An Investigation on the Effect of Contrast Agents in the Chitosan-Nanoclay Shear Thinning Hydrogel for Trans-Catheter Arterial Embolization

Abstract

Shear thinning hydrogels are emerging as a potential candidate of embolic agents for minimally invasive transcatheter arterial embolization because of their advantages over the commonly used conventional solid and liquid embolic agents. The transformation from solid to liquid and vice versa on the application and removal of shear force enables the shear thinning hydrogels to successfully occlude vasculatures of any diameters and thus can be used for the treatment of diseased or injured vasculature, including vascular malformation and malignant or benign tumors. Imageability of the embolic gel is of paramount importance as for successful occlusion of vasculatures requires real-time monitoring of the deployment of the embolic agents. Here, we studied the effect of different contrast agents, including iohexol, Omnipaque^™ and tantalum microparticles, on the radiopacity on an established chitosan-nanoclay hydrogel system. The flow behavior and recoverability of the developed gels are not compromised by the addition of radiopaque contrast agents. The injection force of the gels through a clinically relevant catheter was measured to evaluate the practicality of hand injection. The gels exhibited exceptional contrast under x-ray imaging modality. In addition, the radiopaque gels demonstrated excellent hemocompatibility and cell biocompatibility, highlighting their potential for transcatheter arterial embolization.

INTRODUCTION

Minimally invasive surgical interventions are becoming popular because of their numerous advantages over the conventional open surgeries, including fewer complications, better outcomes, less recovery time and lower cost.^1–4 Any organs in the body can be accessed through an endovascular approach as the blood vessels run through entire body.^1,2 Endovascular embolization refers to the deliberate occlusion of blood vessels using an embolic agent especially for the treatment of acute potentially fatal bleeds to more chronic conditions, such as aneurysms and arteriovenous malformation (AVM), and in some targeted oncologic applications such as embolization of vasculatures feeding certain tumors.² Currently, solid metallic coils and liquid embolic materials are used for endovascular embolization.^2,4,5 Solid metallic coils have a variety of shapes, lengths and thicknesses and are mainly used for embolizing aneurysms and bleeding arteries.^4,6 However, metallic coils cannot be deployed in small vasculatures for the treatment of AVMs because of their size constraints, showing limited efficacy in anticoagulated or coagulopathic patients, producing significant imaging artifact limiting the evaluation of adjacent soft tissues, when deployed they cannot be retrieved, and are not cost effective.^1,6 On the other hand, liquid embolic agents can flow to vasculatures of any diameter and successfully occlude blood vessels. However, the presence of organic solvents (e.g., DMSO) in most of the liquid embolics causes local and systemic cardiovascular toxicity, and also the liquid glue adheres to the catheter resulting in improper embolization.^2,7 Hence, the scientific community strives to develop shear thinning embolic gels which have the ability to act as a solid in the absence of shear stress and can flow like liquid during the application of force, which makes the transcatheter injection easy, thereby overcoming the limitations of the abovementioned conventional embolics.^1,5,7–9 Furthermore, it is necessary to render any newly developed embolic agent radiopacity since successful embolization majorly depends on real-time monitoring during injection using x-ray-based fluoroscopy.^4,8

A number of studies have demonstrated the development of radiopaque shear thinning hydrogels for endovascular embolization. Iodinated contrast media such as iohexol, iodixanol and Omnipaque^™ provide sufficient radiopacity. Hu et al. developed an laponite nanoclay (NC)-silk fibroin gel with Omnipaque^™ for catheter-directed endovascular drug delivery and were successful in imaging under x-ray imaging modality.³ Altun et al. used ethiodized oil in a NC-blood-derived platelet-rich fibrin hydrogel system, which showed good visibility under computed tomography (CT) scan.⁶ Tantalum powder is another common radiopaque agent used in clinics.^4,10,11 Hassan et al. incorporated tantalum microparticles of an average size of 2 μm in an NC-gelatin based hydrogel system for rapid endovascular hemostasis.⁴ In addition, Obsidio^™ conformable embolic agent (Boston Scientific, USA) is the only FDA 510(k) cleared radiopaque semi-solid embolic material using Tantalum microparticles for peripheral embolization.^4,12

In our previous study, we developed a chitosan nanofiber (CH) -NC-based shear thinning hydrogel with different total solid contents (TSC) of 4 wt.%, 6 wt.% and 8 wt.% with varying CH to NC ratio from 0% to 100% of the TSC.¹ We observed that the 6NC50 gel (system with TSC of 6 wt.% and CH content of 50% of TSC) demonstrated excellent mechanical, rheological and injectable properties along with exceptional hemo- and bio-compatibility.¹ Interestingly, the mechanical properties and injection force of 6NC50 gel were the lowest in the group (yield stress—21.1 ± 0.5 Pa and injection force—17.3 ± 0.03 N).¹ However, the yield stress of the gel is four times greater than the maximum shear stress experienced by the arteriole walls (5 Pa),^13,14 and injection force is approximately 4.6 fold less than the maximum comfortable injection force for physicians (79.8 N),¹⁵ making the gel a potential candidate for endovascular embolization. This characteristic property, due to the unique morphology, makes 6NC50 gels easy to inject through a clinically relevant microcatheter and at the same time provides balanced active components and sites in the gel which can be beneficial to carry drugs/contrast agents for therapeutic activities. Therefore, 6NC50 is selected as the pristine material (gel without contrast agents) in this current work to study the effect of contrast agents.

