Biomimetic bright optotheranostics for metastasis monitoring and multimodal image-guided breast cancer therapeutics.

Nanoparticle formulations blending optical imaging contrast agents and therapeutics have been a cornerstone of preclinical theranostic applications. However, nanoparticle-based theranostics clinical translation faces challenges on reproducibility, brightness, photostability, biocompatibility, and selective tumor targeting and penetration. In this study, we integrate multimodal imaging and therapeutics within cancer cell-derived nanovesicles, leading to biomimetic bright optotheranostics for monitoring cancer metastasis. Upon NIR light irradiation, the engineered optotheranostics enables deep visualization and precise localization of metastatic lung, liver, and solid breast tumors along with solid tumor ablation. Metastatic cell-derived nanovesicles (∼80 ± 5 nm) are engineered to encapsulate imaging (emissive organic dye and gold nanoparticles) and therapeutic agents (anticancer drug doxorubicin and photothermally active organic indocyanine green dye). Systemic administration of biomimetic bright optotheranostic nanoparticles shows escape from mononuclear phagocytic clearance with (i) rapid tumor accumulation (3 h) and retention (up to 168 h), (ii) real-time monitoring of metastatic lung, liver, and solid breast tumors and (iii) 3-fold image-guided solid tumor reduction. These findings are supported by an improvement of X-ray, fluorescence, and photoacoustic signals while demonstrating a tumor reduction (201 mm3) in comparison with single therapies that includes chemotherapy (134 mm3), photodynamic therapy (72 mm3), and photothermal therapy (88mm3). The proposed innovative platform opens new avenues to improve cancer diagnosis and treatment outcomes by allowing the monitorization of cancer metastasis, allowing the precise cancer imaging, and delivering synergistic therapeutic agents at the solid tumor site.

Metal coordination micelles for anti-cancer treatment by gene-editing and phototherapy.

CRISPR-Cas9 is a central focus of the emerging field of gene editing and photodynamic therapy (PDT) is a clinical-stage ablation modality combining photosensitizers with light irradiation. But metal coordination biomaterials for the applications of both have rarely been investigated. Herein, Chlorin-e6 (Ce6) Manganese (Mn) coordination micelles loaded with Cas9, termed Ce6-Mn-Cas9, were developed for augmented combination anti-cancer treatment. Manganese played multiple roles to facilitate Cas9 and single guide RNA (sgRNA) ribonucleoprotein (RNP) delivery, Fenton-like effect, and enhanced endonuclease activity of RNP. Histidine (His)-tagged RNP could be coordinated to Ce6 encapsulated in Pluronic F127 (F127) micelles by simple admixture. Triggered by ATP and endolysosomal acidic pH, Ce6-Mn-Cas9 released Cas9 without altering protein structure or function. Dual guide RNAs were designed to target the antioxidant regulator MTH1 and the DNA repair protein APE1, resulting in increased oxygen and enhanced PDT effect. In a murine tumor model, Ce6-Mn-Cas9 inhibited tumor growth with the combination therapy of PDT and gene editing. Taken together, Ce6-Mn-Cas9 represents a new biomaterial with a high degree of versatility to enable photo- and gene-therapy approaches.

Two Laser Treatments Can Improve Tumor Ablation Efficiency of Chemophototherapy.

Chemophototherapy is an emerging tumor ablation modality that can improve local delivery of chemotherapeutic agents. Long circulating doxorubicin (Dox) in porphyrin-phospholipid (PoP) liposomes (LC-Dox-PoP) has previously been developed as an effective chemophototherapy agent. In the present study, we observed that in mice, LC-Dox-PoP showed enhanced accumulation in human pancreatic tumor xenografts even with suboptimal light doses, as assessed by fluorometric analysis of tissue homogenates and microscopic imaging of Dox and PoP in tumor slices. A second laser treatment, at a time point in which tumors had greater drug accumulation as a result of the first laser treatment, induced potent tumor ablation. Efficacy studies were carried out in two human pancreatic cancer subcutaneous mouse tumor models; MIA PaCa-2 or low-passage patient derived pancreatic cancer xenografts. A single treatment of 3 mg/kg LC-Dox-PoP and an initial 150 J/cm2 laser treatment 1 h after drug administration, followed by second laser treatment of 50 J/cm2 8 h after drug administration, was more effective than a single laser treatment of 200 J/cm2 at either of those time points. Thus, this study presents proof-of-principle and rationale for using two discrete laser treatments to enhance the efficacy of chemophototherapy.

