Chitosan-Coated Diatom Silica as Hemostatic ... - ACS Publications

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Chitosan-Coated Diatom Silica as Hemostatic Agent for Hemorrhage Control Chao Feng,*,† Jing Li,† Guang Sheng Wu,‡ Yu Zhi Mu,† Ming Kong,† Chang Qing Jiang,§ Xiao Jie Cheng,† Ya Liu,† and Xi Guang Chen*,† †

College of Marine Life Science, Ocean University of China, 5# Yushan Road, Qingdao 266003, Shandong Province, China Qingdao First Sanatorium, Jinan Military Region, 27# West Hong Kong Road, Qingdao 266071, Shandong Province, China § Qingdao Municipal Hospital, 5# East Sea Road, Qingdao 266003, Shandong Province, China ‡

S Supporting Information *

ABSTRACT: Uncontrolled hemorrhage leads to high death risk both in military and civilian trauma. Current hemostatic agents still have various limitations and side effects. In this study, natural diatom silica obtained from diatomite and diatom culture was purified and developed for hemorrhage control. To improve the biocompatibility and hemostatic performance of diatom silica, a series of chitosan-coated diatom (CS-diatom) was developed. The composition of CSdiatom prepared was optimized by in vitro hemocompatibility and blood coagulation evaluation for that prepared with 0.5%, 1%, 3%, and 5% chitosan. The results demonstrated that the CS-diatom prepared with 1% chitosan exhibited favorable biocompatibility (hemolysis ratio < 5%, no cytotoxicity to MEFs), great fluid absorbility (24.39 ± 1.53 times the weight of liquid), and desirable hemostasis effect (351 ± 14.73 s at 5 mg/ mL, 248 ± 32.42s at 10 mg/mL). Further blood coagulation mechanism study indicated that CS-diatom could provide an ideal interface to induce erythrocyte absorption and aggregation, along with activating the intrinsic coagulation pathway and thus accelerated blood coagulation. Benefitting from the multiple hemostatic performances, CS-diatom showed the shortest clotting time (98.34 ± 26.54 s) and lowest blood loss (0.31 ± 0.11 g) in rat-tail amputation model compare to diatomite and diatom as well as gauze and commercial QuikClot zeolite. The results evidenced that the CS-diatom was a safe and effective hemostatic agent and provided a new understanding of nonsynthetic mesoporous materials for hemorrhage control. KEYWORDS: diatom silica, porous silicon, chitosan, hemostatic agent, hemorrhage control

1. INDRODUCTION Effective hemorrhage control is critical to reduce the risk of trauma death both in military and in civilian medicine.1,2 Nearly 50% of deaths in the military are due to blood loss, 8% of which result from noncompressible injuries. The emergency and medical surgeries such as cardiovascular, hepatic, and orthopedic have a high incidence of severe bleeding, which required hemostatic intervention.3,4 In the past decade, compression with gauze was still prevalent practice for most injuries. Although various materials including inorganic mineral and organic polymer were developed for hemorrhage control, the existing shortcomings limited them in a relatively low efficacy. As a typical hemostatic agent, the widely used QuikClot zeolite generated heat with temperatures increasing from 44 to 95 °C, leading to the secondary tissue burns.5,6 The new generation of QuikClot had already overcome this issue, but the high cost and insufficient provision limited its more extensive application. HemCon chitosan bandage, a lyophilized chitosan derivative, circumvented this problem, yet the definite shape resulted in its incapacity for large or deep wounds.7,8 © XXXX American Chemical Society

The ideal hemostatic agent should rapidly stop blood loss at all scales, minimize collateral damage and be noncytotoxic, have nonimmunogenic properties, as well as be inexpensive. Due to the fast plasma absorbability without notable associated exothermic reactions and efficient contact-activated hemostasis, porous silica has a great significance for the development of hemostatic materials. Inorganic mesoporous bioactive glass microspheres,9 mesoporous silica spheres,10 and mesocellular silicate foams11 were studied for hemorrhage control and showed prominent hemostatic ability in vitro and in vivo. However, an evident drawback for the use of these synthetic mesoporous silica was that their synthesis required toxic chemicals, long time consumption, and high cost.12,13 On the other hand, the cytotoxicity of synthetic mesoporous silica derived from residual toxic or interfacial property should be considered before biomedical application. CetyltrimethylamReceived: September 27, 2016 Accepted: November 30, 2016 Published: November 30, 2016 A

