Chemotherapy Elicits

Subscriber access provided by CMU Libraries - http://library.cmich.edu

Article

Polymeric Nanomedicine for Combined Gene/ chemo-therapy Elicits Enhanced Tumor Suppression Bei Xu, Shan Xia, Fazhan Wang, Quansheng Jin, Ting Yu, Lili He, Yan Chen, Yongmei Liu, Shuangzhi Li, Xiaoyue Tan, Ke Ren, Shaohua Yao, Jun Zeng, and xiangrong Song Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00922 • Publication Date (Web): 23 Dec 2015 Downloaded from http://pubs.acs.org on December 30, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Polymeric Nanomedicine for Combined Gene/chemo-therapy Elicits Enhanced Tumor Suppression Bei Xu1,†, Shan Xia1,2,†, Fazhan Wang1, Quansheng Jin1, Ting Yu1, Lili He3, Yan Chen1, Yongmei Liu1, Shuangzhi Li1, Xiaoyue Tan4, Ke Ren1, Shaohua Yao1, Jun Zeng1,*, Xiangrong Song1,* 1

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan

University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan 610041, China 2

Central Laboratory, Science Education Department, Chengdu Normal University,

Chengdu, Sichuan 610041, China 3

College of Chemistry and Environment Protection Engineering, Southwest University

for Nationalities, Chengdu 610041, Sichuan, China 4

Department of Pathology/Collaborative Innovation Center of Biotherapy, Medical

School of Nankai University, Tianjin 300071, China

† Bei Xu and Shan Xia equally contributed to the work. * Corresponding author at: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, 17 Renmin Nanlu, Chengdu, Sichuan 610041, China. Tel: +86 28 85503817; Fax: +86 28 85503817. E-mail address: [email protected] (Song X.); [email protected] (Zeng J.)

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Combination treatment through simultaneous delivery of DNA and anticancer drugs with nanoparticles has been demonstrated to be an elegant and efficient approach for cancer therapy. Herein, we employed a combination therapy for eliminating both the tumor cells and intratumoral neovascular network based on the nanoplatform we designed. Pigment epithelium-derived factor (PEDF) gene, a powerful antiangiogenic agent, and the clinically widely used chemotherapy agent paclitaxel (PTX) were simultaneously encapsulated in the same nanoparticle by a modified double-emulsion solvent evaporation method. The dual-drug-loaded nanoparticles (D/P-NPs) exhibited a uniform spherical morphology and released PTX and PEDF gene in a sustained manner. D/P-NPs showed an enhanced antitumor effect on C26 and A549 cells and a stronger inhibitory activity on proliferation of HUVECs. Moreover, D/P-NPs could dramatically elevate the PEDF expression levels in both C26 and A549 cells in comparison with PEDF gene loaded nanoparticles and significantly promote the cellular uptake of PTX. Additionally, microtubules were stabilized and G2/M phase arrest along with a higher subG1 cell population was induced by D/P-NPs in contrast to PTX or PTX loaded nanoparticles. Besides, D/P-NPs showed sustained release of PTX and PEDF gene in tumors as well as long term gene expression. A significantly improved anti-cancer effect was also demonstrated in a C26 subcutaneous tumor model using this combinational therapy. D/P-NPs could sharply reduce the microvessel density and significantly promoted tumor cell apoptosis in vivo. More importantly, the in vivo distribution, serological and biochemical analysis and H&E staining revealed that D/P-NPs had no obvious toxicity. Our study suggested that this novel polymeric nanomedicine had great potential for improving the therapeutic efficacy of combined gene/chemo-therapy of cancer. 2


Page 2 of 57

Page 3 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Keywords Pigment epithelium-derived factor; paclitaxel; combination therapy; co-delivery; PLGA nanoparticles

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1. Introduction Combination therapy of multiple therapeutic strategies with synergistic effects is becoming a promising approach for cancer treatment [1] [2]. Chemotherapy is one of the most effective approaches to treat cancers in the clinic, but the problems such as cytotoxicity and low bioavailability limit the future application of chemotherapeutic agents [3]. Currently, gene therapy has obtained great attention over the past two decades as an alternative strategy for the treatment of cancer [2] [3]. Co-delivery of DNA and anticancer drug has shown great promise to achieve the combined effects in cancer therapy [2] [4]. Wiradharma et al. [5] had reported that combination with doxorubicin (DOX) and p53-encoding pDNA by cationic oligopeptide micelles led to a synergistic therapeutic effect in suppressing the proliferation of HepG2 cells. Moreover, Han et al. [6] found that co-delivery of DOX and plasmid DNA by nanostructured lipid carriers inhibited cancer growth more efficiently. However, such combinations still have not been evaluated on clinical trial. Therefore, it's necessary to develop novel combinations with higher antitumor efficacy. A new combination consisting of pigment epithelium-derived factor (PEDF) gene and PTX was designed by our group, which might achieve a synergistic or combined effect in the cancer treatment according to our preliminary investigation. PEDF, a member of the serine protease inhibitor superfamily, is a 50-kDa secreted glycoprotein that belongs to a group of proteins which have a common three-dimensional structure [7] [8]. It has been reported to be the most potent natural antiangiogenic agent associated with the effect of intratumoral vascular network’s 4


Page 4 of 57

Page 5 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


disruption [9]. Numerous studies have shown that PEDF gene has better therapeutic efficacy on many tumors [7], leading to the proposal that PEDF gene therapy may provide a new treatment approach. As one of the most widely used chemotherapeutic agents, PTX has shown extraordinary antitumor effect in clinical applications against ovarian, breast, head and neck, non-small-cell lung [10] and colon cancers [11] [12] 13]. PTX binds to tubulin and promotes tubulin polymerization, leading to G2/M cell cycle arrest and cell death [2]. Furthermore, PTX could enhance gene expression due to its anti-mitotic function [2] [14]. Thus co-delivery of PEDF gene and PTX might display a great potential in cancer therapy. A safe and efficient delivery vector or vehicle is crucial to the combination of PEDF gene and PTX (PEDF/PTX). The clinical application of therapeutic genes is limited by their low stability in biological systems, rapid degradation in cellular cytoplasm, poor cellular uptake and tissue specificity [1]. Meanwhile, the commercial available formulation of PTX (Taxol®) has low therapeutic index and serious side effects because of its poor aqueous solubility and lack of tumor tissue specificity [2]. Nanoparticles made from PEG-PLGA copolymers were used to simultaneously deliver PEDF/PTX to tumor tissues in this study. PEG-PLGA has been approved by FDA and extensively studied for anticancer drug delivery [15] [16]. PEG-PLGA nanoparticles have several unique properties: (1) are biodegradable and biocompatible [15]; (2) have been widely used to encapsulate a variety of chemotherapeutics [15]; (3) have shown its remarkable potential for nucleic acid delivery and expression in vitro and in vivo [9] [17]; (4) could effectively sustain the drug/gene release [9] [17]; (5) are more stable in 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 57

plasma due to the introduction of PEG [10]; (6) could be located in the tumor tissue by the enhanced permeability and retention (EPR) effect [10]. Therefore, the current study aimed to develop PEG-PLGA nanoparticles (D/P-NPs) containing PEDF/PTX to achieve higher antitumor efficacy. D/P-NPs were prepared and characterized firstly, and then the antitumor and antiangiogenic activities were investigated in vitro. Various in vivo studies were subsequently carried out to confirm the combinational effect of PEDF/PTX.

2. Materials and methods 2.1. Materials PEG-PLGA (MW=15 kD; LA/GA=75:25; PEG: MW=2 kD) was purchased from Jinan Daigang Biomaterial Co., Ltd. (Jinan, China). PEDF plasmid pAAV2-PEDF (PEDF gene) and null plasmid pAAV2 were constructed according to our previous report [9]. Paclitaxel (PTX) was purchased from Guilin Huiang Biopharmaceutical Co., Ltd. (Guilin, China) and FITC-labeled PTX (FITC-PTX) was synthesized by our own group. Triton X-100 was obtained from Sanland Chemical Co., Ltd. (Los Angeles, CA, USA).

