Use of gold nanoshells to mediate heating induced perfusion changes in prostate tumors

Use of Gold Nanoshells to Mediate Heating Induced Perfusion Changes in Prostate Tumors

Anil Shetty*a, Andrew M. Elliotta , Jon A. Schwatrzb, James Wangb , Emilio Esparza-Cossa,

Sherry Klumppa , Brian Taylora , John D. Hazlea ,R.Jason Stafforda

aThe University of Texas M.D.Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX, USA 77030; bNanospectra Biosciences Inc. El Rio St., Suite 150, Houston, Texas, U.S.A

ABSTRACT

This study investigates the potential of using gold nanoshells to mediate a thermally induced modulation of tumor vasculature in experimental prostate tumors. We demonstrate that after passive extravasation and retention of the circulating nanoshells from the tumor vasculature into the tumor interstitium, the enhanced nanoshells absorption of near-infrared irradiation over normal vasculature, can be used to increase tumor perfusion or shut it down at powers which result in no observable affects on tissue without nanoshells. Temperature rise was monitored in real time using magnetic resonance temperature imaging and registered with perfusion changes as extrapolated from MR dynamic contrast enhanced (DCE) imaging results before and after each treatment. Results indicate that nanoshell mediated heating can be used to improve perfusion and subsequently enhance drug delivery and radiation effects, or be used to shut down perfusion to assist in thermal ablative therapy delivery.

Keywords: gold nanoshells, prostate cancer, MR temperature imaging, tumor perfusion, contrast uptake

1. INTRODUCTION Prostate cancer is the second most common cancer in American men after melanoma, with an estimated 219,000 cases of diagnosed in the U.S. in 2007 resulting in approximately 27,000 deaths [1]. Prostate cancer rates have continued to rise, although at a slower rate than previous years [2]. While there are a significant number of potential courses for treating prostate cancer, no single treatment has emerged, especially for managing early prostate cancer [3]. The irregularity of tumor vasculature and the treatment resistance of hypoxic cells have hampered the treatment success of many current therapies like chemotherapy and radiation [4]. The current research investigates a method to modulate tumor perfusion via the use of nanoshell mediated heating. 1.1 Treatment options for prostate cancer

Radical prostatectomy and radiotherapy are the most used treatment options for prostate cancer. A study by Fowler et al; found that each type of treatment has associated side effects. Erectile dysfunction, urinary incontinence, painful hemorrhoids, and bowel urgency are common complications after prostatectomy and radiotherapy [5]. Persistent genitourinary complications are more common after radical surgery, and bowel problems are more common after radiation [6]. After transurethral resection of the prostate (TURP), sepsis is common, and blood transfusions are often required [7]. Hematological toxicities such as leucopenia and anemia often occur after chemotherapy, and sexual dysfunction and dizziness caused by a decrease in blood pressure are other side effects of therapy with drugs [8].

1.1.1 Minimally invasive Thermal Therapy A number of minimally invasive locally ablative therapies have been proposed to eliminate the trauma and side effects associated with common methods of treating prostate cancer such as surgical intervention, radiation, and chemotherapy, particularly in the era of early diagnosis where treatment of focal disease could be a viable alternative to current “watch * anil.shetty@mdanderson.org; phone 713-563-8836; fax 713 563-5084;

Photonic Therapeutics and Diagnostics IV, edited by Nikiforos Kollias, Bernard Choi, Haishan Zeng, Reza S. Malek,Brian Jet-Fei Wong, Justus F. R. Ilgner, Kenton W. Gregory, Guillermo J. Tearney, Henry Hirschberg, Steen J. Madsen,

Proc. of SPIE Vol. 6842, 68420S, (2008) · 1605-7422/08/$18 · doi: 10.1117/12.763160

