However, the poor cellular internalization of PEGylated NNPs always leads to intracellular concentration of antitumor drug below the required level, subsequently decrease the efficacy of chemotherapy[8,9]. The positively charged nanoparticles show high cellular internalization due to their high affinity with negatively charged cell membranes. However, the positively charged nanoparticles have a strong interaction with the negatively charged serum components, and can be captured by MPS, which result in a short blood circulation time[10]. On the other hand, the negatively charged nanoparticles show low cellular internalization, but they hardly adsorb serum protein and show a rather long blood circulation[11,12]. In order to solve this dilemma, pH-triggered charge conversion has been proved to be a very efficient strategy to



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improve drug delivery efficiency[13]. Kataoka et al. first reported the concept of charge-conversional polymer based on citraconic amides, which are stable under neutral conditions. In acidic conditions, the negatively charged carboxylate functionalities transformed into positively charged primary amines[14]. After that, many charge-conversional drug delivery systems were reported, including PEG-b-PCL copolymer micelles[15], the PVA nanogels[16], hybrid polymer micelles[17], PEGylated

polymeric

micelles[18]

and

PEG-polypeptide

polyionic

complex

nanoparticles[13]. The imidazole ring in histidine (His) has lone pair electrons on the unsaturated nitrogen atom that can be rapidly and reversibly protonized and deprotonized. The pKa value of histidine is 6.0. Therefore, histidine can provide a negative charge in the normal physiology environment and a positive charge in tumor tissue and endolysosome. In addition, the pKa value of arginine (Arg) is 12.5, the positive charged arginine-rich cell-penetrating peptides (CPPs) and oligoarginine have been reported to penetrate various types of cells efficiently without causing significant cytotoxicity[19-21]. The chemical property of histidine and arginine was used to increase the uptake of drug delivery system by tumor cells and enhance the escape of drug

delivery

system

from

endolysosome[22-27].

Thus,

in

this

paper,

CHO-Arg-His-OMe conjugate was designed and used as charge conversal material. Futhermore, in order to improve the specificity of drug delivery system, it is important to modify NNPs with suitable active-targeting ligands. Folate receptor is highly expressed in a variety of human tumor tissue, but lowly expressed in normal tissue[28,29]. Folic acid (FA) showed high affinity for folate receptor (Kd=0.1 nM)[30]. FA has been used to graft on the nanoparticles surface to selectively increase tumor cellular internalization through receptor-mediated endocytosis [31]. So, in our current study, a new dual-function drug delivery system with high drug delivery efficiency was developed (as showed in figure1). PLGA nanoparticles were coated by FA-PEG-DSPE and CHO-Arg-His-OMe to form lipid-polymer hybrid PLGA nanoparticles (LNPs). LNPs maintained a negatively charged property in the blood circulation and became neutral or slight negatively charged in tumor tissue. FA


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that coated on the surface of nanoparticle guided LNPs to recognize FA receptor over-expressed tumor cells. Finally, the charge reversed components are expected to turn positive in endolysosomes to facilitate the endolysosome escape of the LNPs, consequently, enhance the antitumor activity of DOX-loaded LNPs.

Materials and methods Materials PLGA (lactic/glycolic acid molar ratio is 75/25, and weight average molecular weight is 40-75 kDa) was obtained from EVONIK Industries (Recklinghausen, German). Polyvinyl alcohol (PVA, MW20000-30000) was purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine N-hydroxysuccinimide

(NHS),

(DSPE),

folic

dicyclohexylcarbodiimide

acid

(FA), (DCC),

dimethylaminopyridine (DMAP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), trifluoroacetic acid, and histidine methyl ester (His-OMe) were purchased from

J&K

CHEMICA

(Beijing,

China).

