Ultrasonics 57 (2015) 36–43
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Enhancing laser therapy using PEGylated gold nanoparticles combined with ultrasound and microbubbles Christine Tarapacki, Raffi Karshafian ⇑ Department of Physics, Ryerson University, Toronto, Canada
a r t i c l e
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Article history: Received 13 May 2014 Received in revised form 20 September 2014 Accepted 13 October 2014 Available online 19 October 2014 Keywords: Gold nanoparticles Drug delivery Thermal therapy Microbubbles Sonoporation
a b s t r a c t Background: Gold nanorod (AuNR) laser therapy (LT) is a non-invasive method of increasing the temperature of a target tissue using near infrared light. In this study, the effects of ultrasound and microbubbles (USMB) with AuNR and LT were investigated on cell viability. Methods: MDA-MB-231 cells in suspension were treated with three different treatment combinations of AuNR, LT and USMB (Pneg = 0.6 or 1.0 MPa): (1) AuNR with USMB followed by LT, (2) AuNR and LT followed by USMB, and (3) USMB followed by AuNR and LT. Cells were also exposed to USMB and LT without AuNR. The USMB conditions were: 500 kHz frequency, 16 cycles, 1 kHz pulse repetition frequency for 1 min in the presence of Definity microbubbles (1.7% v/v). AuNR and LT conditions were: mPEG coated AuNR at 3 1011 np/mL and 1.9 W/cm2 for 3 min. Following the treatment, cell viability was assessed using propidium iodide (PI) fluorescent marker and flow cytometry (VPI), and colony assay (VCA). Cell viabilities were compared using a non-parametric Mann–Whitney U-test and synergism was assessed using the Bliss Independence Model. Results and discussion: USMB improved cell death when combined with AuNR and LT. VPI of 17 ± 2% (at 0.6 MPa) and 11 ± 4% (at 1.0 MPa) were observed with combined treatment of AuNR and USMB followed by LT compared to VPI of 60 ± 2% (at 0.6 MPa) and 42 ± 3% (at 1.0 MPa) with USMB alone and VPI of 22 ± 3% for AuNR and LT. The combined effect of AuNR and LT with USMB was additive regardless of treatment order. VCA results agreed with the additive effect caused by combining AuNR and LT with USMB for all treatment orders. In the absence of AuNR, samples exposed to LT prior to USMB at 0.6 MPa increased VPI by 13% (p < 0.01) showing a protective effect. Conclusion: Combining AuNR and LT with USMB resulted in an additive effect on cell viability compared to AuNR and LT, or USMB. In addition, cells exposed to low intensity NIR light appear to be protected against USMB exposure. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Gold nanorod (AuNR) laser therapy (LT) can increase the local temperature in target areas such as solid tumors [6,15,16,25,42]. When AuNR are injected into the body, they preferentially accumulate into tumors with leaky vasculature and can be activated with laser energy to induce local thermal damage. The thermal effect is due to the nanoparticle surface plasmon resonance (SPR) which converts the optical exposure energy into heat and is dependent on the nanoparticle shape and size. Commonly used gold nanorods (AuNR) have an aspect ratio (length/width) of 4 resulting in an SPR peak around 800 nm which is within the ⇑ Corresponding author at: Ryerson University, 350 Victoria Street, KHE 329 E, Toronto, Ontario M5B-2K3, Canada. Tel.: +1 416 979 5000x7536; fax: +1 1 416 979 5343. E-mail address: karshafi[email protected] (R. Karshafian). http://dx.doi.org/10.1016/j.ultras.2014.10.015 0041-624X/Ó 2014 Elsevier B.V. All rights reserved.
optical window of the body (600–1300 nm). This allows for minimum energy absorption by body tissues [3,5], maximizing the absorption by the AuNR. AuNR are favorable as thermal absorption agents because they have absorption cross sections approximately 4–5 orders of magnitude higher than other commonly used agents such as Indocyanine Green (ICG) [20], causing fewer AuNR to induce thermal effects. Gad et al. [12] performed gold nanoparticle toxicity studies on mice, rats, and beagle dogs demonstrating that 150 nm gold nanospheres, coated with polyethalyne glycol (PEG) were retained within the body throughout the 300 days of the study; however, due to the PEG coating they did not appear to affect the animals’ health. The gold nanoparticles were filtered out of the bloodstream and remained inside the organs of the reticuloendothelial system (liver and spleen). Increasing gold nanoparticle localization and concentration within the tumor has the potential to increase the effectiveness of the AuNR assisted LT.
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The application of ultrasound and microbubbles (USMB) has been shown to enhance effectiveness of chemotherapy [11,13,44] and radiotherapy [7]; [35] in both in vitro and in vivo tumor models. USMB induces reversible and irreversible increase in cell membrane permeability through a process known as sonoporation [17,23,22]. The stress can induce a variety of biological pathways that lead to increased endocytosis [31] or increased ceramide production leading to apoptotic pathways [1]. The acoustic mechanism has been associated with biomechanical perturbation of biological membranes induced by microbubbles undergoing non-linear oscillation and inertial cavitation, and generating shear stress on biological membranes in its vicinity [10,37,49]. Membrane disruptions of 30–150 nm range with up to 5 lm were observed in cells allowing intracellular uptake of nanoparticles and small molecules [51,52]. The resealing of the membrane disruptions depends on the size of the pore and generally reseals within 1 min [17]. Small pores (97% viability) and propidium iodide viability (VPI) was reported as mean ± standard error of the mean. In addition, colony assays were performed to measure the cells’ ability to proliferate and form colonies. Cells were plated in polystyrene disposable petri dishes (25384-092, VWR International, Mississauga, ON, CA) with 5 mL RPMI1640 and 10% FBS and placed in a cell culture incubator at 37 °C. After 10 days the media was removed and the cells were stained using methylene blue dye for 15 min. Methylene blue dye was made by mixing 3 g/L of methyl-blue powder (m4159, Sigma–Aldrich, Oakville, ON, CA) with a 50% methanol (179957,
37 C incubator Thermal Camera View 60 C
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810 nm Diode Laser Fig. 2. Laser therapy setup. Cell suspensions were placed in a corner well of a 24-well plate and positioned above a laser fiber connected to an 810 nm continuous wave (CW) diode laser. A thermal camera was positioned perpendicular to the laser fiber with a view shown in the insert on the top right corner. The region of interest (ROI) selected was on the bottom of the well and shown in the insert.