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and wait” strategies [9]. Currently, many of these techniques are gaining acceptance primarily via their continuing success and development as salvage therapies. Such methods include brachytherapy [10], cryotherapy [11] and an assortment of heating therapies, such as laser [8], microwaves [12], ultrasound [13], magnetic fluid hyperthermia [14], and, more recently, use of gold nanoshells [15]. Minimally invasive methods for local treatment offer the potential for a reduction in both trauma and total procedural cost. In addition, image guided interventions, and in particular, minimally invasive MR thermal imaging guided thermal therapies, may reduce the likelihood of the most severe side-effects (impotence, incontinence) by facilitating highly precise targeting of tissue destruction via the use of real-time feedback during treatment procedures. 1.1.2 Gold Nanoshell-mediated Heating of Tumors Nanoshells are a new class of optically tunable nanoparticles composed of a dielectric core (silica) coated with an ultrathin metallic layer (gold). By adjusting the relative core and shell thickness, nanoshells can be manufactured to absorb or scatter light at a desired wavelength across visible and NIR wavelengths. After systemic injection, the nanoshells permeate into the tumor by the extended permeability and retention (EPR) effect. Tumor tissues with high concentration of nanoshells will preferentially heat up when exposed to near infrared (NIR) range lasers. MRTI has been used to monitor the in vivo heating in murine tumors and to validate the thermal confinement, provided by the concentration of nanoshells in the tumors [15]. 1.1.3 Hyperthermia Modification of the tumor microenvironment and the physiological regulation of the pathways has been investigated to assist therapeutic agents in crossing the vascular barrier more efficiently [16]. Hyperthermia has been applied to augment nanoparticle delivery by increasing tumor blood flow and tumor microvascular pore size [17]. The pore cutoff size was increased from 100 nm to > 400 nm, improving the transport of the nanoparticles into the tumor interstitium. Hyperthermic conditions have been use to increase permeability of tumor vessels and improve transport of antibodies [18], ferritin [19] and liposomes [20]. Studies have shown that best results are seen when hyperthermia and therapeutic particles are administered together or temporally as close as possible [21]. Hyperthermia has shown to increase the uniformity of deposition of therapy, especially in the tumor center with poor vasculature [22]. 1.2 Temperature imaging

Magnetic resonance (MR) guidance of therapeutic and interventional procedures is a rapidly growing field of research with a wide array of applications in cancer diagnosis and treatment. MR image guidance offers the opportunity to perform many procedures less invasively, more safely and with a higher degree of accuracy than previously possible, due to the exquisite soft-tissue contrast offered by MRI and the ability to interactively visualize instruments and treatments. MR temperature imaging (MRTI) techniques can provide both high spatial and temperature resolution allowing very rapid ablation procedures to be performed safely and effectively. Several methods have been explored for measuring temperatures using MR imaging including measurement of the apparent diffusion constant, measurement of the spin-lattice (T1) relaxation constant, and measurement of the temperature dependent chemical shift as discussed in Lewa et al;. In particular, a number of investigators have reported successful application of proton resonance frequency (PRF) shift imaging for accurate non-invasive measurement of temperature [23, 24]. This technique is based on the slight change in PRF caused by changes in temperature [25].

1.2.1 Phase-shift MR temperature imaging

In MRI, the frequency at which protons in water molecules resonate has a temperature dependent component [26], this is the basis for the method of temperature measurement used in this work. The temperature dependance causes a ‘chemical shift’ in the magnetic field seen by the local spins [25]. The change in this chemical shift field and hence the change in temperature can be obtained from the phase difference of the complex valued images produced by spoiled gradient echo MRI.

refref

Ε 0

φ(Τ)-φ(Τ )∆Τ=Τ-Τ =αγΤ Β

(1)

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Equation 1 shows the phase of a reference image φ(Tref) is subtracted from the phase of the current image φ(T) to obtain the temperature difference between the two. B0 is the strength of the main magnetic field in Tesla, TE is the echo time for the MR pulse sequence, γ is the gyromagnetic ratio (42.58x106 Hz/Tesla), and α is the temperature sensitivity coefficient of the chemical shift (0.0097 ppm oC-1 for the tissue in this study). 1.3 Tumor Perfusion

