Bioinspired Polydopamine-Coated Hemoglobin as Potential Oxygen

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Bio-inspired Polydopamine Coated Hemoglobin as Potential Oxygen Carriers with Antioxidant Properties Quan Wang, Ruirui Zhang, Mingzi Lu, Guoxing You, Ying Wang, Gan Chen, Caiyan Zhao, Zhen Wang, Xiang Song, Yan Wu, Lian Zhao, and Hong Zhou Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 22, 2017

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Bio-inspired Polydopamine Coated Hemoglobin as Potential Oxygen Carriers with Antioxidant Properties

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Quan Wang,† Ruirui Zhang,‡, §, Mingzi Lu,† Guoxing You,† Ying Wang,† Gan Chen,†

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Caiyan Zhao,‡ Zhen Wang,† Xiang Song,† Yan Wu,*,‡ Lian Zhao,*,† Hong Zhou*,†

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8 9

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†

Beijing Institute of Transfusion Medicine, Beijing Key Laboratory of Blood

Safety and Supply Technologies, 100039 Beijing, P. R. China ‡

National Center for Nanoscience and Technology, 100190 Beijing, P. R. China

§

Beijing Key Laboratory of Ionic Liquids Clean Process, Key Laboratory of Green

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Process and Engineering, Institute of Process Engineering, Chinese Academy of

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Sciences, 100190 Beijing, P. R. China

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ACS Paragon Plus Environment

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ABSTRACT:

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development of hemoglobin-based oxygen carriers (HBOCs). To avoid the oxidative

3

toxicity, we designed and synthesized polydopamine coated hemoglobin (Hb-PDA)

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nanoparticles via simple one-step assemblage without any toxic reagent. Hb-PDA

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nanoparticles showed oxidative protection of Hb by inhibiting the generation of

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methemoglobin (MetHb) and ferryl (Fe IV) Hb, as well as excellent antioxidant

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properties by scavenging free radicals and reactive oxygen species (ROS).

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Interestingly, the scavenging rate of Hb-PDA nanoparticles for ABTS+ radical is at

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most 89% while for DPPH radical it reaches 49%. In addition, Hb-PDA efficiently

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reduced the intracellular H2O2-induced reactive oxygen species (ROS) generation.

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Moreover, Hb-PDA nanoparticles exhibited high oxygen affinity, low effect on blood

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constituents and low cytotoxicity. The results indicate that polydopamine coated

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hemoglobin might be a promising approach for constructing novel oxygen carriers

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with the capacity to reduce oxidative side reaction.

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KEYWORDS:

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properties

Oxidative side reaction is one of major factors hindering the

HBOCs, polydopamine, oxygen transportation, antioxidant

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INTRODUCTION

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Blood transfusion, clinically significant for saving lives and maintaining the

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organisms’ normal physiologic functions, is widely applied in surgical operations,

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nature disasters and battlefield wounds. However, the risks of virus transmission,

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short shelf life and severe shortage seriously affect the availability of blood-based

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products. Hemoglobin (Hb) possesses the ability to transport and transfer oxygen as

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blood substitutes to re-establish oxygen homeostasis in tissues. Nevertheless,

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unmodified hemoglobin is not ideal oxygen carriers since they have two main

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problems: one is the dissociation into dimers resulting in a short circulation time and

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renal toxicity; the other is connected with oxidative stress injury.1 Therefore, various

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approaches have been adopted to explore the possibilities for improvement on

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HBOCs, such as crosslinking,2,3 surface modification4 and encapsulation.5

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Unfortunately, serious adverse effects still occurred in phase II and phase III clinical

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trials.6, 7 For example, the lipid peroxidation related to the oxidative toxicity could be

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hindering the application of liposome-encapsulated Hb (LEH) as oxygen carriers.

