Novel Strategy of Gene Delivery System Based on Dendrimer Loaded

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A novel strategy of gene delivery system based on dendrimer loaded recombinant hirudine plasmid for thrombus targeting therapy Junjie Chen, Yanping Lu, Ying Cheng, Rui Ma, Jiafeng Zou, Hongyue Zheng, Ruwei Wang, Zhihong Zhu, and Fanzhu Li Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b01325 • Publication Date (Web): 25 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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Molecular Pharmaceutics

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Title

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A novel strategy of gene delivery system based on dendrimer loaded recombinant

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hirudine plasmid for thrombus targeting therapy

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Author: Junjie Chena#, Yanping Lua#, Ying Chenga, Rui Maa, Jiafeng Zoua ,Hongyue

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Zhenga, Ruwei Wangb, Zhihong Zhua, Fanzhu Li a*

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a

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Hangzhou, China

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b

College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Zhejiang Conba Pharmaceutical Co.,Ltd.

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#Junjie

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*Corresponding author

Jun and Yanping Lu contributed equally to this work

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Author information：

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Junjie Chen#

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Institution: Zhejiang Chinese Medical University;

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Address: Gaoke Road, Hangzhou, Zhejiang, China;

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Email: [email protected]

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Yanping Lu#

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Ying Cheng

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Rui Ma

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ACS Paragon Plus Environment

Molecular Pharmaceutics 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

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Jiafeng Zou

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Address: Binwen Road, Hangzhou, Zhejiang, China;

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Hongyue Zheng

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Address: Binwen Road, Hangzhou, Zhejiang, China;

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Ruwei Wang

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Institution: Zhejiang Conba Pharmaceutical Co.,Ltd.;

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Address: Binkang Road, Hangzhou, Zhejiang, China;

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Zhihong Zhu

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Fanzhu Li *

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Abstract


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This study proposed a new non-viral gene delivery system for thrombus targeting

2

therapy, based on PEGlyation polyamides dendrimer (PAMAM) modified with

3

RGDyC to condense the pDNA with recombinant hirudine (rHV) gene

4

(RGDyC-rHV-EGFP).

5

characterized by 1H-NMR, PAMAM/pDNA was characterized by particle size, zeta

6

potential, cellular uptake and gel retraction assay. The transfection was carried out

7

between lipofectamine 2000 and PAMAM/pDNA on HUVEC cells at various N/P

8

ratios. The anti-thrombotic effect in vivo was evaluated by venous thrombosis model

9

on Wistar rats. It showed that the drug delivery system of RGDyC modified PAMMA,

10

which entrapped pDNA could significantly improve the transfection efficiency. It was

11

about 7.56 times higher than that of lipofectamine 2000. In addition, the expression

12

level of hirudine fusion protein was the highest at N/P ratio of 0.5. The results of

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anti-thrombotic effect showed that the weight of thrombus was reduced in RGDyC

14

modified group, compared with heparin group, there was no significant difference

15

(P>0.05). Overall, we take the advantage of the unique advantages of hirudine,

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combining the genetic engineering, nano-carriers and targeting technology, to achieve

17

the targeted enrichment and activation the hirudine fusion protein in the thrombus site,

18

to improve the concentration of drugs in the thrombus site, finally increasing the

19

curative effect and reduce the risk of bleeding. The strategy of gene delivery system

20

holds unique properties as a gene delivery system and has great promises in thrombus

21

targeting therapy.

22

Keywords

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recombinant hirudin; dendrimer; RGDyC; gene delivery system; thrombus targeting

24

therapy

The

RGDyC-mPEG-PAMAM

was

synthesized

and

25 26

Introduction

27

Cardiovascular diseases (CVD) play a role in leading the highest mortality worldwide.

28

About 17 million people worldwide die from the CVD every year. In some developed

29

countries, the incidence rate of CVD rank first, among which acute myocardial

30

infarction (AMI) has become the first death disease, and the local ischemic stroke is



1

the third1, 2. Thrombosis is the main cause of AMI and stroke. The current

2

antithrombotic drugs are broadly classified into 3 categories: anti platelet drugs,

3

anticoagulant drugs and thrombolytic drugs. Although these drugs can effectively

4

inhibit the formation of thrombosis, they are lack of selectivity for thrombosis, and

5

have some other side effects. For example, warfarin occasionally elicits disastrous

6

microvascular thrombi and heparin can cause immune mediated thrombocytopenia3.

7

Therefore, searching for the novel anticoagulants with significant efficacy and

8

without such risks is ongoing. And targeting of thrombus may provide a strategy to

9

increase local anticoagulatory potency without systemic bleeding problems.

10

Direct thrombin inhibitors (DTIs) consist by a group of anticoagulants, they could

11

directly obstruct the thrombin activity and do not need cofactors to bind with

12

thrombin active site. DTIs have an advantage that the more predictable anticoagulants

13

over the indirect inhibitors, and they cannot neutralize platelet factor 4 and would not

14

lead to immune mediated thrombocytopenia4.

