Device design and working principle. The patch consists of a piezoelectric transducer and acoustic metamaterials as a plate with periodically distributed sharp metamaterial structures (Fig.1b). The metamaterial structures loaded with therapeutics penetrate the human epidermis. By applying resonant radio frequency (RF) signals, the acoustic metamaterial patch could be excited to generate a localized acoustic field and acoustic streaming around the metamaterial structures. The localized streaming may drive and transport therapeutics from porous sharp metamaterial structures into deeper tissues. By digitally regulating the RF signal parameters, such as power density and treatment time (e.g., timepoint, duration of each burst), the acoustic metamaterial patch could precisely release therapeutics into deeper skin tissues at a controlled dose and rate (Fig.1a). Next, we designed and fabricated acoustic metamaterial patches to penetrate stratum corneum and percutaneous transport. Considering the typical thickness of the epidermis is around 100~400 μm, the patch was designed as a plate with a layer of pyramidal structures (height:600 μm x length:200 μm x width:200 μm) in a square lattice distribution (Fig.S1a). We calculated the acoustic transmission through the metamaterials over frequency (Fig.1c) and identified the resonant frequency of the designed metamaterials as around 1.06 Mhz. With the resonant frequency, we further simulated the acoustic field and acoustic streaming field distribution via the designed smart acoustic metamaterials (Fig.S2). The designed metamaterials split an acoustic beam into multiple localized sub-wavelength acoustic spots around the pyramidal structures (Fig.1d) and induced localized acoustic streaming (Fig.1e). Using the simulation models, we studied the acoustic streaming induced by acoustic metamaterials. We found that four 3D streaming vortices were formed around a sharp pyramidal structure. Theoretical and experimental results both showed that streaming lines flowed down from the pointed tip toward the base of the pyramid structure, then rotated up, and flowed toward the upper space (simulation, Video S1; experiment, Video S2), indicating acoustic streaming may contribute as a driving force to transport therapeutics from the pyramid’s surface toward deeper tissues. We further simulated the distributions of the acoustic field and acoustic streaming above these pyramid tips (Fig.1f). We found that the 3D distribution of released dye (Fig.2e) correlated with the streaming distribution, indicating the dominant effect of streaming in therapeutic transport. Thus, we demonstrate a new mechanism of transdermal delivery via acoustic metamaterials.
Programmable delivery in vitro. We tested and validated the acoustic metamaterial patch-mediated programmable drug release in vitro using the theoretical and experimental approaches. We fabricated the poly (ethylene glycol) diacrylate (PEGDA) based acoustic metamaterial patches with only sharp pyramid tips loaded rhodamine B (RhB) dyes (Fig.S1b). The RhB was used to model the molecular drugs in vitro, ex vivo, and in vivo due to its stability and robust fluorescence.25 We used agar gel phantoms to test drug delivery due to their similar mechano-acoustic properties to human skins.26, 27 We predicted the dynamic processes of dye release into the agar gel from the sharp metamaterial structures with acoustic stimulation (Acoustics (+)) and without acoustic stimulation (Acoustics (-) as passive release) (Fig.2a, and Video S3). Then, we also conducted experiments to track the same dye release process (Fig.2b, and Video S4), which matches well with the simulation. Simulated and experimental results showed that the acoustic materials significantly enhanced dye release compared to passive release over the same time interval. Then, we also quantified the dye release dynamics via acoustic metamaterials with and without acoustic stimulation. Our experimental results (Fig.2c) demonstrated that the released dye via Acoustics (+) (red circles) was significantly greater than that via Acoustics (-) (blue squares), which matches well with our theoretical prediction (black curves). Moreover, we also investigated the dependence of dye release on acoustic power density with the same treatment time (Fig.2d). Simulation and experimental results indicated that we could modulate the dye release rate by tuning power density. Furthermore, to test the programmable delivery, we quantified the rate and accumulative dose of released dye after applying a multi-burst simulation over time (1st burst, 1.2 W/cm2 for 1 min; 2nd burst, 2.4 W/cm2 for 1 min; and 3rd burst, 3.6 W/cm2 for 1 min) (Fig.2e). We found that our acoustic metamaterial patch can precisely regulate the rate and dose of dye release over time, highlighting its potential for on-demand drug delivery with tight control of pharmacokinetics.
