Reducing Interstitial Fluid Pressure and Inhibiting Pulmonary

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Reducing Interstitial Fluid Pressure (IFP) and Inhibiting Pulmonary Metastasis of Breast Cancer by Gelatin Modified Cationic Lipid Nanoparticles Xuan Gao, Jun Zhang, Zun Huang, Tiantian Zuo, Qing Lu, Guangyu Wu, and Qi Shen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b05119 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

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ACS Applied Materials & Interfaces

Reducing Interstitial Fluid Pressure (IFP) and Inhibiting Pulmonary Metastasis of Breast Cancer by Gelatin Modified Cationic Lipid Nanoparticles Xuan Gao#, †, Jun Zhang#, †, Zun Huang†, Tiantian Zuo†, Qing Lu‡, Guangyu Wu‡, Qi Shen*,† #

†

Co-first author School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road,

Shanghai 200240, China ‡

Department of Radiology, Renji Hospital, School of Medicine, Shanghai Jiao Tong

University, 160 Pujian Road, Shanghai 200127, China *To whom correspondence should be addressed

Qi Shen School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China Tel: +86-21-34204049 Fax: +86-21-34204049 E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT Interstitial fluid pressure (IFP) in tumor is much higher than that in normal tissue and it constitutes a great obstacle for the delivery of anti-tumor drugs, thus becoming a potential target for cancer therapy. In this study, cationic nanostructured lipid carriers (NLCs) were modified by low molecular weight gelatin in order to achieve the desirable reduction of tumor IFP and improve the drug delivery. In this way, the chemotherapy of formulations on tumor proliferation and pulmonary metastasis was further improved. The nanoparticles were used to load three drugs - docetaxel (DTX), quercetin (Qu) and imatinib (IMA) - with high encapsulation efficiency of 89.54%, 96.45% and 60.13%, respectively. GNP-DTX/Qu/IMA nanoparticles exhibited an enzyme-sensitive drug release behavior, and the release rate could be mediated by matrix metalloproteinases (MMP-9). Cellular uptake and MTT assays showed that the obtained GNP-DTX/Qu/IMA could be internalized into human breast 4T1 cells effectively and exhibited the strongest cytotoxicity. Moreover, GNP-DTX/Qu/IMA demonstrated obvious advantages in inducing apoptosis and mediating the expression of apoptosis - related proteins (Caspase 3, Caspase 9 and bcl-2). In the wound-healing assay, GNP-DTX/Qu/IMA exhibited evidently inhibition of cell migration. The benefits of tumor IFP reduction induced by GNP-DTX/Qu/IMA were further proved after a continuous administration to 4T1 tumor-bearing mice. Finally, in the in vivo anti-tumor assays, GNP-DTX/Qu/IMA displayed stronger anti-tumor efficiency as well as suppression on pulmonary metastasis. In conclusion, the GNP-DTX/Qu/IMA system might be a promising strategy for metastatic breast cancer treatment.

KEYWORDS: Tumor interstitial fluid pressure, metastasis, MMP-9, imatinib, nanoparticles

1. Introduction The successful anti-tumor drug delivery to tumor sites is the prerequisite of effective cancer therapy. There are three main barriers for drug delivery in vivo transvascular wall transport, interstitial transport and transcellular membrane 2


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transport1. The size advantage of nanoparticles and the special physiological structure in tumor tissues contribute to the successful transvascular transport of nanodrugs through enhanced permeability and retention (EPR) effect2. However, the long-term clinical efficacy of nano-drug delivery system is not clear, and the superiority of EPR effect is still controversial3, 4. The intermediate links – interstitial transport may be the key to solve this problem. S Eetezadi et al5 and Chen et al6 have reported that the nano-delivery system mainly distribute in the vascular area and the limited area close to the blood vessels. Abnormal blood vessels, dense extracellular matrix (ECM) and elevated interstitial fluid pressure (IFP) in the tumor microenvironment hinder the effective distribution and penetration of therapeutic drugs to the tumor area, resulting in undesired therapeutic effect7, 8, 9. In normal tissues, the general IFP is about 0-3 mm Hg, which increases significantly to 5-40 mm Hg in tumor tissues10. High permeability of the blood vessels, poor lymphatic drainage, dense ECM and the rapid proliferation of cancer cells are responsible for the high IFP in tumor sites6, 11, 12. On one hand, high interstitial pressure in tumor region would prevent nanodrugs from reaching the desired location through convection and diffusion. On the other hand, the pressure difference between tumor center and edges might promote the invasion and migration of tumor cells to the surrounding normal tissues and blood vessels8. Therefore, the abnormal tumor microenvironment is considered to be the most promising “target” for improving drug delivery behavior. The treatment strategy based on the optimization of solid tumor IFP might provide a new approach for the development of novel chemotherapeutic drugs and the combination administration strategies. Several strategies have been applied to reduce tumor IFP, such as affecting the contractile function of fiber cells13, direct reducing extracellular matrix14, 15, 16, 17 and inhibiting ECM over-synthesis through some cancer-associated growth factors

6, 8, 12,

18, 19

. Imatinib (IMA, STI571) is a potent tyrosine kinase inhibitor, and could inhibit

the expression of Bcr-Abl gene and PDGF receptors as well20. Previous reports have proved that IMA can inhibit the over-production of ECM through inhibiting 3



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PDGFR-β and disrupting the “CAFs - ECM interaction”21,

