Differences in zwitterionic sulfobetaine and carboxybetaine dextran

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Differences in zwitterionic sulfobetaine and carboxybetaine dextran based hydrogels Xiaofeng Chen, Xia Qiu, Minghong Hou, Xiaotian Wu, Yahao Dong, Yansong Ma, Li-Jun Yang, and Yuping Wei Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01869 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

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Figure 1. Synthesis of the zwitterionic sulfobetaine (SB). 139x52mm (300 x 300 DPI)

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Figure 2. Preparation of the SB-DEX hydrogel. 110x66mm (300 x 300 DPI)


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Figure 3. 1H-NMR spectra of (A) dextran, (B) CB-DEX, (C) SB-DEX, (D) SB. 43x30mm (300 x 300 DPI)


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Figure 4. FT-IR spectra of (A) SB-DEX hydrogel, (B) CB-DEX hydrogel, (C) dextran, (D) SB-DEX. 210x148mm (300 x 300 DPI)


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Figure 5.The swelling ratios of the SB-DEX hydrogels and CB-DEX hydrogels. 204x167mm (300 x 300 DPI)


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Figure 6. The SEM images of the SB-DEX and CB-DEX hydrogels. 88x42mm (300 x 300 DPI)


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Figure 7.The microcalorimetric titration curves for (A) dextran and (B) SB-DEX. 99x64mm (300 x 300 DPI)


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Figure 8. The storage modulus of the CB-G0.20 and SB-G0.20 hydrogels. 210x148mm (300 x 300 DPI)


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Figure 9. The cumulative DOX release from drug loaded SB and CB hydrogels in different pH conditions. (A) SB-G0.20 in pH=5.0, (B) SB-G0.20 in pH=7.4, (C) CB-G0.20 in pH=5.0, (D) CB-G0.20 in pH=7.4. 210x148mm (300 x 300 DPI)


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Figure 10. The cell viability of Dextran, SB-G0.20 and CB-G0.20 hydrogels. 206x143mm (300 x 300 DPI)


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Figure 11. The cell viability of free DOX and DOX loaded hydrogels (SB-G0.20/DOX and CB-G0.20/DOX). 206x143mm (300 x 300 DPI)


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TOC graphic 44x24mm (300 x 300 DPI)


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Differences in zwitterionic sulfobetaine and carboxybetaine dextran based hydrogels Xiaofeng Chena, Xia Qiua, Minghong Houa, Xiaotian Wua, Yahao Donga, Yansong Maa, Lijun Yang*,b, Yuping Wei*,a,c Department of Chemistry, School of Science, Tianjin University, Tianjin 300354, PR

a

China Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine,

b

Institute of Radiation Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192, PR China Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),

c

Tianjin 300072, PR China Corresponding Author. E-mail: [email protected] (L. Yang), [email protected]

*

(Y. Wei). Fax: +86-22-27403475.

Abstract Zwitterionic sulfobetaine (SB) and carboxybetaine (CB) have been extensively investigated for their noticeable antifouling properties. Both SB and CB have cationic and anionic groups in the molecule but they differ in negatively charged groups. Molecular simulations have been conducted to investigate the different properties induced by structure changes. However, few studies have focused on the differences between SB and CB materials especially zwitterionic polysaccharides. In this paper, two zwitterionic sulfobetaine and carboxybetaine dextran hydrogels were designed and used as models to compare their properties. Results showed that the equilibrium 1


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swelling ratios of the SB-DEX hydrogels were much higher than CB-DEX ones and larger interior pores were observed in the SB-DEX hydrogels due to their higher hydrophilicity. Rheological storage modulus of the SB-DEX hydrogels was lower than CB-DEX ones as a result of higher water content of SB-DEX. These results were consistent with molecular modeling. Additionally, both of CB-DEX and SB-DEX had remarkable biocompatibilities. And the in vitro release studies showed the SB-DEX and CB-DEX hydrogels released DOX in a sustained manner in acidic condition (pH 5.0), indicating the promise as effective drug delivery system.

