Salicylic acid analogues as chemical exchange saturation transfer MRI contrast agents for the assessment of brain perfusion territory and blood–brain barrier opening after intra-arterial infusion

Abstract

The blood–brain barrier (BBB) is a major obstacle for drug delivery to the brain. Predicted, focal opening of the BBB through intra-arterial infusion of hyperosmolar mannitol is feasible, but there is a need to facilitate imaging techniques (e.g. MRI) to guide interventional procedures and assess the outcomes. Here, we show that salicylic acid analogues (SAA) can depict the brain territory supplied by the catheter and detect the BBB opening, through chemical exchange saturation transfer (CEST) MRI. Hyperosmolar SAA solutions themselves are also capable of opening the BBB, and, when multiple SAA agents were co-injected, their locoregional perfusion could be differentiated.

Introduction

Effective treatment of brain tumors is challenging due to the inability of many potentially useful drugs and other therapeutics to cross the blood–brain barrier (BBB). ¹ Concurrently, it has also been shown in a mouse model that the BBB is still a functional and limiting factor for solute and drug penetration to and around the tumor. ² Recently, there has been enormous progress in developing catheter-based injection paradigms that allow safe and selective interventional neuroradiology procedures to target selected brain regions including opening the BBB osmotically for improved drug delivery to tumors.^3,4 Cognitive testing has been performed following intra-arterial (IA) osmotic opening of the BBB with no cognitive decline detected, and as such this procedure is considered safe. ⁵ The IA route of drug delivery offers the possibility of high-dose, spatially limited chemotherapy, but to date the reproducibility and predictability of the BBB opening has been disappointing. ⁶ This variability is due to the complex and dynamic vascular supply of the brain, which may result in a variable trans-catheter perfusion area from procedure to procedure despite similar catheter locations. The accuracy of IA injection is of paramount importance for a safe and successful treatment with the goal of targeting the entire area involved by the brain tumor while protecting the surrounding healthy brain tissue from the toxic effects of drugs. The application of imaging techniques for the visualization of trans-catheter perfusion is a logical solution; however, the X-ray fluoroscopy typically used for neurointerventional procedures lacks the sensitivity to determine the territory of parenchymal flow. Performing interventional neuroradiology procedures under MRI guidance would be beneficial and reduce patient exposure to ionizing radiation. Several research groups have previously shown that MR monitoring of the BBB-opening^7,8 and perfusion territory ⁹ can be achieved using iron oxide particles as contrast agents. Gadolinium (Gd)-based MR agents have also been employed for monitoring cerebral blood flow and BBB opening by comparing pre-contrast and post-contrast images at clinical field strengths. However, these relaxation-based agents directly interfere with anatomical and functional MR images, and also persist for a prolonged time in the tissue, making the repetitive scanning necessary to evaluate procedural progress complicated. Furthermore, the safety of Gd-based agents has become a concern for patients with renal dysfunction due to the increased risk of Nephrogenic Systemic Fibrosis (NSF) which has been detected when linear chelates were administered.^10–12 More recently, the FDA issued an alert regarding the potential risk of brain deposits following repeated use of Gd-based agents.^13,14 In this context, it is important to work towards developing alternatives to Gd-based agents. MR-based interventional neuroradiology procedures aimed at opening the BBB would benefit from an MR contrast mechanism that enables differentiation of multiple imaging agents, and which would enable the collection of up-to-date information on perfusion territory changes due to catheter tip repositioning or manipulation of infusion velocity during the BBB opening and subsequent drug delivery. ⁹ Contrast agents of various molecular sizes are also of interest, as access of the drug to tumor or brain parenchyma even after BBB disruption is highly dependent on the molecular weight. ¹⁵

Chemical exchange saturation transfer (CEST) is an emerging contrast mechanism which amplifies the signal from low concentration bioorganic compounds based upon applying frequency-selective saturation pulse(s) on resonance with exchangeable protons on these compounds destroying their signal. Because proton exchange occurs a number of times throughout the saturation period, a large signal loss can be transferred to the surrounding water per exchangeable proton and then detected. This frequency-selective saturation also allows for discrimination of several contrast agents, similar to multicolor fluorescent imaging. ¹⁶ Another benefit of using CEST contrast is that it is “switched on” by the saturation pulse, allowing collection of standard T1/T2 weighted images which are minimally perturbed by the presence of CEST agents in contrast to what occurs when employing relaxation-based agents. There have been a number of pre-clinical and clinical applications using CEST MRI. Endogenous CEST has been used to detect tumors, assess their response to therapy,^17–19 and to monitor glycosaminoglycan ²⁰ and creatine ²¹ levels. The development of specialized CEST agents further strengthens the capabilities of CEST, such as for imaging tissue pH, ²² monitoring transplanted cell viability ²³ and detecting viral infection during oncolytic virotherapy. ²⁴

