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Carbazate modified dextrans as scavengers for carbonylated proteins
Carbohydrate Polymers 232 (2020) 115802
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Carbazate modified dextrans as scavengers for carbonylated proteins a,b,1
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a,b,c,1
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Bo Zhou , Ming Gao * , Xianjing Feng , Lanli Huang , Quanxin Huang Sujit Kootalad, Tobias E. Larssone,f, Li Zhenga,b,*, Tim Bowdend,**
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a Guangxi Engineering Center in Biomedical Materials for Tissue and Organ Regeneration, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, China b Guangxi Collaborative Innovation Center for Biomedicine, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, China c Pharmaceutical College, Guangxi Medical University, Nanning, 530021, China d Department of Chemistry – Ångström Laboratory, Uppsala University, Box 538, SE-751 21, Uppsala, Sweden e Department of Clinical Science, Intervention and Technology, Karolinska Institutet, SE-141 86, Stockholm, Sweden f Department of Nephrology, Karolinska University Hospital, Stockholm, Sweden
A R T I C LE I N FO
A B S T R A C T
Keywords: Oxidative stress Protein carbonyl groups Human blood serum Hydrazide-carbonyl click chemistry Protein gel electrophoresis Carbonyl scavengers
A series of biocompatible and non- toxic polysaccharide molecules have been successfully fabricated and explored their potential application for scavenging the carbonyl species in vitro. These macromolecules were dextrans with different hydrazide substitution ratios determined by TNBS assay, NMR and FTIR characterization. The colorimetric assay had demonstrated that these macromolecules could effectively scavenge acrolein, oxidized bovine serum albumin (BSA) in buffer solutions as well as carbonyl proteins from serum. The scavengers could achieve twice more scavenging effects for modified dextrans with high molecular weight (Mw = 100,000) than those of low ones (Mw = 40,000) with the same substitution ratio. Protein gel electrophoresis confirmed that the formation of the complex between carbonyls and modified dextrans resulted in appearance of slower bands. It also revealed that such macromolecules could protect cultured cells against the toxicity of acrolein or its derivatives. The proposed macromolecules indicated a very promising capability as scavengers for oxidative stress plus its derivatives without side effects.
1. Introduction Chronic kidney disease (CKD) is an expanding public health problem that adversely affects human health and increases costs to healthcare systems worldwide (Small, Coombes, Bennett, Johnson, & Gobe, 2012). However, it can hardly be cured or relieved by current therapeutic strategies such as dialysis and transplantation, which can only improve the quality of patients (Dugbartey, 2018). Therefore, it is imperative to find new therapeutic approaches to retard CKD progression. Reactive oxygen species (ROS) have been generally accepted as one of the major mechanisms for CKD pathogenesis and progression, which may lead to renal cell apoptosis and senescence, as well as fibrosis in the kidney (Krata, Zagozdzon, Foroncewicz, & Mucha, 2018; Liu et al., 2018). ROS can produce the endogenous active aldehydes, such as acrolein, which is highly toxic and reactive with biological nucleophiles (Moghe et al., 2015; Stevens & Maier, 2008). Furthermore, the active
aldehydes can react with proteins, resulting in the condition described as “carbonyl stress” to produce carbonylated proteins. Due to their irreversible non- enzymatic modification of structures, carbonylated proteins can hardly be removed spontaneously and exist in the blood of CKD patients at all stages (Aveles et al., 2010; Costa et al., 2018; Rafael, Renato, & Cleto, 2011). Besides, carbonylated proteins have been widely demonstrated as one of the prominent biomarkers of CKD. Their accumulation further leads to loss of catalytic function, protein degradation, proteolysis and several degrees of denaturation, eventually contributing to tissue damage and organ dysfunction as well as accelerating the process of renal failure (Matsuyama, Terawaki, Terada, & Era, 2009). Hydrazide based macromolecules, such as peptide mimetic analogues of carnosine and polystyrene particles, have been proven to have the strong capability to scavenge aldehydes and carbonylated proteins (Aldini et al., 2011; Burcham & Pyke, 2006; Burcham, Fontaine, Kaminskas, Petersen, & Pyke, 2004). These hydrazide based
Abbreviation: BSA, bovine serum albumin; TNBS, 2, 4, 6- trinitrobenzene sulfonic acid; LPO, lipid peroxidation ⁎ Corresponding authors at: Guangxi Medical University, Nanning, 530021, China. ⁎⁎ Corresponding author at: Division of Polymer Chemistry, Department of Chemistry, Uppsala University, Box 538, SE-751 21, Uppsala, Sweden. E-mail addresses: [email protected] (M. Gao), [email protected] (L. Zheng), [email protected] (T. Bowden). 1 These authors contributed equally. https://doi.org/10.1016/j.carbpol.2019.115802 Received 18 October 2019; Received in revised form 23 December 2019; Accepted 27 December 2019 Available online 28 December 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. A) Schematic formation of hydrazone bonds by the reaction between hydrazine derivatives and aldehydes or ketone groups. B) Schematic illustration of scavenging effects of carbazate modified dextran molecules on carbonylated proteins.
2.2. Characterization of carbazate modified dextran
agents can react with aldehydes and carbonylated proteins by hydrazide- carbonyl coupling quickly and reliably, which generates physiologically stable and inoffensive by products with very high chemical yields (Chang et al., 2010; Lutz & Zarafshani, 2008; Thirumurugan, Matosiuk, & Jozwiak, 2013) (Fig. 1A). However, the above hydrazide based materials have unsatisfied biocompatibility, which limited their application in the clinic. Polysaccharides are natural polymers that have excellent biocompatibility and abundant groups that can be linked with hydrazine, which are the promising backbone for hydrazine. Some polysaccharides, such as alginates and hyaluronic acids, have been reported to be functionalized by hydrazine (Lou et al., 2018; Oommen et al., 2013; Wang et al., 2018). The carboxyl groups directly reacted with the hydrazine group. However, due to the limited amount of carboxyl groups and steric resistance effect, the degrees of substitution (DS) of hydrazine groups are generally only around 10 % or below, which greatly limited the efficiency of coupling (Lou et al., 2018; Oommen et al., 2013). Therefore, higher DS may be obtained by 1, 1′- carbonyl diimidazole (CDI) activation of hydroxyl groups followed by reacted with amino molecules achieving up to 25 %∼55 % DS (Bratusa et al., 2019; Zink, Hotzel, Schubert, Heinze, & Fischer, 2019), which provided a direction of the carbazate modification of polysaccharides. In this study, we innovatively chose dextran macromolecule which was a natural polysaccharide and only had hydroxyl groups after carbazate groups modification for clearance of aldehydes and carbonylated proteins by hydrazine reaction (Fig. 1). Dextran has been widely applied in biomedical fields due to its non- toxicity and intrinsically biodegradability (Sun & Mao, 2012; Van Tomme & Hennink, 2007). It has abundant hydroxyl groups that suitably acted as a backbone for hydrazine groups. Our study may provide a reference for further clinical application.
DS of carbazate groups was investigated by 2, 4, 6- trinitrobenzene sulfonic acid (TNBS, 5 % (w/ v) in H2O, Sigma) assay. 1 mg each of samples were dissolved in 20 ml sodium tetraborate decahydrate (≥ 99.5 %, Sigma) buffer (pH = 9.3, 0.1 M), and then 1 ml sample solution was mixed with 25 μl TNBS solution. After three hours of reaction, the mixture was analyzed by UV–vis spectroscopy at 505 nm and compared to a standard curve based on tert- butyl carbazate (≥ 98.0 %, Sigma) (supporting information, Figure s1). The final samples were additionally characterized by 1H, 13C NMR (Jeol JNM- ECP Series FT NMR) at 40℃ and FT- IR (Perkin Elmer Spectrum One FT- IR spectrometer). 2.3. Scavenging of acrolein Dynamic light scattering (DLS) of dextran samples- acrolein adducts. Modified dextran samples were dissolved in phosphate buffer saline (PBS) buffer (10 mM, pH = 7.4) and filtered by a syringe filter with the pore size of 0.2 μm before mixing with different concentrations of acrolein at dark for 24 h. The size and distribution of adducts were measured by the zeta sizer nano instrument (Malvern Instruments, UK). Spectroscopic analysis of acrolein scavenging efficiency: The modified dextran samples were dissolved in PBS buffer (10 mM, pH = 7.4) and filtered through a syringe filter with the pore size of 0.2 μm to give stock solutions. Stock solutions were charged in dialysis tubes (Slide- ALyzer MINI Dialysis Devices, 3.5 kD MWCO, Fisher) and then incubated in 2 ml different concentrations (100, 200, and 500 μM) of acrolein PBS buffer solution in the dark. After 24 h, the absorbance was measured at 209.5 nm, corresponding to the absorption maxima for acrolein, using a UV–vis spectrometer. Observed values were compared with corresponding acrolein solutions treated under the same condition in the absence of dextran samples. The absorbance of acrolein with different concentrations in PBS buffer can be found in supporting information (Figure s2) as standard curves.
2. Experimental 2.1. Synthesis of carbazate modified dextran
2.4. Spectroscopic analysis of protein carbonyls General procedure (Fig. 2): To a 0.5 g of dextran (Fluka) was 20 ml dimethyl sulfoxide (DMSO, ≥ 99.9 %, Sigma) added. The mixture was heated at 80℃ for 15 min. to dissolve the dextran totally. After cooling to room temperature, 1 g 1, 1′- carbonyldiimidazole (CDI, ≥ 97.0 %) was added and the mixture was stirred for 24 h. 3.2 ml of hydrazine hydrate (80 %, Sigma) was added to the mixture, which was left to stir for another 24 h. The reaction mixture was diluted with water and dialyzed (Spectra/Por ® 6 Dialysis Membrane, MWCO: 3.5 kD) for three days and the product was isolated as white powders after lyophilization.
