Fibrin Clot Degradation by Polyaniline-Coated AuNP Using Laser Photolysis

2.1 Chemicals

The solution of the gold (III) chloride trihydrate (HAuCl₄·3H₂O) was prepared in the laboratory by reacting gold metal with chlorine gas. (King et al., 2015) In detail, Hydrochloric acid (15 mL, 36 %) was added in a pressure-balancing dropping funnel, and slowly added drop-wise to a flask containing potassium permanganate (3.0 g, 19 mmol). This reaction generates chlorine gas which was passed into a two-neck round-bottom flask containing 100 mL ultrapure water and one pellet of gold (∼250 mg). Undissolved chlorine gas was bubbled through a solution of sodium thiosulfate (1.1 g, 7.0 mmol) dissolved in 50 mL of water. The reaction mixture was stirred at the selected temperature (40–60 °C) until all the gold dissolved.

Tri-sodium citrate (Na₃C₆H₅O₇·2H₂O), and glucose anhydrous (C₆H₁₂O₆, 99.0 %) were purchased from Winlab. Copper (II) acetate (Cu (COOCH₃)₂) and aniline (C₆H₅NH₂) were purchased from BDH. Sodium chloride (NaCl) was purchased from lobe chemie. HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Fibrinogen from bovine plasma (≥75 % of protein is clottable), thrombin from bovine plasma (lyophilized powder, ≥2,000 NIH units/mg protein) was purchased from sigma. All solutions were prepared in deionized water and used without further purification steps unless otherwise indicated.

2.2 Sample characterization techniques

The absorption spectra of AuNPs and AuNPs@PANI core/shell were recorded using a UV–Visible Spectrophotometer (UV-2450), Shimadzu Corporation, Kyoto, Japan, in a quartz cell. The functional groups of the prepared samples were confirmed by recording infrared spectroscopy (FT-IR) on the Spectrum BX spectrometer (PerkinElmer, Waltham, USA) using KBr pellets of the samples. Dynamic light scattering (DLS) and zeta potential of the working particles were measured using a device from Malvern. A pH meter (Thermo, S-2STAR-E 0608 RevB) was used for pH measurements.

Additionally, the size and morphology of the samples were characterized using a transmission electron microscope (TEM) (JEOL, JEM 1400 Plus, USA). TEM samples were prepared by dripping the sample onto carbon-coated copper grids and then air-drying overnight. Scanning electron microscope (SEM) was carried out on a JEOL JSM-6060LV. The critical point dryer (Tousimis-Samdri-PVT-3D) was used to completely exchange ethanol in the fibrin samples with liquid CO₂. The chemical composition of the core/shell nanoparticles was found by energy-dispersive X-ray analysis (EDX) connected with the SEM device.

2.3 Synthesis of AuNPs

Following the traditional Turkevich method. (Turkevich et al., 1951; Walker et al., 2011; Ojea-Jiménez et al., 2011) Briefly, in a flask, 100 mL of deionized H₂O and 3 mL of freshly prepared trisodium citrate (1 % w/w) solution were added. Solution then heated up to 100 °C for 15 min. 1 mL of an aqueous solution of HAuCl₄·3H₂O (1 % w/w) was added, and the mixture was kept at the boiling point for 5 min. The solution immediately changed from colorless to light blue and finally brick-red color, indicating the formation of AuNPs.

2.4 Synthesis of AuNPs@PANI core/shell

The AuNPs coated with PANI as a shell were prepared using the hydrothermal synthetic method. (Alshammari et al., 2020) In the capacity of Teflon container were added 8 mL of AuNPs solution, which was prepared in advance, 20 μL of aniline, Cu(OAc)₂·H₂O (0.25 mL, 80 mM), 10 mL of deionized water, and a freshly prepared (2 mL, 2 M) aqueous solution of glucose. The mixture was then added to a steel bomb and heated at 160 °C in an oven for 2 h. Finally, after the steel bomb cooled naturally to room temperature for 1 h, the AuNPs@PANI core/shell nanoparticles were centrifuged at 10000 rpm for 15 min and washed twice with water.