15

Hirudin, a 65-amino-acids peptide, is a small molecule protein secreted by the

16

salivary glands of leech. Its relative molecular mass is about 7 kDa. And there are

17

mainly three subtypes: HV1, HV2, HV3. HV has two main functional domains:

18

N-terminal catalytic domain that inhibits thrombin activity and C-terminal functional

19

domain with strong affinity for thrombin5. At present, HV is considered to be the

20

strongest inhibitor of thrombin, inhibitting the active of thrombin directly without the

21

involvement in other factors of the blood. It not only inhibits the free thrombin, but

22

also inhibits the active of thrombin, which has been combined with thrombus or fibrin

23

degradation products. And it has anti-thrombotic and anticoagulant activities. An

24

important factor affecting the clinical application of hirudine is the relatively short

25

plasma half-life when administered through an intravenous or subcutaneous route6.

26

Coupled with polyethylene glycol (PEG) could significantly prolong the plasma

27

half-life. However, the recent research indicated platelets are largely recruited into

28

arterial thrombi by thrombus bound thrombin rather than by soluble thrombin,

29

because PEG-hirudine has a molecular mass of 17 kDa, which may inhibit

30

thrombus-bound thrombin less effectively than hirudine7-9.


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The recombinant hirudine and the hirulogs are approved as anticoagulants10. They are

2

direct thrombin inhibitor, but they lead to some side effects such as the formation of

3

irreversible hirudin thrombin complex, short half-life of the hirulogs and their dosage

4

needs to be strictly monitored. Therefore, we designed a recombination hirudine

5

plasmid and it could express hirudine fusion protein. The fusion protein of RGD and

6

HV was linked by the identification sequence of coagulation factor Xa (FXa), it could

7

improve the thrombolytic effect and reduce the risk of bleeding11.

8

A successful gene delivery system is largely dependent on the capability of carriers to

9

effectively deliver genetic materials into specific cells. Dendrimers, such as poly

10

lysine (PLL), polyethylene imine (PEI), polyamidoamine (PAMAM) and mesoporous

11

silica nanoparticles12, have been used in gene delivery, due to their excellent

12

biocompatibility, tunable size and multiple functionalities properties13. They can

13

interact with nucleic acids through electrostatic interactions to form dendriplexes.

14

Compared to other nanocarriers, dendrimers are versatile platform for gene delivery

15

owing to its smaller particles and positive charges, which are important for gene

16

transfer14. Also dendrimers have attracted great interest in biomedical application,

17

because of their unique dendritic structures and multiple surface properties. In

18

addition, dendrimers are non-viral vector, which could escape from endosomes

19

rapidly and release pDNA or siRNA into cytosol through proton sponge effect15.

20

However, the dendrimers have the short blood circulation time and the poor targeting

21

efficiency. The previous researches showed that polyethylene glycol (PEG)

22

modification could prolong the blood circulation time and reduce the toxicity of

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dendriners caused by the numerous cationic groups on the surface16, 17. Some attempts

24

have been made to solve the non-targeting problem, such as folate, Angiopep-2, and

25

RGD modification of PAMAM by a covalent linkage, affecting the distribution and

26

tumor site accumulation, significantly 16, 18, 19.

27

Therefore, it is of interest to know whether the recombinant plasmid with hirudine and

28

RGD sequence loaded onto the RGD and PEG modified PAMAM can facilitate the

29

gene transfer and assist the thrombosis therapy. Additionally, the gene drug delivery

30

system of RGDyC-PEG-PAMAM was used to deliver and release the recombinant



1

plasmid at the thrombus site, and the plasmid could express the hirudin fusion protein.

2

Then the hirudin fusion protein with RGDyC expression will target to the thrombus.

3

This study will pave a strategy with safer, high transfer effect and targeting of

4

different therapeutics for thrombosis treatment based on the receptor mediated way.

5 6

Materials and methods

7

Materials

8

The polyamidoamine dendrimer generation 5 (PAMAM G5) with an ethylenediamine

9

core (in methyl alcohol, containing 128 surface primacy amino groups, MW 28826),

10

5-diphenyltetrazoliumbromide (MTT), Fetal bovine serum (FBS), Dulbecco's

11

modified Eagle's medium (DMEM) was purchased from Sigma-Aldrich Co.Ltd. (St.

12

Louis, MO, USA); the cyclization of arginine-glycine-aspartic peptides (RGDyC) was

13

synthesized by the GL Biochem Co. Ltd. (Shanghai,China); DAPI, the 5.745 kbp

14

pSecTag2 A plasmid were provided by Invitrogen (Carlsbad, CA,USA); Methoxy

15

PEG Succinimidyl Carboxymethyl Ester (mPEG–NHS, MW 3000) and Maleimide

16

PEG Succinimidyl Carboxymethyl Ester (MAL-PEG-NHS, MW 3500) were

17

purchased from JenKem Technology Co. Ltd. (Beijing, China).

18

The human umbilical vein endothelial cells (HUVEC) were kindly donated by

19

Zhejiang University and maintained in folate-deficient RPMI 1640 growth medium

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supplemented with 10% FBS and penicillin (100 U/mL)-estreptomycin (100 mg/mL)

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at 37 ℃ in a humidified atmosphere containing 5% CO2.

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Wistar rats (220±20g) and New Zealand white rabbits were purchased by Laboratory

23

Animal Center, Zhejiang Chinese Medical University (Zhejiang, China). All animal

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studies were performed in compliance with the guidelines on the Animal Welfare Act

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and the Guide for the Care and Use of Laboratory Animals by following protocols

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approved by the Institutional Animal Care and Use Committee at Zhejiang Chinese

27

Medical University.

