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Determination of the Cavitation Pressure Threshold in Focused Ultrasound Wave Fields applied to Sonosensitive, Biocompatible Nanoparticles for Drug Delivery Applications

Determination of the Cavitation Pressure Threshold in Focused Ultrasound Wave Fields applied to... DE GRUYTER Current Directions in Biomedical Engineering 2020;6(3): 20203138 Benedikt George*, Markus Lehner, Michael Fink, Stefan J. Rupitsch, Pia T. Hiltl, Ula Savšek, Geoffrey Lee, and Helmut Ermert Determination of the Cavitation Pressure Threshold in Focused Ultrasound Wave Fields applied to Sonosensitive, Biocompatible Nanoparticles for Drug Delivery Applications https://doi.org/10.1515/cdbme-2020-3138 centration of API in the tumorous tissue, while its concentra- tion in healthy tissue is below a certain level to prevent toxic Abstract: Employing sonosensitive nanoparticles as carri- effects. Additionally, this localized drug delivery enables a sig- ers of active pharmaceutical ingredients emerges in ultrasonic nificant reduction of the total amount of administered API. Drug Delivery. Drug release can be initiated by focused ultra- In Drug Targeting (DT), various methods are applied to sound via the effect of inertial cavitation in certain target areas accumulate the drug into the tumorous tissue. These meth- of particle loaded tissue. For stimulating inertial cavitation, a ods are based on physical principles as well as on physio- specific peak rarefaction pressure threshold must be exceeded. logical features of the tumorous tissue, e.g., Passive Target- This pressure threshold has to be determined in order to esti- ing, exploiting abnormalities like the leaky vasculature of cell mate the risk of tissue damage during the drug release proce- membrane to accumulate APIs passively, also known as the dure. Therefore, this study provides a method to reliably verify Enhanced Permeability and Retention Effect (EPR) [1, 2], or the cavitation pressure threshold of sonosensitive and biocom- Thermal Targeting in which hyperthermia is commonly used patible nanoparticles. as stimulus for triggered release of liposomal APIs [3, 4], or Keywords: Drug Delivery, FUS, Cavitation Pressure Thresh- Magnetic Drug Targeting, which employs superparamagnetic old, Nanoparticles iron oxide nanoparticles (SPIONs) as vehicles to guide the API to the tumor controlled by an external magnetic field [5, 6]. An emerging non-invasive treatment method is Ultrasonic 1 Introduction Drug Targeting (UDT). This method is characterized by the fact that the release of the API from their sonosensitive carri- Still one of the most devastating diseases in the world is can- ers can be controlled by targeted sonication with focused ultra- cer, pushing researchers to develop a successful treatment sound (FUS) via the effect of inertial cavitation (IC) [7, 8]. In method for the cure of cancer patients. However, over the this process - as mentioned at the DT methods above - the car- past years, mortality has decreased due to improved treatment rier particles required to transport the API close to the tumor, modalities like radiation therapy, surgical interventions as well vary on their material and size very much. as chemotherapy to contain tumor growth. Though, these cur- In this contribution we focus on newly designed sonosen- rent treatment methods also impair healthy cell tissue and lead sitive and biocompatible poly-L-lactid acid (PLA) formula- to considerable side effects. Especially, the systemically ad- tions (spheres and capsules), which are planned to serve as ministered chemotherapeutic drug has an toxic effect on the drug carriers for drug delivery applications. Drug release is entire body of the patient, which should be reduced. initiated via IC generated by a FUS transducer and is further A suitable approach to minimize toxic side effects is Drug enhanced due to the sonosensitive behavior of the carrier for- Targeting, which is characterized by a selective accumulation mulation. of active pharmaceutical ingredient (API) to increase the con- As the release of the active ingredient is significantly in- fluenced by FUS, a suitable transducer’s excitation signal with *Corresponding author: Benedikt George, Department of corresponding parameters such as amplitude and frequency Sensor