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Meanwhile, either the chemically modified HBOCs or the encapsulated HBOCs

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require the complex procedure to prepare such as emulsification and cross bonding

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using inorganic reagents like glutaraldehyde，imposing restrictions on applying the

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HBOCs. Moreover, oxidative side reactions of HBOCs caused by oxidative state

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formation of Hb are still an important issue which needs to be addressed.8, 9 Hb in its

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reduced state could be auto-oxidized to generate MetHb which is incapable of

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transporting oxygen. The increasing MetHb levels (>10%) significantly decreases the

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oxygen supply to tissues transported by the total Hb.10 In addition, the ROS such as

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hydrogen peroxide (H2O2) would be generated by Hb during autoxidation.11

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Consequently, the ferryl oxidation state through the reaction of Hb with H2O2

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promotes oxidative side reactions because of the heme degradation products or iron

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release.12 Meanwhile when the ischemic tissue is reperfused in hemorrhagic shock

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and other disease states, the over-generation of superoxide (O2-) and H2O2 promotes

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oxidative toxicity of Hb and even oxidative damages. Such oxidative side reactions 3


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might be associated with the lack of protection by redox cycling (e.g. catalase,

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glutathione) in HBOCs.13 Chang et al. reported a strategy has been taken to minimize

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the above-mentioned side reactions based on cross-linking Hb with superoxide

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dismutase (SOD) and catalase (CAT) extracted from RBCs.14 A specific strategy we

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need is necessary not only to reduce the autoxidation of Hb, but also to attenuate the

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oxidative damage. Hb copolymerized with rubrerythrin or binded with haptoglobin

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(Hp) have been recognized as the prevention of Hb auto-oxidation.15,

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bovine Hb crosslinks intra-molecularly with ATP and inter-molecularly with

9

adenosine, and conjugates with glutathione (GSH) as the oxygen carrier,

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HemoTech.17 A new approach of incorporating a Pt nanoparticle (PtNP) embedded

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into the exterior HSA units has showed superoxide dismutase and catalase activities

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as the improvement of a Hb-HSA3 cluster.18, 19 The preparing procedure of the above

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approaches may be relatively complicated because of chemical cross-linking,

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meanwhile adding some inorganic reagents, which may be have influence on Hb

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activities. It is therefore essential to develop simple methods to prepare HBOCs to

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reduce the autoxidation of Hb and attenuate the oxidative damage.

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Purified

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Polydopamine (PDA) as a novel coating material could be strongly attached to

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virtually all types of the substrate surfaces in an efficient and simple process.20, 21

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Recently, Ben Wang et al. reported a catecholic chemistry-based strategy to shelter

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antigenic epitopes on Red Blood Cells (RBCs) using PDA against agglutination.22

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Furthermore, PDA has been showing great promise for biomedical applications with

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excellent hemocompatibility and biocompatibility. Meanwhile, PDA-modified

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surfaces could effectively inhibit platelet adhesion and fibrinogen conformation

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transition.23 In addition, PDA would not affect the viability or proliferation of many

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kinds of mammalian cells and showed no tissue damage, inflammatory action, or

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ﬁbrosis.24-26 Moreover, PDA could be acted as an antioxidant agent, which could

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scavenge free radicals caused by the distinct hydroquinone moiety.27

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In this work, we focused on the desirable properties of the PDA coating to

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encapsulate Hb via a simple one-step process. The morphology and chemical

30

properties were characterized. The oxygen-carrying capacity, effect on blood 4


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constituents and cytotoxicity were evaluated. Moreover, the capacity for oxidative

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protection of hemoglobin and scavenging free radicals and ROS were detected by

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five approaches. Subsequently, oxygen transportation and antioxidant activities of

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Hb-PDA nanoparticles were investigated to confirm as potential oxygen carriers

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with less oxidative side reactions. The synthetic and functional scheme of the

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Hb-PDA nanoparticle is illustrated in Scheme 1.

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8 9

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Scheme 1. Design of Hb-PDA nanoparticles for oxidative protection of Hb,

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antioxidant properties and oxygen-carrying capacity. 5


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EXPERIMENTAL SECTION

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Materials. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and dopamine hydrochloride and

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adenosine 5′-diphosphate sodium (ADP) were purchased from Sigma-Aldrich (USA).

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0.9% physiological saline (0.9% NaCl solution) was provided by Shijiazhuang SiYao

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Co. Ltd (China). Potassium hexacyanoferrate (III) (K3Fe(CN)6) was bought from by

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Sinopharm Chemical Reagent Co. Ltd (China). Cell counting kit-8 (CCK-8) assay

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was bought from by Dojindo (Japan). ROS assay kit was provided by Nanjing

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Jiancheng Bioengineering Institute (Chain). Tris-HCl buffer solution (10 mM pH 8.5)

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was bought from Beijing Leagene Biotech Co. Ltd (China). Phosphate buffer saline

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(1x PBS, pH 7.2-7.4) solution was purchased from Thermo Fisher Scientific. DMEM

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culture

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(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS+) and ferric reducing antioxidant

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power (FRAP) assay kit were purchased from Beyotime Institute of Biotechnology

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(China). Fetal bovine serum (FBS) was purchased from Hyclone (USA). Antibiotics

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were purchased from Excell Biology (Chain). All chemicals were used without further

16

purification.

