Sudden Cell Death Induced by Ca2+ Delivery Via Microbubble Cavitation

biomedicines

Article Sudden Cell Death Induced by Ca2+ Delivery via Microbubble Cavitation

Martynas Maciuleviˇcius* , Diana Navickaite,˙ Sonam Chopra , Baltramiejus Jakštys and Saulius Šatkauskas *

Biophysical Research Group, Faculty of Natural Sciences, Vytautas Magnus University, Vileikos st. 8, LT-44404 Kaunas, Lithuania; [email protected] (D.N.); [email protected] (S.C.); [email protected] (B.J.) * Correspondence: [email protected] (M.M.); [email protected] (S.Š.)

Abstract: Intracellular calcium ion delivery via sonoporation has been validated to be a substitute for classical chemotherapy. However, the mechanism behind calcium sonoporation remains unclear to this day. To elucidate the role of calcium in the process of sonoporation, we aimed to investigate the influence of different calcium concentration on cell membrane permeabilization and cell viability after sonoporation. In this study, we present experimental evidence that extracellular calcium plays a major role in cell membrane molecular transport after applying ultrasound pulses. Ultrasound- microbubble cavitation in the presence of different calcium concentration affects fundamental cell bio-physio-chemical conditions: cell membrane integrity, metabolic activity, and colony formation. Corresponding vital characteristics were evaluated using three independent viability tests: propidium iodide assay (20 min–3 h), MTT assay (48 h), and cell clonogenic assay (6 d). The results indicate instant cell death, as the level of cell viability was determined to be similar within a 20 min–48 h–6 d period. Inertial cavitation activities have been determined to be directly involved in calcium delivery via sonoporation according to high correlation (R2 > 0.85, p < 0.01) of inertial cavitation dose with change in either cell membrane permeabilization, metabolic activity, and colony formation efficiency. In general, calcium delivery via sonoporation induces rapid cell death, occurring within 20 min after treatment, that is the result of ultrasound mediated microbubble cavitation. Citation: Maciuleviˇcius,M.; Navickaite,˙ D.; Chopra, S.; Jakštys, B.; Keywords: ultrasound; sonoporation; microbubbles; calcium; cavitation Šatkauskas, S. Sudden Cell Death Induced by Ca2+ Delivery via Microbubble Cavitation. Biomedicines 2021, 9, 32. https://doi.org/ 1. Introduction 10.3390/biomedicines9010032 Cell sonoporation (SP) is a physical method, developed to attain spatiotemporally Received: 9 November 2020 regulated intracellular transfer of bioactive compounds. SP process is intrinsically related Accepted: 30 December 2020 to microbubble (MB) cavitation engendered by irradiation with ultrasound (US). Simul- Published: 4 January 2021 taneous US–MB interaction leads to temporal increase in cell membrane permeability for exogenous therapeutic agents, such as anticancer drugs, DNA, RNA, etc. The concur- Publisher’s Note: MDPI stays neu- rent application of cytotoxic drug, gemcitabine, and SP has already been well-applied for tral with regard to jurisdictional clai- antitumour therapy of pancreatic cancer in clinical trials [1]. Currently, cellular entry of ms in published maps and institutio- calcium ions (Ca2+), facilitated by physical therapeutic agent delivery techniques, has been nal affiliations. proposed as an option for conventional chemotherapy [2]. Ca2+ intracellular apportionment inside membrane-bound organelles plays an es- sential role in cellular response to diverse stress stimuli as well as coordination of fatal mechanisms. Substantial importance of Ca2+ has been demonstrated to facilitate the reseal- Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. ing rate of cell membrane injuries, caused by MB activity after irradiation with US [3,4]. 2+ This article is an open access article Diffusible Ca rapidly engages numerous feedback mechanisms in respect to specific 2+ distributed under the terms and con- extracellular triggers. Thus, combination of Ca and SP for tumor treatment implies ditions of the Creative Commons At- the enhanced momentum of desirable outcome. Compared to conventional chemother- tribution (CC BY) license (https:// apeutic agent delivery modalities, such as intravascular injection or oral administration, 2+ creativecommons.org/licenses/by/ targeted US-MB enhanced Ca delivery may substantially reduce adverse after-effects 4.0/). while sustaining efficient anticancer activity.

Biomedicines 2021, 9, 32. https://doi.org/10.3390/biomedicines9010032 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 32 2 of 15

The delivery of anticancer drugs, bleomycin (BLM), doxorubicin (DOX), etc. via physical methods is designed to stimulate cancer cell death in vitro and inhibit/reduce tumour growth in vivo. Ueno et al. and Piron et al. have shown that cancer cell death after DOX delivery via SP was significantly initiated after 24 h post exposure [5,6]. Lee et al. have shown that, although there was immediate DOX intracellular delivery via SP, cell viability decrease due to DOX cytotoxicity was achieved only after 48 h with neither immediate nor 24-h effect after SP [7]. Wang et al. have achieved cell death due to SP using mitoxantrone after 24 h [8]. Recently it has been shown by our group that cell death after BLM delivery to cells using electroporation was substantially promoted after 36–48 h, as indicated by MTT test, performed at consistent time periods after treatment [9]. Similar results after BLM delivery via SP were obtained by Sonoda et al., who have shown that melanoma cell viability decreased 48 h after therapy [10]. In addition to this, they have shown that tumor weight was reduced 4 d after SP. Therefore, in most studies, performed using anticancer drugs, cell death is evaluated after 24–48 h [11–13]. These observations imply the duration, necessary to achieve cell death after chemotherapy, to be in the range of hours to days. The development of effective and non-mutagenic intracellular delivery methods for genetic materials is a major challenge for gene therapy. It is difficult for non-viral gene delivery techniques to achieve fast and efficient transfection rate, mainly due to slow plasmid cytoplasmic migration to nucleus, followed by subsequent degradation and expression kinetics [14]. Indeed, in order to obtain maximum DNA expression efficiency, 5–6 h [15], 12 h [16], or even longer periods were necessary, as indicated by increasing GFP expression rate up to 48 or 72 h after SP [17–19]. Fast cellular response after Ca2+ delivery via SP would offer new opportunities for initiating effective and rapid cell death by intrinsic Ca2+ activated mechanisms, associated to supra-physiological Ca2+ concentration, as compared to slow cell death, induced by chemo- or gene therapy. Therefore, deeper understanding of the biological as well as physical mechanisms behind Ca2+ delivery via SP is crucial for the advancement of the current treatment modality. With the aim to determine the role of Ca2+ in cell death after SP, we have exploited a simplified in vitro model—Ca2+ intracellular delivery into Chinese hamster ovary (CHO) cells was examined in the presence of MBs and US. Therefore, in this study we aimed to simultaneously reveal the impact of different Ca2+ concentrations on cell membrane integrity, cell metabolic activity, and cell colony formation efficiency after SP. Propidium iodide (PI) assay (after 20 min–3 h) was used in order to evaluate cell membrane permeability. Cell metabolic activity and cell colony formation efficiency were evaluated using MTT test (after 48 h) and cell clonogenic assay (6 d), respectively. Generally, US induced MB cavitation is considered to be dual-nature and classified into stable cavitation (SC) and inertial cavitation (IC). Open-ended debate has been initiated considering the preeminent cavitation model, since it has been explicitly demonstrated that both SC and IC were efficient to enhance biological compound delivery [20]. Thus, in this research, we have estimated the pattern of inertial cavitation, according to the results of US induced MB cavitation acoustic emissions, obtained using passive cavitation detection.

