Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, and imposes significant morbidity and mortality in this population. The aggressive chemoradiotherapy required to treat high-risk NB results in survival of less than 50%, yet is associated with significant long-term adverse effects in survivors. Boosting efficacy and reducing morbidity are therefore key goals of treatment for affected children. We hypothesize that these may be achieved by developing strategies that both focus and limit toxic therapies to the region of the tumor. One such strategy is the use of targeted image-guided drug delivery (IGDD), which is growing in popularity in personalized therapy to simultaneously improve on-target drug deposition and assess drug pharmacodynamics in individual patients. IGDD strategies can utilize a variety of imaging modalities and methods of actively targeting pharmaceutical drugs, however in vivo imaging in combination with focused ultrasound is one of the most promising approaches already being deployed for clinical applications. Over the last two decades, IGDD using focused ultrasound with “microbubble” ultrasound contrast agents (UCAs) has been increasingly explored as a method of targeting a wide variety of diseases, including cancer. This technique, known as sonopermeation, mechanically augments vascular permeability, enabling increased penetration of drugs into target tissue. However, to date, methods of monitoring the vascular bioeffects of sonopermeation in vivo are lacking. UCAs are excellent vascular probes in contrast-enhanced ultrasound (CEUS) imaging, and are thus uniquely suited for monitoring the effects of sonopermeation in tumors. Methods : To monitor the therapeutic efficacy of sonopermeation in vivo, we developed a novel system using 2D and 3D quantitative contrast-enhanced ultrasound imaging (qCEUS). 3D tumor volume and contrast enhancement was used to evaluate changes in blood volume during sonopermeation. 2D qCEUS-derived time-intensity curves (TICs) were used to assess reperfusion rates following sonopermeation therapy. Intratumoral doxorubicin (and liposome) uptake in NB was evalauted ex vivo along with associated vascular changes. Results : In this study, we demonstrate that combining focused ultrasound therapy with UCAs can significantly enhance chemotherapeutic payload to NB in an orthotopic xenograft model, by improving delivery and tumoral uptake of long-circulating liposomal doxorubicin (L-DOX) nanoparticles. qCEUS imaging suggests that changes in flow rates are highly sensitive to sonopermeation and could be used to monitor the efficacy of treatment in vivo . Additionally, initial tumor perfusion may be a good predictor of drug uptake during sonopermeation. Following sonopermeation treatment, vascular biomarkers show increased permeability due to reduced pericyte coverage and rapid on...
Active targeted delivery of small molecule drugs is becoming increasingly important in personalized therapies, especially in cancer, brain disorders, and a wide variety of other diseases. However, effective means of spatial targeting and delivering high drug payloads in vivo are still lacking. Focused ultrasound combined with superheated phase-shift nanodroplets, which vaporize into microbubbles using heat and sound, are rapidly becoming a popular strategy for targeted drug delivery. Focused ultrasound can target deep tissue with excellent spatial precision and without using ionizing energy, thus can activate nanodroplets in circulation. One of the main limitations of this technology has been poor drug loading in the droplet core or the shell material. To address this need, we have developed a strategy to combine low-boiling point decafluorabutane and octafluoropropane (DFB and OFP) nanodroplets with drug-loaded liposomes, creating phase-changeable droplet-liposome clusters (PDLCs). We demonstrate a facile method of assembling submicron PDLCs with high drug-loading capacity on the droplet surface. Furthermore, we demonstrate that chemical tethering of liposomes in PDLCs enables a rapid release of their encapsulated cargo upon acoustic activation (>60% using OFP-based PDLCs). Rapid uncaging of small molecule drugs would make them immediately bioavailable in target tissue or promote better penetration in local tissue following intravascular release. PDLCs developed in this study can be used to deliver a wide variety of liposome-encapsulated therapeutics or imaging agents for multi-modal imaging applications. We also outline a strategy to deliver a surrogate encapsulated drug, fluorescein, to tumors in vivo using focused ultrasound energy and PDLCs.
