Drug delivery by sonosensitive liposome and microbubble with acoustic-lens attached ultrasound: an in vivo feasibility study in a murine melanoma model

Conventional chemotherapy methods have adverse off-target effects and low therapeutic efficiencies of drug release in target tumors. In this study, we proposed a combination therapy of doxorubicin (DOX)-loaded ultrasound (US)-sensitive liposomal nanocarriers (IMP301), microbubbles (MBs) under focused US exposure using convex acoustic lens-attached US (LENS) to tumor treatment. The therapeutic effects of each treatment in a murine melanoma model were evaluated using contrast-enhanced US (CEUS) and micro-computed tomography (micro-CT) imaging, bioluminescence and confocal microscopy imaging, and liquid chromatography–mass spectroscopy (LC/MS) analysis. Tumor-bearing mice were randomly assigned to one of the following groups: (1) G1: IMP301 only (n = 9); (2) G2: IMP301 + LENS (n = 9); (3) G3: IMP301 + MB + LENS (n = 9); (4) G4: DOXIL only (n = 9); and (5) G5: IMP301 without DOXIL group as a control group (n = 4). Ten days after tumor injection, tumor-bearing mice were treated according to each treatment strategy on 10th, 12th, and 14th days from the day of tumor injection. The CEUS images of the tumors in the murine melanoma model clearly showed increased echo signal intensity from MBs as resonant US scattering. The relative tumor volume of the G2 and G3 groups on the micro-CT imaging showed inhibited tumor growth than the reference baseline of the G5 group. DOX signals on bioluminescence and confocal microscopy imaging were mainly located at the tumor sites. LC/MS showed prominently higher intratumoral DOX concentration in the G3 group than in other treated groups. Therefore, this study effectively demonstrates the feasibility of the synergistic combination of IMP301, MBs, and LENS-application for tumor-targeted treatment. Thus, this study can enable efficient tumor-targeted treatment by combining therapy such as IMP301 + MBs + LENS-application.

The therapeutic effect of the combination of IMP301, MBs, and LENS-applied treatment (G3) was evaluated by using micro-computed tomography (micro-CT) imaging and 3D reconstruction and compared with those of other treatment groups (Fig. 3a-e).Based on the micro-CT representatives, relative tumor growth was calculated by dividing the tumor volume measured from the micro-CT image before treatment by that after the treatment.The G3 group has a smaller relative tumor growth of 210.4% than the other groups, followed by G2 at 239.6%, G4 at 254.2%, G1 at 264.6%, and G5 at 302.4% (Fig. 3ai-ei).When treating tumors with IMP301 and LENS-applied therapies (G2 and G3 groups), the target-specific therapeutic effect and intratumoral necrosis significantly reduced intratumoral vascular perfusion from tumor angiogenesis, especially at the tumor core sites.

Biodistribution of IMP301 and DOX after LENS-applied treatment
As shown in Fig. 5a, the IMP301 signal accumulated in the whole murine body and was primarily distributed in the brain and liver in the non-LENS-treated group (G1).However, IMP301 signals were mainly located at the tumor sites in the LENS-treated groups (G2 and G3).These data demonstrated that the LENS-applied treatment could actively improve IMP301 accumulation at the tumor sites.The IVIS imaging conditions were optimized to ensure precise quantification of IMP301 in treatments involving IMP301 (G1, G2, and G3).However, the DOXIL group (G4) shows limited DOX accumulation in the tumor region due to the imaging conditions not being optimized for DOXIL visualization.The fluorescent signal measurement via IVIS was not conducted for the IMP301 without the DOXIL group (G5) as this aspect of the analysis was specifically designed to evaluate the biodistribution of the drug in conjunction with because the IVIS analysis was to assess the biodistribution of the drug when combined with IMP301 under US exposure.The IMP301 solution without DOXIL (G5) remained transparent due to the absence of DOX.To validate the sonosensitive effect of IMP301 by LENS on organ-dependent targeting efficacy, we selected three groups-G1, G2, and G3.After each treatment process, we harvested major organs and tumors to conduct ex vivo bioluminescence imaging for further evaluation of organ-dependent targeting efficacy (Fig. 5b).Compared with the LENS-treated group, IMP301 was found to accumulate mainly in the liver and kidney without LENS-treatment.In particular, the combination treatment with IMP301 + MB + LENS-applied treatment (G3) achieved better tumor-targeting ability than IMP301 + LENSapplied treatment (G2).When quantifying the corresponding bioluminescence intensities, the IMP301 signals at the tumor sites for the IMP301 + MB + LENS-treated group (G3) were significantly higher than that in the non-LENS-treated group (G1) (Tumor, G1 vs. G3: *p < 0.05) (Fig. 5c).