Technology, Paul-Gordan-Street 3/5, Erlangen, Germany, must be developed. With respect to clinical application, the e-mail: benedikt.george@fau.de signal development is quite challenging, since it is known that Markus Lehner, Michael Fink, Stefan J. Rupitsch, Helmut tissue can be irreparably damaged by FUS due to large peak Ermert, Department of Sensor Technology, Paul-Gordan-Street rarefaction pressures (PRFP) and high US intensities. For a 3/5, Erlangen, Germany Pia T. Hiltl, Ula Savšek, Geoffrey Lee, Department of Pharma- safe application of diagnostic ultrasound, limit values have ceutics, Cauerstraße 4, Erlangen, Germany Open Access. © 2020 Benedikt George et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 License. 2 B. George et al., Sonosensitive Nanoparticles been recommended by the FDA. The maximum applicable Carl Roth) mounted in a bracket containing the nanoparti- PRFP is set by the Mechanical Index (𝑀𝐼 ), which is defined cle suspension is placed in the focal volume of the spheri- as the spatial-peak value 𝑝 ^ of the PRFP in MPa, derated by cally focusing transducer. The sound velocities of water (W) 0.3 dB/MHz cm at each point along the beam axis, divided by and polystyrene (PS) are 𝑐 = 1500 m/s and 𝑐 = 2340 m/s, W PS 6 2 the square root of the ultrasound frequency 𝑓 in MHz [9] and the acoustic impedances are 𝑍 ≈ 1.5 · 10 kg/m s √︂ 6 2 ⧸︂ and 𝑍 ≈ 2.35 · 10 kg/m s. Based on a normal incidence PS 𝑝 ^ 𝑓 𝑀𝐼 = . (1) plane wave analysis [10], the amplitude transmission coeffi- MPa MHz cients of the polystyrene cuvette wall at 𝑓 = 650 kHz; 750 kHz; For our clinical application, the release of the API should be 850 kHz become 𝜏 = (0.93; 0.97; 0.99). Measured values of instigated in the blood vessels leading to the tumor. Hence, we the standing wave ratio (SWR) of the acoustic pressure are establish an 𝑀𝐼 of 1.9 for peripheral vessels complying with (1.11; 1.1; 1.07), representing some inhomogeneity of the FDA regulations for diagnostic ultrasound (Tab. 3 in [9]). In sound field distribution inside the cuvette. During the FUS ex- addition to the 𝑀𝐼 , the spatial-peak pulse-average intensity position the nanoparticles move due to the acoustic radiation 𝐼 is mentioned, which is defined as the pulse-average SPPA.3 pressure, which leads to a certain stirring effect and results intensity at the point in the acoustic field, where the pulse- in a reasonably uniform overall exposure of the particles. The average intensity is a maximum or is a local maximum within a selected ultrasonic frequencies from 650 kHz to 850 kHz are specified region, derated by 0.3 dB/MHz cm to account for the well focusable due to the concave shape of the transducer sur- acoustic attenuation in soft tissues [9]. For peripheral vessels, face. This enables the generation of a focused ultrasonic beam, the FDA sets the 𝐼 to 190 W/cm . SPPA.3 whereby the sound energy is strongly concentrated in a focal Under consideration of these limit values, we stimulate volume shaped like an ellipsoid of 2.01 mm lateral diameter IC at frequencies (650 kHz - 850 kHz) permitting sufficient fo- and 14.7 mm axial size corresponding to the −6 dB area. For cusing to the desired tissue region. Higher ultrasound frequen- medical application, the small size of the focal volume is ap- cies in the MHz-range can be focused even better, but it is propriate, as the location of the API release inside the tumor- known that the cavitation pressure threshold (CPT) is notori- ous tissue has to be accurately addressed by the ultrasound. ously high. Besides, stronger focusing of an ultrasound wave For measuring the cavitation noise signal of the sonosen- concentrates the sound energy in an area, which is too small sitive nanoparticles, we utilize a focusing ultrasonic trans- and poses a greater risk of thermal tissue damage. Based on ducer (V315-SU, Olympus), which is confocally installed in this information, we would like to realize IC excited drug re- a crossed beam orientation with respect to the FUS beam axis. lease in the frequency range mentioned above at the lowest To reduce unwanted cross-coupled frequency components of possible pressures, corresponding to a minimal 𝑀𝐼 . the transmitter signal, the received broadband IC noise signal In this study we describe a method to reliably determine is filtered by a passive highpass filter (2nd order) suppressing the IC pressure threshold employing mono frequent burst se- the FUS transmitter signal. After filtering, the voltage signal quencies. 