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22

. Thus IMA could

effectively reduce tumor IFP and improve the delivery efficiency of therapeutic drugs23. PI3K activation and the associated PI3K/Akt-mediated signaling pathways are implicated in tumor cell invasion through the promotion of cell motility and MMP production24. Kim et al. found that MMPs activity and cell invasion ability were inhibited in CT-26 cells by using a PI3K inhibitor (LY294002)25, which suggesting PI3K-mediated signaling pathway is associated with upregulation of MMPs. Quercetin (Qu) is a member of polyphenolic flavonoid compounds which are widely existed in various herbs and foods26. Studies show that quercetin, as a PI3K inhibitor, can effectively inhibit the phosphorylation of Akt27,

28

and downregulate MMP-9

through PI3K/Akt signaling pathway, resulting in the inhibition of tumor invasion and migration29, 30 as well as the induction of apoptosis31, 32. Gelatin is a kind of denatured protein extracted from the bones, skin or muscle membrane of animals. It has been widely applied in drug and gene delivery systems due to a series of advantages, such as the good biocompatibility, biodegradability and low cost33, 34. Cliff Wong35 prepared a kind of intelligent "contractive" nanoparticles modified with gelatin type B. They found that the particle size decreased from 100 nm to about 10 nm after MMPs (highly expressed in the tumor area) degradation of gelatin layer, so that the drug could be better exposed to the tumor microenvironment and exert therapeutic effect. In this study, DTX and Qu were loaded in the cationic NLC-core, and the outer layer was modified with low molecular gelatin containing IMA for the first time (shown in Figure 1). We performed a series of experiments to verify whether the IFP reducing effect of IMA could help to modulate the tumor microenvironment and promote the penetration of DTX/Qu-loaded cationic NLC-core into tumor. The inhibition of tumor proliferation and pulmonary metastasis by the combination of PI3K inhibitors with chemotherapeutic agents was further investigated as well. The aim of this study was to (i) construct and characterize a gelatin modified cationic NLCs system for intravenous administration of DTX, Qu and IMA, (ii) evaluate the 4


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cytotoxicity, cellular uptake efficiency, apoptosis induction and cell migration inhibiting ability of GNP-DTX/Qu/IMA at cellular level, and (iii) estimate whether GNP-DTX/Qu/IMA could reduce tumor IFP and improve the anti-tumor efficacy on 4T1 human breast cell-bearing nude mice. GNP-DTX/Qu/IMA was prepared by an improved emulsification and low-temperature solidification method36. The in vitro release behaviors of three different drugs loaded in GNP were evaluated using dialysis bags. The cellular uptake of nanoparticles by 4T1 human breast cell was estimated using coumarin 6 as a fluorescence probe. The induction of cell apoptosis was investigated through flow cytometry and western blotting technique. The reduction of tumor IFP was demonstrated using “wick-in-needle” method37. In addition, the anti-tumor efficiency of GNP-DTX/Qu/IMA was also studied in nude mice.

Figure 1. Illustration of inhibiting the over production of ECM to modulate the tumor microenvironment and promote nanodrugs to penetrate deeper into tumor tissues.

2. Materials and methods 2.1 Materials Glycerin monostearate (GMS), egg phosphatidylcholine (EPC), Caprylic/Capric Triglyceride (GTCC), cetrimonium bromide (CTAB) and quercetin were all purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Gelatin type B ≈ 75g bloom as well as Active Human Recombinant MMP-9 (expressed in HEK 293 5



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cells) and fluorescence probe coumarin 6 was provided by Sigma-Aldrich Co. LLC. (Missouri, USA). Docetaxel was purchased from Biocompounds Co., Ltd. (Shanghai, China) and imatinib mesylate was provided by Chembest Research Laboratories Limited (Shanghai, China). Quercetin was obtained from Sinopharm Chemical Reagent Co., Ltd. Ultrafiltration centrifuge tube (MWCO: 10 kDa) was supplemented by Merck Millipore (Darmstadt, Germany). RPMI-1640 medium, trypsin (containing 0.02% EDTA), fetal bovine serum and penicillin-streptomycin mixture (100×) were all from Biosera (Villebon sur Yvette, France). All the antibodies and Kits involved in cell assays were come from Beyotime Biotech. Co. Ltd. (Jiangsu, China). Adhesive slides (positively charged surface) and embedding boxes were from Citoglas. Co. Ltd. (Jiangsu, China). All other chemical reagents were from Sinopharm Chemical Reagent Co., Ltd. and were of analytical or HPLC grade. Human breast cancer cell line 4T1 was kindly provided by Chinese Academy of Sciences (Shanghai, China), and was cultured in RPMI-1640 medium containing 10% fetal

bovine

serum,

2.5

g/L

glucose,

0.11

g/L

sodium

pyruvate

and

penicillin-streptomycin mixture (diluted from 100× into 1×, final concentrations of penicillin and streptomycin were 100 U/mL and 100 µg/mL) in a 5% CO2 incubator at 37 ℃. Female Balb/c mice (nu/nu, 5 weeks, 18-20g) were from Laboratory Animal Center, Shanghai Jiao Tong University (Shanghai, China) and kept under 25 ± 2 ℃ and 50 ± 5% humidity conditions with free access to food and water. All experiments were performed following Care and Use of Laboratory Animals Principles and approved by the Animal Ethics Committee of Shanghai Jiao Tong University.

