DSpace at VNU: Preparation of the Cationic Dendrimer-Based Hydrogels for Controlled Heparin Release

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Preparation of the Cationic Dendrimer-Based Hydrogels for Controlled Heparin Release

Nhat-Anh N Tongab, Thi Hai Nguyena, Dai Hai Nguyenc, Cuu Khoa Nguyenc & Ngoc Quyen Tranac

a

Institute of Research and Development, Duy Tan University, Da Nang City 550000, Vietnam

b

School of Biotechnology, International University, Ho Chi Minh City Vietnam National University, Ward 6, Linh Trung, Thu Duc district Ho Chi Minh City 70000, Vietnam

c

Institute of Applied Materials Science, Vietnam Academy Science and Technology, 01 Mac Dinh Chi, District 1, Ho Chi Minh City 70000, Vietnam

Published online: 28 Aug 2015

To cite this article: Nhat-Anh N Tong, Thi Hai Nguyen, Dai Hai Nguyen, Cuu Khoa Nguyen & Ngoc Quyen Tran (2015)

Preparation of the Cationic Dendrimer-Based Hydrogels for Controlled Heparin Release, Journal of Macromolecular Science, Part A: Pure and Applied Chemistry, 52:10, 830-837, DOI: 10.1080/10601325.2015.1067043

To link to this article: http://dx.doi.org/10.1080/10601325.2015.1067043

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Preparation of the Cationic Dendrimer-Based Hydrogels for Controlled Heparin Release

NHAT-ANH N TONG1,2, THI HAI NGUYEN1, DAI HAI NGUYEN3,

CUU KHOA NGUYEN3, and NGOC QUYEN TRAN1,3,*

1Institute of Research and Development, Duy Tan University, Da Nang City 550000, Vietnam

2School of Biotechnology, International University, Ho Chi Minh City Vietnam National University, Ward 6, Linh Trung, Thu Duc district Ho Chi Minh City 70000, Vietnam

3Institute of Applied Materials Science, Vietnam Academy Science and Technology, 01 Mac Dinh Chi, District 1, Ho Chi Minh City

70000, Vietnam

Received April 2015, Revised and Accepted June 2015

We introduce a cationic polyamidoamine (PAMAM) dendrimers and tetronic (Te) based hydrogels in which precursor copolymers were prepared with simple methods In the synthetic process, tyramine-conjugated tetronic (TTe) was prepared via activation of its four terminal hydroxyl groups by nitrophenyl chloroformate (NPC) and then substitution of tyramine (TA) into the activated product to obtain TTe Cationic PAMAM dendrimers G3.0 functionalized with p-hydroxyphenyl acetic acid (HPA) by use of carbodiimide coupling agent (EDC) to obtain Den-HPA 1H-NMR confirmed the amount of HPA and TA conjugations The aqueous TTe and Den-HPA copolymer solution rapidly formed the cationic hydrogels in the presence of horseradish peroxidase enzyme (HRP) and hydrogen peroxide (H2O2) at physiological conditions The gelation time of the hydrogels could be modulated ranging from 7 to 73 secs, when the concentrations of HRP and H2O2varied The hydrogels exhibited minimal swelling degree and low degradation under physical condition In vitro cytotoxicity study indicated that the hydrogels were highly cytocompatible as prepared at 0.15 mg/mL HRP and 0.063 wt% of H2O2concentration Heparin release profiles show that the cationic hydrogels can sustainably release the anionic anticoagulant drug The obtained results demonstrated a great potential of the cationic hydrogels for coating medical devices or delivering anionic drugs

Keywords: Cationic hydrogels, polyamidoamine (PAMAM) dendrimers, injectable hydrogels, drugs delivery

1 Introduction

In recent years, special attention has been paid to cationic

hydrogels ‘on-off’ controlled drugs and proteins delivery

properties Several kinds of these chitosans, gelatin,

polya-midoamine (PAMAM), polyethylenimine-based hydrogels

have performed potentially biomedical applications

because of their electrostatics interaction with negatively

bioactive molecules, antibacterial activity and

biocompati-bility (1–5) These hydrogels have proven its efficiency in

releasing insulin in order to regulate blood glucose

concentration, or deliver genes (cationic nanogel) or anionic drugs (6–8)