3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). H2N-PEG-NH2 (average molecular weight 4000) was obtained from Shanghai Yare Biotech Inc.(Shanghai, China). Doxorubicin was obtained from Hisun Pharmaceutical Co. (Zhejiang, China). RPMI1640 medium, LysoTracker green and 4’,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen Technology Company (Carlsbad, USA). Cell line and animals Human breast cancer cell line MDA-MB-231 cell (high expression of FA receptor)[32,33], human squamous carcinoma cell line KB cell (high expression of FA receptor)[34-36] and human lung adenocarcinoma epithelial cell line A549 cell (deficiency in FA receptor)[37] were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Science (Shanghai). DOX-resistant cell, MDA-MB-231/ADR, was induced in our lab[38]. BALB/c nude mice (20±2 g) and SD rats (200±15 g) was purchased from Animal Center of the Fourth Military



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Medical University. Animals were feed with water and laboratory chow with a 12-hour light-dark cycle. Synthesis of CHO-Arg-His-OMe The synthetic scheme for CHO-Arg-His-OMe is showed in supplementary figure 1. Fmoc-Arg (1.2 g, 0.0030 mol) was dissolved in 10 ml pyridine, then POCl3 (697.5 mg, 4.5 mmol) and His-OMe (484 mg, 2 mmol) were added and stirred for 2 h at room temperature. The pyridine was removed by using rotary evaporator. The residue was dissolved in methanol, and the same volume of diethylamine (DEA) was added and stirred for 2 h. Then, water and dichloromethane were added and stirred, and the water phase was collected. Then the water was removed by rotary evaporator, and the residue (Arg-His-OMe) was purified by reversed phase column (the eluent was consist of water and methanol, v/v=20:80). Cholesterol hemisuccinate (CHEMS, 2.0 g, 4.1 mmol) was dissolved in dichloromethane, then EDC (1.3 g, 6.8 mmol) and NHS (0.9 g, 7.8 mmol) was added and stirred. After 2 h, Arg-His-OMe (1.0 g, 3.1 mmol) and TEA were added into the reaction mixture and stirred for 24 h at room temperature. The solvent was removed by rotary evaporator. Finally, the residue was purified through silica gel column (the eluent was consist of petroleum ether and dichloromethane, v/v=1:1). The yield of CHO-Arg-His-OME was 21%. Synthesis of FA-PEG-DSPE The synthetic scheme for FA-PEG-DSPE is showed in supplementary figure 2[39]. DSPE (748 mg, 1 mmol) and succinic anhydride (200 mg, 2 mmol) were dissolved in 12 mL chloroform and stirred for 10 h at room temperature. After the solvent was removed, the residue was crystallized in acetone, and the product DSPE hemisuccinate was collected by filtering. DSPE hemisuccinate (50 mg, 0.0577 mmol), DCC (25 mg, 0.121 mmol) and NHS (9 mg, 0.078 mmol) were dissolved in 2 mL chloroform and reacted for 6 h at room temperature. Then FA-PEG-NH2 (130 mg) and 20 µl TEA were added, and the reaction mixture was stirred under nitrogen for 24 h at room temperature. The chloroform was removed by rotary evaporator. Then the residue was dissolved in water and filtered. The filtrate was dialyzed (molecular weight cut off: 1000 Da) in water for 3 days. The solution in dialysis tube was


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lyophilized. The freeze-dried powder (FA-PEG-DSPE) was collected. The yield of FA-PEG-DSPE was 40%. Preparation of DOX-loaded lipid hybrid PLGA nanoparticles A modified water-in-oil-in-water (W/O/W) double nanoemulsion method was used to prepare DOX loaded lipid hybrid PLGA nanoparticles (LNPs)[40]. 4.0 mL water solution containing 3.0 mg DOX was emulsified by adding 8.0 mL dichloromethane containing 16 mg PLGA. The above mixture was further emulsified by adding 15 mL of 3.0% PVA aqueous solution and 4 mL dichloromethane containing 4 mg CHO-Arg-His-OMe and 8 mg FA-PEG-DSPE to produce W/O/W double nanoemulsion. Then the mixture was immediately poured into 50 mL of 0.3% PVA solution. After the mixture solution was stirred for 3 h at room temperature, the solidified LNPs were collected by centrifugation and rinsed four times with deionized water. DOX loaded PLGA nanoparticles (NNPs) was prepared by the same method. Characterization of LNPs The particle size, polydispersity, and zeta potential of nanoparticles were determined at 25 °C by using a Beckman Coulter Particle Analyzer (Fullerton, California, USA). The morphology of nanoparticles was observed using transmission electron microscopy (TEM, JEOL-100CXII, Japan), and the surface compositions of the nanoparticle were investigated by using X-ray photoelectric spectrometer (XPS, RATOS AXIS His system, Shimadzu, Japan) [40]. The drug loading and encapsulation were