Polyvinyl

alcohol

(PVA,

MW=30-70

kD,

HD:

80

%),

3-(4,

5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT), Hochest 33258 and poly-L-lysine (PLL, MW=15-30 kD) were procured from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade and were used as supplied. Free PTX for use in vivo was formulated in a mixture of Cremophor-EL/ethanol (1:1 in volume) similar to Taxol®.

6


Page 7 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.2. Cell culture and animals

Murine colon adenocarcinoma cells (C26) and adenocarcinomic human alveolar basal epithelial cells (A549) were obtained from ATCC and cultured in RPMI-1640 with 10 % calf serum and 10 % fetal bovine serum, respectively. Primary human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cord veins by a standard procedure [18], and grew in EBM-2 medium with Single Quots (Lonza, USA) containing VEGF and other growth factors. HUVECs at passages 2 to 4 were used for the experiments. Balb/c mice (18 ± 2 g) were used for in vivo anti-tumor tests. The animals were purchased from the Laboratory Animal Center of Sichuan University (Sichuan University, Chengdu, Sichuan, China). The mice were housed and maintained under SPF conditions in facilities. All studies were approved and supervised by the State Key Laboratory of Biotherapy Animal Care and Use Committee (Sichuan University, Chengdu, Sichuan, China). 2.3. Preparation of D/P-NPs D/P-NPs were prepared by a modified double-emulsion (W/O/W) solvent evaporation method. Briefly, 200 µg PEDF gene was incubated with equivalent PLL to be condensed to form the inner aqueous phase. The organic phase containing 1 mg PTX and 10 mg PEG-PLGA was emulsified with the inner aqueous phase by probe sonication at 20 W for 20 s in ice bath to form the primary W/O emulsion. The primary emulsion was added to 4 mL 1 % (w/w) PVA solution and future emulsified by sonication at 40 W for 40 s to obtain the multiple emulsion (W/O/W). Then, 2 mL 1 % 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

PVA was added to the resulted W/O/W emulsion and continued to be emulsified at 40 W for 20 s. The final emulsion was evaporated under vacuum to remove the organic solvent and the colloidal solution was then centrifuged (13300 rpm, 40 min) at 4 oC and washed three times with de-ionized (DI) water to remove PVA and the unencapsulated PEDF/PTX. The sediment was redispersed to obtain D/P-NPs. The contrast agents were also prepared using the similar process, including single PEDF gene loaded nanoparticles (D-NPs), single PTX loaded nanoparticles (P-NPs) and single pAAV2 loaded nanoparticles (Dv-NPs). D+P-NPs containing the same amount of PEDF/PTX as D/P-NPs were prepared by mixing D-NPs and P-NPs. All experiments were performed in triplicate and all nanoparticles were stored at 4 oC before use. 2.4. Characterization of D/P-NPs 2.4.1

Entrapment efficiency and drug loading During the preparation of D/P-NPs, the obtained supernatant and sediment by

centrifuging the colloidal solution were stored to determine entrapment efficiency (EE%) and drug loading (DL%). The former was incubated with 0.15 µg/mL Hoechst 33258 for a few minutes to measure the fluorescence intensity at an excitation wavelength of 358 nm and an emission wavelength of 457 nm by LS55 Luminescence Spectrometer (Perkin Elmer, USA), which was further used to calculate the amount of PEDF gene unentrapped into D/P-NPs. Meanwhile, the latter was dissolved in 1 mL acetonitrile to determine the content of PTX entrapped into D/P-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) with a mobile phase consisting of a mixture of acetonitrile/water (60/40, v/v) at a flow rate of 1 mL/min. The detection was performed at 227 nm. EE% and DL% 8


Page 8 of 57

Page 9 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of PEDF/PTX were determined using the following formulae: EE% (PEDF)= ൬

initial PEDF gene content - content of PEDF gene unentrapped ൰ ×100 initial PEDF gene content

initial PEDF gene content - content of PEDF gene unentrapped DL% (PEDF)= ൬ ൰ ×100 weight of nanoparticles content of PTX entrapped in nanoparticles EE% (PTX)= ൬ ൰ ×100 initial PTX content DL% (PTX)= ൬ 2.4.2

content of PTX entrapped in nanoparticles ൰ ×100 weight of nanoparticles

Particle size and zeta potential The mean particle size and size distribution were measured by dynamic laser

scattering (DLS) using a Zetasizer (Zetasizer Nano-ZS 90, Malvern Instruments Ltd., Malvern, UK) at 25 oC. The zeta potential was automatically calculated from the electrophoretic mobility using the same instrument. All results were the mean of three different samples and all the data were expressed as the mean ± SD. 2.4.3

Appearance The morphology of D/P-NPs was examined by transmission electronic

microscopy (TEM). Before analysis, D/P-NPs were first diluted with DI water, and then placed on a copper electron microscopy grids. After the nanoparticles were stained with a 2 % (w/v) phosphotungstic acid solution at room temperature, the excess fluid was removed with a piece of filter paper. 2.4.4

Stability D/P-NPs were stored at 4 oC for one month to investigate the preliminary

stability. The changes in particle size, zeta potential and EE% of PEDF/PTX were examined by the methods described above. 2.4.5

In vitro release The release profiles of PEDF/PTX in D/P-NPs were investigated in pH 7.4 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

phosphate buffer (PBS) containing 1 % (w/v) Tween 80 by comparison with P-NPs and D-NPs. Nanoparticles were first dispersed in PBS and then divided into 7 tubes which were shaken with a gentle rate of 100 rpm at 37 oC. At definite time interval, one tube was taken out and treated with centrifugation. The released PEDF in the supernatant and PTX remained in the sediment were determined by the same methods as described above. Experiments were performed in triplicate. 2.4.6

Hemolysis The red blood cells (RBCs) of healthy rabbit were collected by centrifuging the

diluted whole blood in normal saline (NS) at 1000 rpm for 5 min. The supernatant was removed and the pellet was washed with NS at least three times. The RBCs pellet was re-suspended in NS to obtain a standard 2 % erythrocyte dispersion. For the hemolysis experiment, 2 % erythrocyte dispersion (2.5 mL) was treated with various formulations which had been preliminarily diluted with 2 mL NS. DI water and NS were employed as the positive and negative control, respectively. All the samples were centrifuged after incubation at 37 oC for 3 h. The absorbance (A) of the obtained supernatant was monitored at 545 nm by UV-Vis spectrophotometer (Perkin-Elmer Lambda35, USA). The percentage of the samples-induced hemolysis was calculated as follows. Hemolysis ሺ%ሻ=

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

2.5. In vitro experiments 2.5.1

Growth inhibition of tumor cells and HUVECs Cytotoxicity assay of D/P-NPs was performed on C26, A549 and HUVECs by

contrast with free PTX, P-NPs, D-NPs, D+P-NPs and Dv-NPs. In brief, cells were plated in 96-well plates at the density of 3 × 10

3

cells/well and cultured in 5 % CO2

10


Page 10 of 57

Page 11 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


humidified air at 37 oC for 24 h. Then, cells were exposed to different concentrations of various nanoparticles and free PTX for 48 h. The cell viability was assessed with MTT method as previously described [19]. Besides, cytotoxicity test of polymer material PEG-PLGA was conducted on the three cell lines in the same way [19]. All data were expressed as the mean ± SD. 2.5.2

