Hemostatic material with near-infrared light-controlled shape memory properties and its preparation method and application

Abstract

A hemostatic material with near-infrared light-controlled shape memory properties, its preparation method, and its application. Due to the shape memory effect, the sponge's shape can be restored to 90% within 25 seconds. By switching the near-infrared light source on and off, the sponge's expansion can be controlled and adjusted. The sponge's hemostatic ability has been verified in vitro and in vivo. As expected, compared with commercial medical gelatin sponges with insufficient expansion capacity and commercial medical PVA sponges with excessive expansion capacity, the PDA-TPI-PU shape memory sponge can quickly and effectively control bleeding under near-infrared light irradiation, showing potential application prospects as a hemostatic material. With excellent mechanical properties, blood compatibility, cytocompatibility, and photothermal conversion performance, the PDA-TPI-PU sponge provides a new approach to using light-controlled shape memory materials as coagulation materials for treating incompressible bleeding.

Description

Technical Field

[0001] The present invention relates to the technical field of wound repair materials, and in particular to a hemostatic material with near-infrared light-controlled shape memory properties, and a preparation method and application thereof.

[0002] Background technology theory

[0003] Excessive blood loss from uncontrolled bleeding often leads to high mortality rates. Most commercial hemostatic products remain challenging to treat life-threatening, incompressible bleeding wounds with massive hemorrhage and hypertension. This is primarily due to the uncontrollable expansion capacity of these hemostatic products.

[0004] Excessive blood loss from uncontrolled bleeding often leads to shock, hypotension, impaired consciousness, and multiple organ failure, often resulting in high mortality rates among civilians and military personnel. Timely and effective hemostasis at the wound site is crucial to saving lives. In addition to significantly shortening the time it takes to stop bleeding and reducing the amount of bleeding, ideal hemostatic materials should also have good biocompatibility, blood compatibility, ease of manufacture, and low cost. Currently, the main categories of hemostatic materials include gauze, bandages, zeolite and chitosan-based powders, gelatin-based sponges, etc. However, most of these hemostatic products still face significant challenges in treating life-threatening, incompressible bleeding wounds with massive bleeding and hypertension. The main reason is that the expansion capacity of these hemostatic products is uncontrollable, making it difficult to achieve effective hemostasis.

[0005] In recent years, medical products based on shape memory polymers (SMPs) have been widely used in clinical treatments. For example, SMP foams are used as hemostatic agents because they can recover from a temporary shape to a "memorized" permanent shape in response to water or blood stimulation, and can achieve hemostasis in common wounds by applying appropriate pressure to the bleeding site. However, most SMP foams are not suitable for incompressible bleeding due to their slow water absorption and shape recovery. To overcome these obstacles, a number of hemostatic sponges have been developed. These sponges can rapidly expand to fill the wound with water and apply pressure to the wound, effectively stopping severe bleeding. However, the expansion of most hemostatic sponges is uncontrollable. Excessive expansion of the sponge at the bleeding site often causes damage to surrounding tissues or peripheral nerves, prolonging the wound's healing process and duration. In more serious cases, it can lead to postoperative complications and irreversible damage to the body.

[0006] Cryogels with interconnected macropores can be compressed and quickly recover their shape upon contact with blood. The resulting squeezing force on the wound will achieve effective hemostasis. However, many cryogels generally exhibit poor mechanical properties. The water / blood-triggered shape recovery properties of cryogels are also uncontrollable, which may limit their application in hemostasis. Therefore, the development of shape memory hemostatic materials with fast shape recovery speed and controllable expansion ability for incompressible large-scale bleeding control remains a challenge.

[0007] Different lasers can act as external triggers to control the stimulus-responsive behavior of shape memory materials, enabling remote control, precise focusing, and rapid switching. In recent years, light-triggered SMPs have attracted widespread attention due to their wide range of biomedical applications. Xie developed a biodegradable shape memory implant based on black phosphorus nanofillers, which exhibits excellent near-infrared (NIR) light-responsive shape memory properties. Polyurethane / black phosphorus pillars implanted in the subcutaneous tissue of the back and vagina rapidly changed shape under moderate 808 nm light irradiation. Dai prepared a near-infrared light-triggered shape memory polyurethane based on PCL prepolymer, which has great potential for application in biomedical implants. Under NIR light irradiation, samples under the skin of the mouse's back gradually recovered from their initial recumbent state to a visible contour. Remarkably, during the recovery process, the sample's shape remained stable after the laser irradiation was removed. Therefore, using NIR light irradiation technology to control the expansion properties of shape memory materials is an ideal choice.

[0008] Many materials with photothermal conversion properties have been used in shape memory research. Polydopamine (PDA) is the most common, simplest, and most effective material. PDA is formed by the self-polymerization of dopamine in an alkaline solution and has good bioactivity and biocompatibility. At the same time, PDA has a high molar extinction coefficient and can effectively absorb near-infrared light energy and convert it into heat energy. In addition, polydopamine contains phenolic hydroxyl and amino groups, which can activate the blood coagulation system, increase the possibility of preventing infection, and accelerate wound healing. Summary of the Invention

[0009] To address the technical deficiencies of existing technologies, the present invention provides a hemostatic material with near-infrared light-controlled shape memory properties, as well as its preparation method and application. The sponge's degree of shape recovery can be controlled by adjusting the near-infrared light exposure time according to the bleeding condition of the wound. The speed of shape recovery, temperature changes at different locations on the sponge during the recovery process, and the effects of different near-infrared light intensities on shape recovery were systematically studied.

