Design of Carrageenan-Based Heparin-Mimetic Gel Beads as Self

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Design of Carrageenan-Based Heparin-Mimetic Gel Beads as Self-Anticoagulant Hemoperfusion Adsorbents Xin Song, Kang Wang, Cheng-Qiang Tang, Wen-Wen Yang, Weifeng Zhao, and Changsheng Zhao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01724 • Publication Date (Web): 09 Feb 2018 Downloaded from http://pubs.acs.org on February 12, 2018

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Biomacromolecules

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Design of Carrageenan-Based Heparin-Mimetic

2

Gel Beads as Self-Anticoagulant Hemoperfusion

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Adsorbents

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Xin Song,a Kang Wang,a Cheng-Qiang Tang,a Wen-Wen Yang,b Wei-Feng Zhao,a,c*

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and Chang-Sheng Zhao.a**

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a

College of Polymer Science and Engineering, State Key Laboratory of Polymer

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Materials Engineering, Sichuan University, Chengdu, 610065, People’s Republic of

8

China

9

b

10

11 12

College of Foreign Languages, University of Electronic Science and Technology,

Chengdu, 611731, People’s Republic of China c

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials,

Donghua University, Shanghai, 201620, People’s Republic of China

13 14 15 16 17 18 19

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ACS Paragon Plus Environment

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Abstract: The currently used hemoperfusion adsorbents such as activated carbon and

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ion exchange resin show dissatisfactory hemocompatibility, and a large dose of

22

injected heparin leads to the increasing cost and the risk of systematic bleeding.

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Natural polysaccharide adsorbents commonly have good biocompatibility, but their

24

application is restricted by the poor mechanical strength and low content of functional

25

groups. Herein, we developed an efficient, self-anticoagulant and blood compatible

26

hemoperfusion adsorbent by imitating the structure and functional groups of heparin.

27

Carrageenan and poly (acrylic acid) (PAA) cross-linked networks were built up by the

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combination of phase inversion of carrageenan and post-crosslinking of AA, and the

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formed dual-network structure endowed the beads with improved mechanical

30

properties and controlled swelling ratios. The beads exhibited low protein adsorption

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amounts, low hemolysis ratios, low cytotoxicity and suppressed complement

32

activation

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thromboplastin time, prothrombin time and thrombin time of the gel beads were

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prolonged over 13, 18 and 4 times than those of the control. The self-anticoagulant

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and biocompatible beads showed good adsorption capacities toward exogenous toxins

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(560.34 mg/g for heavy metal ions) and endogenous toxins (14.83 mg/g for creatinine,

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228.16 mg/g for bilirubin and 18.15 mg/g for low density lipoprotein (LDL)), thus

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highlighting their potential usage for safe and efficient blood purification.

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Keywords: Blood purification; carrageenan; heparin-mimicking; anticoagulant;

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dual-network structure.

and contact activation

levels. Especially,

2


the activated

partial

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1. Introduction:

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At present, chronic renal failure (CRF) has become one of the major threats to

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public health after cardiovascular disease, cancer and diabetes. As a terminal

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manifestation of CRF, uremia has aroused increasing attention of researchers.1-3 Renal

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transplantation may be the ultimate therapy for uremia, but donor shortage,

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postoperative immune rejection and expensive medical expenses limit its application.

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Blood purification technologies such as hemodialysis (HD),4 hemofiltration (HF),5

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and hemoperfusion (HP)6 have become crucial approaches for clinical treatment of

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uremia. Hemoperfusion, which can eliminate exogenous or endogenous toxins by

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filtering the blood of patients extracorporeally, has become one of the most effective

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methods for the treatments of heavy metal poisoning,7 hyperlipidaemia,8

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hyperbilirubinemia,9 endotoxemia10 and uremia.11

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Natural polysaccharide materials have been extensively studied for hemoperfusion

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due to their excellent biocompatibility and high hydrophilicity.9, 12, 13 Nevertheless,

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their mechanical strengths and adsorption capacities are unsatisfactory. Traditional

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adsorbents with good mechanical strengths or enough adsorption capacities, such as

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activated carbon14 and synthetic resins15 have been widely used for clinical

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hemoperfusion, but these adsorbents show undesired hemocompatibility. The

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injection of heparin during hemoperfusion increases the cost of treatment, and the

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patients also have the risk of bleeding and/or other side effects.16 Thus, the adsorbents

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composed of natural polymer and synthetic polymer with excellent biocompatibility

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and satisfactory adsorption capacity are highly desired for hemoperfusion.

