Immobilized thrombin on X-ray radiopaque polyvinyl alcohol/chitosan embolic microspheres for precise localization and topical blood coagulation

Abstract

Trans-catheter arterial embolization (TAE) plays an important role in treating various diseases. The available embolic agents lack X-ray visibility and do not prevent the reflux phenomenon, thus hindering their application for TAE therapy. Herein, we aim to develop a multifunctional embolic agent that combines the X-ray radiopacity with local procoagulant activity. The barium sulfate nanoparticles (BaSO₄ NPs) were synthesized and loaded into the polyvinyl alcohol/chitosan (PVA/CS) to prepare the radiopaque BaSO₄/PVA/CS microspheres (MS). Thereafter, thrombin was immobilized onto the BaSO₄/PVA/CS MS to obtain the thrombin@BaSO₄/PVA/CS MS. The prepared BaSO₄/PVA/CS MS were highly spherical with diameters ranging from 100 to 300 μm. In vitro CT imaging showed increased X-ray visibility of BaSO₄/PVA/CS MS with the increased content of BaSO₄ NPs in the PVA/CS MS. The biocompatibility assessments demonstrated that the MS were non-cytotoxic and possessed permissible hemolysis rate. The biofunctionalized thrombin@BaSO₄/PVA/CS MS showed improved hemostatic capacity and facilitated hemostasis in vitro. Additionally, in vivo study performed on a rabbit ear embolization model confirmed the excellent X-ray radiopaque stability of the BaSO₄/PVA/CS MS. Moreover, both the BaSO₄/PVA/CS and thrombin@BaSO₄/PVA/CS MS achieved superior embolization effects with progressive ischemic necrosis on the ear tissue and induced prominent ultrastructural changes in the endothelial cells. The findings of this study suggest that the developed MS could act as a radiopaque and hemostatic embolic agent to improve the embolization efficiency.

Graphical abstract

Highlights

• •Excellent in vitro and in vivo visibility of BaSO₄/PVA/CS MS.
• •Excellent cytocompatibility and hemocompatibility of BaSO₄/PVA/CS MS.
• •Enhanced hemostatic capacity and hemostasis of thrombin@BaSO₄/PVA/CS MS.
• •Potential application of thrombin@BaSO₄/PVA/CS MS for in vivo embolization.

1. Introduction

Minimally invasive procedures to treat a variety of cancerous and noncancerous diseases, such as unresectable hepatocellular carcinoma [1], benign prostatic hyperplasia [2], uterine fibroids [3], and acute hemorrhage [4], have gained much attention in the clinic. Particularly, trans-catheter arterial embolization (TAE) is generally considered safe and effective in interventional therapy, which selectively delivers the embolic agents to the target artery under X–ray guidance. It exerts therapeutic effects by deliberately blocking the blood vessel, thereby cutting off the nutrition and oxygen supply to the target tissue or organ. The characteristics of embolic agents play an important role in the performance of TAE treatment [5,6]. Over the past few years, there have been growing concerns with the radiolucent nature of the embolic agents [7,8]. This issue can be addressed to some extent by physically mixing the embolic agent with iodinated contrast medium (e.g., iohexol) and delivering the mixture to the target arteries [9,10]. However, the main disadvantages of this method include several aspects. First, the contrast medium is easily dissociated from the embolic agent and results in fuzzy imaging and misdiagnosis [11]. Second, the rapid diffusion of the contrast medium causes difficulties in re-examination after TAE [12]. Third, the iodinated contrast medium may cause adverse events in some patients who suffer from thyroid disorder [13]. Besides, there are some other strategies to achieve precise visualization of the embolic agents. For example, the copolymerization method is always employed to prepare the iodine-containing polymeric embolic agents [14,15]; however, the preparation process is complicated, and the iodinated polymers exhibit potential toxicity [16]. Alternatively, the radiopacity of the embolic microspheres (MS) could be achieved by encapsulating heavy metal salts in the polymeric MS [17,18]. Barium sulfate (BaSO₄) has been used as a contrast agent for gastrointestinal examination due to its high X-ray attenuation, biocompatibility, cost-effectiveness, and insolubility [19,20]. However, the commercially available BaSO₄ particles are in the micro-scale size, and show inferior performance of radiographic contrast compared to the nano-sized BaSO₄ particles [21,22]. Therefore, it is crucial to synthesize BaSO₄ nanoparticles (NPs) and load them within the embolic agent, which could provide a radiopaque formulation suitable for clinical use in TAE.

Another challenge associated with the embolization process is the reflux phenomenon in some cases [23,24]. This phenomenon can potentially result in significant complications, including the non-target embolization, catheter entrapment, and even treatment failure [[25], [26], [27]]. Triggered by these complications, two strategies have been proposed to avoid the reflux effect. One option is to use a balloon catheter for controlling the reflux during the embolization. However, there exists the risk of vessel damage caused by the balloon inflation, catheter entrapment, catheter fracturing, and the increased concomitant intranidal pressure and associated bleeding [[28], [29], [30]]. The other option is to pause the injection for a while in the case of reflux until the embolic particles penetrate more distally into the vasculature. Nonetheless, this procedure is mainly limited to liquid embolic agents and undoubtedly increases the operation complexity and fluoroscopy time [29,31].

Thrombin, a serine protease with procoagulant activity, accelerates the conversion of fibrinogen to fibrin and promotes the rapid formation of thrombus and hemostasis [32,33]. Considering the intrinsic hemostatic properties of thrombin, it is of vital importance to develop a biofunctionalized embolic agent via covalently binding thrombin onto the embolic agent to cause adequate hemostatic effects, and prevent the reflux effect.

In recent years, polyvinyl alcohol (PVA) has been extensively used in many biomedical fields due to its excellent biocompatibility, non-immunogenicity, as well as good chemical and thermal stability [34,35]. Specifically, the PVA MS are commercially available in the clinic and regarded as a permanent embolic agent [6,36]. Although PVA MS have been successfully used for treating various diseases, there exist several limitations associated with them, most notably, the lack of X-ray radiopacity and inability to prevent the reflux phenomenon. Such limitations restrict their use and development in TAE applications.