Protein carbonyls measurements were typically performed as follows. (Reznick & Packer, 1994) 1 ml of 2, 4- dinitrophenylhydrazine (DNPH, 97 %, Sigma) (10 mM in 2 M HCl) was added to 200 μl protein samples (6 mg/ml in PBS buffer) and incubated at room temperature in the dark for 45 min. The mixture was chilled for 10 min. using an ice bath and then centrifuged at 3000 rpm for 10 min. after adding 1.2 ml cold trichloroacetic acid (TCA, ≥ 99.0 %, Sigma) (20 wt% in PBS buffer) in order to precipitate the proteins. The protein pellet was washed three times by 2.5 ml ethanol/ ethyl acetate (volume ratio of 1:1) before re- dispersing in 6 M urea (98 %, Sigma) PBS buffer. Pristine 2
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Fig. 2. Synthesis of carbazate modified dextran.
cells were washed with PBS buffer and 100 μl 1 % Alamar blue solution (Life Technologies) was added to each well. The plate was incubated at 37℃, 5 % CO2 for 2 h. The absorbance was measured at 570 nm by a microplate reader (Tecan infinite M200). The results were compared with control wells to determine relative cell viability.
bovine serum albumin (BSA, Sigma) sample only treated with 2 M HCl without DNPH was used as a negative control for carrying the spectroscopic measurements. The absorbance was read by UV–vis spectroscopy at the wavelength of 370 nm. 2.5. Preparation of oxidized and fully reduced BSA
2.8. Protein gel electrophoresis Oxidized BSA and fully reduced BSA were prepared as previously described. (Alamdari et al., 2005; Buss, Chan, Sluis, Domigan, & Winterbourn, 1997) Briefly, 0.35 g of BSA was dissolved in 35 ml PBS, and 1 mM H2O2 and 1 mM ferrous sulfate were added. The mixture was incubated for 20 min., followed by overnight dialysis against PBS buffer at 4℃ to obtain the oxidized BSA solutions. For preparation of fully reduced BSA, 1 g BSA was dissolved in 100 ml PBS and 1 g of solid NaBH4 was added. The mixture was incubated for 30 min. at room temperature before slowly adding HCl (2.5 M) to neutralize the reaction mixture, followed by overnight dialysis against PBS buffer at 4℃. The protein concentrations of the oxidized and reduced BSA solutions were measured by the Bradford assay, then adjusted with PBS to 6 mg/ml and stored at -80℃ before use. Oxidized BSA standard curves were constructed by mixing various different proportions of oxidized BSA with fully reduced BSA to maintain a constant total protein concentration and obtained by the analysis of protein carbonyls measurements (supporting information, Figure s3).
All protein samples were diluted to 1 mg/ml by PBS buffer for gel electrophoresis. The measurements were implemented by running the polyacrylamide gels (mini- protean® TGX™ precast gels, Biorad) at 100 V for 1.5 or 2 h after loading 10 μl protein samples in each of wells. After running, the gels were stained with coomassie blue dyes (Biorad) and then analyzed. 3. Results and discussion 3.1. Synthesis and characterization of carbazate modified dextrans Dextran, a hydroxyl rich hydrophilic polysaccharide, was chosen as a backbone for carbazate nucleophilic groups suitable for scavenging electrophiles such as aldehydes and carbonylated proteins. Hydrazide derivatives have long been used in organic synthesis to form stable hydrazone bonds (Fig. 1A) after reaction with carbonyl compounds and have also been in focus as low molecular weight scavengers. The novelty of combining hydrazone chemistry and hydrophilic polymers has the potential to give macromolecules scavengers with the high local concentration of nucleophiles, the possibility for local delivery and retention, and possible altered pharmacokinetics. We also hypothesized that hydrophilic polymers scavenged proteins and made them less cell toxicity if the polymers acted as a hydrated coating. This would be similar to stealth technology offered for macromolecules, proteins and particles by simple PEGylation. The multiple hydroxyl groups presented in dextran would be the obvious choice for introducing carbazate derivatives with minimal alteration of the native backbone, e.g. the breakage of the pyranose ring structure. Using CDI, a phosgene derivative, (Bethell, Ayers, Hearn, & Hancock, 1987; Lanzillotto, Konnert, Lamaty, Martinez, & Colacino, 2015) and hydrazine, a carbazate group can be introduced in a one- pot two- step procedure. CDI was added to dextran dissolved in DMSO whereupon one imidazole is displaced by a dextran hydroxyl group to form an imidazole carbonate intermediate (alkyl carbamate). After the addition of hydrazine, the second imidazole was displaced to produce the carbazate group (-O-C(O)-NHNH2). Removal of DMSO, liberated imidazole and excess reagents was effectively done with dialysis. The modified dextrans were isolated after lyophilization. The carbazate DS was obtained by TNBS assay based on tert- butyl carbazate standard curves (Figure s1). DS was defined as the number of carbazate groups per glucose units presented in the starting material, e.g. DS 0.2 would mean that 20 % of glucose units carried a carbazate. Four different modified dextrans were synthesized based on DS (low and high) and molecular weight (40,000 g/mol denoted low (L) or 100,000 g/mol denoted high (H)). The products obtained are
2.6. Scavenging effects on carbonylated protein samples BSA was dissolved in PBS buffer at a concentration of 6 mg/ml for used as standards and following measurements. The oxidized BSA samples for scavenging effects were prepared by mixing the fully reduced BSA and oxidized BSA solutions with the volume ratio of 1:1, defined as 50 % oxidized BSA. Additionally, BSA (6 mg/ml in PBS buffer) solutions were mixed with acrolein to respectively obtain 100, 200 and 500 μM acrolein in the final solutions at dark for 24 h, defined as acrolein treated BSA samples. Furthermore, for human blood serum from patients, the protein concentration of serum was measured by Bradford assay and adjusted to 6 mg/ml by addition of PBS buffer to obtain blood serum samples. The all above protein samples (2 ml) were respectively mixed with 10 mg, 20 mg, and 40 mg dextran samples for 24 h at dark before the mixtures were analyzed for protein carbonyls. 2.7. Cell viability and cytotoxicity Mouse myoblast cells (C2C12) were cultured in Dulbecco's modified eagle medium (DMEM, Life Technologies) supplemented with 10 % fetal bovine serum (FBS, Life Technologies) and 0.1 % penicillinstreptomycin to near 80 % confluence. Cells were maintained at 37℃ with 5 % CO2 at 90 % humidity for 24 h and then seeded in 96- well plates with a density of 8000 cells per ml. Cells were replaced with fresh medium and then respectively added the corresponding sample solutions (acrolein- or acrolein treated BSA- dextran samples) to reach total 100 μl medium in each of wells. After incubated for another 24 h, the 3
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no clear changes in the hydrodynamic diameter of the polymers were observed. This indicated that the overall dispersity in PBS of the polymeric system was unaltered by its modifications. Modified polysaccharide molecules below 50 nm in PBS buffer would not be easy to be absorbed by cells and beneficial to the treatment of oxidative stress in vivo.
Table 1 Reaction conditions and DS of carbazate modified dextrans. Product
Dextran (g)
DMSO (ml)
CDI (g)
Hydrazine (80 %, ml)
DS (TNBS)
L1 H1 L2 H2
1 1 1 1
20 20 20 32.5
2 2 2 2
10 10 6.4 6.4
0.2 0.17 0.52 0.57
(Mw = 40000) (Mw = 100000) (Mw = 40000) (Mw = 100000)
3.2. In vitro scavenging effects of carbazate modified dextran The ability of the carbazate functional dextrans to scavenge endogenous electrophiles was tested in four set- ups: acrolein, 50 % oxidized BSA, acrolein treated BSA and human blood serum.
summarized in Table 1. FT- IR results of dextran products were illustrated in Figure s4. For comparison, unmodified dextran (Mw = 40,000) was used and denoted as L0. The characteristic eC]O stretching vibration was detected at 1710 cm−1 (L1, L2, H1 and H2 compared to L0) increasing with DS, while the intensity of the eOH stretching (3499 cm−1) decreased. The peak around 1900∼2200 cm−1 was the background of the instrument. NMR characterization of dextrans was implemented at 40℃ in D2O. For 1H NMR (Fig. 3A), the signal at 4.65∼4.85 ppm is assigned to the solvent (D2O) curve. For α- 1→ 6- glucosyl residue of dextrans it was shifted to 5.16 ppm (1 in Fig. 3A) at 40℃. The extra signals appeared at 5.35 ppm for carbazate modified dextrans (L1, L2, H1 and H2) compared to unmodified dextran (L0) were the protons of -NHNH2 groups while the intensity of sugar ring protons in the range of 3.5∼4.2 ppm (2–6 in Fig. 3A) decreased with the increased DS of dextrans. Furthermore, their 13C NMR was also presented in Fig. 3B. The specific carbons (eC]O) detected at 158∼160 ppm for high DS of modified dextran (L2 and H2) comparing with unmodified dextran (L0). The multi signals for specific carbons meant the modification succeed in different parts of sugar rings. However, no peaks existed at a similar ppm for L1 and H1 due to the low modification of carbazate groups. DLS data revealed that the hydrodynamic sizes of dextran scavenger samples in PBS buffer were around 12 nm (L1 and L2) and 23 nm (H1 and H2) respectively (Figure s5). Increasing the concentration of dextrans in PBS from 10, 20–50 mg/ml, still showed the same dynamic size. Even after reacting the carbazates with acrolein (100, 200 or 500 μM),
Fig. 3. 1H (A) and
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3.2.1. Scavenging effects on acrolein Carbazate modified dextrans were placed inside the dialysis tube (MWCO: 3.5 kD) with a cut- off of 15 ml syringe tube. During the incubation, dextran molecules could not pass through the membrane to acrolein medium, due to their higher molecular weight, while acrolein had the possibility to enter into the tube and react with carbazate groups. Measuring the acrolein concentration by UV absorbance in a medium outside the dialysis tube indirectly related to the amount of acrolein bound to dextrans without the interference from polysaccharides presented in the tube. Results illustrated that 2 mg samples could respectively remove almost 0.18 μmol (L1), 0.26 μmol (L2), 0.23 μmol (H1) and 0.29 μmol (H2) of acrolein when exposure to 2 ml, 200 μM acrolein buffer. Increasing the amount of L2 from 2 mg to 10 mg, 0.31 μmol of added acrolein could be scavenged. If the amount of acrolein was increased to 1 μmol, only 0.33 μmol was removed by 2 mg of L2 (Fig. 4A). From the experiments, it was shown that a higher carbazate DS gave more efficient scavenging of acrolein, which corresponds to the higher concentration of functional groups on the polymer, L1 vs. L2 and H1 vs. H2. Increasing the amount of modified dextran from 2 mg to 10 mg also increased the scavenging effect as a result of increased concentration of functional groups. Interestingly, high molecular weight dextrans seemed to be better scavengers of acrolein than their low molecular counterparts even at comparable DS, L1 vs H1 and
C (B) NMR spectra of dextran samples dissolved in D2O at 40℃. 4
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Fig. 4. Scavenging effects on acrolein. A) Scavenging efficiencies on 2 ml different concentration of acrolein (0.1, 0.2 and 0.5 μM) buffer after exposure to different types of carbazate modified dextrans (L1, L2, H1 and H2 with 2 mg), or different amount of L2 (2, 4 and 10 mg) in PBS buffer. B) Cell viability of C2C12 cells after incubation for 24 h respectively with 2 mg/ml L2 (a), 30 μM acrolein (b), 30 μM acrolein- 2 mg/ml unmodified dextran (L0) (c), 30 μM acrolein- 1 mg/ml L2 (d), 30 μM acrolein- 2 mg/ml L2 (e), 30 μM acrolein- 5 mg/ml L2 (f), and 30 μM acrolein- 2 mg/ml H2 (g).
