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Biological and Medical Applications of Materials and Interfaces

A high-efficiency drug and gene co-delivery system for cancer therapy constructing from a pH-sensitive supramolecular inclusion between oligoethylenimine-graft#-cyclodextrin and hyperbranched polyglycerol derivative Xiaoyan Zhou, Lanqin Xu, Jiake Xu, Jianping Wu, Thomas Brett Kirk, Dong Ma, and Wei Xue ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14517 • Publication Date (Web): 02 Oct 2018 Downloaded from http://pubs.acs.org on October 3, 2018

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ACS Applied Materials & Interfaces

A high-efficiency drug and gene co-delivery system for cancer therapy constructing from

a

pH-sensitive

supramolecular

inclusion

between

oligoethylenimine-graft-β-cyclodextrin and hyperbranched polyglycerol derivative

Xiaoyan Zhou 1,2, Lanqin Xu 3, Jiake Xu 4, Jianping Wu 5, Thomas Brett Kirk 5, Dong Ma 1,*, Wei Xue 1,6,7,*

1

Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of

Biomedical Engineering, Jinan University, Guangzhou 510632, China 2

National Engineering Research Center for Healthcare Devices, Guangdong Key Lab of Medical

Electronic Instruments and Polymer Material Products, Guangdong Institute of Medical Instruments, Guangzhou 510500, China 3 4

School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou, China The School of Pathology and Laboratory Medicine, University of Western Australia, Perth,

Australia 5

3D Imaging and Bioengineering Laboratory, Department of Mechanical Engineering, Curtin

University, Perth, Australia 6

Institute of Life and Health Engineering, Key Laboratory of Functional Protein Research of

Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China 7

The First Affiliated Hospital of Jinan University, Guangzhou 510630, China

E-mail addresses: [email protected] (Dong Ma), [email protected] (Wei Xue)

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Abstract Introducing genes into drug delivery system for a combined therapy has become a promising strategy for cancer treatment. However, how to improve the in vivo therapy effect resulted from the high delivery efficiency, low toxicity and good stability in the blood remains a challenge. For this purpose, the supramolecular inclusion was considered to construct a high-efficiency drug and gene co-delivery system in this work. The oligoethylenimine-conjugated β-cyclodextrin (β-CD-PEI600) and

benzimidazole-modified

4-arms-polycaprolactone-initiated

hyperbranched

polyglycerol

(PCL-HPG-BM) were synthesized as the host and guest molecules respectively, and then the co-delivery carrier of PCL-HPG-PEI600 was formed from the pH-mediated inclusion interaction between β-CD and BM. PCL-HPG-PEI600 showed the improved drug (doxorubicin, DOX) and gene (MMP-9 shRNA plasmid, pMMP-9) delivery ability in vivo, and their cellular uptake and intracellular delivery were investigated. Particularly, PCL-HPG-PEI600 showed excellent pMMP-9 delivery ability with significantly higher transfection efficiency than PEI25k due to its excellent serum resistance. For the combined therapy to breast cancer MCF-7 tumor, the co-delivery system of PCL-HPG-PEI600/DOX/pMMP-9 resulted in much better inhibition effect on MCF-7 cells proliferation and migration in vitro as well as the suppression effect on MCF-7 tumors in vivo compared with those of single DOX or pMMP-9 formulation used. Moreover, PCL-HPG-PEI600 displayed non-toxicity and excellent blood compatibility, suggesting a promising drug and gene co-delivery carrier in combined therapy to tumors.

Keywords: supramolecular inclusion, low-molecular-weight PEI, hyperbranched polyglycerol, drug and gene co-delivery, cancer therapy


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1. Introduction The combination of chemotherapy with gene therapy represents a promising strategy to achieve synergistic therapeutic method for tumor diseases, which allows the administration of chemotherapeutic agents at lower doses and then helps to reduce the adverse effects and improve the treatment efficacy 1-3. For this tactics, a high-efficiency and safe co-delivery carrier is necessary. Some works have reported various co-delivery carriers, such as liposomes, micelles, inorganic nanoparticles an d nanovesicles

4-7

. Among them, the most concern is still how to enhance their in

vivo delivery effect because of the currently lower gene transfection efficiency and instability in the blood, although many attempts have been made in preparing high-efficiency non-viral gene carrier. From the cationic liposomes to the linear cationic polymers, and then to the topological (hyperbranched or dendritic) cationic polymers, generations of non-viral gene vectors have been developed

8-11

. Now, the biggest challenge for non-viral carrier is still that how to balance the

contradiction between the cytotoxicity and the transfection efficiency.

For branched

polyethylenimine (PEI) as an example, the PEI25k is a well-known gene carrier and usually used as the standard for evaluating other non-viral gene carriers

12

. However, its high cytotoxicity and

instability in the blood limit its application in clinical. The low molecular PEI600 shows the low cytotoxicity and good stability in the blood, while its gene transfection efficiency is very low

13-14

.

Therefore, how to design a non-viral vectors with both high gene transfection efficiency and the low cytotoxicity is a critical issue. Our previous works have confirmed that for the low-molecular-weight cationic polymers with low cytotoxicity, the improvement of transfection efficiency can be achieved by conjugating low-molecular-weight cationic polymers to a multifunctional core polymer. The obtained copolymer showed not only the improved gene transfection efficiency similar to the high-molecular-weight cationic polymers, but also the low cytotoxicity just as the low-molecular-weight cationic polymers 15-16

. So, in this work, a number of PEI600 were considered to conjugate with a core molecule to

construct the high-efficiency gene carrier. Compared with the conventional covalent construction, the non-covalent supramolecular host-guest inclusion has attracted much attention to construct the macromolecules

17-21

. The

constructed macromolecules usually responded to the environment and displayed the reversible assembled-disassembled structures, which was conducive to the cellular drug/gene release as well ACS Paragon Plus Environment


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as the degradation and removal of the carriers 22. It was reported that β-cyclodextrin (β-CD) could interact with benzimidazole (BM) to form the pH-sensitive β-CD/BM inclusion, which could disassemble under the acidic conditions (pH < 6), similar to the endosomal/lysosomal conditions 23. This property may help the β-CD/BM-constructing carrier escape from the endosome/lysosome and then enhance its drug/gene delivery ability. In this work, the β-CD/BM interaction was used to construct the drug and gene co-delivery carrier composed of the oligoethylenimine-graft-β-CD (β-CD-PEI600) and BM-modified 4-arms-poly-ε-caprolactone (PCL)-hyperbranched polyglycerol (HPG) (PCL-HPG-BM). For the amphiphilic PCL-HPG-BM segment, HPG was water-soluble and displayed excellent blood compatibility which could enhance the blood stability of the co-delivery system

24

. Moreover, the

abundant hydroxyl groups of HPG provided many reaction sites for BM conjugation and then resulted in many inclusion sites for the co-delivery system construction. The biodegradable PCL core could load hydrophobic drugs and form the stable complexes 25. Doxorubicin (DOX) was first loaded into the hydrophobic core of PCL-HPG-PEI600 to form the PCL-HPG-PEI/DOX complex, and MMP-9 shRNA plasmid (pMMP-9) was then bound through the electrostatic interaction between PEI600 segment and pMMP-9 to obtain the drug and gene co-delivery system (PCL-HPG-PEI/DOX/pMMP-9). This co-delivery system displayed excellent DOX and pMMP-9 delivery abilities to MCF-7 cells and tumors, and its combined anti-tumor effect was much better than only DOX or pMMP-9 formulation used, suggesting a potential application in combined tumor therapy.

2. Materials and methods 2.1 Materials 5-benzimidazolecarboxylic acid (BM) and glycidol were obtained from Macklin Biochemical Co., Ltd (Shanghai, China). 4-Arms polycaprolactone (4-arms PCL, MW = 15000) was procured from Daigang Biomaterial Co., Ltd (Jinan, China). Polyethyleneimine with the molecular weight of 600 (PEI600) and 25000 (PEI25k) were purchased from Thermo Fisher Scientific (USA). β-Cyclodextrin (β-CD), doxorubicin hydrochloride (DOX·HCl) and p-toluenesulfonyl chloride were obtained from Aladdin Industrial Corporation (Shanghai, China). Antibodies against MMP-9 and β-actin were purchased from Proteintech Group, Inc. (Wuhan, China). ACS Paragon Plus Environment

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2.2 Synthesis of amphiphilic hyperbranched polyglycerol derivative (PCL-HPG-BM) For PCL-HPG-BM synthesis, the amphiphilic PCL-HPG with the 4-arms PCL as a core was synthesized by previous method

26

. Typically, 4-arms-PCL (3.0 g, 0.20 mmol) and 0.8 mL

potassium methylate solution were added to a Schlenk flask under the protection of nitrogen. The mixture was heated on oil-bath, and then glycidol (12 g, 162 mmol) was added dropwise to the flask using a syringe pump within 6 h. After that, the product was neutralized by cationic exchange resin. The obtained PCL-HPG was subjected to dialysis in purified water for 3 days (MWCO = 2000, USA) and then lyophilized (yield = 53.3%). BM was then conjugated to the PCL-HPG via an esterification reaction. Briefly, 0.5 g PCL-HPG

and 0.05

g BM were

added

into anhydrous

DMF, and

then

0.02

g

N,N′-dicyclohexylcarbodiimide (DCC) and 0.005 g 4-dimethylaminopyridine (DMAP) were dissolved in the solution. The solution was stirred at 25°C for 48 h. At the end of reaction, the mixture was dialyzed in purified water for 3 days and lyophilized to obtain PCL-HPG-BM (yield = 55.5%). 1H NMR analysis (Bruker DPX-300, Germany) was performed to verify the successful synthesis of PCL-HPG-BM using the DMSO-d6 as the solvent.