Recently, substantial progress has been made in the search for the best natural compound as CEST agents. One important discovery was that compounds which feature IntraMolecular-bond Shifted HYdrogens (IM-SHY) give very strong CEST contrast due to their properly tuned exchange rates and large frequency offsets from water and from endogenous brain metabolites (Figure 1(a)), facilitating their detection on clinical 3 T scanners. Salicylic Acid Analogues (SAA) are members of this group, which are potentially safe in relatively high doses and have well-documented pharmacokinetics.^25,26 Additionally, progress has been made in the refinement of CEST imaging sequences for detecting these compounds. In this work, we have evaluated the performance of these new CEST agents and imaging methods for monitoring IA delivery in rodents at 11.7 T. In particular, we employ three SAA: 2,4-dihydroxybenzoic acid (DHB); sodium salicylate (SA); and 2,5-dihydroxyterephthalic acid (DHTA), which have different characteristic proton frequencies. We have tuned our MeLOVARS method^27,28 for detection of these SAA agents and demonstrate that this methodology enables visualization of perfusion territory without BBB disruption and after mannitol infusion to produce a local disruption of the BBB. Finally, we demonstrate that hyperosmolar preparations of these agents can open the BBB via internal cerebral artery (ICA) infusion, and demonstrate that CEST imaging can differentiate the perfusion territories of DHB and DHTA after serial administration of these agents.

Figure 1.

Dynamic CEST-MR imaging, upon infusion of DHB (Δω = 9.6 ppm). (a) CEST MTR_asym spectra for the 3 Aspirin analogues (25 mM, pH7), with peak frequency offsets easily separated from offsets of tissue endogenous contrast; (b) MR experimental protocol. (c) An example of the dynamic CEST MTR_asym images at multiple time points: pre-infusion, during infusion, Post-3 min and Post 15 min, with significant enhancement only shown during infusion. (ΔMTR_asym between the enhanced region and the control region is > 5%) (d) Upper row: dynamic T2* weighted EPI images with IA infusion of Feraheme®, at three time points: pre-, during- and post-1 min of infusion. The during-infusion image shows the vessel perfusion with hypointensity (white arrows); middle row: the dynamic CEST MTR_asym images pre-infusion, during infusion and post-15 min; bottom row: Two T2w Spin Echo images acquired at pre- and post-22 min of CEST agent infusion, which show no changes; (e) the kinetic time course for three rats with IA infusion of DHB, where at post-45 s there are significant differences of the CEST signal with *P < 0.01. (Blue: perfusion territory during infusion. Black: contralateral hemisphere as a negative control). Note that the CEST contrast quickly dropped to baseline after infusion has been discontinued.

Materials and methods

Animal preparation

Experiments were performed in accordance with guidelines for the care and use of laboratory animals approved by the Institutional Animal Care and Use Committee of Johns Hopkins University, Baltimore, MD and reported according to the ARRIVE (Animal Research: Reporting In Vivo Experiments). The procedure of intra-arterial infusion has been described elsewhere. ¹³ Briefly, rats (Spraguey–Dawley, n = 5) were anesthetized with 1–2% isoflurane and after supine positioning paramedian skin cut was performed to expose carotid arteries unilaterally. Then, pterygopalatine artery (PPA) and external carotid artery (ECA) were ligated, common carotid artery (CCA) was incised, and the catheter was introduced into internal carotid artery (ICA) and fixed to the arterial wall. Immediately later the animals were transferred to the MR scanner and positioned on a temperature controlled bed and underwent imaging. The MRI compatible pump (Harvard apparatus) has been for infusion of compounds. After the procedure, the animals were sacrificed.

