Bioengineered ionic liquid for catheter-directed tissue ablation, drug delivery and embolization

Abstract

Delivery of therapeutics to solid tumors with high bioavailability remains a challenge and is likely the main contributor to the ineffectiveness of immunotherapy and chemotherapy. Here, a catheter-directed ionic liquid embolic (ILE) was bioengineered to achieve durable vascular embolization, uniform tissue ablation, and drug delivery in non-survival and survival porcine models of embolization, outperforming the clinically used embolic agents. To simulate the clinical scenario, rabbit VX2 orthotopic liver tumors were treated showing successful trans-arterial delivery of Nivolumab and effective tumor ablation. Furthermore, similar results were also observed in human ex-vivo tumor tissue as well as significant susceptibility of highly resistant patient-derived bacteria was seen to ILE, suggesting that ILE could prevent abscess formation in embolized tissue. ILE represents a new class of liquid embolic agents that can treat tumors, improve the delivery of therapeutics, prevent infectious complications, and potentially increase chemo- and immunotherapy response in solid tumors.

Graphical Abstract

A catheter-directed ionic liquid embolic (ILE) was bioengineered to achieve durable vascular embolization, uniform tissue ablation, and drug delivery (Nivolumab) in non-survival and survival porcine and rabbit tumor models of embolization, outperforming the clinically used embolic agents. ILE represents a new class of liquid embolic agents that can treat tumors, achieving chemical segmentectomy, improve the delivery of therapeutics, prevent infectious complications, and potentially increase chemo- and immunotherapy response in solid tumors.

Despite decades of research and the promise of immunotherapy, 5-year survival of hepatocellular carcinoma has not significantly changed; it remains to be a death sentence. Alcohol, obesity, and viral hepatitis continue to be major causes of liver cancer. Worldwide 296 million living with chronic viral hepatitis are at risk for developing liver cancer¹. The standard of treatment for cancer is intravenous chemotherapy; however, systemic chemo- and immunotherapy for liver cancer have many challenges^{2, 3}. Low bioavailability of the drug at the tumor site with heterogeneous distribution within the tumor contributes to low treatment efficacy, high relapse rate, and poor survival, especially at advanced stages of cancer^4-7. The pronounced collateral toxicity to non-cancerous liver and systemic chemotherapy’s side effects further impair patients’ quality of life and limit overall efficacy^{8, 9}. Catheter-administered beads mixed with doxorubicin as a locoregional treatment for liver cancer overcome some of the challenges of systemic therapy^{10, 11}. However, precise spatiotemporal control of the embolic material remains a challenge often leading to their unpredictable proximal aggregation preventing deeper penetration for uniform delivery into the tumor¹². Additionally, the inefficiency of drug extravasation from the blood vessel to the interstitial space poses a great clinical challenge. The irregular vasculature of the tumor coupled with poor visualization of the beads leads to uncertainty of tumor ablation at the time of the procedure limiting the overall value and efficacy of chemoembolization procedures. Here, we describe a multi-functional, solvent-free, biocompatible and x-ray visible liquid embolic agent that can stably embolize blood vessels, ablate parenchymal tissue with malignant tumors, and achieve effective trans-arterial delivery of therapeutics while protecting the ablated tissue from infection.

Results

Preparation and characterization of ILE

The molar ratio between the anionic (geranic acid; GA) and cationic (choline; C) monomers of the ionic liquid embolic (ILE) was varied to develop a formulation optimized for tissue ablation and drug delivery. Seven versions of the ILE were synthesized; these varied in the ratio of GA: C ranging from 4:1 to 1:4 (Fig. 1a and Fig. S1, S2). We focused on identifying a formulation that would allow hand-injectability for ease of use; we measured the viscosity and the corresponding injection force of each ILE. The viscosity of the ILEs at 37°C and the force required to inject the ILE through a 2.8 French (F) (length: 110 cm) microcatheter indicated that 4:1, 1:2, 1:3, and 1:4 formulations to be the most suitable for injection using microcatheters (Fig. 1b, c). Next, the diffusivity of the ILEs was tested using an agar-based diffusion assay inspired by an immunodiffusion assay¹³; fluorescent dyes indocyanine green (ICG; used as a drug surrogate) and doxorubicin (DOX), were used to visualize the diffusion capability of each ILE (Fig. 1d, e). At 24 h, ILE ratios of 1:2, 1:3, and 1:4 demonstrated the highest level of circumferential diffusion compared to control samples. To show that diffusion of the fluorescent dye in Fig 1d,e also represented the spreading of ILE, a functional diffusion assay was performed to measure the viability of hepatocellular carcinoma cells (HepG2). Using a Transwell plate with 400 nm-sized membrane pores (Fig. 1f), which is similar to the size of pores in hyperpermeable vessels in solid tumors^{14, 15}, ILE ratios 1:2, 1:3 and 1:4 demonstrated maximal diffusion across the transwell into the cell culture chamber resulting in near zero viability of the HepG2 cells (Fig. 1g, h). To determine the minimum amount of ILEs required for cell death, IC₅₀ values for each ILE were determined (Fig. 1i). Fractional viability assays showed that all three candidate ILEs have potent cytotoxic effects with IC₅₀ values of 0.24 %, 0.39 %, and 0.54 % for the 1:2, 1:3 and 1:4 formulation, respectively (Fig. 1i). The viability of HepG2 cells in Fig. 1h and the IC₅₀ data of HepG2 cells in Fig. 1i collectively indicate that at least 13% of the ILE from the Transwell likely diffused into the lower chamber within 24 h. These data suggest that 1:2, 1:3, and 1:4 formulations of the ILEs have favorable viscosity for microcatheter injection and robust diffusion capability in agar and Transwell diffusion assays mimicking tissue matrix.

Fig. 1: Characterization of ILE formulations.

a, Various formulations of ILE indicating the ratio of GA to C. b, Flow curves of ILEs at 37°C showing various viscosity levels with 4:1 being the most and 1:4 being the least viscous. c, Injection force testing of ILEs. d,e Representative ICG and DOX fluorescence images in tissue-mimicking agar diffusion assay at 0, 4, 24 h. The diffusion coefficient was calculated based on diffusion area at 24 h time-point. f, Schematic of the Transwell diffusion assay conducted using ICG-incorporated ILEs (1:2, 1:3 and 1:4) and its effect on HepG2 cells. g, Measurements of ICG fluorescence intensity in the lower chamber of Transwell suggesting diffusion across the insert pores (400 μm) over 24 h. h, The viability assessment of HepG2 cells in the lower compartment of the Transwell after 24 h incubation. i, Fractional viability of HepG2 cells treated with the ILEs and their corresponding IC₅₀ values are shown. Data are presented as mean ± s.e.m. (n = 4 for c and i; n = 3 for d-f). Statistical significance was determined using one-way ANOVA (c and g), two-way ANOVA (d and e) followed by Tukey’s multiple comparisons test and nonparametric multiple t-tests (h). ns, not significant; ****p < 0.0001.