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Carrageenan,

composed

of

alternate

units

with

D-galactose

and

3,

64

6-anhydro-galactose (3, 6-AG) joined by α-1, 3 and β-1, 4-glycosidiclinkage,17, 18 is a

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natural polysaccharide derived from edible red seaweeds. It has been widely used in

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food additives, pharmaceuticals and cosmetics due to its unique gelling property and

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various biological activities.19-23 Moreover, it has been reported that carrageenan

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shows anticoagulant activity because of its sulfate groups which are similar to the

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structure of heparin.24,

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common anticoagulant used in hemoperfusion process, we aim to prepare a

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heparin-mimetic hemoperfusion adsorbent based on carrageenan with superior

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anticoagulant property, so that thrombosis formation can be prevented and the use of

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heparin can be reduced remarkably. In order to overcome the weaknesses of

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carrageenan, such as poor mechanical strength and low adsorption capacity, poly

75

(acrylic acid) (PAA) was introduced since PAA gels have plenty of carboxyl groups

76

and own excellent adsorption effect.

25

Inspired by its similar structure to heparin, which is a

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Herein, we report carrageenan-based heparin-mimetic gel beads constructed by

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carrageenan and PAA. The blood compatibility of the beads would be improved by

79

imitating the polysaccharide structure and functional groups (the sulfonic groups

80

provided by carrageenan and the carboxyl groups provided by PAA) of heparin. The

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dual-network structure of the beads would endow them with improved mechanical 4


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strengths and restricted swelling ratios. The beads were characterized by Fourier

83

transform infrared spectroscopy (FTIR) and thermo gravimetric analysis (TGA), and

84

the morphologies of the beads were observed by scanning electron microscopy

85

(SEM). Then the mechanical properties and swelling ratios of the beads were

86

measured. The biocompatibility of the beads was evaluated by protein adsorption,

87

hemolysis analysis, cytotoxicity, contact activation and complement activation, as

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well as clotting time experiments. The adsorption capacities for exogenous and

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endogenous toxins were also studied.

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2. Experimental:

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2.1. Materials

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Acrylic acid (AA, containing MEHQ, 180 ppm), κ-carrageenan (κ-CRG), N,

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N’-methylene bisacrylamide (N, N’-MBA, crosslinker), α-ketoglutaric acid (initiator),

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phosphate buffered saline (PBS) and chitosan (CS, 100−200 mPa·s) powder were

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purchased from Aladdin Reagent Co. Ltd. The deacetylation degree and molecular

96

weight of CS were 95% and 100-300 kDa, respectively. Sodium dodecyl sulfate

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(SDS), potassium chloride (KCl), ethanol (C2H5OH), normal saline (0.9%NaCl),

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glutaraldehyde (GA, 50 wt. % in water) and copper sulfate (CuSO4) were purchased

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from Chengdu Kelong Chemical Reagent Co. Ltd. (China). Bovine serum albumin

100

(BSA), bovine serum fibrinogen (FBG) and creatinine were obtained from Sigma

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Chemical Co. Ltd. Human low-density lipoprotein (LDL, 99%) was purchased from

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Absin Bioscience Inc. Micro BCATM Protein Assay Reagent kit was purchased from 5



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PIERCE Inc. Activated partial thromboplastin time (APTT) reagent, prothrombin

104

time (PT) reagent and thrombin time (TT) reagent were purchased from SIEMENS

105

Co. Ltd. Deionized (DI) water was homemade and used during the whole study. All

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the chemicals were used without further purification.

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2.2. Preparation of carrageenan-based heparin-mimetic gel beads

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In this study, a simple method was used to prepare carrageenan-based

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heparin-mimetic gel beads. 1 g κ-carrageenan powder was dissolved in 50 mL DI

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water under 60 °C with magnetic stirring. Then the hot carrageenan solution was

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dropped into 0.3 M KCl solution using a bead production device (See Figure S1) with

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a speed of 100 drops/min. Sodium dodecyl sulfate (SDS) was added to prepare the

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beads with good spherical shape. To prevent the needle occluded by the gelation of

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carrageenan, the whole device was preheated for about 10 min. Then the carrageenan