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Sigma–Aldrich, Oakville, ON, CA) and 50% distilled water solution. For each treatment condition, 6–12 dishes were plated and stained. Following staining, the number of colonies per dish were counted manually and normalized to the untreated control samples. The clonogenic assay viability (VCA) is shown as mean ± standard error of the mean. 2.6. Statistical analysis Statistical differences were analyzed using non-parametric Mann–Whitney U-test and viability differences were considered statistically significant with a p-value less than 0.05. In addition, the Bliss Independence Criteria was used to analyze synergism between treatment conditions [4,14].
(75 ± 2%), and (AuNR + USMB)c + LT (69 ± 3%) compared to the untreated control (100 ± 1%), however, the differences between the ultrasonically treated conditions were not statistically significant. The clonogenic viability (VCA) with the combined treatment of (AuNR + USMB)c + LT (VCA = 38 ± 3%) was statistically lower than that of USMB (VCA = 50 ± 3%) and AuNR + USMB (VCA = 48 ± 3%) (p < 0.05) (Fig. 3B). The combined treatment induced an additive effect on the clonogenic viability of cells. The expected additive effect of the combined treatments (AuNR)c + LT (VCA = 64 ± 4%) and USMB (VCA = 50 ± 3%) on cell viability was 32 ± 3% which was comparable to the experimental (AuNR + USMB)c + LT viability of 38 ± 3%. In addition, the expected additive effect from VCA of LT and AuNR was calculated to be 61 ± 11% which was similar to the experimental viability of (AuNR)c + LT (VCA = 64 ± 4%) indicating an additive effect.
3. Results 3.2. Cells with AuNR in suspension during LT 3.1. Cells centrifuged before LT Cell viability assessed with PI (VPI) and colony assay (VCA) following treatment with laser, AuNR, USMB (Pneg = 0.6 MPa), AuNR + USMB, (AuNR)c + LT and (AuNR + USMB)c + LT, where the subscript c represents centrifugation prior to laser treatment, are shown in Fig. 3A and B, respectively. VPI were not statistically different between (AuNR)c + LT LT, and AuNR (Fig. 3A). A decrease in viability was achieved with USMB (74 ± 4%), AuNR + USMB
A
Cell viability – VPI and VCA – following LT, AuNR, USMB, AuNR + USMB, (AuNR) + LT and (AuNR + USMB) + LT where samples were not centrifuged prior to LT are shown in Fig. 4A and B, respectively. A VPI of 22 ± 3% was observed with AuNR + LT where AuNR were in suspension compared to 98 ± 2% with (AuNR)c + LT (centrifuged). As expected, cells exposed to 1.0 MPa had a significantly lower viability than cells exposed to 0.6 MPa; a VPI of 60 ± 2% and 42 ± 3% were achieved at 0.6 MPa and 1.0 MPa
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Fig. 3. (A) Immediate viability (VPI; n = 3) of centrifuged cells prior to laser irradiation assessed through flow cytometry and (B) clonogenic viability (VCA; n = 9). (AuNR)c + LT represents 3 1011 np/mL of gold nanorods centrifuged prior to laser exposure for 3 min at 1.9 W/cm2, USMB represents ultrasound and microbubbles at 0.6 MPa, AuNR + USMB represents 3 1011 np/mL of gold nanorods present during USMB and (AuNR + USMB)c + LT represents samples exposed to AuNR + USMB and centrifuged prior to LT. A star (⁄) represents a Mann–Whitney Utest significant difference with a p < 0.05 and 2 stars (⁄⁄) represents p < 0.001.
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Fig. 4. (A) Immediate viability (VPI; n = 9) and (B) Clonogenic Viability (VCA; n = 12) of cells exposed to USMB at 0.6 and 1.0 MPa in combination with AuNR and LT. AuNR represents 3 1011 np/mL of gold nanorods present in solution with the cells. One star (⁄) represents a Mann–Whitney U-test significant difference with a p < 0.05, two stars (⁄⁄) represent p < 0.001 and three stars (⁄⁄⁄) represent p < 0.0001.
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3.3. USMB and LT The VPI and VCA of cells treated with LT and USMB (Pneg = 0.6 and 1.0 MPa) are shown in Fig. 5A and B, respectively. Generally, cell viability increased with the combined treatment of USMB and LT compared to USMB alone. A VPI of 60 ± 2% and 73 ± 2% were achieved with USMB alone (Pneg = 0.6 MPa) and LT + USMB, respectively; a statistically significant increase of 13% was observed in cell viability (p < 0.01) (Fig. 5A). However, the effect was only significant for VPI at 0.6 MPa and not for VCA or at the higher ultrasound pressure (Pneg = 1.0 MPa).
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(p < 0.001), respectively, and VCA of 5 ± 1% was observed at 1.0 MPa which is 19% lower than the VCA of USMB at 0.6 MPa (p < 0.0001). At 0.6 MPa, the combined treatment of (AuNR + LT) + USMB showed a significantly lower VPI of 12 ± 1% compared to that of AuNR + LT (p < 0.05). In addition, a statistically significant difference (p < 0.05) was observed between (AuNR + LT) + USMB and (AuNR + USMB) + LT. However, the combined treatment of AuNR, USMB and LT for all treatment orders showed no significant difference compared to the expected additive viability of 13 ± 2% when combining AuNR + LT (VPI = 22 ± 3%) and USMB (VPI = 60 ± 2%; Pneg = 0.6 MPa) which indicates an additive effect. Similarly, VCA of AuNR + LT (Fig. 4B) showed a statistically significant difference between the combined treatment of AuNR, USMB and LT for all treatment orders. VCA with AuNR + LT was 3 ± 2% and with USMB alone (Pneg = 0.6 MPa) was 24 ± 2%. VCA experimental results for (AuNR + USMB) + LT, USMB + (AuNR + LT), and (AuNR + LT) + USMB showed no significant difference compared to the calculated additive value for any of the three treatment orders. The VPI achieved with the combined treatment of AuNR, LT and USMB at Pneg of 1.0 MPa in different permutations was statistically different compared to that of AuNR + LT (VPI = 22 ± 3%) and USMB (VPI = 42 ± 3%; Pneg = 1.0 MPa) individually (Fig. 4A). Similar to 0.6 MPa, USMB at Pneg of 1.0 MPa induced an additive effect when combined with AuNR + LT. The expected VPI of USMB combined with AuNR + LT was 9 ± 2% and for VCA was 0.2 ± 0.2% which was statistically similar to all treatment combinations indicating an additive effect.