L2 vs H2. The carbazate groups in higher Mw dextran with more branched chains were easier to capture the carbonyl groups. Therefore it would be more efficient for high Mw macromolecules (H2) than low Mw macromolecules (L2) to scavenge the acrolein in PBS buffer. Fig. 4B described the viability of C2C12 cells after incubating in different corresponding cell mediums for 24 h. Carbazate modified dextran molecule showed no apparent toxicity so that close to 100 % of cells were metabolically unaffected in cell medium mixing with 2 mg/ ml of L2. However, around 89 % of cells died after the addition of 30 μM acrolein showing the cell toxic properties of acrolein. When dextrans were added subsequently to acrolein, the cell survival increased as a result of scavenging. Cells were only incubated with 30 μM acrolein for 10 min. to make sure that no cell death was induced by acrolein before adding the scavengers (Burcham & Fontaine, 2001). Addition of L2 to cell medium containing acrolein saved 40 % of the cells at 1 mg/ ml and 92 % at 5 mg/ml. The addition of unmodified dextran L0 (2 mg/ ml) had no effect on cell survival. When H2 was added 87 % of cells survived at 2 mg/ml. The results showed a clear dose response where a higher concentration of carbazates gave a higher scavenging effect. Similar to the scavenging experiments in PBS it could be noticed that the high molecular weight dextran (H2) was a better scavenger than the low molecular counterpart (L2) as manifested by the higher survival ratio of cells exposed to H2.
Fig. 5. Scavenging efficiencies on 2 ml, 6 mg/ml 50 % oxidized BSA samples after exposure to different types of carbazate modified dextrans (L1, L2, H1 and H2 with 20 mg) or different amount of L2 (10, 20 and 40 mg) in PBS buffer.
using the amount of 20 mg. A four- fold increase in the amount of modified dextran L2 from 10 mg to 40 mg had almost similar increase in scavenging capacity, from 20 % (2.37 mg) to 70 % (8.41 mg), of acrolein treated BSA, showing a linear dose response. BSA treated by different concentrations of acrolein (200 μM or 100 μM) was scavenged with different capacities using the same amount of dextran samples (20 mg) and different amounts of L2 (Fig. 6A). Using different amounts of acrolein would affect the number of carbonyl groups introduced in the protein and this would, in turn, affect the scavenging. More carbonyl groups would consume more carbazates and from the results, it could be seen that scavenging efficiency decreased from 7.48 mg to 6.01 mg when the BSA became more carbonylated. Fig. 6B described the capability of carbazate modified dextrans to protect C2C12 cells against the toxicity of acrolein treated BSA. Herein, BSA was treated with 500 μM acrolein and incubated for 24 h in the dark to make sure that acrolein totally reacted with BSA. Viability of the cells decreased to 3 % when treated with 3 mg/ml acrolein treated BSA probably as a result of cell death showing the toxicity of carbonylated BSA. When carbazate modified dextran L2 (5 mg/ml) was added to the culture cell, cell survival manifested in the increase of viability (41 %). The unmodified dextran L0 had no rescuing effects since viability was 8 % at 10 mg/ml. If 20 mg/ml L2 or 10 mg/ml H2 was applied to the same mixtures, it was observed that approximately 100 % of cells would be rescued from acrolein treated BSA toxicity. Protein gel electrophoresis was utilized to shine some light on the protein polymer complexes presumably formed when the functional polymer was covalently linked to the protein through the hydrazone linkage. A few different observations could be made from the protein
3.2.2. Scavenging effects on oxidized BSA or acrolein treated BSA 50 % oxidized BSA was utilized as oxidized BSA samples for scavenging effects due to its linear relationship between UV absorbance and carbonylated proteins concentration inside the PBS buffer. The scavenging effects on 50 % oxidized BSA samples were shown in Fig. 5. For unmodified dextran L0, there was no scavenging effect observed with about 98 % carbonylated proteins left in PBS buffer. 20 mg L2 (Mw = 40,000 g/mol, DS 0.52) could scavenge 1.47 mg of oxidized BSA from 2 ml, 6 mg/ml 50 % oxidized BSA while L1 with DS 0.20 only removed 0.80 mg of oxidized BSA. If the amount of L2 was increased to 40 mg, the scavenging efficiency increased to 40 % (2.40 mg). Similar scavenging capacity 2.49 mg could be achieved with H2 (Mw = 100,000 g/mol, DS 0.57) at a lower amount (20 mg) again showing the higher scavenging capacity of high molecular weight dextran. For acrolein treated BSA, differences in the scavenging effect were observed for variations on the degree of carbazate substitution, the concentration of dextran and the concentration of carbonylated BSA (Fig. 5). As a reference unmodified dextran (L0) had no effects on acrolein treated BSA with above 99 % of carbonyl proteins still existed after incubation. In addition, H2 achieved the highest scavenging effects (8.41 mg) on 2 ml, 6 mg/ml 500 μM acrolein treated BSA among all samples, followed by L2 (6.86 mg), H1 (5.99 mg) and L1 (5.04 mg) 5
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Fig. 6. Scavenging effects on acrolein treated BSA. A) Scavenging efficiencies on 2 ml different concentration of acrolein (100, 200 and 500 μM) treated BSA (6 mg/ ml) after exposure to unmodified dextran (L0, 20 mg) or different types of carbazate modified dextrans (L1, L2, H1 and H2 with 20 mg), or different amount of L2 (10, 20 and 40 mg) in PBS buffer. B) Cell viability of C2C12 cells after incubation for 24 h respectively with 10 mg/ml L2 (a), 3 mg/ml 500 μM acrolein treated BSA (b), 3 mg/ml 500 μM acrolein treated BSA- 10 mg/ml unmodified dextran (L0) (c), 3 mg/ml 500 μM acrolein treated BSA- 5 mg/ml L2 (d), 3 mg/ml 500 μM acrolein treated BSA- 10 mg/ml L2 (e), 3 mg/ml 500 μM acrolein treated BSA- 20 mg/ml L2 (f), and 3 mg/ml 500 μM acrolein treated BSA- 10 mg/ml H2 (g).
Fig. 7. Protein gel electrophoresis of 2 ml, 6 mg/ml acrolein treated BSA samples exposure to carbazate modified dextrans. A) gel electrophoresis of protein samples: protein markers (a), pristine BSA (b), pristine BSA- 20 mg L2 (c), 500 μM acrolein treated BSA (d), 500 μM acrolein treated BSA- 20 mg unmodified dextran (L0) (e), 500 μM acrolein treated BSA- 2 mg L2 (f), 500 μM acrolein treated BSA- 4 mg L2 (g), 500 μM acrolein treated BSA- 10 mg L2 (h), and 500 μM acrolein treated BSA4 mg H2 (i). B) gel electrophoresis of protein samples: protein markers (a), pristine BSA (b), 500 μM acrolein treated BSA (c), 500 μM acrolein treated BSA- 20 mg L2 (d), 200 μM acrolein treated BSA (e), 200 μM acrolein treated BSA- 20 mg L2 (f), 100 μM acrolein treated BSA (g), and 100 μM acrolein treated BSA- 20 mg L2 (h). Gel electrophoresis was running at 100 V for 1.5 h.
