Elizabeth Huynh

Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents

Optically active nanomaterials promise to advance a range of biophotonic techniques through nanoscale optical effects and integration of multiple imaging and therapeutic modalities. Here, we report the development of porphysomes; nanovesicles formed from self-assembled porphyrin bilayers that generated large, tunable extinction coefficients, structure-dependent fluorescence self-quenching and unique photothermal and photoacoustic properties. Porphysomes enabled the sensitive visualization of lymphatic systems using photoacoustic tomography. Near-infrared fluorescence generation could be restored on dissociation, creating opportunities for low-background fluorescence imaging. As a result of their organic nature, porphysomes were enzymatically biodegradable and induced minimal acute toxicity in mice with intravenous doses of 1,000 mg kg(-1). In a similar manner to liposomes, the large aqueous core of porphysomes could be passively or actively loaded. Following systemic administration, porphysomes accumulated in tumours of xenograft-bearing mice and laser irradiation induced photothermal tumour ablation. The optical properties and biocompatibility of porphysomes demonstrate the multimodal potential of organic nanoparticles for biophotonic imaging and therapy.

In situ conversion of porphyrin microbubbles to nanoparticles for multimodality imaging

Converting nanoparticles or monomeric compounds into larger supramolecular structures by endogenous or external stimuli is increasingly popular because these materials are useful for imaging and treating diseases. However, conversion of microstructures to nanostructures is less common. Here, we show the conversion of microbubbles to nanoparticles using low-frequency ultrasound. The microbubble consists of a bacteriochlorophyll-lipid shell around a perfluoropropane gas. The encapsulated gas provides ultrasound imaging contrast and the porphyrins in the shell confer photoacoustic and fluorescent properties. On exposure to ultrasound, the microbubbles burst and form smaller nanoparticles that possess the same optical properties as the original microbubble. We show that this conversion is possible in tumour-bearing mice and could be validated using photoacoustic imaging. With this conversion, our microbubble can potentially be used to bypass the enhanced permeability and retention effect when delivering drugs to tumours.

Enzymatic Regioselection for the Synthesis and Biodegradation of Porphysome Nanovesicles

We recently reported that porphyrin–phospholipid conjugates can self-assemble into “porphysome” nanovesicles composed of a dense porphyrin bilayer. Porphysomes exhibit structurally driven nanoscale optical properties and have intrinsic capabilities for multimodal imaging, drug delivery, and photothermal therapy. Previous studies were based on porphysomes formed from a mixture of two chemically similar phospholipid–porphyrin regioisomers. However, use of a mixture of regioisomers is far from ideal for robust nanoparticle characterization in vitro and in vivo. In general, difficulties in synthesizing, detecting, and distinguishing phospholipid regioisomers have prevented examination of their in vivo fate until now. To our knowledge, the results reported herein are the first to demonstrate in vivo enzymatic biodegradability for any intrinsically optically active nanoparticle, a feature that might be important when considering the use of new nanomaterials in human clinical trials. Chemically modified phospholipids have proven useful for diverse biotechnology applications. Phospholipids can be labeled at various positions on their head group or side chain. While head-group modification can readily be achieved using the primary amine group of phosphatidylethanolamine, side-chain modification is less straightforward but is appropriate for conjugating more hydrophobic ligands while maintaining an amphipathic phospholipid character. Recently, phospholipids modified with cholesterol, retinoic acid, and porphyrin side chains have been developed that display useful properties for drug-delivery, immunological, and biophotonic applications. Synthesis of single sidechain-modified phospholipids is often affected by acyl migration of the side chains. The resulting regioisomers (e.g., Figure 1a) have similar structures, which make their separation impractical and their detection challenging or impossible using techniques such as HPLC, NMR spectroscopy, and mass spectrometry. Regioselective phospholipid side-chain modification has been achieved using a number of techniques. Synthesis of modified phospholipids has been performed in multistep reactions using a modified glycerol backbone, with protecting groups sometimes being required. Acylation of lyso-

Methylene blue microbubbles as a model dual-modality contrast agent for ultrasound and activatable photoacoustic imaging