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Fig. 5. (A) Immediate viability (VPI; n = 9) and (B) Clonogenic Viability (VCA; n = 12) of cells exposed to USMB at 0.6 and 1.0 MPa in combination with laser before or after acoustic exposure. One star (⁄) represents a Mann–Whitney U-test significant difference with a p < 0.05, two stars (⁄⁄) represent p < 0.001 and three stars (⁄⁄⁄) represent p < 0.0001.
significant correlation for VPI or VCA at either ultrasound pressure with thermal dose.
3.4. Thermal dose
4. Discussion
The maximum temperature of the cell suspension during LT, which occurred at the 3 min time point, and CEM43 of the combined treatments with AuNR and USMB are shown in Fig. 6A and B, respectively. The maximum temperature ranged from 50 °C to 53 °C with the combined treatment of AuNR, USMB and LT. A statistically lower temperature of 50 ± 1 °C was achieved with (AuNR + USMB) + LT compared to 53 ± 1 °C with USMB + (AuNR + LT) at Pneg of 1.0 MPa. The maximum temperature achieved with AuNR + LT was 51.5 ± 1 °C, whereas cells exposed to LT without AuNR had a maximum temperature between 37.5–39 °C (data not shown). The cumulative equivalent minutes at 43 °C (CEM43) – shown in a box plot with the median and interquartile range of the sample, and error bars representing the 10th and 90th percentile – ranged from a median CEM43 of 60 for samples treated with (AuNR + USMB) + LT at 1.0 MPa to a CEM43 of 349 for samples treated with USMB + (AuNR + LT) at 1.0 MPa (Fig. 6B). No statistical differences were observed between the maximum temperature of the combined treatments (AuNR, USMB and LT) and AuNR + LT with a median CEM43 of 135 and an interquartile range between 86 and 361 (Fig. 6B). Scatter plots of median CEM43 with VPI and VCA for the different treatment conditions are shown in Fig.7A and B, respectively, with error bars representing interquartile range. Although viability decreased with increasing thermal dose (CEM43), Spearman’s rank correlation coefficient showed no
Ultrasound and microbubbles induced an additive effect on cell viability when combined with PEGylated AuNR laser therapy. In general, the effect on cell viability was independent of treatment order. The thermal dose did not appear to be an indicator of cell death for combined treatments with AuNR in suspension during LT. However, when the AuNR were centrifuged out of suspension, the thermal dose was comparable to laser alone; the temperature remained below 39 °C throughout LT. This is the first study to show that in the absence of AuNR, NIR energy caused a protective effect on cell viability from ultrasound and microbubble exposure. Centrifuged cells that were exposed to AuNR also experienced a negligible thermal increase; however, they did not show the same protective effect. The combined treatment of ultrasound-and-microbubble and laser therapy with gold nanoparticles, under the conditions studied, induced an additive effect on cell death suggesting independent mechanisms of killing cells. USMB causes mainly biomechanical effects on cells due to microbubble activity such as creating membrane pores that can either be transient or lead to cell death [17,23,22]. The pores caused by USMB can recover within the order of minutes [17] showing that cellular recovery from physical damage begins to occur quickly. USMB also triggers biological effects such as apoptosis [24,50] and enhanced endocytosis [31]. Although acoustically induced membrane pores can be physically
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Fig. 6. (A) Maximum sample temperature measured with a thermal camera after 3 min of laser therapy at 1.9 W/cm2. (B) Cumulative equivalent minutes at 43 °C (CEM43) during laser exposure.
large enough to allow for the particles to pass through [51,52], larger membrane pores also correlate with increased cell death due to USMB alone [17] limiting the potential for an enhancement due to nanoparticle uptake. In contrast, AuNR + LT cause mainly thermal effects that can lead to protein denaturation, coagulation and necrosis [9]. The thermal dose, or CEM43, is often used to approximate the treatment severity showing an exponential relationship with temperature and linear relationship with time [36,40]. Thermal therapy can also induce bioeffects such as increased heat shock proteins to protect vital cellular proteins [21,32] and can trigger apoptosis [39]. Protective biological pathways that involve more sophisticated protein cascades such as heat-shock protein up-regulation, require a longer time on the order of hours, to induce their effect [32]. Changing the time delay between ultrasound and laser treatments may affect the cell viability due to possible protective effects of chaperone proteins [21,28]. Considering there was an additive effect for both acoustic pressures tested, cells appear to be independently sensitive to thermal damage and mechanical damage under the treated conditions so combining the two treatments creates an enhanced, additive effect. A previous study of ours showed a synergistic enhancement in cell death when AuNR laser thermal therapy was combined with ultrasound and microbubbles using CTAB coated gold nanorods [43]. It is likely that variations in the cell viability between the two studies could be attributed to the toxicity difference between the nanoparticle coatings. CTAB is positively charged and highly cytotoxic so it cannot safely be utilized in in vivo applications [2]. The PEG coating used in this study is biocompatible which limits uptake of AuNR through endocytosis but highly decreases cytotoxicity [30,34]. Other than the nanoparticle coating, another possible
