pH-responsive polydopamine nanoparticles for photothermally promoted gene delivery

Journal Pre-proof pH-responsive polydopamine nanoparticles for photothermally promoted gene delivery

Peng Zhang, Qinan Xu, Xinfang Li, Youxiang Wang PII:

S0928-4931(19)32135-6

DOI:

https://doi.org/10.1016/j.msec.2019.110396

Reference:

MSC 110396

To appear in:

Materials Science & Engineering C

Received date:

11 June 2019

Revised date:

4 November 2019

Accepted date:

4 November 2019

Please cite this article as: P. Zhang, Q. Xu, X. Li, et al., pH-responsive polydopamine nanoparticles for photothermally promoted gene delivery, Materials Science & Engineering C (2019), https://doi.org/10.1016/j.msec.2019.110396

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© 2019 Published by Elsevier.

Journal Pre-proof

pH-responsive polydopamine nanoparticles for photothermally promoted gene delivery Peng Zhang, Qinan Xu, Xinfang Li,Youxiang Wang*

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, 310027, P. R. China. Tel.:

-p

ro

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+8657187953729; E-mail: [email protected]

1 Abstract

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Recently, stimuli-responsive gene carriers have been widely studied to overcome

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the extra- and intracellular barriers in cancer treatment. In this study, we modified polydopamine nanoparticles with low-molecular weight polyethylenimine (PEI1.8k)

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and polyethylene glycol-phenylboronic acid (PEG-PBA) to prepare pH-responsive gene carrier PDANP-PEI-rPEG. PBA and polydopamine could form pH-responsive

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boronate ester bonds. Non-responsive PDANP-PEI-nPEG and non-PEGylated PDANP-PEI were also studied as control. Both PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes remained stable in the pH environment of blood circulation or extracellular delivery (pH 7.4) owing to the PEG modification. And after being internalized into endosomes, the boronate ester bonds could be cleaved. The pH responsive ability of PDANP-PEI-rPEG might facilitate complexes dissociation

and

gene

release

inside

cells.

The

transfection

level

of

PDANP-PEI-rPEG/DNA complexes was about 100 times higher than that of PDANP-PEI-nPEG/DNA complexes with the same mass ratios. Moreover, after NIR light irradiation at the power density of 2.6 W/cm2 for 20 minutes, the good photothermal conversion ability of PDANP resulted in quick endosomal escape. The transfection level of PDANP-PEI-rPEG/DNA complexes doubled, even higher than that of lipofectamine 2000/DNA complexes. This was also confirmed by Bafilomycin 1

Journal Pre-proof A1 inhibition test and CLSM observation. In response to the acidic pH within cancer cells and the NIR light irradiation, the PDANP-PEI-rPEG carrier could overcome multiple obstacles in gene delivery, which was promising for further application in gene therapy. Key Words: Gene delivery; Stimuli responsive; Polydopamine nanoparticles; Enhanced endosomal escape

2 Introduction

of

For gene therapy, non-viral gene vectors have attracted significant attention.

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Compared with viral gene vectors, non-viral gene vectors showed easy modification ability, low cytotoxicity and immunogenicity1-3. In the past few decades, many

-p

non-viral gene vectors have been developed for tumor treatment, such as polycations4,

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liposomes5，6 and polymeric micelles7. However, to realize effective gene delivery,

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those non-viral vectors must overcome several extra- and intracellular barriers: (1) they must remain stable in blood circulation with sufficient time; (2) they should be

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internalized by target cells efficiently; (3) to protect gene from being destroyed by hydrolases, the non-viral vectors need to escape from endosomes effectively after

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cellular uptake; (4) the complexes also require uncoupling to release gene after endosomal escape8-10. Hence, many approaches have been developed to conquer those obstacles.

Stimuli-responsive gene and drug carriers have been proven to be an outstanding candidate to achieve complexes dissociation inside target cells. Those carriers could dissociate in response to the intracellular environment, such as pH11-14, glutathione8, 15 and reactive oxygen species16, 17, resulting in effective gene transfection. For example, the pH-responsive gene vectors could remain stable in the surrounding normal tissues (pH~7.4), but release encapsulated payload inside tumor cells or endosomes (pH < 6.5)18-20. Boronate esters, as pH-responsive bonds formed by phenylboronic acid (PBA) and diol moieties, have been widely studied in drug delivery21, 22 or molecular sensing23, 24. Kim et al. developed a stimuli-responsive gene vector based on the 2

Journal Pre-proof PBA-galactose interaction25. The crosslinked gene complexes composed by PBA-modified polyethylenimine (PBA-PEI), PBA-modified polyethylene glycol (PEG-PBA) and galactose-modified polyethylenimine (Gal-PEI) could release gene effectively at the pH of 5 or at high ATP concentration. They showed over 10 times higher gene transfection level than that of non-crosslinked PBA-PEI or Gal-PEI in MCF-7 cells. To improve endosomal escape, many strategies have been developed, such as using photochemical internalization26, 27 (PCI) and cell-penetrating peptide modification28.

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In comparison to UV or visible light, near-infrared (NIR) light can penetrate into deep

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cells and tissues, thus being beneficial for solid tumor treatment29. Furthermore, NIR light was also investigated to promote endosomal escape. Kim et al. developed a

-p

graphene oxide-based nanocomposite for NIR light enhanced gene delivery30. They

re

modified reduced graphene oxide (rGO) with polyethylenimine, and then grafted

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polyethylene glycol on the polyethylenimine as gene carrier. Due to good photothermal conversion ability, rGO could produce local heat under NIR irradiation

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and provoke quick endosomal escape. After 20-minute NIR light irradiation, the transfection level of gene-loaded complexes tripled in PC-3 cells.

