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Article
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
1
Title
2
A novel strategy of gene delivery system based on dendrimer loaded recombinant
3
hirudine plasmid for thrombus targeting therapy
4 5
Author: Junjie Chena#, Yanping Lua#, Ying Chenga, Rui Maa, Jiafeng Zoua ,Hongyue
6
Zhenga, Ruwei Wangb, Zhihong Zhua, Fanzhu Li a*
7
a
8
Hangzhou, China
9
b
College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Zhejiang Conba Pharmaceutical Co.,Ltd.
10
#Junjie
11
*Corresponding author
Jun and Yanping Lu contributed equally to this work
12 13
Author information:
14
Junjie Chen#
15
Institution: Zhejiang Chinese Medical University;
16
Address: Gaoke Road, Hangzhou, Zhejiang, China;
17
Email:
[email protected] 18 19
Yanping Lu#
20
Institution: Zhejiang Chinese Medical University;
21
Address: Gaoke Road, Hangzhou, Zhejiang, China;
22
Email:
[email protected] 23 24
Ying Cheng
25
Institution: Zhejiang Chinese Medical University;
26
Address: Gaoke Road, Hangzhou, Zhejiang, China;
27
Email:
[email protected] 28 29
Rui Ma
30
Institution: Zhejiang Chinese Medical University;
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1
Address: Gaoke Road, Hangzhou, Zhejiang, China;
2
Email:
[email protected] 3 4
Jiafeng Zou
5
Institution: Zhejiang Chinese Medical University;
6
Address: Binwen Road, Hangzhou, Zhejiang, China;
7
Email:
[email protected] 8 9
Hongyue Zheng
10
Institution: Zhejiang Chinese Medical University;
11
Address: Binwen Road, Hangzhou, Zhejiang, China;
12
Email:
[email protected] 13 14
Ruwei Wang
15
Institution: Zhejiang Conba Pharmaceutical Co.,Ltd.;
16
Address: Binkang Road, Hangzhou, Zhejiang, China;
17
Email:
[email protected] 18 19
Zhihong Zhu
20
Institution: Zhejiang Chinese Medical University;
21
Address: Gaoke Road, Hangzhou, Zhejiang, China;
22
Email:
[email protected] 23 24
Fanzhu Li *
25
Institution: Zhejiang Chinese Medical University;
26
Address: Gaoke Road, Hangzhou, Zhejiang, China;
27
Email:
[email protected] 28 29
Abstract
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Molecular Pharmaceutics
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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,
16
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
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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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Molecular Pharmaceutics
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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
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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
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effectively deliver genetic materials into specific cells. Dendrimers, such as poly
10
lysine (PLL), polyethylene imine (PEI), polyamidoamine (PAMAM) and mesoporous
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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
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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
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tumor site accumulation, significantly 16, 18, 19.
27
Therefore, it is of interest to know whether the recombinant plasmid with hirudine and
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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
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system of RGDyC-PEG-PAMAM was used to deliver and release the recombinant
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plasmid at the thrombus site, and the plasmid could express the hirudin fusion protein.
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Then the hirudin fusion protein with RGDyC expression will target to the thrombus.
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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
20
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.
22
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
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Medical University.
28 29 30
Methods
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Molecular Pharmaceutics
1
Recombinant hirudin plasmid production and purification
2
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
4
(Arg-Gly-Asp) tripeptide sequence and FXa (Ile-Glu-Gly-Arg) recognition sequence
5
were introduced at the N-terminus of Hirudin gene. And FXa (Ile-Glu-Gly-Arg)
6
recognition sequence and Green Fluorescent Protein Gene (EGFP) were introduced
7
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
13
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
21
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
24
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
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(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
26
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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Molecular Pharmaceutics
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
23
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
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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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Molecular Pharmaceutics
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
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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
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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
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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).
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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.
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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.
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Molecular Pharmaceutics
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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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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Molecular Pharmaceutics
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
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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)
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Molecular Pharmaceutics
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)
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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)
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Molecular Pharmaceutics
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)
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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)
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