Herein, we developed thrombin-immobilized and BaSO₄-loaded embolic MS, which combined the X-ray visibility and local blood coagulation activities. The developed MS were used for precise localization and avoidance of the reflux effect during the embolization procedure (Scheme 1). More specifically, BaSO₄ NPs were first synthesized and characterized, followed by adding to PVA/CS polymeric matrix with varied contents to prepare the BaSO₄-loaded PVA/CS (BaSO₄/PVA/CS) MS via inverse suspension crosslinking method. The addition of a small content of CS in the composite MS was beneficial to obtain the well-dispersed MS, where it was positively charged in acidic conditions and acted as a barrier to prevent the aggregation of MS. The prepared BaSO₄/PVA/CS MS were comprehensively characterized for their physicochemical properties, X-ray visibility, cytocompatibility, and hemocompatibility. Thereafter, biofunctionalization of BaSO₄/PVA/CS MS was performed via covalent binding of thrombin on their surface and further evaluated their procoagulant activity. Moreover, in vivo X-ray radiopacity and embolization efficacy were investigated in a rabbit central auricular artery model.

Scheme 1.

Schematic illustration of the thrombin@BaSO₄/PVA/CS MS as an embolic agent that combines X-ray visibility and local blood coagulation.

2. Materials and methods

2.1. Materials

Poly (vinyl alcohol) 124 (PVA-124, Mw~105 kDa, degree of hydrolysis 98.0–99.8 mol%), barium chloride (BaCl₂), and ammonium sulfate ((NH₄)₂SO₄) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Thrombin and chitosan (CS, Mw = 110–150 kDa, 93% degree of deacetylation) were provided by Sigma-Aldrich (Beijing, China). Glutaraldehyde (GA, 50 wt%) and 4-dimethylaminopyridine (DMAP) were received from Macklin Biochemical Co., Ltd. (Shanghai, China). Succinic anhydrate, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS), DMEM medium, and antibiotics (penicillin: 100 IU/mL + streptomycin: 100 mg/mL) were provided by Gibco Life Technology Co., Ltd. (Grand Island, USA). CCK-8 assay kit was produced by Dojindo Laboratories (Kumamoto, Japan).

In vivo animal tests were performed on New Zealand white rabbits supplied by the Experimental Animal Center in our institution, and the experiments were complied with the guidelines established by the Institutional Animal Care and Use Committee.

2.2. Preparation of BaSO₄/PVA/CS MS

The BaSO₄ NPs were prepared by a precipitation method via the reaction between BaCl₂ and (NH₄)₂SO₄ aqueous solutions at appropriate concentrations (See details in the Supporting Information). The prepared BaSO₄ NPs were loaded inside the PVA/CS MS through the solid-in-water-oil (S/W/O) emulsification crosslinking method according to a previous report with some modifications [37]. Briefly, 8.6% (w/v) PVA solution was prepared by dissolving PVA powder in hot water at 80 °C. Similarly, CS was dissolved in 1 wt% acetic acid solution to form a 0.84% (w/v) CS solution. The PVA and CS solutions were mixed and stirred to form a homogeneous blend solution (CS:PVA = 1:19, w/w). The BaSO₄ NPs-containing PVA/CS solution was prepared at 10, 20, and 30 wt% loading by mixing the required contents of BaSO₄ NPs with PVA/CS solution, and subjected to high-speed homogenization. The obtained suspension was then added dropwise into the n-heptane containing 2.8% (w/v) Span 80 with continuous stirring at 480 rpm. After 30 min, a mixture of GA (2.6 mL, 25 wt%) and diethyl ether (DEE, 4.15 mL) was added to PVA/CS solution at a volume ratio of 0.03:1, and continued stirring for 5 min. Afterwards, a small volume of 1 M HCl (catalyst) was added to facilitate the crosslinking process. The reaction was carried out at 55 °C for 5 h. After cooling to room temperature, the resultant BaSO₄/PVA/CS MS were recovered from n-heptane by centrifugation, and rinsed with petroleum ether, followed by rinsing with ethanol for several times, and finally rinsed thoroughly with distilled water. Thereafter, the BaSO₄/PVA/CS MS were immersed in 4 wt% glycine aqueous solution for 12 h to remove the excess of unreacted GA and vacuum-dried. Similarly, the PVA/CS MS were also prepared following the above protocol except the addition of BaSO₄ NPs. Finally, all prepared MS were sieved to the final size of 100–300 μm.

2.3. Immobilization of thrombin on BaSO₄/PVA/CS MS

Considering the syringeability and X-ray detectability, 20 wt% BaSO₄/PVA/CS MS were selected and used in the following experiments. The carboxylated BaSO₄/PVA/CS MS were used to immobilize the thrombin. The detailed modification procedure of BaSO₄/PVA/CS MS is provided in the Supporting Information. The succinylated BaSO₄/PVA/CS MS were suspended in 100 mM EDC and 25 mM NHS in anhydrous dimethyl sulfoxide (DMSO) and gently stirred at 25 °C for 1 h. Then, the MS were thoroughly rinsed with DMSO to remove the unreacted EDC and NHS. Subsequently, the thrombin solution was prepared by dissolving 1 mg of thrombin (1000 U/mg) in HEPES buffer (20 mM, 0.15 M NaCl, pH 8.2), and then added to the above activated MS. After incubation at room temperature for 1.5 h, the resultant thrombin-immobilized BaSO₄/PVA/CS (thrombin@BaSO₄/PVA/CS) MS were extensively rinsed with HEPES buffer, freeze-dried, and stored at −20 °C for further use. In the immobilization experiment, the BaSO₄/PVA/CS MS were used as the control.

2.4. Characterization

The surface and cross-sections of the MS were observed under a field emission scanning electron microscope (FESEM). The elemental analysis of BaSO₄ NPs, PVA/CS, and BaSO₄@PVA/CS MS was carried out by energy dispersive X-ray (EDX). A field emission transmission electron microscope (FETEM) was used to study the morphology of the synthesized BaSO₄ NPs. The size distribution of BaSO₄ NPs was determined from different FETEM images. The chemical compositions of BaSO₄ NPs, PVA/CS, and BaSO₄@PVA/CS MS were determined by Fourier transform infrared (FTIR) spectrometer. The crystalline state of BaSO₄ NPs loaded into the BaSO₄@PVA/CS MS was determined by an X-ray diffractometer (XRD). Thermogravimetric analysis (TGA) was performed at a heating rate of 10 °C/min. The immobilization of thrombin on the surface of BaSO₄@PVA/CS MS was determined through X-ray photoelectron spectra (XPS, Kratos Axis Ultra DLD, Japan) analysis. The X-ray radiopacity test of the MS was performed on a 128-row CT scanner. The images were recorded and then transferred to an external workstation for further analysis. The characterization details are provided in the Supporting Information.

