Measuring Cellular Uptake of Polymer Dots for Quantitative Imaging and Photodynamic Therapy.

There is a great deal of interest in the development of nanoparticles for biomedicine. The question of how many nanoparticles are taken up by cells is important for biomedical applications. Here, we describe a fluorescence method for the quantitative measurement of the cellular uptake of polymer dots (Pdots) and a further estimation of intracellular Pdots photosensitizer for fluorescence imaging and photodynamic therapy. The approach relies on the high brightness, excellent stability, minimal aggregation quenching, and metalloporphyrin doping properties of the Pdots. We correlated the single-cell fluorescence brightness obtained from fluorescence spectrometry, confocal microscopy, and flow cytometry with the number of endocytosed Pdots, which was validated by inductively coupled plasma mass spectrometry. Our results indicated that, on average, ∼1.3 million Pdots were taken up by single cells that were incubated for 4 h with arginine 8-Pdots (40 µg/mL, ∼20 nm diameter). The absolute number of endocytosed Pdots of individual cells could be estimated from confocal microscopy by comparing the single-cell brightness with the average intensity. Furthermore, we investigated the cell viability as a result of an intracellular Pdots photosensitizer, from which the half maximal inhibitory concentration was determined to be ∼7.2 × 105 Pdots per cell under the light dose of 60 J/cm2. This study provides an effective method for quantifying endocytosed Pdots, which can be extended to investigate the cellular uptake of various conjugated polymer carriers in biomedicine.

Ratiometric Fluorescent Detection of Intracellular Singlet Oxygen by Semiconducting Polymer Dots.

Singlet oxygen (1O2) plays important roles in many biological processes. However, it is very difficult to detect 1O2 in the intracellular environment because of its relatively low concentration and short lifetime. Here, we developed a ratiometric probe based on semiconducting polymer dots (Pdots) that can sensitively detect 1O2 in live cells. An organic dye, singlet oxygen sensor green (SOSG), was doped in polyfluorene Pdots, and excitation energy was efficiently transferred from the polymer to the SOSG dye. Accordingly, the Pdots showed constant blue fluorescence as a reference, and increased green fluorescence upon singlet oxygen generation. The ratiometric response of Pdots was examined in the intracellular environment by in situ 1O2 generation with a photosensitizer and light irradiation. Both spectroscopic measurements and confocal imaging were performed to monitor intracellular 1O2 generation during photodynamic therapy using the Pdot probe. Our results indicate that the SOSG-doped Pdots are promising for intracellular 1O2 detection.

Biodegradable Polymer Nanoparticles for Photodynamic Therapy by Bioluminescence Resonance Energy Transfer.

Conventional photodynamic therapy is severely constrained by the limited light-penetration depth in tissue. Here, we show efficient photodynamic therapy (PDT) mediated by bioluminescence resonance energy transfer (BRET) that overcomes the light-penetration limitation. The photosensitizer Rose Bengal (RB) was loaded in biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles, which were then conjugated with firefly luciferase. Spectroscopic characterizations indicated that BRET effectively activated RB to generate reactive oxygen species (ROS). In vitro studies of the cellular cytotoxicity and photodynamic effect indicated that cancer cells were effectively destroyed by BRET-PDT treatment. In vivo studies in a tumor-bearing mouse model demonstrated that tumor growth was significantly inhibited by BRET-PDT in the absence of external light irradiation. The BRET-mediated phototherapy provides a promising approach to overcome the light-penetration limitation in photodynamic treatment of deep-seated tumors.

Therapeutic Considerations and Conjugated Polymer-Based Photosensitizers for Photodynamic Therapy.

Conjugated polymers have recently attracted a great deal of attention for applications in photodynamic therapy (PDT) because of their light-harvesting capability, efficient energy transfer, and singlet oxygen generation properties. This review describes recent advances in PDT development, including therapeutic mechanisms of PDT in cancer treatments, light excitation methods, and especially recent advances of conjugated polyelectrolytes and conjugated polymer nanoparticles as photosensitizers. The future direction on PDT and further development of conjugated polymer photosensitizers are discussed. The aim of this review is to stimulate innovative ideas to synthesize a new generation of conjugated polymer photosensitizers and promote their translation to clinical applications of PDT.