and higher molecular weight of the dextran polymer all leaded to an increased scavenging capacity. Cell culture experiments showed that acrolein treated BSA was cell toxic, but when the BSA was captured by modified dextran, the resulting conjugate was non- toxic. A plausible explanation for the non- toxicity of the BSA- dextran conjugate was that the polymer acted as a hydrophilic coating similar to PEGylated proteins. Introduction of polyethylene glycol chains to surfaces of particles/ proteins formed a hydrated layer that prevented protein adsorption and reduced the immune response. (Michel, Pasche, Textor, & Castner, 2005; Pelaz et al., 2015) The BSA- dextran conjugate was also visualized in the gel electrophoreses experiments, corresponding to the protein bands larger than 150 kD. Some parts of acrolein treated BSA ran a little faster (d in Fig. 7A) compared to pristine BSA (b in Fig. 7A) due to the presence of -C = C and disappearance of -SH or -NH groups in BSA induced by acrolein and not easily trapped by polyacrylamide gel during electrophoresis. Pristine BSA also contained a carbonylated fraction as indicated by dark bands observed at higher molecular weights after treatment with carbazate modified dextran (c in Fig. 7A). BSA treated by 100μM acrolein only formed a few of carbonylated proteins so it showed broader bands after mixing with enough H2 samples than that of 200 or 500 μM acrolein treated BSA at around 50 kD.
bands exhibited in the gel (Fig. 7A). When BSA was treated with acrolein, the protein band at 50 kD became broader than that of pristine BSA (line b and d in Fig. 7A). No clear differences could be observed between the bands' structure of 12 mg 500 μM acrolein treated BSA sample after mixing with 20 mg of unmodified dextran L0 (comparing line d and e in Fig. 7A). When carbazate modified dextran was added to acrolein treated BSA, the high molecular weight bands (above 100 kD) became darker (f, g, h and i in Fig. 7A). Specifically, acrolein treated BSA mixed with 10 mg L2 or 4 mg H2 presented darkest and broadest bands in the range of 150–250 kD but also narrower bands at 50 kD. A similar result was seen for a higher amount of L2 (20 and 40 mg) or H2 (20 mg) (Figure s7). This is a strong indication that the modified dextran reacts with carbonylated BSA to produce a conjugate of higher molecular weight hence the shift from 50 kD to 150–250 kD. Additionally, scavenging effects on BSA treated with different amounts of acrolein were also analyzed by protein gel electrophoresis. From Fig. 7B, narrower bands existed at around 50 kD after exposure to 20 mg L2 (d and f) comparing to 12 mg BSA only treated by 500 and 200 μM acrolein (c and e) while 12 mg 100 μM acrolein treated BSA only presented slightly narrower bands at 50 kD after mixing with the same amount of L2 (g and h). There was a strong agreement between experiments that a higher degree of carbazate substitution, a higher concentration of derivatives 6
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Fig. 8. Scavenging effects on patient blood serum. A) Scavenging efficiencies on 2 ml, 6 mg/ml blood serum P1 respectively exposure to different amount of L2 (10, 20 and 40 mg/ml) or 20 mg H2, and P2 exposure to 20 mg L2. B) gel electrophoresis of protein samples: protein markers (a), pristine BSA (b), serum sample P1 (c), P1- 20 mg unmodified dextran (L0) (d), P1- 10 mg L2 (e), P1- 20 mg L2 (f), P1- 40 mg L2 (g), serum sample P2 (h), P2- 20 mg L2 (i). Gel electrophoresis was running at 100 V for 1.5 h.
Authors’ contributions
3.2.3. Scavenging effects on blood serum Two blood serum samples from patients with CKD were defined as P1 and P2. Levels of carbonylated proteins in serum were determined using the 2, 4- dinitrophenylhydrazine (DNPH) assay as described in the experimental section. The assay was selective towards carbonyl groups but would not be able to discriminate between different oxidized proteins so the readout was a total carbonyl content. Scavenging effects on serum samples were illustrated in Fig. 8A. The same trend as for carbonylated BSA and acrolein was present in the serum study. A clear dose response was observed where increasing the amount of L2 used (10, 20 and 40 mg) increased the amount of scavenged carbonyl groups (2.97, 5.17 and 6.90 mg) from 12 mg serum. Also, the high molecular weight dextran was a better scavenger since 20 mg of H2 captured 7.42 mg of carbonyl groups while 40 mg of L2 was needed for 6.9 mg. For P2, 20 mg L2 could remove 4.55 mg of carbonyl proteins. Fig. 8B displayed the gel electrophoresis of serum samples. It was seen that all serum samples presented the same bands between 37 and 100 kD while serum samples exposure to L2 showed darker bands above 250 kD (e, f, g and i in Fig. 7B). Again this was an indication that protein dextran conjugates were formed. If running the gel electrophoresis a little long time (2 h), darker bands apparently were observed at around 150 kD for serum samples mixing with L2 (supporting information, Figure s11).
Li Zheng provided the funding and designed the study. Bo Zhou, Ming Gao and Xianjing Feng substantially conducted the study and prepare the manuscript. Ming Gao, Xianjing Feng, Lanli Huang, Quanxin Huang, Sujit Kootala, and Tobias E. Larsson contributed to acquisition, analysis and interpretation of data; Ming Gao, Xianjing Feng and Tim Bowden contributed to drafting the article or revising it. Consent for publication All authors agreed on the final approval of the version to be published. Declaration of Competing Interest No potential conflicts of interest were disclosed. Acknowledgments This work was supported by National Key R&D Program of China (2018YFC1105900), the Guangxi Science and Technology Base and Talent Special Project (Grant No. GuikeAD17129012), and the local Science and Technology Development Project leading by the central government (the three-D printing and digital medical platform, Grant No. GuikeZY18164004).
4. Conclusions In this work, we successfully synthesized the carbazate modified dextrans for scavenging the acrolein and carbonylated proteins in PBS buffer, or carbonylated proteins in blood serum. It was observed that the dextran molecular weight had an influence on scavenging capacity where high molecular weight modified dextran H2 (100,000 g/mol, DS 0.57) could most efficiently scavenge acrolein or carbonylated proteins and thereby protect cultured cells against their toxicity. The prepared polysaccharide samples had a dynamic size below 50 nm in PBS buffer and no observed cell toxicity. What is of importance was that the formed conjugate between protein and modified dextran was non- toxic and we believed that the dextran formed a hydrophilic coating around the proteins. From the above, the presented carbazate modified dextran samples had the potential to be utilized as an efficient scavenger for secondary products of the oxidative stress.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.115802. References Alamdari, D. H., Kostidou, E., Paletas, K., Sarigianni, M., Konstas, A. G. P., Karapiperidou, A., et al. (2005). High sensitivity enzyme-linked immunosorbent assay (ELISA) method for measuring protein carbonyl in samples with low amounts of protein. Free Radical Biology & Medicine, 39(10), 1362–1367. Aldini, G., Orioli, M., Rossoni, G., Savi, F., Braidotti, P., Vistoli, G., et al. (2011). The carbonyl scavenger carnosine ameliorates that and renal function in Zucker obese rats. Journal of Cellular and Molecular Medicine, 15(6), 1339–1354. Aveles, P. R., Criminacio, C. R., Goncalves, S., Bignelli, A. T., Claro, L. M., Siqueira, S. S., et al. (2010). Association between biomarkers of carbonyl stress with increased systemic inflammatory response in different stages of chronic kidney disease and after renal transplantation. Nephron Clinical Practice, 116(4), c294–299. Bethell, G. S., Ayers, J. S., Hearn, M. T. W., & Hancock, W. S. (1987). Investigation of the activation of various insoluble polysaccharides with 1,1′-carbonyldiimidazole and of the properties of the activated matrices. Journal of Chromatography A, 219(3), 361–371.
Availability of data and materials The datasets collected, obtained and analyzed during the current study are available from the corresponding online database. 7
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serum protein carbonyl formation are progressively enhanced with advancing stages of chronic kidney disease. Clinical and Experimental Nephrology, 13(4), 308–315. Michel, R., Pasche, S., Textor, M., & Castner, D. G. (2005). Influence of PEG architecture on protein adsorption and conformation. Langmuir, 21(26), 12327–12332. Moghe, A., Ghare, S., Lamoreau, B., Mohammad, M., Barve, S., McClain, C., et al. (2015). Molecular mechanisms of acrolein toxicity: Relevance to human disease. Toxicological Sciences, 143(2), 242–255. Oommen, O. P., Wang, S., Kisiel, M., Sloff, M., Hilborn, J., & Varghese, O. P. (2013). Smart design of stable extracellular matrix mimetic hydrogel: synthesis, characterization, and in vitro and in vivo evaluation for tissue engineering. Advanced Functional Materials, 23(10), 1273–1280. Pelaz, B., del Pino, P., Maffre, P., Hartmann, R., Gallego, M., Rivera-Fernández, S., et al. (2015). Surface functionalization of nanoparticles with polyethylene glycol: Effects on protein adsorption and cellular uptake. ACS Nano, 9(7), 6996–7008. Rafael, M. N., Renato, N. A., & Cleto, A. A. (2011). Protein conjugated with aldehydes derived from lipid peroxidation as an independent parameter of the carbonyl stress in the kidney damage. Lipids in Health and Disease, 10, 7. Reznick, A. Z., & Packer, L. (1994). Oxidative damage to proteins: Spectrophotometric method for carbonyl assay. In P. Lester (Vol. Ed.), Methods in enzymology: Vol. 233, (pp. 357–363). Academic Press. Small, D. M., Coombes, J. S., Bennett, N., Johnson, D. W., & Gobe, G. C. (2012). Oxidative stress, anti-oxidant therapies and chronic kidney disease. Nephrology (Carlton), 17(4), 311–321. Stevens, J. F., & Maier, C. S. (2008). Acrolein: Sources, metabolism, and biomolecular interactions relevant to human health and disease. Molecular Nutrition & Food Research, 52(1), 7–25. Sun, G., & Mao, J. J. (2012). Engineering dextran-based scaffolds for drug delivery and tissue repair. Nanomedicine, 7(11), 1771–1784. Thirumurugan, P., Matosiuk, D., & Jozwiak, K. (2013). Click chemistry for drug development and diverse chemical-biology applications. Chemical Reviews, 113(7), 4905–4979. Van Tomme, S. R., & Hennink, W. E. (2007). Biodegradable dextran hydrogels for protein delivery applications. Expert Review of Medical Devices, 4(2), 147–164. Wang, L., Zhou, W., Wang, Q., Xu, C., Tang, Q., & Yang, H. (2018). An injectable, dual responsive, and self-healing hydrogel based on oxidized sodium alginate and hydrazide-modified poly(ethyleneglycol). Molecules, 23(3). Zink, M., Hotzel, K., Schubert, U. S., Heinze, T., & Fischer, D. (2019). Amino acid-substituted dextran-based non-viral vectors for gene delivery. Macromolecular Bioscience, 19(8), e1900085.
