Surface-enhanced Raman spectroscopy with single cell manipulation by microfluidic dielectrophoresis.

When exposed to an alternating current (AC) electric field, a polarized microparticle is moved by the interaction between the voltage-induced dipoles and the AC electric field under dielectrophoresis (DEP). The DEP force is widely used for manipulation of microparticles in diverse practical applications such as 3D manipulation, sorting, transfer, and separation of various particles such as living cells. In this study, we propose the integration of surface-enhanced Raman spectroscopy (SERS), an extremely sensitive and versatile technique based on the Raman scattering of molecules supported by nanostructured materials, with DEP using a microfluidic device. The microfluidic device combines microelectrodes with gold nanohole arrays to characterize the electrophysiological and biochemical properties of biological cells. The movement of particles, which varies depending on the electrical properties such as conductivity and permittivity of particles, can be manipulated by the cross-frequency change. For proof of concept, Raman spectroscopy using the DEP-SERS integration was performed for polystyrene beads and biological cells and resulted in an improved signal-to-noise ratio by determining the direction of the DEP force applied to the cells with respect to the applied AC power and collecting them on the nanohole arrays. The result illustrates the potential of the concept for simultaneously examining the electrical and biochemical properties of diverse chemical and biological microparticles in the microfluidic environment.

Antibody-Conjugated Polymersomes with Encapsulated Indocyanine Green J-Aggregates and High Near-Infrared Absorption for Molecular Photoacoustic Cancer Imaging.

Imaging plays a critical role in all stages of cancer care from early detection to diagnosis, prognosis, and therapy monitoring. Recently, photoacoustic imaging (PAI) has started to emerge into the clinical realm due to its high sensitivity and ability to penetrate tissues up to several centimeters deep. Herein, we encapsulated indocyanine green J (ICGJ) aggregate, one of the only FDA-approved organic exogenous contrast agents that absorbs in the near-infrared range, at high loadings up to ∼40% w/w within biodegradable polymersomes (ICGJ-Ps) composed of poly(lactide-co-glycolide-b-polyethylene glycol) (PLGA-b-PEG). The small Ps hydrodynamic diameter of 80 nm is advantageous for in vivo applications, while directional conjugation with epidermal growth factor receptor (EGFR) targeting cetuximab antibodies renders molecular specificity. Even when exposed to serum, the ∼11 nm-thick membrane of the Ps prevents dissociation of the encapsulated ICGJ for at least 48 h with a high ratio of ICGJ to monomeric ICG absorbances (i.e., I895/I780 ratio) of approximately 5.0 that enables generation of a strong NIR photoacoustic (PA) signal. The PA signal of polymersome-labeled breast cancer cells is proportional to the level of cellular EGFR expression, indicating the feasibility of molecular PAI with antibody-conjugated ICGJ-Ps. Furthermore, the labeled cells were successfully detected with PAI in highly turbid tissue-mimicking phantoms up to a depth of 5 mm with the PA signal proportional to the amount of cells. These data show the potential of molecular PAI with ICGJ-Ps for clinical applications such as tumor margin detection, evaluation of lymph nodes for the presence of micrometastasis, and laparoscopic imaging procedures.

Volume-rendering three-dimensional image analysis of macular neovascularization in age-related macular degeneration.

BACKGROUND: To visualize and investigate the three-dimensional (3D) images of macular neovascularization (MNV) in eyes with neovascular age-related macular degeneration using optical coherence tomography angiography (OCTA) according to the treatment response to intravitreal aflibercept injection (IVI). METHODS: OCTA images at baseline and 12 weeks (after three loading IVIs) were retrospectively reconstructed as 3D images for patients with type 1 and 2 MNV treated with the "pro-re-nata" regimen. The fluid-free and persistent fluid groups were divided according to the presence of subretinal and intraretinal fluid at 12 weeks after treatment. Using reconstructed 3D images of MNV, the volume, average volume per slice, and z-axis of the volumetric structure were evaluated. RESULTS: Twenty-three and nine were classified into the fluid-free and persistent fluid groups, respectively. The MNV volume decreased significantly from baseline to 12 weeks in the fluid-free group (p = 0.005), not in the persistent fluid group (p = 0.250). The average volume of MNV per slice at 12 weeks correlated with the persistent fluid group in both the univariate and multivariate analyses (p = 0.034, p = 0.039, Exp [B] = 14.005). CONCLUSIONS: This study may provide a perspective on vascular volumetric changes of MNV according to treatment response.