2.3 Synthesis of β-CD-PEI600 For β-CD-PEI600 synthesis, β-CD-OTs was synthesized as the reported method 27. In brief, a solution of p-toluenesulfonyl chloride (5.1 g, 26.4 mmol) in acetonitrile was added dropwise to the solution of β-CD (31.2 g, 26.4 mmol) in aqueous NaOH (8.2 mol/L, 10 mL). Then, the solution was stirred at 25°C for 2 h. After that, HCl was added to adjust its pH value to 6.0-6.5. The resultant white precipitate was collected by filtration. The obtained β-CD-OTs was recrystallized with distilled water and then dried under vacuum with a yield of 19.5%. β-CD-PEI600 was synthesized as the previous report

28

. PEI600 (1.1 g, 1.8 mmol) and

β-CD-OTs (2.1 g, 1.8 mmol) were added into DMSO, and the mixture was stirred for 3 days under the protection of nitrogen. After that, the solution was dialyzed and lyophilized to obtain β-CD-PEI600 (yield = 28.3%).

2.4 Supramolecular PCL-HPG-PEI600 formation PCL-HPG-PEI600 was prepared by the supramolecular interaction between β-CD units from ACS Paragon Plus Environment


β-CD-PEI600 and BM units from PCL-HPG-BM. Briefly, 0.1 g β-CD-PEI600 and 0.02 g PCL-HPG-BM were added into DMSO. Next, the mixture was dissolved in PBS (pH = 7.4). The solution was stirred overnight. At last, the solution was dialyzed to remove DMSO, salts and the unreacted molecules (MWCO = 3000, USA). PCL-HPG-PEI600 was obtained by lyophilization (yield = 55.5%). 1

H NMR spectrum was recorded using D2O as the solvent. Its molecular weight was measured

by a gel permeation chromatography (GPC). aqueous NaNO3 (0.2 mol/L) was set as a mobile phase. Isothermal titration calorimetry (ITC) was performed using a high-sensitivity Nano-ITC instrument (TA Instruments，USA). Prior to use, all solutions (PCL-HPG-BM and β-CD-PEI600) were degassed using an ultrasound bath for 15 minutes. Then, 190 µL of PCL-HPG-BM (2 mg/mL) was placed in the cell, and titrated with 50 µL of β-CD-PEI600 solution (100 mg/mL) placed in the stirring syringe. Experiments were planned to consist of 20 consecutive injections (2.5 µL) each with intervals of 300 seconds at a stirring speed of 300 rpm at 25°C. Fluorescence emission spectrum was recorded by the fluorescence spectrophotometer. The final concentration of PCL-HPG-BM was 0.5 mg/mL and the concentration range of β-CD-PEI600 was 0 to 25 mg/mL (excitation wavelength : 240 nm, emission wavelength : 250 - 650 nm, scan speed : 1200 nm/min).

2.5 DOX loading and delivery DOX was loaded into the hydrophobic core of PCL-HPG-PEI600 to form the DOX-loaded complex through a dialysis method. In brief, 5 mg DOX·HCl and 100 mg PCL-HPG-PEI600 were dissolved in distilled water, and then 5 µL triethylamine in 1 mL methanol was added into the mixture to neutralize the hydrochloric acid. The solution was stirred overnight and dialyzed for 48 h. After that, the solution was filtered and lyophilized to obtain the PCL-HPG-PEI600/DOX complex. The DOX loading in PCL-HPG-PEI600/DOX complex was measured by UV-Vis spectroscopy using a standard curve of DOX (R2 > 0.99). In vitro DOX release. 2 mL aqueous PCL-HPG-PEI600/DOX solution (10 mg/mL) was transferred to a dialysis bag (MWCO = 2000), and then the dialysis bag was immersed in PBS (10 mL, pH = 7.4 or 5.5) and shaken at 37°C. The DOX concentration at different time points was measured by a UV-Vis spectroscopy. Cellular uptake of DOX. The endocytosis of the PCL-HPG-PEI600/DOX in MCF-7 cells were ACS Paragon Plus Environment

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analyzed by the flow cytometry. The cells (5 × 104 cells/well) were plated onto the 24-well plates and incubated overnight. Next, the cells were treated with PCL-HPG-PEI600/DOX or free DOX. At the determined time intervals, the cells were harvested, and then detected using the flow cytometry.· Endocytosis pathway analysis. For this study, MCF-7 cells culture were as described above. Consider the reported method of selecting the dose of each inhibitor 29-30. The cells were precultured in DMEM containing 10 µg/mL chlorpromazine, 200 µM genistein, 200 nM wortmannin or 5 µg/mL cytochalasin B, respectively. After 3 h pretreatment, the culture mediums were removed, and the PCL-HPG-PEI600/DOX complexes containing inhibitors were added for further 2 h incubation. The cells treated with PBS or incubated at 4°C were set as the control. Next, the cells were trypsinized, centrifuged for flow cytometry detect. The mean fluorescence for each group compared to the untreated cells was analyzed using FlowJo software. Cell viability inhibition. To determine the effects of PCL-HPG-PEI600/DOX on MCF-7 cell inhibition, CCK-8 assays were performed at 24, 48 and 72 h. MCF-7 cells (5000 cells/well) were seeded in 96-well plates and incubated for 12 h. Then, cells were treated with PCL-HPG-PEI600/DOX, and the DOX concentration range was from 0.01 to 10 µg/mL. After incubation, 10 µL CCK-8 was added. The cytotoxicity of PCL-HPG-PEI600/DOX was measured by a microplate reader.

2.6 MMP-9 plasmid binding and delivery For pMMP-9 binding, 1 µg pMMP-9 was mixed with aqueous PCL-HPG-PEI600 solution at different weight ratios (w/w). The mixture was incubated at 37°C for 20 min to form the PCL-HPG-PEI600/pMMP-9 complexes. Gel electrophoretic assay. To study the binding ability of PCL-HPG-PEI600 to pMMP-9, gel electrophoretic assay was carried out. The PCL-HPG-PEI600/pMMP-9 complexes with various weight ratios were run under 120 V in TAE buffer containing GoldView II (Sigma). After that, the band was visualized under the UV transilluminator (BioDoc-It® 220 Imaging System, UVP, USA) at a wave length of 365 nm

31

. To assess the pH-sensibility of the complex,

PCL-HPG-PEI600/pMMP-9 (w/w = 20) were firstly incubated in acetate buffer. Next, the appropriate amount heparin were added (heparin/pMMP-9 = 1, 2, 4, 6, 8 and 10, w/w) and then co-incubated for 30 min. Then, the gel electrophoretic assay was carried out as described above. ACS Paragon Plus Environment


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The same formulations of heparin-containing PCL-HPG-PEI600/pMMP-9 complexes in PBS (pH = 7.4) were set as control group. Complex

size,

potential

and

morphology.

The

potentials

and

sizes

of

PCL-HPG-PEI600/pMMP-9 complexes were measured at room temperature by a nano particle analyzer. Data were recorded by triplicate independent experiments. The morphology of PCL-HPG-PEI600/pMMP-9 complexes was observed using a transmission electron microscope (TEM). The aqueous PCL-HPG-PEI600/pMMP-9 complexes (w/w = 60) were prepared and then adsorbed to TEM copper grids prior to photography. In vitro transfection. MCF-7 cells culture were as described of cellular uptake assay. After that, PCL-HPG-PEI600/pMMP-9 complexes were added gently to each plate with Opti-MEM® Reduced Serum Medium or 10% serum-containing DMEM, and pMMP-9 was set to 2.0 µg for each well. For serum-absence transfection assay, the cells were treated with formulations for 4 h. After that, the cells were allowed to proliferate in fresh DMEM containing 10% FBS for another 44 h, Next, the cells were observed for GFP expression by fluorescence microscope (Observer A1, ZEISS, Germany). For serum-presence transfection assay, the cells were incubated with formulations for full time in 10% FBS DMEM. To analyze quantificationally the transfection efficiency of formulations to MCF-7 cells, after GFP observation, the cells were tested using flow cytometry (Beckman Gallios, USA). PEI600/pMMP-9, β-CD-PEI600/pMMP-9 with various weight ratios and PEI25k/pMMP-9 were used as control groups. Western-blot assay. For western-blot analysis, MCF-7 cells (2 × 105 cells/well) plated in 6-well plate and incubated with PBS, blank PCL-HPG-PEI600, PCL-HPG-PEI600/pMMP-9 (w/w = 120) and PEI25k/pMMP-9 (w/w = 1.3) for 48 h. Then, the cells were lysed by Protein Extraction Reagent

(KeyGen

Biotech,

China)

supplemented

with

protease

inhibitors

and

phenylmethanesulfonyl fluoride. The concentrations of protein samples were measured using a BCA Protein Assay Kit. Protein samples were separated on 10% SDS-PAGE gels electrophoresis and transferred to polyvinylidene fluoride membranes (Bio-Rad) for 1.5 h at 230 mA. Membranes were blocked and then incubated with primary antibodies for pMMP-9 and β-actin for 12 h and subsequently immersed into the TBST containing secondary antibodies for 1 h. Then, the sample were visualized by the gel imaging system after treated with ECL reagent. Cellular uptake and endocytosis pathway of complexes. Cellular uptake behaviors and ACS Paragon Plus Environment

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endocytosis pathway analysis of PCL-HPG-PEI600 carrying gene (FAM-labeled siRNA, siRNA-FAM) in MCF-7 cells at the presence of serum were quantitatively examined using the flow cytometry

by

quantifying

FAM

fluorescence

intensity.

The

formulation

of

PCL-HPG-PEI600/siRNA-FAM (w/w = 120) was carried out and the methods were referred to the previous description in Section 2.5. Endosomal

escape

exploration.

