Partner: Krzysztof Bilmin

Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)

Recent publications
1.Secomski W., Bilmin K., Kujawska T., Nowicki A., Grieb P., Lewin P.A., In vitro ultrasound experiments: Standing wave and multiple reflections influence on the outcome, Ultrasonics, ISSN: 0041-624X, DOI: 10.1016/j.ultras.2017.02.008, Vol.77, pp.203-213, 2017
Abstract:

The purpose of this work was to determine the influence of standing waves and possible multiple reflections under the conditions often encountered in examining the effects of ultrasound exposure on the cell cultures in vitro. More specifically, the goal was to quantitatively ascertain the influence of ultrasound exposure under free field (FF) and standing waves (SW) and multiple reflections (MR) conditions (SWMR) on the biological endpoint (50% cell necrosis). Such information would help in designing the experiments, in which the geometry of the container with biological tissue may prevent FF conditions to be established and in which the ultrasound generated temperature elevation is undesirable. This goal was accomplished by performing systematic, side-by-side experiments in vitro with C6 rat glioma cancer cells using 12 well and 96 well plates. It was determined that to obtain 50% of cell viability using the 12 well plates, the spatial average, temporal average (ISATA) intensities of 0.32 W/cm2 and 5.89 W/cm2 were needed under SWMR and FF conditions, respectively. For 96 well plates the results were 0.80 W/cm2 and 2.86 W/cm2 respectively. The corresponding, hydrophone measured pRMS maximum pressure amplitude values, were 0.71 MPa, 0.75 MPa, 0.75 MPa and 0.73 MPa, respectively. These results suggest that pRMS pressure amplitude was independent of the measurement set-up geometry and hence could be used to predict the cells’ mortality threshold under any in vitro experimental conditions or even as a starting point for (pre-clinical) in vivo tests. The described procedure of the hydrophone measurements of the pRMS maximum pressure amplitude at the k/2 distance (here 0.75 mm) from the cell’s level at the bottom of the dish or plate provides the guideline allowing the difference between the FF and SWMR conditions to be determined in any experimental setup. The outcome of the measurements also indicates that SWMR exposure might be useful at any ultrasound assisted therapy experiments as it permits to reduce thermal effects. Although the results presented are valid for the experimental conditions used in this study they can be generalized. The analysis developed provides methodology facilitating independent laboratories to determine their specific ultrasound exposure parameters for a given biological end-point under standing waves and multiple reflections conditions. The analysis also permits verification of the outcome of the experiments mimicking pre- and clinical environment between different, unaffiliated teams of researchers.

Keywords:

Standing wave, Ultrasound pressure, Ultrasound intensity, C6 glioma, Anticancer therapy, Sonodynamic therapy, Ultrasound bio-effects

Affiliations:
Secomski W.-IPPT PAN
Bilmin K.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)
Kujawska T.-IPPT PAN
Nowicki A.-IPPT PAN
Grieb P.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)
Lewin P.A.-Drexel University (US)
2.Bilmin K., Kujawska T., Secomski W., Nowicki A., Grieb P., 5-Aminolevulinic acid-mediated sonosensitization of rat RG2 glioma cells in vitro, FOLIA NEUROPATHOLOGICA, ISSN: 1641-4640, DOI: 10.5114/fn.2016.62233, Vol.54, No.3, pp.1-7, 2016
Abstract:

Sonodynamic therapy (SDT) is a promising technique based on the ability of certain substances, called sonosensitizers, to sensitize cancer cells to non-thermal effects of low-energy ultrasound waves, allowing their destruction. Sonosensitization is thought to induce cell death by direct physical effects such as cavitation and acoustical streaming as well as by complementary chemical reactions generating oxygen free radicals. One of the promising sonosensitizers is 5-aminolevulinic acid (ALA) which upon selective uptake by cancer cells is metabolized and accumulated as protoporphyrin IX. The objective of the study was to describe ALA-mediated sonodynamic effects in vitro on a rat RG2 glioma cell line. Glioma cells, seeded at the bottom of 96-well plates and incubated with ALA (10 μg/ml) for 6 h, were exposed to the sinusoidal US pulses with a resonance frequency of 1 MHz, 1000 μs duration, 0.4 duty-cycle, and average acoustic power varying from 2 W to 6 W. Ultrasound waves were generated by a flat circular piezoelectric transducer with a diameter of 25 mm. Cell viability was determined by MTT assay. Structural cellular changes were visualized with a fluorescence microscope. Signs of cytotoxicity such as a decrease in cell viability, chromatin condensation and apoptosis were found. ALA-mediated SDT evokes cytotoxic effects of low intensity US on rat RG2 glioma cells in vitro. This cell line is indicated for further preclinical assessment of SDT in in vivo conditions.