Optimization of ILE

Ideally, the liquid embolic agent should not cross into the venous circulation so that the intended effect of the liquid embolic agent is isolated to the targeted arterial distribution. In addition, embolic agents in the venous circulation may potentially lead to unwanted complications such as pulmonary embolism. To limit the effect of ILE to the arterial distribution, we increased the viscosity of the ILE formulations by adding glycerol, a biocompatible and viscous additive often used as a therapeutic preservative (Fig. S3)¹⁶. Rheological measurements showed that the final concentration of 40 % (v/v) glycerol significantly increased the viscosity of the ILEs compared to controls (Fig. 2a) without impacting their ability to diffuse (Fig. S4). Next, to achieve precise delivery of the ILE using clinical imaging tools such as fluoroscopy and computed tomography (CT), iohexol (IOH), an FDA-approved aqueous contrast agent, was added to the ILE. The amount of IOH mixed with ILE was balanced by X-ray visibility, material properties, and cytotoxicity. The concentration-dependent increase in IOH corresponded to a proportional increase in radiodensity on fluoroscopic and CT images; 20% (v/v) of IOH was selected as an acceptable amount for visibility using X-ray imaging. Furthermore, the visibility of the ILEs with 20% IOH did not change when mixed with glycerol (Fig. 2b, c). Next, we tested the stability of Nivolumab, which is the most commonly used immunotherapy drug worldwide, when mixed with different ratios of ILE with or without glycerol. Results showed that Nivolumab did not undergo degradation over a 24 h incubation period when mixed with various ILE formulations (Fig. 2d). To ensure the stability of Nivolumab over a longer period, we continued the analysis up to 28 days at 37°C; no significant degradation of the protein was observed (Fig. S5). To understand the charge distribution of the various ILE formulations (Table S1), zeta potential was measured. When glycerol is incorporated, the zeta potential shifts from positive to negative indicating a greater likelihood to diffuse rapidly throughout tissues (Fig. 2e)¹⁷. Next, to examine the capability of the ILE formulation to preserve smaller molecules, near-infrared imaging agent ICG mixed with ILE was tested. Within 24 h, the fluorescent signal from the free ICG was barely detectable and the signal was completely lost within 2 days¹⁸. However, when ICG is mixed with ILE with and without glycerol, the fluorescence signal remained significantly higher up to 28 days when compared to the control group indicating that ILE+glycerol has the capability to stabilize large protein and small molecule structures (Fig. S6). We examined the release of Nivolumab (146 kDa) from the ILE formulation to achieve drug delivery, which is critical for oncology. ILE mixed with Nivolumab inside a dialysis tubing showed rapid release of Nivolumab reaching nearly 90% within 24 h. The release behavior of a smaller protein, bovine serum albumin (BSA; 67 kDa) was also examined showing a burst release reaching 67% within 24 h and subsequent sustained release up to ~86% on day 5 (Fig. S7). Viscosity measurements of the final ILE formulations containing ICG, IOH, and Nivolumab demonstrated a range of 20~30 mPa*s, which is approximately 10-fold higher than the viscosity of whole blood at 37°C¹⁹. The break-loose and injection force of ILEs also increased; however, they were still easily injectable using a 2.8F microcatheter (Fig. 2g and Video. S1). Fractional viability assays of ILEs with ICG, IOH, and Nivolumab showed a slight increase in IC₅₀ values compared to ILE alone, however, still highly lethal to the cancer cells (Fig. 2h). The absorbance spectra of various ILE formulations were also investigated; these revealed that only ILE formulations that contained ICG demonstrated a fluorescence signal (Fig. S8). The impact on viscosity and injection force of ILE under various pH conditions (pH 6.5 and 9) was minimal. This suggests that the mechanical properties of ILE will not significantly change in a lower pH tumor environment (Fig. S9).

Fig. 2: Optimization of ILEs.

a, Viscosity measurements of ILEs mixed with various amounts of glycerol (0%, 10%, 20%, 30%, and 40%). b,c, Fluoroscopic images (b) and micro-CT images (c) of ILE mixed with iohexol and their corresponding intensity in X-ray fluoroscopy and micro-CT images of various concentrations of IOH (0%, 5%, 10%, 20%, 30%) and ILEs (1:2, 1:3, or 1:4)+glycerol (40% v/v)+IOH (20% v/v). d, SDS-PAGE gel image demonstrating the stability of Nivolumab after mixing with various ILEs with or without glycerol for 24 h at 37°C. e, Zeta potential measurements of ILE, ILE+glycerol (40% v/v), and final ILE formulations containing ILE+glycerol+IOH+Nivolumab+ICG. f, Viscosity measurements of final 3 candidates of ILE (ILE12, 13, and 14). g, Break-loose and injection forces of ILEs injected through 2.8F 110cm microcatheter at a constant flow rate of 1 mL/min. h, Fractional viability of HepG2 cells following incubation with neat ILE only, and final formulation of ILEs, i.e., ILE12, 13, and 14, respectively. Data are presented as mean ± s.e.m (n = 3 for a-e; n = 4 for f-h). Statistical significance was determined using one-way ANOVA (a-f) and two-way ANOVA (g) followed by Tukey’s multiple comparison test. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Renal artery embolization in a nonsurvival porcine model

To assess ILE’s ability to embolize, ablate and deliver drugs locally, we used a porcine renal embolization model, which has a hierarchical vascular network similar to tumors. Under real-time fluoroscopic imaging guidance, the lower pole of the kidney was embolized with either ILE(1:2)+glycerol+IOH+ICG+Nivolumab (ILE12), ILE13, or ILE14, allowing the use of the upper pole of the same kidney as a negative control. As the ILEs exited the tip of the microcatheter, ILEs demonstrated excellent X-ray radiodensity reaching smaller distal arteries in the renal parenchyma and allowing real-time visibility, indicating the completion of embolization (Fig. S10 and Fig. 3a, b). These images also showed that the embolization of the lower pole arteries was instant; subsequent DSA imaging of the renal artery showed an absence of flow in the lower pole arteries that received ILEs (Fig. 3b and Fig. S11-13). On necropsy, the kidneys visibly demonstrated the lower half of the kidney that received ILEs to appear darker in color, and on IVIS imaging, ICG distribution showed that the darker areas on gross evaluation corresponded to ILE delivery (Fig. 3c). On sagittal sections of the kidney, IVIS images further indicated uniform detection of the ICG throughout the lower half of the kidney suggesting successful delivery into the renal parenchyma (Fig. 3d, h, i and Fig. S11-13). ILE delivery into the kidneys demonstrated similar ICG detection levels and markedly higher ICG fluorescence in the lower pole when compared to the untreated upper pole (Fig. 3h, i). On histology, lower pole arteries were embolized containing ILE and blood vessels also demonstrated no nuclei implying ablation compared to untreated upper pole arteries (Fig. 3e, j). Immunohistochemical (IHC) staining of cleaved caspase-3 showed apoptosis throughout the lower pole of the ILE-treated kidneys suggesting that the ILE diffused trans-arterially into the renal parenchyma to achieve tissue ablation (Fig. 3f, k)²⁰. To further prove that trans-arterial delivery occurred with ILE, in addition to uniform ICG detection and apoptosis, we immunostained for Nivolumab. Fig. 3g and 3l show that Nivolumab can be trans-arterially delivered throughout the renal parenchyma; however, ILE14 demonstrated a greater level of delivery. Thus, subsequent experiments focused on the ILE14 formulation.