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reason for differences between the two studies is a difference in cell lines used which can affect the bioeffects of USMB and intrinsic thermal resistance. Variation of 3 °C was observed in the temperature of the cell sample for the different treatment conditions. This may be due to the interaction of AuNR with the surrounding medium causing changes in the nanoparticles’ optical properties [2]. Previous studies have shown that the optical absorption of gold nanoparticles in a solution changes with the dielectric properties of the medium and the interaction of the PEG coating with proteins and charges in the solution [2,29]. In addition, the size of the nanoparticle coating, has previously been found to change through time due to the interaction of the media [29,33]. Following USMB, microbubble shell fragments in the media increase the lipid concentration and may affect the nanoparticle coating leading to changes in absorption properties and thermal dose. The VPI and VCA of the combined treatments showed no significant relationship with thermal dose, indicating that the CEM43 differences during the 3 min laser treatment did not appear to significantly affect cell viability. The application of laser therapy enhanced cell viability following exposure to ultrasound-and-microbubbles. At a low intensity, NIR light has been found to stimulate cytochrome c oxidase and other mitochondrial chromophores resulting in increased metabolic activity leading to a protective effect on cells [8]. Low intensity NIR light has previously been used for photobiomodulation to precondition tissues for recovery prior to the application of severe treatments or surgeries [48]. It is hypothesized that with an increased metabolic activity, cells may be able to repair membrane
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damage induced by ultrasound-and-microbubbles. However, the protective effect of laser and USMB may limit the treatment efficacy of the combined treatment of AuNR, USMB and LT. The failure to deliver a sufficient nanoparticle concentration may cause protective effects on cancer cells. However, gold nanoparticles can be monitored using photoacoustic and optical modalities prior to laser therapy [18,19,47]. Although a protective effect of laser and USMB is not desirable in cancer therapy, the NIR light could be beneficial for gene therapy with ultrasound-microbubbles that aim to increase intracellular uptake while minimizing cell death. 5. Conclusion The therapeutic effect of ultrasound-microbubbles with mPEG coated gold nanorod laser therapy is additive with and without gold nanoparticles present in the solution. Under the conditions tested, thermal dose appeared not to be a predictor of cell death with the combined treatment of gold nanorod, ultrasound-microbubble and laser. In the absence of gold nanoparticles, NIR light can induce protective effects on cells minimizing the damage caused by ultrasound and microbubbles. Acknowledgment The research was supported by NSERC Discovery grant. References [1] A.A. Al-Mahrouki, R. Karshafian, A. Giles, G.J. Czarnota, Bioeffects of ultrasound-stimulated microbubbles on endothelial cells: gene expression changes associated with radiation enhancement in vitro, Ultrasound Med. Biol. 38 (2012) 1958–1969. [2] A.M. Alkilany, C.J. Murphy, Toxicity and cellular uptake of gold nanoparticles: what we have learned so far?, J Nanoparticle Res. 12 (2010) 2313–2333. [3] R.R. Anderson, J.A. Parrish, The optics of human skin, J. Investig. Dermatol. 77 (1981) 13–19. [4] C.I. Bliss, The toxicity of poisons applied jointly1, Ann. Appl. Biol. 26 (1939) 585–615. [5] S.-S. Chang, C.-W. Shih, C.-D. Chen, W.-C. Lai, C.R.C. Wang, The shape transition of gold nanorods, Langmuir 15 (1999) 701–709. [6] C.-L. Chen, L.-R. Kuo, C.-L. Chang, Y.-K. Hwu, C.-K. Huang, S.-Y. Lee, K. Chen, S.-J. Lin, J.-D. Huang, Y.-Y. Chen, In situ real-time investigation of cancer cell photothermolysis mediated by excited gold nanorod surface plasmons, Biomaterials 31 (2010) 4104–4112. [7] G.J. Czarnota, R. Karshafian, P.N. Burns, S. Wong, A. Al Mahrouki, J.W. Lee, A. Caissie, W. Tran, C. Kim, M. Furukawa, Tumor radiation response enhancement by acoustical stimulation of the vasculature, Proc. Natl. Acad. Sci. 109 (2012) E2033–E2041. [8] K.D. Desmet, D.A. Paz, J.J. Corry, J.T. Eells, M.T.T. Wong-Riley, M.M. Henry, E.V. Buchmann, M.P. Connelly, J.V. Dovi, H.L. Liang, D.S. Henshel, R.L. Yeager, D.S. Millsap, J. Lim, L.J. Gould, R. Das, M. Jett, B.D. Hodgson, D. Margolis, H.T. Whelan, Clinical and experimental applications of NIR-LED photobiomodulation, Photomed. Laser Surg. 24 (2006) 121–128. [9] F. Despa, D.P. Orgill, J. Neuwalder, R.C. Lee, The relative thermal stability of tissue macromolecules and cellular structure in burn injury, Burns 31 (2005) 568–577. [10] A.A. Doinikov, A. Bouakaz, Theoretical investigation of shear stress generated by a contrast microbubble on the cell membrane as a mechanism for sonoporation, J. Acoust. Soc. Am. 128 (2010) 11–19. [11] M. Duvshani-Eshet, O. Benny, A. Morgenstern, M. Machluf, Therapeutic ultrasound facilitates antiangiogenic gene delivery and inhibits prostate tumor growth, Mol. Cancer Ther. 6 (2007) 2371–2382. [12] S.C. Gad, K.L. Sharp, C. Montgomery, J.D. Payne, G.P. Goodrich, Evaluation of the toxicity of intravenous delivery of auroshell particles (gold–silica nanoshells), Int. J. Toxicol. 31 (2012) 584–594. [13] D.E. Goertz, M. Todorova, O. Mortazavi, V. Agache, B. Chen, R. Karshafian, K. Hynynen, Antitumor effects of combining docetaxel (taxotere) with the antivascular action of ultrasound stimulated microbubbles, PloS One 7 (2012) e52307. [14] M. Goldoni, C. Johansson, A mathematical approach to study combined effects of toxicants in vitro: evaluation of the Bliss independence criterion and the Loewe additivity model, Toxicol. In Vitro 21 (2007) 759–769. [15] J.H. Grossman, S.E. McNeil, Nanotechnology in cancer medicine, Phys. Today 65 (2012) 38–42. [16] T.S. Hauck, T.L. Jennings, T. Yatsenko, J.C. Kumaradas, W.C.W. Chan, Enhancing the toxicity of cancer chemotherapeutics with gold nanorod hyperthermia, Adv. Mater. 20 (2008) 3832–3838.
