fabricating-labeling-microbubbles-with-fluorescent-radioactive

Fabricating and Labeling Microbubbles with Fluorescent and Radioactive Tracers

Microbubbles are lipid-shelled, gas-filled particles that have evolved from vascular ultrasound contrast agents into revolutionary cancer therapy ...

University Health Network

University of Toronto

Ultrasound (US) is one of the most commonly used clinical imaging techniques. However, the use of US and US-based intravenous agents extends far beyond imaging. In particular, there has been a surge in the fabrication of multimodality US contrast agents and theranostic US agents for cancer imaging and therapy. The unique interaction of US waves with microscale and nanoscale agents has attracted much attention in the development of contrast agents and drug-delivery vehicles. The dimensions of the agent not only dictate how it behaves in vivo, but also how it interacts with US for imaging and drug delivery. Furthermore, these agents are also unique due to their ability to convert from the nanoscale to the microscale and vice versa, having imaging and therapeutic utility in both dimensions. Here, we review multimodality and multifunctional US-based agents, according to their size, and also highlight recent developments in size conversion US agents. WIREs Nanomed Nanobiotechnol 2016, 8:796-813. doi: 10.1002/wnan.1398 For further resources related to this article, please visit the WIREs website.

Multimodal micro, nano, and size conversion ultrasound agents for imaging and therapy.

Lipoprotein mimetic nanostructures, which consist of an amphiphilic lipid shell, a hydrophobic core, and an apolipoprotein mimetic peptide, serve as a versatile platform for the design of drug delivery vehicles as well as the investigation of supramolecular assemblies. Porphyrin incorporation into biomimetic lipoproteins allows one to take advantage of the inherent multimodal photophysical properties of porphyrins, yielding various fluorescence, photoacoustic, and photodynamic agents. To facilitate their incorporation into a lipoprotein structure, porphyrins have been conjugated through a variety of strategies. However, the effects of the conjugate structure on the associated nanoparticle's phototherapeutic properties warrants further investigation. Herein, we systematically investigated the effects of two widely utilized porphyrin conjugates, oleylamide and lipid, on biophotonic properties of their resultant porphyrin-lipoprotein nanoparticles in vitro and in vivo. Specifically, we demonstrated that incorporation of the porphyrin moiety as an oleylamide conjugate leads to a highly stable J-aggregate with strong photoacoustic contrast, while incorporation as an ampiphilic lipid moiety into the lipid shell yields an effective fluorescent and photodynamic agent. The current study proposes a rational design strategy for next-generation lipoprotein-based phototheranostic agents, for which nanoassembly-driven biophotonic and therapeutic properties can be tailored through the specific selection of porphyrin conjugate structures.

Tailoring Porphyrin Conjugation for Nanoassembly-Driven Phototheranostic Properties.

Sonodynamic therapy (SDT) is a promising therapeutic platform for minimally invasive cancer treatment in which acoustically susceptible drug agents, sonosensitizers, are activated by deep-tissue-penetrating low frequency ultrasound. Despite growing research in recent years, the field has yet to clearly elucidate broadly applicable mechanisms by which acoustic cavitation triggers sonosensitizer therapeutic activity, creating difficulties in achieving substantial and translatable therapeutic efficacy. In this review, we will critically analyze the proposed mechanisms underlying SDT and overview how nanomedicines can complement and extend these mechanisms to deliver more efficacious SDT. In doing so, we aim to highlight potential avenues toward viable implementation of SDT as a cancer therapy.

Activating Drugs with Sound: Mechanisms Behind Sonodynamic Therapy and the Role of Nanomedicine.

Porphyrin aggregates have attractive photophysical properties for phototherapy and optical imaging, including quenched photosensitization, efficient photothermal conversion, and unique absorption spectra. Although hydrophobic porphyrin photosensitizers have long been encapsulated into liposomes for drug delivery, little is known about the membrane properties of liposomes with large amphiphilic porphyrin compositions. In this paper, a porphyrin-lipid conjugate was incorporated into liposomes formed of saturated or unsaturated lipids to study the membrane composition-dependent formation of highly ordered porphyrin J-aggregates and disordered aggregates. Porphyrin-lipid readily phase-separates in saturated membranes, forming J-aggregates that are destabilized during the ripple phase below the main thermal transition. Porphyrin-lipid J-aggregates are photostable with a photothermal efficiency of 54 ± 6%, comparable to gold. Even at high porphyrin-lipid compositions, porphyrin J-aggregates coexist with a minority population of disordered aggregates, which are photodynamically active despite being fluorescently quenched. For photothermal applications, liposome formulations that encourage porphyrin-lipid phase separation should be explored for maximum J-aggregation.

Photophysics of J-Aggregating Porphyrin-Lipid Photosensitizers in Liposomes: Impact of Lipid Saturation.