2.2 Preparation of different nanoparticles Lipids of GMS/EPC/GTCC were used for the preparation of NLC-core (50:35:15, w/w/w), while Tween-80 and CTAB performed as emulsifiers (50:3, w/w). Briefly, GMS and GTCC were stirred and dissolved in 5 mL methanol under 60 ℃. After that, 2 mg DTX and 1 mg Qu were added into methanol to form organic phase Ⅰ. Meanwhile, organic phase Ⅱ was prepared by dissolving EPC and Tween-80 in 6


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ethanol (5 mL) under ultrasonic wave. Two organic phases were mixed, stirred under 60 ℃ and injected rapidly into 60 ℃ aqueous phase containing 20 mL pure water and 3 mg CTAB, then keep on stirring under 60 ℃, 600 rpm conditions using a magnetic stirrer until solution was concentrated to about 10 mL to form NLCs. In order to prepare GNPs (gelatin-coated nanoparticles), NLCs solution was added drop by drop into 0.1% gelatin aqueous solution (35 mL) containing 2 mg IMA in the water bath at 60 ℃, 600 rpm for 30 min and solidified for another 30 min in ice bath. Both nanoparticles should be certificated under 15000 rpm for 30 min until the clearance of free drug. The same method was applied to prepare the coumarin-6 loaded NLCs and coumarin-6 loaded GNPs in subsequent experiments.

2.3 Characterization of different lipid nanoparticles The average particle size and zeta potential of nanoparticles were estimated using Nano ZS (Malvern Instruments, UK). Transmission electron microscopy was used to identify the morphology of lipid nanoparticles pretreated by negative staining method. Entrapment efficiency (EE%) and drug loading (DL%) of lipid nanoparticles were estimated using centrifugal ultrafiltration method and calculated using the following formula: % =

−

× 100%

% =

−

× 100%

Where WTotal represented the amount of drug existing in the lipid nanoparticle suspension and WFree meant the unentrapped drug in ultrafiltrate, respectively. WCarrier was the weight of carriers in the lipid nanoparticle solution. The in vitro release profile of lipid nanoparticles was estimated using dialysis technique. Briefly, 1 mL GNP-DTX/Qu/IMA suspension was mixed with 2 mL PBS (0.5% Tween-80, pH 7.4) or PBS containing 500 ng (0.35 µM) of activated MMP-9 (Matrix metalloproteinases-9) and put into a dialysis bag (10,000 Da). Then, it was incubated with 50 mL PBS (0.5% Tween-80, pH 7.4) at 37 ℃ with 100 rpm gentle shaking. At determined time points, 30 mL medium was removed for HPLC 7



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quantitative analysis and replaced by the same volume of isothermal PBS. The release profiles of free drug and NLC-DTX/Qu were achieved using the same method as described above and their release systems didn’t contain MMP-9. The released DTX was analyzed by HPLC using a mobile phase consisting of acetonitrile and water (6:4, v/v). The HPLC analysis conditions of Qu and IMA were the same as DTX except changing the mobile phases into methanol: 0.2% phosphoric acid (7:3, v/v) and acetonitrile: 0.2% phosphoric acid (18:82, v/v), respectively.

2.4 Cell viability assays 4T1 cells in the logarithmic growth phase were seeded into 96-well plates (2.5× 104 cells/mL, 200µL per well) and cultured for 24 h under 37 ℃, 5% CO2 conditions. DTX, DTX + Qu, NLC-DTX/Qu and GNP-DTX/Qu/IMA were diluted with complete culture medium into a series of concentrations, and added into 96-well plates to replace the previous medium. After incubated for 24 h, a volume of 20 µL MTT solution (5 mg/mL) was added into each well. Once the 4 h-incubation finished, the corresponding formazan crystal was dissolved in 150 µL DMSO and the optical density was measured at 570 nm using the microplate reader (Thermo Scientific, USA). The inhibitory rate against 4T1 cells could be calculated according to the following formula: Inhibitory rate = （OD570control cells - OD570treated cells）/ OD570control cells × 100% And the IC50 value was calculated using GraphPad Prism 6.0 software to determine the cytotoxicity on 4T1 cells.

2.5 In vitro cellular uptake Cellular uptake of different formulations was investigated using a flow cytometer. Prior to this assay, coumarin-6 was first dissolved in ethanol and loaded into NPs, then diluted to 20 ng/mL with complete culture medium. 4T1 cells were seeded into 6-well plates (2.5 × 105 cells/mL, 2 mL per well) and cultured overnight under 37 ℃, 5% CO2 conditions. Medium containing various coumatin-6 loaded formulations was added to each well and medium without drug was set to the blank control. After 4 h 8


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co-incubation, cells were washed thrice with cold PBS and harvested in 0.5 mL PBS. 10,000 cells/sample were measured through the flow cytometer (FACScan, USA).

2.6 Evaluation of cell apoptosis Annexin V-FITC/PI double channel staining method38 was applied in the detection of 4T1 cells apoptosis induced by different NPs. Briefly, 4T1 cells were seeded into 6-well plates (2.5 × 105 cells/mL, 2 mL per well) to adhere overnight. Fresh medium containing DTX, DTX + Qu, NLC-DTX/Qu and GNP-DTX/Qu/IMA (DTX was 5 µg/mL, Qu was 2.5 µg/mL, IMA in GNPs was 5 µg/mL) was added to each well and cultured in the incubator for 24 h. Then cells were washed with cold PBS and harvested in 0.5 mL Annexin V-FITC binding buffer. In the flow tubes, cells were incubated in dark with 5 µL Annexin V-FITC and 10 µL PI staining solution for 20 min, and then tubes were transferred into ice for another 5 min incubation. 10,000 cells/sample were measured through the flow cytometer (FACScan, USA). All samples should be kept in cold and dark environment and detection was finished within 1 h.