determined

by

using

fluorescence

spectroscopy

(970

CRT

Spectrofluorophotometer, Shanghai Precision and Scientific Instrument Co. Ltd, Shanghai, China). Briefly, the free DOX concentration in DMSO was measured at 470 nm/597 nm (λexcitation/λemission) by using a 970CRT fluorescence spectrophotometer. The regression equation of working curve was y=25.183x-12.917 (R2=0.9959)，and linear ranged from 0.4 to 25 µg/mL. To determine the drug loading, 1 mg of LNPs was dissolved in 3 mL DMSO solution. The fluorescent intensity of DMSO solution was detected by using fluorescence spectrophotometer, and the amount of DOX in DMSO solution was calculated according to regression equation of working curve. Drug loading (DL) and encapsulation efficiency (EE) was expressed as a percentage



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and was calculated by following formula: DL= (weight of drug loaded/weight of drug loaded in nanoparticles)×100% EE= (weight of drug loaded/total weight of drug in feed)×100% DOX release kinetics from LNPs in vitro 10 mg DOX-loaded LNPs was dispersed in 3 mL PBS solution (pH7.4, pH6.4 and pH5.0) in eppendorf tube and shaken at 80 rpm in water bath at 37 °C. At the predetermined time point, the mixture was centrifuged at 8000×g at 4 °C for 15 minutes. Then 20 µL of supernatant was collected and the same volume of fresh PBS solution was added into the tube. The LNPs was re-dispersed by shaking the eppendorf tube. The DOX concentration in PBS solution was measured at 470 nm/597 nm (λexcitation/λemission) by using a 970CRT fluorescence spectrophotometer. The regression equation of working curve was y=24.967x-13.0214 (R2=0.9987)，and linear ranged from 0.4 to 25 µg/ mL. The fluorescent intensity of supernatant was detected by using fluorescence spectrophotometer, and the amount of DOX in supernatant was calculated according to regression equation of working curve. In vitro membrane dissolution experiment In order to determine whether the drug-free LNPs could cause hemolysis and damage to membrane of endolysosomes, blood red cell obtained from rat was used to perform hemolysis test. After washing red cells with PBS for three times, erythrocytes were dispersed in PBS (pH 5.0, 6.5, 7.4) and incubated with drug-free LNPs or NNPs (0.6 mg/mL). Normal saline and 0.2% triton X-100 were used as negative and positive control, respectively. The erythrocyte disperse solution were incubated at 37 ◦C for 2 h and centrifuged at 2000 rpm for 10 min. The supernatant was collected to detect the hemoglobin by using spectrophotometry at 414 nm. The hemolysis ratio (HR%) was calculated

according

to

the

following

formula:

HR

(%)=[(ODsample-ODnegative)/(ODpositive-ODnegative)]×100%. Cytotoxicity of LNPs MTT assay was used to evaluate the cytotoxicity of DOX-loaded LNPs on KB and MBA-MD-231/ADR cells[41]. KB and MBA-MD-231/ADR cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (5% CO2 at 37 ◦C).


