Biodegradable microporous starch with assembled thrombin for rapid

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Biodegradable microporous starch with assembled thrombin for rapid induction of hemostasis Qing Li, Fei Lu, Songmin Shang, Hongli Ye, Kun Yu, Bitao Lu, Yang Xiao, Fangyin Dai, and Guangqian Lan ACS Sustainable Chem. Eng., Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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ACS Sustainable Chemistry & Engineering

Biodegradable Microporous Starch with Assembled Thrombin for Rapid Induction of Hemostasis Qing Lia,1, Fei Lua,b,1, Songmin Shangc, Hongli Yea, Kun Yua, Bitao Lua,, Yang Xiaod, Fangyin Daia,b, Guangqian Lana,b* aCollege

of Textile and Garments, Southwest University, No. 2 Tiansheng Road, BeiBei District, Chongqing 400715, China

bChongqing

Engineering Research Center of Biomaterial Fiber and Modern Textile, No. 2 Tiansheng Road, BeiBei District,

Chongqing 400715, China cInstitute

of Textiles and Clothing, The Hong Kong Polytechnic University, No. 11 Yucai Road, hung hom, Kowloon, Hong

Kong, China dSericulture

and Agri-Food Research Institute of Guangdong Academy of Agriculture Science, No. 133 Yiheng Road,

Dongguan Zhuang, Tianhe District, Guangzhou Province 510610, China *Corresponding author: College of Textile and Garments, Southwest University, Chongqing 400715, China. Phone: +8613594005200; fax: +8602368251228; e-mail: [email protected] 1Equally

contributed.

ABSTRACT: Immediate hemorrhage control is pivotal for saving lives both in civilian life and in the military. In the present study, we developed a three-dimensional carrier based on the enzymolysis of corn starch loaded with thrombin, which efficiently controlled bleeding in a short time. The microporous starch (MS) with a large surface area was modified with sodium trimetaphosphate (STMP) to generate starch phosphate, which showed enhanced thrombin entrapment efficiency. The thrombin-assembled MS-STMP structure (MS-STMP-T) showed excellent hydrophilicity, rapid water absorption, high negative surface charge, and high hemostatic efficiency upon contact with blood in whole blood clotting test, APTT, and PT measurements and animal injury models. Additionally, MS-STMP-T showed no cytotoxicity and was degraded within 14 days in a rabbit model. These results indicate that MS-STMP-T is a safe and effective agent for the control of bleeding.

KEYWORDS: Microporous starch, Hemostasis, Thrombin, Biodegradability, Biocompatibility INTRODUCTION Limiting blood loss following trauma is critical for survival and can reduce the risk of death from coagulopathy, infection, and multisystem organ failure.1, 2 Hemostasis is traditionally achieved using gauze and suture materials mixed with hemostatic agents; however, while these methods can be applied to minor bleeding, they are not always effective for controlling massive hemorrhage, especially in organs such as heart, liver, and kidney. Ideally, hemostatic materials should control different degrees of bleeding within a short time and be bloodcompatible, biocompatible, and biodegradable.3-5 They should also possess high absorption capacity, which is determined by a porous structure or gel performance. A variety of materials have been investigated for their potential as hemostatic agents, including chitosan,6, 7 gelatin,8, 9 cellulose,10-12 starch,13 montmorillonoid,14 zeolite,15 mesoporous silica,16 and alginate.17 The high porosity of mesoporous bioactive glass chitosan composite scaffold containing gallium promotes blood absorption18 and platelet aggregation to stem bleeding, but its efficacy has not been confirmed in vivo. Vancomycin-loaded hydrogels showed excellent mechanical and antibacterial properties for the treatment of critical bleeding but exhibited inadequate biodegradability.19 N-Hydroxysuccinimide-ester functionalized poly(2-oxazoline) developed as hemostatic patches showed high blood uptake to limit hemorrhage during surgical procedures,20 but this

