Abstract
Anti-hemorrhage nanomaterials are emerging as a promising alternative to traditional materials such as cotton and medical gauze, which often exhibit limited hemostatic properties and excessive adhesion to wound surfaces. This study presents the development of hemostatic nanofiber mats fabricated from polyacrylonitrile (PAN) via electrospinning. The incorporation into these mats of cost-effective and eco-friendly hemostatic agents, specifically exfoliated bentonite and calcium chloride, is investigated. Structural characterization using X-ray diffraction (XRD) reveals significant alterations in the bentonite structure, supported by the disappearance of the (001) crystal plane peak following exfoliation. Fourier Transform Infrared Spectroscopy (FTIR) confirms the successful integration of bentonite and calcium chloride into the fiber matrix. Scanning Electron Microscopy (SEM) illustrates a uniform morphology of continuous, randomly distributed fibers free of beads, with bentonite evenly dispersed throughout the mat. The addition of calcium chloride reduces fiber diameter without causing noticeable agglomeration. Water contact angle measurements indicate enhanced adhesion properties of the mats, with reduced hydrophobicity attributed to the water affinity of the incorporated additives. In vitro tests demonstrate that all electrospun mats exhibit superior hemostatic activity, with the most effective formulation -PAN loaded with exfoliated bentonite and calcium chloride-achieving a low blood coagulation index of 44.9% and significantly shortening blood-clotting time from 285 s in control samples to just 105 s. These findings highlight that the developed nanofiber mats outperform traditional hemostatic products in terms of eco-friendliness, blood coagulation efficiency, and clotting time, highlighting their potential for enhanced clinical applications in hemorrhage control at a reduced cost.
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Introduction
Uncontrolled hemorrhage, resulting from severe injury or trauma, accounts for 50% of deaths on the battlefield, 31% of civilian injury fatalities, and 10% of total annual deaths worldwide1. This alarming statistic highlights the urgent need for effective hemostatic products that can aid in bleeding control and significantly reduce mortality associated with hemorrhage. Traditional hemostatic agents, such as creams, solvents, medical gauze, and cotton, exhibit several limitations. They absorb fluids rapidly, leading to a sharp decline in their hemostatic effectiveness. Additionally, their antimicrobial properties are moderate. Consequently, there is a pressing demand for innovative, high-performance hemostatic materials2. A comprehensive understanding of the hemostasis process is essential for the effective development of these materials. Hemostasis is a physiological mechanism that facilitates thrombus formation at the site of vascular injury while maintaining normal blood flow throughout the rest of the body3. The primary stages of natural hemostasis include vascular contraction, platelet adhesion, the conversion of prothrombin to thrombin (which subsequently forms fibrin to stabilize the clot), and the processes of clot retraction and dissolution. Fibrin formation is particularly crucial, as it provides a scaffold for platelets to aggregate, mimicking the extracellular matrix (ECM)4,5. Therefore, it is vital to create hemostatic materials that closely resemble fibrin and effectively retain platelets, thereby promoting clot formation.
The selection of the appropriate morphology is the crucial first step in the development of effective hemostatic materials. Morphologies that resemble the extracellular matrix (ECM) serve as templates for clot formation. However, the choice of materials remains equally critical. The properties of the materials are of great importance for hemostatic applications, particularly concerning low toxicity and biocompatibility.
Among the various fabrication methods, electrospinning stands out as one of the simplest, most cost-effective, and versatile techniques for producing polymer nanofibers. The morphology of electrospun nanofibers resembles that of the extracellular matrix (ECM), making them highly suitable for hemostatic applications. The electrospinning process benefits from an electric field to transform a polymeric solution into nanofibers. The fibers are ejected from a needle connected to the positive electrode and subsequently collected on a substrate linked to the negative electrode. The surface tension of the polymer solution droplets can be precisely controlled by adjusting the molecular weight and concentration of the polymer6,7.
