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.

Barium sulfate and pigment admixture for photoacoustic and x-ray contrast imaging of the gut.

Significance: X-ray imaging is frequently used for gastrointestinal imaging. Photoacoustic imaging (PAI) of the gastrointestinal tract is an emerging approach that has been demonstrated for preclinical imaging of small animals. A contrast agent active in both modalities could be useful for imaging applications. Aim: We aimed to develop a dual-modality contrast agent comprising an admixture of barium sulfate with pigments that absorb light in the second near-infrared region (NIR-II), for preclinical imaging with both x-ray and PAI modalities. Approach: Eleven different NIR-II dyes were evaluated after admixture with a 40% w/v barium sulfate mixture. The resulting NIR-II absorption in the soluble fraction and in the total mixture was characterized. Proof-of-principle imaging studies in mice were carried out. Results: Pigments that produced more uniform suspensions were assessed further for photoacoustic contrast signal at a wavelength of 1064 nm that corresponds to the output of the Nd:YAG laser used. Phantom imaging studies demonstrated that the pigment-barium sulfate mixture generated imaging contrast in both x-ray and PAI modalities. The optimal pigment selected for further study was a cyanine tetrafluoroborate salt. Ex-vivo and whole-body mouse imaging demonstrated that photoacoustic and x-ray contrast signals co-localized in the intestines for both imaging modalities. Conclusion: These data demonstrate that commercially-available NIR-II pigments can simply be admixed with barium sulfate to generate a dual-modality contrast agent appropriate for small animal gastrointestinal imaging.

Intraperitoneal administration for sustained photoacoustic contrast agent imaging.

Photoacoustic (PA) imaging at 1064 nm in the second near-infrared (NIR-II) has attracted recent attention. We recently reported a surfactant-based formulation of a NIR-II dye (BIBDAH) for NIR-II PA contrast. Here, we investigated BIBDAH as a NIR-II PA contrast agent for longitudinal preclinical PA imaging. When administered to mice by the conventional intravenous (I.V.) route, BIBDAH was rapidly cleared from circulation, as indicated by a decrease in NIR-II absorption in sampled plasma. Conversely, when mice were injected with BIBDAH by the intraperitoneal (I.P.) route, peak NIR-II absorption levels in plasma were lower initially, but there was a sustained dye presence that resulted in a more consistent concentration of dye in plasma over 2 days. Increasing the I.P. injection dose and volume resulted in increased NIR-II area under the curve (AUC) in serum. Bimodal PA and ultrasound imaging reflected these results, showing a rapid decrease in PA signal in blood with I.V. administration, but permitting sustained imaging with I.P. administration. These results show that I.P. administration can be considered as an administration route in preclinical animal studies for improved longitudinal observation with more consistent contrast signal intensity.

Three-Dimensional Deep-Tissue Functional and Molecular Imaging by Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT).

Non-invasive small-animal imaging technologies, such as optical imaging, magnetic resonance imaging and x -ray computed tomography, have enabled researchers to study normal biological phenomena or disease progression in their native conditions. However, existing small-animal imaging technologies often lack either the penetration capability for interrogating deep tissues (e.g., optical microscopy), or the functional and molecular sensitivity for tracking specific activities (e.g., magnetic resonance imaging). To achieve functional and molecular imaging in deep tissues, we have developed an integrated photoacoustic, ultrasound and acoustic angiographic tomography (PAUSAT) system by seamlessly combining light and ultrasound. PAUSAT can perform three imaging modes simultaneously with complementary contrast: high-frequency B-mode ultrasound imaging of tissue morphology, microbubble-enabled acoustic angiography of tissue vasculature, and multi-spectral photoacoustic imaging of molecular probes. PAUSAT can provide three-dimensional (3D) multi-contrast images that are co-registered, with high spatial resolutions at large depths. Using PAUSAT, we performed proof-of-concept in vivo experiments on various small animal models: monitoring longitudinal development of placenta and embryo during mouse pregnancy, tracking biodistribution and metabolism of near-infrared organic dye on the whole-body scale, and detecting breast tumor expressing genetically-encoded photoswitchable phytochromes. These results have collectively demonstrated that PAUSAT has broad applicability in biomedical research, providing comprehensive structural, functional, and molecular imaging of small animal models.

Light-Triggered Release of Large Biomacromolecules from Porphyrin-Phospholipid Liposomes.

Liposomes containing small amounts of porphyrin-phospholipid (PoP) have been shown to encapsulate small molecular weight cargos and then release them upon exposure to red light. A putative mechanism involves transient pore formation in the bilayer induced by PoP-mediated photo-oxidation of unsaturated lipids. However, little is known about the properties of such pores. This study assesses whether large carbohydrate and protein molecules could be released from PoP liposomes upon red light exposure. A small fluorophore with ∼0.5 kDa in molecular weight, fluorescently labeled dextrans of ∼5 and ∼500 kDa, and a ∼240 kDa fluorescent protein were passively entrapped in PoP liposomes. When exposed to 665 nm irradiation, liposomes containing PoP, but not liposomes lacking it, released all these cargos in a size-dependent manner that occurred with oxidation of unsaturated lipids included in the bilayer. Thus, this study demonstrates the feasibility of light-triggered release of large biomacromolecules from liposomes.

Cross-linked Histone as a Nanocarrier for Gut Delivery of Hydrophobic Cargos.