Cationic PAMAM dendrimers are well known to exhibit some advantages for drugs delivery such as their high drug-loading capacity or highly electrostatic interac-tion with anionic bioactive molecules, enhancement of hydrophobic drugs solubility, drug slow-release, and easy functionalization of their external groups to prepare drug-loaded biomaterials (9–11) Therefore, the use of versatile dendrimers platforms for preparing many cationic hydro-gels would be significant in drugs delivery and tissue engi-neering In fact, Raghavendra et al reported that the cationic PAMAM dendrimers and multi-arm-polyethylene glycol-based hydrogels could sustainably release amoxicil-lin anionic drug in the intracervical tissue of pregnant guinea pigs (12) Photocurable PAMAM dendrimers hydrogels introduced as a versatile platform for tissue engineering and drug delivery (13) These cationic hydro-gels also exhibited biocompatibility and biodegradation The hydrogels would have much more potential if gel for-mation did not produce by-products or use photocurable

*Address correspondence to: Ngoc Quyen Tran, Institute of

Research and Development, Duy Tan University, Da Nang City

550000, Vietnam; and Institute of Applied Materials Science,

Vietnam Academy Science and Technology, 01 Mac Dinh

Chi, District 1, Ho Chi Minh City 70000, Vietnam E-mail:

tnquyen@iams.vast.vn

Color versions of one or more figures in this article can be

found online at www.tandfonline.com/lmsa

Journal of Macromolecular Science, Part A: Pure and Applied Chemistry (2015) 52, 830 –837

Copyright © Taylor & Francis Group, LLC

ISSN: 1060-1325 print / 1520-5738 online

DOI: 10.1080/10601325.2015.1067043

ultraviolet light As a further matter of clarification,

hemo-lytic toxicity and cell lysis due to a strong interaction of the

positively charged dendrimers and the negatively charged

cell membrane resulting in membrane disruption are weak

points of the original PAMAM dendrimer (9–11)

Our study aims to produce cationic and injectable

TTe-PAMAM-based hydrogels via an enzymatic reaction with

horseradish peroxidase (HRP) and hydrogen peroxide

(H2O2) to carry heparin-anionic drug for coating stent or

blood contacting devices (Fig 1) In a system using an

enzymatic reaction, the degree of crosslinking of hydrogel

can be easily controlled by the feed amount of hydrogen

peroxide and therefore, the stability of hydrogel can be

varied (15) The heparin-encapsulated hydrogels, when

coated to a metal surface, may enhance blood

compatibil-ity and reduce platelet adhesion and prolong the material

existence due to its high stability In the systems, PAMAM

dendrimers (G3.0) were functionalized with p-hydroxy

phenyl acetic acid molecules and tetronic

(Ethylenedi-amine tetrakis(propoxylate-block-ethoxylate) tetrol), a

nontoxic surfactant which was terminated by tyramine

moieties in order to prepare the injectable hydrogel that

could reduce toxicity of PAMAM dendrimer in the hydro-gel and prevent the contact between the polymer with cell membranes (14) Heparin could be dispersed in polymer solutions and then these mixtures are cured in situ form-ing a polymeric three-dimension network under several desired shapes or thin layers of hydrogels by spraying or injecting preparation (16, 17) The cationic TTe-PAMAM enzymatic-based hydrogels systems would be essential materials for medical treatment due to its convenient applications

2 Experimental

2.1 Materials Heparin sodium, 4-Nitrophenyl chloroformate 96% (NPC), tyramine (TA) were purchased from Acros Organics Horse-radish peroxidase (HRP) enzyme (type VI, 298), 1-thyl-3-(-3-(dimethylamino) propyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and 4-Hydroxyphenylacetic acid 98% (HPA) were purchased from Sigma-Aldrich Tetronic 1307 (Te, MW D 18,000) was obtained from Fig 1 In situ formation of cationic hydrogels from heparin-loaded polymer solutions

BASF PAMAM dendrimers at generation 3.0 (G3.0;

MwD6,530) were prepared in the Department of Materials

and Pharmacy Chemistry (Institute of Applied Materials

Science) (18) following the procedure reported by Tomalia

et al (19)