Polyacrylonitrile (PAN), a semi-crystalline thermoplastic polymer, is particularly significant for its remarkable mechanical properties and stability in a wide range of organic solvents. PAN emerges as a promising candidate for hemostatic applications due to its favorable characteristics such as low toxicity and biocompatibility8,9. The strong intramolecular forces between PAN chains, facilitated by hydrogen bonding with nitrogen atoms, contribute to its robustness. Among solvents, dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) are especially effective at dissolving PAN due to their high polarity. In the field of wound healing, PAN has been employed in the development of medical dressings that exhibit antimicrobial properties8,9. Its desirable characteristics -such as low toxicity, impermeability to pathogens, and biocompatibility with blood- make it an ideal candidate for such applications. Additionally, PAN dressings facilitate easy removal after clot formation and help maintain an appropriate moisture level at the wound site, enhancing the healing process10.
Bentonite is a type of rock primarily composed of mineral clays, with Montmorillonite being its main constituent, classified within the Smectite group11. Bentonite, known for its tunable morphology and reduced cost, is well-suited for hemostatic applications12. As a result, bentonite does not have a specific molecular formula. Mineral clays are predominantly made up of hydrated aluminum silicate and possess a unique structure characterized by tetrahedral silicate sheets and octahedral aluminate sheets13,14. Due to its structure, bentonite possesses large surface area and high ability to absorb water and liquids. Additionally, bentonite exhibits a significant cation exchange capacity, allowing it to accommodate both organic cations, such as proteins, and inorganic cations, including metal ions. Bentonite can be exfoliated through the replacement of calcium (II) cations with sodium (I) cations. This process separates the octahedral and tetrahedral sheets, resulting in an even larger specific surface area and an enhanced ability to absorb water14. Thus, exfoliated bentonite is expected to exhibit high hemostasis performance. Furthermore, bentonite is a low-cost clay, making it a suitable choice for inclusion as a cost-effective hemostatic material.
Various metal ions are known for their antimicrobial properties, good dispersion characteristics, and low drug resistance, making them valuable in the development of biological materials15. Calcium chloride plays a significant role in enzyme catalysis during the hemostasis process, making it a valuable additive for enhancing the efficacy of hemostatic materials. In the extrinsic coagulation pathway calcium ions facilitates the enzymatic process for activating factor X. They are essential for prothrombin formation and the activation of factor XIII, which ultimately contributes to fibrin formation, the final step in the coagulation process16.
The global interest in anti-hemorrhage nanomats and blood clotting aids is increasing, prompting the exploration of various materials and methods to fabricate these mats with maximum performance at minimal cost. In 2021, Mishra et al. developed nano-Chitosan-Casein fibers with hemostatic properties using a self-assembly method. Although the mats demonstrated promising results, their production costs were high1. In 2018, Omolola and his team harnessed the antibacterial properties of polyacrylonitrile (PAN) to produce nanofiber mats via electrospinning. Fiber’s properties were further enhanced by incorporating moringa leaf extract, known for its antibacterial effects against Escherichia coli and Staphylococcus aureus10. Earlier, in 2009, Mortazavi et al. conducted studies on the blood-clotting time and blood loss in animals using bentonite, revealing a significant improvement in coagulation time of approximately 50%17. In 2021, Sarah Nasser and her team fabricated nanofiber mats from poly-L-lactic acid (PLLA) via electrospinning, loading them with aluminum chloride, gentamicin, and lidocaine to enhance hemostasis, antimicrobial resistance, and local anesthesia. Their findings indicated an impressive reduction in blood-clotting time by 80%18. In 2022, Amani Sheikhani et al. developed a hydrogel composed of chitosan and polylactic acid, which served as a hemostatic aid, demonstrating a notable Blood Coagulation Index of 3.3%3.