2. Materials and Methods 2.1. Cell Line Chinese hamster ovary (CHO) cells were cultured in DMEM (Sigma Aldrich, St. Louis, MO, USA) growth medium supplemented with 10% heat non-inactivated fetal bovine serum (Sigma Aldrich, St. Louis, MO, USA), 1% L-glutamine solution (Invitrogen Inc., Carlsbad, CA, USA), and 100 U/mL penicillin with 100 µg/mL streptomycin (Sigma Aldrich, St. Louis, MO, USA) solution. The cells were grown in monolayer in 10 cm ◦ Petri dishes (TPP, Switzerland), incubated at 37 C in 5% CO2 atmosphere. The cells were harvested using trypsin-EDTA solution (Sigma Aldrich, St. Louis, MO, USA). Biomedicines 2021, 9, x FOR PEER REVIEW 3 of 16

Biomedicines 2021, 9, 32 Aldrich, St. Louis, MO, USA) solution. The cells were grown in monolayer in 10 cm3 Petri of 15 dishes (TPP, Switzerland), incubated at 37 °C in 5% CO2 atmosphere. The cells were har‐ vested using trypsin‐EDTA solution (Sigma Aldrich, St. Louis, MO, USA). 2.2. Experimental Setup 2.2. Experimental Setup Sonoporation experiments were performed using experimental setup (Figure1), con- Sonoporation experiments were performed using experimental setup (Figure 1), con‐ sisting of experimental chamber ﬁlled with degassed water, US transducers, and signal sisting of experimental chamber filled with degassed water, US transducers, and signal acquisition hardware. acquisition hardware.

Figure 1. Experimental setup designed for sonoporation studies. Figure 1. Experimental setup designed for sonoporation studies.

AnAn arbitraryarbitrary waveformwaveform generator/oscilloscopegenerator/oscilloscope (Picoscope 5242B, Picotech, St Neots,Neots, UK)UK) waswas usedused toto generate/recordgenerate/record US signals. The signals were amplified amplified by signal ampli-ampli‐ fierfier (Kaunas(Kaunas University University of of Technology, Technology, Kaunas, Kaunas, Lithuania), Lithuania), powered powered by high by voltage high voltage power supplypower supply (MCP Lab (MCP Electronics, Lab Electronics, Shanghai, Shanghai, China). China). The unfocused The unfocused transducer transducer (Medelkom, (Medel‐ Vilnius,kom, Vilnius, Lithuania), Lithuania), 18 mm 18 diameter, mm diameter, operating operating at 1 MHz at 1 center MHz center frequency frequency 0.9–1.2 0.9–1.2 MHz, −MHz,6 dB − bandwidth,6 dB bandwidth, was usedwas used for MB for excitation.MB excitation. The The spatial spatial distribution distribution of theof the acoustic acous‐ pressuretic pressure within within of US of beam US beam was evaluatedwas evaluated to be homogenousto be homogenous at 1 cm at distance 1 cm distance from the from ex- citationthe excitation transducer. transducer. The US The acoustic US acoustic peak negative peak negative pressure pressure in the cuvette in the was cuvette determined was de‐ usingtermined hydrophone using hydrophone (HNR 1000, (HNR Onda 1000, Corp, Onda Sunnyvale, Corp, Sunnyvale, CA, USA). CA, In orderUSA). to In decrease order to USdecrease multiple US reflections,multiple reflections, the inner the surface inner of surface the experimental of the experimental chamber waschamber covered was with cov‐ acousticered with absorber acoustic (AptFlex absorber F28, (AptFlex Precision F28, acoustics, Precision Dorchester, acoustics, UK). Dorchester, The experiments UK). The were ex‐ performedperiments were at room performed temperature at room (24 ◦temperatureC), in 1 cm width (24 °C), sonoporation in 1 cm width cuvette sonoporation (Plastibrand, cu‐ Wertheim,vette (Plastibrand, Germany) Wertheim, filled to 1 Germany) mL volume. filled to 1 mL volume. PassivePassive cavitation detection detection was was performed performed using using 8 mm 8 mm diameter, diameter, 5 MHz 5 MHz center center fre‐ frequencyquency 2.1–7.9 2.1–7.9 MHz, MHz, −6− 6dB dB bandwidth, bandwidth, transducer transducer (Doppler (Doppler Electronic Technologies, ◦ Guangzhou,Guangzhou, China),China), referredreferred asas aa receiver,receiver, positioned positioned at at 90 90°angle angle to to the the transmitter. transmitter.

2.3.2.3. Experimental ProcedureProcedure CHO cells were sonoporated with different CaCl (Lachema, Brno, Czech Republic) CHO cells were sonoporated with different CaCl22 (Lachema, Brno, Czech Republic) concentrationconcentration using using US US alonealone oror USUS inin combination withwith SonovueSonovue MBsMBs (Bracco(Bracco diagnosticsdiagnostics Inc,Inc, Manno, Switzerland). Sonovue Sonovue MBs MBs were were prepared prepared in in 0.9% 0.9% NaCl NaCl solution solution according accord- ingto manufacturer’s to manufacturer’s instructions. instructions. Sonoporation Sonoporation experiments experiments were wereperformed performed in 0.9% in NaCl 0.9% NaCl supplemented with different concentration (0–20 mM) CaCl2 solution. MB and supplemented with different concentration (0–20 mM) CaCl2 solution. MB and cell con‐ cell concentrations were 4 × 107 MB/mL and 0.75 × 106 cells/mL, respectively, yielding centrations were 4 × 107 MB/mL and 0.75 × 106 cells/mL, respectively, yielding 53:1 MB to 53:1 MB to cell ratio. MB or cell concentration was evaluated using optical microscope cell ratio. MB or cell concentration was evaluated using optical microscope (Nicon Eclipse (Nicon Eclipse TS100, Tokyo, Japan) and hematocytometer (Assistent, Sondheim, Germany). TS100, Tokyo, Japan) and hematocytometer (Assistent, Sondheim, Germany). The exper‐ The experimental groups were divided depending on the type of experiment. For US alone, experimental series were (1) control group (no treatment); (2) US group (US); (3) Ca2+ + US group (Ca2+ + US). For US and MB combination, experimental series were (1) control group (no treatment); (2) cavitation group (MB + US), refers to therapeutic group at 0 mM Ca2+ concentration; (3) therapeutic group (Ca2+ + MB + US). Cells were sonoporated using Biomedicines 2021, 9, 32 4 of 15

1 MHz central frequency, 1 kHz pulse repetition frequency, 10% duty cycle (100 µs on, 900 µs off), 0–800 kPa peak negative acoustic pressure US, and 6 s exposure duration. After US irradiation, the cells were incubated for 10 min at 37 ◦C, and then transferred to growth medium for subsequent preparation for cell viability assessment using the following assays: PI, MTT, or cell clonogenic. The level of bioeffects (permeabilization, metabolic activity decrease, or colony formation efficiency decrease) in (Ca2+ + MB + US) group can be the result of (i) cell death caused by US alone and/or US induced MB cavitation (MB + US), (ii) the cells killed by alone Ca2+ or MBs or their combination (Ca2+ + MB), (iii) Ca2+ sonotransfer due to reversible cell permeabilization resulting in cell death due to intracellular Ca2+ toxicity. Therefore, in order to reveal the change of membrane permeabilization, metabolic activity, and cell colony formation efficiency, the percentages of cell permeabilization, metabolic activity decrease, and decrease in colony formation efficiency in (MB + US) and (Ca2+ + MB) groups were subtracted from the corresponding percentage obtained in (Ca2+ + MB + US) group. Subtraction is performed in order to evaluate the effect of intracellularly delivered Ca2+ to cell permeability, cell metabolic activity, and cell colony formation efficiency. In the results, (Ca2+ + MB) group refers to (Ca2+ + MB + US) group at 0 kPa acoustic pressure.

2.4. Propidium Iodide (PI) Assay PI assay is a cell viability assay based on the assessment of cell membrane integrity. PI intracellular uptake is due to the loss of cell membrane barrier function. After cell incubation, the medium from cell samples was removed by centrifugation. Subsequently, cells were resuspended in 1 × PBS (Lonza Inc., Rockland, ME, USA) and supplemented with PI (40 µM ﬁnal concentration). The number of PI positive cells was evaluated at different time after SP: 20 min, 1 h, 2 h, and 3 h using ﬂow cytometer (BD Accuri C6, Accuri Cytometers Inc., Ann Arbor, MI, USA).

2.5. MTT Assay MTT assay is a colorimetric cell viability assay based on the assessment of cell metabolic/enzymatic activity. After incubation, 8 × 103 cells were grown in the growth medium (100 µl) in the wells of 96-well microplate (Plastibrand, Wertheim, Germany) for 48 h. At the time of measurement, the growth medium was removed, and the cells in the wells were supplemented with fresh growth medium and MTT salt solution (0.5 mg/mL ﬁnal concentration) and incubated for 2 h. Afterwards the medium was removed from the wells and the wells were washed with 1×PBS. Formazan formed in the cells was dissolved in isopropanol (Chempur, Karlsruhe, Germany). All the resulting content of each well was transferred into the corresponding well of another transparent 96-well microplate for absorbance measurements using microplate reader (Spectrostar Nano, BMG Labtech, Ortenberg, Germany). Optical density of the suspension was evaluated at 550 nm wave- length; the values were corrected by subtracting the background, and then normalized to the control (no treatment).