Systematically quantifying the dynamic characteristics of PM2.5 in multiple indoor environments in a plateau city: Implication for internal contribution.

People generally spend most of their time indoors, making a comprehensive evaluation of air pollution characteristics in various indoor microenvironments of great significance for accurate exposure estimation. In this study, field measurements were conducted in Kunming City, Southwest China, using real-time PM2.5 sensors to characterize indoor PM2.5 in ten different microenvironments including three restaurants, four public places, and three household settings. Results showed that the daily average PM2.5 concentrations in restaurants, public spaces, and households were 78.4 ± 24.3, 20.1 ± 6.6, and 18.0 ± 4.3 µg/m3, respectively. The highest levels of indoor PM2.5 in restaurants were owing to strong internal emissions from cooking activities. Dynamic changes showed that indoor PM2.5 levels increased during business time in restaurants and public places, and cooking time in residential kitchens. Compared with public places, restaurants generally exhibit more rapid increases in indoor PM2.5 due to cooking activities, which can elevate indoor PM2.5 to high levels (5.1 times higher than the baseline) in a short time. Furthermore, indoor PM2.5 in restaurants were dominated by internal emissions, while outdoor penetration contributed mostly to indoor PM2.5 in public places and household settings. Results from this study revealed large variations in indoor PM2.5 in different microenvironments, and suggested site-specific measures for indoor PM2.5 pollution alleviation.

Rapid analysis of charge variants of monoclonal antibodies with capillary zone electrophoresis in dynamically coated fused-silica capillary.

A capillary zone electrophoresis (CZE) method was developed for the rapid analysis of charge heterogeneity of immunoglobulin G (IgG) monoclonal antibodies (mAbs). The separation was carried out in a short, dynamically coated fused-silica capillary. A number of separation parameters were investigated and optimized, including pH, concentration of the separation buffer (ε-amino caproic acid), concentration of the triethylenetetramine (TETA) dynamic coating, the capillary internal diameter and the field strength used for the separation. The effects of between-run flushing of the capillary and the data acquisition rate were also evaluated. Under the optimized conditions, a fast (<5 min), selective and reproducible separation of mAb charge variants was achieved under a very high electric field strength (1000 V/cm). This method also requires only a short conditioning of the capillary, with between-run conditioning completed within 2 min. The method was evaluated for specificity, sensitivity, linearity, accuracy and precision. The same separation conditions were applied to the rapid separation (2-5 min) of charge variants of multiple monoclonal antibodies with pI in the range of 7.0-9.5. Compared with other existing methods for charge variants analysis, this method has several advantages including a short run time, rapid capillary conditioning and simple sample preparation.

Recent advances in semiconducting polymer dots as optical probes for biosensing.

Optical probes that specifically and sensitively change the optical properties upon contact with targets have become irreplaceable tools in fundamental biology and medicine. Semiconducting polymer dots (Pdots) have emerged as popular optical nanoplatforms because of their excellent characteristics, such as tunable luminescence, high brightness, superior stability and biocompatibility, for biological applications. In particular, facile surface and intra-particle modifications enable Pdots to detect various biological parameters, such as reactive oxygen species (ROS), typical metal ions, pH values, temperature and a variety of biomolecules. In this review, we provide a brief overview of the preparation and bio-functionalization strategies of Pdots. This review focuses on the applications of Pdots as optical probes in biosensors and describes the challenges in this field.

Temporal and spatial variation of PM2.5 in indoor air monitored by low-cost sensors.