In this study, we present the preparation of cost-effective polyacrylonitrile (PAN) nanofiber mats using the electrospinning technique. These mats are loaded with hemostatic agents—exfoliated bentonite and calcium chloride—to enhance their performance compared to traditional hemostatic materials. The fiber diameter is adjusted to optimize hemostatic efficacy. We evaluate the hemostatic effectiveness of the prepared mats in vitro through blood coagulation index tests and blood-clotting time test, comparing the results with conventional hemostatic products such as medical gauze and cotton. The overall process is summarized within a comprehensive scheme in Fig. 1.
Polyacrylonitrile electrospun fibrous nanomats are loaded with exfoliated bentonite and calcium chloride. The prepared mats are employed as hemostatic patches after proving their efficiency by the mean of blood coagulation index (BCI) and clotting time.
To the best of the authors’ knowledge, this study represents the first investigation into hemostatic medical dressings that combine exfoliated bentonite, PAN, and calcium chloride as a cost-effective hemostatic material. Furthermore, there is currently no research that has explored the use of exfoliated bentonite specifically for hemostatic applications.
Experimental section
Chemicals
Polyacrylonitrile (PAN) of Mw = 120 kDa, calcium chloride, sodium citrate, and sodium hydroxide were purchased from Merk (Darmstadt—Germany). N-N-dimethylformamide (DMF) was purchased from Sigma Aldrich. Acetone (99.5%) was from Panereac (Barcelona—Spain), and ethanol (99.8%) was from Reidel de haen. Syrian bentonite (Aleppo-belon) was obtained from local market. All chemicals were used as purchased, without any further purification.
Exfoliation of bentonite
The exfoliation process involves stirring 20 g of bentonite in 200 ml of 1 M sodium hydroxide for 2 h. Following this, the exfoliated bentonite is filtered and thoroughly washed with distilled water multiple times to ensure the complete removal of any residual sodium hydroxide. Subsequently, it is rinsed once more with acetone. Next, 10 g of the exfoliated bentonite is combined with 100 ml of acetone. This mixture is then homogenized for 10 min using an ultrasonic probe set at 49 W. After homogenization, the mixture is stirred for an additional hour and allowed to air dry for 24 h19.
Preparation of nanofiber mats
A high-voltage power supply, the ES813-D50.1 Dual Output model (0 to ± 50 kV/1 mA, Electrostatic/HV Generator, Rolla, Missouri, USA), along with a programmable syringe pump (TOP-5300, Japan), was utilized for the electrospinning process.
The electrospinning solutions were prepared by dissolving polyacrylonitrile (PAN) powder at a concentration of 2 wt% in a dimethylformamide (DMF) solution. Hemostatic agents were incorporated into the solutions in varying weight ratios relative to the polymer weight. Exfoliated bentonite powder was added in four different proportions with respect to the polymer: 5, 10, 15, and 30 wt%. Calcium chloride was included at a single ratio of 5 wt%. After the addition of the hemostatic ingredients, all electrospinning solutions underwent ultrasonication for 2 h, followed by stirring for 24 h to ensure homogeneity.
The resulting polymer and hemostatic agent composite solution was transferred into a 5 ml syringe. The needle of the syringe has an inner diameter of 0.6 mm and an outer diameter of 0.8 mm. The programmable syringe pump was employed to regulate the flow of the polymer solution. For the electrospinning process, a collector consisting of a 20 × 20 cm2 aluminum sheet covered with aluminum foil was used. The parameters for electrospinning were consistently set as follows: an applied voltage of 10 ± 2 kV, a flow rate maintained at 1 ml/h, and a fixed distance of 20 cm between the needle tip and the collector. Table 1 lists the sample codes and their corresponding compositions.