Cell cycle distribution Both C26 and A549 cells cultured in 6-well plates were collected and washed

three times by PBS 7.4 after treated with free PTX, P-NPs, D-NPs, D+P-NPs and D/P-NPs for 4 h respectively (Cells without treatment were used as control). They were further fixed with 70 % cold ethanol. After storage at 4 oC for 24 h, the suspension was centrifuged. The obtained sediment was washed by cold PBS 7.4 and then incubated with 100 µL RNase A at 37 oC for 30 min. Subsequently, 400 µL propidium iodide (PI) was added. The mixture was incubated at 4 oC for 30 min in the dark. Finally, the distribution of cells in various phase of cell cycle was measured using flow cytometry (FACS Calibur, BD, USA). 2.5.3

ELISA analysis PEDF concentrations in the media were measured using a sandwich

enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer’s instructions. Briefly, C26 and A549 cells were treated with free PTX, D-NPs, P-NPs, D+P-NPs and D/P-NPs at 37 oC for 48 h. Cells without treatment were used as control. All media were collected and then added to the wells which had been pre-coated with a monoclonal antibody specific for PEDF. After a series of reactions, absorbance was measured at the wavelength of 450 nm with an ELISA reader (Thermo Scientific Multiskan MK3, Thermo Fisher, USA). A standard curve was plotted and the protein concentration in each group was interpolated from the standard curve. 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.5.4

Page 12 of 57

Cellular uptake

PTX was replaced by FITC-PTX to detect the uptake of PTX into C26 and A549 cells by high content screening (HCS) and flow cytometric (FCM) analyses. The nanoparticles co-loaded with PEDF and FITC-PTX (PEDF/FITC-PTX-NPs) were prepared by the same W/O/W solvent evaporation method as described in section 2.3. To evaluate the uptake profiles, C26 and A549 cells at an initial density of 1 × 10 5 cells per well were seeded in 24-well plates. After 24 h of culture at 37 oC, the original medium

was

replaced

by

fresh

medium

containing

free

FITC-PTX

or

PEDF/FITC-PTX-NPs at a final FITC-PTX concentration of 10 µg/mL, respectively. The media was removed after incubation at 37 oC for 1 h, and then cells were washed twice with cold PBS. Fresh prepared 4 % paraformaldehyde was subsequently added into each well, and cells were fixed for 10 min at room temperature. After cells were washed three times by PBS, the nuclei were stained with 1 mL Hoechst 33258 (2.5 µg/mL) for 15 min away from light. The cells were finally washed three times with PBS and imaged using an HCS instrument (Thermo Scientific Cellomics, Thermo, USA) as described previously [20]. For FCM analysis, cells were collected and washed with PBS. Then intracellular FITC-PTX fluorescence was analyzed by FCM after excitation with a 488 nm argon laser. Fluorescence emission at 520-530 nm from 20,000 cells were collected, amplified and scaled to generate single parameter histogram. 2.5.5

α-tubulin immunofluorescence The

microtubules

of

C26

and

A549

cells

were

visualized

by

immunofluorescence microscopy using an antibody against α-tubulin. Cells were seeded at 5 × 10

4

cells/well on microscope cover glass (15 mm round). After treatment with

free PTX and various nanoparticles for 4 h, cells were fixed in 4 % paraformaldehyde

12


Page 13 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for 15 min at room temperature and permeabilized with 0.2 % Triton X-100. They were further incubated with a rabbit anti-α-tubulin antibody (1:200; Abcam, Cambridge, MA, USA) for 2 h at room temperature after blocked with 5 % bovine serum albumin (BSA). Alexa Fluor® 488-conjugated goat anti-rabbit IgG (ZSGB-BIO, Beijing, China) at 1/400 dilution, the secondary antibody, was subsequently added to specifically bind with anti-α-tubulin antibody. Finally, the nuclei were visualized by Hoechst 33258 (2.5 µg/mL). Images were captured using a fluorescence microscopy (Leica). 2.6. In vivo experiments 2.6.1 In vivo distribution of nanoparticles DiD was used as the fluorescent probe to monitor the in vivo distribution of nanoparticles in C26 tumor bearing mice. DiD loaded nanoparticles (DiD-NPs) were prepared by the similar method as described in 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 and DiD-NPs at the same dose for DiD (100 µg/Kg) were injected via tail vein, respectively. The mice were sacrificed at 12 h post-injection and the tumors, hearts, livers, spleens, lungs and kidneys were excised and imaged via a Quick View 3000 Bio-Real in vivo imaging system (Bio-Real, Austria). 2.6.2

Accumulation and release of nanoparticles PTX was replaced by FITC-PTX to investigate the accumulation and release

profiles of PTX in tumors and the resulting nanoparticles were designated as PEDF/FITC-PTX-NPs. In this experiment, C26 tumor bearing mice models were established by subcutaneous injection of a suspension of 1×10 6 C26 cells into the right 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

flank of male Balb/c mice. When the tumor volume reached about 500 mm3, NS, free FITC-PTX and PEDF/FITC-PTX-NPs (FITC-PTX at dose of 3 mg/Kg, PEDF gene at dose of 500 µg/Kg) were injected via tail vein, respectively. The mice were sacrificed at 12 h, 24 h and 48 h post-injection and the tumors were excised and imaged via a Quick View 3000 Bio-Real in vivo imaging system (Bio-Real, Austria). Furthermore, to evaluate the expression of PEDF gene, PEDF in tumor tissues at different time point was further analyzed by western blotting. Briefly, the tumors were grinded and then lysed with RIPA solution. Supernatants were collected after centrifugation and subjected to western blotting analysis. The protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. After incubating with primary PEDF 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 detection kit (Pierce, Rockford, IL, USA). A membrane was tested for β-actin to confirm equal loading. 2.6.3

In vivo antitumor assessments Antitumor activity of D/P-NPs was investigated in C26 tumor-bearing mice

model. Briefly, male Balb/c mice were injected subcutaneously on the right flank with 100 µL of cell suspension containing 5 × 10 5 viable C26 cells. When the tumor reached approximate 50 mm3 in volume, mice were randomly divided into six groups (5 mice per group) and were respectively treated with saline (NS, control), free PTX, P-NPs, D-NPs, D+P-NPs, D/P-NPs and Dv-NPs (PEDF gene at dose of 250 µg/Kg and PTX at dose of 5 mg/Kg) via tail vein every two days for two weeks. Mice weight and tumors sizes were measured every two days. The tumor volumes were calculated by the equation: Volume = 0.5 × L × W2 (L and W represented the length and the width of the 14


Page 14 of 57

Page 15 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tumor, respectively). To further investigate the antitumor efficacy of the D/P-NPs, survival times of the mice were also evaluated (10 mice per group). 2.6.4

CD31 immunohistochemistry CD31 immunohistochemistry was done on fresh frozen sections of C26 tumor

tissues. After fixation with cold acetone for 15 min, samples were incubated with anti-CD31 antibody (1:50 dilution, Abcam, Cambridge, MA) for the identification of endothelial cells. The vessels were revealed with streptavidin-peroxidase followed by chromogenic substrate diaminobenzidine (DAB) and the sections were counterstrained with hematoxylin. Immunostaining images were observed under a light microscope (Olympus, Tokyo, Japan) and microvessel density (MVD) was determined according to the previously reported method [21]. 2.6.5

TUNEL assay Terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL)

staining was performed on paraffin sections using an in situ cell death detection kit (DeadEndTM Fluorometric TUNEL System, Promega, Madison, USA). TUNEL-positive nuclei with dark green fluorescence were monitored using a fluorescence microscope (Olympus, Tokyo, Japan). Four equal-sized fields were randomly chosen and analyzed. The density of apoptotic cells was evaluated as the apoptotic index (AI), which was defined as follow: AI (%) = apoptotic cells/total tumor cells×100. 2.6.6

Serological and biochemical analysis Blood samples were collected from the mice in section 2.6.3 before they were

sacrificed on day 14. Serum was obtained by centrifugation at 13300 rpm for 10 min and was used for biochemical analysis with an automatic analyzer (Hitachi High-Technologies Corp., Minato-ku, Tokyo, Japan) immediately. 2.6.7

H&E staining 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 57

The heart, liver, spleen, lung and kidney were immediately collected after the mice in section 2.6.3 were scarified and fixed in a 4 % paraformaldehyde solution overnight. Then the organs were embedded in paraffin, sectioned and processed for hematoxylin and eosin (H&E) staining. The images for the H&E staining were acquired on a light microscope (Olympus, Tokyo, Japan).