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material cannot be degraded in vivo. Microporous starch (MS) is a biocompatible material with high porosity and, consequently, a large surface area with exposed hydroxyl and aldehyde groups that increase hydrophilicity and water absorption capacity.21-23 Moreover, MS can be enzymatically degraded in vivo into the oligosaccharides maltose and glucose, which are readily absorbed by human tissue.24, 25 Owing to these properties, MS is an excellent carrier of adsorbents,26, 27 catalysts,28-30 and other molecules for various biological applications, including temporary vascular occlusion31 and drug delivery.32 MS can also be used as hemostatic agents,22, 33, 34 such as Arista® (Medafor, Minneapolis, MN, USA), a widely used hemostatic starch that is safe and effective for stopping conventional bleeding. However, Arista® is not useful for rapid and severe hemorrhage; use of MS in conjunction with hemostatic agents may solve this problem. Hemostatic agents such as batroxobin, thrombin, and fibrinogen aggregate blood cells on the wound surface and are routinely applied to stop bleeding.35 Of these, thrombin is the most widely used and is used in various combinations such as in conjugation with graphene sponge.36 Thrombin, also known as activated factor II, is a 39-kDa serine protease extracted from animal blood; it has a near-spherical shape and is used as a topical hemostatic agent. When it contacts blood, thrombin catalyzes the transformation of fibrinogen into fibrin, which induces platelet aggregation.35, 37 It is normally used in conjunction with sponge or gauze in clinical diagnosis; however, when used in this manner, thrombin shows low hemostatic efficiency due to slow blood absorption and insufficient contact with plasma. Thus, its inefficiency in controlling rapid and severe bleeding and the high cost have limited the application of thrombin as a hemostatic agent.38, 39 Herein, we aimed to design a modified hemostatic starch loaded with thrombin, which greatly enhanced the hemostatic capacity through the synergistic actions of the constituents. To increase thrombin entrapment, we used sodium trimetaphosphate (STMP), which shows no adverse effects in humans,40, 41 to modify MS. STMP serves as a polyphosphate and may help enhance the biological activity of MS, such as via activating coagulation factor V and steadying fibrin clot, thereby contributing to halt hemorrhage.42-45 Following the reaction with STMP, MS attains a more stable structure, leading to the formation of the starch phosphate on the MS surface and increasing the hydrophilicity of MS, thereby allowing it to form more hydrogen bonds with thrombin and entrap the thrombin, without obviously decreasing the activity. Moreover, the negative surface potential of MS greatly accelerated the blood clotting time by stimulating the plasmatic contact activating system.46, 47

EXPERIMENTAL SECTION Materials. Native corn was purchased from Jinhui Biotechnology Co. (Shanghai, China). α-Amylase (100 U mg−1) and glucoamylase (100 U mg−1) were from Duly Biotechnology Co. (Nanjing, China). Sodium trimetaphosphate (STMP) was from Beijing Chemical Company (Beijing, China). Bovine thrombin (100 U mg−1) was from Yuanye Biotechnology Co. (Shanghai, China). Thrombin chromogenic substrate S-2238 (H-D-Phe-Pip-ArgpNA·2HCl) was from Chromgeneix (Woburn, MA, USA). Prothrombin time (PT) and activated PT time (APTT) kits were purchased from HEALL Bio-science technology Co. (Qingdao, China). New Zealand white rabbits were obtained from the Animal Laboratory Center of Third Military Medical University. All animal experiments and care were approved by the National Center of Animal Science Experimental Teaching at the College of Animal Science and Technology of Southwest University of China, and were in accordance with the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health. All reagents were of analytical grade and were used without further purification. Deionized water was used in all experiments. Preparation of Thrombin-loaded STMP-MS (MS-STMP-T). MS was prepared by enzymolysis.


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Native corn starch (NS) (40 g) was added to 200 ml sodium acetate buffer (pH 4.6) containing a mixture of α-amylase and glucoamylase at a 1:4 ratio, yielding an enzyme-to-starch ratio of 2:100 (w/w). The slurry was stirred at 250 rpm for 10 h at 40°C. Upon completion of the enzymatic reaction, hydrolysis was terminated by adding 20 ml NaOH solution (4%, w/w). The slurry was centrifuged at 4000 rpm for 5 min, vacuum filtered, and washed three times with deionized water, followed by drying in a vacuum dryer for 24 h to obtain MS. To generate the sodium trimetaphosphate-conjugated microporous starch (MS-STMP), 20 g microporous starch was added to 50 ml distilled water containing 0.6 g Na2CO3 (3 g/100 g dry starch) and 1.2 g STMP (6 g/100 g dry starch). The reaction was allowed to proceed for 24 h at 50°C, with the pH maintained at 11.0 by adding 1.5 M NaOH. After 24 h, 0.1 M HCl was added to stop the reaction. The resultant material was vacuum filtered, washed three times, and dried in a vacuum dryer for 24 h to obtain MS-STMP. For assembling thrombin, 1 g MS-STMP was sterilized with ultraviolet (UV) light and mixed by vibration with 5 ml of different thrombin solutions (activity = 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, and 50 U) at 4°C for 1 h, followed by three cycles of washing and vacuum filtration at 4°C. The residue was freeze dried at -50°C for 48 h to obtain thrombin-entrapped starch (MS-STMP-T). Thrombin-loaded native starch (NS-T) and microporous starch (MS-T) were prepared by the same method as controls for the entrapment efficiency assay.

Determination of Thrombin Entrapment Efficiency. The amount of thrombin assembled onto MSSTMP was determined with the thrombin color substrate assay. Briefly, 25 mg of sample were incubated with 1.5 ml of 4 mmol l−1 S-2238 for 3 min at 37°C. The reaction was terminated by transferring the tube to a 100°C water bath for 2 min. After cooling to room temperature, the sample was centrifuged at 3000 rpm for 10 min, and the thrombin content was determined from a standard curve by measuring the absorbance at 405 nm. This test was performed three times. The entrapment efficiency of NS-T, MS-T, and MS-STMP-T was determined in a similar manner using the following equation: entrapment efficiency(%) =

( ) × 100% Ua

U0

(1)

where Ua is the amount of thrombin assembled onto starch and U0 is the initial amount of thrombin.