Structural and morphological analysis
X-ray diffractograms were obtained for both exfoliated and unexfoliated bentonite using a PHILIPS PW370 X-ray diffractometer (Amsterdam, Netherlands) operating at 40 kV. The radiation was generated from a Cu Kα source (λ = 0.154 nm). The diffractograms were recorded over a 2θ range of 0° to 20° to assess the changes in the crystalline structure of bentonite resulting from the exfoliation process. Additionally, Fourier Transform Infrared (FTIR) spectroscopy was employed to analyze the functional groups present in the polyacrylonitrile (PAN) mats. The morphology of the prepared mats was examined using a VEGA II XMU5136 scanning electron microscope (SEM). The fiber diameter distribution was analyzed using ImageJ, with a total of 50 fibers measured for each sample. The results are presented along with their corresponding standard deviations to provide a comprehensive overview of the fiber size variability.
Density and porosity test
Due to the low thickness of the samples, measurements were conducted using a micrometer (Ger. Nr. 03012.00) with an accuracy of 0.01 mm, following the folding of a specific type number. The thickness was assessed at multiple positions and averaged, then divided by the number of layers to determine the thickness of a single layer. Subsequently, square nanofiber patches measuring 5 cm2 were cut. The mass of these patches was recorded using a sensitive electronic scale (Sartorius, LS1200) with an accuracy of 0.001 g. The density was calculated by dividing the mass by the volume of the patch. Porosity was then determined according to Eq. (1):
where \({\rho }_{exp}\) and \({\rho }_{th}\) are the experimental density and theoretical density respectively. Theoretical density is calculated based on sample ingredients.
Functional analysis
Water absorbency
The test is conducted at room temperature. Initially, the mass of the dry sample (Md) is measured using a sensitive electronic scale (Sartorius, LS1200). The sample is then immersed in 50 ml of distilled water for a duration of 24 h. After immersion, the wet sample is left aside for 5 min to remove excess water, and the wet mass (Mw) is subsequently measured. The water absorbency (Aw), expressed in grams of water per gram of mat, is calculated according to Eq. (2).
Water contact angle
To assess the contact angle, a 20 μl droplet of distilled water is placed on the surface of the mat using a micropipette. The contact angle is determined using Image-J software, which analyzes the droplet image by marking five reference points.
Blood-clotting time
The hemostatic performance of the mats -specifically 2%PAN, 2%PAN-X%exben, 2%PAN-5%CaCl2, and 2%PAN-10%exben-5%CaCl2 (where X represents 5, 10, 15, or 30 wt% relative to the polymer)—is evaluated by measuring the blood-clotting time. For comparison, a standard gauze bandage and a cotton bandage are also tested. The testing procedure is as follows: Fresh human blood is drawn from an informed, healthy volunteer who has not taken any medication for at least 1 week. The blood is collected in evacuated blood tubes containing sodium citrate as an anticoagulant and maintained at 37 °C in a water bath for 3 min. Square patches measuring 1 × 1 cm2 of both the gauze bandage and the prepared mats are placed in separate test tubes. To each tube, 0.1 ml of blood is added, along with an empty tube for comparison of clotting times with natural clotting. Following this, 20 µl of a 0.2 M calcium chloride solution is introduced into each tube. The blood-clotting time is recorded using a chronometer starting from the moment calcium chloride is added. Every 15 s, each tube is carefully tilted at a 45° angle to ensure that the blood has completely clotted. The process continues until complete hemostasis is observed, indicated by the absence of any movement of the blood when the tube is inclined at a 90° angle.
Blood coagulation index
In this experiment, the UV–Vis absorbance of hemoglobin released from uncoagulated red blood cells is measured in the presence of prepared mat samples and compared to a control one. The absorbance of the released hemoglobin is recorded at a wavelength of 545 nm using a Jasco V-530 UV–Vis spectrophotometer (Tokyo, Japan). Fresh human blood is drawn from an informed, healthy volunteer who has not taken any medication for at least a week. The blood is collected in evacuated blood tubes containing sodium citrate as an anticoagulant. The test follows the protocol outlined by Zhang et al.16, with some modifications as detailed below:
Square patches of the mats, gauze, and cotton measuring 1 × 1 cm2 are prepared. These patches are placed into a flat-bottom glass tube and incubated in a water bath at 37 °C for 5 min. Following this, 0.2 ml of the anticoagulated blood is added to the tube, along with 40 µl of a 0.2 M calcium chloride solution. The tube is then returned to the water bath at 37 °C for an additional 5 min. After this incubation period, 25 ml of deionized water is carefully added to the tube without disturbing the clotted blood to facilitate the release of hemoglobin from red blood cells that did not participate in clot formation. A volume of 10 ml of the supernatant is then extracted from the tube and centrifuged at 1000 rpm for 1 min. The same procedure is repeated for the control sample, which does not include any patches. The blood coagulation index is calculated using Eq. (3).