2.5. In vitro biocompatibility studies

The viability of HVSMCs incubated with BaSO₄/PVA/CS MS was quantitatively evaluated by CCK-8 assay based on the indirect contact model. Briefly, the MS were sterilized with Co⁶⁰ radiation for 12 h (9 kGy), and then 10 and 20 mg/mL MS were immersed in DMEM containing 10% FBS and 1% antibiotics, and incubated at 37 °C for 24 h to obtain the cell culture extracts. The positive control was prepared by using the cell culture medium containing 0.64% phenol as recommended by ISO10993-5 [38], while the culture medium was used as the negative control. The HVSMCs at a density of 5 × 10³ cells/well were seeded in 96-well plates and incubated under 5% CO₂, 37 °C, and 95% humidity for 24 h prior to use. The MS extracts and controls were added to the 96-well plates. At predetermined time points (24, 48, and 72 h), the cell viability was quantified via CCK-8 assay. The relative cell viability was calculated relative to the negative control group. All experiments were repeated six times. The cell viability was further evaluated by Live/Dead cell assay, while the cell morphology on the MS was observed under FESEM. The detailed experimental procedures are provided in the Supporting Information.

The hemolysis potential of BaSO₄/PVA/CS MS was evaluated similar to that described by Wang et al. [39]. Briefly, the fresh whole blood was obtained from a New Zealand white rabbit via a marginal ear vein puncture, and collected in medical vacuum tubes containing acid citrate dextrose (ACD) as an anticoagulant (ACD:blood = 1:9, v/v). Thereafter, 10 and 20 mg/mL disinfected MS were separately immersed in tubes filled with 1 mL of normal saline at 37 °C for 1 h. Afterwards, 20 μL of diluted blood (4 mL of fresh blood + 5 mL of normal saline) was added to the MS and incubated at 37 °C for another 1 h. The positive and negative controls were prepared by adding 20 μL of diluted blood in 1 mL of distilled water and saline solution, respectively. After incubation, all samples were centrifuged, and the absorbance of supernatants was measured at 545 nm by a UV–Vis spectrophotometer. The percentage of hemolysis was calculated according to equation (1).

$$\text{Hemolysis}(\text{\%})=\frac{ODtest–ODneg}{ODpos–ODneg}\times 100$$ (1)

where OD_test, OD_pos, and OD_neg are the absorbance of the test samples, positive control, and negative control, respectively. Each experiment was performed in triplicate.

2.6. In vitro hemostatic performance study

The whole blood clotting kinetics tests were performed according to a previously reported method with some modifications [40]. Briefly, the anticoagulant whole blood was re-calcified by adding 100 μL of 0.1 M CaCl₂ to 1 mL of fresh whole blood. Subsequently, 200 μL of activated blood was immediately added to each tube containing 10 mg of MS and incubated at 37 °C for different time intervals of 5, 20, 35, and 50 min. At each time point, 6 mL of distilled water was added to each tube and maintained at 37 °C for 5 min to lyse the red blood cells (RBCs), which were not trapped in the blood clots. The RBCs were hemolyzed, and the absorbance of the resultant hemoglobin solution was measured at 545 nm with a UV–Vis spectrophotometer. The blood sample without mixing with MS was used as an experimental control and tested following the same protocol. Five replicates were run for each sample.

The hemostatic properties of thrombin@BaSO₄/PVA/CS MS were further investigated in terms of the adhesion of whole blood and RBCs on MS, as well as the MS-induced fibrin formation. The anticoagulant whole blood was centrifuged at 250 g for 15 min and the precipitated RBCs were collected for RBCs adhesion assay. The RBCs were added to the MS in a 24-well plate and incubated at 37 °C for 1 h. After that, the MS were rinsed twice with saline solution, and then suspended in 2.5 wt% GA to fix the RBCs for 1 h. The MS-RBCs constructs were subjected to sequential dehydration with gradually ascending concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 95%, and 100%), and then freeze-dried. Similar to the RBCs adhesion assay, the whole blood was incubated with the samples at 37 °C for 1 h. After that, the samples were fixed, dehydrated, and freeze-dried. To evaluate the fibrin formation induced by the MS, the platelet-rich plasma (PRP) was collected by centrifuging the whole blood at 250 g for 15 min, and the concentration of the blood platelets was quantified to be 1,506,040 ± 320,500/μL using a hemocytometer. Subsequently, the obtained PRP was re-calcified by adding 0.1 M CaCl₂ at a volume ratio of 9:1. The MS were incubated with re-calcified PRP in a 24-well plate at 37 °C for 5 min. Thereafter, the samples were fixed in 2.5 wt% GA and allowed to dry in lyophilizer. All samples were vacuum-coated with gold for 300 s prior to FESEM observation.

2.7. In vivo embolization studies

2.7.1. In vivo rabbit ear artery embolization and X-ray visibility tests

The rabbits (2.5 ± 0.5 kg, male) were anesthetized by administering 30 mg/kg pentobarbital sodium from the marginal ear vein. Afterwards, the rabbit ear hair was shaved, and the ears were disinfected with 75% ethanol. Then, an incision was made on the ear skin to identify the central auricular artery. Thereafter, PVA/CS, BaSO₄@PVA/CS, and thrombin@BaSO₄/PVA/CS MS at a concentration of 20 mg/mL were mixed with glycerol, and then infused slowly using a 22-Gauge puncturing needle. The rabbits treated with glycerol alone were designated as the control. After that, the injection site was pressed until it stopped bleeding. The changes in the color and shape of the embolized ears were inspected at 0, 7, 15 days, and recorded by photography. The X-ray visible stability test was performed in vivo after embolization with PVA/CS and BaSO₄@PVA/CS MS for 3, 7, and 15 days. The rabbits were subjected to X-ray imaging using an X-ray machine (General Electric, XR/A, 80 keV, exposure time 0.2 s). All images were obtained from the same animal, and the in vivo X-ray radiopacity of each sample was compared.

2.7.2. Histopathological examination and ultrastructural findings

At 7 and 15 days post-embolization, the rabbits were sacrificed, and the infarcted tissues containing embolic MS were resected and fixed in 4% paraformaldehyde for 48 h prior to evaluation by histopathological analysis. Thereafter, the central auricular artery and the surrounding tissue were dissected, embedded in paraffin, and then cut into 5 μm thick cross-sections. The sections were further stained with hematoxylin-eosin (H&E) and Masson's trichrome, and examined under an optical microscope. After 15 days, the morphological and ultrastructural changes in the vascular endothelial cells in different treated groups were investigated via FETEM. The details are presented in the Supporting Information.

2.8. Statistical analysis

The results obtained were analyzed by student's t-test (SPSS 20.0, IBM, Armonk, USA). The difference between groups was statistically significant at P < 0.05, and P < 0.01 indicated an extraordinary significant difference.