Fig. 3: In vivo characterization of ILEs in a nonsurvival porcine embolization model.

a, Baseline DSA showing normal arterial anatomy of the porcine kidney following contrast injection from a 5 French catheter in the main renal artery. b, DSA following embolization of the lower pole arterial branches of a porcine kidney with ILE12, 13, or 14. c, Gross images of resected kidneys at necropsy showing a discoloration of the lower half of the kidney demarcating the treated kidney tissue. d, Kidneys in (c) were transected along the sagittal plane and imaged using IVIS to detect the ICG; images localize the ICG in the ILE formulation to the treated lower lobe of the kidney. e, Representative H&E images of embolized renal arteries. f, Representative images of renal parenchyma stained with cleaved caspase 3. g, Representative IHC staining for Nivolumab showing detection of diffused Nivolumab in the renal parenchyma, outside the arterial vasculature suggesting trans-arterial delivery. h, Quantification of ICG intensities in the whole kidney indicates no significant difference between the three ILE formulations. i, ICG radiance ratio of embolized (lower)/ control (upper) kidney lobe. j, Nuclei count of the vessel wall from the kidney tissue sections indicates uniform ablation of the vessel wall. k, Quantitative analysis of cleaved caspase 3 and l, Nivolumab detection by IHC significantly greater in ILE14 tissue compared to ILE12 and ILE13. Data are presented as mean ± s.e.m (n = 8 for h-j; n = 4 for k and l). Scale bars: e, 50 μm. f, 200 μm. g, 6 mm. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test. ns, not significant; **p < 0.01, ***p < 0.001 and ****p < 0.0001.

To show that glycerol is necessary to enhance the delivery of the ILE and its components, separate porcine renal embolization experiments were performed with bare ILE (1:4) and glycerol alone. Injection of ILE into the lower pole of the kidneys led to instant but incomplete embolization; glycerol demonstrated immediate recanalization suggesting that they are not effective embolic agents alone. Both also presented reduced levels of tissue ablation and drug delivery when compared to the ILE14 formulation (Fig. S14-17)

ILE14 in blood

Liquid embolic agents are injected into blood vessels leading to significant interactions with blood. To evaluate the effect of ILE14 on blood coagulation potential and hemocyte count, the activated clotting time (ACT) and hemorheology of various ILE14 concentrations and bare ILE (1:4) were tested. ILE14 up to 1% (v/v) and bare ILE at 0.1% (v/v) when mixed with blood demonstrated thrombosis similar to untreated blood. However, the blood remains anticoagulated at ILE14 concentrations greater than 1% and ILE alone greater than 0.1% and did not demonstrate the ability to thrombose (Fig. 4a-d). Furthermore, hemolysis was minimal at concentrations up to 2%; however, lymphocytes demonstrated significant resistance at concentrations up to 10% (Fig. 4e-g and Fig. S18). ILE14 and glycerol alone, demonstrated preservation of various hemocytes up to 10% concentration possibly due to the preservative properties of glycerol (Fig. S19)^{21, 22}. These results indicate that the proximal embolization observed in Fig. 3 is unrelated to thrombosis but to the viscosity of the material, while downstream embolization may have been caused by thrombosis. In addition, these results show that the cellular components of blood are minimally affected by ILE14 suggesting that the delivery of Nivolumab to the ablated renal parenchyma may enhance the chances of achieving immunotherapy in solid tumors.

Fig. 4: ILE14 in blood.

a, Schematic illustration of ILE14 endovascular injection from a microcatheter inside an artery demonstrating deep penetration into distal blood vessels. b, Measurement of blood ACT with different concentrations of ILE (1:4) and ILE14 showing that >1% is required to delay thrombosis. c, Representative hemorheology amplitude sweeps flow curves at 37°C after mixing with ILE (1:4) and ILE14. d, Graph demonstrating storage modulus in blood samples at 37°C 30 min after mixing with ILE (1:4) and ILE14. e, Hemolysis rate after incubation with various concentrations of ILE (1:4) and ILE14. f, Lymphocytes’ count in porcine whole blood after incubation with different concentrations of ILE (1:4) and ILE14 indicating resistance to ablation. g, Consistent with (f), images of blood smear with Wright’s stain showing intact lymphocytes at 10% ILE (1:4) and ILE14. Data are presented as mean ± s.e.m (n = 3 for b and d; n = 5 for e; n = 4 for f). c and g: Representative result from n = 3. Scale bars: g, 150μm and 25μm (inset). Statistical significance was determined using one-way ANOVA (b and d) and two-way ANOVA (e and f) followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

ILE14 in porcine renal embolization survival model

To determine the durability of ILE14 embolization, Nivolumab and ICG delivery and tissue ablation, we performed survival experiments and compared the results to clinically used chemoembolization beads (Bead Block^™) (Fig. S20)²³. Beads were suspended in a mixture that contained an equal amount of Nivolumab and ICG in ILE14. At baseline, both bead and ILE14 were able to achieve embolization (Fig. 5a and Video. S2-4). However, at day 7, the upper pole of the kidneys that received bead embolization all demonstrated significant recanalization; the lower pole of the kidneys that received ILE14 demonstrated persistent embolization with no evidence of any recanalization implying that the vessel occlusions are permanent (Fig. 5a, b and Video. S5). On gross examination of the kidneys, the upper pole that received beads showed heterogeneity with ischemic areas and normally perfused areas when compared to the contralateral normal kidneys. However, the lower pole of the kidneys that received ILE14 demonstrated significant ischemic injury with uniform atrophy and ablation of the renal parenchyma (Fig. 5c). On IVIS imaging, only the lower pole of the kidneys that received ILE14 showed ICG fluorescence suggesting successful small molecule delivery to the tissue parenchyma that did not wash away up to day 7 (Fig. 5d, f and Fig. S21). On histology, marked differences were observed; both the untreated and the bead-embolized kidneys showed similar cellularity with normal endothelial cell (CD31) immunostaining suggesting that tissue ablation was not observed in the sections evaluated (Fig. 5e and Fig. S22). Hypoxia-inducible factor-2α (HIF-2α) was detected at greater levels in the bead-embolized samples when compared to the controls suggesting a state of increased hypoxia from temporary embolization. In contrast to the untreated controls and to the bead-embolized kidneys, ILE14-treated samples demonstrated notable differences; arteries were occluded with no signs of CD31 or HIF-2α immunostaining suggesting persistent embolization and ablation of the arterial wall (Fig. 5e and g-i). Similarly, consistent with gross observations, apoptosis in the bead-embolized kidneys was patchy confined mostly to the glomeruli with no evidence of Nivolumab detection. In the ILE14-treated samples, apoptosis was uniformly detected throughout the renal cortex with significant delivery of Nivolumab. These results suggest that ILE14 achieves durable and permanent embolization and marked tissue ablation with significant drug delivery.