2.7 Cell scratch wound healing assays 5 × 105 cells/well 4T1 cells were seeded into 6-well plates and cultivated in an incubator for over 24 h to achieve 90% confluence. Abandon the medium, and then a 10-µL sterile pipette tip was used to create the horizontal mechanical scratch wound in the bottom of the culture plate. Cells were washed thrice with cold PBS and fresh medium containing DTX, DTX + Qu, NLC-DTX/Qu and GNP-DTX/Qu/IMA (Qu was 1 µg/mL, DTX was 2 µg/mL, IMA was 2 µg/mL) was added to each well. Wound regions of the cells in each well were captured randomly at 0 h and 36 h using a IX51 inverted microscope (10× objective, Olympus). And the distance of wound closure could reflect the inhibitory capacity of different NPs on cell movement39.

2.8 Western blotting analysis of apoptosis and metastasis-related proteins Western blotting analysis were used to evaluate the expression levels of 9



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Caspase-3, Caspase-9, bcl-2 and MMP-9 proteins. In brief, 4T1 cells were seeded at 5

× 105 cells/well in 6-well plates and cultured for 24 h in a 37 ℃, 5% CO2 incubator. Replace the original medium with fresh medium containing various drug-loaded formulations (DTX was 5 µg/mL, Qu was 2.5 µg/mL, IMA in GNPs was 5 µg/mL) and co-incubate with cells for another 24 h. According to the standard Western blotting protocol, proteins were extracted by lysing cells, denatured at 98 ℃ and analyzed immediately. Band density was calculated using ImageJ 1.48u software.

2.9 Effect of GNP formulation on tumor IFP 4T1 tumor-bearing female Balb/c nude mice models were constructed by the following procedures: 4T1 cells were harvested and resuspended in PBS to make the final concentration of cell suspension become 2.5×107 cells/mL, 0.2 mL of cell suspension was inoculated subcutaneously into the right axillary of female Balb/c mice (nu/nu, 5 weeks, 18-20g)40. Seven days after tumor cell inoculation, mice were treated with three continuous administration of saline or GNP suspension (DTX and IMA dosage was 10 mg/kg, Qu was 5 mg/kg) via tail veins, respectively. Tumor IFP was measured before and after administration by “wick-in-needle” method37, 41 using a 23-gauge needle with a side hole about 5 mm away from the sealed tip, and strands of nylon fibers were placed in the needle through a steel wire loop. The needle was connected to the pressure measuring system through a tube filled with saline. Mice were subjected to intraperitoneal anesthesia before IFP measurement. The needle was inserted into the center of the lump on 4T1 tumor-bearing nude mice, which could cause a transient fluctuation in pressure and this fluctuation will return toward stable level within 5 to 10 min. Record data in the computer and calibrate instruments before each measurement. Each sample was measured three times. The pressure signals inside tumors could be transferred into electric signals through a pressure transducer (PowerLab, ADInstruments) and recorded by LabChart 8 software.

2.10 In vivo antitumor efficiency 4T1 tumor-bearing female Balb/c mice models were established as described in 10


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section 2.9. When tumor volume grew to ∼200 mm3, 4T1 tumor-bearing mice were divided randomly into control (saline) group, DTX+Qu+IMA group and GNP-DTX/Qu/IMA group. The two weeks-administration regimen of tail vein injection was adopted and mice were treated with different formulations twice a week (DTX and IMA dosage was 10 mg/kg, Qu was 5 mg/kg). Body weight as well as tumor volume was evaluated every other day after the first administration. Tumor volume was calculated using the following formula: =

!"# "$%& × !%' "$%& 2

(

At the end of the third week, mice were sacrificed, and tumors were weighted to evaluate the in vivo antitumor efficiency. Meanwhile, the lung tissues were removed and soaked in cold PBS, photos were taken in white light to calculate the number of visible pulmonary nodules in each group. H&E staining was performed to observe the formation of tumor micrometastasis in lung tissues.

2.11 Statistical analysis All assays were performed at least three parallel samples. Values were processed using GraphPad Prism 6.0 software and presented as mean ± SD. Statistical analyses were performed using an unpaired, two tailed student t-test. The level of significance was set *p < 0.05, **p < 0.01.

3. Results and discussion 3.1 Preparation and characterization of different nanoparticles NLC-DTX/Qu and GNP-DTX/Qu/IMA nanoparticles were prepared using optimized emulsion evaporation method. Both DTX and Qu were loaded in the NLC core owing to their high hydrophobicity, meanwhile the hydrophilic IMA was entrapped into the gelatin layer. As shown in Table 1, the encapsulation efficiency of DTX and Qu in NLC-DTX/Qu and GNP-DTX/Qu/IMA nanoparticles were almost the same, around 88% for DTX and 95% for Qu. GNP-DTX/Qu/IMA has lower encapsulation efficiency of IMA (about 60%), it might be due to the fact that water 11



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soluble drugs are easy to escape into the aqueous phase. NLC-DTX/Qu exhibited a positive zeta potential (17.21 ± 0.58 mV), and after gelation (type B) modification, this potential turned into -3.27 ± 0.15 mV. The isoelectric point (pI) of gelatin type B (obtained by an alkaline hydrolysis of bovine collagen) was 4.8-542. Therefore, gelatin type B tends to be significantly negative under physiological conditions. This charge reversal certified the successful modification of gelation layer. NLC-DTX/Qu and GNP-DTX/Qu/IMA presented different size distributions with mean diameters around 95 nm and 112 nm, respectively. The change of particle size could also testify the gelatin

layer

modification.

