Radiopaque thermosensitive Pickering emulsion vascular embolist for permanent arterial embolization

doi:10.21203/rs.3.rs-4394607/v1

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Radiopaque thermosensitive Pickering emulsion vascular embolist for permanent arterial embolization

https://doi.org/10.21203/rs.3.rs-4394607/v1

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The emergence of liquid embolic agents has advanced the utilization of transcatheter arterial embolization (TAE). Unlike the conventional solid embolic agent (gelatin sponge/PVA),which lacks developmental properties and necessitates lipiodol combination for efficacy, leading to a cumbersome two-step embolization process, there is a need for an embolic material capable of carrying imaging effects and displaying favorable cell compatibility to enhance clinical treatment outcomes. Wehave developed a novel liquid embolic material, poly (N-isopropylacrylamide)-co-acrylic acid nanogels (PNAs). By blending PNAs with lipiodol using a medical three-way tube, we obtain a thermosensitive lipiodol gel emulsion (TLGE). This material exhibits excellent temperature sensitivity and biocompatibility, with TLGE demonstrating fluidity and X-ray contrast in the decellularized liver model. This embolization technique necessitates only a single injection, allowing for quick mixing and usage, greatly benefiting clinicians. Digital subtraction angiography (DSA)facilitates intraoperative imaging, real-time embolization process monitoring, and prevention of inadvertent embolization and arterial leakage, ensuring precise embolization treatment for arterial vessels. Over a 42-day period of embolization of the right renal artery in New Zealand rabbits, there were no instances of vessel recanalization or damage to other target organs. Consequently, this innovative temperature-sensitive material holds substantial promise in TAE surgery.

Embolization

high-strength nanogels

long-term X-ray imaging

temperature sensitive

decellularization liver

Embolization refers to the intentional blockage of blood vessels through the intravascular placement of embolic agents via catheters for therapeutic purposes [1–3]. Currently, the clinical application of embolic agents primarily includes two dosage forms: solid and liquid. Solid embolic agents commonly include coils, PVA microspheres, and chitosan microspheres [4–7]. Liquid formulations play a significant role in therapeutic vessel embolization, with lipiodol, anhydrous ethanol, and butyl cyanoacrylate adhesive being widely utilized [8–10]. Liquid embolic agents can compensate for defects in which solid embolic agents cannot reach the distal blood vessel and have good vascular casting properties[11, 12]. Liquid embolic agents are frequently employed for transcatheter arterial chemoembolization (TACE), particularly in liver cancer treatment, where they can obstruct blood flow and induce tumor necrosis[13–17]. This approach is less invasive and more readily accepted by patients. However, commonly used liquid embolic agents like lipiodol may lack robustness, leading to potential vascular revascularization and reduced embolization effectiveness [18–20]. While current liquid embolic agents suffice for many clinical applications, they possess several limitations. Hence, there is a critical need to develop novel liquid embolization materials to address shortcomings in development efficacy, biocompatibility, and vascular casting.

Therefore, we examined 2,2 '-azobis(2-methylpropionamidine) dihydrochloride as an initiator, valued for its water solubility and consistent, controllable effects. Utilizing this initiator alongside the ionic emulsifier, we synthesized poly (N-isopropylacrylamide)-co-acrylic acid nanogels (PNAs) through polymerization. In a prior investigation [21], the resulting emulsion displayed a temperature-sensitive sol–gel transition, demonstrating notable thermodynamic stability. It exhibited high yield stress in the gel phase while maintaining favorable flowability and shear-thinning properties in the sol phase. Temperature regulation enabled gelation at 33°C, enhancing clinical usability.

Lipiodol, an embolic material compatible with computed tomography (CT) development, falls short in achieving optimal embolization effects independently and may lead to vascular recanalization[22]. To address this, lipiodol is often combined with a solid embolic agent like gelatin sponge or PVA[23]. However, solid embolic agents have limitations in development and can cause tube blockages. Additionally, the need for dual embolization with lipiodol and solid embolic agents increases the risk of ectopic embolization[24]. Therefore, by using the temperature sensitivity of PNAs and the development properties of lipiodol, we combined the two, offering advantages in development efficacy, biocompatibility, and simplicity of operation. PNAs and lipiodol were separately extracted with syringes, connected via a medical three-way tube, and alternately injected and mixed to produce thermosensitive lipiodol gel emulsion (TLGE), significantly easing clinical application. This method eliminates the need for mixing instruments and allows for quick preparation without lengthy waiting periods.