3. Results and discussion

3.1. Microstructure examination of BaSO₄/PVA/CS MS

The typical FETEM image of as-synthesized BaSO₄ NPs is presented in Fig. 1a. It is evident that the particles are nano-sized, and exhibit an elliptical-shaped structure with a smooth surface, which is further verified by the FESEM observation (Fig. 1b). The size distribution of the BaSO₄ NPs is presented in Fig. S1a, which shows that the mean particle size is 81.7 ± 20.4 nm. EDX analysis of BaSO₄ NPs revealed the compositional distribution of Ba and S elements among the nanocrystals. Besides, a small content of C element was also detected, which could possibly be derived from the resin stub (Fig. S1b).

Fig. 1.

FETEM (a) and FESEM (b) images of as-prepared BaSO₄ NPs, and FESEM surface morphology of (c-c'') PVA/CS MS, (d-d'') 10 wt% BaSO₄/PVA/CS MS, (e-e'') 20 wt% BaSO₄/PVA/CS MS, and (f-f'') 30 wt% BaSO₄/PVA/CS MS, respectively. (g) Is the EDX mapping images of a single 20 wt% BaSO₄/PVA/CS MS.

The surface morphologies of PVA/CS MS with and without the loading of BaSO₄ NPs were observed by FESEM. As shown in Fig. 1c-c'', the blank PVA/CS MS are well-dispersed and possess a perfectly spherical shape with a smooth surface. After loading of various contents of BaSO₄ NPs, the BaSO₄/PVA/CS composite MS exhibited no difference with regard to the dispersity and morphological characteristics as compared to the blank MS (Fig. 1d-f''). It should be noted that, upon loading of the BaSO₄ NPs with different contents into the PVA/CS MS, all the BaSO₄/PVA/CS MS maintained their smooth surface without superfluous BaSO₄ NPs on the MS surface, indicating ideal miscibility between BaSO₄ NPs and PVA/CS matrix. The similarity in the shape and surface features of these MS is consistent with the trends that reported in previous studies [41,42]. In addition, the dense layer of BaSO₄/PVA/CS MS was supposed to prevent the BaSO₄ NPs from leaking out and to maintain a long time radiopacity in vivo. This can be explained by the fact that the chemical covalent crosslinking of PVA/CS polymeric matrix with GA improved the strength of BaSO₄/PVA/CS MS. Moreover, the obtained MS were 100–300 μm in diameter, which was an appropriate size for embolizing the central auricular artery of rabbits. The EDX elemental mapping analysis was further carried out for 20 wt% BaSO₄/PVA/CS MS to study the BaSO₄ NPs distribution in the composite MS. As shown in Fig. 1g, the Ba and S elements were found to be homogeneously distributed throughout the MS, indicating that the BaSO₄ NPs were dispersed well in the interior of the PVA/CS matrix.

To study the internal structure of the composite MS and the miscibility of BaSO₄ NPs within the PVA/CS polymeric matrix, the cross-sections of pure PVA/CS and BaSO₄/PVA/CS MS with 10, 20, and 30 wt% BaSO₄ NPs loading were observed under FESEM. The cross-sectional view shows that the BaSO₄ NPs are entrapped in the dense PVA/CS polymeric matrix (Fig. 2b-d’), unlike the rough texture for pure PVA/CS polymer (Fig. 2a,a’). It is worth noting that, in the higher magnification images, the BaSO₄ NPs showed a tendency to form separate domains and exhibited compacted clusters throughout the MS (indicated by yellow arrows). More importantly, with the increased contents of BaSO₄ NPs in the PVA/CS MS, the composite MS were formed with the loading of more BaSO₄ NPs in a single MS. To further verify the loading of BaSO₄ NPs inside the PVA/CS polymeric matrix, EDX analysis was carried out to determine the elemental compositions on the cross-sections of PVA/CS and BaSO₄/PVA/CS MS containing different contents of BaSO₄ NPs (Fig. 2e–h). Fig. 2e shows the presence of C and O elements, which are derived from the PVA/CS MS. In addition, the Au element was also detected, which came from the sputtered gold layer. The EDX spectra of BaSO₄/PVA/CS MS showed the presence of other elements such as Ba and S were also detected as compared to the PVA/CS MS. This indirectly verified that the BaSO₄ NPs were successfully loaded into the PVA/CS MS. Moreover, the intensity of characteristic peaks of Ba and S elements increased with the increased content of BaSO₄ NPs in the PVA/CS MS. Thus, it could be speculated that the X-ray radiopacity of the MS would be increased with the increase of BaSO₄ NPs, which was favorable for their application as a radiopaque embolic agent for further use in vivo.

Fig. 2.

FESEM images of the cross-sectional morphologies of (a, a’) PVA/CS MS, (b, b’) 10 wt% BaSO₄/PVA/CS MS, (c, c’) 20 wt% BaSO₄/PVA/CS MS, and (d, d’) 30 wt% BaSO₄/PVA/CS MS, respectively. (e)–(h) are the corresponding EDX spectra of the cross-sections of PVA/CS and 10, 20, and 30 wt% BaSO₄/PVA/CS MS. The yellow arrows indicate the presence of BaSO₄ NPs in the cross-sectional structures of the MS.

3.2. Characterization and X-ray visibility of BaSO₄/PVA/CS MS

3.2.1. XRD analysis

The XRD patterns of pure BaSO₄ NPs, blank PVA/CS, and BaSO₄/PVA/CS MS are presented in Fig. 3a. The XRD pattern of BaSO₄ NPs showed that all diffraction peaks are in accordance with the barite structure (JCPDS card No. 24–1035). Particularly, the diffraction peaks at 20.4°, 22.8°, 25.8°, 26.8°, 28.7°, 31.5°, 32.8°, and 42.9° could be indexed as typical (101), (111), (021), (210), (121), (211), (002), and (212) planes of BaSO₄ crystals, respectively. This result confirms that the resultant NPs are pure BaSO₄ with an orthorhombic barite crystal structure. The XRD pattern of PVA/CS MS shows the broad peak appearing at around 19.5°, which can be attributed to the semi-crystalline structure. In the case of BaSO₄ NPs loaded formulations with various contents, the sharp peaks associated with the crystalline state were evident, which could be attributed to the presence of BaSO₄ NPs in the PVA/CS MS. In addition, the intensity of the characteristic peaks increased with the increased content of BaSO₄ NPs in the PVA/CS MS. Moreover, the characteristic peaks remained distinguishable, indicating that BaSO₄ NPs exhibited phase stability during the loading process, which suggests their suitability for embolization applications.

Fig. 3.

Characterization of BaSO₄/PVA/CS MS. (a) XRD patterns, (b) FTIR spectra, and (c) TGA curves of BaSO₄ NPs, PVA/CS, and 10, 20, 30 wt% BaSO₄/PVA/CS MS, and (d) show the radiopacity values of the samples (n = 3).