28 29 30

Methods


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Recombinant hirudin plasmid production and purification

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The recombinant hirudine (rHV) gene (RGD-rHV-EGFP) was designed based on

3

Hirudin gene (NCBI Reference Sequence: M14964.1) in this study. The RGDyC

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(Arg-Gly-Asp) tripeptide sequence and FXa (Ile-Glu-Gly-Arg) recognition sequence

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were introduced at the N-terminus of Hirudin gene. And FXa (Ile-Glu-Gly-Arg)

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recognition sequence and Green Fluorescent Protein Gene (EGFP) were introduced

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into the C-terminus. The EcoR I cleavage site and the initiation codon ATG were

8

introduced into the 5' end. The stop codon and the Xho I cleavage site were

9

introduced into the 3' downstream end. Together with protective bases of both ends,

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the recombinant hirudin gene was synthesized by Shanghai Shenggong Co. Ltd. The

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pSecTag2 A- RGDyC-rHV-EGFP plasmid was constructed with DNA recombination

12

technology and amplified in E.coli Top 10’ and isolated, purified by an endotoxin free

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Plasmid Giga Kit (Promega) according to the manufacture instructions. Concentration

14

and purity of plasmid were measured by Trace nucleic acid protein detector

15

(Nanodrop 2000, ThermoFish). Plasmid integrity was confirmed by 1% agarose gel

16

electrophoresis assay and SDS-PAGE, and stored at -20℃ for further use.

17 18

Synthesis of RGDyC conjugated PEG-PAMAM nanoparticles

19

RGDyC-mPEG-PAMAM was synthesized as literature report17: RGDyC was

20

conjugated to MAL-PEG-NHS by the reaction under slightly acidic conditions

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between the male imide group of PEG and the thiol group of RGDyC. Firstly,

22

MAL-PEG3500-NHS was added into pH 6.0, 0.1 mol/L NaAc-HAc buffer which

23

containing RGDyC, then mixed by vortex for 30 s. Then the solution added into

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borate buffer solution to PAMAM (pH 9.0, 0.05 mol/L) and stirred overnight at room

25

temperature (RGDyC:PEG:PAMAM=45:45:1, molar ratio). Afterward the pH of

26

reaction mixture was adjusted to7.0 and the unreacted male imide group was

27

quenched by 2-mercaptoethanol. Then the mixture was transferred to an ultrafiltration

28

tube (Millipore, USA, MW 30,000) and centrifuged (4000 rpm, 20 min) for 3 times.

29

The concentrated solution after centrifugation was redissolved in 2 mL phosphate

30

buffers (pH 8.0) and mixed with different amount of mPEG-NHS



1

(PEG:PAMAM=64:1, molar ratio). After further stirred for 48 h at room temperature,

2

the unreacted PEG was removed by ultrafiltration and centrifuged. The products were

3

retrieved by freeze drying from the aqueous solution and white solids were obtained.

4

The synthesized of mPEG-PAMAM was similar to RGDyC-mPEG-PAMAM except

5

the adding RGDyC.

6 7

Preparation and characterization of PAMAM/pDNA nanocomplexes

8

mPEG-PAMAM and RGDyC-mPEG-PAMAM were positively charged, and able to

9

interact with negatively charged pDNA by electrostatic interactions to form

10

PAMAM/pDNA nanocomplexes. The PAMAM/pDNA nanocomplexes were

11

prepared with mPEG-PAMAM or RGDyC-mPEG-PAMAM and pDNA at a series of

12

N/P raios (the molar ratio of PAMAM-primary amines to pDNA-phosphate; 0, 0.5, 1,

13

2, 4, 8, 10). The final concentration of pDNA was 5 µg/mL. Briefly, 100 µL pDNA

14

solution was added drop wise to 200 µL mPEG-PAMAM or

15

RGDyC-mPEG-PAMAM solution and stirred at room temperature. The

16

PAMAM/pDNA nanocomplexes were equilibrated in solution for 1 h and washed in

17

250 mmol/L sodium phosphate buffers at pH 7.4 by a series of sonication and gently

18

vortex for several seconds, then centrifuged. The PAMAM/pDNA nanocomplexes

19

were dried under vacuum. The supernatant was collected to determine the pDNA

20

encapsulation efficiency (EE%) and loading efficiency (LE%) were calculated.

21

Particle size and Zeta potential were determined by dynamic light scattering (DLS)

22

(Malvern Nano-ZS90 UK) and the nanocomplexes were prepared at different N/P

23

rations of PEGylation PAMAM with or without RGD were examined. For each

24

sample, 5 μg of pDNA was used to prepare the PAMAM/pDNA nanocomplexes

25

solutions at the different N/P ratios. Then, the PAMAM/pDNA nanocomplexes were

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diluted to a final volume of 1 mL by adding 950 μL PBS. And the morphological

27

evaluation was determined by transmission electron microscope (TEM, HT7700,

28

Hitachi, Japan).

29 30

Agarose gel retardation assay


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1

Agarose gel electrophoresis was performed to check the ability of the PAMAM

2

nanoparticles to induce DNA condensation. Complexation of PAMAM nanoparticles

3

with DNA was induced at various N/P ratios ranging from 0.5:1 to 10:1. The

4

complexes were mixed with 2 μL 6× loading buffer and loaded onto a 1% agarose gel

5

with GoldView and ran with TAE buffer solution at 90 V for 45 min. Retardation of

6

DNA mobility was revealed by irradiation with UV light.