Spherical

shapes

of

NLC-DTX/Qu

and

GNP-DTX/Qu/IMA were imaged through transmission electron microscopy (TEM) as Figure 2 showed. The in vitro behaviors of DTX, Qu and IMA releasing from GNP-DTX/Qu/IMA in two kinds of buffer (with or without MMP-9) were displayed in Figure 3. The release profiles of all drugs from GNP-DTX/Qu/IMA tended to be stable and sustained, and the cumulative release of three drugs in 72 h was no more than 50%, basically maintained between 30-40%. But the release obviously accelerated from 4 h to 12 h after adding the activated MMP-9, and the final cumulative release amount of DTX, Qu and IMA from GNP-DTX/Qu/IMA was 1.86-fold, 1.50-fold and 1.57-fold compared with that in PBS, respectively. The slow release profile of GNP-DTX/Qu/IMA indicated its good stability in pH 7.4, which might prevent drug from rapidly releasing in the bloodstream and promote accumulation of nanoparticles in tumor sites. Meanwhile, the accelerated release rate under pH 7.4 with MMP-9 illustrated that GNP-DTX/Qu/IMA was enzyme sensitive, thus might release drug accurately in tumor tissues (with high expression of MMP-9) and exert therapeutic effect.

12


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Table 1 Physicochemical characteristics of different formula (Mean ± SD, n = 3). EE (%)

Zeta

DL (%)

Formula

Size (nm)

PDI

potential

DTX

Qu

IMA

DTX

Qu

IMA

NLC

88.16±4.72

95.24±2.26

-

1.56±0.41

0.81±0.19

-

95.6±7.15

0.151±0.06

17.21±0.58

GNP

89.54±5.16

96.45±3.22

60.13±6.42

1.62±0.36

0.83±0.11

1.06±0.32

112.8±6.20

0.227±0.10

-3.27±0.15

(mV)

Figure 2. Morphology of the multifunctional lipid nanoparticles by TEM. (A) NLC (B) GNP

13



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Figure 3. In vitro release profile of (A) DTX, (B) Quercetin and (C) Imatinib from GNP-DTX/Qu/IMA in PBS (pH 7.4) and PBS (pH 7.4) with activated enzyme MMP-9 at 37 ℃. Data were presented as mean ± SD (n = 4).

14


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3.2 Cytotoxicity of nanoparticles on 4T1 cells MTT method was applied to investigate the in vitro cytotoxicity of free drug and drug-loaded nanoparticles on 4T1 cells. As shown in Figure 4, after 24 h co-incubation with different formula at various concentration, the growth rate of 4T1 cells reduced in a dose-dependent way. At low concentration (1 µg/mL, calculated as DTX concentration), there was no significant statistical difference between free drug and nanoparticles. When drug concentration increased, differences were presented. At 10 µg/mL concentration (calculated as DTX concentration), the cell viability of each group was 62.43 ± 4.33% ( DTX)、61.94 ± 5.26% (DTX + Qu)、47.85 ± 2.78% (NLC-DTX/Qu) and 39.27 ± 3.35% (GNP-DTX/Qu/IMA), respectively. At higher concentration (25 and 75 µg/mL), cell viability followed the same order: DTX > DTX + Qu > NLC-DTX/Qu > GNP-DTX/Qu/IMA. The IC50 value summarized in Table 2 indicated that the inhibition rate of nanoparticles on breast cancer cell line 4T1 was significantly higher than that of the original DTX group, and GNP-DTX/Qu/IMA showed the highest cytotoxicity. The IC50 of GNP-DTX/Qu/IMA was about 2.3- and 1.4-fold of DTX solution and NLC-DTX/Qu, respectively. There was no significant difference between the DTX group and DTX + Qu group (p < 0.05), which might demonstrate Qu has unapparent cytotoxicity on 4T1 cells. The previous work in our lab had proved that the carrier materials was biocompatible and had no obvious cytotoxicity. On one hand, the increased cytotoxicity of GNP-DTX/Qu/IMA might be due to the increased cellular uptake, which was caused by electrostatic adsorption43 between cationic NLC core and negatively charged membrane after the degradation of gelatin coating. On the other hand, IMA might make a contribution to the raised toxicity as an adjuvant44, 45, 46.

15



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Figure 4. In vitro cytotoxicity determination using MTT method on 4T1 cells. The cell line was co-incubated with free drug or drug-loaded nanoparticles at 37 ℃ for 24 h. Data presented as mean ± SD (n=4). *p < 0.05.

Table 2 IC50 of the experimental groups on 4T1 cells. （mean±SD，n=4） Groups

IC50 (µg/mL)

DTX

14.27±0.58

DTX+Qu

13.43±0.82

NLC-DTX/Qu

8.923±0.49*

GNP-DTX/Qu/IMA

6.181±0.35*

*p < 0.05 vs. free DTX.

3.3 Cellular uptake Coumarin-6 loaded nanoparticles were used in the investigation of formula’s uptake efficiency on 4T1 cells. Compared with blank control, both coumarin-6 solution group and nanoparticles groups exhibited clearly rightward movement (shown in Figure 5). This phenomenon demonstrated that 4T1 cells displayed different absorption efficiency for free coumarin-6, NLC and GNP. These formula were internalized into 4T1 cells following the order: NLC > GNP > coumarin-6 solution. The minimum uptake of coumarin-6 solution was consistent with previous 16


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reports47, which might be owing to the slow passive diffusion. Compared with that, conmarin-6 loaded cationic NLC and GNP exhibited better cellular uptake efficiency after 4 h co-incubation. As expected, cationic NLC showed the highest fluorescence intensity, which might be due to the combination between cationic NLC and the negative charged cell membrane. This combination promoted nanoparticles entering cells through endocytosis mechanism48. The cellular uptake efficiency of GNP nanoparticles was slightly less than that of cationic NLC, this might be because the gelatin coating of GNP hindered the positive charge thus weakening the affinity between nanoparticles and cell membrane. In addition, the increased cellular uptake of NLC and GNP might result in their higher cytotoxicity (Table 2).