3.2.2. FTIR analysis

FTIR analysis was carried out to investigate the loading of BaSO₄ NPs into the PVA/CS MS. As depicted in Fig. 3b, the FTIR spectrum of BaSO₄ NPs reveals that the broad absorption bands at 1073–1192 and 982 cm⁻¹, which were attributed to the symmetrical vibrations of SO₄²⁻. The absorption bands at 609 and 637 cm⁻¹ were assigned to the out-of-plane bending vibration of SO₄²⁻. For PVA/CS MS, a wide absorption band at around 3450 cm⁻¹ was associated with the –OH stretching vibrations. The intense bands at 2898 and 2935 cm⁻¹ were associated with the characteristic –CH₂-.symmetric and asymmetric stretching vibrations. Besides, the characteristic absorption bands at 1713 and 1659 cm⁻¹ were assigned to C O and C C stretching vibrations, respectively. The FTIR spectrum for CS did not exist in the composite MS, which might be due to the insensitivity of the instrument to detect a small content of CS in the MS. Noticeably, the relative intensity of the characteristic bands derived from BaSO₄ NPs could be clearly seen in the spectra of BaSO₄/PVA/CS MS, and increased with the increased content of BaSO₄ NPs in the PVA/CS MS, which further confirms the successful loading of BaSO₄ NPs into the PVA/CS MS.

3.2.3. TGA

The exact content of BaSO₄ NPs in the MS as well as their thermal behavior were analyzed by TGA. As shown in Fig. 3c, the BaSO₄ NPs showed negligible weight loss when the sample was heated from 30 to 600 °C, indicating their excellent thermal stability. The TGA thermograms of PVA/CS and BaSO₄/PVA/CS MS with different BaSO₄ contents showed similar weight loss behavior, where two phases of weight loss were observed. The initial weight loss between 30 and 200 °C was ascribed to the evaporation of physically adsorbed water, while the sharp weight loss between 200 and 500 °C was attributed to the decomposition of the polymer. Beyond 500 °C, there was no considerable weight changes in the TGA curves. The final weight loss of PVA/CS MS and 10, 20, and 30 wt% BaSO₄/PVA/CS MS were 94.8%, 87.5%, 81.3%, and 74.8%, respectively. These results reveal that the thermal stability of BaSO₄/PVA/CS MS was increased with the increased content of BaSO₄ NPs. In addition, the actual contents of BaSO₄ NPs in 10, 20, and 30 wt% BaSO₄/PVA/CS MS were calculated to be 7.3%, 13.5%, and 20.0%, respectively, compared to the PVA/CS MS. Hence, the BaSO₄ NPs content in the MS matrix indicated a relatively high level, which would ensure an excellent X-ray detectability of the prepared MS.

3.2.4. In vitro X-ray visibility study

The X-ray imaging was performed to verify the X-ray visibility performance of BaSO₄/PVA/CS MS. Fig. S2 shows that the X-ray visibility increased gradually with the increased content of BaSO₄ NPs in the PVA/CS MS. Furthermore, the quantitative radiopacity evaluation indicated that the 10 wt% BaSO₄/PVA/CS MS showed a mean radiopacity of 273 Hounsfield Unit (HU) and increased to 607 and 1400 HU when the BaSO₄ NPs content increased to 20 wt% and 30 wt%, respectively (n = 5) (Fig. 3d). Thus, the BaSO₄/PVA/CS MS with a suitable content of BaSO₄ NPs could act as an effective radiopaque embolic agent, which was traceable under X-ray irradiation.

3.3. In vitro biocompatibility evaluations of BaSO₄/PVA/CS MS

3.3.1. Cytocompatibility examination

Considering the fact that the MS to be used as embolic materials should be safe and non-toxic [43], it is therefore important to determine their cytocompatibility. The viability of HVSMCs co-cultured with the extracts of MS for 24, 48, and 72 h was determined using CCK-8 assay (Fig. 4a). The cells co-cultured with 10 and 20 mg/mL extracts of BaSO₄/PVA/CS MS showed a relative cell viability of 95.0% and 92.0% on day 1, 96.0% and 91.5% on day 2, and reached about 96.5% and 92.2% on day 3, respectively. No significant difference was observed between the MS and negative control group throughout the incubation period (P > 0.05), suggesting no cytotoxic effects of the MS towards the HVSMCs. In contrast, the relative cell viability decreased as a function of time, and all values were lower than 10% when in contact with the 0.64% phenol containing medium (positive control). This indicated that the cell viability of the positive control was significantly lower than that of the MS and negative control group (P < 0.01). These results suggest that the MS extracts were able to preserve the cell viability and, thus, have no cytotoxicity. In this study, the presence of residual GA released from the MS might be a concern regarding the cytotoxicity. It is reported that the cytotoxicity may occur at a high concentration of GA [44]. The GA-crosslinked MS were rinsed thoroughly with distilled water, and further treated with 4 wt% glycine solution to react with the aldehyde groups in the residual GA [45]; thus, the potential toxicity was greatly minimized. With regard to the appropriate concentrations of MS in the cytotoxicity studies, most researchers performed the cytotoxicity analysis at a concentration of 10 or 20 mg/mL of the MS [[46], [47], [48], [49]]. A previous report by Campos et al. showed that when increased the concentrations of GA-crosslinked chitosan–PVA microparticles (CCPMS) to 10.0% (v/v), there was no significant difference in the cell viability compared to the 2.0% (v/v) MS [50]. Accordingly, in our experiment, the viability of HVSMCs might decrease slightly but not significantly if the concentration of the MS increased to 30 or 40 mg/mL. The MS may still retain the excellent cytocompatibility at these concentrations. On the other hand, the maximum concentration of MS was maintained at 20 mg/mL in order to maintain the consistency with the following animal tests.

Fig. 4.

In vitro biocompatibility evaluation of BaSO₄/PVA/CS MS. (a) In vitro cell viability of HVSMCs incubated with BaSO₄/PVA/CS MS extracts at various concentrations for a different time period, **P < 0.05 indicates a statistically significant difference with respect to the positive control group (n = 6). (b) CLSM images of HVSMCs after incubation in the control and experimental groups for 1 and 3 days. (c) FESEM observations of HVSMCs on the surface of BaSO₄/PVA/CS MS after incubation for 1 and 3 days. (d) Hemolysis ratios of diluted rabbit blood after incubation with 10 and 20 mg/mL BaSO₄/PVA/CS MS at 37 °C for 1 h. The blood incubated with distilled water was taken as 100% hemolyzed, while that incubated with normal saline was taken as 0% hemolyzed, respectively.