DOI: 10.1021/acsami.6b12317 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustrations of the Preparation and the Hemostatic Mechanism of Diatomite, Diatom, and CS-Diatom

In this study, we set our target to evaluate the potential of diatom silica for hemorrhage control (Scheme 1). Two sources of diatom silica containing diatomite and diatom were comparatively studied. To improve the hemostatic effect and biocompatibility, chitosan, a natural polysaccharide with favorable biocompatibility, biodegradability, and effective hemostatic performance, was coated on diatom (CS-diatom) with different CS loading content. We characterized the physicochemical properties and biocompatibility of diatomite, diatom, and CS-diatom, including chemical composition, surface properties, absorption ability, blood compatibility, and cytotoxicity. Furthermore, the hemostatic efficacy of diatomite, diatom, and CS-diatom was evaluated both in vitro and in vivo.

monium bromide (CTAB), a surfactant commonly used in synthesis of mesoporous silica, had severe cytotoxicity for various cell lines.14,15 Several groups reported that the bare silica and mesoporous silica would cause hemolysis, which was determined by the size, shape, and the interfacial properties of particles, especially at high concentrations.16−20 Nature has developed an elegant biosynthesis route to produce porous silica materials with complex three-dimensional (3D) architecture. The most outstanding example is diatom, the largest group of single cellular microalgae, which has distinct 3D architecture of silica wall (frustules) with highly ordered pore structure and hierarchical pore organization.21,22 In recent years, diatom silica was intensively studied in biomedical fields, especially in the drug delivery field, which demonstrated its favorable biocompatibility and strong potential to replace synthetic silica-based materials.23−28 The diatom silica could be obtained from inexpensive sources such as diatom culture or diatomite. The production is highly environmentally friendly because no toxic byproducts are produced, and low energy consumption is required in the process. Compare to diatom silica obtained from diatomite, the ones obtained from diatom culture possess specific species characteristic patterns and pure component without heavy metal pollution.29,30 More importantly, the higher concentration of polar silanol groups on the surfaces of diatom leads to its negative charges interface, which might be able to effectively promote blood clotting. Up to now, no literature existed in regard to hemostatic properties of the diatom silica.

2. EXPERIMENTAL SECTION 2.1. Materials. Diatomite was purchased from RuiJinTe Chemical Reagent Ltd. Coscinodiscus sp. (CCAP 1013/11) was supplied by Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China. Soluble CS was prepared by our group using heterogeneous degradation method (molecular weight, Mw: 10 kDa, degree of deacetylation: 85%). Hydrochloric acid and hydrogen peroxide (H2O2, 30%) were purchased from Sinopharm Chemical Reagent Co., Ltd. All other chemicals used in this work were of analytical grade. 2.2. Preparation of Purified Diatomite, Diatom, and CSDiatom. Diatomite was purified by a one-step method. Two hundred milligrams of diatomite was suspended in 100 mL mixture solution containing 50 mL HCl (2 mol/L) and 50 mL H2O2 (30%) for 24 h. Then the diatomite was collected by filtration and rinsed with deionized water ten times to completely remove the residual. The B