2.2 Synthesis of Tyramine-Tetronic (TTe)

Tyramine-tetronic was prepared by using a simple and

organic solvent-free procedure First, the four

end-hydroxyl groups of tetronic were activated with NPC

Finally, TA substituted NPC moieties as shown in

Scheme 1

Briefly, tetronic (7.0 g, 1.56 mmol of hydroxyl group)

was molten at 75
C for 2 h under stirring in a vacuum

atmosphere NPC (0.43 g, 2.13 mmol) was added to the

melted tetronic and continuously stirred for 4 h under

medium vacuum with HCl entrapment equipment After

4 h, the reaction mixture was adjusted to room

tempera-ture (20) To solubilize the solid copolymer and decompose

the excess NPC, 5 mL of distilled water and 30 mL THF

were added to the resulting product and then TA (213 mg, 1.56 mmol) added to the copolymer solution The reaction mixture was stirred overnight Finally, the solution was dialyzed in acetone three times with membrane dialysis (8000 MWCO) The dialyzed copolymer solution was pre-cipitated in diethylether to collect a powdery TTe copoly-mer and dried in a vacuum oven.1H-NMR (CDCl3)/ppm

of TTe, d ppm D 6.78 and 7.02 (d, –CHDCH–, TA) According to integrals of methyl tetronic protons and aro-matic TA protons in1H-NMR, 97% of TA moieties were determined to be conjugated into the tetronic

2.3 Synthesis of PAMAM G3.0-HPA (Den-HPA) PAMAM G3.0– Hydroxyphenyl acetic acid (Den-HPA) was prepared based on amide formation (-NHCO-) between some external amine groups of PAMAM and carboxylic group of HPA using an EDC coupling agent (Sch 2) Briefly, 40 mL methanol solution containing 10g PAMAM G3.0 (1.54 mmol) was neutralized to pH 6.0 in cool condition by HCl: Methanol (1:2) After

Sch 1 Synthetic scheme of TTe

neutralization, methanol was removed and then the

neu-tralized PAMAM was dissolved in 20 mL DI water HPA

(1.25 g; 8.22 mmol) and EDC (1.67 g; 8.70 mmol) were

respectively added to the neutralized PAMAM solution

under stirring for 24 h in nitrogen After this time, the

reaction mixture was dialyzed with dialysis membrane

(3500 MWCO) in methanol for 3 days The dialyzed

solu-tion was removed solvent to obtain Den-HPA 1H-NMR

(D2O)/ppm, d ppm D 2.40–2.65 (-CH2CH2CONH-);

2.70–2.85 (-CH2CH2N<); 2.85–3.10 (-CH2CH2CO-);

3.22–3.45 (-CH2NH2-), 3.60–3.70 (-CONHCH2-) and

6.75–7.15 (-CHDCH-, TA) According to the 1H-NMR

result, number of conjugated CHPA was approximately 5

HPA moieties per PAMAM dendrimer molecule

2.4 Preparation of Hydrogels and Gelation Time

Hydrogels (300 mL) containing 6.67% wt/vol of copolymers

was rapidly prepared by mixing solution A containing

100 mL TTe (100 mg/mL) and 50 mL H2O2 (0.25 wt%)

with solution B containing 100 mL Den-HPA (100mg/mL)

and 50 mL HRP (0.02 mg/mL) Final concentrations of

H2O2 and HRP were 0.042 wt% and 0.0067 mg/mL,

respectively The experiments were change H2O2and HRP

concentrations to determine the gelation time The time

taken for the gel to form (denoted by gelation time) was

determined using the test tube inversion method The

gela-tion time was determined when the solugela-tion turned into gel

that did notflow for 1 min after inverting the tube

2.5 Equilibrium Water Content

Water uptake of the hydrogels were determined by

gravi-metric method The hydrogels samples (300 mg)

containing 6.67% wt/vol of copolymers were prepared in

a tube by mixing solution A containing 100 mL TTe (100 mg/mL) and 50 mL H2O2 (0.25 wt%) with solution

B containing 100 mL Den-HPA (100 mg/mL) and 50 mL HRP at different concentrations (0.02 mg/mL; 0.04 mg/ mL; 0.06 mg/mL) Before doing any hydrogels-swelling rate experiments, the hydrogels were lyophilized with freeze-drying and weighted in their dry state (Wd) These dried hydrogel samples were immersed in PBS solution (pH 7.4) and incubated at 37
C At certain points of time, unabsorbed water was completely removed from the swelled hydrogels and quickly weighed to obtain weight (Ws) After weighing the hydrogels, PBS solution (pH 7.4) was introduced into hydrogels tubes and incubated at