2.7. Statistical analysis Statistical analysis was performed using the Statistical Product and Service Solutions software (SPSS). Data were analyzed by one-way analysis of variance (ANOVA). Survival curves were generated based on the Kaplan-Meier method and statistical significance was determined by Mann-Whitney U-tests. P values less than 0.05 and 0.01 were considered indications of statistical difference and statistically significant difference, respectively.

3. Results and discussion 3.1. Preparation of D/P-NPs PEDF gene is highly water-soluble, but PTX is highly hydrophobic. Thus, W/O/W solvent evaporation method was used to prepare the nanoparticles simultaneously loaded with PEDF gene and PTX in order to achieve higher EE% in this study (Fig. 1A). The component of organic phase was optimized to achieve ideal D/P-NPs with higher encapsulation efficiency and better particle size according to preliminary experiments. As shown in Fig. 1B, D/P-NPs with the smallest size and highest EE% were obtained when the organic phase was the mixture of dichloromethane/acetone (DCM/DMK) rather than single organic solvent DCM or the other

two

mixed

solvents

(dichloromethane/methanol 16


[DCM/MeOH]

or

Page 17 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dichloromethane/acetonitrile

[DCM/ACN]).

The

organic

phase

containing

water-miscible solvent contributed to the formation of smaller W/O/W emulsion drops. However, only DCM/DMK mixture resulted in the smallest D/P-NPs probably because DCM/DMK could volatilize quickly due to the boiling point of DMK lower than either of MeOH and ACN. The optimal volume ratio of DCM/DMK was further investigated. As presented in Fig. 1C, the particle size first decreased and then increased with the increase of DMK, while the EE% first increased and then decreased. The introduction of DMK into the organic phase could reduce the oil-aqueous interfacial energy and accelerate the escape of PEG-PLGA from the oil phase, which made smaller nanoparticles quickly form. However, excessive DMK might lead to easier dispersion of the inner aqueous phase into the outer aqueous phase, which made the W/O/W double emulsion unstable and thereby larger particles with lower EE% form [22]. Therefore, the best D/P-NPs were obtained using DCM/DMK mixture (4:1, v/v) as organic phase. 3.2. Characterization of D/P-NPs The characteristics of the optimal D/P-NPs were shown in Table 1 including particle size, polydispersity index (PDI), zeta potential, EE% and DL%. D/P-NPs had average diameters around 180 nm with narrow PDI and were characterized with negative surface charges, high encapsulation efficiencies and high drug loading capacities. D-NPs and P-NPs had the similar characteristics to D/P-NPs, which indicated that both nanoparticles were suitable contrast agents of D/P-NPs. D/P-NPs colloidal solution presented slight blue opalescence as seen in Fig. 1D. The further TEM analysis displayed that D/P-NPs were spherical and well-dispersed (Fig. 1E), but the diameter observed by TEM was smaller than that obtained by DLS (Fig. 1F). This phenomenon was probably due to that the particle size determined by TEM was the mean diameter of dried nanoparticles rather than the hydrodynamic 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

diameter of nanoparticles in aqueous solution measured by DLS [22]. The stability study showed that D/P-NPs remained stable without change of size, zeta potential and EE% for at least 2 weeks (Fig. 2A). This might attribute to the electrostatic repulsions between negatively charged nanoparticles and the stereospecific blockade of the hydrated PEG [23]. When D/P-NPs were stored for 3 weeks, the particle size became larger and EE% of PEDF/PTX got smaller. D+P-NPs had the similar change to D/P-NPs during storage. Interestingly, it was found that D/P-NPs presented smaller increase in diameter and smaller decline in EE% after storage for 3 or 4 weeks, which demonstrated that D/P-NPs had better storage stability apparently because of better quality control than the mechanical mixture of two kinds of nanoparticles.

Fig. 1 Pharmaceutical properties of PEG-PLGA nanoparticles. (A) Schematic diagram for preparation of D/P-NPs. (B) The effect of the organic phase components on diameter and EE% of D/P-NPs. (C) The effect of DCM/DMK volume ratios on diameter and EE% of D/P-NPs. (D) The appearance of different nanoparticles. (E) Transmission electron microscopy (TEM) image of D/P-NPs. (F) Size distribution of D/P-NPs detected by DLS. 18


Page 18 of 57

Page 19 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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

Size (nm)

PDI

EE%/DL% (PTX)

EE%/DL% (PEDF gene)

Zeta potential (mV)

D-NPs

176.9

0.085

-----

82.6/1.63

-23.04

P-NPs

186.7

0.081

87.7/8.06

-----

-20.16

D/P-NPs

186.9

0.051

86.6/7.94

83.9/1.52

-20.44

In vitro release profiles of PEDF/PTX from D/P-NPs were presented in Fig. 2B. Both PEDF gene and PTX loaded in D/P-NPs released slowly without obvious burst release, probably leading to a potent and prolonged therapeutic efficacy. Approximate 100 % of PEDF gene released completely from D/P-NPs in 48 h, while only near 65 % of PTX released from D/P-NPs. The mass ratio of PTX to PEDF gene released from D/P-NPs at time interval examined was about 1/1.5, which might achieve a desirable antitumor effect by combination according to our preliminary experiment. The release rate of PTX was similar to Ap-PTX-NPs [24] which were also made by PEG-PLGA materials and PEDF gene exhibited a slow release profile, indicating that the sustained release was caused by the diffusion of PTX/PEDF gene through PLGA matrix as well as the erosion of polymer [25]. Moreover, the release profile of PEDF gene from D/P-NPs was similar to that from D-NPs, and there was no difference between D/P-NPs and P-NPs in PTX release. It can be concluded that PEDF gene might have no interaction with PTX when both were simultaneously entrapped into the same nanoparticle.

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 2 Stabilities and in vitro release profiles of nanoparticles. (A) Change in size and EE% of D+P-NPs and D/P-NPs in 4 weeks. (B) Release profiles of PEDF gene and PTX in D/P-NPs.

Hemolysis analysis was used to evaluate the blood compatibility of D/P-NPs. No visible hemolytic effect was observed in D/P-NPs treated group as shown in Fig. 3, while free PTX induced 35.25 % hemolysis resulted from the added excipients (Cremophor-EL and ethanol). The percentage of D/P-NPs-induced hemolysis was below 5 %, which demonstrated that D/P-NPs had good blood compatibility and would be safe for intravenous administration. The lower hemolytic effect might be attributed to the stealth PEG layer on the nanoparticulate surface [26]. The other three contrast nanoparticles (D-NPs, P-NPs and D+P-NPs) also didn't cause hemolysis, indicating that PEG-PLGA nanoparticles were one

kind of desirable carriers with good

biocompatibility.

Fig. 3 Hemolysis assay of D/P-NPs compared with free PTX, D-NPs, P-NPs and D+P-NPs. (A) Images of hemolysis on rabbit red blood cells. (B) The percentage of hemolysis (%) in each group.

3.3. Proliferation inhibition of tumor cells and HUVECs As shown in Fig. 4A~4C, D/P-NPs presented concentration-dependent cell growth inhibition activities on C26, A549 and HUVECs. PEG-PLGA constituting the nanoparticles was found to be nontoxic to all the three investigated cells even if its concentration was up to 2 mg/mL (Fig. 4D). The highest concentration of PEG-PLGA used in D/P-NPs was only 0.5 mg/mL, thus it could be inferred that the cytotoxic effect 20


Page 20 of 57

Page 21 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of D/P-NPs resulted from the entrapped agents rather than the carrier itself. D/P-NPs displayed higher growth inhibition activity than either of single drug-loaded nanoparticles (D-NPs and P-NPs) on each cell under study (P < 0.05), indicating that the combination of PTX and PEDF could achieve enhanced antitumor activity. Intriguingly, stronger cytotoxic effect after D/P-NPs treatment was found compared with D+P-NPs (P < 0.05). It might be difficult for D+P-NPs consisting of D-NPs and P-NPs to ensure the simultaneous internalization of PEDF/PTX into cells, thereby dispersing their firepower against the target inevitably. Hence, encapsulating PEDF/PTX into the same nanoparticle was crucial to exert higher antitumor activity.

