R Gupta

WE-AB-BRA-01: Biodistribution of Paclitaxel-Loaded Nanodroplets for Prostate Cancer Management Using Ultrasound-Mediated Drug Delivery Under MRGuidance

PURPOSE To study the biodistribution of paclitaxel-loaded nanodroplets in vivo in order to evaluate the efficacy of focused ultrasound (FUS)-mediated drug delivery under MR-guidance to prostate tumor. METHODS Poly {ethylene oxide}-co-poly {D, L-lactide} (PDLA) nanodroplets loaded with fluorescently labeled-paclitaxel (F-PTX) were synthesized using solid dispersion technique. Human prostate cancer, LNCaP cells were implanted orthotopically in prostates of male nude mice. Tumor-bearing mice (n=3) were injected with 0.1% F-PTX, 2% PDLA nanodroplets using tail vein. At chosen time points (30min; 2h; 4h; 6h; 12h; 24h) animals were anesthetized, blood was collected by eye bleed, animals were sacrificed, tumor and vital organs (liver, spleen, lung and heart) were excised, cut and weighted. Then the organ was homogenized, incubated in lysis buffer and centrifuged. The lysates were read using fluorescence spectrophotometer (Excitation- 496nm, Emission- 524nm). Fluorescence readings were compared with values from the standard calibration curve. Tumor-bearing mice (n=3) were also injected with 0.1% F-PTX solution (fluorescent drug in free form). RESULTS Quantitative analysis showed 20% of the injected drug-loaded nanodroplets in the tumor, 30 min after systemic injection; which increased to 30% after 2h; 35% after 4h; then decreased to 27% after 6h; 11% after 12h and 5% after 24h. When drug in free form was injected, drug accumulation in the tumor was 7% within 30mnt; 8% within 2h; 10% within 4h; 9% within 6h, 3% within 12h and 2% within 24h. Liver showed major drug accumulation for drug-loaded nanodroplets while heart and lung showed less. For drug in free form, liver and spleen showed maximum drug concentrations while heart showed concentrations similar to that in tumor. CONCLUSION Present work indicates that the optimum time for applying focused ultrasound is after 4h of systemically injecting drug-loaded nanodroplets. This would increase treatment efficacy and provide preclinical data for FUS-mediated drug delivery under MR-guidance.

WE-AB-BRA-02: Study of the Therapeutic Effects of Pulsed Focused Ultrasound Combined with Encapsulated Chemotherapeutic Agents for Prostate Cancer in Vivo

PURPOSE To investigate the improvement of prostate cancer inhibition by combining pulsed focused ultrasound (pFUS) exposures and chemotherapeutic agents encapsulated nanodroplets under MR guidance for prostate cancer therapy. METHODS Prostate cancer (LNCaP) cells were implanted orthotopically. First we developed nanodroplets encapsulated with chemotherapeutic agents for both paclitaxel (PTX) and docetaxel (DTX). Secondly, tumor-bearing mice were randomly divided into 5 groups (n=5). Group 1 animals were treated with an i.v injection of DTX-encapsulated nanodroplets (DTX-ND) + pFUS. Group 2 were treated with pFUS alone. Group 3 were injected (i.v) with DTX-ND alone, Group 4 received free DTX and Group 5 was used as control. Ultrasound treatment parameters were 1MHz, 25W acoustic power, 10% duty cycle and 60 seconds for each sonication. After treatment, animals were allowed to survive for 4 weeks. Tumor volumes were measured on MRI. Third, we repeated the experiment with PTX-ND. Finally we performed study on biodistribution of PTX-ND for prostate cancer and the treatment effects on tumor growth delay are being evaluated. RESULTS With DTX-ND, significant tumor growth delay was observed in Group 1 with p=0.039. There was no significant tumor growth delay observed for Group 2 (p=0.477), Group 3 (p=0.209) and Group 4 (p=0.476). The results are consistent with earlier PTX-ND studies in which significant tumor growth delay was observed in Group 1 with p=0.004. There was no significant tumor growth delay observed for Group 2 (p=0.285), Group 3 (p=0.452) and Group 4 (p=0.158). The peak of drug update in tumor appeared at 4h after injection in the biodistribution study. CONCLUSION Our results showed a great potential of targeted nanodroplets for prostate cancer therapy, which could be activated by pFUS. Our study also suggested that the optimal timing for applying pFUS is 4h after i.v injection of PTX-ND and the treatment effect is being evaluated.