𝑢 (𝑡) is measured using an digital oscilloscope (DSO7014B, Agilent). We investigate the CPT at a water temperature of 30 C, 2 Experimental which was kept constant by means of a temperature controller unit. Additionally, a circulating pump was used to achieve a 2.1 Measurement Setup homogeneous temperature distribution inside the water basin. The IC pressure threshold of the sonosensitive and biocom- It should be mentioned, that the cavitation pressures inside the patible nanoparticles was investigated by utilizing a passive cuvette generated by imploding bubbles can not be measured. cavitation detection (PCD) test bench (Fig. 1). The PCD setup These bubble implosions are abrasive and would destroy any is partly placed in a water basin, which is filled with de- sensor surface. gassed, filtered and deionized water. The burst signal is gen- erated via an arbitrary waveform generator (33522B, Agi- lent), and an RF power amplifier (1140LA, ENI) is used to 2.2 Sonosensitive Nanoparticles generate the amplified output signal 𝑢 (𝑡). A matching net- work is installed to maximize the power transfer to the broad- The nanoparticles used for these investigations were produced band FUS transducer (H-231, Sonic Concepts ) as well as to at the Division of Pharmaceutics of the Friedrich-Alexander- transform its electrical impedance at the center frequency of University (FAU) Erlangen-Nuremberg. We designed new 750 kHz from 27 Ω to 50 Ω. A polystyrene cuvette (EXP2.1, sonosensitive, biocompatible structures (spheres and capsules) 12.5 mm x 12.5 mm x 45 mm, wall thickness 𝑏 = 1.25 mm, with dimensions from 120 nm to 250 nm, which also show IC B. George et al., Sonosensitive Nanoparticles 3 −4 The burst length 𝑇 was set to 6 · 10 s and the period 𝑇 Computer 𝐵 𝑃 u (t) lasted 2 s. For each pressure 𝑝 (𝑚), we spectrally ana- PRFP lyzed the digitized signal 𝑢 (𝑡) via FFT to evaluate the dis- Arbitrary waveform Digital generator oscilloscope crete frequency amplitudes U(𝑖). Then, we dynamically calcu- u (t) lated the voltage spectral density (VSD) 𝑆 in the 5.925 MHz- R 𝑟 +55dB RF Amplifier Highpass filter 6.625 MHz domain within a bandwidth 𝐵 = 0.5 MHz to as- Receiving sess the IC activity of the nanoparticles u (t) S transducer ⧸︃ ∑︁ ⎸ √ ⎷ 2 𝑆 = 𝑈 (𝑖) 𝐵 , (5) Matching network 𝑖=𝑖 FUS transducer with Cuvette with 𝐵 = (𝑖 − 𝑖 )· Δ𝑓 (6) E S nanoparticle Water basin suspension and a frequency resolution Δ𝑓 = 1000 Hz. The summation Circulating pump limits 𝑖 and 𝑖 (see Tab. 1) are symmetrically set between the S E Heating rod harmonics 𝑓 of the corresponding excitation frequency 𝑓 and Temperature controller Temperature sensor result from Fig. 1: PCD test bench to investigate the IC pressure threshold. 𝑖 = 𝑓 /Δ𝑓 = (𝑓 + (𝑓 − 𝑓 − 𝐵)/2)/Δ𝑓 , (7) S S h h+1 h in a well focusable frequency range. For the production of the 𝑖 = 𝑓 /Δ𝑓 = (𝑓 − (𝑓 − 𝑓 − 𝐵)/2)/Δ𝑓 . (8) E E h+1 h+1 h nanostructures, an aqueous solution and an organic solution The spectral evaluation domain from 5.925 MHz-6.625 MHz were mixed and then dried by means of a freeze-drying pro- is based on the filter characteristics of the analog highpass fil- cess, resulting in a cake [11]. It is assumed that the nanopar- ter. ticles have rough surfaces produced at the freeze-drying pro- cedure, which yields to small gas pockets acting as IC bubble Tab. 1: Frequency dependent summation limits for the calculation nuclei. Furthermore, it is supposed that the API adheres better of 𝑆 . to a rough surface than to a smooth surface. The PLA-samples 𝑓 in kHz were rehydrated with degassed pure water, before the CPT was 650 750 850 investigated. 𝑓 in MHz 5.925 6.125 6.125 𝑓 in MHz 6.425 6.625 6.625 2.3 Determination of the Cavitation Pressure Threshold For each frequency 𝑓 from 650 kHz to 850 kHz, we measured 𝐿 = 15 nanoparticle samples from 𝑝 to PRFPmin We determine the CPT in a wide pressure range from 𝑝 and determined the mean VSD 𝑆 corresponding PRFPmax 𝑟 𝑝 = 0.2 MPa to 𝑝 = 2 MPa. The 𝑝 (𝑚) PRFPmin PRFPmax PRFP to is incremented in Δ 𝑝 = 0.025 MPa with each measure- PRFP ∑︁ ment 𝑚 from 𝑚 = 1 to 𝑀 with 𝑀 = 73 𝑆 (𝑓, 𝑝 (𝑚)) = 𝑆 (𝑓, 𝑝 (𝑚)) . (9) 𝑟 PRFP 𝑟𝑗 PRFP 𝑗=1 𝑝 (𝑚) = 𝑝 + (𝑚− 1)· Δ 𝑝 . (2) PRFP PRFPmin PRFP Figure 2 shows the mean VSD 𝑆 at the different frequencies A series of sinusoidal burst signals 𝑢 (𝑡) was used to excite as a function of the PRFP. After that, we calculated the stan- IC, according to dard deviation 𝜎 of the VSD 𝑆 with 𝑢 (𝑡) = sin(2𝜋𝑓𝑡) · 𝑊 (𝑡) , (3) S 𝐿 ⃒ ⃒ ⎸ ∑︁ 2 ⃒ ⃒ 𝜎 = ⃒ 𝑆 (𝑓, 𝑝 (𝑚))− 𝑆 (𝑓, 𝑝 (𝑚))⃒ . 