7 8

Cytotoxicity

9

The cytotoxicity of the different formulation of PAMAM/pDNA nanocomplexes was

10

determined by the MTT assay with HUVEC cells. The cells were seeded in 96-well

11

plates at a density of 5×103 cells/well in 200 μL culture medium supplemented with

12

10% FBS without antibiotics, and incubated overnight. Then the culture medium was

13

replaced by fresh medium containing PAMAM/pDNA nanocomplexes (based on

14

PAMAM molar mass). After incubated for 24 h, 100 µL of MTT (0.05mg·mL-1) was

15

added to each well, followed by incubation for 4 h at 37℃. The medium was

16

aspirated and 150 µL DMSO was added. The OD values were measured by the micro

17

plate reader (SpectraMax M2, Molecular Devices, USA) at wavelength 570 nm. The

18

cell viability was calculated by dividing the OD values of samples and OD values of

19

blank. Six replicate were considered for each formulation.

20 21

Anti-platelet aggregation activity assay

22

The New Zealand white rabbits (2.0-2.5 kg in weight) were obtained from the

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Laboratory Animal Centre, Zhejiang Chinese Medical University (Zhejiang, China), it

24

was half male and female, clean around the anus, without soft stools, loose stools.

25

Animals were acclimatized at least 7 days before experimentation with alternating

26

dark/light cycle of each 12 h at 23±2 ℃, and relative humidity: 60%-70%. Water and

27

stand laboratory food were available ad libitum. All experiments were performed in

28

accordance with the Guidelines and Policies of Ethical and Regulatory for Animal

29

Experiments as approved by Animal Ethics Committee of Zhejiang Chinese Medical

30

University (Zhejiang, China). All animals got humane treatment throughout the



1

experiment.

2

The blood drawn from the rabbit ear vein was placed in a centrifuge tube containing

3

1/10 volume of 0.109 mol/L sodium citrate anticoagulants (10 % anticoagulant and 90

4

% whole blood) and centrifuged at 700 rpm for 10 min. The supernatant was collected

5

to obtain platelets rich plasma (PRP). The remaining portion was centrifuged at 4000

6

rpm for 15 min, the supernatant was collected for platelet poor plasma (PPP). The

7

PRP was diluted with PPP to a platelet count of 25×104-30×104/μL as PRP reagent.

8

The undigested fusion protein or fusion protein digested by FXa was added into 0.2

9

mL of PRP reagent to get final concentrations of 0.5 μg/mL, 1.0 μg/mL, 5.0 μg/mL,

10

10.0 μg/mL, 25.0 μg/mL, 50.0 μg/mL, resting for 15min at room temperature.

11

Subsequently, 5 μL of 200 μmol/L adenosine diphosphates (ADP) preheated at 37℃

12

was added. The natural hirudine, hirudine fusion protein undigested by FXa and saline

13

were used as controls, the aggregation rate was measured with a platelet aggregation

14

apparatus.

15 16

Cellular uptake and intracellular disposition of PAMAM/pDNA nanocomplexes

17

PAMAM fluorescently labeled was described elsewhere16. To evaluate whether the

18

presence of RGDyC could affect transfection efficiency, the cellular uptake and

19

intracellular of PAMAM/pDNA nanocomplexes assays were carried out.

20

To evaluate the amount of PAMAM/pDNA nano particles uptake by the cells, flow

21

cytometry experiments were conducted by using fluorescently labeled FITC-PAMAM

22

conjugates. Briefly, HUVEC cells were seeded in 6-well plates at an initial density of

23

2×105 cells/well and incubated overnight. Then cells were transfected with

24

fluorescently labeled PAMAM/pDNA nanoparticles. After transfection 4h, the cells

25

were harvested and measured by BD FACSCalibur flow cytometer and the acquisition

26

of the data was made in the CellQuestTM Pro software.

27

For confocal laser scanning microscopy analysis, HUVEC cells were seeded at a

28

density of 2×105 cells/well on a petri dish and cultured for 24 h. The media was then

29

exchanged with 1 mL of FITC-labeled samples including PAMAM/pDNA

30

nanoparticles (at the final concentration of FITC 10 µmol·L-1), respectively. The


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1

medium was removed and the cells were washed with PBS for three times after

2

incubation for 4h. Then cells were fixed by 4% paraformaldehyde for 20 min, and

3

DAPI was used to stain the nuclei for another 10 min. Finally, confocal images were

4

obtained with Zeiss LSM 710 laser scanning confocal microscope (Carl Zeiss SMT

5

Inc., USA).

6 7

In vitro transfection studies

8

For pDNA transfection, 1×105 HUVEC cells were plated into each well of a 24-well

9

plate without FBS and allowed to 60-70% confluence. Then the medium was removed

10

and 0.5 mL medium containing lipofectamine 2000 (10 µg/mL, pDNA: lipofectamine

11

2000=1:2), mPEG-PAMAM/pDNA and RGDyC-mPEGPAMAM/pDNA, or

12

PAMAM/pDNA nanocomplexes at the N/P of 0.5, 1, 2, 4, 8 and 10 was added (the

13

final concentration of pDNA was 5 µg/mL). The medium was replaced by fresh

14

medium containing 10% FBS after 4h incubation at 37℃, and HUVEC cells were

15

further incubated for 48 h. Each transfection assay was carried out in triplicate. The

16

transfect cells were observed by fluorescence microscopy.