37
C for the next measurement This process was repeated continuously for 30 days Swelling degrees were calculated from the following equation:

Degree of swelling.%/ DWs¡ Wd

Wd £ 100 20/:

2.6 Cytotoxicity Assay Human Foreskin Fibroblast (cell line designation: HFF-1; SCRC-1041TM; USA) was used for cytotoxicity evalua-tion To encapsulate the cells, TTe and Den-HPA solu-tions werefirst exposed to UV light for 30 min HRP and

H2O2solutions in PBS were sterilized byfiltration through 0.2 mm syringe filters Cellular hydrogels containing 6.67% wt/vol of copolymer was prepared in 24 wells plate

by mixing of H2O2-contained PAMAM G3.0-HPA solu-tion with cell and HRP-contained TTe solusolu-tion Final con-centration of H2O2 was 0.042 wt%) The cell seeding density in the gel was 1 £ 106 cell/mL The cellular

Sch 2 Synthetic scheme of Den-HPA

hydrogels were immersed in cell culture medium and

incu-bated at 37
C and 5% CO2 for 6 h and 24 h Viability

assessment of the encapsulated cells in the hydrogels were

performed by using a commercially available Viability/

Cytotoxicity kit (Molecular ProbesTM, USA) in which

composed Calcein AM and Ethidium homodimer-1 After

incubating 24 h, the cellular hydrogels was rinsed with

PBS and stained for 45 min with the combined Live/Dead

reagents at 37
C in darkness The stained cells in hydrogels

were visualized usingfluorescence microscopes

2.7 Evaluation of Drug Releasing Behavior

The PAMAM-based hydrogels samples (300 mg)

contain-ing 6.67% wt/vol of copolymers and 10 mg heparin drug

were prepared with a different amount of the PAMAM

cationic polymer (weight ratios of TTe over Den-HPA

was 3:1 and 2:1) Final concentrations of H2O2and HRP

were 0.042 wt%) and 0.0067 mg/mL, respectively For

control sample, TTe-based hydrogels containing 10 mg

heparin drugs was also prepared at a same formulation

1 mL PBS solution (pH 7.4) was respectively added into

the hydrogel-contained tubes, sharked well for 3 min

After interval times of 2 h; 6 h; 14 h; 24 h; 48 h; 72 h;

120 h; 7 days; 14 days; 4 weeks, 200 mL solution was

drawn from these tubes and fresh PBS solution (200 mL)

was added to these tubes Released heparin from the

hydrogels were determined by Toluidine blue assay

3 Results and Discussion

3.1 Characterizations of the TTe and Den-HPA

Figure 2 shows1H-NMR spectrum of the synthesized TTe

in which appears signals indicating aromatic protons of

TA at d D 6.95–7.02 ppm, methylene protons in

ethoxylate at d D 3.76 ppm and methyl proton in

block-propoxylate at d D 1.20 ppm Conjugation of TA resulted

in appearing a signal at 4.31 ppm, which was referred to

as terminated methylene protons of tetronic bound directly

to TA moieties According to integrals of methyl tetronic protons and aromatic TA protons in 1H-NMR, 97%

of TA moieties were determined to conjugate into the tetronic

Figure 3 shows spectrum of HPA-functionalized of PAMAM dendrimers in which typical peaks corresponds

to resonance signals of protons in the PAMAM den-drimers structure New signals (d D 6.75 and 7.15 ppm) of aromatic protons (HPA) could be observed PAMAM dendrimers are symmetric molecules, in which every kind

of proton in the molecules has a theoretically identify num-ber In1H-NMR spectrum of high purity dendrimers, inte-gral ratios of the resonance signals in every spectrum are equal to their ratios of these protons in the dendrimers structure According to this characteristic, it could be easy

to calculate the substitution degree of synthesized den-drimers derivatives by using formula 1 (18) In the study, percentages of the grafted HPA was easily calculated based on the integral ratio between the signals of

Fig 2.1H-NMR spectra of TTe

Fig 3.1H-NMR spectrum of Den– HPA (D2O)