Fig. 5: ILE14 in a survival porcine renal embolization model compared to clinically used bead-embolization.

a, Baseline and post-embolization (D+0) DSA of the porcine kidney. The upper lobe arteries of the kidney received the clinically used microbeads and the lower lobe arteries received ILE14. Both images indicate technically successful embolization. b, DSA of control and embolized kidney at 7-day post-embolization. The upper lobe arteries that received microbeads demonstrate recanalization, which is expected based on clinical experience. The lower lobe arteries that received ILE14 demonstrate the desired, persistent embolization of the arteries indicating that the effect is durable. c, At necropsy, sagittal sections of the kidneys indicate greater atrophy in the lower lobe of the kidney that received ILE14 treatment (black arrow). Only the renal arteries embolized with ILE14 showed occlusion (red arrows). d, IVIS imaging of the kidneys in (c) for ICG showed a persistent signal in the lower lobe of the kidneys that received ILE14 indicating delivery and persistence of the ICG suggesting that it did not wash away. The upper pole of the kidney that received microbeads did not show any ICG signal suggesting that it washed away. e, Representative histology images of H&E and immunostaining for CD31, HIF-2α, cleaved caspase 3 and Nivolumab. Images of slides from kidney tissue that received microbeads demonstrated a viable vessel wall, normal CD31 signal in the intima layer, and significant HIF-2α signal indicating a state of hypoxia. Cleaved Cas-3 showed mild, scattered evidence for apoptosis with beads detected in vessel walls (arrow) and the absence of any Nivolumab detection. In contrast to the untreated control and the microbead-treated kidney sections, the ILE14-treated samples demonstrated complete embolization of the arteries and the absence of any CD31 signal, consistent with the absence of HIF-2α indicating that there is no viable vasculature. Corresponding cleaved Cas-3 signal showed diffuse apoptosis and uniform delivery of the Nivolumab throughout the treated kidney tissue. f, Quantitative analysis of ICG signal in the kidney 7 days post-embolization. g, Nuclei count of the vessel wall from kidney sections. h, Quantitation of CD31, i, HIF-2α, j, cleaved caspase 3, and k, Nivolumab stained area. Data are presented as mean ± s.e.m (n = 8 for f; n = 10 for g; n = 5 for h-k). Scale bars: e, 200 μm (control), 75 μm (beads) and 150 μm (ILE14) for H&E, CD31 and HIF-2α staining. 6 mm (control), 4 mm (beads) and 6 mm (ILE14) for cleaved caspase 3 and Nivolumab staining. Statistical significance was determined using paired t-test (f, h-k) and one-way ANOVA followed by Tukey’s multiple comparison test (g). ns, not significant; **p < 0.01, ***p < 0.001 and ****p < 0.0001.

ILE14 demonstrated no signs of toxicity when compared to untreated controls, immediately post-embolization, and to animals at day 7. Complete blood cell (CBC) counts, biochemical analysis of serum (Fig. S23), serum cytokine array (Table S2), and histological evaluation of major organs (Fig. S24) showed no signs of abnormality. Furthermore, an estimation of the potential pharmacodynamics of ILE14 based on the calculation of physiologic concentrations showed that the systemic amount of ILE will be too low to cause any detectable toxicity (Fig. S25).

The effect of sub-lethal doses of ILE14 on hepatocellular carcinoma cells

Since tumor margins are often the sites of tumor recurrence, we examined whether 5 to 25-fold dilution of the ILE14 from its IC₅₀ (0.57%) will have an effect on the migration of HepG2 cells. ILE14 as low as 0.025% (v/v) was able to suppress the migration of HepG2 (Fig. S26). This suggests that while the local concentration of ILE14 may be under the threshold of cell death, ILE14 may still exert a therapeutic benefit by reducing cellular migration and potentially reduce recurrence.

Assessment of ILE14 in a rabbit VX2 liver tumor model

To determine whether ILE14 can embolize, ablate an arterial distribution, and deliver therapeutics to this ablated zone, we used the highly malignant rabbit VX2 orthotopic liver tumor model. Once the liver tumors reached a certain size based on ultrasound imaging (Fig. 6a), a vascular embolization procedure was performed using the ILE14. Similar to clinical cases, a microcatheter was used in combination with a microwire to super-select the segmental artery that perfused the VX2 tumor (Video. S6). With the angiography image magnified to help visualize the material exiting the catheter tip, ILE14 was slowly injected using a 1 cc syringe, as would be done clinically, until stasis of flow was achieved (Video. S7). DSA imaging following ILE14 injection showed immediate embolization (Fig. 6b and Fig. S27a, b, and Video. S8). At 1 h post-embolization, gross and IVIS images of the explanted liver were obtained, which showed preferential delivery and accumulation of the small molecule ICG into the VX2 liver tumor tissue (Fig. 6c-f and Fig. S27c, d). A histology analysis of the tumor was performed to determine whether ICG accumulation at the liver tumor also implied delivery of the ILE and Nivolumab. On H&E and proliferating cell nuclear antigen (PCNA) immunostained sections, control tumors showed obvious cellularity with no evidence of Nivolumab delivery. In stark contrast, the ILE14 embolized tumors demonstrated marked tissue ablation and Nivolumab delivery resulting from transarterial diffusion as evidenced by hypocellularity, significantly reduced PCNA staining, and a marked increase in Nivolumab detection surrounding the embolized tumor arteries (Fig. 6g-l and Fig. S28, S29). An off-target ILE14 injection demonstrated viable VX2 tumor and the absence of any Nivolumab detection, indicating that therapeutic delivery and tissue ablation are specific to targeted ILE14 injection (Fig. S30).

Fig. 6: Evaluation of ILE14 in rabbit VX2 liver tumor model.

a, Representative color ultrasound image of a rabbit VX2 liver tumor (yellow dotted outline) showing blood flow. b, Angiograms at baseline and post-ILE14 embolization to the segmental artery that feeds the rabbit tumor. The blue arrow indicates the tip of the microcatheter where ILE14 was slowly infused until stasis. The red arrow indicates the absence of blood flow in the embolized segment of the rabbit liver. c, The gross image of the VX2 liver tumor post-embolization. d, IVIS imaging of the excised liver 1 h post-embolization demonstrating specific ICG signal accumulation in a tumor region. e, Sagittal transection of the tumor in (d) shows the ICG signal predominantly localized to the tumor, in comparison to a control tumor that did not receive ILE14. f, The quantitative analysis of total ICG radiance within the tumor tissue. g, Representative H&E images of VX2 tumor showing tissue ablation and vessel occlusion in the ILE14 embolized tumor compared to control (black arrows indicate tumor microvasculature). h, nuclei count within the tumor tissue sections. These images demonstrate trans-arterial diffusion of ILE14 and marked ablation of the adjacent cancer tissue. i, Representative PCNA immunostaining and j, quantification of PCNA positively stained area. k, Representative immunostained section showing marked Nivolumab delivery in the tumor that received ILE14 and l, Quantification of Nivolumab detection within the tumor tissues. Data are presented as mean ± s.e.m (n = 4 for f; n = 3 (control) and n = 2 (embolized) for h, j, and l). Scale bars: a, 1 cm. g, i and k, 75 μm. Statistical significance was determined using an unpaired t-test (f, h, j, and l). *p < 0.05 and ***p < 0.001.