2.5 Protein stock solution

The thrombin and fibrinogen solutions were separately synthesized in HBS buffer, pH = 7.3 (20 mM HEPES, 150 mM NaCl). First, A fibrinogen stock solution of 3.5 mg/mL in HBS buffer was prepared, and every 150 μL was placed in small plastic tubes and frozen until later use. In a similar method, 50 U/mL thrombin was dissolved, partitioned into small plastic tubes, and frozen until use.

2.6 Fibrin clot preparation

Thrombin (12 μL, 50 U/mL) and fibrinogen (300 μL, 3.5 mg/mL) were mixed, and then quickly, 50 μL of the mixture was injected into short glass tubes, where these tubes were made with open ends, 4 cm in length and 4 mm in diameter, and one of their ends was closed using parafilm before adding the solution to make fibrin clot removal easier for later characterization. And then, the fibrin clots were left in the tubes for 1 h without moving to ensure complete formation of the fibrin network.

2.7 Photolysis of fibrin clot

After the complete formation of a fibrin clot, 50 μL of the previously prepared nanoparticle solution (AuNPs@PANI core/shell or AuNPs) was added over the fibrin clot. Then photolysis of the samples was carried out for 1:30 h using laser light of a specific wavelength of 520 nm with 300 mW. After that, the modified clot was separated from the tube for later characterization.

2.8 Sample preparation for SEM

Fibrin clots were washed with different concentrations of ethanol and water for 45 min each step (20 %, 40 %, 60 %, 80 %, and 100 % of ethanol). 2 % glutaraldehyde was added and left overnight at 4 °C to maintain the crosslinking of the fibrin clot obtained for SEM imaging. After warming the clot to room temperature, it was washed again with 100 % ethanol and dried using a CO₂ critical point dryer. Create critical conditions for CO₂; the chamber containing the samples and liquid CO₂ was heated to 36 °C at a pressure above 1072 psi. Such conditions were then maintained for about 4 min. The sample was sputter-coated with Au to prepare it for SEM imaging.

2.9 Statistical calculations of the gaps area

Image J software was used to analyze and process the SEM microscopy image of the fibrin network for characterization and measure the gaps at three different locations for each sample. The threshold feature in the Image J software was used to determine the appropriate threshold for the SEM image. An area of approximately 500 gaps was measured for each SEM image. In particular, the gaps were measured in three SEM images per sample (i.e., the total number of gaps measured for each sample is at least 1500). (Morais et al., 2021; Feng et al., 2019; Hartig, 2013) Gaps smaller than 1 nm were excluded. After that, the percentage was calculated for the gaps. All SEM micrographs used in the statistical analysis of the sample were at × 15,000 magnifications. After calculating the percentage of the total gap area, use the same method for calculating the gaps with areas larger than 100 nm.

3.1 Preparation procedure of AuNPs@PANI core/shell

The present work illustrates the synthesis of gold nanoparticles coated with polyaniline (AuNPs@PANI) as a core/shell using a two-step method. Initially, citrate-stabilized AuNPs obtained via the reduction of HAuCl₄ by sodium citrate were prepared. According to previous studies, the formation of dicarboxyacetone can result from the thermal decomposition of a sodium citrate solution, this will improve the final morphology of AuNPs and result in a significant increase in the rate of nucleation and growth, resulting in the complete formation of AuNPs in less time. (Ojea-Jiménez et al., 2011; Sivaraman et al., 2011) AuNPs were completely formed in less than 5 min after adding HAuCl₄, where a rapid change in color occurred from colorless to the distinctive characteristic red of gold particles.

The second step involves coating the prepared AuNPs with PANI by the one-pot hydrothermal method at 160 °C for 2 h in the presence of aniline, copper (II) acetate, and glucose as a reducing agent of Cu²⁺ ions. The hydrothermal approach uses an aqueous solution as a reaction method in a specialized closed reaction system Tevlon container to heat and pressurize the reaction to create a high temperature and pressure reaction environment (Yang and Park, 2019). Covering gold particles with a layer of polymer aims to increase their biocompatibility and obtain a positive surface charge that is able to bind with negatively charged biopolymers through electrostatic interactions (Hou et al., 2015; Alshammari et al., 2020).