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Preparation of Hb. Hb was extracted from bovine red cells. Packed fresh bovine

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whole blood (acquired from JinXiu DaDi Agricultural Park, China) was centrifuged at

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4000 g for 10 min at 4 oC. The Hb solutions were obtained by hypotonic hemolysis

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following the ultrafiltration and concentration.28 The Hb at a concentration of 22 g/dL

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was prepared via anion-exchange chromatography assay and the MetHb concentration

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was less than 5% by a blood gas analyzer (Radiometer ABL90COOX, Denmark). The

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Hb solutions were stored at -80 oC.

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Preparation of Hb-PDA. The Hb was prime-coated with PDA by the oxidative

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polymerization of dopamine. Hb was incubated with dopamine hydrochloride solution

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in Tris-HCl buffer (10 mM, pH 8.5) for 210 min at 4 oC under the slight stirring. The

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total volume of reaction system was 2 mL and then was dialyzed in PBS solution to

media

was

purchased

from

Gibco

(USA).

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2,

2-azio-bis

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remove excess dopamine hydrochloride.29 Dopamine concentration was fixed at 4.88

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mg/mL unless specified otherwise.

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Characterization of Hb-PDA. The average size of Hb and Hb-PDA nanoparticles

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were detected by dynamic light scattering (DLS) via a Zeta Sizer Nano series

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Nano-ZS (Malvern Instruments Ltd, Malvern, UK). Determinations were detected at

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633 nm with a constant angle of 90o at room temperature after the samples were

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appropriately diluted in distilled water.30 The morphology of the Hb-PDA was

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observed by transmission electron microscopy (TEM) (EM-200CX, JEOL Ltd,

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Japan).

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Chemical Structure. Fourier transform infrared spectroscopy (FTIR) was recorded

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on a FTIR spectrometer (Spectrum One, PerkinElmer). The circular dichroism (CD)

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spectra of samples was acquired on a Jasco J-800 spectropolarimeter (Japan) with a

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path length of 1cm, a bandwidth of 2 nm and a wavelength range of 250-190 nm. The

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UV-vis spectra of oxygenated Hb (OxyHb) and Hb-PDA suspensions were scanned

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using a UV–vis absorption spectrometer at room temperature (Helios, Thermo).

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Oxygen-carrying Capacity of Hb-PDA. The oxygen dissociation curves (ODCs) of

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the oxyHb and Hb-PDA suspensions were required by a Hemox analyzer (TCS

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Scientific, PA, USA). The samples (6 mg Hb) were suspended in Hemoscan buffer

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(pH 7.4, 4 mL). The temperature controller of the instrument maintained the reaction

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system

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spectrophotometer was measured at the range of the partial oxygen pressure from 150

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mmHg to 2 mmHg. The P50 was defined as the partial oxygen pressure when oxygen

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saturation reached 50% according to the Adair equation.

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Reducing Ferric Power. FRAP was measured according to the method of Beyotime

25

Institute of Biotechnology. 9 mL FRAP working solution containing 2, 4,

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6-tripyridyl-s-triazine (TPTZ) dilution, detective buffer and TPTZ solution in a ratio

at

37±0.2

o

C.

The

oxygen

saturation

using

7


a

dual-wavelength

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of 10:1:1 (v/v) was warmed to 37 oC. 10 µL Hb-PDA (0.1-0.5 mM), Hb and trolox

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was incubated with 200 µL working solution for 5 min. Trolox was regarded as a

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reference compound. The absorption peak at 598 nm revealed the presence of

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reducing product of Fe3+-TPTZ (Fe2+-TPTZ ). The samples were scanned via a

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UV-vis spectrometer with a wavelength range of 570-690 nm.

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Oxidative Protection Activity. 1 µL of 5% (w/v) K3Fe(CN)6 was added to 1 mL Hb

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solution to obtain MeHb solution.31 8 µL of Hb and Hb-PDA solution were incubated

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with 1 mL MetHb solution for 30 min at room temperature. The absorption of MetHb

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incubated with Hb-PDA, MetHb and Hb suspensions were detected on a UV-vis

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spectrometer with a wavelength range of 370-670 nm.