ILE14 in a survival rabbit VX2 liver tumor model

To demonstrate the efficacy of ILE14 as a tumor ablation and drug delivery modality, a survival study in a rabbit VX2 liver tumor model was performed. At baseline angiography, the main segmental branch hepatic artery was identified, and ILE14 was infused to embolize the liver tumor. At day 7, angiography demonstrated a durable and persistent embolization of the hepatic arteries (Fig. 7a), consistent with the survival porcine experiments in Fig. 5. Ultrasound imaging of the VX2 tumors revealed a substantially smaller tumor volume compared to untreated control (Fig. 7b), also consistent with successful embolization of the tumor blood vessels. At necropsy, gross and IVIS images of the explanted whole liver and transected tumors were obtained, which showed preferential uptake and retention of ICG in the VX2 liver tumor (Fig. 7c). Histology analysis of the VX2 tumors showed that tumors that received ILE14 demonstrated significant hypocellularity and significant TUNEL signal indicating that the tumors have been ablated due to the combined embolization and potent ablation effects of ILE14. In addition, consistent with the porcine studies, Nivolumab was shown to localize into the tumor parenchyma and did not wash away and remain within the ablation zone (Fig. 7d-g). These results show that ILE14 can embolize and ablate the tumor tissue in a geographic manner and the possibility of combinatorial therapy when loaded with functional drugs.

Figure 7. Evaluation of ILE14 embolization in a survival rabbit VX2 liver tumor model.

a, Angiogram at baseline and at day 7 post-ILE14 embolization of the feeding tumor arteries. The red arrow indicates the embolized main segmental branch of the tumor artery. b, Representative ultrasound imaging of untreated (control) and embolized rabbit VX2 liver tumors (white dotted line). Tumor volume growth of individual rabbits was monitored by ultrasound imaging. c, IVIS imaging of harvested whole liver (left) and IVIS imaging and gross view of medial transection of the tumor (right) at day 7. Whole liver and transected liver tissue IVIS imaging demonstrates the persistent ICG signal localized to the tumor. d, Representative histology images of H&E and immunostaining for TUNEL and Nivolumab. Control tumor slides showed viable tumor tissues, minimal TUNEL staining, and no detection of Nivolumab. In contrast, tumors treated with ILE14 showed significantly reduced tumor size and cellularity, high TUNEL detection within the treated zone, and localized Nivolumab detection within the ablation zone (black arrows). e, Medial cross-sectional area of tumors. f, Quantification of TUNEL (apoptotic index), and g, Immunohistochemical detection of Nivolumab. Data are presented as mean ± s.e.m (n = 2 for e-g). Scale bars: b, 5 mm. d, 4 mm. Statistical significance was determined using an unpaired t-test (e-g). *p < 0.05 and **p < 0.01.

Assessment of ILE14 in ex vivo human tumor tissue

To demonstrate whether ILE14 can diffuse and ablate human tumor matrix, freshly resected human renal cell carcinoma tumors were injected with ILE14 within one hour of resection. IVIS imaging and quantitative analysis showed increased fluorescence radiance and diffusion area over 24 h (Fig. S31a-c) with histology showing a near-complete ablation of the tumor tissue, compared to the untreated samples (Fig. S31d). Furthermore, Nivolumab immunostaining of the tissues showed uniform distribution of Nivolumab across large tissue area suggesting that ILE14 is also capable of delivering drugs throughout the human tumor matrix similar to the rabbit VX2 tumor (Fig. S31e).

The bacteriocidal effect of ILE14

In clinical practice, abscess formation in the ablation zone is a highly undesired and potentially fatal complication of embolization procedures²⁴. To determine whether the unique tissue ablation capability of ILE14 extends to infections, susceptibility testing using antibiotic-resistant patient-derived pathogens was performed. ILE14 demonstrated major bactericidal effects on highly drug-resistant strains including, methicillin-resistant Staphylococcus aureus (MRSA; minimum inhibitory concentration: 6.25%), vancomycin-resistant Enterococcus faecium (VRE; minimum inhibitory concentration: 6.25%), and carbapenem-resistant Enterobacterales (CRE; minimum inhibitory concentration: 12.5%), Clostridioides difficile (C. difficile; minimum inhibitory concentration: 6.25%), and Candida auris (C. auris; minimum inhibitory concentration: 3.13%) (Fig. S32). These results suggest that while currently used embolic agents may be prone to procedural infections²⁵, ILE14 can achieve embolization, tissue ablation, and drug delivery and, at the same time, protect the patient against abscess formation and potentially fatal sepsis even at very low concentrations.

Discussion

Despite recent advances in oncology, specifically immunotherapy, the 5-year survival rate for liver cancer has remained largely unchanged over the past few decades. Taking advantage of the tissue permeation enhancing property of IL that facilitates drug infiltration²⁶, ILE provides a unique biomaterial-based platform technology to enable the delivery of therapeutics of various sizes to tissues including to tumor matrix. With the capability to embolize and ablate tissue coupled with the delivery of large molecules, i.e. Nivolumab, we show that ILE has the potential to lead to better clinical outcomes. ILE-based embolization can achieve deep penetration to the capillary level, achieve a uniform and sustained drug delivery, and allow real-time visualization of ILE under X-ray imaging so that accumulation within the target can be seen. ILE is biocompatible, easily injectable, and does not require any prior preparation for use. Our study focused primarily on the technology demonstrating the development of the formulation, and its optimization in non-survival and survival large animal models and testing in an orthotopic liver tumor model. Future studies could include analysis of the immune response in animal models and clinical trials.

Methods

Preparation and characterization of ILE in different ratios

ILE was prepared from a mixture of geranic acid and choline bicarbonate using salt metathesis. Neat geranic acid (Sigma-Aldrich, St. Louis, MO) was purified five times via recrystallization method at −80°C in acetone in a 500mL round-bottom flask (Thermo Fisher Scientific, Waltham, MA). Purified geranic acid was then added with choline bicarbonate (Sigma-Aldrich) at predetermined molar ratios (ranging from 4:1 to 1:4) and stirred at ambient temperature until the CO₂ byproduct was no longer detected with a handheld carbon dioxide probe (GM70, VAISALA, Vantaa, Finland). Subsequently, residual H₂O was removed by rotary evaporator (R-300, Buchi, New Castle, DE) at 60°C for 1 h. The synthesized ILE was characterized using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy (Lumos II with Alpha II add-on, Bruker, Kontich, Belgium).