A decellularized liver model was generated to study the performance and embolization effect of TLGE emulsions. We designed a simple model of decellularized liver tissue for basic laboratory research. Decellularization involves employing a combination of physical and chemical treatments to effectively eliminate cellular contents from tissue or organs while preserving extracellular matrix (ECM) components and functional proteins [25–29]. The resulting decellularized liver yields a translucent bioscaffold comprising an acellular natural matrix, maintaining both vascular system integrity and mechanical strength [30–33]. Injecting TLGE into the decellularized liver enabled observation of X-ray imaging and vascular fluidity, confirming TLGE emulsion's favorable biocompatibility and ease of use. We applied this material to embolize a normal rabbit kidney model, achieving complete embolization of the main rabbit renal artery for up to 42 days without vascular recanalization at the embolization site. This underscores the advantages of this thermosensitive nanogel embolization material with X-ray development in intraoperative imaging and long-term embolization post-surgery, offering significant potential for future clinical TAE treatment (Scheme 1).

2.1. Materials and animals

N-Isopropylacrylamide (NIPAM) and acrylic acid (AA) were procured from Tokyo, Japan. Poppy iodine oil was obtained from Jiangsu Hengrui Pharmaceutical Co., Ltd. Medical three-way tubes were sourced from Yangzhou Yangsheng Pharmaceutical Technology Co., Ltd. N, N'-methylene bisacrylamide (MBA), octadecyl trimethyl ammonium chloride (STAC), 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH), and rhodamine-B were purchased from Aladdin Chemical Reagent Co., Ltd. 4% paraformaldehyde and pentobarbital sodium were acquired from McLean Reagent Co., Ltd. Lidocaine hydrochloride and gentamicin sulfate were obtained from Huabei Pharmaceutical Co., Ltd. Heparin sodium injection was sourced from Changzhou Qianhong Biochemical Pharmaceutical Co., Ltd. Sodium chloride injection was purchased from Wuhan Binhu Shuanghe Pharmaceutical Co., Ltd. Sodium lauryl sulfate (SDS) and ethylene glycol-bis-(2-aminoethyl) tetraacetic acid (EGTA) were procured from Aladdin Biochemical Technology Co., Ltd. Milli-Q ultrapure water (18.2 MΩ) was utilized for experimental verification. DNA reagent extraction kits were obtained from QIAGEN.

Adult New Zealand white rabbits (males, weighing 2.5–3.0 kg) and Sprague Dawley rats (males, weighing 300–400 g) were obtained from the Laboratory Animal Center of Hubei University of Science and Technology. All animal experiments adhered to the Guide for the Care and Use of Laboratory Animals issued by the Hubei Provincial Department of Science and Technology and were approved by the Animal Ethics Committee of Hubei University of Science and Technology (approval number: 2024-02-002).

2.2. Synthesis of PNAs

First, the formula was designed by emulsion polymerization, and the optimal formula ratio for the preparation of the hydrogel was determined. Briefly, MBA (0.03 g, 0.19 mmol), STAC (0.05 g, 0.29 mmol), NIPAM (1.7 g, 15 mmol), and AAPH (0.08 mg, 0.14 mmol) were dissolved in 150 mL of ultrapure water, added to a three-necked flask, and stirred in an oil bath at 80°C and 250 rpm until the solution turned blue, followed by the addition of AA (215 L, 3 mmol). After the solution changed from light blue to milky white, the reaction was continued for 4 h. The reaction-completed gel solution was dialyzed against mobile water using a Molecular Weight Cut-Off Point (14,000 Da) for 3 days. On completion of dialysis, the samples were freeze-dried in a -80°C refrigerator for 4 h and then freeze-dried for 2–3 days until powdered ready for use.

2.3. Preparation of TLGE

A mixture of 6 wt.% and 8 wt.% PNAs and lipiodol was prepared for blending. Lipiodol volumes of 0.1 mL, 0.3 mL, 0.5 mL, 0.7 mL, and 0.9 mL were chosen, while corresponding volumes of 6 wt.% and 8 wt.% PNAs were added to yield a total volume of 1 mL. This ensured lipiodol concentration ratios of 10%, 30%, 50%, 70%, and 90%. The combined solutions were transferred into a 1 mL syringe connected to a medical three-way tube and alternately pushed and mixed for 100 cycles to create TLGE, which was then transferred to a 1 mL glass bottle for storage. Due to excessive lipiodol content, the 70% and 90% lipiodol blends did not gel and were discarded. Therefore, experiments comparing 10%, 30%, and 50% lipiodol mixed with 6 wt.% and 8 wt.% PNAs by weight were conducted.

2.4. Characterization of the PNAs

The PNAs were diluted to 1 wt.% by content, heated at 1°C/min, and the change in transmittance at 500 nm was measured using an ultraviolet spectrophotometer (Beijing General Analytical Instruments, China) from 25°C to 40°C. The morphology of the PNA nanogels was observed by transmission electron microscopy at 200 kV (Tecnai G2 20 FEI Corp, Netherlands). The nanogel aqueous dispersion was diluted with ultrapure water to 1 mg/mL. After 10 min of ultrasonication, 10 µL of the suspension was dropped on a TEM copper net coated with carbon film (300 mesh) at 25°C, dried, and then stained with phosphotungstic acid solution (0.1 wt.%) for observation.