To

explore

the

endosomal

escape

behavior

of

PCL-HPG-PEI600 carrying gene, siRNA-Cy3 was replaced by pMMP-9 for visual observation by a confocal laser scanning microscope (CLSM). The cells (2 × 105 cells/well) were plated in the glass bottom dish for 24 h, and then the PCL-HPG-PEI600/siRNA-Cy3 (w/w = 120) complexes were added into each well. Endosomes were stained with LysoTracker Green probe for 1 h. At the certain time intervals (2, 4 and 8 h), cells were fixed in 4% paraformaldehyde. Thereafter, the cells were incubation with Hoechst 33342 for 20 min to stain the cell nucleus. After washed again and incubated with 2 mL PBS for further observation, the cells were observed by CLSM (Hoechst 33342: 405 nm diode, Cy3: 555 nm laser, Lysotracker Green probe: 488 nm laser). Ex vivo western-blot analysis. To detect the MMP-9 protein expressed in tumors, the BALB/C nude mice (female, 4-5 weeks old, 16-18 g) were purchased from Huafukang Bioscience Co. (Beijing, China). At first, the tumor model of breast cancer in the mice was generated. The mice were injected by PCL-HPG-PEI600/pMMP-9 (w/w = 120) every 2 days when the tumor volume reached 150-200 mm3. After 15 days feeding, the mice were sacrificed, and then the tumors were collected and firstly shredded into pieces, and then the tumor pieces was homogenized in 1 mL of homogenizing buffer (with protease inhibitors and PMSF) by glass homogenizer. The suspension was centrifuged, and then the debris was removed. Subsequently, western-blot assay was performed as the previous description. Tumor imaging observation. 200 µL PBS, free RNA-Cy5, PEI25k/siRNA-Cy5 (2 mg/kg of siRNA-Cy5) and PCL-HPG-PEI600/siRNA-Cy5 (2 mg/kg of siRNA-Cy5) were intravenously injected. After 8 h, the mice were sacrificed, and the tumors were collected and subjected to imaging with IVIS Lumina system.

2.7 Co-delivery of DOX and pMMP-9 in vitro CLSM observation. MCF-7 cells were plated in the glass bottom dish and incubated overnight. ACS Paragon Plus Environment


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Next, the cells were cultured with various formulations (DOX: 4.5 µg/well; pMMP-9: 3 µg/well), and blank PCL-HPG-PEI600 as well as free pMMP-9 and DOX were set as control groups. After 6 h incubation, the cells were fixed with 4% paraformaldehyde, and the cell nucleus were stained with Hoechst 33342. Next, the treated cells were observed and captured by CLSM. Endocytosis pathway. The endocytosis pathway analysis of the co-delivery system of PCL-HPG-PEI600/DOX/pMMP-9 in MCF-7 cells was examined using the flow cytometry by quantifying DOX fluorescence intensity, which was referred to the previous description in Section 2.5. Cytotoxicity. To evaluate the effect of the co-delivery PCL-HPG-PEI600/DOX/pMMP-9 on tumor cell viability, CCK-8 assay was performed as described above. The MCF-7cells were cultured

onto

a

96-well

plate

for

12

h.

Then,

cells

were

treated

with

the

PCL-HPG-PEI600/DOX/pMMP-9 with a series of DOX/pMMP-9 concentrations. The cells treated with only pMMP-9 (PCL-HPG-PEI600/pMMP-9) and DOX (PCL-HPG-PEI600/DOX) were used as the control groups. Scratch/wound healing assay. The effect of the combined DOX and pMMP-9 on MCF-7 cell migration was assessed using an in vitro cellular migration assay according to the reported method 32

. MCF-7 cells culture were as described of western-blot assay. Next, the cells were treated with

the

media

containing

blank

PCL-HPG-PEI600,

PCL-HPG-PEI600/DOX,

PCL-HPG-PEI600/pMMP-9 and PCL-HPG-PEI600/DOX/MMP-9 respectively (w/w = 120; DOX: 9.6 µg/well; pMMP-9: 8 µg/well). The cells were incubated at 37°C for 24 h and reached to 90% confluency, and then scratch wounds were created by a pipet tip. Subsequently, the cells were cultured in fresh medium without serum. At the determined time intervals (0, 24, 48 and 72 h), the cells were observed and captured by microscope. Transwell assay. In vitro invasion assay was performed with BD Falcon cell culture inserts. Briefly, MCF-7 cells culture were as described of cellular uptake assay. Then, the cells were precultured

in

DMEM

containing

blank

PCL-HPG-PEI600,

PCL-HPG-PEI600/DOX,

PCL-HPG-PEI600/pMMP-9 and PCL-HPG-PEI600/DOX/MMP-9 respectively (w/w = 120; DOX: 9.6 µg/well; pMMP-9: 8 µg/well). After pretreatment, the cells (2.5 × 104 cells/well) were carefully trypsinized and re-plated into the cell culture insert. Then, the insert was placed into 24-well plate, and the medium with 10% FBS was added to each well external to the insert. After another 24 h, the ACS Paragon Plus Environment

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unmigrated cells were removed by scrubbing with a cotton swab. Migrating cells were stained with 1% (wt/vol) crystal violet at room temperature before observation. To quantify the migration ratio, the cells were counted in five randomly selected fields by a microscope. Cell cycle assay. Briefly, MCF-7 cells culture were as described of cellular uptake assay. Then, the cells were treated with various formulations (PCL-HPG-PEI600, PCL-HPG-PEI600/DOX, PCL-HPG-PEI600/pMMP-9 and PCL-HPG-PEI600/DOX/MMP-9) with DOX content of 2.4 µg/well and pMMP-9 of 2 µg/well. After 24 h, the cells were fixed with 70% ethanol. Before analysis, the fixed cells were stained with RNase A and propidium iodide (PI). Next, the cells were analyzed using the flow cytometry and the cell cycle was analyzed by the cell cycle analysis software.

2.8 In vivo assay Tumor inhibition assay. For tumor inhibition assay, the mice were randomly divided into seven treatment groups: PBS control, blank PCL-HPG-PEI600 (240 mg/kg), free DOX (1.2 mg/kg), PEI25k/pMMP-9 (2 mg/kg of pMMP-9), PCL-HPG-PEI600/DOX (1.2 mg/kg of DOX), PCL-HPG-PEI600/pMMP-9 (2 mg/kg of pMMP-9) and PCL-HPG-PEI600/DOX/pMMP-9 (1.2 mg/kg of DOX and 2 mg/kg of pMMP-9). Each treatment group consisted of 5 mice and received injection every 2 days. The tumor volume was measured using an electronic caliper and calculated as : 0.5 × shortest diameter2 × longest diameter. The weight of mice was weighted at the same time. After 15 days feeding, the tumors were photographed and weighted. Histologic and immunohistochemical analysis. Hematoxylin and eosin (H&E) staining of the isolated major organs and tumor was performed as described before

33

. Briefly, the samples were

fixed in 4% (w/v) paraformaldehyde for 24 h and then dehydrated in graded ethanol. Next, all samples were embedded in paraffin. Then, the section was stained with H&E. For immunohistochemical analysis, the apoptosis level of the tumor was assessed by TUNEL assays. Furthermore, the tumor sections were also detected for the expressions of CD31 and Ki67.

2.9 Biocompatibility Cytotoxicity. The cytotoxicity of the blank PCL-HPG-PEI600 on MCF-7 cells was examined by CCK-8 assay. MCF-7 cells culture were as described of cytotoxicity assay in Section 2.7. Then, ACS Paragon Plus Environment


the cells were treated with media containing various concentrations of PCL-HPG-PEI600, and PBS and PEI25k were used as the control groups. After incubation, 10 µL CCK-8 was added. The cytotoxicity of blank PCL-HPG-PEI600 was measured by a microplate reader. Hemolysis assay in vitro. The hemolysis ratio was calculated according to the previously reported method 34. Briefly, 4 mL of PCL-HPG-PEI600 and PEI25k (0.01, 0.1, 0.5 and 1 mg/mL) was added into 0.2 mL of 16% red blood cells suspension and incubation for 6 h. After that, the supernatant were measured after centrifuged by a microplate reader at 540 nm. The distilled water and PBS were used as control. Hemolysis ratio was calculated according to the following formula: Hemolysis (%) = (A-C)/(B-C) × 100 (A: the absorbance of the sample, B: the absorbance of the distilled water, C: the absorbance of the PBS) Morphology of red blood cells (RBCs). The blank PCL-HPG-PEI600 was mixed with 20 µL RBCs for 1 h incubation, and 0.01 mg/mL PEI25k and PBS were employed as the control. Subsequently, the RBCs were collected and then fixed by 4% paraformaldehyde for 1 h and dehydrated with ethanol. The RBCs were smeared on clean coverslips and observed and captured under the scanning electron microscope (SEM, Philips XL-30). Serum biochemical analysis. The blood samples were collected from the mice at 15 days after injection of different formulations (PBS, PEI25k and PCL-HPG-PEI600). Then, the supernatant of blood were collected after centrifuged at 3000 rpm/min. Various parameters including albumin (ALB) and lactate dehydrogenase (LDH) for hepatic function, urea nitrogen (BUN) and creatinine (CRE) for kidney function, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total cholesterol (T-CHO), glucose (GLU), low-density lipoprotein (LDL) were examined by the biochemical analyzer (Rayto, China).

2.10 Statistical analysis The data were expressed as mean values ± standard deviation from at least three replicates. Differences between different experimental groups were analyzed using ordinary one-way ANOVA of SPSS 16.0. Significance was claimed with P values.


