Cavitation-enhanced extravasation for drug delivery

A flow-through tissue-mimicking phantom composed of a biocompatible hydro-gel with embedded tumour cells was used to assess and optimize the role of ultrasound-induced cavitation on the extravasation of a macromolecular compound from a channel mimicking vessel in the gel, namely a non-replicating luciferase-expressing adenovirus (Ad-Luc). Using a 500 KHz therapeutic ultrasound transducer confocally aligned with a focussed passive cavitation detector, different exposure conditions and burst mode timings were selected by performing time and frequency domain analysis of passively recorded acoustic emissions, in the absence and in the presence of ultrasound contrast agents acting as cavitation nuclei. In the presence of Sonovue, maximum ultraharmonic emissions were detected for peak rarefactional pressures of 360 kPa, and maximum broadband emissions occurred at 1250 kPa. The energy of the recorded acoustic emissions was used to optimise the pulse repetition frequency and duty cycle in order to maximize either ultraharmonic or broadband emissions while keeping the acoustic energy delivered to the focus constant. Cell viability measurements indicated that none of the insonation conditions investigated induces cell death in the absence of a therapeutic agent (i.e. virus). Phase contrast images of the tissue-mimicking phantom showed that short range vessel disruption can occur when ultra-harmonic emissions (nf0/2) are maximised whereas formation of a micro-channel perpendicular to the flow can be obtained in the presence of broadband acoustic emissions. Following Ad-Luc delivery, luciferase expression measurements showed that a 60-fold increase in its bioavailability can be achieved when broadband noise emissions are present during insonation, even for modest contrast agent concentrations. The findings of the present study suggest that drug delivery systems based on acoustic cavitation may help enhance the extravasation of anticancer agents, thus increasing their penetration distance to hypoxic regions and poorly vascularised tumour regions.     

Identifying the Inertial Cavitation Threshold and Skull Effects in a Vessel Phantom Using Focused Ultrasound and Microbubbles

Focused ultrasound (FUS) in combination with microbubbles has been shown capable of delivering large molecules to the brain parenchyma through opening of the blood-brain barrier (BBB). However, the mechanism behind the opening remains unknown. To investigate the pressure threshold for inertial cavitation of preformed microbubbles during sonication, passive cavitation detection in conjunction with B-mode imaging was used. A cerebral vessel was simulated by generating a cylindrical hole of 610 μm in diameter inside a polyacrylamide gel and saturating its volume with microbubbles. Definity microbubbles (Mean diameter range: 1.1-3.3 μm, Lantheus Medical Imaging, N. Billerica, MA, USA) were injected prior to sonication (frequency: 1.525 MHz; pulse length: 100 cycles; PRF: 10 Hz; sonication duration: 2 s) through an excised mouse skull. The acoustic emissions due to the cavitation response were passively detected using a cylindrically focused hydrophone, confocal with the FUS transducer and a linear-array transducer with the field of view perpendicular to the FUS beam. The broadband spectral response acquired at the passive cavitation detector (PCD) and the B-mode images identified the occurrence and location of the inertial cavitation, respectively. Findings indicated that the peak-rarefactional pressure threshold was approximately equal to 0.45 MPa, with or without the skull present. Mouse skulls did not affect the threshold of inertial cavitation but resulted in a lower inertial cavitation dose. The broadband response could be captured through the murine skull, so the same PCD set-up can be used in future in vivo applications. (E-mail: ek2191@columbia.edu)     

Loss of Echogenicity and Onset of Cavitation from Echogenic Liposomes: Pulse Repetition Frequency Independence

Echogenic liposomes (ELIP) are being developed for the early detection and treatment of atherosclerotic lesions. An 80% loss of echogenicity of ELIP has been found to be concomitant with the onset of stable and inertial cavitation. The ultrasound pressure amplitude at which this occurs is weakly dependent on pulse duration. It has been reported that the rapid fragmentation threshold of ELIP (based on changes in echogenicity) is dependent on the insonation pulse repetition frequency (PRF). The study described here evaluates the relationship between loss of echogenicity and cavitation emissions from ELIP insonified by duplex Doppler pulses at four PRFs (1.25, 2.5, 5 and 8.33 kHz). Loss of echogenicity was evaluated on B-mode images of ELIP. Cavitation emissions from ELIP were recorded passively on a focused single-element transducer and a linear array. Emissions recorded by the linear array were beamformed, and the spatial widths of stable and inertial cavitation emissions were compared with the calibrated azimuthal beamwidth of the Doppler pulse exceeding the stable and inertial cavitation thresholds. The inertial cavitation thresholds had a very weak dependence on PRF, and stable cavitation thresholds were independent of PRF. The spatial widths of the cavitation emissions recorded by the passive cavitation imaging system agreed with the calibrated Doppler beamwidths. The results also indicate that 64%–79% loss of echogenicity can be used to classify the presence or absence of cavitation emissions with greater than 80% accuracy.     

