Localization of unlabeled bepirovirsen antisense oligonucleotide in murine tissues using in situ hybridization and CARS imaging.

Current methods for detecting unlabeled antisense oligonucleotide (ASO) drugs rely on immunohistochemistry (IHC) and/or conjugated molecules, which lack sufficient sensitivity, specificity, and resolution to fully investigate their biodistribution. Our aim was to demonstrate the qualitative and quantitative distribution of unlabeled bepirovirsen, a clinical stage ASO, in livers and kidneys of dosed mice using novel staining and imaging technologies at subcellular resolution. ASOs were detected in formalin-fixed paraffin-embedded (FFPE) and frozen tissues using an automated chromogenic in situ hybridization (ISH) assay: miRNAscope. This was then combined with immunohistochemical detection of cell lineage markers. ASO distribution in hepatocytes versus nonparenchymal cell lineages was quantified using HALO AI image analysis. To complement this, hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) imaging microscopy was used to specifically detect the unique cellular Raman spectral signatures following ASO treatment. Bepirovirsen was localized primarily in nonparenchymal liver cells and proximal renal tubules. Codetection of ASO with distinct cell lineage markers of liver and kidney populations aided target cell identity facilitating quantification. Positive liver signal was quantified using HALO AI, with 12.9% of the ASO localized to the hepatocytes and 87.1% in nonparenchymal cells. HS-CARS imaging specifically detected ASO fingerprints based on the unique vibrational signatures following unlabeled ASO treatment in a totally nonperturbative manner at subcellular resolution. Together, these novel detection and imaging modalities represent a significant increase in our ability to detect unlabeled ASOs in tissues, demonstrating improved levels of specificity and resolution. These methods help us understand their underlying mechanisms of action and ultimately improve the therapeutic potential of these important drugs for treating globally significant human diseases.

Weakly Supervised Identification and Localization of Drug Fingerprints Based on Label-Free Hyperspectral CARS Microscopy.

Understanding drug fingerprints in complex biological samples is essential for the development of a drug. Hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy, a label-free nondestructive chemical imaging technique, can profile biological samples based on their endogenous vibrational contrast. Here, we propose a deep learning-assisted HS-CARS imaging approach for the investigation of drug fingerprints and their localization at single-cell resolution. To identify and localize drug fingerprints in complex biological systems, an attention-based deep neural network, hyperspectral attention net (HAN), was developed. By formulating the task to a multiple instance learning problem, HAN highlights informative regions through the attention mechanism when being trained on whole-image labels. Using the proposed technique, we investigated the drug fingerprints of a hepatitis B virus therapy in murine liver tissues. With the increase in drug dosage, higher classification accuracy was observed, with an average area under the curve (AUC) of 0.942 for the high-dose group. Besides, highly informative tissue structures predicted by HAN demonstrated a high degree of similarity with the drug localization shown by the in situ hybridization staining results. These results demonstrate the potential of the proposed deep learning-assisted optical imaging technique for the label-free profiling, identification, and localization of drug fingerprints in biological samples, which can be extended to nonperturbative investigations of complex biological systems under various biological conditions.

Measuring and Predicting the Internal Structure of Semiconductor Nanocrystals through Raman Spectroscopy.

Nanocrystals composed of mixed chemical domains have diverse properties that are driving their integration in next-generation electronics, light sources, and biosensors. However, the precise spatial distribution of elements within these particles is difficult to measure and control, yet profoundly impacts their quality and performance. Here we synthesized a unique series of 42 different quantum dot nanocrystals, composed of two chemical domains (CdS:CdSe), arranged in 7 alloy and (core)shell structural classes. Chemometric analyses of far-field Raman spectra accurately classified their internal structures from their vibrational signatures. These classifications provide direct insight into the elemental arrangement of the alloy as well as an independent prediction of fluorescence quantum yield. This nondestructive, rapid approach can be broadly applied to greatly enhance our capacity to measure, predict and monitor multicomponent nanomaterials for precise tuning of their structures and properties.

Defined Host-Guest Chemistry on Nanocarbon for Sustained Inhibition of Cancer.