While imageability is critical for the development of any embolic agents for tracking on clinically used imaging modalities, there is a lack of systematic investigation on the effect of contrast agents on the behavior of shear thinning embolic gels. Hence, in this study, we incorporated different radiopaque contrast agents such as iohexol (IHX), Omnipaque^™ (IHX OMNI) and tantalum powder (Ta) into 6NC50 gel system, an already established shear thinning hydrogel system consisting of chitosan nanofibers and laponite nanoclay, aiming to access the radiopacity and physical/mechanical behavior of gels with varying contrast agents. The rheological properties of the radiopaque gels were investigated, suggesting the preservation of shear thinning properties, whereas the strength was either preserved or enhanced compared to their pristine counterpart. In addition, transcatheter injectability of the gels was assessed through a clinically relevant microcatheter used for endovascular embolization to confirm a comfortable injection process experienced by the physicians. The imageability of the gels was studied under x-ray to confirm the radiopacity, making them an excellent embolic candidate for during and after surgical examinations. In addition, the gels also exhibited excellent hemocompatibility, thrombogenicity and biocompatibility, suggesting their potential to serve as embolic agents for transcatheter interventions.

MATERIALS AND METHODS

Materials

Laponite-XLG nanoclay (NC) was purchased from BYK USA Inc., Texas, USA (lot no. 0002303785), and chitosan nanofiber (CH) was purchased from Sugino Machine Ltd., Japan. For contrast agents, iohexol was purchased from TCI America, Portland, OR, USA (lot no. IPT4N-PQ), Omnipaque^™ (350 mg I/mL) was obtained from GE Healthcare, Norway (lot no. 16388187), and Ta powder of 2 μm average particle size was purchased from Thermo Scientific, USA (lot no. Y23H019). Gels were engineered using molecular-grade water from Inter-mountain Life Sciences (Utah, USA). Phosphate-buffered saline (PBS) tablet was purchased from Sigma Aldrich (St. Louis, MO, USA).

Preparation of Radiopaque Embolic Gels

Radiopaque gels were engineered using a previously used protocol.¹ Briefly, NC, CH, molecular grade water and contrast agents (IHX or IHX OMNI) were mixed at predetermined weight ratios using a high-speed mixer (DAC 330–100 SE, Flack-Tek Speed Mixer, USA) at 3000 rpm for 5 min and three times. For Ta-based embolic gels, the mixing was performed at a lower speed of 800 rpm for 3 min and repeated three times. This ensures the homogeneous distribution of Ta powder in the CH-NC gel. The gels were named 6NC50-x% CA, where 6NC50 represents gel with 6 wt.% TSC, where 50% of the TSC is NC (3 wt.%) and the remaining 50% of the TSC is CH nanofibers (3 wt.%), x is the percentage of the contrast agent in the gel, and CA represents the specific contrast agents including IHX, IHX OMNI or Ta. The percentage of the contrast agent in the gel is calculated using the following equation (Eq. 1):

$$\text{CA}\%=\frac{\text{Total}\text{weight}\text{of}\text{active}\text{component}\text{in}\text{the}\text{contrast}\text{agent}}{\text{Total}\text{weight}\text{of}\text{the}\text{gel}}*100$$ (1)

The active components of IHX and Ta were powders and hence were added into the gel directly. For IHX OMNI (an aqueous contrast agent), the CA represents the active ingredient, which is iohexol, in the gel. Pristine 6NC50, gel without any contrast agent, will be hereafter mentioned as 6NC50-Control.

Rheology

All rheological studies were conducted at 25°C (MCR 302e, Anton Paar, Austria), unless otherwise noted, since it is the ambient temperature at which an injection would be performed in clinics.¹⁶ A sandblasted 8-mm upper plate and a sandblasted lower plate were used, with a 1-mm gap in between for all tests. A solvent trap was used to maintain the humidity and prevent the gels from drying. The flow curves were performed to evaluate the shear thinning behavior of the gels by varying shear rate between 0.001 s⁻¹ and 1000 s⁻¹. Amplitude sweeps were carried out with shear strain from 0.01% to 100% to assess the linear viscoelastic (LVE) region of gel, as well as its storage modulus (G’) and loss modulus (G”). The angular frequency was kept at 10 rad/s. Flow curves and amplitude sweeps were conducted three times for each test. Frequency sweeps were conducted with increasing angular frequency from 0.1 to 100 rad/s at 0.1% shear strain (LVE region). Lastly, thixotropic test was performed to evaluate gel’s recoverability by oscillating between 0.1% (low shear rate) and 100% (high shear rate) for 2 min each for a total of 18 min. Selected gels (6NC50-25% IHX, 6NC50-25% IHX OMNI and 6NC50-25% Ta) were also tested at 37°C to study the effect of body temperature on the viscosity and other rheological properties of the gel.