Abstract. Ultrasound and photoacoustic imaging are highly complementary modalities since both use ultrasonic detection for operation. Increasingly, photoacoustic and ultrasound have been integrated in terms of hardware instrumentation. To generate a broadly accessible dual-modality contrast agent, we generated microbubbles (a standard ultrasound contrast agent) in a solution of methylene blue (a standard photoacoustic dye). This MB2 solution was formed effectively and was optimized as a dual-modality contrast solution. As microbubble concentration increased (with methylene blue concentration constant), photoacoustic signal was attenuated in the MB2 solution. When methylene blue concentration increased (with microbubble concentration held constant), no ultrasonic interference was observed. Using an MB2 solution that strongly attenuated all photoacoustic signal, high powered ultrasound could be used to burst the microbubbles and dramatically enhance photoacoustic contrast (>800-fold increase), providing a new method for spatiotemporal control of photoacoustic signal generation.

Aggregate enhanced trimodal porphyrin shell microbubbles for ultrasound, photoacoustic, and fluorescence imaging.

Microbubbles (MBs) are currently used as ultrasound (US) contrast agents and as delivery vehicles for site-specific US-triggered drug and gene delivery. Multimodal US-based imaging methods have been applied preclinically to assess and validate the effectiveness and fate of MBs in imaging and therapy. Here we present the first intrinsically trimodal MBs by incorporating a dense concentration of porphyrin molecules within a MB shell, enabled by the use of a single porphyrin-lipid component. These MBs possess US, photoacoustic, and fluorescence properties that are demonstrated in solution and in a mouse tumor xenograft model. They also have potential to be extended to other imaging modalities such as magnetic resonance imaging and nuclear imaging.

Porphyrin Nanodroplets: Sub‐micrometer Ultrasound and Photoacoustic Contrast Imaging Agents

A novel class of all-organic nanoscale porphyrin nanodroplet agents is presented which is suitable for multimodality ultrasound and photoacoustic molecular imaging. Previous multimodality photoacoustic-ultrasound agents are either not organic, or not yet demonstrated to exhibit enhanced accumulation in leaky tumor vasculature, perhaps because of large diameters. In the current study, porphyrin nanodroplets are created with a mean diameter of 185 nm which is small enough to exhibit the enhanced permeability and retention effect. Porphyrin within the nanodroplet shell has strong optical absorption at 705 nm with an estimated molar extinction coefficient >5 × 10(9) m(-1) cm(-1) , allowing both ultrasound and photoacoustic contrast in the same nanoparticle using all organic materials. The potential of nanodroplets is that they may be phase-changed into microbubbles using high pressure ultrasound, providing ultrasound contrast with single-bubble sensitivity. Multispectral photoacoustic imaging allows visualization of nanodroplets when injected intratumorally in an HT1080 tumor in the chorioallantoic membrane of a chicken embryo. Intravital microscopy imaging of Hep3-GFP and HT1080-GFP tumors in chicken embryos determines that nanodroplets accumulated throughout or at the periphery of tumors, suggesting that porphyrin nanodroplets may be useful for enhancing the visualization of tumors with ultrasound and/or photoacoustic imaging.

Biodegradable star polymers shine for cancer drug delivery.

