Human Biomolecular Corona of Liposomal Doxorubicin: The Overlooked Factor in Anticancer Drug Delivery

Abstract

More than 20 years after its approval by the Food and Drug Administration (FDA), liposomal doxorubicin (DOX) is still the drug of choice for the treatment of breast cancer and other conditions such as ovarian cancer and multiple myeloma. Yet, despite the efforts, liposomal DOX did not satisfy expectations at the clinical level. When liposomal drugs enter a physiological environment, their surface gets coated by a dynamic biomolecular corona (BC). The BC changes liposome's synthetic identity, providing it with a new one, referred to as "biological identity" (size, aggregation state, and BC composition). Today, the concept is emerging that specific BCs may determine either success (e.g., stealth effect and accumulation at the target site) or failure (e.g., rapid blood clearance and off-target interactions) of liposomal drugs. To get a comprehensive investigation of liposome synthetic identity, biological identity, and cellular response as a function of human plasma (HP) concentration, here we used a straightforward combination of quantitative analytical and imaging tools, including dynamic light scattering, microelectrophoresis, synchrotron small-angle X-ray scattering, transmission electron microscopy (TEM), fluorescence lifetime imaging microscopy (FLIM), nano-liquid chromatography tandem mass spectrometry/mass spectrometry (nano-LC-MS/MS), confocal microscopy, flow cytometry, and cell viability assays. Doxoves was selected as a reference. Following exposure to HP, Doxoves was surrounded by a complex BC that changed liposome's synthetic identity. Observations made with nano-LC-MS/MS revealed that the BC of Doxoves did not evolve as a function of HP concentration and was poorly enriched of typical "opsonins" (complement proteins, immunoglobulins, etc.). This provides a possible explanation for the prolonged blood circulation of liposomal DOX. On the other hand, flow cytometry showed that protein binding reduced the internalization of DOX in MCF7 and MDA-MB-435S human breast carcinoma. Combining FLIM and TEM experiments, we clarified that reduction in DOX intracellular content was likely due to the frequent rupture of the liposome membrane and consequent leakage of the cargo. In light of reported results, we are prompted to speculate that a detailed understanding of BC formation, composition, and effects on liposome stability and uptake is an indispensable task of future research in the field, especially along the way to clinical translation of liposomal drugs.

Protein denaturation caused by heat inactivation detrimentally affects biomolecular corona formation and cellular uptake

“…19,20 Understanding and controlling this process has become increasingly important. 21,22 In cell culture fetal bovine or human serum are commonly used for cell maintenance and in vitro experiments. 23 Mirshafiee et al 24 and Schöttler et al 25 have already reported that protein sources such as fetal calf serum, human serum, human heparin plasma or human citrate plasma can significantly influence cellular uptake of nanocarriers and their protein adsorption patterns.…”

The Crown and the Scepter: Roles of the Protein Corona in Nanomedicine

“…In addition, the protein corona formed in a complete cell culture medium inhibits the release of camptothecin (CPT, a hydrophobic antitumoral drug) from the pores of mesoporous silica NPs (MSNs) . However, for another important lipid‐based nanodrug, Doxove, which is similar to the commercial product Doxil containing the same lipid composition for doxorubicin (Dox) delivery, exposure to human plasma and protein corona formation may interfere with its integrity, possibly leading to a fraction of DOX leakage . In addition to establishing the drug release profile, acquiring a detailed understanding of the effect of the protein corona on uptake by targeted cells is an indispensable task for desirable drug delivery ( Figure ).…”

“…In addition to establishing the drug release profile, acquiring a detailed understanding of the effect of the protein corona on uptake by targeted cells is an indispensable task for desirable drug delivery ( Figure ). Human protein binding reduces internalization of Doxoves in MCF7 and MDA‐MB‐435S human breast carcinoma cells because the opsonins, such as complements and immunoglobulins, are poorly enriched, but the apolipoproteins are well‐enriched. That liposome‐based nanocarriers prefer apolipoproteins has been verified by Pozzi et al as well.…”

Artificial Phospholipids and Their Vesicles

“…95 It has become clear that once in contact with blood, a liposome is coated with a dynamic corona, which changes the "biological identity" of the liposome such as its size and aggregation state. 96 Additionally, the interaction between ions and headgroups can lead to significant physical changes on the liposomes, such as vesicle fusion. 97 Alternative, clever methods are being explored and are entering clinical trials, such as the use of red blood cells as drug delivery devices.…”