𝑟𝑗 PRFP 𝑟 PRFP with 𝑗=1 (10) (︂ )︂ ∑︁ 𝑡− 𝑇 /2 (𝑚− 1)· 𝑇 𝐵 𝑃 Finally, we employed a Savitzky-Golay filter (SG) with a poly- 𝑊 (𝑡) = 𝑝 (𝑚)· rect − . PRFP 𝑇 𝑇 𝐵 𝐵 nomial order 𝑝 = 6 and an impulse response length 𝑙 = 17 for 𝑚=1 (4) smoothing the standard deviation data. The first maximum of the filtered standard deviation 𝜎 indicates the correspond- max ing CPT. 4 B. George et al., Sonosensitive Nanoparticles −7 −6 · 10 · 10 1.4 𝑓 =650 kHz 𝑓 =650 kHz 1.2 𝑓 =650 kHz, SG filtered 𝑓 =750 kHz max 𝑓 =850 kHz 0.5 0.8 0.6 0.4 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0.2 𝑝 (𝑚) in Pa 6 PRFP · 10 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 𝑝 (𝑚) in Pa 6 PRFP Fig. 3: Unfiltered and filtered standard deviation 𝜎 of the VSD ·10 𝑆 at a frequency 𝑓 = 650 kHz. The first maximum 𝜎 = 𝑟 max −7 0.273· 10 V/ Hz indicates the CPT at 𝑝 = 0.425 MPa. PRFP Fig. 2: Progression of the mean VSD 𝑆 in a pressure range from 0.2 MPa to 2 MPa at frequencies 𝑓 from 650 kHz to 850 kHz. References 3 Results and Conclusion [1] Yuan F et al.: Vascular permeability in human tumor xenograft: molecular size dependence and cutoff size. Figure 3 depicts the standard deviation (STD) 𝜎 of the VSD 𝑆 AACR 1995. from 0.2 MPa to 2 MPa at a frequency 𝑓 = 650 kHz, in which [2] Prabhakar U et al.: Challenges and Key Considerations of the Enhanced Permeability and Retention Effect for 𝜎 indicates the corresponding CPT (0.425 MPa). The STD max Nanomedicine Drug Delivery in Oncology. AACR 2013. increases until the first maximum is reached, then it decreases [3] Ponce AM et al.: Hyperthermia mediated liposomal drug as the pressure increases. At low pressures, the STD of the delivery. Int. J. Hyperthermia 2006. VSD is little because IC can not be generated. Close to the [4] Meyer DE et al.: Drug targeting using thermally respon- CPT, IC is either excited or not resulting in a large STD 𝜎 of sive polymers and local hyperthermia. J. Controlled Re- lease 2001. the VSD 𝑆 . Pressures higher than the CPT lead to a slight de- [5] Tietze R et al.: Efficient drug-delivery using magnetic crease of the STD, because IC can be excited reliably. Above a nanoparticles-biodistribution and therapeutic effects in tu- certain pressure, the STD increases again, which is caused by mour bearing rabbits. Nanomed. 2013. the high non-linearity of IC at large PRFPs. The results of this [6] Alexiou C et al.: Cancer therapy with drug loaded magnetic investigation at different FUS frequencies are listed in Tab. 2. nanoparticles—magnetic drug targeting. J. Magn. Magn. For the used measurement configurations, the lowest CPT of Mater. 2011. [7] Pitt WG, Husseini GA, Staples BJ.: Ultrasonic drug delivery – 0.375 MPa as well as the lowest 𝑀𝐼 of 0.43 are obtained at a general review. Expert Opin. Drug Deliv. 2004. 𝑓 = 750 kHz, which establishes this frequency as the most [8] Coussios CC, Roy RA.: Applications of Acoustics and Cavi- suited one for the excitation of IC complying with the FDA tation to Noninvasive Therapy and Drug Delivery. Annu. Rev. regulations for diagnostic ultrasound. Fluid Mech. 2008. [9] Marketing Clearance of Diagnostic Ultrasound Systems and Transducers: Guidance for Industry and Food and Drug Ad- Tab. 2: Results of the CPT investigations at different frequencies. ministration Staff. FDA-2017-D-5372. U.S. Department of Health and Human Services. Rockville, USA. 2019 𝑓 in kHz [10] Kuttruff H.: Physik und Technik des Ultraschalls. S. Hirzel 650 750 850 Verlag Stuttgart 1988. 𝜎 in nV/ Hz 27.3 19.6 24.9 [11] Hiltl PT. et al.: Inertial cavitation of lyophilized and rehydrated CPT in MPa 0.425 0.375 0.45 nanoparticles of poly(L-lactic acid) at 835 kHz and 1.8 MPa 𝑀𝐼 0.53 0.43 0.49 ultrasound. Scientific reports 9(1) 2019. Acknowledgment: The authors gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (DFG) - project no. ER 94/33-1, LE 626/16-1, RU 1656/2-1. 𝑆 in V/ Hz in V/ Hz http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Current Directions in Biomedical Engineering de Gruyter