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ACS Applied Materials & Interfaces purified diatomite was vacuum-dried at 35 °C for 24 h and stored in a glass vial. Diatom frustule (diatom) was obtained from diatom culture. Coscinodiscus sp. was cultured in Guillard’s medium (f/2 + Si) at 20 °C using a 12/12 light/dark cycle for 10 days. The cells were collected by filtration and washed with deionized water three times and then purified using the same process as that of diatomite. Chitosan-coated diatom (CS-diatom) was prepared by a simple coating process. Two hundred milligrams diatom was suspended in 10 mL chitosan solution at different concentration (0.5%, 1%, 3%, and 5%) under magnetic stirring for 3 h. The precipitate was collected by filtration and washed by deionized water and ethyl alcohol three times, respectively and then vacuum-dried at 35 °C for 24 h. CS-diatom was denoted as 0.5-CS-diatom, 1-CS-diatom, 3-CS-diatom, and 5-CSdiatom corresponding to the chitosan concentrations, respectively. 2.3. Characterization. The morphologies of diatomite, diatom, and CS-diatom were observed via the scanning electron microscope (SEM, JSM-6010LA, JEOL Ltd., Japan). At the same time, the surface element was analyzed by energy dispersive X-ray spectroscopy (EDXS, SEM, JSM-6010LA, JEOL Ltd., Japan). The thermogravimetrydifferential scanning calorimetry thermograms (TG-DSC, Netzsch Ltd. STA449F3, Germany) were used to analyze the thermostability of materials and determined the amount of absorbed CS on diatom with different composition (300−500 °C, refer to ref 8). The dye adsorption test was performed to measure the specific surface area of diatomite, diatom, and CS-diatom by the methylene blue (MB) method as previous reports.31,32 The chemical structures were characterized by Fourier Transform Infrared Spectroscopy (FTIR, Nicolet Ltd., 5DX/550II) with a disk of KBr. The zeta potential was measured on a Zetasizer Nano Instrument (Malvern Ltd.) in deionized water to compare the content of silanol groups on diatomite and diatom and analyze the modification efficiency of CS on the surface of diatom. The liquid absorbability of diatomite, diatom, and CS-diatom with simulated body fluid (SBF) was tested according to a previous report.8,10 The dry sample was placed on a prewetted filter paper in a funnel. SBF was slowly added dropwise and stopped until the first drop of SBF dripped from the funnel. The absorption ratio (AR, %) of the sample was calculated using eq 1: AR (%) = (Wwet − Wdry )/Wdry × 100

The coagulation tests inculuding prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed using a semiautomatic coagulation analyzer (TS6000, MD PACIFIC Ltd., China). Blood was taken from the heart of anesthetized New Zealand White Rabbit and mixed with a 1/10 volume of 3.8% sodium citrate and then centrifuged at 3000 rpm/min for 15 min at 37 °C to get the platelet poor plasma (PPP). For PT measurement, 100 μL of PPP was preincubated at 37 °C for 3 min and mixed with 100 μL of PT reagent and the samples (preincubated at 37 °C for 3 min, separately) and then measured PT (s) by semiautomatic coagulation analyzer. APTT test was performed by mixed 100 μL of aPTT reagent with 100 μL of citrated plasma. After incubation at 37 °C for 3 min, 100 μL of 0.025 mol/L CaCl2 and samples were added in the mixture, and then aPTT (s) was measured by semiautomatic coagulation analyzer. 2.6. Rat-Tail Amputation. Sprague−Dawley (SD) rats (weight of 200−250 g, purchased from QINGDAO Food and Drug Administration, Qingdao, China) were anesthetized by an intraperitoneal injection of 40 mg/kg sodiumpentobarbital. Fifty percent length of the tail was cut by surgical scissors and then placed in air for 15 s to ensure normal blood loss. Subsequently, the wound was covered with the 200 mg of diatomite, diatom, CS-diatom, commercial gauze, or Quikclot zeolite. The clotting time (s) and blood loss (g) were recorded during the hemostatic process.34,35 All above-mentioned experimental animals including the New Zealand White Rabbits and SD rats were cared and treated in accordance with the National Research Council’s Guide for the care and use of laboratory animals. 2.7. Cytotoxicity Evaluation. The mouse embryonic fibroblasts (MEFs) cell line was used to assess the cytotoxicity of diatomite, diatom, and CS-diatom. Briefly, MEFs were seeded in 96-well plates at 1−3 × 105 cells/well and allowed to attach for 24 h. Then, the culture media was respectively replaced with media containing diatomite, diatom, and CS-diatom at different concentration (0.625, 1.25, 2.5, 5, 10 mg/mL). The cells treated with DMEM were taken as the control. After 24, 48, and 72 h incubation, the MTT assay was tested. The cell viability was calculated by eq 3.