Agents preparation

Three IM-SHY agents were tested via intra-arterial delivery in rats: DHB, a metabolite found in human plasma after cranberry juice consumption with characteristic frequency from H₂O (Δω) = 9.6 ppm; SA, the main metabolite of Aspirin, Δω = 9.3 ppm; DHTA, with two exchangeable protons per molecule, Δω = 8.3 ppm (Figure 1(a) illustrates their frequency spectra). The agents were dissolved in ultrapure water at a concentration of 25 mM for DHB and SA, and 12.5 mM DHTA, and titrated using high concentration HCl/NaOH to pH = 6.9–7.2 and osmolality of 290 mosmol/kg. The total dose which was injected was ∼11 mg/kg bodyweight. For BBB opening, we used two agents: the clinical grade 25% mannitol (APP) (∼1.5 g/kg body weight via IA infusion) and another batch of concentrated DHB (100 mM) and DHTA (50 mM) was prepared at a osmolality of ∼ 650 mosmol/kg through dissolving these in sterilized PBS at 100 mM and titrating to pH = 6.9–7.2.

MR imaging

Animals were placed in a Bruker Biospec 11.7T scanner, with a 72 mm volume coil as transmitter and a 4-channel phase-array surface coil receiver. Following acquisition of baseline T₁ and anatomical T₂ MR scans, Feraheme® particles containing 0.03 mg Fe (a T2 agent) were infused at different flow rates (from 200 µl/min up to 600 µl/min with increment of 50 µl/min) for 20 s each, to predict IA transcatheter perfusion territory in rat brain. The wash-in and wash-out of agents was monitored for up to 3 min using a dynamic fast GE-EPI sequence (two-segment EPI, FOV 31 × 23 mm, matrix 96 × 64, slice thickness 1 mm, number of averages = 1, TR/TE = 1000 ms/100 ms, flip angle = 60°). Based on the reduction of signal intensity, an optimized flow rate with efficient distribution to the brain parenchymal was chosen for infusion of the chosen SAA.

Upon clear-up of the Feraheme®, DHB, and SA (25 mM, with a total dose of ∼11 mg/kg) were infused in series with the optimized infusion flow rate for 60–90 s. For each agent, Figure 1(b) illustrates the imaging protocol including two T2w images and several blocks of dynamic CEST (Dyn-CEST) monitoring from pre-infusion to post-infusion for > 15 min. CEST images were rapidly acquired using MeLOVARS sequences which contains four modules with each Sat. Pulses of 0.6 s in length (B₁ = 4 uT), followed by an EPI readout. With each image costing 7 s to 14 s, the DynCEST block collects images at six saturation offsets (Δω): [±9.6, ± 9.3, ± 9.0] ppm for DHB and SA, [±8.4, ±8.1, ±7.9] ppm for DHTA. This allows higher temporal resolution during and right after injection. To clearly illustrate the frequency-specific profiles of CEST spectra, at both pre- and > 7 min post-infusion, two longer blocks were also acquired, with more images at 12 offsets of [±9.9, ±9.6, ±9.3, ±9, ±8.7, ±8.4] ppm for DHB and SA, while with [±9, ±8.7, ±8.4, ±8.1, ±7.8, ±7.5] ppm for DHTA. For the multi-color imaging, 16 images were acquired with the frequency offsets ranging from 7.5 ppm to 9.9 ppm. The other parameters are: TR/TE = 3.6 s/5 ms, α = 25°, 2 - or 4-segment EPI (7.07 ms per segment), α = 25°, FOV = 30 × 22 × 1 mm, matrix size = 96 × 64. A WASSR image-set (80 s) was also collected for B0 mapping and corrections.

At the end of experiments, 400 µl Gd-DTPA (Magnevist®, 1:10 diluted) was injected i.v. to check the BBB disruption, using a T1w RARE sequence with TR/TE = 250 ms/9 ms and the same geometric parameters as before.