[0010] The technical solution adopted by the present invention is: a hemostatic material with near-infrared light-controlled shape memory properties, wherein the hemostatic material includes a composite material PDA-TPI-PU sponge made of trans-1,4-polyisoprene (TPI), polyurethane (PU) sponge and polydopamine (PDA) material.

[0011] The trans-1,4-polyisoprene (TPI) and polydopamine (PDA) are coated on the surface of the PU sponge.

[0012] A method for preparing a hemostatic material having near-infrared light-controlled shape memory properties comprises the following steps:

[0013] (1) Preparation of TPI-PU coating: TPI was added to a chloroform solution with a concentration of 1 mg / mL. The PU sponge was then immersed in acetone and ethanol at 24°C. After ultrasonic cleaning with deionized water, the PU sponge was immersed in chloroform containing TPI to form a TPI coating on the surface of the PU sponge.

[0014] (2) Preparation of PDA-TPI-PU sponge: Dopamine (DA) and NaIO4 were added to sodium hydroxide solution, and the TPI-PU coated sponge was immersed in the prepared solution. The beaker was then wrapped with aluminum foil and shaken at 37°C for 24 h. Finally, the sponge was washed with deionized water until the washing solution became transparent and dried for 4 h to obtain the PDA-TPI-PU sponge.

[0015] In the step (3), the mass ratio of dopamine (DA) to NaIO4 is 4:1.

[0016] The pH of the sodium hydroxide solution in the step (3) is 8.

[0017] Application of PDA-TPI-PU composite materials in the preparation of hemostatic materials with controllable expansion.

[0018] The hemostatic material is a hemostatic sponge material controlled by near-infrared light.

[0019] The near infrared light control adopts 808nm 0.18-0.32W / cm 2 of near-infrared radiation.

[0020] The present invention provides a hemostatic material with near-infrared light-controlled shape memory properties, as well as its preparation method and application. Due to the shape memory effect, the sponge's shape can be restored to 90% within 25 seconds. By switching the near-infrared light source on and off, the sponge's expansion can be controllably adjusted. The sponge's hemostatic ability has been verified in vitro and in vivo. As expected, compared to commercial medical gelatin sponges with insufficient expansion capacity and commercial medical PVA sponges with excessive expansion capacity, the PDA-TPI-PU shape memory sponge can quickly and effectively control bleeding under near-infrared light irradiation, demonstrating potential application as a hemostatic material. The material possesses excellent mechanical properties, hemocompatibility, cytocompatibility, and photothermal conversion performance. The PDA-TPI-PU sponge provides a new approach for utilizing light-controlled shape memory materials as coagulation materials for treating incompressible bleeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 (a) FT-IR spectra of different sponges. (bd) SEM images and magnified images of PU sponge, TPI-PU sponge, and PDA-TPI-PU sponge, respectively. (e) Porosity of different sponges. (f) Statistical quantitative data of pore size of different sponges. (g) Density of different sponges. (hj) Water absorption capacity over time, water absorption capacity, and water absorption rate of different sponges. (km) Blood absorption capacity over time, absorption capacity, and absorption rate of different sponges. (*p < 0.05, **p < 0.01, ***p < 0.001).

[0022] Figure 2 Mechanical properties of sponges. (a) Compressive stress-strain curves of different sponges. (b) Maximum compressive stress of different sponges. (c) Stress-strain curves (10 cycles) of a PDA-TPI-PU sponge at 90% compressive strain. (d) Tensile stress-strain curves of different sponges. (e) Tensile strength at break and (f) Young's modulus of different sponges. (g) Tensile images of a PDA-TPI-PU sponge. (*p < 0.05, **p < 0.01, ***p < 0.001).

[0023] Figure 3 The photos show the PDA-TPI-PU sponge in air (a) and blood (b) at 0.32W / cm 2 NIR-triggered shape memory behavior under NIR irradiation. (ce) Different power densities (0.18 W / cm 2 and 0.32W / cm 2 ) near-infrared light irradiation, the shape recovery rate curve of the sponge in air, the maximum recovery rate, and the recovery rate within 25s. (fh) Different power densities (0.18W / cm2 and 0.32W / cm 2 ) under near-infrared light irradiation, the shape recovery rate curve, maximum recovery rate and recovery rate within 25s of the sponge in the blood.

[0024] Figure 4 (a) Thermal infrared image of PDA-TPI-PU sponge irradiated by 808 nm laser with an irradiation power of 0.18 W / cm 2 and 0.32 W / cm 2 (bd) Temperature profiles of the core (the position where the sponge is in direct contact with the near-infrared light), bottom (the bottom position of the sponge), and environment (the side position of the sponge) of the PDA-TPI-PU sponge during 808 nm laser irradiation at 0.18 W / cm 2 and 0.32 W / cm 2 (e) Under laser irradiation of 0.18 W / cm 2 and 0.32 W / cm 2 The maximum-minimum temperature difference (ΔT) at different positions of the sponge under strong 808 nm near-infrared irradiation. (f) Compressed PDA-TPI-PU sponge at 0.32 W / cm 2 Temperature change under near-infrared irradiation, irradiation turned off after 25 s. (g) 0.32 W / cm 2 Volume recovery rate under 808 nm laser irradiation (*p<0.05, **p<0.01, ***p<0.001).

