CAGW Modified Polymeric Micelles with Different Hydrophobic Cores

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CAGW Modified Polymeric Micelles with Different Hydrophobic Cores for Efficient Gene Delivery and Capillary-Like Tube Formation Xuefang Hao, Qian Li, Huaning Wang, Khan Muhammad, Jintang Guo, XiangKui Ren, Changcan Shi, Shihai Xia, Wencheng Zhang, and Yakai Feng ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00529 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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ACS Biomaterials Science & Engineering

CAGW Modified Polymeric Micelles with Different Hydrophobic Cores for Efficient Gene Delivery and Capillary-Like Tube Formation Xuefang Hao1, Qian Li1, Huaning Wang1, Khan Muhammad1, Jintang Guo1,2, Xiangkui Ren*1,2,3, Changcan Shi4,5, Shihai Xia6, Wencheng Zhang7, Yakai Feng*1,2,3 Corresponding Author: Y. Feng, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China Email: [email protected] (Y. Feng) 1 School of Chemical Engineering and Technology, Tianjin University, Yaguan Road 135, Tianjin 300350, China 2 Collaborative Innovation Center of Chemical Science and Chemical Engineering (Tianjin), Tianjin 300350, China 3 Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, China 4 School of Ophthalmology & Optometry, Eye Hospital, School of Biomedical Engineering, Wenzhou Medical University, Wenzhou, Zhejiang 325011, China 5 Wenzhou Institute of Biomaterials and Engineering, CNITECH, CAS, Wenzhou, Zhejiang 325011, China 6 Department of Hepatopancreatobiliary and Splenic Medicine, Affiliated Hospital, Logistics University of People’s Armed Police Force, 220 Chenglin Road, Tianjin 300162, China 7 Department of Physiology and Pathophysiology, Logistics University of Chinese

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People’s Armed Police Force, Tianjin 300309, China

Abstract Recently, polymeric micelles with different biodegradable hydrophobic cores, such

as

poly(lactide-co-glycolide)

(PLGA)

and

poly(lactide-co-3(S)-methyl-morpholine-2,5-dione) (PLMD), have been used for gene delivery. The biodegradable hydrophobic cores should play an important role in gene delivery. However, fewer researches focused on selectively promoting proliferation and migration of endothelial cells (ECs) as well as vascularization by altering hydrophobic cores of polymeric micelles. Herein, we prepared two kinds of CAGW peptide (selective adhesion for ECs) modified micelles with PLGA and PLMD as hydrophobic

cores,

respectively,

and

poly(ethylene

glycol)

(PEG)

and

polyethylenimine (PEI) as mixed hydrophilic shell. Their ability of condensing pEGFP-ZNF580 (pZNF580) to form gene complexes was proved by agarose gel electrophoresis assay. MTT results showed that the relative cell viability of the micelles with PLMD cores was higher than control groups and the micelles with PLGA cores. The cellular uptake ability of these CAGW modified gene complexes was higher than the complexes without CAGW target function. A similar trend was also found in transfection tests in vitro, which further demonstrated the effect of CAGW peptide and different hydrophobic cores on gene delivery. The number of migrated cells treated by the gene complexes with PLGA cores was 82 (non-target


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group) and 115 (target group), while the complexes with PLMD cores was 88 (non-target group) and 120 (target group). Capillary-like tube formation of CAGW peptide modified complexes with PLMD core group was much higher (about 6 times) than the PEI(10 kDa)/pZNF580 group. These results demonstrated that transfection efficiency, cell proliferation, migration and vascularization could be promoted by altering hydrophobic cores and CAGW modification. Keywords: Gene delivery, hydrophobic, CAGW, endothelial cells, transfection efficiency 1. Introduction The reconstruction of blood vessels was the key point for neovascularization in ischemic diseases. Some specific factors affect this process, such as the proliferation and migration of endothelial cells (ECs), and the potential of tube-like structure formation etc.1-5 Human umbilical vein endothelial cells (HUVECs) are widely investigated for in vitro angiogenesis due to their ability to form capillary-like structure.6 Recently, many studies have focused on gene-mediated therapy of ischemic diseases.7,8 The development of safe and efficient gene carriers is very important for gene therapy, and it has attracted much attention for efficacious gene therapy. 9-13 In order to obtain high transfection efficiency and good treatment effect, different gene carriers based on cationic polymers have been prepared owing to their specific characteristics, such as multifunctionality for convenient modification and intelligent design, and their convertible surface properties by altering the ratio of polymers and DNA.14-16 Moreover, amphiphilic copolymers are one of the most widely-developed



