The excessive localized fat is a matter of great concern among subjects from current society. It affects image and body shape negatively; and results in dissatisfaction on individual (de Gusmao et al., 2020). Body sculpture refers to the use of either surgical or non-invasive techniques to modify the body for those who desire to fat reduction for specific problem areas, such as, abdomen, hips, thighs (Jewell et al., 2012). In 2018, the global market to body sculpture reached to US$ 6.1 billion; that might increase to $16.5 billion by 2025 (Michon, 2021).

Generally, the diet and exercise are the first suggestion to keep body shape in normal or as so-called attraction (Kordi et al., 2015). However, strict diets and intense daily exercise are difficult to maintain routinely for much longer time; that may result to fail (Mason et al., 2018). Liposuction is a surgical technique used to remove fat tissue to make people have a desired contour, which is among the top five cosmetic surgical procedures performed in United States (Jalian and Avram, 2012). Unfortunately, the side effects of liposuction include lidocaine toxicity, infections, numbness, fat embolism, or even death. Furthermore, the skin may locally appear contour irregularities, for instance, bumpy, wavy or withered due to uneven fat removal, poor skin elasticity and unusual healing (Mrad et al., 2019; Witte et al., 2020; O’Neill et al., 2021). Along with safety concerns, several noninvasive nonsurgical approaches have been developed for body sculpting, which have drawn more attentions in recent years (Jalian and Avram, 2012; Shek et al., 2014).

Ultrasound is one of powerful tools in medical image for diagnosis and very popular in rehabilitation as a therapeutic modality (Moreno-Moraga et al., 2007). Over the last decade, ultrasound has been developed to a commercial set in plastic surgery as physical lipolysis for body sculpture by specific ultrasonic parameters to break down fat tissue around the patients’ waist.

As known, low-intensity ultrasound (0.5–17.5 W/cm²) would increase the inertial cavitation and then go through the bubble growth, finally to bubble implosion to generate the heat and stress to destroy the fat tissue for lipolysis (Zhou et al., 2017); however, the result of breaking down the fat tissue is not so promising, and it could only serve as the side treatment along with the liposuction (Tonucci et al., 2014). Alternatively, high-intensity focused ultrasound (HIFU) was developed to burn-down subcutaneous adipose tissue by high intensity (1,000 W/cm²) with a special focusing plate to converge the ultrasonic waves to the intended ablation area. HIFU has been reported to induce rapid cell necrosis by the high energy and temperature generated from cavitation explosion; that might effectively dissipate adipose tissue. However, HIFU has been reported to burn the surface skin and charred surrounding tissues, causing a serious inflammatory response.

In summary, lipolysis by low-intensity ultrasonic provides a good method for non-invasive and low-risk body sculpturing, without requiring a recovery period. However, non-invasive ultrasonic lipolysis still has some potential shortcomings that need to be improved and to skip the shortages from HIFU. In the study, a sonodynamic microparticles of europium-doped calcium carbonate would be synthesized to combine with low-intensity ultrasound for lipolysis on body sculpture by a mild and non-invasive way.

Calcium carbonate (CaCO₃) is the candidate material selected for the study due to its excellent biocompatibility and stability (Xiao et al., 2021). CaCO₃ is a biodegradable material that can decompose into carbon dioxide (CO₂) and calcium ions (Ca²⁺) in the acidic environment of endosome-lysosome complex. It is also one of materials with the property of sonoluminescence; where the particle could absorb the energy from the explosion of ultrasonic cavitation to generate heat to react with oxygen or biomolecules to induce reactive oxygen species (ROS) generation, and then convert into different free radicals to de-nature the proteins for cell necrosis (Jonnalagadda et al., 2021). In addition, CO₂ decomposed from CaCO₃ may serve as bomb to make cell damage under explosive stress, that could further kill the adipocyte (Yang et al., 2019). Ca²⁺ released from the breaking down of CaCO₃ at the acidic endosome-lysosome complex would increase the local calcium level around the adipose tissue; that might inhibit the differentiation of mesenchymal stem cells toward adipogenesis (Li et al., 2018). We believe that ROS from sonoluminescence, CO₂-bomb and locally increasing Ca²⁺ level would be three major mechanisms to effectively remove away adipo-tissue for body sculpture.