Fig. 4 Growth inhibition of C26, A549 and HUVECs after treatment with D/P-NPs, D+P-NPs, D-NPs, P-NPs, Dv-NPs, free PTX and PEG-PLGA copolymer for 48 h. * represented the statistical difference.

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3.4. Cell cycle distribution The cycle distribution of C26 and A549 treated with D/P-NPs was investigated by flow cytometry. D/P-NPs could induce G2/M cell cycle arrest compared with cells without treatment as presented in Fig. 5 and Fig 6. G2/M fractions in D/P-NPs-treated C26 and A549 were 43.67 % and 29.74 % respectively, which were significantly higher than the untreated cells (24.61 % in C26 and 12.19 % in A549). Moreover, D/P-NPs partially abrogated G2/M cycle delay, leading to higher proportion of cells containing sub-G1 DNA than control group. The sub-G1 phase population was relatively smaller in C26 and A549 treated by either of single drug-loaded nanoparticles (D-NPs and P-NPs), which indicated that D/P-NPs could increase cell death and explained the phenomenon that PEDF/PTX combination achieved enhanced tumor cell growth inhibition activity in vitro. D+P-NPs group also had fewer cells in sub-G1 phase, revealing that D/P-NPs would bring out better antitumor activity.

22


Page 22 of 57

Page 23 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 5 Cycle distribution of C26 and A549 which were either untreated or treated with various formulations (free PTX, D-NPs, P-NPs, D+P-NPs or D/P-NPs) after 4 h of treatment at 37 oC by flow cytometry.

Fig.6. Statisctical data of cycle distribution of G2/M and subG1 phase on C26 cells and A549 cells.

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3.5. PEDF expression in tumor cells treated by D/P-NPs The ELISA analysis showed that D/P-NPs could result in higher expression of PEDF which secreted into the culture media in both C26 and A549. As shown in Fig.7, similar expression levels of PEDF gene were observed on the two cell lines which were untreated or treated by free PTX or P-NPs, whereas D-NPs upregulated the expression of PEDF (P = 0.009 on C26 cells and P = 0.002 on A549 cells, D-NPs versus control), indicating that PTX alone would not enhance the cells’ own PEDF expression. However, the combination of PEDF/PTX elevated the PEDF concentration (P = 0.007 on C26 cells and P = 0.002 on A549 cells, D+P-NPs versus D-NPs; P = 0.002 on C26 cells and P = 0.002 on A549 cells, D/P-NPs versus D-NPs), probably because PTX enhanced transfection of the exogenous PEDF gene, which was consistent with the previous reports that co-delivery of PTX and gene (such as DNA and siRNA) resulted in elevated gene transfer [27] [28]. This was most likely due to the anti-mitotic function of PTX [2]. Hence, the combination of PTX and PEDF in our study was advantageous.

Fig. 7 The concentration of PEDF secreted into the culture media of C26 and A549 which were either untreated or treated with various formulations (free PTX, P-NPs, D-NPs, D+P-NPs or D/P-NPs) after 48 h of treatment at 37 oC by ELISA analysis. * represented the statistical significant difference.

24


Page 24 of 57

Page 25 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.6. Cellular uptake of of PTX To investigate the mechanism of improved cytotoxicity and enhanced gene expression of D/P-NPs, the cellular uptake study of PEDF/FITC-PTX-NPs was evaluated in C26 and A549 cells. No fluorescence was observed in cells treated with blank medium. In free FITC-PTX group, a dim fluorescence was observed in the cytosol of C26 and A549 cells. In contrast, PEDF/FITC-PTX-NPs could rapidly accumulate in the cytosol of cells, which revealed by bright green fluorescence (Fig. 8A). In addition, the enhanced cellular uptake of PEDF/FITC-PTX-NPs was also confirmed by FCM analysis. Fig. 8B showed that the fluorescence intensity of cells treated with PEDF/FITC-PTX-NPs was much stronger than that in free FITC-PTX group. All the data demonstrated that more drug molecules entrapped in the nanoparticles entered cells than free drug on both cell lines, which might be due to different endocytosis pathways [29].

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 8 Cellular uptake assay of FITC-PTX on C26 and A549 cells. (A) Fluorescent images of cells treated with free FITC-PTX and PEDF/FITC-PTX-NPs for 1 h. Nuclei were stained blue with Hoechst 33258 and cellular distribution of FITC-PTX was shown as green fluorescence in the cytosol. (B) Flow cytometeric histograms for free FITC-PTX and PEDF/FITC-PTX-NPs.

3.7. Effect of D/P-NPs on microtubule disassembly The α-tubulin immunofluorescence assay demonstrated that D/P-NPs could lead to a disruption of the microtubule network. There were several irregular and short filaments around the nuclei of C26 and A549 treated by D/P-NPs (Fig. 9). Similar appearance of microtubule was also found in both cells after treatment with D+P-NPs or P-NPs, whereas D-NPs treated cells exhibited a well-organized microtubule network in accordance with control group without treatment. PTX could inhibit microtubule disassembly and make microtubules be locked in polymerized state according to previous reports [30]. The microtubule breakdown activity of free PTX was also verified in C26 and A549 in this study. Thus, it could be concluded that D/P-NPs-induced tubulin assembly originated from entrapped PTX instead of PEDF gene.

26


Page 26 of 57

Page 27 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 9 α-tubulin immunofluorescence assay of C26 and A549 which were either untreated or treated with various formulations (free PTX, D-NPs, P-NPs, D+P-NPs or D/P-NPs) after 4 h of treatment at 37 oC. α-tubulin was immunolabeled with an Alexa Fluor® 488-conjugated secondary antibody (green) and cells’ nuclei were stained with Hoechst 33258 (blue).

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3.8. In vivo distribution of nanoparticles In vivo distribution of nanoparticles had employed DiD as the fluorescence probe for its good in vivo imaging capability [31]. Its emission wavelength is above 700 nm, which effectively eliminates emission interference from the animals or organs used [32]. As seen in Fig. 10, the DiD-NPs group showed a much higher fluorescence in the tumor site compared with that of the free DiD group. The result reflected that PEG-PLGA nanoparticles could be largely accumulated in the tumor region, possibly benefiting from the EPR effect. Otherwise, free DiD showed relatively higher accumulation in liver, lung, spleen and kidney than DiD-NPs, suggesting that the high free drug content in normal tissue might be contributed to the high systematic toxicity. In contrast to free DiD, pegylated DiD-NPs escaped the capture of the RES at least to some extent as lower fluorescence were observed in main organs. The results further confirmed PEG-PLGA would be a safe carrier for drug delivery.

Fig. 10 In vivo distribution of nanoparticles. (A) Representative ex vivo fluorescent images of tumors and various organs at 12 h after intravenous injection of the free DiD and DiD-NPs. (B) Quantitative analysis of the average fluorescence signal from free DiD and DiD-NPs formulations in each organ.