Rheological study

All rheological measurements of samples were performed using a rheometer (MCR 302, Anton Paar, Torrance, CA) at 37°C. For rheological measurements, a sandblasted 25-mm aluminum shaft and aluminum plate were used, keeping the gap in between at 1 mm. Flow curve measurements of all samples were performed at a shear rate range from 10⁰ to 10³ s⁻¹. During the measurements, a water trap was used to prevent samples from drying out. Quantification of viscosity was calculated based on the values from the 500 1/s shear rate. All measurements were performed at least 3 times.

Injection force measurements

The injectability of all samples were determined using a mechanical tester equipped with a 100N load cell (Instron 5942, Instron, Norwood, MA). Briefly, all material was loaded in 3 cc syringes (Medallion, Merit Medical, South Jordan, UT) and injected through a 110 cm 2.8 F microcatheter (PROGREAT^®, Terumo Interventional Systems, Somerset, NJ) at a constant flow rate of 1 mL min⁻¹. The injection force was recorded using the Bluehill version 3 software (Instron).

Agar diffusion assay

Tissue-mimicking matrix was created to assess the diffusion of small molecules loaded in ILEs. Agarose (11685660001, Roche, Mannheim, Germany) was measured and dissolved in pH 7.4 1x phosphate buffered saline (PBS) (Life Technologies, Bleiswijk, Netherlands) to the final concentration of 2%. The 3D-printed well-casting inserts (5-mm diameter) were placed in 6-well plates (CELLTREAT Scientific Products, Pepperell, MA) and 11 mL of warm 2% agarose solution was poured into each well and left to cure in a sterile hood. Once cured, inserts were carefully removed and 250 μL of free dyes and ILEs incorporated with equal amounts of dyes were transferred into the center reservoir to investigate the diffusion. Indocyanine green (ICG) (USP, Rockville, MD) and doxorubicin (DOX) (Pfizer, New York, NY) were used at 0.25 and 1 mg/mL, respectively. The samples were incubated in a 37°C humidified chamber and at predetermined time points (0, 4, 24 h), the distribution of fluorescence radiating from the dyes was imaged using the IVIS^® spectrum in vivo imaging system (PerkinElmer Inc., Waltham, MA) at 740/850nm (ICG) and 460/560nm (DOX) wavelengths. The diffusion coefficient of each ILE sample was calculated at 24 h timepoint with the following formula:

$$Diffusioncoefficient({cm}^2{s}^{-1})=\frac{Diffusedarea}{Time}$$

Cell culture

HepG2 hepatocellular carcinoma cell line (CRL10741, ATCC, Manassas, VA) was cultured using an Iscove’s Modified Dulbecco’s Medium (IMDM; HyClone, Logan, UT) supplemented with 10% heat-inactivated bovine serum albumin (BSA; HyClone), 100 IU penicillin and 10 μg/mL streptomycin (Thermo Fisher Scientific), and 0.1mM non-essential amino acids (Lonza, Walkersville, MD). Cells were cultured in a 37 °C, 5% CO₂ humidified chamber and handled aseptically.

Transwell diffusion assay

In a 12-well permeable transwell culture plate with 400 nm pore-sized polycarbonate membrane (230615, CELLTREAT), 5 × 10⁵ cells/ well of HepG2 cells were seeded in the lower compartment and incubated for 16 h at 37 °C, 5% CO₂ humidified chamber. Upon incubation, HepG2 cells were washed and replenished with a fresh growth medium. 500 μL of samples (1:2 or 1:3 or 1:4 ILE+ICG) were transferred into the inserts and incubated for 24 h (n= 3). After incubation, inserts were removed and ICG that has been diffused to the lower chamber through the pores was quantified using a microplate reader using 780/820 nm wavelengths (SpectraMax iD5, Molecular Devices, San Jose, CA). The viability of HepG2 cells was assessed using WST-1 cell assay kit (Cayman, Ann Arbor, MI) according to the manufacturer’s instructions.

In vitro fractional viability assay

3 × 10⁴ cells/ well of HepG2 were seeded in 96-well tissue culture plates (229196, CELLTREAT) and incubated overnight in a 37 °C, 5% CO₂ humidified chamber. Followed by incubation, cells were washed with a pre-warmed Dulbecco’s modified phosphate buffer (DPBS; Sigma-Aldrich) and treated with a fresh IMDM growth medium contained with serially 2-fold diluted samples (Maximum concentration: 25%). The samples were incubated for 24 h followed by three repeats of washing with DPBS to remove dead cells. After the washing step, each well was added with 100 μL of growth medium and 10 μL of WST-1 reagent and incubated for 1.5 h. The absorbance of samples was measured using a microplate reader (Molecular Devices) at 450 nm and the percentage of viable cells in each treated well was calculated relative to the control. The dose-response factional viability plot and corresponding IC₅₀ values were analyzed using Prism Software (GraphPad, San Diego, CA).

Optimization of ILE

To increase the viscosity of ILEs, different amount of glycerol (Sigma-Aldrich) ranging from 0 ~40% (v/v) was added. The viscosity of the resulting mixture at 37°C was measured with a rheometer (Instron). For real-time visualization during the embolization procedure, different amount of FDA-approved contrast agent iohexol (Omnipaque^™, GE Healthcare Systems, Chicago, IL) was incorporated and examined using fluoroscopy (OEC Elite C-Arm, GE Healthcare Systems) and micro-CT scanner (Skyscan 1276, Bruker). The quantification of fluoroscopic intensities and radiodensities of different concentrations of IOH was quantified using ImageJ (National Institutes of Health, Bethesda, MD) and CTAn (Bruker) software. Next, the stability of Nivolumab (Bristol Myers Squibb, Princeton, NJ) in ILE was investigated. 10 mg/mL stock of Nivolumab was diluted to 1 mg/mL in ILE and ILE+glycerol samples and incubated at 37 °C. At predetermined time points up to day 28, the samples were evaluated by running sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-denaturing condition. Each sample was mixed with Laemmli protein sample buffer (1610747, Bio-Rad, Hercules, CA), loaded (5 μg/ lane) onto stain-free gels (4568084, Bio-Rad), and electrophoresed at 100V for 70 min. The samples in the gel were transferred to a 0.2 μm polyvinylidene fluoride (PVDF) membrane (Bio-Rad) using a trans-blot turbo transfer system (Bio-Rad). Following a successful transfer, the samples in the membrane were visualized using a gel imaging system (Gel Dox XR+, Bio-Rad).

Zeta potential measurements

The zeta potential of ILE, ILE+glycerol, and ILE+glycerol+IOH+ICG+Nivolumab samples (ILE12, 13, and 14) were loaded in capillary cells (DTS1070, Malvern Panalytical Ltd., Malvern, United Kingdom) and analyzed using a Zetasizer ultra (Malvern Panalytical Ltd.) at ambient temperature. The data was collected and processed with ZS Xplorer software (Malvern Panalytical Ltd.).

In vitro ICG degradation and fluorescence intensity over time

ICG dissolved in an aqueous solution (control) and other ILE samples with various formulations were loaded into black solid 96-well plates and visualized every 24 h for 28 days using IVIS. The radiance efficiency of ICG was quantified with Living Image^® software (PerkinElmer Inc.). The change in radiance efficiency values were calculated relative to the values of day 0 as a control.