Introduction Hydrogels are soft and moisty materials consisting of three-dimensional network cross-linked via covalent or non-covalent bonds, swelling rapidly to equilibrium without dissolution.1,2 Hydrogels have been extensively investigated in biomedical applications such as biosensing,3 tissue engineering,4,5 wound-dressing6 and drug delivery,7-9 due to their high water content, excellent biocompatibility and great potential for drug loading. Despite their advantages, however, most hydrogel implants suffer from the non-specific protein adsorption and immunological response,10-12 triggering bacterial adhesion and chronic inflammation.11,12 Biomaterial-associated fouling accounts for ~45% of the nosocomial infections.13 Therefore, it’s critical to develop new materials with desired antifouling property as well as excellent biocompatibility. Recently, zwitterionic polymers have been found to be a new generation of

2


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antifouling materials. Zwitterionic materials have both cationic and anionic groups in one molecule while neutrally charged as a whole. The antifouling ability of zwitterionic polymers was attributed to the static induced hydration layer.12,14 The unique structure facilitates their increasing applications in biological fields.15,16 For example, zwitterionic polymer brushes have been demonstrated as promising stealth coatings due to the excellent antifouling performance in human blood.12 Previously, most studies focused on the zwitterionic co-polymers based on polymethacrylate or polyacrylamide backbone.17-19 However, more biocompatible materials are required for sustainable development. Polysaccharides have attracted increasing research interests because of their non-toxicity, protein-resistant feature, tunable structure, and abundance in nature.8,20 Particularly, zwitterionic polysaccharides were able to activate CD4+ T cells and thus prevent from abscess formation.21,22 Recently, a series of zwitterionic polysaccharides have been developed. For example, zwitterionic SB starch based hydrogels were fabricated for cell encapsulation and showed high resistance to nonspecific protein and cell adhesion.23 Cyclodextrin hydrogels with zwitterionic SB guest were reported to retain the beneficial nature of native cyclodextrin.24 Among these, dextran and its derivatives have been extensively exploited in biomedical applications owing to their good water solubility and antifouling properties. For example, zwitterionic CB was introduced into dextran backbone to endow the hydrogel platform with excellent antifouling property and minimized cell attachment.11 All these studies indicated that both CB and SB modified polysaccharides perform well in resisting protein

3


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adsorption although they differ in negatively charged headgroups (COO- for CB and SO3- for SB). Further, the difference of negative charges may cause varying hydration properties as predicted in Jiang group’s works.25-28 Therefore, to distinguish SB and CB functionalized polysaccharides on the molecular level is an very important issue needed to be solved. In this paper, two zwitterionic SB and CB dextran hydrogels were designed and synthesized as models. In order to figure out if the two hydrogels will show different properties as predicted in previous simulations, various properties including equilibrium swelling ratio, surface zeta potential, interior morphology and rheological properties were investigated. Furthermore, the potential for drug delivery was also studied using doxorubicin (DOX) as a model drug. Additionally, the cytotoxicity experiments were performed.

Experimental section Materials Dextran (Mn=40000) was purchased from Sinopharm Chemical Reagent Co., Ltd. (99%) and used as received. Dimethylsulfoxide (DMSO) was obtained from Tianjin Fuchen Chemical Industry Co., Ltd., distilled under reduced pressure and stored over molecular sieves. Di-tert-butyldicarbonate and propane sultone were purchased from Tianjin Heowns Biochem Technologies Co., Ltd. and used as received. N,N  -dimethylethylenediamine and N,N  -carbonyldiimidazole (CDI) were obtained

from

Shanghai

Dibai

Biological

4


Technology

Co.,

Ltd.

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4-(N,N-dimethylamino)-pyridine (DMAP) was obtained from Beijing HWRK Chem Co., Ltd. (99%) and used as received. Chloroform, methanol and ethyl acetate were purchased from Tianjin Yuanli Chemical Industry Co., Ltd. and used without further purification. Fibrinogen was provided by Shanghai Yuanye Biological Technology Co., Ltd. and stored in 4 °C environment before used. Doxorubicin hydrochloride (DOX•HCl) was obtained from Dalian Meilun Biology Technology Co., Ltd. (98%) and stored in 4 °C environment. Dialysis tubes (MWCO 7000) were purchased from Sigma. Synthesis of SB Zwitterionic sulfobetaine (SB) was synthesized as shown in Figure 1. N-Boc-dimethylethylenediamine (a). Di-tert-butyldicarbonate (9.17 g, 42 mmol) was added to the solution of N,N-dimethylethylenediamine (3.61 g, 40 mmol) in 50 mL chloroform at 0 °C dropwise and then the mixture was stirred overnight at room temperature. The solvent was removed by evaporation under vacuum. Thereafter, the same volume of water and ethyl acetate was added for extraction. The oil-like product was obtained (6.07 g, yield 80.68%) by concentrating the extracts under reduced pressure. The 1H-NMR spectrum is shown in Figure S1. 3-((3-((tert-Butoxycarbonyl)amino)ethyl)-dimethylammonio)propane-1-sulfonate (Boc-SB, b). Propane sultone (5.91 g, 48.41 mmol) was dissolved in dimethylformamide