3.9. Accumulation and sustained release/expression of PTX/PEDF gene in tumors Accumulation and sustained release/expression manners of PTX/PEDF gene in 28


Page 28 of 57

Page 29 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


D/P-NPs were investigated by ex vivo image using the FITC-PTX as the probe and western blotting assay in C26 xenografts tumors, respectively. As shown in Fig. 11A, the stronger signal appeared in PEDF/FITC-PTX-NPs group at 12 h, confirming that PEDF/FITC-PTX-NPs could significantly accumulate in tumor region by the EPR effect compared to free FITC-PTX. This observation was consistent with the previous result of in vivo distribution of free DiD and DiD-NPs. In addition, strong fluorescence signals of PEDF/FITC-PTX-NPs group were still observed with the time increased to 24h and 48 h, while the fluorescence signals in free FITC-PTX group were eliminated at 24 h and even disappeared at 48 h. The results might be caused by the long circulation time of PEDF/FITC-PTX-NPs as well as the sustained release of the FITC-PTX from the PEDF/FITC-PTX-NPs in tumor site. Otherwise, to confirm the sustained expression of PEDF in tumors, western blotting assay of the tumors at different time points were shown in Fig. 11B. PEDF expression was maintained by PEDF/FITC-PTX-NPs over 48 h. The results might be caused by the sustained PEDF gene release [33]. It was noteworthy that majority of the reported co-delivery systems of DNA and anticancer drug did not investigate their sustained release property in vivo. Our present results demonstrated PEG-PLGA nanoparticles’ capability for sustained release of PTX and PEDF gene in tumors as well as long term gene expression. This sustained-release platform could be of interest in both fundamental biological studies and clinical applications.

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 11 Accumulation and sustained release/expression of PTX/PEDF gene in tumors. (A) Representative ex vivo fluorescent images of tumors at 12 h, 24 h and 48 h after intravenous injection of free FITC-PTX and PEDF/FITC-PTX-NPs. (B) Sustained PEDF expression in tumors after PEDF/FITC-PTX-NPs treatments for 12 h,24 h and 48 h.

3.10. In vivo antitumor efficacy The results described above stimulated us to further explore the in vivo antitumor activity of D/P-NPs. Administration of D/P-NPs significantly inhibited the tumor growth and prolonged the survival time of mice compared with D+P-NPs (Fig. 12A~12D). The median survival of mice was 61 days in D/P-NPs group but only 48 days in D+P-NPs group (Fig. 12D). A common reason should be the highly efficient co-delivery of PEDF/PTX encapsulated simultaneously in D/P-NPs [34]. Dv-NPs had no influence in tumor size similar to NS, while D-NPs markedly inhibited the tumor growth (P < 0.01, D-NPs versus NS). This phenomenon indicated that the carrier itself was nontoxic and verified that PEG-PLGA nanoparticles were indeed good vectors for PEDF gene transfection by systemic administration in vivo. Moreover, P-NPs displayed dramatically higher tumor growth inhibition activity than free PTX (P < 0.05), demonstrating that the tumor tissue specificity was improved by PEG-PLGA nanoparticles through EPR effect [16]. As discussed above, it can be concluded that PEG-PLGA nanoparticles were superior carriers for simultaneous delivery PTX and PEDF to tumor tissues and the enhanced antitumor activity of D/P-NPs originated from the combinational therapy of PEDF/PTX. Furthermore, a slight increase in the body weight of the mice was found during the treatment (Fig. 12E). Both NS and Dv-NPs treated groups had higher body weight, mainly attributing to the higher tumor mass. All the investigated nanoparticles caused no weight loss, attesting that PEG-PLGA nanoparticles were safer carriers for the 30


Page 30 of 57

Page 31 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


systemic administration of gene or chemical drug.

Fig. 12 In vivo antitumor effect of NS, free PTX and various nanoparticles (Dv-NPs, D-NPs, P-NPs, D+P-NPs or D/P-NPs) on the C26 tumor subcutaneous xenograft mouse model after intravenous administration. (A) The photographs of tumors collected from the mice in each group (n=5). (B) Tumor volume growth curves (n=5). (C) Weight of subcutaneous tumors after the treatments in each group (n=5). (D) Survival curves of tumor-bearing mice (n=10). (E) Body weight of tumor-bearing mice in each group (n=5). * represented the statistical significant difference.

3.11. Effect of D/P-NPs on tumor angiogenesis and apoptosis The potential mechanism underlying the efficacy of D/P-NPs-based therapy was investigated by CD31 staining and TUNEL assay. CD31 is used primarily to demonstrate the presence of endothelial cells in histological tissue sections, which can evaluate the degree of tumor angiogenesis and imply a rapidly growing tumor. Thus, CD31 expression was examined by IHC staining to study the effect of D/P-NPs on 31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

angiogenesis in this study. As seen in Fig. 13A, D-NPs significantly decreased the MVD (14.57 %, P < 0.001, D-NPs versus NS) due to their anti-angiogenic effects, which was associated with the disruption of intratumoural vascular network [7]. The MVD in PTX (23.88 %, P < 0.001, PTX versus NS) and P-NPs (18.63 %, P < 0.001, P-NPs versus NS) treated groups was much lower than that in NS group (49.50 %). This inhibitory effects on angiogenesis mainly resulted from the ability of inhibiting endothelial cell functions [35]. Moreover, significant fewer immunoreactive microvessels were observed after D+P-NPs (11.02 %, P < 0.001, D+P-NPs versus NS) and D/P-NPs (7.69 %, P < 0.001, D/P-NPs versus NS) treatment than NS group, suggesting combination PEDF gene with PTX had enhanced inhibition effect on angiogenesis. D/P-NPs had the lowest MVD than free PTX (P < 0.001) and single drug-loaded nanoparticles (P = 0.012, D/P-NPs versus D-NPs; P < 0.001, D/P-NPs versus P-NPs), indicating the potential in vivo anti-angiogenic activity of D/P-NPs. TUNEL could detect DNA fragmentation resulting from apoptotic signaling cascades. This assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. Hence, apoptosis of tumor cells was examined by immunofluorescent TUNEL assay in this study. D-NPs induced 5.87 % (P < 0.001, D-NPs versus NS) apoptosis as seen in Fig. 13B, probably because the PEDF successfully expressed in the tumor tissue increased the levels of p53 and BAX and concomitantly inhibited BCL-2 [7]. PTX and P-NPs also provoked 5.02 % (P < 0.001, PTX versus NS) and 8.58 % (P < 0.001, P-NPs versus NS) of cell death, possibly through disrupting the normal tubule dynamics required for cellular division [36]. Additionally, D+P-NPs and D/P-NPs showed a higher rate of cell apoptosis (P < 0.001, D+P-NPs versus NS; P < 0.001, D/P-NPs versus NS) which suggested that a 32


Page 32 of 57

Page 33 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


combination therapy of PEDF gene and PTX would enhance tumor apoptosis. In particular, D/P-NPs produced the strongest cell apoptosis induction especially compared with D+P-NPs (P = 0.002), pointing to the essential of encapsulating PEDF/PTX into the same nanoparticle to exert a higher apoptosis rate. According to the results of CD31 staining and TUNEL assay, it was suggested that the better tumor growth inhibition activity of D/P-NPs in vivo was probably caused by increased antiangiogenesis and enhanced tumor apoptosis of PEDF/PTX combinational therapy.

Fig. 13. Effect of D/P-NPs on tumor angiogenesis and apoptosis determined by CD31 staining and TUNEL analysis, respectively. (A) Representative CD31 immunohistochemical image of tumors and the mean microvessel density (MVD) in each group (×400). (B) Representative TUNEL immunofluorescent image of tumors and the mean apoptotic index in each group (×400). * represented the statistical significant difference.

3.12. Preliminary safety evaluation of D/P-NPs In order to further study the effects of nanoparticles on the physiology of mice treated by various formulations, the serological and biochemical analysis were carried

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

out. As shown in Fig. 14, although there were individual differences, all biochemical indexes suggested that the functions of nanoparticles-treated mice’s vital organs were present at the same levels according to the data of control mice. Meaningfully, several biochemical indicators of these treated mice including TG, CK, Glu and AST were approaching to the normal ranges. The results indicated that nanoparticles treated mice had no obvious side effects compared to the group treated by NS. Hence, P-NPs, D-NPs, and D/P-NPs were all safe formulations by systemic administration. H&E staining was performed to investigate the pathological changes of mice organs. No significantly toxic pathological changes in the heart, liver, spleen, lung and kidney were detected as displayed in Fig. 15. Together, D/P-NPs were demonstrated to be safe for i.v. injection.