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3. Results and discussion 3.1 Formation of supramolecular PCL-HPG-PEI600 To construct a high-efficiency co-delivery system using the amphiphilic HPG derivative and low-molecular-weight PEI (PEI600), β-CD-PEI600 and PCL-HPG-BM were synthesized respectively and then host-guest interact to form the supramolecular inclusion. As shown in Scheme 1, a 4-arms-PCL (terminal OH groups) was used to initiate the glycidol monomers by anionic ring-opening polymerization to form PCL-HPG in the presence of potassium methoxide, and then BM was conjugated with PCL-HPG to synthesize the guest molecule PCL-HPG-BM through an esterification reaction. Its chemical structure was confirmed by 1H NMR and determined the molar ratio of PCL/HPG was 1:5 and the amount of BM in PCL-HPG-BM was 0.171 mmol/g (Fig. S1, Supporting Information). For the host molecule β-CD-PEI600 synthesis, β-CD was first activated by p-toluenesulfonyl chloride to form the mono-functionalization β-CD-OTs, and then PEI600 was conjugated to synthesize β-CD-PEI600 through a nucleophilic substitution reaction. Its chemical structure was also confirmed by 1H NMR and calculated that each PEI600 molecule conjugated with 1.5 β-CD moieties in average (Fig. S2, Supporting Information). Then, the co-delivery carrier of PCL-HPG-PEI600 formed through the supramolecular inclusion between β-CD and BM moieties in aqueous solution. To confirm the successful inclusion of PCL-HPG-BM with β-CD-PEI600, the fluorescence spectra of the mixed aqueous PCL-HPG-BM/β-CD-PEI600 were recorded with different weight ratios. As shown in Fig. 1A, the pure aqueous PCL-HPG-BM showed the typical fluorescence peak at 483 nm due to its BM moieties. After β-CD-PEI600 was mixed, the fluorescence intensity of BM moieties decreased obviously which was ascribed to the inclusion of BM with β-CD moieties 35. It is noteworthy that the fluorescence intensity of the mixture decreased with the increase of β-CD-PEI600 concentration in the early stage and then changed little beyond 5 mg/mL, implying that all BM moieties were included with β-CD moieties when β-CD-PEI600 concentration reached to 5 mg/mL. By calculating the concentration of PCL-HPG-BM and β-CD-PEI600 in the mixture, the saturated inclusion weight ratio between PCL-HPG-BM and β-CD-PEI600 was less than 1:10. The formed supramolecular PCL-HPG-PEI600 was analyzed by 1H NMR. As shown in Fig. 1B, all signals have been identified and it displayed the characterized signals from PCL, HPG, PEI600 and β-CD respectively. In particular, the signals of BM ranging from 7.5 to 8.5 ppm (shown ACS Paragon Plus Environment


in Fig. S1, Supporting Information) disappeared in PCL-HPG-PEI600 due to the interaction between BM and β-CD moieties

23

, which indicated further the successful inclusion of

PCL-HPG-BM with β-CD-PEI600. Moreover, by calculating the integral areas of the characterized peaks, it was found that each PCL-HPG-PEI600 molecule contained 10.3 β-CD-PEI600 molecules in average. To determine the real molar ratio between PCL-HPG-BM and β-CD-PEI600 in PCL-HPG-PEI600, ITC analysis was carried out and the representative calorimetric titration profile was shown in Fig. 1C. Each peak in the binding isotherm represented a single injection of aqueous β-CD-PEI600 into the aqueous PCL-HPG-BM, and β-CD-PEI600 interacted with PCL-HPG-BM through the supramolecular inclusion of β-CD and BM. It was found that there was no calorie recorded after 2800 s titration of β-CD-PEI600, suggesting that all PCL-HPG-BM in the solution had been consumed to interact with β-CD-PEI600. By calculating the consumption of β-CD-PEI600, it was determined that the saturated inclusion weight ratio between β-CD-PEI600 and PCL-HPG-BM was 7.9. For the pH-sensitive analysis, GPC was carried out and the result was shown in Fig. 2A. PCL-HPG-PEI600 displayed the elution time at 10.1 min under the condition of pH 7.4, earlier than PCL-HPG-BM of 10.3 min and β-CD-PEI600 of 11.5 min, indicating that PCL-HPG-BM interacted with β-CD-PEI600 to form the PCL-HPG-PEI600 with a higher molecular weight. After incubated under an acid condition (pH 5.5), PCL-HPG-PEI600 sample was recorded the decreased molecular weight and both peaks were shown at 10.3 and 11.5 min, which were in accordance with those of PCL-HPG-BM and β-CD-PEI600. This result showed that PCL-HPG-PEI600 disassembled as PCL-HPG-BM and β-CD-PEI600 under an acid condition, suggesting a pH sensitive β-CD/BM inclusion. The fluorescence spectrum of aqueous PCL-HPG-PEI600 at different pH values was also recorded and the result was shown in Fig. 2B. The fluorescence intensity of BM peak at 483 nm changed little when the pH decreased from 7.4 to 6.5, while decreased significantly when the pH decreased further to 6.0. Such a saltation may be attributed from the disassembly of the β-CD/BM inclusion, and the disassembled pH of 6.0 was just accordance with the pH value of intracellular endosome 36. This result suggested that the supramolecular PCL-HPG-PEI600 could disassemble in intracellular endosome.


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3.2 DOX delivery 3.2.1 In vitro DOX release and inhibition to MCF-7 cells Due to the amphiphilic PCL-HPG segments, the hydrophobic DOX could be encapsulated in the core of PCL-HPG-PEI600, and the loading amount of DOX in PCL-HPG-PEI600/DOX complex was 5.0 mg/g. The DOX release profiles were investigated through immersing the PCL-HPG-PEI600/DOX complexes into PBS at pH of 5.5 and 7.4, mimicking the physiological pH value of endosome and normal condition, respectively. There was obvious difference in DOX release profiles at different pH conditions (Fig. 3A). At the pH value of 7.4, DOX was released gently and about 45% encapsulated DOX was released from the PCL-HPG-PEI600/DOX complex within 72 h. On the contrary, DOX was released much faster in the endosome-mimicking condition of pH 5.5, and more than 75% DOX was released within 72 h. This pH-sensitivity from β-CD/BM inclusion may help accelerate DOX release in endosome after cellular uptake and enhance the inhibition effect to tumor cells. Its inhibition effect on MCF-7 cells was performed and the result show that PCL-HPG-PEI600/DOX complex displayed the obvious inhibition effect on cells with a concentration-dependent manner (Fig 3B). Moreover, the longer incubation time also resulted into the higher inhibition effect, and there were obvious differences in cell viability among them at different incubation time.

3.2.2 Cellular uptake and pathway The endocytosis of the PCL-HPG-PEI600/DOX complexes was explored by the flow cytometry depending on the autofluorescence of DOX. As shown in Fig. 3C and Fig. S3 (Supporting Information), the red fluorescence intensity recorded increased significantly with the incubation time, and there were no obvious differences in fluorescence intensity between PCL-HPG-PEI600/DOX and free DOX. This result indicated that the PCL-HPG-PEI600/DOX complexes could be uptaken efficiently by MCF-7 cells and the cellular uptake of complexes was time-dependent. For nanocarriers, endocytosis is a prerequisite for entering into cells, and the endocytosis occurs mechanisms usually including phagocytosis, macropinocytosis, clathrin-dependent endocytosis and clathrin-independent endocytosis

30

. To explore the pathway of the

PCL-HPG-PEI600/DOX complexes entering the MCF-7 cells, several specific pharmacological ACS Paragon Plus Environment


Page 16 of 48

inhibitors, such as chlorpromazine (inhibitor to clathrin-dependent endocytosis), genistein (inhibitor to caveolin-mediated endocytosis), wortmannin (specific inhibitors of macropinocytosis) and cytochalasin B (specific inhibitors of phagocytosis) were added to block some endocytosis pathways

29

. The results of endocytic pathway were shown in Fig. 3D and Fig. S4 (Supporting

Information), similar to other reports, the low temperature (4°C) inhibited the cellular uptake significantly compared with normal 37°C, indicating the endocytosis process was energy dependent. Besides, the uptake of PCL-HPG-PEI600/DOX by MCF-7 cells was also inhibited obviously by chlorpromazine with a 20% reduction in fluorescence intensity compared to control group, suggesting that the clathrin-dependent endocytosis was the main pathway of endocytosis for PCL-HPG-PEI600/DOX complexes into MCF-7 cells.

3.3 pMMP-9 delivery 3.3.1 PCL-HPG-PEI600/pMMP-9 complexes formation For pMMP-9 delivery, the gel electrophoresis assay was performed to test the gene binding ability

of

PCL-HPG-PEI600.

PCL-HPG-PEI600/pMMP-9

The

weight

pMMP-9

ratios

(lower

migration

was

observed

than

Fig.

4A).