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Although those strategies could enable the gene carriers to overcome single barrier in delivery, the demand for gene vector with the ability to pass multiple obstacles still remains. In this work, we synthesized phenylboronic acid modified polyethylene glycol (PEG-PBA). Because the PBA could react with polydopamine to form boronate ester bond under alkaline environment, then we modified polydopamine nanoparticles (PDANP) with low-molecular weight polyethylenimine (PEI1.8k) and PEG-PBA to prepare pH-responsive nanoparticles PDANP-PEI-rPEG as gene carriers (Scheme 1). PEI segments could condense gene effectively via electrostatic forces and PEI was commonly used as gold standard due to its proton sponge effect. Those carriers were designed to remain stable in blood circulation and extracellular delivery as a result of the PEG modification. After internalized into endosomes (pH<6.5), the boronate ester bonds could be cleaved and the PEG chains might detach from the nanoparticles, which was facilitated the gene release. The excellent photothermal 3

Journal Pre-proof conversion ability of PDANP might promote quick endosomal escape for gene transfection under NIR irradiation. In the meanwhile, non-responsive nanoparticles PDANP-PEI-nPEG and non-PEGylated nanoparticles PDANP-PEI were also studied as reference in this work.

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Scheme 1.

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3 Materials & Methods 3.1 Materials

-p

Methoxypolyethylene glycol amine (PEG-NH2), 4-carboxyphenylboronic acid,

(EDC•HCl)

and

3-(4,

5-dimethyl-2-thiazolyl)-2,

lP

hydrochloride

re

N-hydroxysuccinimide (NHS), N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide

5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Aladdin Industrial

na

Corporation (Shanghai, China). Branched polyethylenimine (PEI1.8k) and dopamine hydrochloride were obtained from Sigma-Aldrich LLC (Shanghai, China).

Jo ur

Lipofectamine 2000 was purchased from Thermal Scientific (Shanghai, China). pGL-3 gene was obtained from Genscript (Nanjing, China). Cy3-DNA, bafilomycin A1 and hoechst 33342 were from Sangon Biotech (Shanghai, China). Luciferase reporter gene assay kit was obtained from Promega (Shanghai, China). BCA protein assay kit was purchased from KEYGEN (Nanjing, China). Lyso Tracker Green was from Invitrogen (USA). 3.2 Synthesis and characterization of PDANP-PEI-rPEG, PDANP-PEI-nPEG and PDANP-PEI 3.2.1 Synthesis of PEG-PBA PEG-PBA was synthesized using the reaction between the amino groups in methoxypolyethylene

glycol

amine

and

the

carboxyl

groups

in

4-carboxyphenylboronic acid. Carbodiimide hydrochloride (1.1590 g, 6.0 mmol), N-hydroxysuccinimide (0.6993 g, 6.0 mmol) and 4-carboxyphenylboronic acid 4

Journal Pre-proof (0.2020 g, 1.2 mmol) were dissolved in 15 mL methanol, after which this solution was stirred at room temperature for 1 h. Then methoxypolyethylene glycol amine (0.2954 g, 0.06 mmol) in 15 mL methanol was added into the above solution. After 24 h, the obtained product was purified through dialysis (Mw=3500) against water for 3 days and lyophilized for further study. The structure of PEG-PBA was characterized by 1H NMR (500 MHz, Varian Spectrometer, USA). 3.2.2 Preparation of PDANP Polydopamine nanoparticles (PDANP) were prepared according to previous report

of

with minor modification31. Briefly, dopamine hydrochloride (322.1 mg) was dissolved

ro

in 180 mL water and heated up to 50 °C while stirring. Then, NaOH (1.35 mL, 1 M) was added into the solution, followed by a 5-hour reaction. After adding NaOH, the

-p

color of the solution turned dark brown gradually. Those PDANP were collected by

re

ultrafiltration at 4500 rpm with ultrafiltration tubes (Millipore, 15 mL, 50k) and

lyophilized.

lP

washed with water for several times. For further characterization, PDANP were

na

3.2.3 Synthesis of PDANP-PEI-rPEG, PDANP-PEI-nPEG and PDANP-PEI For the preparation of pH-responsive nanoparticles PDANP-PEI-rPEG, PEG-PBA

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(20 mg) and PDANP (2.5 mg) were initially added into 8 mL Tris-HCl buffer (50 mM, pH=8.5) and reacted for 24 h. PEG was coated onto PDANP through the reversible covalent interaction between PBA and polydopamine. Then PEI1.8k (30 mg) was added and stirred for another 24 h, during which the amino groups in PEI1.8k reacted with polydopamine. The resulted nanoparticles were purified using dialysis (Mw=14000) against Hepes buffer (pH=7.4, 20 mM) for 3 days. The non-responsive nanoparticles PDANP-PEI-nPEG were synthesized in a similar method. Briefly, methoxypolyethylene glycol amine (20 mg) was reacted with PDANP (2.5 mg) in 8 mL Tris-HCl for 24 h, and then PEI1.8k (30 mg) was added. After another 24 h, the solution was dialyzed against Hepes buffer (pH=7.4, 20 mM) for 3 days. Non-PEGylated nanoparticles PDANP-PEI were also prepared similarly. PDANP (2.5 mg) and PEI1.8k (30 mg) reacted in 8 mL Tris-HCl for 24 h, following by a 3-day 5