In vitro drug release test

The release profiles of Nivolumab (146 kDa) and BSA (67 kDa) from ILE14 were examined using the Float-A-Lyzer^®G2 dialysis device with 300 kDa-sized pores (Repligen Corporation, Rancho Dominguez, CA). 2mL of ILE(1:4)+glycerol+IOH+nivolumab and ILE(1:4)+glycerol+IOH+BSA were transferred into the dialysis tube and incubated at 37°C while being immersed in 6mL of pH 7.4 1x PBS (Life Technologies) as they resemble the pH of the blood. At predetermined time points, 25 μL of 1x PBS was taken out and the amount of protein was quantified using a Pierce^™ BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Renal embolization in a porcine model

All animal studies were performed according to animal protocols approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC). The renal embolization procedure was performed as previously described ^{42, 43}. Briefly, Yorkshire/Landrace pigs (Premier BioSource, Ramona, CA) weighing 50~55 kg were acclimated for 7 days under the supervision of a veterinarian. On the day of the procedure, anesthesia of pigs was induced by intramuscular injection of 5 mg/kg tiletamine-zolazepam (Telazol^®, Zoetis, Parsippany-Troy Hills, NJ), 2 mg/mL xylazine, and 0.02 mg/kg glycopyrrolate and maintained with 1.5~2% of isoflurane inhalation. The iliac artery was percutaneously accessed under ultrasound guidance (Butterfly iQ+, Butterfly Network Inc., Guilford, CT) and a Bentson guidewire (Cook Medical, IN, USA) was placed. 150 U/kg of heparin (Mylan, Canonsburg, PA) was intravenously administered to prevent coagulation during the procedure. Real-time digital substraction angiography (DSA) was used to navigate a 2.8 F microcatheter (Terumo Interventional Systems) and positioned inside the targeted renal artery branch. ILE (1:4), glycerol, ILE12, 13, 14, and beads (Bead Block^™, Boston Scientific, Marlborough, MA) were injected into the targeted blood vessels under real-time fluoroscopy. After embolization, DSA was repeated to confirm vessel occlusion. The animals were euthanized at 1 h post-embolization for the nonsurvival study and at 7 days post-embolization for the survival study, respectively. Blood samples were collected before embolization, after embolization, and at the terminal time-point for analysis. At necropsy, all kidneys and other major organs (heart, lung, liver, muscle, and spleen) were resected for analysis. Images of intact and bisected kidneys were taken immediately after the organ collection. Subsequently, ex vivo fluorescence imaging of cross-sectioned whole kidneys was performed using IVIS and radiance efficiency was calculated using Living Image^® software.

Histological and Immunohistochemical (IHC) analysis

Tissues were fixed in 10% buffered formalin (Fisher Scientific, Waltham, MA) for at least 7 days then processed for paraffin-embedding. All tissue sections were sliced in 5μm thickness and hydrated by serially immersing in xylene (twice, Fisher Scientific), 100% (three times), 95%, 80, 70, and 50% ethanol (Fisher Scientific) and distilled water for hematoxylin and eosin (H&E, Thermo Fisher Scientific) staining. For immunostaining, hydrated slides were subsequently immersed in 10mM sodium citrate buffer and boiled to 95°C for 30 min followed by 30 min of cooling down in a water bath for antigen retrieval. After washing the slides in distilled water for 5 min, endogenous peroxidase quenching was performed with 3% hydrogen peroxide (Sigma-Aldrich) in 60% methanol (Fisher Scientific) for 30 min at ambient temperature. The slides were then washed with 0.05% PBS-T for 5 min and blocked using 5% goat serum (50197Z, Thermo Fisher Scientific) in 1x PBS for 1 h to prevent non-specific binding. After the blocking step, cleaved caspase-3 (ab13847, 1:250, Abcam, Cambridge, MA), CD31 (ab182981, 1:200, Abcam), HIF-2α (ab109616, 1:1000, Abcam), PCNA (NB500-106, 1:1000, Novus Biologicals, Centennial, CO) primary antibodies diluted in 5% goat serum were added to each slide and incubated overnight at 4°C in a humidified chamber. The following day, the slides were washed three times with 0.05% PBS-T for 5 min each and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG H&L (ab97051, 1:500, Abcam) was added to the tissues and incubated at room temperature for 1 h. The samples were then washed three times with 0.05% PBS-T and developed using 3,3'-Diaminobenzidine (DAB) substrate (Vector Laboratories, Newark, CA) for 1 to 10 min. Once the color development is visible, the slides were rinsed in distilled water to halt further color development, counterstained in hematoxylin for 5 sec, and mounted using a mounting medium (Richard-Allan Scientific, Fisher Scientific). For Nivolumab detection in the porcine tissue, peroxidase-quenched slides were blocked using an avidin-biotin blocking buffer (004303, Invitrogen, Waltham, MA) for 1 h at room temperature. After, a biotin-conjugated rabbit anti-human IgG4 secondary antibody (ab238617, 5 μg/mL, Abcam) diluted in a universal blocking reagent (FP1020, Akoya Biosciences, Marlborough, MA) was added to the slides and incubated for 1 h at room temperature. For detecting Nivolumab in the rabbit tissue, peroxidase-quenched slides were blocked in a universal blocking reagent overnight at 4°C in a humidified chamber. After, a biotin-conjugated rabbit anti-human IgG4 secondary antibody (1 μg/mL) was blocked in 5% rabbit serum for 1 h. The blocked secondary antibody was directly added to the slides and incubated for 1 h at room temperature. Streptavidin-HRP solution (51-75477E, BD Biosciences, Franklin Lakes, NJ) was later applied to the slides and incubated for another 1 h at room temperature. DAB staining, counterstaining, and mounting were performed as previously described. Endothelial cell counting was performed using Qupath software (University of Edinburgh) and IHC staining quantification was performed using ImageJ software.

ILE (1:4) and ILE14 in blood

For activated clotting time (ACT) measurements, anti-coagulated citrated blood (Innovative Research Inc. Novi, MI) was mixed with ILE (1:4) or ILE14 to the final concentrations of 0, 0.1, 1, 2, and 10% (v/v), respectively. Next, 0.2 M calcium chloride (30:1, v/v, Sigma-Aldrich) was added to the samples and gently mixed to induce coagulation. The samples were instantly loaded in the Celite ACT cartridge (Abbott laboratories, Chicago, IL) and inserted into the i-STAT handheld critical blood analyzer (Abbott laboratories) for ACT analysis. The sample preparation for the hemorheology study was performed in the same preparation as in the ACT samples and flow curves were recorded as described in the “rheological study” section.