10,

at

low

Beyond

that,

PCL-HPG-PEI600 could entirely delay the mobility of pMMP-9, which illustrated the effective electrostatic complexation of PCL-HPG-PEI600 to pMMP-9. The potentials and sizes of the PCL-HPG-PEI600/pMMP-9 complexes were shown in Fig. 4B. It was found that the complexes displayed positive charges and stable particle sizes after the ratio was higher than 20. Moreover, their particle sizes were around 300 nm, which was suitable for cellular internalization

37

. The

morphology of PCL-HPG-PEI600/MMP-9 complex (w/w = 60) was visualized by TEM and the complex showed a spherical morphology with a mean size of about 80 nm (Fig. 4C). These results showed that PCL-HPG-PEI600 could form the compact complexes with pMMP-9, and their positive charges and particle sizes were conducive to the efficient cellular endocytosis. The pH-sensitivity of PCL-HPG-PEI600/pMMP-9 complex was explored by the gel electrophoresis assay and the result was shown in Fig. 4D. For this performance, the negative charged heparin was added, which were able to compete with pMMP-9 to bind the positive charged PCL-HPG-PEI600. As shown, the PCL-HPG-PEI600/pMMP-9 complex remained stable in the heparin-containing solution at a pH value of 7.4 when the weight ratio of heparin/pMMP-9 was 1, ACS Paragon Plus Environment

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and no pMMP-9 migration was observed. In contrast, the PCL-HPG-PEI600/pMMP-9 complex at a pH value of 5.5 showed an obvious pMMP-9 migration, suggesting pMMP-9 was released from the complexes. This result was attributed from the disassembly of β-CD/BM inclusion and the resultant low molecular weight PEI could not bind pMMP-9 entirely. Such a pH-sensitivity may help the PCL-HPG-PEI600/pMMP-9 complexes escape from the endosome and then release pMMP-9 readily in cytoplasm, which was much important for the high-efficiency transfection.

3.3.2 In vitro transfection To study the gene transfection efficiency of the supramolecular construction strategy, PEIs with different molecular weight (PEI600, PEI1.8k and PEI25k) was conjugated with β-CD and then interacted with PCL-HPG-BM, the obtained all supramolecular inclusions of PCL-HPG-PEI showed significantly higher transfection efficiency than PEI25k. In particular, there was no obvious difference among them with different molecular weight of PEIs in transfection efficiency, while the PCL-HPG-PEI600 showed the lowest cytotoxicity and hemolysis effect (shown in Fig. S5, Supporting Information). The optimal transfection quality ratio of different PCL-HPG-PEIs is different. The optimum transfection quality ratio of PCL-HPG-PEI25k、PCL-HPG-PEI1800 and PCL-HPG-PEI600 is 3:1, 50:1 and 120:1, respectively. This result indicated that the supramolecular construction strategy could realize the purpose of obtaining an ideal gene carrier with the high efficiency and low cytotoxicity using the low-molecular-weight PEI600. The in vitro transfection efficiency of PCL-HPG-PEI600/pMMP-9 in the absence of serum was shown in Fig. 5A. PEI25k displayed good pMMP-9 delivery ability and about 32% cells were transfected under its best transfection condition (PEI25k/pMMP-9 = 1.3)

34

. For PEI600 and

β-CD-PEI600, both of them showed the poor transfection efficiency due to their low molecular weights and were useless in gene delivery applications. After interacted with PCL-HPG-BM, the PCL-HPG-PEI600 showed good pMMP-9 delivery ability and its gene transfection efficiency with increasing PCL-HPG-PEI600/pMMP-9 weight ratio increased (ranging from 50 to 150). Particularly, at PCL-HPG-PEI600/pMMP-9 weight ratio of 150, about 35% MCF-7 cells were transfected, significantly higher than PEI25k. It has been reported that the serum components would interact with the polycations and greatly affect the stability of the gene-loaded complexes, and then resulted in a significant decrease of gene ACS Paragon Plus Environment


transfection efficiency

38-39

Page 18 of 48

. So, the serum-containing transfection assay has explored for the real

gene delivery ability. As shown in Fig. 5B, compared to the absence of serum condition, for PEI25k, only 17% MCF-7 cells were transfected, which decreased about 52% in transfection efficiency. However, PCL-HPG-PEI600 showed the excellent serum tolerance and much better gene delivery result in the presence of serum. Moreover, the transfection efficiency increased with the increase of PCL-HPG-PEI600/pMMP-9 weight ratios. Particularly, PCL-HPG-PEI600/pMMP-9 (w/w = 120:1) displayed higher than 50% transfection efficiency while the cell viability remained higher than 80%, much better than those of PEI25k with the transfection efficiency of 17% and cell viability of only 38%. The excellent gene delivery ability of PCL-HPG-PEI600 in the blood may be ascribed to its HPG segment, low molecular weight of PEI and the pH-sensitivity. HPGs are the well-known blood compatible polymers and used widely as the blood substitutes in clinical. Their abundant hydroxyl groups outside the PCL-HPG-PEI600/pMMP-9 complexes played the “hydroxylation effects” that effectively inhibit the adsorption by macromolecular biospecies and then resulted in the good stability in the presence of serum 40. The low molecular weight of PEI600 displayed lower charge density than PEI25k, which endowed it a better blood compatibility and lower cytotoxicity. After constructing through this supramolecular strategy, PEI600 resulted in the better gene delivery ability than PEI25k

41

. The pH-sensitivity of β-CD/BM inclusion may help the complexes escape

from endosome and then disassociated to release pMMP-9 in cytoplasm, overcoming the biggest barrier (how to escape from endosome) in gene transfection process for the non-viral carriers 42. Western blot assay was carried out to quantificationally determine the MMP-9 protein expression, which could further verify the transfection effect of PCL-HPG-PEI600/pMMP-9 into MCF-7 cells. MMP-9 protein is a type of matrix metalloproteinase that is highly expressed in tumor cells and plays an important role in tumor growth and migration

43

. In this work, MMP-9 siRNA

was used to silence the MMP-9 protein expression and β-actin was set as the internal control. The blank PCL-HPG-PEI600 did not affect the expression of MMP-9 protein in cells compared to the PBS control, while the pMMP-9-containing groups (both PCL-HPG-PEI600/pMMP-9 and PEI25k/pMMP-9) induced the obvious reduction of MMP-9 protein expression (Fig. 5C). Specifically, PCL-HPG-PEI600/pMMP-9 showed the more significant regulation effect than PEI25k/pMMP-9, and more than 75% of the MMP-9 protein expression quantity was inhibited. The result was consistent with the results of transfection assay and showed that PCL-HPG-PEI600 ACS Paragon Plus Environment

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displayed much better pMMP-9 delivery ability than PEI25k. Moreover, the stability assay further indicates

that

the


complex

has

better

stability

than

the

PEI25k/pMMP-9 complexes in the presence of serum (Fig. S6, Supporting Information).

3.3.3 Cellular uptake and endosome escape The cellular uptake of the PCL-HPG-PEI600/gene complexes was explored by the flow cytometry using the FAM-labeled siRNA. The fluorescence intensity of PCL-HPG-PEI600/siRNA increased obviously with the incubation time and was significantly higher than that of PEI25k/siRNA, suggesting the PCL-HPG-PEI600/siRNA displayed the excellent blood stability and could be uptaken efficiently by MCF-7 cells (Fig. 6A and Fig. S7 , Supporting Information). Its endocytosis pathway exploration was performed and the results were shown in Fig. 6B and Fig. S8 (Supporting Information). As the same to PCL-HPG-PEI600/DOX complexes, the cellular uptake of PCL-HPG-PEI600/siRNA complexes were also affected by the temperature and chlorpromazine, meaning that the clathrin-dependent endocytosis was also the main pathway of endocytosis for PCL-HPG-PEI600/siRNA complexes into MCF-7 cells. For non-vital carriers, the biggest challenge to improve the transfection efficiency may be how to help the gene-loaded complexes escape from the endosome efficiently (avoid gene to be degraded in lysosome) and then release gene in cytoplasm. Then, a pH-sensitive PCL-HPG-PEI600 containing low-molecular-weight PEI was constructed in this work. PCL-HPG-PEI600 complexes may consume the protons in endosome due to its pH-sensitivity and then disassemble. The consumption of the protons could make the endosome rupture and then release the gene-loaded complexes

due

to

the

osmotic

pressure

change

of

endosome.

The

disassemble

low-molecular-weight PEI could not bind gene entirely and then release free gene in cytoplasm. So, the pH-sensitive PCL-HPG-PEI600/gene was expected to escape from endosome successfully to improve the transfection efficiency, A time-dependent intracellular localization was monitored by CLSM

using

Cy3-labeled

siRNA to

assess

the

endosomal

escape

ability

of

the

PCL-HPG-PEI600/gene complexes. The red fluorescence (siRNA) was weak within the first 2 h (Fig. 7), denoting only a small amount of PCL-HPG-PEI600/siRNA were trapped in lysosome (green). After 4 h incubation, it was found that the green and red fluorescence were overlapped well and the large area of yellow fluorescence was observed, indicating that the majority of ACS Paragon Plus Environment


PCL-HPG-PEI600/siRNA complexes were trapped in endosome. Particularly, after 8 h incubation, many red fluorescence dots were observed and the isolated distribution of the green and red fluorescence indicated that most complexes escaped from the endosome. These results demonstrated that PCL-HPG-PEI600 could deliver gene into cells and help gene escape from the endosome effectively, which played an important role in the high-efficiency transfection. 3.3.4 In vivo transfection Furthermore, the in vivo pMMP-9 transfection assay was carried out by injecting PCL-HPG-PEI600/pMMP-9 complexes into the mice bearing the MCF-7 tumor, and MMP-9 protein expression in tumors was analyzed by western-blot assay. Accordance with the result of in vitro transfection, the blank PCL-HPG-PEI600 mediated little in MMP-9 protein expression of tumor, which was similar to PBS control (Fig. 8A). And pMMP-9-loaded complexes induced the decrease of MMP-9 protein expression significantly. Particularly, the tumor treated with PCL-HPG-PEI600/pMMP-9 showed much lower MMP-9 protein expression than that treated with PEI25k/pMMP-9, and the difference in reducing protein expression was more obvious than in vitro assay. This result indicated that PCL-HPG-PEI600 displayed much better pMMP-9 delivery in vivo than PEI25k, which was attributed from the excellent blood biocompatibility of PCL-HPG-PEI600. The accumulation and distribution of the gene-loaded complexes in tumor tissue was studied by ex vivo imaging using siRNA-Cy5.5 instead of pMMP-9. The tumors treated with both PCL-HPG-PEI600/siRNA and PEI25k/siRNA showed the obvious fluorescence signal, indicating siRNA was delivered into tumors (Fig. 8B). The much higher fluorescence signal was observed in PCL-HPG-PEI600/siRNA-treated tumor, which suggested that PCL-HPG-PEI600 delivered much more siRNA into tumor than PEI25k.

3.4 Co-delivery of DOX and pMMP-9 3.4.1 In vitro assays To investigate the effect of PCL-HPG-PEI600 co-deliver DOX and pMMP-9, MCF-7 cells were incubated with the co-delivery system (PCL-HPG-PEI600/DOX/siRNA) and then observed using the CLSM. The cell nucleus showed the blue fluorescence, and DOX showed its characterized red fluorescence. A FAM-labeled siRNA was used to replace pMMP-9 and showed the green fluorescence. Free DOX could enter into cells and its red fluorescence was found around ACS Paragon Plus Environment

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nucleus(Fig. 9). Nevertheless, free siRNA was hard to enter into MCF-7 cells with none of green fluorescence observed. After delivered by PCL-HPG-PEI600, the dramatically green fluorescence appeared intracellularly indicating PCL-HPG-PEI600 could deliver siRNA into MCF-7 cells. For the co-delivery system of PCL-HPG-PEI600/DOX/siRNA, the dramatically red and green fluorescence were observed simultaneously, and both fluorescence profiles overlapped with the blue fluorescence of cell nucleus. The above results suggested that the PCL-HPG-PEI600 could efficiently co-deliver drug and gene into the cells. The endocytosis pathway of the co-delivery system was also explored and found that the clathrin-dependent endocytosis was still the main endocytosis pathway for PCL-HPG-PEI600/DOX/pMMP-9 complexes into MCF-7 cells (Fig. S9, Supporting Information). The inhibition effect of the co-delivery system on MCF-7 cells was evaluated through CCK-8 assay. The co-delivery formulation showed significantly better cell proliferation inhibition effect than that of only DOX or pMMP-9 used alone (Fig. 10). Moreover, the 50% inhibitive concentration (IC50) of DOX was also calculated. It was found that the IC50 of DOX in PCL-HPG-PEI600/DOX was 2.61 µg/mL, while the IC50 decreased to 1.95 µg/mL when using the co-delivery system of PCL-HPG-PEI600/DOX/pMMP-9, the dose of used DOX could reduce 25.2%. This result suggested that the co-delivery may be a promising strategy to reduce the dose of drugs, reduce side effects and improve the treatment effect. The inhibition effect on MCF-7 cells migration of PCL-HPG-PEI600/DOX/pMMP-9 was performed by transwell and wound healing assays. As shown in Fig. 11A and B, the blank PCL-HPG-PEI600 showed no significant difference with PBS control in cell invasion ratios, while both DOX and pMMP-9 could reduce MCF-7 cells invasion ratios significantly and there were only 19.2% and 39.8% invasive cells for DOX and pMMP-9 respectively. Particularly, for the co-delivery system, only 14.6% MCF-7 cells invaded through transwell, which was much lower than that of DOX or MMP-9 used only. The inhibition effect on MCF-7 cells migration of the different formulations (PCL-HPG-PEI600/pMMP-9 and PEI25k/pMMP-9) was performed by transwell

assays

(Fig.

S10,

Supporting

Information).

The

results

confirmed

PCL-HPG-PEI600/pMMP-9 complexes was more effective in decreasing the MCF-7 cell migration than that of PEI25k/pMMP-9 complexes. For wound healing assay as shown in Fig. 11C and D, the complete wound closure was observed for the blank PCL-HPG-PEI600 after 72 h of incubation. ACS Paragon Plus Environment


Page 22 of 48

However, the cells treated with either DOX or pMMP-9 remained the large wound areas uncovered. For the co-delivery system, the largest uncovered wound area was observed, indicating that PCL-HPG-PEI600/DOX/pMMP-9 could inhibit MCF-7 cells migration effectively which would help to improve the antitumor efficacy. Moreover, the co-delivery strategy was more effective in decreasing the MCF-7 cell migration than that of only DOX or pMMP-9 treatment. The effect of the PCL-HPG-PEI600/DOX/pMMP-9 on the cell cycle progression by analyzing DNA content was carried out by the flow cytometry. As reflected by the Sub-G1 cell populations shown in Fig. 11E and F, no obvious differences were observed in cells treated between the blank PCL-HPG-PEI600 and the PBS control. Nevertheless, the cells treated with either DOX or pMMP-9 showed a significantly larger cell population at Sub-G1 with 12.6% and 16.6% respectively, much higher than 4.83% of PBS control, indicating that both DOX and pMMP-9 could inhibit

the

intracellular

DNA

synthesis

and

induce

cells

apoptosis.

For

the

PCL-HPG-PEI600/DOX/pMMP-9, it showed a much larger cell population at Sub-G1 of 23.3%, suggesting the co-delivery strategy was more effective in inhibiting MCF-7 cell than that of only DOX or pMMP-9 used.

3.4.2 In vivo therapy To confirm the antitumor effect of PCL-HPG-PEI600/DOX/pMMP-9 complex in vivo, the anticancer treatments with a serious of formulations were carried out when the tumors were around 200 mm3. As shown in Fig. 12A, the tumor growth profiles during 15 days was recorded and it was found that the tumors treated with both PBS and blank PCL-HPG-PEI600 grew up rapidly and their volumes reached approximately 2500 mm3 within 15 days. On the contrary, all other formulations containing either DOX or pMMP-9 showed the obvious inhibitory effects on tumor growth. Particularly, the DOX loaded in PCL-HPG-PEI600 (PCL-HPG-PEI600/DOX) showed better tumor inhibition effect compared with free DOX after 15 days incubation, which implying the advantage and necessity of the PCL-HPG-PEI600 carriers. The PCL-HPG-PEI600 carrier could improve the stability and solubility of DOX in the blood because of the excellent blood compatibility of HPG segment, resulting in the longer half-life for DOX in the blood. For pMMP-9 therapeutic effect, the tumor treated with PCL-HPG-PEI600/pMMP-9 showed significantly smaller than that of PEI25k/pMMP-9, which was accordance with their in vitro transfection results and indicated that ACS Paragon Plus Environment

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PCL-HPG-PEI600

could

deliver

pMMP-9

efficiently

in

vivo.

Most

especially,

the

PCL-HPG-PEI600/DOX/pMMP-9 displayed the most distinct suppressive effect on MCF-7 tumor among all formulations, and the tumor volume was only 21% of that PBS control after 15 days incubation. The tumor image and tumor weight data of 15 days later were shown in Fig. 12B and C, these results further showed the effective in vivo anticancer effect of DOX and pMMP-9 on MCF-7 tumor, and the co-delivery system of PCL-HPG-PEI600/DOX/pMMP-9 displayed much better therapeutic effect among them. The change of mice body weights during in vivo assay was recorded and considered as a key factor for reflecting the safety and side effects of the used individual formulations. As shown in Fig. 12D, all groups except of the free DOX group showed the gain in weight of mice. Only free DOX resulted in the obvious decrease in body weight of mice. This result showed that free DOX induced an obvious side effect when in vivo tumor therapy while PCL-HPG-PEI600/DOX formulation was safe to mice, suggesting the necessity and safety of the PCL-HPG-PEI600 carrier.

3.4.3 Histologic and immunohistochemical analysis The antitumor efficacy of the co-delivery PCL-HPG-PEI600/DOX/pMMP-9 was further evaluated by the histopathological analysis of H&E-stained MCF-7 tumor sections. As shown in Fig. 13, the results are highly supportive of the tumor inhibition data. The tumor cells treated with the PBS and blank PCL-HPG-PEI600 displayed intact structures with more chromatin, meaning that the tumor was in rapid growth. On the contrary, the other groups treated with DOX or pMMP-9 displayed less tumor cells with the wider intracellular spaces at different degrees, suggesting these groups generated the effective treatment response in tumors. Particularly, it is noteworthy that the tumor treated with PCL-HPG-PEI600/DOX/pMMP-9 displayed the widest intracellular spaces and the

fewest

tumor

cells

among

all

the

tested

groups,

indicating

that

the

PCL-HPG-PEI600/DOX/pMMP-9 performed the best therapeutic effect. TUNEL assay was also performed to observe the apoptosis of the tumor sections and the dark brown color represents the apoptotic cells, accompanying with chromatin condensed, nuclear membrane cracked and some other signals

44-45

. Compared with the PBS and blank

PCL-HPG-PEI600, the groups containing either DOX or pMMP-9 exhibited the distinct degrees of MCF-7 tumor cell apoptosis, and the co-delivery system showed the most significant effect. ACS Paragon Plus Environment


Page 24 of 48

The significantly higher antitumor effect of PCL-HPG-PEI600/DOX/pMMP-9 was further supported by immunohistochemical staining of CD31 assay. CD31 immunohistochemical staining was used for staining microvessel to evaluate the suppression of angiogenesis. The light yellow represents the positive expression of the CD31 in normal cells. The immunoreactive microvessel treated with either DOX and pMMP-9 groups clearly decreased or disrupted compared with those treated with PBS or blank PCL-HPG-PEI600 (Fig. 13). As the same to other antitumor results, PCL-HPG-PEI600/DOX/pMMP-9 complexes exerted the most significant angiogenesis inhibition in tumors. Additionally, the expression of Ki67 was detected to further evaluate the antitumor efficacy of the various groups. Ki67 is a marker of cell proliferation, and the pale brown and blue colors represented the positive and negative expression of Ki67 protein respectively. Consistent with the in vivo antitumor efficacy results, the treatment by PCL-HPG-PEI600/DOX/pMMP-9 was the most effective in reducing tumor cell proliferation, which resulted in the lowest Ki67 expression in MCF-7

cells.