2.6. Cell Clonogenic Assay Cell clonogenic assay is a cell viability assay based on live cell ability to proliferate— form colonies. After cell incubation, 330 cells were loaded into 4.1 cm2 tissue culture dishes (TPP, Switzerland) containing 2 mL of growth medium. The cells were allowed to grow for 6 days, and then ﬁxed in 1 mL of 96% ethanol for 10 min and stained using crystal violet solution (Sigma Aldrich, St. Louis, MO, USA) that contained 2.3% crystal violet, 0.1% ammonium oxalate, and 20% ethanol. The number of cell colonies was assessed using light microscope (MBS 9, LOMO, St. Petersburg, Russia) and then normalized to the control (no treatment). Biomedicines 2021, 9, x FOR PEER REVIEW 5 of 16

Biomedicines 2021, 9, 32 0.1% ammonium oxalate, and 20% ethanol. The number of cell colonies was assessed5 ofus 15‐ ing light microscope (MBS 9, LOMO, St. Petersburg, Russia) and then normalized to the control (no treatment).

2.7.2.7. Cavitation Cavitation Signal Signal Analysis Analysis USUS induced induced MB MB acoustic acoustic emission emission signals signals were were recorded recorded using using passively passively coupled coupled US US transducertransducer in order toto provideprovide information information about about MB MB cavitation cavitation activity. activity. Passive Passive cavitation cavita‐ tiondetection detection system system was was used used for for MB MB cavitation cavitation signal signal detection detection and and recording. recording. Acoustic emissionemission signals signals were were acquired acquired in in two two cases: cases: (i) (i) with with MBs MBs present present (+MB (+MB group) group) and and (ii) (ii) absentabsent ( (−−MBMB group/background group/background group). group). Cavitation Cavitation signals signals were were recorded recorded at 31.25 MS/sMS/s samplingsampling rate, rate, 8 8 bits bits resolution. resolution. The The signals signals from from 6 s 6overall s overall exposure exposure duration duration were were rec‐ ordedrecorded in 102 in 102frames, frames, corresponding corresponding to the to frame the frame rate of rate 17 of frames/s. 17 frames/s. US signals US signals in every in frameevery were frame transformed were transformed to a frequency to a frequency spectrum spectrum using usingfast Fourier fast Fourier transform transform (FFT). (FFT). Fig‐ ureFigure 2A 2representsA represents the the FFT FFT of acoustic of acoustic emissions, emissions, recorded recorded in the in the 18th 18th frame frame at 400 at 400 kPa kPa US − excitationUS excitation in two in twoseparate separate +MB +MB and − andMB groups.MB groups.

FigureFigure 2. 2. FFTFFT of of the the acoustic acoustic emission emission pulses, pulses, recorded recorded in in the the 18th 18th frame frame (1–1.01 (1–1.01 s) s) ( (AA),), corresponding corresponding estimate estimate “Acoustic “Acoustic emissionRMS (AERMS)”, quantified in 1.5–1.75 MHz frequency range (B), after sample exposure to 400 kPa acoustic pressure emission (AE )”, quantiﬁed in 1.5–1.75 MHz frequency range (B), after sample exposure to 400 kPa acoustic pressure US. RMS RMS US.

InIn order order to to quantitatively quantitatively evaluate evaluate the the amount amount of of broadband broadband noise, noise, the the root root mean squaresquare ( (RMSRMS)) values values were were calculated calculated in in a a particular particular frequency frequency range range (Equation (Equation (1)) (1)) of of FFT FFT spectrumspectrum of of US US acoustic acoustic emissions: emissions: r 1 1 (1) RMS𝑅𝑀𝑆= x𝑥2+𝑥x2+⋯𝑥··· + x2 (1) n𝑛 1 2 n wherewhere nn isis the the number number of of values values in in frequency frequency spectrum, spectrum, obtained obtained after after FFT; FFT; xx—the—the ampli ampli-‐ tudetude value value corresponding corresponding to to particular particular frequency frequency value ( (n).). WithWith the the aim aim to to analyze analyze acoustic acoustic emission emission signals signals only only from from MBs, MBs, background background RMS RMS valuesvalues were subtracted from +MB+MB group.group. Thus, the estimateestimate “Acoustic“Acoustic emissionemissionRMSRMS ((AEAERMSRMS)”) ”was was obtained: obtained: 𝐴𝐸 𝑅𝑀𝑆 𝑅𝑀𝑆 (2) AERMS= RMS+MB − RMS−MB (2)

where RMS+MB is RMS, obtained from +MB group and RMS−MB—from background group. where RMS+MB is RMS, obtained from +MB group and RMS−MB—from background group. AERMS, evaluated in the 1.5–1.75 MHz frequency range, contributed to the highest AE , evaluated in the 1.5–1.75 MHz frequency range, contributed to the highest differenceRMS between +MB and background groups; therefore, it was shown in Figure 2B. difference between +MB and background groups; therefore, it was shown in Figure2B. Inertial cavitation dose (ICD) is the integral of differential RMS in exposure duration Inertial cavitation dose (ICD) is the integral of differential RMS in exposure duration scale [21,22]: scale [21,22]: t Z F ICD = AERMS (t) dt (3) 0 where ICD is inertial cavitation dose, t-time, 0 indicates 0 s (the beginning of the exposure), and tF indicates the exposure duration when the integration is ﬁnished. Biomedicines 2021, 9, x FOR PEER REVIEW 6 of 16

𝐼𝐶𝐷 𝐴𝐸 𝑡 𝑑𝑡 (3) Biomedicines 2021, 9, 32 6 of 15 where ICD is inertial cavitation dose, t‐time, 0 indicates 0 s (the beginning of the exposure), and tF indicates the exposure duration when the integration is finished.

2.8.2.8. Statistical Statistical Analysis Analysis TheThe data data are are presentedpresented as the the mean mean ± ±standardstandard error error of the of themean mean of at of least at leastsix exper six ‐ experimentalimental replicates. replicates. Statistical Statistical significance significance between between the two the twogroups groups was wasevaluated evaluated using usingStudent’s Student’s t‐test,t-test, and multiple and multiple comparison comparison was performed was performed using usingone‐way one-way ANOVA. ANOVA. Corre‐ Correlationlation analysis analysis was wasperformed performed to determine to determine the dependence the dependence between between ICD ICDand andthe sono the ‐ sonoporationporation results; results; the the strength strength of correlation of correlation was was defined defined according according to tothe the correlation correlation de‐ 2 determinationtermination coefficient coefficient (R (R2). ).Data Data analysis analysis was was performed performed using using Matlab Matlab (Mathworks, (Mathworks, Na‐ Natick,tick, MA, MA, USA) USA) and and Origin Origin (OriginLab (OriginLab Co, Co, Northampton, Northampton, MA, MA, USA) USA) software. software. 3. Results 3. Results 3.1. Ca2+ Delivery Using US Alone 3.1. Ca2+ Delivery Using US Alone At the initial stage of the research, Ca2+ cytotoxicity to CHO cells was evaluated using At the initial stage of the research, Ca2+ cytotoxicity to CHO cells was evaluated using cell clonogenic assay after cell treatment with US alone, in the absence of MBs. The impact cell clonogenic assay after cell treatment with US alone, in the absence of MBs. The impact of different Ca2+ concentration (0–20 mM) on cell viability (without US treatment, 0 kPa) of different Ca2+ concentration (0–20 mM) on cell viability (without US treatment, 0 kPa) as well as after US treatment (400 and 800 kPa) is presented in Figure3. as well as after US treatment (400 and 800 kPa) is presented in Figure 3.