The nanogel was diluted to 0.2 wt.% in ultrapure water, and 2 mL was removed and placed into a particle size sample pool for testing. The temperatures were set at 25°C and 37°C. A helium-neon laser (λ = 633 nm) was used as the light source, and the laser was balanced at a 90° scattering angle for 200 s before being circulated four times. Particle size analysis was conducted using a dynamic light scattering particle size analyzer, which produced a particle size distribution curve. Following this, 2 mL of gel dispersion liquid was transferred into the sample cell, with careful removal of bubbles, and then subjected to Zeta potential measurement using a dynamic scattering particle size analyzer.

2.5 Characterization of the TLGE emulsion

Lipiodol concentrations of 10%, 30%, and 50% were combined with PNAs at 6 wt.% and 8 wt.% content. Subsequently, 1 mL of the mixture was placed into a clear glass bottle. The bottle was then kept at room temperature (25°C) and photographed continuously for 1 h, 2 h, 3 h, 1 day, 3 days, and 10 days to evaluate its in vitro stability, oil phase separation, and emulsion migration. This procedure aimed to determine the optimal formulation ratio.

Following the mixing of 10%, 30%, and 50% lipiodol with 6 wt.% PNAs, the resulting emulsions were transferred into 2 mL glass bottles. Subsequently, the emulsions were photographed using a fluorescence electron microscope at room temperature (25°C) to examine their morphology.

The PNAs nanogel was stained with rhodamine-B and then mixed with 50% lipiodol for an in vitro injection experiment. Morphological changes were observed at both 25°C and 37°C. Various types of catheters were utilized for injection to investigate the impact of TLGE on vascular casting.

TLGE was subjected to CT scanning to observe its developability and corresponding CT value. The rheological properties of TLGE and PNAs nanogels were analyzed using a high-speed rotating rheometer (Anton Paar MCR92, Austria) under the following test conditions: parallel plates (PP50, Φ = 40 mm) with a gap of 0.5 mm, stress of 0.5 Pa, heating rate of 2.0°C/min, and frequency of 1.0 Hz. The G' and G" moduli of the PNAs and TLGE were determined over the temperature range of 25°C to 40°C. The viscosities of the PNA and TLGE nanogels were measured at 25°C, and the shear rates ranged from 150 s^–1 to 2580 s^–1.

2.6 Evaluation of the ex vivo live model

Twenty male SD rats, aged 8–10 weeks and weighing 300–400 g, were selected with SPF grade. They were housed under standard conditions with a 12-h light-dark cycle, having unrestricted access to water, and kept at a temperature of 22–25°C with a relative humidity of 50–60%. Our investigation into the liquid fluidity and imaging of TLGE utilized an ex vivo model of an acellular liver. The livers of male Sprague Dawley rats were euthanized by carbon dioxide asphyxia, and the whole liver was removed after being rinsed with 20 mL of PBS through the portal vein and then quickly placed at -80°C for 24 h. After 24 h, the liver was removed and slowly thawed at room temperature. Following this, a syringe pump (Mindray BeneFusion VP3 infusion pump) was employed to administer normal saline at a rate of 120 drops/min, along with 0.25% EGTA for 3 h, and 1% SDS for 6 h into the portal vein. Both the normal liver and the decellularized liver were subsequently fixed in 4% paraformaldehyde for 24 h, dehydrated using a tissue gradient ethanol, cleared with chloroform, and processed into liver specimens using the standard paraffin embedding technique. After the cells were removed, the sections were cut at a thickness of 4 µm. Hematoxylin and eosin (H&E) staining was used for histological analysis. For comparison with the decellularized liver, the nuclei and surrounding tissues were observed. Normal liver and acellular liver specimens were selected for immunofluorescence evaluation using rabbit polyclonal anti-type I collagen antibodies (Abcam, ab254349), rabbit polyclonal anti-laminin antibodies (Abcam, ab11575), and rabbit polyclonal anti-fibronectin antibodies (Servicebio, GB13091). Additionally, the liver morphology was examined using a scanning electron microscope (SU8100). Normal and decellularized liver tissue will be fixed with 2.5% glutaraldehyde for 24 h, dehydrated in an alcohol solution for 15–20 min, frozen at − 80°C for 2 h, and freeze-dried for 24 h. Normal and decellularized liver tissue will be sprayed with a platinum layer sputtering at 10 nm, after which scanning electron microscopy (SEM) images will be acquired at an accelerating voltage of 3 kV. A digital medical X-ray radiography system (DP528-B) was used at 55 kV, a current of 320.0 mA, a time of 12.00 ms, an EI of 57338, and 161.86 µGy. The acellular liver underwent imaging using a digital medical X-ray system. Lipiodol was then injected into the liver through the portal vein, and immediate imaging was conducted using DR. The decellularized liver was observed using an SZ680 stereoscopic microscope, with adjustments made to position and focal length. Intravenous injection of TLGE through the portal vein was performed at a rate of 0.5 mL/s, with vessel access observed at 1 s, 2 s, 3 s, 4 s, and 5 s intervals. DNA content analysis was carried out on six rats, comparing acellular and normal livers. After 10 h of irrigation, the decellularized liver underwent freeze-drying using a vacuum freeze-drying machine, alongside the normal liver. DNA extraction from both types of liver followed the kit's instructions, and the DNA concentration and purity were assessed using Nanodrop 2000. Agarose (0.75 g) was weighed into a conical bottle, and 50 mL of buffer solution was added. The samples were then microwaved and boiled until the agarose melted. The mixture was shaken well to form a 1.5% agarose gel. An equal volume of loading buffer was added to the sample (3–5 µL), and then the sample was added to the small tank of the rubber plate with a 10 µL micropipette. After the sample was added, the gel plate was immediately subjected to electrophoresis at 170 V, and the sample was moved from the negative electrode (black) to the positive electrode (red). After electrophoresis, the gel was removed, and the gel was observed under an ultraviolet lamp. The fluorescence bands were displayed in the presence of DNA, and a gel imaging system was used to take and save the images.