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ABTS+ Radical Scavenging Activity. ABTS+ radical scavenging activity was

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assayed according to the instruction of Beyotime Institute of Biotechnology. The

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ABTS+ working solution containing ABTS+ and oxidant solution reacted for 14 h at

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room temperature in the dark and was diluted by PBS solution to obtain an

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absorbance of 0.7 ± 0.05 at 734 nm. 10 µL Hb-PDA (0.1-0.5 mM), Hb and trolox

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solution was incubated with 200 µL working solution for 5 min. The absorption peak

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at 734 nm revealed the presence of ABTS+ in solution. The absorbance at 734 nm of

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the samples was read as At. Under the same conditions, A- is the absorbance of the

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samples solution in PBS buffer without ABTS+ and A is the absorbance of ABTS+

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solution without samples. The samples were scanned via a UV-vis spectrometer with

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a wavelength range of 690-810 nm. The ABTS+ radical scavenging activity was

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calculated as follows:

A – (Aｔ-A-) Scavenging Ratio (%) =

A

×100%

23

8


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DPPH Radical Scavenging Activity. 10 µL Hb-PDA (0.1-0.5 mM), Hb and trolox

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solution was incubated with 200 µL 0.1 mM DPPH solution in 95% ethanol for 30

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min.27 The absorbance at 520 nm of the samples was read as At. Under the same

4

conditions, A- is the absorbance of the samples solution without DPPH and A is the

5

absorbance of DPPH solution without samples. The absorption peak at 520 nm reveals

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the presence of DPPH in solution. The samples were scanned via a UV-vis

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spectrometer with a wavelength range of 500-620 nm. The ABTS+ radical scavenging

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activity was calculated as follows:

A – (Aｔ-A-) Scavenging Ratio (%) =

A

×100%

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Intracellular ROS Scavenging Activity．．The rat cardiomyocytes cells (H9c2) were

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cultured in DMEM media with 10% FBS and 1% antibiotics in 6-well plates at a

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density of about 1.5 × 106 cells per the culture dish surface. Cells were cultured at 37

13

o

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serum-free medium with 5 µM Hb-PDA for 12 h. After treated, 50 µM H2O2 was

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added to each well for 2 h. Wells were then rinsed with PBS and DCFH-DA of 1 µM

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was added to each dish for 20 min. Each system was washed three times with PBS.

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The samples were viewed via confocal laser microscopy with excitation wavelength

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at 488 nm (Leica DMI 4000B, Germany). To quantify the fluorescent intensity, flow

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cytometry was conducted with a Flow Cytometry Caliber（BD FACS Caliber, U.S.A.）

C with 5% CO2 for 24 h. After 24h of incubation, cells were incubated in fresh

20

9


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In vitro Platelets Aggregation. The washed human platelets were obtained from

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Blood Center of Affiliated Hospital of Academy of Military Medical Sciences. The

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washed human platelets were centrifuged at 3000 rpm for 10 min. The supernatant

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was collected as platelet-poor plasma (PPP). 1 mL the washed human platelets was

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diluted with 2 mL PPP as platelet-rich plasma (PRP). 500 µL PRP was incubated with

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10 µL different concentration of Hb-PDA for 30 min at 22 oC with shaking culture.

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The Platelets aggregation was detected with the platelet aggregometer (Chrono-log,

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USA) before and after adding 25 µL ADP（5 µM）.

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Coagulation Assays. The normal plasma was obtained from the arterial blood of the

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male Wistar rats (Vital River Laboratories, Beijing, China) with 3.2% sodium citrate

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as anticoagulant (10%, v/v) after centrifugation at 3000 rpm for 10 min at 4 oC. 1 mL

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PPP was incubated with Hb-PDA and 0.9% NaCl solution. Aliquots (1 ml) of PPP

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were incubated with particles at 37 OC for 30 min. The time of coagulation was

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performed using automatic coagulometer (Beijing Center for Drug Safety Evaluation

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and Research) by Siemens reagents. Coagulation assays were performed to evaluate

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material-induced abnormalities in the extrinsic and intrinsic coagulation pathways.

