Ultrasound-induced microbubble (USMB) cavitation is widely used to promote drug delivery. Our previous study investigated USMB targeting the round window membrane by applying the ultrasound transducer to the tympanic bulla. In the present study, we further extended the use of this technology to enhance drug delivery to the inner ear by introducing the ultrasound transducer into the external auditory canal (EAC) or applying it to the skull. Using a 3-dimensional–printed diffusion apparatus mimicking the pathway for ultrasound passing through and reaching the middle ear cavity in vitro, the models simulating the transcanal and transcranial approach demonstrated 4.8-fold– and 3.7-fold–higher delivery efficiencies, respectively. In an in vivo model of guinea pigs, by filling tympanic bulla with microbubbles and biotin-FITC, USMB applied transcanally and transcranially induced 2.8-fold and 1.5-fold increases in biotin-FITC delivery efficiencies, respectively. In addition, the gentamicin uptake by cochlear and vestibular hair cells and gentamicin-induced hair cell loss were significantly enhanced following transcanal application of USMB. On the 28th day after transcanal USMB, safety assessment showed no significant changes in the hearing thresholds and the integrity of cochlea. These are the first results to our knowledge to demonstrate the feasibility and support the potential clinical application of applying USMB via EAC to facilitate drug delivery into the inner ear.

Minoxidil (Mx) is a conventional drug for treating androgenetic alopecia, preventing hair loss, and promoting hair growth. The solubility of Mx has been improved using chemical enhancement methods to increase its skin permeability over the long term. This study created a new ultrasound (US) contrast agent—albumin-shelled microbubbles (MBs) that absorb chitosan oligosaccharide lactate (COL) and Mx—and combined it with sonication by US energy in the water phase to enhance hair growth while shortening the treatment period. COL and Mx grafted with MBs (mean diameter of 1480 nm) were synthesized into self-assembled complexes of COL-MBs and Mx-COL-MBs that had mean diameters of 4150 and 4500 nm, respectively. The US was applied at 3 W/cm² for 1 min, and combined with Mx-COL-MBs containing 0.3% Mx. The diffusion of Mx through the dialysis membrane from Mx-COL-MB during US (US+Mx-COL-MB) was more rapid at pH 4 than at pH 7.4, which is favorable given that the environment of the scalp is mildly acidic (pH=4.5-5.5). In Franz diffusion experiments performed in vitro, the release rates at 18 hours in the US+Mx-COL-MBs and US+MBs+Mx groups resulted in 2.3 and 1.7 times the penetration and deposition, respectively, of Mx relative to the group with Mx alone. During 21 days treatment in animal experiments, the growth rates at days 10 and 14 in the US+Mx-COL-MBs group increased by 22.6% and 64.7%, respectively, and there were clear significant differences (p<0.05) between the US+Mx-COL-MBs group and the other four groups. The use of US+Mx-COL-MB in the water phase can increased the effects of Mx so as to shorten the telogen phase, and also increase both the diameter of keratinized hair shafts and the size of hair follicles without causing skin damage.

The transdermal delivery of a wide range of high-molecular-weight drugs is limited by the stratum corneum layer of the epidermis representing a significant barrier to penetration across the skin. This study first determined the different effects of different-size ultrasound (US) contrast agents and microbubbles (MBs) for enhancing the transdermal delivery of high-molecular-weight drugs. The effects of US-mediated different-size (1.4, 2.1, and 3.5 μm) MBs (as a contrast agent) and ascorbyl tetraisopalmitate (VC-IP) on enhancing skin transdermal delivery were demonstrated both in vitro and in vivo. The results indicated that at a power density of 3 W/cm² the penetration depth in group US combined with 3.5-μm MBs and penetrating VC-IP (U+3.5) was 34% and 14% higher than those in groups US combined with 1.4-μm MBs and penetrating VC-IP (U+1.4) and US combined with 2.1-μm MBs and penetrating VC-IP (U+2.1), respectively, for the agarose phantoms, while the corresponding increases for pigskin were 37% and 19%.In terms of the skin permeation of VC-IP, the VC-IP concentration in group U+3.5 was 23% and 10% higher in than those in groups U+1.4 and U+2.1, respectively. The whitening effect (luminosity index) of mice skin in group U+3.5 had increased (significantly) by 28% after 1 week, by 34% after 2 weeks, and tended to stabilize after 3 weeks (45%) in C57BL/6J mice over a 4-week experimental period. The results obtained in this study indicate that combining US with MBs of different sizes can produce different degrees of skin permeability so as to enhance the delivery of VC-IP to inhibit melanogenesis, without damaging the skin in mice.

The feasibility of ultrasound (US) controlled cavitation for transdermal drug delivery (TDD) using gas-filled microbubbles (MBs) has been explored. However, liquid or gel-type MBs is not easy used for TDD. The present study investigated a new treatment model for evaluating the US-mediated liquid-type epidermal growth factor (EGF)-coated lysozyme microbubble (LYMB) cavitation in a wound dressing for enhancing wound healing. The maximum loading efficacy of EGF onto LYMBs was 19.40 ± 0.04%. In terms of the in vitro treatment efficacy, the growth of Staphylococcus aureus was inhibited by 97.50 ± 1.50% in the group with LYMBs exposed to 3 W/cm² US. During 21 days in vivo wound healing experiments, the recovery rate during the first 6 days was significant higher in the group with EGF-LYMB dressings and US exposure (day 6: 54.28 ± 3.26%) than in the control group (day 6: 26.36 ± 3.34%) (p < 0.05). Our results show that the new model can significantly reduce the treatment duration during wound healing.

Ultrasound (US) has been found to rejuvenate and invigorate the hair follicles, increase the size of hair shafts, and promote new hair growth. Our present study found that dual-frequency US-mediated microbubble (MB) cavitation significantly enhanced minoxidil (Mx) delivery in both in vitro and in vivo models, while increasing the hair growth efficacy compared to single-frequency US sonication. The in vitro experiments showed that cavitation activity was enhanced more significantly during dual-frequency sonication than single-frequency sonication in higher concentration of MBs. The pigskin penetration depth in the group in which dual-frequency US was combined with MBs was 1.54 and 2.86 times greater than for single-frequency US combined with MBs and in the control group, respectively; the corresponding increases in the release rate of Mx at 18 hours in in vitro Franz-diffusion-cell experiments were 24.9% and 43.7%. During 21 days of treatment in C57BL/6J mice experiments, the growth rate at day 11 in the group in which dual-frequency US was combined with MBs increased by 2.07 times compared to single-frequency US combined with MBs. These results indicate that dual-frequency US-mediated MB cavitation can significantly increase both skin permeability and transdermal drug delivery. At the same US power density, hair growth was greater in the group with dual-frequency US plus MBs than in the group with single-frequency US plus MBs, without damaging the skin in mice.