Cavitational mechanisms in ultrasound-accelerated fibrinolysis

The role of both inertial and stable cavitation was investigated during in vitro ultrasound-accelerated fibrinolysis by recombinant tissue plasminogen activator (rt-PA) in the presence and absence of Optison. A unique treatment configuration applied ultrasound, rt-PA and Optison to the interior of a plasma clot. Lysis efficacy was measured as clot weight reduction. Cavitational mechanisms were investigated by monitoring subharmonic and broadband noise. In the absence of Optison, 1.7 MHz pulsed ultrasound with 1.5 MPa peak-negative pressure applied for 30 min resulted in 45 +/- 19% lysis enhancement relative to rt-PA alone. Cavitation was not detected, indicating a role of noncavitational effects of ultrasound. The addition of Optison increased lysis enhancement to 88 +/- 25%. Inertial cavitation was present only at the start of the exposure, while low-amplitude subharmonic emissions persisted throughout. Additional protocols suggested a possible correlation between the increased lysis in the presence of Optison and the subharmonic emission, indicating a potentially important role of stable rather than inertial cavitation in microbubble-enhanced ultrasound-accelerated rt-PA-mediated thrombolysis.     

Comparison of molecular strategies for breast cancer virotherapy using oncolytic adenovirus

Oncolytic viruses are regulated by the tumor phenotype to replicate and lyse cancer cells selectively. To identify optimal strategies for breast cancer we compared five adenoviruses with distinct regulatory mechanisms: Ad-dl922-947 (targets G1-S checkpoint); Ad-Onyx-015 and Ad-Onyx-017 (target p53/mRNA export); Ad-vKH1 (targets Wnt pathway), and AdEHE2F (targets estrogen receptor/G1-S checkpoint/hypoxic signaling). The quantity of virus required to kill 50% of breast cancer cells after 6 days (EC(50), plaque-forming units per cell) was measured. The most potent virus was Ad-dl922-947 (EC(50), 0.01-5.4 in SkBr3, MDA-231, MDA-468, MCF7, and ZR75.1 cells), followed by wild-type (Ad-WT; EC(50), 0.3-5.5) and AdEHE2F (EC(50), 1.4-3.9). Ad-vKH1 (EC(50), 7.2-72.1), Ad-Onyx-017 (EC(50), 8.4-167), and Ad-Onyx-015 (EC(50), 17.7-377) showed less activity. Most viruses showed limited cytotoxicity in normal human cells, including breast epithelium MCF10A (EC(50), >722) and fibroblasts (EC(50), >192) and only moderate cytotoxicity in normal microvascular endothelial cells (HMVECs; EC(50), 42.8-149), except Ad-dl922-947, which was active in HMVECs (EC(50), 1.6). After injection into MDA-231 xenografts, Ad-WT, AdEHE2F, and Ad-dl922-947 showed replication, assessed by hexon staining and quantitative polymerase chain reaction measurement of viral DNA, and significantly inhibited tumor growth, leading to extended survival. After intravenous injection Ad-dl922-947 showed DNA replication (233% of the injected dose was measured in liver after 3 days) whereas AdEHE2F did not. Overall, AdEHE2F showed the best combination of low toxicity in normal cells and high activity in breast cancer in vitro and in vivo, suggesting that molecular targeting using estrogen response elements, hypoxia response elements, and a dysregulated G1-S checkpoint is a promising strategy for virotherapy of breast cancer.     

