Folate-Targeted Nanoliposomal Chemophototherapy.

Light-responsive liposomes have been developed for the on-demand release of drugs. However, efficient delivery of chemotherapeutic drugs to tumor for cancer theranostics remains a challenge. Herein, folic acid (FA), an established ligand for targeted drug delivery, was used to decorate light-sensitive porphyrin-phospholipid (PoP) liposomes, which were assessed for FA-targeted chemophototherapy (CPT). PoP liposomes and FA-conjugated PoP liposomes were loaded with Doxorubicin (Dox), and physical properties were characterized. In vitro, FA-PoP liposomes that were incubated with FA receptor-overexpressing human KB cancer cells showed increased uptake compared to non-targeted PoP liposomes. Dox and PoP contributed towards chemophototherapy (CPT) in vitro, and PoP and FA-PoP liposomes induced cell killing. In vivo, mice bearing subcutaneous KB tumors treated with PoP or FA-PoP liposomes loaded with Dox, followed by 665 nm laser treatment, had delayed tumor growth and improved survival. Dox delivery to tumors increased following laser irradiation for both PoP and FA-PoP liposomes. Thus, while Dox-FA-PoP liposomes were effective following systemic administration and local light irradiation in this tumor model, the FA targeting moiety did not appear essential for anti-tumor responses.

Chemophototherapeutic Ablation of Doxorubicin-Resistant Human Ovarian Tumor Cells.

Porphyrin-phospholipid (PoP) liposomes loaded with Doxorubicin (Dox) have been demonstrated to be an efficient vehicle for chemophototherapy (CPT). Multidrug resistance (MDR) of cancer cells is a problematic phenomenon in which tumor cells develop resistance to chemotherapy. Herein, we report that Dox-resistant tumor cells can be ablated using our previously described formulation termed long-circulating Dox loaded in PoP liposomes (LC-Dox-PoP), which is a PEGylated formulation containing 2 mol. % of the PoP photosensitizer. In vitro studies using free Dox and LC-Dox-PoP showed that human ovarian carcinoma A2780 cells were more susceptible to Dox compared to the corresponding Dox-resistant A2780-R cells. When CPT was applied with LC-Dox-PoP liposomes, effective killing of both nonresistant and resistant A2780 cell lines was observed. An in vivo study to assess the efficiency of LC-Dox-PoP showed effective tumor shrinkage and prolonged survival of athymic nude mice bearing subcutaneous Dox-resistant A2780-R tumor xenografts when they were irradiated with a red laser. Biodistribution analysis demonstrated enhanced tumoral drug uptake in Dox-resistant tumors with CPT, suggesting that increased drug delivery was sufficient to induce ablation of resistant tumor cells.

Trans-illumination intestine projection imaging of intestinal motility in mice.

Functional intestinal imaging holds importance for the diagnosis and evaluation of treatment of gastrointestinal diseases. Currently, preclinical imaging of intestinal motility in animal models is performed either invasively with excised intestines or noninvasively under anesthesia, and cannot reveal intestinal dynamics in the awake condition. Capitalizing on near-infrared optics and a high-absorbing contrast agent, we report the Trans-illumination Intestine Projection (TIP) imaging system for free-moving mice. After a complete system evaluation, we performed in vivo studies, and obtained peristalsis and segmentation motor patterns of free-moving mice. We show the in vivo typical segmentation motor pattern, that was previously shown in ex vivo studies to be controlled by intestinal pacemaker cells. We also show the effects of anesthesia on motor patterns, highlighting the possibility to study the role of the extrinsic nervous system in controlling motor patterns, which requires unanesthetized live animals. Combining with light-field technologies, we further demonstrated 3D imaging of intestine in vivo (3D-TIP). Importantly, the added depth information allows us to extract intestines located away from the abdominal wall, and to quantify intestinal motor patterns along different directions. The TIP system should open up avenues for functional imaging of the GI tract in conscious animals in natural physiological states.

Photoactivation of sulfonated polyplexes enables localized gene silencing by DsiRNA in breast cancer cells.

Translation potential of RNA interference nanotherapeutics remains challenging due to in vivo off-target effects and poor endosomal escape. Here, we developed novel polyplexes for controlled intracellular delivery of dicer substrate siRNA, using a light activation approach. Sulfonated polyethylenimines covalently linked to pyropheophorbide-α for photoactivation and bearing modified amines (sulfo-pyro-PEI) for regulated endosomal escape were investigated. Gene knock-down by the polymer-complexed DsiRNA duplexes (siRNA-NPs) was monitored in breast cancer cells. Surprisingly, sulfo-pyro-PEI/siRNA-NPs failed to downregulate the PLK1 or eGFP proteins. However, photoactivation of these cell associated-polyplexes with a 661-nm laser clearly restored knock-down of both proteins. In contrast, protein down-regulation by non-sulfonated pyro-PEI/siRNA-NPs occurred without any laser treatments, indicating cytoplasmic disposition of DsiRNA followed a common intracellular release mechanism. Therefore, sulfonated pyro-PEI holds potential as a unique trap and release light-controlled delivery platform for on-demand gene silencing bearing minimal off target effects.