Characterizations. The morphology of NS, MS, STMP-MS, and STMP-MS-T was examined by scanning electron microscopy (SEM) (S-4800, Hitachi, Tokyo, Japan). The chemical structure was determined by Fourier transform infrared (FTIR) spectroscopy (Bruker Alpha; Bruker, Bremen, Germany). The zeta potential was measured on a Zetasizer Nano instrument (Malvern Instruments, Malvern, UK). Xray diffraction (XRD) was performed using a Bruker X-ray diffractometer. X-ray photoelectron spectroscopy (XPS) measurements were carried out on a Thermo ESCALAB 250XI spectrometer to determine the chemical composition. Porosity of samples was measured with Automatic Mercury Porosimeter (AutoPore IV 9500, Micromeritics, USA). Elemental analysis was applied to detect the phosphorus content for calculation of substitution degree (SD) on a PerkinElmer ICP 2100 (Parkin Elmer, USA) instrument. The SD value was calculated as the following equation: SD(%) =

× Ppercentage content (1005.23 ― 3.87 × P percentage content) × 100%

(2)

Water Absorption Ratio. Phosphate-buffered saline (PBS; pH 7.4) was used to evaluate the absorption of the starch samples. The samples were dried at 50°C for 24 h, and their initial weight was recorded (Wa).



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They were then immersed in PBS for 30 min at 37°C. After suction filtration, the weight of each sample was recorded (Wb). The same sample was measured three times. The absorption ratio was determined using the following equation: absorption ratio(%) =

(

Wb ― Wa Wa

) × 100%

(3)

Wettability Measurement. The wettability of NS, MS, STMP-MS, and STMP-MS-T was measured by the sessile drop contact angle method using a DSA100 drop shape analyzer (Krüss, Hamburg, Germany) at 37°C. Before tests, sample powder was heaped into a cylindrical shape with a diameter of 10 mm and a thickness of 5 mm with flat surface. A PBS (pH 7.4) droplet of 3 μl was dropped onto the surface of the sample and contact angle was measured. Five different positions of the same sample were evaluated to obtain average contact angle value. Whole Blood Clotting Test. In vitro blood coagulation capacity of NS, MS, STMP-MS, NS-T, MS-T, and STMP-MS-T was evaluated with the whole blood clotting test. Briefly, 2 ml of recalcified New Zealand white rabbit blood were mixed with 50 μl of 0.2 mol l−1 CaCl2 and 0.1 g sample; clotting time was recorded when blood ceased flowing. The recalcified blood without sample and 0.1 ml thrombin solution (activity = 10 U) were used as negative and positive controls, respectively. Each sample was tested three times. The change in thrombin activity with time after assembling onto MS-STMP-T was tested by whole blood clotting method as described above. Before testing, MS-STMP-T was stored in a vacuum drying oven at 4°C for 1, 10, 30, 90, and 180 days. Each sample was evaluated three times. Assessment of Plasmatic Coagulation. Prothrombin time (PT) and activated PT time (APTT) were determined with the coagulation test using an automated coagulation analyzer (Sysmex CA-1500; Siemens, Tokyo, Japan) at the Ninth People’s Hospital of Chongqing. The test was repeated three times. Blood from New Zealand White rabbits mixed with 1/10 volume of 3.8% sodium citrate was centrifuged at 3000 rpm/min for 15 min at 37°C to obtain platelet-poor plasma (PPP). The APTT test was performed by mixing 100 μl APTT reagent with 100 μl PPP, followed by incubation at 37°C for 3 min. After adding 100 μl of 0.025 mol/l CaCl2 and 2 mg sample to the mixture, APTT (in seconds) was measured. For PT measurement, 100 μl of PPP was mixed with 2 mg samples at 37°C for 3 min, followed by addition of 100 μl PT reagent; PT (in seconds) was then measured. 4 mg sample was evaluated as above method, and six replicates of each sample were prepared for each test. After blood coagulation, samples were rinsed three times with PBS (pH 7.4) to remove attached cells and immobilized with 2.5% glutaraldehyde for 2 h; blood cells were dehydrated in a graded series of alcohol and dried in a critical point dryer before SEM analysis. Platelet Adhesion. Platelet adhesion was evaluated with the lactate dehydrogenase (LDH) assay. After adjusting the number of platelets to 108 ml−1, platelet-rich plasma (PRP) was incubated with NS, MS, MSSTMP, and MS-STMP-T for 30 min at 37°C. Non-adherent platelets were removed by washing three times with PBS, and adherent platelets were lysed with 0.25 ml 1% Triton X-100 in PBS at 37°C for 1 h. LDH activity was measured using a kit (Sigma-Aldrich, St. Louis, MO, USA), and the number of adherent platelets was determined from the calibration curve by measuring the optical density (OD) at 490 nm, with the final value calculated as the average of six measurements for each sample. Samples with aggregated platelets were also immobilized with 2.5% glutaraldehyde for 2 h and then dehydrated in a graded series of alcohol


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for SEM analysis.