A review of in vitro bioeffects of inertial ultrasonic cavitation from a mechanistic perspective

This selective review of the biological effects of ultrasound presents a synopsis of our current understanding of how cells insonated in vitro are affected by inertial cavitation from the standpoint of physical and chemical mechanisms. The focus of this review is on the physical and chemical mechanisms of action of inertial cavitation which appear to be effective in causing biological effects. There are several fundamental conditions which must be satisfied before cavitation-related bioeffects may arise. First, bubbles must be created and then brought into proximity to cells. Exposure methods are critical in this regard, and simple procedures such as rotation of a vessel containing the cells during exposure can drastically alter the results. Second, once association is achieved between bubbles and cells, the former must interact with the latter to produce a bioeffect. It is not certain that the inertial event is the prime mechanism by which cells are lysed; there is evidence that the turbulence associated with bubble translation may cause lysis. Additionally, there appear to be chemical and other physical mechanisms by which inertial cavitation may affect cells; these include the generation of biologically effective sonochemicals and the apparent emission of ultraviolet (UV) and soft X-rays. The evidence for inertial cavitation occurring within cells is critically reviewed.     

Interstitial Matrix Prevents Therapeutic Ultrasound From Causing Inertial Cavitation in Tumescent Subcutaneous Tissue

We search for cavitation in tumescent subcutaneous tissue of a live pig under application of pulsed, 1-MHz ultrasound at 8 W cm^?2 spatial peak and pulse-averaged intensity. We find no evidence of broadband acoustic emission indicative of inertial cavitation. These acoustic parameters are representative of those used in external-ultrasound-assisted lipoplasty and in physical therapy and our null result brings into question the role of cavitation in those applications. A comparison of broadband acoustic emission from a suspension of ultrasound contrast agent in bulk water with a suspension injected subcutaneously indicates that the interstitial matrix suppresses cavitation and provides an additional mechanism behind the apparent lack of in-vivo cavitation to supplement the absence of nuclei explanation offered in the literature. We also find a short-lived cavitation signal in normal, non-tumesced tissue that disappears after the first pulse, consistent with cavitation nuclei depletion in vivo.     

Cavitation threshold of microbubbles in gel tunnels by focused ultrasound

The investigation of inertial cavitation in micro-tunnels has significant implications for the development of therapeutic applications of ultrasound such as ultrasound-mediated drug and gene delivery. The threshold for inertial cavitation was investigated using a passive cavitation detector with a center frequency of 1 MHz. Micro-tunnels of various diameters (90 to 800 microm) embedded in gel were fabricated and injected with a solution of Optison(trade mark) contrast agent of concentrations 1.2% and 0.2% diluted in water. An ultrasound pulse of duration 500 ms and center frequency 1.736 MHz was used to insonate the microbubbles. The acoustic pressure was increased at 1-s intervals until broadband noise emission was detected. The pressure threshold at which broadband noise emission was observed was found to be dependent on the diameter of the micro-tunnels, with an average increase of 1.2 to 1.5 between the smallest and the largest tunnels, depending on the microbubble concentration. The evaluation of inertial cavitation in gel tunnels rather than tubes provides a novel opportunity to investigate microbubble collapse in a situation that simulates in vivo blood vessels better than tubes with solid walls do.     

A Study of Bubble Activity Generated in Ex Vivo Tissue by High Intensity Focused Ultrasound

Cancer treatment by extracorporeal high-intensity focused ultrasound (HIFU) is constrained by the time required to ablate clinically relevant tumour volumes. Although cavitation may be used to optimize HIFU treatments, its role during lesion formation is ambiguous. Clear differentiation is required between acoustic cavitation (noninertial and inertial) effects and bubble formation arising from two thermally-driven effects (the vapourization of liquid into vapour, and the exsolution of formerly dissolved permanent gas out of the liquid and into gas spaces). This study uses clinically relevant HIFU exposures in degassed water and ex vivo bovine liver to test a suite of cavitation detection techniques that exploit passive and active acoustics, audible emissions and the electrical drive power fluctuations. Exposure regimes for different cavitation activities (none, acoustic cavitation and, for ex vivo tissue only, acoustic cavitation plus thermally-driven gas space formation) were identified both in degassed water and in ex vivo liver using the detectable characteristic acoustic emissions. The detection system proved effective in both degassed water and tissue, but requires optimization for future clinical application. (E-mail: jmclaughlan7@gmail.com)     

Correlation Between Brain Tissue Damage and Inertial Cavitation Dose Quantified Using Passive Cavitation Imaging

