Hang-Kang Chen

Ultrasound-aided microbubbles facilitate the delivery of drugs to the inner ear via the round window membrane

The round window membrane (RWM) acts as a barrier between the middle ear and cochlea and can serve as a crucial route for therapeutic medications entering the inner ear via middle ear applications. In this study, we targeted the practical application of microbubbles (MBs) ultrasound on increasing the RWM permeability for facilitating drug or medication delivery to the inner ear. Using biotin-fluorescein isothiocyanate conjugates (biotin-FITC) as delivery agents and guinea pig animal models, we showed that MB ultrasound exposure can improve the inner ear system use of biotin-FITC delivery via the RWM by approximately 3.5 to 38 times that of solely soaking biotin-FITC around the RWM for spontaneous diffusion. We also showed that there was significant enhancement of hair cell uptake of gentamicin in animals whose tympanic bullas were soaked with MB-mixed gentamicin-Texas Red or gentamicin and exposed to ultrasound. Furthermore, increased permeability of the RWM from acoustic cavitation of MBs could also be visualized immediately following ultrasound exposure by using Alexa Fluor 488-conjugated phalloidin as a tracer. Most importantly, such applications had no resulting damage to the integrity of the RWM or deterioration of the hearing thresholds assessed by auditory brainstem responses. We herein provide a basis for MB ultrasound-mediated techniques with therapeutic medication delivery to the inner ear for future application in humans.

Effects of Microbubble Size on Ultrasound-Induced Transdermal Delivery of High-Molecular-Weight Drugs.

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.

Hypoxia Induces a Metabolic Shift and Enhances the Stemness and Expansion of Cochlear Spiral Ganglion Stem/Progenitor Cells

Previously, we demonstrated that hypoxia (1% O2) enhances stemness markers and expands the cell numbers of cochlear stem/progenitor cells (SPCs). In this study, we further investigated the long-term effect of hypoxia on stemness and the bioenergetic status of cochlear spiral ganglion SPCs cultured at low oxygen tensions. Spiral ganglion SPCs were obtained from postnatal day 1 CBA/CaJ mouse pups. The measurement of oxygen consumption rate, extracellular acidification rate (ECAR), and intracellular adenosine triphosphate levels corresponding to 20% and 5% oxygen concentrations was determined using a Seahorse XF extracellular flux analyzer. After low oxygen tension cultivation for 21 days, the mean size of the hypoxia-expanded neurospheres was significantly increased at 5% O2; this correlated with high-level expression of hypoxia-inducible factor-1 alpha (Hif-1α), proliferating cell nuclear antigen (PCNA), cyclin D1, Abcg2, nestin, and Nanog proteins but downregulated expression of p27 compared to that in a normoxic condition. Low oxygen tension cultivation tended to increase the side population fraction, with a significant difference found at 5% O2 compared to that at 20% O2. In addition, hypoxia induced a metabolic energy shift of SPCs toward higher basal ECARs and higher maximum mitochondrial respiratory capacity but lower proton leak than under normoxia, where the SPC metabolism was switched toward glycolysis in long-term hypoxic cultivation.

Effectiveness of a Layer-by-Layer Microbubbles-Based Delivery System for Applying Minoxidil to Enhance Hair Growth

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.

Effects of Microbubble Size on Ultrasound-Mediated Gene Transfection in Auditory Cells

Gene therapy for sensorineural hearing loss has recently been used to insert genes encoding functional proteins to preserve, protect, or even regenerate hair cells in the inner ear. Our previous study demonstrated a microbubble- (MB-)facilitated ultrasound (US) technique for delivering therapeutic medication to the inner ear. The present study investigated whether MB-US techniques help to enhance the efficiency of gene transfection by means of cationic liposomes on HEI-OC1 auditory cells and whether MBs of different sizes affect such efficiency. Our results demonstrated that the size of MBs was proportional to the concentration of albumin or dextrose. At a constant US power density, using 0.66, 1.32, and 2.83 μm albumin-shelled MBs increased the transfection rate as compared to the control by 30.6%, 54.1%, and 84.7%, respectively; likewise, using 1.39, 2.12, and 3.47 μm albumin-dextrose-shelled MBs increased the transfection rates by 15.9%, 34.3%, and 82.7%, respectively. The results indicate that MB-US is an effective technique to facilitate gene transfer on auditory cells in vitro. Such size-dependent MB oscillation behavior in the presence of US plays a role in enhancing gene transfer, and by manipulating the concentration of albumin or dextrose, MBs of different sizes can be produced.

