Gellan gum particle synthesis
Gellan gum nanoparticles (npGG) were produced by the double emulsion water-in-oil-in-water technique [41]. Briefly, a 1% (w/V) of gellan gum (Sigma-Aldrich, Germany) solution was prepared in deionized water, under stirring at 90 °C for 30 min. After dissolution, the solution was allowed to stabilize at 50 °C. A drop of Tween-20 (Sigma-Aldrich, Germany) was then added to the GG solution (W1) and each mL of the GG solution was emulsified in 3 mL of 0.5% of Span®80 (Sigma-Aldrich, Germany) in chlorophorm (Sigma-Aldrich, Germany)(O) while under stirring with a T18 BASIC Ultraturrax (IKA, NC, USA) for 3 min. W1/O was then added dropwise to a solution of 3.5% poly(vinyl alcohol) (PVA)(W2) (Sigma-Aldrich, Germany) in water under continuous stirring and then crosslinked by adding gradually W1/O/W2 into 5 mL of a 60% w/v solution of calcium chloride (Merck Millipore, Germany) under stirring. Chlorophorm was removed using a rotary vaccum evaporator at 37 °C (Stuart, Staffordshire, UK). The particles were recovered by centrifugation for 1 h at 13,000 × g (Eppendorf, Germany) and by passing through a series of strainers (pluriSelect, Leipzig, Germany) ranging from 10 μm down to 1 μm to limit size dispersion. In order to remove PVA, 3 additional washing steps were performed by centrifugation at 13,000 × g for 10 min with deionized water.
GG particles were then frozen [-196 °C in liquid nitrogen (N2)] and then freeze-dried (LyoQuest, Telstar, Spain) to obtain dried GG particles for further modification upon rehydration.
npGG characterization
Chemical characterization of npGG
The presence of specific functional groups in the npGG was assessed by Fourier transform infrared spectroscopy (FTIR). To obtain the FTIR spectra of npGG, transparent potassium bromide (Sigma-Aldrich, Germany) pellets were prepared containing the samples to be analyzed. The readings were performed in an infrared spectroscope (IR Prestige-21, Shimadzu, Japan) with 4 cm−1 resolution and the results are presented as the average of 32 scans.
npGG morphology
The npGG size distribution and surface charge were analyzed by dynamic light scattering after preparing a 1 mg.mL−1 suspension of rehydrated GG particles in deionized water (Zetasizer Nano ZS, Malvern Instruments, UK). Acquisition was performed with the detector positioned at a scattering angle of 173°.
Particle size and morphology were analyzed with a High-Resolution Field Emission Scanning Electron Microscope with Focused Ion Beam (FIB – SEM) (AURIGA COMPACT, ZEISS, Germany). For this effect, the samples were diluted at 0.5 mg.mL−1 in ultrapure water followed by a further dilution of 1:20 and were dispersed onto the surface of a 400-mesh carbon-coated copper grid (Ted Pella, USA) for further observation.
Particle stability upon rehydration
To determine the stability of the produced GG particles over time, a 1 mg.mL−1 suspension of npGG was prepared in different solutions (culture medium, phosphate buffer saline (PBS), and deionized water) and filtered through a 0.8 µm filter (Millex, Millipore, France). Particle size and dispersity was evaluated over 7 days by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments, UK). Between data acquisition times, samples were stored at room temperature.