Signal transducer and activator of transcription factor 3 (STAT-3) is known to be overexpressed in cancer stem cells. Poor solubility and variable drug absorption are linked to low bioavailability and decreased efficacy. Many of the drugs regulating STAT-3 expression lack aqueous solubility; hence hindering efficient bioavailability. A theranostics nanoplatform based on luminescent carbon particles decorated with cucurbit[6]uril is introduced for enhancing the solubility of niclosamide, a STAT-3 inhibitor. The host-guest chemistry between cucurbit[6]uril and niclosamide makes the delivery of the hydrophobic drug feasible while carbon nanoparticles enhance cellular internalization. Extensive physicochemical characterizations confirm successful synthesis. Subsequently, the host-guest chemistry of niclosamide and cucurbit[6]uril is studied experimentally and computationally. In vitro assessments in human breast cancer cells indicate approximately twofold enhancement in IC50 of drug. Fourier transform infrared and fluorescence imaging demonstrate efficient cellular internalization. Furthermore, the catalytic biodegradation of the nanoplatforms occur upon exposure to human myeloperoxidase in short time. In vivo studies on athymic mice with MCF-7 xenograft indicate the size of tumor in the treatment group is half of the controls after 40 d. Immunohistochemistry corroborates the downregulation of STAT-3 phosphorylation. Overall, the host-guest chemistry on nanocarbon acts as a novel arsenal for STAT-3 inhibition.

Rapid visualization of macromolecular orientation by discrete frequency mid-infrared spectroscopic imaging.

Infrared (IR) spectroscopic imaging has been used to measure the composition and orientation of polymeric systems for decades. IR microscopy can provide detailed views of microscopic regions, allowing the observation of both morphology and molecular properties of a sample, but involves a trade-off between the spatial extent and details of molecular content. Here we describe an approximately two orders of magnitude faster approach to measure the spherulitic structure and molecular orientation in large semi-crystalline polymer samples compared to extant Fourier transform infrared (FT-IR) spectroscopic imaging. This discrete frequency approach utilizes individual narrowband emission lines of a quantum cascade laser (QCL) source to spectrally image large areas rapidly. The inherent polarization of the laser beam is employed to measure orientation, enabling calculation of Hermans in-plane orientation function along with molecular chain angles distribution.

Tunable Luminescent Carbon Nanospheres with Well-Defined Nanoscale Chemistry for Synchronized Imaging and Therapy.

In this work, we demonstrate the significance of defined surface chemistry in synthesizing luminescent carbon nanomaterials (LCN) with the capability to perform dual functions (i.e., diagnostic imaging and therapy). The surface chemistry of LCN has been tailored to achieve two different varieties: one that has a thermoresponsive polymer and aids in the controlled delivery of drugs, and the other that has fluorescence emission both in the visible and near-infrared (NIR) region and can be explored for advanced diagnostic modes. Although these particles are synthesized using simple, yet scalable hydrothermal methods, they exhibit remarkable stability, photoluminescence and biocompatibility. The photoluminescence properties of these materials are tunable through careful choice of surface-passivating agents and can be exploited for both visible and NIR imaging. Here the synthetic strategy demonstrates the possibility to incorporate a potent antimetastatic agent for inhibiting melanomas in vitro. Since both particles are Raman active, their dispersion on skin surface is reported with Raman imaging and utilizing photoluminescence, their depth penetration is analysed using fluorescence 3D imaging. Our results indicate a new generation of tunable carbon-based probes for diagnosis, therapy or both.

Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging.

