Use Of Fluorescent Labels Research Articles

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes.

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

Machine learning-based motion tracking reveals an inverse correlation between adhesivity and surface motility of the leptospirosis spirochete

Bacterial motility is often a crucial virulence factor for pathogenic species. A common approach to study bacterial motility is fluorescent labeling, which allows detection of individual bacterial cells in a population or in host tissues. However, the use of fluorescent labeling can be hampered by protein expression stability and/or interference with bacterial physiology. Here, we apply machine learning to microscopic image analysis for label-free motion tracking of the zoonotic bacterium Leptospira interrogans on cultured animal cells. We use various leptospiral strains isolated from a human patient or animals, as well as mutant strains. Strains associated with severe disease, and mutant strains lacking outer membrane proteins (OMPs), tend to display fast mobility and reduced adherence on cultured kidney cells. Our method does not require fluorescent labeling or genetic manipulation, and thus could be applied to study motility of many other bacterial species.

Deep Semantic Segmentation of Cell Painted Nuclei Images using UNet++

Abstract Fluorescence microscopy based cell painting technique profiles the morphological characteristics of specific cell organelles with high resolution. However, photo toxicity, photo bleaching and advanced instrumentation limits its utility for comprehensively annotating the cell structure. Generating cell painted organelles from simple and least invasive transmitted light microscopy provides a surrogate for clinical applications. In this study, the employability of semantic segmentation model for delineating the nuclei from composite image using UNet++ is investigated. For this, a public dataset consisting of 3456 composite images are considered from Broad Bioimage Benchmark collection. The binary masks of endoplasmic reticulum (ER), nuclei and cytoplasm are generated for pixel wise labelling. The composite images and their labelled dataset are fed to the UNet++ model for segmenting the cell painted nuclei. The performance of the deep semantic network is analysed for 50 epochs and the segmentation results are validated with mean intersection-over-union (IoU). The UNet++model provides an accuracy of 95.8% with a minimum loss of 0.1. Mean IoU of 0.91 is achieved for the prediction of nuclei from the composite images. The obtained results suggest that this study could be employed for predicting the subcellular components from transmitted light microscopy without the use of fluorescent labelling.

Investigating ionizing radiation-induced changes in breast cancer cells using stimulated Raman scattering microscopy.

Altered lipid metabolism of cancer cells has been implicated in increased radiation resistance. A better understanding of this phenomenon may lead to improved radiation treatment planning. Stimulated Raman scattering (SRS) microscopy enables label-free and quantitative imaging of cellular lipids but has never been applied in this domain. We sought to investigate the radiobiological response in human breast cancer MCF7 cells using SRS microscopy, focusing on how radiation affects lipid droplet (LD) distribution and cellular morphology. MCF7 breast cancer cells were exposed to either 0 or 30Gy (X-ray) ionizing radiation and imaged using a spectrally focused SRS microscope every 24hrs over a 72-hr time period. Images were analyzed to quantify changes in LD area per cell, lipid and protein content per cell, and cellular morphology. Cell viability and confluency were measured using a live cell imaging system while radiation-induced lipid peroxidation was assessed using BODIPY C11 staining and flow cytometry. The LD area per cell and total lipid and protein intensities per cell were found to increase significantly for irradiated cells compared to control cells from 48 to 72hrs post irradiation. Increased cell size, vacuole formation, and multinucleation were observed as well. No significant cell death was observed due to irradiation, but lipid peroxidation was found to be greater in the irradiated cells than control cells at 72hrs. This pilot study demonstrates the potential of SRS imaging for investigating ionizing radiation-induced changes in cancer cells without the use of fluorescent labels.

Post-translational modifications and glycoprofiling of palivizumab by UHPLC-RPLC/HILIC and mass spectrometry.

