Integration of In Vitro and In Vivo Models to Predict Cellular and Tissue Dosimetry of Nanomaterials Using Physiologically Based Pharmacokinetic Modeling.

Nanomaterials (NMs) have been increasingly used in a number of areas, including consumer products and nanomedicine. Target tissue dosimetry is important in the evaluation of safety, efficacy, and potential toxicity of NMs. Current evaluation of NM efficacy and safety involves the time-consuming collection of pharmacokinetic and toxicity data in animals and is usually completed one material at a time. This traditional approach no longer meets the demand of the explosive growth of NM-based products. There is an emerging need to develop methods that can help design safe and effective NMs in an efficient manner. In this review article, we critically evaluate existing studies on in vivo pharmacokinetic properties, in vitro cellular uptake and release and kinetic modeling, and whole-body physiologically based pharmacokinetic (PBPK) modeling studies of different NMs. Methods on how to simulate in vitro cellular uptake and release kinetics and how to extrapolate cellular and tissue dosimetry of NMs from in vitro to in vivo via PBPK modeling are discussed. We also share our perspectives on the current challenges and future directions of in vivo pharmacokinetic studies, in vitro cellular uptake and kinetic modeling, and whole-body PBPK modeling studies for NMs. Finally, we propose a nanomaterial in vitro to in vivo extrapolation via physiologically based pharmacokinetic modeling (Nano-IVIVE-PBPK) framework for high-throughput screening of target cellular and tissue dosimetry as well as potential toxicity of different NMs in order to meet the demand of efficient evaluation of the safety, efficacy, and potential toxicity of a rapidly increasing number of NM-based products.

Tracking of fluorescent antibiotic conjugate in planta utilizing fluorescence lifetime imaging.

MAIN CONCLUSION: Excited state lifetime-based separation of fluorophore-tagged antibiotic conjugate emission from the spectrally broad plant autofluorescence enables in planta tracking of the translocation of systemic cargo such as antibiotics via fluorescence lifetime imaging. The efficacy of antibiotic treatments in citrus crops is uncertain due to mixed results from in-field experiments and a lack of study on their systemic movement. As of yet there has been an inability to track treatments using traditional fluorescence microscopy due to treatments having little fluorescence characteristics, and signal convolution due to plant autofluorescence. In this study, we used streptomycin sulfate, a commercially available antibiotic, and conjugated it to a modified tris(bipyridine) ruthenium (II) chloride, a dye with an excited state lifetime magnitudes higher than other commonly used organic fluorescent probes. The resultant is a fluorescence lifetime imaging (FLIM) trackable antibiotic conjugate, covalently attached via an amide linkage that is uniquely distinguishable from plant autofluorescence. Characterization of the fluorescent antibiotic conjugate showed no mitigation of excited state lifetime, and a distinct IR peak not found in any synthetic components. Subsequent tracking using FLIM in citrus tissue was achieved, with identification of movement through citrus plant vasculature via tissue localization in xylem and phloem. Results indicated upwards systemic movement of the conjugate in both xylem and phloem after 48 h of incubation. However, the conjugate failed to move down towards the root system of the plant by 168 h. Mechanistically, it is likely that xylem contributes heavily in the translocation of the conjugate upwards; however, phloem led flow due to growth changes could act as a contributor. This proof-of-concept sets groundwork for subsequent studies regarding antibiotic localization and movement in citrus.

Animal simulations facilitate smart drug design through prediction of nanomaterial transport to individual tissue cells.

Smart drug design for antibody and nanomaterial-based therapies allows optimization of drug efficacy and more efficient early-stage preclinical trials. The ideal drug must display maximum efficacy at target tissue sites, with transport from tissue vasculature to the cellular environment being critical. Biological simulations, when coupled with in vitro approaches, can predict this exposure in a rapid and efficient manner. As a result, it becomes possible to predict drug biodistribution within single cells of live animal tissue without the need for animal studies. Here, we successfully utilized an in vitro assay and a computational fluid dynamic model to translate in vitro cell kinetics (accounting for cell-induced degradation) to whole-body simulations for multiple species as well as nanomaterial types to predict drug distribution into individual tissue cells. We expect this work to assist in refining, reducing, and replacing animal testing, while providing scientists with a new perspective during the drug development process.

Ligand assisted swelling-deswelling microencapsulation (LASDM) for stable, color tunable perovskite-polymer composites.