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Enhancing laser therapy using PEGylated gold nanoparticles combined with ultrasound and microbubbles Christine Tarapacki, Raffi Karshafian ⇑ Department of Physics, Ryerson University, Toronto, Canada
a r t i c l e
i n f o
Article history: Received 13 May 2014 Received in revised form 20 September 2014 Accepted 13 October 2014 Available online 19 October 2014 Keywords: Gold nanoparticles Drug delivery Thermal therapy Microbubbles Sonoporation
a b s t r a c t Background: Gold nanorod (AuNR) laser therapy (LT) is a non-invasive method of increasing the temperature of a target tissue using near infrared light. In this study, the effects of ultrasound and microbubbles (USMB) with AuNR and LT were investigated on cell viability. Methods: MDA-MB-231 cells in suspension were treated with three different treatment combinations of AuNR, LT and USMB (Pneg = 0.6 or 1.0 MPa): (1) AuNR with USMB followed by LT, (2) AuNR and LT followed by USMB, and (3) USMB followed by AuNR and LT. Cells were also exposed to USMB and LT without AuNR. The USMB conditions were: 500 kHz frequency, 16 cycles, 1 kHz pulse repetition frequency for 1 min in the presence of Definity microbubbles (1.7% v/v). AuNR and LT conditions were: mPEG coated AuNR at 3 1011 np/mL and 1.9 W/cm2 for 3 min. Following the treatment, cell viability was assessed using propidium iodide (PI) fluorescent marker and flow cytometry (VPI), and colony assay (VCA). Cell viabilities were compared using a non-parametric Mann–Whitney U-test and synergism was assessed using the Bliss Independence Model. Results and discussion: USMB improved cell death when combined with AuNR and LT. VPI of 17 ± 2% (at 0.6 MPa) and 11 ± 4% (at 1.0 MPa) were observed with combined treatment of AuNR and USMB followed by LT compared to VPI of 60 ± 2% (at 0.6 MPa) and 42 ± 3% (at 1.0 MPa) with USMB alone and VPI of 22 ± 3% for AuNR and LT. The combined effect of AuNR and LT with USMB was additive regardless of treatment order. VCA results agreed with the additive effect caused by combining AuNR and LT with USMB for all treatment orders. In the absence of AuNR, samples exposed to LT prior to USMB at 0.6 MPa increased VPI by 13% (p < 0.01) showing a protective effect. Conclusion: Combining AuNR and LT with USMB resulted in an additive effect on cell viability compared to AuNR and LT, or USMB. In addition, cells exposed to low intensity NIR light appear to be protected against USMB exposure. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Gold nanorod (AuNR) laser therapy (LT) can increase the local temperature in target areas such as solid tumors [6,15,16,25,42]. When AuNR are injected into the body, they preferentially accumulate into tumors with leaky vasculature and can be activated with laser energy to induce local thermal damage. The thermal effect is due to the nanoparticle surface plasmon resonance (SPR) which converts the optical exposure energy into heat and is dependent on the nanoparticle shape and size. Commonly used gold nanorods (AuNR) have an aspect ratio (length/width) of 4 resulting in an SPR peak around 800 nm which is within the ⇑ Corresponding author at: Ryerson University, 350 Victoria Street, KHE 329 E, Toronto, Ontario M5B-2K3, Canada. Tel.: +1 416 979 5000x7536; fax: +1 1 416 979 5343. E-mail address: karshafi[email protected] (R. Karshafian). http://dx.doi.org/10.1016/j.ultras.2014.10.015 0041-624X/Ó 2014 Elsevier B.V. All rights reserved.
optical window of the body (600–1300 nm). This allows for minimum energy absorption by body tissues [3,5], maximizing the absorption by the AuNR. AuNR are favorable as thermal absorption agents because they have absorption cross sections approximately 4–5 orders of magnitude higher than other commonly used agents such as Indocyanine Green (ICG) [20], causing fewer AuNR to induce thermal effects. Gad et al. [12] performed gold nanoparticle toxicity studies on mice, rats, and beagle dogs demonstrating that 150 nm gold nanospheres, coated with polyethalyne glycol (PEG) were retained within the body throughout the 300 days of the study; however, due to the PEG coating they did not appear to affect the animals’ health. The gold nanoparticles were filtered out of the bloodstream and remained inside the organs of the reticuloendothelial system (liver and spleen). Increasing gold nanoparticle localization and concentration within the tumor has the potential to increase the effectiveness of the AuNR assisted LT.
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The application of ultrasound and microbubbles (USMB) has been shown to enhance effectiveness of chemotherapy [11,13,44] and radiotherapy [7]; [35] in both in vitro and in vivo tumor models. USMB induces reversible and irreversible increase in cell membrane permeability through a process known as sonoporation [17,23,22]. The stress can induce a variety of biological pathways that lead to increased endocytosis [31] or increased ceramide production leading to apoptotic pathways [1]. The acoustic mechanism has been associated with biomechanical perturbation of biological membranes induced by microbubbles undergoing non-linear oscillation and inertial cavitation, and generating shear stress on biological membranes in its vicinity [10,37,49]. Membrane disruptions of 30–150 nm range with up to 5 lm were observed in cells allowing intracellular uptake of nanoparticles and small molecules [51,52]. The resealing of the membrane disruptions depends on the size of the pore and generally reseals within 1 min [17]. Small pores (97% viability) and propidium iodide viability (VPI) was reported as mean ± standard error of the mean. In addition, colony assays were performed to measure the cells’ ability to proliferate and form colonies. Cells were plated in polystyrene disposable petri dishes (25384-092, VWR International, Mississauga, ON, CA) with 5 mL RPMI1640 and 10% FBS and placed in a cell culture incubator at 37 °C. After 10 days the media was removed and the cells were stained using methylene blue dye for 15 min. Methylene blue dye was made by mixing 3 g/L of methyl-blue powder (m4159, Sigma–Aldrich, Oakville, ON, CA) with a 50% methanol (179957,
37 C incubator Thermal Camera View 60 C
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810 nm Diode Laser Fig. 2. Laser therapy setup. Cell suspensions were placed in a corner well of a 24-well plate and positioned above a laser fiber connected to an 810 nm continuous wave (CW) diode laser. A thermal camera was positioned perpendicular to the laser fiber with a view shown in the insert on the top right corner. The region of interest (ROI) selected was on the bottom of the well and shown in the insert.