Page 4 of 37

carriers due to their stability, high cellular uptake and endosome escape.14-18 Kataoka et

al.

prepared

amphiphilic

catiomer

glycol)-b-poly{N’-[N-(2-aminoethyl)-2-aminoehtyl]

of

poly(ethylene

aspartamide}-cholesteryl

and

developed a strategy of cyclic (Arg-Gly-Asp) (cRGD) peptide ligand conjugation at the α-terminus for specific affinity to the targeted cells. Their results showed that the cRGD conjugated carriers achieved potent tumor growth suppression by efficient gene expression of anti-angiogenic protein (sFlt-1) at the tumor site in an intractable pancreatic cancer mice model.19 The hydrophobic modification of polycations has been proved to be a useful route for efficient gene delivery via simultaneously promoting internalization and endo/lysosomal escape so as to improve transfection efficiency.20-22 Many researches focused on drug or gene delivery in tumor models by adjusting the structures of hydrophobic segments, while few research investigated the effect of hydrophobic segments on EC transfection.23 In our previous studies, polyethylenimine (PEI) was grafted onto hydrophobic biodegradable polymers as gene carriers for EC transfection.14,24-28 These amphiphilic copolymers with different biodegradable hydrophobic and hydrophilic segments exhibited low cytotoxicity and high gene delivery efficiency. REDV and CAG peptides were immobilized on gene carriers to improve the efficiency of gene delivery and the vascularization ability via selective adhesion to the targeted ECs relative to smooth muscle cells (SMCs). These targeting peptides modified gene carriers possessed high internalization and transfection efficiency than the non-targeting carriers.17,18, 29-33 It is well known that REDV peptide is recognized by the integrin


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α4β1 on ECs.31 CAG peptide is derived from extracellular matrix (ECM) in the basement membrane of blood vessels. It showed highly selectivity for ECs other than SMCs, and the selectivity ratio of ECs was about 2.4 times higher than SMCs.34 In addition, gene carriers with high content of CAG peptide presented efficient gene delivery and high gene expression, which highlighted the effect of CAG peptide on cellular uptake.18,35 In order to investigate the effect of hydrophobic cores of polymeric micelles on EC transfection,

we

synthesized

methoxy-poly(ethylene

two

kinds

of

polymeric

carriers,

i.e.

glycol)-b-poly(lactide-co-glycolide)-g-polyethylenimine

(mPEG-PLGA-PEI)

and

methoxy-poly(ethylene

glycol)-b-poly(lactide-co-3(S)-methyl-morpholine-2,5-dione)-g-polyethylenimine (mPEG-PLMD-PEI)

with

poly(lactide-co-glycolide)

biodegradable

hydrophobic

segments

(PLGA)

poly(lactide-co-3(S)-methyl-morpholine-2,5-dione)

(PLMD),

of and

respectively.

For

improving selective cellular uptake, CAGW peptide was conjugated to PEI via click reaction. These amphiphilic polymers self-assembled into the micelles with different hydrophobic cores, and then complexed with pZNF580 plasmid to form gene complexes. These complexes could protect pZNF580 from enzyme degradation in the process of gene delivery and expression (Scheme 1). The cytotoxicity was characterized by MTT assay. The internalization efficiency of the gene complexes was quantitatively evaluated by flow cytometry. Furthermore, the gene delivery efficiency was evaluated by EC transfection in vitro. To testify the effect of gene complexes on



EC proliferation and vascularization, wound healing assay, transwell assay and in vitro tube formation assay were investigated for these gene complexes.

Scheme 1 Preparation process of pZNF580 complexes and selective gene delivery in ECs