The COVID-19 pandemic triggered the resurgence of synthetic RNA vaccine platforms allowing rapid, scalable, low-cost manufacturing, and safe administration of therapeutic vaccines. Self-amplifying mRNA (SAM), which self-replicates upon delivery into the cellular cytoplasm, leads to a strong and sustained immune response. Such mRNAs are encapsulated within lipid nanoparticles (LNPs) that act as a vehicle for delivery to the cell cytoplasm. A better understanding of LNP-mediated SAM uptake and release mechanisms in different types of cells is critical for designing effective vaccines. Here, we investigated the cellular uptake of a SAM-LNP formulation and subsequent intracellular expression of SAM in baby hamster kidney (BHK-21) cells using hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy and multiphoton-excited fluorescence lifetime imaging microscopy (FLIM). Cell classification pipelines based on HS-CARS and FLIM features were developed to obtain insights on spectral and metabolic changes associated with SAM-LNPs uptake. We observed elevated lipid intensities with the HS-CARS modality in cells treated with LNPs versus PBS-treated cells, and simultaneous fluorescence images revealed SAM expression inside BHK-21 cell nuclei and cytoplasm within 5 h of treatment. In a separate experiment, we observed a strong correlation between the SAM expression and mean fluorescence lifetime of the bound NAD(P)H population. This work demonstrates the ability and significance of multimodal optical imaging techniques to assess the cellular uptake of SAM-LNPs and the subsequent changes occurring in the cellular microenvironment following the vaccine expression.

Differential Uptake of Antisense Oligonucleotides in Mouse Hepatocytes and Macrophages Revealed by Simultaneous Two-Photon Excited Fluorescence and Coherent Raman Imaging.

Antisense oligonucleotides (ASOs), a novel paradigm in modern therapeutics, modulate cellular gene expression by binding to complementary messenger RNA (mRNA) sequences. While advances in ASO medicinal chemistry have greatly improved the efficiency of cellular uptake, selective uptake by specific cell types has been difficult to achieve. For more efficient and selective uptake, ASOs are often conjugated with molecules with high binding affinity for transmembrane receptors. Triantennary N-acetyl-galactosamine conjugated phosphorothioate ASOs (GalNAc-PS-ASOs) were developed to enhance targeted ASO delivery into liver through the hepatocyte-specific asialoglycoprotein receptor (ASGR). We assessed the kinetics of uptake and subsequent intracellular distribution of AlexaFluor 488 (AF488)-labeled PS-ASOs and GalNAc-PS-ASOs in J774A.1 mouse macrophages and primary mouse or rat hepatocytes using simultaneous coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence (2PF) imaging. The CARS modality captured the dynamic lipid distributions and overall morphology of the cells; two-photon fluorescence (2PF) measured the time- and dose-dependent localization of ASOs delivered by a modified treatment of suspension cells. Our results show that in macrophages, the uptake rate of PS-ASOs did not significantly differ from that of GalNAc-PS-ASOs. However, in hepatocytes, GalNAc-PS-ASOs exhibited a peripheral uptake distribution compared to a polar uptake distribution observed in macrophages. The peripheral distribution correlated with a significantly larger amount of internalized GalNAc-PS-ASOs compared to the PS-ASOs. This work demonstrates the relevance of multimodal imaging for elucidating the uptake mechanism, accumulation, and fate of different ASOs in liver cells that can be used further in complex in vitro models and liver tissues to evaluate ASO distribution and activity.

Gating mechanism of the influenza A M2 channel revealed by 1D and 2D IR spectroscopies.

The pH-controlled M2 protein from influenza A is a critical component of the virus and serves as a target for the aminoadamantane antiflu agents that block its H+ channel activity. To better understand its H+ gating mechanism, we investigated M2 in lipid bilayers with a new combination of IR spectroscopies and theory. Linear Fourier transform infrared (FTIR) spectroscopy was used to measure the precise orientation of the backbone carbonyl groups, and 2D infrared (IR) spectroscopy was used to identify channel-lining residues. At low pH (open state), our results match previously published solid-state NMR and X-ray structures remarkably well. However, at neutral pH when the channel is closed, our measurements indicate that a large conformational change occurs that is consistent with the transmembrane alpha-helices rotating by one amino acid register--a structural rearrangement not previously observed. The combination of simulations and isotope-labeled FTIR and 2D IR spectroscopies provides a noninvasive means of interrogating the structures of membrane proteins in general and ion channels in particular.

Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H.