Thromboxane (TX) B2 Release. Samples (0.1 g) were incubated for 5 min at 37°C and then cultured with 2 ml PRP for 30 min at 37°C. The supernatant containing TXB2 was obtained, and detected from the calibration curve by measuring the optical density (OD) at 490 nm. Each sample was measured three times. Evaluation of In Vivo Hemostatic Performance. New Zealand white rabbits were used to evaluate the hemostatic performance of these samples. All animal experiments were performed according to previously reported methods.13, 42 The animals were anesthetized (xylazine hydrochloride injection, 0.2 ml kg-1), and their limbs were affixed on an anatomy set. Wounds were photographed before, during, and after surgery. Six parallel groups (6 rabbits per parallel group) of each model were tested, and the average time of hemostasis was recorded . Gauze without and with absorption of 10 U thrombin solution were used as negative and positive controls, respectively. After surgery, each rabbit was housed in its own cage at room temperature. The ear edge hairs were shaved off and the skin disinfected with ethanol; a 1-cm perpendicular line was made with the scalpel on the rabbit ear to transect the artery. After bleeding for the first 5 s, blood on injury site was removed using gauze, sample (1 g) was sprinkled directly onto the bleeding site, and compressed with medical gauze. The gauze was slightly lifted every 5 s to examine the bleeding. Hemostatic time was recorded when the bleeding stopped. The abdominal cavity of disinfected rabbits was cut open, and a wound approximately 1 cm long and 0.5 cm deep was made to the left medial lobe of the liver. After allowing of bleeding for the first 5 s, and removing blood, 1 g sample was directly sprinkled over the bleeding liver, medical gauze was used to compress the wound. Clotting time was recorded when the bleeding stopped. The femoral skin and overlying muscles were transected and the femoral artery was clipped. After bleeding for 5 s, the blood was cleaned, and the sample (1 g) was sprinkled over the injury. Manual compression was used as described above, and hemostatic time was recorded when the bleeding stopped. In Vitro Hemolysis Test. The hemolysis ratio of NS, MS, STMP-MS, and STMP-MS-T at different concentrations (0.25, 0.5, 1, 2, 4, and 8 μg/ml) was evaluated in vitro. A 5-ml volume of fresh, anticoagulated rabbit blood (mixed with 1/10 volume of 3.8% sodium citrate) was added to 10 ml of PBS (pH 7.4), followed by centrifugation at 1200 rpm/min for 5 min to collect red blood cells (RBCs). The sample was dissolved in PBS (pH 7.4) and pre-warmed at 37°C for 5 min. RBC dispersion (60 μl) was added to 3 ml of sample suspension followed by culturing at 37°C for 1 h. The sample mixture was centrifuged at 2000 rpm/min for 5 min, and absorbance at 545 nm was determined with a UV–visible light spectrophotometer (UV-2550; Hitachi). Distilled water and PBS without sample were used as positive control and negative controls, respectively. The same sample was measured three times. Hemolysis rate was determined with the following equation: hemolysis ratio(%) =

(

As ― Ap Ad ― Ap

) × 100%

(4)

where As, Ap, and Ad are the absorbance values of the sample, PBS, and distilled water, respectively.

Cytotoxicity Evaluation. L929 murine fibroblasts were used in the cytotoxicity assay. NS, MS, STMPMS, and STMP-MS-T were immersed in sterilized Dulbecco’s modified Eagle medium (DMEM) for equilibration and the mixture was cultured for 72 h. Cells were incubated with 100 μl DMEM containing



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10% fetal bovine serum for 24 h at 37°C and 5% CO2, then seeded in a 96-well plate with a density of 5 × 103 cells/well. A 10-μl volume of leaching liquor was added to each well, followed by culturing for 24, 48, and 72 h at 37°C and 5% CO2. Cell proliferation was evaluated with the (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) tetrazolium reduction assay with absorbance measured at 490 nm on a Multiskan MK3 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Each measurement was repeated five times. Samples were immersed in DMEM and laid on the surface of the L929 cells in a 24-well plate for 24 h. After removing the samples, the medium was replaced with 100 μl of fresh DMEM and the cells were examined and photographed using a fluorescence microscope (Nikon, Tokyo, Japan). Cell viability was evaluated using a calcein-AM/propidium iodide double staining kit (40747ES76; Yeasen Biological Technology Co., Shanghai, China) according to the manufacturer’s protocol, and imaged with a fluorescence microscope.

