Synthesis and characterization of dual-functionalized core-shell fluorescent microspheres for bioconjugation and cellular delivery

The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins, as described in the accompanying paper

Polymeric microspheres as protein transduction reagents

Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere-protein linkage was stable for ≥90 h at 37°C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake

Eyes on the future – evidence for trade‐offs between growth, storage and defense in Norway spruce

Carbon (C) allocation plays a central role in tree responses to environmental changes. Yet, fundamental questions remain about how trees allocate C to different sinks, for example, growth vs storage and defense. In order to elucidate allocation priorities, we manipulated the whole‐tree C balance by modifying atmospheric CO2 concentrations [CO2] to create two distinct gradients of declining C availability, and compared how C was allocated among fluxes (respiration and volatile monoterpenes) and biomass C pools (total biomass, nonstructural carbohydrates (NSC) and secondary metabolites (SM)) in well‐watered Norway spruce (Picea abies) saplings. Continuous isotope labelling was used to trace the fate of newly‐assimilated C. Reducing [CO2] to 120 ppm caused an aboveground C compensation point (i.e. net C balance was zero) and resulted in decreases in growth and respiration. By contrast, soluble sugars and SM remained relatively constant in aboveground young organs and were partially maintained with a constant allocation of newly‐assimilated C, even at expense of root death from C exhaustion. We conclude that spruce trees have a conservative allocation strategy under source limitation: growth and respiration can be downregulated to maintain ‘operational’ concentrations of NSC while investing newly‐assimilated C into future survival by producing SM.Supplementary material: Fig. S1 Concentrations of soluble sugars, starch and NSC (soluble sugars + starch) expressed as percentage of control (400 ppm [CO2]) at the whole‐tree level. Fig. S2 Concentrations of soluble sugars, starch and NSC (soluble sugars + starch) at the whole‐tree level. Fig. S3 Concentrations of phenolic compounds, monoterpenes and total secondary metabolites expressed as percentage of control (400 ppm [CO2]) at the whole‐tree level. Fig. S4 Concentrations of phenolic compounds, monoterpenes and total secondary metabolites (phenolic compounds + monoterpenes) at the whole‐tree level. Fig. S5 δ13C (‰) of bulk tissue, water soluble C and phenolic compounds at the whole‐tree level. Methods S1 TD‐GC‐MS conditions for BVOC analysis. Table S1 Internal standards, weight‐based response factors and methods used for the measurements of secondary metabolites. Table S2 A rough estimation of allocation of newly‐assimilated carbon.JH was funded by the Chinese Scholarship Council and Max Planck Institute for Biogeochemistry, and acknowledges support from the International Max Planck Research School for Global Biogeochemical Cycles.http://www.newphytologist.com2020-04-01hj2019Forestry and Agricultural Biotechnology Institute (FABI)Zoology and Entomolog

Versatile workflow for cell-type resolved transcriptional and epigenetic profiles from cryopreserved human lung

Complexity of lung microenvironment and changes in cellular composition during disease make it exceptionally hard to understand molecular mechanisms driving development of chronic lung diseases. Although recent advances in cell type–resolved approaches hold great promise for studying complex diseases, their implementation relies on local access to fresh tissue, as traditional tissue storage methods do not allow viable cell isolation. To overcome these hurdles, we developed a versatile workflow that allows storage of lung tissue with high viability, permits thorough sample quality check before cell isolation, and befits sequencing-based profiling. We demonstrate that cryopreservation enables isolation of multiple cell types from both healthy and diseased lungs. Basal cells from cryopreserved airways retain their differentiation ability, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing and EPIC Array, we show that gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for omics profiling of lung cells. Moreover, we obtained high-quality single-cell RNA-sequencing data of cells from cryopreserved human lungs, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our workflow is well suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to viable human tissue

Research data supporting "Targeted β-phase formation in poly(fluorene)-ureasil grafted organic-inorganic hybrids"