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Prothrombin time (PT) is a measure of the integrity of the extrinsic and final common

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pathways of the coagulation cascade following activation of thromboplastin (Factor

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III, volume ratio to PPP 2:1) containing Ca2+. Thrombin time (TT) is a screening

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coagulation test designed to assess fibrin formation from fibrinogen following

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activation of thrombin (volume ratio to PPP 2:1). Fibrinogen (Fbg) is known as a

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soluble protein in the plasma that is broken down to fibrin by the enzyme thrombin to

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form clots following activation of thrombin (volume ratio to PPP 2:1).

10


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Hemolysis Rate Test. Hemolysis rate test was determined according to previously

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introduction.32 The normal RBCs were drawn from the heparin-stabilized blood of the

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male Wistar rats (Vital River Laboratories, Beijing, China). 0.2 mL Hb-PDA

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solutions at different concentration were incubated for 60 min in 0.8 mL 0.9% NaCl

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solution. Then, 0.2 mL RBCs solution was added into 0.8ml Hb-PDA or water

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solution and incubated at 37 oC for another 1 h. After centrifugation at 3000 rpm for

7

10 min, the absorbance at 545 nm of the supernatant was read as At. The absorbance

8

at 545 nm of the Hb-PDA and 0.9% NaCl solution mixture was read as A. Under the

9

same conditions, a RBCs and 0.9% NaCl solution mixture solution was used as a

10

negative reference A- and a RBCs/water mixture as a positive reference A+。The

11

hemolysis rate was calculated via the following formula:

Hemolytic Ratio (%) = 12

A -A t

A - A+

×100%

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Cytotoxicity Assays. The cytotoxicity of Human Umbilical Vein Endothelial Cell

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(HUVEC) cells was detected according to a CCK-8 assay. The HUVEC cells were

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plated in 96-well plates when the density of the cells reached 80% on the culture dish

16

surface and incubated at 37 oC with 5% CO2 for 24 h. Wells were then treated with

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100 µL new culture media containing different nanoparticles concentrations of 10 µL

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0 µg/mL，250 µg/mL，500 µg/mL，1000 µg/mL，2000 µg/mL. After 24 h of incubation,

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10 µL CCK-8 solutions were added to each well. The plates were incubated at 37 oC

20

with 5% CO2 for 3 h and then determinations were performed at 450 nm. The

21

absorbance of PBS wells was read as At. Under the same conditions, cell free wells

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were used as a negative reference A-. The cell viability was calculated via the

23

following formula:

Cell Viability (%) = 24

A -A t

A - A+

×100%

25 11


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Statistical Analysis. Data are expressed in terms of the mean ± standard deviation

2

(SD) and analyzed statistically with Student’s t-test and ANOVA by SAS 9.2 (SAS

3

Institute Inc., Cary, USA).

4

5

RESULTS AND DISCUSSION

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Figure 1. (A) Photographic images of Tris-HCl buffer, Hb and Hb-PDA solution. (B) Size

8

distribution curves of Hb and Hb-PDA. (C) Size stability of Hb-PDA during 7 days. (D)

9

TEM images of Hb-PDA. The scale bar for TEM is 20 nm.

10

Fabrication and Characterization. To successfully synthesize the Hb-PDA, Hb was

11

incubated with dopamine hydrochloride in Tris-HCl buffer solution (pH 8.5) at 4 oC

12

under slight stirring. Color of the solution quickly changed from red to brown and

13

eventually became black on account of the polymerization of dopamine (Figure 1A). 12


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Unlike glutaraldehyde cross-linked Hb by reactions of amino groups33 and

2

PEG-conjugated Hb by reactions of sulfhydryl groups34 as oxygen carriers, PDA has

3

scarcely any chemical reaction with the groups of Hb amino acids and closely bonds

4

on Hb surfaces. For the attachment mechanism, PDA contains catechol function

5

groups can form covalent or strong noncovalent interactions, such as p-electron,

6

hydrogen-bonding, and other interactions.20 Compared with particle-encapsulated

7

hemoglobin using liposomes35 and polymeric particles,5 the method to prepare

8

Hb-PDA is via one-step assemblage without any toxic reagent and easy to enlarge the

9

synthetic system, avoiding liposome peroxidation and the complex preparation. The

10

size of Hb-PDA nanoparticles were approximately 6-8 nm (Figure 1B) and showed no

11

obvious changes for at least 7 days (Figure 1C). The transmission electron

12

microscopy (TEM) image also revealed that the PDA deposited on the Hb surface and

13

developed uniform nanoparticles (Figure 1D).