In Vitro and In Vivo Degradation. The degradability in vitro was investigated by calculating the weight loss ratio of samples after soaking in simulated body fluid (SBF, pH 7.4). Briefly, 1 g of sample (W0) was immersed in 10 ml SBF and then placed on an orbital shaker at 37°C. The SBF was changed once a day. The sample was separated from solution by filtration at different time points (12, 24, 48, 72, 96, 120, 144, and 168 h) and dried at 60°C to obtain a constant weight (Wa). The weight loss ratio was calculated with the equation below: weight loss ratio(%) =

(

W0 ― Wa W0

) × 100%

(5)

For in vivo experiments, samples were subcutaneously implanted into the back muscle of New Zealand white rabbits. Briefly, the animals were anaesthetized and their backs were shaved and disinfected with ethanol, and 2-cm incisions were made in the muscle for implantation of 0.1 g NS and MS-STMP-T. After 3, 7, 14, and 21 days, three animals in each group at each time point were euthanized, and implants along with surrounding tissue were removed for histological evaluation under a light microscope.

RESULTS AND DISCUSSION The preparation of MS-STMP-T is illustrated in Figure 1a. Micropores formed on native starch after enzymatic hydrolysis, which significantly enlarged the surface area to facilitate reaction with STMP, leading to starch phosphate formation on the MS surface (Fig. 1b), thereby enhancing the entrapment ratio of thrombin on MS via hydrogen bonding (Fig. 1c).


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Figure 1. Scheme of MS-STMP-T preparation in three steps. (a) Enzymolysis, (b) formation of starch phosphate, and (c) thrombin assembly.

Thrombin Loading and Entrapment Efficiency. The amount of assembled thrombin was determined using the thrombin color substrate assay (Fig. 2a). To evaluate activity retention of assembled thrombin, an exponential fitting curve was generated (y = −1069.6 × e−x/1.5 + 82.9; R2 = 0.998). The use of increasing thrombin amount for loading the particles resulted in an exponential increase in the amount of assembled thrombin. When the amount of thrombin added to 1 g MS-STMP was 10 U, the amount of thrombin assembled onto MS-STMP-T was 81.53%; however, there was no increase in the amount of assembled thrombin, when the activity of added thrombin was increased to 40 U (82.89%). MS-STMP-T was therefore prepared using 10 U thrombin for 1 g MS-STMP. The entrapment efficiency of NS, MS, and MS-STMP was 17.3±5.8%, 39.6±4.4%, and 81.53±8.3%, respectively (Fig. 2b); the thrombin entrapment efficiency of MSSTMP was four times higher than that of NS, which may be attributed to the larger surface area and the function of starch phosphate. Due to the increased thrombin entrapment, MS-STMP-T showed the shortest blood clotting time in vitro as 50±5s compared to NS-T (110±5s) and MS-T (83±6s). Thus, MS-STMP was chosen to assemble thrombin as a hemostatic agent; the amount of thrombin added in the preparation procedure was 10 U for 1 g MS-STMP, and the activity of entrapped thrombin was about 8.15 U for 1 g MSSTMP.



Figure 2. Assembly ratio of thrombin on MS-STMP. (a) The assembly ratios of added thrombin onto MSSTMP-T. (b) Entrapment efficiency and blood blotting time of NS-T, MS-T, and MS-STMP-T. (c) SEM images of NS, MS, MS-STMP, and MS-STMP-T and EDS maps of C, N, O, and P elements on the surface of MS-STMP-T. Scale bar: 5 µm. (d) XRD patterns and (e) FTIR spectra of NS, MS, MS-STMP, and MSSTMP-T. *P < 0.05.

Characterization of MS-STMP-T. MS-STMP-T structure was evaluated by SEM and energy dispersive spectroscopy (EDS) (Fig. 2c). NS had an axiolytic shape with a smooth surface, whereas the surface of MS, MS-STMP, and MS-STMP-T was covered with micropores about 2 μm in size. The pore size was observed to be larger for MS-STMP and MS-STMP-T than for MS, and this difference could be due to