Yield strain was determined from the intersection point of tangents drawn from the linear viscoelastic (LVE) region of G’ and from the section with low G’ at high shear strain. Yield stress was then obtained from the stress corresponding to the yield strain following previously established protocol.¹

Transcatheter Injectability

The transcatheter injectability of the gels was measured using a mechanical tester (Univert, CellScale, Canada). The catheter was connected to a 1 mL syringe (BD Luer-Lok 1 mL syringe, BD, USA) filled with the gel using the Luer lock. The force required to pass the gel through a 150-cm 1.8-F microcatheter (SuperCross microcatheter, Teleflex, USA) at a flow rate of 1 mL/min was recorded. Each test was run in triplicate.

Imageability of Gels

The imageability of the gels under x-ray was studied using AMI HTX (Spectral Instruments Imaging, USA). Commercially used contrast agent, Omnipaque^™ (350 mgI mL⁻¹), was used as positive control. PBS and 6NC50-Controls were used as negative controls.

Hemolysis

The hemolysis rate of selected radiopaque gels was tested according to ISO 10993–4.¹⁷ Citrated fresh porcine blood (Lampire Biological Laboratories, Pipersville, PA, USA. Lot no. 24B52034) was diluted with 1X PBS in 4:5 ratio. Approximately 1 mL of the embolic gels was taken in a centrifuge tube containing 9 mL PBS. After pre-warming the PBS containing gels at 37°C for 30 min, 0.2 mL of diluted blood was added. The samples were incubated for an additional 1 h at 37°C followed by centrifugation at 3000 rpm for 5 min. The supernatant was carefully transferred into a 96-well plate, and the absorbance (A) was measured using a microplate reader (GENios, TECAN, Crailsheim, Germany) at wavelength 545 nm. 10 mL of PBS and deionized water incubated with 0.2 mL of diluted blood were used as negative and positive controls, respectively. The hemolysis percentage was calculated using the following formula (Eq. 2):

$$\text{Hemolysis}\left(\%\right)=\frac{A_{\text{sample}}-A_{\text{negative}\text{control}}}{A_{\text{positive}\text{control}}-A_{\text{negative}\text{control}}}\times 100$$ (2)

Four independent experiments with four replicates in each experiment were conducted.

Thrombogenicity

The interaction of the embolic gels and blood was studied through thrombogenicity assay. Clotting time was quantified using a previously developed protocol.^3,4 Briefly, 100 μL of 6NC50-Control, 6NC50-25% IHX, 6NC50-25% IHX OMNI and 6NC50-25% Ta gels was deposited at the bottom of 96-well plate, which was then centrifuged at 1000 rpm to standardize the blood interaction surface. Uncoagulated citrated whole porcine blood was activated by adding 10% (v/v) 0.1 M CaCl₂. A 100 μL of activated porcine blood was added to each sample and allowed to react for 2 min, 4 min, 6 min, 8 min and 10 min. At each time point, clotting was stopped by the addition of 100 μL of 0.109 M sodium citrate solution. Residual liquid was removed to isolate the blood clot. Porcine blood alone and clinically used coils (2D Helical-35, Boston Scientific, Ireland) were used as controls.

In Vitro Cell Viability

The in vitro cytotoxicity of gels was studied using L929 fibroblast cells (ATCC, CCL-1, Lot no. 70008726) according to ISO 10993–5.¹⁸ The L929 cells were grown in medium comprising Dulbecco’s modified Eagle Medium (DMEM, Gibco BRL, Grand Island, NY), 10% heat-inactivated fetal bovine serum (Cytiva, Marlborough, MA) and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37°C and 5% CO₂. The L929 cells were seeded in their culture media in 96-well plates at a density of 5000 cells per wall followed by incubation at 37°C and 5% CO₂ for 24 h. Then, the growth medium was aspirated out and replaced with extraction media of embolic gel at 37°C for another 24 h. Different concentrations of extraction media of embolic gels were prepared such as 100%, 50%, 25% and 12.5% in respective media. Cell viability was analyzed using WST-1 (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s protocol. 10% DMSO and non-treated cells served as positive and negative controls, respectively. Finally, the absorbance (A) was measured using a microplate reader at wavelength 450 nm. Three independent experiments with four replicates in each experiment were conducted.