14

15

Figure 2. (A) FTIR spectra of Hb and Hb-PDA. (B) Uv-vis spectra ranging 220 nm from

16

650 nm of OxyHb and Hb-PDA. (C) CD spectra of Hb and Hb-PDA through scanning 190 13


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nm to 250 nm. (D) Oxygen dissociation curves of oxyHb and Hb-PDA.

2

The integrity of Hb structure in HBOCs is fundamental for transporting oxygen.

3

FTIR was used to identify the influence of the preparation process on Hb structure

4

(Figure 2A). The C-O stretching band (amide I) was located at 1643 cm-1 and the N-H

5

bending band (amide II) at 1528 cm-1 in the spectrum of the Hb and Hb-PDA, which

6

demonstrated a negligible change in the chemical structure of the Hb after PDA

7

coated.36 The peaks at 809 cm-1and 875 cm-1 corresponding to the three and five

8

substituted benzene stretching vibrations showed the modification of PDA layer. In

9

addition, the Hb with a PDA coating was also monitored through scanning 220 nm to

10

650 nm using a UV-vis spectrometer. The absorption peaks at 414 nm is regarded to

11

as the Soret band and two further peaks between 500 and 600 nm as the Q-band,

12

varying with the ligand state of the heme and oxygenation of Hb. It can be seen from

13

Figure 2B, three maxima bands at 414, 540 and 574 nm were the characteristic peaks

14

of Hb-PDA in accordance with OxyHb, which reveals integrity of oxyHb structure.

15

The absorbance spectra of oxidation products from dopamine exhibiting a

16

eumelanin-like character a characteristic band around 280 nm and a broad-band

17

absorption throughout the visible region.37 After the encapsulation, Hb-PDA showed

18

the additional characteristic peak at 279 nm, which was also proved that the

19

generation of PDA coating. To confirm whether the encapsulation process has

20

influence on the bioactivity of Hb, we detected the secondary structures via CD. As

21

shown in Figure 2C, comparing the CD spectrum of Hb-PDA with that of Hb, the

22

characteristic absorption peaks of the α-helix at 194, 210 and 224 nm were almost

23

identical and the content of α-helix about Hb and Hb-PDA was 94.8% and 93.1%,

24

respectively, which illustrated that the preparation process rarely influence on the

25

secondary structure of Hb.38

26

As an oxygen carrier, the primary biological function of Hb-PDA is maintaining

27

the capacity to bind and release oxygen. Figure 2D shows ODCs of Hb-PDA and Hb.

28

Compared to the P50 (the oxygen partial pressure at 50% oxygen saturation) value of 14


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Biomacromolecules

1

approximately 25.38 mmHg for Hb suspension, the P50 value for Hb-PDA was 13.86

2

mmHg. A high oxygen affinity (low P50) could contribute to the release of oxygen in

3

the capillaries of the ischemic tissue and is necessary to prevent vasoconstriction and

4

oxygen oversupply in the pre-capillary arterioles.39

5 6

Figure 3. Oxidative protection activity of Hb-PDA. (A) Diagram showing the auto-oxidation

7

and oxidative side reactions of hemoglobin. (B) Absorption spectra for monitoring change of

8

absorption peaks at 598 nm after treated with Hb-PDA (0.1-0.5 mM). The inset is absorption

9

spectra of iron after treated with Hb, trolox and Hb-PDA. (C) Uv-vis absorption spectra

10

about the effect of Hb-PDA on oxidation protection of Hb.

11

Oxidative Protection of Hb. The iron in Hb molecule is easy to bind and release

12

oxygen and converts into MetHb, which impairs the oxygen-carrying capacity and

13

leads to inflammation (Figure 3A). Redox character about PDA is related to the

14

quinone groups because of release of electrons during the spontaneous oxidation

15

progress.20 To confirm whether PDA could protect Hb against the formation of

16

MetHb, the evaluation of the oxidation protection activity was conducted via FRAP

17

assay kit. The insert of Figure 3B reveals that Hb has no reducing power on Fe (III), 15


Biomacromolecules

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

1

while Hb-PDA could convert Fe (III) into Fe (II). Moreover, the reducing ability was

2

improved with the increasing concentration of PDA (Figure 3B). The functionality of

3

the oxidation protection activity of Hb was monitored via UV-vis spectrum (Figure

4

3C). The oxygenation of oxyHb exhibited typical absorption peaks at 414, 540 and

5

574 nm, whereas MetHb showed the characteristic peaks at 406 and 500 nm caused

6

by oxidation of iron.40 There was a red shift in the absorption spectra of MetHb

7

solution after incubation with the Hb-PDA and the absorbance peaks at 500 nm of

8

MetHb could be not scarcely observed in MetHb and Hb-PDA mixture solution,

9

which exhibit slightly protective activity on Fe(II) because of PDA. Therefore, the

10

PDA coating is beneficial for oxidative protection of Hb to maintain its

11

oxygen-carrying capacity.