Keywords:

5-aminolevulinic acid, sonodynamic therapy, rat RG2 glioma cells, cell viability

Affiliations:
Bilmin K.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)
Kujawska T.-IPPT PAN
Secomski W.-IPPT PAN
Nowicki A.-IPPT PAN
Grieb P.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)
3.Kujawska T., Secomski W., Bilmin K., Nowicki A., Grieb P., Impact of thermal effects induced by ultrasound on viability of rat C6 glioma cells, Ultrasonics, ISSN: 0041-624X, DOI: 10.1016/j.ultras.2014.02.002, Vol.54, pp.1366-1372, 2014
Abstract:

In order to have consistent and repeatable effects of sonodynamic therapy (SDT) on various cancer cells or tissue lesions we should be able to control a delivered ultrasound energy and thermal effects induced. The objective of this study was to investigate viability of rat C6 glioma cells in vitro depending on the intensity of ultrasound in the region of cells and to determine the exposure time inducing temperature rise above 43°C, which is known to be toxic for cells. For measurements a planar piezoelectric transducer with a diameter of 20 mm and a resonance frequency of 1.06 MHz was used. The transducer generated tone bursts with 94 μs duration, 0.4 duty-cycle and initial intensity ISATA (spatial averaged, temporal averaged) varied from 0.33 W/cm2 to 8 W/cm2 (average acoustic power varied from 1 W to 24 W). The rat C6 glioma cells were cultured on a bottom of wells in 12-well plates, incubated for 24 h and then exposed to ultrasound with measured acoustic properties, inducing or causing no thermal effects leading to cell death. Cell viability rate was determined by MTT assay (a standard colorimetric assay for assessing cell viability) as the ratio of the optical densities of the group treated by ultrasound to the control group. Structural cellular changes and apoptosis estimation were observed under a microscope. Quantitative analysis of the obtained results allowed to determine the maximal exposure time that does not lead to the thermal effects above 43°C in the region of cells for each initial intensity of the tone bursts used as well as the threshold intensity causing cell death after 3 min exposure to ultrasound due to thermal effects. The averaged threshold intensity was found to be about 5.7 W/cm2.

Keywords:

Cancer cells, Photo-sensitizers, Sonodynamic therapy, Thermal effects, Ultrasonic beam properties

Affiliations:
Kujawska T.-IPPT PAN
Secomski W.-IPPT PAN
Bilmin K.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)
Nowicki A.-IPPT PAN
Grieb P.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)
4.Secomski W., Bilmin K., Kujawska T., Nowicki A., Grieb P., Rat cancer cells necrosis induced by ultrasound, HYDROACOUSTICS, ISSN: 1642-1817, Vol.17, pp.179-186, 2014
Abstract:

Sonodynamic therapy is the ultrasound dependent enhancement of the cytotoxic activities of certain drugs called sonosensitizers. The study of therapeutic efficacy of ultrasound is always preceded by in-vitro tests. In this work, two in-vitro sonication procedures were compared. One with the transducer positioned bellow the cell colony, radiating upward, with standing wave reflected from the water-air surface, the second, in the free field conditions. Efficiency of the cancer cells necrosis caused by ultrasound was compared with acoustical field intensity ISPTA measured by a hydrophone. The standing wave conditions effectively increased the intensity of the ultrasonic wave at the level of cells. To achieve 50% of cell viability, the intensity ISATA, decreased from 5.8 W/cm2 to 0.3 W/cm2. In summary, sonication in the standing wave conditions can effectively and reproducibly destroy cells by ensuring the sterility and without the risk of overheating.

Keywords:

ultrasound, sonodynamic therapy, cancer cells, necrosis

Affiliations:
Secomski W.-IPPT PAN
Bilmin K.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)
Kujawska T.-IPPT PAN
Nowicki A.-IPPT PAN
Grieb P.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)

Conference papers
1.Secomski W., Bilmin K., Kujawska T., Nowicki A., Grieb P., Niszczenie komórek nowotworowych glejaka C6 za pomocą fali ultradźwiękowej i kwasu aminolewulinowego (ALA), 59th Open Seminar on Acoustics, 2012-09-10/09-14, Boszkowo (PL), pp.225-228, 2012
Abstract:

Nową metodą terapii antynowotworowej glejaków jest sonoterapia polegająca na jednoczesnym podawaniu leku kumulującego się w tkankach nowotworowych i napromieniowanie falą akustyczną. Skuteczność metody została sprawdzona na komórkach glejaka C6, poddanych działaniu kwasu aminolewulinowego (ALA) i fali ultradźwiękowej 1MHz o natężeniu 0.94, 1.88 i 3.77W/cm2 przez 3 min. Jednoczesne działanie fali akustycznej i ALA było skuteczniejsze (pozostało 15%-13% żywych komórek) niż samej fali akustycznej (41%-27%) dla natężeń 0.94 i 1.88W/cm2. Natężenie 3.77W/cm2 było wystarczające do zniszczenia większości komórek niezależnie od obecności ALA. Podczas eksperymentu zaobserwowano powstawanie fali stojącej, zwiększającej natężenie 7x-10x. Otrzymane wyniki wskazują na przydatność nowej techniki sonodynamicznej do terapii nowotworów.