Live Dead Cell Assay

Live dead cell assay was performed with L929 fibroblast cells to understand the viability of the cells when they come in contact with the gel extract. The L929 cells were grown in a medium comprising DMEM, 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin at 37°C and 5% CO₂ in a six-well plate for 24 h. Then, the medium was aspirated out and replaced with the extraction media of 6NC50-Control, 6NC50-25% IHX, 6NC50-25% IHX OMNI and 6NC50-25% Ta. 10% DMSO and non-treated cells served as positive and negative controls, respectively. Approximately two drops of the staining reagent [Invitrogen ReadyProbes^™ Cell Viability Imaging Kit (Blue/Green), Thermo Fischer Scientific, Eugene, OR, USA] per mL of the extract was added into the six-well plate followed by incubation at 37°C for 15 min. Finally, the cells are imaged using a microscope (Leica DMi8, Germany). Three independent experiments were conducted with six randomly selected images taken per each experiment. The live and dead cells were counted using ImageJ software (National Institutes of Health, Bethesda, MD).¹⁹

Statistical Analysis

Statistical differences between multiple groups were calculated using analysis of variance (ANOVA) with Tukey post test using GraphPad Prism 10 (GraphPad Software, CA, USA). P< 0.05 was considered significant. Data are reported as average ± standard deviation (SD) unless otherwise stated.

RESULTS AND DISCUSSIONS

Rheology of the Hydrogels

Based on our previous studies, we found that a 6NC50 gel exhibited excellent mechanical and biological properties that can serve as a suitable pristine system for further investigation on the effect of contrast agents. An optimum storage modulus and injection force were observed for yNC50 samples (y is the total solid content in the gel) because of their unique characteristic morphology, which aids adequate mechanical properties and comfortable injectability.¹ In addition, the 6NC50 samples have an ample amount of active sites for drug or contrast agent conjugations because of the high chitosan content (50% of TSC is chitosan). Here, we engineered 6NC50 gel with three different contrast agents including IHX, IHX OMNI and Ta with concentrations of 10%, 20% and 25% in each system. The radiopaque gels were subjected to various tests such as flow tests, amplitude sweeps, frequency sweeps and thixotropic tests to access their rheological behavior.

Flow Curve Test

The flow curve of all IHX, IHX OMNI and Ta gels at 25°C (Fig. 1a, b and c) showed drastic reduction in the viscosity with increase in shear rate, suggesting their shear thinning property. This unique property facilitates easy flow of the embolic gel through the catheter because of the liquid-like behavior upon the application of force during the trans-catheter injection and solidifies back occluding the vasculature upon the removal of force. Compared to the control (6NC50), a reduction in viscosity with increasing IHX content was observed, whereas the viscosity increased with increasing amount of IHX OMNI or Ta.

Fig. 1.

Rheology of 6NC50 with varying concentrations (0–25 wt.%) of IHX, IHX OMNI and Ta microparticles. (a)–(c) Representative flow curves indicating the shear thinning behavior of the gels; (d)–(f) representative amplitude sweep curves indicating gel like behavior of the engineered radiopaque materials; (g)–(i) summary of storage modulus at 0.1% shear strain; (j)–(l) summary of yield stress and yield strain. Data are presented as mean ± standard deviation (sd) (n = 3 for (g)–(l)).

Amplitude Sweep Test

Amplitude sweep tests were carried out to determine the storage (G’) and loss modulus (G”) of the gels, which correspond to their elastic and viscous components, respectively. The dominance of G’ over G” suggests that the material behaves as solid at that particular shear strain. Furthermore, high G” compared to G’ indicates the dominance of the viscous component resulting in the flow of the embolic gel. The amplitude sweep curves of all the gels (Fig. 1d, e and f) showed that the G’ was higher than G” at lower shear strains, suggesting solid-like behavior. However, G“ dominated G’ at higher shear strain, indicating the dominance of the viscous component.