-CH2CH2N< (a) and HPA According to the calculation,

number of conjugated CHPA was approximately 5 HPA

moieties per PAMAM dendrimer molecule

X%D

SH

¡ CHHPA

S ð Þa

H ð ¡ CH2 ¡ Þ

X​ H

¡ CHHPA

X​

H ð Þa

¡ CH2 ¡

£ 100%

SH ¡ CHð HPAÞ; SH ¡ CHð ð Þa2¡Þ: The peak area of protons in

–CHHPA and (a) positions appeared in the 1H-NMR

spectrum.P​

H ¡ CH HPA /; P​ H .a/

¡ CH 2 ¡ /: The sum of protons in –CHHPA (100% of theoretical HPA conjugation on 32

amine groups of PAMAM dendrimers) and (a) positions

in the molecular formula of the PAMAM dendrimers’

derivatives

x%: Degree of conjugation

3.3 Characterizations of Hydrogels

In situ forming cationic and injectable TTe-PAMAM

enzymatic-based hydrogels were prepared via an

enzy-matic reaction In an embodiment of the present invention,

4-arm-PPO-PEO (Tetronic) is conjugated with a phenol

derivative to synthesize Tyramine-Tetronic (TTe) then

mixing with modified Den-HPA which is then converted

into in situ-forming, bioadhesive hydrogel in the presence

of HRP and H2O2 (21) The gelation time changed as

varying H2O2 or HRP concentration An increment of

H2O2 concentration in ranging from 0.042 to 0.16 wt%)

(or from 4.1 to 15mM) at a constant HRP concentration

(0.0033 mg/mL) leaded to prolong hydrogels formation

(Fig 4a) This is the same behavior as with some previous

studies because an excess of H2O2could produce reversible

inactivity of HRP (22, 23, 24) The PAMAM-based

hydro-gels formation didn’t occur when the used H2O2

concen-tration was below 0.042 wt%) (Fig 4b) indicates that an

increment of HRP concentration ranging from 0.0033 to

0.0167 mg/mL and the fixed concentration of H2O2

(0.042 wt%) resulted in reducing the gelation time signi

fi-cantly It took about 7 sec to form the hydrogels at the

highest concentration of HRP (0.0167 mg/mL) The

dependence on HRP concentration could be explained by

the general theory of enzyme kinetics in which the

cross-linking reaction rate increases with increasing enzyme

con-centration With the obtained results, the polymer

solutions could be versatile to modulate its gelation time

for suitable applications such as injectable or spraying

materials Control over the gelation time was possible by

altering the HRP and H2O2concentrations, hydrogel with

0.042 wt% H O and 0.067 mg/mL HRP and gelation

within 30 s, which enables usage as injectable materials was achieved

3.4 Degree of Hydrogels Swelling The degree of hydrogels swelling was measured gravimet-rically by comparing its weight ratios in the dry and water-swollen hydrogels over the time interval The study indi-cated that maximum swelling of these hydrogels obtained after 2 days as shown in Figure 5 Hydrogels that contain HRP (0.04 mg/mL) and HRP (0.02 mg/mL) have highest degree of swelling; however, it was decreased after 48 h The degree of swelling hydrogels with HRP (0.06 mg/mL) was lower (100.51%–102.57%), and came into degradation stage after 360 h The hydrogels was prepared with a low HRP concentration exhibiting a higher degree of swelling

in early stages This is seems to be a low or high cross-linkers density formed in different HRP concentration affecting to the swelling degree The degree of swelling in hydrogels influences the pore size or crosslinking density,

Fig 4 Dependence of gelation time on catalytic concentrations: (a) effect of H2O2concentrations with 0.033 mg/mL HRP and (b) effect of HRP concentrations with 0.042 wt%) H2O2