2.7 Application value of TLGE in renal artery embolization in normal rabbits

Embolization experiments were carried out in New Zealand rabbits involving their normal right kidneys. Twelve adult rabbits (males, weighing 3.0–3.5 kg) were specifically chosen for the study. Feeding conditions entailed maintaining temperatures between 25–27°C, humidity levels at 50–60%, ventilation frequencies ranging from 10–15 times/h, an air flow rate of 0.1 m/s, colony counts of ≤ 30/dish, noise levels below 50 dB, and 12-h illumination periods during both day and night. Feeding sessions were conducted three times daily.

Normal New Zealand rabbits were selected and euthanized immediately after 42 days of renal embolization with TLGE. Following a two-week adaptation period and a 12-h fast, pentobarbital sodium (30 mg/kg) was administered intravenously through the rabbit ears. Anesthesia took effect 15 min later, immobilizing them in a supine position. The inguinal fur was shaved and disinfected, and the femoral artery was separated using eye forceps after local anesthesia with lidocaine. Inserting an 18 g needle into the proximal artery along with a 4f coaxial microcatheter (Terumo, Tokyo, Japan), renal arteries underwent arterial angiography with iodixanol (300 mgI/mL, 0.5 mL/s) to confirm vessel patency. TLGE was injected into the rabbit renal artery, and real-time observation of emboli entering the renal artery at 1, 3, 5, and 7 s post-injection was performed using Digital Subtraction Angiography (DSA, Siemens BicorTop, Germany). Follow-up assessments were conducted seven days, 21 days, and 42 days post-embolization using Somatom Sensing 64 Slice (Siemens, Germany) with ioxatriol (300 mgI/mL^–1) as the contrast agent. Scanning parameters were set at tube voltage: 80 kV and tube current: 60 mA. Renal artery embolization and vascular recanalization were monitored. After 42 days of daily treatment with intramuscular injection of 1–20 000 IU penicillin sodium, administered three days after surgery, rabbits were euthanized. Liver, spleen, lung, and kidney tissues were processed with H&E staining and immersed in 4% paraformaldehyde for 24 h. The tissues were then embedded in paraffin and sectioned. The sections were hydrated with graded ethanol and stained with H&E to analyze pathological changes after embolization. The left and right kidneys were removed and sectioned along the longitudinal axis for analysis of differences.

2.8 Evaluation of Biocompatibility

Blood (2 mL) from New Zealand rabbits was combined with normal saline (1,500 rpm × 10 min), and centrifuged (1,500 rpm × 10 min) until the supernatant cleared, yielding a 2% suspension of red blood cells. The experimental group consisted of TLGE emulsion containing 6 wt.% PNAs mixed with 50% lipiodol, diluted to concentrations of 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, and 0.625 mg/mL. Normal saline served as the negative control, while ultra-pure water acted as the positive control for treatment.