Surfactant-Stripped Pheophytin Micelles for Multimodal Tumor Imaging and Photodynamic Therapy.

Porphyrin-based nanomaterials can inherently integrate multiple contrast imaging functionalities with phototherapeutic capabilities. We dispersed pheophytin (Pheo) into Pluronic F127 and carried out low-temperature surfactant-stripping to remove the bulk surfactant. Surfactant-stripped Pheo (ss-Pheo) micelles exhibited a similar size, but higher near-infrared fluorescence, compared to two other nanomaterials also with high porphyrin density (surfactant-stripped chlorophyll micelles and porphysomes). Singlet oxygen generation, which was higher for ss-Pheo, enabled photodynamic therapy (PDT). ss-Pheo provided contrast for photoacoustic and fluorescence imaging, and following seamless labeling with 64Cu, was used for positron emission tomography. ss-Pheo had a long blood circulation and favorable accumulation in an orthotopic murine mammary tumor model. Trimodal tumor imaging was demonstrated, and PDT resulted in delayed tumor growth.

Surfactant-Stripped Micelles for NIR-II Photoacoustic Imaging through 12 cm of Breast Tissue and Whole Human Breasts.

Surfactant-stripped micelles are formed from a commercially available cyanine fluoroalkylphosphate (CyFaP) salt dye and used for high contrast photoacoustic imaging (PAI) in the second near-infrared window (NIR-II). The co-loading of Coenzyme Q10 into surfactant-stripped CyFaP (ss-CyFaP) micelles improves yield, storage stability, and results in a peak absorption wavelength in the NIR-II window close to the 1064 nm output of Nd-YAG lasers used for PAI. Aqueous ss-CyFaP dispersions exhibit intense NIR-II optical absorption, calculated to be greater than 500 at 1064 nm. ss-CyFaP is detected through 12 cm of chicken breast tissue with PAI. In preclinical animal models, ss-CyFaP is visualized in draining lymph nodes of rats through 3.1 cm of overlaid chicken breast tissue. Following intravenous administration, ss-CyFaP accumulates in neoplastic tissues of mice and rats bearing orthotopic mammary tumors without observation of acute toxic side effects. ss-CyFaP is imaged through whole compressed human breasts in three female volunteers at depths of 2.6-5.1 cm. Taken together, these data show that ss-CyFaP is an accessible contrast agent for deep tissue PAI in the NIR-II window.

A malaria vaccine adjuvant based on recombinant antigen binding to liposomes.

Pfs25 is a malaria transmission-blocking vaccine antigen candidate, but its apparently limited immunogenicity in humans has hindered clinical development. Here, we show that recombinant, polyhistidine-tagged (his-tagged) Pfs25 can be mixed at the time of immunization with pre-formed liposomes containing cobalt porphyrin-phospholipid, resulting in spontaneous nanoliposome antigen particleization (SNAP). Antigens are stably presented in uniformly orientated display via his-tag insertion in the cobalt porphyrin-phospholipid bilayer, without covalent modification or disruption of antigen conformation. SNAP immunization of mice and rabbits is well tolerated with minimal local reactogenicity, and results in orders-of-magnitude higher functional antibody generation compared with other 'mix-and-inject' adjuvants. Serum-stable antigen binding during transit to draining lymph nodes leads to enhanced antigen uptake by phagocytic antigen-presenting cells, with subsequent generation of long-lived, antigen-specific plasma cells. Seamless multiplexing with four additional his-tagged Plasmodium falciparum polypeptides induces strong and balanced antibody production, illustrating the simplicity of developing multistage particulate vaccines with SNAP immunization.

Ingestible roasted barley for contrast-enhanced photoacoustic imaging in animal and human subjects.