The proposed polymerization of PANI around AuNPs to formation core/ shell includes several stages: Assuming that the reaction between copper (II) acetate and aniline does not take place without additional chemical reagents that help complete the reaction, Therefore, the occurrence of the reaction was enhanced by the addition of glucose, which initially acts as a reducing agent for Cu²⁺ ions to produce Cu⁺ through the formation of cuprous oxide (Han et al., 2018). Aniline possesses an NH₂ group that is able to form electrostatic bonds with Cu⁺ (Mallick et al., 2006). The additional amount of copper leads to the loss of a proton from the individual aniline under the Cu/O₂ catalytic system, which leads to the formation of active aniline radicals with high activity, which are responsible for the initiation of the polymerization process (Ji et al., 2017).

In addition, the oxidative polymerization process involves the release of an electron in each step, which is then used to reduce copper ions to copper atoms (Fig. 1) (Mallick et al., 2006).

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Fig. 1

Schematic representation of hydrothermal method for synthesis of AuNPs@PANI core/shell and the chemical reaction process.

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3.2 Characterization of AuNPs@PANI Core/Shell

The surface plasmon resonance (SPR) properties of nanoparticles were analyzed by using ultraviolet–visible (UV–vis) absorption spectroscopy. Fig. 2 shows the UV–vis absorption spectrum of synthesized AuNPs. Significantly, the SPR peak appear to have one major absorbance peak at 520 nm, which indicates the formation of AuNPs with a spherical shape (Ojea-Jiménez et al., 2011). Additionally, when comparing the SPR properties between the AuNPs and AuNPs@PANI, we notice that after covering the AuNPs with PANI shell, there was a red shift in the position of the SPR peak from 520 to 610 nm (Fig. 2). This red shift occurs as a result of the change in the surrounding environment around the AuNPs after coating them with the PANI polymer layer (Yang et al., 2015; Lin et al., 2020). Worth noting that PANI absorbs at 420 nm as shown in figure S1.

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Fig. 2

UV–vis spectra of the LSPR peak position of AuNPs and AuNPs@PANI.

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FT-IR measurements (Fig. 3) provide chemical structural information, surface functionalization, and confirmation of the interaction between the AuNPs and citrate ions. The structure of the citrate ion molecule has three groups of carboxylates (COO⁻). The vibration modes of (COO⁻) confirm citrate ion adsorption on AuNPs. The characteristic peaks of (COO⁻), asymmetric and symmetric, appeared respectively at1617 and 1401 cm⁻¹ (COO⁻ stretching). In addition, FT-IR spectra have an O–H stretching peak at 3395 (broad). At low frequencies regions, have (C–O stretching) of 1265 cm⁻¹ and 1079 cm⁻¹ (Wulandari et al., 2015; Mohan et al., 2013). The FT-IR spectrum shows a significant change in the position of the peaks after coating the AuNPs with a layer of PANI. The vibrational bands observed for the resultant compound can be reasonably explained on the basis of the basic structure of PANI. The absorption peak is at 3395 and 3238 cm⁻¹ (N–H stretching). The peaks are at 2923 and 2854 cm⁻¹ (aromatic C–H stretching). Additionally, two distinct bands appear in the FT-IR spectrum, one at 1571 cm⁻¹ representing C = C stretching on the quinoid ring in PANI and the other at 1498 cm⁻¹ representing C = C stretching on the benzenoid ring. The peak at 1294 cm⁻¹ corresponds to the CN stretching. A band shows up at around 1075 cm⁻¹ for PANI and can be characterized as a vibrational mode structure of the (–NH⁺=), indicating the PANI chain has positive charges and a distribution of structure between benzenoid rings and quinone ring, in agreement with PANI spectrum as in Figure S2 (Mondal et al., 2014; Mallick et al., 2006; Aboelkheir et al., 2022; Yi et al., 2024; Dong et al., 2007; Abdelghany et al., 2021).