[0025] Figure 5 Evaluation of the blood and cytocompatibility of the sponges. (a) Hemolysis rates of different samples and photos taken during the hemolysis test. Numbers 1, 2, 3, 4, 5, and 6 represent DW, NS, Medical PVA, PU, ​​TPI-PU, and PDA-TPI-PU, respectively. (b) Cell viability of different sponge samples at 24 and 48 hours (*p < 0.05, **p < 0.01, ***p < 0.001).

[0026] Figure 6 (a) BCI test results. (b) BCT test results. (c) SEM images of RBC and platelet adhesion on different sponges. (*p < 0.05, **p < 0.01, ***p < 0.001).

[0027] Figure 7(a) Schematic diagram of the in vivo SD rat liver penetrating wound model. (b) Photographs of the hemostatic effects of gelatin sponge, medical PVA sponge, and PDA-TPI-PU sponge. (c) and (d) Total blood loss and hemostasis time of gelatin sponge, medical PVA sponge, and PDA-TPI-PU sponge. (eh) Photographs of the hemostatic effects of gelatin sponge, medical PVA sponge, and PDA-TPI-PU sponge in the SD rat liver penetrating wound model. The red circle and yellow oval represent the bleeding site and the size of the sponge after expansion, respectively. n = 3 rats per group. (*p < 0.05, **p < 0.01, ***p < 0.001).

[0028] Figure 8 Schematic diagram of the hemostatic mechanism of the PDA-TPI-PU sponge. (a) Incompressible, heavy bleeding. (b) Hemostatic effect of an underexpanded hemostat. (c) Hemostatic effect of a PDA-TPI-PU sponge after controlled and effective expansion. (d) Hemostatic effect of an overexpanded hemostat. (e) Illustration of the controllable and effective expansion of a PDA-TPI-PU sponge in an incompressible wound under near-infrared light. DETAILED DESCRIPTION

[0029] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0030] Material

[0031] Trans-1,4-polyisoprene (TPI) was purchased from Wuhan Provi Biotechnology Co., Ltd. Dopamine, sodium periodate, and sodium hydroxide (NaOH) were purchased from Aladdin Industries. Anhydrous ethanol was purchased from Kelong Chemical Co., Ltd. (Chengdu, China). Polyurethane (PU) sponge was purchased from Yimeijia Technology Co., Ltd. (Henan, China). Acetone was obtained from Zhejiang Zhongxing Chemical Reagent Co., Ltd. The Calcein / PI cell viability / cytotoxicity assay kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Sprague-Dawley (SD) rats (480–550 g) were provided by the First Affiliated Hospital of Wenzhou Medical University. Fresh rabbit blood was provided by the Experimental Animal Center of the Wenzhou Institute of the University of Chinese Academy of Sciences (Wenzhou, China). Deionized water was used in all experiments.

[0032] Preparation of TPI-PU coating

[0033] The TPI coating was formed using the following steps. First, TPI was added to a chloroform solution at a concentration of 1 mg / mL. Next, the polyurethane (PU) sponge was immersed in acetone and then ethanol at 24°C for 15 minutes. After ultrasonic cleaning with deionized water for 15 minutes, the PU sponge was immersed in chloroform containing TPI for approximately 30 minutes. Finally, the TPI coating was formed on the surface of the PU sponge.

[0034] Preparation of PDA-TPI-PU sponge

[0035] DA and NaIO4 (4:1, w / w) were added to a sodium hydroxide solution (pH = 8). A TPI-PU-coated sponge was immersed in the prepared solution. The beaker was then wrapped in aluminum foil and shaken at 37°C for 24 hours. Finally, the resulting sponge was rinsed with deionized water until the rinse solution became transparent. After drying for 4 hours, the PDA-TPI-PU sponge was obtained.

[0036] Chemical and physical properties

[0037] FTIR

[0038] FTIR (Magna-560, Nicolet) was used to confirm the successful coating of TPI and PDA on the PU sponge and record the spectra of the sponge samples. All FT-IR samples were characterized by attenuated total reflectance (ATR).

[0039] Morphological observation

[0040] The macrostructure and microstructure of the sponge samples were characterized using a field emission scanning electron microscope (SU8010, HITACHI, Japan). Before observation, the samples were freeze-dried and sputtered with platinum.

[0041] Porosity and density

[0042] To calculate the density (ρ) of a sponge, first cut the sponge into a cylindrical shape and measure the mass (M) and dimensions (V) (length and diameter) of the cylindrical sponge using a balance and a digital caliper, respectively. The formula for calculating density (ρ) is as follows (1):

[0043]

[0044] The porosity of the sponge sample was evaluated using the liquid displacement method. The initial weight of the sponge sample was called M0. Next, the sponge sample was soaked in a certain amount of ethanol for 30 minutes, then removed from the ethanol and weighed again as M1. The formula for calculating the porosity (P) is as follows (2):

[0045]

[0046] V0 is the volume of the sponge. ρ eis the density of ethanol (0.785 g / cm 3 ).

[0047] Water / blood absorbing ability

[0048] Before testing, the sponge samples were cut into the same shape and placed in a freeze dryer for freeze drying. The volume of the sponge was recorded as V (cm 3 The weight of the freeze-dried sponge sample was recorded as M dry (g). The freeze-dried sponges were then soaked in deionized water / blood at 37°C. At different time points, the samples were removed and gently wiped on filter paper to remove excess surface water. The wet weight of the samples was immediately measured and recorded as M. wet (g) The fluid absorption capacity is given by the following formula (3):

[0049]

[0050] The water / blood absorption rate was calculated by measuring the slope of the water / blood absorption capacity-time curve within 5 s.

