Laser-activated perfluorocarbon nanodroplets for intracerebral delivery and imaging via blood-brain barrier opening and contrast-enhanced imaging.

BACKGROUND: Ultrasound and photoacoustic (US/PA) imaging is a promising tool for in vivo visualization and assessment of drug delivery. However, the acoustic properties of the skull limit the practical application of US/PA imaging in the brain. To address the challenges in targeted drug delivery to the brain and transcranial US/PA imaging, we introduce and evaluate an intracerebral delivery and imaging strategy based on the use of laser-activated perfluorocarbon nanodroplets (PFCnDs). METHODS: Two specialized PFCnDs were developed to facilitate blood‒brain barrier (BBB) opening and contrast-enhanced US/PA imaging. In mice, PFCnDs were delivered to brain tissue via PFCnD-induced BBB opening to the right side of the brain. In vivo, transcranial US/PA imaging was performed to evaluate the utility of PFCnDs for contrast-enhanced imaging through the skull. Ex vivo, volumetric US/PA imaging was used to characterize the spatial distribution of PFCnDs that entered brain tissue. Immunohistochemical analysis was performed to confirm the spatial extent of BBB opening and the accuracy of the imaging results. RESULTS: In vivo, transcranial US/PA imaging revealed localized photoacoustic (PA) contrast associated with delivered PFCnDs. In addition, contrast-enhanced ultrasound (CEUS) imaging confirmed the presence of nanodroplets within the same area. Ex vivo, volumetric US/PA imaging revealed PA contrast localized to the area of the brain where PFCnD-induced BBB opening had been performed. Immunohistochemical analysis revealed that the spatial distribution of immunoglobulin (IgG) extravasation into the brain closely matched the imaging results. CONCLUSIONS: Using our intracerebral delivery and imaging strategy, PFCnDs were successfully delivered to a targeted area of the brain, and they enabled contrast-enhanced US/PA imaging through the skull. Ex vivo imaging, and immunohistochemistry confirmed the accuracy and precision of the approach.

Coupling Gold Nanospheres into Nanochain Constructs for High-Contrast, Longitudinal Photoacoustic Imaging.

Structural parameters play a crucial role in determining the electromagnetic and thermal responses of gold nanoconstructs (GNCs) at near-infrared (NIR) wavelengths. Therefore, developing GNCs for reliable, high-contrast photoacoustic imaging has been focused on adjusting structural parameters to achieve robust NIR light absorption with photostability. In this study, we introduce an efficient photoacoustic imaging contrast agent: gold sphere chains (GSCs) consisting of plasmonically coupled gold nanospheres. The chain geometry results in enhanced photoacoustic signal generation originating from outstanding photothermal characteristics compared to traditional gold contrast agents, such as gold nanorods. Furthermore, the GSCs produce consistent photoacoustic signals at laser fluences within the limits set by the American National Standards Institute. The exceptional photoacoustic response of GSCs allows for high-contrast photoacoustic imaging over multiple imaging sessions. Finally, we demonstrate the utility of our GSCs for molecular photoacoustic cancer imaging, both in vitro and in vivo, through the integration of a tumor-targeting moiety.

Magnetically Steered Cell Therapy For Functional Restoration Of Intraocular Pressure Control In Open-Angle Glaucoma.

Trabecular meshwork (TM) cell therapy has been proposed as a next-generation treatment for elevated intraocular pressure (IOP) in glaucoma, the most common cause of irreversible blindness. Using a magnetic cell steering technique with excellent efficiency and tissue-specific targeting, we delivered two types of cells into a mouse model of glaucoma: either human adipose-derived mesenchymal stem cells (hAMSCs) or induced pluripotent cell derivatives (iPSC-TM cells). We observed a 4.5 [3.1, 6.0] mmHg or 27% reduction in intraocular pressure (IOP) for nine months after a single dose of only 1500 magnetically-steered hAMSCs, associated with restoration of function to the conventional outflow pathway, as judged by increased outflow facility and TM cellularity. iPSC-TM cells were also effective, but less so, showing only a 1.9 [0.4, 3.3] mmHg or 13% IOP reduction and increased risk of tumorigenicity. In both cases, injected cells remained detectable in the iridocorneal angle three weeks post-transplantation. Based on the locations of the delivered cells, the mechanism of IOP lowering is most likely paracrine signaling. We conclude that magnetically-steered hAMSC cell therapy has potential for long-term treatment of ocular hypertension in glaucoma. One Sentence Summary: A novel magnetic cell therapy provided effective intraocular pressure control in a mouse model of glaucoma, motivating future translational studies.

In Vivo Photoacoustic Monitoring of Stem Cell Location and Apoptosis with Caspase-3-Responsive Nanosensors.

Stem cell therapy has immense potential in a variety of regenerative medicine applications. However, clinical stem cell therapy is severely limited by challenges in assessing the location and functional status of implanted cells in vivo. Thus, there is a great need for longitudinal, noninvasive stem cell monitoring. Here we introduce a multidisciplinary approach combining nanosensor-augmented stem cell labeling with ultrasound guided photoacoustic (US/PA) imaging for the spatial tracking and functional assessment of transplanted stem cell fate. Specifically, our nanosensor incorporates a peptide sequence that is selectively cleaved by caspase-3, the primary effector enzyme in mammalian cell apoptosis; this cleavage event causes labeled cells to show enhanced optical absorption in the first near-infrared (NIR) window. Optimization of labeling protocols and spectral characterization of the nanosensor in vitro showed a 2.4-fold increase in PA signal from labeled cells during apoptosis while simultaneously permitting cell localization. We then successfully tracked the location and apoptotic status of mesenchymal stem cells in a mouse hindlimb ischemia model for 2 weeks in vivo, demonstrating a 4.8-fold increase in PA signal and spectral slope changes in the first NIR window under proapoptotic (ischemic) conditions. We conclude that our nanosensor allows longitudinal, noninvasive, and nonionizing monitoring of stem cell location and apoptosis, which is a significant improvement over current end-point monitoring methods such as biopsies and histological staining of excised tissue.

pH-responsive ratiometric photoacoustic imaging of polyaniline nanoparticle-coated needle for targeted cancer biopsy.

Cancer microenvironment exhibits lower pH compared to healthy tissues, a characteristic which can be exploited using a pH-responsive needle to increase the accuracy of cancer biopsy. A needle, coated with pH-responsive polyaniline (PANI) nanoparticles (PANI-needle), is developed for the minimally invasive and quantitative pH analysis of tissue based on ratiometric photoacoustic (PA) imaging. The ratiometric PA signal from the PANI-needle within the 850-700 nm wavelength range shows a linear response as pH changes from 7.5 to 6.5. Owing to the high surface area of nanostructured PANI, the PA signal of PANI-needle exhibits a fast and reversible response of less than a few seconds. In a tissue-mimicking hydrogel phantom composed of two regions with different pH, PA ratios of PANI-needle successfully differentiate the local pH. The PANI-needle coupled with ultrasound-guided PA imaging is a promising technology for detection of malignant tissue through quantitative pH analysis during needle biopsy.

Tunable Interparticle Connectivity in Gold Nanosphere Assemblies for Efficient Photoacoustic Conversion.

Manipulating matter at the nanometer scale to create desired plasmonic nanostructures holds great promise in the field of biomedical photoacoustic (PA) imaging. We demonstrate a strategy for regulating PA signal generation from anisotropic nano-sized assemblies of gold nanospheres (Au NSs) by adjusting the inter-particle connectivity between neighboring Au NSs. The inter-particle connectivity is controlled by modulating the diameter and inter-particle spacing of Au NSs in the nanoassemblies. The results indicate that nanoassemblies with semi-connectivity, i.e., assemblies with a finite inter-particle spacing shorter than the theoretical limit of repulsion between nearby Au NSs, exhibit 3.4-fold and 2.4-fold higher PA signals compared to nanoassemblies with no connectivity and full connectivity, respectively. Furthermore, due to the reduced diffusion of Au atoms, the semi-connectivity Au nanoassemblies demonstrate high photodamage threshold and, therefore, excellent photostability at fluences above the current American National Standards Institute limits. The exceptional photostability of the semi-connectivity nanoassemblies highlights their potential to surpass conventional plasmonic contrast agents for continuing PA imaging. Collectively, our findings indicate that semi-connected nanostructures are a promising option for reliable, high-contrast PA imaging applications over multiple imaging sessions due to their strong PA signals and enhanced photostability.

Copper Sulfide Nanodisks and Nanoprisms for Photoacoustic Ovarian Tumor Imaging.

Transvaginal ultrasound is widely used for ovarian cancer screening but has a high false positive rate. Photoacoustic imaging provides additional optical contrast to supplement ultrasound and might be able to improve the accuracy of screening. Here, we report two copper sulfide (CuS) nanoparticles types (nanodisks and triangular nanoprisms) as the photoacoustic contrast agents for imaging ovarian cancer. Both CuS nanoprisms and nanodisks were ~6 nm thick and ~26 nm wide and were coated with poly(ethylene glycol) to make them colloidally stable in phosphate buffered saline (PBS) for at least 2 weeks. The CuS nanodisks and nanoprisms revealed strong localized surface plasmon resonances with peak maxima at 1145 nm and 1098 nm, respectively. Both nanoparticles types had strong and stable photoacoustic intensity with detection limits below 120 pM. The circular CuS nanodisk remained in the circulation of nude mice (n=4) and xenograft 2008 ovarian tumors (n=4) 17.9-fold and 1.8-fold more than the triangular nanoprisms, respectively. Finally, the photoacoustic intensity of the tumors from the mice (n=3) treated with CuS nanodisks was 3.0-fold higher than the baseline. The tumors treated with nanodisks had a characteristic peak at 920 nm in the spectrum to potentially differentiate the tumor from adjacent tissues.

A Mechanistic Investigation of Methylene Blue and Heparin Interactions and Their Photoacoustic Enhancement.

We recently reported a real-time method to measure heparin in human whole blood based on the photoacoustic change of methylene blue (MB). Intriguingly, the MB behaved unlike other "turn on" photoacoustic probes-the absorbance decreased as the photoacoustic signal increased. The underlying mechanism was not clear and motivated this study. We studied the binding mechanism of MB and heparin in water and phosphate buffer saline (PBS) with both experimental and computational methods. We found that the photoacoustic enhancement of the MB-heparin mixture was a result of MB-heparin aggregation due to charge neutralization and resulting sequestration of MB in these aggregates. The sequestration of MB in the MB-heparin aggregates led to decreased absorbance-there was simply less free dye in solution to absorb light. The highest photoacoustic signal and aggregation occurred when the number of negatively charged sulfate groups on heparin was approximately equal to the number of positively charged MB molecule. The MB-heparin aggregates dissociated when there were more sulfated groups from heparin than MB molecules because of the electrostatic repulsion between negatively charged sulfate groups. PBS facilitated MB dimer formation regardless of heparin concentration and reprecipitated free MB in aggregates due to ionic strength and ionic shielding. Further molecular dynamics experiments found that binding of heparin occurred at the sulfates and glucosamines in heparin. Phosphate ions could interact with the heparin via sodium ions to impair the MB-heparin binding. Finally, our model found 3.7-fold more MB dimerization upon addition of heparin in MB solution confirming that heparin facilitates MB aggregation. We conclude that the addition of heparin in MB decreases the absorbance of the sample because of MB-heparin aggregation leading to fewer MB molecules in solution; however, the aggregation also increases the PA intensity because the MB molecules in the MB-heparin aggregate have reduced degrees of freedom and poor heat transfer to solvent.

In vivo photoacoustic imaging of chorioretinal oxygen gradients.

Chorioretinal imaging has a crucial role for the patients with chorioretinal vascular diseases, such as neovascular age-related macular degeneration. Imaging oxygen gradients in the eye could better diagnose and treat ocular diseases. Here, we describe the use of photoacoustic ocular imaging (PAOI) in measuring chorioretinal oxygen saturation (CR - sO2) gradients in New Zealand white rabbits (n = 5) with ocular ischemia. We observed good correlation (R2 = 0.98) between pulse oximetry and PAOI as a function of different oxygen percentages in inhaled air. We then used an established ocular ischemia model in which intraocular pressure is elevated to constrict ocular blood flow, and notice a positive correlation (R2 = 0.92) between the injected volume of phosphate buffered saline (PBS) and intraocular pressure (IOP) as well as a negative correlation (R2 = 0.98) between CR - sO2 and injected volume of PBS. The CR - sO2 was measured before (baseline), during (ischemia), and after the infusion (600-µL PBS). The ischemia-reperfusion model did not affect the measurement of the sO2 using a pulse oximeter on the animal's paw, but the chorioretinal PAOI signal showed a nearly sixfold decrease in CR - sO2 (n = 5, p = 0.00001). We also observe a sixfold decrease in CR - sO2 after significant elevation of IOP during ischemia, with an increase close to baseline during reperfusion. These data suggest that PAOI can detect changes in chorioretinal oxygenation and may be useful for application to imaging oxygen gradients in ocular disease.