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Fig. 3

The FTIR spectra of AuNPs and AuNPS@PANI.

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The morphology of the resultant nanoparticles AuNPs@PANI at the optimum synthesis (80 mM, Cu (OAC)₂ and 2 M, glucose) was characterized by different techniques. Transmission electron microscope (TEM) images (Fig. 4 a and b) show AuNPs before and after the PANI coating, which clearly display the nanosphere shapes of AuNPs and the shell of the PANI polymer around the surface of AuNPs, with average size of 20 nm. The AuNPs@PANI surface was studied using scanning electron microscopy (SEM) (Fig. 4c), where it showed the spherical shape of the PANI shell, which supports the results obtained in TEM images. Fig. 4d shows the statistical diameters and the distribution of the particles depending on TEM images of the AuNPs.

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Fig. 4

The TEM image for (a) AuNPs, and (b) AuNPs@PANI. (c) SEM image for AuNPs@PANI. (d) The statistic diameters based on TEM images of AuNPs. (e) Show Zeta potential for AuNPs and AuNPs@PANI core/shell.

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The surface charge of the prepared nanoparticles was confirmed by dynamic light scattering and Zeta potential analysis. (Fig. 4e) shows that AuNPs@PANI exhibit positive zeta potential. However, an increase in zeta-potentials from −51.8 mV for AuNPs to 17.8 mV for AuNPs@PANI indicates a shift to a more positive surface potential when coating the surface with PANI.

The chemical composition of the core/shell was found by energy-dispersive X-ray analysis (EDX). The SEM-EDX spectra show the electron microscope mapping analysis of the AuNPs@PANI (Fig. 5), which showed a good distribution of Au, Cu, N, C, and O elements in the spectrum. The percentage of elements confirms the presence of core/shell structure through the presence of carbon (43.07 %), oxygen (24.37 %), and copper (17.71 %). Along with TEM images, EDX results indicates the formation of polymer chains on the surfaces. The lower percentage of gold (8.61 %) indicates its presence in the core.

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Fig. 5

(a) EDX pattern, (b) The percentage of elements, SEM-EDX element mappings image (d) N, (e) Au, (f) Cu, (g) O, and (h) C of AuNPs@PANI core/ shell.

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3.3 Fibrin degradation

The polymerization of fibrin initiates upon contact of the thrombin enzyme with the fibrinogen, which leads to the formation of a fibrin clot network. The fibrin polymerization takes place in several steps: (1) The thrombin enzyme solution works to release the fibrinopeptides from the fibrinogen, which leads to the formation of the fibrin monomer. (2) Through knob-hole interactions, the fibrin monomer self-assembled and formed a fibrin polymer (Weisel and Litvinov, 2013). Therefore, the thrombin and fibrinogen solutions in the laboratory were prepared separately using HBS buffer at pH 7.3, after that, thrombin enzyme was mixed with fibrinogen solution to prepare the clot. With the passage of time, it began to form a gel-like phase which indicates the formation of the fibrin clot network, and the clot was left at room temperature for an hour to ensure its complete formation. The effect of the prepared AuNPs and AuNPs@PANI solutions was studied by placing them in an outer layer over the network fibrin. After that, one of the samples was subjected to photolysis, and the other was placed in the dark at room temperature. Then, the modified fibrin clot was separated from the tube after completion of the reaction. Fig. 6 illustrates the protocol used to study the effect of the prepared nanoparticles on the fibrin clot.

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Fig. 6

Illustrates the protocol used to study the effect of the prepared nanoparticles core/shell on the fibrin clot.

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SEM images were used to verify the collapse of fibrin framework. The effect of adding a nanoparticle solution on the morphology of the formed fibrin network was studied by SEM on several spots. The change in the morphology of the fibrin polymer can be tracked by the formation of gabs on the surface. The appearance of gaps or an increase in the number of gaps, and the appearance of a fibril terminus is an indicating of degradation in the fibrin clots.