[0051] Mechanical testing

[0052] The compression, recovery, and tensile strength of the sponge were measured using a UTM2102 electronic universal testing machine through compression, cyclic compression, and tensile tests. In the compression test, the wet sponge was formed into a cylinder with a height of 10 mm and a diameter of 10 mm and compressed at a rate of 10 mm / min to a maximum strain of 90%. In the cyclic compression test, a compressive strain of 90% was applied to the sponge sample. A cylindrical sponge of the same dimensions as above was compressed at a rate of 10 mm / min to the strain and then recovered to 0% strain at the same rate. This cyclic experiment was repeated 10 times. In the tensile test, a cylindrical sponge with a height of 10 mm and a diameter of 10 mm was stretched at a rate of 50 mm / min until it broke. The tensile strength was defined as the maximum stress during the tensile test. Each set of tests was repeated three times.

[0053] Shape memory properties

[0054] To measure the photothermally induced shape memory properties of a PDA-TPI-PU sponge, the sponge was compressed at 70°C and then placed at 4°C to stabilize its shape. After the temporary shape was fixed, the sponge was returned to room temperature and irradiated with laser light. The shape memory behavior was filmed using a mobile phone, and the thermal response time was recorded. The shape recovery rate was calculated by measuring the volume change. The measurement was repeated three times.

[0055] Photothermal experiments

[0056] A fiber-coupled continuous semiconductor diode laser (808 nm, FC-808-2000-MM) was used as the light source. In order to study the photothermal effect of the sponge, a power density of 0.18 W / cm 2 and 0.32W / cm 2 The sponge was irradiated with a laser of varying power output, causing it to gradually recover. Temperature changes were monitored using an infrared thermal imager. Photothermal images and temperatures of the sponge were also recorded using treatments with different output powers.

[0057] In vitro hemostatic properties

[0058] Hemolysis test

[0059] First, 0.2 mL of fresh rabbit blood was mixed with 1.8 mL of normal saline. Next, 5 mg of each sponge sample was immersed in the above mixed blood. After incubation at 37 ° C for 8 h, all samples were centrifuged at 3000 rpm for 10 min. Finally, 100 μL of supernatant was carefully transferred to a 96-well culture plate. The absorbance of the supernatant was measured at 540 nm using a microplate reader (Varioskan LUX, ThermoFisher). Ultrapure water and normal saline were used as the positive control group and negative control group, respectively. The hemolysis rate was calculated according to the following formula (4):

[0060] Among them A S 、A n and A p The absorbance of sponge samples, normal saline and ultrapure water at 540 nm are shown in Table 1. All experiments were repeated five times (n=5).

[0061] Blood coagulation index (BCI)

[0062] BCI was evaluated using a previously reported method. With the authorization of the Clinical Research Professional Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University, a certain amount of pure blood (9:1 whole blood and 3.8% sodium citrate) was collected from healthy blood donors. First, the prepared sponge samples were cut into 8 mm diameter circles, placed in a culture dish, and preheated at 37 °C for 5 min. Then, 200 μL of anticoagulated whole blood was dropped on the surface of each sponge sample. Subsequently, 20 μL of 0.2 mol / L CaCl2 solution was added to initiate coagulation and incubated at 37 °C for 5 min. After 5 min, 25 mL of deionized water was gently added to wash away the uncoagulated blood while avoiding disturbing the blood clot. Finally, the absorbance of the hemoglobin solution was recorded at 540 nm using a microplate reader (Varioskan LUX, ThermoFisher) (n = 5 per group). The culture dish without sponge served as the blank control group. BCI was calculated using the following formula (5):

[0063]

[0064] Among them A bs1 Represents the absorbance of the sample, A bs0 Represents the absorbance of the blank control group.

[0065] Blood clotting time (BCT)

[0066] Samples were tested for BCT according to previous literature. Since fresh human blood was unavailable, fresh rabbit blood was used for the BCT assay. Briefly, 10 mg of sponge was weighed and placed in a 2.5 mL centrifuge tube, which was then preheated at 37°C for 5 minutes. After adding 1 mL of fresh rabbit blood to each tube, 20 μL of CaCl2 (0.2 mol / L) was added. The tubes were inverted every 15 seconds to check the fluidity of the blood. The clotting time from the addition of the CaCl2 solution to complete clotting was recorded (n = 3).

[0067] Adhesion of red blood cells and platelets

[0068] Red blood cell suspension and platelet-rich plasma (PRP) were obtained by centrifuging whole blood from healthy donors (9:1 whole blood to 3.8% sodium citrate) at 1500 rpm for 10 minutes. Next, 10 mg of sponges were placed in a 24-well culture plate, and 100 μL of PRP and 250 μL of RBCs suspension were added to the plate. After incubation at 37°C for 30 minutes, the sponges were washed three times with PBS. Then, all sponges were fixed in 2.5% glutaraldehyde and then dehydrated with a series of graded ethanol solutions (50%, 75%, 80%, 90% and 100%). Finally, the dehydrated samples were fixed on a sample stage. After a thin layer of platinum was splashed on its surface, they were characterized using SEM.