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beads were immersed in a mixture solution of 0.3 M KCl and 0.3 wt. % chitosan to

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enhance the mechanical strength and thermostability of the beads.26 Then the harden

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carrageenan beads were immersed into a mixture solution of monomer (acrylic acid),

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crosslinker (N, N’-methylene bisacrylamide) and initiator (α-ketoglutaric acid) with

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continuous oscillation under dark condition for 24 h. Then, the beads were put into an

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ultraviolet analyzing equipment (ZF-20D, Shanghai glory instrument & equipment

121

Co., Ltd.) to carry out the UV-induced polymerization (wavelength: 365 nm,

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intensity: 24 W, distance of lamp from the samples: 11 cm). Subsequently, the

6


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prepared beads were washed with DI water to remove the unreacted molecules

124

thoroughly.

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To investigate the effect of carboxyl content in the beads on properties, the beads

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with different carboxyl content were prepared by varying the AA content of the

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monomer solution. As shown in Table 1, the beads prepared by 10, 15, 20 and 25 wt.

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% of AA content were named as CRG-PAA10, CRG-PAA15, CRG-PAA20 and

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CRG-PAA25, respectively. The pristine carrageenan beads were named as CRG. Table 1. The compositions of the monomer solution.

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131

Samples

CRG (wt. %)

AA (g)

MBA (g)

initiator (g)

H2O (g)

CRG

2

-

-

-

-

CRG-PAA10

2

3

0.24

0.06

27

CRG-PAA15

2

4.5

0.36

0.09

25.5

CRG-PAA20

2

6

0.48

0.12

24

CRG-PAA25

2

7.5

0.6

0.15

22.5

2.3. Characterization of the gel beads

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For Fourier transform infrared spectroscopy (FTIR) analysis, carrageenan beads and

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carrageenan-based heparin-mimetic gel beads were completely dried, and then a FTIR

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spectrometer (Nicolet 560, USA) was used to obtain the FTIR spectra between 1000

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and 4000 cm−1, using the KBr disk method. The thermogravimetric analysis (TGA)

136

curves of the beads were obtained by using a Q500 thermogravimetric analyzer

137

(TG209F1, Netzsch, Germany) under a dry nitrogen atmosphere from 50 to 750 °C at

138

the heating speed of 10 °C/min. 7



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Scanning electron microscope (SEM, JSM-7500F, JEOL, Japan) was used to

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observe the cross-section and surface morphologies of the beads. To prepare SEM

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samples, the beads were freeze-dried overnight and then cut off after being immersed

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in liquid nitrogen for 30 s. Subsequently, the samples were attached to a support and

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then coated with a gold layer under vacuum. Finally, the SEM observation was

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carried out at an accelerating voltage of 5 kV.

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In order to test the mechanical properties of the beads preliminarily, the beads were

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placed between two glass plates, on which a weight bar was held for 1 min. Then the

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beads were removed to observe if they were deformed. To further investigate the

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mechanical properties of the heparin-mimicking carrageenan gel, a cylindrical gel was

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prepared by the exactly same procedure as previously described and pressed for 5 min

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using a 200 g weight and a 500 g weight. In order to systematically study the

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compressive capacity of the heparin-mimicking carrageenan gel, the gel was

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thoroughly swollen in deionized water and then applied to a universal tensile testing

153

machine (SANS CMT4000) with a constant speed of 3 mm/min under a 200 kg load

154

mechanical sensor.

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The swelling ratios of the beads were measured by a gravimetric method.27 A

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certain amount of the wet beads were weighted after gently removing excess water

157

with a filter paper. Afterwards, the wet beads were dried at 60 °C more than 2 days to

158

get a constant weight. Then the swelling ratios of the beads were calculated by the

159

following formula (1): 8


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/ =

(1)

where We and Wd are the weights of the wet and dried beads, respectively.

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To evaluate the level of chitosan and the concentration of free sulfate groups after

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complexation in the beads, energy dispersive spectrometer (EDS) and ion exchange

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capacity (IEC) were performed. The swelling ratios and mechanical properties of the

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beads before and after hardening were also studied. Furthermore, the weight

166

percentage of the carboxyl groups in the beads was also analyzed by acidimetry. The

167

experimental details are shown in Supporting Information.