Results and discussion
X-ray diffraction patterns
Figure 2(1) displays the X-ray diffractogram for the unexfoliated bentonite sample, while Fig. 2(2) illustrates the X-ray diffractogram for the exfoliated bentonite. Notably, the diffractogram in Fig. 2(1) bears a medium peak at a 2θ value of 5.9 degrees, which corresponds to the (001) planes with a d-spacing of 497 nm. After exfoliation, this peak is significantly diminished, as shown in Fig. 2(2). The disappearance of this peak can be attributed to the loss of layered structure that occurs due to the exfoliation process19,20.
X-ray diffractograms of: 1-unexfoliated bentonite, and 2-exfoliated bentonite.
FTIR spectra
To ensure the good affinity and distribution of loaded materials within the polymer matrix during electrospinning, FTIR spectra of the prepared samples were recoded and are shown in Fig. 3. Pure PAN exhibits a sharp peak with high intensity at 1454 cm⁻1, corresponding to the scissoring vibration mode of the CH2 group. Additionally, a sharp peak at 2244 cm⁻1 is associated with the stretching vibration of the C≡N bond, while a weak peak at 1068 cm-1 is attributed to the bond stretching C–CN. The sharp peak at 2920 cm⁻1 is linked to sp3 C–H stretching. In contrast, a broad and weak band at 3550 cm⁻1, and peak around 1630 cm-1 are attributed to the stretching of the O–H bond and O–H deformation, respectively. This hydroxyl group arises from PAN’s high affinity for moisture21. The above-mentioned peaks remain clear when loading PAN mats with exfoliated bentonite and/or calcium chloride. This indicates that the electrospinning process does not significantly impact the chemical groups present in PAN and PAN composite. When loading PAN with exfoliate bentonite, a strong large peak appears at 1030 cm-1. This peak is attributed to the Si–O–Si and Si–O– stretching22,23, while, the peaks observed at 3614 cm−1 and 3547 cm−1 are for the OH groups stretching in Al–OH and Fe3+-OH, respectively. This indicates a successful loading of bentonite within the PAN mat. On the other hand, the spectrum of 2%PAN-5%CaCl2 reveals that loading PAN with calcium chloride leads to larger and broader O–H peak. This is due to the high affinity of CaCl2 to water molecule24. Finally, both of Si–O– peak at 1030 cm−1 and broad O–H band at 3420 cm−1 are present in the spectrum of 2%PAN-10%exben-5%CaCl2. These results indicate the successful loading process of hemostatic components onto PAN electrospun mats.
FTIR spectra of 2%PAN-R, 2%PAN-10%exben, 2%PAN-5%CaCl2 and 2%PAN-10%exben-5%CaCl2 mats.
SEM micrographs
The morphological structure of the four electrospun mats was examined using scanning electron microscopy (SEM). As illustrated in Fig. 4, the SEM images depict the mats labeled 2%PAN-R, 2%PAN-10%exben, 2%PAN-5%CaCl2, and 2%PAN-10%exben-5%CaCl2. The images reveal a network of continuous and randomly distributed fibers, characterized by the absence of beads and a homogeneous distribution of the incorporated hemostatic agents.
SEM images of 2%PAN-R, 2%PAN-10%exben, 2%PAN-5%CaCl2 and 2%PAN-10%exben-5%CaCl2 mats at magnification of × 10,000.