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Cells were seeded in 96-well plates (1×104 cells per well) for 12 h. After that, the cell culture medium was removed, and cells were incubated with fresh medium containing series concentration of free DOX and DOX-loaded LNPs for 48 h. Then, 20 µL of MTT (5 mg/mL) was added to 96-well plates and incubated for 4 h. The formazan crystals were solubilized with DMSO, and the absorbance of DMSO solution was determined at 493 nm by using an automated computer-linked microplate reader (Bio-Rad Laboratories, Richmond, California, USA). Measurement of caspase-3 activity KB cells and MDA-MD-231/ADR cells were seeded in dishes and incubated for 24 h. The culture medium was replaced with fresh medium containing DOX-loaded LNPs. After 12 h or 24 h, cells were harvested and washed with PBS. After centrifugation for 15 min at 4 ºC, cells were collected and re-suspended in cell lysis buffer. The cell lysis buffer was centrifuged. 40 µL supernatant was collected and incubated with 10 µL Ac-DEVD-pNA（2 mmol/L）for 12 h. The absorbance of culture mixture was detected at 405 nm by using Bio-Rad Microplate Reader (Bio-Rad Laboratories, Richmond, California, USA). Cellular uptake of LNPs[42] KB cells and MDA-MD-231/ADR cells were seeded into 24-well plates (104 cells/well) containing a cover glass in each well. After 24 h, cells were incubated with fresh medium containing DOX-loaded LNPs (2 µmol DOX/L) for 30 min and 4 h, respectively. Cells were washed 3 times with PBS (pH7.4) and stained with 500 µL DAPI (100 µg/mL) for 15 min. After washing with PBS for three times, cells were fixed with 1.5% formaldehyde and imaged by a Zeiss 510 LSMNLO confocal microscope (Carl Zeiss Microscope systems, Jena, Germany). Because the main target of DOX is localized in nucleus, thus confocal laser scanning microscopy was used to investigate the traffic of DOX in tumor cells. KB cells and MDA-MD-231/ADR cells were grown on glass coverslips and allowed to adhere for 24 h. Then they were exposed to the fresh cell culture medium containing 2 µmol DOX/L DOX-loaded LNPs. The cells were incubated for 4 h. After that, the drug-containing medium was replaced by the fresh cell culture medium containing



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DAPI (10 µg/mL), and cells were incubated for another 10 min to stain the nucleus of the cells. The cells were rinsed with PBS three times and incubated with 50 nmol/L LysoTracker green for 30 min at 37 °C. After removing the staining solution, cells were rinsed with PBS three times and fixed with formaldehyde for 15 min at 4 °C. A Zeiss 510 LSMNLO confocal microscope was used to obtain the cell image. In vivo anti-tumor activity Balb/c nude female mice (4-6 weeks old) were subcutaneously implanted with KB cells in dorsal ﬂank (3×106 cells/0.2 mL/mice). After the tumor volume reached 70-100 mm3, animals were randomly divided into five groups. There were five mice in each group. The tumor-bearing mice were treated with normal saline, free DOX (5 mg/kg) or DOX-loaded LNPs (equivalent dose of doxorubicin was 1.5, 3 and 5 mg/kg) by tail vein injection every 7th day (day 1, 7, and 14). Body weight of mice was detected as an index of toxicity. Tumor size was measured every 3 days with a caliper and calculated using the formula: volume= LW2/2 (L is the long diameter and W is the short diameter of tumor)[43]. DOX distribution in tumor-bearing mice For vivo DOX distribution study, free DOX and DOX-loaded LNPs (1 mmol DOX/kg) were administered to the tumor-bearing nude mice via the tail vein. After 24 h, mice were sacrificed. The organs and tumor tissues were collected. The fluorescence intensity in different organs and tumor tissues was detected by the Caliper IVIS Lumina Ⅱ in vivo image (Caliper Life Science, USA).