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2.4. Biocompatibility

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The biocompatibility of the beads were evaluated by protein adsorption, hemolysis

170

analysis, cytotoxicity, platelet adhesion, contact activation and complement

171

activation, as well as blood clotting time experiments; and the experimental details are

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shown in Supporting Information. The blood used for hemocompatibility tests was

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obtained from 3 donors (24-year-old males) and then centrifuged to get the platelet

174

poor plasma (PPP) and platelet rich plasma (PRP). The experiments were approved by

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West China Hospital, Sichuan University, and all experiments were performed

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according to the relevant laws and national guidelines.

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2.5. Adsorption experiments

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Cu2+ was set as the model of heavy metal ions to investigate the adsorption capacity

179

for exogenous toxins; while creatinine, low density lipoprotein (LDL) and bilirubin

180

were selected as the representatives of endogenous toxins. At least 3 parallel sample 9



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groups were applied to get a reliable value, and the results were expressed as mean ±

182

SD (n = 3).

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For Cu2+ adsorption, briefly, 2 beads (about 6 mg) were immersed into 10 mL of 40

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mmol/L CuSO4 solution at room temperature under continuously oscillation for 72 h

185

to reach the adsorption equilibrium. Then the concentrations of the solutions were

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measured by an atomic absorption spectroscopy (SPCA-626D, Shimadzu).

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To study the adsorption of creatinine, firstly, creatinine was dissolved in phosphate

188

buffered saline (PBS, pH 7.4) and the final creatinine concentration was set at 50

189

mg/L. Two beads (about 6 mg) were then added to 10 mL creatinine solution and

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incubated at 37 °C with shaking at 200 rpm. The concentrations of the creatinine

191

solutions were determined by an UV-Vis spectrophotometer (UV-1750, Shimadzu) at

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232 nm. To evaluate the utility of the beads, the removal of creatinine under patient

193

concentration (177 µmol/L) by the beads was also studied.

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In order to investigate the adsorption of LDL, two beads (about 6 mg) were

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pre-immersed in PBS overnight. To prepare LDL solution, LDL was diluted with PBS

196

and the concentration of LDL was controlled at 7.4 mmol/L. Then, the beads were

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immersed in 1 mL LDL solution at 37 °C for 3 h with constant mild shaking. The

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concentrations of the LDL solution after adsorption were determined by Cobas 8000

199

autobiochemical analyzer.

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For bilirubin adsorption, bilirubin was firstly dissolved in NaOH solution (2 mL,

201

0.1 mol/L), and then diluted with PBS to a concentration of 150 mg/L. Subsequently, 10


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two beads (about 6mg) were applied to 10 mL of bilirubin solution at 37 °C with

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constant mild shaking. All the experiments were processed under dark condition. The

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concentrations of bilirubin solution were determined by an UV–Vis spectrometer at

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the wavelength of 438 nm.

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2.6. Statistical analysis

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The sizes of all bead types as well as the pore sizes from SEM analysis were

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measured and analyzed using Image-Pro Plus 6.0 software package. Statistical

209

analyses were performed using the SPSS 11.0 software package and the data were

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expressed as means± SD. The statistical significance of differences between groups

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was determined as *P 0.05).

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3.2.4. Complement activation and contact activation levels

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To track the host defense mechanism of blood,53 the complement activation levels

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of the beads were evaluated by the determination of generated anaphylatoxins:

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Human Complement Fragment 3a (C3a) and Human Complement Fragment 5a

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(C5a).54, 55 The concentrations of C3a and C5a were determined after incubating the 24


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beads with whole blood, and the results are showed in Figure 7 (a) and (b). The

438

concentrations of C3a for the beads showed obvious decrease comparing with control

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group; while the concentrations of C5a showed significant decrease. Moreover, it has

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been reported that carrageenan might incite unwanted immune responses;56-58

441

however, the CRG-PAA beads showed decreased C3a and C5a concentrations, which

442

indicated that the inflammation responses and the complement activation would be

443

suppressed when contacted blood.

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The contact activation system is a pathophysiological surface defense mechanism

445

for foreign materials. It has been reported that platelet factor 4 (PF4) is released after

446

platelet activation when the materials contact with blood,59, 60 hence being chosen to

447

detect platelet activation for the beads. As shown in Figure 7 (c), the PF4

448

concentrations for the beads decreased obviously comparing with the plasma.