Post-processing

All data were processed using custom-written MATLAB scripts. CEST images were produced by using asymmetry in magnetization transfer (MTR_asym) with the contrast quantified for the Nth module in MeLOVARS, by MTR_asym (N, Δω) = (S^N_−Δω–S^N_+Δω)/ S^N_−Δω. The final contrast was calculated by averaging Module 2 to Module 4 in order to maximize the CNR. Furthermore, the dynamic CEST map at each time point was generated by averaging the MTR_asym values of three offsets around the characteristic Δω of the agent. The pseudo-colored CEST MTR_asym images were also overlapped on the gray-scaled anatomical image to further illustrate the enhanced region. For ROI analysis in Figure 1(e), the perfused regions were determined on the during-infusion CEST images, which includes the ‘highlighted’ voxels with MTR_asym >4% larger than the averaged MTR_asym_pre. The control regions were then drawn in the contralateral side with the same anatomy as the perfused regions. For the final plot, the averaged CEST contrast of the perfused region = pre-perfusion was normalized to zero. The CEST images during infusion and the T2*w EPI image were then co-registered (MATLAB function ‘imregister’), with only the brain region selected and intensity normalized in the range from 0 to 1. To convert the negative contrast to a positive one, the intensity of each pixel on T2*w image was transformed to its reciprocal. The correlation between enhancements by the CEST image and the T2*w image were then estimated using MATLAB function ‘corr2’.

Results

Depiction of brain territory supplied by endovascular catheter

In our initial experiments, we were interested in determining the amount of contrast that would be produced after IA of these SAA (Figure 1(a)). Pre-infusion CEST images were collected which displayed very low contrast at the characteristic frequencies of these agents and were also fairly homogeneous using the protocol shown in Figure 1(b). During IA infusion, a portion of the ipsilateral hemisphere was highlighted by >5% CEST signal at a dosage of ∼11 mg/kg of DHB (Figure 1(c)), and the signal decayed to the pre-injection level between 3 and 15 min after the infusion was discontinued. This confirms that the agent has sufficient sensitivity and that CEST MRI is capable of depicting the brain territory supplied by the catheter. To further evaluate the specificity of the proposed method, we performed a control experiment, with infusion of 0.9% saline and there was no CEST enhancement (Supplementary Figure 1). In the subsequent experiments, we infused the iron oxide-based agent, Feraheme®, and acquired dynamic T2*-weighted EPI images to determine trans-catheter perfusion territory as we previously published. ⁹ DHB were then infused for dynamic CEST imaging to compare the territories visualized by Feraheme® and by DHB. As indicated by the white arrows in Figure 1(d), infusion of Feraheme and infusion of DHB from the same catheter resulted in similar perfusion regions. We further performed a quantitative comparison (methods are described in the “post-processing” session in Supplementary. Information), which further confirmed the strong spatial correlation (r = 0.861, P < 0.05) of the brain territories enhanced by CEST and by T2* MRI. In addition, the administration of DHB did not affect the T1/T2 contrast, as indicated by T2w spin-echo-based images acquired pre- and post-CEST MRI which are almost identical with both displaying thin streaks generated by the remaining Feraheme®. This property is an advantage of SAA for repeated interventional radiological procedures. Furthermore, the fast wash-out of the agent is confirmed by the kinetic time course (Figure 1e) of the averaged MTR_asym, which also clearly showed for three rats that the CEST signal first reached 5% during the infusion of DHB (*P < 0.01 compared to control region) and then quickly decayed to baseline. We also tested infusion of a 25 mM SA solution with a neutralized pH, which showed decay patterns similar to those with DHB (Supplementary Figure 2).

Detection of BBB-opening territory with IM-SHY CEST agents

Next, we moved to evaluating whether we could detect local BBB disruption. The experimental protocol is shown in Figure 2(a), with mannitol employed for opening the BBB. CEST MRI shows that there is an enhancement in the ipsilateral hemisphere during DHB-infusion and that enhancement at the lateral region persisted at 5 min, and also 22 min post DHB infusion (Figure 2(b)). For comparison, there was almost no enhancement after infusion at these timepoints without mannitol (Figure 1). After completing the CEST imaging protocol, Gd-DTPA was subcutaneously injected and the post-injection T1w image showed hyperintensity in the ipsilateral hemisphere regionally, corresponding to the enhancement on CEST MRI and validating the BBB opening. Figure 2(b) also displays two high-resolution T2w images of the same slice, which were acquired pre- and post-20 min of DHB infusion, respectively, indicating that there was no tissue damage.

Figure 2.