and

added

dropwise

to

the

solution

of

N-Boc-dimethylethylenediamine (6.07 g, 32.27 mmol), and this mixture was kept stirring for 2 days at room temperature. The solvent was evaporated and the

5


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precipitate was rinsed with ethyl acetate for three times and collected by filtration, then it was dried under vacuum for 12 h. The resulting white solid was obtained (7.05 g, yield 70.50%). The 1H-NMR spectrum is shown in Figure S2. 3-((2-aminoethyl)-dimethylammonio)propane-1-sulfonate (sulfobetaine, c). 10% HCl in methanol was utilized in the removal of the Boc groups. The solid of b (7.05 g, 22.75 mmol) was dissolved in 20 ml methanol, and excess amount of HCl/MeOH was dropped into the solution at 0 °C, then the mixture was warmed to room temperature and left stirring for overnight. The final product was obtained as white powders (4.72 g, yield 98.86%) by centrifugation and drying.

Figure 1. Synthesis of the zwitterionic sulfobetaine (SB).

Synthesis of SB-DEX The sulfobetaine (SB) was covalently attached onto the dextran backbone via the amidation of the amine and hydroxyl groups using CDI as the activation agent. Dextran (0.16 g, 1 mmol) was dissolved in 15 ml dried DMSO. After dissolving DMAP (0.012 g), CDI (0.48 g, 3 mmol) was added to activate the hydroxyl group of dextran. The reaction mixture was stirred at 50 °C for 1 h under nitrogen atmosphere. Then SB (0.21 g, 1 mmol) was added, and the solution was left stirring at 50 °C for 48 6


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h under nitrogen atmosphere. The polymer solution was dialyzed against distilled water and then lyophilized to give the sulfobetaine conjugated dextran (SB-DEX) as white powders (0.19 g, yield 47.38%). Synthesis of CB-DEX CB-DEX was prepared as described in our previous work.29 Dextran (1.50 g, 9.23 mmol) was dissolved in 50 ml NaOH solution (0.08 g/ml), then 5 ml of epichlorohydrin (63.77 mmol) was added and the solution was stirred for 16 h at 30 °C, and 2 g NaOH was dissolved in the mixture before the addition of N,N-dimethylglycine (5.00 g, 48.49 mmol), and the solution was then allowed to stir for 3 days at 55 °C. Afterwards, the resulting mixture was dialyzed against distilled water for 2 days and then lyophilized. The CB-DEX was obtained as white powders (1.95 g, yield 33.68%). Preparation of SB and CB dextran based hydrogels Hydrogels were prepared following the previously reported method.2 As shown in Figure 2, SB-DEX (or CB-DEX) was dissolved in 2 M NaOH aqueous solution, and then different amounts of crosslinking agent was added to the polymer solution, and the mixture was kept at 50 °C for 1 h to give three dimensional reticulated structures. The molar ratios of MBA/SB-DEX (or CB-DEX) were 0.15, 0.20, 0.25, respectively. The detailed dosage of regents used in hydrogel preparation was shown in Table 1. The formation of hydrogels was characterized by the vial-tilting method. The resulting hydrogels were immersed in distilled water for 7 days to remove the unreacted chemicals and then lyophilized.

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Table1. The dosage of regents used in hydrogel preparation Hydrogel

Mass of zwitterionics (g)

Mass of

Molar ratio of

Volume of

samples

SB-DEX

CB-DEX

MBA (g)

MBA/zwitterionics

NaOH (mL)

SB-G0.15

0.282

0

0.016

0.15

1.0

SB-G0.20

0.282

0

0.022

0.20

1.0

SB-G0.25

0.282

0

0.028

0.25

1.0

CB-G0.15

0

0.262

0.016

0.15

1.0

CB-G0.20

0

0.262

0.022

0.20

1.0

CB-G0.25

0

0.262

0.028

0.25

1.0

Figure 2. Preparation of the SB-DEX hydrogel.