2. Experimental section 2.1 Synthesis of mPEG-PLGA-PEI-CAGW and mPEG-PLMD-PEI-CAGW micelles


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mPEG-PLGA-PEI-DA and mPEG-PLMD-PEI-DA copolymers were prepared and purified as recently described in our previous study.29 They were dissolved in DMSO to prepare 5.0 mg mL−1 solutions, respectively. 0.1 mg DMPA and 4.0 mg CAGW peptide were added into 2.0 mL of the above solutions, and then the mixed solution was treated by UV-light for 10 min. The reacted solutions were added dropwise into 20 mL of phosphate buffer saline (PBS, pH=7.4) in a beaker and sonicated for 0.5 h. The micelles were obtained by dialyzing. Then, the micelles loaded pZNF580 via electrostatic interaction with different N/P ratios (5, 10, 15, 20) to form gene complexes. 2.2 Cellular Uptake The cellular uptake of the complexes was quantitatively evaluated by a flow cytometry. EA.hy926 cells were seeded into 6-well plates at a density of 3 × 105 cells/well for 24 h and then transfected with micelle/Cy5-oligonucleotide complexes at an N/P molar ratio of 20. After 4 h incubation, the transfected cells were washed two times with PBS to remove the residual complex suspensions and trypsinized with 0.25% trypsin. Subsequently, the cells were collected and resuspended in 500 µL PBS, and then they were analyzed by a flow cytometer (Beckman MoFlo XDP, USA). Non-transfected cells were used as a negative control to identify viable cells. 2.3 Tube Formation Assay In vitro tube formation assay was performed using transfected HUVECs. Briefly, 50 µL volume of growth factor-reduced Matrigel was added into each well of a pre-cooling 96-well plate and incubated at 37 °C for 1 h. Subsequently, the transfected



Page 8 of 37

HUVECs by micelle/pZNF580 complexes were trypsinized and seeded on the solidified Matrigel at the density of 4 × 104 cells per well and cultured in low glucose DMEM medium for 6 h. The HUVECs treated by PEI (10 kDa)/pZNF580 complexes were used as positive control, and the cells treated by naked pZNF580 were used as negative control. The formed capillary-like structures in five randomly selected fields were photographed using a microscope. Tubular structures were quantified by manually counting the numbers of connected cells in the randomly selected fields. 2.4 Statistical analysis Each experiment was performed three times at least, and the data were shown as mean ± standard deviation (SD). The one-way analysis of variance (ANOVA) was used to determine the statistical significance of difference between any two groups. P values less than 0.05 were considered to be statistically significant. 3. Results and discussion 3.1

Characterization

of

mPEG-PLGA-PEI-CAGW

and

mPEG-PLMD-PEI-CAGW micelles In order to investigate the effect of different hydrophobic cores of polymeric micelles on cellular uptake and gene delivery efficiency in ECs, the diblock copolymers of mPEG-PLGA and mPEG-PLMD were prepared as previous studies.29,31 The molecular weight of mPEG was 5 kDa, and the hydrophobic segments of PLGA and PLMD were designed to be 5 kDa. As measured by GPC, the number and weight average molecular weights of mPEG-PLGA were 8073 and 10491, and mPEG-PLMD showed Mn = 10003 and Mw =10970 (Table S1, supporting information). PEI(10 kDa)


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was grafted to these diblock copolymers in order to condense DNA, and then targeting peptide of CAGW was conjugated to PEI.29 The representative structures of these amphiphilic polymers and the intermediate products were characterized by 1H NMR spectra as shown in Figure S1 and Figure S2 (supporting information). The results proved that these amphiphilic polymers were successfully synthesized. CAGW peptide content was 18.6% and 15.0% for mPEG-PLGA-PEI-CAGW and mPEG-PLMD-PEI-CAGW by fluorescence emission spectrum, respectively (Figure S3 in supporting information). Generally, the particle size and zeta potential of gene complexes are two important factors for cellular uptake. These amphiphilic polymers could self-assemble to form the stable micelles with hydrophobic and degradable cores. The outermost PEI of these micelles condensed pZNF580 and formed gene complexes. The particle size and zeta potential values of the micelles and their pZNF580 complexes at various N/P ratios were summarized in Table 1 and shown in Figure 1. The size of the micelles and complexes with PLMD cores was smaller than that with PLGA cores at the same N/P molar ratio. According to our previous study, the micelles with biodegradable cores were spherical, and the PLMD cores were more compact than the PLGA cores due to the intermolecular hydrogen bonding in the PLMD core.24 So, the micelles assembled from mPEG-PLMD-PEI-CAGW were smaller than those assembled from mPEG-PLGA-PEI-CAGW, which benefited for their stability via reducing clearance rate by reticuloendothelial system (RES).16 From Table 1 and Figure 1, we can see that the hydrophobic cores didn’t play a significant role on the zeta potential of