17 18

Westen blot

19

The HUVEC cells were harvested 72 h after transfected by mPEG-PAMAM/pDNA

20

or RGD-mPEG-PAMAM/pDNA at different N/P and lysed in RIPA buffer with

21

protease inhibitors (PMSF). BCA kit was used for quantitative of protein. Equal

22

amounts of the total proteins were separated in a polyacrylamide gel containing 12%

23

sodium dodecylsulfate and transferred to a 0.2 μm nitrocellulose filter membrane

24

(GE Healthcare, Buckinghamshire, UK). Then the membranes were blocked in 5%

25

nonfat powder and incubated with primary antibodies at 4℃ overnight. After

26

washed with TBS containing 0.1% Tween 20, the membranes were incubated with

27

secondary antibodies for 2 h at room temperature. The protein bands were visualized

28

by dual color infrared laser imaging system (Odyessey Clx, USA). The image

29

analyses were performed with the Image Studio Ver 5.2.

30



1

In vivo thrombin therapy

2

The venous thrombosis model was established by platinum wire insertion method20.

3

All rats were intraperitoneal anesthetized by thiopental (100 mg/kg) and the left

4

external jugular vein was separated and cannulated for administration or blood

5

collection. Then exposed the inferior vena cava and inserted a platinum wire (2 cm)

6

into the vein just caudal to the left renal vein to induce thrombus formation. The

7

thrombus was removed and weighed after 1h given the different antithrombotic drug.

8

The rats given the saline and heparin were negative and positive controls, respectively.

9

All thrombus were fixed with 10% formaldehyde solution for 24 h. For morphometry,

10

cross cryosections were prepared and stained routinely with hematoxylin and eosin

11

(H&E) and Masson’s trichrome for collagen. Immunostaining was used to identify

12

vascular endothelium specific marker by CD34 antibody (Dako Cytomation), smooth

13

muscle cell actin by ɑ-SMC actin (Sigma-Aldrich). The thrombus inhibition rate was

14

obtained by the following formula,

15

Inhibition rate= (1-weight of experimental group)/weight of control group×100%

16 17

Statistical analysis

18

Data was expressed as mean ± standard deviations (SD). Significant differences in the

19

mean values were evaluated by one-way analysis of variance (ANOVA). A statistical

20

test of a value of P < 0.05 was considered to be statistically significant.

21 22

Results and discussion

23 24

Recombinant hirudin plasmid production and purification

25

The recombinant hirudine plasmid of pSecTag2 A- RGDyC-rHV-EGFP was

26

successfully constructed and identified by 1.0% agarose gel electrophoresis, as shown

27

in Fig.1A. The target band is appeared at 5000 kb, which accords with the theoretical

28

value. While the result of SDS -PAGE about the recombinant proteins expression was

29

showed in Fig.1B. And it suggested that the recombinant hirudin plasmid was

30

synthesised successfully.

31


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1

Characterization of RGDyC conjugated PEG-PAMAM nanoparticles

2

The modification of PAMAM with RGDyC was confirmed by 1H-NMR spectroscopy

3

(Fig. 1C). The peaks between 2.5 and 3.5 ppm were assigned to the methylene proton

4

peaks of PAMAM, the appearance of characteristic peaks at 3.60-3.66 and 3.99 ppm

5

indicated the existence of PEG in the conjugates, while the characteristic proton peaks

6

at 6.7 and 7.0 ppm were assigned to the RGDyC. The result of 1H-NMR spectroscopy

7

suggested RGDyC-mPEG-PAMAM was synthesized successfully. And it also was

8

proved in the previous study, there were about 32 PEG chains and 7 RGDyC moieties

9

were grafted on the periphery of each G5 PAMAM molecule.

10

The FT-IR spectrum of mPEG-PAMAM and RGDyC-mPEG-PAMAM were given in

11

Fig. 1D. The peaks of PEG were about 2,886.3 cm-1 and 1,108.1 cm-1. Additionally,

12

peaks appeared both at 843 cm-1 and 842.7 cm-1, and presumably it was the C-H

13

bending vibration on the binary substituted benzene ring of tyrosine, by the RGDyC

14

structure, according to the IR and 1H-NMR of RGDyC.

15 16

Preparation and characterization of PAMAM/pDNA nanocomplexes

17

The formulations of pDNA loaded nanocomplexes with RGDyC modification were

18

synthesized based on the electrostatic interactions between positively charged

19

PAMAM and negative charged pDNA. Zeta potential and particle size of different

20

PAMAM/pDNA nanocomplexes at various N/P ratios were summarized in Table 1.

21

All PAMAM nanoparticles after incubated with plasmid at different N/P ratios

22

showed positive zeta potential and the particle size was ranged between 19 from 40

23

nm. With the increasing N/P ratio, the size of PAMAM/pDNA nanocomplexes getting

24

smaller, it may be caused by the increasing DNA compaction ability.