Focused ultrasound (FUS)-induced cavitation-mediated brain therapies have become emerging therapeutic modalities for neurologic diseases. Cavitation monitoring is essential to ensure the safety of all cavitation-mediated therapeutic techniques as inertial cavitation can be associated with tissue damage. The objective of this study was to reveal the correlation between the inertial cavitation dose, quantified by passive cavitation imaging (PCI), and brain tissue histologic-level damage induced by FUS in combination with microbubbles. An ultrasound image-guided FUS system consisting of a single-element FUS transducer (1.5 MHz) and a co-axially aligned 128-element linear ultrasound imaging array was used to perform FUS treatment of mice. Mice were sonicated by FUS with different peak negative pressures (0.5 MPa, 1.1 MPa, 4.0 MPa and 6.5 MPa) in the presence of systemically injected microbubbles. The acoustic emissions from the FUS-activated microbubbles were passively detected by the imaging array. The pre-beamformed channel data were acquired and processed offline using the frequency-domain delay, sum and integration algorithm to generate inertial cavitation maps. All the mice were sacrificed after the FUS treatment, and their brains were harvested and processed for hematoxylin and eosin staining. The obtained inertial cavitation maps revealed the dynamic changes of microbubble behaviors during FUS treatment at different pressure levels. It was found that the inertial cavitation dose quantified based on PCI had a linear correlation with the scale of histologic-level tissue damage. Findings from this study suggested that PCI can be used to predict histologic-level tissue damage associated with the FUS-induced cavitation.     

Low-Intensity Ultrasound Promotes Clathrin-Dependent Endocytosis for Drug Penetration into Tumor Cells

Ultrasound-mediated drug delivery is a field of research with promising results, although the exact mechanisms underlying intracellular delivery of therapeutic compounds remain to be elucidated. Many studies use drug carriers and cavitation to enhance drug uptake into tumor cells. However, cavitation could induce cell lysis and remain difficult to control and predict in vivo. In this study, low-intensity ultrasound was delivered using two transducers working at 2.9 and 1.3 MHz. The maximal peak negative pressure was 0.29 MPa to avoid cavitation. Low-intensity ultrasound induced clathrin-mediated endocytosis and forced the penetration of a bisphosphonate (zoledronic acid) into MCF-7 human breast cancer cells potentially as a result of mechanical stresses. When sonication parameters were adjusted to create mild hyperthermia in addition to the mechanical stress, further significant accumulation of ZOL was observed. These results provide better insight into the role of acoustic parameters in drug uptake.     

Non-linear Acoustic Emissions from Therapeutically Driven Contrast Agent Microbubbles

Non-linear emissions from microbubbles introduced to the vasculature for exposure to focused ultrasound are routinely monitored for assessment of therapy and avoidance of irreversible tissue damage. Yet the bubble-based mechanistic source for these emissions, under subresonant driving at typical therapeutic pressure amplitudes, may not be well understood. In the study described here, dual-perspective high-speed imaging at 210,000 frames per second (fps), and shadowgraphically at 10 Mfps, was used to observe cavitation from microbubbles flowing through a 500-µm polycarbonate capillary exposed to focused ultrasound of 692 kHz at therapeutically relevant pressure amplitudes. The acoustic emissions were simultaneously collected via a broadband calibrated needle hydrophone system. The observations indicate that periodic bubble-collapse shock waves can dominate the non-linear acoustic emissions, including subharmonics at higher driving amplitudes. Contributions to broadband emissions through variance in shock wave amplitude and emission timings are also identified. Possible implications for in vivo microbubble cavitation detection, mechanisms of therapy and the conventional classification of cavitation activity as stable or inertial are discussed.     

Vascular effects induced by combined 1-MHz ultrasound and microbubble contrast agent treatments in vivo