Antibiotic Cross-linked Micelles with Reduced Toxicity for Multidrug-Resistant Bacterial Sepsis Treatment.

One potential approach to address the rising threat of antibiotic resistance is through novel formulations of established drugs. We designed antibiotic cross-linked micelles (ABC-micelles) by cross-linking the Pluronic F127 block copolymers with an antibiotic itself, via a novel one-pot synthesis in aqueous solution. ABC-micelles enhanced antibiotic encapsulation while also reducing systemic toxicity in mice. Using colistin, a hydrophilic, potent ″last-resort" antibiotic, ABC-micelle encapsulation yield was 80%, with good storage stability. ABC-micelles exhibited an improved safety profile, with a maximum tolerated dose of over 100 mg/kg colistin in mice, at least 16 times higher than the free drug. Colistin-induced nephrotoxicity and neurotoxicity were reduced in ABC-micelles by 10-50-fold. Despite reduced toxicity, ABC-micelles preserved bactericidal activity, and the clinically relevant combination of colistin and rifampicin (co-loaded in the micelles) showed a synergistic antimicrobial effect against antibiotic-resistant strains of Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. In a mouse model of sepsis, colistin ABC-micelles showed equivalent efficacy as free colistin but with a substantially higher therapeutic index. Microscopic single-cell imaging of bacteria revealed that ABC-micelles could kill bacteria in a more rapid manner with distinct cell membrane disruption, possibly reflecting a different antimicrobial mechanism from free colistin. This work shows the potential of drug cross-linked micelles as a new class of biomaterials formed from existing antibiotics and represents a new and generalized approach for formulating amine-containing drugs.

Co-delivery of Bee Venom Melittin and a Photosensitizer with an Organic-Inorganic Hybrid Nanocarrier for Photodynamic Therapy and Immunotherapy.

Photodynamic therapy (PDT) is a clinical cancer treatment modality based on the induction of therapeutic reactive oxygen species (ROS), which can trigger immunogenic cell death (ICD). With the aim of simultaneously improving both PDT-mediated intracellular ROS production and ICD levels, we designed a serum albumin (SA)-coated boehmite ("B"; aluminum hydroxide oxide) organic-inorganic scaffold that could be loaded with chlorin e6 (Ce6), a photosensitizer, and a honey bee venom melittin (MLT) peptide, denoted Ce6/MLT@SAB. Ce6/MLT@SAB was anchored by a boehmite nanorod structure and exhibited particle size of approximately 180 nm. Ce6/MLT@SAB could significantly reduce hemolysis relative to that of free MLT, while providing MLT-enhanced PDT antitumor effects in vitro. Compared with Ce6@SAB, Ce6/MLT@SAB improved Ce6 penetration of cancer cells both in vitro and in vivo, thereby providing enhanced intracellular ROS generation with 660 nm light treatment. Following phototreatment, Ce6/MLT@SAB-treated cells displayed significantly improved levels of ICD and abilities to activate dendritic cells. In the absence of laser irradiation, multidose injection of Ce6/MLT@SAB could delay the growth of subcutaneous murine tumors by more than 60%, compared to controls. When combined with laser irradiation, a single injection and phototreatment with Ce6/MLT@SAB eradicated one-third of subcutaneous tumors in treated mice. The addition of an immune checkpoint blockade to Ce6/MLT@SAB phototreatment further augmented antitumor effects, generating increased numbers of CD4+ and CD8+ T cells in tumors with concomitant reduction of myeloid-derived suppressor cells.

Indocyanine green binds to DOTAP liposomes for enhanced optical properties and tumor photoablation.

Indocyanine green (ICG) is a clinically-approved near infrared (NIR) dye used for optical imaging. The dye is only slightly soluble in water and is prone to aggregation in saline solutions, so that alternative formulations can improve photophysical performance. Numerous nanoscale formulations of ICG have been described in the literature, but we sought to develop an approach that does not require additional purification steps. Pre-formed liposomes incorporating 45 mol% of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) rapidly bind ICG, resulting in enhanced NIR optical properties. ICG binding is dependent on the amount of DOTAP incorporated in the liposomes. A dye-to-lipid mass ratio of [0.5 : 25] is sufficient for full complexation, without additional purification steps following mixing. NIR absorption, fluorescence intensity, and photoacoustic signals are increased for the liposome-bound dye. Not only is the optical character enhanced by simple mixing of ICG with liposomes, but retention in 4T1 mammary tumors is observed following intratumor injection, as assessed by fluorescence and photoacoustic imaging. Subsequent photothermal therapy with 808 nm laser irradiation is effective and results in tumor ablation without regrowth for at least 30 days. Thus, ICG optical properties and photothermal ablation outcomes can be improved by mixing the dye with pre-formed DOTAP liposomes in conditions that result in full dye-binding to the liposomes.