H-NMR and FT-IR characterization

1

H-NMR spectra of the synthesized SB, SB-DEX and CB-DEX were measured

1

by a Bruker Avance III 400 MHz spectrometer in D2O using TMS as the internal standard. The samples for FT-IR characterization were prepared in KBr pellets. The FT-IR tests were performed using a Nicolet 5DXC FTIR spectrometer with a scanning range of 4000-400 cm-1 and a resolution of 4 cm-1.

8


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Swelling characterization The swelling behavior of the hydrogels were measured gravimetrically2,23 as the following procedures. The prepared hydrogels were immersed in distilled water for 7 days to remove the uncrosslinked polymers, and then the samples were taken out, followed by removing excess water on the surface using filter papers, the swollen hydrogels were weighed immediately and the equilibrium swelling ratio was calculated based on the equilibrium weight ( We ) and the freeze-dried weight ( W 0 ). All tests were performed in triplicate. Equilibrium Swelling Ratio = We  W0  W0

(1)

Scanning electron microscope (SEM) characterization The morphology of the hydrogels was investigated using a scanning electron microscope (S-4800, Hitachi, Japan). The swollen hydrogels were lyophilized at −40 °C for 48 h. Then the freeze-dried samples were putter-coated with a thin layer of gold to enhance conductivity for the investigation of internal morphology. Zeta potential measurements The hydrogels prepared were crushed and soaked in distilled water for overnight. The zeta potentials of the hydrogel samples were then measured using the zeta/nano particle analyzer (Micromeritics, Co., China) with 20 scans for each sample. The average zeta potentials were directly calculated and recorded by the instrument. Isothermal titration calorimetry (ITC) The ITC200 microcalorimeter was utilized to evaluate the resistance of the zwitterionic SB-DEX against protein. Fibrinogen was chosen as a representative

9


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hydrophobic protein. Dextran and SB-DEX were dissolved in PBS buffer solution (pH = 7.4, 0.01 M) with the concentration of 1.82  M. Titration was performed by injecting the fibrinogen solution with the concentration of 7.35  M into a 280  L polymer solution. For each time, 2  L fibrinogen solution was injected, and 120 second pauses between injections were allowed for the solution to reach equilibrium. The generated calories for the interactions between the polymer and protein solutions were recorded by the instrument. Dynamic rheology Rheological experiments were carried out using a modular advanced rheometer system, (HAAKE MARS, Germany). The storage modulus ( G ' ) and loss modulus ( G ' ' ) were determined by time-sweep mode at a constant oscillator frequency (0.5 Hz) and strain (0.1%) using a 35 mm diameter parallel plate geometry and a 0.5 mm gap. All measurements were performed at 50 °C. DOX loading and in vitro release For DOX loading, 6.5 mg of the dried SB-G0.20 (or CB-G0.20) hydrogels were immersed in 2.0 mL DOX aqueous solution (0.5 mg/mL) in a tube, and the mixture was spinning at 100 rpm for 48 h at 37 °C in dark. Then the hydrogels were taken out and washed with distilled water for three times to remove the unloaded DOX on the surface. The supernatant was collected for UV-Vis spectral analysis (481 nm) of the unloaded DOX.20 The drug loading efficiency in the hydrogels was calculated based on the decrease of the original amount of DOX before drug loading.30 The standard curve of DOX is shown in Figure S5. The encapsulation efficiency ( EE ) and loading

10


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capacity ( LC ) were calculated as follows: DOX weight in the hydrogels × 100 % Feed DOX weight DOX weight in the hydrogels × 100 % LC (%) = Total weight of hydrogels EE (%) =

(2) (3)

For the controlled release, DOX-loaded hydrogels (6.5 mg) were immersed in PBS buffer (5.0 mL) with different pH, 5.0 and 7.4 respectively. The in vitro process was carried out at 37 °C. At desired time intervals, 3.0 mL DOX contained buffer was taken out to measure the DOX concentration by UV-Vis spectrophotometer (481 nm). The equal volume of corresponding fresh PBS buffer was added to maintain the total volume of the release buffer. The cumulative drug release percentage (RP) was calculated as follows: n 1

RP (%) =

Va Ci  VoCn 1

M

× 100 %

(4)