Tumor vessels are structurally and functionally abnormal [27]. In contrast to normal vessels, tumour vasculature is highly disorganized and vessels are tortuous and dilated. Tumor vasculature is severely compromised owing to rapid growth of tumors cells and angiogenesis, which is regulated by hypoxia [28]. They have uneven diameter, excessive branching and shunts. The chaotic tumor blood flow leads to hypoxic and acidic pockets in the tumors, which tend to be treatment resistant. Many chemotherapeutic agents, which have shown effectiveness in vitro, are not effective in vivo [29]. The prime reason is due to the ineffective distribution due to the chaotic vascular distribution. 1.3.1 Dynamic Contrast-enhanced MR imaging (DCE-MRI) DCE-MRI has been used to study the microenvironment within the tumour [30]. Factors such as blood flow, microvessel permeability, vessel size, tissue oxygenation, and metabolism can be characterized with this technique [31]. Pharmacokinetic modeling of the DCE-MRI signal has been used to derive estimates of factors related to blood volume and permeability that are hallmarks of the angiogenic phenotype associated with aggressive tumors [32]. More recently, it has been used to study treatment effects on the tumor vascular parameter. For example, preclinical testing using DCE-MRI in a prostate cancer xenograft model showed decrease in volume transfer constant (ktrans) consistent with a reduction in tumor vascular permeability and perfusion [33]. Heavily T1-weighted gradient echo images are obtained with 5 seconds temporal sampling. After a baseline acquisition for several time frames, an intravenously contrast injection gives rise to an increase in signal intensity. Time-signal intensity curves, or “uptake curves,” are obtained from a region of interest (ROI) on the tumor. Uptake curves are examined by means of measuring parameters such as Time to Peak Enhancement, Maximum Rate of Uptake (Maximum Slope) and Initial Area Under the Curve (IAUC) after fitting to a sigmoidal curve. To partially account for inter-scan variability, these measures are normalized to “normal tissue” measures, e.g., muscle. or the signal intensity data from the vessel, commonly called the “arterial input function”. Semi-quantitative measures approximate “real” quantitative measures when normal values are assumed for the reference tissue. The dysfunctional tumor vasculature could lead to drug treatment ineffectiveness and tumor recurrence after radiation. Based on the known inter-relation of blood supply/oxygenation and radiation response, it has been reported that hypoxic cells in poorly perfused tumor regions are responsible for radiation therapy failure [34]. There is a large body of evidence that hypoxic cells that are radioresistant and can quickly repopulate the tumor after radiotherapy [35]. Researchers have also demonstrated that distribution of many anticancer drugs in tumor tissue is incomplete [36]. If the anticancer drugs are unable to access all the cells within a tumor, then whatever their mode of action or potency, their effectiveness will be compromised. We propose non-invasive nanoshell mediated heating under control of real time MRTI to improve drug penetration into tumor tissue and to reduce radioresistance of hypoxic tumors. In this study, we will use DCE-MRI to confirm the vascular changes in a prostate tumor xenograft caused by gold nanoshell mediated heating at different temperature ranges.

2. METHODS 2.1 Gold Nanoshells The nanoshells were generously provided by Nanospectra, Inc. (Houston, Texas) and fabricated as described [37]. Very small gold colloid (1–3 nm) are grown by using the method of Duff et al ; 1993[38]. This colloid is aged for 2 weeks at 4°C. Aminated silica particles are then added to the gold colloid suspension. Gold colloid adsorbs to the amine groups on the silica surface, resulting in a silica particle covered with gold colloid as nucleating sites. Gold–silica nanoshells are then grown by reacting HAuCl4 with the silica-colloid particles in the presence of formaldehyde. This process reduces additional gold onto the adsorbed colloid, which act as nucleation sites, causing the surface colloid to grow and coalesce with neighboring colloid, forming a complete metal shell. Particle formation was assessed by using a UV-Vis