2+ FigureFigure 3. 3.The The effect effect of of different different Ca Ca2+ concentration concentration on on cell cell colony colony formation formation efficiency: efficiency: without without US US exposure (0 kPa) and with US exposure (400 and 800 kPa acoustic pressure). * indicates statistical exposure (0 kPa) and with US exposure (400 and 800 kPa acoustic pressure). * indicates statistical significance (p < 0.05) between 0 and 20 mM Ca2+ concentration groups at 800 kPa acoustic pres‐ significance (p < 0.05) between 0 and 20 mM Ca2+ concentration groups at 800 kPa acoustic pressure. sure. Clonogenic assay results have shown that CHO cell incubation with Ca2+ without Clonogenic assay results have shown that CHO cell incubation with Ca2+ without US US irradiation (0 kPa) in the range of concentration of 0–20 mM had no effect on cell irradiation (0 kPa) in the range of concentration of 0–20 mM had no effect on cell colony colony formation efficiency. Similarly, the combined 1–20 mM Ca2+ and US treatment 2+ offormation 400 kPa acoustic efficiency. pressure Similarly, had the no significantcombined 1–20 effect mM on cellCa colony and US formation treatment efficiency, of 400 kPa comparedacoustic pressure to cell to exposurehad no significant to US (400 effect kPa) on alone cell with colony no administeredformation efficiency, Ca2+ (0 mM compared Ca2+ 2+ 2+ concentration).to cell to exposure The to only US significant(400 kPa) alone difference with no (p administered< 0.05) between Ca (US) (0 mM and Ca (Ca concentra2+ + US) ‐ 2+ groupstion). The was only observed significant at 20 mMdifference Ca2+ concentration (p < 0.05) between using (US) 800 and kPa (Ca US acoustic + US) groups pressure. was This indicates the negative effect of Ca2+ and US combination for cell biological conditions. Since 800 kPa, US had reduced cell colony formation efficiency due to US alone (at 0 mM Ca2+ concentration), lower Ca2+ concentrations, 0.1 and 0.5 mM, were explored at 800 kPa. However, they had no positive effect for cell colony formation efficiency as compared to (US) group. Although, there is evidence that low Ca2+ concentrations facilitate pore closure, therefore increasing cell viability [3,4]. Overall, 400 kPa acoustic pressure had no significant effect on the decrease in cell colony formation efficiency as compared to control (0 mM) in either Ca2+ concentration combined with US treatment. Therefore, Biomedicines 2021, 9, x FOR PEER REVIEW 7 of 16

observed at 20 mM Ca2+ concentration using 800 kPa US acoustic pressure. This indicates the negative effect of Ca2+ and US combination for cell biological conditions. Since 800 kPa, US had reduced cell colony formation efficiency due to US alone (at 0 mM Ca2+ concentration), lower Ca2+ concentrations, 0.1 and 0.5 mM, were explored at 800 kPa. However, they had no positive effect for cell colony formation efficiency as compared Biomedicines 2021, 9, 32 to (US) group. Although, there is evidence that low Ca2+ concentrations facilitate pore7 of clo 15 ‐ sure, therefore increasing cell viability [3,4]. Overall, 400 kPa acoustic pressure had no significant effect on the decrease in cell colony formation efficiency as compared to control (0 mM) in either Ca2+ concentration combined with US treatment. Therefore, 400 kPa was 400 kPa was selected for further investigation with MBs, as it would better explicit the selected for further investigation with MBs, as it would better explicit the effect of MB effect of MB cavitation for Ca2+ intracellular delivery. cavitation for Ca2+ intracellular delivery. 3.2. Ca2+ Delivery Using US and MB Combination 3.2. Ca2+ Delivery Using US and MB Combination The impact of different Ca2+ concentration on cell colony formation efﬁciency was The impact of different Ca2+ concentration on cell colony formation efficiency was evaluated after MB + US application using 400 kPa acoustic pressure treatment (Figure4). evaluated after MB + US application using 400 kPa acoustic pressure treatment (Figure 4).

FigureFigure 4. 4.Cell Cell colony colony formationformation efﬁciency,efficiency, evaluatedevaluated after cell treatment with US US (400 (400 kPa) kPa) in in the the 2+ 2+ 2+ presencepresence of of MBs MBs at at different different Ca Ca2+ concentration.concentration. (MB), (Ca2+++ MB) MB) and and (Ca (Ca2++ US)+ US) experimental experimental groups (A); (Ca2+ + MB + US) experimental groups (B). ** p < 0.01, *** p < 0.001 indicate statistical groups (A); (Ca2+ + MB + US) experimental groups (B). ** p < 0.01, *** p < 0.001 indicate statistical significance between cavitation [(Ca2+ + MB +US) at 0 mM Ca2+ concentration] and therapeutic signiﬁcance between cavitation [(Ca2+ + MB +US) at 0 mM Ca2+ concentration] and therapeutic [(Ca2+ + MB +US) at 1–20 mM Ca2+ concentration] groups. [(Ca2+ + MB +US) at 1–20 mM Ca2+ concentration] groups.

2+ TheThe results results of of cell cell clonogenic clonogenic assay assay have have revealed revealed that that 5–20 5–20 mM mM Ca2+ Caconcentration concentration in combinationin combination with with MBs MBs after after US treatment US treatment have signiﬁcantlyhave significantly reduced reduced cell colony cell colony formation for‐ 2+ efﬁciencymation efficiency as compared as compared to the cavitation to the cavitation (MB + US) (MB group, + US) which group, refers which to (Ca refers2+ + MB to +(Ca US)+ 2+ groupMB + atUS) 0 mMgroup Ca at2+ 0concentration. mM Ca concentration. In comparison In comparison to the cavitation to the cavitation group, lower group, 0.5 lower and 2+ 10.5 mM and Ca 2+1 mMconcentrations Ca concentrations in combination in combination with MBs with and MBs US neither and US reduced neither cell reduced viability cell norviability had positive nor had effect positive on cell effect viability. on cell viability. InIn order order to to gain gain deeper deeper understanding understanding in in cell cell death death dynamics dynamics after after Ca Ca2+2+delivery delivery via via SP,SP, the the impact impact of of different different Ca Ca2+2+concentrations concentrations on on cell cell membrane membrane permeabilization permeabilization and and cell cell

metabolic activity were evaluated after (MB + US) application (Figures 5 and 6). metabolic activity were evaluated after (MB + US) application (Figures5 and6). PI assay represents the increase in cell membrane permeability induced by SP (Figure5 ). PI was added at a different time after SP: 20 min, 1 h, 2 h, and 3 h. The experimental results show that MBs without US or MB and Ca2+ (20 mM) coadministration without US as well as cell exposure to US in the presence of Ca2+ (20 mM) have only insignificant effect to cell membrane permeabilization (Figure5A). The obtained results in (Ca 2+ + MB + US) groups (Figure5B) have shown that in the presence of 5–20 mM Ca 2+ concentration, cell membrane was significantly permeabilized in comparison to cavitation (MB + US) group. US assisted MB cavitation increases membrane permeabilization to ~35–40%, while increasing Ca2+ concentration to 5 mM leads to ~60–70% permeabilization, which is similar at higher Ca2+ concentrations. The measurements, performed at different PI administration times, indicate (Ca2+ + MB + US) induced membrane permeabilization to reach the state of maximal permeabilization within 20 min after treatment, with no significant increase at later time Biomedicines 2021, 9, 32 8 of 15

points. The latter ﬁnding suggests rapid distribution of highly diffusible calcium ions Biomedicines 2021, 9, x FOR PEER REVIEW 8 of 16 within the cell interior, eventually leading to directly or indirectly induced cell membrane Biomedicines 2021, 9, x FOR PEER REVIEWpermeability increase. 8 of 16

FigureFigure 5.5. CellCell membranemembrane permeabilizationpermeabilization (PI(PI assayassay afterafter 2020 min–3min–3 h),h), evaluatedevaluated afterafter USUS (400(400 kPa)kPa) treatment in the presence of MBs at different Ca2+2+ concentration. (MB), (Ca2+2+ + MB) and (Ca2+2+ + US) treatmentFigure 5. in Cell the membrane presence of permeabilization MBs at different Ca(PI assayconcentration. after 20 min–3 (MB), h), (Ca evaluated+ MB) after and (CaUS (400+ US)kPa) experimental groups (A); (Ca2+ + MB + US) experimental groups (B). ** p < 0.01, *** p < 0.001 indi‐ experimentaltreatment in the groups presence (A); (Caof MBs2+ + at MB different + US) Ca experimental2+ concentration. groups (MB), (B). (Ca ** 2+p +< MB) 0.01, and *** (Cap <2+ + 0.001 US) cate statistical significance between cavitation [(Ca2+ + MB +US) at 0 mM Ca2+ concentration] and indicateexperimental statistical groups signiﬁcance (A); (Ca2+ between + MB + US) cavitation experimental [(Ca2+ groups+ MB +US) (B). ** at p 0 < mM 0.01, Ca ***2+ pconcentration] < 0.001 indi‐ therapeutic [(Ca2+ + MB +US) at 1–20 mM Ca2+ concentration] groups. andcate therapeutic statistical significance [(Ca2+ + MB between +US) at cavitation 1–20 mM Ca [(Ca2+2+concentration] + MB +US) at groups.0 mM Ca2+ concentration] and therapeutic [(Ca2+ + MB +US) at 1–20 mM Ca2+ concentration] groups.