Fig.14. Serological and biochemical analysis of the mice treated with saline (Control), PTX, P-NPs, D-NPs, D+P-NPs and D/P-NPs. ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; TG, triglycerides; CK, creatine kinase; GLU, glucose; TC, total cholesterol; AST, 34


Page 34 of 57

Page 35 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


aspartate transaminase; TP, total protein; HDL-C, high density lipoprotein-cholesterol; LDL-C, low density lipoprotein-cholesterol; UA, urea.

Fig.15. Representative H&E images (×200) after treatment with saline (NS), PTX, D-NPs, P-NPs, D+P-NPs and D/P-NPs.

4. Conclusion PEDF gene and PTX co-encapsulated PEG-PLGA nanoparticles (D/P-NPs) were successfully developed by a modified double-emulsion solvent evaporation method for the first time. The anticancer effects were systemically investigated in vitro and in vivo. D/P-NPs exhibited a superior antitumor effect than the cocktail combination of PEDF/PTX, possibly due to simultaneous transportation of PEDF/PTX into the same tumor cells. Furthermore, PEDF/PTX combination indeed resulted in enhanced G2/M phase arrest, higher PEDF gene transfection, improved antiangiogenesis and apoptosis. Altogether, D/P-NPs offered a compelling strategy for combined gene/chemo-therapy of cancer. In our future study, a targeting moiety will be modified on D/P-NPs to further improve the targeting properties and antitumor activities.

35



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 57

Acknowledgements This research has been received financial support from the National Natural Science Foundation of China (No. 81302729 and 30901868), the National High Technology Research and Development Program of China (No. 2012AA020803) and the Fundamental Research Funds for the Central Universities (No. 2013SCU04A19).

References [1] Gandhi, N. S.; Tekade, R. K.; Chougule, M. B. Nanocarrier mediated delivery of siRNA/miRNA in combination with chemotherapeutic agents for cancer therapy: current progress and advances. J. Control. Release. 2014, 194, 238-256. [2] Zhao, F.; Yin, H.; Li, J. Supramolecular self-assembly forming a multifunctional synergistic system for targeted co-delivery of gene and drug. Biomat. 2014, 35(3), 1050-1062. [3] Wang, H. J.; Zhao, P. Q.; Su, W. Y.; Wang, S.; Liao, Z. Y.; Niu, R. F.; Chang, C. PLGA/polymeric liposome for targeted drug and gene co-delivery. Biomat. 2010, 31(33), 8741-8748. [4] Shi, S.; Zhu, X. C.; Guo, Q. F.; Wang, Y. J.; Zuo, T.; Luo, F.; Qian, Z. Y. Self-assembled

mPEG–PCL-g–PEI

micelles

for

simultaneous

codelivery

of

chemotherapeutic drugs and DNA: synthesis and characterization in vitro. Int. J. Nanomed. 2012, 7, 1749-1759. [5] Wiradharma, N.; Tong, Y. W.; Yang, Y. Y. Self-assembled oligopeptide nanostructures for co-delivery of drug and gene with synergistic therapeutic effect. Biomat. 2009, 30(17), 3100-3109. [6] Han, Y. Q.; Zhang, Y.; Li, D. N.; Chen, Y. Y.; Sun, J. P.; Kong, F. S. Transferrin-modified nanostructured lipid carriers as multifunctional nanomedicine for 36


Page 37 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


codelivery of DNA and doxorubicin[J]. Int. J. Nanomed. 2014, 9, 4107-4115. [7] Becerra, S. P.; Notario, V. The effects of PEDF on cancer biology: mechanisms of action and therapeutic potential. Nat. Rev. Cancer. 2013, 13(4), 258-271. [8] Wu, Q. J.; He, S. S.; Wei, X. W.; Shao, B.; Luo, S. T.; Guo, F. C.; Zhang, H. L.; Wang, Y. S.; Gong, C. Y.; Yang, L. Synergistic antitumor effect of recombinant adeno-associated virus-mediated pigment epithelium-derived factor with hyperthermia on solid tumor. Hum. Gene. Ther. 2014, 25(9), 811-823. [9] Cui, F. Y.; Song, X, R.; Li, Z. Y.; Li, S. Z.; Mu, B.; Mao, Y. Q.; Wei, Y. Q.; Yang, L. The pigment epithelial-derived factor gene loaded in PLGA nanoparticles for therapy of colon carcinoma. Oncol. Rep. 2010, 24(3), 661-668. [10] Ma, Q.; Li, B.; Yu, Y. Y.; Zhang, Y.; Wu, Y.; Ren, W.; Zheng, Y.; He, J.; Xie, Y. M.; Song, X. R.; He, G. Development of a novel biocompatible poly (ethylene glycol)-block-poly (γ-cholesterol-l-glutamate) as hydrophobic drug carrier. Int. J. Pharm. 2013, 445(1), 88-92. [11] Chang, Y. F.; Li, L. L.; Wu, C. W.; Liu, T. Y.; Lui, W. Y.; Peng, F. K.; Chi, C. W. Paclitaxel-induced apoptosis in human gastric carcinoma cell lines. Cancer. 1996, 77(1), 14-18. [12] Xu, R.; Sato, N.; Yanai, K.; Akiyoshi, T.; Nagai, S.; Wada, J.; Koga, K.; Mibu, R.; Nakamura, M.; Katano, M. Enhancement of paclitaxel-induced apoptosis by inhibition of mitogen-activated protein kinase pathway in colon cancer cells. Anticancer. Res. 2009, 29(1), 261-270. [13] Smitha, K. T.; Anitha, A.; Furuike, T.; Tamura, H.; Nair, S. V.; Jayakumar, R. In vitro evaluation of paclitaxel loaded amorphous chitin nanoparticles for colon cancer drug delivery. Colloids. Surf. B. Biointerfaces. 2013, 104, 245-253. [14] Nair, R. R.; Rodgers, J. R. Schwarz, L. A. Enhancement of transgene expression by 37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

combining glucocorticoids and anti-mitotic agents during transient transfection using DNA-cationic liposomes. Mol. The. 2002, 5(4), 455-462. [15] Xu, X. Y.; Xie, K.; Zhang, X. Q.; Pridgen, E. M.; Park, G. Y.; Cui, D. S.; Shi, J. J.; Wu, J.; Kantoff, P. W.; Lippard, S. J.; Langer, R.; Walker, G. C.; Farokhzad, O. C. Enhancing tumor cell response to chemotherapy through nanoparticle-mediated codelivery of siRNA and cisplatin prodrug. Proc. Natl. Acad. Sci. U. S. A. 2013, 110(46), 18638-18643. [16] Devulapally, R.; Sekar, N. M.; Sekar, T. V.; Foygel, K.; Massoud, T. F.; Willmann, J. K.; Paulmurugan, R. Polymer nanoparticles mediated codelivery of antimiR-10b and antimiR-21 for achieving triple negative breast cancer therapy. ACS. Nano. 2015, 9(3), 2290-2302. [17] Qi, X. R.; Song, X. R.; Liu, P.; Yi, T.; Li, S. Z.; Xie, C.; Zheng, Y.; Bai, Y.; Sun, C. T.; Wei, Y. Q.; Zhao, X. Antitumor effects of PLGA nanoparticles encapsulating the human PNAS-4 gene combined with cisplatin in ovarian cancer. Oncol. Rep. 2011, 26(3), 703-710. [18] Jaffe, E. A.; Nachman, R. L.; Becker, C. G.; Minick, C. R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. Inves. 1973, 52(11), 2745-2756. [19] Song, X. R.; Zheng, Y.; He, G.; Yang, L.; Luo, Y. F.; He, Z. Y.; Li, S. Z.; Li, J. M.; Yu, S.; Luo, X.; Hou, S. X.; Wei, Y. Q. Development of PLGA nanoparticles simultaneously loaded with vincristine and verapamil for treatment of hepatocellular carcinoma. J. Pharm. Sci. 2010, 99(12), 4874-4879. [20] Song, Q. X.; Huang, M.; Yao, L.; Wang, X. L.; Gu, X.; Chen, J.; Chen, J.; Huang, J. L.; Hu, Q. Y.; Kang, T.; Rong, Z. X.; Qi, H.; Zheng, G.; Chen, H. Z.; Gao, X. L. Lipoprotein-based nanoparticles rescue the memory loss of mice with Alzheimer’s 38