Journal Pre-proof dialysis against Hepes buffer (pH=7.4, 20 mM). For further characterization, all those obtained nanoparticles were lyophilized. 3.2.4 Characterization of PDANP, PDANP-PEI-rPEG, PDANP-PEI-nPEG and PDANP-PEI Fourier-transform infrared spectroscopy (FT-IR, VECTOR22, BRUKER CO., Germany) was carried out to confirm the PEG and PEI modification. The sizes and zeta-potentials of nanoparticles were investigated using dynamic light scattering (DLS) (Malvern Inst. Ltd. UK). Those samples were tested in triplicate at 25 °C with the

of

scattering angle of 173°. The size and morphology of those nanoparticles were also

ro

observed by transmission electron microscopy (TEM, JEM-1200EX, NEC, Tokyo, Japan) with an accelerating voltage of 80 kV. To prepare TEM samples, 15 μL

-p

nanoparticles’ solution was dropped onto 200-mesh carbon coated copper grid and

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settled for 15 min, after which the residual solution was removed by filter paper. This

lP

process was carried out for another 2 times before observation. 3.3 Preparation and characterization of gene-loaded complexes

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3.3.1 Formulation of gene-loaded complexes For the preparation of gene-loaded complexes, PEI1.8k, PEI25k，PDANP-PEI-rPEG,

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PDANP-PEI-nPEG or PDANP-PEI in HEPEs buffer solution (pH 7.4, 20 mM) were mixed with equal volume of DNA solution using a 30-second vortex, after which the mixed solution was incubated for 30 min at room temperature. PEI1.8k/DNA and PEI25k/DNA complexes were formulated with the N/P ratio (molar ratio of nitrogen in PEI to phosphate in DNA) of 10. PDANP-PEI-rPEG/DNA, PDANP-PEI-nPEG/DNA and PDANP-PEI/DNA complexes were prepared at various mass ratios of PDANP to DNA. Lipofectamine 2000/DNA complexes were also prepared at the mass ratio of 2.5 according to the user guide as reference. 3.3.2 Characterization of gene-loaded complexes The size and zeta-potential of gene-loaded complexes were studied by DLS (Malvern

Inst.

Ltd.

UK).

The

morphology

6

of

PDANP-PEI-rPEG/DNA,

Journal Pre-proof PDANP-PEI-nPEG/DNA was further investigated by TEM (JEM-1200EX, NEC, Tokyo, Japan) at the mass ratio of PDANP to DNA of 1.5. For gel retardation experiment, pGL-3 gene was used. 5 μL of loading buffer and 25 μL of PDANP-PEI-rPEG/DNA or PDANP-PEI-nPEG/DNA (containing 300 ng gene) were mixed and added into 1% agarose gel. Then the gel was treated with 110 V electrophoresis for 50 min, after which 0.5 μg/mL ethidium bromide was used to treat the gel. UV transilluminator (Gel-Doc, Bio-Rad, USA) was used for observation. 3.3.3 Complexes stability under different pH conditions

of

PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes were prepared

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at the mass ratio of PDANP to DNA of 1.5. Both complexes were incubated in Dulbecco Modified Eagle Medium (DMEM, containing 10% FBS) with the pH value

re

Inst. Ltd. UK) every 10 minutes.

-p

of 6.0 or 7.4 for 1 h. The sizes of those complexes were measured with DLS (Malvern

The

lP

3.3.4 Photothermal conversion ability test photothermal

conversion

ability

of

PDANP-PEI-rPEG/DNA

and

na

PDANP-PEI-nPEG/DNA complexes was further investigated. Those complexes were prepared at the mass ratio of PDANP to DNA at 1.5 with various concentrations. The

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complexes were then irradiated by 808 nm laser (LSR-PS-FA, LASEVER INC., China) with a power density of 2.6 W/cm2. The temperature values were measured using a thermal imager (FLIR E60, FLIR Systems OÜ, Estonia). 3.4 In vitro experiments 3.4.1 Cell culture

For in vitro study, human hepatoblastoma cell line (HepG2 cells) was utilized. And for in vivo experiment, mouse skin melanoma cell line (B16F10 cells) was used. Cells were incubated in DMEM with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin under an atmosphere of 5 % CO2 at 37 °C. 3.4.2 Cell cytotoxicity assay MTT assay was carried out to study the cell cytotoxicity of different complexes. Cells were seeded into a 96-well plate with a density of 1×104 cells/well and incubated for 24 h, after which the medium was replaced by fresh medium. 7

Journal Pre-proof PDANP-PEI-rPEG/DNA (or PDANP-PEI-nPEG/DNA) complexes containing 1 μg DNA was added. PEI25K/DNA, PEI1.8k/DNA with the N/P ratio of 10 and lipofectamine 2000/DNA complexes with the mass ratio of 2.5 were chosen as reference. After 4 h incubation, cells were treated with or without 808 nm laser (2.6 W/cm2) irradiation for 20 minutes, following by another 20 h incubation. Then culture media was replaced by 120 μL DMEM containing 100 μg MTT. After 4 h incubation, cells were treated in 200 μL/well dimethyl sulphoxide for 15 minutes and the absorbance of 100 μL such solution was measured with a microplate reader