25

Generally, zeta potential also could assess the surface potential of the formed

26

polyplexes. The positive zeta potential of PAMAM/pDNA nanocomplexes could

27

protect DNA from enzymatic degradation by strong complex with nucleic acids on the

28

surface of the PAMAM nanoparticles, facilitate cellular uptake and particle wrapping

29

because of strong electrostatic interaction between cationic PAMAM/pDNA

30

nanocomplexes and the negatively charged cell surface membrane. The results of zeta



1

potential showed that at the N/P ratios from 0.5 to 10, the complexation of pDNA

2

changed the surface potential of the PAMAM/pDNA nanocomplexes. Some

3

researches showed that small particle size and positive surface of nanocomplexes had

4

much more potential for gene transfection applications21.

5 6

Agarose gel retardation assay

7

The negative of pDNA could be completely or partially neutralized by the vector

8

upon the formation of nanocomplexes, due to the retardation of pDNA upon gel

9

electrophoresis. The results of agarose gel retardation (Fig.1E) could confirm the

10

pDNA complexation ability of PAMAM nanoparticles, it showed that RGDyC

11

modification had the ability to completely compact pDNA at the N/P ratio above 0.5,

12

indicating that the partial modified with RGDyC did not appreciably compromise the

13

DNA compaction ability of PAMAM.

14 15

##Insert Fig.1##

16

##Insert Tab.1##

17 18

Cytotoxicity

19

For effective and safe gene delivery, low cytotoxicity of gene delivery vector is

20

important. The cytotoxicity of PAMAM/pDNA via MTT assay on HUVEC cells at

21

different concentrations was explored (Fig.2A). The results showed that the cell

22

viability of different N/P ratios of PAMAM/pDNA nanocomplexes was gradually

23

decreases with the increase of the PAMAM concentration. It may be caused by the

24

cytotoxicity of PAMAM nanoparticles and the increased positive charge of

25

PAMAM/pDNA nanocomplexes. Nevertheless, when the N/P ratio below 1, there

26

were still more than 85% cells survival. For security and efficiency considerations, the

27

N/P ratio lower than 1 was selected for further study.

28 29

Anti-platelet aggregation activity assay

30

The results of platelet aggregation were showed in Fig.2B. The recombinant hirudine


Page 14 of 26

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1

fusion protein had good anti-platelet aggregation activity. However the fusion protein

2

was not active when it was undigested by FXa. Thereby it suggested the recombinant

3

hirudine fusion protein had site-specific activation after FXa digested. The result

4

showed it could reduce the risk of bleeding caused by large-scale use from the

5

injection sites to the thrombus site. The results were consistent with the purpose that

6

the hirudine fusion protein only after FXa digestion. When PAMAM/pDNA delivery

7

system was injected intravenously, the hirudine fusion protein would be expressed

8

and released into the blood. Then targeting to the thrombus site, and the specific

9

enzyme digestion of the fusion protein of hirudin at the thrombus site was realized, so

10

that it had antithrombotic activity.

11 12

Cellular uptake and intracellular disposition of PAMAM/pDNA nanocomplexes

13

To further evaluate whether the presence of RGDyC could affect the transfection

14

efficiency, the cellular uptake and intracellular disposition of PAMAM/pDNA

15

nanocomplexes were carried out. As the Fig.2C shown, the intensity of green

16

fluorescence was higher in RGDyC-mPEG-PAMAM group, and much more green

17

fluorescence appeared near the nucleus, it suggested that modification RGDyC could

18

facilitate the cell uptake. The result of intracellular disposition in Fig.2D showed that

19

both mPEG-PAMAM and RGDyC-mPEG-PAMAM group could be uptake by

20

HUVEC cells. But modified with RGDyC (RGDyC-mPEG-PAMAM group) could

21

remarkably improve the cell uptake compared to the mPEG-PAMAM group (P0.05).


Page 16 of 26

Page 17 of 26 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

The modification RGDyC had favorable targeting effect, thus the gene could be

2

delivered to the thrombin site and express the hirudine, which had an obvious

3

inhibition thrombin effect compared to the non-modification.

4 5

##Insert Fig.3##

6 7

Immunohistochemistry

8

The results of HE staining showed the condition of inferior vena cava thrombosis in

9

each group after administration24. In saline group (Fig.4), a large amount of

10

cellulose-like components were observed, the platelet trabeculae were formed and

11

aggregated into lamellae, and trabecular bone was filled with red blood cells.

12

However, in the heparin control group and RGDyC-mPEG-PAMAM group, the

13

situation above was significantly improved.

14

In the Masson staining, green part represents collagen fibers and mucus stains, and

15

red indicates patina, muscle, cellulose, black indicates nucleus25. At the conditions of

16

slow blood flow, intimal damage or hypercoagulability, the platelets can adhere to the

17

intima of the valve or the wall of the tube, and cellulose deposits to form a white

18

thrombus that can be seen by the naked eye. And the platelets continue to aggregate

19

and accumulate. Before the white thrombus does not completely block the lumen, the

20

distally stagnant plasma, red blood cells and white blood cells will entangle and

21

coagulate on the thrombus. After the thrombus completely blocks the lumen, the

22

stagnant blood solidifies as if it was placed in a test tube, forming a red thrombus

23

composed of red blood cells, platelets, and cellulose.

24

The Masson staining of the saline group showed a large amount of collagen

25

deposition in the thrombus. Compared to saline group, the collagen area in the

26

thrombus was lesser in heparin group. However, in RGDyC-mPEG-PAMAM group,

27

the collagen deposition area decreased, and there was a significantly different between

28

mPEG-PAMAM and RGDyC-mPEG-PAMAM group (P < 0.05). Compared to

29

another three groups, the collagen deposition area of RGDyC-mPEG-PAMAM group

30

gradually spreads from the inside of the thrombus to the outside and decreases.