Five blood compatibility experiments were conducted in each group. In each group, 300 µL of solution was added to a 1.5 mL EP tube, along with 300 µL of red blood cell suspension. After incubation at a constant temperature for 1 h, the samples were centrifuged at 3000 rpm for 25 min, and 100 µL of supernatant was transferred into a 96-well plate. The hemolysis rate (Hr%) was calculated using a microplate reader (λ = 540 nm, 1420 MultiLabel Counter, Perkin Elmer).

$$Hr\%=\frac{{OD}_{s}-{OD}_{n}}{{OD}_{n}-{OD}_{b}}\times 100\%$$

OD_s represent the absorbance of the experimental group, OD_n represents the absorbance of the negative control group, and OD_b represents the absorbance of the blank group.

The MTT method was employed to assess the cytotoxicity of HepG2 cells. Once the HepG2 cells reached the logarithmic growth phase, they were harvested following trypsin digestion. The cells were diluted to 1 × 10⁵ cells/mL in DMEM and incubated for 24 h in 96-well plates filled with 4–5 × 10³ cells/well. The TLGE emulsions of 6 wt.% PNAs mixed with 50% lipiodol and PNAs at different concentrations were diluted to 0.4 mg/mL, 0.2 mg/mL, 0.1 mg/mL, 0.05 mg/mL, and 0.025 mg/mL in DMEM. After that, the old medium was discarded. One hundred microliter samples of different concentrations were added to a 96-well plate and incubated with HepG2 cells for 24 h. DMEM containing cells was used as the negative control, and DMEM without cells was used as the blank control. Then, 50 mg of MTT powder and 10 mL of PBS were added, and the final concentration was 5 mg/mL. Then, 20 µL of 5 mg/mL MTT solution was added to each well, the plate was cultured in the dark for 4 h, the supernatant was discarded, 150 µL of DMSO solution was added to each well, the plate was shaken with a shaker at a constant temperature for 10 min, and the absorbance was measured with an automatic enzyme-labeled reader at 570 nm. The cell viability was calculated according to the following formula:

$$Cv\%= \frac{{OD}_{s}-{OD}_{b}}{{OD}_{n}-{OD}_{b}}\times 100\%$$

OD_s, OD_b, and OD_n represent the absorbance values of the sample group, blank group, and negative control group, respectively.

3.1 Structure and morphology characterization of the PNA nanogels

The infrared absorption functional groups of the nanogel were examined using an infrared spectrometer. Key findings included the N-H vibration peak at 3300 cm^–1, C-H stretching vibration peaks for methyl and methylene at 2972 cm^–1 and 2934 cm^–1 respectively, and N-H bending vibration peaks of amide I and amide II at 1655 cm^–1 and 1550 cm^–1 respectively. The presence of the carboxyl group of acrylic acid was identified at 1705 cm^–1, indicating successful integration into the hydrogel system (Fig. S1). Particle size and Zeta potential were determined using 0.2 wt.% PNAs in a dynamic light scattering particle size analyzer. Results showed a Zeta potential of -4.2 mV for PNAs gel at 25°C and − 9.0 mV at 37°C (Fig. 1a). Zeta potential serves as an indicator of mutual repulsion or attraction between particles. Higher absolute values of Zeta potential, whether positive or negative, indicate smaller molecules or dispersed particles and contribute to system stability by resisting aggregation during dissolution or dispersion. The presence of carboxyl groups (-COOH) in acrylic acid increases the negative charge density in the nanogel system, resulting in the observed negative Zeta potential of PNAs nanogel. As the temperature increases, the negative value of the zeta potential increases. The results Surface: At 25°C, the average particle size of the PNAs was 102 nm, and the PDI was 0.279 (Fig. 1b). At 37°C, the particle size was 50.7 nm, and the PDI was 0.332, indicating strong temperature sensitivity. When the temperature increased from 25°C to 37°C, the size of the nanogel decreased by half. The main reason was that with increasing temperature, the hydrophobicity of the nanogel increased, and increasing the temperature decreased the size of the gel. As a result, the size of the nanogel decreases, and the overall particle size decreases with good dispersion at 25°C and 37°C. The changes in the light transmittance of the PNA gels at 25°C and 40°C were measured using a UV‒Vis spectrophotometer. With increasing temperature, the transmittance of the PNAs decreased continuously. The temperature of the PNA nanogels was approximately 28°C when the light transmittance was reduced to 50%. The transmittance is stable at approximately 0.4% from 37°C to 40°C (Fig. 1c). TEM images showed that the micromorphology of the PNA nanogel was spherical, and the distribution was uniform and flat (Fig. 1d).