Figure 5. Flow cytometry analysis of coumarin-6 loaded nanoparticles by 4T1 cells. The cell line was co-incubated with free coumarin-6 or coumarin-6 loaded nanoparticles for 4 h before submitted to FACS analysis.

3.4 Cell apoptosis The ability of nanoparticles inducing 4T1 cell apoptosis was analyzed by flow cytometer and Western blotting. Annexin V-FITC/PI dual staining method was used to quantitatively investigate the relation between NPs-mediated cell inhibitory effect and the induction of apoptosis. As shown in Figure 6, compared with blank control, the apoptotic cells (include early and late apoptotic cells) of both free drug and nanoparticles groups increased by different degrees and the order was as follows: 17



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GNP-DTX/Qu/IMA > NLC-DTX/Qu > DTX + Qu > DTX. Cell populations of different stages during apoptosis were displayed in Figure 7 and Table 3, an obvious increase in apoptotic cells was detected in 4T1 cells by GNP-DTX/Qu/IMA (85.4%) and NLC-DTX/Qu (52.8%), compared with DTX (21.6%), DTX + Qu (25.6%) and blank control (0.1%). These results indicated that the above two kinds of nanoparticles could effectively induce 4T1 cells apoptosis than free DTX at the same concentration. And GNP-DTX/Qu/IMA exhibited the strongest ability to induce apoptosis in 4T1 cells. Cell apoptosis has passed through a complicated procedure, involving a series of protein expression, such as Caspase family protein, bcl-2 family protein, etc. The zymogen of Caspase family can be cleaved and activated through extraneous signals thus leading to programmed cell death eventually49, 50. Additionally, bcl-2 family concluding pro-apoptotic proteins (Bad, Bid, Bax, etc.) and anti-apoptotic proteins (bcl-2, bcl-x, etc.)51, is mainly responsible for regulating the stability of mitochondrial structure and function, and plays the role as a "main switch" in the process of cell apoptosis52,

53

. The expressions of Caspase-9 (initiator caspase), Caspase-3 (key

executer caspase) and bcl-2 (a kind of anti-apoptotic protein) in 4T1 cells were measured by Western blotting method. Results were displayed in Figure 8, compared with control, bcl-2 showed a decreased expression in 4T1 cells with whether free drug or nanoparticles treatment (Figure 8B). Among these, GNP-DTX/Qu/IMA exhibited the strongest ability to inhibit bcl-2 expression. That was to say, GNP-DTX/Qu/IMA had the strongest ability to promote apoptosis. Figure 8C and Figure 8D showed the gray value histogram of full length Caspase-3 and -9 expression. The expression levels of both proteins followed the same order: Control > DTX + Qu > NLC-DTX/Qu > GNP-DTX/Qu/IMA. This order indicated that the GNP nanoparticles could promote more potent caspase activation than other formula. In general, as important substrates in the downstream of PI3K/Akt signalling pathways, the expression of Caspase - 9 and Bad were modulated by the phosphorylation of Akt54, 55. Briefly, the inhibition of Akt phosphorylation will lead to (i) the promotion of Caspase - 9, thus activating the downstream Caspase - 3 and enhancing cell 18


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apoptosis, (ii) the dephosphorylation of Bad residues, thus reducing the expression of free bcl-2 and promoting apoptosis. Therefore we speculated that the down-regulation of bcl-2 and up-regulation of activated Caspase - 9 and - 3 (Figure 8) might result from the inhibition of Akt phosphorylation. These results demonstrated that GNP-DTX/Qu/IMA could effectively induce the expression of apoptosis-related proteins, which was consistent with cytotoxicity (Figure 4) and AnnexinV/PI staining assays (Figure 6). The reasons of the apoptosis results might be summarized as follows. When drug-loaded GNP nanoparticles dispersing around 4T1 cells, MMPs separated by tumor cells could degrade the gelatin layer of GNP56. The exposed cationic NLC-core could promote the cellular uptake efficiency through the combination between cationic NLC and negative charged membrane48, thus increase the accumulation amount of DTX and Qu in tumor cells and improve the ability of nanoparticles to induce cell apoptosis.

Figure 6. Quantitative cell apoptosis assay of 4T1 using a flow cytometer. 4T1 cells treated with different experimental groups for 24 h. Then the cells were harvested and stained with the Annexin V/PI dual staining kit. The first line from left to right is Control, DTX, DTX +Qu, second line from left to right is NLC-DTX/Qu and 19



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GNP-DTX/Qu/IMA. Viable cells are shown in the lower-left quadrant (Annexin V−, PI−). The early and late apoptotic cells are shown in the lower-right quadrant (Annexin V+, PI−) and upper-right quadrant (Annexin V+, PI+), respectively.