12

13

Figure 4. Absorption spectra of (A) ABTS+ and (C) DPPH after treated with 10 µL Hb,

14

trolox and Hb-PDA. Uv-vis absorption spectra about the effect of 10 µL Hb-PDA (0.1-0.5

15

mM) on (B) ABTS+ and (D) DPPH redical moleculars. The inset is the scavenging effect with

16

different concentration of PDA.

16


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Biomacromolecules

1

Antioxidant properties of Hb-PDA. Free radicals keep a balance between

2

production and elimination in the body. During severe injury and shock, the increase

3

of intracellular H2O2 accounts for the excessive generation of free radicals. Thus,

4

HBOCs with free radicals scavenging ability is significant in practical application.

5

PDA was recognized as an efficient antioxidant in the biological system for the

6

distinct hydroquinone moiety. To verify whether PDA could maintain the antioxidant

7

properties in Hb-PDA nanoparticles, in vitro antioxidant activities of Hb-PDA were

8

studied in detail.

9

The potential antioxidant activity of synthesized Hb-PDA nanoparticles was

10

extensively evaluated by DPPH and ABTS+ radical-scavenging assay. In Figure 4A,

11

after incubation with Hb-PDA nanoparticles, the absorbance at 734 nm of the all

12

samples decreased because of the scavenging ABTS+ radical. According to Figure 4B,

13

the ABTS+ radical scavenging effect of Hb-PDA nanoparticles enhanced at most up to

14

89% in dose-dependent manner, which involved electron-transfer process.41 As shown

15

in Figure 4C, the absorbance of DPPH at 520 nm was decreased, which represented

16

the ability of Hb-PDA with DPPH radical scavenging activity. In Figure 4D, the

17

scavenging ability of Hb-PDA nanoparticles on DPPH in dose-dependent manner and

18

reached 49% at the concentration of 0.5 mM due to the hydrogen-donating ability.

19

Taken together, Hb-PDA nanoparticles have an excellent antioxidant capacity through

20

scavenging a variety of free radicals. In addition, a higher concentration of Hb-PDA

21

nanoparticles shows a stronger antioxidant activity.

17


Biomacromolecules

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

1

2

Figure 5. Intracellular ROS scavenging activity of Hb-PDA in the H2O2-induced damaged

3

H9c2 cells. Cells were incubated with Hb-PDA nanoparticles for 12 h at 37 °C, and after that

4

damaged with H2O2 for 120 min and then observed by confocal microscopy and conducted

5

by flow cytometry via the probe of DCFH-DA.

6

In pathological conditions, the over-generation H2O2, an essential oxygen

7

metabolite and a source of oxidative stress, is considered as a marker of

8

ROS-associated inflammatory diseases, leading to the functional decline of the living

9

cells.42 The antioxidant capacity of Hb-PDA nanoparticles to reduce H2O2-mediated

10

oxidative damage was investigated in H9c2 cells which were incubated with Hb-PDA

11

and then damaged with H2O2 resulting in the generation of intracellular ROS. The

12

probe of dichlorofluorescein-diacetate (DCFH-DA) could enter the cells and become

13

dichlorofluoresection (DCF) to fluorescence in the presence of ROS-mediated

14

oxidation. The confocal laser fluorescence micro-scope was used to observe the

15

fluorescence intensity of cells. To measure the intracellular ROS level, the fluorescent

16

intensity of cells treated Hb-PDA is conducted by flow cytometry. As shown in

17

Figure 5, the fluorescent intensity of cells treated by Hb-PDA is weaker than that of

18

H2O2 group, which indicated Hb-PDA nanoparticles have the suppressive effects on 18


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Biomacromolecules

1

ROS generation in the H2O2-induced damaged H9c2 cells..