Results and Discussion Characterization of synthesized material 1

H NMR (CD3Cl) spectrum of CHO-Arg-His-OME is showed in supplementary

figure 3, δ=7.58 (1H, s, -N-CH=C-)，6.81 (1H, s, -N=CH-)，5.38-5.39 (1H, d, HC=C-, J=4.0Hz)，4.79-4.83 (1H, m,-CH-)，4.61-4.68 (1H, m, -CH-)，4.12-4.14 (1H, m, -CH-)， 3.75 (3H, s, -OCH3)，3.49-3.53 (2H, m, -CH2-CON-)，3.19-3.20 (2H, d, -CH2-CON-, J=4.0Hz)，2.51-2.62 (4H, m, -CH2-)，2.34-2.35 (2H, d, -CH2-N-, J=4.0Hz), 0.69-1.70 (48H, m, aliphatic group). MS spectrum of CHO-Arg-His-OME is showed in


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supplementary figure 4, [M+H]+ was 793. IR (KBr) spectrum of CHO-Arg-His-OME is showed in supplementary figure 5, 1653 (C=O, st), 1736 (C=O, st), 1169 (C-N, st). 1

H NMR (CD3Cl) spectrum of FA-PEG-DSPE is showed in supplementary figure 6,

δ=6.9, 7.0, 7.5 were characteristic peaks of FA, δ=3.6 was the characteristic peak of PEG backbone, and δ=0.7 was characteristic peak of DSPE. IR (KBr) spectrum of FA-PEG-DSPE is showed in supplementary figure 7, 1637 (C=O, st), 1618 (C=O, st). Particle characterization The particle size, polydispersity, zeta potential and DOX-loaded NNPs and DOX-loaded LNPs are showed in table 1. The results indicated that the drug loading and encapsulation efficiency of LNPs was significantly higher than that of NNPs. The particle size distribution and TEM image of DOX-loaded LNPs are showed in figure 2. DOX-loaded LNPs exhibited core-shell structure, and the morphology of DOX-loaded LNPs was spherical in shape. The mean diameter of DOX-loaded LNPs determined by the transmission electron microscopy (TEM) was smaller than that measured by DLS. This is because the diameter obtained from the DLS was detected in water solution, whereas the diameter observed by TEM was detected in the form of dried LNPs. Therefore, the decrease of particle size obtained from TEM is probably resulted from the dehydration and shrinkage of particles during drying. Similar result was also found in the previous papers[44,45]. The surface chemistry of LNPs is showed in figure 2. There is no nitrogen atom in chemical structure of PLGA, so no N 1s signal was observed on the surface of NNPs (figure 2C). There are 10 nitrogen atoms in FA-PEG-DSPE, the chemical composition weight percentages of nitrogen on the surface of FA-PEG-DSPE modified PLGA nanoparticles were 0.29% (figure 2D). The chemical composition weight percentages of nitrogen on the surface of LNPs were 2.31% (figure 2E). This result indicated that folate and CHO-Arg-His-OME were coated on the surface of LNPs, whereby guide LNPs to recognize the FA receptor over-expression tumor cells. Because zeta potential indicates the surface charge of nanoparticle, it has been usually used to predict dispersion stability. The dissociation degree of carboxyl groups and amino groups in polymers is significantly influenced by environmental pH[46]. The