[0069] Cytocompatibility

[0070] L929 fibroblasts were maintained in DMEM medium, and the medium was changed every 2 days. First, 100 μL of cell suspension (density 1×10 5 Cells / well) were seeded in 96-well plates and cultured in an incubator at 37°C and 5% CO2 for 24 hours. At the same time, the sponge samples that had been sterilized by ultraviolet light for 4 hours were immersed in DMEM, and the extract (concentration of 1 mg / mL) was prepared at 37°C for 24 hours. Then, the culture medium in the 96-well plate was replaced with 100 μL of sample extract. After 24 hours and 48 hours, 10 μL of CCK-8 reagent was added to each 96-well plate, and the well plate was placed in the incubator and cultured for another 2 hours. The test was repeated three times. Finally, the optical density (OD) value of the sample in the well plate at 450 nm was measured using a microplate reader. The cell viability obtained from the CCK-8 test was calculated using formula (6):

[0071]

[0072] Among them A s is the optical density of the cell suspension contacting the sponge sample; A c and A b is the optical density value of the control group and the blank group.

[0073] In vivo hemostatic properties

[0074] Sprague-Dawley (SD) rats were randomly divided into four groups, namely, a blank group, a gelatin sponge group, a medical PVA sponge group, and a PDA-TPI-PU sponge group. The wound without any treatment was used as the blank group. First, the rats were anesthetized by injecting 10wt% chloral hydrate (dose = 1mL / 250g). The abdomen of the rats was then opened to expose the liver. After placing a pre-weighed filter paper under the liver, a circular perforated wound (8mm in diameter) was created on the liver to form incompressible massive bleeding. The cylindrical sponge was compressed and filled into the bleeding site. In particular, the PDA-TPI-PU group needed to be irradiated with infrared laser in time. The amount of blood loss was measured by determining the total weight of blood absorbed by the filter paper. The hemostasis time was recorded when the bleeding stopped.

[0075] Statistical analysis

[0076] In this study, at least three samples were included in the experiment. Data were expressed as mean ± SD (standard deviation of the mean) and compared using one-way analysis of variance (ANOVA) using SPSS software. Differences were considered statistically significant when the P value was less than 0.05.

[0077] Results and discussion

[0078] Fabrication and characterization of sponges

[0079] To verify the structure of the sponges, different sponges were characterized by FT-IR spectroscopy ( Figure 1 (a)). Compared with the spectrum of PU sponge, 839cm -1 and 879cm -1 The characteristic peak is attributed to the out-of-plane bending vibration of =CH, 1665cm -1 The absorption peak at 879 cm was attributed to the stretching vibration of C=C, which indicated that TPI had been successfully coated on the PU sponge. When PDA was coated on the TPI-PU sponge, the absorption peak at 879 cm was still observed. -1 The peak of TPI coated on the sponge was 0.04, which proved that the TPI coated on the sponge had good stability. The above results showed that TPI and PDA were coated on the PU sponge and the PDA-TPI-PU sponge was successfully prepared.

[0080] The porous structure and rough surface of the hemostatic sponge play a key role in absorbing blood and concentrating the main blood components. The structural morphology of the sponge was evaluated by SEM. Figure 1 As shown in (b)-(d), the SEM images show that all sponges have interconnected and irregularly shaped macroporous three-dimensional structures, which can expand the surface area for absorbing blood. The partially enlarged SEM images show that the surface of the PU sponge is smooth and flat ( Figure 1 (b)). After coating with TPI, a layer of obvious wrinkles appeared on the surface of PU sponge ( Figure 1 (c)). After coating with PDA, the color of the sponge changed from white to black. From the partially enlarged SEM image, it can be observed that there are large particles on the rough surface of the PDA-TPI-PU sponge, which indicates that PDA is successfully coated on the surface of the TPI-PU sponge ( Figure 1 (d)).

[0081] The porosity measurement results are shown in Figure 1 (e) The porosity of PU sponge, TPI-PU and PDA-TPI-PU sponge are 95%, 94% and 94% respectively. The pore size distribution is shown in Figure 1 (f) The densities of PU sponge, TPI-PU and PDA-TPI-PU sponge are 3.38 g / cm 3 、3.45g / cm 3 and 3.59g / cm 3 ( Figure 1 (g)). These results indicate that the coating of TPI and PDA does not affect the porosity and density of PU sponge.

[0082] The properties of rapid absorption and concentration of blood components can help hemostatic sponges control heavy bleeding in a short period of time and promote the accumulation of blood coagulation components at the wound site, especially for the treatment of incompressible bleeding. Therefore, we measured the water and blood absorption properties of different sponges. The maximum water / blood absorption capacity of all sponges reached water / blood saturation within 5 seconds, indicating their rapid water / blood absorption capacity ( Figure 1 (h) and Figure 1 (k)). This is mainly attributed to its interconnected macroporous structure and high porosity, which allows water / blood to quickly penetrate into the sponge. After coating the surface with TPI and PDA, the maximum water absorption capacity of the sponge ( Figure 1 (i)) and blood-sucking ability ( Figure 1 (l)) decreased significantly. The water absorption rate of PDA-TPI-PU sponge ( Figure 1 (j)) and blood sucking rate ( Figure 1The PDA-TPI-PU sponge's m / s ratio (m) was reduced to approximately two-fifths that of the PU sponge. This is likely due to the cross-linked structure formed on the surface of the polyurethane sponge. Although the water and blood absorption rates of the PDA-TPI-PU sponge were lower than those of the PU sponge, the weight of the PDA-TPI-PU sponge after absorbing water was at least seven times its dry weight. This helps the PDA-TPI-PU sponge concentrate coagulation factors, thereby improving hemostasis efficiency.