Metal halide perovskite nanocrystals (PNCs), with excellent electronic and optical properties, are promising for a variety of optoelectronic and photonic applications. However, the instability issue still impedes their practical applications. Here a ligand-assisted swelling-deswelling microencapsulation (LASDM) strategy is proposed and evaluated for improving the stability and photoluminescence (PL) performance of PNCs. With ligand assistance, well dispersed and intimately passivated PNCs in polymer matrices are obtained. Compared with the previously reported swelling-deswelling microencapsulation (SDM) strategy, the proposed method can provide better nanocrystal size control and surface coordination. Thus, full-color perovskite-polymer composites (PPCs) with unprecedented environmental stability can be achieved and concentration quenching can be avoided in polymer matrices. The excellent color purity, color tunability, optical density variability and environmental stability make PPCs highly promising for a range of PL applications, such as tailored lighting and transparent projection displays. Moreover, the simple, low cost, scalable process and the compatibility of this method with a group of polymer matrices should pave the way for PPCs to meet the requirements for practical use.

Author Correction: An in vitro assay and artificial intelligence approach to determine rate constants of nanomaterial-cell interactions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ultrastable and Biofunctionalizable Conjugated Polymer Nanoparticles with Encapsulated Iron for Ferroptosis Assisted Chemodynamic Therapy.

We report the development of novel tumor-targeted conjugated polymer nanoparticles (CPNPs) carrying iron for chemodynamic therapy (CDT). Tumor cell killing proceeds through ferroptosis, a reactive oxygen species (ROS) mechanism that is not dependent on external activation by, for example, light, as is the case in photodynamic therapy (PDT). The ferroptosis mechanism is also not heavily reliant on oxygen availability and is, therefore, promising for the treatment of hypoxic tumors. In this work, we apply this development to the case study of melanoma, a difficult to treat cancer in advanced stages due to resistance to chemotherapy. The iron-carrying CPNPs reported here are targeted to endothelin-B receptors (EDNRB) through endothelin-3 surface moieties (EDN3-CPNPs). Our results show excellent targeting to tumor cells that overexpress EDNRB, specifically for melanoma and bladder tumor cells. In these cases, efficient cell killing, over 80% at higher doses, was found. Conversely, tumor cells not targeted by the EDN3-CPNPs show little effects of CDT, with tumor cell death under 20% in most cases. The outcomes of our work demonstrate that EDN3-CPNPs enable ferroptosis-assisted CDT and present a new therapeutic avenue for tumor treatment.

An in vitro assay and artificial intelligence approach to determine rate constants of nanomaterial-cell interactions.

In vitro assays and simulation technologies are powerful methodologies that can inform scientists of nanomaterial (NM) distribution and fate in humans or pre-clinical species. For small molecules, less animal data is often needed because there are a multitude of in vitro screening tools and simulation-based approaches to quantify uptake and deliver data that makes extrapolation to in vivo studies feasible. Small molecule simulations work because these materials often diffuse quickly and partition after reaching equilibrium shortly after dosing, but this cannot be applied to NMs. NMs interact with cells through energy dependent pathways, often taking hours or days to become fully internalized within the cellular environment. In vitro screening tools must capture these phenomena so that cell simulations built on mechanism-based models can deliver relationships between exposure dose and mechanistic biology, that is biology representative of fundamental processes involved in NM transport by cells (e.g. membrane adsorption and subsequent internalization). Here, we developed, validated, and applied the FORECAST method, a combination of a calibrated fluorescence assay (CF) with an artificial intelligence-based cell simulation to quantify rates descriptive of the time-dependent mechanistic biological interactions between NMs and individual cells. This work is expected to provide a means of extrapolation to pre-clinical or human biodistribution with cellular level resolution for NMs starting only from in vitro data.

In situ synthesis and macroscale alignment of CsPbBr3 perovskite nanorods in a polymer matrix.

We report an in situ catalyst-free strategy to synthesize inorganic CsPbBr3 perovskite nanorods in a polymer matrix (NRs-PM) with good dimensional control, outstanding optical properties and ultrahigh environmental stability. Polarization photoluminescence (PL) imaging with high spatial resolution was carried out for the first time on single nanorod (NR) and shows a relatively high local polarization ratio (∼0.4) consistent with theoretical predictions based on a dielectric contrast model. We further demonstrate that macroscale alignment of the CsPbBr3 nanorods can be achieved through mechanically stretching the NRs-PM films at elevated temperature, without deteriorating the optical quality of the NRs. A polarization ratio of 0.23 is observed for these aligned NRs-PM films, suggesting their potential as polarized down-converters to increase the light efficiency in liquid crystal display (LCD) backlights.

