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Combined tumor- and neovascular-“dual targeting” gene/ chemo-therapy suppresses tumor growth and angiogenesis Bei Xu, Quansheng Jin, Jun Zeng, Ting Yu, Yan Chen, Shuangzhi Li, Daoqiong Gong, Lili He, Xiaoyue Tan, Li Yang, Gu He, Jinhui Wu, and Xiang-Rong Song ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b08603 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 14, 2016

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ACS Applied Materials & Interfaces

Combined tumor- and neovascular-“dual targeting” gene/chemo-therapy suppresses tumor growth and angiogenesis Bei Xu 1, a, Quansheng Jin 1, a, Jun Zeng 1, a, Ting Yu1, Yan Chen1, Shuangzhi Li1, Daoqiong Gong1, Lili He 2, Xiaoyue Tan 3, Li Yang 1, Gu He 1, Jinhui Wu1, *, Xiangrong Song1, * (1 State Key Laboratory of Biotherapy/Geriatrics and Cancer Center, West China Hospital, and Collaborative Innovation Center for Biotherapy, Sichuan University, Chengdu 610041, China; 2 College of Pharmacy, Southwest University for Nationalities, Chengdu 610041, China; 3 Department of Pathology/Collaborative Innovation Center of Biotherapy, Medical School of Nankai University, Tianjin 300071, China)

a

These three authors contributed equally to this article.

* Corresponding authors: Jinhui Wu, Xiangrong Song Email: [email protected] (X. Song); [email protected] (J. Wu)

1

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Abstract A rational combination is critical to achieve efficiently synergistic therapeutic efficacy for tumor treatment. Hence, we designed novel anti-tumor combinations (T-NPs) by integrating the tumor vascular and tumor cells dual-targeting ligand with anti-angiogenesis/anti-tumor agents. The truncated bFGF peptide (tbFGF), which could effectively bind to FGFR1 overexpressed on tumor neovasculature endothelial cells and tumor cells, was selected to modify PLGA nanoparticles (D/P-NPs) simultaneously loaded with PEDF gene and paclitaxel in this study. The obtained T-NPs with better pharmaceutical properties had elevated cytotoxicity and enhanced expression of PEDF and α-tubulin on FGFR1-overexpressing cells. The uptake of T-NPs increased in C26 cells, probably mediated by tbFGF via specific recognization of the overexpressed FGFR1. T-NPs dramatically disrupted the tube formation of primary human umbilical vein endothelial cells (HUVECs), and displayed improved anti-angiogenic activity in transgenic zebrafish model and alginate-encapsulated tumor cell model. More importantly, T-NPs achieved a markedly higher antitumor efficacy in C26 tumor-bearing mice model. The anti-tumor effect involved the inhibition of tumor cell proliferation and angiogenesis, induction of apoptosis and down-regulation of FGFR1. The enhanced anti-tumor activity of T-NPs probably resulted from the raised distribution in tumor tissues. In addition, T-NPs had no obvious toxicity through the weight monitoring, serological and biochemical analysis, as well as H&E staining. These results revealed that T-NPs, an active targeting gene/chemo-therapy, indeed had superior antitumor efficacy and negligible side effect, suggesting that this novel combination was potential for tumor therapy as a new treatment strategy and the tbFGF modified nanoparticles could be applied to a wide range of tumor-genetic therapies and/or tumor-chemical therapies.

Keywords FGF receptors; Truncated bFGF peptide; Pigment epithelium-derived factor gene; Paclitaxel; Co-delivery; Active targeting nanoparticles 2


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1. Introduction Recently, synergistic therapy based on co-administration of gene medicine with small molecular chemotherapeutic drug has emerged rapidly as a promising treatment modality against cancer 1. A rational combination is critical for high antitumor efficacy and negligible side effect. We have previously developed novel combinations of pigment epithelium-derived factor (PEDF) gene and paclitaxel (PTX) 2

. PEDF is a 50-kDa secreted glycoprotein, which has been reported to be one of the

most powerful endogenous antiangiogenic fators to date

3,4,5

. The in vivo transfer of

PEDF gene has been proved to suppress neovascularization and limit tumor growth 6,7. Paclitaxel (PTX), one of the most effective chemotherapeutic drugs, has been widely used to treata variety of cancers

8,9

. It works by interfering with normal microtubule

breakdown during cell division 10 and leads to G2/M cell cycle arrest and cell death 11. Moreover, PTX could enhance gene expression due to its anti-mitotic function

11,12

.