Page 38 of 57

Page 39 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


disease by accelerating the clearance of amyloid-beta. ACS. Nano. 2014, 8(3), 2345-2359. [21] He, Z. Y.; Wei, X. W.; Luo, M.; Luo, S. T.; Yang, Y.; Yu, Y. Y.; Chen, Y.; Ma, C. C.; Liang, X.; Guo, F. C.; Ye, T. H.; Shi, H. S.; Shen, G. B.; Wang, W.; Gong, F. M.; He, G.; Yang, L.; Zhao, X.; Song, X. R.; Wei, Y. Q. Folate-linked lipoplexes for short hairpin RNA targeting Claudin-3 delivery in ovarian cancer xenografts. J. Control. Release. 2013, 172(3), 679-689. [22] Song, X. R.; Zhao, Y.; Hou, S. X.; Xu, F. Y.; Zhao, R.; He, J. Y.; Cai, Z.; Li, Y. B.; Chen, Q. H. Dual agents loaded PLGA nanoparticles: systematic study of particle size and drug entrapment efficiency. Eur. J. Pharm. Biopharm. 2008, 69(2), 445-453. [23] Lv, L.; Shen, Y. Y.; Li, M.; Xu, X. F.; Li, M. N.; Guo, S. R.; Huang, S. T. Novel 4-arm poly (ethylene glycol)-block-poly (anhydride-esters) amphiphilic copolymer micelles loading curcumin: preparation, characterization, and in vitro evaluation. Biomed. Res. Int. 2013, 2013. [24] Guo, J. W.; Gao, X. L.; Su, L. N.; Xia, H. M.; Gu, G. Z.; Pang, Z. Q.; Jiang, X. G.; Yao, L.; Chen, J.; Chen, H. Z. Aptamer-functionalized PEG–PLGA nanoparticles for enhanced anti-glioma drug delivery. Biomat. 2011, 32(31), 8010-8020. [25] Tahara, K.; Sakai, T.; Yamamoto, H.; Takeuchi, H.; Kawashima, Y. Establishing chitosan coated PLGA nanosphere platform loaded with wide variety of nucleic acid by complexation with cationic compound for gene delivery. Int. J. Pharm. 2008, 354(1), 210-216. [26] Gupta, M.; Chashoo, G.; Sharma, P. R.; Saxena, A. K.; Gupta, P. N.; Agrawal, G. P.; Vyas, S. P. Dual Targeted Polymeric Nanoparticles Based on Tumor Endothelium and Tumor Cells for Enhanced Antitumor Drug Delivery. Mol. Pharm. 2014, 11(3), 697-715. 39



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

[27] Zhu, C. H.; Jung, S.; Luo, S. B.; Meng, F. H.; Zhu, X. L.; Park, T. G.; Zhong, Z. Y. Co-delivery of siRNA and paclitaxel into cancer cells by biodegradable cationic micelles based on PDMAEMA–PCL–PDMAEMA triblock copolymers. Biomat. 2010, 31(8), 2408-2416. [28] Zhan, C. Y.; Wei, X. L.; Qian, J.; Feng, L. L.; Zhu, J. H.; Lu, W. Y. Co-delivery of TRAIL gene enhances the anti-glioblastoma effect of paclitaxel in vitro and in vivo. J. Control. Release. 2012, 160(3), 630-636. [29] Wei, T.; Liu, J.; Ma, H. L.; Cheng, Q.; Huang, Y. Y.; Zhao, J.; Huo, S. D.; Xue, X. D.; Liang, Z. C.; Liang, X. J. Functionalized nanoscale micelles improve drug delivery for cancer therapy in vitro and in vivo. Nano Lett. 2013, 13(6), 2528-2534. [30] Jordan, M. A.; Kamath, K. How do microtubule-targeted drugs work? An overview. Curr. Cancer. Drug. Targets. 2007, 7(8), 730-742. [31] Ran, R.; Liu, Y. Y.; Gao, H. L.; Kuang, Q. F.; Zhang, Q. Y.; Tang, J.; Huang, K.; Chen, X. X.; Zhang, Z. R.; He, Q. Enhanced gene delivery efficiency of cationic liposomes coated with PEGylated hyaluronic acid for anti P-glycoprotein siRNA: A potential candidate for overcoming multi-drug resistance. Int. J. Pharm. 2014, 477(1), 590-600. [32] Zheng, N., Dai, W., Du, W., Zhang, H., Lei, L., Zhang, H., Wang, X., Wang, J., Zhang, X., Gao, J., Zhang, Q., A novel lanreotide-encoded micelle system targets paclitaxel to the tumors with overexpression of somatostatin receptors. Mol. Pharm. 2012, 9(5), 1175–1188. [33] Shi, J. J.; Xu, Y. J.; Xu, X. Y.; Zhu, X.; Pridgen, E.; Wu, J.; Votruba, A. R.; Swami, A.; Zetter, B. R.; Farokhzad, O. C. Hybrid lipid-polymer nanoparticles for sustained siRNA delivery and gene silencing. Nanomed-Nanotechnol. 2014, 10(5), 897-900. [34] Devulapally, R.; Sekar, T. V.; Paulmurugan, R. Formulation of anti-miR-21 and 40


Page 40 of 57

Page 41 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4-hydroxytamoxyfen

co-loaded

biodegradable

polymer

nanoparticles

and

its

anti-proliferative effect on breast cancer cells. Mol. Pharm. 2015, 12(6), 2080-2092. [35] Luo, L. M.; Huang, Y.; Zhao, B. X.; Zhao, X.; Duan, Y.; Du, R.; Yu, K. F.; Song, P.; Zhao, Y.; Zhang, X.; Zhang, Q. Anti-tumor and anti-angiogenic effect of metronomic cyclic NGR-modified liposomes containing paclitaxel. Biomat. 2013, 34(4), 1102-1114. [36] Shi, S. J.; Han, L.; Deng, L.; Zhang, Y. L.; Shen, H. X.; Gong, T.; Zhang, Z. R.; Sun, X. Dual drugs (microRNA-34a and paclitaxel)-loaded functional solid lipid nanoparticles for synergistic cancer cell suppression. J. Control. Release. 2014, 194, 228-237.

41



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents Graphic (TOC)

42


Page 42 of 57

Page 43 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


149x111mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

30x11mm (600 x 600 DPI)


Page 44 of 57

Page 45 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


22x6mm (600 x 600 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

540x407mm (300 x 300 DPI)


Page 46 of 57

Page 47 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80x91mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

30x11mm (600 x 600 DPI)


Page 48 of 57

Page 49 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


56x39mm (600 x 600 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

190x152mm (300 x 300 DPI)


Page 50 of 57

Page 51 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


122x301mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

81x25mm (300 x 300 DPI)


Page 52 of 57

Page 53 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


53x21mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

91x104mm (600 x 600 DPI)


Page 54 of 57

Page 55 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


47x28mm (600 x 600 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

40x40mm (600 x 600 DPI)


Page 56 of 57

Page 57 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


33x18mm (600 x 600 DPI)


Chemotherapy Elicits

Recommend Documents