Figure 6. Cell metabolic activity (MTT assay after 48 h), evaluated after US (400 kPa) treatment in the presence of MBs at different Ca2+ concentration. (MB), (Ca2+ + MB) and (Ca2+ + US) experimental Figure 6. Cell metabolic activity (MTT assay after 48 h), evaluated after US (400 kPa) treatment in Figuregroups 6. (ACell); (Ca metabolic2+ + MB + activity US) experimental (MTT assay groups after 48 (B h),). evaluated** p < 0.01, after *** p US < 0.001 (400 indicate kPa) treatment statistical in the the presence of MBs at different Ca2+ concentration. (MB), (Ca2+ + MB) and (Ca2+ + US) experimental presencesignificance of MBs between at different cavitation Ca2+ [(Caconcentration.2+ + MB +US) (MB),at 0 mM (Ca Ca2+ 2++ concentration] MB) and (Ca2+ and+ US) therapeutic experimental groups (A); (Ca2+ + MB + US) experimental groups (B). ** p < 0.01, *** p < 0.001 indicate statistical groups[(Ca2+ + ( MBA); (Ca+US)2+ at+ MB1–20 + mM US) Ca experimental2+ concentration] groups groups. (B). ** p < 0.01, *** p < 0.001 indicate statistical significance between cavitation [(Ca2+ + MB +US) at 0 mM Ca2+ concentration] and therapeutic 2+ 2+ signiﬁcance[(Ca2+ + MB between+US) at 1–20 cavitation mM Ca [(Ca2+ concentration]+ MB +US) groups. at 0 mM Ca concentration] and therapeutic [(Ca2+PI+ assay MB +US) represents at 1–20 mM the Ca increase2+ concentration] in cell membrane groups. permeability induced by SP (Figure 5). PI PIwas assay added represents at a different the increase time after in cell SP: membrane 20 min, 1 permeability h, 2 h, and 3 induced h. The experimental by SP (Figure 2+ results5). PI was show added that MBsat a differentwithout US time or afterMB and SP: Ca20 min, (20 mM)1 h, 2 coadministration h, and 3 h. The experimental without US 2+ asresults well as show cell exposurethat MBs towithout US in theUS presenceor MB and of Ca2+ (20 mM) havecoadministration only insignificant without effect US 2+ toas cell well membrane as cell exposure permeabilization to US in the (Figurepresence 5A). of Ca The2+ (20 obtained mM) have results only in insignificant (Ca + MB + effect US) 2+ groupsto cell membrane(Figure 5B) permeabilization have shown that (Figure in the presence 5A). The ofobtained 5–20 mM results Ca inconcentration, (Ca2+ + MB + cellUS) membranegroups (Figure was significantly5B) have shown permeabilized that in the inpresence comparison of 5–20 to cavitationmM Ca2+ concentration,(MB + US) group. cell membrane was significantly permeabilized in comparison to cavitation (MB + US) group. Biomedicines 2021, 9, x FOR PEER REVIEW 9 of 16

US assisted MB cavitation increases membrane permeabilization to ~35–40%, while in‐ creasing Ca2+ concentration to 5 mM leads to ~60–70% permeabilization, which is similar at higher Ca2+ concentrations. The measurements, performed at different PI administra‐ tion times, indicate (Ca2+ + MB + US) induced membrane permeabilization to reach the

Biomedicines 2021, 9, 32 state of maximal permeabilization within 20 min after treatment, with no significant9 of 15in‐ crease at later time points. The latter finding suggests rapid distribution of highly diffus‐ ible calcium ions within the cell interior, eventually leading to directly or indirectly in‐ duced cell membrane permeability increase. InIn order order to to find find out out how how these these conditions conditions affect affect cell cell metabolic metabolic activity, activity, MTT MTT assay assay 2+ waswas performed performed after after 48 48 h h (Figure (Figure6). 6). Similarly, Similarly, MBs MBs without without US US or or MB MB and and Ca Ca2+(20 (20 mM) mM) 2+ coadministrationcoadministration without without US US as wellas well as cellas cell exposure exposure to US to in US the in presence the presence of Ca of(20 Ca mM)2+ (20 havemM) only have insignificant only insignificant effect on effect cell metabolic on cell metabolic activity (Figure activity6A). (Figure However, 6A). theHowever, combina- the 2+ tioncombination of US, MB, of and US, Ca MB, inand the Ca range2+ in the of 5–20 range mM of 5–20 concentration mM concentration has significantly has significantly reduced cellreduced metabolic cell activitymetabolic down activity to ~40% down (Figure to ~40%6B). The(Figure results 6B). also The indicate results thatalso the indicate increase that 2+ inthe Ca increaseconcentration in Ca2+ concentration has gradual negative has gradual effect negative for cell effect metabolic for cell activity. metabolic activity. TheThe overall overall experimental experimental results results of of PI, PI, MTT, MTT, and and colony colony formation formation assays assays are are given given inin Figure Figure7 .7. The The results results of of membrane membrane permeabilization permeabilization efficiency, efficiency, observed observed after after 20 20 min, min, areare in in agreement agreement with with the the results results of of decrease decrease in cellin cell metabolic metabolic activity activity (48 h)(48 and h) and cell colonycell col‐ formationony formation efficiency efficiency (6 d). (6 Since d). Since there there was was no no significant significant difference difference (NS) (NS) in in the the results, results, obtained by different cell viability assays (based on different cell viability characteristics), obtained by different cell viability assays (based on different cell2+ viability characteristics), itit implies implies instant instant cell cell death death (up (up to to 20 20 min) min) to to be be dominant dominant in in Ca Ca2+delivery delivery via via SP SP with with no no additionaladditional cell cell death death occurring occurring within within 6 6 d. d.

FigureFigure 7. 7.Cell Cell membrane membrane permeabilization,permeabilization, decrease in metabolic activity activity and and decrease decrease in in colony colony formation efficiency, evaluated after SP for different experimental groups and compared within. formation efﬁciency, evaluated after SP for different experimental groups and compared within. “NS” “NS” indicates no significance between experimental groups (one‐way ANOVA). indicates no signiﬁcance between experimental groups (one-way ANOVA).

3.3.3.3. Acoustic Acoustic Emissions Emissions at at 400 400 kPa kPa Acoustic Acoustic Pressure Pressure AcousticAcoustic emission emission signals, signals, obtained obtained at at 400 400 kPa kPa acoustic acoustic pressure, pressure, were were transformed transformed to frequencyto frequency spectrum spectrum (Figure (Figure8A) for 8A) subsequent for subsequent AE RMS AEquantificationRMS quantification in different in different frequency fre‐ rangesquency from ranges 1.5–1.75 from MHz 1.5–1.75 up toMHz 10.5–10.75 up to 10.5–10.75 MHz (Figure MHz8B). (Figure 8B). Figure8A represents the temporal evolution of the amplitude of broadband noise in the frequency domain. As the pronounced frequency components are observed in whole frequency spectrum, reaching their maximal values at 0.647–0.657 s, broadband noise is observed. The latter phenomenon is directly associated to MB inertial cavitation [21–25]. Correspondingly, the curves of AERMS, quantified in different frequency ranges throughout whole frequency spectrum, indicate high above-background acoustic emissions in all of the tested frequency ranges up to 8.5–8.75 MHz (Figure8B). AE RMS values, quantified in 9.5–9.75 and 10.5–10.75 MHz frequency bands, had minor increases above background level, which is in line with the frequency spectrum. The highest difference in acoustic emissions between +MB and −MB groups was determined in the 1.5–1.75 MHz frequency range. In addition to this, in all the frequency ranges tested, AERMS decrease to ~0 V Biomedicines 2021, 9, x FOR PEER REVIEW 10 of 16

Biomedicines 2021, 9, 32 10 of 15

Biomedicines 2021, 9, x FOR PEER backgroundREVIEW level occurred within 3 s (Figure8B), indicating complete MB sonodestruction.10 of 16 Thus, the adjusted exposure duration of 6 s is sufﬁcient for maximal sonoporation efﬁciency. Figure 8. Broadband noise temporal evolution in frequency spectrum (A), AERMS dynamics of the corresponding signal, evaluated in different frequency ranges (B), at 400 kPa acoustic pressure US.