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zeta potential of LNPs in different pH medium is showed in figure 3. The zeta potential of LNPs in pH 7.4 medium (mimicking the blood circulation environment) was about -23.4 mV. The results implied that the modification of PLGA with FA-PEG-DSPE and CHO-Arg-His-OMe could attenuate the absorption of opsonin in blood circulation. In pH 6.5 medium (mimicking the microenvironment of tumor tissue), zeta potential was close to zero. However, in pH 5.0 medium (mimicking the microenvironment of endolysosome), zeta potential was +11.9 mV. This was because the dissociation degree of carboxyl groups was decreased and dissociation degree of amino groups was increased when the environment pH was changed from neutral to acidic. The pH is about 6.2 to 6.8 in the microenvironment of tumor tissue where the surface charge of LNPs was changed from -23.4 mV to zero, subsequently increased the uptake of LNPs by tumor cells. When LNPs got into endolysosome, the surface charge of LNPs could reverse to be positive, which resulted in the enhancement of the interaction between LNPs and endolysosome membrane, consequently enhanced the escape of LNPs from endolysosome. Drug release profiles of LNPs DOX release characteristic from DOX-loaded LNPs is showed in figure 4A. DOX was released out relatively fast from DOX-loaded LNPs in 10 h, which was mainly resulted from the release of DOX adsorbed in the outer layer of LPNs. DOX-loaded LPNs released about 40%, 57% and 80% of the total loaded DOX in 10 h in pH 7.4, pH 6.4 and pH 5.0 medium, respectively. About 55%, 71% and 98% of the loaded drug were released in pH 7.4, pH 6.4 and pH 5.0 medium in 96 h, respectively. The relative slow release of DOX from LPNs in the neutral medium was resulted from the gradual hydrolysis and corrosion of PLGA core

[6]

. The hydrolysis of PLGA is faster

in acidic medium than in neutral medium, consequently LPNs released DOX in pH-dependent manner. The stability of LNPs in PBS (pH=7.4) is showed in figure 4B. The results indicated that LNPs could be stable more than 8 days. Hemolysis effect of LNPs Hemolysis experiment reflects the effect of nanoparticles on the biomembrane[47]. As showed in table 2, the hemolytic effect of LNPs was dependent on the pH of medium.


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The results indicated that LNPs did not induce any significant hemolysis in pH 7.4 medium. The pH is 7.4 in the microenvironment of blood circulation where LNPs did not cause any damage to the membrane of boold red cell. This implied that LNPs was safe for blood red cell in the blood circulation. LNPs caused significant hemolysis in pH 6.4 medium. LNPs exhibited the strongest hemolytic effect in pH 5.0 medium. This is because the surface charge of LNPs was +11.9 mV in pH 5.0 medium, which enhanced the interaction between LNPs and the membrane of blood red cell, and subsequently led to fracture of red cell membrane. The pH is about 4 to 5 in the microenvironment of lysosome in which the LNPs could cause the obvious damage to the membrane of lysosome, consequently enhanced the escape of LNPs from lysosomes. In vitro cytotoxicity on tumor cells The cytotoxicity of drug-free LNPs on KB cells is showed in figure 5A. The results indicated that the cytotoxicity of drug-free LNPs on KB cells was negligible. The cytotoxicity of free DOX, DOX-loaded NNPs, DOX-loaded LNPs on KB cells and MDA-MB-231/ADR cells are showed in figure 5B and figure 5C, respectively. DOX-loaded

LNPs

exhibited

obvious

cytotoxicity

on

KB

cells

and

MDA-MB-231/ADR cells, while free DOX and DOX-loaded NNPs showed no significant cytotoxicity on MDA-MB-231/ADR cells. In addition, as shown in figure 5D, the exogenous FA decreased the cytotoxicity of DOX-loaded LNPs on KB cells in dose dependent manner. The above results indicated that DOX-loaded LNPs could enhance the antitumor activity on wild type of tumor cell and overcome the DOX-resistance on MDA-MB-231/ADR cell. This implied that modification with FA-PEG-DSPE and CHO-Arg-His-OMe played an important role in the cytotoxicity of DOX-loaded LNPs. The overexpression of p-glycoprotein (p-gp) is related to the mechanisms of multidrug resistance (MDR)

[48]

. P-glycoprotein can efflux a wide

ranges of chemotherapeutic drugs with different structures from tumor cells and decrease the accumulation of drugs in MDR cancer cells. Some studies have showed that nano-sized particles, such as liposome, micelle and inorganic hybrid particle, can bypass the p-gp efflux pumps, and show significant cytotoxicity on drug resistant