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(Multiskan FC, Thermo Scientific, USA). All the samples were prepared in

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quintuplicate. 3.4.3 Cellular uptake test

-p

For cellular uptake test, all the complexes were prepared with Cy3-labelled DNA as

re

mentioned. PEI25k/DNA, PEI1.8k/DNA and lipofectamine 2000/DNA complexes were

lP

used as reference. HepG2 cells were planted into a 24-well plate at a density of 5×104 cells/well. After 24 h incubation, the culture media was replaced and complexes

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containing 0.4 μg DNA were added and incubated for 4 h. Then, the cells were washed with PBS buffer (pH=7.4) for 3 times and removed from plate. After the cells

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were diluted with 330 μL DMEM, they were analyzed with a flow cytometry (Becton and Dickinson, BD FACSCalibur Flow, USA). 3.4.4 In vitro gene transfection HepG2 cells were seeded into a 24-well plate with a density of 5×104 cells/well and incubated for 24 h. Then, PDANP-PEI-rPEG/DNA, PDANP-PEI-nPEG/DNA, PEI25k /DNA (N/P=10), PEI1.8k/DNA (N/P=10) and lipofectamine 2000/DNA (mass ratio of 2.5) complexes containing 2 μg pGL-3 were added and incubated for 4 h. For the investigation of the influence of NIR irradiation on the transfection efficiency, cells with PDANP-PEI-rPEG/DNA were irradiated with 808 nm laser at the power density of 2.6 W/cm2 for various times. The temperature was measured using a thermal imager (FLIR E60, FLIR Systems OÜ, Estonia). After additional 20 h incubation, cells were washed with PBS buffer for 3 times. Then, 200 μL PBS was added into each well and cells were frozen at -80 °C and thawed for 3 times. Then the relative 8

Journal Pre-proof light unit (RLU) was measured using a microplate reader (Fluoroskan ascent FL, Thermo Scientific, USA) and BCA protein assay was carried out for normalization. 3.4.5 Bafilomycin A1 inhibition test To further study the possible mechanism of NIR-induced gene transfection promotion, bafilomycin A1 (Baf A1) was utilized to block the proton sponge effect. HepG2 cells were planted into 24-well plate with a density of 5×104 cells/well and incubated for 24 h, after which the culture media was replaced with fresh media (DMEM containing 10% FBS and 1% penicillin-streptomycin) with or without 200 Baf

A1.

The

cells

were

incubated

for

30

minutes

of

nM

and

then

ro

PDANP-PEI-rPEG/DNA complexes with the mass ratio of PDANP to DNA of 1.5 were added, and PEI25k/DNA complexes (N/P=10) were used as reference. After 4 h

-p

incubation, the media was replaced and cells were irradiated with 808 nm laser at the

re

power density of 2.6 W/cm2 for 20 minutes. After another 20 h incubation, cells were

lP

washed with PBS buffer for 3 times. Then, 200 μL PBS was added into each well and cells were frozen at -80 °C and thawed for 3 times. Then the relative light unit (RLU)

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was measured using a microplate reader (Fluoroskan ascent FL, Thermo Scientific, USA) and BCA protein assay was carried out for normalization.

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3.4.6 Confocal laser scanning microscope observation HepG2 cells were seeded with a density of 5×104 cells/dish into glass base dishes. After 24 h, the cells were incubated with DMEM containing 10% FBS, 1% penicillin-streptomycin and 10 μg/mL hoechst 33342 for 20 minutes, then washed with PBS buffer. Afterward, cells were cultured with PDANP-PEI-rPEG/Cy3-DNA complexes (0.4 μg gene, mass ratio of PDANP to DNA of 1.5) for 4 h. Subsequently, the culture media was replaced, above cells were irradiated with or without 808 nm laser with the power density of 2.6 W/cm2 for 20 minutes. After this, cells were cultured in DMEM with LysoTracker Green DND for 15 minutes and washed with PBS buffer. Confocal laser scanning microscopy (Leica TS SP5, Germany) was used for the observation.

9

Journal Pre-proof 3.5 In vivo photothermal effect test All animal experiments were performed in according with the guidelines for Animal Care and Use Committee of Zhejiang University and the “Principles of Laboratory Animal Care” (NIH publication no. 86-23, revised 1985). Female nude mice (5 weeks old, average weight around 20 g) were provided by animal center of Zhejiang Academy of Medical Sciences and Shanghai SLAC Laboratory Animal CO. Ltd. Mice were subcutaneously injected with 5×105 B16F10 cells/site to establish tumor-bearing model. After the tumor volume reached about 100 mm3, the mice were

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randomly divided into two groups. Then they were intratumorally injected with 50 μL

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PDANP-PEI-rPEG/pGL-3 complexes (mass ratio of PDANP to pGL-3 of 1.5, 30 μg gene/mouse) or not. After 2 hours, the tumor sites were irradiated with NIR light at

-p

the power density of 0.3 W/cm2 for 2 minutes, and the temperature of tumor sites was

re

measured using a thermal imager (FLIR E60, FLIR Systems OÜ, Estonia).