Comparing to previous studies25, the electrospun fibers of 2%PAN-R were significantly continuous with no appeared breaks or beads. However, the electrospun fibers prepared by Furlan25 with 2% PAN concentration were remarkably thin and short with high concentration of beads. These major drawbacks were significantly disappeared when increasing the polymer concentration. The successful electrospinning of the nanomats in this study can be attributed to the higher molecular weight of PAN. Furthermore, the fibers maintain these attractive characteristics after loading process. Homogeneous distribution of the incorporated hemostatic agents can be noticed with effect on fiber diameter.
Figure 5 illustrates the distribution of fiber diameters in the prepared mats, highlighting the mean fiber diameter for each sample. The fiber diameters for the 2%PAN and 2%PAN-5%CaCl2 mats exhibit a unimodal distribution, measuring 263 ± 49 nm and 187 ± 37 nm, respectively. This indicates a uniformity in the fiber structure, with the 2%PAN-R mat demonstrating a narrower fiber diameter distribution compared to the unloaded samples. In contrast, the introduction of loading particles into the nano mats tends to distort the fibers, resulting in a broader range of fiber diameters. Specifically, the incorporation of exfoliated bentonite leads to the formation of two distinct types of fibers: those containing bentonite particles within them and those that are surrounded by these particles. Consequently, a bimodal distribution of fiber diameters is observed in both the 2%PAN-10%exben and 2%PAN-10%exben-5%CaCl2 samples. The bimodal distribution reveals two primary fiber diameter ranges: (244 ± 32 nm and 313 ± 73 nm) for the 2%PAN-10%exben mat, and (190 ± 58 nm and 250 ± 29 nm) for the 2%PAN-10%exben-5%CaCl2 mat.
Distribution of fibers’ diameters of 2%PAN-R (2%PAN-10%exben, 2%PAN-5%CaCl2 and 2%PAN-1s0%exben-5%CaCl2 mats.
In addition, the fibers in the 2%PAN-10%exben mat have a larger diameter compared to those in the 2%PAN-R mat. This can be attributed to the incorporation of exfoliated bentonite within the fibers. Similar effect can be seen when loading PAN mats with nanomaterials such as nano lead oxide26. A significant reduction in fiber diameter is evident in the 2%PAN-5%CaCl2 mat, where the presence of calcium ions in the electrospinning solution enhances conductivity19. This increase in solution conductivity results in greater electrostatic repulsive forces between the fibers, leading to a decrease in diameter. Moreover, the 2%PAN-10%exben-5%CaCl2 mat displays average fiber diameters that fall between those of mats. This is attributed to the synergistic effects of both exfoliated bentonite and calcium chloride.
Density and porosity calculation
A higher porosity of the mat means higher area-to-volume ratio, which enhances gas permeability, increases blood absorption, and improves hemostatic properties27,28. The density and porosity results for the prepared samples are presented in Table 2. Most of the mats obtain higher porosity than the ideal porosity of hemostatic materials (at least 90%)29. Both density and porosity are influenced by the diameter of the fibers. Specifically, as the average diameter of the fibers increases, the density of the mat also increases, while porosity rises as the average diameter decreases30,31. The porosity of the 2%PAN-R mat in this study is very close to the findings of Al-Okaidy et al.32. The porosity of the 2%PAN-15%exben mat is lower compared to the mats containing exfoliated bentonite. This reduction is attributed to the fact that the average fiber diameter reaches its maximum at this loading percentage. Higher concentrations lead to the formation of aggregates, which reduces fiber diameter due to increased electrostatic repulsive forces. This is consistent with the work of Hashmi et al.33, as increasing the percentage of the loaded ingredients leads to formation of beads and short nanofibers. Similarly, electrostatic repulsion among calcium ions contributes to a decrease in fiber diameter and an increase in porosity19, as observed in the 2%PAN-5%CaCl2 sample. When exfoliated bentonite is added at a concentration of 10% to the previous mat, there is a slight decrease in porosity, by 0.4% in the 2%PAN-10%exben-5%CaCl2 mat. This change results from the synergistic effects of exfoliated bentonite and calcium ions; however, the influence of repulsive forces between calcium ions remains the dominant factor.