The first set of samples (Fig. 6, step 1) shows AuNPs added to fibrin clot under two conditions: (a) photolysis and (b) in dark, as shown in SEM Fig. 7. When comparing the sample of fibrin network in the dark (Fig. 7a) with the sample (Fig. 7b) under photolysis conditions by laser light at λ = 520 nm in the interface area between the clot and the AuNPs solution at room temperature for 1.5 h, the photolysis of the sample has very little effect on the fiber clot, which includes the appearance of some clusters in the fibrin network. That may be due to the absence of a strong bond between the AuNPs before coating with PANI polymer and fibrin.

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Fig. 7

SEM images of the fibrin network morphologies after placing the AuNPs: (a) represent the dark control, and (b) represent the fibrin clot after photolysis by laser light at λ = 520 nm. Scale bar 100 nm. (onset show low magnification for large area of fibrin).

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On the other hand, the other set of samples (Fig. 6, step 2) showing the effect of adding the AuNPs@PANI to fibrin. The characteristic strong blackish green color of AuNPs@PANI solution allows their penetration into the fibrin network to be monitored visually, where it leads to a clearly visible change in the color of the clot after the photolysis. When the sample was photoionized, there were clear differences in the structure of the SEM images. Severe morphological degradation sustained by the fibrin network structure (Fig. 8 a). In the dark control, no significant change was observed in the network shape, (Fig. 8 b). This result is very comparable to the shape of the fibrin network after adding AuNPs before coating with the PANI layer (Fig. 7 a and b) with the same approach.

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Fig. 8

SEM images of the fibrin network morphologies after placing the AuNPs@PANI: (a) represent the dark control, and (b) represent the fibrin clot after photolysis with different magnifications. Scale bar 100 nm. (onset show high magnification for large area of fibrin).

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The obtained results can be interpreted from several angles. First, the effectiveness of irradiation at 520 nm in the interface between the solution of the AuNPs coated with PANI polymer and the fibrin clot. As shown in SEM images the change in the morphology of the network is significant (Fig. 8 b). We were able to achieve fibrin degradation by photolysis AuNPs@PANI using the green laser (λ = 520 nm, 1:30 h) (Alshammari et al., 2020). Our approach offers more specific laser light and less time for degradation comparing to previous studies used 6 h and broad band light (Walker and Zaleski, 2014).

Additionally, the appearance of gaps and fibrous endings indicates the beginning of the degradation of the fibrin network in different sites of the clot. Second, According to zeta potential results, the surface PANI has a positive charge (17.8 mV) of AuNPs@PANI (Fig. 4e). This may lead to a bonding between the $\gamma$ chain in fibrin and the AuNPs@PANI as a result of the electrostatic interactions between the positive charge in the PANI and the negative charge in the $\gamma$ chain in fibrin newtwork (Kattula et al., 2017; Alshammari et al., 2020). Also, the thermal effects of AuNPs can impact the formed fibrin polymer. The fibrin clots were irradiated after adding the AuNPs@PANI by using an external source of laser light at 520 nm with high photothermal conversion efficiencies to heat. As a result, there is a temperature difference between the biological medium and the hot surface of AuNPs, which can cause an abrupt local hyperthermia and has an effect on fibrin clots (Hou et al., 2015). Significantly, the resonance wavelength makes the AuNPs able to efficiently absorb radiation, which leads to degradation and melting in different positions of the fibrin clots. In addition, the shells of PANI and Cu₂O induce the production of electrons, which stimulate the formation of reactive oxygen species (ROS). In the presence of laser light irradiation and O₂, the interaction of ¹O₂ with the fibrin network could be responsible for the degeneration and formation of gaps in clots. (Alshammari et al., 2020).