Indoor air pollution has significant adverse health impacts, but its spatiotemporal variations and source contributions are not well quantified. In this study, we used low-cost sensors to measure PM2.5 concentrations in a typical apartment in Beijing. The measurements were conducted at 15 indoor sites and one outdoor site on 1-minute temporal resolution (convert to 10-minute averages for data analysis) from March 14 to 24, 2020. Based on these highly spatially-and temporally-resolved data, we characterized spatiotemporal variations and source contributions of indoor PM2.5 in this apartment. It was found that indoor particulate matter predominantly originates from outdoor infiltration and cooking emissions with the latter contributing more fine particles. Indoor PM2.5 concentrations were found to be correlated with ambient levels but were generally lower than those outdoors with an average I/O of 0.85. The predominant indoor source was cooking, leading to occasional high spikes. The variations observed in most rooms lagged behind those measured outdoors and in the studied kitchen. Differences between rooms were found to depend on pathway distances from sources. On average, outdoor sources contributed 36% of indoor PM2.5, varying extensively over time and among rooms. From observed PM2.5 concentrations at the indoor sites, source strengths, and pathway distances, a multivariate regression model was developed to predict spatiotemporal variations of PM2.5. The model explains 79% of the observed variation and can be used to dynamically simulate PM2.5 concentrations at any site indoors. The model's simplicity suggests the potential for regional-scale application for indoor air quality modeling.

Analysis of identity, charge variants, and disulfide isomers of monoclonal antibodies with capillary zone electrophoresis in an uncoated capillary column.

A set of related capillary zone electrophoresis (CZE) methods have been developed for the analysis of identity, charge variants, and disulfide isoforms of IgG monoclonal antibodies (mAbs). These methods utilize an uncoated capillary column. The combined use of concentrated zwitterionic (e-amino-caproic acid) buffer and acid flushing was effective in minimizing the adsorption of protein to the inner wall of a bare capillary. Under these conditions, a selective and reproducible separation of multiple IgG1 and IgG2 monoclonal antibodies (mAbs) was obtained with a long capillary column (40 cm effective length), allowing the reliable identification of different mAbs by migration time. A rapid ( approximately 10 min) and selective separation of charged variants of IgG mAbs was attained using a short capillary column (10 cm effective length). Finally, the addition of urea in the separation buffer resulted in the separation of disulfide isoforms of IgG2 mAbs by CZE. CZE methods using an uncoated capillary column offer a versatile, generic, and economical approach to the evaluation of identity, charge heterogeneity, and disulfide isoforms of IgG antibodies.

In vivo dynamic cell tracking with long-wavelength excitable and near-infrared fluorescent polymer dots.

Development of cell-based therapeutic systems has attracted great interest in biomedicine. In vivo cell tracking by fluorescence provides indispensable information for further advancing cell therapy in clinical applications. However, it is still challenging in many cases because of the limited light penetration depth as well as the variations in fluorescent probes, cell lines, and labeling brightness. Here, we designed highly fluorescent polymer dots (Pdots) with far-red-light absorption and near-infrared (NIR) emission for cell tracking. The Pdots consisted of a donor-acceptor polymer blending system where intra-particle energy transfer yielded a narrow-band emission at 800 nm with a high quantum yield of ~0.22. We investigated biocompatibility and cell labeling brightness of the Pdots coated with cell penetrating peptides. Flow cytometry indicated that the cell-labeling brightness of both stem cells and cancer cells increased as much as ~4 orders of magnitude comparing the intensity measurements of labeled cells and controls. Yet, in vivo cell tracking results revealed distinctive fluorescence distribution for the same number of cells that were administered into mice through the tail vein. The stem cells initially accumulated in the lung and remained for seven days, whereas the cancer cells tended to be cleared by the liver in four days. The difference is likely due to the fact that cancer cells are easily attacked by the immune system, whereas stem cells have low immunogenicity. Results obtained herein confirm that NIR-fluorescent Pdots are promising platforms for in vivo cell tracking in small animals.

Sensitive quantitation of low level free polysaccharide in conjugate vaccines by size exclusion chromatography-reverse phase liquid chromatography with UV detection.

