Electrostatic Conjugation of Nanoparticle Surfaces with Functional Peptide Motifs

Abstract

We report the surface functionalization of anionic layer by layer nanoparticles (LbL NPs) with cationic tumor-penetrating peptides (TPPs) via electrostatic adsorption while retaining particle stability and charge characteristics. This strategy eliminates the need for structural modifications of the peptide and enables facile functionalization of surface chemistries difficult to modify or inaccessible via covalent conjugation strategies. We show that both carboxylated and sulfated LbL NPs are able to accommodate linear and cyclic TPPs and used fluorescence-based detection assays to quantify peptide loading per NP. We also demonstrate that TPP activity is retained upon adsorption, implying sufficient numbers of peptides take on the appropriate surface orientation, enabling efficient uptake of functionalized NPs in vitro, as characterized via flow cytometry and deconvolution microscopy. Overall, we believe that this strategy will serve as a broadly applicable approach to impart electrostatically assembled NPs with bioactive peptide motifs.

Figure 1.

(A) Electrostatic adsorption of cationic peptides enables surface functionalization of anionic LbL NPs. Structures of (B) LyP-1 and (C) LinTT1, with cationic residues indicated in red, anionic residues indicated in blue, and the disulfide bond indicated in green.

Figure 2.

(A) Effects of LyP-1 titration in 1 mM HEPES onto the LbL NP library on particle size and zeta potential were monitored using dynamic light scattering. The −30 mV charge threshold for colloidal stability is indicated by a dashed line. (B) The zeta potential was measured before and after adsorption of 0.25 wt equiv of LyP-1 onto the LbL NP library in 1 mM HEPES as well as after purification of the modified NPs via tangential flow filtration. (C) Zeta potential measurements of PLE NPs after LyP-1 and LinTT1 adsorption (0.2 wt equiv) in 1 mM HEPES and purification to remove unadsorbed peptide indicate similar interactions between the two peptides and the NP. Error bars represent the standard deviation of three measurements. *p < 0.05, ***p < 0.005, determined using Student’s t test.

Figure 3.

(A) ABD-F assay scheme depicting LyP-1 reduction with TCEP and subsequent detection of free thiols with ABD-F, resulting in a fluorescent signal. (B) Quantification of LyP-1 retained on NPs after purification. Peptide retention was calculated as the post-TFF LyP-1 concentration divided by the pre-TFF concentration (0.2 wt equiv). The number of LyP-1 molecules per particle is indicated above each bar. Error bars represent standard deviation of three independent repeats.

Figure 4.

Stability of PLE NPs and DXS NPs adsorbed with 0.5 wt equiv of (A) LyP-1 and (B) LinTT1 to increasing concentrations of NaCl as determined via DLS. Arrows denote thresholds for particle stability. (C) Peptide sequences of LinTT1 analogues are shown. Residues are colored red to denote cationic charge and blue to denote anionic charge. (D) Effect of peptide charge on stability of PLE NPs and DXS NPs adsorbed with LinTT1 analogues (0.5 wt equiv) to increasing concentrations of NaCl. A line placed at 200 nm denotes the threshold for NPs of stable size. Error bars represent the standard deviation of three repeat measurements. In panels A and B the error bars are smaller than the graph symbols and therefore omitted. Adsorption of the peptides tested in this figure was carried out in 25 mM HEPES + 20 mM NaCl.

Figure 5.

(A) Flow cytometry was used to assess NP–cell association of 0.2 wt equiv of LyP-1 and LinTT1 functionalized LbL NPs with OVCAR8 cells at 24 h, also represented as (B) median fluorescence intensity. For these experiments, peptides were adsorbed in water. *p < 0.05, as determined using the Wilcoxon rank-sum test.

Figure 6.

Deconvolution microscopy analysis of OVCAR8 cells treated for 24 h with (A) PLE NPs, (B) PLE NPs^+LyP‑1 (C), pPLE LbL NPs^+iRGD, (D) PLE NPs^+LinTT1, and (E) PLE NPs^+ScrTT1. Images are pseudocolored with red to represent membranes (wheat germ agglutinin-AF647), cyan to represent nanoparticles (sulfoCy3), and blue for the nuclei (Hoechst 33342). Scale bars = 10 μm. 0.3 wt equiv of each peptide was adsorbed, with the exception of panel C, in which case an equivalent amount of iRGD was utilized. Peptide adsorption was carried out in 25 mM HEPES + 20 mM NaCl.