Viral infections are progressively becoming a global health burden, as witnessed in the ongoing COVID-19 pandemic. Respiratory Syncytial Virus (RSV) is another highly contagious negative-sense RNA virus that causes lower respiratory tract infections and high mortality in infants. Palivizumab (Synagis®) is the only humanized monoclonal antibody (mAb) approved by the FDA against RSV. The virus neutralization efficacy often depends on the nature and abundance of the glycoforms in therapeutic mAbs. Therefore, a thorough estimation of their PTM profile, especially glycosylation, is relevant. Here, we describe the intact and released glycan analysis of palivizumab (Synagis®) using HILIC chromatography and mass spectrometry. We detected five glycoforms (Man5/G0FB, G0F/G1F, G1F/G1F, G0FB/G0FB, and G2F/G2F) in deconvoluted MS spectra of intact glycosylated palivizumab. The mapping of the peptide and glycopeptides using LC–ESI–MS led to the detection of associated PTMs and the direct identification of a glycopeptide, GlcNAc3Man2. EEQYNSTYR, derived from the heavy chain of palivizumab.Release glycan analysis using UHPLC–HILIC revealed a typical glycan profile consisting of major glycans, G0F (33.94%), G1F (35.50%), G2F (17.24%) also reported previously and minor G1F’ (5.81%), Man5 (3.96%) and G0FB (2.26%) forms with the superior resolution of isomeric G1F/G1F’. Next, we provide the first experimental evidence of Neu5Gc in the commercial palivizumab formulation using DMB labelling. The estimated monosaccharide composition was consistent with previous studies. The findings of the study highlight the efficiency of the release glycan method in providing a correct measure of the total palivizumab glycan pool compared to the intact glycoprotein/glycopeptide approach. The UHPLC–RPLC/HILIC and MS combinations provide a more comprehensive glycoprofile assessment due to the parallel use of fluorescent labels for the analysis of the release of N-glycan, sialic acid, and monosaccharide composition. This approach is suitable for quick quality testing and market surveillance of therapeutic mAbs. Alongside a well-perceived need for cost-effective immunoprophylaxis and the ongoing fast-paced development of next-generation variants of palivizumab, such as MEDI8897, the study reiterates glycosylation as a critical parameter that needs monitoring for drug characterization and quality control.Supplementary InformationThe online version contains supplementary material available at 10.1007/s42485-022-00086-1.

Development of Mechanical Stability in Late-Stage Embryonic Erythroid Cells: Insights From Fluorescence Imaged Micro-Deformation Studies.

The combined use of fluorescence labeling and micro-manipulation of red blood cells has proven to be a powerful tool for understanding and characterizing fundamental mechanisms underlying the mechanical behavior of cells. Here we used this approach to study the development of the membrane-associated cytoskeleton (MAS) in primary embryonic erythroid cells. Erythropoiesis comes in two forms in the mammalian embryo, primitive and definitive, characterized by intra- and extra-vascular maturation, respectively. Primitive erythroid precursors in the murine embryo first begin to circulate at embryonic day (E) 8.25 and mature as a semi-synchronous cohort before enucleating between E12.5 and E16.5. Previously, we determined that the major components of the MAS become localized to the membrane between E10.5 and E12.5, and that this localization is associated with an increase in membrane mechanical stability over this same period. The change in mechanical stability was reflected in the creation of MAS-free regions of the membrane at the tips of the projections formed when cells were aspirated into micropipettes. The tendency to form MAS-free regions decreases as primitive erythroid cells continue to mature through E14.5, at least 2 days after all detectable cytoskeletal components are localized to the membrane, indicating continued strengthening of membrane cohesion after membrane localization of cytoskeletal components. Here we demonstrate that the formation of MAS-free regions is the result of a mechanical failure within the MAS, and not the detachment of membrane bilayer from the MAS. Once a “hole” is formed in the MAS, the skeletal network contracts laterally along the aspirated projection to form the MAS-free region. In protein 4.1-null primitive erythroid cells, the tendency to form MAS-free regions is markedly enhanced. Of note, similar MAS-free regions were observed in maturing erythroid cells from human marrow, indicating that similar processes occur in definitive erythroid cells. We conclude that localization of cytoskeletal components to the cell membrane of mammalian erythroid cells during maturation is insufficient by itself to produce a mature MAS, but that subsequent processes are additionally required to strengthen intraskeletal interactions.