Limited tumor nanoparticle accumulation remains one of the main challenges in cancer nanomedicine. Here, we demonstrate that subtherapeutic photodynamic priming (PDP) enhances the accumulation of nanoparticles in subcutaneous murine prostate tumors ∼3-5-times without inducing cell death, vascular destruction, or tumor growth delay. We also found that PDP resulted in an ∼2-times decrease in tumor collagen content as well as a significant reduction of extracellular matrix density in the subendothelial zone. Enhanced nanoparticle accumulation combined with the reduced extravascular barriers improved therapeutic efficacy in the absence of off-target toxicity, wherein 5 mg/kg of Doxil with PDP was equally effective in delaying tumor growth as 15 mg/kg of Doxil. Overall, this study demonstrates the potential of PDP to enhance tumor nanomedicine accumulation and alleviate tumor desmoplasia without causing cell death or vascular destruction, highlighting the utility of PDP as a minimally invasive priming strategy that can improve therapeutic outcomes in desmoplastic tumors.

Subtherapeutic Photodynamic Treatment Facilitates Tumor Nanomedicine Delivery and Overcomes Desmoplasia.

Theranostic nanoparticles aim to integrate diagnostic imaging and therapy to facilitate image-guided treatment protocols. Herein, we present a theranostic nanotexaphyrin for prostate-specific membrane antigen (PSMA)-targeted radionuclide imaging and focal photodynamic therapy (PDT) accomplished through the chelation of metal isotopes (In, Lu). To realize nanotexaphyrin's theranostic properties, we developed a rapid and robust In/Lu-nanotexaphyrin radiolabeling method using a microfluidic system that achieved a high radiochemical yield (>90%). The optimized metalated nanotexaphyrin displayed excellent chemical, photo, and colloidal stabilities, potent singlet oxygen generation, and favorable plasma circulation half-life  ( = 6.6 h). Biodistribution, including tumor accumulation, was characterized by NIR fluorescence, SPECT/CT imaging, and γ counting. Inclusion of the PSMA-targeting ligand enabled the preferential accumulation of In/Lu-nanotexaphyrin in PSMA-positive (PSMA+) prostate tumors (3.0 ± 0.3%ID/g) at 48 h with tumor vs prostate in a 2.7:1 ratio. In combination with light irradiation, the PSMA-targeting nanotexaphyrin showed a potent PDT effect and successfully inhibited PSMA+ tumor growth in a subcutaneous xenograft model. To the best of our knowledge, this study is the first demonstration of the inherent metal chelation-driven theranostic capabilities of texaphyrin nanoparticles, which, in combination with PSMA targeting, enabled prostate cancer imaging and therapy.

Targeted Theranostic In/Lu-Nanotexaphyrin for SPECT Imaging and Photodynamic Therapy.

Despite the substantial burden posed by osteoarthritis (OA) globally, difficult challenges remain in achieving early OA diagnosis and adopting effective disease-modifying treatments. In this study, we use a biomolecular approach to address these limitations by creating an inherently theranostic molecular beacon whose imaging and therapeutic capabilities are activated by early pathological changes in OA. This platform comprised (1) a peptide linker substrate for metalloproteinase-13 (MMP-13), a pathological protease upregulated in OA, which was conjugated to (2) a porphyrin moiety with inherent multimodal imaging, photodynamic therapy, and drug delivery capabilities, and (3) a quencher that silences the porphyrin's endogenous fluorescence and photoreactivity when the beacon is intact. In diseased OA tissue with upregulated MMP-13 expression, this porphyrin molecular beacon (PPB) was expected to undergo sequence-specific cleavage, yielding porphyrin fragments with restored fluorescence and photoreactivity that could, respectively, be used as a readout of MMP-13 activity within the joint for early OA imaging and disease-targeted photodynamic therapy. This study focused on the synthesis and characterization of PPB, followed by a proof-of-concept evaluation of its OA imaging and drug delivery potential. In solution, PPB demonstrated 90% photoactivity quenching in its intact form and robust MMP-13 activation, yielding a 13-fold increase in fluorescence post-cleavage. In vitro, PPB was readily uptaken and activated in an MMP-13 cell expression-dependent manner in primary OA synoviocytes without exuding significant cytotoxicity. This translated into effective intra-articular cartilage (to a 50 μm depth) and synovial uptake and activation of PPB in a destabilization of the medial meniscus OA mouse model, yielding strong fluorescence contrast (7-fold higher signal than background) at the diseased joint site. These results provide the foundation for further exploration of porphyrin molecular beacons for image-guided OA disease stratification, effective articular delivery of disease-modify agents, and OA photodynamic therapy.

Protease-Activatable Porphyrin Molecular Beacon for Osteoarthritis Management.