Figure 8A represents the temporal evolution of the amplitude of broadband noise in the frequency domain. As the pronounced frequency components are observed in whole frequency spectrum, reaching their maximal values at 0.647–0.657 s, broadband noise is observed. The latter phenomenon is directly associated to MB inertial cavitation [21–25]. Correspondingly, the curves of AERMS, quantified in different frequency ranges through‐ out whole frequency spectrum, indicate high above‐background acoustic emissions in all of the tested frequency ranges up to 8.5–8.75 MHz (Figure 8B). AERMS values, quantified in 9.5–9.75 and 10.5–10.75 MHz frequency bands, had minor increases above background level, which is in line with the frequency spectrum. The highest difference in acoustic emissions between +MB and −MB groups was determined in the 1.5–1.75 MHz frequency range. In addition to this, in all the frequency ranges tested, AERMS decrease to ~0 V back‐ ground level occurred within 3 s (Figure 8B), indicating complete MB sonodestruction. Figure 8. Broadband noise temporal evolution in frequency spectrum (A), AERMS dynamics of the corresponding signal, Figure 8. Broadband noise temporalThus, the evolution adjusted in exposure frequency duration spectrum (ofA ),6 AE s isRMS sufficientdynamics for of themaximal corresponding sonoporation signal, effi‐ evaluated in different frequency ranges (B), at 400 kPa acoustic pressure US. evaluated in different frequencyciency. ranges (B), at 400 kPa acoustic pressure US. Figure 8A represents the temporal evolution of the amplitude of broadband noise in 3.4. Sonoporation Results in Acoustic Pressure Scale 3.4.the Sonoporation frequency domain. Results As in the Acoustic pronounced Pressure frequency Scale components are observed in whole frequencyIn order spectrum, to investigate reaching the their role maximal of cavitation values at in 0.647–0.657 Ca2+ intracellular s, broadband delivery noise isvia SP, 2+ acousticobserved.In order pressure The to latter investigate was phenomenon varied thein the role is directlyrange of cavitation of associated 0–500 kPa in to Ca MBat constant inertialintracellular cavitation 5 mM deliveryCa [21–25].2+ concentra via SP,‐ 2+ tion.acousticCorrespondingly, The pressure latter concentration was the varied curves in ofwas theAE selectedRMS range, quantified of according 0–500 in kPa different to at constantthe frequency data of 5 mM previous ranges Ca throughconcentration. experiments,‐ Theout latter whole concentration frequency2+ spectrum, was selected indicate according high above to‐background the data of previousacoustic emissions experiments, in all as the as the increase 2+in Ca concentration from 5 to 10 or 20 mM had practically no influence increaseof the tested in Ca frequencyconcentration ranges up from to 8.5–8.75 5 to 10 MHz or 20(Figure mM 8B). had AE practicallyRMS values, noquantified inﬂuence on on inducing additional cell death in (Ca2+ + MB + US) group (Figures 4–6). The results of inducingin 9.5–9.75 additional and 10.5–10.75 cell death MHz in frequency (Ca2+ + bands, MB + US)had groupminor increases (Figures 4above–6). The background results of cell cell permeabilization, metabolic activity decrease, and colony formation activity decrease permeabilization,level, which is in metabolic line with the activity frequency decrease, spectrum. and colonyThe highest formation difference activity in acoustic decrease at at constant 5 mM Ca2+ concentration and varied acoustic pressure are presented in Figure constantemissions 5 mM between Ca2+ +MBconcentration and −MB groups and varied was determined acoustic pressurein the 1.5–1.75 are presented MHz frequency in Figure 9. 9. range. In addition to this, in all the frequency ranges tested, AERMS decrease to ~0 V back‐ ground level occurred within 3 s (Figure 8B), indicating complete MB sonodestruction. Thus, the adjusted exposure duration of 6 s is sufficient for maximal sonoporation effi‐ ciency.

3.4. Sonoporation Results in Acoustic Pressure Scale In order to investigate the role of cavitation in Ca2+ intracellular delivery via SP, acoustic pressure was varied in the range of 0–500 kPa at constant 5 mM Ca2+ concentra‐ tion. The latter concentration was selected according to the data of previous experiments, as the increase in Ca2+ concentration from 5 to 10 or 20 mM had practically no influence on inducing additional cell death in (Ca2+ + MB + US) group (Figures 4–6). The results of cell permeabilization, metabolic activity decrease, and colony formation activity decrease at constant 5 mM Ca2+ concentration and varied acoustic pressure are presented in Figure 9. Figure 9. CellCell membrane membrane permeabilization permeabilization (A (),A ),metabolic metabolic activity activity decrease decrease (B) ( andB) and decrease decrease in cell in cellcolony colony formation formation effi‐ ciency (C), evaluated in cavitation (MB + US) and therapeutic (Ca2+ + MB + US) groups at different acoustic pressure. efﬁciency (C), evaluated in cavitation (MB + US) and therapeutic (Ca2+ + MB + US) groups at different acoustic pressure.

Similar dynamics have been observed in the results of cell death, evaluated by different cell viability assays. PI assay results, obtained by PI test at different time after SP (Figure9A), indicate increasing permeability for both cavitation (MB + US) and therapeutic (Ca2+ + MB + US) groups up to 300 kPa with the subsequent plateau of ~35% and ~65%, respectively, achieved at higher acoustic pressures. At constant acoustic pressure, the results of membrane permeabilization, obtained at different times, indicate similar permeabilization efﬁciency, which remains unchanged from 20 min up to 3 h. The results of decrease in cell metabolic activity (Figure9B) indicate a similar tendency with increasing Figure 9. Cell membraneacoustic permeabilization pressure, (A), followedmetabolic activity by subsequent decrease (B) saturation and decrease at in 300–500cell colony kPa formation for both effi‐ cavitation 2+ ciency (C), evaluated in cavitation(~35%) and (MB + therapeutic US) and therapeutic (~60%) (Ca groups. + MB + US) The groups results at different of cell clonogenicacoustic pressure. assay (Figure9C) are in accordance with the results of previous tests, indicating a decrease in cell colony Biomedicines 2021, 9, x FOR PEER REVIEW 11 of 16