Transdermal delivery ex vivo. We validated the acoustic metamaterial patches using fresh mouse skin tissues to evaluate stratum corneum penetration and control drug release. We applied the RhB dye-loaded patch device to the freshly harvested mouse skin. After applying acoustic waves for 3 minutes with a power density of 2.4W/cm2, we characterized the dye release within the histologically sectioned mouse skin tissues using fluorescent imaging. As shown in Fig.3a, our patch device penetrated the mouse skin with a maximal penetration depth of ~500 μm and released dye into the epidermis and deeper tissues with a maximal diffusion depth of ~1,000 μm highlighting its ability for transdermal delivery in vivo. Moreover, histological section images also showed deeper tissue transport and a higher dose of released dye surrounding the pyramidal structure after the patch treatment with acoustic stimulation (acoustics (+)) compared to the same treatment condition without acoustic treatment (acoustics (-) or passive diffusion). Therefore, these results clearly showed that (i) our metamaterial structures penetrate mouse skin, and (ii) acoustics actively releases the dye inside mouse skin tissues and significantly enhance percutaneous transport compared to the passive dye diffusion. Furthermore, to achieve transdermal drug delivery with precise dose control, we explored the ability of the patch devices to quantitatively release dye into mouse skin by tuning acoustic simulation (e.g., adjusting treatment time while maintaining the power density at 2.4W/cm2). After applying the RhB dye-loaded patch device into the fresh mouse skin tissues, we observed transdermal dye release under different treatment times (e.g., 30 s, 60 s, and 180 s in Fig.3b). We also quantified the released dye over additional treatment times (Fig.3c). We found that the patch device could control the dose of transdermal RhB by tuning acoustic treatment time. The acoustic metamaterial patch (Acoustics (+)) released about 9.3 times as much transdermal RhB dye as released passively (Acoustics (-)) during the same treatment time. Thus, we demonstrated that the acoustic metamaterial patch could achieve transdermal drug delivery with precise control over drug dose and release rate.
Transdermal delivery in vivo and biodistribution. We tested the acoustic metamaterials meditated transdermal delivery in vivo using live C57BL/6 mice and compared the delivery efficiency with subcutaneous injection (S.C. injection). We conducted the transdermal delivery of RhB dye with the same dose (~ 4.5 μg) to the mice via the acoustic metamaterial patch with (Acoustics (+)) or without acoustic stimulation (Acoustics (-)) and S.C. injection methods, respectively. After applying the different techniques, the transdermally delivered RhB dyes will diffuse away from the administration area within the mouse skin tissue and be taken up by the circulatory system via systemic delivery. We visualized and analyzed the dynamic diffusion and absorption process of transdermally delivered dyes within each living animal using an In Vivo Imaging Systems (IVIS) system, where the photon counts represent the intensity of RhB dyes. The IVIS images of a representative mouse from four different treatment groups (Acoustics (+), Acoustics (-), S.C. injection, and blank) were taken at pre-determined time intervals (10 min, 60 min, or 240 min in Fig.3d). The IVISR images showed that the mouse treated by the Acoustics (+) treatment showed higher photon intensity, a larger area of diffused dye and that the dye was retained for a longer time within the skin tissue than the Acoustics (-) or S.C. injection treatment groups. In contrast, the mouse without treatment (blank) showed the lowest photon intensity. Moreover, we also quantified these dynamic processes by counting the average photon intensity over time in the different treatment groups (mean ± s.e.m., n= 3 mice per group, independent experiments, Fig.3e). The results also showed that the Acoustics (+) group had a remarkedly higher dose of transdermally delivered dyes within one hour post the transdermal delivery compared to the Acoustics (-) (passive diffusion) or S.C. injection treatment groups, which is possibly caused by the acoustically enhanced dye diffusion within the superficial dermis and deeper tissues.
To further explore the diffusion and uptake of transdermal delivery in vivo, we investigated the biodistribution of transdermally delivered dyes within different treatment groups (Acoustics (+), Acoustics (-), S.C. injection, and Blank). Twenty-four hours after transdermal delivery, we harvested and imaged mouse organs (e.g., muscle, liver, and kidney) from the different treatment groups using the IVISR system (Fig.S3). We quantified the averaged photon intensity of the three organs from each treatment group (mean ± s.e.m., n= 3 mice per group, independent experiments, Fig.3f). The results showed that the dosage of RhB dyes in muscle tissues of the acoustic patch treatment group was significantly greater than that in the microneedle patch treatment group (or S.C. injection group). The reason may be that the acoustic waves enhanced the diffusion and uptake of transdermally delivered dyes into subcutaneous blood vessels, which further transferred them from the administration site into distal muscle tissues. Similarly, the dose of RhB dyes in the liver and kidney tissues from the Acoustic (+) group was slightly higher than the Acoustics (-) or S.C. injection group. Taken together, the acoustic metamaterials-mediated transdermal delivery method exhibited rapid and enhanced transdermal delivery in vivo, enabling a better means than the S.C. injection and passive diffusion methods.