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Moreover, to further investigate the effect of the heparin-mimicking structure on

450

platelet activation, CRG-PAA gel slices were prepared and platelet adhesion tests

451

were performed. As shown in Figure S6, comparing with other blood-contacting

452

materials (such as polyethersulfone membranes), the CRG-PAA gels showed nearly

453

no platelet adhesion, pseudopodia and deformation. Combined with the suppressed

454

platelet adhesion, the beads showed inhibitory effects on platelet activation.

455

Moreover, thrombin-antithrombin (TAT) concentration is another important index to

456

evaluate the hemocompatibility of materials, since the formation of TAT complex has

457

become a surrogate marker for thrombin generation.61 As shown in Figure 7 (d), the 25



458

concentrations of TAT for the CRG-PAA beads decreased compared with that of the

459

CRG beads, indicating that the TAT generation could be suppressed.

460 461

Figure 7. The generated concentrations of C3a (a), C5a (b), PF4 (c) and TAT (d) after

462

incubating the beads with whole blood. Values are expressed as mean ± SD, n = 3.

463

3.2.5. Anticoagulant activity

464

As a kind of blood-contacting materials, hemoperfusion adsorbents should have

465

good hemocompatibility, especially anticoagulant activity, otherwise it will lead to

466

blood coagulation even thrombosis during hemoperfusion treatment.62 We expect that

467

the anticoagulant properties of carrageenan beads can be dramatically improved by

468

imitating the structure and functional groups of heparin. 26


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The anticoagulant activities of the CRG-PAA beads were evaluated by activated

470

partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT).

471

In general, APTT is used to determine the inhibitory effect of endogenous and

472

common plasma coagulation pathways63, while PT is used to evaluate the inhibitory

473

effect of exogenous and common pathways.64 TT is mainly used to measure the time

474

taken for the conversion of fibrinogen into fibrin.65 The longer clotting times mean the

475

lower possibility of thrombus formation. As shown in Figure 8 (a), the pristine CRG

476

beads could prolong the APTT value slightly. When two beads (about 14 µL) were

477

added into 200 µL PPP, the APTT value could be prolonged from 44.5 s to 50.3 s.

478

The anticoagulant activities of the CRG-PAA beads were improved significantly with

479

increasing the content of PAA cross-linked network. The APTT value of one

480

CRG-PAA25 bead could reach 413.9 s, which was over 12 times longer than that of

481

PPP. Moreover, the blood was incoagulable (APTT exceed 600 s) when two

482

CRG-PAA25 beads were added into 200 µL PPP. As shown in Figure 8 (b) and (c),

483

for the CRG, CRG-PAA10, CRG-PAA15 and CRG-PAA20 beads, the prolongations

484

of PT and TT were not as obvious as APTT. The reasons might be that Ca2+, which

485

could interact with the carboxyl groups, was more involved in the coagulation

486

pathway of APTT (Factor IX−IXa, Factor X−Xa, and prothrombin to thrombin).66

487

However, the CRG-PAA25 beads still showed excellent anticoagulant activities:

488

exceeding 250 s for PT (no coagulation) and 73.3 s for TT. In conclusion, the

489

CRG-PAA beads showed excellent anticoagulant activities, so that thrombus 27



490

formation could be prevented during the process of removing toxins from blood.

491

Thus, the use of heparin during hemoperfusion using the beads could be greatly

492

reduced, which meant that the cost of the treatment and the side effects of heparin

493

could reduce.

494 495

Figure 8. APTT (a), PT (b) and TT (c) values for the CRG and CRG-PAA beads. As

496

for the control groups, 5 µL of PBS was added into platelet poor plasma (PPP). The

497

concentrations of 7 µL/100 µL PPP and 14 µL/100 µL PPP meant that one bead and

498

two beads used in 200 µL PPP. All values are expressed as mean ± SD (n = 3).

499

3.3. Toxin removal by the gel beads

500

The discharge of heavy metal ions has become a major threat to public health.