The viability of HVSMCs incubated with 10 and 20 mg/mL MS, the medium (negative control), and 0.64% phenol containing medium (positive control) were determined via Live/Dead cell assay. As illustrated in Fig. 4b, the cells showed good attachment and normal growth morphology, with predominantly alive cells (stained green) and almost no dead red spots (dead cells) both on the negative control and MS group on day 1. After three days, the cells had grown and spread uniformly and extensively, and distributed more densely in the negative control and MS groups. In addition, the cell numbers in the MS groups were comparable to the negative control, indicating that the MS were non-cytotoxic and biocompatible. However, in the positive control group, massive dead cells (stained red) were observed both on day 1 and day 3, which could be due to the cytotoxic effect of phenol towards the cells. These observations were consistent with the CCK-8 assay results (Fig. 4a).

The characteristic cell morphology of HVSMCs on the surface of BaSO₄/PVA/CS MS was further investigated via FESEM. As shown in Fig. 4c, after incubation for one day, it was observed that the MS supported the cell adhesion and growth, where the cells tended to spread and maintained physical contact with each other through the filopodia. On day 3, the cells proliferated further and stretched to form confluent monolayers on the MS surface.

Overall, the results of cytocompatibility analysis imply that the MS are nontoxic towards the HVSMCs, and could be safe for in vivo embolization.

3.3.2. In vitro hemolysis analysis

One of the critical prerequisites of the embolic agents to be used in contact with the blood should not induce hemolysis upon implantation [51,52]. Thus, the hemolysis behavior of the BaSO₄/PVA/CS MS was evaluated by incubating the MS with fresh rabbit blood. As represented in Fig. 4d, the hemolysis ratios of BaSO₄/PVA/CS MS at the concentration of 10 and 20 mg/mL were 0.75% and 1.16%, respectively, which was significantly lower than that of the positive control (p < 0.01). Generally, the hemolysis ratio of less than 5% is considered as an acceptable value [53], thus, the hemolysis results indicate the non-hemolytic nature of BaSO₄/PVA/CS MS. The hemolysis photograph further confirm that the BaSO₄/PVA/CS MS were hemocompatible (Fig. S3).

3.4. Characterization of thrombin@BaSO₄/PVA/CS MS

The comparative FESEM morphology studies between the free and thrombin-immobilized BaSO₄/PVA/CS MS with different magnifications are shown in Fig. 5a-b''. Apparently, both the BaSO₄/PVA/CS MS without or with thrombin display a similar spherical morphology. The surface features of BaSO₄/PVA/CS MS before thrombin immobilization indicate their relatively compact and smooth surface (Fig. 5a-a''). Contrarily, the MS immobilized with thrombin indicate that the coverage of thrombin occurred on the MS surface (Fig. 5b-b''). The magnified FESEM images show that the thrombin@BaSO₄/PVA/CS MS possessed a relatively rough layer with enzyme evenly distributed, as compared to the smooth surface of BaSO₄/PVA/CS MS. These morphological features confirm the immobilization of thrombin on the MS surface.

Fig. 5.

FESEM surface morphology of (a-a'') BaSO₄/PVA/CS MS and (b-b'') thrombin@BaSO₄/PVA/CS MS. (c) FTIR spectra of BaSO₄/PVA/CS, succinated BaSO₄/PVA/CS, and thrombin@BaSO₄/PVA/CS MS. (d) XPS spectra of BaSO₄/PVA/CS and thrombin@BaSO₄/PVA/CS MS.

The carboxylation of BaSO₄/PVA/CS MS and immobilization of thrombin onto the carboxylated BaSO₄/PVA/CS MS were confirmed through ATR-FTIR analysis. As represented in Fig. 5c, the FTIR spectrum of BaSO₄/PVA/CS MS showed that both the characteristic absorption peaks of BaSO₄ and PVA/CS. After carboxylation with succinic anhydride, it is clear that the grafted BaSO₄/PVA/CS MS showed an increased peak intensity at 1713 cm⁻¹ and introduced a new peak at 1559 cm⁻¹, both of which were derived from the carbonyl groups that were formed during the ring-opening of the anhydride on the MS surface [54]. After the biofunctionalization of the grafted BaSO₄/PVA/CS MS with thrombin, there was no appreciable peak change compared to the grafted BaSO₄/PVA/CS MS, except the decreased peak intensity at 1713 and 1559 cm⁻¹. This result might be due to the reaction between the carbonyl groups on the grafted BaSO₄/PVA/CS MS and the amide groups on the thrombin, which decreased the number of carbonyl groups on the MS surface. On the other hand, the peak for –CO–NH-groups did not appear in the FTIR spectra, which could be due to the insensitivity of ATR-FTIR to detect the thin layer of thrombin.

The introduction of thrombin on the surface of BaSO₄/PVA/CS MS was further verified by XPS analysis. Fig. 5d presents the XPS spectra of BaSO₄/PVA/CS and thrombin@BaSO₄/PVA/CS MS. The most significant difference between the spectra is the N1s at 398 eV, and could be due to the presence of thrombin after the surface biofunctionalization. The quantitative evaluation results of XPS demonstrated that the mass ratio of N atoms in the thrombin@BaSO₄/PVA/CS MS was 8.6%, which indirectly reflected the content of thrombin immobilized on the MS surface. Thus, the XPS data provided additional evidence for the introduction of thrombin after the surface modification. The peaks for Ba centered at 777 and 793 eV were not presented in the spectrum of thrombin@BaSO₄/PVA/CS MS, which might account for the coverage of thrombin minimize the Ba signal. All these results confirm the successful immobilization of thrombin on the carboxylated BaSO₄/PVA/CS MS, which would enable fast local coagulation of blood following embolization.