Page 10 of 37

micelles and gene complexes. The zeta potential of CAGW modified micelles was a little lower than the micelles without CAGW because of targeting peptide conjugation. What’s more, the gene complexes showed much lower zeta potential than their corresponding micelles due to charge neutralization. The zeta potential of the gene complexes was raised with the increase of N/P molar ratios. To reveal the effect of CAGW modification and hydrophobic cores on condensing and loading DNA ability, agarose gel electrophoresis was performed. In Figure 2, the gene complexes without CAGW modification could inhibit pZNF580 migration at N/P molar ratio of 10, and different hydrophobic cores had little effect on pZNF580 condensation. The CAGW modified gene carriers condensed and loaded pZNF580 at N/P molar ratio of 20, which was higher than the carriers without CAGW modification. It indicated that the CAGW modification reduced the pZNF580 condensation ability because of their low zeta potential values.

Table 1 Size and zeta potential of mPEG-PLMD-PEI, mPEG-PLMD-PEI-CAGW, mPEG-PLGA-PEI and mPEG-PLGA-PEI-CAGW micelles.

a

Sample ID

Size (nm)

PDIa

Zeta Potential (mV)

mPEG-PLMD-PEI

68.4 ± 1.6

0.23 ± 0.04

25.5 ± 2.1

mPEG-PLMD-PEI-CAGW

65.3 ± 0.5

0.09 ± 0.01

24.4 ± 0.7

mPEG-PLGA-PEI

71.5 ± 3.8

0.31 ± 0.09

24.5 ± 0.5

mPEG-PLGA-PEI-CAGW

93.4 ± 7.2

0.33 ± 0.07

21.6 ± 1.9

PDI: Polydispersity index.


Page 11 of 37

(A)

160 mPEG-PLMD-PEI-CAGW/pZNF580 mPEG-PLGA-PEI-CAGW/pZNF580

Size (nm)

140 120 100 80 60

5

10

15

20

N/P (B) mPEG-PLMD-PEI-CAGW/pZNF580 mPEG-PLGA-PEI-CAGW/pZNF580

25 Zeta potential (mV)

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


20 15 10 5 0

5

10

15

20

N/P Figure 1 Size (A) and zeta potential (B) of mPEG-PLMD-PEI-CAGW/pZNF580 and mPEG-PLGA-PEI-CAGW/pZNF580 complexes at various N/P molar ratios (5, 10, 15 and 20).



Page 12 of 37

Figure 2 Agarose gel retardation assay of pZNF580 complexes at various N/P ratios ranging

from

2

to

40.

mPEG-PLGA-PEI-CAGW/pZNF580,

(a)

mPEG-PLGA-PEI/pZNF580,

(b)

(c)

mPEG-PLMD-PEI/pZNF580,

(d)

mPEG-PLMD-PEI-CAGW/pZNF580. 3.3 In Vitro Cytotoxicity To evaluate the cytotoxicity of different micelles and gene complexes for ECs, MTT


Page 13 of 37

assay was performed and the result was shown in Figure 3. Compared with the control groups of PEI(10 kDa) and PEI(10 kDa)/pZNF580, the micelles and gene complexes displayed markedly high relative cell viability. Moreover, the relative cell viability of the gene complexes was higher than the micelles at the same concentration due to the lower positive charge after binding with pZNF580 and the proliferation of ECs promoted by pZNF580. Besides, the micelles and gene complexes with PLMD cores showed slightly high cell viability compared with that comprising PLGA cores. The reason may be that the degradation product of the PLMD hydrophobic core might motivate the proliferation of ECs, which was advantageous for the development of low cytotoxic gene delivery systems.28 At the concentration of 60 µg mL-1, except control groups, all of the micelles and their gene complexes displayed more than 70% relative cell viability. Therefore, these gene carriers with biodegradable cores were suitable and safe for gene delivery in ECs.

Relative cell viability (%)

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


110 100 90 80 70 60 50 40 30 20 10 0

#

#

* # *# *# # * # * * * #

*

5

#

#

#

#

*#* *# * # * * *

*

#

10

#

##

#

*

**

*

# # #* #**

*

## #

# #

# *# ** * ** **

20

-1

#

40

a c e

a' c' e' #

#

b d #

b' d'

#

# #*# # * * ** ** *

60

Concentration (µg mL ) Figure

3

Relative

cell

viability

of


mPEG-PLGA-PEI

(a),


mPEG-PLGA-PEI/pZNF580

(a’),

mPEG-PLGA-PEI-CAGW/pZNF580 mPEG-PLMD-PEI/pZNF580

mPEG-PLGA-PEI-CAGW (b’),

(c’),

mPEG-PLMD-PEI-CAGW/pZNF580

Page 14 of 37

(d’),

(b),

mPEG-PLMD-PEI

(c),

mPEG-PLMD-PEI-CAGW

(d),

PEI(10

kDa)