1

CD34 is expressed as a yellow stained area in the thrombus, and CD34 is a specific

2

marker of vascular endothelium. CD34 expression increased with the increase of

3

neovascularization, the new endothelial cells appeared in the thrombus and they

4

would participate in neovasculaqrization to dissolve the thrombus. In the saline group,

5

CD34 expression was less, and its expression significantly increased in the

6

RGDyC-mPEG-PAMAM group.

7

ɑ-SMA is expressed in the thrombus as a yellow stained area and expressed in the

8

cytoplasm. ɑ-SMA is a smooth actin that is expressed in fibroblasts during

9

thrombosis26. In the saline group, the expression of ɑ-SMA was positively expressed

10

at the edge of the thrombus, and the expression was significantly decreased in the

11

heparin group. While the expression of ɑ-SMA was much more at the saline group,

12

and significantly reduced at RGDyC-mPEG-PAMAM group.

13 14

##Insert Fig.4##

15 16

Conclusion

17

In summary, PEGylation PAMAM G5 modified with RGDyC by covalent bond was

18

successfully used to delivery recombinant hirudine gene plasmid for thrombus

19

targeting therapy. This strategy of PAMAM gene delivery is able to condense pDNA

20

effectively and has good transfection efficiency to HUVEC cells in vitro. Additionally,

21

the gene delivery system of PAMAM/pDNA has good thrombus therapy effect. With

22

good cytocompatibility, the designed PEGylation PAMAM G5 modified with

23

RGDyC gene delivery system has great potential in the application of delivering the

24

recombinant hirudine plasmid for thrombus targeting therapy in clinic effectively.

25 26

Acknowledgements

27

This research is financially supported by the National Natural Science Foundation of

28

China (No. 81603303 and 81673607). The authors would like to thank Dr. Gao jianli

29

(Department of traditional Chinese Medicine, Zhejiang Chinese Medical University,

30

China) for providing us with technical assistance.


Page 18 of 26

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1 2

Disclosure

3

The authors report no conflicts of interest in this work.

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

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cardiovascular and cerebrovascular diseases: cardiovascular actions, molecular mechanisms, and therapeutic potential. Pharmacol. Res 2018, 139 ,62-75. 3.

Sheng, G. H.; Aronowitz, P. The lesser known side-effect of warfarin: warfarin-induced venous

limb gangrene. Mayo Clinic proc 2014, 89 (5), e47. 4.

Lee, C. J.; Ansell, J. E. Direct thrombin inhibitors. Br J Clin Pharmacol 2011, 72 (4), 581-592.

5.

Shabareesh, P. R. V.; Kaur, K. J. Structural and Functional Characterization of Hirudin P6

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Evaluating prodrug

characteristics of a novel anticoagulant fusion protein neorudin, a prodrug targeting release of hirudin variant 2-Lys47 at the thrombosis site, by means of in vitro pharmacokinetics. Eur J Pharm Sci 2018, 121, 166-177. 12. Zhou, Y.; Quan, G.; Wu, Q.; Zhang, X.; Niu, B.; Wu, B.; Huang, Y.; Pan, X.; Wu, C. Mesoporous silica nanoparticles for drug and gene delivery. Acta Pharm Sin B 2018, 8 (2), 165-177. 13. Guo Z.; Lin L; Chen J.; Sun P.; Wu J.; Xu C.; Tian H.; Chen X. Molecular Strings Significantly Improved the Gene Transfection Efficiency of Polycations. J. Am. Chem. Soc. 2018, 140(38), 11992-12000. 14. Li, J.; Liang, H.; Liu, J.; Wang, Z. Poly (amidoamine) (PAMAM) dendrimer mediated delivery of drug and pDNA/siRNA for cancer therapy. Int J Pharm 2018, 546 (1-2), 215-225. 15. Varkouhi, A. K.; Scholte, M.; Storm, G.; Haisma, H. J. Endosomal escape pathways for delivery of biologicals. J Control Release 2011, 151 (3), 220-228. 16. Han, S.; Zheng, H.; Lu, Y.; Sun, Y.; Huang, A.; Fei, W.; Shi, X.; Xu, X.; Li, J.; Li, F. A novel