Water uptake and water content quantification
The dried GG particles were rehydrated with PBS up to 7 days at 37 ºC, to determine the water uptake profile. Samples were weighed prior (Wd) and after each time point (Ww) and the percentage of water uptake over time was calculated based on the Eq. (1) below. The water content of the hydrogel particles was determined through of the particles in their wet state (Ww) versus the weight in their dried state (Wd):
$$\mathrm{Water}\;\mathrm{uptake}/\mathrm{content}\;(\%)=({\mathrm W}_{\mathrm w}-{\mathrm W}_{\mathrm d})/{\mathrm W}_{\mathrm d}\times100$$
Particle functionalization with antibodies
npGG in suspension were functionalized through chemical coupling by one of two methods. Briefly, 1 mg of npGG was dissolved in MES buffer at 50 mM and pH 6.5. Carboxylic groups were activated through the addition of N-(3_Dimethylaminopropyl)-N´- ethylcarbodiimide hydrochloride and N-hydroxysulfosuccinimide to achieve a final concentration of 17.3 μM and 18 μM respectively and left to stir for 1 h. The particles were then washed 3 × by centrifugation at 18,000 × g and afterwards either i) NeutrAvidin was added first and left to react over 4 h at RT under agitation after which the particles were washed again and 10 µg/20 µg of biotinylated α-CD3/CD28 antibodies (MyBioSource™, California, USA) were added or ii) 10 µg/20 µg of functional grade α-CD3/CD28 (Tonbo Biosciences™, California, USA) antibodies were added directly to the EDC/NHS activated particles and incubated overnight at 4 ºC. The following day, the produced activator-npGG were washed again 3 × by centrifugation at 18,000 × g to remove unbound antibodies and particles were suspended in adequate medium.
Density of NeutrAvidin
The density of NeutrAvidin protein on the particles surface was determined using the micro bicinchoninic acid assay (Pierce, Rockford, IL) in accordance with the manufacturer’s instructions. For this effect, 1 mg of GG particles was functionalized as stated above with either 0.5 mg or 1 mg of avidin by conjugation through EDC/NHS. Experimental replicates were performed for reproducibility of the chemical conjugation and elution’s resulting from washing step were stored for quantification of unbound NeutrAvidin.
Assessment of npGG functionalization
To evaluate the binding of functional grade antibodies to npGG’s, SDS-PAGE (Sodium, Dodecyl, Sulfate, Polyacrylamide Gel Electrophoresis) analysis was used (Sigma-Aldrich, Germany). To achieve protein separation, two separate gels were used, a 4,7% stacking gel and a 12% resolving gel. In each well, an equal amount (10 µg) of either control or modified particles were loaded after being prepared by heating at 60 ºC for 30 min prior. A calibration curve of the biotinylated antibody of 0.5 µg, 1 µg and 2 µg was performed. The direct binding of the antibody to the particles was also performed and again a calibration curve of the standard antibody was equally performed 0.5 µg, 1 µg and 2 µg. The bands in each well were observed by soaking the gel with Coomassie Blue (National Diagnostics, Atlanta, USA) followed by destaining.
Functionality evaluation
Functionality assays were carried out to determine the in vitro performance of the produced activator-npGG in terms of both surface interaction with host immune cells, as well as, the capacity to trigger T cell proliferation.
Cell lines and culture conditions
Primary human dermal fibroblasts (hDFbs) (Gibco, UK) were obtained from Thermo Fischer and cultured in α-MEM, supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA) and 1% antibiotic/antimycotic. Cells were kept in culture at 37 °C in a humidified atmosphere with 5% CO2.
Cell isolation
All procedures were approved by the Direcção Geral de Alimentação Veterinária, the Portuguese National Authority for Animal Health, and respected the national regulations and international animal welfare rules. Murine splenocytes cells were obtained from Balb/c mice. Briefly, mouse spleens were excised and sliced into small pieces. The fragments were placed onto a strainer attached to a 50 mL conical tube. The excised spleen fragments were pressed through the strainer using the plunger end of a 5 mL syringe. The cells were washed through the strainer using excess cell culture medium. Cells were collected by centrifugation at 400 × g for 5 min. The cell pellet was resuspended in 1 mL of pre-warmed (37 ºC) red blood cell lysis buffer and incubated at 37 ºC for 5 min. Murine splenocytes were then washed with excess PBS and centrifuged at 400 × g for 5 min.
Cells were cultured from hereon with RPMI 1640 medium (Sigma-Aldrich, Germany) supplemented with 10% FBS (Gibco, ThermoFisher Scientific, Paisley, UK) 1% HEPES (Gibco, ThermoFisher Scientific, Paisley, UK), 1% Sodium Pyruvate (Gibco, ThermoFisher Scientific, Paisley, UK) and 0.05 mM Mercaptoethanol (Gibco, ThermoFisher Scientific, Paisley, UK) at 37 °C in an incubator equilibrated with 5% CO2.