(e)

and

PEI(10

kDa)/pZNF580 (e’) treated EA.hy926 cells at different polymer concentrations ranging from 5 to 60 mg mL-1. (n = 3, mean ± SD, *statistically different from e group (p< 0.05), #statistically different from e’ group (p< 0.05)). 3.4 Cellular Uptake It’s well known that the incorporation of hydrophobic moiety potentially increases the interaction of gene complexes with cell membrane, beneficial for efficient cellular uptake.22 The internalization of the CAGW modified gene complexes was evaluated by a flow cytometry, and Cy5-oligonucleotide was used as a reporter gene. The results were shown in Figure 4. The mean fluorescence intensity (MFI) of the gene complexes with different hydrophobic cores was much higher than the PEI(10 kDa)/Cy5-oligonucleotide group. These results demonstrated that the incorporation of hydrophobic moieties was in favor of cellular uptake. Moreover, the complexes with PLMD hydrophobic cores (MFI = 235 for group without CAGW and MFI = 312 for group with CAGW, respectively) showed higher MFI value than the complexes with PLGA hydrophobic cores (MFI = 164 for group without CAGW and MFI = 276 for group with CAGW, respectively). These results indicated that PLMD hydrophobic cores were beneficial for cellular uptake. PLMD hydrophobic cores were better than PLGA cores, even though PLGA copolymers were biocompatible and FDA-approved


Page 15 of 37

biodegradable polymer.36 Besides, the MFI values of the CAGW modified gene complexes were significantly higher than the complexes without CAGW modification owing to the CAGW-mediated selective cellular uptake in ECs. The CAGW modified complexes with PLMD hydrophobic cores demonstrated the highest internalization efficiency. The CAGW peptide content in mPEG-PLMD-PEI-CAGW/pZNF580 group (15.0%) was lower than that in mPEG-PLGA-PEI-CAGW/pZNF580 group (18.6%). It exhibited superiority as pZNF580 carrier for internalization in ECs.

(A)

300 250

a c e

b d

200 Counts

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


150 100 50 0

100

104 102 103 101 Fluorescence intensity


105


(B) Mean fluorescence intensity

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

Page 16 of 37

#

400

#

#

*

#

*

300

* 200

*

100 0

a

b

c

d

e

Figure 4 Cellular uptake of gene complexes in ECs (A) and mean fluorescence intensity measured by flow cytometry (B). (a) mPEG-PLGA-PEI/Cy5-oligonucleotide, (b)

mPEG-PLGA-PEI-CAGW/Cy5-oligonucleotide,

mPEG-PLMD-PEI/Cy5-oligonucleotide, mPEG-PLMD-PEI-CAGW/Cy5-oligonucleotide,

(c) (d)

(e)

PEI(10

kDa)/Cy5-oligonucleotide. (n = 3, mean ± SD, *p < 0.05, statistically significant difference with e group, #p < 0.05, statistically significant difference between two groups).

3.5 In Vitro Transfection The gene delivery efficiency in ECs and SMCs was evaluated at the level of protein expression by transfection in vitro. In Figure 5, green fluorescence was observed in the images of different gene complexes groups. This verified that the pZNF580 plasmid was delivered into ECs and expressed into green fluorescence protein. All of the gene complexes with hydrophobic cores showed significantly higher protein


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expression than the pZNF580 group and the PEI(10 kDa)/pZNF580 group. In addition, the

mPEG-PLMD-PEI-CAGW/pZNF580

group

with

CAGW

and

PLMD

hydrophobic cores displayed the highest transfection efficiency, which was about 1.4 times as high as that of mPEG-PLGA-PEI-CAGW/pZNF580 group, and 6.1 times higher than the PEI(10 kDa)/pZNF580 group. These results can be explained by the optimal PLMD hydrophobic cores, which benefited for low cytotoxicity and high internalization efficiency. Besides, the transfection efficiency of the CAGW modified gene complexes groups was much higher than that of the groups without CAGW modification due to their optimal cellular uptake efficiency. However, no green fluorescence protein was observed in HUASMCs (Figure 5(A)g), indicating the mPEG-PLMD-PEI-CAGW/pZNF580 complexes exhibited well selectivity for ECs other than HUASMCs. Therefore, hydrophobic cores and CAGW modification affected gene delivery and expression. The PLMD hydrophobic cores were superior to PLGA hydrophobic cores for gene delivery and expression in ECs.