Microbubble-enabled focused ultrasound (MB-FUS) has revolutionized nano and molecular drug delivery capabilities. Yet, the absence of longitudinal, systematic, quantitative studies of microbubble shell pharmacokinetics hinders progress within the MB-FUS field. Microbubble radiolabeling challenges contribute to this void. This barrier is overcome by developing a one-pot, purification-free copper chelation protocol able to stably radiolabel diverse porphyrin-lipid-containing Definity® analogues (pDefs) with >95% efficiency while maintaining microbubble physicochemical properties. Five tri-modal (ultrasound-, positron emission tomography (PET)-, and fluorescent-active) [ Cu]Cu-pDefs are created with varying lipid acyl chain length and charge, representing the most prevalently studied microbubble compositions. In vitro, C16 chain length microbubbles yield 2-3x smaller nanoprogeny than C18 microbubbles post FUS. In vivo, [ Cu]Cu-pDefs are tracked in healthy and 4T1 tumor-bearing mice ± FUS over 48 h qualitatively through fluorescence imaging (to characterize particle disruption) and quantitatively through PET and γ-counting. These studies reveal the impact of microbubble composition and FUS on microbubble dissolution rates, shell circulation, off-target tissue retention (predominantly the liver and spleen), and FUS enhancement of tumor delivery. These findings yield pharmacokinetic microbubble structure-activity relationships that disrupt conventional knowledge, the implications of which on MB-FUS platform design, safety, and nanomedicine delivery are discussed.

Quantitative Pharmacokinetics Reveal Impact of Lipid Composition on Microbubble and Nanoprogeny Shell Fate.

: Multimodal imaging provides important pharmacokinetic and dosimetry information during nanomedicine development and optimization. However, accurate quantitation is time-consuming, resource intensive, and requires anatomical expertise. : We present NanoMASK: a 3D U-Net adapted deep learning tool capable of rapid, automatic organ segmentation of multimodal imaging data that can output key clinical dosimetry metrics without manual intervention. This model was trained on 355 manually-contoured PET/CT data volumes of mice injected with a variety of nanomaterials and imaged over 48 hours. : NanoMASK produced 3-dimensional contours of the heart, lungs, liver, spleen, kidneys, and tumor with high volumetric accuracy (pan-organ average %DSC of 92.5). Pharmacokinetic metrics including %ID/cc, %ID, and SUV achieved correlation coefficients exceeding R = 0.987 and relative mean errors below 0.2%. NanoMASK was applied to novel datasets of lipid nanoparticles and antibody-drug conjugates with a minimal drop in accuracy, illustrating its generalizability to different classes of nanomedicines. Furthermore, 20 additional auto-segmentation models were developed using training data subsets based on image modality, experimental imaging timepoint, and tumor status. These were used to explore the fundamental biases and dependencies of auto-segmentation models built on a 3D U-Net architecture, revealing significant differential impacts on organ segmentation accuracy. : NanoMASK is an easy-to-use, adaptable tool for improving accuracy and throughput in imaging-based pharmacokinetic studies of nanomedicine. It has been made publicly available to all readers for automatic segmentation and pharmacokinetic analysis across a diverse array of nanoparticles, expediting agent development.

Article	Year
Multimodal micro, nano, and size conversion ultrasound agents for imaging and therapy. Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology\| PubMed ID: 27006001	2016
Tailoring Porphyrin Conjugation for Nanoassembly-Driven Phototheranostic Properties. ACS nano\| PubMed ID: 30916932	2019
Activating Drugs with Sound: Mechanisms Behind Sonodynamic Therapy and the Role of Nanomedicine. Bioconjugate chemistry\| PubMed ID: 32129984	2020
Photophysics of J-Aggregating Porphyrin-Lipid Photosensitizers in Liposomes: Impact of Lipid Saturation. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 32338515
Subtherapeutic Photodynamic Treatment Facilitates Tumor Nanomedicine Delivery and Overcomes Desmoplasia. Nano letters\| PubMed ID: 33301689	2021
Targeted Theranostic In/Lu-Nanotexaphyrin for SPECT Imaging and Photodynamic Therapy. Molecular pharmaceutics\| PubMed ID: 34965727	2021
Protease-Activatable Porphyrin Molecular Beacon for Osteoarthritis Management. Chemical & biomedical imaging\| PubMed ID: 37122828	2023
Quantitative Pharmacokinetics Reveal Impact of Lipid Composition on Microbubble and Nanoprogeny Shell Fate. Advanced science (Weinheim, Baden-Wurttemberg, Germany)\| PubMed ID: 38032129	2024
Deep learning for automatic organ and tumor segmentation in nanomedicine pharmacokinetics. Theranostics\| PubMed ID: 38250039	2024

Maneesha A. Rajora

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