Monitoring of BBB opening and multi-color imaging. Panel a. the CEST experimental protocol, with mannitol for inducing BBB disruption . Panel b. Rows 1and 2: the four pseudo-colored CEST MTR_asym contrast maps overlapped on anatomical image, at time point of pre-infusion, during-infusion and post-5 min, post-22 min of 25 mM DHB, respectively. Row 3: High-resolution T2w images acquired pre- and post-20 min of DHB infusion. Row 4: pre-Gd T1 image and post-Gd T1w acquired after CEST imaging, with the region of hyperintensity indicating the BBB disruption. Panel c. Two-color CEST images and spectra at Time Point (TP) 1. Upper: Two-color CEST images at 9.6 ppm (frequency of DHB) and 8.4 ppm (frequency of DHTA), respectively, which monitor the two agents simultaneously using two different color coding. Lower: the MTR_asym spectra for 3 ROIs as marked on e, where both ROI 1&2 peaks at 9.6 ppm with ROI 1 show > 20% signal and there were no peaks for the Left control region (ROI 3). Note that ROI 2 has almost no signal at 8.4 ppm. Panel d. Two-color CEST images and spectra at TP 2. Same layout as d, at TP2 after infusion of DHTA, ROI 1 still peaks at 9.6 ppm while ROI 2 peaks at 8.4 ppm with a ∼16% signal. Panel e. The kinetic time-course of CEST signals, following infusion with DHB (100 mM) and DHTA (50 mM) with high osmolality, where arrows show infusion time points of two agents. The prolonged stay of agents within brain parenchyma indicates the BBB disrutpion. Panel f. pre-Gd T1w and post-Gd_T1w images acquired after CEST imaging, validating the opened BBB.

While hyperosmolar mannitol has previously been employed for BBB opening, it would be advantageous to use a contrast agent, as this would eliminate the need for two separate infusions (the first to open and the second to visualize). We calculated that a higher concentration IM-SHY agent solution (100 mM DHB, equivalent to ∼40 mg/kg, still far below an 800 mg/kg toxic dosage ²⁹ ) could result in a suitable solution of hyperosmolality (∼650 mosmol/kg) that should enable BBB opening. In contrast to the fast wash-out of the agents seen in Figure 1, the CEST contrast images (Supplementary Figures 2(c) and (d) and 3) and the ROI time course data (Figure 2(e)) revealed a prolonged persistence with both DHB and DHTA, with the opened BBB region validated by a Gd-enhanced T1w image (Figure 2(f)).

Furthermore, although utilization of a single contrast agent to demonstrate the BBB opening is useful as it reports BBB status at a given moment, it does not reflect the dynamic changes occurring within the BBB. For that purpose, multiple agents with distinct enhancement patterns would be required. We tested the feasibility of that approach by the injection of two CEST agents sequentially, i.e. DHTA was infused 23 min post-DHB infusion. We used the blue-tone images to display CEST contrast at 9.6 ppm (characterized frequency for DHB), and the red-tone images for 8.4 ppm (Characterized frequency for DHTA). CEST images along with the spectra for three regions of interest (ROI) demonstrated that specific detection of two agents is feasible, which was the case after DHB infusion (Figure 2(c), Time Point (TP) 1) and after DHTA infusion (TP 2). The different accumulation of each of these agents could be easily visualized on the CEST images, where there was predominantly DHB enhancement at TP 1 and both DHB and DHTA appeared at TP 2 with a distinct enhanced region. The differences are more evident when comparing the frequency-dependent CEST spectra for the three ROIs, with ROI1 displaying peak contrast at the characteristic frequency for DHB, ROI2 displaying peak contrast at the characteristic frequency for DHTA at TP2 and ROI3 without contrast (Figure 2(c) and (d)). More quantitatively, at TP 1 both ROI 1 and 2 peaked at 9.6 ppm, with >20% contrast present at ROI 1 and only ∼6% present at ROI 2 indicating different concentrations; at TP 2, ∼23 min after DHTA infusion, ROI 1 still peaked at 9.6 ppm, while ROI 2 peaked at 8.4 ppm with a ∼16% signal. The width of the contrast curves was such that there was some contamination of DHB at 8.4 ppm and DHTA at 9.6 ppm; however, general trends can still be determined. Furthermore, it is also worth mentioning that the post-Gd T1 in Figure 2(f) shows an enhancement pattern similar to that of the right cortex in the CEST image at TP 2 (Figure 2(d) at 8.4 ppm), which may reflect an evolution of the opened-BBB region during this procedure. There is also a third region responding to the location of the lateral ventricle, which highlighted after administration of both CEST contrast agents and Gadolinium, we postulate that this is due to “stealing” of blood by plexus choroideus with subsequent BBB opening and leak of contrast agents to CSF.

