[Paper Review] The effects of aggregation and protein corona on the cellular internalization of iron oxide nanoparticles

Muhammad Asim Safi, Jérémie Courtois+3 more|arXiv (Cornell University)|Aug 24, 2011

Nanoparticle-Based Drug Delivery44 references4 citations

TL;DR

This study investigates how nanoparticle aggregation and protein corona formation influence cellular uptake of iron oxide nanoparticles. By replacing citrate coating with poly(acrylic acid), the authors demonstrate enhanced colloidal stability, reduced protein corona formation, and altered internalization dynamics—showing that polymer-coated particles avoid rapid surface adsorption and instead enable more controlled endocytic uptake, significantly reducing non-specific membrane binding compared to citrate-coated particles.

ABSTRACT

Engineered inorganic nanoparticles are essential components in the development of nanotechnologies. For applications in nanomedicine, particles need to be functionalized to ensure a good dispersibility in biological fluids. In many cases however, functionalization is not sufficient : the particles become either coated by a corona of serum proteins or precipitate out of the solvent. In the present paper, we show that by changing the coating of iron oxide nanoparticles from a low-molecular weight ligand (citrate ions) to small carboxylated polymers (poly(acrylic acid)), the colloidal stability of the dispersion is improved and the adsorption/internalization of iron towards living mammalian cells is profoundly affected. Citrate-coated particles are shown to destabilize in all fetal-calf-serum based physiological conditions tested, whereas the polymer coated particles exhibit an outstanding dispersibility as well as a structure devoid of protein corona. The interactions between nanoparticles and human lymphoblastoid cells are investigated by transmission electron microscopy and flow cytometry. Two types of nanoparticle/cell interactions are underlined. Iron oxides are found either adsorbed on the cellular membranes, or internalized into membrane-bound endocytosis compartments. For the precipitating citrate-coated particles, the kinetics of interactions reveal a massive and rapid adsorption of iron oxide on the cell surfaces. The quantification of the partition between adsorbed and internalized iron was performed from the cytometry data. The results highlight the importance of resilient adsorbed nanomaterials at the cytoplasmic membrane.

Motivation & Objective

• To understand how nanoparticle aggregation and protein corona formation influence cellular internalization of iron oxide nanoparticles in physiological conditions.
• To evaluate the impact of surface coating chemistry (citrate vs. poly(acrylic acid)) on colloidal stability in serum-containing media.
• To compare the kinetics and mechanisms of nanoparticle interaction with human lymphoblastoid cells.
• To quantify the partitioning between surface adsorption and endocytic internalization of iron oxide nanoparticles.
• To assess the role of resilient surface-adsorbed nanoparticles in modulating cellular uptake dynamics.

Proposed method

• Synthesized iron oxide nanoparticles coated with citrate or poly(acrylic acid) to compare colloidal stability.
• Assessed dispersion stability in fetal calf serum and physiological buffers using dynamic light scattering and zeta potential measurements.
• Performed transmission electron microscopy (TEM) to visualize nanoparticle localization on cell membranes and within endocytic compartments.
• Used flow cytometry to quantify iron content in cells and distinguish between surface-adsorbed and internalized nanoparticles.
• Applied cytometry data to calculate the partition ratio between adsorbed and internalized iron over time.
• Compared interactions under identical physiological conditions to isolate the effects of coating chemistry.

Experimental results

Research questions

• RQ1 How does the choice of surface coating (citrate vs. poly(acrylic acid)) affect the colloidal stability of iron oxide nanoparticles in serum-containing media?
• RQ2 To what extent does protein corona formation differ between citrate-coated and polymer-coated iron oxide nanoparticles?
• RQ3 What are the kinetics and mechanisms of nanoparticle interaction with human lymphoblastoid cells?
• RQ4 How does the partitioning between surface adsorption and endocytic internalization vary between the two nanoparticle types?
• RQ5 What is the role of surface-adsorbed nanoparticles in modulating cellular uptake dynamics?

Key findings

• Citrate-coated iron oxide nanoparticles rapidly aggregate and destabilize in fetal calf serum, leading to massive, rapid adsorption on cell membranes.
• Poly(acrylic acid)-coated nanoparticles exhibit superior colloidal stability and remain dispersed in serum without significant aggregation.
• The polymer-coated nanoparticles show minimal protein corona formation, preserving their surface identity in biological environments.
• Flow cytometry revealed that citrate-coated particles had a significantly higher proportion of iron adsorbed on the cell surface compared to internalized iron.
• In contrast, poly(acrylic acid)-coated particles showed a higher fraction of internalized iron, indicating more efficient endocytic uptake.
• TEM imaging confirmed that citrate-coated particles predominantly accumulated on the plasma membrane, while polymer-coated particles were observed within membrane-bound endocytic compartments.