Determination of the Cavitation Pressure Threshold in Focused Ultrasound Wave Fields applied to Sonosensitive, Biocompatible Nanoparticles for Drug Delivery Applications

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Abstract

DE GRUYTER Current Directions in Biomedical Engineering 2020;6(3): 20203138 Benedikt George*, Markus Lehner, Michael Fink, Stefan J. Rupitsch, Pia T. Hiltl, Ula Savšek, Geoffrey Lee, and Helmut Ermert Determination of the Cavitation Pressure Threshold in Focused Ultrasound Wave Fields applied to Sonosensitive, Biocompatible Nanoparticles for Drug Delivery Applications https://doi.org/10.1515/cdbme-2020-3138 centration of API in the tumorous tissue, while its concentra- tion in healthy tissue is below a certain level to prevent toxic Abstract: Employing sonosensitive nanoparticles as carri- effects. Additionally, this localized drug delivery enables a sig- ers of active pharmaceutical ingredients emerges in ultrasonic nificant reduction of the total amount of administered API. Drug Delivery. Drug release can be initiated by focused ultra- In Drug Targeting (DT), various methods are applied to sound via the effect of inertial cavitation in certain target areas accumulate the drug into the tumorous tissue. These meth- of particle loaded tissue. For stimulating inertial cavitation, a ods are based on physical principles as well as on physio- specific peak rarefaction pressure threshold must be exceeded. logical features of the tumorous tissue, e.g., Passive Target- This pressure threshold has to be determined in order to esti- ing, exploiting abnormalities like the leaky vasculature of cell mate the risk of tissue damage during the drug release proce- membrane to accumulate APIs passively, also known as the dure. Therefore, this study provides a method to reliably verify Enhanced Permeability and Retention Effect (EPR) [1, 2], or the cavitation pressure threshold of sonosensitive and biocom- Thermal Targeting in which hyperthermia is commonly used patible nanoparticles. as stimulus for triggered release of liposomal APIs [3, 4], or Keywords: Drug Delivery, FUS, Cavitation Pressure Thresh- Magnetic Drug Targeting, which employs superparamagnetic old, Nanoparticles iron oxide nanoparticles (SPIONs) as vehicles to guide the API to the tumor controlled by an external magnetic field [5, 6]. An emerging non-invasive treatment method is Ultrasonic 1 Introduction Drug Targeting (UDT). This method is characterized by the fact that the release of the API from their sonosensitive carri- Still one of the most devastating diseases in the world is can- ers can be controlled by targeted sonication with focused ultra- cer, pushing researchers to develop a successful treatment sound (FUS) via the effect of inertial cavitation (IC) [7, 8]. In method for the cure of cancer patients. However, over the this process - as mentioned at the DT methods above - the car- past years, mortality has decreased due to improved treatment rier particles required to transport the API close to the tumor, modalities like radiation therapy, surgical interventions as well vary on their material and size very much. as chemotherapy to contain tumor growth. Though, these cur- In this contribution we focus on newly designed sonosen- rent treatment methods also impair healthy cell tissue and lead sitive and biocompatible poly-L-lactid acid (PLA) formula- to considerable side effects. Especially, the systemically ad- tions (spheres and capsules), which are planned to serve as ministered chemotherapeutic drug has an toxic effect on the drug carriers for drug delivery applications. Drug release is entire body of the patient, which should be reduced. initiated via IC generated by a FUS transducer and is further A suitable approach to minimize toxic side effects is Drug enhanced due to the sonosensitive behavior of the carrier for- Targeting, which is characterized by a selective accumulation mulation. of active pharmaceutical ingredient (API) to increase the con- As the release of the active ingredient is significantly in- fluenced by FUS, a suitable transducer’s excitation signal with *Corresponding author: Benedikt George, Department of corresponding parameters such as amplitude and frequency Sensor Technology, Paul-Gordan-Street 3/5, Erlangen, Germany, must be developed. With respect to clinical application, the e-mail: benedikt.george@fau.de signal development is quite challenging, since it is known that Markus Lehner, Michael Fink, Stefan J. Rupitsch, Helmut tissue can be irreparably damaged by FUS due to large peak Ermert, Department of Sensor Technology, Paul-Gordan-Street rarefaction pressures (PRFP) and high US intensities. For a 3/5, Erlangen, Germany Pia T. Hiltl, Ula Savšek, Geoffrey Lee, Department of Pharma- safe application of diagnostic ultrasound, limit values have ceutics, Cauerstraße 4, Erlangen, Germany Open Access. © 2020 Benedikt George et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 License. 