Pegylated Gold Nanoparticles Conjugated with siRNA: Complexes Formation and Cytotoxicity

Abstract

Drug delivery systems such as dendrimers, liposomes, polymers or gold/silver nanoparticles could be used to advance modern medicine. One significant pharmacological problem is crossing biological barriers by commonly used drugs, e.g., in the treatment of neurodegenerative diseases, which have a problem of the blood-brain barrier (BBB) restricting drug delivery. Numerous studies have been conducted to find appropriate drug carriers that are safe, biocompatible and efficient. In this work, we evaluate pegylated gold nanoparticles AuNP14a and AuNP14b after their conjugation with therapeutic siRNA directed against APOE4. This genetic risk factor remains the strongest predictor for late-onset Alzheimer's disease. The study aimed to assess the biophysical properties of AuNPs/siAPOE complexes and to check their biological safety on healthy cells using human brain endothelial cells (HBEC-5i). Techniques such as fluorescence polarization, circular dichroism, dynamic light scattering, ζ-potential measurements and gel retardation assay showed that AuNPs form stable complexes with siRNA. Subsequently, cytotoxicity assays proved the biological safety of formed conjugates. Obtained results enabled us to find effective concentrations of AuNPs when complexes are formed and non-toxic for healthy cells. One of the studied nanoparticles, AuNP14b complexed with siRNA, displayed lower cytotoxicity (MTT assay, cells viability -74.8 ± 3.1%) than free nanoparticles (44.7 ± 3.6%). This may be promising for further investigations in nucleic acid delivery and could have practical use in treating neurodegenerative diseases.

Keywords: biophysical interaction; complex formation; cytotoxicity; gold nanoparticles; siRNA.

Figure 1

Zeta potential (A), hydrodynamic diameter (B) and polydispersity index (C) of siRNA alone and in the presence of increasing amounts of studied gold nanoparticles. Measurements were performed by the titration method. siRN concentration, 1 µM. The results represent mean values with standard deviation (n = 3).

Figure 2

Changes in siRNA-FL fluorescence polarization after titration with (A) AuNP14a and (B) AuNP14b. Results of samples containing AuNPs presented as [%] of siRNA values without nanoparticles. siRNA concentration 1 μmol/L in sodium phosphate buffer 10 mmol/L, pH 7.4. λexc = 485 nm, λem = 516 nm. Results are mean ± standard deviation (SD), n = 3.

Figure 3

3% Agarose gel electropherograms of ApoE4 siRNA complexed with gold nanoparticles, (A) AuNP14a and (B) AuNP14b. Samples containing 1 µmol/L siRNA per line and nanoparticles applied in the corresponding concentrations were prepared in sodium phosphate buffer 10 mmol/L in the presence of GelRed stain. Gels were visualized upon transillumination at 525 nm.

Figure 4

Effect of gold nanoparticles on siRNA’s secondary structure (θ—ellipticity). (A) Spectra CD of siRNA at the presence of AuNP14a, and (B) AuNP14b. (C) Changes in mean ellipticity at λ = 265 nm. Measurements were conducted in λ = 200–320 nm in phosphate buffer, pH = 7.4. The concentration of siRNA 1 µmol/L. The values are the mean ± standard error of the mean (SEM), n = 3.

Figure 5

Cytotoxicity effect of AuNPs alone and their complexes with siRNA toward HBEC-5i cells, MTT assay. (A) The viability of HBEC-5i cells in the presence of AuNPs at the concentrations varied from 1.56 to 200 µg/mL. Statistical significance showed when compared with the control, non-treated cells. (B) Calculated from the results shown on panel A, IC₅₀, values of noncomplexed nanoparticles vs. control (100% viability). (C) The viability of HBEC-5i cells in the presence of AuNPs/siRNA complexes. Significant differences compared to the negative control. (D) Comparison of the cytotoxic effects of naked AuNPs and their complexes with siRNA. Statistical significance was marked for the same concentrations between AuNPs alone and in the complex with siRNA. To form the AuNP/siRNA complex, the concentration of siRNA was always 1 µmol/L, whereas the concentration of nanoparticles was varied—incubation time for all samples 24 h. Data points represent means ± SD obtained from min. 3 separate experiments. Statistical significance was assessed using a two-way ANOVA test (n = 3, * p < 0.05; ** p < 0.01, *** p < 0.0001).

Figure 6

Cytotoxicity effect of AuNPs alone and complexes with siRNA toward HBEC-5i cells, LDH assay. The nanoparticles concentrations varied from 50 to 200 µg/mL. All results were compared to positive control with Triton X-100: incubation time, 24 h. Data points represent means ± SD obtained from min. 3 separate experiments.

Figure 7

Viability of PBMC cells after incubating with AuNPs/siRNA complexes, Alamar Blue assay. Statistical effects showed in comparison to control cells. The concentration of siRNA is 1 µmol/L, incubation time 24 h. Data points represent means ± SD obtained from min. 3 separate experiments. Statistical significance was assessed using a two-way ANOVA test (n = 3, *** p < 0.0001).

Figure 8

Structure of tested dendronized AuNPs modified with PEG. AuNPs were synthesized in water by the reaction of HAuCl₄·3H₂O with a mixture of two ligands containing a thiol moiety: (1) the cationic dendrons HSG₂(SNMe₃⁺)₄ and (2) commercial PEG ligand CH₃O(CH₂CH₂O)_nCH₂CH₂SH, HS-PEG. In addition, NaBH₄ was used as a reducing agent [21].