効率的なナノ粒子検出に向けた振動誘起マイクロ混合デバイスの開発

Detection of nanoparticles (NPs) plays an important role in a wide variety of fields such as medical diagnostics and environmental monitoring. We have been developing a simple system for detecting NPs in small samples using vibration-induced flow (VIF), in which a mean flow is induced around a micropillar by applying small periodic vibrations. The system can evaluate the presence and concentration of specific NPs in a minute sample from the extent of aggregation of affinity capture beads agitated by VIF. This technique eliminates the use of fluorescent labeling or expensive image analysis equipment. In this study, we investigated the relationship between various flow patterns and capture bead aggregation. It was found that large vortex flows induced by circular and elliptical vibration produce large aggregations, whereas small vortex flows induced by rectilinear vibration produce a large number of small aggregations with relatively uniform sizes. This result indicates that the size of aggregations depends on the size of the vortices in the flow field and that can be readily controlled by the flow pattern tuned with vibration conditions.

Quantification of ATP in cell by fluorescence spectroscopy based on generalized ratio quantitative analysis model

In this contribution, fluorescence spectroscopy based on G-quadruplex formation was developed for the quantification of ATP in cell. In the absence of ATP, thioflavin T (ThT) dye can combine with the G-rich ATP aptamer to form an ATP aptamer-ThT G-quandruplex complex, resulting in the enhancement of fluorescence intensity; otherwise, fluorescence intensity of the system will weaken with the addition of ATP, because ATP has a strong affinity with G-rich ATP aptamer and can replace ThT to form an ATP aptamer-ATP complex. A calibration model based on generalized ratio quantitative analysis model was employed to mitigate the influence of scatterers and background absorbers in cell suspensions. The proposed fluorescence method was applied to the quantitative analysis of ATP in two type of cell lines, and achieved satisfactory quantitative results with accuracy comparable to that of the reference method–ATP detection kit. The limit of detection and limit of quantification of the proposed method for ATP in cell were estimated to be 0.22 and 0.66 μM, respectively. This proposed fluorescence method is highly simple and rapid, and does not require the use of fluorescent labeling.

Machine learning based approach to pH imaging and classification of single cancer cells.

The ability to identify different cell populations in a noninvasive manner and without the use of fluorescence labeling remains an important goal in biomedical research. Various techniques have been developed over the last decade, which mainly rely on fluorescent probes or nanoparticles. On the other hand, their applications to single-cell studies have been limited by the lengthy preparation and labeling protocols, as well as issues relating to reproducibility and sensitivity. Furthermore, some of these techniques require the cells to be fixed. Interestingly, it has been shown that different cell types exhibit a unique intracellular environment characterized by specific acidity conditions as a consequence of their distinct functions and metabolism. Here, we leverage a recently developed pH imaging modality and machine learning-based single-cell segmentation and classification to identify different cancer cell lines based on their characteristic intracellular pH. This simple method opens up the potential to perform rapid noninvasive identification of living cancer cells for early cancer diagnosis and further downstream analyses.

Fluorescence-Based and Fluorescent Label-Free Characterization of Polymer Nanoparticle Decorated T Cells.

Cells are attractive carriers for the transport and delivery of nanoparticulate cargo. The use of cell-based carriers allows one to enhance control over the biodistribution of drug-loaded polymers and polymer nanoparticles. One key element in the development of cell-based delivery systems is the loading of the cell-based carrier with the nanoparticle cargo, which can be achieved either by internalization of the payload or by immobilization on the cell surface. The surface modification of cells with nanoparticles or the internalization of nanoparticles by cells is usually monitored with fluorescence-based techniques, such as flow cytometry and confocal microscopy. In spite of the widespread use of these techniques, the use of fluorescent labels also poses some risks and has several drawbacks. Fluorescent dyes may bleach, or leach from, the nanoparticles or alter the physicochemical properties of nanoparticles and their interactions with and uptake by cells. Using poly(d,l-lactic acid) nanoparticles that are loaded with Coumarin 6, BODIPY 493/503, or DiO dyes as a model system, this paper demonstrates that the use of physically entrapped fluorescent labels can lead to false negative or erroneous results. The use of nanoparticles that contain covalently tethered fluorescent dyes instead was found to provide a robust approach to monitor cell surface conjugation reactions and to quantitatively analyze nanoparticle-decorated cells. Finally, it is shown that optical diffraction tomography is an attractive, alternative technique for the characterization of nanoparticle-decorated cells, which obviates the need for fluorescent labels.