Ultrastable, Highly Luminescent Organic-Inorganic Perovskite-Polymer Composite Films.

A simple yet general swelling-deswelling microencapsulation strategy has been developed to achieve well dispersed and intimately passivated crystalline organic-inorganic perovskites nanoparticles within polymer matrixes and results in a series of highly luminescent CH3 NH3 PbBr3 (MAPbBr3 )-polymer composite films with unprecedented water and thermal stabilities and superior color purity.

Probing Ternary Solvent Effect in High V(oc) Polymer Solar Cells Using Advanced AFM Techniques.

This work describes a simple method to develop a high V(oc) low band gap PSCs. In addition, two new atomic force microscopy (AFM)-based nanoscale characterization techniques to study the surface morphology and physical properties of the structured active layer are introduced. With the help of ternary solvent processing of the active layer and C60 buffer layer, a bulk heterojunction PSC with V(oc) more than 0.9 V and conversion efficiency 7.5% is developed. In order to understand the fundamental properties of the materials ruling the performance of the PSCs tested, AFM-based nanoscale characterization techniques including Pulsed-Force-Mode AFM (PFM-AFM) and Mode-Synthesizing AFM (MSAFM) are introduced. Interestingly, MSAFM exhibits high sensitivity for direct visualization of the donor-acceptor phases in the active layer of the PSCs. Finally, conductive-AFM (cAFM) studies reveal local variations in conductivity in the donor and acceptor phases as well as a significant increase in photocurrent in the PTB7:ICBA sample obtained with the ternary solvent processing.

Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers.

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next generation photosensitizers for Photodynamic Therapy (PDT) is reported. The nanoparticles are formed by hydrophobic interaction of the semiconducting polymer MEH-PPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]) with the fullerene PCBM (phenyl-C61-butyric acid methyl ester) in the presence of a non-compatible solvent. MEH-PPV has a high extinction coefficient that leads to high rates of triplet formation, and efficient charge and energy transfer to the fullerene PCBM. The latter processes enhance the efficiency of the PDT system through fullerene assisted triplet and radical formation, and ultrafast deactivation of MEH-PPV excited stated. The results reported here show that this nanoparticle PDT sensitizing system is highly effective and shows unexpected specificity to cancer cell lines.

Development and characterization of conducting polymer nanoparticles for photodynamic therapy in vitro.

Conducting polymer nanoparticles (CPNPs), composed of the conducting polymer poly[2-methoxy-5-(2-ethylhexyl-oxy)-p-phenylenevinylene] (MEH-PPV) were studied for applications in biophotonics and therapeutics. The extent of cellular uptake, cytotoxicity, and effectiveness of these nanoparticles in photodynamic therapy (PDT) was investigated for four cell lines, namely TE-71, MDA-MB-231, A549 and OVCAR3. Confocal fluorescence imaging and flow cytometry show that CPNPs are taken up only in limited quantities by TE-71, while they are taken up extensively by the cancer cell lines. The uptake among the cancer cell lines was observed to vary with cell line, with CPNPs uptake increasing from MDA-MB-231 to A549 to OVCAR3. Fluorescence imaging experiments show that the CPNPs have high brightness and appear stable in the intracellular environment. No cytotoxicity of non-photoactivated CPNPs (in dark) was observed from MTT assay. After completion of PDT, the quantitative data on cell viability suggest that cell death scales across the cell lines with CPNP uptake, is light dose dependent, and is complete for OVCAR3. In addition, for OVCAR3 apoptotic cell death is observed after PDT. The reported work illustrates the potential of the intrinsically fluorescent and photoactivateable CPNPs for application in biophotonics and therapeutics.

Caveolae-mediated endocytosis of conjugated polymer nanoparticles.

Understanding the cellular entry pathways of synthetic biomaterials is highly important to improve overall labeling and delivery efficiency. Herein, cellular entry mechanisms of conjugated polymer nanoparticles (CPNs) are presented. CPNs are intrinsic fluorescent materials used for various biological applications. While CPNs cause no toxicity, decreased CPN uptake is observed from cancer cells pretreated with genistein, which is an inhibitor of caveolae-mediated endocytosis (CvME). CvME is further confirmed by high co-localization with caveolin-1 proteins found in the caveolae and caveosomes. Excellent photophysical properties, non-toxicity, and non-destructive delivery pathways support that CPNs are promising multifunctional carriers minimizing degradation of contents during delivery.