Where M represents the weight of DOX in the hydrogels, and Va

is the

withdrawn buffer volume per time ( Va = 3.0 mL), and Vo represents the whole volume of the release buffer ( Vo = 5.0 mL), and Ci is the concentration of DOX in the specific withdrawn buffer. All tests were performed in triplicate. In vitro cytotoxicity assays The cytocompatibility of the blank hydrogels was evaluated using NIH3T3 cells. The hydrogels were sterilized by 75% ethanol before the cytotoxicity assays. The NIH3T3cells suspension was added into a 96-well culture plate at a density of 8000 per well in 100 L of DMEM and incubated at 37 °C, 5% CO2 for 24 h. The original culture medium was replaced by the same volume of fresh medium containing dextran,

11


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CB-G0.20 or SB-G0.20 hydrogels with different concentrations (0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mg/mL). Cells without hydrogels treatment were used as the control. After being incubated for 48 h, 20  L of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) stock solution (5 mg/mL in PBS) was added to each well, and the cells were incubated for another 4 h. Then, the medium was aspirated, and 150  L DMSO was added to dissolve the formed blue formazan crystals. The absorbance were measured at 570 nm with a microplate reader (Varioskan Flash, USA). The experiments were performed three times separately. The antiproliferative activity of the DOX loaded hydrogels against Hela tumor cells was also examined by MTT assays in a similar way. Hela cells were incubated with free DOX, DOX loaded CB-G0.20 or SB-G0.20 hydrogels (CB-G0.20/DOX or SB-G0.20/DOX) with different DOX concentrations (0.5, 1, 2.5, 5, 10, 20 and 50 g/mL) for 48 h. The cell viability was normalized to the control group cultured with complete DMEM and each sample was measured in triplicate.

Results and discussion H-NMR and FT-IR characterization

1

12


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O

f

O

CB-DEX

O

HO

HO O

H 3C

HO

N

O

-

O

k

j a

i

g,h

l

O

i

D2O

SB

O

O S

OH

CH 3

O

b

c

O

H 3C

g

+

N

j

CH 3

h

l

m

SB-DEX

a

OH

i

O

HO

e

HO

OH

+

-

O

SB-DEX

HO

m O

d

O

O HO O

NH

OH O

k l

(D)

k

(C)

CB-DEX

(B)

Dextran

(A)

5.0

4.5

4.0 3.5 3.0 Chemical Shift (ppm)

2.5

2.0

Figure 3. 1H-NMR spectra of (A) dextran, (B) CB-DEX, (C) SB-DEX, (D) SB.

In Figure 3, the 1H-NMR spectra of natural dextran, CB-DEX, SB-DEX and SB are presented. As shown in Figure 3(D), the remarkable peak centered at 3.16 ppm (i, 6H) is assigned to the methyl groups connected to the quaternary ammonium. The peaks of propane sultone residues appear at 2.93 ppm (l, 2H), 2.19 ppm (k, 2H) and 3.66 ppm (j, 2H), and the peak at 3.50 ppm (g, h, 4H) is assigned to methylene protons of N,N  -dimethylethylenediamine. As indicated in Figure 3(C), the peaks at 4.88 ppm (a, 1H) and at the range of 4.0-3.5 ppm are assigned to dextran backbone, and the new peaks observed at 3.08 ppm (i, 6H), 2.90 ppm (l, 2H) and 2.15 ppm (k, 2H) are assigned to the corresponding protons of SB, indicating the introduction of SB onto the dextran backbone. The grafting ratio determined by 1H-NMR integration is 16%, DS = Ii/6Ia. In addition to the original signals of natural dextran, the new peak 13


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at 3.19 ppm (m, 6H) in Figure 3(B) is attributed to the methyl groups adjacent to the quaternary ammonium in CB-DEX. Based on the integration data from the anomeric protons of dextran (a, 4.88 ppm) and the methyl groups of carboxybetaine (m, 3.19 ppm), the calculated grafting ratio is 15.5%, DS = Im/6Ia.

(A)

SB-DEX hydrogel

(B)

CB-DEX hydrogel Dextran

(C)

SB-DEX

(D)

1710 3407 4000

3500

2936 3000

2500

1545 1249

2000

1500

1000

500

-1

wavenumber (cm )

Figure 4. FT-IR spectra of (A) SB-DEX hydrogel, (B) CB-DEX hydrogel, (C) dextran, (D) SB-DEX.