cell viability = OD(test)/OD(control) × 100

The qualitative cytotoxicity evaluation was performed using calceinam cell viability assay. Briefly, MEFs were seeded in 24-well plates at 1−3 × 105 cells/well and allowed to attach for 24 h. Then, the culture media was respectively replaced with media containing diatomite, diatom, and CS-diatom at different concentrations (5 and 10 mg/mL). The cells treated with DMEM and DMSO were taken as the positive and negative control, respectively. After 24 h incubation, the media was removed and the live cells were stained with 4 mM calcein AM (Sigma-Aldrich) and then observed by fluorescence microscopy (Nikon TS 100, Japan). 2.8. Statistical Analysis. The data collected in this work was presented as mean ± SD and statistically analyzed by one way analysis of variance with Sigma Plot, version 11.0 (Systat Software Inc.). The differences were considered to be statistically significant when the p values were less than 0.05.

(1)

where Wdry and Wwet were the weight of dry sample and wet sample. 2.4. In Vitro Hemolysis Test. The hemolysis ratio of diatomite, diatom, and CS-diatom at different concentration was tested in vitro. The sample was dissolved in saline and prewarmed to 37 °C. Then, 60 μL erythrocyte stock dispersions were added into sample suspensions (3 mL) and incubated at 37 °C for 1 h. Then the mixtures were centrifuged at 2000 rpm/min for 5 min. The absorbance of the supernatant was determined at 545 nm by UV−vis spectrophotometer (UV-1200 MAPADA, China). Distilled water and saline without sample treated groups were used as positive control and negative control, respectively. The hemolysis rate (HR, %) was calculated by eq 2.

HR (%) = (Ds − Dn)/(Dp − Dn) × 100

(3)

3. RESULTS AND DISCUSSION 3.1. Characterization. Two sources of diatom silica from diatomite and diatom culture were purified by a simple onestep method. High concentration of HCl and H2O2 guaranteed the complete removal of organic matter in diatomite and diatom. CS-diatom was prepared by mixed diatom frustule with CS solution under stirring for 3 h. The appropriate concentration of chitosan must be controlled by no more than 5%. High CS concentration (>5%) led to high viscosity, which prevented the homodispersion of diatom in CS solution. The purified diatomite consisted of various broken fossilized diatom with irregular shape structures and broad size range from several micrometers to several hundred micrometers (Figure 1a1). The porous structure on the diatomite surface was

(2)

where Ds, Dn, and Dp were the absorbance of the sample, the saline, and the distilled water, respectively. 2.5. In Vitro Blood Clotting Evaluation. The whole blood clotting test was based on reported literature.33,34 Diatomite, diatom, CS-diatom, and commercial gauze were prewarmed to 37 °C, respectively. One milliliter recalcified New Zealand White Rabbit blood solution (per 1 mL blood added 25 μL CaCl2, 0.2 M) was added into the sample. The recalcified blood without sample was used as the control. The clotting time (s) was recorded until the blood completely ceased to flow. After blood coagulation, the samples were rinsed with phosphate buffer solution (PBS, pH 7.4) three times to remove the physically adhered cells and immobilized with 2.5% glutaraldehyde for 2 h, and then the blood cells were dehydrated with gradient alcohol and dried with critical point drier, subsequently, observed by SEM. C


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Figure 1. (A) SEM images of diatomite, diatom, and CS-diatom and corresponding EDXS analysis of (B) diatomite, (C) diatom, and (D) CSdiatom.

Figure 2. (A) FTIR spectra of diatomite, diatom, and CS-diatom. (B) Zeta-potential and (C) liquid absorbability of diatomite, diatom, 0.5-CSdiatom (0.5-CS), 1-CS-diatom (1-CS), 3-CS-diatom (3-CS), and 5-CS-diatom (5-CS). Data represents the mean ± SD (n = 5).