[0083] Mechanical properties of sponge

[0084] The ideal hemostatic sponge should have excellent mechanical properties, form a physical barrier at the wound site, and act as a stable hemostatic plug to stop bleeding, especially for fatal bleeding in deep wounds. Therefore, compression stress-strain and cyclic compression stress-strain tests were used to evaluate the compressive strength and fatigue resistance of the sponge. After coating with TPI and PDA, the maximum compressive stress of the sponge increased from 40 kPa to 55 kPa and 71 kPa ( Figure 2 (b)). The compressive stress-strain curve of PDA-TPI-PU sponge after 10 cycles at 90% strain is also shown ( Figure 2 (c) After multiple high-strain compression cycles, the PDA-TPI-PU sponge can still maintain its original shape with only a slight recovery loss, indicating that the sponge has good mechanical properties.

[0085] The tensile strength of the sponge was evaluated by uniaxial tensile test. Figure 2 (d)- Figure 2 (f) shows the tensile stress-strain curve, Young's modulus and tensile strength at break of the sponge. The PDA-TPI-PU sponge shows better tensile properties than the PU sponge and TPI-PU sponge. For example, the tensile strength at break and the Young's modulus of the PDA-TPI-PU sponge are 855.46 kPa and 580 kPa, respectively, which are higher than those of the PU sponge and TPI-PU sponge. The reason can be explained by the fact that the interfacial bonding formed between the TPI-PU sponge and the PDA coating increases the crack propagation resistance of the TPI-PU sponge, and the interfacial stress transferred between the PDA and TPI-PU sponges during the compression test. The PDA-TPI-PU sponge has good reversibility during elongation and relaxation ( Figure 2 (g)). The above experimental results ensure that the PDA-TPI-PU sponge has good mechanical properties and can be easily removed from the wound.

[0086] NIR photothermal induced shape memory behavior

[0087] Since the sponge has good mechanical properties, we evaluated the shape memory behavior induced by near-infrared photothermal irradiation. The experiment found that the sponge has a fixation rate of 79%. After the shape is fixed, the sponge Figure 3 (a)) and blood( Figure 3 (b) PDA-TPI-PU sponge was irradiated with 808nm laser. The results showed that PDA-TPI-PU sponge had a good recovery effect not only in air but also in blood. In addition, the surrounding blood was absorbed during the sponge's recovery process. Further research found that in air, PDA-TPI-PU sponge had a good recovery effect at 0.18W / cm 2 The maximum recovery rate under near-infrared irradiation is 67%, and the recovery rate within 25s is 0.042cm / s. When the power density of near-infrared light increases to 0.32W / cm 2 When the maximum recovery rate of PDA-TPI-PU sponge is 91%, the recovery rate within 25s is 0.058cm / s( Figure 3 (c)-(e)).

[0088] In blood, at 0.18W / cm 2 Under near-infrared irradiation of 0.32W / cm, the maximum recovery rate of the sponge was 60%, and the recovery rate within 25s was 0.036cm / s. 2 Under near-infrared irradiation, the maximum recovery rate of the sponge is 80%, and the recovery rate within 25s is 0.048cm / s( Figure 3 (f)-(h)). Notably, the recovery time of the NIR-induced PDA-TPI-PU sponge is significantly shorter than that of the shape memory polymer foam used as a hemostatic agent (approximately 8 min).

[0089] The structural morphology of the PDA-TPI-PU sponges before and after recovery was then evaluated using SEM. The results revealed that the PDA-TPI-PU sponges exhibited a collapsed and closed porous structure after compression, yet they maintained the integrity of their network structure. After near-infrared light-induced recovery, the sponges retained their original interconnected macroporous structure, demonstrating that these sponges possess stable classical elastic properties suitable for hemostasis in incompressible hemorrhages.

[0090] Next, the temperature of the sponge surface under near-infrared light was further studied using an infrared thermal imager. The photothermal imaging of the compressed PDA-TPI-PU sponge under 808nm laser of different intensities was evaluated and analyzed in detail. Figure 4 As shown in (a), the photothermal image shows that the temperatures of the core, bottom and environment of the PDA-TPI-PU sponge are different. 2 Under the 808nm laser, the core temperature of the sponge was as high as 55℃, the ambient temperature was as high as 47℃, and the bottom temperature was as high as 38℃. Figure 4 (b)-(d)). At an intensity of 0.32W / cm 2Under the 808nm laser, the highest core temperature of the sponge was 77℃, the highest ambient temperature reached about 49℃, and the highest bottom temperature was 40℃ ( Figure 4 (b)-(d)). Although at 0.32W / cm 2 Under the irradiation intensity, the ΔT of the center temperature, ambient temperature and bottom temperature is higher than 0.18W / cm 2 The ΔT of the temperature at each position of the sponge under the irradiation intensity, but the highest temperature of the environment where the sponge is in direct contact with the tissue is 49°C, which will not cause damage to the tissue ( Figure 4 (e)). Although the temperature of the core part rises rapidly from 26°C to 77°C within 25 seconds, it can drop rapidly to 31°C within 12 seconds after the near-infrared light irradiation is stopped ( Figure 4 (f)).

[0091] Based on the above experiments, we found that PDA-TPI-PU sponge has good shape stability and photothermal stability. Figure 4 As shown in (g), during the recovery process, the shape of the PDA-TPI-PU sponge can remain unchanged after the light is turned off, and it can continue to recover when irradiated again. The results show that the PDA-TPI-PU sponge has controllable shape recovery ability under the triggering of near-infrared light.