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spectrophotometer (Hitachi U-0080D) and a DLS (Zetasizer Nano-ZS, Malvern Instruments Ltd.). Particles for this project are designed to have a 120-nm core diameter and a 14-nm-thick shell resulting in an absorption peak between 780 and 800nm. For passive targeting, an SH-PEG (Laysan Bio, Huntsville, AL) is assembled onto nanoshell surfaces by combining 5 µM SH-PEG and 1.5 x1010 particles per mL in DI H2O for 12 hrs, followed by diafiltration to remove excess SH-PEG. Resulting particles are coated with an average of 3.2x105 SH-PEG molecules. Before injection, nanoshells are suspended in 10% trehalose solution to create an iso-osmotic solution for injection. The dose given was 150µ l /20 gram mouse weight. 2.2 Mouse Model and Tumor Cell Line Female non-obese diabetic CB17-Prkd c SCID/J mice were handled in accordance with the Institutional Animal Care and Use Committee of the University of Texas M. D. Anderson Cancer Center. A human prostate cancer cell line, PC3, (provided by Dr. Andrei Volgin, The University of Texas M.D. Anderson Cancer Center) was used in the study. The lower back of the mice were debilitated with Nair cream (CVS pharmacy), and 5 × 10 6 tumors cells were inoculated sc. in a volume of 100µ l. Tumors were allowed to grow 5-6 mm in diameter before starting treatment. Prior to nanoshell injection, the animals were placed in a 38°C warming chamber in order to dilate peripheral blood vessels to enhance nanoshell circulation in the tumor. The mice were placed under anesthesia (2% isoflurane). Each mouse was injected with nanoshells suspended in 10% trehalose in either lateral tail vein at a dosing level of 8x108 nanoshells/g animal weight. Following injection, the mice were removed from anesthesia and allowed to awaken. Then, the mice were again placed in the warming chamber for another 3 hours 2.3 Nanoshell mediated heating with MR temperature and dynamic imaging 2.3.1 Treatment and Imaging parameters The treatment was planned on the next day (24 hrs post-injection). SCID mice (n=9) were anesthetized with isoflurane, and the skin over the tumor was swabbed with PEG diacrylate (M x 600, Sartomer, West Chester, PA) as an index-matching agent. The tumor sites were exposed to NIR light (808 nm, 7mm spot size, 3 min) at different laser power levels of 0.8W/cm 2 , 2W/cm 2 and 4W/cm 2 . The temperature was monitored in real time with fast SPGR sequence. Imaging was done on a MR scanner (Signa Echospeed, General Electric Medical Systems, Milwaukee, WI) equipped with high performance gradients (23 mT/m maximum amplitude and 120 T per m per sec maximum slew rate) and fast receiver hardware (bandwidth, +/- 500MHz). Mice were placed on a 3 –inch receive – only surface coil specially designed for small animal imaging. Pretreatment T1- and T2-weighted images were acquired. MRTI (Magnetic Resonance temperature imaging) was performed by a complex phase-difference technique with a fast 2-D RF-spoiled gradient-recalled echo sequence (TR/TE = 49.5 ms/ 20 ms, flip angle = 30 o , bandwidth = 9.62 kHz). Temperature maps were generated off-line, with the use of MATLAB (Mathworks, Natick, MA). 24 hours after the treatment, the mice were sacrificed by cervical dislocation, after use of CO2 chamber. The tumors were excised and cut through the middle, to match the plane of MR imaging. Half the tumor was fixed in paraffin for hematoxylin/ eosin staining and the other half was frozen for immunostaining. 2.3.2 Dynamic Contrast –enhanced MR imaging For dynamic studies, an axial 2D fast RF-spoiled gradient-echo acquisition with gadolinium-DTPA (Gd-DTPA Magnevist, Schering, 1:5 dilution in saline, dose: 2 l/gram body wt.) was used. Images were acquired every 5 seconds. Dynamic imaging with contrast was performed before the laser heating, to estimate the pre-heating tumor perfusion parameters. Post-heating, the dynamic imaging was repeated in the same slice as before to compare the perfusion changes caused by the nanoshell mediated heating. IDL software was used to estimate the vascular parameters, using a semi-quantitative method. ROIs were drawn along the tumor perimeter and also in the tumor center. Contrast uptake curves were plot for each ROI, and the initial area under the curve (IAUC) was calculated. 5-6 baseline pre-contrast images were acquired and averaged to estimate the reference intensity. The injection dephase spike in the uptake curve from an arterial ROI allowed to clearly depict the starting point for enhancement. Semi-quantitative pharmokinetic analysis was done to estimate parameters related to the vascular permeability and the tumor vascular volume.