The heterogeneous nature of extracellular vesicles (EVs) creates the need for single EV characterization techniques. However, many common biochemical and functional EV analysis techniques lack single EV resolution. Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to functionally characterize the reduced form of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H) in cells and tissues. Here, we demonstrate that FLIM can also be used to image and characterize NAD(P)H in single isolated EVs. EVs were isolated using standard differential ultracentrifugation techniques from multiple cell lines and imaged using a custom two-photon FLIM system. The presented data show that the NAD(P)H fluorescence lifetimes in isolated cell-derived EVs follow a wide Gaussian distribution, indicating the presence of a range of different protein-bound and free NAD(P)H species. EV NAD(P)H fluorescence lifetime distribution has a larger standard deviation than that of cells and a significantly different fluorescence lifetime distribution than the nuclei, mitochondria, and cytosol of cells. Additionally, changes in the metabolic conditions of cells were reflected in changes in the mean fluorescence lifetime of NAD(P)H in the produced EVs. These data suggest that FLIM of NAD(P)H could be a valuable tool for EV research.

Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy.

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.

Real-time investigations of Pt(111) surface transformations in sulfuric acid solutions.

We present the first broadband sum-frequency generation (SFG) spectra of adlayers from sulfuric acid solutions on Pt(111) surfaces and reveal surface transformations of (bi)sulfate anions in unprecedented detail. SFG amplitudes, bandwidth, and electrochemical Stark tuning of (bi)sulfate vibrational bands centered at 1250-1290 cm(-1) strongly depend on the applied potential and are correlated with prominent voltammetric features. (Bi)sulfate adlayers on Pt(111) are important model systems for weak, specific adsorption of anions on catalytically active surfaces. Although the existence of surface transformations on Pt(111) in dilute H(2)SO(4) solutions has been established by previous studies, so far they have not been observed with surface vibrational spectroscopy. Our results confirm previous reports of a surface transformation at 0.21 V and provide new information on a second transformation at 0.5 V due to surface hydroxyl formation and rearrangement of the electric double layer.

Sum-frequency generation of acetate adsorption on Au and Pt surfaces: molecular structure effects.

The reversible adsorption of acetate on polycrystalline Au and Pt surfaces was investigated with broadband sum-frequency generation (SFG) and cyclic voltammetry. Specifically adsorbed acetate as well as coadsorbed sulfuric acid anions are observed for the first time with SFG and give rise to dramatically different SFG intensities on Au and Pt surfaces. While similar coverages of acetate adlayers on Au and Pt surfaces are well established by previous studies, an identification of the interfacial molecular structure has been elusive. However, we have applied the high sensitivity of SFG for interfacial polar ordering to identify different acetate structures at Au and Pt surfaces in contact with HClO(4) and H(2)SO(4) electrolytes. Acetate competes with the formation of surface oxides and shifts the oxidation threshold of both Au and Pt electrodes anodically. Effects of the supporting electrolyte on the formation of acetate adlayers are revealed by comparing SFG spectra in HClO(4) and H(2)SO(4) solutions: Sulfuric acid anions modify the potential-dependent acetate adsorption, compete with adsorbed acetate on Au and coadsorb with acetate on Pt surfaces.

On-demand Milifluidic Synthesis of Quantum Dots in Digital Droplet Reactors.

Colloidal quantum dots (QDs) offer dramatic potential due to their size-dependent optical properties. Lack of facile synthesis methods for precise and reproducible size and composition, however, present an extant barrier to their widespread use. Here we report the use of droplet microfluidics for the simple and highly reproducible synthesis of cadmium sulfide (CdS) and cadmium selenide (CdSe) QDs without the use of harsh solvents and in ambient conditions. Our approach uses a liquid-liquid barrier between two immiscible liquids to generate a digital droplet reactor. This reaction droplet is easily controlled and manipulated and offers enhanced mixing when coupled to a helical mixer, resulting in a significant reduction in size distribution compared to benchtop procedures. Furthermore, QD characteristics have modeled and predicted based on the parameters of the microfluidic device. We believe this method overcomes the current manufacturing challenges with synthesizing nanostructures, which is required for the next generation of nanosensors.

Assessing the severity of psoriasis through multivariate analysis of optical images from non-lesional skin.