(B) Transfection efficiency (%)

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

Page 18 of 37

+

+

16

*# +

12

+

*# *#

8 4

*#

0 a

b

c

d

e

f

g

Figure 5 Fluorescence images (A) and transfection efficiency (B) of EA.hy926 cells transfected

for

24

h

by

(a)

mPEG-PLGA-PEI/pZNF580,

(b)

mPEG-PLGA-PEI-CAGW/pZNF580,

(c)

mPEG-PLMD-PEI/pZNF580,

(d)


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


mPEG-PLMD-PEI-CAGW/pZNF580, (e) PEI(10 kDa)/pZNF580, (f) pZNF580 and fluorescence

images

of

HUASMCs

transfected

for

24

h

by

mPEG-PLMD-PEI-CAGW/pZNF580 (g). (mean ± SD, n= 3, *statistically different from the control group f with P＜0.05 and #statistically different from group g with P

＜0.05, +statistically different between two groups with P＜0.05).

3.6 Cell Migration Assay The migration ability of ECs is essential for wound healing and the treatment of vascular diseases.18,37,38 In the present study, wound healing assay and transwell migration assay were performed to investigate the migration ability of the transfected ECs. As shown in Figure 6, the gene complexes groups showed much larger relative recovered area than the PEI(10 kDa)/pZNF580 complexes group. Moreover, the relative recovered area of the gene complexes with PLMD hydrophobic core group was higher than the gene complexes with PLGA hydrophobic core group. Hence, the PLMD hydrophobic cores benefited for the pZNF580 plasmid delivery so as to improve the transfection and promote the migration of ECs. Meanwhile, transwell assay was also conducted to evaluate the migration ability of transfected ECs. In Figure 7, the gene complexes groups showed significantly high number of migrated cells compared with the pZNF580 control group and the PEI(10 kDa)/pZNF580 group. Moreover, the EC migrated numbers of a, b, d and e groups were 82, 115, 88 and 120, respectively. The migrated cell number of the CAGW modified gene complexes group was much higher than the complexes without



Page 20 of 37

CAGW modification. Overall, the ECs transfected by CAGW modified gene complexes with PLMD cores showed the highest promotion of migration and proliferation.

Figure 6 Wound recovery of EA.hy926 cells at 0, 6 and 12 h time points (A) and the relative recovered area after 12 h calculated by the Image-J software (B). (a) mPEG-PLGA-PEI/pZNF580

complexes

mPEG-PLGA-PEI-CAGW/pZNF580 mPEG-PLMD-PEI/pZNF580

treated

complexes

complexes

treated treated


group, group, group,

(b) (c) (d)

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


mPEG-PLMD-PEI-CAGW/pZNF580 complexes treated group and (e) PEI(10 kDa)/pZNF580 complexes treated group (mean ± SD, n= 3, *statistically different from the control group (PEI(10 kDa)/pZNF580) with P＜0.05 and #statistically different between two groups with P＜0.05).

Figure 7 Transwell migration assay of EA.hy926 cells after 6 h (A) and the average migrating cell number (B). (a) mPEG-PLGA-PEI/pZNF580 complexes treated group, (b)

mPEG-PLGA-PEI-CAGW/pZNF580

complexes


treated

group,

(c)


mPEG-PLMD-PEI/pZNF580

treated

group

Page 22 of 37

and

(d)

mPEG-PLMD-PEI-CAGW/pZNF580 treated group, (e) PEI(10 kDa)/pZNF580 complexes treated group and (f) pZNF580 treated group. (mean ± SD, n = 3, *

statistically different from (f) group with p < 0.05 and #statistically different between

two groups with p < 0.05). 3.8 In Vitro Tube Formation Assay The formation of tubule-like structure plays an important role in the neovascularization for the treatment of vascular diseases.18,37,38 The in vitro tube formation assay was performed to characterize the tubule-like formation ability of HUVECs transfected by the gene complexes with different hydrophobic cores. In Figure 8, the gene complexes with hydrophobic cores showed larger number of tubes than the control groups of pZNF580 and PEI(10 kDa)/pZNF580, demonstrating HUVECs treated by the gene complexes with hydrophobic cores possessed high vascularization ability. The tube number of the CAGW modified gene complexes groups was much higher than that of the groups without CAGW modification. The gene complexes groups with PLMD hydrophobic cores formed much more tubule-like structures than the groups with PLGA hydrophobic cores. Moreover, the CAGW peptide modified complexes with PLMD core group showed much higher number of tubule-like structures (about 6 times) than the PEI(10 kDa)/pZNF580 group. These results of the in vitro tube formation assay further proved that the hydrophobic cores of gene carriers had important effect on the promotion of tubule-like structure formation for transfected HUVECs.