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2.4 Silver Staining 5-micron thick paraffin sections were dewaxed and hydrated to water, then treated with the HQ Silver Enhancement Kit (Nanoprobes, Yaphank, New York) for 20 minutes. Sections were washed with water, dried, and then premounted with coverslips. The sample was examined in a fluorescence microscope equipped with a polarizing filter (immunogold silver staining - IGSS filter, Nikon Inc., Mellville, New York). 2.5 CD31 staining for vascular endothelial cells Frozen sections were stained with 5 mg/ml rat antimouse CD31 (Pharmingen) at 37° for 1 h, rinsed, and subsequently stained with 2.5 mg/ml biotinylated goat antirat antibody (Pharmingen) at 37° for 30 min. Sections were rinsed and incubated at room temperature for 30 min with streptavidin-alkaline phosphatase conjugate (Vector Labs, Burlingame, CA) at a 1:500 dilution. Then, sections were rinsed and developed with Vector Labs Alkaline Phosphatase Kit III for 15 min at room temperature in the dark. Slides were then rinsed, counterstained for 20 min with nuclear Fast Red (Vector Labs), rinsed, dried, and mounted. Two high power fields (40x) were photographed. Apoptotic cells were visualized using a commercially available TUNEL kit (Intergen Company, Purchase, NY).

3. RESULTS 3.1 MR temperature imaging Temperature images were generated in real-time using algorithms developed in MATLAB (Mathworks, Natick, MA), based on complex phase-subtraction. The temperature rise was very low (approximately 5-6˚C, fig.1a) and tumor temperature reached 36 ˚C, at laser exposure of 0.8W/cm2 for 3 minutes. At the mid-power of 2W/cm2, a temperature rise of approximately 20˚C (fig.1b) and tumor temperature of 50 ˚C was observed. At the high laser power of 4W/cm2, the temperature rose by approximately 34-35˚C (fig. 1c) and tumor temperature of 64-65 ˚C, at which level substantial tissue damage occurs.

Fig 1.The estimated MR temperature maps for a representative mouse from each group is displayed, along with a temperature

plot from a region of interest (box) in each tumor. The heating period was 300 seconds, with a 808 nm laser spot size of 7mm

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low

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e)high

Time (seconds)Pre-heating DCE Post-heating DCE

3.2 DCE-MR imaging and semi-quantitative pharmacokinetic analysis DCE images with contrast Gd-DTPA were acquired before and after each heating protocol, and the contrast uptake curves were plotted for comparison. Initially, the signal intensity was plotted against time (figure 2) to understand the dynamic response to the temperature rise in each group. It demonstrated that there was drop in contrast uptake in the low and marked decrease in the high group, whereas there was increase in the uptake curve in the mid group. In this group, there was rise in temperature of ~ 20 ˚C.