3.2 Structure and morphology characterization of the TLGE

In the study, 2 wt.% and 4 wt.% PNAs were discarded due to poor gelling of the 2 wt.% and 4 wt.% PNAs and incomplete coagulation at 37°C. We also tested 6 wt.% and 8 wt.% PNAs, which can achieve gelation at 33°C (Fig. S2). 6 wt.% PNAs and 8 wt.% PNAs were chosen and combined with varying amounts of lipiodol (0.1 mL, 0.3 mL, 0.5 mL, 0.7 mL, and 0.9 mL) to achieve a total volume of 1 mL, resulting in lipiodol concentrations of 10%, 30%, 50%, 70%, and 90%, respectively (Table S1). When 0.7 mL and 0.9 mL of lipiodol were mixed with 0.3 mL and 0.1 mL of PNAs, it was observed that they failed to gel at 37°C. Consequently, these two mixing configurations were discarded (Fig. S3 and S4). Mixtures of 6 wt.% PNA and 8 wt.% PNA with 10%, 30% and 50% lipiodol were mixed, and the stabilities were compared at 1 h, 2 h, 3 h, 1 d, 3 d and 10 d at room temperature (25°C) (Fig. S5). The mixtures of 6 wt.% PNAs and 8 wt.% PNAs with 10% and 30% lipiodol exhibited slight delamination within 1 h and noticeable delamination after 3 h, indicating instability of the emulsion in these mixing configurations. However, slight stratification was observed by the tenth day after adding 50% lipiodol, suggesting a relatively stable trend (Fig. 2a). Due to the relatively high viscosity of the 8 wt.% emulsion, which tended to adhere to the catheter and had poor liquidity, it was deemed unsuitable for catheter delivery. Consequently, the 8 wt.% synthetic TLGE was abandoned, and the 6 wt.% TLGE mixed with 50% lipiodol was utilized for the rabbit kidney model embolization experiment. The development properties of the materials were assessed using digital radiography (DR) and CT (Fig. S6). The CT values of the 6 wt.% PNAs mixed with 10%, 30%, and 50% lipiodol were 2328.6 ± 31.26 Hu, 3063.31 ± 11.42 Hu, and 3067.73 ± 7.41 Hu, respectively (Fig. 2b). Similarly, the CT values of 8 wt.% PNAs mixed with 10%, 30%, and 50% lipiodol were 2020.67 ± 21.56 Hu, 3068.65 ± 1.38 Hu, and 3068.96 ± 1.08 Hu, respectively. These CT values confirmed the suitability of the material for the experiment (Fig. S7).

Images of the mixture of 6 wt.% PNAs with 10%, 30%, and 50% lipiodol were compared using a fluorescence microscope. The results revealed varying degrees of increase in particle sizes for the PNAs and lipiodol mixtures (Fig. 2c). Specifically, the particle sizes of the mixtures of 6 wt.% PNAs and 50% lipiodol exhibited uniform distribution and good morphology, indicating greater stability compared to the other two mixtures. The particle sizes of 6 wt.% PNAs mixed with 10%, 30%, and 50% lipiodol were quantified, yielding values of 112.8 ± 9.7 µm, 122.4 ± 27.4 µm, and 123.2 ± 7.8 µm, respectively (Fig. 2d).

The sol-gel phase transitions sensitive to temperature of PNAs and TLGE were examined using a high-speed rotating rheometer. Upon reaching approximately 33°C, both TLGE and PNAs nanogels experienced an increase in G' and G", ultimately reaching a point where G' equals G" (Fig. 3a and 3b). This suggests that the critical gelation temperature of TPGEL and nanogel aqueous dispersions is approximately 33°C, indicating a temperature-dependent sol-gel phase transition. Beyond 33°C, G" continued to rise, surpassing G' significantly, indicating the transition of TLGE and PNAs into solid gels, exhibiting solid characteristics. To examine the flow properties of PNAs nanogels and TLGE, changes in viscosity were analyzed with increasing shear rate. As the shear rate increased from 105 s^–1 to 2580 s^–1, the viscosity of PNAs nanogel decreased from 14 MPa·s to 9.8 MPa·s (Fig. 3c), while that of TLGE decreased from 240 MPa·s to 28 MPa·s (Fig. 3d). Notably, TLGE exhibited nearly an 8-fold decrease in viscosity, indicating significant shear thinning of the non-Newtonian fluid. Coagulation effects at 25°C and 37°C were compared using rhodamine-B-stained TLGE, which was introduced into a 1 mL syringe, connected to the catheter, and submerged in water at 25°C and 37°C to evaluate its performance. The results revealed homogeneous dispersion of TLGE in water at 25°C without gelation. However, at 37°C, noticeable gelation occurred in the catheter, and various catheter sizes (2 mm, 0.55 mm, and 0.2 mm) were connected to analyze their vascular moldability. It was observed that all catheters could induce gelation, demonstrating TLGE's ability to mold for various types of blood vessels to optimize its performance (Fig. 3e).