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the effect of STMP. EDS mapping of P and N confirmed the presence of starch phosphate and successful assembly of thrombin on the MS-STMP surface, respectively. Corn starch had the four characteristic peaks at 14.8°, 16.9°, 17.8°, and 22.7° of an A-type starch structure (Fig. 2d). MS-STMP had a similar A-type crystalline pattern with higher intensity and sharper peaks, suggesting that enzymatic hydrolysis has not altered the physical structure of starch. The paired peaks of MS-STMP and MS-STMP-T at 16.9° and 17.8° gradually merged into a single peak, and the crystallinity of MS-STMP and MS-STMP-T was higher than that of MS and NS, possibly because amorphous region was easily destroyed by external force from the reaction with STMP. Thrombin assembly had no effect on the ordered crystalline structure. In the IR spectrum of NS and MS (Fig. 2e), similar absorption peaks were observed, indicating there were no differences between NS and MS, since they share the same basic chemical structure. The modification of MS with STMP was indicated by the peaks observed at 1150–1330 cm−1 and peaks at 1000 cm−1. The IR spectrum of MS-STMP and MS-STMP-T showed higher peaks compared to those of NS and MS within 1150–1330 cm−1 range, and this result corresponded to the symmetric stretching of the P=O. Additionally, a new peak was obvious in the IR spectrum of MS-STMP and MS-STMP-T at 1000 cm−1, indicating the existence of a P-O-C absorption peak. The formation of starch phosphate was further supported by determining the detail bonding configuration with X-ray photoelectron spectroscopy (XPS) analyses as shown in Fig. 3a. Two obvious peaks at 285.1 and 531.9 eV were observed in the full XPS survey of MS and MS-STMP corresponding to C 1s and O 1s, respectively, and a peak at 133.1 eV corresponded to P 2p can be find in the full XPS survey of MS-STMP. The high resolution C 1s spectrum of MS and MS-STMP can be fitted with three component peaks at 284.4, 286, and 286.6 eV, corresponding to the C-C, C-OH, and C-O bondings, respectively. The area percentage of C-OH in the C 1s spectrum of MS-STMP was 50.7 %, which was lower than that of MS (60.3 %), suggesting that phosphate groups replaced the C-OH from starch molecules. The high resolution O 1s and P 2p spectrum also provided the evidences of the chemical conjunction. Two fitted peaks can be observed at 531.6 and 532.8 eV in the O 1s spectrum of MS and MS-STMP corresponding to C-OH and -O- bondings, respectively, and a new peak at 532.1 eV appeared for MS-STMP attributed to the P-O bonding. Additionally, two fitted peaks in the high resolution P 2p spectrum were observed at 132.6 and 133.9 eV corresponding to P=O and P-O bondings, respectively. The mass percentage of P element in MS-STMP was determined as 0.4 %, the substitution degree was calculated with ICP as 0.02 % (Fig. 3b). These results well indicated the formation of starch phosphate.

Porosity, Wettability, Water Absorption Ratio, and Zeta Potential. The porosity of NS, MS, MSSTMP, and MS-STMP-T was about 6.1±3.2%, 63.8±3.5%, 77.3±4.2%, and 76.4±2.3%, respectively (Fig. 3c). Thus, the porosity of MS was over 10 times that of NS due to enzymolysis, which created micropores that were further enlarged by the reaction with STMP in the amorphous region; this result was consistent with the XRD analysis. Thrombin had no obvious effect on the porosity of MS-STMP-T. As shown in Fig. 3d, the contact angle decreased with the increased in porosity for the same contact time, which corresponded to the enhanced hydrophilicity. All samples showed good hydrophilicity, with water droplets absorbed within 1 s. However, at 0.5 s, water drops on NS, MS, MS-STMP, and MS-STMP-T showed different contact angles (Fig. 3e). For NS and MS, the contact angles were 57.3° and 30.7°, respectively, indicating that the latter had higher hydrophilicity owing to a larger surface area with more exposed hydroxyl and aldehyde groups. There were no water drops observed on MS-STMP and MS-STMP-T—that is, the drops were absorbed within 0.5 s—indicating superior hydrophilicity due to the presence of starch phosphate. The water absorption ratio of MS was nearly two times higher than that of NS (134.6±10.6% vs. 66.3±6.4%) due to the



high porosity, while introduction of starch phosphate further increased this ratio in MS-STMP to 236.3±11.2%, which was about 75% higher than the ratio of MS (Fig. 3f). After thrombin loading, the change in absorption was negligible. Thus, a combination of high porosity, excellent hydrophilicity and water absorption capacity endowed MS-STMP-T with the ability to rapidly absorb large amounts of blood.

Figure 3. Physical properties of the carrier. (a) Full XPS spectrums and high resolution C 1s, O 1s, and P 2p spectrum of MS and MS-STMP. (b) The mass percentage of P element in MS-STMP and the substitution degree. (c) Porosity, (d) the contact angle changed with porosity increase at contact times of 0, 0.25, 0.5, 0.75, and 1 s, (e) hydrophilicity, and (f) water absorption of NS, MS, and MS-STMP-T. *P < 0.05.

Whole Blood. Gauze and NS were unable to coagulate blood in vitro (Fig. 4a). The clotting time of thrombin was 87±18 s, which was much lower than that of MS (182±6 s) and MS-STMP (151±20 s) whereas MS-STMP-T coagulated blood in 50±5 s, indicating that thrombin significantly enhanced the blood coagulation capacity of MS-STMP. The average retention of thrombin activity for MS-STMP-T after 1, 10, 30, 90, and 180 days was 93.6±2.4%, 92.7±2.3%, 93.1±1.8%, 93.9±2.3%, and 93.2±0.7%, respectively (Fig.