Functional analysis
Water absorbency
Water absorbency is a critical factor in determining the hemostatic properties of a mat. Higher water absorbency facilitates the concentration of coagulation factors in blood, thereby accelerating the clotting process3. Table 3 presents the water absorbency results for the prepared mats. The findings indicate that the 2%PAN-R mat exhibits a slightly higher absorbency compared to medical cotton. This increased absorbency can be attributed to its nanofiber structure34 and the hydrophilic terminal group of polyacrylonitrile (C≡N), which has a strong affinity for water. Exfoliated bentonite is known for its high specific surface area and excellent water absorbency; however, its absorbency is lower than that of the 2%PAN-R mat due to high level of water absorbency34. Consequently, the incorporation of exfoliated bentonite reduces the overall absorbency of the mat. This justifies that the absorbency of mats containing exfoliated bentonite is inversely proportional to the loading percentage. Calcium chloride (CaCl2), being a salt, also exhibits a strong affinity for water, resulting in the 2%PAN-5%CaCl2 mat having greater absorbency than the 2%PAN-R mat35. The absorbency of the 2%PAN-10%exben-5%CaCl2 mat falls between that of the 2%PAN-10%exben and 2%PAN-5%CaCl2 mats. Table 3 shows that the absorbency of cotton is higher than the absorbency of gauze. When compared to cotton, the final mat shows slightly greater absorbency; however, it is important to note that less mass of the final mat is required to effectively cover a wound compared to cotton. This characteristic contributes to reduced blood loss when applying the final mat. In contrast, gauze demonstrates significantly lower absorbency than both cotton and the prepared mats, which adversely affects its hemostatic capabilities.
Water contact angle
The water contact angle test assesses the affinity between the mat surface and water. This angle is influenced by the surface structure of the mat, including fiber diameter and the presence of loaded particles36. Although polyacrylonitrile (PAN) is inherently a hydrophobic polymer, its nanofiber architecture exhibits hydrophilic properties34, resulting in contact angles of less than 90 degrees, as indicated in Table 4. Table 4 shows also the taken pictures during the measurement, verifying the observed hydrophilicity. 2%PAN-R mat possesses a water contact angle of 59.2 degree, which is close to the findings of Kim et al.37. The hydrophilic behavior of PAN fibers can be attributed to the terminal groups in their structure; specifically, nitrogen atoms in the cyanide groups form hydrogen bonds with water, enhancing the fibers’ wettability38. Furthermore, the variation of surface morphology (e.g., Roughness and porosity) also contributes to the high wettability37. When PAN fibers are loaded with exfoliated bentonite, the contact angle of the mat decreases37. Therefore, loading exfoliated bentonite lowers the contact angle of the mat. This reduction is due to the various cations present in exfoliated bentonite, which improve the fibers’ affinity for water. As the percentage of exfoliated bentonite in the mat increases, a corresponding increase in contact angle is observed in the 2%PAN-X%exben mats, where x represents (5, 10, 15 wt%). This trend is attributed to an increase in fiber diameter that occurs with the percentage of exfoliated bentonite increase, as bentonite is loaded within the fibers38. However, when the loading of exfoliated bentonite reaches 30%, aggregates begin to form, leading to a decrease in fiber diameter. Additionally, incorporating calcium chloride into the mat enhances its water affinity and reduces its contact angle. The contact angle of the 2%PAN-10%exben-5%CaCl2 mat falls between those of the 2%PAN-10%exben and 2%PAN-5%CaCl2 mat, as expected.
