The adsorption of proteins and their layering onto nanoparticle surfaces has been called the "protein corona". This dynamic process of protein adsorption has been extensively studied following in vitro incubation of many different nanoparticles with plasma proteins. However, the formation of protein corona under dynamic, in vivo conditions remains largely unexplored. Extrapolation of in vitro formed protein coronas to predict the fate and possible toxicological burden from nanoparticles in vivo is of great interest. However, complete lack of such direct comparisons for clinically used nanoparticles makes the study of in vitro and in vivo formed protein coronas of great importance. Our aim was to study the in vivo protein corona formed onto intravenously injected, clinically used liposomes, based on the composition of the PEGylated liposomal formulation that constitutes the anticancer agent Doxil. The formation of in vivo protein corona was determined after the recovery of the liposomes from the blood circulation of CD-1 mice 10 min postinjection. In comparison, in vitro protein corona was formed by the incubation of liposomes in CD-1 mouse plasma. In vivo and in vitro formed protein coronas were compared in terms of morphology, composition and cellular internalization. The protein coronas on bare (non-PEGylated) and monoclonal antibody (IgG) targeted liposomes of the same lipid composition were also comparatively investigated. A network of linear fibrillary structures constituted the in vitro formed protein corona, whereas the in vivo corona had a different morphology but did not appear to coat the liposome surface entirely. Even though the total amount of protein attached on circulating liposomes correlated with that observed from in vitro incubations, the variety of molecular species in the in vivo corona were considerably wider. Both in vitro and in vivo formed protein coronas were found to significantly reduce receptor binding and cellular internalization of antibody-conjugated liposomes; however, the in vivo corona formation did not lead to complete ablation of their targeting capability.

Nanoparticles (NPs) are instantly modified once injected in the bloodstream because of their interaction with the blood components. The spontaneous coating of NPs by proteins, once in contact with biological fluids, has been termed the 'protein corona' and it is considered to be a determinant factor for the pharmacological, toxicological and therapeutic profile of NPs. Protein exposure time is thought to greatly influence the composition of protein corona, however the dynamics of protein interactions under realistic, in vivo conditions remain unexplored. The aim of this study was to quantitatively and qualitatively investigate the time evolution of in vivo protein corona, formed onto blood circulating, clinically used, PEGylated liposomal doxorubicin. Protein adsorption profiles were determined 10 min, 1 h and 3 h post-injection of liposomes into CD-1 mice. The results demonstrated that a complex protein corona was formed as early as 10 min post-injection. Even though the total amount of protein adsorbed did not significantly change over time, the fluctuation of protein abundances observed indicated highly dynamic protein binding kinetics.

The present study describes leucine zipper peptide-lipid hybrid nanoscale vesicles engineered by self-assembled anchoring of the amphiphilic peptide within the lipid bilayer. These hybrid vesicles aim to combine the advantages of traditional temperature-sensitive liposomes (TSL) with the dissociative, unfolding properties of a temperature-sensitive peptide to optimize drug release under mild hyperthermia, while improving in vivo drug retention. The secondary structure of the peptide and its thermal-responsiveness after anchoring onto liposomes were studied with circular dichroism. In addition, the lipid-peptide vesicles (Lp-peptide) showed a reduction in bilayer fluidity at the inner-core as observed with DPH anisotropy studies, while the opposite effect was observed with ANS probe, indicating peptide interactions with both the head group region and the hydrophobic core. A model drug molecule, doxorubicin, was successfully encapsulated in the Lppeptide vesicles at higher than 90% efficiency following the remote loading, pH-gradient methodology. The release of doxorubicin from Lp-peptide hybrids in vitro indicated superior serum stability at physiological temperatures compared to lysolipid-containing temperaturesensitive liposomes (LTSL) without affecting the overall thermo-responsive nature of the vesicles at 42 °C. A similar stabilizing effect was observed in vivo after intravenous administration of the Lp-peptide vesicles by measuring 14 C-doxorubicin blood kinetics that also led to increased tumor accumulation after 24 hours. We conclude that Lp-peptide hybrid vesicles present a promising new class of TSL that can offer previously unexplored opportunities for the development of clinically-relevant mild hyperthermia-triggered therapeutic modalities. KeywordsTemperature-sensitive liposomes (TSL); leucine zipper peptide; hyperthermia (HT); doxorubicin (DOX); cancer; nanomaterials * k.kostarelos@ucl.ac.uk.Supporting Information Available Supporting Data include: Physicochemical characterization of DOX loaded Lp-peptide hybrids; Hydrodynamic diameter and zeta potential of the DPPC:DSPC:DSPE-PEG liposomes with and without the peptide; Thermal reversibility of unbound leucine zipper peptide and Lp-peptide hybrids (200:1); Differential scanning calorimetric scan of unmodified DPPC:DSPC:DSPE-PEG2000 liposomes, Lp-peptide hybrids, Lp-CHOL and LTSL; The effect of CHOL on liposome fluidity, lipid packing and DOX release; Solid-state NMR study of leucine zipper temperature-sensitive peptides effect on the average order parameters of the DPPC lipid acyl chains as a function of temperature; Temperature-sensitivity of Lp-peptide hybrids at 45°C and 50°C. Wheel diagram amino acids arrangement of leucine zipper peptide II (VSSLESK)6, its temperature dependent conformational changes with and without liposomes and blood profile of 14 C-Doxorubicin loaded liposomes with and without peptide and peptide II in C57BL/6 mice after intravenous administration without hyperthermia. These materials are available free of charge via...

The self‐assembled layered adsorption of proteins onto nanoparticle (NP) surfaces, once in contact with biological fluids, is termed the “protein corona” and it is gradually seen as a determinant factor for the overall biological behavior of NPs. Here, the previously unreported in vivo protein corona formed in human systemic circulation is described. The human‐derived protein corona formed onto PEGylated doxorubicin‐encapsulated liposomes (Caelyx) is thoroughly characterized following the recovery of liposomes from the blood circulation of ovarian carcinoma patients. In agreement with previous investigations in mice, the in vivo corona is found to be molecularly richer in comparison to its counterpart ex vivo corona. The intravenously infused liposomes are able to scavenge the blood pool and surface‐capture low‐molecular‐weight, low‐abundance plasma proteins that cannot be detected by conventional plasma proteomic analysis. This study describes the previously elusive or postulated formation of protein corona around nanoparticles in vivo in humans and illustrates that it can potentially be used as a novel tool to analyze the blood circulation proteome.