Bratusa, A., Elschner, T., Heinze, T., Frohlich, E., Hribernik, S., Bozic, M., et al. (2019). Functional dextran amino acid ester particles derived from N-protected S-trityl-Lcysteine. Colloids and Surfaces B, Biointerfaces, 181, 561–566. Burcham, P. C., & Fontaine, F. (2001). Extensive protein carbonylation precedes acroleinmediated cell death in mouse hepatocytes. Journal of Biochemical and Molecular Toxicology, 15(6), 309–316. Burcham, P. C., Fontaine, F. R., Kaminskas, L. M., Petersen, D. R., & Pyke, S. M. (2004). Protein adduct-trapping by hydrazinophthalazine drugs: mechanisms of cytoprotection against acrolein-mediated toxicity. Molecular Pharmacology, 65(3), 655–664. Burcham, P. C., & Pyke, S. M. (2006). Hydralazine inhibits rapid acrolein-induced protein oligomerization: role of aldehyde scavenging and adduct trapping in cross-link blocking and cytoprotection. Molecular Pharmacology, 69(3), 1056–1065. Buss, H., Chan, T. P., Sluis, K. B., Domigan, N. M., & Winterbourn, C. C. (1997). protein carbonyl measurement by a sensitive ELISA method. Free Radical Biology & Medicine, 23(3), 361–366. Chang, P. V., Prescher, J. A., Sletten, E. M., Baskin, J. M., Miller, I. A., Agard, N. J., et al. (2010). Copper-free click chemistry in living animals. Proceedings of the National Academy of Sciences, 107(5), 1821–1826. Costa, N. A., Gut, A. L., Azevedo, P. S., Tanni, S. E., Cunha, N. B., Fernandes, A. A. H., et al. (2018). Protein carbonyl concentration as a biomarker for development and mortality in sepsis-induced acute kidney injury. Bioscience Reports, 38(1). Dugbartey, G. J. (2018). The smell of renal protection against chronic kidney disease: Hydrogen sulfide offers a potential stinky remedy. Pharmacological Reports, 70(2), 196–205. Krata, N., Zagozdzon, R., Foroncewicz, B., & Mucha, K. (2018). Oxidative stress in kidney diseases: the cause or the consequence? Archivum Immunologiae et Therapiae Experimentalis (Warsz), 66(3), 211–220. Lanzillotto, M., Konnert, L., Lamaty, F., Martinez, J., & Colacino, E. (2015). Mechanochemical 1,1′-carbonyldiimidazole-mediated synthesis of carbamates. ACS Sustainable Chemistry & Engineering, 3(11), 2882–2889. Liu, M., Sun, Y., Xu, M., Yu, X., Zhang, Y., Huang, S., et al. (2018). Role of mitochondrial oxidative stress in modulating the expressions of aquaporins in obstructive kidney disease. American Journal of Physiology-Renal Physiology, 314(4), F658–F666. Lou, J., Liu, F., Lindsay, C. D., Chaudhuri, O., Heilshorn, S. C., & Xia, Y. (2018). Dynamic hyaluronan hydrogels with temporally modulated high injectability and stability using a biocompatible catalyst. Advanced Materials, 30(22), e1705215. Lutz, J.-F., & Zarafshani, Z. (2008). Efficient construction of therapeutics, bioconjugates, biomaterials and bioactive surfaces using azide-alkyne “click” chemistry. Advanced Drug Delivery Reviews, 60(9), 958–970. Matsuyama, Y., Terawaki, H., Terada, T., & Era, S. (2009). Albumin thiol oxidation and
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Carbazate modified dextrans as scavengers for carbonylated proteins a,b,1
a,b, ,1
a,b,c,1
b,c
Bo Zhou , Ming Gao * , Xianjing Feng , Lanli Huang , Quanxin Huang Sujit Kootalad, Tobias E. Larssone,f, Li Zhenga,b,*, Tim Bowdend,**
a,b
T
,
a Guangxi Engineering Center in Biomedical Materials for Tissue and Organ Regeneration, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, China b Guangxi Collaborative Innovation Center for Biomedicine, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, China c Pharmaceutical College, Guangxi Medical University, Nanning, 530021, China d Department of Chemistry – Ångström Laboratory, Uppsala University, Box 538, SE-751 21, Uppsala, Sweden e Department of Clinical Science, Intervention and Technology, Karolinska Institutet, SE-141 86, Stockholm, Sweden f Department of Nephrology, Karolinska University Hospital, Stockholm, Sweden
A R T I C LE I N FO
A B S T R A C T
Keywords: Oxidative stress Protein carbonyl groups Human blood serum Hydrazide-carbonyl click chemistry Protein gel electrophoresis Carbonyl scavengers
A series of biocompatible and non- toxic polysaccharide molecules have been successfully fabricated and explored their potential application for scavenging the carbonyl species in vitro. These macromolecules were dextrans with different hydrazide substitution ratios determined by TNBS assay, NMR and FTIR characterization. The colorimetric assay had demonstrated that these macromolecules could effectively scavenge acrolein, oxidized bovine serum albumin (BSA) in buffer solutions as well as carbonyl proteins from serum. The scavengers could achieve twice more scavenging effects for modified dextrans with high molecular weight (Mw = 100,000) than those of low ones (Mw = 40,000) with the same substitution ratio. Protein gel electrophoresis confirmed that the formation of the complex between carbonyls and modified dextrans resulted in appearance of slower bands. It also revealed that such macromolecules could protect cultured cells against the toxicity of acrolein or its derivatives. The proposed macromolecules indicated a very promising capability as scavengers for oxidative stress plus its derivatives without side effects.
1. Introduction Chronic kidney disease (CKD) is an expanding public health problem that adversely affects human health and increases costs to healthcare systems worldwide (Small, Coombes, Bennett, Johnson, & Gobe, 2012). However, it can hardly be cured or relieved by current therapeutic strategies such as dialysis and transplantation, which can only improve the quality of patients (Dugbartey, 2018). Therefore, it is imperative to find new therapeutic approaches to retard CKD progression. Reactive oxygen species (ROS) have been generally accepted as one of the major mechanisms for CKD pathogenesis and progression, which may lead to renal cell apoptosis and senescence, as well as fibrosis in the kidney (Krata, Zagozdzon, Foroncewicz, & Mucha, 2018; Liu et al., 2018). ROS can produce the endogenous active aldehydes, such as acrolein, which is highly toxic and reactive with biological nucleophiles (Moghe et al., 2015; Stevens & Maier, 2008). Furthermore, the active
aldehydes can react with proteins, resulting in the condition described as “carbonyl stress” to produce carbonylated proteins. Due to their irreversible non- enzymatic modification of structures, carbonylated proteins can hardly be removed spontaneously and exist in the blood of CKD patients at all stages (Aveles et al., 2010; Costa et al., 2018; Rafael, Renato, & Cleto, 2011). Besides, carbonylated proteins have been widely demonstrated as one of the prominent biomarkers of CKD. Their accumulation further leads to loss of catalytic function, protein degradation, proteolysis and several degrees of denaturation, eventually contributing to tissue damage and organ dysfunction as well as accelerating the process of renal failure (Matsuyama, Terawaki, Terada, & Era, 2009). Hydrazide based macromolecules, such as peptide mimetic analogues of carnosine and polystyrene particles, have been proven to have the strong capability to scavenge aldehydes and carbonylated proteins (Aldini et al., 2011; Burcham & Pyke, 2006; Burcham, Fontaine, Kaminskas, Petersen, & Pyke, 2004). These hydrazide based
Abbreviation: BSA, bovine serum albumin; TNBS, 2, 4, 6- trinitrobenzene sulfonic acid; LPO, lipid peroxidation ⁎ Corresponding authors at: Guangxi Medical University, Nanning, 530021, China. ⁎⁎ Corresponding author at: Division of Polymer Chemistry, Department of Chemistry, Uppsala University, Box 538, SE-751 21, Uppsala, Sweden. E-mail addresses: [email protected] (M. Gao), [email protected] (L. Zheng), [email protected] (T. Bowden). 1 These authors contributed equally. https://doi.org/10.1016/j.carbpol.2019.115802 Received 18 October 2019; Received in revised form 23 December 2019; Accepted 27 December 2019 Available online 28 December 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. A) Schematic formation of hydrazone bonds by the reaction between hydrazine derivatives and aldehydes or ketone groups. B) Schematic illustration of scavenging effects of carbazate modified dextran molecules on carbonylated proteins.