In order to increase the sonoluminescent effect, a rare element Eu, would be doped into the crystal lattice to partially replace the Ca²⁺ in the lattice site of CaCO₃. A green method was developed to synthesize the particle of Eu-doped CaCO₃ (CaCO₃: Eu) at relative-lower temperature without organic solvent involved in.

In this study, x-ray diffractometer (XRD) was used for the crystal structure identification of the synthesized CaCO₃: Eu. The morphology of the developed particle was observed by scanning electron microscope (SEM). The semi-quantitative chemical composition of the developed particle was examined and evaluated by energy-dispersed spectrophotometer (EDS) and inductively coupled plasma mass spectrometer (ICP-MS). The electronic diffraction pattern was to further check the crystal structure of the synthesized individual grain by transmission electron microscope (TEM). The particle size was determined using a Zeta-sizer. The water-soluble tetrazolium salt (WST-1) on L-929 cells were used to evaluate the cell viability of the developed material; that would be in terms of in vitro cytotoxicity. Chloromethyl-2′,7′-dichlorofluorescein diacetate (CM-H₂DCFDA) and live/dead stain were used to evaluate how the combination of CaCO₃: Eu and low-intensity ultrasound works on 3T3-L1; the results would serve as first screening in vitro. Finally, SD-rat was used as the target animal to evaluate the safety and efficacy in vivo; where the body weight, body temperature, waist line, the weight of subcutaneous adipose tissue on ultrasonic area, histological sectioning, blood element analysis, and serological analysis would be measured and checked to prove the concept.

The scenario of the study was firstly to synthesize a high-sonoluminescent CaCO₃: Eu particles by a new developed method. Secondly, the synthesized particles would be injected to abdomen area and then locally applied with low-intensity ultrasound to prevent from the skin burning and charred surrounding tissue. The combination of sonoluminescent CaCO₃: Eu and low-power ultrasound would generate ROS to damage the adipo-tissue under the stress of free radicals. The CO₂ and Ca²⁺ decomposed from CaCO₃: Eu would serve as CO₂-bomb and increase local Ca²⁺ level to further break-down the adipo-tissue and to inhibit local adipogenesis. The overall process would be schemed in Figure 1.

CaCO₃, comprises more than 4% of the mineral on earth’s crust and is found throughout the world. Its most common natural forms are chalk, limestone, and marble, produced by the sedimentation of the shells of small fossilized snails, shellfish, and coral over millions of years (Mar and Phyo, 2013; Castro-Alonso et al., 2019). CaCO₃ has been widely used in medical applications, such as bone graft for tissue repair, biodegradable vehicle for drug and gene delivery etc. (Maleki et al., 2015; Song et al., 2018). In this study, we used Eu-doped calcium carbonate as sonodynamic reagent to combine with ultrasonic irradiation for body sculpture. Eu is a non-toxic rare earth element with an atomic number of 63, which belongs to the trivalent ion (Li et al., 2020). Eu could replace the calcium ion position of calcium carbonate to promote defects in calcium carbonate and increase the number of electron-hole pairs. Compared to divalent Ca ions, the doped Eu ions can obtain additional electrons, which creates a new energy level near the conduction band to reduce the energy gap effectively (Han et al., 2014; Wang et al., 2014). This makes the sonosensitizer more susceptible to ultrasonic irradiation and stimulates the generation of singlet oxygen and ROS in adipocytes for increasing the effective on lipolysis.

The CaCO₃: Eu was successfully synthesized using the eco-friendly method. The crystal structure was identified by XRD, which was matched with the standard pattern of calcite CaCO₃ (Figure 2). Zeta-sizer was used to analyze the particle size and distribution of the synthesized particles. The average particle size of CaCO₃: Eu was 2.1 μm, which fall in the range of optimum particle size for cellular endocytosis (0.5–10 μm) (Hirota and Ter, 2012; Foroozandeh and Aziz, 2018). The particle size was further evaluated by TEM and SEM, those supposedly larger than that of Zeta-sizer due to the aggregation during the sample preparation before examined under electronic microscope. The real grain size was around 100–300 nm as shown in the electron-penetrated edge of the TEM picture (Figure 3A).