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4 Results and discussion

4.1 Preparation and characterization of nanoparticles

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To prepare pH-responsive nanopartoicles PDANP-PEI-rPEG, we synthesized PEG-PBA first. As shown in Figure 1a, 4-carboxyphenylboronic acid was grafted

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onto methoxypolyethylene glycol amine through amide bonds. The structure of PEG-PBA was determined by 1H NMR (Figure 1b). The peak at 7.8 ppm and 7.7 ppm were assigned to the aromatic ring in phenylboronic acid and the peak at 3.6 ppm was assigned to the -CH2- in polyethylene glycol. The molar ratio of PEG to PBA was calculated to be 1:1, indicating the successful synthesis of PEG-PBA.

Figure 1

Thereafter, polydopamine nanoparticles (PDANP) were synthesized according to the previous report31. During the reaction, the alkaline condition provided by NaOH triggered the self-polymerization of dopamine32. The obtained PDANP were characterized by DLS and TEM. Those PDANP showed a spherical morphology with the size of 154.4 nm (Figure 1c). 10

Journal Pre-proof Then

pH

responsive

nanoparticles

PDANP-PEI-rPEG,

non-responsive

nanoparticles PDANP-PEI-nPEG and non-PEGylated nanoparticles PDANP-PEI were prepared. PDANP-PEI-rPEG were synthesized by two steps under the alkaline condition provided by Tris-HCl buffer (pH=8.5). PEG-PBA was first grafted onto PDANP through the reaction between PBA and the catechol structure in polydopamine (Figure 1a). Those obtained boronate ester bonds could dissociate under acidic pH when they were internalized inside endosomes21,

22

. After PEG

modification, polyethylenimine with the molecular weight of 1800 (PEI1.8k) was

of

modified onto PDANP through the Michael addition or Schiff base reaction between

ro

the amino groups in PEI and the polydopamine33 (Figure 1a). Similarly, PDANP-PEI-nPEG were prepared using the conjugation between the amino groups in

re

-p

PEG-NH2 or PEI with polydopamine. And PDANP-PEI were synthesized likewise.

lP

Figure 2.

FT-IR was further used to confirm the structure of those nanoparticles. As depicted

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in Figure 2a, the peak of C=N vibration at 1645 cm-1 appeared in PDANP-PEI-rPEG,

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PDANP-PEI-nPEG and PDANP-PEI, suggesting the successful conjugation of polydopamine with PEI1.8k. The peak at 1235 cm-1 and 1115 cm-1 assigned to C-O-C bonds in PEG, indicating that PEG was modified onto those nanoparticles. DLS was used to determine the particle size. After modification, the particle sizes of PDANP-PEI-rPEG, PDANP-PEI-nPEG and PDANP-PEI were 167.0 nm, 190.8 nm and 179.2 nm respectively, which were higher than that of PDANP (154.4 nm) (Figure 2b). All those nanoparticles demonstrated narrow size distributions (PDI<0.2). Besides, the zeta-potential of those nanoparticles was also determined (Figure 2c). After modification of PEI only, the zeta-potential of PDANP changed from -17 mV to +37 mV. However, comparing with PDANP-PEI nanoparticles, the zeta-potential of PDANP-PEI-rPEG and PDANP-PEI-nPEG was significantly reduced due to the shielding effect of PEG shell. All the results proved the successful conjugation of 11

Journal Pre-proof PEG and PEI1.8k segments. TEM images showed all those nanoparticles were spherical and well-dispersed (Figure 2d-2f). 4.2 Chemo-physical characterization of gene-loaded complexes The physical property of the gene-loaded complexes could affect their in vitro behaviors, such as cellular uptake and gene transfection34. Thus, the sizes and zeta-potentials

of

PDANP-PEI-rPEG/DNA,

PDANP-PEI-nPEG/DNA

and

of

PDANP-PEI/DNA complexes with various mass ratios of PDANP to DNA were

ro

studied using DLS. As shown in Figure 3a, at the mass ratio above 0.3, the sizes of

-p

PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes maintained around

re

200-300 nm. And as depicted in Figure 3b, the zeta-potential of gene complexes increased with their mass ratios. At the mass ratio of 1.5, the zeta-potential of

lP

PDANP-PEI-rPEG/DNA complexes was similar to that of PEI1.8k/DNA complexes

na

(21.1 mV). The positive zeta-potential was beneficial for interacting with cellular

1000

nm,

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membranes and cell uptake. However, PDANP-PEI/DNA complexes were above which

were

not

suitable

for

gene

delivery.

Therefore,

PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes were chosen for further investigation.

To realize effective gene delivery, excellent gene condense ability is essential for gene vectors35. Thus, gel retardation experiment was carried out to investigate the gene condense ability of PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes. Those complexes with various mass ratios of PDANP to DNA were applied in the experiment. PEI1.8k and PEI25k were used as references. As polycations, PEI1.8k and PEI25k could condense gene through electrostatic forces, and the fluorescent band of free DNA would disappear after gene was fully condensed (Figure

3c).

Meanwhile,

although

the 12

gene

condensation

ability

of

Journal Pre-proof PDANP-PEI-rPEG and PDANP-PEI-nPEG was slightly different, they could both condense gene completely at the mass ratio above 0.3. Therefore, those complexes with the mass ratios above 0.3 were used in the following study.

Figure 3.