501

When heavy metal ions enter into human body and accumulate in tissues, it will result 28


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in anemia, cancer and renal injury.67, 68 Hemoperfusion combined with drug therapy

503

had become one of the most common methods for treating heavy metal poisoning.7, 69,

504

70

505

adsorption capacity for exogenous toxins. As shown in Figure 9 (a), the Cu2+ solution

506

changed from light blue to colorless while the beads changed from transparent to blue,

507

indicating the great adsorption capacities of the beads. The adsorption amounts

508

increased with increasing the content of cross-linked PAA, and the CRG-PAA25

509

beads showed the maximum adsorption amounts of 560.34 mg/g. The robust

510

adsorption capacity to heavy metal ions was ascribed to the existence of a large

511

number of negative and oxygen-containing functional groups, which could interact

512

with heavy metal ions.71, 72

In this study, Cu2+ was selected as a model heavy metal ion to investigate the

513

Creatinine, one of the major uremic toxins, whose accumulation could cause a

514

series of neuromuscular system abnormalities and accelerate kidney failure,73 was

515

also selected as a representative toxin to investigate the adsorption capacities of the

516

beads. As shown in Figure 9 (b), the adsorption amounts increased with increasing

517

the content of PAA. However, unlike the adsorption of Cu2+, the adsorption capacity

518

for creatinine of the CRG-PAA20 (13.467 mg/g) was close to that of CRG-PAA25

519

(14.832 mg/g), which might be due to the steric hindrance of creatinine molecules.

520

Creatinine molecules showed larger size comparing with Cu2+, so the adsorption sites

521

could reach adsorption saturation easily when the content of carboxyl groups was

522

almost the same.74 Furthermore, to investigate the adsorption kinetics systematically, 29



523

the effect of contacting time on the adsorption amounts was studied and then

524

pseudo-first-order, pseudo-second-order and intraparticle diffusion kinetic models

525

were performed, as shown in Figure S7 and Table S4. As shown in Figure S7 (a),

526

almost all the adsorption processes of beads reached equilibrium after about 10 h, and

527

the adsorption amount of the CRG-PAA25 beads could reach 12.09 mg/g after about

528

4 h, which indicated that the beads could meet the demand of actual application. As

529

shown in Figure S7 (b) and (c), the adsorption process fitted intraparticle diffusion

530

kinetic model well, and agreed with the pseudo-second-order kinetic model better

531

than the pseudo-first-order kinetic model, indicating that there are diffusion steps

532

during the adsorption process,75 and the chemical process plays a leading role during

533

the adsorption process.76 The removal ratio of creatinine under patient concentration

534

was also studied, as shown in Figure S8. The removal ratio of creatinine increased

535

with increasing PAA cross-linked content, and the CRG-PAA25 could remove

536

83.89% of creatinine from the solution under patient concentration, indicating that the

537

beads showed great advantages for hemoperfusion.

538

Moreover, the adsorption capacity for bilirubin (a typical endogenous toxin which

539

could cause hyperbilirubinemia at high level77) and low density lipoprotein (LDL,

540

whose clearance was of great importance for treating familial hyperlipidemia and

541

serious cardiovascular diseases78) were also investigated. As shown in Figure 9 (c)

542

and (d), the same trends with creatinine adsorption were observed since the major

543

adsorption mechanisms were also the electrostatic interactions between the beads and 30


Page 30 of 58

Page 31 of 58 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

Biomacromolecules

544

toxin molecules. The CRG-PAA25 beads showed the maximum adsorption amounts

545

of 228.16 mg/g for bilirubin and 18.15 mg/g for LDL, respectively. The versatile

546

adsorption capacities of the beads indicated that the beads showed great potential

547

applications in hemoperfusion.

548 549

Figure 9. The Cu2+, creatinine, low density lipoprotein (LDL) and bilirubin

550

adsorption amounts per unit mass of the beads (CRG, CRG-PAA10, CRG-PAA15,

551

CRG-PAA20 and CRG-PAA25). All values are expressed as mean ± SD (n = 3).

552

3.4. State-of-the-art comparison on the hemoperfusion adsorbents.

553

In the past decades, various adsorbents for blood purification were reported.

554

However, some of them, such as activated carbon, showed high adsorption capacities 31



Page 32 of 58

555

but poor hemocompatibility, which might cause thrombus and other side effects.

556

Some adsorbents owned good biocompatibility but showed insufficient adsorption

557

capacities. Herein, for further state the advancements of this study, a systematical

558

comparison of some previous studies was made and shown in Table 2.

559

The carrageenan-based heparin-mimetic gel beads showed good adsorption

560

capacities towards exogenous and endogenous toxins comparing with other

561

adsorbents. Moreover, the beads exhibited excellent biocompatibility, especially the

562

remarkable anticoagulant property, which meant that thrombus generation could be

563

prevented during the adsorption process. Thus, the dosage of heparin could be

564

effectively reduced so that the treatment cost and the side effects of heparin could

565

decrease. Overall, the beads showed great advantages in hemoperfusion.