3.3 Ex vitro liver evaluation results

There is no standardized protocol for eluting decellularized liver. We aimed to simplify the process to evaluate embolization material performance [34–36]. Following a 10-h perfusion, H&E staining revealed that the acellular liver had removed most nuclei and cytoplasm, while immunofluorescence confirmed the retention of type I collagen, fibronectin, and laminin (Fig. 4a, 4b, and S8). Laminin, a non-collagenous sugar found in the intercellular substance, is primarily synthesized by liver endothelial and lipid storage cells, constituting the basement membrane alongside collagen. Collagen, a structural protein of the extracellular matrix (ECM), is a polysaccharide protein containing small amounts of galactose and glucose, serving as the primary ECM component. Fibronectin, another non-collagenous sugar, commonly exists in the ECM and basement membrane, contributing to cell repair. SEM images revealed that the acellular liver maintained intact vascular morphology, with the vascular wall periphery appearing loose and porous due to hepatocyte removal (Fig. 4c). We investigated TLGE mobility within the liver. Rhodamine-B staining followed by injection into the blood vessel demonstrated TLGE's ability to reach thinner blood vessel ends, indicating good mobility and vascular casting (Fig. 4d and S9). The decellularized liver underwent evaluation under a digital medical X-ray imaging system, revealing TLGE's favorable imaging properties (Fig. 4e). This process mimics TACE embolization. Before clinical application, the ex vivo liver model can be used for testing. By simulating human body temperature through changing physical conditions, it verifies the fluidity of the embolization agent and its ability to develop under X-ray guidance. To verify the extent of decellularization, we performed the following tests, which revealed that the brightness of the DNA bands in the agarose gel-digested decellularized liver was lower than that in the normal liver (Fig. 4f). DNA tests revealed that more than 90% of the DNA in decellularized liver tissue was removed compared to that in normal liver tissue, which is lower than the 50 ng/mg required by international standards (Fig. 4g) [37].

3.4 Evaluation of the embolization effect of TLGE on the blood vessels of normal rabbit renal arteries at all levels

Renal artery embolization is commonly utilized to assess the effectiveness of embolic materials[38–41]. New Zealand white rabbits underwent a 14-day adaptive feeding regimen and were assessed via CT and HE within 42 days post-surgery. CT scans demonstrated sustained excellent visualization of TLGE at the 42-day mark. HE staining revealed significant necrosis and atrophy in the embolized kidney (Fig. 5a). To evaluate TLGE's embolization efficacy, we embolized the right kidney of a rabbit, chosen for its plentiful renal vessels. The nanogel emulsion had good liquidity and strong viscosity, and it could embolize the rabbit right renal aorta and bulbar arteriole. Compared with the use of a solid embolism agent, it has the advantage of a wide embolism area. Following the injection of 2 mL of TLGE into the rabbit renal artery, the TLGE was distributed to all vascular branches of the right renal artery via a 4F microcatheter. A comparison with DSA at 1 s, 3 s, 5 s, and 7 s indicated that TLGE exhibited good fluidity and low viscosity on the surface, facilitating its reach to the distal cortex of the kidney. Consequently, TLGE demonstrates an optimal embolization effect for vascular casts at all levels of the renal aorta, interlobular artery, and lesser bulbar artery, showcasing the significant potential for TAE treatment (Fig. 5b).

TLGE caused complete occlusion of the rabbit renal vessels within 4 min, in sharp contrast to the left kidney. The experimental rabbits were subjected to femoral artery incision suturing, disinfection of the wound with iodophor, daily injection of gentamicin sulfate and penicillin sodium for three days, and continued feeding (Fig. 5c). Follow-up monitoring of CT images was performed at 7 days, 21 days and 42 days. According to CT scans, TLGE gel displayed no vascular recanalization on days 7 and 21, while the right kidney appeared significantly smaller than the left on day 42, suggesting necrosis and calcification in the right kidney (Fig. 5d). Cross-sectional images indicated TLGE visibility on days 7, 21, and 42, with the emulsion persisting without recanalization despite blood flow over time (Fig. 5e). The embolized emulsion remained distributed within the vascular lumen, with no signs of demulsification observed. In summary, TLGE exhibits effective imaging capabilities and embolic efficacy, achieving complete arterial occlusion for up to 42 days in New Zealand rabbits, yielding promising outcomes.

Gross images showed that the right embolized kidney was smaller in size and markedly more atrophic than the normal left kidney. Pathology of the right kidney revealed karyocyte collapse, glomerular atrophy, unclear renal parenchyma, fibrosis and calcification. In addition, the glomerular and renal tubular tissues were necrotic and indistinct (Fig. 5f).