Pharmacokinetics and pharmacodynamics of liposomal chemophototherapy with short drug-light intervals.

Chemophototherapy (CPT) merges photodynamic therapy with chemotherapy and can substantially enhance drug delivery. Using a singular liposomal formulation for CPT, we describe a semi-mechanistic pharmacokinetic-pharmacodynamic (PK/PD) model to investigate observed antitumor effects. Long-circulating, sterically-stabilized liposomes loaded with doxorubicin (Dox) stably incorporate small amounts of a porphyrin-phospholipid (PoP) photosensitizer in the bilayer. These were administered intravenously to mice bearing low-passage, patient-derived pancreatic cancer xenografts (PDX). Dox PK was described with a two-compartment model and tumor drug disposition kinetics were modeled with first-order influx and efflux rates. Tumor irradiation with 665 nm laser light (200 J/cm2) 1 h after liposome administration increased tumor vascular permeabilization and drug accumulation, which was accounted for in the PK/PD model with increased tumor influx and efflux rates by approximately 12- and 4- fold, respectively. This modeling approach provided an overall 7-fold increase in Dox area under the curve in the tumor, matching experimental data (7.4-fold). A signal transduction model based on nonlinear direct cell killing accounted for observed tumor growth patterns. This PK/PD model adequately describes the CPT anti-PDX tumor response based on enhanced drug delivery at the short drug-light interval used.

Short Drug-Light Intervals Improve Liposomal Chemophototherapy in Mice Bearing MIA PaCa-2 Xenografts.

Chemophototherapy (CPT) is an emerging tumor treatment that combines phototherapy and chemotherapy. Long-circulating (LC) liposomes can stably incorporate 2 mol % porphyrin-phospholipid (PoP) in the bilayer and load doxorubicin (Dox) to generate LC-Dox-PoP liposomes, for single-agent CPT. Following intravenous administration to mice, LC-Dox-PoP liposomes (2 mg/kg Dox) circulated with similar blood concentration ranges produced by a typical human clinical dose of DOXIL (50 mg/m2 Dox). This dosing approach aims to achieve physiologically relevant Dox and PoP concentrations as well as CPT vascular responses in mice bearing subcutaneous human pancreatic MIA PaCa-2 xenografts. Phototreatment with 2 mg/kg LC-Dox-PoP induced vascular permeabilization, leading to a 12.5-fold increase in Dox tumor influx estimated by a pharmacokinetic model, based on experimental data. Shorter drug-light intervals (0.5-3 h) led to greater tumoral drug deposition and improved treatment outcomes, compared to longer drug-light intervals. At 2 mg/kg Dox, CPT with LC-Dox-PoP liposomes induced tumor regression and growth inhibition, whereas chemotherapy using several other formulations of Dox did not. LC-Dox-PoP liposomes were well tolerated at the 2 mg/kg dose.

Intrabilayer 64Cu Labeling of Photoactivatable, Doxorubicin-Loaded Stealth Liposomes.

Doxorubicin (Dox)-loaded stealth liposomes (similar to those in clinical use) can incorporate small amounts of porphyrin-phospholipid (PoP) to enable chemophototherapy (CPT). PoP is also an intrinsic and intrabilayer 64Cu chelator, although how radiolabeling impacts drug delivery has not yet been assessed. Here, we show that 64Cu can radiolabel the stable bilayer of preformed Dox-loaded PoP liposomes with inclusion of 1% ethanol without inducing drug leakage. Dox-PoP liposomes labeled with intrabilayer copper behaved nearly identically to unlabeled ones in vitro and in vivo with respect to physical parameters, pharmacokinetics, and CPT efficacy. Positron emission tomography and near-infrared fluorescence imaging visualized orthotopic mammary tumors in mice with passive liposome accumulation following administration. A single CPT treatment with 665 nm light (200 J/cm2) strongly inhibited primary tumor growth. Liposomes accumulated in lung metastases, based on NIR imaging. These results establish the feasibility of CPT interventions guided by intrinsic multimodal imaging of Dox-loaded stealth PoP liposomes.