2.2. Characterization of carbazate modified dextran
agents can react with aldehydes and carbonylated proteins by hydrazide- carbonyl coupling quickly and reliably, which generates physiologically stable and inoffensive by products with very high chemical yields (Chang et al., 2010; Lutz & Zarafshani, 2008; Thirumurugan, Matosiuk, & Jozwiak, 2013) (Fig. 1A). However, the above hydrazide based materials have unsatisfied biocompatibility, which limited their application in the clinic. Polysaccharides are natural polymers that have excellent biocompatibility and abundant groups that can be linked with hydrazine, which are the promising backbone for hydrazine. Some polysaccharides, such as alginates and hyaluronic acids, have been reported to be functionalized by hydrazine (Lou et al., 2018; Oommen et al., 2013; Wang et al., 2018). The carboxyl groups directly reacted with the hydrazine group. However, due to the limited amount of carboxyl groups and steric resistance effect, the degrees of substitution (DS) of hydrazine groups are generally only around 10 % or below, which greatly limited the efficiency of coupling (Lou et al., 2018; Oommen et al., 2013). Therefore, higher DS may be obtained by 1, 1′- carbonyl diimidazole (CDI) activation of hydroxyl groups followed by reacted with amino molecules achieving up to 25 %∼55 % DS (Bratusa et al., 2019; Zink, Hotzel, Schubert, Heinze, & Fischer, 2019), which provided a direction of the carbazate modification of polysaccharides. In this study, we innovatively chose dextran macromolecule which was a natural polysaccharide and only had hydroxyl groups after carbazate groups modification for clearance of aldehydes and carbonylated proteins by hydrazine reaction (Fig. 1). Dextran has been widely applied in biomedical fields due to its non- toxicity and intrinsically biodegradability (Sun & Mao, 2012; Van Tomme & Hennink, 2007). It has abundant hydroxyl groups that suitably acted as a backbone for hydrazine groups. Our study may provide a reference for further clinical application.
DS of carbazate groups was investigated by 2, 4, 6- trinitrobenzene sulfonic acid (TNBS, 5 % (w/ v) in H2O, Sigma) assay. 1 mg each of samples were dissolved in 20 ml sodium tetraborate decahydrate (≥ 99.5 %, Sigma) buffer (pH = 9.3, 0.1 M), and then 1 ml sample solution was mixed with 25 μl TNBS solution. After three hours of reaction, the mixture was analyzed by UV–vis spectroscopy at 505 nm and compared to a standard curve based on tert- butyl carbazate (≥ 98.0 %, Sigma) (supporting information, Figure s1). The final samples were additionally characterized by 1H, 13C NMR (Jeol JNM- ECP Series FT NMR) at 40℃ and FT- IR (Perkin Elmer Spectrum One FT- IR spectrometer). 2.3. Scavenging of acrolein Dynamic light scattering (DLS) of dextran samples- acrolein adducts. Modified dextran samples were dissolved in phosphate buffer saline (PBS) buffer (10 mM, pH = 7.4) and filtered by a syringe filter with the pore size of 0.2 μm before mixing with different concentrations of acrolein at dark for 24 h. The size and distribution of adducts were measured by the zeta sizer nano instrument (Malvern Instruments, UK). Spectroscopic analysis of acrolein scavenging efficiency: The modified dextran samples were dissolved in PBS buffer (10 mM, pH = 7.4) and filtered through a syringe filter with the pore size of 0.2 μm to give stock solutions. Stock solutions were charged in dialysis tubes (Slide- ALyzer MINI Dialysis Devices, 3.5 kD MWCO, Fisher) and then incubated in 2 ml different concentrations (100, 200, and 500 μM) of acrolein PBS buffer solution in the dark. After 24 h, the absorbance was measured at 209.5 nm, corresponding to the absorption maxima for acrolein, using a UV–vis spectrometer. Observed values were compared with corresponding acrolein solutions treated under the same condition in the absence of dextran samples. The absorbance of acrolein with different concentrations in PBS buffer can be found in supporting information (Figure s2) as standard curves.
2. Experimental 2.1. Synthesis of carbazate modified dextran
2.4. Spectroscopic analysis of protein carbonyls General procedure (Fig. 2): To a 0.5 g of dextran (Fluka) was 20 ml dimethyl sulfoxide (DMSO, ≥ 99.9 %, Sigma) added. The mixture was heated at 80℃ for 15 min. to dissolve the dextran totally. After cooling to room temperature, 1 g 1, 1′- carbonyldiimidazole (CDI, ≥ 97.0 %) was added and the mixture was stirred for 24 h. 3.2 ml of hydrazine hydrate (80 %, Sigma) was added to the mixture, which was left to stir for another 24 h. The reaction mixture was diluted with water and dialyzed (Spectra/Por ® 6 Dialysis Membrane, MWCO: 3.5 kD) for three days and the product was isolated as white powders after lyophilization.
Protein carbonyls measurements were typically performed as follows. (Reznick & Packer, 1994) 1 ml of 2, 4- dinitrophenylhydrazine (DNPH, 97 %, Sigma) (10 mM in 2 M HCl) was added to 200 μl protein samples (6 mg/ml in PBS buffer) and incubated at room temperature in the dark for 45 min. The mixture was chilled for 10 min. using an ice bath and then centrifuged at 3000 rpm for 10 min. after adding 1.2 ml cold trichloroacetic acid (TCA, ≥ 99.0 %, Sigma) (20 wt% in PBS buffer) in order to precipitate the proteins. The protein pellet was washed three times by 2.5 ml ethanol/ ethyl acetate (volume ratio of 1:1) before re- dispersing in 6 M urea (98 %, Sigma) PBS buffer. Pristine 2
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Fig. 2. Synthesis of carbazate modified dextran.
cells were washed with PBS buffer and 100 μl 1 % Alamar blue solution (Life Technologies) was added to each well. The plate was incubated at 37℃, 5 % CO2 for 2 h. The absorbance was measured at 570 nm by a microplate reader (Tecan infinite M200). The results were compared with control wells to determine relative cell viability.
bovine serum albumin (BSA, Sigma) sample only treated with 2 M HCl without DNPH was used as a negative control for carrying the spectroscopic measurements. The absorbance was read by UV–vis spectroscopy at the wavelength of 370 nm. 2.5. Preparation of oxidized and fully reduced BSA
2.8. Protein gel electrophoresis Oxidized BSA and fully reduced BSA were prepared as previously described. (Alamdari et al., 2005; Buss, Chan, Sluis, Domigan, & Winterbourn, 1997) Briefly, 0.35 g of BSA was dissolved in 35 ml PBS, and 1 mM H2O2 and 1 mM ferrous sulfate were added. The mixture was incubated for 20 min., followed by overnight dialysis against PBS buffer at 4℃ to obtain the oxidized BSA solutions. For preparation of fully reduced BSA, 1 g BSA was dissolved in 100 ml PBS and 1 g of solid NaBH4 was added. The mixture was incubated for 30 min. at room temperature before slowly adding HCl (2.5 M) to neutralize the reaction mixture, followed by overnight dialysis against PBS buffer at 4℃. The protein concentrations of the oxidized and reduced BSA solutions were measured by the Bradford assay, then adjusted with PBS to 6 mg/ml and stored at -80℃ before use. Oxidized BSA standard curves were constructed by mixing various different proportions of oxidized BSA with fully reduced BSA to maintain a constant total protein concentration and obtained by the analysis of protein carbonyls measurements (supporting information, Figure s3).
All protein samples were diluted to 1 mg/ml by PBS buffer for gel electrophoresis. The measurements were implemented by running the polyacrylamide gels (mini- protean® TGX™ precast gels, Biorad) at 100 V for 1.5 or 2 h after loading 10 μl protein samples in each of wells. After running, the gels were stained with coomassie blue dyes (Biorad) and then analyzed. 3. Results and discussion 3.1. Synthesis and characterization of carbazate modified dextrans Dextran, a hydroxyl rich hydrophilic polysaccharide, was chosen as a backbone for carbazate nucleophilic groups suitable for scavenging electrophiles such as aldehydes and carbonylated proteins. Hydrazide derivatives have long been used in organic synthesis to form stable hydrazone bonds (Fig. 1A) after reaction with carbonyl compounds and have also been in focus as low molecular weight scavengers. The novelty of combining hydrazone chemistry and hydrophilic polymers has the potential to give macromolecules scavengers with the high local concentration of nucleophiles, the possibility for local delivery and retention, and possible altered pharmacokinetics. We also hypothesized that hydrophilic polymers scavenged proteins and made them less cell toxicity if the polymers acted as a hydrated coating. This would be similar to stealth technology offered for macromolecules, proteins and particles by simple PEGylation. The multiple hydroxyl groups presented in dextran would be the obvious choice for introducing carbazate derivatives with minimal alteration of the native backbone, e.g. the breakage of the pyranose ring structure. Using CDI, a phosgene derivative, (Bethell, Ayers, Hearn, & Hancock, 1987; Lanzillotto, Konnert, Lamaty, Martinez, & Colacino, 2015) and hydrazine, a carbazate group can be introduced in a one- pot two- step procedure. CDI was added to dextran dissolved in DMSO whereupon one imidazole is displaced by a dextran hydroxyl group to form an imidazole carbonate intermediate (alkyl carbamate). After the addition of hydrazine, the second imidazole was displaced to produce the carbazate group (-O-C(O)-NHNH2). Removal of DMSO, liberated imidazole and excess reagents was effectively done with dialysis. The modified dextrans were isolated after lyophilization. The carbazate DS was obtained by TNBS assay based on tert- butyl carbazate standard curves (Figure s1). DS was defined as the number of carbazate groups per glucose units presented in the starting material, e.g. DS 0.2 would mean that 20 % of glucose units carried a carbazate. Four different modified dextrans were synthesized based on DS (low and high) and molecular weight (40,000 g/mol denoted low (L) or 100,000 g/mol denoted high (H)). The products obtained are
2.6. Scavenging effects on carbonylated protein samples BSA was dissolved in PBS buffer at a concentration of 6 mg/ml for used as standards and following measurements. The oxidized BSA samples for scavenging effects were prepared by mixing the fully reduced BSA and oxidized BSA solutions with the volume ratio of 1:1, defined as 50 % oxidized BSA. Additionally, BSA (6 mg/ml in PBS buffer) solutions were mixed with acrolein to respectively obtain 100, 200 and 500 μM acrolein in the final solutions at dark for 24 h, defined as acrolein treated BSA samples. Furthermore, for human blood serum from patients, the protein concentration of serum was measured by Bradford assay and adjusted to 6 mg/ml by addition of PBS buffer to obtain blood serum samples. The all above protein samples (2 ml) were respectively mixed with 10 mg, 20 mg, and 40 mg dextran samples for 24 h at dark before the mixtures were analyzed for protein carbonyls. 2.7. Cell viability and cytotoxicity Mouse myoblast cells (C2C12) were cultured in Dulbecco's modified eagle medium (DMEM, Life Technologies) supplemented with 10 % fetal bovine serum (FBS, Life Technologies) and 0.1 % penicillinstreptomycin to near 80 % confluence. Cells were maintained at 37℃ with 5 % CO2 at 90 % humidity for 24 h and then seeded in 96- well plates with a density of 8000 cells per ml. Cells were replaced with fresh medium and then respectively added the corresponding sample solutions (acrolein- or acrolein treated BSA- dextran samples) to reach total 100 μl medium in each of wells. After incubated for another 24 h, the 3
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no clear changes in the hydrodynamic diameter of the polymers were observed. This indicated that the overall dispersity in PBS of the polymeric system was unaltered by its modifications. Modified polysaccharide molecules below 50 nm in PBS buffer would not be easy to be absorbed by cells and beneficial to the treatment of oxidative stress in vivo.