2 B. George et al., Sonosensitive Nanoparticles been recommended by the FDA. The maximum applicable Carl Roth) mounted in a bracket containing the nanoparti- PRFP is set by the Mechanical Index (𝑀𝐼 ), which is defined cle suspension is placed in the focal volume of the spheri- as the spatial-peak value 𝑝 ^ of the PRFP in MPa, derated by cally focusing transducer. The sound velocities of water (W) 0.3 dB/MHz cm at each point along the beam axis, divided by and polystyrene (PS) are 𝑐 = 1500 m/s and 𝑐 = 2340 m/s, W PS 6 2 the square root of the ultrasound frequency 𝑓 in MHz [9] and the acoustic impedances are 𝑍 ≈ 1.5 · 10 kg/m s √︂ 6 2 ⧸︂ and 𝑍 ≈ 2.35 · 10 kg/m s. Based on a normal incidence PS 𝑝 ^ 𝑓 𝑀𝐼 = . (1) plane wave analysis [10], the amplitude transmission coeffi- MPa MHz cients of the polystyrene cuvette wall at 𝑓 = 650 kHz; 750 kHz; For our clinical application, the release of the API should be 850 kHz become 𝜏 = (0.93; 0.97; 0.99). Measured values of instigated in the blood vessels leading to the tumor. Hence, we the standing wave ratio (SWR) of the acoustic pressure are establish an 𝑀𝐼 of 1.9 for peripheral vessels complying with (1.11; 1.1; 1.07), representing some inhomogeneity of the FDA regulations for diagnostic ultrasound (Tab. 3 in [9]). In sound field distribution inside the cuvette. During the FUS ex- addition to the 𝑀𝐼 , the spatial-peak pulse-average intensity position the nanoparticles move due to the acoustic radiation 𝐼 is mentioned, which is defined as the pulse-average SPPA.3 pressure, which leads to a certain stirring effect and results intensity at the point in the acoustic field, where the pulse- in a reasonably uniform overall exposure of the particles. The average intensity is a maximum or is a local maximum within a selected ultrasonic frequencies from 650 kHz to 850 kHz are specified region, derated by 0.3 dB/MHz cm to account for the well focusable due to the concave shape of the transducer sur- acoustic attenuation in soft tissues [9]. For peripheral vessels, face. This enables the generation of a focused ultrasonic beam, the FDA sets the 𝐼 to 190 W/cm . SPPA.3 whereby the sound energy is strongly concentrated in a focal Under consideration of these limit values, we stimulate volume shaped like an ellipsoid of 2.01 mm lateral diameter IC at frequencies (650 kHz - 850 kHz) permitting sufficient fo- and 14.7 mm axial size corresponding to the −6 dB area. For cusing to the desired tissue region. Higher ultrasound frequen- medical application, the small size of the focal volume is ap- cies in the MHz-range can be focused even better, but it is propriate, as the location of the API release inside the tumor- known that the cavitation pressure threshold (CPT) is notori- ous tissue has to be accurately addressed by the ultrasound. ously high. Besides, stronger focusing of an ultrasound wave For measuring the cavitation noise signal of the sonosen- concentrates the sound energy in an area, which is too small sitive nanoparticles, we utilize a focusing ultrasonic trans- and poses a greater risk of thermal tissue damage. Based on ducer (V315-SU, Olympus), which is confocally installed in this information, we would like to realize IC excited drug re- a crossed beam orientation with respect to the FUS beam axis. lease in the frequency range mentioned above at the lowest To reduce unwanted cross-coupled frequency components of possible pressures, corresponding to a minimal 𝑀𝐼 . the transmitter signal, the received broadband IC noise signal In this study we describe a method to reliably determine is filtered by a passive highpass filter (2nd order) suppressing the IC pressure threshold employing mono frequent burst se- the FUS transmitter signal. After filtering, the voltage signal quencies. 