Targeted Nanomaterials for Phototherapy.

Phototherapies involve the irradiation of target tissues with light. To further enhance selectivity and potency, numerous molecularly targeted photosensitizers and photoactive nanoparticles have been developed. Active targeting typically involves harnessing the affinity between a ligand and a cell surface receptor for improved accumulation in the targeted tissue. Targeting ligands including peptides, proteins, aptamers and small molecules have been explored for phototherapy. In this review, recent examples of targeted nanomaterials used in phototherapy are summarized.

Vessel-Targeted Chemophototherapy with Cationic Porphyrin-Phospholipid Liposomes.

Cationic liposomes have been used for targeted drug delivery to tumor blood vessels, via mechanisms that are not fully elucidated. Doxorubicin (Dox)-loaded liposomes were prepared that incorporate a cationic lipid; 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), along with a small amount of porphyrin-phospholipid (PoP). Near-infrared (NIR) light caused release of entrapped Dox via PoP-mediated DOTAP photo-oxidation. The formulation was optimized to enable extremely rapid NIR light-triggered Dox release (i.e., in 15 seconds), while retaining reasonable serum stability. In vitro, cationic PoP liposomes readily bound to both MIA PaCa-2 human pancreatic cancer cells and human vascular endothelial cells. When administered intravenously, cationic PoP liposomes were cleared from circulation within minutes, with most accumulation in the liver and spleen. Fluorescence imaging revealed that some cationic PoP liposomes also localized at the tumor blood vessels. Compared with analogous neutral liposomes, strong tumor photoablation was induced with a single treatment of cationic PoP liposomes and laser irradiation (5 mg/kg Dox and 100 J/cm2 NIR light). Unexpectedly, empty cationic PoP liposomes (lacking Dox) induced equally potent antitumor phototherapeutic effects as the drug loaded ones. A more balanced chemo- and phototherapeutic response was subsequently achieved when antitumor studies were repeated using higher drug dosing (7 mg/kg Dox) and a low fluence phototreatment (20 J/cm2 NIR light). These results demonstrate the feasibility of vessel-targeted chemophototherapy using cationic PoP liposomes and also illustrate synergistic considerations. Mol Cancer Ther; 16(11); 2452-61. ©2017 AACR.

Multifunctional Liposomes for Image-Guided Intratumoral Chemo-Phototherapy.

Intratumoral (IT) drug injections reduce systemic toxicity, but delivered volumes and distribution can be inconsistent. To improve IT delivery paradigms, porphyrin-phospholipid (PoP) liposomes are passively loaded with three hydrophilic cargos: sulforhodamine B, a fluorophore; gadolinium-gadopentetic acid, a magnetic resonance (MR) agent; and oxaliplatin, a colorectal cancer chemotherapeutic. Liposome composition is optimized so that cargo is retained in serum and storage, but is released in less than 1 min with exposure to near infrared light. Light-triggered release occurs with PoP-induced photooxidation of unsaturated lipids and all cargos release concurrently. In subcutaneous murine colorectal tumors, drainage of released cargo is delayed when laser treatment occurs 24 h after IT injection, at doses orders of magnitude lower than systemic ones. Delayed light-triggering results in substantial tumor shrinkage relative to controls a week following treatment, although regrowth occurs subsequently. MR imaging reveals that over this time frame, pools of liposomes within the tumor migrate to adjacent regions, possibly leading to altered spatial distribution during triggered drug release. Although further characterization of cargo loading and release is required, this proof-of-principle study suggests that multimodal theranostic IT delivery approaches hold potential to both guide injections and interpret outcomes, in particular when combined with chemo-phototherapy.

Surfactant-stripped naphthalocyanines for multimodal tumor theranostics with upconversion guidance cream.

Surfactant-stripped, nanoformulated naphthalocyanines (nanonaps) can be formed with Pluronic F127 and low temperature membrane processing, resulting in dispersed frozen micelles with extreme contrast in the near infrared region. Here, we demonstrate that nanonaps can be used for multifunctional cancer theranostics. This includes lymphatic mapping and whole tumor photoacoustic imaging following intradermal or intravenous injection in rodents. Without further modification, pre-formed nanonaps were used for positron emission tomography and passively accumulated in subcutaneous murine tumors. Because the nanonaps used absorb light beyond the visible range, a topical upconversion skin cream was developed for anti-tumor photothermal therapy with laser placement that can be guided by the naked eye.