4.3 pH-responsive property of gene-loaded complexes Ideal gene delivery system should remain stable in extracellular delivery, and be

of

able to dissociate after cellular internalization in order to promote gene release36. A 37,38

.

ro

common strategy is to construct stimuli-responsive PEGylated gene complexes

Since the pH value inside the endosome and in the tumor tissue are usually more

-p

acidic (pH 5.5-6.5) than the physiological or blood pH (pH 7.4)18,39, the pH difference

DLS

was

carried

out

to

investigate

the

size

changes

of

lP

research.

re

was utilized for the development of pH-responsive PEGylated gene complexes in this

PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes in DMEM

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containing 10% FBS with the pH of 6.0 and 7.4. Herein, the pH of 7.4 was used to mimic the physiological pH. And the pH of 6.0 was chosen to mimic the pH condition

Figure 4

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inside endosome and in tumor tissue.

Figure 4a indicated both PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes remained stable at the pH of 7.4. The size of gene complexes at pH value of 6.0 showed obvious difference. PDANP-PEI-nPEG/DNA complexes kept unchanged after 1 h treatment, which was similar to that at the pH of 7.4. However, the size of PDANP-PEI-rPEG/DNA complexes reached around 350 nm after 1 h incubation at pH of 6.0. We speculated that at pH of 6.0, the cleavage of boronate ester bonds led to PEG detachment from the surface of PDANP-PEI-rPEG/DNA complexes. The protein adsorption from the culture medium might attribute to the size 13

Journal Pre-proof increase. This result indicated that PDANP-PEI-rPEG/DNA complexes could response to the acidic pH environment. 4.4 Photothermal conversion ability test Photothermal conversion agents can absorb NIR light and produce heat40-42. Therefore, the photothermal conversion ability of PDANP-PEI-rPEG/DNA, PDANP-PEI-nPEG/DNA complexes was investigated next. Both complexes were prepared with various concentrations at the mass ratios of PDANP to DNA of 1.5. Water was used for reference. For photothermally enhanced gene delivery, the power

of

density from 1 W/cm2 to 6 W/cm2 was commonly used in the NIR light irradiation30, 43.

ro

And in our work, the power density of 2.6 W/cm2 was chosen.

As shown in Figure 4b, PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA

-p

complexes demonstrated similar trend after NIR light irradiation at the same PDANP

re

concentrations. And their temperatures increased about 35°C after 20-minute

lP

irradiation at PDANP concentration of 48 μg/mL. At the same time, the temperature of water merely grew 13°C. The photothermal conversion efficiencies of

na

PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes were 54.9% and 52.8% respectively, which was sufficient to apply for the promotion of endosomal

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escape. 4.5 In Vitro tests

4.5.1 Cytotoxicity assay

The cytotoxicity of PDANP-based gene complexes was investigated using MTT method in HepG2 cells. PEI25k, PEI1.8k and commercial transfection reagent lipofectamine 2000 were used as reference. Lipofectamine 2000 was mixed with DNA at the mass ratio of 2.5 according to the user guide. Both PEI25k/DNA complexes and PEI1.8k/DNA complexes were prepared with the N/P ratio of 10. And the gene concentration was 10 μg/mL. As shown in Figure 5a, the cell viability of PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes maintained above 80% at various mass ratios, higher than that of PEI25k/DNA (66%) and lipofectamine 2000/DNA complexes (61%). Those results indicated both PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA 14

Journal Pre-proof complexes had better biocompatibility, which attributed to the biocompatible polydopamine and low-toxic PEI1.8k in the preparation. In this research, due to the effective photothermal conversion ability of PDANP, NIR-promoted gene transfection was expected to achieve. However, the temperature increase might influence the cell viability and overhigh temperature could even lead to cell apoptosis. So, in this test, the power density of 2.6 W/cm2 and the irradiation time of 20 min were chosen. As shown in Figure 5a, after 20-minute irradiation, the cell viability for all the complexes demonstrated no obvious change, which meant that

ro

of

NIR irradiation at the experiment condition showed little effect on cell cytotoxicity.

-p

Figure 5

re

4.5.2 Cellular uptake test

lP

In gene delivery, the gene-loaded complexes should be internalized by target cells effectively to achieve ideal gene transfection. To measure the cellular uptake

na

efficiency of different complexes, Cy3-DNA was used and the proportion of Cy3-positive cells and mean fluorescence intensity was determined by flow cytometry

Jo ur

After 4 h uptake, the percentages of Cy3-positive cells for PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes at different mass ratios were all around 70% (Figure 5b). And the mean fluorescence intensity of those complexes was similar to that of PEI25k/DNA complexes, suggesting they could be effectively internalized by HepG2 cells. Their good cellular uptake might attribute to the positive surface zeta-potential of those complexes (Figure 3b), which could help them to contact with negative-charged

cellular

membranes.

Those

results

indicated

that

PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes were promising for gene transfection. 4.5.3 Gene transfection assay Luciferase reporter gene (pGL-3) was utilized to investigate the gene transfection. To quantitatively measure the gene transfection level, relative luciferase activity per mg of protein (RLU/mg Protein) was presented. PDANP-PEI-rPEG/DNA and 15

Journal Pre-proof PDANP-PEI-nPEG/DNA complexes were prepared with various mass ratios. PEI25k, PEI1.8k and lipofectamine 2000 were used as reference. Lipofectamine 2000/DNA complexes were prepared at the mass ratio of 2.5. And PEI25k/DNA, PEI1.8k/DNA complexes were prepared.