Keywords:

ultradźwięki, sonoterapia, terapia antynowotworowa, glejaki

Affiliations:
Secomski W.-IPPT PAN
Bilmin K.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)
Kujawska T.-IPPT PAN
Nowicki A.-IPPT PAN
Grieb P.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)

Conference abstracts
1.Secomski W., Olszewski R., Bilmin K., Nowicki A., Kujawska T., Grieb P., Terapeutyczne wykorzystanie ultradźwięków – wspomaganie procesu rozpuszczania skrzeplin oraz stymulacja apoptozy komórek nowotworowych, XIX Krajowa Konferencja Biocybernetyka i Inżynieria Biomedyczna, 2015-10-14/10-16, Warszawa (PL), pp.59, 2015
Abstract:

Ultradźwięki są stosowane w medycynie zarówno w diagnostyce - ultrasonografii jak i w terapii. Działanie terapeutyczne sprowadza się do efektów termicznych, uśmiercania komórek przez apoptozę oraz efektów sonomechanicznych. Terapie in-vivo są poprzedzane testami in-vitro komórek umieszczonych na szalce Petriego. W tej pracy zostaną przedstawione dwa efekty oddziaływania fali akustycznej na komórki: wspomaganie rozpuszczania skrzepliny oraz apoptoza komórek nowotworowych.
Do badań przygotowano zbiornik wypełniony odgazowaną wodą destylowaną z termostatem i mieszadłem elektromagnetycznym. W zbiorniku umieszczono płaski przetwornik ultradźwiękowy, promieniujący do góry, w kierunku zanurzonej w wodzie szalki Petriego ze skrzepliną lub komórkami.
W pierwszej części oddziaływano falą ultradźwiękową o częstotliwości 1.0 MHz i natężeniu Isata = 0.2 W/cm2 na skrzeplinę uzyskaną z kropli krwi 30 min po pobraniu. Skrzeplinę umieszczono w roztworze soli fizjologicznej z dodatkiem leku rozpuszczającego skrzeplinę tPA typu Actilyse 50 w stężeniu 10μg/ml i nadźwiękawiano przez 20 min. Eksperyment powtórzono z kolejnymi skrzeplinami stosując same ultradźwięki oraz sam lek Actilyse. Współdziałanie leku i fali ultradźwiękowej w ciągu 20 min spowodowało całkowite rozpuszczenie skrzepliny. W pozostałych przypadkach skrzeplina pozostała zmniejszając swoją objętość o 10-20%.
W drugiej części badano wpływ fali ultradźwiękowej 1.0 MHz na komórki nowotworowe szczurzego glejaka C6. Komórki wysiano na dnie szalki i hodowano w pożywce DMEM w temperaturze 37ºC. Następnie do pożywki dodano 1mM kwasu 5-aminolewulinowego ALA. Po 24h inkubacji, komórki poddano działaniu ultradźwięków o natężeniu Isata = 0.4, 0.8 oraz 1.2 W/cm2 przez 3 min. Po kolejnych 24h, żywotność komórek zbadano spektrofotometrem po dodaniu do pożywki odczynnika MTT assay. Badania powtórzono dla komórek bez ALA oraz bez ultradźwięków. Dla natężenia Isata = 1.2 W/cm2, żywotność komórek wynosiła 39% dla współdziałania ALA i ultradźwięków, 78% dla ALA oraz 99% dla ultradźwięków.
Przygotowane przez autorów stanowisko pomiarowe umożliwia prowadzenie badań in-vitro nad terapeutycznym oddziaływaniu ultradźwięków na komórki. Uzyskano pozytywne rezultaty w wykorzystaniu ultradźwięków zarówno do rozpuszczania skrzeplin – sonotrombolizy jak i uśmiercania komórek nowotworowych – terapii sonodynamicznej. Stosowano ultradźwięki małej mocy, niepowodujące zniszczenia komórek przez przegrzanie lub kawitację.

Keywords:

ultradźwięki terapeutyczne, sonotromboliza, tromboliza, leczenie nowotworów

Affiliations:
Secomski W.-IPPT PAN
Olszewski R.-other affiliation
Bilmin K.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)
Nowicki A.-IPPT PAN
Kujawska T.-IPPT PAN
Grieb P.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)