[0092] In summary, the shape recovery of the sponge can be flexibly controlled by turning the near-infrared light on and off. This is attributed to the TPI and PDA coatings on the sponge surface. Trans-1,4-polyisoprene (TPI) is a typical SMP. Its shape memory ability is achieved through the formation and melting of crystalline regions. Compared with PU sponge, TPI has a relatively high modulus, which helps the sponge to fix its shape. Polydopamine (PDA) has excellent photothermal conversion ability. Under near-infrared light irradiation, the PDA coating on the sponge surface can undergo photothermal conversion, which can make the sponge surface temperature reach the melting temperature of TPI (T m ,55°C), causing its crystalline regions to melt. At this point, the TPI releases entropic energy due to the activation of molecular chain mobility, driving the molecular chains back to their original shape. Combined with the elastic force of the PU sponge, the sponge's shape is restored. However, once NIR illumination ceases, the material rapidly cools through thermal conduction, and crystalline domains form in the TPI, reducing molecular chain mobility, locking the deformed chain conformation, and allowing the system to store entropic energy. At this point, the shape recovery process ceases. Therefore, when the external force is removed and the temperature is below 55°C, the fixed sponge can overcome the elastic force and maintain its shape. After repeated NIR irradiation, the sponge eventually returns to its permanent shape. Given the reproducible formation and reversibility of the TPI domain formation and melting and the short cooling time to the melting temperature, the volume and shape of the PDA-TPI-PU sponge are controllable.

[0093] Hemocompatibility and cytocompatibility

[0094] The hemolysis rate is very important for evaluating the blood compatibility of sponges. Figure 5 (a) Neither the normal saline group (negative control group) nor the sponge-treated group showed hemolysis. The hemolysis rate of the sponges met the American Society for Testing and Materials standard (<5%), indicating that these sponges have good compatibility with human red blood cells and can be used as a potential hemostatic agent.

[0095] Non-cytotoxicity is one of the prerequisites for applying materials as wound dressings. As fibroblasts play a key role in wound reconstruction, L929 cells were used to evaluate the cytotoxicity of sponges using the CCK-8 assay. Figure 5 As shown in (b), cell viability results indicate that the sponge is non-cytotoxic. Furthermore, the in vitro cytocompatibility of the PDA-TPI-PU sponge was assessed using a contact assay. LSCM images showed that L929 cells adhered and grew well on the sponge surface, consistent with the CCK-8 assay. These results demonstrate that the PDA-TPI-PU sponge has excellent hemocompatibility and cytocompatibility.

[0096] In vitro coagulation performance

[0097] In vitro coagulation tests were performed to evaluate the coagulation ability of the sponges, including BCI, BCT, and adhesion of red blood cells and platelets. First, the BCI of all sponges was evaluated by measuring the absorbance value of the collected hemoglobin solution. Lower BCI values ​​indicate faster coagulation. Figure 6 As shown in (a), the BCI values ​​of commercial PVA sponge, PU, ​​TPI-PU, and PDA-TPI-PU sponges were 41.11%, 36.83%, 35.76%, and 39.61%, respectively, which were significantly lower than those of the blank control group (100%, untreated blood served as the blank control). Therefore, the PDA-TPI-PU sponge exhibited excellent coagulation performance.

[0098] BCT is defined as the time required for plasma to transform from a heavily calcified solution into a solid clot. Figure 6 As shown in (b), in the blank control group (untreated blood), the BCT of whole blood was 639 s. The BCTs of the commercial PVA sponge, PU sponge, TPI-PU sponge, and PDA-TPI-PU sponge were 362 s, 377 s, 358 s, and 287 s, respectively. Clearly, the PDA-TPI-PU sponge exhibited superior hemostatic efficacy compared to the blank control. This is attributed to the fact that polydopamine with phenolic hydroxyl and amino groups improves the adhesion properties of the sponge surface, activates the extrinsic coagulation system, and thus induces platelet adhesion and promotes blood cell aggregation.

[0099] Blood coagulation and platelet plug formation at the bleeding site play an important role in hemostasis and are the main steps in controlling bleeding. The coagulation effect of the sponge was further evaluated by the adhesion test of red blood cells and platelets ( Figure 6 (c)). Images of red blood cells attached to the surface of the PDA-TPI-PU sponge show that most red blood cells maintain their normal disc shape, with only a few migrating to spherical and spherical echinocytes. Therefore, the PDA-TPI-PU sponge exhibits good red blood cell compatibility. Furthermore, platelets on the PDA-TPI-PU sponge adopt a unique, irregular shape. Pseudopodia are visible, indicating that platelets are activated and participate in the hemostatic process. This is because the -N-group-containing PDA may activate platelets through charge, causing platelet aggregation and enhancing the sponge's hemostatic ability. These results demonstrate the significant ability of the PDA-TPI-PU sponge to promote coagulation through blood cell adhesion and activation.

[0100] Incompressible heavy bleeding control

[0101] The control of incompressible massive bleeding was further verified using a rat liver penetrating wound model. Figure 7 As shown in (a), untreated rats served as positive controls, and commercial gelatin sponges and medical PVA sponges served as negative controls. Blood loss and hemostasis time were used as the measurement criteria for hemostasis. Figure 7 (b) shows the bloodstain images of different groups. Figure 7 (c) shows that the hemostasis time in the blank group was 310 seconds. After treatment with the commercial gelatin sponge, medical PVA sponge, and PDA-TPI-PU sponge, the hemostasis times were 280 seconds, 221 seconds, and 186 seconds, respectively. Clearly, the hemostasis time of the PDA-TPI-PU sponge was significantly shorter than that of the blank group.