All

above

results

demonstrated

that

the

co-delivery

of

PCL-HPG-PEI600/DOX/pMMP-9 complexes displayed the superior antitumor efficacy in vivo compared to the other formulations. Furthermore, the major organs were excised from the BALB/C nude mice for further histological analyses to assess the safety of the formulations. The representative photographs of H&E-stained samples were shown in Fig. 14, the organs treated with most formulations do not show any visible damage compared with the PBS control, indicating that most formulations had the good biological safety. Only free DOX induced the obvious pathological changes in the lung tissue, and the alveolar edema and lung hemorrhage were observed. This result confirmed again the safety and necessity of the PCL-HPG-PEI600 carrier.

3.5 Biocompatibility The biocompatibility of PCL-HPG-PEI600 was explored to confirm the safety and practicability of the co-delivery system. CCK-8 assay was firstly performed to evaluate the cytotoxicity of the PCL-HPG-PEI600 carrier to MCF-7 cells. As shown in Fig. 15A, as expected, PCL-HPG-PEI600 showed little toxicity to the MCF-7 cells; even its concentration was 500 µg/mL, the cell viability was still more than 80%. However, the PEI25k displayed the obvious cytotoxicity ACS Paragon Plus Environment

Page 25 of 48 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


and just 20% cells were viable after treated with 50 µg/mL PEI25k. The low cytotoxicity of PCL-HPG-PEI600 may be originated from the inherent good biocompatibility of HPG and PCL as 46-47

well as the low molecular weight of PEI600

. Additionally, the cytotoxicity of

PCL-HPG-PEI600 and the degradation products (PCL-HPG-BM and β-CD-PEI600) to 3T3 cells also has been studied (Fig. S11, Supporting Information), PCL-HPG-PEI600, PCL-HPG-BM and β-CD-PEI600 showed no significant toxicity to 3T3 cells at concentrations below 500 µg/mL. Additionally, the blood compatibility of PCL-HPG-PEI600 was also conducted by hemolysis assay and morphological changes of RBCs. Shown from in Fig. 15B, 1 mg/mL of PCL-HPG-PEI600 did not cause any hemolysis effect (the hemolytic ratio was lower than 5%) 48. On the contrary, the serious hemolysis was found when PEI25k was used at a concentration of only 0.5 mg/mL, and the hemolysis ratio was more than 40%. Moreover, the effect of PCL-HPG-PEI600 on RBCs was also further evaluated. As shown in Fig. 15C, the excellent blood biocompatibility of PCL-HPG-PEI600 was further confirmed. The results showed the morphology of the RBCs has not changed after treated with PCL-HPG-PEI600 at the concentration range from 0.01 and 1 mg/mL. However, the RBCs showed the obviously change in morphology when treated with 0.01 mg/mL PEI25k. The reason for the excellent blood biocompatibility of PCL-HPG-PEI600 was that the presence of HPG segment and low molecular weight of PEI600. Moreover, the serum biochemical analysis was studied to further evaluate the safety of PCL-HPG-PEI600 after injecting aqueous PCL-HPG-PEI600 on mice. A series of parameters including ALT, AST, ALB and LDH for hepatic function, BUN and CRE for kidney function, and T-CHO, LDL as well as GLU are examined

49-50

. As shown in Fig. 16, all indicators of

PCL-HPG-PEI600 group were at normal levels, and there was no obvious difference in biochemical indicators between PCL-HPG-PEI600 and PBS control. However, for PEI25k, the biochemical indicators of ALB, LDH and T-CHO showed the obvious difference with PBS group, suggesting that PEI25k affected the hepatic function obviously. This result confirmed again the safety of PCL-HPG-PEI600. The above results indicated that the concentration of PCL-HPG-PEI600 in vitro and in vivo assays still falls into the safe concentration range, the maximum concentration studied is just 0.48 mg/mL.



Page 26 of 48

4. Conclusion In this work, a high-efficiency drug and gene co-delivery carrier (PCL-HPG-PEI600) was constructed from the supramolecular inclusion between β-CD-PEI600 and PCL-HPG-BM, and then DOX and pMMP-9 was loaded. The supramolecular PCL-HPG-PEI600 showed good DOX and pMMP-9 delivery ability as well as pH-sensitivity. For DOX delivery, PCL-HPG-PEI600/DOX displayed the pH-controlled DOX release and concentration-dependent inhibition effect on MCF-7 cells. For pMMP-9 delivery, PCL-HPG-PEI600 showed excellent pMMP-9 delivery ability with significantly higher transfection efficiency than PEI25k in vitro and in vivo, especially in the presence of serum. It was also found that PCL-HPG-PEI600 could help pMMP-9 escape from the endosome due to its pH-sensitivity. The cellular uptake pathway was also explored and found that the clathrin-dependent endocytosis was the main endocytosis pathway for PCL-HPG-PEI600 complexes into MCF-7 cells. For the co-delivery of PCL-HPG-PEI600/DOX/pMMP-9, it was found that the co-delivery strategy was much better than single DOX or pMMP-9 formulation used. PCL-HPG-PEI600/DOX/pMMP-9 resulted in the better inhibition effect on MCF-7 cells proliferation and migration in vitro as well as the suppression effect on MCF-7 tumors in vivo. Moreover, PCL-HPG-PEI600 displayed non-toxicity and excellent blood compatibility, suggesting a promising drug and gene co-delivery carrier in combined therapy to tumors.

Supporting Information Characterization of PCL-HPG-BM and β-CD-PEI600, cellular uptake analyses of PCL-HPG-PEI600/DOX, transfection efficiency and biocompatibility of PCL-HPG-PEIs, the stability

of

the


PCL-HPG-PEI600/gene

complexes,

the

complexes,

cellular

endocytosis

uptake

pathway

analyses of

of the

PCL-HPG-PEI600/DOX/pMMP-9 complexes, the inhibition effect on MCF-7 cells migration of PCL-HPG-PEI600/pMMP-9 and PEI25k/pMMP-9, the cytotoxicity of the blank PCL-HPG-PEI600 , PCL-HPG-BM and β-CD-PEI600 in 3T3 cells.

Acknowledgements This work is financially supported by the National Natural Science Foundation of China


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(51573071,

31870943),

Guangzhou

Science

and

Technology

Program

(201607010127,

2017B030314174), the Chinese Fundamental Research Funds for the Central Universities as well as Guangdong Academy of Sciences Program (2017GDASCX-0103).

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TOC



Page 32 of 48

Scheme 1. Schematic showing the synthesis routes to PCL-HPG-BM (A) and β-CD-PEI600 (B). (C) Schematic illustration of a supramolecular inclusion of PCL-HPG-PEI600.


Page 33 of 48

-CD-PEI600

Emissiom intensity

8000 6000

Adding -CD-PEI600

(mg/mL) 0 0.25 0.5 2.5 5 10 25

4000 2000

-CH-,-CH2- of HPG H of  -CD -(NHCH2CH2)nof PEI600 4-arms-PCL

H1 of  -CD

0 450

475

500

525

Wavelength (nm)

8

7

6

5

4

3

2

1

0

Chemical Shift (ppm) -20 Raw heat rate (μJ/s)

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


-60

-100

-140 1500

2500

3500

4500

Time (sec)

Fig. 1. (A) Fluorescence emission spectra of PCL-HPG-BM/β-CD-PEI600 mixture (the concentration of PCL-HPG-BM was fixed at 0.5 mg/mL). (B) 1H NMR spectrum of PCL-HPG-PEI600 (D2O, 25°C). (C) Typical ITC heat flow trace of the titration of β-CD-PEI600 into PCL-HPG-BM at 25°C.



8000

Emissiom Intensity

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 34 of 48

PCL-HPG-BM  -CD-PEI600

PCL-HPG-PEI600 (pH 7.4) PCL-HPG-PEI600 (pH 5.5)

pH=7.4 pH=6.8 pH=6.5 pH=6.0 pH=5.5

6000 4000 2000 0

2

4

6

8

10

12

14

450

475

500

525

Wavelength (nm)

Elution time (min)

Fig. 2. (A) The GPC chromatogram of PCL-HPG-BM, β-CD-PEI600 and PCL-HPG-PEI600 (pH of 7.4 and 5.5). (B) Fluorescence emission spectra of PCL-HPG-PEI600 at different pH values.


Page 35 of 48

120

pH=7.4 pH=5.5

80

Cell viablity (%)

Cumulative Release (%)

100

60 40 20 0 0

12

24

36

48

60

24 h 48 h 72 h

100 80 60 40 20 0

72

0.01 0.1 0.5 1 2 5 10 Concentration of DOX (g/mL)

Time (h)

0.5

4

0.4 4

1.0

DOX PCL-HPG-PEI600/DOX

MFI ( 10 )

1.2

MFI ( 10 )

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


0.8 0.6 0.4

0.3 0.2 0.1

0.2 0.0

0.0 Control 0.5 2 4 Incubation time (h)

8

e B in ol ol 4°C ein in zin nn ntr ntr ist las ma co co ma en a t o e e r r h G p iv tiv Wo toc sit lor ga Cy Po Ch Ne

Fig. 3. (A) In vitro DOX release profiles of PCL-HPG-PEI600/DOX complexes at a pH of 7.4 and 5.5. (B) The cytotoxicity of PCL-HPG-PEI600/DOX to MCF-7 cells. (C) Intracellular mean fluorescence intensity (MFI) of DOX in MCF-7 cells incubated with free DOX or PCL-HPG-PEI600/DOX for different time. The equivalent dose of DOX was 5 μg/mL. (D) The effect of various inhibitors on the endocytosis of PCL-HPG-PEI600/DOX in MCF-7 cells.