SYNTHESIS OF MODYFIED FLUORESCEINE FOR CLICK REACTIONS

Reactions of azide-alkilic cycloaddition are well known since 1893. At the same time, with the elaboration of click chemistry, the techniques of labelling biological objects, particularly by fluorescent dyes, were widely developed. The use of fluorescent labels provides the ability to visually monitor the process streamlines hardware load and reduces research time. Fluorescein is among the fluorophores mostly used for labelling of biomolecules due to its high quantum yields and good stability in biological media. However, despite of commercial availability of fluorescein derivatives functionalized for use in click reactions, methods for their synthesis are virtually absent in the literature. Therefore, we have developed a reliable and effective methodic for the synthesis of functionalized fluoresceines for the use in the click reactions. Synthesis of diacetyl N-(4-azidobutyl)-fluoresceine-5(6)-carboxamide was performed in six stages, starting form resorcinol and trimellitic acid anhydride. The diacetylated analogue was synthesized for the click modifications in the “soft” conditions, since N-(4-azidobutyl)-fluorescein-5(6)-carboxamide is poorly soluble in the classic organic solvents. Proposed synthetic protocol allows to increase the yield of the final and intermediate compounds and to optimized the procedure of their isolation and purification.

Malaria-Infected Red Blood Cell Analysis through Optical and Biochemical Parameters Using the Transport of Intensity Equation and the Microscope's Optical Properties.

The accuracy, reliability, speed and cost of the methods used for malaria diagnosis are key to the diseases’ treatment and eventual eradication. However, improvement in any one of these requirements can lead to deterioration of the rest due to their interdependence. We propose an optical method that provides fast detection of malaria-infected red blood cells (RBCs) at a lower cost. The method is based on the combination of deconvolution, topography and three-dimensional (3D) refractive index reconstruction of the malaria-infected RBCs by use of the transport of intensity equation. Using our method, healthy RBCs were identified by their biconcave shape, quasi-uniform spatial distribution of their refractive indices and quasi-uniform concentration of hemoglobin. The values of these optical and biochemical parameters were found to be in agreement with the values reported in the literature. Results for the malaria-infected RBCs were significantly different from those of the healthy RBCs. The topography of the cells and their optical and biochemical parameters enabled identification of their stages of infection. This work introduces a significant method of analyzing malaria-infected RBCs at a lower cost and without the use of fluorescent labels for the parasites.

Fluorophore Labeling Affects the Cellular Accumulation and Gene Silencing Activity of Cholesterol-Modified siRNAs In Vitro.

The objective of this study was to analyze the effects of fluorophores on the intracellular accumulation and biological activity of small interfering RNA (siRNA) and its cholesterol conjugates. In this study, we used stem-loop real-time PCR and calibration curves to quantitate cellular siRNA accumulation. Attachment of fluorophores significantly affected both the accumulation and biological activity of siRNA conjugates. The severity of this effect depended significantly on the structure of the conjugate; fluorophores (Cy5.5 or Alexa-488) attached to siRNA, facing the side of the duplex opposite to cholesterol, enhanced the unproductive intracellular accumulation of the conjugate when delivered in carrier-free mode. Enhanced cellular accumulation of siRNA conjugates did not result in enhanced biological activity of the conjugate. Moreover, the attachment of a hydrophobic fluorophore, such as Cy5.5, to conventional siRNA also enhanced its apparent intracellular accumulation, but not its biological activity. Thus, the use of fluorescent labels for the study of the intracellular accumulation of siRNA and its conjugates formed with different molecules is possible only for a limited range of structures, and requires verification using alternative methods.

Abstract 5030: Development and optimization of Matrigel-based multi-spheroid 3D tumor assays using real-time live-cell analysis