Chemophototherapy: An Emerging Treatment Option for Solid Tumors.

Near infrared (NIR) light penetrates human tissues with limited depth, thereby providing a method to safely deliver non-ionizing radiation to well-defined target tissue volumes. Light-based therapies including photodynamic therapy (PDT) and laser-induced thermal therapy have been validated clinically for curative and palliative treatment of solid tumors. However, these monotherapies can suffer from incomplete tumor killing and have not displaced existing ablative modalities. The combination of phototherapy and chemotherapy (chemophototherapy, CPT), when carefully planned, has been shown to be an effective tumor treatment option preclinically and clinically. Chemotherapy can enhance the efficacy of PDT by targeting surviving cancer cells or by inhibiting regrowth of damaged tumor blood vessels. Alternatively, PDT-mediated vascular permeabilization has been shown to enhance the deposition of nanoparticulate drugs into tumors for enhanced accumulation and efficacy. Integrated nanoparticles have been reported that combine photosensitizers and drugs into a single agent. More recently, light-activated nanoparticles have been developed that release their payload in response to light irradiation to achieve improved drug bioavailability with superior efficacy. CPT can potently eradicate tumors with precise spatial control, and further clinical testing is warranted.

A Phosphorus Phthalocyanine Formulation with Intense Absorbance at 1000 nm for Deep Optical Imaging.

Although photoacoustic computed tomography (PACT) operates with high spatial resolution in biological tissues deeper than other optical modalities, light scattering is a limiting factor. The use of longer near infrared wavelengths reduces scattering. Recently, the rational design of a stable phosphorus phthalocyanine (P-Pc) with a long wavelength absorption band beyond 1000 nm has been reported. Here, we show that when dissolved in liquid surfactants, P-Pc can give rise to formulations with absorbance of greater than 1000 (calculated for a 1 cm path length) at wavelengths beyond 1000 nm. Using the broadly accessible Nd:YAG pulse laser emission output of 1064 nm, P-Pc could be imaged through 11.6 cm of chicken breast with PACT. P-Pc accumulated passively in tumors following intravenous injection in mice as observed by PACT. Following oral administration, P-Pc passed through the intestine harmlessly, and PACT could be used to non-invasively observe intestine function. When the contrast agent placed under the arm of a healthy adult human, a PACT transducer on the top of the arm could readily detect P-Pc through the entire 5 cm limb. Thus, the approach of using contrast media with extreme absorption at 1064 nm readily enables high quality optical imaging in vitro and in vivo in humans at exceptional depths.

Doxorubicin encapsulated in stealth liposomes conferred with light-triggered drug release.

Stealth liposomes can be used to extend the blood circulation time of encapsulated therapeutics. Inclusion of 2 molar % porphyrin-phospholipid (PoP) imparted optimal near infrared (NIR) light-triggered release of doxorubicin (Dox) from conventional sterically stabilized stealth liposomes. The type and amount of PoP affected drug loading, serum stability and drug release induced by NIR light. Cholesterol and PEGylation were required for Dox loading, but slowed light-triggered release. Dox in stealth PoP liposomes had a long circulation half-life in mice of 21.9 h and was stable in storage for months. Following intravenous injection and NIR irradiation, Dox deposition increased ∼ 7 fold in treated subcutaneous human pancreatic xenografts. Phototreatment induced mild tumor heating and complex tumor hemodynamics. A single chemophototherapy treatment with Dox-loaded stealth PoP liposomes (at 5-7 mg/kg Dox) eradicated tumors while corresponding chemo- or photodynamic therapies were ineffective. A low dose 3 mg/kg Dox phototreatment with stealth PoP liposomes was more effective than a maximum tolerated dose of free (7 mg/kg) or conventional long-circulating liposomal Dox (21 mg/kg). To our knowledge, Dox-loaded stealth PoP liposomes represent the first reported long-circulating nanoparticle capable of light-triggered drug release.

Metal Chelation Modulates Phototherapeutic Properties of Mitoxantrone-Loaded Porphyrin-Phospholipid Liposomes.