566

Table 2. Systematical comparison of the results of the adsorbents for blood

567

purification. Adsorbents

Adsorption capacities

Lysine-immobilized 107.2 mg/g for bilirubin chitin/carbon nanotube beads79

Biocompatibility Cell viability value: almost 100% Hemolysis ratio: less than 2% Blood clotting: PT and APTT: significant changes

no

Fe3O4@SiO2@DMSA 37.8% of removal ratio Blood clotting: nanomaterials69 for Pb(II) APTT: prolonged 100% comparing with the control 32


Page 33 of 58 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

Biomacromolecules

PT: prolonged 66.67% comparing with the control FIB: no significant changes Effect on cellular immunity and inflammation: no significant changes. Zwitterionic hydrogel 8 mg/g for bilirubin coating activated 80 carbon

Hemolysis ratio: less than 1%

Polymer brush grafted 1268 µg/g for creatinine carbon nanotube/PES composite membranes73

Blood clotting: APTT: prolonged 116.5% comparing with the control TT: prolonged 32.98% comparing with the control Contact activation: TAT: decreased 13.6% comparing with the control Platelets adhesion: decreased 94.6% comparing with the control

Non-ionic macroporous polystyrene adsorbents81

Hemolysis ratio: less than 1%

68 mg/g for bilirubin 45 mg/g for bile acid

Blood routine test: significant changes.

Dextran coated 166.90 mg/g 82 activated carbons methylene blue 7.4 mg/g for bilirubin

no

for Platelets activation: no appreciable platelet activation in blood samples

Granulocyte and monocyte 54.11 mg/g for vitamin activation: relatively small B12 increase in activation 625.0 mg/g for albumin

Blood clotting: significant changes

33


no for


Page 34 of 58

APTT, PT and TT Complement activation: reduction in the levels of complement activation for C3a, C4a and C5a Hexanediamine 60 mg/g for bilirubin functionalized poly (glycidyl methacrylate-co-Nvinylpyrrolidone) particles83

Blood clotting:

This study

Blood clotting:

560.34 mg/g for Cu(II)

APTT: prolonged 44.44% comparing with the control TT: no significant changes

14.832 mg/g creatinine

for APTT: prolonged 1348.31% comparing the control 228.16 mg/g for PT: prolonged bilirubin 1879.70% comparing 18.15 mg/g for LDL the control

over with

over with

TT: prolonged 404.9% comparing with the control Platelets adhesion: no platelets adhesion observed Hemolysis ratio: less than 2% Complement activation: C3a: decreased 23.16% comparing with the control C5a: decreased 52.52% comparing with the control Contact activation: TAT: decreased 4.83% comparing with the control PF4: 34


decreased

17.49%

Page 35 of 58 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

Biomacromolecules

comparing with the control 568

4. Conclusions

569

The carrageenan-based heparin-mimetic gel beads with dual-network structure were

570

successfully prepared through a UV-induced free radical polymerization. The results

571

of FTIR, TGA and SEM proved the introduction of PAA cross-linked network. The

572

dual-network structure endowed the beads with improved mechanical properties

573

(from 0.47 MPa to 1.25 MPa) and restricted swelling ratios (from 10.66 g/g to 1.42

574

g/g). Moreover, the beads exhibited low protein adsorption amounts, low hemolysis

575

ratios, low cytotoxicity, suppressed complement activation and contact activation

576

levels and excellent anticoagulant property. The beads showed satisfied adsorption

577

capacities towards exogenous (560.34 mg/g for Cu (II)) and endogenous toxins

578

(14.832 mg/g for creatinine, 228.16 mg/g for bilirubin, and 18.15 mg/g for LDL).

579

Thus, it is believed that carrageenan-based heparin-mimetic gel beads with superior

580

performances show strong advantages in hemoperfusion.