A reduction in G’ was observed for IHX gels compared to the 6NC50-Control (Fig. 1g). Addition of 10% IHX (G’ of 6NC50 - 10% IHX is 709.7 ± 117.9 Pa) reduced the G’ to approximately half of the 6NC50-Control (G’ of 6NC50-Control is 1359.3 ± 21.5 Pa). At the same time, a further increase in the IHX content resulted in drastic reduction of the G’. In particular, the G’ of 6NC50-20% IHX (244.3 ± 58.8 Pa) and 6NC50-25% IHX (237.8 ± 52.3 Pa) were approximately one fifth of the 6NC50-Control gel. However, G’ increased with the addition of IHX OMNI and Ta microparticles (Fig. 1h and i). Approximately 1.8-fold increase in the G’ was observed for 6NC50-10% Ta (2485.8 ± 328.9 Pa) and 6NC50-20% Ta (2492.2 ± 228.6 Pa), whereas a 1.6-fold increase in G’ was observed for 6NC50-25% Ta (2197.8 ± 170.5 Pa). A comparable G’ with respect to 6NC50-Control was observed for 6NC50-10% IHX OMNI (1441.1 ± 61.6 Pa). However, a 2- and 2.7-fold increase in G’ was observed for 6NC50-20% IHX OMNI (2786.2 ± 206.3 Pa) and 6NC50-25% IHX OMNI (3673.9 ± 629.2 Pa). The yield stress of the gels was studied to evaluate the stress required to overcome to initiate steady material flow. Previous studies have shown that the mean shear stress experienced by the endothelial cells in the artery walls varies between 0.3 Pa in femoral artery to 1.3 Pa in carotid artery.^13,14 In addition, a higher shear stress up to 5 Pa is experienced in the arteriole.^13,14 The gels engineered with IHX OMNI and Ta exhibited a yield stress of 5 to 19 times the shear stress experienced by the artery walls (Fig. 1k and l). On the other hand, yield stresses of 6NC50-20% IHX and 6NC50-25% IHX are 6.2 ± 0.3 Pa and 4.7 ± 1.4 Pa, respectively (Fig. 1j). These results suggested that there might be a slight compromise in the deployment of these two gel systems in practical clinical applications since the values are very close to the shear stress experienced by the artery walls (5 Pa).^13,14

Frequency Sweep Test

Frequency sweep tests were carried out at 0.1% shear strain (LVE region) to predict the structural integrity and mechanical strength of the embolic gels with respect to frequency of oscillations. The results demonstrated the dominance of G’ over G”, suggesting elastic behavior of the gel at all tested angular frequencies (0.1–100 rad/s) (Fig. 2a, b and c). A fluctuation in modulus is observed for most of the samples at higher angular frequency which might be due to the slipping of the gels.

Fig. 2.

(a)–(c) Representative frequency sweep curves of 6NC50 with varying concentration (0–25 wt.%) of IHX, IHX OMNI and Ta microparticles respectively indicating the stability of the gels at 0.1% shear strain at varying frequency of oscillations; (d)–(f) representative thixotropic curves of 6NC50 with varying concentration (0–25 wt.%) of IHX, IHX OMNI and Ta microparticles respectively indicating their excellent recoverable properties.

Thixotropic Test

Thixotropic studies were performed to understand the recoverability and stability of the gels when subjected to multiple oscillatory shearing. The test mimicked intermittent injections in real case clinical setup, showing instantaneous recovery of the gel to its initial state once the shear was reduced (Fig 2d, e and f). A slight reduction in the storage modulus was observed after the first cycle for every gels. However, a 90% recovery was observed for all samples in all the cycles indicating stability and excellent recoverability of the gel after shearing.

Effect of Temperature on the Rheological Properties

Rheological studies at 37°C were performed for selected gels (6NC50-25% IHX, 6NC50-25% IHX OMNI and 6NC50-25% Ta), which exhibited maximum deviation from 6NC50-Control, to study the effect of body temperature on the gel’s rheological properties. The viscosity profile with varying shear rate (supplementary Fig. S-1 (a)–(c)) at 37°C is comparable to flow curve test performed at room temperature (25°C). Solidification of 6NC50-25% IHX was observed for the amplitude sweep test at 37°C, which is indicated by the dominance of the storage modulus over the loss modulus even at the higher shear rates (supplementary Fig. S-1 (d)). However, the amplitude sweep profile of 6NC50-25% IHX OMNI and 6NC50-25% Ta is similar to room temperature (supplementary Fig. S-1 (e)–(f)). Any significant change in the storage modulus of 6NC50-25% IHX (94.08 ± 19.42 Pa), 6NC50-25% IHX OMNI (2510.60 ± 257.68 Pa) and 6NC50-25% Ta (1820.17 ± 488.67 Pa) was not observed at 37°C (supplementary Fig. S-1 (g)–(i)). The gels demonstrated stability at different angular frequencies (supplementary Fig. S-2 (a)–(c)) and recoverability after cyclic oscillatory shearing (supplementary Fig. S-2 (d)–(f)) at 37°C comparable to the room temperature properties. Overall, an increase in temperature did not influence the flow, mechanical and recoverable properties of the embolic gels, indicating the stability of the gel.