Figure 6

PEI25k was often used as a gold standard in gene transfection44, 45. In our experiment,

of

the PEI25k/DNA complexes at the N/P ratio of 10 demonstrated the best transfection

ro

result. After cellular internalization, the proton sponge effect of PEI25k would facilitate endosomal escape, which would result in high gene transfection46. Whereas in

-p

cytotoxicity test, only 66% HepG2 cells survived after treated with PEI25k/DNA

re

complexes, indicating they had poor biocompatibility, which constrained their further

lP

utilization. For PDANP-PEI-rPEG/DNA complexes, their transfection efficiency increased as the rise of PDANP to DNA mass ratio. As shown in Figure 6a, at the

na

mass ratio of 1.5, PDANP-PEI-rPEG/DNA complexes showed similar transfection result as the commercial transfection reagent lipofectamine/2000 complexes. At the

Jo ur

meantime, PDANP-PEI-rPEG/DNA complexes demonstrated lower cytotoxicity than lipofectamine/2000 complexes, which suggested that our PDANP-PEI-rPEG/DNA complexes were promising for further application. Besides that, the value of RLU/mg protein of PDANP-PEI-rPEG/DNA complexes was almost 100 times higher than that of PDANP-PEI-nPEG/DNA complexes at the same mass ratio. Since both complexes showed

similar

cellular

uptake,

the

higher

gene

transfection

of

PDANP-PEI-rPEG/DNA complexes might result from the pH-responsive ability. After

cellular

internalization,

the

PEG

layer

would

detach

from

the

PDANP-PEI-rPEG/DNA complexes, which facilitated the dissociation of the complexes for better gene transfection47-49. The effect of NIR light irradiation on gene transfection was then investigated. After 20-minute irradiation, the value of RLU/mg protein doubled and became even higher than that of lipofectamine2000/DNA complexes (Figure 6b). NIR light irradiation 16

Journal Pre-proof promoted the gene transfection of PDANP-PEI-rPEG/DNA complexes. Many researches have reported this phenomenon and discussed the mechanism50, 51. When the gene-loaded complexes were internalized by the cells and then entered into the endosomes, the local temperature increase produced by photothermal conversion of PDANP might disrupt the endosomal membranes and lead to quick escape, which avoided the gene enzymolysis in the endosomes and increased the gene transfection. To confirm it, Bafilomycin A1 inhibition test was studied in the following. 4.5.4 Bafilomycin A1 inhibition test

of

Bafilomycin A1 can restrict the acidification of endosomes, block the proton

ro

sponge effect of PEI to restrict endosomal escape52. As shown in Figure 6c, the transfection level of PEI25k/DNA dropped sharply after Bafilomycin A1 treatment,

-p

suggesting the successful inhibition of proton sponge effect. Similar phenomenon was

re

also observed in the transfection of PDANP-PEI-rPEG/DNA complexes. After Bafilomycin A1 treatment, the transfection level of PDANP-PEI-rPEG/DNA

lP

complexes plunged from 4.6×1010 to 1.2×108 RLU/mg protein. But after NIR

na

irradiation at the power density of 2.6 W/cm2 for 20 minutes, their transfection level increased to 4.1 times, while the transfection level of PDANP-PEI-rPEG/DNA

Jo ur

complexes without Bafilomycin A1 treatment merely doubled. We speculated that for PDANP-PEI-rPEG/DNA

complexes,

the

higher

transfection

increase

after

Bafilomycin A1 treatment vindicated that the NIR light irradiation promoted the endosomal escape of PDANP-PEI-rPEG/DNA complexes, thus led to better gene transfection50.

4.5.5 Intracellular trafficking study Confocal laser scanning microscope (CLSM) was further used to confirm the mechanism of NIR irradiation-promoted gene transfection. PDANP-PEI-rPEG/DNA complexes with the mass ratio of 1.5 were utilized. The gene-loaded complexes were marked in red using Cy3-DNA. Endosomes were stained in green by Lyso Tracker, and cell nuclei were labeled in blue using hoechst 33342. The CLSM observation was carried out with or without 808 nm laser irradiation (Figure 7). Without NIR light irradiation, the colocalization of red and green indicated that PDANP-PEI-rPEG/DNA 17

Journal Pre-proof complexes were sited inside endosomes even after 4-hour uptake. But with NIR light irradiation, the color of PDANP-PEI-rPEG/DNA complexes (red) and endosomes (green) became separated, showing the successful endosomal escape. This bore out that the local heat generated by PDANP-PEI-rPEG/DNA complexes under NIR irradiation promoted the endosomal escape, giving rise to higher gene transfection result.

of

Figure 7

ro

4.6 In vivo photothermal conversion test

The photothermal conversion ability of PDANP-PEI-rPEG/DNA complexes was

-p

further investigated in vivo. Considering the treatment compliance of mice, the power

re

density of 0.3 W/cm2 was utilized. The temperature change and thermal images of

lP

tumor sites were recorded by a thermal imager.

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Figure 8

Jo ur

As shown in Figure 8a, after treated with PDANP-PEI-rPEG/pGL-3 complexes, the temperature of tumor site increased 15°C after 2-minute NIR irradiation. Meanwhile, the temperature of tumor site for untreated mice merely rose 8°C. The same results were also observed in the thermal images (Figure 8b). These results proved the excellent photothermal conversion ability of PDANP-PEI-rPEG/pGL-3 complexes in vivo, even in the low power density and short irradiation time. As we all know, photothermal therapy (PTT) is one promising route for cancer therapy.

By

taking

advantage

of

excellent

photothermal

conversion

of

PDANP-PEI-rPEG, the synergistic gene/photothermal therapy is more attractive in cancer therapy. The relative research is ongoing in our lab by using the p53 tumor suppressor gene (p53 DNA) instead of pGL-3.