Particle size (nm)

700

20 Particle size

600

Zeta potential

10

500

0

400 -10

300

-20

200 100

Zeta potential (mV)

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 48

-30 0

20

40 60 Weight ratio

80

Fig. 4. (A) Agarose gel electrophoresis retardation assay of PCL-HPG-PEI600/pMMP-9 complexes at various weight ratios. (B) The particle sizes and zeta potentials of PCL-HPG-PEI600/pMMP-9 complexes formed at various weight ratios. (C) Typical TEM image of the PCL-HPG-PEI600/pMMP-9 complex (w/w = 60). (D) Agarose gel electrophoresis of PCL-HPG-PEI600/MMP-9 complexes formed at a weight ratio of 20 in the presence of heparin incubation at pH 7.4 and 5.5 (the weight ratios of heparin/pMMP-9 was 1, 2, 4, 6, 8 and 10).


Page 37 of 48

120

PEI25k PEI600 -CD-PEI600 PCL-HPG-PEI600

40 30 20 10 0

1.3

50

80

Transfection efficiency

Cell viability

100

Percentage (%)

Transfected cells (%)

50

100

120

150

80 60 40 20 0

PEI25k

60

80

100

120

PCL-HPG-PEI600/pMMP-9 (w/w)

Polymer/DNA (w/w)

Relative protein expression

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


1.0 0.8 0.6 0.4 0.2 0.0 1

2

3

4

Fig. 5. (A) Transfection efficiency of pMMP-9 mediated by PEI600, β-CD-PEI600 and PCL-HPG-PEI600 at various weight ratios in the absence of serum. (B) Transfection efficiency and the relative cytotoxicity mediated by PCL-HPG-PEI600/pMMP-9 complexes at various weight ratios in the presence of serum (Explained specially: pEGFP as a model plasmid in the cytotoxicity experiment). (C) Western blot analysis and the relative MMP-9 protein expression in MCF-7 cells treated with different formulations (1: PBS control, 2: blank PCL-HPG-PEI600, 3: PEI25k/pMMP-9, 4: PCL-HPG-PEI600/pMMP-9).



4 PEI25k/siRNA PCL-HPG-PEI600/siRNA

1.2

MFI ( 10 )

3

1.5

4

4

MFI ( 10 )

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 38 of 48

2 1

0.9 0.6 0.3

0

0.0 ol NA ntr siR o C e e Fr

0.5

2

4

Incubation time (h)

8

l l B ein ine nin tro tro 4°C sin az ist an on on en ala om ec ec rtm r h G v v o c p i i t W to sit lor ga Cy Po Ch Ne

Fig. 6. (A) Mean fluorescence intensity (MFI) of siRNA-FAM in MCF-7 cells incubated with PEI25k/siRNA and PCL-HPG-PEI600/siRNA for different time. (B) The effect of various inhibitors on the endocytosis of PCL-HPG-PEI600/siRNA in MCF-7 cells.


Page 39 of 48 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. 7. The endosomal escape observation of the PCL-HPG-PEI600/siRNA-Cy3 complexes in MCF-7 cells after 2, 4 and 8 h incubation (600×). Endosomes were stained with Lysotracker (green); siRNA was labeled with Cy3 (red); and the nuclei were stained with Hoechst33342 (blue).



1 Relative protein expression

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 40 of 48

2

1.0 0.8

3

0.6 0.4

4

0.2 0.0 1

2

3

4

Fig. 8. (A) Western blot analysis and the relative expression of MMP-9 protein in MCF-7 tumors after the last treatment with different formulations (1: PBS control, 2: blank PCL-HPG-PEI600, 3: PEI25k/pMMP-9, 4: PCL-HPG-PEI600/pMMP-9). (B) Ex vivo fluorescence imaging of the MCF-7 tumor-bearing nude mice at 8 h after intravenous injection of different formulations (1: PBS control, 2: siRNA-Cy5, 3: PEI25k/siRNA-Cy5, 4: PCL-HPG-PEI600/siRNA-Cy5).


Page 41 of 48 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. CLSM observation of the MCF-7 cells after 6 h treatment with different formulations (400×). The nuclei were stained with Hoechst33342 (blue), siRNA was labeled with FAM (green) and DOX showed red.



150

Blank PCL-HPG-PEI600 PCL-HPG-PEI600/pMMP-9 PCL-HPG-PEI600/DOX PCL-HPG-PEI600/DOX/pMMP-9

120

Cell Viability (%)

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 42 of 48

Free pMMP-9 Free DOX

90 60 30 0

1

2

3

4

5

6

Fig. 10. The cytotoxicity to MCF-7 cells of blank PCL-HPG-PEI600, free pMMP-9, free DOX, PCL-HPG-PEI600/pMMP-9, PCL-HPG-PEI600/DOX and PCL-HPG-PEI600/DOX/pMMP-9 (1: 0.1 μg/well pMMP-9 or/and 0.6 μg/mL DOX, 2: 0.2 μg/well pMMP-9 or/and 1.2 μg/mL DOX, 3: 0.4 μg/well pMMP-9 or/and 2.4 μg/mL DOX, 4: 0.6 μg/well pMMP-9 or/and 3.6 μg/mL DOX, 5: 0.8 μg/well pMMP-9 or/and 4.8 μg/mL DOX, 6: 1 μg/well pMMP-9 or/and 6 μg/mL DOX).


Page 43 of 48

120

Invasive cells (% of control)

90 60 30 0 1

2

Migration rate (% of control)

120

1

1

3

2

4

3

4

90 60 30 0

24

48 Time (h)

72

2

25 Sub G1=12.6

3 Sub G1=16.6

4 Sub G1=23.3

Sub G1 (%)

Sub G1=5.1

Cell Number

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


20 15 10 5 0 1

2

3

4

DNA Content

Fig. 11. Inhibition and the relative quantitative results of cell invasion/migration after treatment with different formulations in MCF-7 cells. (A and B) Transwell assay (200×); (C and D) wound healing assay (100×); (E and F) flow cytometry analysis of Sub G1 of cell cycle. (1: blank PCL-HPG-PEI600; 2: PCL-HPG-PEI600/DOX; 3: PCL-HPG-PEI600/pMMP-9; 4: PCL-HPG-PEI600/DOX/pMMP-9).



3000 3

Tumor volume (mm )

1 2 3 4 5 6 7

2500 2000 1500

* * *

* *

1000 500 0 0

2

4

6

8

10 12 14 16 18

Time (day) 24 3.0

21

Body weight (g)

Tumor weight (g)

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 44 of 48

2.5 2.0 1.5 1.0 0.5

18 15

1 2 3 4 5 6 7

12 9 6

0.0 1

2

3

4

5

6

7

0

2

4

6

8

10

12

14

16

Time (day)

Fig. 12. (A) In vivo tumor growth curves of MCF-7 tumor-bearing mice treated with different formulations. (B) Representative image of MCF-7 tumors at the 15st day. (C) The tumor weights excised from different groups after 15 days treatment. (D) Body weight changes of mice treated with different formulations during the treatment (1: PBS control; 2: blank PCL-HPG-PEI600; 3: free DOX; 4: PEI25k/pMMP-9; 5: PCL-HPG-PEI600/DOX; 6: PCL-HPG-PEI600/pMMP-9; 7: PCL-HPG-PEI600/DOX/pMMP-9).


Page 45 of 48 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. 13. Immunohistochemical analyses of H&E, TUNEL, CD31 and Ki67 for MCF-7 tumor tissues after the last treatment with different formulations in vivo (200×). (1: PBS control; 2: blank PCL-HPG-PEI600; 3: free DOX; 4: PEI25k/pMMP-9; 5: PCL-HPG-PEI600/DOX; 6: PCL-HPG-PEI600/pMMP-9; 7: PCL-HPG-PEI600/DOX/pMMP-9).



Page 46 of 48

Fig. 14. Histologic assessments of major organs in mice (100×) treated with different formulations. (1: PBS control; 2: blank PCL-HPG-PEI600; 3: free DOX; 4: PEI25k/pMMP-9; 5: PCL-HPG-PEI600/DOX; 6: PCL-HPG-PEI600/pMMP-9; 7: PCL-HPG-PEI600/DOX/pMMP-9).


Page 47 of 48

Cell viability (%)

120

PCL-HPG-PEI600

PEI25k

100 80 60 40 20 0 5

10 50 100 200 500 Concentration (g/mL)

60

Hemolysis (%)

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


PCL-HPG-PEI600 PEI25k

40

20

0 0.01

0.1 0.5 1 Concentration (mg/mL)

Fig. 15. (A) CCK-8 results of PCL-HPG-PEI600 and PEI25k at different concentrations on MCF-7 cells. (B) Effect of PCL-HPG-PEI600 and PEI25k with different concentrations on the hemolysis. (C) Effect of PCL-HPG-PEI600 with different concentrations on the aggregation and morphology of RBCs.



40

120

AST (U/L)

ALT (U/L)

50

150

PBS PEI25k PCL-HPG-PEI600

30 20

50


ALB (g/L)

60

90 60

40


30

***

20 10

0 1200

0 40

0 120

LDH (U/L)

1000 800


**

600 400

30


20 10

CRE (mol/L)

30

BUN (mg/dl)

10

90


60 30

200

5 4 3 2


*

2.5

LDL (mmol/L)

6

0

2.0


1.5 1.0

0 50

GLU (mmol/L)

0

T-CHO (mmol/L)

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 48 of 48

40


30 20

1

0.5

10

0

0.0

0

Fig. 16. Blood biochemistry analysis of mice after intravenous injection of PCL-HPG-PEI600 (PBS and PEI25k were used as the control groups).


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