D


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Figure 3. (A) Hemolysis rate of diatomite, diatom, 0.5-CS-diatom, 1-CS-diatom, 3-CS-diatom, and 5-CS-diatom at different concentrations. Data represents the mean ± SD (n = 5). (B) Photographs of RBCs treated with diatomite, diatom 0.5-CS-diatom, 1-CS-diatom, 3-CS-diatom, and 5-CSdiatom at different concentrations.

method, the diatom exhibited the highest specific surface area (152.6 ± 4.5 m2/g), followed by 0.5-CS-diatom (131.5 ± 5.8 m2/g), 1-CS-diatom (121.4 ± 3.8 m2/g), 3-CS-diatom (115.6 ± 2.1 m2/g), 5-CS-diatom (111.8 ± 8.1 m2/g), and diatomite (42.5 ± 5.8 m2/g), which were consistent with the results of the morphology. FTIR analysis demonstrated the chemical structure on the diatomite, diatom, and CS-diatom surfaces (Figure 2A). The characteristic peaks of the asymmetric Si−O−Si bonds at 1081 and 465 cm−1 occurred in all samples. A broad overlapping band between 3100 and 3700 cm−1 was found in diatom spectra, attributing to the abundant Si−OH on its surface. For CS-diatom, the more intense composite band between 3100 cm−1 and 3700 cm−1 was due to the overlapping of Si−OH on the diatom and N−H of chitosan stretching vibrations. Meanwhile, new characteristic peaks including the amide I band of CS at 1645 cm−1 and the amide II band of CS at 1539 cm−1 were observed in the CS-diatom spectra, which indicated that CS was absorbed on the surface of the diatom. The successful modification of diatom with CS was also confirmed by zeta potential detection (Figure 2B). The zeta potential of diatom drastically increased from −25.31 ± 1.56 mV to +30.56 ± 2.31 mV for the 0.5-CS-diatom, +32.45 ± 1.34 mV for the 1-CS-diatom, +31.98 ± 1.67 mV for the 3-CSdiatom, and +33.24 ± 2.09 mV for the 5-CS-diatom. In

not obvious owing to the mineral sedimentation during the long period of the petrochemical process (Figure 1, panels a2a4). In contrast, the purified diatoms exhibited uniform disc shape with about 100 μm diameter (Figure 1a5). The highly ordered pore structure was clearly observed on the valves and girdle bands (Figure 1a6). The pores of diatoms were regular circle with hierarchical organization: several small pores (about 200 nm) were nested in a big pore (about 1 μm) (Figure 1, panels a7 and a8). After a series of CS modification, the shape and size of CS-diatom with different composition were similar to that of diatom, but the pore structure was filled with chitosan (Figure 1, panels a9−a12, and Figure S1). Corresponding EDXS analysis showed the differences in surface element among diatomite, diatom, and CS-diatom (Figure 1, panels B, C, and D). The surface of diatomite and diatom exhibited O and Si peaks with similar intensity. The new peak of carbon was observed in the CS-diatom, and the higher intensity signal of the O element was found in the CSdiatom than that of diatom and diatomite, attributing to the polysaccharide molecular skeleton structure of chitosan on the surface of the diatom. Thermogravimetric analysis indicated that the amount of absorbed CS on 0.5-CS-diatom, 1-CS-diatom, 3-CS-diatom, and 5-CS-diatom were 5.5%, 18.97%, 16.25%, and 17.03% (w/ w), respectively (Figure S2). In accordance with the MB E


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Figure 4. (A) In vitro blood clotting time of diatomite, diatom 0.5-CS-diatom, 1-CS-diatom, 3-CS-diatom, and 5-CS-diatom. Data represents the mean ± SD (n = 5). SEM images of interfacial interaction between RBCs and (B) diatomite, (C) diatom, (D) 0.5-CS-diatom, and (E) 1-CS-diatom.