3.5. Biocompatibility evaluation

Forty-two days after embolization, the New Zealand White rabbits were euthanized, and gross images and H&E images of heart, liver, spleen, lung, and kidney tissue sections were taken for analysis. Gross examination of all organs showed no notable findings except for necrosis detected in the right kidney due to embolization. The heart, liver, spleen, lungs, and H&E staining of tissue sections exhibited normal appearances, with no signs of pathological changes. No evident abnormalities were noted in the renal vessels, renal tubules, and glomerular tissue of the left kidney (Fig. 6a and 6b). TLGE's biocompatibility was assessed through evaluations of hemolysis rate and cytotoxicity in rabbits. The hemolysis rate was < 5% in the TLGE and PNA groups at concentrations of 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, and 0.625 mg/mL (Fig. 6c). The hemolysis rates in the TLGE group were 0.2 ± 0.95%, 1.1 ± 1.34%, 0.4 ± 0.32% and 0.7 ± 1.01%, respectively. To determine cytotoxicity, TLGE and PNA nanogels at concentrations ranging from 0.4–0.025 mg/mL were tested by using the MTT method (Fig. 6d). The cell viability of all experimental groups exceeded 80%, indicating low cytotoxicity for both TLGE and PNAs. These findings validate the favorable biocompatibility of TLGE.

In this study, we utilized the polymerization method to create PNAs nanogel by polymerizing NIPAM and AA, resulting in a double-layer core-shell structure. PNAs possess characteristics such as small particle size, good dispersibility, and effective mobility, allowing for embolization of small peripheral vessels and gelation at around 33°C. Subsequently, TLGE was synthesized by a straightforward process, exhibiting ease of operation and good fluidity when mixed with lipiodol via a medical tee tube. Despite its common use in surgery, lipiodol tends to revert easily, often requiring additional procedures when combined with granule embolic agents; thus, increasing the risk of ectopic embolization. Moreover, the granule embolic agent does not develop and easily blocks the tube. Moreover, lipiodol has its own ability to prevent vascular recanalization during embolization. Solid embolic agents typically consist of large particles that struggle to enter small vessels. Therefore, the combination of PNAs and lipiodol addresses the shortcomings of lipiodol in vascular recanalization and the inability of PNAs to develop. In decellularized liver, TLGE exhibited excellent fluidity, enabling it to access terminal vascular branches inaccessible to solid embolic agents, while also providing good X-ray visualization. Decellularized liver tissue is highly valuable for future studies of liquid embolic agents. This study demonstrated that TLGE has good efficacy and can reach thinner vascular branches to better block the blood vessels supplying the tumor. In vivo studies demonstrated that TLGE exhibited excellent mobility and embolic effects in rabbit kidney models. The embolic material reached the cortical layer, and after 42 days of embolization, the rabbit kidneys showed signs of atrophy, necrosis, and calcification, with TLGE covering the surface to achieve complete vessel occlusion without evidence of recanalization. Biocompatibility experiments further confirmed TLGE's low toxicity, making it suitable for clinical treatment. Therefore, TLGE demonstrates outstanding long-term stability and effective temperature-sensitive sol-gel phase transformation, suggesting significant potential for application in TACE.

Acknowledgements

This work was supported by Hubei Province Nature Science Foundation of China (2023AFB1077), Doctoral Start-up Fund Project of Hubei University of Science and Technology (BK202118), Innovation team and Medical research program of Hubei University of Science and Technology (2023T10, 2022YKY05), Hubei Province Key R&D Plan Big Health Local Special Project (2022BCE042).

Author contributions

Ling Li and Yanyan Cao conceived the project and designed the experiments. Ling Li, Ning Chen and Guotao Cheng carried out the experiments and analyzed the data. Ning Chen, Jun Xing, Anna Liu and Ling Zhang assisted in some experiments. Houqiang Yu and Ling Li wrote the manuscript. Yanyan Cao and Ling Li contributed to the revision.

Ethics approval and consent to participate

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of the Science and Technology Department (Hubei Province, China) and approved by the Animal Ethics Committee of Hubei University of Science and Technology.

Consent for publication

All authors agreed to publish this manuscript.

Competing interests

The authors declare no competing fnancial interests.

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Scheme 1 is available in the Supplementary Files section.

No competing interests reported.

Scheme1.jpg
Scheme 1 Diagram of thermosensitive lipiodol gel emulsion with long-acting arterial embolization and long-term X-ray imaging ability
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Radiopaque thermosensitive Pickering emulsion vascular embolist for permanent arterial embolization

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Materials and animals

2.2. Synthesis of PNAs

2.3. Preparation of TLGE

2.4. Characterization of the PNAs

2.5 Characterization of the TLGE emulsion

2.6 Evaluation of the ex vivo live model

2.7 Application value of TLGE in renal artery embolization in normal rabbits

2.8 Evaluation of Biocompatibility

3. Results and discussion

3.1 Structure and morphology characterization of the PNA nanogels

3.2 Structure and morphology characterization of the TLGE

3.3 Ex vitro liver evaluation results

3.5. Biocompatibility evaluation

4. Conclusions

Declarations

References

Scheme 1

Additional Declarations

Supplementary Files

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