3.5. Hemolytic properties of thrombin@BaSO₄/PVA/CS MS

3.5.1. Whole blood clotting kinetics

To evaluate the effectiveness of thrombin@BaSO₄/PVA/CS MS on activation of blood coagulation, the whole blood clotting kinetics as a function of time was conducted in vitro. In view of the main component of the blood clot was RBCs, the absorbance values of the blood are inversely proportional to the hemostatic efficiency of the materials assessed [55]. Fig. 6a shows the whole blood clotting kinetics of control, BaSO₄/PVA/CS, and thrombin@BaSO₄/PVA/CS MS. It is clear that at 5 min, the thrombin@BaSO₄/PVA/CS MS show significantly lower absorbance values than the BaSO₄/PVA/CS MS, suggesting that the thrombin@BaSO₄/PVA/CS MS induced a rapid blood clotting. In the following time points measured from 20 to 50 min, the absorbance values of both the groups showed a similar tendency to decrease, indicating that the blood clots were gradually formed on the MS surface. Moreover, in the serial time point, the thrombin@BaSO₄/PVA/CS MS exhibited a significantly faster whole blood clotting rate as compared to the BaSO₄/PVA/CS MS. These results demonstrate that the BaSO₄/PVA/CS MS with immobilized thrombin would lead to the enhanced hemostatic efficiency. Compared to the control group, the BaSO₄/PVA/CS MS group showed a faster whole blood clotting rate, which could be due to the intrinsic physicochemical properties of the MS. On one hand, the polymeric BaSO₄/PVA/CS MS absorbed water from the blood and swelled, and led to the concentration and adhesion of RBCs on the MS, followed by initiation of blood clotting. On the other hand, the presence of a small content of CS in the BaSO₄/PVA/CS MS endowed them with a positively charged surface, which absorbed more RBCs (negatively charged) electrostatically and accelerated the blood clotting [56,57]. Fig. S4 shows that the blood gelled in contact with the thrombin@BaSO₄/PVA/CS MS for 100 s, while the blood still retained flowability in the presence of BaSO₄/PVA/CS MS, suggesting that the thrombin@BaSO₄/PVA/CS MS could effectively promote the blood coagulation.

Fig. 6.

(a) Whole blood clotting kinetics of the control (blood without MS), BaSO₄/PVA/CS, and thrombin@BaSO₄/PVA/CS MS. FESEM images of whole blood clotting on the surface of (b-b'') BaSO₄/PVA/CS and (c-c'') thrombin@BaSO₄/PVA/CS MS; fibrin network formed on the surface of (d-d'') BaSO₄/PVA/CS and (e-e'') thrombin@BaSO₄/PVA/CS MS (White arrow: RBCs; Red arrow: platelets; Yellow arrow: fibrin).

3.5.2. Adherence of whole blood, RBCs, and fibrin on MS

The whole blood coagulation upon incubation with the thrombin@BaSO₄/PVA/CS MS was examined via FESEM. As shown in Fig. 6c-c'', it is observed that the thrombin@BaSO₄/PVA/CS MS were aggregated with blood clots. More specifically, the fibrin network covered the MS surface, with RBCs and deformed platelets entrapped. This is due to that the thrombin immobilized on the MS surface led to the activation of the intrinsic blood coagulation cascade [58]. Compared to the thrombin@BaSO₄/PVA/CS MS, the BaSO₄/PVA/CS MS demonstrated a lower hemostatic activity with respect to few RBCs and platelets captured on their surface (Fig. 6b-b''). It is therefore believed that the BaSO₄/PVA/CS MS immobilized with thrombin showed better blood clot formation. Similarly, the thrombin@BaSO₄/PVA/CS MS enhanced the RBCs adhesion on the surface compared to the BaSO₄/PVA/CS MS (Fig. S5).

As the immobilized thrombin acted as a coagulant by stimulating the conversion of fibrinogen into fibrin, the fibrin formation on the MS was investigated after incubation with the PRP. As shown in Fig. 6e-e'', the fibrin network combined with the platelets was formed on the surface of thrombin@BaSO₄/PVA/CS MS, forming a clot to entrap the MS. In contrast, the BaSO₄/PVA/CS MS did not show a fibrin network on their surface, the MS were well dispersed with few adhered platelets (Fig. 6d-d''). These results indicate that the thrombin@BaSO₄/PVA/CS MS possessed excellent hemostatic activities and, could be a potential hemostatic embolic agent.

3.6. In vivo embolization performance

3.6.1. Macroscopic observations of vessel embolization

The embolization behavior and efficacy of BaSO₄/PVA/CS MS were inspected by macrography. The results are shown in Fig. 7a. It is evident that there were no abnormalities in those injected with glycerol in the whole course. It was likely that the glycerol solution flew into the branches without blocking the ear arteries. Therefore, the rabbit ears treated with glycerol did not show ischemic necrosis or related complications. Conversely, as indicated by the arrows, the ears immediately became pale along the arterial course upon injection with BaSO₄/PVA/CS or thrombin@BaSO₄/PVA/CS MS, which was attributed to that the vascular occlusion effectively prevented the blood flow into the posterior arteries. After embolization for 7 days, a small area of blackened skin necrosis was observed, suggesting that the injection of MS was sufficient to impede the blood flow at the injection site and induced the tissue ischemic necrosis after a certain period of embolization. On day 15, the ischemic necrosis seemed to be more pronounced as large areas of blackened ear tissue were formed and a definite demarcation became apparent between the damaged and normal tissues. In the present study, we attempted to perform the embolization to an extended period up to 21 days to evaluate the treatment efficacy. Nevertheless, the acute damaged ear tissue became dried and separated from the normal tissue, and the macroscopic appearance of the treated ears could not be taken. The results demonstrate that the developed BaSO₄/PVA/CS and thrombin@BaSO₄/PVA/CS MS could achieve obvious vascular embolization effects, suggesting their great potential in endovascular embolization treatments of various diseases.

Fig. 7.

(a) The gross observations of rabbit ear necrotic changes after treatment with glycerol, BaSO₄/PVA/CS, and thrombin@BaSO₄/PVA/CS MS for 0, 7, and 15 days. The yellow arrows indicate that the blood flow was interrupted and the vessel occlusion was achieved. (b) The X-ray images of rabbit ears treated with PVA/CS and BaSO₄/PVA/CS MS at predetermined time intervals (3, 7, and 15 days). The yellow arrows show the distribution of radiopaque MS within the central auricular arteries and side branches.

3.6.2. X-ray visibility in vivo

In the clinic, it is desirable to monitor the embolic agents by imaging equipment to allow their precise control and facilitate a convenient post-embolization diagnosis [14,18,59]. The radiopaque BaSO₄/PVA/CS MS were assayed for their detectability in vivo with fluoroscopy at day 3, day 7, and day 15, respectively. As depicted in Fig. 6b, the PVA/CS MS in the left ear were not visible under the X-ray, whereas the ear artery treated with BaSO₄/PVA/CS MS showed clear radiopacity, which was derived from the BaSO₄ NPs loaded in the MS. More importantly, it is evident that the radiopacity of MS persisted for the duration of 7 and 15 days, without a reduction in visibility. These results demonstrate that the BaSO₄/PVA/CS MS could be detected by fluoroscopy in vivo and the X-ray detectability could be maintained for at least 15 days.