It was reported that the hemolytic activity of mesoporous silica had a positive correlation with the content of the surface silanol groups.15,16 Adem Yildirim et al. found that the organic surface modification was able to dramatically reduce hemolytic activity of mesoporous silic.15 The steric hindrance of the bulky functional groups could reduce the quantity of accessible surface silanol groups, thus improving the compatibility of modified mesoporous silic with red blood cells (RBCs). Our findings strongly support this conclusion. From the results of FTIR and zeta- potential, diatom possessed more surface silanol groups than diatomite, thus exhibiting higher hemolytic activity. CS was a natural polysaccharide with favorable hemocompatibility whose hemolysis ratio at different concentration approximately equaling the loading content of CS on 0.5-CSdiatom, 1-CS-diatom, 3-CS-diatom, and 5-CS-diatom was shown in Figure S3. After CS surface modification, the hemolytic activity of diatom could be prevented almost completely. 3.3. In Vitro Blood Clotting Evaluation. Recalcification test was evaluated to compare the coagulation efficiency among diatomite, diatom, and CS-diatom (Figure 4A). Diatom and CS-diatom with different compositions exhibited significant concentration-dependent coagulation effects compared to gauze (p < 0.05). With CS loading content increasing from 0% (diatom) to 18.97% (1-CS-diatom), the coagulation effect was significantly enhanced. The shortest clotting time was found in the 1-CS-diatom group (351 ± 14.73 s at 5 mg/mL, 248 ± 32.42 s at 10 mg/mL), nearly 150 and 250 s shorter than that of diatom and diatomite at each concentration. Due to the similar CS-loading content, no significant difference was found among 1-CS-diatom, 3-CS-diatom, and 5-CS-diatom. To figure out the effect of CS composition in the CS-diatom-induced blood coagulation, we tested the coagulation efficiency of CS powder at different concentrations approximate equal to the loading content of CS on 0.5-CS-diatom, 1-CS-diatom, 3-CS-

addition, diatom exhibited more surface negative charge compared to that of diatomite (zeta potential equal to −12.14 ± 1.58). This phenomenon was due to the fact that more silanol groups existed on the surface of the diatom. The liquid absorbability of the hemostatic agent is a key point for hemorrhage control. The rapid and efficient liquid absorption was beneficial to stop blood loss and concentrate platelet and clotting factors for accelerating blood coagulation.8 The hierarchical pore structure endowed diatom with great specific surface area, leading to the high liquid absorbability (37.9 ± 2.48 times the weight of liquid), which was 9.26 folds and 7.04 folds as that of gauze and diatomite, respectively (Figure 2C). The modification of CS decreased specific surface area of diatom, leading to a slight decrease in absorption ratio of CS-diatom. No significant differences were found among that of 0.5-CS-diatom, 1-CS-diatom, 3-CS-diatom, and 5-CS-diatom (p > 0.05). 1-CS-diatom could absorb 24.39 ± 1.53 times as the weight of liquid, which was close to that of the diatom. Most importantly, a droplet of SBF liquid could be absorbed within 30 ms by 1-CS-diatom, indicating its excellent liquid absorbability. 3.2. In Vitro Hemolysis Test. Hemolysis evaluation was found to be concentration-dependent as treated with diatomite, diatom, and CS-diatom with different composition (Figure 3, panels A and B). The hemolysis ratio basically rose up with the increase of sample concentration. Diatom exhibited the highest hemolysis ratio at each concentration (16.05 ± 0.87% at 5 mg/ mL, 17.76 ± 1.16% at 10 mg/mL). Hemolysis for diatomite at 13.83 ± 0.11% was observed when its concentration reached 5 mg/mL, which manifested the inadequate hemocompatibility of diatomite. On the other hand, CS modification could effectively prevent hemolytic activity of diatom; the hemolysis in 0.5-CSdiatom, 1-CS-diatom, 3-CS-diatom, and 5-CS-diatom was no more than 2.71 ± 0.91%, which demonstrated the favorable hemocompatibility of the CS-diatom. F


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Figure 5. (A) aPTT and (B) PT measurement of d diatomite, diatom, 0.5-CS-diatom, 1-CS-diatom, 3-CS-diatom, and 5-CS-diatom. Data represents the mean ± SD (n = 5).

diatom, and 5-CS-diatom (Figure S4). CS could not act out the obvious coagulation effect at low concentration (from 0.3125 to 2.5 mg/mL). The shortest clotting time (587.31 ± 28.47s) was achieved when CS concentration reached 5 mg/mL (equal to the CS content of 10 mg/mL of 1-CS-diatom), which was significantly longer than that of the 1-CS-diatom at 10 mg/mL. It could be concluded that the enhanced coagulation effect 1CS-diatom was attributed to the combined effect of CS and diatom core. The SEM images showed the morphologies of diatomite, diatom 0.5-CS-diatom, and 1-CS-diatom contacted with the whole blood. Unlike diatomite, on whose surface only a few RBSs were absorbed (Figure 4B), a lot of RBCs were found to be aggregated and adhered strictly on diatom (Figure 4C). For 0.5-CS-diatom treated group, pseudopodia-like RBCs were found to be aggregated and adhered strictly on the surface of diatom (Figure 4D). On contrast, a large amount of pseudopodia-like RBCs were piled up on the surface of 1-CSdiatom, which demonstrated the superiority performance of 1CS-diatom for blood clotting (Figure 4E). The contact activation of the blood coagulation cascade induced by diatomite, diatom, and CS-diatom with different compositions were investigated by the measurements of aPTT

and PT (Figure 5, panels A and B). In the clinical, aPTT, and PT were two key indexes to check the intrinsic- and extrinsiccoagulation pathway, respectively. The results showed that diatomite, diatom, 0.5-CS-diatom, 1-CS-diatom, 3-CS-diatom, and 5-CS-diatom were able to significantly reduce aPTT compared to the negative control (p < 0.05), while no significant change was observed in the PT test. Previous reports demonstrated that the hemostatic activity of chitosan was mainly due to the electrostatic interaction between the positivecharged chitosan and the negative-charged RBCs,36 which had no effect on the coagulation factors and did not bring any changes of aPTT and PT (Figure S5).37 But for mesoporous silica, its negative charged surface could remarkably promote the intrinsic blood coagulation by activation of coagulat factors XI and XII, along with cofactors HWK-kininogen and prekallikrein.38−40 In this study, all samples including diatomite, diatom, and CS-diatom exhibited similar reduction effect on aPTT values, where no statistically significant difference was found among them (p > 0.05). It indicated that the intrinsic blood coagulation could be activated by CS-diatom at the same level as that of the pure diatom. The CS surface modification would not block the procoagulant activity of silicon core in CSdiatom. G


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Figure 6. Data from the rat-tail amputation model among the diatomite, diatom, 1-CS-diatom, gauze, and commercial Quikclot zeolite. (A) Photograph of hemostatic effect by contacting with the wound. (B) Clotting time. (C) Blood loss. Data represents the mean ± SD (n = 6).

Figure 7. Cytotoxicity of diatomite, diatom, and CS-diatom exposure on MEFs at different concentrations after (A) 24, (B) 48, and (C) 72 h incubation. Data represent the mean ± SD (n = 6). (D) Fluorescence microscopy images of MEFs treated with diatomite, diatom, and 1-CS-diatom at 5 and 10 mg/mL for 24 h incubation.

3.4. In Vivo Hemostatic Performance. Rat-tail amputation model was employed to comparably evaluate in vivo hemostatic effect of diatomite, diatom, and 1-CS-diatom. Gauze and commercial hemostatic agent, Quikclot zeolite, were used

as control groups. After hemostasis, a big blood clot was formed in Quikclot zeolite and diatomite-treated wounds. In contrast, no apparent blood clot was observed in diatom and 1-CSdiatom treated group, except that only a few materials were H


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hemolysis and cytotoxicity of the diatom could be almost completely prevented by chitosan surface modification. We believed that this study determined the hemostatic property of natural diatom silica for the first time and provided an alternative approach for hemorrhage control by using nonsynthetic porous silica materials.