Influence of backbone rigidness on single chain conformation of thiophene-based conjugated polymers.

Structural order of conjugated polymers at different length scales directs the optoelectronic properties of the corresponding materials; thus it is of critical importance to understand and control conjugated polymer morphology for successful application of these materials in organic optoelectronics. Herein, with the aim of probing the dependence of single chain folding properties on the chemical structure and rigidness of the polymer backbones, single molecule fluorescence spectroscopy was applied to four thiophene-based conjugated polymers. These include regioregular poly(3-hexylthiophene) (RR-P3HT), poly(2,5-bis(3-tetradecylthiophen-2-yl)thieno[3,2-b]thiophene) (PBTTT-14), poly(2,5-bis(3-tetradecylthiophen-2-yl)thiophene-2-yl)thiophen-2-ylthiazolo[5,4-d]thiazole) (PTzQT-12), and poly(3,3-didodecylquaterthiophene)] (PQT-12). Our previous work has shown that RR-P3HT and PBTTT-14 polymer chains fold in their nanostructures, whereas PQT-12 and PTzQT-12 do not fold in their nanostructures. At the single molecule level, it was found that RR-P3HT single chains almost exclusively fold into loosely and strongly aggregated conformations, analogous to the folding properties in nanostructures. PQT-12 displays significant chain folding as well, but only into loosely aggregated conformations, showing an absence of strongly aggregated polymer chains. PBTTT-14 exhibits a significant fraction of rigid polymer chain. The findings made for single molecules of PQT-12 and PBTTT-14 are thus in contrast with the observations made in their corresponding nanostructures. PTzQT-12 appears to be the most rigid and planar conjugated polymer of these four polymers. However, although the presumably nonfolding polymers PQT-12 and PTzQT-12 exhibit less folding than RR-P3HT, there is still a significant occurrence of chain folding for these polymers at the single molecule level. These results suggest that the folding properties of conjugated polymers can be influenced by the architecture of the polymer backbones; however, other factors such as intermolecular stacking interactions, solvent environment, and side chain interactions in corresponding materials should also be taken into account to predict conjugated polymer material morphology.

Linker-induced anomalous emission of organic-molecule conjugated metal-oxide nanoparticles.

Semiconductor nanoparticles conjugated with organic- and dye-molecules to yield high efficiency visible photoluminescence (PL) hold great potential for many future technological applications. We show that folic acid (FA)-conjugated to nanosize TiO(2) and CeO(2) particles demonstrates a dramatic increase of photoemission intensity at wavelengths between 500 and 700 nm when derivatized using aminopropyl trimethoxysilane (APTMS) as spacer-linker molecules between the metal oxide and FA. Using density-functional theory (DFT) and time-dependent DFT calculations we demonstrate that the strong increase of the PL can be explained by electronic transitions between the titania surface oxygen vacancy (OV) states and the low-energy excited states of the FA/APTMS molecule anchored onto the surface oxygen bridge sites in close proximity to the OVs. We suggest this scenario to be a universal feature for a wide class of metal oxide nanoparticles, including nanoceria, possessing a similar band gap (∼3 eV) and with a large surface-vacancy-related density of electronic states. We demonstrate that the molecule-nanoparticle linker can play a crucial role in tuning the electronic and optical properties of nanosystems by bringing optically active parts of the molecule and of the surface close to each other.

An activatable multimodal/multifunctional nanoprobe for direct imaging of intracellular drug delivery.

Multifunctional nanoparticles integrated with imaging modalities (such as magnetic resonance and optical) and therapeutic drugs are promising candidates for future cancer diagnostics and therapy. While targeted drug delivery and imaging of tumor cells have been the major focus in engineering nanoparticle probes, no extensive efforts have been made towards developing sensing probes that can confirm and monitor intracellular drug release events. Here, we present quantum dot (Qdot)-iron oxide (IO) based multimodal/multifunctional nanocomposite probe that is optically and magnetically imageable, targetable and capable of reporting on intracellular drug release events. Specifically, the probe consists of a superparamagnetic iron oxide nanoparticle core (IONP) decorated with satellite CdS:Mn/ZnS Qdots where the Qdots themselves are further functionalized with STAT3 inhibitor (an anti-cancer agent), vitamin folate (as targeting motif) and m-polyethylene glycol (mPEG, a hydrophilic dispersing agent). The Qdot luminescence is quenched in this nanocomposite probe ("OFF" state) due to combined electron/energy transfer mediated quenching processes involving IONP, folate and STAT3 agents. Upon intracellular uptake, the probe is exposed to the cytosolic glutathione (GSH) containing environment resulting in restoration of the Qdot luminescence ("ON" state), which reports on uptake and drug release. Probe functionality was validated using fluorescence and MR measurements as well as in vitro studies using cancer cells that overexpress folate receptors.