Abstract The tumor-associated extracellular matrix (ECM) provides critical biochemical micro-environment cues, as well as an essential structural scaffold, for solid tumors to survive and grow (see Pickup et al. 2014 for review). With a view to enabling more translational and turnkey 3D in vitro assays for cancer biology, we have developed and optimized techniques for seeding, growing and automatically quantifying the properties of multiple tumor spheroids in ECMs in 96-well format using real-time live-cell analysis. Matrigel (Corning) was dispensed across a range of volumes (20 - 50 μL) and concentrations (1 - 5 mg mL-1) into flat-bottomed 96-well TC micro-plates to form a solidified base layer. Tumor cells (A549, MCF-7, SKOV-3, MDA-MB-231) were seeded on top (1 - 2K cells per well), and in some experiments a full ECM sandwich was created by addition of a further volume of Matrigel (2 - 25%, 0.2 - 5 mg mL-1). Using a custom autofocusing method, phase contrast, bright-field and fluorescence images (10x) were captured every 6h for 7 days from within the cell incubator (IncuCyte S3 live-cell analysis system). Typically, 20 - 80 spheroids were analyzed in each well. All four cell types formed multiple cell aggregates within the first 3 days, ranging in diameter from 30 - 80 μM. A549, SKOV-3 and MCF-7 multi-spheroids grew as round aggregates while MDA-MB-231 spheroids displayed stellate branching characteristic of an invasive morphology. At Matrigel volumes less than 40 μL or concentrations less than 3 mg mL-1, cells penetrated to the base of the plate and grew as ‘flat 2D' cultures. Using a novel bright-field image analysis algorithm, the number, area and average size of the spheroids could be computed over time non-invasively and without the use of fluorescent labels. Once formed, A549, SKOV-3, MCF-7 and MDA-MB-231 multi-spheroids increased 3.0-, 1.6-, 3.8- and 3.3-fold in size over 4 days, respectively. Treatment of A549 multi-spheroids with the DNA enzyme topo-isomerase inhibitor camptothecin (1μM) inhibited growth with comparable spheroid size at day 0 and day 7 post treatment (average brightfield area 1.4 x104 μM2). Using fluorescent protein reporters for apoptosis (Annexin V) and cell viability (IncuCyte CytoTox Green) we could verify camptothecin-induced cell death (fluorescence values 149±16% of control (Annexin V) and 243±51% of control (CytoTox). A concomitant decrease of stably expressed RFP (to 3±1% of control) was observed. The combination of protocol developments, novel image acquisition/analysis algorithms and cell health reporters creates an integrated solution for measuring growth and vitality of multiple small spheroids in a relevant and 3D bio-matrix over time. This approach should be applicable to primary- and patient-derived organoid tumor samples as well as cancer cell lines. Pickup, MW, Muow, JK, Weaver, MW (2014), EMBO Rep. 15(12): 1243-1253 Citation Format: Kalpana Patel, Miniver Oliver, Nevine Holtz, Tim Jackson, Nicholas Dana, Gillian Lovell, Nicola J. Bevan, Tim J. Dale, Derek J. Trezise. Development and optimization of Matrigel-based multi-spheroid 3D tumor assays using real-time live-cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5030.

Optical projection tomography for rapid whole mouse brain imaging.

In recent years, three-dimensional mesoscopic imaging has gained significant importance in life sciences for fundamental studies at the whole-organ level. In this manuscript, we present an optical projection tomography (OPT) method designed for imaging of the intact mouse brain. The system features an isotropic resolution of ~50 µm and an acquisition time of four to eight minutes, using a 3-day optimized clearing protocol. Imaging of the brain autofluorescence in 3D reveals details of the neuroanatomy, while the use of fluorescent labels displays the vascular network and amyloid deposition in 5xFAD mice, an important model of Alzheimer's disease (AD). Finally, the OPT images are compared with histological slices.

Microfluidic Programming of Compositional Hydrogel Landscapes.

A droplet microfluidics strategy to rapidly synthesize, process, and screen up to hundreds of thousands of compositionally distinct synthetic hydrogels is presented. By programming the flow rates of multiple microfluidic inlet channels supplying individual hydrogel building blocks, microgel compositions and properties are systematically modulated. The use of fluorescent labels as proxies for the physical and chemical properties of the microgel permits the rapid screening and discovery of specific formulations through fluorescence microscopy or flow cytometry. This concept should accelerate the discovery of new hydrogel formulations for various novel applications.