SU-F-T-674: In Vitro Study of 5-Aminolevulinic Acid-Mediated Photo Dynamic Therapy in Human Cancer Cell Lines

PURPOSE Photodynamic therapy (PTD) is a promising cancer treatment modality. 5-sminolevulinic acid (ALA) is a clinically approved photosensitizer. Here we studied the effect of 5-ALA administration with irradiation on several cell lines in vitro. METHODS Human head and neck (FaDu), lung (A549) and prostate (LNCaP) cancer cells (104/well) were seeded overnight in 96-well plates (Figure 1). 5-ALA at a range from 0.1 to 30.0mg/ml was added to confluent cells 3h before irradiation in 100ul of culture medium. 15MV photon beams from a Siemens Artiste linear accelerator were used to deliver 2 Gy dose in one fraction to the cells. Cell viability was evaluated by WST1 assay. The development of orange color was measured 3h after the addition of WST-1 reagent at 450nm on an Envision Multilabel Reader (Figure 2) and directly correlated to cell number. Control, untreated cells were incubated without 5-ALA. The experiment was performed twice for each cell line. RESULTS The cell viability rates for the head and neck cancer line are shown in Figure 3. FaDu cell viability was reduced significantly to 36.5% (5-ALA) and 18.1% (5-ALA + RT) only at the highest concentration of 5-ALA, 30mg/ml. This effect was observed in neither A549, nor LNCaP cell line. No toxicity was detected at lower 5-ALA concentrations. CONCLUSION Application of 5-ALA and subsequent PDT was found to be cytotoxic at the highest dose of the photosensitizer used in the FaDu head and neck cell line, and their effect was synergistic. Further efforts are necessary to study the potential therapeutic effects of 5-ALA PTD in vitro and in vivo. Our results suggest 5-ALA may improve the efficacy of radiotherapy by acting as a radiomediator in head and neck cancer.

SU‐F‐J‐225: Histology Study of MR Guided Pulsed Focused Ultrasound On Treatment of Prostate Cancer in Vivo

PURPOSE Our previous study demonstrated significant tumor growth delay in the mice treated with pulsed high intensity focused ultrasound (pHIFU). The purpose of this study is to understand the cell killing mechanisms of pHIFU. METHODS Prostate cancer cells (LNCaP), were grown orthotopically in 17 nude mice. Tumor-bearing mice were treated using pHIFU with an acoustic power of 25W, pulse width 100msec and 300 pulses in one sonication under MR guidance. Mutiple sonications were used to cover the whole tumor volume. The temperature (less than 40 degree centigrade in the focal spot) was monitored using MR thermometry. Animals were euthanized at pre-determined time points (n=2) after treatment: 0 hours; 6 hrs; 24 hrs; 48 hrs; 4 days and 7 days. Two tumorbearing mice were used as control. Three tumor-bearing mice were treated with radiation (RT, 2 Gy) using 6 MV photon beams. RT treated mice were euthanized at 0 hr, 6 hrs and 24 hrs. The tumors were processed for immunohistochemical (IHC) staining for PARP (a surrogate of apoptosis). A multispectral imaging analysis system was used to quantify the expression of PARP staining. Cell apoptosis was calculated based on the PARP expression level using the DAB analysis software. RESULTS Our data showed that PARP related apoptosis peaked at 48 hrs and 7 days in pHIFU treated mice, which is comparable to that for the RT group at 24 hrs. The preliminary results from this study were consistent with our previous study on tumor growth delay using pHIFU. CONCLUSION Our results demonstrated that non-thermal pHIFU increased apoptotic tumor cell death through the PARP related pathway. MR guided pHIFU may have a great potential as a safe, noninvasive treatment modality for cancer therapy. This treatment modality may synergize with PARP inhibitors to achieve better therapeutic result.