Discussion
The study found that the combination of US-responsive liposomal DOX and FUS exposure enhanced by LENS application efficiently delivered antitumor drugs into melanoma.This approach results in their qualitative therapeutic effects (G2 and G3) compared with IMP301 alone (G1) and DOXIL (G4), which does not reveal any significant differences in the tumor volume and body weight with IMP301 without DOXIL (G5).When comparing to the G5 group (IMP301 without DOXIL), we found significant differences in the relative tumor volume for both the G2 (IMP301 + LENS) and G3 (IMP301 + MB + LENS) groups.Upon examining the intratumoral drug concentration results, we observed a significant difference exclusively in the G3 group compared to the results obtained from the G4 and G5 groups.A comparison of the relative radiant efficiency in tumors between the G1 and G3 groups revealed a substantially higher tumor accumulation ratio in the G3 group.Additionally, in the CLSM imaging analysis, we confirmed a significantly higher rate of DOX penetration to the tumor nucleus over time in both the G2 and G3 groups compared to the other treatment groups.These findings highlight the significant feasibility of our proposed drug delivery system in cancer therapy, showing the enhanced tumortargeting capabilities and the potential for therapeutic effects.The enhanced drug delivery strategy involves the There are two main advantages of inducing drug release using US and MB.It enables tumor characterization utilizing the CEUS system, providing valuable insights into tumor characteristics and morphology.Another notable aspect of our study is a target-specific drug delivery approach achieved by modulating the power and shape of the US beam.This capability can allow for precise and controlled drug delivery to the intended target, further enhancing the efficacy and accuracy of the treatment.
CEUS enables tumor imaging by visualizing intratumoral blood perfusion where the MBs flow.Following LENS-applied treatment, the treated tumor sites exhibit a more significant and continuous reduction in normalized US intensity than untreated sites.This is because FUS exposure by LENS-applied treatment can promote MB cavitation, leading to MB destruction [12][13][14][15] .These results indicated that LENS-applied CEUS imaging enabled tumor imaging and real-time treatment monitoring simultaneously.
MB oscillation by cavitation leads to microstreaming and acoustic pressure, generating high shear stress on the liposomal carrier and cell membrane.Encapsulated drugs in IMP301 can be released by FUS exposure alone, but FUS-mediated MB cavitation enhances drug release and intratumoral drug penetration 14,16 .The mechanical stress promotes the disruption of the phospholipid shell of IMP301 and improves the dispersive transport and diffusivity of DOX to tumor cells 17,18 .The combination of LENS-induced MB cavitation and liposomal drug carriers can improve therapeutic efficacy by providing high vascular permeability, increased interstitial transport, enhanced cellular uptake, hydrostatic pressure reduction, and loosened intercellular junctions in terms of biophysics [19][20][21][22] .This study confirms the feasibility of our proposed approach through observations of the tumor growth rate over time and the increased intratumoral DOX concentration by the IMP301 + MB + LENS-applied treatment (G3).However, weight loss might be regarded as an adverse effect of DOX as an antitumor drug.Without FUS,  drug-loaded liposomes are hindered by the high interstitial fluid pressure [23][24][25] .Moreover, liposomal formulations enable intratumoral concentration due to their characteristics, including longer half-life and slower clearance 26,27 .DOX accumulated mainly in the liver and kidney without the LENS, compared with the LENS-applied group.It is consistent with previous studies due to the hepatic metabolic conversion and renal clearance 28,29 .In particular, the combination treatment with IMP301 + MB + LENS-applied treatment (G3) achieved better tumortargeting ability than IMP301 + LENS-applied treatment (G2) due to the FUS-mediated MBs cavitation effect.In contrast to the tumor-targeting efficacy of the combination of IMP301, MBs, and LENS-applied treatment as shown in Fig. 6, CLSM images of the major organs of each treated group (G1, G2, and G3) led to no significant increase in DOX signal intensity over time after the treatments (Supplementary Figs.S1-S3).These results also indicate that FUS exposure, combined with MBs, can enhance the permeability of the intratumoral vasculature and tumor tissues, facilitating target-specific drug delivery comparable to previous studies 30,31 .
This study is subject to certain limitations.Specifically, when performing statistical analyses to evaluate the therapeutic effects of each treatment, we did not observe statistically significant differences among them directly in the results of relative tumor volume and body weight.We attribute this limitation to the highly aggressive growth rate exhibited by the melanoma tumor used as the target in our study.Consequently, the tumor size in the 2nd and 3rd treatment stages was considerably more extensive compared to the area of the treated region.In future investigations, we will closely examine significant differences in the tumor progression delay effect on the model exhibiting less aggressive growth rates.It will result when the treated tumor size remains relatively small, enabling the comprehensive targeting of the entire tumor region during the treatment process.Secondly, the bioluminescence imaging conditions used in this study for IVIS analysis posed challenges when attempting to compare all treatment methods quantitatively.Usually, DOX exhibits bioluminescence when excitation/emission wavelengths (Ex/Em) are 488/530 nm.However, in this study, we employed a near-infrared wavelength range with Ex/Em of 640/710 nm to detect IMP301.This imaging condition was chosen to reduce autofluorescence signals in vivo but simultaneously resulted in relatively low absorbance.Furthermore, since DOXIL is a passively targeted liposomal drug, achieving a precise observation of tumor accumulation may be more challenging than treatments based on IMP301.Therefore, to accurately assess the biodistribution of each treatment using the IVIS analysis, optimized imaging conditions for both DOXIL and IMP301 are necessary.These limitations warrant further exploration and consideration in future studies.Third limitations include a lack of in-depth discussion on pathological changes and adverse effects on surrounding tissues.Although we examined the efficacy of our therapeutic approach using micro-CT imaging and bioluminescence imaging, it would be necessary to elucidate the therapeutic effects of our proposed treatment on tumor cells and nearby healthy cells with histopathologic analysis and deal with their in-depth discussion, compared to previous studies 14,30 .Furthermore, large animal models should consider unintentional acoustic reflection and scattering.With future animal and tumor model studies, we will demonstrate the capability of US beam control using a LENS system based on acoustic lens geometry.Pharmacodynamic analysis of IMP301 is also needed to understand drug metabolism and tumor metastasis.Future clinical studies will confirm the treatment's combined therapeutic effects on various tumors.
We propose combining US-responsive liposomal drug carriers, MBs, and LENS-applied treatment for effective tumor progression delay and monitoring.FUS exposure by LENS leads to on-demand drug release from liposomal carriers and improved tumor penetration and cellular uptake, demonstrating the potential for tumor progression delay effect.Our approach showed the feasibility and potential for the tumor progression delay effect in a murine melanoma model, offering a promising strategy for precise tumor treatment and drug release control, and could extend to potential benefits for tumor diagnosis.Considering the perspective of an in vivo feasibility study introducing the synergistic combination treatment, these findings hold significant importance due to expansion into diverse treatment modalities employing various acoustic lens conditions.