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Sigma–Aldrich, Oakville, ON, CA) and 50% distilled water solution. For each treatment condition, 6–12 dishes were plated and stained. Following staining, the number of colonies per dish were counted manually and normalized to the untreated control samples. The clonogenic assay viability (VCA) is shown as mean ± standard error of the mean. 2.6. Statistical analysis Statistical differences were analyzed using non-parametric Mann–Whitney U-test and viability differences were considered statistically significant with a p-value less than 0.05. In addition, the Bliss Independence Criteria was used to analyze synergism between treatment conditions [4,14].
(75 ± 2%), and (AuNR + USMB)c + LT (69 ± 3%) compared to the untreated control (100 ± 1%), however, the differences between the ultrasonically treated conditions were not statistically significant. The clonogenic viability (VCA) with the combined treatment of (AuNR + USMB)c + LT (VCA = 38 ± 3%) was statistically lower than that of USMB (VCA = 50 ± 3%) and AuNR + USMB (VCA = 48 ± 3%) (p < 0.05) (Fig. 3B). The combined treatment induced an additive effect on the clonogenic viability of cells. The expected additive effect of the combined treatments (AuNR)c + LT (VCA = 64 ± 4%) and USMB (VCA = 50 ± 3%) on cell viability was 32 ± 3% which was comparable to the experimental (AuNR + USMB)c + LT viability of 38 ± 3%. In addition, the expected additive effect from VCA of LT and AuNR was calculated to be 61 ± 11% which was similar to the experimental viability of (AuNR)c + LT (VCA = 64 ± 4%) indicating an additive effect.
3. Results 3.2. Cells with AuNR in suspension during LT 3.1. Cells centrifuged before LT Cell viability assessed with PI (VPI) and colony assay (VCA) following treatment with laser, AuNR, USMB (Pneg = 0.6 MPa), AuNR + USMB, (AuNR)c + LT and (AuNR + USMB)c + LT, where the subscript c represents centrifugation prior to laser treatment, are shown in Fig. 3A and B, respectively. VPI were not statistically different between (AuNR)c + LT LT, and AuNR (Fig. 3A). A decrease in viability was achieved with USMB (74 ± 4%), AuNR + USMB
A
Cell viability – VPI and VCA – following LT, AuNR, USMB, AuNR + USMB, (AuNR) + LT and (AuNR + USMB) + LT where samples were not centrifuged prior to LT are shown in Fig. 4A and B, respectively. A VPI of 22 ± 3% was observed with AuNR + LT where AuNR were in suspension compared to 98 ± 2% with (AuNR)c + LT (centrifuged). As expected, cells exposed to 1.0 MPa had a significantly lower viability than cells exposed to 0.6 MPa; a VPI of 60 ± 2% and 42 ± 3% were achieved at 0.6 MPa and 1.0 MPa
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Fig. 3. (A) Immediate viability (VPI; n = 3) of centrifuged cells prior to laser irradiation assessed through flow cytometry and (B) clonogenic viability (VCA; n = 9). (AuNR)c + LT represents 3 1011 np/mL of gold nanorods centrifuged prior to laser exposure for 3 min at 1.9 W/cm2, USMB represents ultrasound and microbubbles at 0.6 MPa, AuNR + USMB represents 3 1011 np/mL of gold nanorods present during USMB and (AuNR + USMB)c + LT represents samples exposed to AuNR + USMB and centrifuged prior to LT. A star (⁄) represents a Mann–Whitney Utest significant difference with a p < 0.05 and 2 stars (⁄⁄) represents p < 0.001.
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Fig. 4. (A) Immediate viability (VPI; n = 9) and (B) Clonogenic Viability (VCA; n = 12) of cells exposed to USMB at 0.6 and 1.0 MPa in combination with AuNR and LT. AuNR represents 3 1011 np/mL of gold nanorods present in solution with the cells. One star (⁄) represents a Mann–Whitney U-test significant difference with a p < 0.05, two stars (⁄⁄) represent p < 0.001 and three stars (⁄⁄⁄) represent p < 0.0001.
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3.3. USMB and LT The VPI and VCA of cells treated with LT and USMB (Pneg = 0.6 and 1.0 MPa) are shown in Fig. 5A and B, respectively. Generally, cell viability increased with the combined treatment of USMB and LT compared to USMB alone. A VPI of 60 ± 2% and 73 ± 2% were achieved with USMB alone (Pneg = 0.6 MPa) and LT + USMB, respectively; a statistically significant increase of 13% was observed in cell viability (p < 0.01) (Fig. 5A). However, the effect was only significant for VPI at 0.6 MPa and not for VCA or at the higher ultrasound pressure (Pneg = 1.0 MPa).
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(p < 0.001), respectively, and VCA of 5 ± 1% was observed at 1.0 MPa which is 19% lower than the VCA of USMB at 0.6 MPa (p < 0.0001). At 0.6 MPa, the combined treatment of (AuNR + LT) + USMB showed a significantly lower VPI of 12 ± 1% compared to that of AuNR + LT (p < 0.05). In addition, a statistically significant difference (p < 0.05) was observed between (AuNR + LT) + USMB and (AuNR + USMB) + LT. However, the combined treatment of AuNR, USMB and LT for all treatment orders showed no significant difference compared to the expected additive viability of 13 ± 2% when combining AuNR + LT (VPI = 22 ± 3%) and USMB (VPI = 60 ± 2%; Pneg = 0.6 MPa) which indicates an additive effect. Similarly, VCA of AuNR + LT (Fig. 4B) showed a statistically significant difference between the combined treatment of AuNR, USMB and LT for all treatment orders. VCA with AuNR + LT was 3 ± 2% and with USMB alone (Pneg = 0.6 MPa) was 24 ± 2%. VCA experimental results for (AuNR + USMB) + LT, USMB + (AuNR + LT), and (AuNR + LT) + USMB showed no significant difference compared to the calculated additive value for any of the three treatment orders. The VPI achieved with the combined treatment of AuNR, LT and USMB at Pneg of 1.0 MPa in different permutations was statistically different compared to that of AuNR + LT (VPI = 22 ± 3%) and USMB (VPI = 42 ± 3%; Pneg = 1.0 MPa) individually (Fig. 4A). Similar to 0.6 MPa, USMB at Pneg of 1.0 MPa induced an additive effect when combined with AuNR + LT. The expected VPI of USMB combined with AuNR + LT was 9 ± 2% and for VCA was 0.2 ± 0.2% which was statistically similar to all treatment combinations indicating an additive effect.
