Evaluation of Peptide Nucleic Acid Encapsulation in Ferritin Nanocages for Gene Silencing Applications

Abstract

Peptide nucleic acids (PNAs) feature a neutral peptide-like backbone, providing nuclease resistance and potential for precision medicine and diagnostics through specific DNA and RNA binding. However, their therapeutic use is hindered by poor solubility and cell permeability. In this study, we demonstrated that negatively charged PNAs can be readily loaded into the polycationic Humanized Archaeoglobus Ferritin, namely, PA3.2-HumAfFt bioconjugate system, following a divalent-cation-triggered oligomerization technique. The versatility of PNA chemistry enabled the production of synthetic nucleic acid homologues with varying lengths and charges, ranging from positive to negative. We evaluated the loading performance of HumAfFt with and without chemical modifications and investigated the release dynamics of PNAs under conditions simulating the intracellular environment. Our findings demonstrated the effective uptake, release, and biological activity of PNAs in cancer cells, notably silencing the GAPDH gene with good efficiency. This evaluation paves the way for optimizing PNA-based therapeutics and broadening their applications.

1

Schematic representation of the PA3.2-HumAfFt delivery system (orange spheres indicate the bioconjugation positions) (structural figures were prepared with UCSF Chimera).

2

Schematic representation of PNA oligomers conjugated to differently charged amino acid residues.

3

Schematic drawing of the encapsulation process of 10-mer FITC-PNA. The FITC-PNAs were added to the open conformation, and physical entrapment followed the closure of the nanocage by increasing salt concentration.

4

Encapsulation of 10-mer and 19-mer PNAs with the desired excess fold compared to the HumAfFt (24-mer) structure. The x-axis represents different complexes of HumAfFt with their respective excesses of PNA. The y-axis indicates the number of PNA molecules encapsulated within HumAfFt relative to the treated excesses. Measurements are conducted in triplicate. (A) The loading capacity of PNA _10‑mer in PA3.2-HumAfFt is shown. In blue, PNA _10‑mer K6 (+); in orange, PNA _10‑mer E4 (−); in gray, PNA _10‑mer E8 (−). (B) The loading capacity of PNA _19‑mer E4 (−) in PA3.2-HumAfFt is shown. Error bars are lower than 4%.

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Spectra of FITC-PNA behavior under various pH conditions. Spectra of FITC-PNA _10‑mer 5 μM at pH 7.4 (dots), FITC-PNA _10‑mer at pH 5.0 (dashed line), and FITC-PNA _10‑mer adjusted from 5.0 to 7.4 (black line) showed the decrease of absorption intensity at 494 nm with the lowering of pH.

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PNA-loaded PA3.2-HumAfFt uptake: (A) flow cytometry analysis of the negative control (black line), FITC-PNA _10‑mer E4 (−) alone (blue line), and FITC-PNA _10‑mer E4 (−) loaded into PA3.2-HumAfFt (green line) after 6 h of incubation in MEG-01 cells. Representative data of three independent experiments with similar results. (B) Mean fluorescence intensities of cellular uptake are depicted as histograms showing mean values ± SD from 3 independent measurements. ** = p < 0.001.

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Real-time PCR analysis of GAPDH expression. Cells were treated with PNA _10‑mer E4 (−) alone (PNA), PNA _10‑mer E4 (−) loaded into PA3.2-HumAfFt nanocages (PNA–PA-Ft), or siRNA targeting GAPDH (RNAi-TX), and transfected with a jetPRIME transfection agent. Data represent the mean of three independent experiments. *p < 0.05; **p < 0.001; n.s., not significant. Statistical significance was assessed using one-way ANOVA, followed by the Bonferroni post hoc test.