LENS system with sonosensitive liposome for drug delivery
A 3D-printed polylactic acid mold was created for a convex acoustic lens using acoustic modeling software (COMSOL Multiphysics 5.3a).The lens was then replicated using polydimethylsiloxane and attached to a 64-element phased-array transducer (3Sp-D, Humanscan Co. Ltd.) with a center frequency of 3.3 MHz.This configuration enabled the creation of FUS beams, where the US waves converged to facilitate localized US exposure treatment specifically for US-induced drug delivery.The custom US pulser system, controlled by a field-programmable gate array (FPGA) device (Spartan-6 FPGA, Xilinx), enabled simultaneous control of pulse repetition frequency (PRF), duty cycle, and acoustic power.FUS exposure was performed at 2.34 MPa acoustic pressure, 100 kHz PRF, and a 9% duty cycle, which was optimized by an MB destruction experiment setup and analysis method in www.nature.com/scientificreports/our previous study 13 .The acoustic lens-attached US system allows for precise control of the treated region based on the size and location of the tumor tissue.It can manipulate the US exposure area (both its size and depth) by modifying the geometry of the acoustic lens attached to the US transducer 13 .The newly developed LENS system enhanced target-specific drug delivery and selective release of US-sensitive liposomes encapsulating DOX (IMP301; IMGT Co. Ltd, Seongnam, Korea) chemotherapeutics dependent on MB cavitation under FUS exposure (Fig. 1).The US-sensitive liposome encapsulating DOX, IMP301, was designed for DOX release under specific conditions of FUS pressure based on the composition and ratio of phospholipids.Previous studies have described the fabrication process, physicochemical characteristics, and sonosensitivity of IMP301 14 .Briefly, the complex lipid composition of IMP301 was fabricated into multilamellar vesicles, DOX was encapsulated into the intraliposomal dispersion, and the final DOX concentration of IMP301 was 2 mg/ mL and stored at 2-8 °C.For a quantitative comparison of therapeutic effects across the treatments, we ensured that the final DOX concentration of IMP301 corresponded to the DOX concentration (2 mg/mL) of the DOXIL product (CAELYX™) employed in this study.In the fabrication process of multilamellar vesicles using complex lipids, we utilized the sonoresponsive liposome without DOX loaded to create the comparison group for IMP301, distinct from DOXIL.

