Intracranial Gene Delivery Mediated by Albumin-Based Nanobubbles and Low-Frequency Ultrasound

Abstract

Research in the field of high-intensity focused ultrasound (HIFU) for intracranial gene therapy has greatly progressed over the years. However, limitations of conventional HIFU still remain. That is, genes are required to cross the blood-brain barrier (BBB) in order to reach the neurological disordered lesion. In this study, we introduce a novel direct intracranial gene delivery method, bypassing the BBB using human serum albumin-based nanobubbles (NBs) injected through a less invasive intrathecal route via lumbar puncture, followed by intracranial irradiation with low-frequency ultrasound (LoFreqUS). Focusing on both plasmid DNA (pDNA) and messenger RNA (mRNA), our approach utilizes LoFreqUS for deeper tissue acoustic penetration and enhancing gene transfer efficiency. This drug delivery method could be dubbed as the "Spinal Back-Door Approach", an alternative to the "front door" BBB opening method. Experiments showed that NBs effectively responded to LoFreqUS, significantly improving gene transfer in vitro using U-87 MG cell lines. In vivo experiments in mice demonstrated significantly increased gene expression with pDNA; however, we were unable to obtain conclusive results using mRNA. This novel technique, combining albumin-based NBs and LoFreqUS offers a promising, efficient, targeted, and non-invasive solution for central nervous system gene therapy, potentially transforming the treatment landscape for neurological disorders.

Keywords: central nervous system; drug delivery system; low-frequency ultrasound; nanobubble.

Figure A1

Skin damage caused by LoFreqUS irradiation. (A) Immediately after sonication. (B) After 24 h.

Figure 1

Schematic representation of preparation of albumin-based NBs solutions. (A) Gas replacement with perfluoropropane gas (white arrow 1) and injection of distilled water or opti-MEM containing 0.12% HSA (white arrow 2) into vial. (B) Pressurization by addition of perfluoropropane gas into a vial sealed with a rubber stopper and aluminum cap (white arrow). (C) Bubbling by high-speed shaking. (D) Cooling for bubble stabilization. (E) Pressure relief inside the vial (white arrow). (F) Centrifugation (gray arrow) to float and burst microbubbles (white arrow). (G) Recovery of solution containing NBs from opened vials. (H) Schematic of the structure of albumin-based NBs. NBs: nanobubbles. HSA: human serum albumin. C₃F₈: perfluoropropane gas.

Figure 2

Schematic representation of ultrasound treatment of albumin-based NBs or sonoporation in 96-well plates. (A) Sonication treatment of NBs by applying ultrasound to the medium. (B–F) Sonoporation method. (B) Incubation medium is removed from the well (white arrow) of a 96-well plate seeded with malignant glioma cells (U-87 MG). (C) Each well is filled with medium containing NBs (white arrow). (D) Transfection by ultrasound. pDNA or mRNA is transferred into the cytoplasm (white arrow). (E) Aspiration of sonicated medium (white arrow 1) and (E) addition of fresh incubation medium (white arrow 2). (F) After 24 or 48 h of incubation, the supernatant was collected for reporter assay. (G) Arrangement of wells with seeded cells (indicated by color) on a 96-well plate. NBs: nanobubbles.

Figure 3

Overview of in vitro ultrasound imaging of albumin-based NBs. (A) LoFreqUS irradiation into A-NBs solution. (B) Acoustic evaluation using a diagnostic US imaging system. NBs: nanobubbles, US: ultrasound.

Figure 4

Overview of intrathecal lumbar puncture and in vivo sonoporation. (A) Posture of a mouse in which a lumbar puncture was performed. (B) Schema of sonication method to the cranial region of the mouse.

Figure 5

Physical properties of NBs at different ultrasound exposure times. (A) Size distribution of NBs on NTA. (B) FCM measurement NBs size distribution and concentration after 40-fold dilution. Yellow or gray shaded area indicates NBs size less than 200 nm or more than 500 nm, respectively. NBs: nanobubbles.

Figure 6

Visualization of albumin-based NBs using ultrasound diagnostic equipment. (A) Image of NBs solution before LoFreqUS irradiation. (B) Image of NBs solution after LoFreqUS irradiation. (C) Image of control solution. NBs: nanobubbles.

Figure 7

pDNA or mRNA transfection efficiency by sonoporation using different irradiation times. (A) Ultrasonic irradiation time-dependent profile of pDNA luciferase expression in the condition with or without albumin-based NBs. (B) Ultrasonic irradiation time-dependent profile of mRNA luciferase expression in condition with or without NBs. Data are presented as mean ± standard error of the mean (s.e.m.). Statistical significance was assessed using unpaired t-test (* p < 0.05, ** p < 0.01, *** p < 0.001) (n = 4). NBs: nanobubbles, RLU: relative luminescence units.

Figure 8

Cell viability after sonication. Cell viability with and without NBs and after different ultrasound exposure times, data are presented as mean ± standard error of the mean (s.e.m.). Statistical significance was assessed by unpaired t-test (n.s. not significant) (n = 4). NBs: nanobubbles.

Figure 9

Distribution of intrathecal dye after administration via lumbar puncture. (A) Overall view of the cranial and nasal cavity with the skull removed (100 μL dye injected; arrow indicates the turbinates). (B) Whole brain viewed from below. (C) Coronal section of the cerebrum in the region anterior to the hypothalamus (50 μL dye injected; arrow indicates the anterior cerebral artery). (D) Coronal section of the cerebrum in the center of the hypothalamus (50 μL dye injected; arrow 1 indicates the lateral ventricle, arrow 2 indicates the hypothalamus).

Figure 10

Intracranial luciferase expression in mice after intrathecal injection of pDNA. (A) Representative IVIS images of mice one day after sonoporation. (B) Quantification of luciferase expression of the cranial region over time based on IVIS images. Data are presented as mean ± standard error of the mean (s.e.m.). Statistical significance was determined using a one-way ANOVA (** p < 0.01, *** p < 0.001), NBs+US− (n = 4), NBs−US+ (n = 6), NBs+US+ (n = 5). NBs: nanobubbles, US: ultrasound.

Figure 11

Intracranial Luciferase expression in mice after intrathecal injection of mRNA. (A) Representative IVIS images of mice one day after sonoporation. (B) Quantification of luciferase expression of the cranial region on IVIS images. Data are presented as mean ± standard error of the mean (s.e.m.). Statistical significance was determined using a one-way ANOVA (n.s. not significant), NBs+US− (n = 3), NBs−US+ (n = 4), NBs+US+ (n = 4). NBs: nanobubbles, US: ultrasound.