Treating anaphylaxis through dynamic transdermal delivery of epinephrine. As a proof-of-concept, we evaluated the management of acute and heterogeneous anaphylaxis events through the dynamic transdermal delivery of epinephrine via the acoustic metamaterial patch device. We established a mouse model of anaphylaxis using a passive systemic anaphylaxis protocol (details in methods). We then tested the on-demand delivery of epinephrine through the acoustic metamaterial patch for treating anaphylaxis (Fig.4a). About 10 mins post-induction of anaphylaxis, the anaphylactic mice received the first burst of epinephrine (2.5 μg). If needed, the anaphylactic mouse received a second burst (or multiple bursts) of epinephrine (2.5 μg per burst) until anaphylaxis was reversed. Before conducting the epinephrine delivery for anaphylaxis treatment in vivo, we fabricated the acoustic metamaterial patches with only the pyramidal structures loaded with epinephrine and tested the controllable delivery of epinephrine in vitro (Fig.S4). We demonstrated that the single patch device could enable the precise delivery of epinephrine with the desired dose (e.g., 0, 2.5, 5, 7.5, or 10 μg) by applying one or more bursts over time. The device can be used on freely-moving mice in a minimally invasive manner (Fig.4b, and Video S5).
To validate the efficacy of our acoustic metamaterial patch in treating acute anaphylaxis, we tracked the dynamic progression and treatment response of anaphylactic mice using body temperature, respiration, behavior score, and serum histamine level (Fig.S5). Right after the injection of the allergen DNP-HSA, the behavior score, body temperature, and respiration of three mice dropped dramatically, indicating the successful induction of anaphylaxis (Fig.4c). The anaphylactic mouse received a multi-burst delivery of epinephrine via the acoustic metamaterial patch (red squares and red arrows show the first burst 10 min post anaphylaxis and the second burst 45 min post anaphylaxis). The reversal of anaphylaxis was demonstrated by the normalized behavior score, body temperature, and respiration. In contrast, anaphylactic mice without any treatment (black triangles) died. These results indicated that the multi-burst epinephrine delivery via the acoustic metamaterial patch could effectively treat acute anaphylaxis.
Compared with the current ‘epi-pen strategy’ that relies on the needle injection of a fixed dosage of epinephrine, the acoustic metamaterial patch could precisely adjust the dose of epinephrine administered via dynamic delivery, highlighting its potential for personalizing the treatment of acute anaphylaxis of varying severity. To mimic anaphylaxis of different severity (e.g., mild and severe conditions), we induced anaphylaxis in mice of varying severity by modifying the allergen dosage. After triggering variable anaphylaxis responses in mice (12 for mild; 12 for severe), we tested and compared the acoustic metamaterial patch method (a multi-burst delivery of epinephrine with 2.5 μg per burst) with the classic ‘epi-pen strategy’ (fixed 5 μg S.C. dose of epinephrine). The representative body temperature curves (Fig.4d, find the whole dataset at Fig.S6) showed that the acoustic metamaterial patch method effectively rescued the mice with mild and severe anaphylactic reactions via a multi-burst delivery of epinephrine (in red). In contrast, the ‘epi-pen strategy’ only succeeded in routinely saving mice with mild anaphylactic reactions but failed to rescue 25% of the mice with severe anaphylactic reactions (in green). Moreover, we also found that compared to the ‘epi-pen strategy’, the acoustic metamaterial patch significantly reduced the maximal drop in body temperature (> 4 oC) shortened the average recovered time (> 35 mins) of mice with severe conditions (Fig.4e). Moreover, the acoustic metamaterial patch method showed a slight, but significantly better performance than the fixed-dose injection of epinephrine for mice with severe anaphylaxis. Thus, we demonstrated that the acoustic metamaterial patch provides an alternative, convenient, and effective means for treating an acute disease of varying severity such as anaphylaxis, highlighting its potential for personalized therapy.