Sonosensitizers can be divided into organic-based compounds and inorganic-based particles (Rosenthal et al., 2004; Chen et al., 2014). The organic-based materials, such as porphyrin-based structures, were reported to have short life span under ultrasound irradiation and showed great cytotoxicity. The inorganic-based particles, such as Ag, Au, Pt, TiO₂, and quantum dots, etc., have been used as sonosensitizer on sonodynamic therapy (SDT) for tumor/cancer treatment with better biostability and much longer life span (Xu et al., 2016). However, this kinds of material produce too much of ROS after exposed to ultrasonic stimulation, that is too strong and may result to the higher cytotoxicity (Serpe et al., 2012). In addition, the inorganic-based materials are not biodegradable in the human body. In the study, we develop a mild sonosensitizer CaCO₃: Eu for body sculpture after exposed to ultrasound. It is a biodegradable particle that can be decomposed in endosome-lysosome complex, and then turn into carbon dioxide (CO₂) and calcium ions (Ca²⁺), as described in the following series of reactions (Yang et al., 2019). CaCO 3 ( s ) → Ca 2 + + CO 3 2 − (1) CO 3 2 − + H + → HCO 3 − (2) HCO 3 − + H + → H 2 CO 3 (3) H 2 CO 3 → H 2 O + CO 2 (4) where the CO₂ could serve as bomb to break down the endosome-lysosome complex and as one of mechanisms to kill the adipocytes in the fat tissue. The high concentration of Ca²⁺ ions, decomposed from the CaCO₃: Eu, could create a osmotic pressure to quickly escape from the complex environment. The adipose tissue with locally high level of Ca²⁺ ions would have the effect to the inhibition on the conversion of pre-adipocyte to adipocyte as following discussions.

Ca²⁺ ion has been investigated that was in association with adipocyte lipid metabolism, such as lipid synthesis and catabolism (Shapses et al., 2004; Duncan et al., 2007). Extracellular Ca²⁺ is also involved in the modulation of adipogenesis. It has been reported that high extracellular Ca²⁺ inhibits adipogenesis in 3T3-L1 pre-adipocytes (Jensen et al., 2004; Zhai et al., 2020). The process of pre-adipocyte differentiation of mature adipocytes is regulated by complex transcription factors, which can regulate the expression of hundreds of proteins responsible for establishing mature adipocyte phenotypes (Lowe et al., 2011). The two major adipogenic factors are peroxisome proliferator-activated receptor (PPARγ) and cytosine-cytosine-adenosine-adenosine-thymidine/enhancer-binding protein (C/EBP) (Farmer, 2006; Payne et al., 2009). Once the CaCO₃: Eu is decomposed by cells, calcium ions diffuse into the interstitial space, turning the entire local environment into a high-calcium environment. A high-calcium concentration in the microenvironment activates the preadipocyte factor 1 (PREF1) expression, which causes the up-regulation of the transcription factor SOX9 (Wang and Sul, 2009), that could inhibit the formation of sterol regulatory element-binding protein (SREBP), C/EBP, and PPARγ for pre-adipogenic cell maturation (Jensen et al., 2004; Vergara et al., 2016; Das and Choudhuri, 2017; Pramme-Steinwachs et al., 2017). In this study, we cultured the cells with different concentration of calcium ions in the cell culture medium, this result was verified that, 3T3-L1 cells under high-calcium ion environment were inhibited the differentiation of fat precursor cells into adipocytes (Supplementary Figure S2).