Fig 2. Dynamic contrast-enhanced images acquired before and after each heating protocol. 2a) and 2c) are the pre and post

heating images at laser power of 0.8W/cm2 and the plot (2b) shows that there is no significant difference between the 2 uptake curves. 2d) and 2f) are the pre and post heating DCE images at laser power of 2W/cm2 and the graph (2e) demonstrates that after heating the contrast uptake in the tumor increases. 2g) and 2i) are the pre and post-heating DCE images at laser power of 4W/cm2 and the graph (2h) illustrates that the high temperature reached, shuts down the tumor vasculature, resulting in decreased contrast uptake

Fig 3.Semi-quantitative pharmacokinetic analysis of the contrast uptake gives an insight into vascular parameters like initial area under the curve (IAUC) , a(0)/a(2) (related to vascular permeability) and max dI/dt ( related to tumor vascular volum e). In the low group (fig 3a & 3d), the IAUC reduced (from 0.108 to 0.045) in the post-heating group. The a(0)/a(2) reduced from 0.529 to 0.134 and the max dI/dt reduced from 0.141 to 0.037. In the mid group ( fig 3b & 3e), the IAUC increased from 0.117 to 0.179, the a(0)/a(2) increased from 0.413 to 0.811 and the max dI/dt increased from 0.110 to 0.216. In the high group ( fig 3c and 3f), the IAUC decreased from 0.590 to 0.112 and the max dI/dt decreased from 0.731 to 0.675.

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3.3 Silver staining for gold nanoshells The staining method was used to visualize the spread of the nanoshells in the tumor, after intravenous infusion. As shown in figure 3, the nanoshells permeate out of the blood vessels, but stay in close proximity to the vessels.

Fig 4. Silver staining done on the paraffin sections of the fixed tumor tissue gives an insight into the distribution of the gold

nanoshells in the tumor. It was noted that the nanoshells had a high concentration in the perivascular region, in proximity to the blood vessels.

3.4 CD31 staining for vascular endothelial cells

Fig 5. CD 31 Immunostaining for vascular endothelial cells was done to visualize the effect of heating on the tumor

vasculature. At the high temperatures reached in the 4W/cm2 group, it was observed that damage to the endothelial cells was non-apoptotic.

4. CONCLUSION

A visible and measureable change in the vascular properties was noted after nanoshell mediated heating, as detected by the DCE-MRI parameters. In the mid group (2W/cm2), the semi-quantitative parameters of IAUC, a(0)/a(2) and max dI/dt increased. These factors indicate an increase in the permeability and vascular volume of the tumor. The parameters decreased in the low (0.8Wcm2) and high (4W/cm2) groups, possibly as a result of low temperature rise and vascular damage respectively. A frequent complaint against hyperthermia is that it could actually increase radioresistance, if the uncontrolled heating caused vascular injury. But as shown in our study, the controlled nanoshell mediated heating with

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real time MRTI monitoring and follow up with DCE-MRI to rule out damage could solve this dilemma. DCE-MRI is been used increasingly, as a non-invasive measure for the prediction of treatment outcome and followup of therapy [39]. Tumors with higher mean dynamic enhancement values showed better response and were postulated to have an overall better blood and oxygen supply [40]. In addition, DCE-MRI could be used to help predict the altered drug distribution after hyperthermia. The altered pharmacokinetic parameters could guide the drug dose and schedule selection. This might also benefit the implementation of combinatorial treatments incorporating both ablation or hyperthermia with radiation and/or chemotherapy by indicating where enhanced permeability exists immediately subsequent to treatment within the tumor as well as indicating regions of inflammatory response outside the tumor. A similar study could be devised to select hyperthermia responders and treatments could be individualized. Once the timeline for the post-heating perfusion changes is observed, the radiation could be appropriately timed to potentiate the reperfusion injury to the tumor cells. These enrichment studies could substantially reduce the number of patients required to demonstrate drug effectiveness. Therefore, these techniques could be used as tools to enhance the efficacy of drugs and radiation in tumors. ACKNOWLEDGEMENTS: We would like to thank Dr. Andrei Volgin from the department of Experimental Diagnostic Imaging, the University of M.D. Anderson Cancer Center for supplying the PC3 cells. We also want to thank Charlie Kingsley and Jorge Delacerda for their assistance with the animal imaging. We want to acknowledge funding support from the NIH grant for the small animal imaging facility CA 16672. REFERENCES 1. Jemal, A., et al., Cancer statistics, 2007. CA Cancer J Clin, 2007. 57(1): p. 43-66. 2. Jemal, A., et al., Cancer statistics, 2006. CA Cancer J Clin, 2006. 56(2): p. 106-30. 3. Klimberg, I., et al., Early prostate cancer: is there a need for new treatment options? Urol Oncol, 2003. 21(2):