Similar dynamics have been observed in the results of cell death, evaluated by differ‐ ent cell viability assays. PI assay results, obtained by PI test at different time after SP (Fig‐ ure 9A), indicate increasing permeability for both cavitation (MB + US) and therapeutic (Ca2+ + MB + US) groups up to 300 kPa with the subsequent plateau of ~35% and ~65%, respectively, achieved at higher acoustic pressures. At constant acoustic pressure, the re‐ sults of membrane permeabilization, obtained at different times, indicate similar permea‐ bilization efficiency, which remains unchanged from 20 min up to 3 h. The results of de‐ Biomedicines 2021, 9, 32 crease in cell metabolic activity (Figure 9B) indicate a similar tendency with increasing11 of 15 acoustic pressure, followed by subsequent saturation at 300–500 kPa for both cavitation (~35%) and therapeutic (~60%) groups. The results of cell clonogenic assay (Figure 9C) are in accordance with the results of previous tests, indicating a decrease in cell colony for‐ 2+ mationformation efficiency efficiency for both for both cavitation cavitation (MB (MB+ US) + and US) therapeutic and therapeutic (Ca2+ + (Ca MB + +US) MB groups + US) upgroups to 300 up kPa, to with 300 kPa, further with saturation further saturation in the range in of the 300–500 range kPa of 300–500 at (~35–40%) kPa at and (~35–40%) (~65%) forand the (~65%) respective for the groups. respective As groups.PI, MTT, As and PI, MTT,cell clonogenic and cell clonogenic assay results assay have results similar have tendenciessimilar tendencies and values and in values the acoustic in the acoustic pressure pressure range, we range, again we observe again observe that cell that death cell achieveddeath achieved after 20 after min 20 sustains min sustains the same the after same the after 48 h the as 48 well h as as well 6 d period. as 6 d period. TheThe results results of of PI, PI, MTT, MTT, and and cell cell clonogenic clonogenic assays assays were were used used to to calculate calculate the the changes changes inin cell cell membrane membrane permeability, permeability, obtained obtained after after 20 20 min min ( (Δ ∆Permeabilisation)Permeabilisation) (Figure (Figure 10A), 10A), metabolicmetabolic activity activity (Δ (∆MetabolicMetabolic activity) activity) (Figure (Figure 10B), 10B), and and colony colony formation formation efficiency efficiency (Δ Colony(∆ Colony formation formation efficiency) efficiency) (Figure (Figure 10C). 10C). Either Either change change was was evaluated evaluated as as the the difference difference in cell membrane permeabilization, decrease in metabolic activity, or decrease in colony in cell membrane permeabilization, decrease in metabolic activity, or decrease in colony formation efficiency of three groups: (Ca2+ + MB + US)−(MB + US)−(Ca2+ + MB). formation efficiency of three groups: (Ca2+ + MB + US)−(MB + US)−(Ca2+ + MB).

FigureFigure 10. 10. TheThe change change ( (∆∆) )in in cell cell permeabilization permeabilization (A (A),), cell cell metabolic metabolic activity activity (B (B),), and and cell cell colony colony formation formation efficiency efﬁciency (C (C),), evaluated as permeabilization, decrease in metabolic activity, and decrease in colony formation efficiency between groups: evaluated as permeabilization, decrease in metabolic activity, and decrease in colony formation efﬁciency between groups: (Ca2+ + MB + US)−(MB + US)−(Ca2+ + MB). (Ca2+ + MB + US)−(MB + US)−(Ca2+ + MB).

Δ ∆Permeabilization,Permeabilization, evaluated at standardstandard time time after after SP, SP, 20 20 min min (Figure (Figure 10 10A),A), gradually gradu‐ allyincreases increases following following the the increment increment in acousticin acoustic pressure pressure until until it reaches it reaches saturation saturation level level of of~25–30% ~25–30% at at 300 300 kPa kPa acoustic acoustic pressure. pressure.∆ Δ MetabolicMetabolic activity activity (Figure (Figure 10 B)10B) and and∆ Δ cellcell colony col‐ onyformation formation efﬁciency efficiency (Figure (Figure 10C) have10C) similarhave similar absolute absolute values andvalues dynamics and dynamics in acoustic in acousticpressure pressure scale as ∆scalepermeabilization. as Δ permeabilization.

3.5.3.5. Acoustic Acoustic Emission Emission Quantification Quantiﬁcation in in Acoustic Acoustic Pressure Pressure Scale Scale SinceSince the the increase increase in in US US acoustic acoustic pressure pressure is is associated associated to to intensification intensiﬁcation in in cavitation cavitation activity,activity, MB MB acoustic acoustic emission emission signals, signals, detected detected at at different different acoustic acoustic pressures pressures (0–500 (0–500 kPa), kPa), Biomedicines 2021, 9, x FOR PEER REVIEW 12 of 16 werewere used used to to evaluate evaluate AE AERMSRMS in 1.5–1.75in 1.5–1.75 MHz MHz frequency frequency range. range. The obtained The obtained AERMS curves AERMS incurves exposure in exposure duration duration scale are scale given are in given Figure in 11A. Figure 11A.

Figure 11. AERMS curves in exposure duration scale at different acoustic pressure (A). ICD in acoustic pressure scale (B). Figure 11. AERMS curves in exposure duration scale at different acoustic pressure (A). ICD in acoustic pressure scale (B).

AERMS, plotted in exposure duration scale, represents both the intensity and duration of MB cavitation activity. Thus, the integral combining both parameters is considered to be a direct estimate of MB cavitation. In order to estimate ICD, the integral of AERMS was calculated during the entire US exposure duration for each acoustic pressure value using Equation (3). The form of ICD curve, plotted in the acoustic pressure range, is sigmoidal (Figure 11B).

3.6. ICD and Biological Effect Correlation The results of Δ permeabilization, Δ cell metabolic activity, and Δ cell colony for‐ mation efficiency were plotted in the scale of ICD in order to establish the quantified re‐ lation to MB inertial cavitation (Figure 12).

Figure 12. The correlation of ICD and the change (Δ) in cell permeabilization (A), cell metabolic activity (B), and cell colony formation efficiency (C).

The correlation results of ICD and the change in permeabilization (Figure 12A), met‐ abolic activity (Figure 12B), and cell colony formation efficiency (Figure 12C) indicate strong (R2 > 0.85) significant (p < 0.05) correlation. This shows that the obtained results, represented by three different cell viability assays, based on different cell viability charac‐ teristics: membrane permeabilisation, metabolic activity, and cell colony formation effi‐ ciency, are linearly dependent on the “amount” of broadband noise, produced by cavitat‐ ing MBs, and are directly associated to US induced MB IC.

4. Discussion In the current study, we have employed three different assays to evaluate cell viabil‐ ity dynamics after SP‐mediated calcium delivery into CHO cells. They all indicate that Ca2+ increase in combination with US assisted MB cavitation negatively affects all three different vital parameters associated to cell viability: cell membrane integrity, cell meta‐ bolic activity, and cell colony formation efficiency, in a similar fashion. PI assay has indi‐ cated decrease in membrane integrity after 20 min, which sustained similar for up to 3 h; Biomedicines 2021, 9, x FOR PEER REVIEW 12 of 16

Biomedicines 2021, 9, 32 12 of 15

Figure 11. AERMS curves in exposure duration scale at different acoustic pressure (A). ICD in acoustic pressure scale (B). AERMS, plotted in exposure duration scale, represents both the intensity and duration

of MBAE cavitationRMS, plotted activity. in exposure Thus, duration the integral scale, combining represents both the parameters intensity isand considered duration of to MBbe acavitation direct estimate activity. of Thus, MB cavitation. the integral In ordercombining to estimate both parameters ICD, the integral is considered of AERMS to bewas a calculated during the entire US exposure duration for each acoustic pressure value using direct estimate of MB cavitation. In order to estimate ICD, the integral of AERMS was calculated duringEquation the (3).entire The US form exposure of ICD duration curve, plotted for each in acoustic the acoustic pressure pressure value range,using Equation is sigmoidal (3). The(Figure form 11 ofB). ICD curve, plotted in the acoustic pressure range, is sigmoidal (Figure 11B). 3.6. ICD and Biological Effect Correlation 3.6. ICD and Biological Effect Correlation The results of ∆ permeabilization, ∆ cell metabolic activity, and ∆ cell colony formation The results of Δ permeabilization, Δ cell metabolic activity, and Δ cell colony for‐ efﬁciency were plotted in the scale of ICD in order to establish the quantiﬁed relation to mation efficiency were plotted in the scale of ICD in order to establish the quantified re‐ MB inertial cavitation (Figure 12). lation to MB inertial cavitation (Figure 12).

FigureFigure 12. 12. TheThe correlation correlation of of ICD ICD and and the the change change ( (Δ∆)) in in cell cell permeabilization permeabilization ( (AA),), cell cell metabolic metabolic activity activity ( (BB),), and and cell cell colony colony formationformation efficiency efﬁciency ( (CC).).

TheThe correlation correlation results results of of ICD ICD and and the thechange change in permeabilization in permeabilization (Figure (Figure 12A), 12metA),‐ abolicmetabolic activity activity (Figure (Figure 12B), 12 andB), andcell cellcolony colony formation formation efficiency efficiency (Figure (Figure 12C) 12 indicateC) indi- strongcate strong (R2 > (R 0.85)2 > 0.85) significant significant (p < ( p0.05)< 0.05) correlation. correlation. This This shows shows that that the the obtained obtained results, results, representedrepresented by three different cellcell viabilityviability assays, assays, based based on on different different cell cell viability viability character- charac‐ teristics:istics: membrane membrane permeabilisation, permeabilisation, metabolic metabolic activity, activity, and and cell colonycell colony formation formation efficiency, effi‐ ciency,are linearly are linearly dependent dependent on the “amount”on the “amount” of broadband of broadband noise, produced noise, produced by cavitating by cavitat MBs,‐ ingand MBs, are directly and are associated directly associated to US induced to US MBinduced IC. MB IC.