Transcatheter Injectability of the Gels

The force required to inject the embolic gels through a 1.8-F, 150-cm-long microcatheter was measured to simulate the experience of physicians during clinical practice. For all the gels, the force built up and then plateaued to a constant value after few seconds. The average of the constant force is called injection force (IF). In a few cases (6NC50-Control and 6NC50-20% Ta), a peak force was achieved at first before the force plateaued. This peak force, namely the break loose force (BLF), is the force required to achieve the initial movement of the syringe plunger. A maximum injection force of 79.8 N is considered optimum for the physicians to comfortably inject the gel through the catheter.¹⁵

All the engineered gels, except 6NC50-25% Ta, exhibit BLF and IF below the acceptable force, suggesting their excellent transcatheter deliverability. The high concentration of Ta microparticles in 6NC50-25% Ta occluded the catheter, making the injection through an 1.8-F, 150-cm-long microcatheter impossible. The addition of IHX decreased the IF compared to 6NC50-Control (25.7 ± 1.5 N) (Fig. 3a). Contrarily, addition of IHX OMNI and Ta microparticles increased the IF (Fig. 3b and c). Particularly, a slight reduction in the IF, from 21.1 ± 0.1 N for 6NC50-10% IHX to 19.4 ± 0.3 N for 6NC50-25% IHX, was observed with the increase in IHX content (Fig. 3d). However, IF increased from 35.4 ± 1.2 N (6NC50-10% IHX OMNI) to 59.7 ± 0.5 N (6NC50-25% IHX OMNI) with increased IHX OMNI content (Fig. 3e). In addition, IF increased from 28.4 ± 0.8 N (6NC50-10% Ta) to 37.2 ± 3.8 N (6NC50-20% Ta) with the addition of Ta microparticles into the 6NC50 gel (Fig. 3f).

Fig. 3.

Transcatheter injection forces of 6NC50 with varying concentration (0–25 wt.%) of IHX, IHX OMNI or Ta microparticles through a 1.8-F, 150-cm microcatheter. (a)–(c) Representative injection force curves; (d)–(f) summary of injection forces. NA indicates no reported force for 6NC50-25% Ta. Data are presented as mean ± standard deviation (s.d.) (n = 3 for (d)–(f)).

Imageability of the Gels

The addition of contrast agents such as IHX, IHX OMNI or Ta microparticles in the CH-NC gel system aids the visualization the embolic agents on x-ray-based imaging modality, which is critical for precise and targeted delivery. Figure 4a, b and c compared the gray scale intensity of 6NC50 with varying concentrations (0–25 wt.%) of IHX, IHX OMNI or Ta microparticle, respectively. Clinically used contrast agent Omnipaque^™ 350 was included as the control. It is observed that an increase in the concentration of any contrast agent leads to an increase in the gray scale intensity compared to 6NC50-Control. The gels with 25% contrast agent exhibited the maximum intensity in their respective systems. The gray scale intensity of 6NC50-25% IHX and 6NC50-25% IHX OMNI (182.8 ± 2.5 and 196.1 ± 0.9, respectively) was less than that of 100% Omnipaque^™ 350 (233.4 ± 2.3), showing a statistical difference (****p< 0.0001). Contrarily, 6NC50-20% Ta and 6NC50-25% Ta (234.7 ± 1.4 and 232.1 ± 2.6, respectively) were comparable to the gray-scale intensity of clinically used contrast agent (p = 0.9994, and p = 0.999, respectively). This suggested that the contrast offered by 6NC50-20% Ta and 6NC50-25% Ta exhibited similar radiopacity compared to the commercially used contrast agent Omnipaque^™ 350.

Fig. 4.

X-ray intensity of 6NC50 with varying concentrations (0–25 wt.%) of (a) IHX, (b) IHX OMNI and (c) Ta microparticles, respectively, compared to 100% concentration of Omnipaque^™ 350. 6NC50 and PBS were used as controls. The corresponding x-ray image of each gel is provided on top. Data are presented as mean ± standard deviation (s.d.) (n = 3). Statistical significance was determined using one-way analysis of variance (ANOVA) with multiple comparisons against respective controls. ns – not significant, ****p< 0.0001.

Thrombogenicity

The thrombogenicity of 6NC50-Control, 6NC50-25% IHX, 6NC50-25% IHX OMNI and 6NC50-25% Ta was investigated using porcine blood by estimating the clotting time of the blood when it comes in contact with the gels. Figure 5a shows the control (pure blood) clots at 6 min. The blood in contact with the 6NC50-25% IHX OMNI and 6NC50-25% IHX fully coagulated at 8 min, comparable to the clinically used coils. A slightly faster coagulation was observed for 6NC50-Control and 6NC50-25% Ta, which completely coagulated at 6 min. These results suggested that the thrombogenicity of the engineered radiopaque gels were not compromised compared to the clinically used metallic coils.

Fig. 5.