SU-F-T-610: Comparison of Output Factors for Small Radiation Fields Used in SBRT Treatment

PURPOSE In order to fundamentally understand our previous dose verification results between measurements and calculations from treatment planning system (TPS) for SBRT plans for different sized targets, the goal of the present work was to compare output factors for small fields measured using EDR2 films with TPS and Monet Carlo (MC) simulations. METHODS 6MV beam was delivered to EDR2 films for each of the following field sizes; 1×1 cm2 , 1.5×1.5 cm2 , 2×2 cm2 , 3×3 cm2 , 4×4 cm2 , 5×5 cm2 and 10×10 cm2 . The films were developed in a film processer, then scanned with a Vidar VXR-16 scanner and analyzed using RIT113 version 6.1. A standard calibration curve was obtained with the 6MV beam and was used to get absolute dose for measured field sizes. Similar plans for all fields sizes mentioned above were generated using Eclipse with the Analytical Anisotropic Algorithm. Similarly, MC simulations were carried out using the MCSIM, an in-house MC code for different field sizes. Output factors normalized to 10×10 cm2 reference field were calculated for different field sizes in all the three cases and compared. RESULTS For field sizes ranging from 1×1 cm2 to 2×2 cm2 , the differences in output factors between measurements (films), TPS and MC simulations were within 0.22%. For field sizes ranging from 3×3cm2 to 5×5cm2 , differences in output factors were within 0.10%. CONCLUSION No clinically significant difference was obtained in output factors for different field sizes acquired from films, TPS and MC simulations. Our results showed that the output factors are predicted accurately from TPS when compared to the actual measurements and superior dose calculation Monte Carlo method. This study would help us in understanding our previously obtained dose verification results for small fields used in the SBRT treatment.

SU-E-J-01: MR Guided Pulsed Focused Ultrasound Mediated Targeted Chemotherapeutic-Agent Delivery in Management of Prostate Cancer

Purpose: The purpose of this study is to investigate an innovative approach to prostate cancer therapy using nanodroplet-encapsulated drugs combined with pulsed high intensity focused ultrasound (pFUS) treatment. Pulsed FUS treatment improves localized drug release from the carrier and enhances intracellular uptake, which ensures temporal and spatial control of local drug delivery while reducing systemic toxicity from the drugs. Methods: LNCaP cells were implanted into the prostates of nude mice. Tumor growth was monitored using MRI. The tumor—bearing mice were randomly divided into 5 groups (n=5 for each group). Group 1, animals were treated with an IV injection of docetaxel (DTX)-encapsulated nanodroplets (ND-DTX) + pFUS. Animals in Group 2 were treated with pFUS alone. Animals in Group 3 were injected (IV) with DTX-encapsulated nanodroplets alone, Group 4 received free DTX alone and Group 5 was used as a control group. After treatment, animals were allowed to survive for 3 weeks. Tumor growth delay was monitored by MRI (resolution: <0.2mm). The formulation of the DTX-encapsulated nanodroplets was: DTX dose 20 mg/kg, Nanoemulsion composition 0.5% PTX, 1% perfluorocarbon and 2% stabilizing block copolymer. Ultrasound treatment parameters were 1MHz, 25W acoustic power, 10% duty cycle and 60 sec for each sonication. Results: Our results showed that Group 1, in which animals were treated with an IV injection of ND-DTX + pFUS, exhibited the most tumor growth control (50%–30%) among all groups. All other groups showed similar tumor growth to that of the control group after the treatment (within statistical uncertainties). Conclusion: Our preliminary results showed a great potential for prostate cancer therapy using targeted DTX+ nanodroplets, which could be activated by pFUS. Further animal studies are warranted to confirm the results. The enhancement effect of pFUS on targeted drug delivery needs to be investigated quantitatively.