Figure 7. Stacked bars of 4T1 cell apoptosis determined by flow cytometry using Annexin V-FITC and PI. Table 3 Effect of free drug and nanoparticles on apoptotic induction in 4T1 cells. Groups

Viable cells (%)

Early apoptotic + Late apoptotic cells (%)

Control

99.9

0.1

DTX

78.2

21.6

DTX+Qu

74.3

25.6

NLC-DTX/Qu

46.4

52.8*

GNP-DTX/Qu/IMA

14.6

85.4**

*p < 0.05 vs. free DTX. **p < 0.01 vs. free DTX.

20


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Figure 8. Western blotting analysis of apoptosis related proteins expressed in 4T1 cells after incubating with free drug or drug-loaded nanoparticles for 24 h. (A) Protein bands a.control b.DTX + Qu c.NLC-DTX/Qu d.GNP-DTX/Qu/IMA. (B), (C), (D) the gray value histogram of bcl-2, Caspase-3 and Caspase-9 proteins. *p < 0.05, **p < 0.01 vs. control.

3.5 Inhibition effect on 4T1 cell migration Wound healing method is a simple technique for determining cell migration and repairing ability. The inhibition effect of drugs on the migration ability of tumor cells can be determined by the healing degree of scratches39, 57. Although this method cannot perfectly simulate the migration behavior of tumor cells in vivo, it mimics the migration of cells in wound healing to some degree58. As shown in Figure 9A and 9B, the wound closure followed the order: control > DTX > DTX + Qu > NLC-DTX/Qu > GNP-DTX/Qu/IMA, which could indirectly reflect the migration ability of 4T1 cells after co-incubating with different formula. Compared with the complete healing of wound in blank control, the wound was still open with GNP-DTX/Qu/IMA treatment after 36 h. The wound distance in GNP group was about twice of that in free DTX group, indicating that GNP nanoparticles worked the best on inhibiting cell migration. 21



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Compared with DTX group, the wound distance increased obviously after the treatment with DTX + Qu (Figure 9A.c and d), and the PI3K/Akt inhibition of Qu could account for this phenomenon. Qu can effectively inhibit Akt phosphorylation thus reduce the expression of downstream MMPs and further inhibit cell migration30. Moreover, the distance in NLC-DTX/Qu group was longer than that in DTX + Qu group, which proved the better anti-migration effect of cationic nanoparticles (Figure 9A.d and e). It was worth mentioning that the better inhibitory effect on cell migration of GNP-DTX/Qu/IMA compared with NLC-DTX/Qu (Figure 9A.e and f) might result from the cell growth inhibition of IMA. Apart from reducing tumor IFP, IMA itself could also inhibit tumor growth and is often co-administrated with chemotherapy drugs as an adjuvant in the clinical59. To further confirm the inhibition effect of nanoparticles on cell migration, Western blotting assay was applied to measure the expression of MMP-9 in 4T1 cells. Matrix metallopeptidase family (MMPs) plays an important role in the degradation of the extracellular matrix and basal lamina, as well as improving the ability of tumor invasion

and

migration60,

61

.

Akt

phosphorylation

and

the

associated

PI3K/Akt-mediated signalling pathway are implicated in cell migration and invasion through the promotion of MMP production24. Western blotting analysis (results were shown in Figure 10) indicated that NLC-DTX/Qu and GNP-DTX/Qu/IMA nanoparticles could effectively inhibit the expression of MMP-9 protein compared with free drug, and GNP-DTX/Qu/IMA exhibited the strongest inhibition on MMP-9 expression. This could certify the inhibition effect of GNP-DTX/Qu/IMA on 4T1 cell migration indirectly.

22


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Figure 9. Inhibitory effect of different formula on 4T1 cell migration. (A) Microscopy images of 4T1 cells wound scratch assay. a. Control (0h) b. Control (36h) c. DTX (36h) d. DTX + Qu (36h) e. NLC-DTX/Qu (36h) f. GNP-DTX/Qu/IMA (36h). (B) Histogram of scratch distance. **p < 0.01 vs. control.

Figure 10. Western blotting analysis of MMP-9 proteins expressed in 4T1 cells after incubating with free drug or nanoparticles for 24 h. (a) Control (b) DTX (c) DTX + Qu (d) NLC-DTX/Qu (e) GNP-DTX/Qu/IMA. *p < 0.05 vs. control.

23



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3.6 Effect of formula on the tumor IFP The dense ECM (extracellular matrix) in tumor microenvironment is one of the main reasons responsible for exorbitant IFP in tumor site. IMA could inhibit the overproduction of extracellular matrix effectively, adjusting tumor IFP and priming tumor microenvironment6,

23

. To estimate the effect of GNP-DTX/Qu/IMA

nanoparticles on reducing interstitial pressure, a consecutive administration regimen was carried out. After three days administration, the interstitial pressure of both saline and nanoparticle group rose at different extent, probably due to the tumor growth. As shown in Figure 11, at the end of administration, the IFP of tumors treated with saline was 29.18 mm Hg, which was consistent with previous report10. Compared with saline group, GNP-DTX/Qu/IMA exhibited an obvious inhibitory effect on tumor IFP. In this group the tumor IFP reached 17.20 mm Hg. This significant reduction of tumor IFP might be because IMA could inhibit the PDGF/PDGFR-β signaling pathway and hinder the “CAFs - ECM interaction”21, 22 thus inhibiting the over-production of ECM in tumor microenvironment. Once the pressure inside the solid tumors reduced, nanoparticles were easier to delivery and accumulate into the center of tumors. Fan et al.23 designed a combined therapeutic strategy using liposomal imatinib and liposomal doxorubicin. They found that tumor IFP reduced obviously after intravenous injection of imatinib-loaded liposomal. Meanwhile the decrease of tumor IFP could effectively improve the anti-tumor efficacy of liposomal doxorubicin. What’s more, the antihypertensive effect of imatinib after single dose administration could last more than 50 h. Chen et al.