Endogenous Neural Stem Cell Activation after Low-Intensity Focused Ultrasound-Induced Blood-Brain Barrier Modulation.

Endogenous neural stem cells (eNSCs) in the adult brain, which have the potential to self-renew and differentiate into functional, tissue-appropriate cell types, have raised new expectations for neurological disease therapy. Low-intensity focused ultrasound (LIFUS)-induced blood-brain barrier modulation has been reported to promote neurogenesis. Although these studies have reported improved behavioral performance and enhanced expression of brain biomarkers after LIFUS, indicating increased neurogenesis, the precise mechanism remains unclear. In this study, we evaluated eNSC activation as a mechanism for neurogenesis after LIFUS-induced blood-brain barrier modulation. We evaluated the specific eNSC markers, Sox-2 and nestin, to confirm the activation of eNSCs. We also performed 3'-deoxy-3'[18F] fluoro-L-thymidine positron emission tomography ([18F] FLT-PET) to evaluate the activation of eNSCs. The expression of Sox-2 and nestin was significantly upregulated 1 week after LIFUS. After 1 week, the upregulated expression decreased sequentially; after 4 weeks, the upregulated expression returned to that of the control group. [18F] FLT-PET images also showed higher stem cell activity after 1 week. The results of this study indicated that LIFUS could activate eNSCs and induce adult neurogenesis. These results show that LIFUS may be useful as an effective treatment for patients with neurological damage or neurological disorders in clinical settings.

Clinically translatable quantitative molecular photoacoustic imaging with liposome-encapsulated ICG J-aggregates.

Photoacoustic (PA) imaging is a functional and molecular imaging technique capable of high sensitivity and spatiotemporal resolution at depth. Widespread use of PA imaging, however, is limited by currently available contrast agents, which either lack PA-signal-generation ability for deep imaging or their absorbance spectra overlap with hemoglobin, reducing sensitivity. Here we report on a PA contrast agent based on targeted liposomes loaded with J-aggregated indocyanine green (ICG) dye (i.e., PAtrace) that we synthesized, bioconjugated, and characterized to addresses these limitations. We then validated PAtrace in phantom, in vitro, and in vivo PA imaging environments for both spectral unmixing accuracy and targeting efficacy in a folate receptor alpha-positive ovarian cancer model. These study results show that PAtrace concurrently provides significantly improved contrast-agent quantification/sensitivity and SO2 estimation accuracy compared to monomeric ICG. PAtrace's performance attributes and composition of FDA-approved components make it a promising agent for future clinical molecular PA imaging.

Fate of Antibody-Targeted Ultrasmall Gold Nanoparticles in Cancer Cells after Receptor-Mediated Uptake.