Metabolic activity in response to npGG
To determine the metabolic activity of cells in response to npGG, hDFbs were stimulated with different npGG concentrations (2, 20, 100, 200 µg.mL-1) for 72 h. Metabolic activity and cell proliferation were quantified after this period using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, USA) and Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, UK), respectively as recommended by the manufacturer. Metabolic activity data was expressed as the percentage of cell metabolic activity of the stimulated cells normalized to the non-stimulated hDFbs. The concentration of DNA present in each condition was determined against a standard curve.
Visualization of activator npGG-cell binding
For visualizing activator npGG-splenocyte interactions, murine splenocytes were stimulated for 5 days with varying concentrations of the developed system. At different time-points, cells were collected, washed and growth medium supernatant was aspirated and replaced with PBS at a concentration of ~ 1 × 106 cells.mL−1. A pipette was used to gently mix and transfer 1 mL of the cell suspension into a culture plate well holding a coverslip. The culture plate was then left stationary for 30 min at room temperature to allow for the sedimentation and adhesion of cells to the coverslip. Afterwards, the PBS suspension containing remaining non-adherent cells was gently aspirated. The adhered cells were then fixed for 15 min at room temperature with ice cold methanol (Sigma-Aldrich, Germany) pipetted along the side of the well. Fixed cells were then washed with PBS and stored at 4 ºC until immunolabeling.
Primary antibody CD4 (Rabbit anti-Mouse, 1:200, NovusBiologicals, U.K.) and Alexa Fluor 488-tagged Goat anti-rabbit secondary antibody (1:500, ThermoFisher Scientific, Paisley, UK.) were used for cell visualization (Supplementary Table S1, Additional file 1). Nuclei were stained with DAPI (Invitrogen, U.S.) For particle visualization, a Donkey anti-rat AF594 secondary antibody (1:500, ThermoFisher Scientific, Paisley, UK) was used. An AXIOIMAGER Z1M (Carl Zeiss Microscopy GmbH, Jena, Germany) widefield microscope or a TCS SP8 Leica laser confocal microscope (Leica Microsystems, Wetzlar, Germany) were used for visualization of the samples.
Quantification of activator npGG-cell binding
To quantitively determine the percentage of binding of the activator-npGG system to T cells, freshly isolated murine splenocytes were placed in culture with varying concentrations of α-CD3 antibody-functionalized GG particles (1 μg, 10 μg, 50 μg and 100 μg) produced either by directly coupling the antibodies to the particles or through the NeutrAvidin/Biotin immobilization. Cultures proceeded over 5 days and, ultimately, cell-bound particles were labelled in a first step with a rabbit anti-rat IgG AF488 secondary antibody (1:500, ThermoFisher Scientific, Paisley, UK) followed by a wash step. T cells were then labelled with a rat anti-mouse AF647-conjugated CD4+ antibody (2.5 μg.mL-1, Biolegend, London, UK) and conjugation was assessed through flow cytometery.
T cell proliferation and activation in response to functionalized npGG
To monitor T cell expansion when stimulated with functionalized GG particles, previously isolated murine splenocytes were labeled with CFSE. In brief, 2–5 × 107 cells were incubated with 2.5 µM of CFSE at 37 ºC for 10 min. Cells were then washed three times with complete medium to quench and remove unbound CFSE and were then distributed (1 × 106/well) in 96 well flat bottom culture plate. As controls for T cell expansion, 3 conditions were set up: Dynabead Mouse T-Activator CD3/CD28 for T cell Expansion and Activation (Gibco, ThermoFisher Scientific, Paisley, UK), plate bound functional grade anti-CD3 antibody (eBioscience™, ThermoFisher Scientific, Paisley, UK) (2 μg.mL−1) or no additional stimulator.
For the coating of the 96 well plates (Cellstar M3687, Greiner Bio-One, Austria), anti-CD3 antibodies were dissolved to a concentration of 2 µg.mL−1 and 50 µL were added per well and left to incubate for 1 h at RT. The following day, culture plate wells were washed with complete medium to block unspecific binding of antibodies still to be added.