2 3

Figure 6. (A) Platelets aggregation rate with or without ADP after 1, 10, 100 µM Hb-PDA

4

was incubated with the PRP. (B) Hemolysis ratio and photographic image of the positive

5

control (100% lysis), the negative control (0% lysis) and groups with different

6

concentrations. (C) The effects of Hb-PDA on plasma coagulation times, including PT, Fbg

7

and TT. Aliquots (1 ml) of PPP were incubated with particles at 37 OC for 30 min. (D) The

8

cell viability of HUVEC after treated for 24 h with the different concentrations.

9

Interaction with blood constituents. Blood compatibility, a crucial property of

10

HBOCs, is important for the body’s normal physiological function. Therefore, it is

11

imperative to investigate the interaction and effects of Hb-PDA on the blood

12

compositions.

13

The platelets aggregation was examined by incubating platelets with Hb-PDA.

14

Figure 6A demonstrated Hb-PDA could not induce platelets aggregation. In addition

15

normal aggregation process was observed after adding ADP. Thus, it was concluded 19


Biomacromolecules

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

1

that Hb-PDA nanoparticles have scarcely influence on the ADP induced platelets

2

aggregation process. Furthermore, the radios of the platelets aggregation were no

3

difference in groups with different concentrations. The above-mentioned results might

4

be related to the rich in quinone of Hb-PDA or poor in amino groups.23

5

The evaluation of Hb-PDA impact on RBCs was also performed by a hemolysis

6

assay. Hemolytic activity of Hb-PDA is critical to the intravenous injection. If the

7

extent of hemolysis via the quantitation of free hemoglobin in the mixed solution is

8

higher than 5%, the sample would induce hemolytic.28 As shown in Figure 6B,

9

Hb-PDA nanoparticles with different concentrations showed a minor impact on the

10

hemolysis radio of RBCs.

11

Nanoparticles-induced clotting is still a main barrier in the clinical application. Fan

12

et al. showed that PDA, as a surface modifier, can inhibit alteration of plasma proteins

13

which triggers the coagulation of blood.30 The influences of control substrates on the

14

duration time of coagulation pathways were investigated. According to Figure 6C, the

15

results indicated no significant differences between the PT, TT and Fbg values of

16

control (0.9% NaCl solution) group and those of Hb-PDA group. Thus, the

17

coagulation assay results suggest that Hb-PDA nanoparticles had lower effect on

18

coagulation process relative to control.

19

Cytotoxicity of Hb-PDA. In vitro cytotoxicity of the Hb-PDA on HUVEC was

20

assessed by a CCK-8 assay. Figure 6D demonstrates that different concentrations of

21

Hb-PDA had no significant cytotoxic activity on the HUVEC after co-incubation for

22

24 h. Therefore, Hb-PDA nanoparticles would be a potential oxygen carrier because

23

of its excellent biocompatibility of the nanoparticles.

24

20


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Biomacromolecules

1

CONCLUSION

2

In summary, we have presented a novel HBOC employing the bio-inspired PDA to

3

encapsulate Hb with oxygen-carrying and antioxidant activities. Due to the simple

4

preparation method, Hb-PDA could maintain the integrity of Hb chemical structure

5

without introducing any toxic reagent. And the oxidative action of Hb to MetHb and

6

ferryl was decreased during the PDA coating. Excitingly, Hb-PDA exhibited excellent

7

antioxidant activity. The DPPH radical scavenging effect of Hb-PDA was almost 1.44

8

times as that of trolox and the ABTS+ radical scavenging effect reached 2 times. In

9

addition, Hb-PDA nanoparticles show the suppressive effects on intracellular ROS

10

generation. Moreover, Hb-PDA nanoparticles have low influen on blood constituents

11

and cell viability. These excellent characteristics suggested that polydopamine coated

12

hemoglobin represented a promising progress for novel oxygen carriers.

13

AUTHOR INFORMATION

14

Corresponding Authors

15

*E-mail: [email protected]

16


17


18

Notes

19

The authors declare no competing financial interest.

20

21

ACKNOWLEDGMENT The work was supported by grants from the National Natural Science 21


Biomacromolecules

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1

Foundation of China (No. 31271001) and Beijing Natural Science Foundation

2

(No. 2174085).

3

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For Table of Contents Use Only

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6 7 8 9 10 11 12 13 14 15 16 17 18

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Bioinspired Polydopamine-Coated Hemoglobin as Potential Oxygen

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