Photoacoustic computed tomography (PACT) is an emerging imaging modality. While many contrast agents have been developed for PACT, these typically cannot immediately be used in humans due to the lengthy regulatory process. We screened two hundred types of ingestible foodstuff samples for photoacoustic contrast with 1064 nm pulse laser excitation, and identified roasted barley as a promising candidate. Twenty brands of roasted barley were further screened to identify the one with the strongest contrast, presumably based on complex chemical modifications incurred during the roasting process. Individual roasted barley particles could be detected through 3.5 cm of chicken-breast tissue and through the whole hand of healthy human volunteers. With PACT, but not ultrasound imaging, a single grain of roasted barley was detected in a field of hundreds of non-roasted particles. Upon oral administration, roasted barley enabled imaging of the gut and peristalsis in mice. Prepared roasted barley tea could be detected through 2.5 cm chicken breast tissue. When barley tea was administered to humans, photoacoustic imaging visualized swallowing dynamics in healthy volunteers. Thus, roasted barley represents an edible foodstuff that should be considered for photoacoustic contrast imaging of swallowing and gut processes, with immediate potential for clinical translation.

Assessing Photosensitizer Targeting Using Meso-Tetra(Carboxyphenyl) Porphyrin.

Mesotetra(4-carboxyphenyl)porphyrin (mTCPP) is a commercially available small molecule fluorophore and photosensitizer with four free carboxylic acid groups. mTCPP can readily be conjugated with amines for facile attachment of functional groups. In this work, we synthesized and assessed tetravalent, lysine-conjugated mTCPP, for its potential applications in targeted imaging and photodynamic therapy. Fmoc-protected d-lysine or l-lysine was conjugated to mTCPP via amide coupling with the epsilon amine group of lysine, followed by Fmoc deprotection. The resulting compounds did not dissolve well in aqueous solvent, but could be solubilized with the assistance of surfactants, including cholic acid. The l-amino acid transporter (LAT1) can uptake diverse neutral l-amino acids. In vitro studies with U87 cells revealed a non-specific uptake of the hydrophobic Fmoc-protected lysine-conjugated mTCPP precursors, but not d- or l-lysine mTCPP. Likewise, only the Fmoc-protected compounds induced substantial phototoxicty in cells following incubation and irradiation with blue light. These experimental results do not provide evidence to suggest that lysine-mTCPP is able to specifically target cancer cells. However, they do highlight mTCPP as a convenient and accessible framework for assessing molecular targeting of photosensitizers.

Multicolor Liposome Mixtures for Selective and Selectable Cargo Release.

Many approaches exist for stimuli-triggered cargo release from nanocarriers, but few can provide for on-demand release of multiple payloads, selectively. Here, we report the synthesis of purpurin-phospholipid (Pur-P), a lipid chromophore that has near-infrared absorbance red-shifted by 30 nm compared to a structurally similar pyropheophorbide-phospholipid (Pyr-P). Liposomes containing small amounts of either Pur-P or Pyr-P exhibited similar physical properties and fluorescence self-quenching. Loaded with distinct cargos, Pur-P and Pyr-P liposomes were mixed into a single colloidal suspension and selectively released cargo depending on irradiation wavelength. Spatiotemporal control of distinct cargo release was achieved by controlling multicolor laser placement. Using basic orange and doxorubicin anthraquinones, multidimensional cytotoxicity gradients were established to gauge efficacy against cancer cells using light-released drug. Wavelength selectivity of cargo release was maintained following intramuscular administration to mice.

Naphthalocyanines as contrast agents for photoacoustic and multimodal imaging.

Naphthalocyanines (Ncs) are a family of aromatic small molecule with large near infrared extinction coefficients, making them appealing contrast agent candidates for photoacoustic imaging (PAI). Depending on the substitutions on the Nc periphery or metal center, different spectrally-resolved absorption peak wavelengths are possible, which can enable photoacoustic contrast multiplexing. Owing to their generally poor aqueous solubility, approaches have been developed to modify Ncs or formulate them as biocompatible contrast agents for PAI. Due to their inherent capacity for metal ion chelation, Ncs hold potential for complementary multimodal contrast imaging techniques such as 64Cu positron emission tomography. In this research perspective, we highlight some recent reports involving the use of Ncs in PAI.

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.

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.

Deep tissue photoacoustic computed tomography with a fast and compact laser system.

Photoacoustic computed tomography (PACT) holds great promise for biomedical imaging, but wide-spread implementation is impeded by the bulkiness of flash-lamp-pumped laser systems, which typically weigh between 50 - 200 kg, require continuous water cooling, and operate at a low repetition rate. Here, we demonstrate that compact lasers based on emerging diode technologies are well-suited for preclinical and clinical PACT. The diode-pumped laser used in this study had a miniature footprint (13 × 14 × 7 cm3), weighed only 1.6 kg, and outputted up to 80 mJ per pulse at 1064 nm. In vitro, the laser system readily provided over 4 cm PACT depth in chicken breast tissue. In vivo, in addition to high resolution, non-invasive brain imaging in living mice, the system can operate at 50 Hz, which enabled high-speed cross-sectional imaging of murine cardiac and respiratory function. The system also provided high quality, high-frame rate, and non-invasive three-dimensional mapping of arm, palm, and breast vasculature at multi centimeter depths in living human subjects, demonstrating the clinical viability of compact lasers for PACT.