The FT-IR spectrum also confirms the chemical structures of the zwitterionic dextran hydrogels. As shown in Figure 4, the broad band ranging from 3000 to 3600 cm-1 is due to the -OH stretching vibration of the dextran backbone, and the band at 2936 cm-1 is assigned to -CH2 stretching vibration. Compared with unmodified dextran, the peaks at 3407 cm-1 are weakened in Figure 4(D), indicating the participation of the dextran hydroxyl groups. Besides, the remarkable absorption band at 1710 cm-1 in the SB-DEX spectrum is attributed to C=O stretching vibration, and the absorption at 1545 cm-1 is assigned to the in-plane bending vibration of N-H, 14


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suggesting the formation of the COONH carbamate. Furthermore, the appearance of the absorption at 1249 cm-1 corresponding to the C-O bond of the carbamate group also suggests the grafting of sulfobetaine onto the dextran backbone. The FT-IR spectra of the SB-DEX hydrogel and CB-DEX hydrogel are similar in the weakened signal at 3407 cm-1, substantiating the consumption of the hydroxyl groups during hydrogel formation. Additionally, the absorptions of the SB-DEX hydrogel at 1710 cm-1 and 1545 cm-1 represent the characteristic bands of sulfobetaine. Swelling characterization. Plots of the swelling ratios of the prepared SB-DEX and CB-DEX hydrogels versus crosslinking degrees are shown in Figure 5. The swelling ratio values decrease with increasing crosslinking degrees for both SB-DEX and CB-DEX hydrogels. Interestingly, the equilibrium ratios of the SB-DEX hydrogels are much higher than the CB-DEX system at the same crosslinking degree. The swelling ratio is 29.39 for SB-G0.15 compared to 10.28 for CB-G0.15. The SB-G0.20 has the swelling value of 16.34 while for CB-G0.20 it is 9.45. The swelling ratio is decreased to 8.48 for CB-G0.25 compared to 9.89 for SB-G0.25. The difference was probably because the SB-DEX was more hydrophilic thus enhanced the affinity of hydrogels for water. As described in previous work by the Jiang’s group,25 the volume of first coordination shell of sulfobetaine is larger than that of carboxybetaine because of the extra oxygen atoms in the negatively charged groups. The coordination numbers (N) of water molecules in the first coordination shell showed that these two zwitterionic molecules have similar N of the positively charged groups, however varying N of the negatively

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charged groups, indicating that sulfobetaine has more water molecules around it than carboxybetaine. This is in accord with the remarkable difference of swelling ratios of the SB-DEX and CB-DEX hydrogels.

Figure 5.The swelling ratios of the SB-DEX hydrogels and CB-DEX hydrogels.

SEM characterization The morphologies of the SB-DEX and CB-DEX hydrogels are shown in Figure 6. All samples exhibit interconnected porous network structure. The pore size of SB-DEX hydrogels was larger than the CB-DEX hydrogels for three crosslinking degrees, due to the different hydrophilicity of sulfobetaine and carboxybetaine. Sulfobetaine can absorb more water25 and thus the SB-DEX hydrogels possessed higher water content than CB-DEX ones, facilitating the formation of larger ice crystals during the lyophilization process.20,31 In contrast, the CB-DEX hydrogels with lower water content tended to form smaller ice crystals, leading to smaller pore size after lyophilization, which was consistent with the swelling capacity of the two 16


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zwitterionic hydrogels.

Figure 6. The SEM images of the SB-DEX and CB-DEX hydrogels.

Zeta potential measurements The surface charge of materials was reported to influence many properties, including protein resistance and cell adhesion.32 Zwitterionic biomaterials are neutrally charged as a whole, leading to better antifouling ability. The zeta potentials of the hydrogels were measured to determine the surface charge of the ionic dextran based hydrogels. As a control, the native dextran hydrogel was measured to be neutral with a zeta potential of -0.03 mV. The SB-DEX and CB-DEX hydrogels had zeta potentials of -0.58 mV and -0.53 mV, respectively, suggesting that the SB-DEX and CB-DEX hydrogels are zwitterionic as expected. ITC test The ITC titration curves for dextran and SB-DEX were measured with a fibrinogen solution titrant (Figure 7). It is observed that the generated heat during the titration is different between dextran and SB-DEX solution at the same concentration. 17