Image acquisition using MeLOVARS only requires 14 s per offset, with a spatial resolution of 0.3 mm. In addition, four modules of MeLOVARS readout clearly show the feature of the increasing CEST peaks as saturation lengths (Supplemental Figure S4), where the CEST image from modules 2, 3 and 4 could be added up and increase the CNR by√3 times.

Discussion

In this study, we have shown that SAAs can be used as CEST contrast agents along with the MeLOVARS sequence to visualize of the territory of parenchymal flow supplied by an IA catheter at 11.7 Tesla. The combination was shown to provide sufficient sensitivity and speed in detection of focal BBB opening. Three SAA solutions were infused, each with pH = ∼7.4 to avoid the unionized molecules crossing BBB in acidic environment (with their cLogD numbers shown in Supplementary Information Table S1).^30,31 For both DHB, a fruit metabolite, and SA, an aspirin metabolite, the ∼11 mg/kg dose is far below the reported toxic dose for rodents as well as for humans (800 mg/kg oral DHB for rodents ²⁹ and 700 mg/kg oral sodium salicylate for humans ³² ). For DHB, the dosage in hyper-osmolar formulations (∼40 mg/kg) is also well below the reported toxic doses. Only very high doses >200 mg/kg may lead to some changes in cerebral blood flow and metabolism. ³³ The dynamic CEST imaging could be further accelerated to several seconds per time-point by acquiring only one offset, ³⁴ which will enable more accurate kinetic analysis. In addition, we have demonstrated recently that, SAA possess suitable exchange properties allowing their detection at similar concentrations to those used in this study on 3 Tesla clinical scanners. ³⁵

Currently, there is no clinically approved MR contrast agent for IA delivery, which is one obstacle to the transfer from X-ray-based to MR-based interventional neuroradiology. Gadolinium is registered for intravenous delivery, but due to toxicity and hyperosmolarity, it does not appear to be an optimal agent for IA applications to the brain. We have observed respiratory depression and deaths of experimental animals after administration to vertebral and basilar arteries (unpublished data). Several clinical formulations of iron oxide nanoparticles have been discontinued and are no longer commercially available, although one formulation (Feraheme) is approved for intravenous treatment of patients with anemia at high doses. A major advantage of Feraheme is the isoosmolarity and high sensitivity that provides sufficient contrast at 0.03 mg Fe/ml, which is 1/1000 of the original preparation. Also, iron is naturally metabolized by humans, and thus small doses are not toxic. However, its presence (if not cleared) disturbs both anatomical and functional images of the brain. Thus, CEST contrast agents, which are safe and invisible on regular T1/T2 weighted images, are very promising for evaluating the brain territory supplied by catheters as well as the territory with an open BBB. IM-SHY agents can also be prepared as hyperosmotic solutions and used alone for BBB opening, but the safety profile of such formulations needs to be confirmed. Last, various IM-SHY compounds exchange protons with water at different offset frequencies, thus allowing for differentiation via CEST imaging. In our case, we observed the BBB opening mostly in striatal regions after ICA infusion of DHB, and subsequently a delayed infusion of DHTA displayed additional CEST contrast in cortical regions. This is probably due to changes in blood flow between particular arterial branches. One might even speculate that BBB opening could restrict the blood flow within an area and redirect blood flow to cortex. Gd-DTPA showed enhancement that corresponded to a mostly DHTA distribution, which might indicate that the BBB has already been re-sealed in the striatal area with entrapment of DHB in the brain parenchyma. Based on these results, 3-4 IM-SHY compounds might be detected simultaneously, giving an additional flexibility in monitoring the time-course of BBB status during an interventional radiology procedure, capitalizing on the sequential injection of multiple agents with different characteristic frequencies.

Conclusion

In conclusion, we have described a new method with which to visualize the perfusion territory after IA infusions using MRI and small organic compounds. We have identified three agents that perform well for this purpose: SA; DHB; and DHTA; and have evaluated these agents with and without BBB opening. Based on these results, CEST MRI may facilitate the widespread application of MR-guided BBB opening using interventional neuroradiology techniques followed by immediate super-selective, high-dose drug administration for the treatment of a wide range of diseases including oncological and neurodegenerative disorders.