3.7. Histopathological and ultrastructural analysis

3.7.1. Histopathological analysis

To further determine the embolization efficacy of BaSO₄/PVA/CS and thrombin@BaSO₄/PVA/CS MS, the histopathological examinations were performed via H&E staining at 7 and 15 days post-embolization. Fig. 8a shows the histological features of the cross-sections of rabbit ear tissues with different treatments. Microscopically, the morphology of ear arteries embolized with glycerol exhibited empty in the vascular lumen and did not show pathological changes after embolization for 7 and 15 days. It was speculated that the glycerol had spread away with the blood into the distal branch arteries and, thus, no evidence of tissue damage or inflammatory reactions was observed around the ear arteries. In contrast, the auricular embolization with free and thrombin-immobilized BaSO₄/PVA/CS MS showed that a few individual MS were integrated within the ear arteries, and encapsulated within the coagulative thrombus to lead to mechanical vascular occlusion after embolization for 7 days. A mild foreign body reaction with neutrophils and lymphocytes infiltration was found to be provoked around the embolized MS (black arrows), which is in accordance with the histological findings in previous studies [60,61]. After 15 days, the embolic MS were still retained in the embolized arteries and the inflammatory response was eliminated. It should be noted that the ear parenchyma showed large areas of diffuse infarction on the periphery of embolized ears (indicated by yellow arrows), which accounted for the persistent vascular occlusion by the MS. The results of the histology examination coincide well with the macroscopic observations (Fig. 7a).

Fig. 8.

(a) Macrographs of H&E stained cross-sectional ear arteries treated with glycerol, BaSO₄/PVA/CS, and thrombin@BaSO₄/PVA/CS MS at different periods (7 and 15 days). Black arrows indicate the inflammatory cells infiltration around the embolized blood vessels, while yellow arrows indicate the diffuse infarction on the periphery of embolized vessels, (b) is the corresponding Masson's trichrome staining images, the black arrows show the BaSO₄/PVA/CS MS, while red arrows show the thrombin@BaSO₄/PVA/CS MS. (c) FETEM images showing the effects of ischemia induced by the persistent embolization with MS for 15 days, which displays severe degeneration of endothelial cells with extensive ultrastructural changes compared to the glycerol group. [Nucleus (N); Ribosome (R); Endoplasmic reticulum (E); Mitochondria (M)].

As extended in vivo embolization could result in infarct evolution and deposition of the collagenous matrix surrounding the embolized arteries, the Masson's trichrome staining was performed to evaluate the necrotic changes after treatments with the embolic materials. As displayed in Fig. 8b, the histopathological assessment of the glycerol injection group carried out for 7 and 15 days revealed no evidence of foreign body reaction or collagen deposition in the ear parenchyma. Compared to the control group, the Masson's trichrome-stained sections in the BaSO₄/PVA/CS MS group showed that the visible regions of mild fibrotic tissue could be distinguished from the infarct border zone with collagen bundles formation, which correlated with the thrombin@BaSO₄/PVA/CS MS group after embolization for 7 days. After 15 days, there were extensive infarcts with abundant interstitial fibrosis in the perivascular tissues, which could be due to the persistent occlusion of the ear-feeding arteries and induced ischemia. The progressive fibrosis formation further confirms that the BaSO₄/PVA/CS and thrombin@BaSO₄/PVA/CS MS prepared in this study could obstruct the blood and achieve effective artery occlusion.

3.7.2. Ultrastructural changes

The ultrastructural changes in the nucleus and cytoplasm of endothelial cells in the rabbit ear arteries, treated with glycerol and MS for 15 days, were investigated via FETEM. As illustrated in Fig. 8c, in the glycerol group (control), the endothelial cells preserved their integrity and normal appearance of the intracellular structures, where the cytoplasmic organelles including mitochondria, ribosome, and endoplasmic reticulum were well developed without structural changes. Conversely, in the BaSO₄/PVA/CS and thrombin@BaSO₄/PVA/CS MS groups, the endothelial cells experienced severe degeneration with structural variations as compared to the control group. The FETEM observations showed that the nucleus was degenerated with a pleomorphic shape and an indistinct nuclear membrane. In addition, a severely condensed and unevenly distributed chromatin was observed. Moreover, the prominent changes in the cytoplasm were also discerned, which included mitochondria swelling, depletion of the ribosome and endoplasmic reticulum, and cytoplasmic vacuolations. These results are consistent with the previous studies [[62], [63], [64]]. Altogether, the thorough disintegrative necrosis of nucleus and cytoplasm could be due to the endothelial cell damage caused by the persistent MS embolization, which would result in dysfunction of blood perfusion and continued hypoxia due to the alternations in endothelial permeability [65].

4. Conclusions

In this work, the X-ray radiopaque BaSO₄@PVA/CS MS were successfully prepared via S/W/O emulsification crosslinking method, where BaSO₄ NPs were first synthesized as imaging contrast agents, and then mixed with the PVA/CS polymeric matrix. The sieved MS were spherical in shape with desirable particle size ranging from 100 to 300 μm. In vitro cytocompatiblity and hemocompatibility tests demonstrated the non-cytotoxic nature and permissible hemolysis rate of the prepared MS. Importantly, in vitro and in vivo evaluations revealed that the MS were clearly visible on X-ray fluoroscopy by virtue of the loaded BaSO₄ NPs. The surface modification with thrombin endowed the MS with excellent hemostatic activities. The results indicated that the thrombin@BaSO₄/PVA/CS MS could promote a rapid thrombus formation, as well as facilitate whole blood coagulation, RBCs adhesion, and fibrin generation. Moreover, in vivo embolization in a rabbit ear model indicated that both the BaSO₄/PVA/CS and thrombin@BaSO₄/PVA/CS MS could lead to progressive ischemic necrosis on ears as a result of the effective vascular occlusion. Furthermore, the embolization effect also resulted in abnormal changes of endothelial cells in the embolized rabbit ears. Taken together, the thrombin@BaSO₄/PVA/CS MS provide the basis for precise embolization and localized coagulation stimulation, which would enhance the embolization efficiency for various applications.

CRediT authorship contribution statement

Xiaohong Li: Conceptualization, Methodology, Data curation, Writing - original draft. Xiongfa Ji: Methodology, Data curation, Investigation. Kun Chen: Software, Formal analysis. Muhammad Wajid Ullah: Writing - review & editing, Data curation. Basen Li: Methodology, Data curation, Resources. Jiameng Cao: Formal analysis, Investigation. Lin Xiao: Data curation, Validation, Software, Writing - review & editing. Jun Xiao: Conceptualization, Resources, Methodology, Data curation. Guang Yang: Supervision, Conceptualization, Writing - review & editing, Funding acquisition.

Declaration of competing interest

The authors have no conflicts of interest to declare.

Appendix A. Supplementary data

The following is the Supplementary data to this article: