Modification of Living Diatom, Thalassiosira weissflogii, with a Calcium Precursor through a Calcium Uptake Mechanism: A Next Generation Biomaterial for Advanced Delivery Systems

Abstract

The diatom's frustule, characterized by its rugged and porous exterior, exhibits a remarkable biomimetic morphology attributable to its highly ordered pores, extensive surface area, and unique architecture. Despite these advantages, the toxicity and nonbiodegradable nature of silica-based organisms pose a significant challenge when attempting to utilize these organisms as nanotopographically functionalized microparticles in the realm of biomedicine. In this study, we addressed this limitation by modulating the chemical composition of diatom microparticles by modulating the active silica metabolic uptake mechanism while maintaining their intricate three-dimensional architecture through calcium incorporation into living diatoms. Here, the diatom Thalassiosira weissflogii was chemically modified to replace its silica composition with a biodegradable calcium template, while simultaneously preserving the unique three-dimensional (3D) frustule structure with hierarchical patterns of pores and nanoscale architectural features, which was evident by the deposition of calcium as calcium carbonate. Calcium hydroxide is incorporated into the exoskeleton through the active mechanism of calcium uptake via a carbon-concentrating mechanism, without altering the microstructure. Our findings suggest that calcium-modified diatoms hold potential as a nature-inspired delivery system for immunotherapy through antibody-specific binding.

Keywords: Thalassiosira weissflogii; antibody-binding delivery system; calcium uptake; chemical modification; diatoms.

Figure 1

Schematic representation of the experimental design and procedure. Various calcium precursors were examined that play significant roles in the biomineralization process. The two main criteria that need to be fulfilled are that the precursor must be soluble in artificial seawater and should not increase the pH of the seawater. Once identified, the diatom cultures were fed the precursor, followed by monitoring of the cell density and characterization of the morphology and amount of calcium deposition. To characterize the mechanism underlying this phenomenon, an inhibitor study was performed to inhibit the respiratory processes that are responsible for silica incorporation and calcium, along with proteomic analysis to monitor protein regulation and gene analysis using RT-qPCR to determine the gene(s) responsible for calcium deposition. Diatoms were functionalized with polydopamine to allow the immobilization of fluorescent antibodies of CD3 and CD28 onto the surface of diatoms as a proof-of-concept for a delivery system.

Figure 2

Modification of T. weissflogii with a calcium precursor. (a) Representative images showing the whole structure of a single frustule after the cleaning procedure. (b,c) Complex silica walls showing hierarchical patterns of pores of different sizes and shapes. (d) The growth profile of T. weissflogii grown in the presence of Na₂SiO₃ or Ca(OH)₂ was added at 48 h intervals and revealed a significant difference at 144 h. (e) SEM-EDX confirmed a higher carbonate species in the 640 μM Ca(OH)₂ treated diatoms compared to unmodified diatoms. (f) FTIR showed the carbonate species in the Ca(OH)₂ treated sample at the peak of 900 cm^–1. (g) XPS spectra of T. weissflogii with Ca(OH)₂ showed the presence of a Ca peak with higher peak counts (au). (h) Chemically distinguishable footprints of the modified diatoms indicate that Ca²⁺ was deposited as CaCO₃. n = 3, *P < 0.05, significant differences between the Na₂SiO₃ or Ca(OH)₂ treated T. weissflogii with two-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis for (d). Data are means ± SEM. Scale bars 10, 1, and 2 μm for (a), (b), and (c), respectively.

Figure 3

Ca(OH)₂-modified T. weissflogii retains nanoscale architectural features. TEM micrographs of (a) unmodified T. weissflogii and (b) Ca(OH)₂-modified T.weissflogii showing the unaltered nanostructure of the fultoportulae feature in the center and peripheral region. (c) TEM images of the cross-section of unmodified and (d) Ca(OH)₂-modified diatoms indicated that the average thickness of the frustule was not significantly changed at 192 h postinoculation following multiple additions of Na₂SiO₃ or Ca(OH)₂ at 0, 48, 96, and 144 h postinoculation. A typical frustule contains vacuoles, mitochondria, and Golgi bodies. Surface topography of the valve surface of (e) unmodified T. weissflogii and (f) Ca(OH)₂-modified T. weissflogii using AFM, illustrating that the characteristic features of the gross morphology were retained. (g) Quantification data from AFM images revealed rib width, rib-to-rib distance, nodule depth, rib height, ironed surface area, and nodule width. n = 3 for (a,b) and (c,d), n = 6 for (e,g), *P < 0.05, significant differences between the Na₂SiO₃ or Ca(OH)₂ treated T. weissflogii with t test for (g). Data are means ± SEM. Scale bars are 10 μm for (a,b) and 500 nm for (c,d), respectively.