3.4 Statistical analysis of fibrin degradation

Quantitative analysis of fibrin degradation would support the SEM images. We applied this analysis for deeper evaluation of the gap formation on fibrin network. ImageJ software was used to process the SEM microscopy image of the fibrin network to measure the gaps at three different SEM images per sample and calculate the average area by using the threshold feature (Hartig, 2013). Fig. 9 shows the threshold image of the fibrin clot network and the drawing gap area reconstructed from the SEM image after adding the AuNPs solution under green laser light at 520 nm and a control sample in the dark. For AuNPs, the area of the holes in the dark control and photolysis were 11.9 % and 14.2 %, respectively. On the other hand, the same method was used to calculate the internal gaps between the fibers of the fibrin network after adding the prepared AuNPs@PANI. As shown in Fig. 10, the area of gaps were 10.1 % (dark control) and 30.0 % (photolysis). Specifically, when comparing the statistical analysis results (as in Table 1), the fibrin clot network after the photolysis of AuNPs@PANI had the highest gap area, which was 30.0 %. The distribution histograms give an overview of the area of all gaps and the detailed distribution of the gaps smaller than 100 nm (Fig. 11).

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Fig. 9

Gap analysis of the SEM image by ImageJ software of the fibrin clot network after adding AuNPs. The SEM image of the fibrin clot (a) in the dark; (d) after photolysis; (b, e) the threshold images reconstructed from the SEM images above; and (c, f) represent the drawing gap area of each image above, respectively. Scale bar 1 $\mathrm{\mu }$ m.

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Fig. 10

Gap analysis of the SEM image by ImageJ software of the fibrin clot network after adding AuNPs@PANI. The SEM image of the fibrin clot (a) in the dark; (d) after photolysis; (b, e) the threshold images reconstructed from the SEM images above; and (c, f) represent the drawing gap area of each image above, respectively.

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Fig. 11

The gap area and area distribution histograms were measured from the SEM images via analysis of a sample image by ImageJ software (a) AuNPs (Dark),(b) AuNPs (photolysis), (c) AuNPs@PANI (Dark), and AuNPs@PANI (photolysis).

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Additionally, Figures S3 and S4 show the threshold image reconstructed from the SEM image in another different image after adding the AuNPs, where the average value of the gap was 10.402 % (control sample) and 14.838 % (photolysis), as shown in Table S1, which shows a slight increase in the percentage of gaps formed after the photolysis. Figures S5 and S6 show the threshold image reconstructed from the SEM image in another position after adding the AuNPs@PANI solution. When comparing the statistical analysis results as shown in Table S1, the average value of the gaps larger than 100 nm after adding the AuNPs@PANI under photolysis of fibrin clots was 40.803 %. In other words, the average percentage area value of total gapes was ∼ 30.218 %, of which 12.330 % have holes that have an area larger than 100 nm. In contrast, the area percentage of the gaps larger than 100 nm of the control sample AuNPs@PANI (in the dark) were ∼ 4.86 % (meaning ∼ 10.872 % represent total gapes, of which 0.529 % of holes had an area larger than 100 nm). These statistical results clearly show the effect of the photolysis of the sample after adding the AuNPs coated with PANI, which led to a clear increase in the area ratio of the gaps. The reason for the increase in the area of the gaps and the degradation of the fibrin network in some areas is probably due only to the fact that the AuNPs@PANI core/shell structure was placed above the fibrin clot.

Potential future applications of this methodology should focus on the fact that remodeling and changing of the fibrin network could have utility to effect perfusion through the clot network, which increases the probability of the arrival of therapeutics (e.g., tPA) inside the clot (Walker and Zaleski, 2014). Furthermore, treatments may bind to the surfaces of AuNPs, due to the fact that AuNPs have emerged as nanocarriers in the biomedical domain and are diversely used in drug delivery and photo-irradiation-based therapy. Despite the fact that drugs cannot be entrapped within inorganic particles, several studies have undertaken to bind treatments to the surfaces of AuNPs due to their large surface area-to-volume ratio and bioinertness. Chemotherapeutics and photodynamic medicines have therefore been immobilized on AuNPs for disease targeting and therapy in vivo (Mao et al., 2020). Photoactivation of AuNPs coated with PANI could be achieved by using external irradiation, which should match the surface plasmon resonance localized on the nanoparticles and excitation wavelengths with appropriate penetration depth (Walker and Zaleski, 2014).