The combinations consisting of PEDF gene and PTX (PEDF/PTX) in PLGA nanoparticles (D/P-NPs) indeed achieved a higher anti-cancer activity with no obvious toxicity. However, this novel polymeric nanomedicine widely distributed in liver and spleen except at the tumor site. Thus, to specifically transport PEDF/PTX combinations to the same tumor cells might further enhance the anti-cancer effect. The ligand-mediated active targeting, utilizing affinity ligands on the surface of nanoparticles to select to bind surface molecules or receptors overexpressed in tumor cells, might be hopeful to improve the distribution of therapeutic agents in tumor tissues

13,14

enhanced uptake

, as well as the anti-tumor activity by specific retention and

14,15

. However, it remains difficult to deliver drugs to tumor cells that

are distant from tumor vessels due to the lack of binding affinity to tumor endothelial cells

16

. To address the predicament to improve therapeutic efficacy, various

innovative strategies have been reported. The promising drug delivery systems for tumor cells and neovasculature dual targeting delivery might be more advantageous for promoting particle penetration and improving treatment efficacy through the introduction of two ligands

16,17

. These dual targeting delivery systems might be

practically problematic. For example, the length of linkers for the two ligands should be taken into consideration, because one ligand might influence the interaction of the other with its receptor 18. On the other hands, the ligand density, orientation and ratio 3



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between two ligands also might influence the targeting efficiency 18. Compared with utilizing multiple ligands, the ligand which could realize a dual-targeted delivery to both tumor cells and angiogenic vasculature might overcome these barriers. Fibroblast growth factor receptors (FGFRs), which are overexpressed on the surface of a variety of tumor cells

19,20,21

and tumor neovasculature in situ

19

, have

been found to be potential targets for tumor- and vascular-targeting therapy 22,23. As a member of fibroblast growth factors (FGFs) family, basic fibroblast growth factor (bFGF) could interact with FGFR1. Hence, some researchers designed actively targeted liposomes modified with a peptide KRTGQYKLC (bFGFp) which could interact with FGFR1 via binding to bFGF reduce nonspecific binding expression of FGFR1

20

20,21,24

. These liposomes were proved to

and increased uptake by tumor cells with high

21

. The bFGFp had some issues as a ligand: (1) the targeted

delivery efficiency mainly depended on the amount of bFGF in vivo; (2) the direct interaction of bFGF with FGFR1 would induce the proliferation of targeted cells 25,26. Taking into account these information, our group produced a truncated bFGF peptide (tbFGF) which could effectively bind to FGFR1 but not stimulate cell proliferation 23,26

. It could self-assemble onto the surface of liposomes and guide the small

molecular chemotherapeutic drugs (doxorubicin or PTX) to FGFR1-overexpressing tumor cells and tumor neovasculature endothelial cells in vitro and in vivo 23,27. Herein, we designed novel anti-tumor combinations for the first time by organically integrating the tumor vascular/tumor cells dual-targeting ligand with anti-angiogenesis/anti-tumor agents. Specifically, a novel tbFGF-mediated active targeting delivery system simultaneously loaded with PEDF gene and PTX (T-NPs) was firstly fabricated by a self-assembly procedure. The pharmaceutical properties were subsequently evaluated. Furthermore, the anti-tumor effect and the mechanism were also investigated systemically. The current study demonstrated that the introduction of tbFGF significantly improved the anti-tumor activity of PEDF/PTX combinations, mainly attributing to the elevated distribution of nanoparticles in tumor tissues via the specific interaction of tbFGF on the surface of T-NPs with the overexpressed FGFR1 on tumor neovasculature endothelial cells and tumor cells.

4


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2. Materials and Methods 2.1 Materials PEG-PLGA (MW=15 kD; LA/GA=75:25; PEG: MW=2 kD) was obtained from Jinan Daigang Biomaterial Co., Ltd. (Jinan, China). PEDF plasmid and null plasmid were constructed according to our previous report 4. Paclitaxel (PTX) was purchased from Guilin Huiang Biopharmaceutical Co., Ltd. (Guilin, China). All other reagents were of analytical grade and were used without further purification. Free

PTX

for

use

in

vivo

was

formulated

in

a

mixture

of

Cremophor-EL/ethanol (1:1, v/v) similar to Taxol® 28 and then diluted 10 times with normal saline (NS) for intravenous (i.v.) injection. 2.2 Cell culture and animals Human embryonic kidney 293 cells (HEK293), adenocarcinomic human alveolar basal epithelial cells (A549), murine Lewis lung carcinoma cells (LL2) and murine colon adenocarcinoma cells (C26) were all obtained from American Type Culture Collection (ATCC). HEK293 cells were cultured in DMEM with 10 % fetal bovine serum. A549, LL2 and C26 cells were cultured in RPMI-1640 with 10 % fetal bovine serum. Primary human umbilical vein endothelial cells (HUVECs) were isolated from the newborn’s umbilical cord

29

, and then maintained in complete

EGM-2 medium. HUVECs were used for the experiments at passages 2-4. Balb/c mice (18 ± 2 g) were purchased from the Laboratory Animal Center of Sichuan University (Chengdu, Sichuan, China). All animal procedures were approved and supervised by the Institutional Animal Care and Treatment Committee of Sichuan University (Chengdu, Sichuan, China). 2.3 Preparation of T-NPs T-NPs were fabricated via simple self-assembly of tbFGF with D/P-NPs. D/P-NPs were first prepared by the W/O/W double emulsion-solvent evaporation 5



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method according to our previous report

2

. Briefly, the organic phase

(dichloromethane/acetone) containing PTX and PEG-PLGA was emulsified with the inner aqueous phase (PEDF gene) by sonication at 20 W for 20 s to obtain the primary W/O emulsion. Then the primary emulsion was added to 4 mL 1 % PVA solution and further emulsified by sonication at 40 W for 40 s to produce the multiple emulsion (W/O/W). 2 mL 1 % PVA was added to the obtained W/O/W emulsion and continued to be emulsified by sonication at 40 W for 20 s. The organic solvent was then evaporated under vacuum at 37 oC. The obtained colloidal solution was centrifuged (13300 rpm, 40 min) at 4 oC and washed with de-ionized (DI) water three times to remove unencapsulated PEDF/PTX and PVA. The washed pellet was redispersed in pH 7.4 phosphate buffer (PBS) to get D/P-NPs. Finally, to produce T-NPs, the attachment of tbFGF to D/P-NPs surface was carried out by incubating tbFGF with D/P-NPs in pH 7.4 PBS at 4 oC overnight. In addition, the contrast agents were prepared using the similar process, including blank targeting nanoparticles (T-B-NPs), single PEDF gene loaded nanoparticles (D-NPs), single PTX loaded nanoparticles (P-NPs) and single pAAV2 loaded nanoparticles (Dv-NPs). All experiments were performed in triplicate and all nanoparticles were stored at 4 oC. 2.4 Characterization of T-NPs 2.4.1

Binding efficiency of tbFGF Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was

performed to determine the binding efficiency of tbFGF on the surface of D/P-NPs. T-NPs were prepared with different mass ratios of tbFGF to D/P-NPs (0.62 %, 1.24 %, 2.48 % and 4.96 %), and then centrifuged (13300 rpm, 40 min) at 4 oC to separate the supernatants and precipitations. The latters were mixed with 15 % SDS and sonicated to dissociate the adsorbent tbFGF from T-NPs. The obtained samples including the supernatants and dissociated colloidal solution were boiled for 10 min after the addition of SDS loading buffer. Finally, the resulting extracts were subjected to polyacrylamide gel electrophoresis with a constant voltage (80 V) until the dye front reached the bottom edge. The gel was then removed and placed in a staining solution 6


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of 0.1 % Coomassie blue. The proteins in the gel were visualized by destaining the gel overnight with a destaining solution. Commercial protein markers were used as molecular weight standards and free tbFGF was used as control. 2.4.2

Entrapment efficiency and drug loading capacity Entrapment efficiency (EE%) and drug loading capacity (DL%) of T-NPs

were calculated as previously described 2 with minor modification. T-NPs were firstly centrifuged (13300 rpm, 40 min) at 4 oC to obtain supernatant and sediment. The former was mixed with Hoechst 33258 (0.15 µg/mL). Then, the fluorescence intensity was determined by LS55 Luminescence Spectrometer (Perkin Elmer, USA) at an excitation wavelength of 358 nm and an emission wavelength of 457 nm, and was further used to calculate the amount of PEDF gene released from D/P-NPs during the preparation process of T-NPs. The content of PEDF gene unentrapped in D/P-NPs (presented in the centrifugation supernatant as described in the section 2.3) was determined in the same way. Meanwhile, the latter was dissolved in 1 mL acetonitrile to determine the content of PTX entrapped into T-NPs by high performance liquid chromatography (HPLC, Waters Alliance 2695). The chromatographic separation was carried out on a reverse-phase C18 column (150 mm × 4.6 mm, pore size 5 µm, Cosmosil, Nacalai, Japan) using acetonitrile : water (60/40, v/v) as the mobile phase. The flow rate was 1 mL/min. The detection was performed at 227 nm. EE% and DL% of PEDF/PTX were determined using the following formulae: initial PEDF gene content – content of PEDF gene unentrapped in D/P-NPs -content of PEDF gene released from D/P-NPs ൲ ×100 EE% (PEDF)= ൮ initial PEDF gene content initial PEDF gene content – content of PEDF gene unentrapped in D/P-NPs -content of PEDF gene released from D/P-NPs DL% (PEDF)= ൮ ൲ ×100 weight of nanoparticles

2.4.3

content of PTX entrapped in nanoparticles EE% (PTX)= ൬ ൰ ×100 initial PTX content content of PTX entrapped in nanoparticles DL% (PTX)= ൬ ൰ ×100 weight of nanoparticles

Particle size and zeta potential

The mean diameter and zeta-potential of T-NPs were measured at 25 oC by a 7



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Zetasizer Nano ZS (Malvern Instruments, Ltd., Malvern, Worcestershire, UK). All the tests were run 3 times and all data were expressed as the mean ± SD. 2.4.4

Appearance The morphological characteristic was examined by a transmission electron

microscope (TEM, H-600, Hitachi, Ltd, Japan) and an atomic force microscopy (AFM, Veeco Instruments, CA, USA). For TEM images, a drop of the T-NPs dispersion was placed on copper electron microscopy grids and negatively stained with a 2 % (w/v) phosphotungstic acid solution. The excess fluid was removed with a piece of filter paper. TEM analysis was done for the dried samples. AFM images were recorded in tapping mode at 20-25 oC in air on an atomic force microscopy (SPA400, Seiko, Japan). 2.4.5

In vitro PEDF/PTX release The in vitro release behaviors of PEDF gene and PTX from T-NPs were

investigated in pH 7.4 PBS containing 1 % Tween 80 by contrast with D/P-NPs. Both nanoparticles which were dispersed in release media preliminarily were divided into 8 tubes and shaken at 37oC with a gentle rate of 100 rpm. At appropriate time intervals, one tube was taken out and centrifuged (13300 rpm, 40 min) at 4 oC. The amount of released PEDF gene in the supernatant and PTX remained in the sediment were determined by the same methods as described in the section 2.4.2. Experiments were performed in triplicate. 2.4.6

Hemolysis assay Whole rabbit blood was centrifugated and re-suspended in NS to obtain the

standard 2 % erythrocyte dispersion solution. 0.5 mL free PTX solution and various nanoparticles were diluted in 2 mL NS and then added to 2.5 mL erythrocyte dispersion solution (2 %). After incubation at 37 oC for 3 h, all the samples were centrifuged. NS and DI-water were employed as the negative and positive control, 8


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respectively.

The

absorbance

(A)

of

the

supernatants

was

measured

spectrophotometrically at 545 nm. The percentage of the samples-induced hemolysis was calculated according to the following equation: Hemolysis ሺ%ሻ=

2.4.7

A of sample - A of negative control ×100 A of positive control - A of negative control

Stability A preliminary study on the stability was performed. The optimal T-NPs were

stored at 4 oC for one month and the changes in DL% of PEDF/PTX and particle size were examined by the methods described in the section 2.4.2 and 2.4.3, respectively. The samples were assessed in triplicate. 2.5 In vitro experiments 2.5.1

FGFR1 assay HEK293 cells at 80 % confluence were trypsinized using 0.25 %

trypsin-EDTA. After counted, they were fixed with 80 % methanol (5 min) and incubated with 10 % normal goat serum to block non-specific protein-protein interactions. The treated cells were then washed three times with pH 7.4 PBS and incubated with the anti-FGFR1 antibody (1/100 dilution in PBS) for 30 min at room temperature. Alexa Fluor® 488-conjugated goat anti-mouse IgG at 1/400 dilution was used as the secondary antibody. Cells were washed with pH 7.4 PBS to remove excess secondary antibody after incubation for 2 h in the dark and analyzed by FACS Calibur flow cytometer. Unlabelled sample was also used as a control. HUVECs, A549, LL2 and C26 cells were analyzed by the same procedure. 2.5.2

Growth inhibition of normal cells and tumor cells Cytotoxicity of T-NPs was evaluated on HEK293, HUVECs, A549, LL2 and

C26 cells by contrast with free PTX, D-NPs, P-NPs and D/P-NPs. In brief, cells were seeded in 96-well plates and cultured in a humidified atmosphere containing 5 % CO2 9



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at 37 °C for 24 h. Then these cells were incubated with various formulations with different concentrations for 48 h. Cell viability was assessed by MTT assay as previously described

30

. Cytotoxicity of T-B-NPs was also tested on all the five cell

lines. 2.5.3

Expression of PEDF and α-tubulin A549 and C26 cells were respectively treated with free PTX, D-NPs, P-NPs,

D/P-NPs and T-NPs for 48 h, then the media and cells were collected. The former was used to measure the concentrations of the secreted PEDF according to the protocol of human pigment epithelium-derived factor (PEDF) ELISA KIT (R&D Systems, Boston, MA, USA). Briefly, the collected media were added into the monoclonal antibody (McAb)-coated pore plate. After a series of reactions, the absorbance of the suspension was measured by an ELISA reader (Thermo Scientific Multiskan MK3, Thermo Fisher, USA) at the wavelength of 450 nm. Meanwhile, A549 and C26 cells were lysed in RIPA Lysis buffer (Beyotime, Jiangsu, China) and centrifuged at 13000 rpm for 15 min. Supernatants were respectively collected and subjected to western blotting analysis. The protein was separated by 10% SDS-PAGE under reducing conditions and transferred to a PVDF membrane. After incubating with primary anti-alpha tubulin antibody (1:1000, Abcam, Cambridge, MA, USA), the membrane was probed with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000; ZSGB-BIO, Beijing, China) and detected by an enhanced chemiluminescence (ECL) detection kit (Pierce, Rockford, IL, USA). The expression of β-actin was detected as an internal control. 2.5.4

Cellular uptake of T-NPs In order to investigate the cellular uptake of T-NPs, a fluorescent lipophilic

dye coumarin-6 (Cou-6) was incorporated into T-NPs (T-C-NPs) by the same double-emulsion solvent evaporation method as described in the section 2.3. Meanwhile, Cou-6 loaded non-targeting nanoparticles (C-NPs) were used as the contrast agent. C26 and HEK293 cells were seeded into 24-well plates at an initial density of 10