The level of free polysaccharide is a critical quality attribute of polysaccharide-protein conjugate vaccines. The work presented describes a simple and sensitive method for the determination of low level free polysaccharides in multiple polysaccharide-protein conjugates. The method utilizes a reverse phase (RP) column to perform a size exclusion chromatography (SEC) separation of free polysaccharide and a reverse phase liquid chromatography (RPLC) separation of free protein and protein-polysaccharide conjugate. The use of phosphate buffer in the mobile phase enables the universal and sensitive detection of low level free polysaccharides at UV 200 nm. The method has been validated to monitor low level free polysaccharide (<1 %) in multiple polysaccharide-protein conjugates. The limit of quantitation is 2 µg/ml or 0.3 % free polysaccharide in 0.6 mg/ml polysaccharide-protein conjugate. The accuracy is in the range of 94.1.0-108.5 %.

Quantifying source contributions for indoor CO2 and gas pollutants based on the highly resolved sensor data.

Household air pollution is the dominant contributor to population air pollutant exposure, but it is often of less concern compared with ambient air pollution. One of the major knowledge gaps in this field are detailed quantitative source contributions of indoor pollutants, especially for gaseous compounds. In this study, temporally, spatially, and vertically resolved monitoring for typical indoor gases including CO2, CO, formaldehyde, methane, and the total volatile organic compounds (VOCs) was conducted to address pollution dynamics and major sources in an urban apartment. The indoor concentrations were significantly higher than the simultaneously measured outdoor concentrations. A new statistic approach was proposed to quantitatively estimate contributions of different sources. It was estimated that outdoor CO2 contributed largely to the indoor CO2, while main indoor sources were human metabolism and cooking. Outdoor infiltration and cooking contributed almost equally to the indoor CO. The contribution of outdoor infiltration to methane was much higher than that to formaldehyde. Cooking contributed to 24%, 19%, and 25% of indoor formaldehyde, methane, and VOCs, whereas the other unresolved indoor sources contributed 61%, 19%, and 35% of these pollutants, respectively. Vertical measurements showed that the uplifting of hot air masses led to relatively high concentrations of the pollutants in the upper layer of the kitchen and in the other rooms to a lesser extent.

A novel model for regional indoor PM2.5 quantification with both external and internal contributions included.

PM2.5 (particulate matter with an aerodynamic size ≤ 2.5 µm) of indoor origins is a dominant contributor to the overall air pollution exposure. Although some sophisticated models have been developed to simulate indoor air quality for individual households, it is still challenging to quantify indoor PM2.5 on a regional scale, which is critical for health impact assessments. In this study, a new model was developed to predict indoor PM2.5 concentrations by quantifying the external penetration, as well as the internal contributions. The model was parameterized based on a set of simultaneously measured indoor and outdoor PM2.5 concentrations at five-second temporal resolution for 53 households in Beijing. This study found that indoor PM2.5 concentrations were significantly correlated with those in the outdoor environment with an approximately 1-h lag-time on average. Outdoor-to-indoor penetration dominated the contribution to indoor PM2.5 during polluted hours with relatively high ambient PM2.5 concentrations, while the indoor PM2.5, during clean hours, was contributed by internal sources, including smoking, cooking, incense burning, and human disturbance. The influence of windows, house area, and air purifier use was addressed in the new model. The model was applied to evaluate indoor PM2.5 concentrations in six urban districts of Beijing via an uncertainty analysis. The model was developed based on and applied to households using clean residential energy, and it would be interesting also important to evaluate it in households using solid fuels.

Analysis of variation in mouse TPMT genotype, expression and activity.