Page 23 of 37

(B) HUVEC tube numbers per field

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


20

+

+ #

+

*

+ #

15

#

*

*

b

c

#

10

*

5 0

a

d

e

f

Figure 8 The effect of different complexes on pZNF580-mediated HUVECs tube formation in vitro. (A) Microscopy images of HUVECs after incubation on Matrigel for 6 h at 37 °C. (a) mPEG-PLGA-PEI/pZNF580 treated HUVECs, (b) mPEG-PLGA-PEI-CAGW/pZNF580 mPEG-PLMD-PEI/pZNF580

treated treated

HUVECs, HUVECs,

(c) (d)

mPEG-PLMD-PEI-CAGW/pZNF580 treated HUVECs, (e) PEI(10 kDa)/pZNF580 treated HUVECs and (f) pZNF580 treated HUVECs. (B) Tube number corresponding



to different groups. (*statistically significant difference from e group with p < 0.05, #

statistically significant difference from f group with p < 0.05 and +statistically

significant difference between two groups with p < 0.05). 4 Discussion For successful gene delivery in ECs, gene carriers should cross multiple cell barrier systems and tolerate various physiological environments.39,40 The development of stable gene carriers with high gene delivery efficiency is very critical for gene therapy. It’s well known that gene delivery efficiency can be influenced by the hydrophobic cores of polymeric micelle carriers.23 PLA, PGA and PLGA can be used as biomaterials owing to their well biocompatibility and biodegradability. PLA exhibits high crystallinity, low hydrophilicity and slow degradation rate. The PLGA copolymers with the advantages of both PLA and PGA are widely used as biomaterials and display good properties as one of the biocompatible FDA-approved biodegradable polymers.29 Besides, the copolymers of CL and LA or GA are also used to improve the degradation rate of PCL.31 3(S)-Methyl-morpholine-2,5-dione (MMD) is one monomer to prepare biodegradable polydepsipeptide.41 Its homopolymer and copolymers possess high biodegradability and well biocompatibility. The formation of hydrogen bonds between amide bonds and the existence of ester bonds in the main chain endow these polymers with good mechanical, thermal and biodegradable properties.26-28 Moreover, the degradation products are generally non-toxic to organisms and may be advantageous for the promotion of cell viability.26-28 In our previous studies, the effect of different biodegradable hydrophobic cores including


Page 24 of 37

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


poly(lactide-co-ɛ-caprolactone)

(P(LA-co-CL)),

PLA,

PLGA,

PLMD

and

poly(lactide-co-3(S)-methyl-2,5-morpholinedione) (PLMD) with different monomer ratios on gene delivery in ECs was investigated.26-29,31 The degradation rate of PLGA was faster than P(LA-co-CL) and PLA, and the degradation rate of PLMD was faster than PLA and poly(3(S)-methyl-2,5-morpholinedione).26-28 Besides, the micelles with biodegradable hydrophobic cores of PLMD (w/w ratio of LA and MMD = 8:2) demonstrated to have suitable properties as gene carriers.26 Therefore, the gene carriers with PLGA (w/w ratio of LA and GA = 8:2) and PLMD (w/w ratio of LA and MMD = 8:2) hydrophobic cores were chosen for gene delivery in ECs in present study. Compared with polymers with PLGA segments, polymers with PLMD segments can form small micelles due to the intermolecular hydrogen bonding in the PLMD core, which is propitious to the stability and cellular uptake.24 In order to develop targeting gene carriers, we synthesized amphiphilic block copolymers with biodegradable PLGA and PLMD hydrophobic segments, and grafted CAGW peptide to modify the amphiphilic polymers. The number molecular weight of the hydrophobic segments was about 5 kDa, which was optimal for gene delivery in our previous study.42 The CAGW modified gene carriers with different hydrophobic cores, i.e., PLGA and PLMD, were prepared via self-assembly method for the delivery of pZNF580 plasmid in ECs. These gene carriers and their gene complexes had suitable size (less than 150 nm) and adjustable zeta potential by altering N/P molar ratios for cellular uptake. CAGW modification slightly affected the particle size of the micelles and complexes, while different hydrophobic cores