p. 105-16. 4. Jain, R.K., Molecular regulation of vessel maturation. Nat Med, 2003. 9(6): p. 685-93. 5. Bott, S.R., et al., Prostate cancer management: 2. An update on locally advanced and metastatic disease.

Postgrad Med J, 2003. 79(937): p. 643-5. 6. Wasson, J., et al., The treatment of localized prostate cancer: what are we doing, what do we know, and what

should we be doing? The Prostate Patient Outcome Research Team. Semin Urol, 1993. 11(1): p. 23-6. 7. Roos, N.P., et al., Mortality and reoperation after open and transurethral resection of the prostate for benign

prostatic hyperplasia. N Engl J Med, 1989. 320(17): p. 1120-4. 8. Chen, Q. and F.W. Hetzel, Laser dosimetry studies in the prostate. J Clin Laser Med Surg, 1998. 16(1): p. 9-12. 9. Eggener, S.E., et al., Focal therapy for localized prostate cancer: a critical appraisal of rationale and

modalities. J Urol, 2007. 178(6): p. 2260-7. 10. Merrick, G.S., et al., Initial analysis of Pro-Qura: a multi-institutional database of prostate brachytherapy

dosimetry. Brachytherapy, 2007. 6(1): p. 9-15. 11. Huang, W.C., et al., The anatomical and pathological characteristics of irradiated prostate cancers may

influence the oncological efficacy of salvage ablative therapies. J Urol, 2007. 177(4): p. 1324-9; quiz 1591. 12. Servadio, C., Z. Leib, and A. Lev, Diseases of prostate treated by local microwave hyperthermia. Urology,

1987. 30(2): p. 97-9. 13. Hazle, J.D., et al., MRI-guided thermal therapy of transplanted tumors in the canine prostate using a directional

transurethral ultrasound applicator. J Magn Reson Imaging, 2002. 15(4): p. 409-17. 14. Johannsen, M., et al., Magnetic fluid hyperthermia (MFH)reduces prostate cancer growth in the orthotopic

Dunning R3327 rat model. Prostate, 2005. 64(3): p. 283-92. 15. O'Neal, D.P., R.J. Stafford, and J.D. Hazle, Magnetic resonance temperature imaging of nanoshell photo-

thermal therapy. Proceedings of SPIE -- Volume 6095 Nanobiophotonics and Biomedical Applications III, 2006.

16. Monsky, W.L., et al., Augmentation of transvascular transport of macromolecules and nanoparticles in tumors using vascular endothelial growth factor. Cancer Res, 1999. 59(16): p. 4129-35.

17. Kong, G., et al., Efficacy of liposomes and hyperthermia in a human tumor xenograft model: importance of triggered drug release. Cancer Res, 2000. 60(24): p. 6950-7.

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18. Hosono, M.N., et al., Effect of hyperthermia on tumor uptake of radiolabeled anti-neural cell adhesion molecule antibody in small-cell lung cancer xenografts. J Nucl Med, 1994. 35(3): p. 504-9.

19. Fujiwara, K. and T. Watanabe, Effects of hyperthermia, radiotherapy and thermoradiotherapy on tumor microvascular permeability. Acta Pathol Jpn, 1990. 40(2): p. 79-84.