Table 1 Reaction conditions and DS of carbazate modified dextrans. Product
Dextran (g)
DMSO (ml)
CDI (g)
Hydrazine (80 %, ml)
DS (TNBS)
L1 H1 L2 H2
1 1 1 1
20 20 20 32.5
2 2 2 2
10 10 6.4 6.4
0.2 0.17 0.52 0.57
(Mw = 40000) (Mw = 100000) (Mw = 40000) (Mw = 100000)
3.2. In vitro scavenging effects of carbazate modified dextran The ability of the carbazate functional dextrans to scavenge endogenous electrophiles was tested in four set- ups: acrolein, 50 % oxidized BSA, acrolein treated BSA and human blood serum.
summarized in Table 1. FT- IR results of dextran products were illustrated in Figure s4. For comparison, unmodified dextran (Mw = 40,000) was used and denoted as L0. The characteristic eC]O stretching vibration was detected at 1710 cm−1 (L1, L2, H1 and H2 compared to L0) increasing with DS, while the intensity of the eOH stretching (3499 cm−1) decreased. The peak around 1900∼2200 cm−1 was the background of the instrument. NMR characterization of dextrans was implemented at 40℃ in D2O. For 1H NMR (Fig. 3A), the signal at 4.65∼4.85 ppm is assigned to the solvent (D2O) curve. For α- 1→ 6- glucosyl residue of dextrans it was shifted to 5.16 ppm (1 in Fig. 3A) at 40℃. The extra signals appeared at 5.35 ppm for carbazate modified dextrans (L1, L2, H1 and H2) compared to unmodified dextran (L0) were the protons of -NHNH2 groups while the intensity of sugar ring protons in the range of 3.5∼4.2 ppm (2–6 in Fig. 3A) decreased with the increased DS of dextrans. Furthermore, their 13C NMR was also presented in Fig. 3B. The specific carbons (eC]O) detected at 158∼160 ppm for high DS of modified dextran (L2 and H2) comparing with unmodified dextran (L0). The multi signals for specific carbons meant the modification succeed in different parts of sugar rings. However, no peaks existed at a similar ppm for L1 and H1 due to the low modification of carbazate groups. DLS data revealed that the hydrodynamic sizes of dextran scavenger samples in PBS buffer were around 12 nm (L1 and L2) and 23 nm (H1 and H2) respectively (Figure s5). Increasing the concentration of dextrans in PBS from 10, 20–50 mg/ml, still showed the same dynamic size. Even after reacting the carbazates with acrolein (100, 200 or 500 μM),
Fig. 3. 1H (A) and
13
3.2.1. Scavenging effects on acrolein Carbazate modified dextrans were placed inside the dialysis tube (MWCO: 3.5 kD) with a cut- off of 15 ml syringe tube. During the incubation, dextran molecules could not pass through the membrane to acrolein medium, due to their higher molecular weight, while acrolein had the possibility to enter into the tube and react with carbazate groups. Measuring the acrolein concentration by UV absorbance in a medium outside the dialysis tube indirectly related to the amount of acrolein bound to dextrans without the interference from polysaccharides presented in the tube. Results illustrated that 2 mg samples could respectively remove almost 0.18 μmol (L1), 0.26 μmol (L2), 0.23 μmol (H1) and 0.29 μmol (H2) of acrolein when exposure to 2 ml, 200 μM acrolein buffer. Increasing the amount of L2 from 2 mg to 10 mg, 0.31 μmol of added acrolein could be scavenged. If the amount of acrolein was increased to 1 μmol, only 0.33 μmol was removed by 2 mg of L2 (Fig. 4A). From the experiments, it was shown that a higher carbazate DS gave more efficient scavenging of acrolein, which corresponds to the higher concentration of functional groups on the polymer, L1 vs. L2 and H1 vs. H2. Increasing the amount of modified dextran from 2 mg to 10 mg also increased the scavenging effect as a result of increased concentration of functional groups. Interestingly, high molecular weight dextrans seemed to be better scavengers of acrolein than their low molecular counterparts even at comparable DS, L1 vs H1 and
C (B) NMR spectra of dextran samples dissolved in D2O at 40℃. 4
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Fig. 4. Scavenging effects on acrolein. A) Scavenging efficiencies on 2 ml different concentration of acrolein (0.1, 0.2 and 0.5 μM) buffer after exposure to different types of carbazate modified dextrans (L1, L2, H1 and H2 with 2 mg), or different amount of L2 (2, 4 and 10 mg) in PBS buffer. B) Cell viability of C2C12 cells after incubation for 24 h respectively with 2 mg/ml L2 (a), 30 μM acrolein (b), 30 μM acrolein- 2 mg/ml unmodified dextran (L0) (c), 30 μM acrolein- 1 mg/ml L2 (d), 30 μM acrolein- 2 mg/ml L2 (e), 30 μM acrolein- 5 mg/ml L2 (f), and 30 μM acrolein- 2 mg/ml H2 (g).
L2 vs H2. The carbazate groups in higher Mw dextran with more branched chains were easier to capture the carbonyl groups. Therefore it would be more efficient for high Mw macromolecules (H2) than low Mw macromolecules (L2) to scavenge the acrolein in PBS buffer. Fig. 4B described the viability of C2C12 cells after incubating in different corresponding cell mediums for 24 h. Carbazate modified dextran molecule showed no apparent toxicity so that close to 100 % of cells were metabolically unaffected in cell medium mixing with 2 mg/ ml of L2. However, around 89 % of cells died after the addition of 30 μM acrolein showing the cell toxic properties of acrolein. When dextrans were added subsequently to acrolein, the cell survival increased as a result of scavenging. Cells were only incubated with 30 μM acrolein for 10 min. to make sure that no cell death was induced by acrolein before adding the scavengers (Burcham & Fontaine, 2001). Addition of L2 to cell medium containing acrolein saved 40 % of the cells at 1 mg/ ml and 92 % at 5 mg/ml. The addition of unmodified dextran L0 (2 mg/ ml) had no effect on cell survival. When H2 was added 87 % of cells survived at 2 mg/ml. The results showed a clear dose response where a higher concentration of carbazates gave a higher scavenging effect. Similar to the scavenging experiments in PBS it could be noticed that the high molecular weight dextran (H2) was a better scavenger than the low molecular counterpart (L2) as manifested by the higher survival ratio of cells exposed to H2.
Fig. 5. Scavenging efficiencies on 2 ml, 6 mg/ml 50 % oxidized BSA samples after exposure to different types of carbazate modified dextrans (L1, L2, H1 and H2 with 20 mg) or different amount of L2 (10, 20 and 40 mg) in PBS buffer.
using the amount of 20 mg. A four- fold increase in the amount of modified dextran L2 from 10 mg to 40 mg had almost similar increase in scavenging capacity, from 20 % (2.37 mg) to 70 % (8.41 mg), of acrolein treated BSA, showing a linear dose response. BSA treated by different concentrations of acrolein (200 μM or 100 μM) was scavenged with different capacities using the same amount of dextran samples (20 mg) and different amounts of L2 (Fig. 6A). Using different amounts of acrolein would affect the number of carbonyl groups introduced in the protein and this would, in turn, affect the scavenging. More carbonyl groups would consume more carbazates and from the results, it could be seen that scavenging efficiency decreased from 7.48 mg to 6.01 mg when the BSA became more carbonylated. Fig. 6B described the capability of carbazate modified dextrans to protect C2C12 cells against the toxicity of acrolein treated BSA. Herein, BSA was treated with 500 μM acrolein and incubated for 24 h in the dark to make sure that acrolein totally reacted with BSA. Viability of the cells decreased to 3 % when treated with 3 mg/ml acrolein treated BSA probably as a result of cell death showing the toxicity of carbonylated BSA. When carbazate modified dextran L2 (5 mg/ml) was added to the culture cell, cell survival manifested in the increase of viability (41 %). The unmodified dextran L0 had no rescuing effects since viability was 8 % at 10 mg/ml. If 20 mg/ml L2 or 10 mg/ml H2 was applied to the same mixtures, it was observed that approximately 100 % of cells would be rescued from acrolein treated BSA toxicity. Protein gel electrophoresis was utilized to shine some light on the protein polymer complexes presumably formed when the functional polymer was covalently linked to the protein through the hydrazone linkage. A few different observations could be made from the protein
3.2.2. Scavenging effects on oxidized BSA or acrolein treated BSA 50 % oxidized BSA was utilized as oxidized BSA samples for scavenging effects due to its linear relationship between UV absorbance and carbonylated proteins concentration inside the PBS buffer. The scavenging effects on 50 % oxidized BSA samples were shown in Fig. 5. For unmodified dextran L0, there was no scavenging effect observed with about 98 % carbonylated proteins left in PBS buffer. 20 mg L2 (Mw = 40,000 g/mol, DS 0.52) could scavenge 1.47 mg of oxidized BSA from 2 ml, 6 mg/ml 50 % oxidized BSA while L1 with DS 0.20 only removed 0.80 mg of oxidized BSA. If the amount of L2 was increased to 40 mg, the scavenging efficiency increased to 40 % (2.40 mg). Similar scavenging capacity 2.49 mg could be achieved with H2 (Mw = 100,000 g/mol, DS 0.57) at a lower amount (20 mg) again showing the higher scavenging capacity of high molecular weight dextran. For acrolein treated BSA, differences in the scavenging effect were observed for variations on the degree of carbazate substitution, the concentration of dextran and the concentration of carbonylated BSA (Fig. 5). As a reference unmodified dextran (L0) had no effects on acrolein treated BSA with above 99 % of carbonyl proteins still existed after incubation. In addition, H2 achieved the highest scavenging effects (8.41 mg) on 2 ml, 6 mg/ml 500 μM acrolein treated BSA among all samples, followed by L2 (6.86 mg), H1 (5.99 mg) and L1 (5.04 mg) 5
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Fig. 6. Scavenging effects on acrolein treated BSA. A) Scavenging efficiencies on 2 ml different concentration of acrolein (100, 200 and 500 μM) treated BSA (6 mg/ ml) after exposure to unmodified dextran (L0, 20 mg) or different types of carbazate modified dextrans (L1, L2, H1 and H2 with 20 mg), or different amount of L2 (10, 20 and 40 mg) in PBS buffer. B) Cell viability of C2C12 cells after incubation for 24 h respectively with 10 mg/ml L2 (a), 3 mg/ml 500 μM acrolein treated BSA (b), 3 mg/ml 500 μM acrolein treated BSA- 10 mg/ml unmodified dextran (L0) (c), 3 mg/ml 500 μM acrolein treated BSA- 5 mg/ml L2 (d), 3 mg/ml 500 μM acrolein treated BSA- 10 mg/ml L2 (e), 3 mg/ml 500 μM acrolein treated BSA- 20 mg/ml L2 (f), and 3 mg/ml 500 μM acrolein treated BSA- 10 mg/ml H2 (g).