Nanoparticles with ultrasmall sizes (less than 10 nm) offer many advantages in biomedical applications compared to their bigger counterparts, including better intratumoral distribution, improved pharmacokinetics (PK), and efficient body clearance. When functionalized with a biocompatible coating and a target-specific antibody, ultrasmall nanoparticles represent an attractive clinical translation platform. Although there is a tremendous body of work dedicated to PK and the biological effects of various nanoparticles, little is known about the fate of different components of functionalized nanoparticles in a biological environment such as in live cells. Here, we used luminescence properties of 5 nm gold nanoparticles (AuNPs) to study the intracellular trafficking and fate of the AuNPs functionalized with an organic layer consisting of a polyethylene glycol (PEG) coating and epidermal growth factor receptor (EGFR)-targeting antibody. We showed that intracellular uptake of the targeted 5 nm AuNPs results in a strong two-photon luminescence (TPL) that is characterized by broad emission and very short lifetimes compared to the fluorescence of the nanoparticle-conjugated fluorophore-tagged antibody, thereby allowing selective imaging of these components using TPL and two-photon excited fluorescence lifetime microscopy (2P-FLIM). Our results indicate that the nanoparticle's coating is detached from the particle's surface inside cells, leading to formation of nanoparticle clusters with a strong TPL. Furthermore, we observed an optically resolved spatial separation of the gold core and the antibody coating of the particles inside cells. We used data from two-photon microscopy, 2P-FLIM, electron microscopy, and in vitro assays to propose a model of interactions of functionalized 5 nm AuNPs with live cells.

Suspended scattering particles in motion using OCT angiography in branch retinal vein occlusion disease cases with cystoid macular edema.

We aimed to investigate the clinical implication of suspended scattering particles in motion (SSPiM) using optical coherence tomography angiography (OCTA) among branch retinal vein occlusion disease (BRVO) cases with macular edema (ME). Medical records of BRVO patients were reviewed. Central retinal thickness (CRT), ME type, and cyst size on optical coherence tomography images were evaluated before and after intravitreal bevacizumab injection. Nonperfusion area, SSPiM, and microvascular abnormalities in OCTA images were evaluated using a Heidelberg machine. SSPiM was identified in 24 of 56 cases. There were no differences in baseline characteristics between groups with and without SSPiM. Disease duration, disease-free duration, previous injection number, microaneurysms in the superficial vascular complex, and microaneurysms in the deep vascular complex (DVC) (p = 0.003, 0.013, 0.028, 0.003, < 0.001, respectively) differed significantly between the two groups. After multivariate logistic analysis, microaneurysms in the DVC were the only different factor between the two groups (odds ratio [OR]: 0.091; p = 0.001). Furthermore, SSPiM in the DVC (OR 10.908; p = 0.002) and nonperfusion grade (OR 0.039; p < 0.001) were significantly associated with cyst response after intravitreal injection. SSPiM may be correlated with microaneurysms in the DVC and a poor anatomical response after intravitreal injection.

Clinical Implications of Suspended Scattering Particles in Motion Observed by Optical Coherence Tomography Angiography.

The objective of this study was to investigate the relationship between suspended scattering particles in motion (SSPiM) in optical coherence tomography angiography (OCTA) and treatment response in diabetic macular edema (DME). We retrospectively reviewed the medical records of patients diagnosed with DME who had undergone intravitreal injection. The optical density ratio (ODR) of the intraretinal cyst and the numbers of hyperreflective foci from OCT images and SSPiM from OCTA images were compared, and their association with treatment response was analyzed. Forty-five eyes from 45 patients were included in this study. Twenty-four patients were treated with anti-vascular endothelial growth factor, and 21 patients were treated with a steroid. Binary logistic regression model showed that SSPiM in OCTA images was associated with hyperreflective foci numbers (P = 0.038) and mean ODR of the intraretinal cyst (P = 0.006). Linear regression model showed that SSPiM in the inner nuclear layer was related to treatment response (P = 0.006). SSPiM on OCTA images is related to the poor structural response to treatment in DME.

Indocyanine Green J Aggregates in Polymersomes for Near-Infrared Photoacoustic Imaging.