6

found that reducing tumor IFP could promote drug delivery

systems penetrating deeper into tumor spheroids in vitro and tumor tissues in vivo. Both combined therapy and single nanoparticle administration proved the fact that reducing tumor IFP could be beneficial for anti-tumor drug delivery.

24


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Figure 11. Tumor IFP before and after three consecutive administration of saline or GNP-DTX/Qu/IMA suspension through tail veins (n = 5). **p < 0.01 vs. saline control.

3.7 In vivo antitumor efficiency The apoptosis and wound-healing assays demonstrated that GNP-DTX/Qu/IMA nanoparticles obtained effective inhibition of 4T1 cell growth and migration in vitro. We speculated that GNP-DTX/Qu/IMA could also have the same effect in vivo, and further inhibiting tumor growth and metastasis. Therefore, the antitumor efficiency of GNP-DTX/Qu/IMA nanoparticles was estimated in the following studies. Firstly, the tumor growth inhibitory effect was investigated through pharmacodynamics experiments. As shown in Figure 12B, 20th days after tumor inoculation, compared with saline group, both the free drug and GNP nanoparticles showed anti-tumor effect to a certain extent. The final tumor volume of saline group was (1622.4 ± 272.6) mm3, meanwhile the tumor volume of GNP-DTX/Qu/IMA group reached (568.4 ± 73.6) mm3 – which is about 35% of the saline group. The tumor volume in free drug group was 75% of that in saline group. In addition to reflecting the changes of tumor growth, 25



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the body weight changes of model animals could reflect the toxicity or side effects of formula as well62, 63. Results shown in Figure 12A demonstrated that there was no significant difference in the weight of nude mice in each group (p > 0.05), and this might indicate GNP-DTX/Qu/IMA nanoparticles did not cause severe in vivo toxicity. Additionally, H&E staining method was carried out to investigate the formation of pulmonary metastatic nodules in 4T1-bearing nude mice. As shown in Figure 12C and E, there was hardly any micrometastasis in the lung tissue slice of GNP-DTX/Qu/IMA group, and the number of pulmonary metastatic nodules (shown in Figure 12C) followed the order: control > DTX+Qu+IMA > GNP-DTX/Qu/IMA. Compared with saline group, the number of visible lung metastatic nodules in free drug and GNP groups was reduced by 43.2% and 72.5%, respectively. These outcomes indicated that GNP-DTX/Qu/IMA nanoparticles demonstrated excellent ability in inhibiting tumor growth and metastasis. The general mechanism of anti-tumor activity of GNP-DTX/Qu/IMA was summarized in Figure 1. After GNP-DTX/Qu/IMA accumulating in tumor sites through EPR effect, the highly expressed MMP-9 could degrade the gelatin coating and lead to the rapid release of interstitial pressure reversal agents IMA. IMA in conjunction with chemotherapeutic drugs had been reported to reduce tumor IFP through inhibiting PDGFR-β and blocking “CAFs - ECM interaction”23, 64, which was conducive to the delivery of chemotherapeutic drugs penetrating into deeper tumor tissues. In addition, the exposed cationic NLC-DTX/Qu could promote the cellular uptake through the combination between cationic NLC and negative charged membrane, thus increase the accumulation amount of DTX and Qu in tumor cells and achieve the excellent ability in inhibiting tumor proliferation and pulmonary metastasis.

26


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Figure 12. In vivo antitumor effect of saline, free drug and GNP nanoparticles on nude mice bearing 4T1 tumors (n = 5). (A) Body weight changes of 4T1-bearing nude mice with different formula treatment. (B) Tumor volume changes after intravenous injection of different formula. (C) Quantitative analysis of pulmonary metastatic nodules. (D) Pictures of tumors of each group removed from the sacrificed mice. (E) H&E staining and optical images of lungs separated from 4T1-bearing nude mice treated with saline (a), free DTX+Qu+IMA (b) and GNP-DTX/Qu/IMA nanoparticles (c) at different magnifications. Circles indicate the lung metastases. *p < 0.05, **p < 0.01 vs. control.

4. Conclusion In this study, GNP-DTX/Qu/IMA was successfully prepared to achieve the desirable reduction effect of tumor IFP. This formula could improve the drug delivery as well as inhibit breast cancer proliferation and pulmonary metastasis. This study has shown that the obtained GNP-DTX/Qu/IMA could incorporate three drugs with high 27



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encapsulation efficiency and exhibited enzyme sensitivity. Cellular uptake and MTT assays indicated that GNP-DTX/Qu/IMA could effectively enhance uptake efficiency and increase the cytotoxicity. Moreover, through mediating apoptosis and metastasis related proteins, GNP-DTX/Qu/IMA demonstrated significant advantages in inducing cell apoptosis and migration. GNP-DTX/Qu/IMA exhibited excellent efficacy on reducing tumor IFP after single administration. In addition, GNP-DTX/Qu/IMA displayed stronger suppression on tumor proliferation and pulmonary metastasis. Conclusively, GNP-DTX/Qu/IMA might be a promising strategy for anti-tumor drug delivery and improve the efficacy of metastatic tumor therapeutics.

AUTHOR INFORMATION Corresponding Author *Qi Shen School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China Tel: +86-21-34204049. Fax: +86-21-34204049. E-mail: [email protected].

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Abstract Graphic

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Reducing Interstitial Fluid Pressure and Inhibiting Pulmonary

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