(M1130-09-56) Nanoparticle Corona Composition Differs Due to Lifestyle Factors

Purpose: Nanoparticles (NP) are often engineered to target specific organ and cell types.^1,2 However, successful clinical translation of nanoparticle (NP) drug delivery has been hindered by limitations associated with state-of-the-art targeting functionalities. Recent evidence suggests serum biomolecules are adsorbed onto the NP surface in circulation to create a unique biomolecule corona (bio-corona), and this bio-corona may impact NP circulation time, biodistribution, and cellular uptake.^3,4 Significantly, the bio-corona alters the NPs’ anticipated interaction with intended targets obstructing precise drug delivery and treatment outcomes.5< To effectively improve NP drug delivery, effects of the bio-corona must be taken into account, however, there still exists a significant gap of comprehensive and systemic knowledge surrounding bio-corona formation and interactions during systemic drug delivery. Current investigations on bio-corona use assumptions that undermine critical in vivo conditions and processes. Bio-corona is commonly characterized using standardized serum samples in vitro without accounting for the heterogenous nature of individuals, crucially neglecting patient variables, particularly those stemming from diet and disease. Diet habits differ between patients and elicit changes in serum composition impacting bio-corona and hence drug delivery efficacy. This project’s objective is to explore the effect of diet and stress-related lifestyles in serum protein profile, which in turn is expected to affect NP bio-corona composition and resulting changes in NP behavior.

Methods: C57BL/6NTa mice were used in developing models for obesity, overcrowding, or isolation conditions to simulate human lifestyles. After one week of acclimation, lifestyle conditions were initiated. The control groups were fed standard chow. Obese groups were fed a 60% kcal from fat, soft pellet diet. Social crowding groups were housed at double the standard density. Social isolation groups were housed alone, surrounded by a gap between themselves and other groups. Blood was collected from each mouse via submandibular venipuncture to obtain the serum, which was stored in 4°C refrigerator prior to use. Liposomes were prepared in chloroform with the following lipid compositions: cholesterol (20 mg/mL) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (25 mg/mL). Following thin film hydration liposomal synthesis technique, the lipids were mixed in a round bottom flask before being dried using a rotary evaporator under a vacuum. Phosphate buffered saline was used to hydrate the thin film in a 50°C water bath with sonication. The resulting solution was manually extruded step-wise through reducing sizes of polycarbonate filters. Bio-corona adsorption was achieved via blood serum incubation. Liposomes were incubated with blood serum at 0.25 mg/mL in 50% serum for one hour. The solution was then centrifuged, supernatant was removed, and 0.09% saline was added. Pipette mixing was used to disturb the pellet before centrifuging again. The supernatant was removed and saline was added to resuspend the proteins. ELISA kits were completed in accordance with manufacturer protocols. Serum dilutions were performed as needed using accompanying dilution media. All resulting absorbances were measured on a Cytation 7 plate reader at wavelengths provided by the kit manufacturers. Absorbances obtained were converted to concentration and adjusted for dilution.

Results: Concentrations of blood serum (BS) proteins were first compared between controls and lifestyle groups. Then the same pairwise comparison was made with the samples of bio-corona from serum incubated liposomes. BS and bio-corona data were normalized to determine the changes in protein concentrations from one lifestyle condition to another. Results obtained from ELISAs indicate variations in BS concentrations of proteins per cohort. Obese mice show expected elevated BS concentrations of leptin as compared to the control group (Figure 1). That trend follows in the bio-corona as obese mice also show higher concentrations of leptin in the bio-corona. Intriguingly, the bio-corona concentration of leptin does not increase at the same rate as the BS. Opposite results are indicated in social cohorts. Both isolation groups and crowding groups demonstrate lower concentrations of leptin in BS and bio-corona as compared to control. This indicates BS protein concentration alone does not define bio-corona leptin concentration. Similarly to leptin findings, obese groups demonstrate higher concentrations of Immunoglobulin G (IgG) in both BS and bio-corona (Figure 2). However, unlike leptin findings, the fold changes were unsignificant. Opposite results are again indicated in social cohorts with statistically significant multi-fold increases of bio-corona concentrations of IgG. Unrelated to BS concentration, and contrary to control groups, socially affected groups have a higher bio-corona concentration of IgG.

Conclusion: Results suggest variations in BS protein concentrations and bio-corona protein concentrations per lifestyle cohort. Notably, concentrations of a protein on the bio-corona can vary between cohorts, independent of BS concentration. Further research including additional proteins and more subjects is needed to supplement these findings. This preliminary study indicates the existence of a lifestyle impact on the bio-corona of nanoparticles and necessitates further exploration.

References: 1. Francia V, Yang K, Deville S, Reker-Smit C, Nelissen I, Salvati A. Corona Composition Can Affect the Mechanisms Cells Use to Internalize Nanoparticles. ACS Nano. 2019;13(10):11107-11121. doi:10.1021/acsnano.9b03824
2. Salvati A, Pitek AS, Monopoli MP, et al. Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface. Nature Nanotech. 2013;8(2):137-143. doi:10.1038/nnano.2012.237
3. Hadjidemetriou M, McAdam S, Garner G, et al. The Human In Vivo Biomolecule Corona onto PEGylated Liposomes: A Proof-of-Concept Clinical Study. Advanced Materials. 2019;31(4):1803335. doi:10.1002/adma.201803335
4. Caracciolo G, Farokhzad OC, Mahmoudi M. Biological Identity of Nanoparticles In Vivo: Clinical Implications of the Protein Corona. Trends in Biotechnology. 2017;35(3):257-264. doi:10.1016/j.tibtech.2016.08.011
5. Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson KA. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proceedings of the National Academy of Sciences. 2008;105(38):14265-14270. doi:10.1073/pnas.0805135105

Grouped by lifestyle condition. Left: Leptin concentrations of blood serum and bio-corona. Right: Concentration fold change in leptin between blood serum and bio-corona.

Grouped by lifestyle condition. Left: Immunoglobulin G concentrations of blood serum and bio-corona. Right: Concentration fold change in Immunoglobulin G between blood serum and bio-corona.