(a) Images of thrombogenicity assay showing comparable clotting time when porcine blood was in contact with 6NC50-25% IHX, 6NC50-25% IHX OMNI and 6NC50-25% Ta compared to clinically used embolic coil; (b) hemolysis rate of 6NC50-25% IHX and 6NC50-25% IHX OMNI indicating hemocompatibility of the gel; (c) hemolysis rate of Ta incorporated gels (6NC50-10% Ta, 6NC50-20% Ta and 6NC50-25% Ta) indicating concentration-dependent hemocompatibility; (d) representative cell viability of L-929 fibroblasts suggesting biocompatibility the gels; (e) live-dead cell assay of L-929 fibroblast cells suggesting biocompatibility of the material (live, blue; dead, green). The scale bar represents 300 μm; (f) L929 cell viability summary obtained from live-dead cell assay. Data are presented as mean ± standard deviation (s.d.) (n = 4 for (b) and (c), n = 3 for (d), and n = 6 for (f)). Statistical significance was determined using one-way analysis of variance (ANOVA) with multiple comparisons against respective controls. ns, non-significant, *p< 0.05, ***p< 0.001, ****p< 0.0001.

Hemocompatibility

The hemocompatibility of the gels was evaluated since all embolic agents will be in direct contact with the blood. The gels 6NC50-Control, 6NC50-25% IHX and 6NC50-25% IHX OMNI showed a hemolysis rate of 2.34 ± 0.43%, 0.91 ± 1.26% and − 0.31 ± 0.40%, respectively (Fig. 5b). According to ISO standard 10993–4,¹⁷ a hemolysis rate< 5% is acceptable for biomaterials. Therefore, all tested gels demonstrated excellent hemocompatible properties. No statistical significance was observed between 6NC50-Control and 6NC50-25% IHX (p = 0.9062) and 6NC50-25% IHX OMNI (p = 0.2827).

Since an initial high hemolysis rate was observed for 6NC50-25% Ta, a concentration-dependent hemolysis study of Ta gels was carried out. The gels 6NC50-10% Ta, 6NC50-20% Ta and 6NC50-25% Ta showed a hemolysis rate of 0.82 ± 0.29%, 1.49 ± 0.30% and 6.32 ± 0.30%, respectively (Fig. 5c). A statistical significance was observed between 6NC50-Control and 6NC50-10% Ta (***p = 0.0001), 6NC50-20% Ta (*p = 0.0229) and 6NC50 - 25% Ta (****p< 0.0001). Also, statistical significance was observed within the group (6NC50-10% Ta and 6NC50-20% Ta (*p = 0.0395), 6NC50-10% Ta and 6NC50-25% Ta (****p< 0.0001) and 6NC50-20% Ta and 6NC50-25% Ta (****p< 0.0001)) indicating the hemolysis rate is dependent on the concentration of the Ta microparticles present in the gel system. The hemolysis rate of 6NC50-25% Ta (6.32 ± 0.30%) is beyond the acceptable limit of 5% according to ISO 10993–4 standard.¹⁷

In Vitro Biocompatibility

In vitro biocompatibility studies were carried out to evaluate the interaction between the engineered radiopaque gels and L929 fibroblast cells. L929 cell was chosen for the testing of cytotoxicity of biomedical devices according to ISO standards.¹⁸

Cell Viability

The biocompatibility of 6NC50-25% IHX, 6NC50-25% IHX OMNI and 6NC50-25% Ta engineered gels was investigated using L929 fibroblast cells following the ISO 10993–5 standard.¹⁸ The viability for 100%, 50%, 25% and 12.5% concentration of 6NC50-25% IHX extract was 115.1 ± 4.5, 107.8 ± 6.2, 110.2 ± 9.5 and 112.6 ± 11.4, respectively (Fig. 5d). Similarly, L929 viability for 100%, 50%, 25% and 12.5% concentration of 6NC50-25% IHX OMNI extract was 104.4 ± 3.5, 101.1 ± 3.8, 101.6 ± 3.1 and 99.4 ± 3.0, respectively. For 100%, 50%, 25% and 12.5% concentration of 6NC50-25% Ta extract, the viability was measured to be 87.5 ± 11.2, 90.3 ± 2.1, 97.0 ± 7.8 and 98.2 ± 9.3, respectively. There is no statistical significance observed between the groups of the gel and also within the 6NC50-25% IHX, 6NC50-25% IHX OMNI and 6NC50-25% Ta groups (p > 0.05). However, a slight significance is observed between 100% and 12.5% extract of 6NC50-Control (*p = 0.0148). The cell viability values indicate that all the engineered gels are biocompatible since the cell viability values are > 70%.¹⁸

Live-Dead Cell Assay

The biocompatibility of the engineered gels was visualized and counted using live-dead cell assay (Fig. 5e). L929 cell viability in the extraction medium of the engineered 6NC50-Control, 6NC50-25% IHX, 6NC50-25% IHX OMNI and 6NC50-25% Ta gels were 99.7 ± 0.14, 99.57 ± 0.06, 99.35 ± 0.25 and 99.85 ± 0.13, respectively (Fig. 5f). No statistical significance was observed between 6NC50-Control and radiopaque gels (p > 0.05). The high cell viability in the extracts further suggested that newly engineered radiopaque gels were biocompatible.