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Fig. 5. (A) Immediate viability (VPI; n = 9) and (B) Clonogenic Viability (VCA; n = 12) of cells exposed to USMB at 0.6 and 1.0 MPa in combination with laser before or after acoustic exposure. One star (⁄) represents a Mann–Whitney U-test significant difference with a p < 0.05, two stars (⁄⁄) represent p < 0.001 and three stars (⁄⁄⁄) represent p < 0.0001.
significant correlation for VPI or VCA at either ultrasound pressure with thermal dose.
3.4. Thermal dose
4. Discussion
The maximum temperature of the cell suspension during LT, which occurred at the 3 min time point, and CEM43 of the combined treatments with AuNR and USMB are shown in Fig. 6A and B, respectively. The maximum temperature ranged from 50 °C to 53 °C with the combined treatment of AuNR, USMB and LT. A statistically lower temperature of 50 ± 1 °C was achieved with (AuNR + USMB) + LT compared to 53 ± 1 °C with USMB + (AuNR + LT) at Pneg of 1.0 MPa. The maximum temperature achieved with AuNR + LT was 51.5 ± 1 °C, whereas cells exposed to LT without AuNR had a maximum temperature between 37.5–39 °C (data not shown). The cumulative equivalent minutes at 43 °C (CEM43) – shown in a box plot with the median and interquartile range of the sample, and error bars representing the 10th and 90th percentile – ranged from a median CEM43 of 60 for samples treated with (AuNR + USMB) + LT at 1.0 MPa to a CEM43 of 349 for samples treated with USMB + (AuNR + LT) at 1.0 MPa (Fig. 6B). No statistical differences were observed between the maximum temperature of the combined treatments (AuNR, USMB and LT) and AuNR + LT with a median CEM43 of 135 and an interquartile range between 86 and 361 (Fig. 6B). Scatter plots of median CEM43 with VPI and VCA for the different treatment conditions are shown in Fig.7A and B, respectively, with error bars representing interquartile range. Although viability decreased with increasing thermal dose (CEM43), Spearman’s rank correlation coefficient showed no
Ultrasound and microbubbles induced an additive effect on cell viability when combined with PEGylated AuNR laser therapy. In general, the effect on cell viability was independent of treatment order. The thermal dose did not appear to be an indicator of cell death for combined treatments with AuNR in suspension during LT. However, when the AuNR were centrifuged out of suspension, the thermal dose was comparable to laser alone; the temperature remained below 39 °C throughout LT. This is the first study to show that in the absence of AuNR, NIR energy caused a protective effect on cell viability from ultrasound and microbubble exposure. Centrifuged cells that were exposed to AuNR also experienced a negligible thermal increase; however, they did not show the same protective effect. The combined treatment of ultrasound-and-microbubble and laser therapy with gold nanoparticles, under the conditions studied, induced an additive effect on cell death suggesting independent mechanisms of killing cells. USMB causes mainly biomechanical effects on cells due to microbubble activity such as creating membrane pores that can either be transient or lead to cell death [17,23,22]. The pores caused by USMB can recover within the order of minutes [17] showing that cellular recovery from physical damage begins to occur quickly. USMB also triggers biological effects such as apoptosis [24,50] and enhanced endocytosis [31]. Although acoustically induced membrane pores can be physically
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large enough to allow for the particles to pass through [51,52], larger membrane pores also correlate with increased cell death due to USMB alone [17] limiting the potential for an enhancement due to nanoparticle uptake. In contrast, AuNR + LT cause mainly thermal effects that can lead to protein denaturation, coagulation and necrosis [9]. The thermal dose, or CEM43, is often used to approximate the treatment severity showing an exponential relationship with temperature and linear relationship with time [36,40]. Thermal therapy can also induce bioeffects such as increased heat shock proteins to protect vital cellular proteins [21,32] and can trigger apoptosis [39]. Protective biological pathways that involve more sophisticated protein cascades such as heat-shock protein up-regulation, require a longer time on the order of hours, to induce their effect [32]. Changing the time delay between ultrasound and laser treatments may affect the cell viability due to possible protective effects of chaperone proteins [21,28]. Considering there was an additive effect for both acoustic pressures tested, cells appear to be independently sensitive to thermal damage and mechanical damage under the treated conditions so combining the two treatments creates an enhanced, additive effect. A previous study of ours showed a synergistic enhancement in cell death when AuNR laser thermal therapy was combined with ultrasound and microbubbles using CTAB coated gold nanorods [43]. It is likely that variations in the cell viability between the two studies could be attributed to the toxicity difference between the nanoparticle coatings. CTAB is positively charged and highly cytotoxic so it cannot safely be utilized in in vivo applications [2]. The PEG coating used in this study is biocompatible which limits uptake of AuNR through endocytosis but highly decreases cytotoxicity [30,34]. Other than the nanoparticle coating, another possible
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VPI (%)
0 MPa
0 MPa AuNR+LT 0.6 MPa USMB+(AuNR+LT)
1000
(AuNR+USMB)+LT (AuNR+LT)+USMB
1.0 MPa 100
USMB+(AuNR+LT) (AuNR+USMB)+LT (AuNR+LT)+USMB
10 0
1
2
3
4
5
VCA (%) Fig. 7. (A) VPI and (B) VCA relationship with CEM43 for samples containing AuNR at 3 1011 np/mL in suspension during laser treatment with USMB at 0, 0.6 and 1.0 MPa and varying treatment order.