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1 × 10 5 cells per well and incubated for 24 h at 37 oC. Then, the original medium was replaced with fresh medium containing free Cou-6, C-NPs and T-C-NPs at a final Cou-6 concentration of 25 ng/mL, respectively. After incubation at 37 oC for 0.5 h, 1 h, 2 h, 3 h and 4 h, the media was removed and the cells were washed twice with cold pH 7.4 PBS. Then, 4 % paraformaldehyde was added into each well to fix cells for 10 min at room temperature, followed by washing three times using PBS. Nuclei were stained with 1 mL Hoechst 33258 (2.5 µg/mL) for 15 min away from light. The cells were finally washed with cold PBS three times. A high content screening (HCS) instrument (Thermo Scientific Cellomics, Thermo, USA) was used to quantitatively analyze the cellular uptake of each group as described previously 31. 2.5.5

Competitive inhibition assay C26 and HEK293 cells were preliminarily incubated with free tbFGF (0 µg, 2

µg, 4 µg, 6 µg and 8 µg) at 37 oC for 0.5 h, followed by co-incubation with T-C-NPs at a final Cou-6 concentration of 25 ng/mL, respectively. After incubation for another 3 h, the cells were washed with PBS, fixed with 4 % paraformaldehyde, and stained with 2.5 µg/mL Hoechst 33258. Finally, the cellular uptake of T-C-NPs was quantitatively detected under the HCS instrument. Free Cou-6 was used as control. 2.5.6

Endocytosis inhibition C26 and HEK293 cells were pretreated with chlorpromazine (10 µg/mL),

filipin (10 µg/mL) and cytochalasin D (5 µg/mL) at 37 oC for 0.5 h, respectively. After removal of the inhibitor-containing medium, C-NPs- or T-C-NPs-containing medium with the final Cou-6 concentration at 25 ng/mL was added for another 3 h incubation. Cells were then treated as described in the section 2.5.4 for quantitative analysis using the HCS instrument. Cells untreated with any inhibitors were regarded as control group and the cellular uptake was set as 100 %.

11



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2.5.7

HUVEC tube-formation assay The HUVEC tube-formation assay was carried out as previously described 32.

Matrigel (BD Biosciences, Bedford, MA, USA) was thawed overnight on ice at 4 oC and each well of prechilled 96-well plates was coated with 50 µL of Matrigel. After incubation and solidification at 37 oC for 30 min, 100 µL of trypsinized HUVECs (3 × 10 4 cells/well) containing free PTX, D-NPs, P-NPs, D/P-NPs or T-NPs were then seeded to Matrigel-coated wells, respectively. Capillary-like tube formation was observed after 6 h incubation at 37 oC. Tube formation was quantified by manually counting the length of tube-like structures formed in each group. Untreated cells were used as the control and the length in the control group was designated as 100 %. 2.6 Antiangiogenesis in transgenic zebrafish model FLK-1 promoter transgenic EGFP (Tg (FLK-1: EGFP)) zebrafish line (provided by Shuo Lin, UCLA, Los Angeles, CA) was used in antiangiogenesis assay. Healthy embryos were arrayed into 24-well cell culture plates (6-8 embryos per well) containing 1 mL Holtfreter’s solution and further maintained at 28 oC for 15 h. Then embryos were respectively treated with Holtfreter’s solution (control), D-NPs, P-NPs, D/P-NPs and T-NPs for another 12 h. Zebrafish blood vessels were examined using a fluorescence microscope (Olympus, Tokyo, Japan). 2.7 Alginate-encapsulated tumor cell assay An alginate-encapsulated tumor cell assay was performed as previously described 33. Briefly, Balb/c mice were subcutaneously implanted with alginate beads containing 5 × 10

4

C26 cells in the both dorsal sides at day 0 and injected

intravenously with NS (control), free PTX, D-NPs, P-NPs, D/P-NPs and T-NPs (PEDF gene at dose 250 µg/kg and PTX at dose 5 mg/kg) every other day. 12 days later, 0.2 % FITC-dextran solution (0.1 mL) was injected into the tail vein of the mice. Alginate implants were rapidly photographed and removed within 20 min after FITC-dextran injection. The content of FITC-dextran in the beads was quantified as 12


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described previously 33. 2.8 In vivo antitumor assessment 2.8.1

In vivo distribution study DiD was used as the fluorescent probe to monitor the in vivo distribution of

nanoparticles in C26 tumor bearing mice. DiD loaded DiD-NPs and T-DiD-NPs were prepared by the same method as described in the section 2.3. C26 tumor bearing mice models were established by subcutaneous injection of a suspension of 1 × 10 6 C26 cells into the right flank of male Balb/c mice. When the tumor volume reached about 500 mm3, free DiD, DiD-NPs and T-DiD-NPs (DiD at dose 100 µg/kg) were injected via tail vein. The living fluorescent images were acquired at 12 h post-injection via a Quick View 3000 Bio-Real in vivo imaging system (Bio-Real, Austria). Then, the mice were sacrificed. The tumors and the major organs (hearts, livers, spleens, lungs and kidneys) were excised and imaged with the same system. The fluorescent intensity of each organ was calculated by the same instrument. 2.8.2

In vivo therapeutic experiment The antitumor efficacy of T-NPs was investigated in C26 tumor-bearing mice

model. Briefly, male Balb/c mice weighting 18-20 g were subcutaneously injected 100 µL cell suspension containing 5 × 10 5 viable C26 cells on the right flank. When the tumor in the mice exceeded approximately 50 mm 3, mice were assigned randomly into six groups (5 mice per group) and were respectively injected intravenously with NS (control), free PTX, D-NPs, P-NPs, D/P-NPs, T-NPs and Dv-NPs (PEDF gene at dose 250 µg/kg and PTX at dose 5 mg/kg) every two days for two weeks. Throughout the study, tumor volumes were monitored every other day with a caliper and calculated by using the standard formula: Volume=0.5×length×width2 34. The weight of the mice was measured regularly as an indicator of toxicity. After mice were scarified on day 14, tumors in each group were weighed and the tumor growth inhibition rate was calculated by the following formula: 13



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Tumor inhibition rate (%)=

Wc-Wx ×100 Wc

Wc represented the average weight of tumors in control group, while Wx represented the average weight of the tumors in treatment group. To further investigate the antitumor activity of the T-NPs, the survival time of the mice after treatments were observed (10 mice per group). 2.8.3

Immunohistochemical assay CD31 immunohistochemistry was done on frozen sections of C26 tumor

tissues. Samples were incubated with anti-CD31 antibody (1:50 dilution, Abcam, Cambridge, MA) overnight at 4 oC, followed by incubating with biotinylated goat anti rabbit IgG and then streptavidin-biotin-horseradish peroxidase complex at 37 oC. The sections were counterstrained with hematoxylin and the immunostaining images were obtained using a light microscope (Olympus, Tokyo, Japan). Microvessel density (MVD) was determined according to the method reported previously 35. The immunohistochemical assay of Ki67 antigen and FGFR1 expression on the tumor sections (3-4 µm) of paraffin-embedded specimens was done with rabbit anti-mouse Ki67 (Abcam, Boston, MA, USA) and mouse anti-mouse FGFR1 (Abcam, Boston, MA, USA) antibodies using the labeled streptavidin-biotin method as described previously 35. To quantify Ki67 expression, the number of positive cells was counted in 10 random fields at 200 × magnification. Image quantification of FGFR1 expression was performed with Image Pro Plus software (Mediacybernetics, MD, USA), using a customized macro to count the total traced area and integrated option density (IOD) of FGFR1 stained area in each group. A quantitative value of the mean density of FGFR1 was then presented as the IOD value to the total area. 2.8.4

TUNEL assay Apoptotic tumor cells were detected on paraffin sections according to the

manufacturer’s protocol of terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining (DeadEndTM Fluorometric TUNEL System, Promega, 14


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Madison, USA). TUNEL-positive nuclei, which were stained with dark green fluorescence, were monitored using a fluorescence microscope (Olympus, Tokyo, Japan). To quantify TUNEL-positive cells, green-fluorescence-positive cells were counted in four equal-sized fields which were randomly chosen. The density of apoptotic cells was evaluated as the apoptotic index (AI), which was defined as follow: AI (%) = apoptotic cells/total tumor cells×100. 2.8.5

Serological and biochemical analysis Blood samples from the mice which were sacrificed on day 14 were collected

for serological and biochemical analysis. Serum was obtained by centrifugation at 13300 rpm for 10 min and was immediately used for biochemical analysis with an automatic analyzer (Hitachi High-Technologies Corp., Minato-ku, Tokyo, Japan). 2.8.6

H&E staining For the histopathological analysis, the excised vital organs (heart, liver, spleen,

lung and kidney) were fixed in a 4 % paraformaldehyde solution, embedded in paraffin, sectioned and processed for hematoxylin and eosin (H&E) staining. Images were acquired on a light microscope (Olympus, Tokyo, Japan). 2.9 Statistical analysis Statistical analysis was performed by One-Way ANOVA. Survival curves were generated using the Kaplan-Meier method. p values less than 0.05 were considered statistically significant, and extreme significance were set at p < 0.01 and p < 0.001.

3. Results and discussion 3.1 Preparation and characterization of T-NPs W/O/W double emulsion-solvent evaporation method was used to prepare D/P-NPs simultaneously loaded with PEDF gene and PTX in this study, which was suitable for the encapsulation of highly hydrophilic genes. D/P-NPs subsequently 15



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interacted with tbFGF to form T-NPs. When the mass ratios of tbFGF to D/P-NPs was set at less than or equal to 1.24 %, tbFGF could completely bind to D/P-NPs by the analysis of SDS-PAGE (Lane c and Lane d) as seen in Fig. 1A. However, unbound tbFGF was detected in Lane e and Lane f with increase in the amount of tbFGF. The strips in Lane g-j revealed that 15 % SDS could dissociate the adsorbent tbFGF from T-NPs, demonstrating that tbFGF successfully adsorbed on the surface of nanoparticles by the electrostatic interaction. Such an assembly process of ligand was superior to chemical conjugation which was difficult in quality control and scale-up production 36. The optimal T-NPs were finally prepared by fixing the mass ratios of tbFGF to D/P-NPs at 1.24 %. As shown in Fig.1B, T-NPs presented slightly blue opalescence and evident Tyndall phenomenon similar to D/P-NPs. The introduction of the targeting ligand tbFGF into T-NPs neutralized the zeta potential which was significantly improved to -5 mV in contrast to -20 mV of D/P-NPs (Table 1). This phenomenon might result from the electrostatic interaction of the positively charged tbFGF with negative D/P-NPs in pH 7.4 PBS which is lower than the isoelectric point (pH 8.77) of tbFGF

27

. DLS data presented in Fig.1C and Table 1 showed that the

particle size had no obvious change after tbFGF attachment on the surface of D/P-NPs. T-NPs were also similar to D/P-NPs in TEM image (Fig.1D). Moreover, AFM observations (Fig.1E) showed that the T-NPs with the average diameter of 191 nm were spherical and well-dispersed. Limiting the size of particles to less than 200 nm could promote extravasation through the leaky blood vessels and accumulate in tumor tissues 37. Therefore, T-NPs had the potential of enhanced accumulation in tumor sites. T-NPs, consistent with D/P-NPs, exhibited high encapsulation efficiencies and drug loading capacities of both PEDF gene and PTX (Table 1), indicating that the process of tbFGF attachment had no influence on the drug encapsulating capacity. In vitro release profiles of PEDF/PTX from T-NPs were presented in Fig.1F. Both PEDF gene and PTX loaded in T-NPs released slowly without obvious burst release. PEDF/PTX might diffuse gradually from PLGA matrix with erosion of the polymer 38, which would be conducive to concentrate in the tumor tissue and lead to a 16


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potent and prolonged therapeutic efficacy. The sustained drug release profile of T-NPs was similar to D/P-NPs, indicating that bound tbFGF had no impact on PEDF/PTX release. PEDF gene displayed more rapid release than PTX probably due to the higher drug loading capacity (up to 1.5%) than other gene loaded PLGA formulations 39,40. Moreover, the high hydrophilicity of PEDF gene would also lead to relatively weaker interaction with PLGA molecules than PTX. Hemolysis assay was carried out to evaluate the blood compatibility of T-NPs. As shown in Fig. 1G and Fig. 1H, no visible hemolytic effect was observed in T-NPs treated group, while free PTX caused 35.25 % hemolysis due to the excipients for solubilization (Cremophor-EL and ethanol). The percentage of T-NPs-induced hemolysis was below 5 %, demonstrating that T-NPs had good blood compatibility and would be safe for i.v. injection. In addition, T-NPs also had good stability comparable to D/P-NPs when stored at 4 oC. The size, DL% and zeta potential remained stable in 2 weeks (Fig.1I and Fig.S1), probably attributing to the stereospecific blockade of the hydrated PEG 41. In sum, T-NPs displayed similar pharmaceutical properties to D/P-NPs despite of the introduction of the targeting ligand tbFGF, proving that the self-assembly process of tbFGF and D/P-NPs to develop T-NPs was feasible.

Fig. 1 Pharmaceutical properties of T-NPs. (A) Binding efficiency of tbFGF tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). a, commercial protein maker; b, free tbFGF; c-f, unbound tbFGF in supernatants when D/P-NPs were incubated with tbFGF at different mass ratios of 0.62 %, 1.24 %, 2.48 %, and 4.96 %; g-j, the adsorbent tbFGF dissociated from T-NPs obtained from the corresponding group (c-f). (B) The appearance and Tyndall phenomenon of D/P-NPs and T-NPs. (C) Size distribution and zeta potential of T-NPs. (D) Transmission electron microscope (TEM) micrographs of D/P-NPs and T-NPs. (E) Atomic force microscopy (AFM) micrograph of T-NPs. (F) Release profiles of PEDF gene and PTX in D/P-NPs and T-NPs. (G) Images of hemolysis on rabbit red blood cells. (H) The percentage of hemolysis in each group. (I) Change in size and DL% of D/P-NPs and T -NPs when stored at 4 oC for 4 weeks.

17



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Table 1 Characteristics of T-NPs compared with D/P-NPs NPs

Size (nm)

PDI

EE%/DL%

EE%/DL%

(PTX)

(PEDF gene)

Zeta potential (mV)

D/P-NPs

186.9

0.051

86.6/7.9

83.9/1.5

-20.4

T-NPs

191.7

0.082

86.3/7.8

83.7/1.5

-5.2

3.2 Expression of FGFR1 on normal cells and tumor cells The detection assay of FGFR1 was carried out on normal cells (HEK293 and HUVECs) and tumor cells (A549, LL2 and C26) according to the literature

42

. As

shown in Fig. 2A, only 7.11 ± 0.24 % HEK293 cells were positive in FGFR1 expression. HEK293 cells were reported to be low on the expression of FGFR1

42

,

which might be suitable to be the negative control cells to evaluate the biological activities of targeting preparations mediated by FGFR1. HUVECs have been widely applied as model cells to estimate the anti-angiogenesis of tumor-therapeutic agents 43. Fig. 2B displayed that 96.75 ± 0.13 % HUVECs were FGFR1-positive. We previously reported that tbFGF could promote the internalization of liposomes in HUVECs

20

,

probably because of the high affinity of the targeting peptide with FGFR1. A549 cells were recognized as FGFR1-overexpressing cells

20,27

. We found that 98.58 ± 0.31 %

A549 cells expressed FGFR1 positively (Fig. 2C), indicating that A549 cells could be used to assess FGFR1-targeting preparations as one of human tumor cells. Our previous study showed that LL2 and C26 cells had enhanced uptake of tbFGF-modified micelles

27

. Further investigation in this study revealed that the

FGFR1 positive percentages of LL2 and C26 cells were 98.94 ± 0.19 % (Fig. 2D) and 80.53 ± 0.21 % (Fig. 2E), respectively. All the data pointed out that HEK293 cells might be desirable FGFR1-negative model cells and the other four cells would be fit for the FGFR1-targeting assessment of T-NPs as FGFR1-positive model cells.

Fig. 2 Expression of FGFR1 on normal cells and tumor cells. The representative FCS of FGFR1 expressed on HEK293 cells (A), HUVECs (B) and various tumor cells (C, A549; D, LL2; E, C26). 18


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FGFR1 was labeled by anti-FGFR1 antibody and Alexa Fluor® 488-conjugated secondary antibody. The purple and green curve respectively represented untreated control and antibody-treated cells.

3.3 Elevated cytotoxicity of T-NPs on FGFR1-overexpressing cells As shown in Fig. 3, T-NPs displayed the strongest cytotoxic effect on HUVECs, A549, LL2 and C26 cells compared to the other three nanoparticles (D-NPs, P-NPs and D/P-NPs) and free PTX. Moreover, the blank nanoparticles T-B-NPs were found to be nontoxic to all the five investigated cells even if the concentration was up to 1 mg/mL (Fig. 3F) which was 2.5 times the concentration of the corresponding nanoparticles carrier encapsulating 8 µg / mL PEDF/PTX, revealing that the cytotoxic effect of T-NPs resulted from the entrapped agents rather than the carrier itself. The IC50 values of T-NPs were significantly lower than D/P-NPs on the four FGFR1-overexpressing cells, while there were no significant difference between the two kind of nanoparticles on HEK293 with low expression of FGFR1 (Table 2). These results indicated that the increased cytotoxicity of T-NPs might be relevant to the enhanced uptake mediated by the interaction of T-NPs with the overexpressed FGFR1. Additionally, the combination of PEDF and PTX indeed displayed stronger growth inhibition effects on HUVECs and tumor cells than either of single drug-loaded nanoparticles. It would be hopeful that T-NPs achieved anti-tumor efficacy with high efficiency not only due to the integration of the tumor vascular and tumor cellsdual-targeting ligand but also theanti-angiogenesis / anti-tumor agents.

Fig. 3 T-NPs increased the cytotoxicity on the cells overexpressed FGFR1. Growth inhibition of HEK293 (A), HUVECs (B), A549 (C), LL2 (D) and C26 (E) cells after treatment with T-NPs, D/P-NPs, D-NPs, P-NPs and free PTX for 48 h, respectively. (F) The cell viability of T-B-NPs on HEK293, HUVECs, A549, LL2 and C26 cells after 48 h of treatment.

Table 2 IC50 value (µg/mL) of PEDF gene/PTX in each formulation in various cell lines 19



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IC50 (µg/mL)

PEDF gene

PTX

P-NPs

D/P-NPs

T-NPs

HEK293

--

0.66 ± 0.022

0.65 ± 0.021

0.43 ± 0.031

0.40 ± 0.026

HUVECs

--

3.59 ± 0.051

2.16 ± 0.033

0.84 ± 0.075

0.11 ± 0.027

A549

--

4.52 ± 0.065

3.58 ± 0.057

0.68 ± 0.051

0.17 ± 0.018

LL2

--

3.78 ± 0.047

1.68 ± 0.068

0.27 ± 0.016

0.17 ± 0.020

C26

--

3.45 ± 0.042

1.04 ± 0.034

0.16 ± 0.020

0.067 ± 0.024

(For D/P-NPs and T-NPs, IC50 was referred to the concentration of both PTX and PEDF gene.)

3.4 Enhanced expression of PEDF and α-tubulin in T-NPs treated cells ELISA analysis was carried out to assess the in vitro expression of PEDF in A549 and C26 cells treated by T-NPs. T-NPs showed the highest expression level of PEDF on both tumor cells than the other investigative preparations including D/P-NPs, D-NPs, P-NPs and free PTX (p < 0.001, T-NPs versus control, PTX, P-NPs and D-NPs, respectively. p < 0.01, T-NPs versus D/P-NPs.) (Fig. 4A). The enhanced expression of PEDF in T-NPs groups than D/P-NPs (p < 0.01) probably attributed to the enhanced internalization mediated by FGFR1 overexpressed on the membrane of A549 and C26 cells. PTX was reported to enhance gene expression because of its anti-mitotic function

11,12

. The introduction of PTX increased PEDF expression (p

Chemo

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