[0102] The blood loss of different experimental groups was then recorded. The results showed that the blood loss and hemostasis time showed a similar trend ( Figure 7 (d)). The blood loss in the blank group was 2.36 g, while the blood loss in the commercial gelatin sponge group, the medical PVA sponge group, and the PDA-TPI-PU sponge group was 2.04 g, 0.71 g, and 0.51 g, respectively. Notably, the blood loss in the PDA-TPI-PU group was approximately one-quarter that of the commercial gelatin group. These results demonstrate that the PDA-TPI-PU sponge exhibits excellent procoagulant properties both in vitro and in vivo.

[0103] Schematic diagram of the hemostatic effects of gelatin sponge, medical PVA sponge and PDA-TPI-PU sponge in the SD rat liver trauma model. Figure 7 (eh). There was a lot of bleeding in the penetrating wound model ( Figure 7(e)). When the gelatin sponge is filled into the wound cavity, it cannot properly seal the wound due to poor shape recovery, so blood flows out, resulting in massive blood loss and prolonged hemostasis time ( Figure 7 (f)). In the medical PVA group, due to excessive expansion, the wound was over-compressed, resulting in secondary bleeding ( Figure 7 (g)). On the contrary, when the PDA-TPI-PU sponge is filled into the wound cavity, it can recover quickly under the timely irradiation of near-infrared light, fit the wound cavity in time, and exert pressure on the wound wall to form a physical barrier to prevent bleeding ( Figure 7 (h)).

[0104] In previous reports, shape memory foam, a hemostatic agent commonly used for incompressible wounds, has a long time to recover its shape. Macroporous cryogels can recover quickly but have poor mechanical properties. The shape recovery properties of these hemostatic agents are difficult to control. Figure 8 As shown in (b), the under-expanded hemostatic sponge cannot exert pressure on the incompressible, heavily bleeding wound in time, resulting in blood overflow and prolonged hemostasis time. Excessive expansion of the hemostatic sponge usually leads to damage to the surrounding tissues and postoperative complications ( Figure 8 (d)).

[0105] In this study, we explored the PDA-TPI-PU sponge with controllable and efficient expansion triggered by near-infrared light ( Figure 8 (c)). When the sponge is placed in an incompressible wound, under the irradiation of the 808nm laser, the surface of the PDA-TPI-PU sponge undergoes photothermal conversion, causing the surface temperature to rise rapidly, promoting the sponge's ability to recover from the compressed state. When the 808nm laser is turned off, the surface temperature of the sponge quickly drops below the melting temperature, at which time the PDA-TPI-PU sponge can maintain its temporary shape. When the laser is turned on again, the sponge can continue to recover until it fully fits the wound ( Figure 8 (e)).

[0106] in conclusion

[0107] In this study, we report a PDA-TPI-PU shape-memory hemostatic sponge with excellent properties such as mechanical properties, hemocompatibility, cytocompatibility, and photothermal conversion. Due to the shape memory effect, the sponge's shape can be restored by 90% within 25 seconds. By switching a near-infrared light source on and off, the sponge's degree of expansion can be controllably adjusted. Furthermore, the sponge's hemostatic ability has been validated in vitro and in vivo. As expected, compared with a commercial medical gelatin sponge with insufficient expansion capacity and a commercial medical PVA sponge with excessive expansion capacity, the PDA-TPI-PU shape-memory sponge can quickly and effectively control bleeding under near-infrared light irradiation, demonstrating potential application as a hemostatic material. We believe that the PDA-TPI-PU sponge provides a new approach for utilizing light-controlled shape-memory materials as hemostatic materials for treating bleeding from incompressible wounds.

[0108] Technical personnel should note: Although the present invention has been described according to the above specific implementation methods, the inventive concept of the present invention is not limited to this invention. Any modification using the inventive concept will be included in the scope of protection of this patent.

[0109] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. Application of hemostatic materials with near-infrared light-controlled shape memory properties in the preparation of hemostatic materials with controllable expansion, characterized in that The hemostatic material includes a composite material PDA-TPI-PU sponge made of trans-1,4-polyisoprene (TPI), polyurethane (PU) sponge and polydopamine (PDA). The trans-1,4-polyisoprene (TPI) and polydopamine (PDA) are coated on the surface of the PU sponge. The near-infrared light control adopts 808nm 0.18-0.32W / cm 2 The hemostatic material with near-infrared light-controlled shape memory properties is prepared by the following steps: (1) Preparation of TPI-PU coating: TPI is added to a chloroform solution with a concentration of 1 mg / mL, and then the PU sponge is immersed in acetone and ethanol at 24°C in sequence, and after ultrasonic cleaning with deionized water, the PU sponge is immersed in chloroform containing TPI to form a TPI coating on the surface of the PU sponge; (2) Preparation of PDA-TPI-PU sponge: Dopamine (DA) and NaIO4 were added to a sodium hydroxide solution with a mass ratio of dopamine (DA) to NaIO4 of 4:

1. The TPI-PU coated sponge was immersed in the prepared solution, and then the beaker was wrapped with aluminum foil and shaken at 37 °C for 24 h. Finally, the sponge was washed with deionized water until the washing solution became transparent and dried for 4 h to obtain the PDA-TPI-PU sponge.

2. The use according to claim 1, characterized in that , the pH of the sodium hydroxide solution in step (2) is 8.

Images