Previous in vivo studies have demonstrated that microvessel hemorrhages and alterations of endothelial permeability can be produced in tissues containing microbubble-based ultrasound contrast agents when those tissues are exposed to MHz-frequency pulsed ultrasound of sufficient pressure amplitudes. The general hypothesis guiding this research was that acoustic (viz., inertial) cavitation, rather than thermal insult, is the dominant mechanism by which such effects arise. We report the results of testing five specific hypotheses in an in vivo rabbit auricular blood vessel model: (1) acoustic cavitation nucleated by microbubble contrast agent can damage the endothelia of veins at relatively low spatial-peak temporal-average intensities, (2) such damage will be proportional to the peak negative pressure amplitude of the insonifying pulses, (3) damage will be confined largely to the intimal surface, with sparing of perivascular tissues, (4) greater damage will occur to the endothelial cells on the side of the vessel distal to the source transducer than on the proximal side and (5) ultrasound/contrast agent-induced endothelial damage can be inherently thrombogenic, or can aid sclerotherapeutic thrombogenesis through the application of otherwise subtherapeutic doses of thrombogenic drugs. Auricular vessels were exposed to 1-MHz focused ultrasound of variable peak pressure amplitude using low duty factor, fixed pulse parameters, with or without infusion of a shelled microbubble contrast agent. Extravasation of Evans blue dye and erythrocytes was assessed at the macroscopic level. Endothelial damage was assessed via scanning electron microscopy (SEM) image analysis. The hypotheses were supported by the data. We discuss potential therapeutic applications of vessel occlusion, e.g., occlusion of at-risk gastric varices.     

Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: 2. Medium dissolved gas (pO2) content

The data collected in this project supported the a priori hypothesis that the concentration of dissolved oxygen in whole human blood in vitro affected the extent of ultrasound (US)-induced hemolysis under conditions conducive to the occurrence of inertial cavitation. Aliquots of whole human blood in vitro with a relatively high O(2) level had statistically significantly more 1-MHz US-induced hemolysis than aliquots with a relatively low O(2) level in the presence of controlled gas nucleation (Albunex or ALX, supplementation), with US-induced hemolytic yields being substantially less at 2.2- and 3.5-MHz exposures or in the absence of ALX-supplementation at otherwise comparable acoustic pressures, pulse lengths and duty factors. Passive cavitation detection (pcd) measures indicated a linear relationship for hemolysis up to about 70% and pcd values (R(2) = 0.99).     

Cancer treatment using an optically inert Rose Bengal derivative combined with pulsed focused ultrasound

Pulsed high intensity focused ultrasound (HIFU) has been combined with a photo-insensitive Rose Bengal derivative (RB2) to provide a synergistic cytotoxicity requiring the presence of both ultrasonic cavitation and drug. In vitro tests have shown that a short treatment (less than 30 s) of pulsed HIFU with peak negative pressure > 7 MPa (~ 27 W acoustic power at 1.4 MHz) destroys > 95% of breast cancer cells MDA-MB-231 in suspension with > 10 μM of the compound. Neither the pulsed HIFU nor the RB2 compound was found to have any significant impact on the viability of the cells when used alone. Introducing an antioxidant (N-acetylcysteine) reduced the effectiveness of the treatment. In vivo tests using these same cells growing as a xenograft in nu/nu mice were also done. An ultrasound contrast agent (Optison) and lower frequency (1.0 MHz) was used to help initiate cavitation at the tumor site. We were able to demonstrate tumor regression with cavitation alone, however, addition of RB2 compound injected i.v. yielded a substantial synergistic improvement.     

Polymeric Cups for Cavitation-mediated Delivery of Oncolytic Vaccinia Virus

Oncolytic viruses (OV) could become the most powerful and selective cancer therapies. However, the limited transport of OV into and throughout tumors following intravenous injection means their clinical administration is often restricted to direct intratumoral dosing. Application of physical stimuli, such as focused ultrasound, offers a means of achieving enhanced mass transport. In particular, shockwaves and microstreaming resulting from the instigation of an ultrasound-induced event known as inertial cavitation can propel OV hundreds of microns. We have recently developed a polymeric cup formulation which, when delivered intravenously, provides the nuclei for instigation of sustained inertial cavitation events within tumors. Here we report that exposure of tumors to focused ultrasound after intravenous coinjection of cups and oncolytic vaccinia virus , leads to substantial and significant increases in activity. When cavitation was instigated within SKOV-3 or HepG2 xenografts, reporter gene expression from vaccinia virus was enhanced 1,000-fold (P < 0.0001) or 10,000-fold (P < 0.001), respectively. Similar increases in the number of vaccinia virus genomes recovered from tumors were also observed. In survival studies, the application of cup mediated cavitation to a vaccinia virus expressing a prodrug converting enzyme provided significant (P < 0.05) retardation of tumor growth. This technology could improve the clinical utility of all biological therapeutics including OV.     