The hemocompatibility of ILE (1:4) and ILE14 was evaluated via hemolysis assay. Briefly, anti-coagulated citrated blood was centrifuged at 2,000 rpm for 10 min and serum was aspirated. The remaining red blood cell (RBC) layer was resuspended in 150 mM sodium chloride to the original volume and further diluted to 5% (v/v) in 1x PBS. The prepared RBC samples were mixed with either ILE (1:4) or ILE14 to the final concentrations of 0, 0.1, 1, 2, 10% (v/v) and ammonium-chloride-potassium (ACK) lysing buffer (KD Medical, Columbia, MD) was used to prepare positive control. All sample was incubated at 37°C for 1 h and centrifuged at 1,500 rcf for 5 min. 150 μL of supernatant was transferred to a 96-well plate and the absorbance was measured at 450 nm. The hemolysis rate was calculated with the following formula:

$$Hemolysisrate(\%)=\frac{A_{sample}-A_{negative}}{A_{positive}-A_{negative}}\ast 100$$

To assess the oxidation of blood mixed with different concentrations of embolic agents, the samples were prepared in the same way as the hemolysis assay except for the final centrifugation step. The samples were transferred to a 96-well plate and the absorbance spectrum (600~1,000 nm) was measured using a microplate reader. To investigate the fate of innate and recruited immune cells in the presence of ILE, the blood sample was mixed with either ILE (1:4), glycerol or ILE14 to the final concentrations of 0.1, 1, 2 and 10% (v/v). The mixture was incubated at 37°C for 1 h and analyzed using a veterinary hematology analyzer (HemaTrue^®, Heska, Loveland, CO).

Biocompatibility test

Complete blood count (CBC), biochemical analysis and cytokine array of serum were performed using collected blood samples. CBC was analyzed with a veterinary hematology analyzer (Heska) and biochemical analysis was performed using a veterinary chemistry analyzer (DRI-CHEM 4000, Heska). Serum cytokine arrays were performed using the porcine multiplex cytokine array/ chemokine array 13-plex (Eve Technologies, Calgary, CA).

In vitro migration assay

The migration assay was performed using a 35 mm μ-Dish with 2 well culture-insert (ibidi, Grafelfing, Germany). 4 × 10⁴ HepG2 cells in 70 μL of supplemented IMDM growth medium were seeded in each insert well and further incubated until complete confluency. Once the cells have reached the confluency, the insert was carefully removed. The cells were washed twice with fresh 1x PBS and replenished with fresh supplemented IMDM growth medium containing sub-lethal doses (0.025, 0.05, 0.1% v/v) of ILE14 and incubated in a 37 °C, 5% CO₂ humidified chamber. Every 24 h, migration of the cells was observed using a cell imaging microscope (EVOS FL Auto 2, Thermo Fisher Scientific) until the area was completely closed in the control group. The experiments were done in triplicates.

Embolization of the rabbit VX2 liver tumor arteries

To generate a rabbit VX2 liver tumor model, an aliquot of a rabbit VX2 tumor homogenate that was kept in liquid nitrogen was thawed and resuspended in 1 mL of Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific). 1mL of VX2 tumor homogenate suspension was injected into the calf muscle of a female New Zealand white rabbit (Charles River Laboratories, Wilmington, MA). After the injection, donor rabbits were left for 2~3 weeks for tumor growth. Once the tumor growth was confirmed with periodic ultrasound imaging, calf muscle tissue lesion with the tumor was harvested, and donor rabbits were euthanized. Harvested tumor tissue was immediately immersed in DMEM on ice and disassociated to ~1 mm³. 2 pieces of tumor section were implanted in a liver medial segment of recipient rabbits using aseptic surgical techniques. After tumor implantation, a liver incision was compressed for at least 3 min using a gelatin sponge (Ethicon, Summerville, NJ) for hemostasis and wound closure. The tumor was left to grow until it reached 1~2 cm³ based on ultrasound imaging. On the day of the procedure, the rabbits were anesthetized, the iliac artery was percutaneously accessed and a 0.014-inch REFLEX^™ steerable guidewire (Cordis, Miami Lakes, FL) was placed. Under the guidance of real-time DSA, a 2.4 F microcatheter with a 45° tip shape (Medtronic, Dublin, Ireland) was navigated to the hepatic artery. The microcatheter was further guided to the hepatic artery branch feeding the tumor-bearing liver segment and 1cc of ILE14 was carefully injected into the targeted vessels for embolization. After embolization, DSA was repeated to confirm the vascular occlusion. The animals were euthanized at 1h post-embolization for the non-survival study and at 7 days post-embolization for the survival study, respectively. At necropsy, the VX2 tumor-bearing liver was exposed, photographed and liver tissues were harvested with other major organs (liver, heart, lung, kidney, and spleen) for fluorescence imaging and histological analysis. Ex vivo IVIS imaging of intact and bisected tumor-bearing livers was performed and analyzed using Living Image^® software.

Ex vivo evaluation of diffusion and ablation in human cancer tissue

Freshly explanted human kidney cancer tissues were injected with 20-30 μL of ILE14 into the central area of the tumor mass using a 30-gauge needle. The treated tissue samples were then placed in a humidified chamber and incubated in a complete RPMI 1640 medium at 37°C. After 1, 12, and 24 h of injection, the samples were subjected to IVIS imaging and then processed for histological analysis.

Bacteria susceptibility test

The bactericidal efficacy of ILE14 was tested using a green fluorescent protein (GFP) reporter-labeled Escherichia coli (E. coli) (25922GFP, ATCC) and patient blood culture isolates. GFP-E. coli was cultured overnight in a Luria-Bertani (LB) broth (Fisher Scientific) at 37°C/ 230 rpm. Cultured GFP-E. coli was diluted in fresh LB broth to ~0.7 OD₆₀₀ and serially diluted ILE14 was added to the bacteria solution for further incubation. After 24 h incubation at 37°C/ 230 rpm, the samples were collected, and the fluorescence of bacteria suspension was evaluated using IVIS and a microplate reader. For patient-derived pathogens, the stock was diluted to 0.5 McFarland standard absorbance (1.5 x 10⁸ CFU/mL) in 0.45% saline solution tubes (Remel, San Diego, CA). In a 96-well plate, 100 μL of bacteria solution and 100 μL of serially diluted (2⁰ to 2¹⁰–fold) ILE14 were mixed (1:1 v/v). Following incubation at 37 °C (C. difficile for 48 h; the rest for 24 h), the mixes were directly plated on sheep blood agar plate (Remel) using cotton-tipped applicators (MEDLINE, Northfield, IL) and further incubated to assess bacterial susceptibility. Susceptible ILE14 concentrations exhibited no bacterial growth, intermediate ILE14 concentrations showed decreased bacterial colonies compared to the control, and resistant ILE14 concentrations showed comparable bacterial growth to the control.

Cover suggestion:

The image demonstrates the Ionic Liquid Embolic exiting the tip of a catheter filling the arteries of the tumor. The purple color depicts the ablation of the vessel wall and the adjacent tumor tissue as the ionic liquid diffuses. As the ionic liquid diffuses trans-arterially, it carries its payload, which in this case is Nivolumab, an immunotherapy drug. Along the margins of the tumor ablation, Nivolumab is seen interacting with cells depicting its inhibition of tumor suppression of the immune system.