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As shown in Figure 7, the released heat of the dextran and fibrinogen mixture is ~0.14  cal/sec, whereas only 0.025  cal/sec of heat is generated for the SB-DEX and protein mixture, indicating that SB-DEX exhibits better resistance to fibrinogen compared to the natural dextran. The anti-protein ability of CB-DEX has been proved to be better than unmodified dextran in our previous work.29 The low-fouling performance of dextran is attributed to the hydrogen bonds induced hydration originating from the muti-hydroxyl groups on polysaccharide backbone. Furthermore, ionic solvation in the zwitterionic polymer promotes the formation of the hydration water layer which hinders the hydrophobic proteins reaching the surface of the hydrogels.29 Therefore, the protein antifouling property of SB-DEX is much better than that of natural dextran. But there is no significant improvement for both SB-DEX and CB-DEX in comparison to natural dextran when the lysozyme is used as protein model.

Figure 7.The microcalorimetric titration curves for (A) dextran and (B) SB-DEX. 18


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Dynamic rheology The rheology measurements were conducted, revealing the difference of strength between two zwitterionic hydrogels. The rheology behaviors of the CB-G0.20 and SB-G0.20 hydrogels are shown in Figure 8. The storage modulus ( G ' ) of the CB-DEX hydrogel is higher than the SB-DEX hydrogel at the fixed crosslinking degree of G0.20. The negatively charged group of CB polymer showed a higher percentage of dipole orientation distribution in theoretical studies.25,28 It implied the water molecules were more orderly oriented in CB than SB. The stronger interactions between water molecules and CB groups probably resulted in a higher strength of the CB-G0.20 hydrogel. The coordination number of CB and SB was another important factor that could account for the difference of their storage modulus. As shown in swelling tests, SB has larger coordination number of water molecules according to a previous simulation study.25 The SB-G0.20 hydrogels showed a higher water content than the CB-G0.20 hydrogels, which in turn led to a significant decrease of strength in the SB-G0.20 hydrogels. In addition, the rheological experiments of the SB-DEX and CB-DEX hydrogels with different masses of MBA were performed (see Figure S3, S4). The storage modulus was improved with the increase of crosslinking degree for both CB-DEX and SB-DEX hydrogels, which is in accord with previous work.20

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Figure 8. The storage modulus of the CB-G0.20 and SB-G0.20 hydrogels.

DOX loading and in vitro release The porous structure of hydrogels was considered to facilitate the drug loading.6 The SB-G0.20 and CB-G0.20 hydrogels were chosen to investigate the DOX loading efficiency. The EE of the SB-G0.20 hydrogels was 55.08±3.90%, and the LC was 7.55±1.06%, while the EE and LC of the CB-G0.20 hydrogels was 46.85±6.03% and 6.72±0.80%, respectively. It indicates that both SB-DEX and CB-DEX hydrogels had effective drug loading ability. As illustrated in Figure 9, the DOX release behaviors from the drug-loaded SB-G0.20 and CB-G0.20 hydrogels were pH dependent. Both of them showed a sustained release behavior in acidic condition (pH 5.0). The cumulative release ratio reached ~20% after 168 h for SB-G0.20 hydrogels, but only ~2% DOX was released in physiological condition (pH 7.4) during the whole process. And similar results were observed for CB-G0.20 hydrogels. It was probably due to the protonation of the -NH2 20


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group and the resulting solubility increase of DOX in acidic environment,20 which was favorable for the drug release. In physiological condition, however, the strong interactions between the negatively charged groups with the hydrophobic DOX impeded the drug release from the SB-DEX and CB-DEX hydrogels.29

25

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RP(%)

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A B C D

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20

40

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80

100

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140

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Figure 9. The cumulative DOX release from drug loaded SB and CB hydrogels in different pH conditions. (A) SB-G0.20 in pH=5.0, (B) SB-G0.20 in pH=7.4, (C) CB-G0.20 in pH=5.0, (D) CB-G0.20 in pH=7.4.