𝑢 (𝑡) is measured using an digital oscilloscope (DSO7014B, Agilent). We investigate the CPT at a water temperature of 30 C, 2 Experimental which was kept constant by means of a temperature controller unit. Additionally, a circulating pump was used to achieve a 2.1 Measurement Setup homogeneous temperature distribution inside the water basin. The IC pressure threshold of the sonosensitive and biocom- It should be mentioned, that the cavitation pressures inside the patible nanoparticles was investigated by utilizing a passive cuvette generated by imploding bubbles can not be measured. cavitation detection (PCD) test bench (Fig. 1). The PCD setup These bubble implosions are abrasive and would destroy any is partly placed in a water basin, which is filled with de- sensor surface. gassed, filtered and deionized water. The burst signal is gen- erated via an arbitrary waveform generator (33522B, Agi- lent), and an RF power amplifier (1140LA, ENI) is used to 2.2 Sonosensitive Nanoparticles generate the amplified output signal 𝑢 (𝑡). A matching net- work is installed to maximize the power transfer to the broad- The nanoparticles used for these investigations were produced band FUS transducer (H-231, Sonic Concepts ) as well as to at the Division of Pharmaceutics of the Friedrich-Alexander- transform its electrical impedance at the center frequency of University (FAU) Erlangen-Nuremberg. We designed new 750 kHz from 27 Ω to 50 Ω. A polystyrene cuvette (EXP2.1, sonosensitive, biocompatible structures (spheres and capsules) 12.5 mm x 12.5 mm x 45 mm, wall thickness 𝑏 = 1.25 mm, with dimensions from 120 nm to 250 nm, which also show IC B. George et al., Sonosensitive Nanoparticles 3 −4 The burst length 𝑇 was set to 6 · 10 s and the period 𝑇 Computer 𝐵 𝑃 u (t) lasted 2 s. For each pressure 𝑝 (𝑚), we spectrally ana- PRFP lyzed the digitized signal 𝑢 (𝑡) via FFT to evaluate the dis- Arbitrary waveform Digital generator oscilloscope crete frequency amplitudes U(𝑖). Then, we dynamically calcu- u (t) lated the voltage spectral density (VSD) 𝑆 in the 5.925 MHz- R 𝑟 +55dB RF Amplifier Highpass filter 6.625 MHz domain within a bandwidth 𝐵 = 0.5 MHz to as- Receiving sess the IC activity of the nanoparticles u (t) S transducer ⧸︃ ∑︁ ⎸ √ ⎷ 2 𝑆 = 𝑈 (𝑖) 𝐵 , (5) Matching network 𝑖=𝑖 FUS transducer with Cuvette with 𝐵 = (𝑖 − 𝑖 )· Δ𝑓 (6) E S nanoparticle Water basin suspension and a frequency resolution Δ𝑓 = 1000 Hz. The summation Circulating pump limits 𝑖 and 𝑖 (see Tab. 1) are symmetrically set between the S E Heating rod harmonics 𝑓 of the corresponding excitation frequency 𝑓 and Temperature controller Temperature sensor result from Fig. 1: PCD test bench to investigate the IC pressure threshold. 𝑖 = 𝑓 /Δ𝑓 = (𝑓 + (𝑓 − 𝑓 − 𝐵)/2)/Δ𝑓 , (7) S S h h+1 h in a well focusable frequency range. For the production of the 𝑖 = 𝑓 /Δ𝑓 = (𝑓 − (𝑓 − 𝑓 − 𝐵)/2)/Δ𝑓 . (8) E E h+1 h+1 h nanostructures, an aqueous solution and an organic solution The spectral evaluation domain from 5.925 MHz-6.625 MHz were mixed and then dried by means of a freeze-drying pro- is based on the filter characteristics of the analog highpass fil- cess, resulting in a cake [11]. It is assumed that the nanopar- ter. ticles have rough surfaces produced at the freeze-drying pro- cedure, which yields to small gas pockets acting as IC bubble Tab. 1: Frequency dependent summation limits for the calculation nuclei. Furthermore, it is supposed that the API adheres better of 𝑆 . to a rough surface than to a smooth surface. The PLA-samples 𝑓 in kHz were rehydrated with degassed pure water, before the CPT was 650 750 850 investigated. 𝑓 in MHz 5.925 6.125 6.125 𝑓 in MHz 6.425 6.625 6.625 2.3 Determination of the Cavitation Pressure Threshold For each frequency 𝑓 from 650 kHz to 850 kHz, we measured 𝐿 = 15 nanoparticle samples from 𝑝 to PRFPmin We determine the CPT in a wide pressure range from 𝑝 and determined the mean VSD 𝑆 corresponding PRFPmax 𝑟 𝑝 = 0.2 MPa to 𝑝 = 2 MPa. The 𝑝 (𝑚) PRFPmin PRFPmax PRFP to is incremented in Δ 𝑝 = 0.025 MPa with each measure- PRFP ∑︁ ment 𝑚 from 𝑚 = 1 to 𝑀 with 𝑀 = 73 𝑆 (𝑓, 𝑝 (𝑚)) = 𝑆 (𝑓, 𝑝 (𝑚)) . (9) 𝑟 PRFP 𝑟𝑗 PRFP 𝑗=1 𝑝 (𝑚) = 𝑝 + (𝑚− 1)· Δ 𝑝 . (2) PRFP PRFPmin PRFP Figure 2 shows the mean VSD 𝑆 at the different frequencies A series of sinusoidal burst signals 𝑢 (𝑡) was used to excite as a function of the PRFP. After that, we calculated the stan- IC, according to dard deviation 𝜎 of the VSD 𝑆 with 𝑢 (𝑡) = sin(2𝜋𝑓𝑡) · 𝑊 (𝑡) , (3) S 𝐿 ⃒ ⃒ ⎸ ∑︁ 2 ⃒ ⃒ 𝜎 = ⃒ 𝑆 (𝑓, 𝑝 (𝑚))− 𝑆 (𝑓, 𝑝 (𝑚))⃒ . 