Bioeffects considerations for diagnostic ultrasound contrast agents.

Diagnostic ultrasound contrast agents have been developed for enhancing the echogenicity of blood and for delineating other structures of the body. Approved agents are suspensions of gas bodies (stabilized microbubbles), which have been designed for persistence in the circulation and strong echo return for imaging. The interaction of ultrasound pulses with these gas bodies is a form of acoustic cavitation, and they also may act as inertial cavitation nuclei. This interaction produces mechanical perturbation and a potential for bioeffects on nearby cells or tissues. In vitro, sonoporation and cell death occur at mechanical index (MI) values less than the inertial cavitation threshold. In vivo, bioeffects reported for MI values greater than 0.4 include microvascular leakage, petechiae, cardiomyocyte death, inflammatory cell infiltration, and premature ventricular contractions and are accompanied by gas body destruction within the capillary bed. Bioeffects for MIs of 1.9 or less have been reported in skeletal muscle, fat, myocardium, kidney, liver, and intestine. Therapeutic applications that rely on these bioeffects include targeted drug delivery to the interstitium and DNA transfer into cells for gene therapy. Bioeffects of contrast-aided diagnostic ultrasound happen on a microscopic scale, and their importance in the clinical setting remains uncertain.     

Temporal and Spatial Detection of HIFU-Induced Inertial and Hot-Vapor Cavitation with a Diagnostic Ultrasound System

The onset and presence of inertial cavitation and near-boiling temperatures in high-intensity focused ultrasound (HIFU) therapy have been identified as important indicators of energy deposition for therapy guidance. Passive cavitation detection is commonly used to detect bubble emissions, where a fixed-focus single-element acoustic transducer is typically used as a passive cavitation detector (PCD). This technique is suboptimal for clinical applications, because most PCD transducers are tightly focused and afford limited spatial coverage of the HIFU focal region. A Terason 2000 Ultrasound System was used as a PCD array to expand the spatial detection region for cavitation by operating in passive mode, obtaining the radiofrequency signals corresponding to each scan line and filtering the contribution from scattering of the HIFU signal harmonics. This approach allows for spatially resolved detection of both inertial and stable cavitation throughout the focal region. Measurements with the PCD array during sonication with a 1.1-MHz HIFU source in tissue phantoms were compared with single-element PCD and thermocouple sensing. Stable cavitation signals at the harmonics and superharmonics increased in a threshold fashion for temperatures >90°C, an effect attributed to high vapor pressure in the cavities. Incorporation of these detection techniques in a diagnostic ultrasound platform could result in a powerful tool for improving HIFU guidance and treatment. (E-mail: cfarny@bwh.harvard.edu)     

Sound waves and antineoplastic drugs: The possibility of an enhanced combined anticancer therapy

Kremkau wrote a historical review of the use of ultrasound in cancer therapy in 1979⁽¹⁾ In 1990, Kondo and Kano published a Japanese review of the implications of the thermal and nonthermal effects of ultrasound in the treatment of cancer²⁾. Again in 2000, Kondo et al reviewed the therapeutic applications of ultrasound and shock wave, emphasizing their thermal and cavitational effects³⁾. Here we focus on the effects of ultrasound or shock waves in combination with anticancer agents, emphasizing their mechanisms of action and interaction. Most of the studies cited here reported promising results. Although the extent of the augmented combined effects in vivo is limited, synergism is the rule in vitro. In addition to the thermal effect of ultrasound, cavitational effects undoubtedly played a major role in both ultrasound and, more prominently, in shock wave therapy. Although the mechanism of the nonthermal noncavitational effects on biological processes is obscure, several factors, including temperature and the occurrence of cavitation and inertial cavitation, probably coexist and blend with these other effects. Magnification of anticancer activity results mainly from increased localization of drugs or other agents in vivo and increased intracellular permeabilisation both in vivo and in vitro. On the other hand, sublethal damage caused by ultrasound or shock waves may render cells more susceptible, to the effects of the agents, and both may act together, further amplifying these effects. We thus conclude that proper combination of an appropriate agent and ultrasound or shock wave should help improve cancer therapy by minimizing the side effects of drugs by lowering the effective dose and reducing the systemic concentration while increasing the efficiency of the therapy as a whole. Future studies should reveal specific conditions in this combined therapy that will lead to optimal outcome.