reason for differences between the two studies is a difference in cell lines used which can affect the bioeffects of USMB and intrinsic thermal resistance. Variation of 3 °C was observed in the temperature of the cell sample for the different treatment conditions. This may be due to the interaction of AuNR with the surrounding medium causing changes in the nanoparticles’ optical properties [2]. Previous studies have shown that the optical absorption of gold nanoparticles in a solution changes with the dielectric properties of the medium and the interaction of the PEG coating with proteins and charges in the solution [2,29]. In addition, the size of the nanoparticle coating, has previously been found to change through time due to the interaction of the media [29,33]. Following USMB, microbubble shell fragments in the media increase the lipid concentration and may affect the nanoparticle coating leading to changes in absorption properties and thermal dose. The VPI and VCA of the combined treatments showed no significant relationship with thermal dose, indicating that the CEM43 differences during the 3 min laser treatment did not appear to significantly affect cell viability. The application of laser therapy enhanced cell viability following exposure to ultrasound-and-microbubbles. At a low intensity, NIR light has been found to stimulate cytochrome c oxidase and other mitochondrial chromophores resulting in increased metabolic activity leading to a protective effect on cells [8]. Low intensity NIR light has previously been used for photobiomodulation to precondition tissues for recovery prior to the application of severe treatments or surgeries [48]. It is hypothesized that with an increased metabolic activity, cells may be able to repair membrane
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damage induced by ultrasound-and-microbubbles. However, the protective effect of laser and USMB may limit the treatment efficacy of the combined treatment of AuNR, USMB and LT. The failure to deliver a sufficient nanoparticle concentration may cause protective effects on cancer cells. However, gold nanoparticles can be monitored using photoacoustic and optical modalities prior to laser therapy [18,19,47]. Although a protective effect of laser and USMB is not desirable in cancer therapy, the NIR light could be beneficial for gene therapy with ultrasound-microbubbles that aim to increase intracellular uptake while minimizing cell death. 5. Conclusion The therapeutic effect of ultrasound-microbubbles with mPEG coated gold nanorod laser therapy is additive with and without gold nanoparticles present in the solution. Under the conditions tested, thermal dose appeared not to be a predictor of cell death with the combined treatment of gold nanorod, ultrasound-microbubble and laser. In the absence of gold nanoparticles, NIR light can induce protective effects on cells minimizing the damage caused by ultrasound and microbubbles. Acknowledgment The research was supported by NSERC Discovery grant. References [1] A.A. Al-Mahrouki, R. Karshafian, A. Giles, G.J. Czarnota, Bioeffects of ultrasound-stimulated microbubbles on endothelial cells: gene expression changes associated with radiation enhancement in vitro, Ultrasound Med. Biol. 38 (2012) 1958–1969. [2] A.M. Alkilany, C.J. Murphy, Toxicity and cellular uptake of gold nanoparticles: what we have learned so far?, J Nanoparticle Res. 12 (2010) 2313–2333. [3] R.R. Anderson, J.A. Parrish, The optics of human skin, J. Investig. Dermatol. 77 (1981) 13–19. [4] C.I. Bliss, The toxicity of poisons applied jointly1, Ann. Appl. Biol. 26 (1939) 585–615. [5] S.-S. Chang, C.-W. Shih, C.-D. Chen, W.-C. Lai, C.R.C. Wang, The shape transition of gold nanorods, Langmuir 15 (1999) 701–709. [6] C.-L. Chen, L.-R. Kuo, C.-L. Chang, Y.-K. Hwu, C.-K. Huang, S.-Y. Lee, K. Chen, S.-J. Lin, J.-D. Huang, Y.-Y. Chen, In situ real-time investigation of cancer cell photothermolysis mediated by excited gold nanorod surface plasmons, Biomaterials 31 (2010) 4104–4112. [7] G.J. Czarnota, R. Karshafian, P.N. Burns, S. Wong, A. Al Mahrouki, J.W. Lee, A. Caissie, W. Tran, C. Kim, M. Furukawa, Tumor radiation response enhancement by acoustical stimulation of the vasculature, Proc. Natl. Acad. Sci. 109 (2012) E2033–E2041. [8] K.D. Desmet, D.A. Paz, J.J. Corry, J.T. Eells, M.T.T. Wong-Riley, M.M. Henry, E.V. Buchmann, M.P. Connelly, J.V. Dovi, H.L. Liang, D.S. Henshel, R.L. Yeager, D.S. Millsap, J. Lim, L.J. Gould, R. Das, M. Jett, B.D. Hodgson, D. Margolis, H.T. Whelan, Clinical and experimental applications of NIR-LED photobiomodulation, Photomed. Laser Surg. 24 (2006) 121–128. [9] F. Despa, D.P. Orgill, J. Neuwalder, R.C. Lee, The relative thermal stability of tissue macromolecules and cellular structure in burn injury, Burns 31 (2005) 568–577. [10] A.A. Doinikov, A. Bouakaz, Theoretical investigation of shear stress generated by a contrast microbubble on the cell membrane as a mechanism for sonoporation, J. Acoust. Soc. Am. 128 (2010) 11–19. [11] M. Duvshani-Eshet, O. Benny, A. Morgenstern, M. Machluf, Therapeutic ultrasound facilitates antiangiogenic gene delivery and inhibits prostate tumor growth, Mol. Cancer Ther. 6 (2007) 2371–2382. [12] S.C. Gad, K.L. Sharp, C. Montgomery, J.D. Payne, G.P. Goodrich, Evaluation of the toxicity of intravenous delivery of auroshell particles (gold–silica nanoshells), Int. J. Toxicol. 31 (2012) 584–594. [13] D.E. Goertz, M. Todorova, O. Mortazavi, V. Agache, B. Chen, R. Karshafian, K. Hynynen, Antitumor effects of combining docetaxel (taxotere) with the antivascular action of ultrasound stimulated microbubbles, PloS One 7 (2012) e52307. [14] M. Goldoni, C. Johansson, A mathematical approach to study combined effects of toxicants in vitro: evaluation of the Bliss independence criterion and the Loewe additivity model, Toxicol. In Vitro 21 (2007) 759–769. [15] J.H. Grossman, S.E. McNeil, Nanotechnology in cancer medicine, Phys. Today 65 (2012) 38–42. [16] T.S. Hauck, T.L. Jennings, T. Yatsenko, J.C. Kumaradas, W.C.W. Chan, Enhancing the toxicity of cancer chemotherapeutics with gold nanorod hyperthermia, Adv. Mater. 20 (2008) 3832–3838.
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