SU-E-T-323: Dosimetric Evaluation of Small Fields for SBRT Treatment

Purpose: Stereotactic body radiation therapy (SBRT) is commonly employed to treat small targets for effective tumor control with radiation beams of small field sizes. The goal of this work was to evaluate dosimetrically a treatment planning system (TPS) by comparing the calculated dose for SBRT treatment with ion-chamber measurements. Methods: 3D images of a solid-water phantom with a pinpoint ion-chamber (0.015cm3) inside were acquired with a CT scanner. Active volume of the ion-chamber was delineated on CT images. Targets with a diameter of 1.5cm, 2cm, 3cm, 4cm and 5cm were drawn around the chamber. 3DCRT plans were generated for each target size with centrally opened 6MV beams and off-axis beams by changing the isocenter location, respectively, using a TPS with the Analytical Anisotropic Algorithm. A 21iX linear accelerator was employed for plan delivery. The measured and calculated doses were compared. To evaluate the dose calculations in heterogeneity for small fields SBRT treatment, similar plans were also generated and delivered on a heterogeneous thoracic phantom for 5 different size targets in the lung. Results: Dose comparisons between measurements and calculations showed 5.2%, 1.88%, 1.34%, 1.01% and 0.85% difference for SBRT plans with small central axis beams and 0.96%, 0.15%, 0.58%, 0.22% and 0.77% difference for plans with off-axis beams for five different size targets. For the thoracic phantom, the differences on dose between measurements and calculations are bigger, which are 8%, 5.9%, 4.5%, 3.9% and 4.5%, respectively. Conclusion: Dose verification for small fields used in the SBRT treatment has been performed based on ion-chamber measurements in both homogenous and heterogeneous phantoms. More than a 5% difference has been observed in the heterogeneous phantom, especially for very small fields. To meet the ICRU recommendation on a dose difference of no more than 5%, some corrections on the commissioning parameters of the TPS are needed.

SU-E-U-04: Evaluation of Normal Tissue Toxicity of Drug-Loaded Nanodroplets Used for Treating Prostate Cancer with MR-Guided Focused-Ultrasound

Purpose: Our prior studies have showed significant prostate tumor growth-delay when pulsed focused ultrasound (pFUS) is applied under MR-guidance in combination with docetaxel-encapsulated nanodroplets (DTX-ND). The purpose of the present work was to investigate normal tissue toxicity of DTX-ND in order to study true efficacy of a novel prostate cancer treatment approach combining DTX-ND injections and pFUS exposures under MR guidance. Methods: Poly {ethylene oxide}-co-poly {D, L-lactide} (PDLA) nanodroplets loaded with docetaxel were synthesized in our lab using solid dispersion technique with 0.5% docetaxel, 2% perfluorocarbon and 2% PDLA. The mean diameter of the nanodroplets was 220 ± 30nm. Human prostate cancer, LNCaP cells were implanted orthotopically in prostates of male nude mice. Tumor growth was monitored using MRI. Tumor–bearing mice were randomly divided into 5 groups. Group 1 animals were treated with DTX-ND and pFUS. Ultrasound treatment parameters were 1MHz, 25W acoustic power, 10% duty cycle and 60 seconds for each sonication. Group 2 mice were treated with pFUS. Group 3 mice were injected with DTX-ND. Group 4 received free docetaxel and Group 5 mice were used as control (no treatment). Mice weights were monitored for toxicity. Results: Docetaxel-loaded nanodroplets showed no normal tissue toxicity as average mice weights (27.12 ±0.75 g) from group 1 were statistically, no different when compared with average mice weights (24.39 ±0.62 g) from control group (p >0.05). Also, average mice weights from group2 (24.95 ±1.2 g), group3 (26.16 ± 0.66 g) and group4 (27.10 ±0.65 g) showed no significant difference in weights when compared to control group (p >0.05). Conclusion: This study demonstrates that our formulation of DTX-ND shows no normal tissue toxicity in mice. These findings help us in evaluating the true efficacy of our hypothesized prostate cancer treatment strategy using focused-ultrasound activated drug-delivery under MR guidance without incurring additional toxicity.