Figure 3

Ca(OH)₂-modified T. weissflogii retains nanoscale architectural features. TEM micrographs of (a) unmodified T. weissflogii and (b) Ca(OH)₂-modified T.weissflogii showing the unaltered nanostructure of the fultoportulae feature in the center and peripheral region. (c) TEM images of the cross-section of unmodified and (d) Ca(OH)₂-modified diatoms indicated that the average thickness of the frustule was not significantly changed at 192 h postinoculation following multiple additions of Na₂SiO₃ or Ca(OH)₂ at 0, 48, 96, and 144 h postinoculation. A typical frustule contains vacuoles, mitochondria, and Golgi bodies. Surface topography of the valve surface of (e) unmodified T. weissflogii and (f) Ca(OH)₂-modified T. weissflogii using AFM, illustrating that the characteristic features of the gross morphology were retained. (g) Quantification data from AFM images revealed rib width, rib-to-rib distance, nodule depth, rib height, ironed surface area, and nodule width. n = 3 for (a,b) and (c,d), n = 6 for (e,g), *P < 0.05, significant differences between the Na₂SiO₃ or Ca(OH)₂ treated T. weissflogii with t test for (g). Data are means ± SEM. Scale bars are 10 μm for (a,b) and 500 nm for (c,d), respectively.

Figure 4

Mechanism of uptake of the Ca(OH)₂-modified T. weissflogii. Two respiratory inhibitors were used to determine whether calcium uptake occurred due to active uptake or from surface phenomena. (a) Sodium azide and (b) iodoacetamide were shown to block both silica and calcium uptake from the surrounding environment into the cell membrane. (c) SEM analysis indicated no change in the surface microstructure of the Ca(OH)₂-modified T. weissflogii in the presence of sodium azide or iodoacetamide. (d) SEM-EDX of Ca(OH)₂-modified T. weissflogii analysis indicated the absence of a Ca²⁺ peak and an increased sulfur peak following the addition of sodium azide or iodoacetamide. (e) Radioactive uptake rate of calcium deposition in elevated treatment for six consecutive days indicated by LCS analysis. (f) Proteomic analysis showed differentially expressed proteins in Ca(OH)₂-modified diatoms as shown in the heatmap of log2-transformed abundance generated by Peak Studio, with red for high expression and green for low expression. Gene expression analysis indicated upregulation of (g) CAX3 and (h) AEL1, and downregulation of (i) ATPVc′ was monitored to confirm the involvement of these genes in calcium deposition in diatoms. n = 3, *P < 0.05, significant differences between the groups with two-way ANOVA followed by Bonferroni posthoc analysis for (a,b), (e), and (g–i). Data are means ± SEM. Scale bar 10 and 5 μm for (c).

Figure 4

Mechanism of uptake of the Ca(OH)₂-modified T. weissflogii. Two respiratory inhibitors were used to determine whether calcium uptake occurred due to active uptake or from surface phenomena. (a) Sodium azide and (b) iodoacetamide were shown to block both silica and calcium uptake from the surrounding environment into the cell membrane. (c) SEM analysis indicated no change in the surface microstructure of the Ca(OH)₂-modified T. weissflogii in the presence of sodium azide or iodoacetamide. (d) SEM-EDX of Ca(OH)₂-modified T. weissflogii analysis indicated the absence of a Ca²⁺ peak and an increased sulfur peak following the addition of sodium azide or iodoacetamide. (e) Radioactive uptake rate of calcium deposition in elevated treatment for six consecutive days indicated by LCS analysis. (f) Proteomic analysis showed differentially expressed proteins in Ca(OH)₂-modified diatoms as shown in the heatmap of log2-transformed abundance generated by Peak Studio, with red for high expression and green for low expression. Gene expression analysis indicated upregulation of (g) CAX3 and (h) AEL1, and downregulation of (i) ATPVc′ was monitored to confirm the involvement of these genes in calcium deposition in diatoms. n = 3, *P < 0.05, significant differences between the groups with two-way ANOVA followed by Bonferroni posthoc analysis for (a,b), (e), and (g–i). Data are means ± SEM. Scale bar 10 and 5 μm for (c).

Figure 5

Proof-of-concept of Ca(OH)₂-modified diatoms through antibody binding on the surface of diatoms. The fluorescence microscopy images revealed immobilization of PE-labeled CD3 (red) and FITC-labeled CD28 (green) antibodies (Ab) on the surface of Ca(OH)₂-modified diatoms following surface functionalization with a polydopamine coating. Scale bar 5 μm.

Figure 6

Schematic representation of the mechanism of calcium uptake in T. weissflogii. Calcium carbonate (CaCO₃) precipitation requires the production of carbonates (CO₃^2–) from bicarbonate (HCO₃^–) and results in the net production of H⁺. Calcium uptake into cells involves Ca²⁺ transporters; Ca²⁺ ions are concentrated into a compartment distinct from the vesicle–reticular body system.