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. 2022 Sep 30;26(1):48.
doi: 10.1186/s40824-022-00297-z.

Highly tailorable gellan gum nanoparticles as a platform for the development of T cell activator systems

Daniel B Rodrigues  1   2 , Helena R Moreira  1   2 , Mariana T Cerqueira  1   2 , Alexandra P Marques  1   2 , António G Castro  2   3 , Rui L Reis  1   2 , Rogério P Pirraco  4   5
Affiliations

Highly tailorable gellan gum nanoparticles as a platform for the development of T cell activator systems

Daniel B Rodrigues et al. Biomater Res. .

Abstract

Background: T cell priming has been shown to be a powerful immunotherapeutic approach for cancer treatment in terms of efficacy and relatively weak side effects. Systems that optimize the stimulation of T cells to improve therapeutic efficacy are therefore in constant demand. A way to achieve this is through artificial antigen presenting cells that are complexes between vehicles and key molecules that target relevant T cell subpopulations, eliciting antigen-specific T cell priming. In such T cell activator systems, the vehicles chosen to deliver and present the key molecules to the targeted cell populations are of extreme importance. In this work, a new platform for the creation of T cell activator systems based on highly tailorable nanoparticles made from the natural polymer gellan gum (GG) was developed and validated.

Methods: GG nanoparticles were produced by a water in oil emulsion procedure, and characterized by dynamic light scattering, high resolution scanning electronic microscopy and water uptake. Their biocompatibility with cultured cells was assessed by a metabolic activity assay. Surface functionalization was performed with anti-CD3/CD28 antibodies via EDC/NHS or NeutrAvidin/Biotin linkage. Functionalized particles were tested for their capacity to stimulate CD4+ T cells and trigger T cell cytotoxic responses.

Results: Nanoparticles were approximately 150 nm in size, with a stable structure and no detectable cytotoxicity. Water uptake originated a weight gain of up to 3200%. The functional antibodies did efficiently bind to the nanoparticles, as confirmed by SDS-PAGE, which then targeted the desired CD4+ populations, as confirmed by confocal microscopy. The developed system presented a more sustained T cell activation over time when compared to commercial alternatives. Concurrently, the expression of higher levels of key cytotoxic pathway molecules granzyme B/perforin was induced, suggesting a greater cytotoxic potential for future application in adoptive cancer therapy.

Conclusions: Our results show that GG nanoparticles were successfully used as a highly tailorable T cell activator system platform capable of T cell expansion and re-education.

Keywords: Cytotoxic T cells; Gellan gum; Nanoparticles; T cell stimulation.

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Conflict of interest statement

Daniel Barreira Rodrigues and several other authors have the patent hydrogel-like particles, methods ans uses thereof pending to association for the advancement of tissue engineering and cell based technologies & therapies a4tec – associação.

Figures

Fig. 1
Fig. 1
npGG production and morphological characterization. A Schematics of the production of npGG based on a double emulsion method. B FTIR spectra of (a) PVA, (b) npGG, (c) GG. C I. Particles size distribution of npGG according to intensity when produced by a double emulsion. C II. Representative STEM images of npGG. Images were obtained with an accelerating voltage of 20 kV. The scale bars represent a size of 300 nm (left) and 100 nm (right)
Fig. 2
Fig. 2
Physical and biological characterization of npGG. A Water uptake of dried npGG during 72 h of rehydration in PBS. B Effect of different solutions on npGG regarding I. Polydispersity index II. Average size distribution. C Biological performance of human dermal fibroblasts (hDFbs) stimulated with different npGG concentrations (2, 20, 100, 200 µg.mL−1) for 72 h I. Metabolic activity was assessed by MTS and presented as the percentage of control non-stimulated cells. II. Cell proliferation evaluated by DNA quantification. D Morphological analysis of hDFbs by rhodamine-phalloidin/DAPI after stimulation with varying concentrations of npGG (red = rhodamine and blue = DAPI). Original magnification 10 × , scale bar = 100 μm
Fig. 3
Fig. 3
Bio-functionalization of npGG. A Schematic illustration of the fabrication of npGG aAPCS modified with either functional grade α-CD3 and α-CD28 or biotinylated α-CD3 and α-CD28. B I. Quantification by densitometric analysis of the amount of both functional grade or biotinylated antibodies bound to the surface of GG particles by NeutrAvidin (NaV) or by EDC/NHS. C II. Surface functionalization of GG particles with α-CD3 antibody and biotinylated α-CD3 were measured by SDS-PAGE
Fig. 4
Fig. 4
Characterization of the interaction of npGG aAPCs with murine T cells. A npGG aAPCs interaction and T cell interaction schematics. B Confocal images of 100 μg of npGG-α-CD3 surface-bound to CD4+ T cells after 3 days of culture. Green fluorescence shows CD4+ T cells, while red represents npGG surface bound α-CD3, scale bar = 50 μm
Fig. 5
Fig. 5
α-CD3/α-CD28 npGG can stimulate T cells activation A Activation profile of murine splenocytes when stimulated with varying concentrations of npGG aAPCs. I. Measurement of CD4+ T-cell proliferation in vivo by CFSE dilution. Murine splenocytes were labelled CFSE and then stimulated with both α-CD3 and α-CD28 npGG in a ratio of 1:1 for a period of 7 days. After this period cells were labelled and gated for anti-CD4 and analyzed on a BD FACSAria™ III. Results are presented as the percentage of cells in the final population that have divided. Data are presented as means ± standard error. *, #,γ p < 0.05; **, ##, γγ p < 0.01; ***, ###,γγγ p < 0.001 relatively to unmodified particles at 10 μg, 50 μg or 100 μg respectively. II. IL-2 production by murine splenocytes when stimulated over 7 days with both anti-CD3 and anti-CD28 GG particles in a ratio of 1:1. Control (CTRL) corresponds to the conditions in which unmodified particles were used to stimulate the cell cultures. The IL-2 contents of the control media was undetectable. Data are presented as means ± standard error. *, ϕ, Δ, γ p < 0.05; **, ϕ ϕ, Δ Δ, γγ p < 0.01; ***, ϕ ϕ ϕ, Δ Δ Δ, γγγ p < 0.001; ****, ϕ ϕ ϕ ϕ, Δ Δ Δ Δ, γγγγ p < 0.0001. * was used when pairwise comparisons are performed relative to unmodified particles at 10 μg, 50 μg or 100 μg respectively. ϕ was used when comparisons are performed relative to activator beads, Δ relative to unstimulated cells and γ relatively to EDC at comparative 10 μg or 20 μg of antibody at 10 μg, 50 μg or 100 μg respectively. B qPCR mRNA fold change of (i) immune checkpoint genes PDCD1 and CTLA4 (ii) cytotoxic genes PRF1 and GZMB in murine splenocytes stimulated with activator-npGG over 7 days. Quantitative results are expressed as the mean ± standard deviation where n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. One-way ANOVA with Kruskal–Wallis multiple comparison post-test were used
Fig. 6
Fig. 6
Cytotoxic T cell responses of murine splenocytes stimulated with npGG aAPCs A T cell activation profile of freshly isolated splenocytes stimulated with activator-npGG over 24 h, 3 d and 7 d. Activated cells were gated as CD45+CD4+CD69+ and CD45+CD8+CD69+ T cells on a BD FACSAria™ III. Summarized data is shown as bar graphs. B Flow cytometric plots of intracellular staining and frequencies of expression of Perforin (i) and Granzyme B (ii) in CD45+CD8+CD69+ T cells are shown. Quantitative results are expressed as the mean ± standard deviation where n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, two-way ANOVA with Tukey multiple comparison post-test
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