Sonoluminescence is a sonosensitizer absorb energy from inertial cavitation followed bubble rapture after ultrasound applied to the local tissue to produce ROS. The ROS include superoxide ions (O₂ ⁻), peroxide ions (O₂ ²⁻), hydroxyl radicals (OH), and singlet oxygen (¹O₂), which can cause to cell death in fat tissue (Kuroki et al., 2007; Trendowski, 2014, 2015; Pang et al., 2016). The CMH₂-DCFDA fluorescent dye was used to detect hydroxyl, peroxyl, and other ROS-active oxides in the cells. In this study, ROS production in the US group was 1.12 times higher than that in the control group. It is speculated that under the action of ultrasound, the generation of inertial cavitation finally causes the bubble to rupture, it could release strong energy that causes pyrolysis of surrounding water molecules, and producing hydroxyl groups in adipocytes. The production of ROS in the CaCO₃: Eu group was not observed. In addition, compare with US-CaCO₃: Eu and bare CaCO₃ under ultrasound irradiation (US-CaCO₃) group, ROS production of US-CaCO₃: Eu was 1.24 times high than US-CaCO₃ (Supplementary Figure S3). Meanwhile, in the US-CaCO₃: Eu group, the ROS production is 1.61 times higher than control, which is presumed to be inertial cavitation and the generation of sonoluminescence, causing the acoustic-sensitive materials to be excited and produce singlet oxygen and superoxide. The results show that the combination of CaCO₃: Eu and ultrasound treatment could produce more ROS free radicals on adipocytes. In addition, we also used the WST-1 and live/dead assays to verify the in vitro carving effect of CaCO₃: Eu under ultrasound irradiation. The US group showed that only inertial cavitation acts on the pyrolysis of water molecules to produce hydroxyl, which has limited oxidative damage capacity in adipocyte. When ultrasound is applied to activate CaCO₃: Eu, inertial cavitation pyrolysis produces hydroxyl and sonoluminescence excitation material, causing sonosensitizers to be excited to produce singlet oxygen and ROS. These results indicated that combination of CaCO₃: Eu and ultrasound treatment could cause significant damage to the adipocyte.

The results of animal study did not remarkably change between the groups at the beginning. Nevertheless, at a specific time, the US-CaCO₃: Eu sonodynamic treatment groups had a change in waistline within 4 weeks, and a statistical difference was reached in the fourth week. As the animal model used in this study was Sprague Dawley rats, the abdominal viscera and muscle tissue were removed, and the subcutaneous fat was measured based on the actual waist circumference. The results indicated the US-CaCO₃: Eu on SD rats could significantly decrease the growth rate of body weight and waistline and reduce the storage of adipose tissue by the weight of subcutaneous fats. In addition, the reduction in subcutaneous fat cell volume was observed from fat tissue section between Control and US-CaCO₃: Eu group (Supplementary Figure S4). Body temperature changes (Supplementary Figure S5), tissue sections (Supplementary Figure S6), and blood analysis (Supplementary Table S2) of the above animal experiments showed that the injection of acoustically sensitive materials in animals and the effects of ultrasound of the rats are safe, and does not affect the physiological condition and organs of the rats by the ultrasound effect. the CaCO3: Eu exposed to ultrasound irradiation on SD rats could significantly decrease body weight, waistline, and subcutaneous adipose tissue. In summary, the US-CaCO₃: Eu sonodynamic treatment is demonstrated that has a great potential in the application of body sculpture.

In the study, a sonosensitizer of Eu-doped CaCO₃ was successfully synthesized to combine with low-intensity ultrasound for body sculpture. The results showed that the CaCO₃: Eu had good biocompatibility and could produce ROS in adipocytes for lipolysis. In addition, the results showed that developed sonosensitizer could effectively inhibit the adipogenesis after treated with low-intensity ultrasound. After 4-weeks animal study, the developed CaCO₃: Eu exposed to ultrasound irradiation on SD rats could significantly decrease the growth rate of body weight and waistline; and could reduce the storage of adipose tissue by the weight of subcutaneous fats. We could say that the combination of the developed Eu-doped CaCO₃ and low-intensity ultrasound could effectively inhibit the adipogenesis without skin burning and charred sounding tissue; that would be a mild and non-invasive treatment for the body sculpture.