Liposomes incorporating porphyrin-phospholipid (PoP) can be formulated to release entrapped contents in response to near-infrared (NIR) laser irradiation. Here, we examine effects of chelating copper or zinc into the PoP. Cu(II) and Zn(II) PoP liposomes, containing 10 molar % HPPH-lipid, exhibited unique photophysical properties and released entrapped cargo in response to NIR light. Cu-PoP liposomes exhibited minimal fluorescence and reduced production of reactive oxygen species upon irradiation. Zn-PoP liposomes retained fluorescence and singlet oxygen generation properties; however, they rapidly self-bleached under laser irradiation. Compared to the free base form, both Cu- and Zn-PoP liposomes exhibited reduced phototoxicity in mice. When loaded with mitoxantrone and administered intravenously at 5 mg/kg to mice bearing human pancreatic cancer xenografts, synergistic effects between the drug and the light treatment (for this particular dose and formulation) were realized with metallo-PoP liposomes. The drug-light-interval affected chemophototherapy efficacy and safety.

Emerging applications of porphyrins in photomedicine.

Biomedical applications of porphyrins and related molecules have been extensively pursued in the context of photodynamic therapy. Recent advances in nanoscale engineering have opened the door for new ways that porphyrins stand to potentially benefit human health. Metalloporphyrins are inherently suitable for many types of medical imaging and therapy. Traditional nanocarriers such as liposomes, dendrimers and silica nanoparticles have been explored for photosensitizer delivery. Concurrently, entirely new classes of porphyrin nanostructures are being developed, such as smart materials that are activated by specific biochemicals encountered at disease sites. Techniques have been developed that improve treatments by combining biomaterials with photosensitizers and functional moieties such as peptides, DNA and antibodies. Compared to simpler structures, these more complex and functional designs can potentially decrease side effects and lead to safer and more efficient phototherapies. This review examines recent research on porphyrin-derived materials in multimodal imaging, drug delivery, bio-sensing, phototherapy and probe design, demonstrating their bright future for biomedical applications.

One minute, sub-one-watt photothermal tumor ablation using porphysomes, intrinsic multifunctional nanovesicles.

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide α, was conjugated to a phospholipid and then self-assembled to liposome-like spherical vesicles. Due to the extremely high density of porphyrin in the porphyrin-lipid bilayer, porphysomes generated large extinction coefficients, structure-dependent fluorescence self-quenching, and excellent photothermal efficacy. In our formulation, porphysomes were synthesized using high pressure extrusion, and displayed a mean particle size around 120 nm. Twenty-four hr post-intravenous injection of porphysomes, the local temperature of the tumor increased from 30 °C to 62 °C rapidly upon one minute exposure of 750 mW (1.18 W/cm(2)), 671 nm laser irradiation. Following the complete thermal ablation of the tumor, eschars formed and healed within 2 weeks, while in the control groups the tumors continued to grow and all reached the defined end point within 3 weeks. These data show how porphysomes can be used as potent photothermal therapy (PTT) agents.

Self-assembled porphyrin nanodiscs with structure-dependent activation for phototherapy and photodiagnostic applications.

The abilities to deliver and subsequently activate a therapeutic at the intended site of action are two important challenges in the synthesis of novel nanoparticles. Poor tumor permeability as a result of a dense microenvironment can impede the delivery of nanoparticles to the site of action. The design of a sub-40 nm activatable porphyrin nanodisc, based on protein-induced lipid constriction, is described. The biophotonic nanoparticle, self-assembled from aggregated porphyrin-lipid, is stabilized by an amphipathic alpha helical protein and becomes photoactive when its structure is perturbed. Enzymatic cleavage of the constricting protein leads to conversion of the particle from a disc- to a vesicle-shaped structure and provides further evidence that the apolipoprotein serves a functional role on the nanodisc. Fluorescence measurements of these nanodiscs in a detergent show that fluorescence is over 99% quenched in the intact state with a 12-fold increase in singlet oxygen generation upon disruption. Cellular fluorescence unquenching and dose-dependent phototoxicity demonstrate that these nanodiscs can be internalized and unquenched intracellularly. Finally, nanodiscs were found to display a 5-fold increase in diffusion coefficient when compared with the protein-free control ((3.5±0.1)×10(-7) vs (0.7±0.03)×10(-7) cm2 s(-1)). The ability to incorporate large amounts of photosensitizer drugs into its compact structure allows for phototherapeutic action, fluorescence diagnostic applications, and the potential to effectively deliver photosensitizers deep into poorly permeable tumors.