In vitro cytotoxicity The cytotoxicity of blank hydrogels was evaluated by the commonly used MTT method. The normal NIH3T3 cells were cultured with medium containing different hydrogels concentrations for 48 h. CB-G0.20 and SB-G0.20 hydrogels were tested using natural dextran as control. As shown in Figure 10, the cell viability showed no obvious dependence on hydrogels’ concentration. And the cell viability remained above 80% even when the concentrations of hydrogels were up to 1 mg/mL,

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indicating excellent biocompatibility of the two zwitterionic hydrogels. Additionally, the toxicity of drug loaded hydrogels (CB-G0.20/DOX and SB-G0.20/DOX) against Hela tumor cells was compared to free DOX as shown in Figure 11. The DOX concentrations in the hydrogels were fixed the same value as that of free DOX. The cell viability of DOX loaded hydrogels decreased gradually from 100% to ~20% with the concentration increasing from 0 to 50 g/mL due to the slow and sustained drug release manner. But it dropped rapidly to 1.8% for free DOX at the concentration of 50 g/mL. The notable difference indicated much lower cytotoxicity of drug loaded CB-DEX and SB-DEX hydrogels compared to free DOX, which facilitated to prolong the drug circulation time and reduce its damage to normal cells. The cytotoxicity assays suggested the SB and CB modified hydrogels had remarkable biocompatibility and great potential for drug carriers.

Dextran CB-G0.20 SB-G0.20

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Cell Viability (%)

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80 60 40 20 0

0

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Figure 10. The cell viability of Dextran, SB-G0.20 and CB-G0.20 hydrogels.

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Free DOX CB-G0.20/DOX SB-G0.20/DOX

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Cell Viability (%)

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80 60 40 20 0

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Figure 11. The cell viability of free DOX and DOX loaded hydrogels (SB-G0.20/DOX and CB-G0.20/DOX).

Conclusions In this work, two zwitterionic sulfobetaine and carboxybetaine dextran hydrogels were designed and prepared via a Michael-addition reaction using MBA as the crosslinking agent. The effects of negatively charged groups of SB and CB on their properties were investigated on molecular level using the two hydrogels as models. Results indicated that the swelling ratio of the SB-DEX hydrogel was higher than CB-DEX ones owing to the higher hydrophilicity of SB-DEX. The larger pore sizes and lower strength of the SB-DEX hydrogel were ascribed to its higher water content. These results were well in accord with previous simulations. Furthermore, both CB-DEX and SB-DEX hydrogels had great biocompatibility. More importantly, both drug loaded hydrogels exhibited obviously pH dependent and sustained DOX release behavior, making them promising candidates for drug delivery system. 23


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Acknowledgements This work was supported by the National Youth Science Foundation of China (No. 51403151) and National Natural Science Foundation of China (No. 21708048). Reference (1) Amornwachirabodee, K.; Okajima, M. K.; Kaneko, K. Uniaxial Swelling in LC Hydrogels Formed by Two-Step Cross-Linking. Macromolecules 2015, 48, 8615−8621. (2) Denizlia, B. K.; Cana, H. K.; Rzaevb, Z. M. O.; Gunera, A. Preparation Conditions and Swelling Equilibria of Dextran Hydrogels Prepared by Some Crosslinking Agents. Polymer 2004, 45, 6431−6435. (3) Vaisocherova, H.; Yang, W.; Zhang, Z,; Cao, Z. Q.; Cheng, G.; Piliarik, M.; Homola, J.; Jiang, S. Y. Ultralow Fouling and Functionalizable Surface Chemistry Based on a Zwitterionic Polymer Enabling Sensitive and Specific Protein Detection in Undiluted Blood Plasma. Anal. Chem. 2008, 80, 7894−7901. (4) Drury, J. L.; Mooney, D. J. Hydrogels for Tissue Engineering: Scaffold Design Variables and Applications. Biomaterials 2003, 24, 4337−4351. (5)Zhao, X.; Li, P.; Guo, B. L.; Peter, X. M. Antibacterial and Conductive Injectable Hydrogels Based on Quaternized Chitosan-graft-Polyaniline/Oxidized Dextran for Tissue Engineering. Acta Biomater. 2015, 26, 236−48. (6) Tang, Y. Q.; Cai, X. Q.; Xiang, Y. Y.; Zhao, Y.; Zhang, X. G.; Wu, Z. M. Cross-Linked Antifouling Polysaccharide Hydrogel Coating as Extracellular Matrix Mimics for Wound Healing. J. Mater. Chem. 2017, 5, 2989−2999. (7) Qu, J.; Zhao, X.; Peter, X. Ma.; Guo, B. L. pH-Responsive Self-Healing Injectable Hydrogel

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Hydrogels for the Prevention of Nonspecific Protein Adsorption. Carbohydr. Polym. 2015, 117, 384−391.

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Differences in zwitterionic sulfobetaine and carboxybetaine dextran

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