The folder “Figure 2” contains data for the 1H NMR spectra of PF-PTES (3), ICPTES (2) and PFO-OH (1), recorded in CDCl3, using 400 MHz. The folder “Figure 3” contains data for FTIR spectrum and gaussian curve fitting results of the amide I region of DU-PF-0.01, to quantitively analyse the hydrogen-bonding structure in the ureasil hybrids. The folder “Figure 4” contains data for 29Si MAS NMR spectra recorded for DU-PF-0.05, DU-PF-0, TU-PF-0.05 and TU-PF-0, using cross-polarised (CP) and directly excited (DE) magic angle spinning (MAS) solid-state NMR spectrum, operating at 79.44 MHz. The folder “Figure 5” contains the data for absorption, excitation, and emission spectra of PFO-OH in THF solution, DU-PF-0.01 and TU-PF-0.01. The excitation and emission spectra of PFO-OH in THF solution were recorded with emission and excitation wavelength of 480 and 350 nm, respectively. The excitation and emission spectra of DU-PF-0.01 and TU-PF-0.01 were recorded with emission and excitation wavelength of 500 and 360 nm, respectively. The folder “Figure 6” contains data for (a) emission (λex = 360 nm) and (b) excitation (b, λem = 500 nm) spectra of DU-PF-0.01 recorded as a function of time after the initiation of the sol-gel reaction (t = 0 to 143 h). The folder “Figure 7” contains data for (a) area-normalised excitation spectrum of DU-PF-0.01 (λem = 480 nm) and the corresponding Gaussian fits of the spectral components associated with the vibronic modes of PFO-OH and (b) %β-contribution and photoluminescence quantum yields as a function of the PFO-OH wt% for the DU-PF-x and TU-PF-x sample series. The folder “Figure S1” contains the data for the thermograms of ICPTES, PFO and PF-PTES. The data was recorded at air atmosphere, with heat rate = 10 oC min-1. The folder “Figure S2” contains the data for the FTIR spectra of (a) ICPTES, Jeffamine ED-600 and d-UPTES and (b) ICPTES, Jeffamine T-403 and t-UPTES. The FTIR spectra were recorded at a resolution of 4 cm-1, over a range of 4000-650 cm-1 to monitor the completion of the coupling between ICPTES and Jeffamine during the first step of the sol-gel reaction. The folder “Figure S3” contains the data for the FTIR spectra and Gaussian curve-fittings for the Amide I region of (a) DU-PF-0 (b) DU-PF-0.05 (c) DU-PF-0.1 (d) TU-PF-0 (e) TU-PF-0.01 (f) TU-PF-0.05 and (g) TU-PF-0.1. The data are used to quantitively analyse the hydrogen bonding structure of the ureasil hybrids. The folder “Figure S4” contains the data for the 13C NMR spectra of (a) TU-PF-0.05, (b) TU-PF-0, (c) DU-PF-0.05 and (d) DU-PF-0, recorded with cross-polarised (CP) magic angle spinning (MAS) solid state NMR, with 100.56 MHz. The folder “Figure S5” contains the data for powder X-ray diffraction measurements for (a) DU-PF-x and (b) TU-PF-x samples, where x stands for the concentration of the conjugated polymer in the ureasil matrixes. The folder “Figure S6” contains the data for the thermograms of (a) DU-PF-0, DU-PF-0.01, DU-PF-0.05 and DU-PF-0.1 and (b) TU-PF-0, TU-PF-0.01. TU-PF-0.05 and TU-PF-0.1, measured in air atmosphere, with heating rate of 10 oC min-1. The folder “Figure S7” contains the data for the emission spectra of (a) DU-PF-0.01, (b) DU-PF-0.05, (c) DU-PF-0.1, (d) TU-PF-0.01 (e) TU-PF-0.05 and (f) TU-PF-0.1 as a function of excitation wavelengths (320, 330 and 340 nm). The folder “Figure S8” contains the data for the excitation spectra of (a) DU-PF-0.01, (b) DU-PF-0.05, (c) DU-PF-0.1, (d) TU-PF-0.01 (e) TU-PF-0.05 and (f) TU-PF-0.1 as a function of emission wavelengths (430, 440, 460, 470, 500 and 520 nm). The folder “Figure S9” contains the data for the area-normalised excitation spectrum of PFO-OF (10-6 mol × dm-3, λem = 480 nm) and the corresponding Gaussian-fits of the spectral components associated with the 0-0, 0-1, 0-2, 0-3 vibronic transition of PFO-OH and the blue and purplish components of the ureasil. The folder “Figure S10” contains the data for the excitation spectra (λem = 480 nm) of (a) DU-PF-0.05, (b) DU-PF-0.05 and (d) TU-PF-0.1 and the corresponding Gaussian-fits of the spectral components 0-0, 0-1, 0-2, 0-3 and the ureasil blue and purplish components

Photopatterning of self assembled monolayers on oxide surfaces for the selective attachment of biomolecules

The immobilization of functional biomolecules to surfaces is a critical process for the development of biosensors for disease diagnostics. In this work we report the patterned attachment of single chain fragment variable (scFv) antibodies to the surface of metal oxides by the photodeprotection of self-assembled monolayers, using near-UV light. The photodeprotection step alters the functionality at the surface; revealing amino groups that are utilized to bind biomolecules in the exposed regions of the substrate only. The patterned antibodies are used for the detection of specific disease biomarker proteins in buffer and in complex samples such as human serum. (C) 2013 Elsevier B.V. All rights reserved