Different np-GG α-CD3/ α-CD28 concentrations were added (1 µg.mL−1, 10 µg.mL−1, 50 µg.mL−1 and 100 µg.mL−1) over a period of 7 days of stimulation in a ratio of 1:1. For plate bound antibody stimulation, anti-CD28 was added to a final concentration of 2 µg.mL−1 to each respective well. All quantifications were performed in triplicate. The proportion of dividing cells was detected by flow cytometry based on CFSE levels of gated CD4+ T cells [42].
Enzyme-linked immunosorbent assay (ELISA) IL-2 secretion assay
ELISA (Elabscience, Hubei, CN) was used to quantify the levels of IL-2 released upon stimulation with the developed activator-npGG system. Conditioned medium was collected post 7 days of stimulation with varying concentration of the npGG activator system to assess total IL-2 produced over the complete stimulation period. The procedure was carried out according to the manufacturer’s instructions and the absorbance was read at 450 nm using a Synergy HT (BioTek, Vermont, USA) microplate reader. The quantity of IL-2 protein was determined by means of a standard curve. IL-2 within the control media was undetectable during the experiments and therefore was omitted from the figures.
T cell exhaustion and cytotoxic capacity
Quantitative Real-Time Polymerase Chain Reaction (qPCR) was used to detect the expression of T cell exhaustion-related genes after exposure to 7 days of stimulation with either the activator-npGG system or the commercial activator beads. Primers for the tested genes and the housekeeping gene ACTβ were designed using Primer-Blast database (NCBI, Bethesda, MD, USA) (Supplementary Table S2, Additional file 1).
qPCR reactions were carried out in a MasterCycler Realplex4 (Eppendorf, Hamburg, Germany) and primer efficiency was tested out using serial dilutions of cDNA (1, 1:10, 1:100, 1:1000). For qPCR reactions, 1 μL of synthesized cDNA was used in a 20 μL reaction containing 10 μL of PerfeCTa® SYBR Green FastMix (Quanta Biosciences, Beverly, MA, USA) and forward and reverse primers at 300 nM. Reaction conditions comprised a 2 min denaturation at 95 ºC, followed by 45 cycles of 95 ºC for 10 s, a specific annealing temperature as described in Table S2 for 30 s and 72 ºC for 10 s. Products obtained from real-time PCR were subjected to melting curve analysis to check for the correct amplicon length and the absence of unspecific products. Transcript abundances were normalized to the expression of ACTB. Samples were run in triplicate in each PCR assay. Normalized expression values were calculated following the mathematical model proposed by Pfaffl using the formula: 2 −ΔΔCt (Pfaffl, 2001).
Analysis of helper and cytotoxic T cell subsets
To monitor T cell activation profile, splenocytes were incubated with functionalized npGG over 7 days of stimulation. Cells were then washed, permeabilized, and stained with the following antibodies: CD45-PerCP/Cy5.5, CD4-APC-H7, CD8-APC-H7 (from BD Biosciences) and Granzyme B-PE-Cy7, Perforin-APC, CD69-PE (from Biolegend) (Supplementary Table S1, Additional file 1). For intracellular staining, cells were first incubated and fixed with Reagent A-Fix and Perm™ (Thermo-scientific, Netherlands) for 15 min, and stained with the respective conjugated antibodies diluted in Reagent B- Fix and Perm™ (Thermo-scientific, Netherlands) for 30 min at RT. After washing with PBS, cells were fixed in 1% paraformaldehyde (Sigma, USA)/PBS and then analyzed on a FACSCalibur flow cytometer (BD Biosciences, Belgium) and data was treated using Cell Quest Pro version 4.0.2 (BD Biosciences, Belgium). Cells were gated according to (Supplementary Figure S1, Additional file 1).
Statistical analysis
Graphpad software version 7.03 was used to perform statistical analysis. To determine if data sets fell in a normal distribution the Shapiro–Wilk normality test was performed. When a normal distribution was verified a one-way analysis of variance with a Tukey post-test was performed. Otherwise, data was analyzed with the Kruskal–Wallis test followed by the Dunn’s multiple comparison post-test. Results are presented as mean ± standard deviation (SD) and the significance levels between experimental groups was set for *p < 0.05, **p < 0.01, ***p < 0.001.