20. Gaber, M.H., et al., Thermosensitive liposomes: extravasation and release of contents in tumor microvascular networks. Int J Radiat Oncol Biol Phys, 1996. 36(5): p. 1177-87.

21. Meyer, D.E., et al., Drug targeting using thermally responsive polymers and local hyperthermia. J Control Release, 2001. 74(1-3): p. 213-24.

22. Kong, G., R.D. Braun, and M.W. Dewhirst, Characterization of the effect of hyperthermia on nanoparticle extravasation from tumor vasculature. Cancer Res, 2001. 61(7): p. 3027-32.

23. De Poorter, J., et al., Noninvasive MRI thermometry with the proton resonance frequency (PRF) method: in vivo results in human muscle. Magn Reson Med, 1995. 33(1): p. 74-81.

24. Sherar, M.D., et al., Comparison of thermal damage calculated using magnetic resonance thermometry, with magnetic resonance imaging post-treatment and histology, after interstitial microwave thermal therapy of rabbit brain. Phys Med Biol, 2000. 45(12): p. 3563-76.

25. Ishihara, Y., et al., A precise and fast temperature mapping using water proton chemical shift. Magn Reson Med, 1995. 34(6): p. 814-23.

26. Hindman, J., Proton Resonance Shift of Water in the Gas and Liquid States. The Journal of Chemical Physics, 1966. 44(12): p. 4582-4592.

27. Carmeliet, P. and R.K. Jain, Angiogenesis in cancer and other diseases. Nature, 2000. 407(6801): p. 249-57. 28. Cao, Y., et al., Impact of hypoxia and hypercapnia on calcium oxalate toxicity in renal epithelial and interstitial

cells. Urol Res, 2006. 34(4): p. 271-6. 29. Liu, P., et al., Study of non-uniform nanoparticle liposome extravasation in tumour. Int J Hyperthermia, 2005.

21(3): p. 259-70. 30. Yuh, W.T., An exciting and challenging role for the advanced contrast MR imaging. J Magn Reson Imaging,

1999. 10(3): p. 221-2. 31. Padhani, A.R., Dynamic contrast-enhanced MRI in clinical oncology: current status and future directions. J

Magn Reson Imaging, 2002. 16(4): p. 407-22. 32. Hylton, N., Dynamic contrast-enhanced magnetic resonance imaging as an imaging biomarker. J Clin Oncol,

2006. 24(20): p. 3293-8. 33. Checkley, D., et al., Use of dynamic contrast-enhanced MRI to evaluate acute treatment with ZD6474, a VEGF

signalling inhibitor, in PC-3 prostate tumours. Br J Cancer, 2003. 89(10): p. 1889-95. 34. Gray, L.H., et al., The concentration of oxygen dissolved in tissues at the time of irradiation as a factor in

radiotherapy. Br J Radiol, 1953. 26(312): p. 638-48. 35. Dean, M., T. Fojo, and S. Bates, Tumour stem cells and drug resistance. Nat Rev Cancer, 2005. 5(4): p. 275-84. 36. Minchinton, A.I. and I.F. Tannock, Drug penetration in solid tumours. Nat Rev Cancer, 2006. 6(8): p. 583-92. 37. Oldenberg, S.J. and e. al;, Infrared extinction properties of gold nanoshells. Applied Physics Letters, 1999.

75(19): p. p. 2897-2899. 38. Duff, D., A New Hydrosol of Gold Clusters. 1. Formation and Particle Size Variation. Langmuir,, 1993(9): p.

2301-2309. 39. Padhani, A.R. and J.E. Husband, Dynamic contrast-enhanced MRI studies in oncology with an emphasis on

quantification, validation and human studies. Clin Radiol, 2001. 56(8): p. 607-20. 40. Mayr, N.A., et al., Tumor perfusion studies using fast magnetic resonance imaging technique in advanced

cervical cancer: a new noninvasive predictive assay. Int J Radiat Oncol Biol Phys, 1996. 36(3): p. 623-33.

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