𝑟𝑗 PRFP 𝑟 PRFP with 𝑗=1 (10) (︂ )︂ ∑︁ 𝑡− 𝑇 /2 (𝑚− 1)· 𝑇 𝐵 𝑃 Finally, we employed a Savitzky-Golay filter (SG) with a poly- 𝑊 (𝑡) = 𝑝 (𝑚)· rect − . PRFP 𝑇 𝑇 𝐵 𝐵 nomial order 𝑝 = 6 and an impulse response length 𝑙 = 17 for 𝑚=1 (4) smoothing the standard deviation data. The first maximum of the filtered standard deviation 𝜎 indicates the correspond- max ing CPT. 4 B. George et al., Sonosensitive Nanoparticles −7 −6 · 10 · 10 1.4 𝑓 =650 kHz 𝑓 =650 kHz 1.2 𝑓 =650 kHz, SG filtered 𝑓 =750 kHz max 𝑓 =850 kHz 0.5 0.8 0.6 0.4 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0.2 𝑝 (𝑚) in Pa 6 PRFP · 10 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 𝑝 (𝑚) in Pa 6 PRFP Fig. 3: Unfiltered and filtered standard deviation 𝜎 of the VSD ·10 𝑆 at a frequency 𝑓 = 650 kHz. The first maximum 𝜎 = 𝑟 max −7 0.273· 10 V/ Hz indicates the CPT at 𝑝 = 0.425 MPa. PRFP Fig. 2: Progression of the mean VSD 𝑆 in a pressure range from 0.2 MPa to 2 MPa at frequencies 𝑓 from 650 kHz to 850 kHz. References 3 Results and Conclusion [1] Yuan F et al.: Vascular permeability in human tumor xenograft: molecular size dependence and cutoff size. Figure 3 depicts the standard deviation (STD) 𝜎 of the VSD 𝑆 AACR 1995. from 0.2 MPa to 2 MPa at a frequency 𝑓 = 650 kHz, in which [2] Prabhakar U et al.: Challenges and Key Considerations of the Enhanced Permeability and Retention Effect for 𝜎 indicates the corresponding CPT (0.425 MPa). The STD max Nanomedicine Drug Delivery in Oncology. AACR 2013. increases until the first maximum is reached, then it decreases [3] Ponce AM et al.: Hyperthermia mediated liposomal drug as the pressure increases. At low pressures, the STD of the delivery. Int. J. Hyperthermia 2006. VSD is little because IC can not be generated. Close to the [4] Meyer DE et al.: Drug targeting using thermally respon- CPT, IC is either excited or not resulting in a large STD 𝜎 of sive polymers and local hyperthermia. J. Controlled Re- lease 2001. the VSD 𝑆 . Pressures higher than the CPT lead to a slight de- [5] Tietze R et al.: Efficient drug-delivery using magnetic crease of the STD, because IC can be excited reliably. Above a nanoparticles-biodistribution and therapeutic effects in tu- certain pressure, the STD increases again, which is caused by mour bearing rabbits. Nanomed. 2013. the high non-linearity of IC at large PRFPs. The results of this [6] Alexiou C et al.: Cancer therapy with drug loaded magnetic investigation at different FUS frequencies are listed in Tab. 2. nanoparticles—magnetic drug targeting. J. Magn. Magn. For the used measurement configurations, the lowest CPT of Mater. 2011. [7] Pitt WG, Husseini GA, Staples BJ.: Ultrasonic drug delivery – 0.375 MPa as well as the lowest 𝑀𝐼 of 0.43 are obtained at a general review. Expert Opin. Drug Deliv. 2004. 𝑓 = 750 kHz, which establishes this frequency as the most [8] Coussios CC, Roy RA.: Applications of Acoustics and Cavi- suited one for the excitation of IC complying with the FDA tation to Noninvasive Therapy and Drug Delivery. Annu. Rev. regulations for diagnostic ultrasound. Fluid Mech. 2008. [9] Marketing Clearance of Diagnostic Ultrasound Systems and Transducers: Guidance for Industry and Food and Drug Ad- Tab. 2: Results of the CPT investigations at different frequencies. ministration Staff. FDA-2017-D-5372. U.S. Department of Health and Human Services. Rockville, USA. 2019 𝑓 in kHz [10] Kuttruff H.: Physik und Technik des Ultraschalls. S. Hirzel 650 750 850 Verlag Stuttgart 1988. 𝜎 in nV/ Hz 27.3 19.6 24.9 [11] Hiltl PT. et al.: Inertial cavitation of lyophilized and rehydrated CPT in MPa 0.425 0.375 0.45 nanoparticles of poly(L-lactic acid) at 835 kHz and 1.8 MPa 𝑀𝐼 0.53 0.43 0.49 ultrasound. Scientific reports 9(1) 2019. Acknowledgment: The authors gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (DFG) - project no. ER 94/33-1, LE 626/16-1, RU 1656/2-1. 𝑆 in V/ Hz in V/ Hz

Journal

Current Directions in Biomedical Engineering – de Gruyter

Published: Sep 1, 2020

Keywords: Drug Delivery; FUS; Cavitation Pressure Threshold; Nanoparticles

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