Charge trapping and storage by composite P3HT/PC60BM nanoparticles investigated by fluorescence-voltage/single particle spectroscopy.

Fluorescence-voltage/single particle spectroscopy (F-V/SPS) was employed to study exciton-hole polaron interactions and interfacial charge transfer processes for pure poly(3-hexylthiophene) (P3HT) nanoparticles (NPs) and composite P3HT/PC(60)BM NPs in functioning hole-injection devices. F-V/SPS data collected on a particle-by-particle basis reveal an apparent bistability in the fluorescence-voltage modulation curves for composite NPs of P3HT and [6,6]-phenyl-C(61)-butyric acid methyl ester (PC(60)BM) that is absent for pure P3HT NPs. A pronounced deep trapping of free electrons photogenerated from the composite P3HT/PC(60)BM NPs at the NP/dielectric interface and hole trapping by fullerene anions in composite P3HT/PC(60)BM NPs under photoexcitation lies at the basis of this finding. The deep electron trapping effect reported here for composite conjugated polymer/fullerene NPs presents an opportunity for future application of these NPs in nanoscale memory and imaging devices.

Boojum and stripe textures in long-range orientationally ordered monolayers on solid substrates.

Long-range organization of molecular tilt azimuth is a striking feature in monolayers at the air-water interface. We show that the boojum and stripe textures of pentadecanoic acid (PDA) with the continuous variations of molecular tilt azimuth formed at the air-water interface at temperatures lower than room temperature can be preserved after being transferred to glass substrates at low dipping speeds. The long-range tilt order in the transferred boojums and stripes is resolved by frictional force microscopy at room temperature, suggesting that the tilt order is "frozen" through the interaction of PDA molecules with the glass surface. The transferred stripe structure can be used as a unique alignment layer to induce a continuously azimuthal orientation of nematic liquid crystals.

Effect of PCBM concentration on photoluminescence properties of composite MEH-PPV/PCBM nanoparticles investigated by a Franck-Condon analysis of single-particle emission spectra.

The emission of composite conjugated polymer (MEH-PPV)/fullerene (PCBM) nanoparticles is investigated by single particle spectroscopy (SPS), and changes in vibronic structure with nanoparticle composition are evaluated by means of a detailed Franck-Condon analysis. Consistent with previous reports we find that the emission spectra can be modeled as the superposition of two types of emitters, one with aggregate character and one with molecular character. Major findings from the fitting of the SPS data to a Franck-Condon model are that 1) the occurrence of each of the two types of emitters changes with nanoparticle composition to the point that no aggregate emitters are detected (at 50 wt% PCBM), 2) at the highest PCBM doping levels (75 wt% PCBM) aggregate emitters reappear due to nanoscale phase separation in the composite nanoparticles, 3) the molecular emitters show small Huang-Rhys factors that increase with PCBM doping, indicative of extensive delocalization and exciton migration that is reduced by the disorder introduced in the polymer material by PCBM doping and 4) the aggregate emitters show large Huang-Rhys factors, indicative of the localized nature of these energy trap sites, with a broad distribution of values of these Huang-Rhys factors. The latter observation suggests a broad heterogeneous distribution of aggregate morphologies in blended conducting polymer materials, which can be attributed to variations in polymer chain folding and stacking at the aggregate sites. The reported results obtained by the SPS approach show how blending conjugated polymers with fullerenes at various doping levels induces changes in interchain interactions and aggregate site density even at length scales below a few tens of nanometers that affect conjugated polymer material properties, an observation that has gone unnoticed in bulk studies of blended conjugated polymer films.

Single molecule spectroscopy of organic dye nanoparticles.

Organic dye nanoparticles 1-13 nm in height and 10-45 nm in width were prepared by the reprecipitation method. With single-molecule/nanoparticle spectroscopy, two distinct types of nanoparticles were found: particles with blue emission and particles with red emission. The difference in spectral characteristics is attributed to the presence of two morphological types of particles in the samples. The presence of two types of nanoparticles in the samples was further corroborated by our ability to separate blue nanoparticles from red nanoparticles by centrifugation.