Design of a dynamic biofilm imaging cell for white-light interferometric microscopy

In microbiology research, there is a strong need for next-generation imaging and sensing instrumentation that will enable minimally invasive and label-free investigation of soft, hydrated structures, such as in bacterial biofilms. White-light interferometry (WLI) can provide high-resolution images of surface topology without the use of fluorescent labels but is not typically used to image biofilms because there is insufficient refractive index contrast to induce reflection from the biofilm’s interface. The soft structure and water-like bulk properties of hydrated biofilms make them difficult to characterize in situ, especially in a nondestructive manner. We build on our prior description of static biofilm imaging and describe the design of a dynamic growth flow cell that enables monitoring of the thickness and topology of live biofilms over time using a WLI microscope. The microfluidic system is designed to grow biofilms in dynamic conditions and to create a reflective interface on the surface while minimizing disruption of fragile structures. The imaging cell was also designed to accommodate limitations imposed by the depth of focus of the microscope’s objective lens. Example images of live biofilm samples are shown to illustrate the ability of the flow cell and WLI instrument to (1) support bacterial growth and biofilm development, (2) image biofilm structure that reflects growth in flow conditions, and (3) monitor biofilm development over time nondestructively. In future work, the apparatus described here will enable surface metrology measurements (roughness, surface area, etc.) of biofilms and may be used to observe changes in biofilm structure in response to changes in environmental conditions (e.g., flow velocity, availability of nutrients, and presence of biocides). This development will open opportunities for the use of WLI in bioimaging.

Sensitive detection of malachite green and crystal violet by nonlinear laser wave mixing and capillary electrophoresis.

An ultrasensitive label-free antibody-free detection method for malachite green and crystal violet is presented using nonlinear laser wave-mixing spectroscopy and capillary zone electrophoresis. Wave-mixing spectroscopy provides a sensitive absorption-based detection method for trace analytes. This is accomplished by forming dynamic gratings within a sample cell, which diffracts light to create a coherent laser-like signal beam with high optical efficiency and high signal-to-noise ratio. A cubic dependence on laser power and square dependence on analyte concentration make wave mixing sensitive enough to detect molecules in their native form without the use of fluorescent labels for signal enhancement. A 532 nm laser and a 635 nm laser were used for malachite green and crystal violet sample excitation. The use of two lasers of different wavelengths allows the method to simultaneously detect both analytes. Selectivity is obtained through the capillary zone electrophoresis separation, which results in characteristic migration times. Measurement in capillary zone electrophoresis resulted in a limit of detection of 6.9 × 10(-10)M (2.5 × 10(-19) mol) for crystal violet and 8.3 × 10(-11)M (3.0 × 10(-20) mol) for malachite green at S/N of 2.

Derivatisable Cyanobactin Analogues: A Semisynthetic Approach

Many natural cyclic peptides have potent and potentially useful biological activities. Their use as therapeutic starting points is often limited by the quantities available, the lack of known biological targets and the practical limits on diversification to fine‐tune their properties. We report the use of enzymes from the cyanobactin family to heterocyclise and macrocyclise chemically synthesised substrates so as to allow larger‐scale syntheses and better control over derivatisation. We have made cyclic peptides containing orthogonal reactive groups, azide or dehydroalanine, that allow chemical diversification, including the use of fluorescent labels that can help in target identification. We show that the enzymes are compatible and efficient with such unnatural substrates. The combination of chemical synthesis and enzymatic transformation could help renew interest in investigating natural cyclic peptides with biological activity, as well as their unnatural analogues, as therapeutics.

Multifunctional drug carriers comprised of mesoporous silica nanoparticles and polyamidoamine dendrimers based on layer-by-layer assembly

To overcome serious limitations encountered by mesoporous silica nanoparticle (MSN) and dendrimers used alone, and to further enhance their performance for application in cancer treatment, multifunctional mesoporous silica nanoparticles as nanocarriers were prepared by layer-by-layer assembly method, with biocompatible Polyamidoamine (PAMAM) dendrimers and chondroitin sulfate (CS). Due to being incorporated to MSN, PAMAM dendrimers can avoid the fast clearance. Attributing to the multilayer coating of PAMAM dendrimers and CS, the nanocarriers possess self-fluorescent property, avoiding the use of fluorescent labels and enabling to release drugs responded to pH. Furthermore, chondroitin sulfate, a natural polysaccharide found in bone, cartilage, and connective tissue, can be served as linking points to bring dendrimers together, and enhance the delivery performance via its targeting ability.