Patients with psoriasis represent a heterogeneous population with individualized disease expression. Psoriasis can be monitored through gold standard histopathology of biopsy specimens that are painful and permanently scar. A common associated measure is the use of non-invasive assessment of the Psoriasis Area and Severity Index (PASI) or similarly derived clinical assessment based scores. However, heterogeneous manifestations of the disease lead to specific PASI scores being poorly reproducible and not easily associated with clinical severity, complicating the efforts to monitor the disease. To address this issue, we developed a methodology for non-invasive automated assessment of the severity of psoriasis using optical imaging. Our analysis shows that two-photon fluorescence lifetime imaging permits the identification of biomarkers present in both lesional and non-lesional skin that correlate with psoriasis severity. This ability to measure changes in lesional and healthy-appearing skin provides a new pathway for independent monitoring of both the localized and systemic effects of the disease. Non-invasive optical imaging was conducted on lesions and non-lesional (pseudo-control) skin of 33 subjects diagnosed with psoriasis, lesional skin of 7 subjects diagnosed with eczema, and healthy skin of 18 control subjects. Statistical feature extraction was combined with principal component analysis to analyze pairs of two-photon fluorescence lifetime images of stratum basale and stratum granulosum layers of skin. We found that psoriasis is associated with biochemical and structural changes in non-lesional skin that can be assessed using clinically available two-photon fluorescence lifetime microscopy systems.

Bulk Protein and Oil Prediction in Soybeans Using Transmission Raman Spectroscopy: A Comparison of Approaches to Optimize Accuracy.

Rapid measurements of protein and oil content are important for a variety of uses, from sorting of soybeans at the point of harvest to feedback during soybean meal production. In this study, our goal is to develop a simple protocol to permit rapid and robust quantitative prediction of soybean constituents using transmission Raman spectroscopy (TRS). To develop this approach, we systematically varied the various elements of the measurement process to provide a diverse test bed. First, we utilized an in-house-built benchtop TRS instrument such that suitable optical configurations could be rapidly deployed and analyzed for experimental data collection for individual soybean grains. Second, we also utilized three different soybean varieties with relatively low (33.97%), medium (36.98%), and high protein (41.23%) contents to test the development process. Third, samples from each variety were prepared using whole bean and three different sample treatments (i.e., ground bean, whole meal, and ground meal). In each case, we modeled the data obtained using partial least squares (PLS) regression and assessed spectral metric-based multiple linear regression (metric-MLR) approaches to build robust prediction models. The metric-MLR models showed lower root mean square errors (RMSEPs), and hence better prediction, compared to corresponding classical PLS regression models for both bulk protein and oil for all treatment types. Comparing different sample preparation approaches, a lower RMSEPs was observed for whole meal treatment and thus the metric-MLR modeling with ground meal treatment was considered to be optimal protocol for bulk protein and oil prediction in soybean, with RMSEP values of 1.15 ± 0.04 (R2 = 0.87) and 0.80 ± 0.02 (R2 = 0.87) for bulk protein and oil, respectively. These predictions were nearly two- to threefold better (i.e., lower RMSEPs) than the corresponding NIR spectroscopy measurements (i.e., secondary gold standards in grain industry). For content prediction in whole soybean, incorporating physical attributes of individual grains in metric-MLR approach show up to 22% improvement in bulk protein and a relatively mild (up to ∼5%) improvement in bulk oil prediction. The unique combination of metric-MLR modeling approach (which is rare in the field of grain analysis) and sample treatments resulted in improved prediction models; using the physical attributes of individual grains is suggested as a novel measure for improving accuracy in prediction.

Enzyme-catalysed biodegradation of carbon dots follows sequential oxidation in a time dependent manner.

Carbon dots (CDs) have recently garnered significant attention owing to their excellent luminescence properties, thereby demonstrating a variety of applications in in vitro and in vivo imaging. Understanding the long-term metabolic fate of these agents in a biological environment is the focus of this work. Here we show that the CDs undergo peroxide catalysed degradation in the presence of lipase. Our results indicate that differently charged CD species exhibit unique degradation kinetics upon being subjected to enzyme oxidation. Furthermore, this decomposition correlates with the relative accessibility of the enzymatic molecule. Using multiple physico-chemical characterization studies and molecular modelling, we confirmed the interaction of passivating surface abundant molecules with the enzyme. Finally, we have identified hydroxymethyl furfural as a metabolic by-product of the CDs used here. Our results indicate the possibility and a likely mechanism for complete CD degradation in living systems that can pave the way for a variety of biomedical applications.