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4b), indicating that thrombin activity was retained.

Figure 4. In vitro blood clotting time of MS-STMP-T. (a) Clotting time. Insets show a macroscopic view of whole blood clot formation for gauze control, thrombin control, NS, MS, MS-STMP, and MS-STMP-T. (b) Change in thrombin activity with time after loading onto MS-STMP-T for 1, 10, 30, 90, and 180 days. (c) APTT, and (d) PT measurements for NS, MS, MS-STMP, and MS-STMP-T. *P < 0.05.

In Vitro Hemostatic Performance. The APTT of NS, MS, MS-STMP, and MS-STMP-T was shorter relative to that of the negative control (Fig. 4c, d); for 2 mg of sample, the APTT ratio was 92.3±2.3%, 72.2±0.8%, 58.5±3.6%, and 46.6±3.7%, respectively, with a slight decrease observed for 4 mg of sample. The PT of the four samples showed a similar trend. There was no dose dependence observed for PT and APTT, suggesting that MS-STMP-T promotes hemostasis by both intrinsic and extrinsic coagulation pathways.



Figure 5. Hemostatic capacity of MS-STMP-T. (a) Zeta potential analysis. (b) Platelet adhesion. (c) TXB2 release from platelets. (d) SEM images of NS, MS, MS-STMP, and MS-STMP-T activation of RBCs and platelets. (e) Hemostatic mechanisms of MS-STMP-T involving rapid absorption, a negatively charged surface, and thrombin stimulation. NS showed a low negative charge surface potential (−2.3 ± 2.6 mV), which may be due to the weak hydrolysis of starch molecules (Fig. 5a). The larger surface area was associated with a lower negative charge potential for MS (−6.4 ± 1.9 mV). After reaction with STMP, the zeta potential was −43.6 ± 2.1 mV due to the formation of starch phosphate. Thrombin did not alter the negative charge (zeta potential of MS-STMP-T: −42.4 ± 1.8 mV). Since induction of RBC and platelet aggregation by charge stimulation is a hemostatic mechanism, these results indicate that MS-STMP-T had the highest hemostatic efficiency among the tested materials. Following contact with PRP, MS-STMP showed increased platelet adhesion as compared to NS and MS (Fig. 5b), likely due to the greater negative potential, which promoted platelet activation and aggregation.46 However, MS-STMP-T showed the highest degree of platelet adhesion due to the synergistic effect of thrombin (Fig. 5d), which could catalyze the transformation of fibrinogen into fibrin, thereby inducing platelet aggregation. After contacting hemostatic agents, platelets release TXB2, which can in turn promote platelet aggregation in turn (Fig. 5c). MS-STMP-T stimulated TXB2 release, resulting in greater platelet aggregation. Furthermore, after contacting blood, no RBCs were observed on NS and only a few were detected on MS, whereas many RBCs were observed on MS-STMP, especially on MS-STMP-T (Fig. 5d). Additionally, some RBCs had transformed and penetrated the pores of MS-STMP-T, suggesting that this


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material had a stimulant effect on the cells. MS-STMP-T showed superior performance by combining rapid blood absorption, negative charge-induced RBC and platelet stimulation with thrombin stimulation, thus combining passive and active hemostatic mechanisms (Fig. 5e).

Evaluation of The Hemostatic Performance In Vivo. In a rabbit ear injury model, the hemostasis time of gauze control, thrombin control, MS, MS-STMP, and MS-STMP-T was 167 ± 4, 88 ± 3, 107 ± 4, 58 ± 2, and 25 ± 3 s, respectively (Fig. 6a). The bleeding times were significantly reduced by MS, MS-STMP, and MS-STMP-T relative to the gauze control, with MS-STMP-T showing a 15% reduction. The bleeding time was longer with MS than with the thrombin control, but was reduced to 29% and 66% of the control value with MS-STMP and MS-STMP-T, respectively. In the femoral artery injury model, bleeding in femoral artery injury was abundant and could not be stemmed by the gauze or thrombin alone (Fig. 6b). MS, MS-STMP, and MS-STMP-T stopped the bleeding after 149 ± 3, 121 ± 4, and 67 ± 3 s. Additionally, MSSTMP-T stopped bleeding in injured liver in 16 ± 4 s (Fig 6c), which was 20% faster than the thrombin control. These results demonstrate the high in vivo efficiency of MS-STMP-T as a hemostatic agent.

Figure 6. Hemostatic effect and hemostasis time of MS-STMP-T in vivo in (a) rabbit ear, (b) femoral, and (c) liver artery injury models. Continuous bleeding indicates that the material was not able to control bleeding successfully. *P < 0.05.