581

ASSOCIATED CONTENT

582

Supporting Information

583

The detailed procedures for determination of IEC of the beads before and after

584

hardening, determination of the weight percentage of the carboxyl groups in the

585

beads,

586

contact activation and complement activation, as well as blood clotting time

587

experiments, the picture of the bead production device, the results of EDS analysis,

protein adsorption, hemolysis analysis, cytotoxicity, platelet adhesion,

35



588

the swelling ratios and mechanical properties before and after hardening, the

589

calculated percentage of the carboxyl groups in the beads, ATR-FTIR spectra of the

590

beads, the results of calculated ratios of carboxyl group content to sulfate group

591

content in the beads, the calculated Young’s modulus of the beads, surface

592

morphologies, EDX mapping analysis, platelet adhesion for the beads, the adsorption

593

kinetics of the beads and the removal ratios of creatinine under patient concentration

594

by the beads.

595

AUTHOR INFORMATION

596

Corresponding Author

597

*E-mail: [email protected]

598

*E-mail:[email protected]; Tel.: +86-28-85400453; Fax: +86-28-85405402.

599

Author Contributions

600 601

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

602

Notes

603

The authors declare no competing financial interest.

604

ACKNOWLEDGMENT

605

This work was financially sponsored by the National Natural Science Foundation of

606

China (No. 51503125, 51673125 and 51773127), Program for Changjiang Scholars

607

and Innovative Research Team in University (IRT_15R48), State Key Laboratory of

36


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Biomacromolecules

608

Polymer Materials Engineering (No. sklpme2017-3-07), State Key Laboratory for

609

Modification of Chemical Fibers and Polymer Materials (No. LK1619), the State Key

610

Research

611

2016YFC1103001), and the Younth Science and Technology Innovation Team of

612

Sichuan Province (Grant No. 2015TD0001). We should also thank our laboratory

613

members for their generous help.

614

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Biomacromolecules

Figure 1. (a) The process for preparing CRG beads. (b) The graphic symbols of (c) the preparation process of the CRG-PAA beads. 388x347mm (150 x 150 DPI)



Figure 2. (a) The digital pictures of the CRG and CRG-PAA25 beads. (b) FTIR spectra for the CRG and CRGPAA25 beads. The TGA curves (c) and the DTG curves (d) for the CRG, CRG-PAA10, CRG-PAA15, CRGPAA20 and CRG-PAA25 beads. 112x89mm (300 x 300 DPI)


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Biomacromolecules

Figure 3. The cross-section morphologies of the beads. 341x134mm (150 x 150 DPI)



Figure 4. (a) The photos of the CRG-PAA25 gel compressed by a weight bar. (b) The photos of the CRGPAA25 bead compressed by a 500 g weight bar. (c) The compressive stress-strain curves of the gel beads. (d) The cyclic compressive stress-strain curves of the gel. (e) The swelling ratios of the beads. (f) Digital pictures of the beads in swollen state and dried state. 246x106mm (300 x 300 DPI)


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Biomacromolecules

Figure 5. (a) The BSA and BFG adsorption amounts of the beads. (b) The hemolysis ratios of the beads. All values are expressed as mean ± SD (n = 3). 61x25mm (300 x 300 DPI)



Figure 6. Viability of cells cultured with the samples measured by CCK-8 assay. The results were expressed as means ±SD, n=6. The CRG-PAA samples showed significant difference compared with TCP (*P < 0.05); while CRG showed no significant difference compared with TCP at 72 h (&P> 0.05). 240x165mm (150 x 150 DPI)


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Biomacromolecules

Figure 7. The generated concentrations of C3a (a), C5a (b), PF4 (c) and TAT (d) after incubating the beads with whole blood. Values are expressed as mean ± SD, n = 3. 110x85mm (300 x 300 DPI)



Figure 8. APTT (a), PT (b) and TT (c) values for the CRG and CRG-PAA beads. As for the control groups, 5 µL of PBS was added into platelet poor plasma (PPP). The concentrations of 7 µL/100 µL PPP and 14 µL/100 µL PPP meant that one bead and two beads used in 200 µL PPP. All values are expressed as mean ± SD (n = 3). 108x81mm (300 x 300 DPI)


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Biomacromolecules

Figure 9. The Cu2+, creatinine, low density lipoprotein (LDL) and bilirubin adsorption amounts per unit mass of the beads (CRG, CRG-PAA10, CRG-PAA15, CRG-PAA20 and CRG-PAA25). All values are expressed as mean ± SD (n = 3). 121x95mm (300 x 300 DPI)



Table of Contents 445x254mm (150 x 150 DPI)


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Design of Carrageenan-Based Heparin-Mimetic Gel Beads as Self

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