18

Journal Pre-proof 5 Conclusion In

this

study,

we

PDANP-PEI-rPEG, non-PEGylated

successfully

non-responsive

nanoparticles

prepared

pH-responsive

nanoparticles

PDANP-PEI.

nanoparticles

PDANP-PEI-nPEG

and

PDANP-PEI-rPEG

and

Both

PDANP-PEI-nPEG could condense gene to form stable nano-sized complexes with the

size

of

200-300

nm.

And

the

PDANP-PEI-rPEG/DNA

and

PDANP-PEI-nPEG/DNA complexes demonstrated good photothermal conversion ability under NIR light irradiation. Both complexes could remain stable in the pH

of

condition of blood circulation or extracellular delivery (pH 7.4). Due to the cleavage

ro

of boronate ester bonds, the PEG layer could detach from PDANP-PEI-rPEG/DNA complexes at the pH of 6.0. The pH responsive ability of PDANP-PEI-rPEG might

-p

facilitate complexes dissociation and gene release inside cells. The gene transfection

re

level of PDANP-PEI-rPEG/DNA complexes was almost 100 times higher than that of

lP

PDANP-PEI-nPEG/DNA complexes at the same mass ratios in HepG2 cells. Moreover, the transfection level of PDANP-PEI-rPEG/DNA complexes was double

na

after NIR light irradiation, even higher than that of lipofectamine 2000/DNA complexes. Such promotion in gene transfection was resulted from the local heat

Jo ur

produced by PDANP under NIR irradiation, which led to quick endosomal escape and gene transfection. By modify polydopamine nanoparticles with acidically detachable PEG-PBA and low toxic PEI1.8k, the obtained PDANP-PEI-rPEG nanoparticles could overcome multiple obstacles in gene delivery and realize photothermally promoted gene therapy. Furthermore, by taking advantage of excellent photothermal conversion of PDANP-PEI-rPEG in vivo, the synergistic gene/photothermal therapy is more attractive in

cancer therapy. The relative research is ongoing in our lab by using the p53 tumor suppressor gene (p53 DNA) instead of pGL-3.

6 Acknowledgements This work was financially supported by the National Natural Science Foundation of China (51873186) and The Science and Technology Program of Zhejiang Province (2016C04002). 19

Journal Pre-proof

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Scheme 1. A) Schematic representation of PDANP-PEI-rPEG/DNA complexes preparation. B) Possible mechanism of the PEG dissociation under acidic pH value. C) Illustration of enhanced gene delivery for cancer treatment by PDANP-PEI-rPEG nanoparticles.

27

of

Journal Pre-proof

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Figure 1. Synthesis route of PEG-PBA and modification of PDANP by PEG-PBA or PEI (a). 1

H NMR spectrum of PEG-PBA in D2O (b), TEM images of PDANP (c). The scale bar was

Jo ur

na

lP

re

-p

200 μm.

28

of

Journal Pre-proof

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Figure 2. FT-IR spectra (a), sizes (b) and zeta-potentials (c) of PDANP, PDANP-PEI-rPEG,

-p

PDANP-PEI-nPEG and PDANP-PEI nanoparticles. TEM images of PDANP-PEI-rPEG (d),

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PDANP-PEI-nPEG (e) and PDANP-PEI (f). The bar was 200 nm.

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Figure 3. Particle size (a), zeta-potential (b) and gel retardation test (c) of different gene complexes at various mass ratios of PDANP to DNA. PEI1.8k and PEI25k were used in the gel

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retardation test as references.

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Figure 4 (a) Stability test of PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes with the mass ratio of 1.5 (PDANP to DNA) at the pH of 6.0 and 7.4. (b)

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Temperature changes of PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes with the mass ratio of 1.5 (PDANP to DNA) after NIR irradiation. The power density was 2.6

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W/cm2.

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Figure 5. Cytotoxicity (a) and cellular uptake efficiency (b) of gene-loaded complexes in

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0.001, ns denoted no statistically significant difference.)

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HepG2 cells. (* denoted statistically significant difference at p < 0.05, ** p < 0.01, *** p <

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Figure 6. (a) Gene transfection of different gene-loaded complexes. Gene transfection study

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of PDANP-PEI-rPEG/DNA complexes with the mass ratio (PDANP to DNA) of 1.5 after NIR irradiation for various times (b) and after proton sponge effect inhibition using Bafilomycin

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A1 (c). Those data were demonstrated as mean ± SD (n=3). * denoted statistically significant difference at p < 0.05. * denoted statistically significant difference at p < 0.05, ** p < 0.01, ns

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denoted no statistically significant difference.

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Figure 7. Confocal laser scanning microscopy images of PDANP-PEI-rPEG/DNA complexes

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with the mass ratio of 1.5 (PDANP to DNA) with and without NIR irradiation.

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Figure 8 (a) In vivo temperature changes and (b) thermal images of mice treated with PDANP-PEI-rPEG/DNA complexes after NIR irradiation. The power density was 0.3

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W/cm2.

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Conflict of interest No known conflict of interest

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Graphical abstract

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Highlights:  Polydopamine-based nanocarriers were prepared for gene delivery without cytotoxicity.  Nanocarriers could form stable complexes with gene and showed pH-responsive character.

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 Owing to quick endosomal escape after NIR irradiation, higher gene

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transfection was achieved

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