exhibited a remarkably effect on their particle size. The size of the micelles and complexes with PLMD cores was smaller than that comprising PLGA cores. The small-sized particles were advantageous for the promotion of internalization in ECs. The result of agarose gel electrophoresis demonstrated that the polymeric micelles could condense and load pDNA efficiently, and no significant difference was observed between gene carriers with PLGA and PLMD hydrophobic cores. Furthermore, the micelles without CAGW modification could condense and load pZNF580 completely at N/P molar ratio of 10, while the CAGW modified micelles could inhibit pZNF580 migration at a high N/P molar ratio of 20. We have investigated the pDNA releasing profile, micelle stability and degradation profile in our previous study.27,28 The gene complexes with different hydrophobic cores exhibited a sequential pDNA releasing profile. Moreover, the micelles were very stable even at very low concentration (~ 0.01 µg mL-1 for mPEG-PLMD-PEI) owing to their low critical micelle concentration (CMC).27 They showed fast degradation in 5 days in PBS (pH = 7.4) at 37 °C.26,27 Besides, the micelles and complexes with PLMD hydrophobic cores showed higher cell viability than that with PLGA hydrophobic cores. The relative cell viability of the CAGW modified micelles and complexes was much higher than the control groups, indicating they were low cytotoxic for ECs. The cell internalization result of gene complexes demonstrated that the cellular uptake efficiency of the CAGW modified gene complexes was higher than the complexes without CAGW modification due to their selective internalization in ECs. Besides,


Page 26 of 37

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the gene complexes with PLMD hydrophobic cores displayed higher EC cellular uptake than the complexes with PLGA hydrophobic cores. In vitro transfection assay results also proved the CAGW modified gene complexes with PLMD hydrophobic cores exhibited the highest protein expression because of their low cytotoxicity, targeting function and high cellular uptake. The EC migration and proliferation of the mPEG-PLMD-PEI-CAGW/pZNF580 complexes were also testified by the wound healing and transwell migration assays. The results of in vitro tube formation assay showed that the CAGW modified gene complexes groups had high vascularization ability. Moreover, the mPEG-PLMD-PEI-CAGW/pZNF580 complexes group exhibited the best vascularization ability, much higher (6 times) than the PEI(10 kDa)/pZNF580

group.

These

results

proved

that

the

mPEG-PLMD-PEI-CAGW/pZNF580 complexes had great potential in gene delivery for promoting EC migration and proliferation as well as vascularization ability of HUVECs. 5 Conclusions In the present study, aiming for high gene delivery efficiency, CAGW-modified polymeric micelles with different biodegradable hydrophobic cores (PLGA and PLMD) were prepared to investigate their effect on gene delivery, EC migration and vascularization ability. Results showed that the gene delivery systems with targeting moieties possessed low cytotoxicity, high cellular uptake, good gene expression, high EC migration and vascularization ability, especially the groups with PLMD hydrophobic cores. Therefore, the CAGW modified gene complexes with



biodegradable hydrophobic cores exhibited tremendous future perspective as a gene delivery platform for the treatment of vascular diseases by selectively promoting the migration and proliferation of ECs as well as vascularization ability of HUVECs. 6 Acknowledgements This project was supported by National Key R&D Program of China (Grant No. 2016YFC1100300), National Natural Science Foundation of China (Grant No. 51673145 and 31370969), International Science & Technology Cooperation Program of China (Grant No. 2013DFG52040). 7 Supporting Information Experimental section and some characterizations of the prepared intermediate products and final polymers.

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For Table of Contents Use Only

CAGW Modified Polymeric Micelles with Different Hydrophobic Cores for Efficient Gene Delivery and Capillary-Like Tube Formation Xuefang Hao1, Qian Li1, Huaning Wang1, Khan Muhammad1, Jintang Guo1,2, Xiangkui Ren*1,2,3, Changcan Shi4,5, Shihai Xia6, Wencheng Zhang7, Yakai Feng*1,2,3


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TOC Preparation process of pZNF580 complexes and their promotion of HUVECs vascularization


CAGW Modified Polymeric Micelles with Different Hydrophobic Cores

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