Blood-clotting time
Table 5 presents the blood-clotting times for the prepared samples, as well as for commercial cotton and gauze, showing the final step of the test, where a stable clot forms in the test tube. The 2%PAN-R sample demonstrates an improved blood-clotting time, decreasing from 285 to 210 s, attributed to its excellent water absorbency. Among the samples loaded with exfoliated bentonite, the 2%PAN-10%exben exhibited the shortest clotting time of 165 s, outperforming the other samples loaded with bentonite (2%PAN-X%exben, where X = 5, 15, 30 wt%). This improvement can be linked to the uniform distribution of exfoliated bentonite in this sample, which minimizes the formation of beads and aggregates. The 2%PAN-5%CaCl2 sample recorded a blood-clotting time of 195 s, showing a notable enhancement compared to 2%PAN-R. This improvement is mainly due to the role of calcium ions in promoting hemostasis13. However, the addition of 10% exfoliated bentonite could yield even better results.
The 2%PAN-10%exben-5%CaCl2 mat achieved the shortest clotting time of just 105 s. This significant reduction is attributed to the synergistic effects of the PAN nanofibers, the nano-clay structure of exfoliated bentonite, and the presence of calcium chloride. In comparison to the blood-clotting times of commercial cotton and gauze bandages (both at 270 s), the performance of our samples clearly demonstrates superiority.
Blood coagulation index
Table 6 presents the blood coagulation indices for the prepared samples, as well as for commercial cotton and gauze bandages. The results from this test closely align with those obtained from the clotting time test. The 2%PAN-10%exben-5%CaCl2 sample exhibits the highest coagulation index at 44.9%. In comparison, the commercial cotton and gauze bandages recorded indices of 95.4% and 90.0%, respectively. Despite these values, the superior performance of our prepared sample is evident, highlighting its potential as a more effective alternative to conventional products.
Conclusion
In this study, we successfully developed novel electrospun mat of polyacrylonitrile (PAN) incorporated with various hemostatic agents. The prepared mat outperformed the conventional hemostatic materials in Blood Coagulation Index BCI and Clotting time test and demonstrated high hemostatic efficiency. The use of local low-cost hemostatic agents achieved the cost-effectiveness of the mat.
Exfoliated Syrian bentonite (Aleppo-belon) and Calcium chloride were effectively integrated into the PAN nanofibers through the electrospinning technique. Scanning Electron Microscopy (SEM) analysis revealed a continuous fiber structure without beads. Additionally, the electrospun PAN mats exhibited hydrophilic characteristics and excellent absorbency, both of which are critical for effective hemostasis. Functional tests conducted on human blood confirmed the efficacy of these mats in promoting rapid clot formation. Furthermore, the best composite mat (2%PAN-10%exben-5%CaCl2) exhibited an impressive clotting time of just 105 s, compared to 285 s for control blood samples, and demonstrated a low blood coagulation index of 44.9%. These findings highlight the potential of our electrospun PAN mats as a superior alternative for hemostatic applications in medical settings.
Data availability
All data generated or analyzed during this study are included in this published article.
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Badr Saqr: experimental work, data curation, formal analysis, investigation, validation, writing—original draft. Rama Alqassar Bani Almarjeh: conceptualization, formal analysis, validation, writing—review and editing. Yomen Atassi: conceptualization, formal analysis, supervision, writing—review and editing.
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This study involving human participants was conducted in accordance with the Declaration of Helsinki and was approved by the Biotherapeutic Research Center—Damascus University. Informed consent was obtained from all participants prior to the collection of blood samples. All procedures were performed in compliance with relevant guidelines and regulations.
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Saqr, B., Alqassar Bani Almarjeh, R. & Atassi, Y. Development of an innovative cost-effective hemostatic material based on electrospun polyacrylonitrile/exfoliated bentonite/calcium chloride nanocomposite. Sci Rep 15, 15795 (2025). https://doi.org/10.1038/s41598-025-00569-3
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DOI: https://doi.org/10.1038/s41598-025-00569-3