4.4. Discussion Discussion InIn the the current current study, study, we have employed threethree different different assays assays to to evaluate evaluate cell cell viability viabil‐ 2+ itydynamics dynamics after after SP-mediated SP‐mediated calcium calcium delivery delivery into into CHO CHO cells. cells. They They all indicate all indicate that Cathat Caincrease2+ increase in combination in combination with USwith assisted US assisted MB cavitation MB cavitation negatively negatively affects all affects three all different three differentvital parameters vital parameters associated associated to cell viability: to cell cellviability: membrane cell membrane integrity, cell integrity, metabolic cell activity, meta‐ bolicand cellactivity, colony and formation cell colony efficiency, formation in a efficiency, similar fashion. in a similar PI assay fashion. has indicated PI assay decrease has indi in‐ catedmembrane decrease integrity in membrane after 20 min, integrity which after sustained 20 min, similar which for sustained up to 3 h; similar therefore, for up estimated to 3 h; pore lifetime was at least—3 h. The results of MTT and cell clonogenic assays have shown similar tendency and level as PI values. Therefore, we have obtained the same cell death level after 20 min (PI assay) compared to 24 h (MTT assay) as well as to 6 d (cell clonogenic assay). This indicates rapid cell death due to Ca2+ delivery via SP. In contrast to other studies [3,4] we have not observed a positive effect of low (up to 3 mM) Ca2+ concentration for cell viability neither for pore resealing nor cell viability. The detailed reasoning behind this is to be determined in future research. The viability dynamics of CHO cells after Ca2+ delivery via SP are very similar to those obtained in our previous works on BLM delivery [26,27]. Similarly, we have attained efficient Ca2+ delivery via SP only after combined US–MB employment, as we have observed no significant changes in cell viability after 6 d due to the application of US alone, except at 800 kPa using 20 mM Ca2+ concentration. In addition to this, the efficiency of BLM delivery was achieved ~30%, evaluated by cell clonogenic assay, and it is line with the current results using Ca2+. What is more, the mechanism for both BLM and Ca2+ Biomedicines 2021, 9, 32 13 of 15

intracellular delivery was associated to MB IC. These findings indicate a similar anticancer agent delivery mechanism leading to analogous outcome, and indirectly imply successful future feasibility of Ca2+ delivery via SP to initiate anticancer activity in tumor cells. The activity of anticancer chemotherapy and gene therapy is rather slow in cell death, since the effect of anticancer drug use was achieved after 24–48 h [5–8,10] and plasmid DNA internalization to nucleus and expression takes long durations so that the maximum level of gene expression efficiency was generally obtained after 12–72 h [16–19]. In contrast to the conventional therapy techniques, rapid Ca2+ distribution within the cell interior via SP alters the vital state of the cell by intrinsic processes and induces a rapid cell killing effect. Therefore, Ca2+ application for anticancer treatment would offer new opportunities for initiating the rapid cell killing effect, occurring within 20 min. This also reduces high systemic cytotoxicity imposed by anticancer drugs. SC is characterized by linear volumetric oscillations of MBs at lower driving pressures, while IC dominates at higher pressures and signifies in non-linear MB vibrations culminating in implosion. The spectral content of emitted US waves during SC consists of harmonic, subharmonic, and ultraharmonic constituents of US central frequency. Intense MB collapse generates white noise with components plainly observed in all the frequencies of the spectrum of analyzed output US signals. Therefore, broadband noise is considered to be an unambiguous confirmation of MB collapse and, respectively, IC [25,28,29]. IC has been substantiated to be involved in SP according to high correlation of IC based metrics, MB sonodestruction rate [26] and inertial cavitation dose [21], with the efficacy of therapeutic agent sonotransfer [23,24,26,30] and cell viability [21,23,26]. Therefore, passively registered acoustic emissions from MBs, induced by US, were analyzed in order to acquire valuable information about cavitation pattern. After initial analysis, the amount of broadband noise was quantified as differential RMS in different frequency ranges with the aim to obtain the estimate “AERMS”. IC presence (at 400 kPa acoustic pressure) was indicated by temporal curves of AERMS estimate, ranging above 0 V baseline in all frequency ranges 1.5–1.75 to 8.5–8.75 MHz. The 1.5–1.75 MHz frequency range was empirically associated to the highest broadband noise emissions according to the highest difference between +MB and −MB groups. Therefore, the integral metric, ICD, was evaluated in this range for the subsequent correlation analysis in the acoustic pressure scale. Since the beginning of investigation in the mechanism behind SP, an ongoing discourse has been initiated by the researchers about SC vs. IC [20]. SC employment for SP is considered to be very convenient, as it allows the attainment of maximal site-specificity of the method without imposing damage to nearby healthy cells. Low bioeffect efficiency, obtained at low acoustic pressure, 100 kPa, was the preliminary indication of the necessity of IC in order to induce effective SP. Specific evidence of IC presence in Ca2+ delivery via SP are the AERMS curves, which quantitatively indicate the presence of broadband noise in the 1.5–1.75 to 8.5–8.75 MHz frequency ranges. The registered broadband noise emissions of cavitating MBs are considered to be the distinctive feature of IC and allow the detection and quantification of IC activities [21–25]. The cumulated amount of broadband noise, ICD, highly correlates (R2 > 0.85) with the results, provided by different cell viability assays: (i) membrane permeabilization (PI assay), metabolic activity (MTT assay), and colony formation efficiency (cell clonogenic assay). These relations indicate the cellular bioeffects of different origin, produced by Ca2+ delivery via SP, to be directly associated to IC activity. It also implies that Ca2+ delivery via MB IC alters all three different cell viability criteria, independently estimated by three different cell viability tests. The role of SC in SP becomes dominant when MBs are positioned near or bound to the cell surface; thereby, the distance between the cell and nearby MBs is reduced [31,32]. Since the impact of microstreaming is significantly downscaled with the increment in cell–MB separation, SC intensity is reduced and, therefore, IC prevails [22–24,26,27,30]. The as- sumption is reinforced by effective SP, obtained at SC regime, conducted on monolayers, where cells are tightly embedded and come into close contact with adjacent MBs [33,34]. Biomedicines 2021, 9, 32 14 of 15

In general, sudden cell death by Ca2+ delivery via SP is associated to MB IC, as it was empirically revealed by high ICD correlation with change in either permeabilization, metabolic activity, or colony formation efficiency. Therefore, findings, obtained in our study, indicate that in order to initiate rapid cell killing via Ca2+ delivery via SP, the infliction of MB IC activity is preferred.

5. Conclusions 1. The increase in Ca2+ concentration during sonoporation negatively affects the fundamental characteristics of cell viability: (i) cell membrane integrity, (ii) cell metabolic activity, and (iii) cell colony formation efﬁciency, which were independently evaluated using different cell viability assays. 2. Ca2+ delivery via sonoporation induces instantly occurring cell death—within 20 min after treatment, with no additional cell death observed within the 2-d to 6-d period. 3. High correlation (R2 > 0.85, p < 0.01) of inertial cavitation dose with the results of different-criterion-based cell viability assays implies inertial cavitation activities to be directly involved in calcium delivery via sonoporation.

Author Contributions: M.M., D.N., S.C., B.J., and S.Š. conceived and designed the experiments; M.M., D.N., S.C., and B.J. performed the experiments; M.M., D.N., S.C., B.J., and S.Š. analyzed the data; M.M., D.N., and S.Š. wrote the paper; M.M. and S.Š. reviewed and edited the paper. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the European Social Fund according to the activity ‘Im- provement of researchers’ qualification by implementing world-class R&D projects’ of Measure No. 09.3.3-LMT-K-712-01-0188. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data supporting the findings of this study are available within the article. Conflicts of Interest: The authors declare no competing interests.

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