Clinical translation of photoacoustic imaging (PAI) has been limited by the lack of near-infrared (NIR) contrast agents with low toxicity required for regulatory approval. Herein, J aggregates of indocyanine green (ICG) with strong NIR absorbance were encapsulated at high loadings within small 77 nm polymersomes (nanocapsules) composed of poly(lactide-co-glycolide-b-poly(ethylene glycol)) (PLGA-b-PEG) bilayers, thus enabling PAI of of breast and ovarian cancer cells with high specificity and a sensitivity at the level of ∼100 total cells. All of the major components of the polymersomes are FDA approved and used in the clinic. During formation of polymersomes with a water-in-oil-in-water double emulsion process, loss of ICG from the ICG J aggregates was minimized by coating them with a layer of branched polyethylenimine and by providing excess "sacrificial" ICG to adsorb at the oil-water interfaces. The encapsulated J aggregates were protected against dissociation by the polymersome shell for 24 h in 100% fetal bovine serum, after which the polymersomes biodegraded and the J aggregates dissociated to ICG monomers.

Molecular photoacoustic imaging with ultra-small gold nanoparticles.

Gold nanoparticles (AuNPs) below 10 nm in size can undergo renal clearance, which could facilitate their clinical translation. However, due to non-linear, direct relationship between their absorption and size, use of such "ultra-small" AuNPs as contrast agents for photoacoustic imaging (PAI) is challenging. This problem is complicated by the tendency of absorption for ultra-small AuNPs to be below the NIR range, which is optimal for in vivo imaging. Herein, we present 5-nm molecularly activated plasmonic nanosensors (MAPS) that produce a strong photoacoustic signal in labeled cancer cells in the NIR, demonstrating the feasibility of sensitive PAI with ultra-small AuNPs.

Nobiletin attenuates neurotoxic mitochondrial calcium overload through K+ influx and ΔΨm across mitochondrial inner membrane.

Mitochondrial calcium overload is a crucial event in determining the fate of neuronal cell survival and death, implicated in pathogenesis of neurodegenerative diseases. One of the driving forces of calcium influx into mitochondria is mitochondria membrane potential (ΔΨm). Therefore, pharmacological manipulation of ΔΨm can be a promising strategy to prevent neuronal cell death against brain insults. Based on these issues, we investigated here whether nobiletin, a Citrus polymethoxylated flavone, prevents neurotoxic neuronal calcium overload and cell death via regulating basal ΔΨm against neuronal insult in primary cortical neurons and pure brain mitochondria isolated from rat cortices. Results demonstrated that nobiletin treatment significantly increased cell viability against glutamate toxicity (100 µM, 20 min) in primary cortical neurons. Real-time imaging-based fluorometry data reveal that nobiletin evokes partial mitochondrial depolarization in these neurons. Nobiletin markedly attenuated mitochondrial calcium overload and reactive oxygen species (ROS) generation in glutamate (100 µM)-stimulated cortical neurons and isolated pure mitochondria exposed to high concentration of Ca2+ (5 µM). Nobiletin-induced partial mitochondrial depolarization in intact neurons was confirmed in isolated brain mitochondria using a fluorescence microplate reader. Nobiletin effects on basal ΔΨm were completely abolished in K+-free medium on pure isolated mitochondria. Taken together, results demonstrate that K+ influx into mitochondria is critically involved in partial mitochondrial depolarization-related neuroprotective effect of nobiletin. Nobiletin-induced mitochondrial K+ influx is probably mediated, at least in part, by activation of mitochondrial K+ channels. However, further detailed studies should be conducted to determine exact molecular targets of nobiletin in mitochondria.

Tunable Keratin Hydrogels for Controlled Erosion and Growth Factor Delivery.