TU‐CD‐210‐05: A Drug Delivery Technique Employing Pulsed Focused Ultrasound for Prostate Cancer Therapy

Purpose: A number of factors have been identified in the microenvironment of solid tumors that are responsible for non-uniform and insufficient levels of anti-cancer agents being delivered to tumor cells. The purpose of our study is to investigate the improvement of prostate cancer treatment by a novel targeted drug delivery technique combining pulsed high-intensity focused ultrasound (pHIFU) exposures and PTX-encapsulated nanodroplets under MR image guidance. Methods: Human prostate cancer cells were implanted into the prostates of mice. Three weeks after the implantation tumor growth was monitored using 1.5 T MRI. The tumor–bearing mice were randomly divided into 5 groups (n=3 for each group): Group 1, animals were treated with paclitaxel (PTX)-encapsulated nanodroplets + pHIFU. The formulation of the PTX-encapsulated nanodroplets was: PTX dose 20 mg/kg, Nanoemulsion composition 0.5 percent PTX, 1 percent perfluorocarbon and 2 percent stabilizing block copolymer. Ultrasound treatment parameters were 1MHz, 25W acoustic power, 10% duty cycle and 60 sec for each sonication point. Animals in group 2 were treated with pHIFU alone. Animals in group 3 were injected with PTX-encapsulated nanodroplets alone, Group 4 received pFUS+empty nanodroplets and group 5 was used as control. After treatment, animals were allowed to survive for 3 weeks. Results: Compared with the control group tumor growth delay was observed with P=0.001 in group 1, p=0.292 in group2 and P=0.158 in group 4, respectively. There was no significant difference in the tumor volume between the control group and the group received PTX-encapsulated nanodroplets alone (group 3). Conclusion: Our preliminary results showed a great potential for prostate cancer therapy using targeted PTX+ nanodroplets, which could be activated by pHIFU. More experimental studies are warranted to confirm the results and for pHIFU parameter optimization. The role of pHIFU in targeted drug delivery needs to be investigated.

TH-A-BRF-06: Optimal Timing for Ultrasound-Activated Drug Delivery for Treatment of Prostate Cancer Under MR Guidance.

PURPOSE To determine optimal timing for applying tumor targeted focused ultrasound (FUS) under MR guidance to release drugs from nanodroplets (ultrasound responsive, drug delivery vehicles) for effective treatment of prostate cancer. METHODS Poly lactide (PDLA) nanodroplets loaded with model drug {fluorescently labeled paclitaxel (FPTX)} were prepared using solid dispersion technique. Human prostate cancer, LNCaP cells were implanted orthotopically in prostates of male nude mice. Tumor bearing mice (n=3) were injected with 125 microlitre of 0.1% FPTX, 2% PDLA nanodroplets via tail-vein. At 30min, 2h, 6h and 24h animals were anesthetized, blood was collected by eye bleed, then animals were sacrificed, tumor and organs were excised and weighted. One cut portion of the organ was homogenized, incubated in lysis buffer and centrifuged. Lysates were read using fluorescence spectrophotometer. Other portion was cut into sections for qualitative analysis using fluorescence microscope. Mice (n=3) were also injected with 0.1% FPTX solution (non encapsulated drug form) and mice (n=3) with no injections were used as controls. RESULTS Drug-loaded nanodroplets shows more drug accumulation in tumor with maximum concentration of one third of injected dose after 2h of injection when compared with maximum concentration of less than one tenth of injected dose after 2h of injection for free drug. The time range 2h to 6h is considered optimum for applying FUS to activate drug release from nanodroplets, when maximum drug concentration is seen in tumor while low concentration is found in blood thus minimizing potential damage to blood vessels. Microscopy studies show consistency with quantitative data. CONCLUSION Our prior in vivo studies have showed significant tumor growth delay when FUS is applied under MR imaging in combination with docetaxel loaded nanodroplets. Present work with model drug loaded nanodroplets indicates the potential for increasing prostate cancer treatment efficacy and providing preclinical data for using FUS-mediated drug delivery under MR guidance.