A Wearable Colorimetric Dosimeter to Monitor Sunlight Exposure.

The personal ultraviolet (UV) dosimeter is a useful measurement tool to prevent UV induced dermal damages; however, conventional digital dosimeters are either bulky or require external power sources. Here, a wearable, colorimetric UV film dosimeter that provides color transition, from purple to transparent, is reported to indicate the UV dose. The film dosimeter is made of a purple photodegradable dye ((2Z,6Z)-2,6-bis(2-(2,6-diphenyl-4H-thiopyran-4-ylidene)ethylidene)cyclohexanone or DTEC) blended in low density polyethylene film. The DTEC film discolored 3.3 times more under the exposure of UV light (302 nm) than visible light (543 nm), and a UV bandpass filter is developed to increase this selectivity to UV light. The DTEC film completely discolors to transparency in 2 h under an AM 1.5 solar simulator, suggesting the potential as an indicator for individuals with types I-VI skin to predict interventions to avoid sunburn. Finally, the DTEC film is integrated with the UV bandpass filter on a wristband to function as a wearable dosimeter for low cost and convenient monitoring of sunlight exposure.

Switchable Photoacoustic Intensity of Methylene Blue via Sodium Dodecyl Sulfate Micellization.

The interaction between methylene blue (MB) and sodium dodecyl sulfate (SDS) has been widely studied spectroscopically, but details about their interactions remain unclear. Here, we combined photoacoustic (PA) imaging with nanoparticle tracking analysis (NTA) and spectroscopy to further elucidate this interaction. PA imaging of 0.05 mM MB showed a 492-fold increase in intensity upon the addition of 3.47 mM SDS. Higher concentrations above SDS's critical micelle concentration (CMC) at 8.67 mM decreased the PA intensity by 54 times. Relative quantum yield measurements indicated that PA intensity increased as a result of fluorescence quenching. Meanwhile, NTA indicated an increased number of nonmicellar MB/SDS clusters at SDS concentrations below the CMC varying in size from 80 to 400 nm as well as a decreased number above the CMC. This trend suggested that MB/SDS clusters are responsible for the PA intensity enhancement. Comparison of PA intensities and spectral shifts with MB/hexadecyltrimethylammonium bromide, MB/sodium octyl sulfate, and MB/sodium chloride demonstrated that MB was bound to the sulfate moiety of SDS before and after micellization. Our observations suggest that MB forms aggregates with SDS at premicellar concentrations, and the MB aggregates disassociate as monomers that are bound to the sulfate moiety of SDS at micellar concentrations. These findings further clarify the process by which MB and SDS interact and demonstrate the potential for developing MB-/SDS-based contrast agents.

Exosome-like silica nanoparticles: a novel ultrasound contrast agent for stem cell imaging.

Ultrasound is critical in many areas of medicine including obstetrics, oncology, and cardiology with emerging applications in regenerative medicine. However, one critical limitation of ultrasound is the low contrast of target tissue over background. Here, we describe a novel cup-shaped silica nanoparticle that is reminiscent of exosomes and that has significant ultrasound impedance mismatch for labelling stem cells for regenerative medicine imaging. These exosome-like silica nanoparticles (ELS) were created through emulsion templating and the silica precursors bis(triethoxysilyl)ethane (BTSE) and bis(3-trimethoxysilyl-propyl)amine (TSPA). We found that 40% TSPA resulted in the exosome like-morphology and a positive charge suitable for labelling mesenchymal stem cells. We then compared this novel structure to other silica structures used in ultrasound including Stober silica nanoparticles (SSN), MCM-41 mesoporous silica nanoparticles (MSN), and mesocellular foam silica nanoparticles (MCF) and found that the ELS offered enhanced stem cell signal due to its positive charge to facilitate cell uptake as well as inherently increased echogenicity. The in vivo detection limits were <500 cells with no detectable toxicity at the concentrations used for labelling. This novel structure may eventually find utility in applications beyond imaging requiring an exosome-like shape including drug delivery.