Delivering hydrophobic molecules through the intestine can be challenging due to limited cargo solubility and the harsh biochemical environment of the stomach. Here, we show that a protein-based nanocarrier system based on the abundant protein histone and the natural cross-linker genipin can deliver hydrophobic cargos, such as dyes and therapeutic molecules, through the gastrointestinal tract. Using hydrophobic near-infrared dyes as model cargos, a panel of potential protein carriers was screened, and histone was identified as the one with the best loading capability. The resulting nanoparticles had a positive ζ potential and were mucoadhesive. Cross-linking of the amine-rich nanocarrier with genipin was particularly effective relative to other proteins and increased the stability of the system during incubation with pepsin. Cross-linking was required for successful delivery of a hydrophobic dye to the colon of mice after oral gavage. To assess the platform for therapeutic delivery, another hydrophobic model compound, curcumin, was delivered using cross-linked histone nanoparticles in a murine colitis model and significantly alleviated the disease. Taken together, these results demonstrate that histone is a cationic, mucoadhesive, and cross-linkable protein nanocarrier that can be considered for oral delivery.

Sound Out the Deep Colors: Photoacoustic Molecular Imaging at New Depths.

Photoacoustic tomography (PAT) has become increasingly popular for molecular imaging due to its unique optical absorption contrast, high spatial resolution, deep imaging depth, and high imaging speed. Yet, the strong optical attenuation of biological tissues has traditionally prevented PAT from penetrating more than a few centimeters and limited its application for studying deeply seated targets. A variety of PAT technologies have been developed to extend the imaging depth, including employing deep-penetrating microwaves and X-ray photons as excitation sources, delivering the light to the inside of the organ, reshaping the light wavefront to better focus into scattering medium, as well as improving the sensitivity of ultrasonic transducers. At the same time, novel optical fluence mapping algorithms and image reconstruction methods have been developed to improve the quantitative accuracy of PAT, which is crucial to recover weak molecular signals at larger depths. The development of highly-absorbing near-infrared PA molecular probes has also flourished to provide high sensitivity and specificity in studying cellular processes. This review aims to introduce the recent developments in deep PA molecular imaging, including novel imaging systems, image processing methods and molecular probes, as well as their representative biomedical applications. Existing challenges and future directions are also discussed.

Facile formulation of a long-wavelength cyanine for optical imaging in the second near-infrared window.

The second near-infrared window (NIR-II) beyond 1000 nm has attracted attention for optical contrast imaging in small animals. We sought to assess whether commercially available NIR-II dyes could be easily formulated for this purpose. 13 hydrophobic NIR-II dyes were purchased and screened by formulating them in simple solubilizing agents with established use in humans: propylene glycol, Cremaphor EL, Kolliphor HS15 (HS15), Tween 80, and cyclodextrin. Based on the absorption at 1064 nm (matching the Nd:YAG laser output commonly used in photoacoustic imaging), three of the dyes were further assessed at varying dye and surfactant concentrations. Of these, benzo indole butyl diphenylaminocyclopentene heptamethine (BIBDAH) tetrafluoroborate in HS15 generally showed the most favorable NIR-II character. 1 mg mL-1 BIBDAH in 25% HS15 exhibited a single absorption peak at 1030 nm with a calculated intensity greater than 100, which was relatively stable for weeks in storage. Following intravenous administration to mice, determination of BIBDAH pharmacokinetics was possible by absorption measurements of sampled plasma, revealing a circulating half-life of about one hour. Most of the dye was taken up by the liver. BIBDAH was used in vitro and in vivo as a photoacoustic contrast imaging agent and its accumulation could be detected in subcutaneous tumors in mice. BIBDAH was used for fluorescence imaging of blood vessels in mice, including in the brain (through intact skull), and dye clearance from blood to the liver was visualized. Taken together, this study confirms that accessible, strongly-absorbing dye can readily be formulated for injection by simply dissolving them in biocompatible surfactants and used for high-contrast preclinical optical imaging in the second NIR window.

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.

Highly-Soluble Cyanine J-aggregates Entrapped by Liposomes for In Vivo Optical Imaging around 930 nm.

Near infrared (NIR) dyes are useful for in vivo optical imaging. Liposomes have been used extensively for delivery of diverse cargos, including hydrophilic cargos which are passively loaded in the aqueous core. However, most currently available NIR dyes are only slightly soluble in water, making passive entrapment in liposomes challenging for achieving high optical contrast. Methods: We modified a commercially-available NIR dye (IR-820) via one-step Suzuki coupling with dicarboxyphenylboronic acid, generating a disulfonated heptamethine; dicarboxyphenyl cyanine (DCP-Cy). DCP-Cy was loaded in liposomes and used for optical imaging. Results: Owing to increased charge in mildly basic aqueous solution, DCP-Cy had substantially higher water solubility than indocyanine green (by an order of magnitude), resulting in higher NIR absorption. Unexpectedly, DCP-Cy tended to form J-aggregates with pronounced spectral red-shifting to 934 nm (from 789 nm in monomeric form). J-aggregate formation was dependent on salt and DCP-Cy concentration. Dissolved at 20 mg/mL, DCP-Cy J-aggregates could be entrapped in liposomes. Full width at half maximum absorption of the liposome-entrapped dye was just 25 nm. The entrapped DCP-Cy was readily detectable by fluorescence and photoacoustic NIR imaging. Upon intravenous administration to mice, liposomal DCP-Cy circulated substantially longer than the free dye. Accumulation was largely in the spleen, which was visualized with fluorescence and photoacoustic imaging. Conclusions: DCP-Cy is simple to synthesize and exhibits high aqueous solubility and red-shifted absorption from J-aggregate formation. Liposomal dye entrapment is possible, which facilitates in vivo photoacoustic and fluorescence imaging around 930 nm.