adhered around the wound (Figure 6A). These phenomena might be attributed to the excellent fast fluid absorption of 1CS-diatom and diatom. The shortest clotting time was achieved by 1-CS-diatom (98.34 ± 26.54 s), followed by Quikclot zeolite (133.66 ± 21.84 s), diatom (160.78 ± 28.56 s), diatomite (237.75 ± 37.67 s), and gauze (510.26 ± 63.22 s) (Figure 6B). The clotting times of 1-CS-diatom and Quikclot zeolite was similar (p > 0.05), but only a half weight of blood loss was found in the 1-CS-diatom treatment group (0.31 ± 0.11 g) than that of the Quikclot zeolite treatment group (Quikclot 0.63 ± 0.09 g). Meanwhile, the weight of blood loss in the diatom treatment group was also less (0.45 ± 0.07 g) than that of Quikclot zeolite (Figure 6C). Above mentioned experiments proved that the combination of chitosan and diatom had integrated the advantages of both materials and provided efficient hemorrhage control by multiple hemostasis performance (Scheme 1). The fast fluid absorption of CS-diatom was conducive to concentrate platelet and clotting factors, thereby shortening the time lag for initial thrombin generation and the time to peak thrombin generation. On the other hand, hemadsorption induced by chitosan on the surface of CS-diatom was able to lead to the aggregation of RBCs and the formation of the blood clot. Meanwhile, the silanol group rich surface of diatom core could act as polar framework with “catalytic potential” and provide interface stimulation for activation of coagulation factors, further strengthening the efficiency of hemostasis. Cytotoxicity Evaluation. The cytotoxicity of diatomite, diatom, and 1-CS-diatom exposure on MEFs was investigated at different concentrations and incubation times (Figure 7, panels A, B, and C). No significant cytotoxicity was detected in the diatomite treatment group. The cell viability was still higher than 80% during 72 h incubation. Diatom exhibited slight cytotoxicity after 24 h incubation. The cell viability was lower than 60%. Subsequently, time-dependent cell viability, which increased with incubation time was observed and the cell viability was above 80% from 48−72 h. The highest cell viability was obtained by 1-CS-diatom, whose viability remained 100− 120% at each concentration and incubation time. The qualitative study cytotoxicity of diatomite, diatom, and 1-CSdiatom at 5 and 10 mg/mL was carried out using the calcienAM assay after a 24 h incubation (Figure 7D). The results showed that diatom exhibited significant cytotoxicity against MEFs at each concentration, while desired cell viability was observed in 1-CS-diatom treated groups, which consisted of the MTT assay. Previous studies reported that chitosan could significantly promote proliferation of MEFs.41,42 Therefore, we speculated that the high cell viability in 1-CS-diatoms treatment groups was attributed by the proliferation promotion effect of CS composition in 1-CS-diatom. The above results indicated that 1-CS-diatom had no cytotoxicity and was able to be a potential hemostatic agent.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b12317.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected] (X. G. C.). ORCID

Chao Feng: 0000-0003-2327-1981 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by China Postdoctoral Science Foundation (no. 2016M592246), Promotive Research Fund for Young and Middle-aged Scientists of Shandong Province (no. BS2015SW010), Applied Basic Research Plan of Qingdao (no. 16-5-1-70-jch), Public Science and Technology Research Founds Projects of Ocean (no. 2015418022), and the Taishan Scholar Program.

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REFERENCES

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4. CONCLUSION In the present study, chitosan-coated diatom had been developed for hemorrhage control. The prepared CS-diatom with optimized composition (1-CS-diatom) could not only quickly and effectively absorb fluid but also induce adsorption and aggregation of RBCs. In addition, the intrinsic pathway of coagulation cascade was also significantly activated by the silicon core of the CS-diatom to promote the blood clotting and achieve hemorrhage control in vitro and in vivo. More importantly, CS-diatom showed excellent biocompatibility; the I


Research Article


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