Experimental design for drug delivery activated by LENS
This study was approved by the Institutional Animal Care and Use Committee (IACUC; No. 20-0101-S1A0) and was performed under the Guide for the IACUC and the National Institute of Health Guide for the Care and Use of Laboratory Animals.All experiments were performed according to relevant regulations and the ARRIVE guidelines.
B16-F10 melanoma cells were obtained from the Korea Cell Line Bank and cultured in Dulbecco's modified Eagle's medium (Welgene, Gyeongsan, Korea) containing 10% fetal bovine serum (Welgene, Gyeongsan, Korea) and 1% fetal bovine serum.A murine melanoma model was created by injecting melanoma cells (2 × 10 5 /100 μL) subcutaneously into the backs of immunodeficient mice (Balb/c, Orient Bio., Seongnam, Korea).For tumor implantation and imaging examinations, mice were sedated with an intraperitoneal injection of a mixture of zolazepam (Zoletil; Virbac, Carros, France) 5 mg/kg and xylazine hydrochloride (Rompun 2%; Bayer Korea, Seoul, Korea).Ten days after tumor injection, tumor-bearing mice were selected for at least a tumor volume of 200 mm 3 and then treated according to each treatment strategy on 10, 12, and 14 days from the day of tumor injection (i.e., three treatments in total) (Fig. 7).For all mice in the LENS-treated groups, each tumor was immediately exposed to LENS for 5 min after injection of the treatment material.The G3 group with MB injection was treated with IMP301 injection and immediately exposed to LENS for 5 min following MB injection (0.3 mL through the tail vein).On the 15th day after the tumor injection, all mice were euthanized with a lethal dose of sodium pentobarbital (200 mg/kg body weight, intraperitoneal), and the tumors were carefully removed.

Figure 1 .
Figure 1.Targeted Drug Delivery System using Acoustic Lens-attached US (LENS) and Sonoresponsive Liposomes.Schematic of the target-specific drug delivery system by acoustic lens-attached US system (LENS) with the antitumor drug-loaded sonoresponsive liposome carrier (IMP301).Upon reaching the target tumor region through the microvasculature, disrupted IMP301 releases an antitumor drug by FUS exposure and MB oscillation.

Figure 2 .
Figure 2. Contrast-Enhanced Ultrasound Imaging of Subcutaneous Tumors under FUS Exposure.(a) Timedependent in vivo representative subcutaneous tumor US images after intravenous injection of the contrast agent (MB) under FUS exposure (Upper panel: B-mode; lower panel: CEUS-mode) (n = 1).Green, red, and cyan dotted boxes in the CEUS image at 13 min indicate region of interests (ROIs) of background, LENS-applied treatment, and tumor areas for the normalized US intensity calculation.(b) The normalized US intensity value of CEUS imaging, which is obtained by dividing US intensity in the ROI by the maximum US intensity in the entire CEUS image after injection of the contrast agent over time (I tumor /I max vs.I background /I max : ***p < 0.0001; I tumor /I max vs.I LENS-applied treatment /I max : ***p < 0.0001).The spot and shaded area represent the mean and standard deviation of the calculated values.

Figure 5 .
Figure 5. Biodistribution of IMP301 with LENS-Enhanced Treatment in Murine Model.(a) Representative qualitative in vivo body biodistribution profiles of IMP301 with each treatment (G1: IMP301, G2: IMP301 + LENS-applied treatment, and G3: IMP301 + MB + LENS-applied treatment) by fluorescence imaging.DOXIL group (G4) is excluded from the bioluminescence analysis due to the unoptimized imaging condition for DOXIL visualization.Additionally, IMP301 without the DOXIL group (G5) is excluded from the analysis since our analysis focused on assessing drug biodistribution in combination with IMP301 under US exposure.This group also remained transparent due to the absence of DOX.The scale bar indicates 1 cm.(b) Ex vivo fluorescence imaging of dissected organ biodistribution of antitumor drug with each treatment (G1, G2, and G3) to validate the sonosensitive effect of IMP301 by LENS.(c) Relative radiant efficiency of tumor, brain, heart, liver, spleen, lung, and kidney in the corresponding organ biodistribution images after the treatments (Tumor, G1 vs. G3: *p < 0.05).

Figure 7 .
Figure 7. Experimental Timeline for Targeted Drug Delivery using LENS in Murine Melanoma Model.Timeline of the experimental procedure with murine subcutaneous melanoma model to verify target-specific drug delivery treatment efficacy and toxicity of IMP301 with LENS-applied treatment.Each treatment following antitumor drug administration and the subsequent drug delivery evaluation by micro-CT, mice weight, CEUS, CFM, IVIS, and LC/MS.

Table 1 .
Assessing the efficacy and safety of the drug delivery treatment in murine subcutaneous melanoma model.