Neuroblastoma (NB) is a pediatric malignancy that accounts for 15% of cancer-related childhood mortality. High-risk NB requires an aggressive chemoradiotherapy regimen that causes significant off-target toxicity. Despite this invasive treatment, many patients either relapse or do not respond adequately. Recent studies suggest that improving tumor perfusion can enhance drug accumulation and distribution within the tumor tissue, potentially augmenting treatment effects without inflicting systemic toxicity. Accordingly, methods that transiently increase tumor perfusion prior to treatment may help combat this disease. Here, we show the use of gene therapy to confer inducible nitric oxide synthase (iNOS) expression solely in the tumor space, using focused ultrasound targeting. NOS catalyzes the reaction that generates nitric oxide (NO), a potent endogenous vasodilator. This study reports the development of a targeted non-viral image-guided platform to deliver iNOS-expressing plasmid DNA (pDNA) to vascular endothelial cells encasing tumor blood vessels. Following transfection, longitudinal quantitative contrast-enhanced ultrasound (qCEUS) imaging revealed an increase in tumor perfusion over 72 h, attributed to elevated intratumoral iNOS expression. Methods : To construct a gene delivery vector, cationic ultrasound-responsive agents (known as “microbubbles”) were employed to carry pDNA in circulation and transfect tumor vascular endothelial cells in vivo using focused ultrasound (FUS) energy. This was followed by liposomal doxorubicin (L-DOX) treatment. The post-transfection tumor response was monitored longitudinally using qCEUS imaging to determine relative changes in blood volumes and perfusion rates. After therapy, ex vivo analysis of tumors was performed to examine the bioeffects associated with iNOS expression. Results : By combining FUS therapy with cationic ultrasound contrast agents (UCAs), we achieved selective intratumoral transfection of pDNA encoding the iNOS enzyme. While transitory, the degree of expression was sufficient to induce significant increases in tumoral perfusion, to appreciably enhance the chemotherapeutic payload and to extend survival time in an orthotopic xenograft model. Conclusion : We have demonstrated the ability of a novel targeted non-viral gene therapy strategy to enhance tumor perfusion and improve L-DOX delivery to NB xenografts. While our results demonstrate that transiently increasing tumor perfusion improves liposome-encapsulated chemotherapeutic uptake and distribution, we expect that our iNOS gene delivery paradigm can also significantly improve radio and immunotherapies by increasing the delivery of radiosensitizers and immunomodulators, potentially improving upon current NB treatment without concomitant adverse effects. Our findings further suggest that qCEUS imaging can effectively monitor changes in tumor perfusion ...
Background: Neuroblastoma (NB) is the second most common malignancy diagnosed in infants, accounting for 15% of pediatric tumor deaths. Half of children with NB receive an intensive regimen including high-dose chemotherapy with 50% survival, resulting in acute and long term toxicities. One of the challenges of chemotherapy is irregular tumor vasculature. Thus, increasing targeted drug delivery without increasing drug dosage can result in enhanced drug efficacy and improved patient outcomes. We have shown that sonoporation (focused ultrasound-guided gas-filled microbubbles) increases high dose liposomal doxorubicin (L-DOX) uptake in NB xenografts by increasing tumor perfusion. However, these studies used polydisperse microbubbles (PMB), which were developed for imaging purposes. We hypothesized that MB size restriction would control their response to ultrasound pressure, yielding a higher L-DOX payload despite using using lower L-DOX dosages. Methods: Nude mice received 1x10^6 NGP cells (NB cells) intrarenally. When tumors reached 1 gram, NB xenografts received an intravenous polydisperse (PMB) or 4-5uM (SIMB) microbubble infusion with or without 1mg/kg liposomal doxorubicin (L-DOX) under focused ultrasound. Tumors were measured over 7 days with calipers, others sacrificed 24 hours after treatment for histology and immunohistochemistry. We assessed endomucin and isolectin-B4 (endothelium), Zona occludens-1 (ZO-1) (tight junction), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, apoptosis). Results: Tumors receiving low dose L-DOX alone or PMB sonoporation with L-DOX were not different from untreated controls after 7 days. SIMB alone resulted in a slower tumor growth than control tumors (20 vs 100% p<0.05); L-DOX coupled with SIMB resulted in further tumor growth restriction 7 days after treatment (0 vs 100% p<0.05). SIMB increased tumor apoptosis (TUNEL staining) in the absence of L-DOX compared to controls (7 vs 58% of area p=0.003) as well as in the presence of L-DOX (7 vs 78% of area p<0.001). PMBs did not change TUNEL levels regardless of L-DOX. Tumor vascular lumens (widest axis within the endothelial marker endomucin) confirmed PMB duplicates lumen diameter compared to controls (p<0.05), and revealed SIMB triplicates lumen diameter (p<0.01) regardless of L-DOX. SIMB resulted in loss of tight junction protein ZO-1 both in vasculature and tumor cells and widespread L-DOX uptake. Together, our data shows SIMB sonoporation increases tumor blood volume and vascular permeability leading to higher chemotherapy uptake and apoptosis. Conclusions: Together, our data shows SIMB sonoporation enables chemotherapy uptake in poorly perfused NB xenografts by increasing perfusion and permeability, potentiating apoptotic effects. SIMB sonoporation could reduce acute and long term toxicities. Citation Format: Sonia L. Hernandez, Rachael Sundland, Donia Ballan, Aditi Bellary, Jessica J. Kandel, Jameel Feshitan, Shashank Sirsi. Sonoporated size selected microbubblesand liposomal doxorubicinadditively induce apoptosis in neuroblastoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 376.
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