Although the mouse has great potential for pharmacogenomic discovery, little is known about variation in drug response or genetic variation in pharmacologically relevant genes between inbred mouse strains. We therefore assessed variation in gene sequence, mRNA expression and protein activity of thiopurine methyltransferase (TPMT) in multiple inbred mouse strains. TPMT activity was measured by high-performance liquid chromatography detection of 6-MMP produced by incubation of liver homogenates with 6-MP. Genetic variation was assessed by resequencing and single nucleotide polymorphism (SNP) genotyping using pyrosequencing technology. mRNA expression was measured by real-time polymerase chain reaction. We observed an almost five-fold variation in TPMT activity, with strains falling into distinct low and high activity groups. This pattern of TPMT activity was highly correlated with expression of TPMT mRNA among strains, and high TPMT expression is dominant in F1 hybrids. To correlate genotype with phenotype, 29 SNPs and one insertion/deletion were genotyped throughout the TPMT gene and upstream 10 kb. Only two haplotypes were observed across all 30 polymorphisms, corresponding to the low and high activity groups. These results suggest that differential mouse TPMT activity is due to variation in mRNA expression. In addition, the identified pattern of low haplotype diversity suggests that the mouse is likely to be useful for pharmacogenomic discovery by associating haplotype blocks with drug response phenotypes among inbred strains.

Simple and rapid docetaxel assay in plasma by protein precipitation and high-performance liquid chromatography-tandem mass spectrometry.

A simple, rapid and low cost sample preparation method was developed for quantification of docetaxel in mouse plasma by high-performance liquid chromatography/tandem mass spectrometry with paclitaxel as the internal standard. A small volume of plasma (40 microl) and one-step protein precipitation using methanol and acetonitrile (1:1 (v/v)) were used for sample preparation. The calibration curve for docetaxel in mouse plasma was linear over the range 25-2500 nM. The detection limit was 8 nM. The lower limit of quantitation is 25 nM. The intra- and inter-day precisions (CV) of analysis were 9.5 and 9.7% for the low quality control (LQC), 5.5 and 4.9% for the medium quality control (MQC) and 3.9 and 6.3% for the high quality control (HQC), respectively. The accuracy was 102.5% for LQC, 97.9% for MQC and 108.8% for HQC. This assay has now been applied to evaluation of mouse pharmacogenetics and other clinical pharmacology applications.

Relationship between red blood cell thiopurine methyltransferase activity and myelotoxicity in dogs receiving azathioprine.

Thiopurine methyltransferase (TPMT) produces inactive metabolites of azathioprine and, in humans, has a variable amount of activity. Humans with low TPMT activity commonly develop myelotoxicity when receiving azathioprine. Our study sought to characterize the distribution of TPMT activity in a population of dogs and to determine whether the pretreatment knowledge of red blood cell (RBC) TPMT activity could predict myelotoxicity in dogs receiving azathioprine. RBC TPMT activity was measured in 299 healthy dogs, and 9 dogs that represented a wide range of enzyme activity received azathioprine at a standard therapeutic dose for 30 days. TPMT activity in healthy dogs was log normally distributed and varied over an approximately 7-fold range. Geometric mean, minimum, and maximum RBC TPMT activities were 37.1, 16.3, and 115 nmol per gram of hemoglobin (gHb) per hour, respectively. TPMT deficiency was not identified. Two populations of TPMT activity in dogs were detected by statistical modeling (commingling analysis). Dogs with intermediate TPMT activity (14-38 nmol/gHb/h) receiving azathioprine had significantly lower neutrophil counts during week 4 than during weeks 0-3, whereas those with high activity (>39 nmol/gHb/h) did not have a significant change in neutrophil count. An analysis of TPMT activity in 6 dogs with a history of azathioprine-associated myelotoxicity in a clinical setting revealed either intermediate or high TPMT enzyme activity in all dogs, suggesting that TPMT activity, as measured in RBCs, is not the sole cause of severe azathioprine-associated myelosuppression in dogs.

Size exclusion chromatography of polysaccharides with reverse phase liquid chromatography.

This work describes the use of a reverse phase (RP) column for size-based separation of hydrophilic polysaccharides. The entire separation window (before and after void volume) is used to provide unique separation of polysaccharides from other components in a glycol-conjugate vaccine or complex media of fermentation broth. The technique has also been applied to the separation of polysaccharides of different sizes. The effect of chromatographic parameters including type of packing material (CN, C8 and C18), pore size (80 and 300Å) of stationary phase, concentration of organic solvent on the separation was investigated. In addition, characterization of size-based separation was evaluated by coupling of RP column with multi-angle laser light scattering (MALLs) detector.