IMPLICATIONS AND LIMITATIONS OF THIS STUDY

Hydrogels exhibit minimal signal over background tissues using x-ray imaging modalities because of their high-water content and absence of high electron density elements.²⁰ To render intrinsic radiopacity, higher atomic number elements such as iodine, barium, bismuth or tantalum, also known as contrast agents, are incorporated into the embolic agents by either chemical modification or direct embedding.^4,20–22 These chemicals with higher densities and k-edges than the surrounding tissues provide enhanced image contrast, thus enabling real-time monitoring for the interventional procedure.^20,23

In this study, we evaluated the influence of three contrast agents on CH-NC embolic system including mechanical property, transcatheter injectability, radiopacity and biocompatibility. We investigated the influence of two iodine-based contrast agents, namely, IHX powder (pure) and IHX OMNI, whose main active component is IHX. The incorporation of pure IHX powder reduced the mechanical properties and IF, whereas IHX OMNI enhanced the mechanical properties (increased modulus and IF). The reduction of the G’ of the IHX incorporated embolic gels might be due to the interaction of hydroxyl groups (−OH groups) of IHX with the oxygen of NC forming hydrogen bond and thereby weakening the interaction between CH and NC. The bulky aromatic amine group in IHX⁷ can hinder the interaction between the CH and NC. In addition, IHX itself is naturally charged,²⁴ which can interfere with the electrostatic interactions between heterogeneously charged NC and positively charged CH, reducing the entanglement in the formed network (G’). Contrarily, IHX OMNI compound contains IHX along with many other additives as the stabilizers. We hypothesize that the presence of these organic additives in IHX OMNI results in the complicated interaction between the radiopaque contrast agent and gel system. In particular, multiple donor and acceptor sites in IHX OMNI components can form noncovalent crosslinks, such as hydrogen bonding and ionic interactions between CH fibers and NC disks, thereby strengthening the overall nanocomposite structure.⁷ Lastly, Ta microparticles also enhanced the mechanical properties and also increased the injection force of the gels compared to control gel. An increase in the G’ and injection force was observed with increasing Ta concentration. This might be because Ta microparticles served as reinforcing fillers that further enhance the nanocomposite structure.

Our results suggest that different contrast agents interact differently with the gel system, which aids to tailor the properties of the gel to specific applications. The 6NC50 gels with 20% and 25% IHX content (6NC50-20% IHX and 6NC50-25% IHX) demonstrated sufficient radiopacity for monitoring through an x-ray imaging modality. The reduction in rheological properties, mechanical properties and IF of 6NC50-20% IHX and 6NC50-25% IHX, compared to 6NC50-Control gel, can be utilized for occluding fine vasculatures where the gel needs to flow easily, thus making them a potential candidate to treat diseases such as AVM or tumor beds. On the other hand, 20% and 25% IHX OMNI gels (6NC50-20% IHX OMNI and 6NC50-25% IHX OMNI) and Ta gel (6NC50-20% Ta and 6NC50-25% Ta) have adequate radiopacity. However, 6NC50-25% Ta gel occluded the microcatheter, making injection difficult. Its hemolysis rate was also found to be higher than the acceptable upper bound (5%) according to the ISO 10993–4 standard.¹⁷ This makes the application of 6NC50-25% Ta gel impracticable and thus can be excluded. Notably, even though the active component in both IHX and IHX OMNI is IHX, the corresponding engineered radiopaque gels behave differently. The enhanced mechanical properties of the 6NC50-20% IHX OMNI, 6NC50-25% IHX OMNI and 6NC50-20% Ta can be used to embolize large cavities, such as aneurysms, where high mechanical strength is required for embolic agents to avoid distal migration. However, in vivo studies are required to investigate the performance of the gels for specific vascular applications.

CONCLUSION

In this work, we studied the effect of different radiopaque contrast agents such as IHX, IHX OMNI or Ta on a CH-NC embolic system. The radiopaque gels exhibited excellent imageability compared to the clinically used contrast agents. In addition, the addition of radiopaque contrast agents such as IHX OMNI and Ta powder into the CH-NC embolic system does not compromise its shear thinning property, mechanical strength and recoverable properties at both room temperature and body temperature and thus can be considered a potential embolic agent to occlude large vascular cavities such as aneurysm. A slight reduction in the rheological and mechanical properties was observed after the presence of IHX, which is beneficial for the occlusion of finer vasculatures. The injection force of engineered radiopaque gels (except 6NC50-25% Ta) was found to be within the comfortable range for physicians, indicating the excellent transcatheter injectability. Lastly, the radiopaque gels demonstrated biocompatibility, thrombogenicity and hemocompatibility. These findings highlighted their potential as embolic agents, which can be imaged under x-ray imaging modality, for transcatheter-based minimally invasive surgery.

Supplementary Material

The online version contains supplementary material available at https://doi.org/10.1007/s11837-024-06729-6.