Tunable erosion of polymeric materials is an important aspect of tissue engineering for reasons that include cell infiltration, controlled release of therapeutic agents, and ultimately to tissue healing. In general, the biological response to proteinaceous polymeric hydrogels is favorable (e.g., minimal inflammatory response). However, unlike synthetic polymers, achieving tunable erosion with natural materials is a challenge. Keratins are a class of intermediate filament proteins that can be obtained from several sources, including human hair, and have gained increasing levels of use in tissue engineering applications. An important characteristic of keratin proteins is the presence of a large number of cysteine residues. Two classes of keratins with different chemical properties can be obtained by varying the extraction techniques: (1) keratose by oxidative extraction and (2) kerateine by reductive extraction. Cysteine residues of keratose are "capped" by sulfonic acid and are unable to form covalent cross-links upon hydration, whereas cysteine residues of kerateine remain as sulfhydryl groups and spontaneously form covalent disulfide cross-links. Here, we describe a straightforward approach to fabricate keratin hydrogels with tunable rates of erosion by mixing keratose and kerateine. SEM imaging and mechanical testing of freeze-dried materials showed similar pore diameters and compressive moduli, respectively, for each keratose-kerateine mixture formulation (∼1200 kPa for freeze-dried materials and ∼1.5 kPa for hydrogels). However, the elastic modulus (G') determined by rheology varied in proportion with the keratose-kerateine ratios, as did the rate of hydrogel erosion and the release rate of thiol from the hydrogels. The variation in keratose-kerateine ratios also led to tunable control over release rates of recombinant human insulin-like growth factor 1.

Alkylation of human hair keratin for tunable hydrogel erosion and drug delivery in tissue engineering applications.

Polymeric biomaterials that provide a matrix for cell attachment and proliferation while achieving delivery of therapeutic agents are an important component of tissue engineering and regenerative medicine strategies. Keratins are a class of proteins that have received attention for numerous tissue engineering applications because, like other natural polymers, they promote favorable cell interactions and have non-toxic degradation products. Keratins can be extracted from various sources including human hair, and they are characterized by a high percentage of cysteine residues. Thiol groups on reductively extracted keratin (kerateine) form disulfide bonds, providing a more stable cross-linked hydrogel network than oxidatively extracted keratin (keratose) that cannot form disulfide crosslinks. We hypothesized that an iodoacetamide alkylation (or "capping") of cysteine thiol groups on the kerateine form of keratin could be used as a simple method to modulate the levels of disulfide crosslinking in keratin hydrogels, providing tunable rates of gel erosion and therapeutic agent release. After alkylation, the alkylated kerateines still formed hydrogels and the alkylation led to changes in the mechanical and visco-elastic properties of the materials consistent with loss of disulfide crosslinking. The alkylated kerateines did not lead to toxicity in MC3T3-E1 pre-osteoblasts. These cells adhered to keratin at levels comparable to fibronectin and greater than collagen. Alkylated kerateine gels eroded more rapidly than non-alkylated kerateine and this control over erosion led to tunable rates of delivery of rhBMP-2, rhIGF-1, and ciprofloxacin. These results demonstrate that alkylation of kerateine cysteine residues provides a cell-compatible approach to tune rates of hydrogel erosion and therapeutic agent release within the context of a naturally-derived polymeric system.

Surface plasmon-enhanced light-emitting diodes using silver nanoparticles embedded in p-GaN.

We demonstrate the surface plasmon-enhanced blue light-emitting diodes (LEDs) using Ag nanoparticles embedded in p-GaN. A large increase in optical output power of 38% is achieved at an injection current of 20 mA due to an improved internal quantum efficiency of the LEDs. The enhancement of optical output power is dependent on the density of the Ag nanoparticles. This improvement can be attributed to an increase in the spontaneous emission rate through resonance coupling between the excitons in multiple quantum wells and localized surface plasmons in Ag nanoparticles embedded in p-GaN.

Improvement of light output power of InGaN/GaN light-emitting diode by lateral epitaxial overgrowth using pyramidal-shaped SiO(2).

We report on the improvement of light output power of InGaN/GaN blue light-emitting diodes (LEDs) by lateral epitaxial overgrowth (LEO) of GaN using a pyramidal-shaped SiO(2) mask. The light output power was increased by 80% at 20 mA of injection current compared with that of conventional LEDs without LEO structures. This improvement is attributed to an increased internal quantum efficiency by a significant reduction in threading dislocation and by an enhancement of light extraction efficiency by pyramidal-shaped SiO(2) LEO mask.