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

synergetic targeting strategy for glioma therapy employing borneol combination with angiopep-2-modified, DOX-loaded PAMAM dendrimer. J Drug Target 2018, 26 (1), 86-94. 17. Lu, Y.; Han, S.; Zheng, H.; Ma, R.; Ping, Y.; Zou, J.; Tang, H.; Zhang, Y.; Xu, X.; Li, F. A novel RGDyC/PEG co-modified PAMAM dendrimer-loaded arsenic trioxide of glioma targeting delivery system. Int J Nanomedicine 2018, 13, 5937-5952. 18. Wang, M.; Hu, H.; Sun, Y.; Qiu, L.; Zhang, J.; Guan, G.; Zhao, X.; Qiao, M.; Cheng, L.; Cheng, L.; Chen, D. A pH-sensitive gene delivery system based on folic acid-PEG-chitosan PAMAM-plasmid DNA complexes for cancer cell targeting. Biomaterials 2013, 34 (38), 10120-10132. 19. Xu, X.; Li, J.; Han, S.; Tao, C.; Fang, L.; Sun, Y.; Zhu, J.; Liang, Z.; Li, F. A novel doxorubicin loaded folic acid conjugated PAMAM modified with borneol, a nature dual-functional product of reducing PAMAM toxicity and boosting BBB penetration. Eur J Pharm Sci 2016, 88, 178-190. 20. Morishima, Y.; Honda, Y.; Kamisato, C.; Shibano, T. Comparison of antithrombotic and hemorrhagic effects of edoxaban, a novel factor Xa inhibitor, with unfractionated heparin, dalteparin, lepirudin and warfarin in rats. Thromb. Res. 2013, 132 (2), 234-239. 21. Xiao, T.; Cao, X.; Hou, W.; Peng, C.; Qiu, J.; Shi, X. Poly(amidoamine) Dendrimers Modified with 1,2-Epoxyhexane or 1,2-Epoxydodecane for Enhanced Gene Delivery Applications. J Nanosci Nanotechnol 2015, 15 (12), 10134-10140. 22. Wang, G.H.; Chen, H.; Cai, Y.Y.; Li, L.; Yang, H.K.; Li, Q.; He, Z.J.; Lin, J.T. Efficient gene vector with size changeable and nucleus targeting in cancer therapy. Mater Sci Eng C Mater Biol Appl 2018, 90, 568-575. 23. Zarei, H.; Kazemi Oskuee, R.; Hanafi-Bojd, M. Y.; Gholami, L.; Ansari, L.; Malaekeh-Nikouei, B. Enhanced gene delivery by polyethyleneimine coated mesoporous silica nanoparticles. Pharm Dev Technol 2019, 24(1),127-132. 24. Lam T; Boudreau NJ; Bollen AW; Lawton MT;

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33


Page 20 of 26

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1

Tab.1 The characterization of RGDyC-mPEG-PAMAM/pDNA and

2

mPEG-PAMAM/pDNA nanocomplexes.

3 N/P

0.5

1

2

4

8

10

EE%

12.40

19.00

22.40

36.60

67.20

82.20

/

RGDyC-mPEG-

LE%

1.78

1.36

0.80

0.66

0.60

0.59

/

PAMAM/pDNA

Particle size (nm)

22.81

21.95

19.24

18.21

16.29

15.46

23.30

Zeta potential (mV)

0.46

2.39

2.85

3.51

4.88

5.07

5.36

EE%

7.00

14.80

21.80

31.20

41.00

67.80

/

LE%

1.05

1.11

0.82

0.58

0.38

0.51

/

Particle size (nm)

19.73

19.40

17.28

17.05

16.56

16.23

20.11

Zeta potential (mV)

0.58

3.62

4.91

6.04

6.95

8.24

9.27

mPEG-PAMAM/pDNA


Nanoparticles


Fig.1 (A) Identification of the recombinant pSecTag2 A- RGD-rHV-EGFP plasmid Lane1: DNA marker; lane2-6: pSecTag2 A- RGD-rHV-EGFP plasmid. (C) 1H-NMR spectra of PAMAM, RGDyC, mPEG-PAMAM and RGDyC-mPEG-PAMAM. (D) FT-IR spectra of mPEG-PAMAM and RGDyC-mPEG-PAMAM (E)The agarose gel retardation of mPEG-PAMAM (a) and RGDyC-mPEG-PAMAM (b) at different N/P ratios. 31x25mm (600 x 600 DPI)


Page 22 of 26

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Fig.2 A The cytotoxicity of mPEG-PAMAM/pDNA and RGDyC-mPEG-PAMAM/pDNA at different N/P ratios.B The anti-platelet aggregation activity assay of nature hirudin, fusion protein of digested, fusion protein of undigested and saline. The cellular uptake (C) and intracellular (D)of mPEG-PAMAM/pDNA and RGDyCmPEG-PAMAM/pDNA. 27x26mm (600 x 600 DPI)



Fig. 3 (A) The in vitro transfection of Lipofectamin 2000, mPEG-PAMAM/pDNA and RGDyC-mPEGPAMAM/pDNA at different N/P ratios, bar 400 µm. (B-C) The Westen blot of RGDyC-mPEG-PAMAM/pDNA and mPEG-PAMAM/pDNA at different N/P ratios. (D) The inhibition rates of heparin, mPEG-PAMAM/pDNA, RGDyC-mPEG-PAMAM/pDNA . 27x42mm (600 x 600 DPI)


Page 24 of 26

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Fig. 4 The thrombus H&E, Masson, CD34 and ɑ-SMA histology images of the mice after administration of (a) saline, (b) heparin, (c) mPEG-PAMAM/pDNA and (d) RGDyC-mPEG-PAMAM/pDNA, Scale bar, 100 μm. 45x33mm (600 x 600 DPI)



For Table of Contents Use Only, A novel strategy of gene delivery system based on dendrimer for thrombus targeting therapy, Junjie Chen, Yanping Lu, Ying Cheng, Rui Ma, Jiafeng Zou ,Hongyue Zheng, Ruwei Wang, Zhihong Zhu, Fanzhu Li 88x34mm (600 x 600 DPI)


Page 26 of 26

Novel Strategy of Gene Delivery System Based on Dendrimer Loaded

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