Mapping Solvation Environments in Porous Metal-Organic Frameworks with Infrared Chemical Imaging.

We report here the first mesoscale characterization of solvent environments in the metal-organic framework (MOF) Cu3(BTC)2 using infrared imaging. Two characteristic populations of the MOF structures corresponding to the carboxylate binding to the Cu(II) (metal) ions were observed, which reflect a regular solvated MOF structure with axial solvents in the binuclear copper paddlewheel and an unsolvated defect mode that lacks axial solvent coordination. Infrared imaging also shows strong correlation between solvent localization and the spatial distribution of the solvated population within the MOF. This is a vital result as any remnant solvent molecules adsorbed inside of MOFs can render them less effective. We propose fast IR imaging as a potential characterization technique that can measure adsorbate and defect distributions in MOFs.

Structural disorder of the CD3zeta transmembrane domain studied with 2D IR spectroscopy and molecular dynamics simulations.

In a recently reported study [Mukherjee, et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3528] we used 2D IR spectroscopy and 1-(13)C=(18)O isotope labeling to measure the vibrational dynamics of 11 amide I modes in the CD3zeta transmembrane domain. We found that the homogeneous line widths and population relaxation times were all nearly identical, but that the amount of inhomogeneous broadening correlated with the position of the amide group inside the membrane. In this study, we use molecular dynamics simulations to investigate the structural and dynamical origins of these experimental observations. We use two models to convert the simulations to frequency trajectories from which the mean frequencies, standard deviations, frequency correlation functions, and 2D IR spectra are calculated. Model 1 correlates the hydrogen-bond length to the amide I frequency, whereas model 2 uses an ab initio-based electrostatic model. We find that the structural distributions of the peptidic groups and their environment are reflected in the vibrational dynamics of the amide I modes. Environmental forces from the water and lipid headgroups partially denature the helices, shifting the infrared frequencies and creating larger inhomogeneous distributions for residues near the ends. The least inhomogeneously broadened residues are those located in the middle of the membrane where environmental electrostatic forces are weakest and the helices are most ordered. Comparison of the simulations to experiment confirms that the amide I modes near the C-terminal are larger than at the N-terminal because of the asymmetric structure of the peptide bundle in the membrane. The comparison also reveals that residues at a kink in the alpha-helices have broader line widths than more helical parts of the peptide because the peptide backbone at the kink exhibits a larger amount of structural disorder. Taken together, the simulations and experiments reveal that infrared line shapes are sensitive probes of membrane protein structural and environmental heterogeneity.

Multi-functionality Redefined with Colloidal Carotene Carbon Nanoparticles for Synchronized Chemical Imaging, Enriched Cellular Uptake and Therapy.

Typically, multiplexing high nanoparticle uptake, imaging, and therapy requires careful integration of three different functions of a multiscale molecular-particle assembly. Here, we present a simpler approach to multiplexing by utilizing one component of the system for multiple functions. Specifically, we successfully synthesized and characterized colloidal carotene carbon nanoparticle (C(3)-NP), in which a single functional molecule served a threefold purpose. First, the presence of carotene moieties promoted the passage of the particle through the cell membrane and into the cells. Second, the ligand acted as a potent detrimental moiety for cancer cells and, finally, the ligands produced optical contrast for robust microscopic detection in complex cellular environments. In comparative tests, C(3)-NP were found to provide effective intracellular delivery that enables both robust detection at cellular and tissue level and presents significant therapeutic potential without altering the mechanism of intracellular action of ß-carotene. Surface coating of C(3) with phospholipid was used to generate C(3)-Lipocoat nanoparticles with further improved function and biocompatibility, paving the path to eventual in vivo studies.