Effect of Protein Corona on Mitochondrial Targeting Ability and Cytotoxicity of Triphenylphosphonium Conjugated with Polyglycerol-Functionalized Nanodiamond

Abstract

Functionalization of nanoparticles (NPs) with targeting moieties has a high potential to advance precision nanomedicine. However, the targeting moieties on a NP surface are known to be masked by a protein corona in biofluids, lowering the targeting efficiency. Although it has been demonstrated at the cellular level, little is known about the influence of the protein corona on the subcellular targeting. Herein, we adopted triphenylphosphonium (TPP) as a mitochondrial targeting moiety and investigated the effects of protein coronas from fetal bovine serum and human plasma on its targeting ability and cytotoxicity. Specifically, we introduced TPP in low (l) and high (h) densities on the surface of nanodiamond (ND) functionalized with polyglycerol (PG). Despite the "corona-free" PG interface, we found that the TPP moiety attracted proteins to form a corona layer with clear linearity between the TPP density and the protein amount. By performing investigations on human cervix epithelium (HeLa) and human lung epithelial carcinoma (A549) cells, we further demonstrated that (1) the protein corona alleviated the cytotoxicity of both ND-PG-TPP-l and -h, (2) a smaller amount of proteins on the surface of ND-PG-TPP-l did not affect its mitochondrial targeting ability, and (3) a larger amount of proteins on the surface of ND-PG-TPP-h diminished its targeting specificity by restricting the NDs inside the endosome and lysosome compartments. Our findings will provide in-depth insights into the design of NPs with active targeting moiety for more precise and safer delivery at the subcellular level.

Probing the Role of Charged Functional Groups on Nanoparticles Grafted with Polyglycerol in Protein Adsorption and Cellular Uptake

“…This is consistent with our previous studies revealing that PG grafting shields NPs from non‐specific cellular uptake almost completely. [ 20,21,26,44 ] Based on this “transparent” NP‐PG background, we found that the cellular uptake and intracellular distribution of ND‐PG‐Y are correlated with their affinity to serum proteins. Both ND‐PG‐OSO 3 − ‐ h and ND‐PG‐NH 3 + with higher protein affinities (Figures 1 and 2) were taken up by the cells under FBS (+) (Figure 4f–h; Figures S7f–h and S8f–h, Supporting Information) and exhibited different intracellular patterns from those of FBS (−) conditions (Figures S10d–f and S11d–f, Supporting Information).…”

“…To probe the role of the charged functional groups in protein corona formation, the dispersions of NP‐PG‐COOH, NP‐PG‐OSO 3 Na, NP‐PG‐NH 2 and NP‐PG were incubated with 10% and 55% fetal bovine serum (FBS) as well as 55% human plasma (HP) in phosphate buffer saline (PBS) for 1 h. The incubation time was optimized to reach equilibrium according to the kinetic study of protein corona formation in our previous work. [ 26 ] The weights of NP‐PG‐Y were adjusted (Table S3, Supporting Information), so that the total number of each functional group on the surface of ND‐PG and SPION‐PG was in the same range at l and h contents. Since the typical p K a values of the carboxylic acid, sulfuric acid and ammonium are around 5, −3, and 11, respectively, these functional groups on NP‐PG surface should be fully ionized as carboxylate (COO − ), sulfate (OSO 3 − ), and ammonium (NH 3 + ) in PBS.…”

“…The cytotoxicity of NP‐PG, NP‐PG‐COOH, NP‐PG‐OSO 3 Na and NP‐PG‐NH 2 (the core concentration of NDs and SPIONs were adjusted to be 100 and 50 µg mL −1 , respectively) in the culture medium supplemented with and without 10% FBS (FBS (+) and FBS (−), respectively) was assayed according to the previous method. [ 26 ]…”

Intracellular Thermal Probing Using Aggregated Fluorescent Nanodiamonds

“…More importantly, it is very difficult for such micrometer-sized solid dielectric spheres to enter a living cell via endocytosis. Thus, to obtain a given fluorescence emission intensity for the detection of multiple regions, a lower excitation power could be applied to the aggregated microspheres compared with dispersed single nanoprobes, [42][43][44][45][46] thereby reducing photodamage to the cells (Section S3.2 and Figure S12, Supporting Information).…”

“…[39][40][41] Despite being a simple and reliable way to build superstructures using low optical power, thermal conversion requires a gold film, which limits its application to single cells. Nanoparticles with chemical or protein modifications enable imaging with a high spatial resolution, [42][43][44][45][46] but their assembly and detection can only be implemented at specific locations because organelle binding is required.…”