which affects the mechanical strength of the hydrogels and

the drug release properties Moreover, the study could

predicate stability of the hydrogels network or its

degrada-tion stage In general, these hydrogels are high stability

over one month that could be a well scaffold for delivering

drugs or regenerating tissue in long time

3.5 Cytocompatibility of the Cationic Hydrogels

According to some previous reports on HRP enzyme and

H2O2-mediated hydrogels, it is necessary to consider the

amount of the used H2O2 It was reported that a highly

cytocompatible tetronic-based hydrogels prepared at

0.063 wt%) (6.2 mM) H2O2 concentration and all cells

were viable, but all cells were dead in the hydrogels

pre-pared at 0.25 wt%) (25 mM) H2O2 concentration The

study also indicated that the hydrogels were highly

cyto-compatible as prepared at 0.15 mg/mL HRP and

0.063 wt%) of H2O2concentration (18) Figure 6 indicates

that the Den-HPA and TTe-based hydrogels was prepared

at 0.042 wt%) of H2O2and 0.0067 mg/mL HRP exhibited

around 90% of cells viability According to the indicated

evidence and our obtained result, the Den-HPA and

TTe-based hydrogels with the used H2O2concentration could

be good for several biomedical applications

3.6In-Vitro Heparin Drug Release

Due to the electrostatic interaction between the loaded

heparin and the cationic hydrogels, so the hydrogels

matrix has been expected to modulate the fast or slow

release of the heparin In vitro, heparin release profiles

were obtained from three different hydrogels formulations

with a different amount of the cationic PAMAM polymer

Heparin was burst release from TTe hydrogels in threefirst

days (reaching 40% of loaded heparin) After the time, the

heparin was sustainable release and reached over 58% released heparin for one month as shown in Figure 7 This could be explained that ethylenediamine moiety in tetronic (ethylenediamine tetrakis(propoxylate-block-ethoxylate) tetrol) was protonized by hydrochloride that produced in the NPC-activated process, formed positive charges on the TTe structure Therefore, the TTe-based hydrogels could partially control the release of heparin In the presence of Den-HPA, released heparin was much slower than that of the TTe hydrogels Den-HPA could reduce burst release and increase a sustained release of heparin This is reason-able because Den-HPA resulted in increasing cationic

Fig 6 Fluorescent microscopy images offibroblasts in the cat-ionic hydrogels prepared at 0.042 wt%) of H2O2and 0.0067 mg/mL HRP after 24 h incubation (scale bar 100 mm)

Fig 7 Heparin release profile from hydrogels with a different polymer compositions: : TTe C heparin hydrogel, : TTe/Den-HPA (3:1 wt/wt) C heparin hydrogel and : TTe/Den-HPA (2:1 wt/wt)

Fig 5 The degree of swelling of hydrogels

charge density in the hydrogels that contributed to a strong

electrostatic interaction between the hydrogels matrix and

positively charged heparin That is a reason why 35%

hep-arin released over one month from the cationic hydrogels

that was prepared from Te/Den-HPA (ratio weight 3:1)

and 30% heparin released from the cationic hydrogels that

was prepared from Te/Den-HPA (ratio weight 2:1) A

slightly difference of heparin released profile between these

two hydrogels, it was due to the different of Den-HPA

amount Hydrogels with higher amount of Den-HPA

increased cationic charge density For that reason, the

hydrogels matrix formed stronger electrostatic interactions

with positively charged heparin and released smaller

amount of heparin of PAMAM-based hydrogels These

above-obtained results differ from report’s Gutowska A in

which thermo-sensitive and nonionic hydrogels composed

of N-isopropyl acrylamide (NiPAAm) copolymerized with

butyl methacrylate (BMA) or acrylic acid (AAc)

comono-mers showed 100% heparin release over 10 h for

NiPAAm/BMA and 5 days for NiPAAm/AAc (25, 26)

Other hydrogels systems also reported in which heparin

was sustainable release from the cationic gelatin-based

hydrogels or heparin-containing poly(ethylene glycol)

hydrogels However, the hydrogels were shown instability

and all hydrogels matrices were degraded after twenty

days (5, 27) A high stability of the cationic TTe and

Den-HPA-based hydrogels and ability in a sustained release of

heparin from the matrix give potential applications such

as for coating blood-contacting devices and delivering

other anionic drugs in a long term

4 Conclusions

The study successfully synthesized the cationic TTe and

Den-HPA-based hydrogels The hydrogels could control

gelation time by the modulating amount of used HRP

con-centration The cationic hydrogels exhibited a high

stabil-ity and biocompatibilstabil-ity as well as abilstabil-ity in controlling

heparin release Studies should be conducted further such

as fibrinogen adsorption and platelet adhesion on the

hydrogels surface in order to confirm its applications for

coating the blood-contacting devices or delivering anionic

drugs in a long period

Funding

This work wasfinancially supported by Vietnam National

Foundation for Science and Technology Development

(NAFOSTED) under grant number 106-YS.99-2013.29

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