Selective delivery of drugs to target cells and tissues is a central challenge in cancer chemotherapy. Practically speaking, drug delivery is often just as important as the drug itself. Of the numerous drug-delivery systems available, nanoscale polymers are a prominent class. There are endless possible permutations of shape, size and chemical properties of polymeric drug-delivery systems. Developing an understanding of how these properties affect pharmacokinetics, biodegradability, drug release from the polymer and accumulation in tumors and other organs is essential to expand and improve clinical applications of polymer-based nanomedicine. Some previous studies have examined the effect of size on tumor accumulation and circulation times for some polymers, such as p olyvinyl acid [1]. Bo Chen and colleagues from the Universities of California in San Francisco and Berkeley recently reported a methodical examination of drug-loaded star-comb polymers formed by atom transfer radical polymerization. Varying amounts of polyethylene glycol were grafted onto a multifunctional core with eight reactive sites to generate nanoparticles of varying sizes. As multiple ester linkages were incorporated into the design, the star polymers were hydrolyzed over a period of weeks in physiological conditions. The nanoparticles had varying pharmacokinetic profiles; those between 30 kDa and 100 kDa had long b half-lives ranging from 24 to 37 h, while those ~20 kDa and smaller were cleared rapidly due to renal filtration with b halflives of less than 10 h. This is comparable to another recent study that determined that PEGylated quantum dots smaller than 5.5 nm could be rapidly cleared by the kidneys, whereas PEGylated quantum dots of at least 15 nm could more effectively remain in circulation [2]. In that study, the b half-life increased from ~1 h to 20 h as the size increased from ~4 to ~9 nm. The star polymer nanoparticles that avoided renal clearance accumulated in tumors with a remarkable avidity of over 15% injected dose per gram (% ID/g) after two days. Star polymers of different sizes were then conjugated to doxorubicin via pH sensitive hydrazone linkages, which are conducive for lysosomal release of drugs. The drug-loaded nanopolymers of varying sizes from 8 nm to 56 nm all accumulated favorably in tumors (~6% ID/g); however, the smaller nanoparticles avoided entrapment in the reticuloendothelial system of the spleen and liver. This demonstrates an advantage of using PEGylated nanoparticles that are only slightly larger than the renal clearance size threshold to achieve favorable biodistributions. As new polymer nanoparticles are increasingly being explored for in vivo applications, fundamental studies, such as this one, that rigorously examine the functional effects of basic polymer properties, will become indispensible to develop rational approaches to nanoengineering.

Multimodal Image-Guided Surgical and Photodynamic Interventions in Head and Neck Cancer: From Primary Tumor to Metastatic Drainage

Purpose: The low survival rate of head and neck cancer (HNC) patients is attributable to late disease diagnosis and high recurrence rate. Current HNC staging has inadequate accuracy and low sensitivity for effective diagnosis and treatment management. The multimodal porphyrin lipoprotein-mimicking nanoparticle (PLP), intrinsically capable of positron emission tomography (PET), fluorescence imaging, and photodynamic therapy (PDT), shows great potential to enhance the accuracy of HNC staging and potentially HNC management. Experimental Design: Using a clinically relevant VX-2 buccal carcinoma rabbit model that is able to consistently develop metastasis to regional lymph nodes after tumor induction, we investigated the abilities of PLP for HNC diagnosis and management. Results: PLPs facilitated accurate detection of primary tumor and metastatic nodes (their PET image signal to surrounding muscle ratios were 10.0 and 7.3, respectively), and provided visualization of the lymphatic drainage from tumor to regional lymph nodes by both preoperative PET and intraoperative fluorescence imaging, allowing the identification of unknown primaries and recurrent tumors. PLP-PDT significantly enhanced cell apoptosis in mouse tumors (73.2% of PLP-PDT group vs 7.1% of PLP alone group) and demonstrated complete eradication of primary tumors and obstruction of tumor metastasis in HNC rabbit model without toxicity in normal tissues or damage to adjacent critical structures. Conclusions: PLPs provide a multimodal imaging and therapy platform that could enhance HNC diagnosis by integrating PET/computed tomography and fluorescence imaging, and improve HNC therapeutic efficacy and specificity by tailoring treatment via fluorescence-guided surgery and PDT. Clin Cancer Res; 22(4); 961–70. ©2015 AACR.

Optically Controlled Pore Formation in Self‐Sealing Giant Porphyrin Vesicles

Efforts to develop self-contained microreactors and artificial cells have been limited by difficulty in generating membranes that can be robustly and repeatedly manipulated to load and release cargo from phospholipid compartments. Here we describe a purely optical method to form pores in a membrane generated from porphyrin-phospholipid conjugates electro-assembled into microscale giant porphyrin vesicles and manipulated using confocal microscopy. The pores in the membrane resealed within a minute allowing for repeated pore formation with precise spatial and temporal control and optical gating to allow selective diffusion of biomolecules across the membrane. Temporal control of pore formation was illustrated by performing sequential DNA hybridization reactions. A biotin-avidin based strategy was developed to selectively attach enzymes to the interior of the vesicle, demonstrating spatial control and the potential of giant porphyrin vesicles as versatile microreactors.

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

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.