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tumor cells[49-51]. Apoptosis induced by DOX and LNPs After being treated with free DOX, DOX-loaded NNPs and DOX-loaded LNPs, the caspase 3 level in tumor cells is showed in figure 6. Free DOX, DOX-loaded NNPs and DOX-loaded LNPs increased the caspase 3 level in A549 cells, KB cells and MDA-MB-231 cells in time-dependent and dose-dependent manner. Compared with A549 cells, the same dose of DOX-loaded LNPs induced much higher caspase 3 level in KB cells and MDA-MB-231 cells. This was probably because A549 cell was FA receptor deficient cell and it was not sensitive to DOX. In addition, when KB cell was cultured with DOX-loaded LNPs at the dose 10 µg/mL for 24 h, the caspase 3 level was lower than that in 12 h. This was because some KB cells were dead when they were cultured with DOX-loaded LNPs for 24 h, consequently resulted in the decrease of the caspase 3 level[52]. Compared with DOX-loaded NNPs, DOX-loaded LNPs induced much higher caspase 3 level in KB cells and MDA-MB-231 cells. When MDA-MB-231/ADR cells were treated with free DOX for 12 h, the caspase 3 level did not significantly increase as compared with control group. However, after being treated with DOX-loaded LNPs for 12 h, the caspase 3 level in MDA-MB-231/ADR cells was significantly increased as compared with free DOX. The above results were well consistent with the cytotoxicity of free DOX, DOX-loaded NNPs and DOX-loaded LNPs on KB cells and MDA-MB-231/ADR cells. The cellular uptake and subcellular distribution of DOX loaded LNPs The action target of DOX is nuclear DNA of tumor cells, so the cellular uptake of free DOX, DOX loaded NNPs and DOX loaded LNPs in KB cells were investigated by using LSMNLO confocal microscope, the results are showed in figure 7. The cellular uptake of free DOX, DOX-loaded NNPs and DOX-loaded LNPs by KB cells exhibited time-dependent manner. Compared with free DOX and DOX-loaded NNPs, larger amount of DOX-loaded LNPs was uptaken by KB cells in 30 min. When free DOX, DOX-loaded NNPs and DOX-loaded LNPs were cultured with KB cells for 4 h, there were not significant difference in the cellular uptake between DOX, DOX-loaded NNPs and DOX-loaded LNPs. This result indicated that FA coated on


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the surface of LNPs accelerated the cellular uptake of LNPs by KB cells in short time. As culture time was prolonged, the uptake of free DOX, DOX-loaded NNPs and DOX-loaded LNPs by KB cells tended to be saturated. Consequently, the cellular uptake of DOX exhibited no difference between different formulations. Furthermore, as showed in figure 8A, a large amount of DOX was localized in the cytoplasma, and there was little amount of DOX in the nucleus after MDA-MB-231/ADR cells were cultured with free DOX in 4 h. However, after MDA-MB-231/ADR cells were cultured with DOX-loaded LNPs in 4 h, a large amount of DOX was accumulated in the nucleus (figure 8B). This was the reason that free DOX showed almost no cytotoxicity on MDA-MB-231/ADR cells, and DOX-loaded LNPs exhibited high cytotoxicity on MDA-MB-231/ADR cells. This result also implied that DOX-loaded LNPs could escape the efflux of p-gp and delivered more DOX to nucleus of MDA-MB-231/ADR cells. Figure 9 shows the traffic of DOX in the KB cells when KB cells were cultured with DOX-loaded LNPs. When KB cells were cultured with DOX-loaded LNPs for 30 min, DOX was mainly localized in lysosome, and a liittle amount of DOX was accumulated in the nucleus. A large amount of DOX transported to nucleus, and a liittle amount of DOX was accumulated in the lysosome when KB cells were cultured with DOX-loaded LNPs for 4 h. The above data indicated that DOX-loaded LNPs could enhance the escape of DOX from lysosome and deliver more amount of DOX to nucleus, subsequently resulted in the enhancement of antitumor activity of DOX-loaded LNPs. Xiong XB et al. found that when DOX-resistant cells (MDA-435/LCC6MDR) were treated with free DOX, a large amount of DOX was accumulated in the cytoplasma because there was p-gp expressed on the nulear membrane of MDA-435/LCC6MDR cell[53]. The exact mechanism that DOX-loaded LNPs could deliver more DOX to the nucleus need further investigate. Antitumor activity of DOX-loaded LNPs in vivo The antitumor activity of DOX-loaded LNPs on tumor-bearing mice is showed in figure 10. The tumor volume in normal saline treated mice increased rapidly and reached nearly 840 mm3 at the end of the experiment. When tumor-bearing mice were