DNA demethylation of the TIM-3 promoter is critical for its stable expression on T cells

The T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) is selectively expressed on terminally differentiated T helper 1 (Th1) cells and acts as a negative regulator that terminates Th1 responses. The dysregulation of TIM-3 expression on T cells is associated with several autoimmune phenotypes and with chronic viral infections; however, the mechanism of this regulation is unclear. In this study, we investigated the effect of DNA methylation on the expression of TIM-3. By analyzing the sequences of TIM-3 promoter regions in human and mouse, we identified a CpG island within the TIM-3 promoter and demonstrated that the promoter activity was controlled by DNA methylation. Furthermore, treatment with 5-aza-2′-deoxycytidine enhanced TIM-3 expression on mouse primary CD4+ T cells under Th0-, Th1- or Th2-polarizing conditions. Finally, pyrosequencing analysis revealed that the methylation level of the TIM-3 promoter gradually decreased after each round of T-cell polarization, and this decrease was inversely correlated with TIM-3 expression. These data suggest that the DNA methylation of the TIM-3 promoter cooperates with lineage-specific transcription factors in the control of Th-cell development. In conclusion, DNA methylation-based regulation of TIM-3 may provide novel insights into understanding the dysregulation of TIM-3 expression under pathogenic conditions.

TRPC1 is required for survival and proliferation of cochlear spiral ganglion stem/progenitor cells

OBJECTIVE The present studies were designed to test the hypothesis that canonical transient receptor potential channel 1 (TRPC1) is required for the proliferation of cochlear spiral ganglion stem/progenitor cells (SPCs). METHODS AND MATERIALS TRPC1 were detected and evaluated in postnatal day 1 CBA/CaJ mice pups derived-cochlear spiral ganglion SPCs by reverse transcription-polymerase chain reaction, Western blot, immunocytochemistry, and calcium imaging. The cell viability and proliferation of the spiral ganglion SPCs following si-RNA mediated knockdown of TRPC1 or addition of TRPC channel blocker SKF9635 were compared to controls. RESULTS In spiral ganglion SPCs, TRPC1 was found to be the most abundantly expressed TRPC subunit and shown to contribute to store-operated calcium entry. Silencing of TRPC1 or addition of TRPC channel blockers significantly decreased the rate of cell proliferation. CONCLUSION The results suggest that TRPC1 might serve as an essential molecule in regulating the proliferation of spiral ganglion SPCs.

Treatment effects of lysozyme-shelled microbubbles and ultrasound in inflammatory skin disease

Acne vulgaris is the most common skin disorder, and is caused by Propionibacterium acnes (P. acnes) and can induce inflammation. Antibiotic therapy often needs to be administered for long durations in acne therapy, which results in extensive antibiotic exposure. The present study investigated a new treatment model for evaluating the antibacterial effects of lysozyme (LY)-shelled microbubbles (MBs) and ultrasound (US)-mediated LY-shelled MBs cavitation against P. acnes both in vitro and in vivo, with the aims of reducing the dose and treatment duration and improving the prognosis of acne vulgaris. In terms of the in vitro treatment efficacy, the growth of P. acnes was inhibited by 86.08 ± 2.99% in the LY-shelled MBs group and by 57.74 ± 3.09% in the LY solution group. For US power densities of 1, 2, and 3 W/cm2 in the LY-shelled MBs group, the growth of P. acnes was inhibited by 95.79 ± 3.30%, 97.99 ± 1.16%, and 98.69 ± 1.13%, respectively. The in vivo results showed that the recovery rate on day 13 was higher in the US group with LY-shelled MBs (97.8 ± 19.8%) than in the LY-shelled MBs group (90.3 ± 23.3%). Our results show that combined treatments of US and LY-shelled MBs can significantly reduce the treatment duration and inhibit P.-acnes-induced inflammatory skin diseases.

Ultrasound-Mediated EGF-Coated-Microbubble Cavitation in Dressings for Wound-Healing Applications

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.

Comparison of changes in mitochondrial bioenergetics between keratinocytes in human external auditory canal skin and cholesteatomas from normoxia to hypoxia

Cholesteatoma has attracted many studies seeking to uncover its nature and the pathogenesis of related diseases. However, no researchers have explored the mitochondrial bioenergetics of cholesteatoma. The aim of this study was to investigate the energy demand and differential mitochondrial respiration profiles between keratinocytes in external auditory canal (EAC) skin and cholesteatoma samples cultured in normoxic (20% O2) and hypoxic (5% O2) conditions. Enhanced cellular proliferation of both types of keratinocytes was found in hypoxia compared to normoxia. In 20% O2 conditions, cholesteatoma keratinocytes exhibited less mitochondrial mass, lower ATP levels, and significantly lower basal oxygen consumption rate (OCR) and reserve capacity compared to normal skin keratinocytes. In contrast, in hypoxic conditions, cholesteatoma keratinocytes showed markedly higher levels in maximal OCR and reserve capacity, as well as lower proton leak OCRs, compared to normal skin keratinocytes. Hypoxia induced the reverse mitochondrial bioenergy profile from that in normoxia between these two types of keratinocytes, implying that an adaptive change of mitochondrial respiration to oxygen fluctuations may develop in cases of cholesteatoma. Such adaptation in response to hypoxic conditions may play a role in explaining the pathogenesis of acquired cholesteatoma.