In Vitro Hemolysis. The hemolysis ratio increased with NS, MS, MS-STMP, and MS-STMP-T concentration (Fig. 7a, b). NS had the highest hemolysis ratio at each concentration among the four samples: at concentrations of 4 and 8 μg/ml, the ratios were 6.7 ± 0.32% and 7.28 ± 0.47%, respectively, suggesting poor hemocompatibility. In contrast, the hemolysis ratios of MS, MS-STMP, and MS-STMP-T were all < 5%



at each concentration,48 with the lowest ratio being observed for MS-STMP-T. The insignificant hemolysis indicated good hemocompatibility, suggesting that MS-STMP-T can be applied as safe hemostatic material.

a

c

b

d

Figure 7. Hemocompatibility of MS-STMP-T. (a) Hemolysis ratio. (b) Representative light micrographs of RBCs in the presence of NS, MS, MS-STMP, and MS-STMP-T as compared to positive (P) and negative (N) controls. (c) Cytotoxicity of NS, MS, MS-STMP, MS-STMP-T to L929 cells after 1, 2, and 3 days. (d) Fluorescence micrographs of L929 cells treated with NS, MS, MS-STMP, or MS-STMP-T for 1 day. *P < 0.05.

Evaluation of Cytotoxicity. To investigate the biological functions of the hemostatic material, L929 cells were cultured with NS, MS, MS-STMP, and MS-STMP-T for 1, 2, or 3 days (Fig. 7c). After incubation for 1 day, cell viability was > 80% in all groups as compared to 72.6±7.8% in the presence of NS, indicating that the materials had relatively low toxicity. After incubation for 3 days, cell viability was over 100% in the MS, MS-STMP, and MS-STMP-T groups. After 1 day, L929 were spindle-shaped and proliferating (Fig. 7d). Strong green fluorescence signal corresponding to live cells were observed in all groups; however, red fluorescence was higher in the NS than in the other groups, indicating increased cell death. These results indicate that MS-STMP-T is not cytotoxic and is therefore safe as a hemostatic agent. In Vitro and In Vivo Degradation. We determined the weight loss ratio of NS, MS-STMP, and MSSTMP-T as a function of SBF immersion time to evaluate in vitro degradation (Fig. 8a). Weight loss ratio increased over time for all four samples, reaching an equilibrium on day 7. After immersion in SBF for 7 days, the weight loss ratio of NS, MS, MS-STMP, and MS-STMP-T was 59.1±2.3%, 71.2±2.5%, 84.7±1.3%, and 81.5±1.8%, respectively. MS-STMP-T and MS-STMP-T showed high degradation, likely due to the porous structure and the effect of STMP. An in vivo analysis by hematoxylin and eosin staining of muscle wounds that healed showed that NS and MS-STMP-T degraded over time (Fig. 8b). At 3 days, slight inflammation was observed at the site of implantation in both groups, but this disappeared after 7 days, and no starch granules were detected in tissue sections at 14 days, indicating that the materials had been completely absorbed by histiocytes. These results provide further evidence for the safety of MS-STMP-T as


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a hemostatic material.

Figure 8. (a) Weight loss ratio of NS, MS, MS-STMP, and MS-STMP-T after immersion in SBF. (b) Photographs of MS-STMP-T and NS with surrounding tissue at 3, 5, 8, and 14 days after implantation, and corresponding hematoxylin and eosin-stained tissue sections.

CONCLUSION MS-STMP-T was prepared by enzymolysis of corn starch, STMP modification and thrombin-assembly. Our results demonstrate that MS-STMP-T has high porosity and the capacity for rapid water absorption and plasma and platelets aggregation. It also has a high negative surface charge, which can promote hemostasis via charge-induced activation of blood coagulation. These hemostatic mechanisms synergize with thrombin to enhance hemostasis in whole blood clotting test, APTT and PT measurements, and animal injury models. Moreover, MS-STMP-T exhibits low hemolysis and cytotoxicity and is degraded within 14 days, suggesting excellent hemocompatibility, biocompatibility, and biodegradability. Thus, MS-STMP-T has excellent potential as a hemostatic agent in the treatment of massive hemorrhage, for which traditional approaches may be insufficient. AUTHOR INFORMATION Corresponding Author *College of Textile and Garments, Southwest University, Chongqing 400715, China. Phone: +8613594005200. E-mail: [email protected]. ORCID

Guangqian Lan: 0000-0002-5878-8289 Author Contributions Q.L. and F.L. contributed equally.

1

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 51703185), the Social development project of Guangdong province (No. 2017A020211015), and the Fundamental Research Funds



for the Central Universities (nos. XDJK2017B041 and XDJK2017C012), and this work was also funded by National Under-graduate Training Programs for Innovation and Entrepreneurship (201710635019).

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For Table of Contents Use Only

Synopsis: Biodegradable microporous starch with negative charged surface and assembled thrombin was developed as rapid and safe hemostatic agent.


Biodegradable microporous starch with assembled thrombin for rapid

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