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treated with free DOX and DOX-loaded LNPs, the tumor volume in tumor-bearing mice increased slowly. After being treated with DOX in 5 mg/kg, the tumor volume reached about 103 mm3 and 164 mm3 on 15th day and 24th day respectively. The tumor volume in DOX-loaded LNPs (equivalent to 5 mg/kg DOX) treated mice was about 80 mm3 and 50 mm3 on 15th day and 24th day respectively. The results indicated that as compared with free DOX, same dose of LNPs significant delayed the growth of tumor in tumor-bearing mice. Meanwhile, LNPs delayed the growth of tumor in dose-dependent manner. The above data implied that DOX-loaded LNPs greatly increased the antitumor activity of DOX in tumor-bearing mice. In addition, the body weight of the mice treated with 5 mg/kg DOX was 18.2 g and 16.8 g on 15th day and 24th day respectively. The body weight of the mice treated with DOX-loaded LNPs (equivalent to 5 mg/kg DOX) was 18.6 g and 19.2 g on 15th day and 24th day respectively. The results indicated that the body weight of the mice treated with 5 mg/kg DOX decreased faster than the weight of the mice treated with same dose of DOX-loaded LNPs. This implied that systemic toxicity of DOX-loaded LNPs was lower than that of the same dose of free DOX. Biodistribution of DOX in tumor-bearing mice The biodistribution of DOX in tumor-bearing mice is showed in figure 11. DOX was distributed through the whole body at 24 h after free DOX was administered to the tumor-bearing mice by the tail vein. However, DOX was mainly distributed in tumor tissue at 24 h after DOX loaded-LNPs were administered to the tumor-bearing mice. This result indicated that more DOX was delivered to tumor tissue by LNPs, and the distribution of DOX in normal tissue was greatly reduced. Consequently, the antitumor activity of DOX was enhanced, and the systemic toxicity of DOX was significantly attenuated. Conclusion Compared with NNPs, CHO-Arg-His-OME and FA-PEG-DSPE modified PLGA nanoparticles (LNPs) increased the drug loading of DOX. The surface charge of LNPs reversed from negative to positive in acidic environment. Compared with free DOX, DOX-loaded LNPs enhanced the cytotoxicity of DOX on tumor cells, increased the


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accumulation of DOX in tumor tissues, and reduced the distribution of DOX in normal tissue obviously. Thus, pH-triggered surface charge reversed DOX-loaded LNPs significantly enhanced the antitumor activity in vitro and vivo, and had potential in tumor targeted therapy.

Source of funding: This work was supported by Shaanxi Science and Technology Innovation Project (2012KTCL03-18) and Shaanxi Natural Science Foundation (2013JQ4026) Conflicts of Interest/Disclosures: None

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Fig.1 Schematic representation of doxorubicin delivery by pH-triggered surface charge reversed nanoparticle with active targeting in KB cells grafted cancer. Fig.2 Characterization of LNPs. Particle size distribution (panel A). TEM image of LNPs (panel B). XPS results of NNPs (panel C), PLGA nanoparticle modified with FA-PEG-DSPE (panel D) and LNPs (panel E). Fig.3 The zeta potential of LNPs in different pH medium.

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pH-Triggered Surface Charge Reversed Nanoparticle with Active

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