Fig. 7. Protein gel electrophoresis of 2 ml, 6 mg/ml acrolein treated BSA samples exposure to carbazate modified dextrans. A) gel electrophoresis of protein samples: protein markers (a), pristine BSA (b), pristine BSA- 20 mg L2 (c), 500 μM acrolein treated BSA (d), 500 μM acrolein treated BSA- 20 mg unmodified dextran (L0) (e), 500 μM acrolein treated BSA- 2 mg L2 (f), 500 μM acrolein treated BSA- 4 mg L2 (g), 500 μM acrolein treated BSA- 10 mg L2 (h), and 500 μM acrolein treated BSA4 mg H2 (i). B) gel electrophoresis of protein samples: protein markers (a), pristine BSA (b), 500 μM acrolein treated BSA (c), 500 μM acrolein treated BSA- 20 mg L2 (d), 200 μM acrolein treated BSA (e), 200 μM acrolein treated BSA- 20 mg L2 (f), 100 μM acrolein treated BSA (g), and 100 μM acrolein treated BSA- 20 mg L2 (h). Gel electrophoresis was running at 100 V for 1.5 h.
and higher molecular weight of the dextran polymer all leaded to an increased scavenging capacity. Cell culture experiments showed that acrolein treated BSA was cell toxic, but when the BSA was captured by modified dextran, the resulting conjugate was non- toxic. A plausible explanation for the non- toxicity of the BSA- dextran conjugate was that the polymer acted as a hydrophilic coating similar to PEGylated proteins. Introduction of polyethylene glycol chains to surfaces of particles/ proteins formed a hydrated layer that prevented protein adsorption and reduced the immune response. (Michel, Pasche, Textor, & Castner, 2005; Pelaz et al., 2015) The BSA- dextran conjugate was also visualized in the gel electrophoreses experiments, corresponding to the protein bands larger than 150 kD. Some parts of acrolein treated BSA ran a little faster (d in Fig. 7A) compared to pristine BSA (b in Fig. 7A) due to the presence of -C = C and disappearance of -SH or -NH groups in BSA induced by acrolein and not easily trapped by polyacrylamide gel during electrophoresis. Pristine BSA also contained a carbonylated fraction as indicated by dark bands observed at higher molecular weights after treatment with carbazate modified dextran (c in Fig. 7A). BSA treated by 100μM acrolein only formed a few of carbonylated proteins so it showed broader bands after mixing with enough H2 samples than that of 200 or 500 μM acrolein treated BSA at around 50 kD.
bands exhibited in the gel (Fig. 7A). When BSA was treated with acrolein, the protein band at 50 kD became broader than that of pristine BSA (line b and d in Fig. 7A). No clear differences could be observed between the bands' structure of 12 mg 500 μM acrolein treated BSA sample after mixing with 20 mg of unmodified dextran L0 (comparing line d and e in Fig. 7A). When carbazate modified dextran was added to acrolein treated BSA, the high molecular weight bands (above 100 kD) became darker (f, g, h and i in Fig. 7A). Specifically, acrolein treated BSA mixed with 10 mg L2 or 4 mg H2 presented darkest and broadest bands in the range of 150–250 kD but also narrower bands at 50 kD. A similar result was seen for a higher amount of L2 (20 and 40 mg) or H2 (20 mg) (Figure s7). This is a strong indication that the modified dextran reacts with carbonylated BSA to produce a conjugate of higher molecular weight hence the shift from 50 kD to 150–250 kD. Additionally, scavenging effects on BSA treated with different amounts of acrolein were also analyzed by protein gel electrophoresis. From Fig. 7B, narrower bands existed at around 50 kD after exposure to 20 mg L2 (d and f) comparing to 12 mg BSA only treated by 500 and 200 μM acrolein (c and e) while 12 mg 100 μM acrolein treated BSA only presented slightly narrower bands at 50 kD after mixing with the same amount of L2 (g and h). There was a strong agreement between experiments that a higher degree of carbazate substitution, a higher concentration of derivatives 6
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Fig. 8. Scavenging effects on patient blood serum. A) Scavenging efficiencies on 2 ml, 6 mg/ml blood serum P1 respectively exposure to different amount of L2 (10, 20 and 40 mg/ml) or 20 mg H2, and P2 exposure to 20 mg L2. B) gel electrophoresis of protein samples: protein markers (a), pristine BSA (b), serum sample P1 (c), P1- 20 mg unmodified dextran (L0) (d), P1- 10 mg L2 (e), P1- 20 mg L2 (f), P1- 40 mg L2 (g), serum sample P2 (h), P2- 20 mg L2 (i). Gel electrophoresis was running at 100 V for 1.5 h.
Authors’ contributions
3.2.3. Scavenging effects on blood serum Two blood serum samples from patients with CKD were defined as P1 and P2. Levels of carbonylated proteins in serum were determined using the 2, 4- dinitrophenylhydrazine (DNPH) assay as described in the experimental section. The assay was selective towards carbonyl groups but would not be able to discriminate between different oxidized proteins so the readout was a total carbonyl content. Scavenging effects on serum samples were illustrated in Fig. 8A. The same trend as for carbonylated BSA and acrolein was present in the serum study. A clear dose response was observed where increasing the amount of L2 used (10, 20 and 40 mg) increased the amount of scavenged carbonyl groups (2.97, 5.17 and 6.90 mg) from 12 mg serum. Also, the high molecular weight dextran was a better scavenger since 20 mg of H2 captured 7.42 mg of carbonyl groups while 40 mg of L2 was needed for 6.9 mg. For P2, 20 mg L2 could remove 4.55 mg of carbonyl proteins. Fig. 8B displayed the gel electrophoresis of serum samples. It was seen that all serum samples presented the same bands between 37 and 100 kD while serum samples exposure to L2 showed darker bands above 250 kD (e, f, g and i in Fig. 7B). Again this was an indication that protein dextran conjugates were formed. If running the gel electrophoresis a little long time (2 h), darker bands apparently were observed at around 150 kD for serum samples mixing with L2 (supporting information, Figure s11).
Li Zheng provided the funding and designed the study. Bo Zhou, Ming Gao and Xianjing Feng substantially conducted the study and prepare the manuscript. Ming Gao, Xianjing Feng, Lanli Huang, Quanxin Huang, Sujit Kootala, and Tobias E. Larsson contributed to acquisition, analysis and interpretation of data; Ming Gao, Xianjing Feng and Tim Bowden contributed to drafting the article or revising it. Consent for publication All authors agreed on the final approval of the version to be published. Declaration of Competing Interest No potential conflicts of interest were disclosed. Acknowledgments This work was supported by National Key R&D Program of China (2018YFC1105900), the Guangxi Science and Technology Base and Talent Special Project (Grant No. GuikeAD17129012), and the local Science and Technology Development Project leading by the central government (the three-D printing and digital medical platform, Grant No. GuikeZY18164004).
4. Conclusions In this work, we successfully synthesized the carbazate modified dextrans for scavenging the acrolein and carbonylated proteins in PBS buffer, or carbonylated proteins in blood serum. It was observed that the dextran molecular weight had an influence on scavenging capacity where high molecular weight modified dextran H2 (100,000 g/mol, DS 0.57) could most efficiently scavenge acrolein or carbonylated proteins and thereby protect cultured cells against their toxicity. The prepared polysaccharide samples had a dynamic size below 50 nm in PBS buffer and no observed cell toxicity. What is of importance was that the formed conjugate between protein and modified dextran was non- toxic and we believed that the dextran formed a hydrophilic coating around the proteins. From the above, the presented carbazate modified dextran samples had the potential to be utilized as an efficient scavenger for secondary products of the oxidative stress.
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Availability of data and materials The datasets collected, obtained and analyzed during the current study are available from the corresponding online database. 7
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