Design of Hydrated Porphyrin-Phospholipid Bilayers with Enhanced Magnetic Resonance Contrast.

Computer simulations are used to design more hydrated bilayers, formed from amine-modified porphyrin-phospholipids (PoPs). Experiments confirm that the new constructs give rise to bilayers with greater water content. When chelated with manganese, amine-modified PoPs provide improved contrast for magnetic resonance and are safely used for imaging in vivo.

Bimodal Targeting Using Sulfonated, Mannosylated PEI for Combined Gene Delivery and Photodynamic Therapy.

Photodynamic therapy (PDT) and gene delivery have both been used to target both cancer cells and tumor-associated macrophages (TAMs). Given the complex nature of tumor tissue, there could be merit in combining these strategies simultaneously. In this study, we developed a bimodal targeting approach to both cancer cells and macrophages, employing materials conducive to both gene delivery and PDT. Polymers libraries were created that consisted of cationic polyethyleneimine (PEI) conjugated to the photosensitizer pyropheophorbide-a, with sulfonation (to target selectin-expressing cells) and mannosylation (to target TAMs). Polyplexes, consisting of these polymers electrostatically bound to DNA, were analyzed for transfection efficacy and cytotoxicity toward epithelial cells and macrophages to assess dual-targeting. This study provides preliminary proof of principle for using modified PEI for targeted gene delivery and PDT.

Surfactant-Stripped Frozen Pheophytin Micelles for Multimodal Gut Imaging.

Edible, surfactant-stripped, frozen micelles are formed from pheophytin (demetallated chlorophyll), a pigment that is naturally consumed in human diets. Pheophytin nanoparticles pass completely and safely through the gastrointestinal tract and enable trimodal gut contrast imaging via photoacoustic, fluorescence, and positron emission tomography techniques.

Axial PEGylation of Tin Octabutoxy Naphthalocyanine Extends Blood Circulation for Photoacoustic Vascular Imaging.

Attachment of polyethylene glycol (PEG) can prolong blood circulation of biological molecules, a useful trait for a vascular imaging agent. Here, we present a route for modifying octabutoxy naphthalocyanine (ONc) with PEG, via axial conjugation following ONc chelation with Sn(IV) chloride (Sn-ONc). Tin chelation caused ONc absorbance to shift from 860 to 930 nm. Hydroxy terminated PEG was treated with sodium and then was axially attached to the tin, generating PEG-Sn-ONc. Unlike ONc or Sn-ONc, PEG-Sn-ONc was soluble in methanol. ONc and PEG-Sn-ONc were dissolved in polysorbate solutions and administered to mice intravenously. PEG-Sn-ONc demonstrated substantially longer blood circulation time than ONc, with a 4 times longer half-life and a nearly 10 times greater area under the curve. PEG-Sn-ONc gave rise to photoacoustic contrast and could be used for noninvasive brain vessel imaging even 24 h following injection. This work demonstrates that nonmetallic naphthalocyanines can be chelated with tin, and be axially modified with PEG for enhanced circulation times for long-term vascular imaging with photoacoustic tomography.

Therapeutic surfactant-stripped frozen micelles.

Injectable hydrophobic drugs are typically dissolved in surfactants and non-aqueous solvents which can induce negative side-effects. Alternatives like 'top-down' fine milling of excipient-free injectable drug suspensions are not yet clinically viable and 'bottom-up' self-assembled delivery systems usually substitute one solubilizing excipient for another, bringing new issues to consider. Here, we show that Pluronic (Poloxamer) block copolymers are amenable to low-temperature processing to strip away all free and loosely bound surfactant, leaving behind concentrated, kinetically frozen drug micelles containing minimal solubilizing excipient. This approach was validated for phylloquinone, cyclosporine, testosterone undecanoate, cabazitaxel and seven other bioactive molecules, achieving sizes between 45 and 160 nm and drug to solubilizer molar ratios 2-3 orders of magnitude higher than current formulations. Hypertonic saline or co-loaded cargo was found to prevent aggregation in some cases. Use of surfactant-stripped micelles avoided potential risks associated with other injectable formulations. Mechanistic insights are elucidated and therapeutic dose responses are demonstrated.

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.