Diffusion and Protein Corona Formation of Lipid-Based Nanoparticles in the Vitreous Humor: Profiling and Pharmacokinetic Considerations

The vitreous humor is the first barrier encountered by intravitreally injected nanoparticles. Lipid-based nanoparticles in the vitreous are studied by evaluating their diffusion with single-particle tracking technology and by characterizing their protein coronae with surface plasmon resonance and high-resolution proteomics. Single-particle tracking results indicate that the vitreal mobility of the formulations is dependent on their charge. Anionic and neutral formulations are mobile, whereas larger (>200 nm) neutral particles have restricted diffusion, and cationic particles are immobilized in the vitreous. PEGylation increases the mobility of cationic and larger neutral formulations but does not affect anionic and smaller neutral particles. Convection has a significant role in the pharmacokinetics of nanoparticles, whereas diffusion drives the transport of antibodies. Surface plasmon resonance studies determine that the vitreal corona of anionic formulations is sparse. Proteomics data reveals 76 differentially abundant proteins, whose enrichment is specific to either the hard or the soft corona. PEGylation does not affect protein enrichment. This suggests that protein-specific rather than formulation-specific factors are drivers of protein adsorption on nanoparticles in the vitreous. In summary, our findings contribute to understanding the pharmacokinetics of nanoparticles in the vitreous and help advance the development of nanoparticle-based treatments for eye diseases.

. Effect of charge, particle size and surface modification (PEG and ICG) on vitreal mobility of lipid-based formulations based on the corresponding Dv, Dw and Dw/Dv ratios at a time scale of 1s. A: anionic, N: neutral, C: cationic formulation; L: light-activated liposomes and controls; R: rigidmembrane liposomes; H: hexosomes; and N: nanostructured lipid carriers (NLCs).

Vitreal corona protein composition of anionic light-activated liposomes
In total, 535 non-redundant proteins were identified in the porcine vitreous used as source and at least one sample of the liposome corona subsections by nLC-ESI-MS/MS. These included 101 (18.9%) annotations that have not been previously reported in proteomic studies of the human vitreous, [1][2][3][4] and 88 (16.4%) previously unknown annotations when reports on dog, rabbit, and mouse vitreal proteomes were included in addition to human ( Figure S2 and S4). [5][6][7] Only 24 common annotations were identified in the corona and human vitreal proteomes, and three when the other species were included (Table S2). Functional analysis with gene sets in the human group identified glycolysis (FDR 1.88E-11) and glucose catabolic process (FDR 5.87E-11) as significantly enriched pathways, while the common genes in the interspecies group were linked by their association with visual perception and sensory perception of light stimulus (both FDR 2.57E-2) ( Figure S3).   Figure S3 Functional analysis of the set of 24 genes common to all common with liposome corona and human vitreous studies shows glycolysis (FDR 1.88 e-11, red), glucose catabolic process (FDR 5.87 e-11, blue) and blood microparticle (FDR 6.77 e-2, purple) as enriched pathways.  As demonstrated in Figure 3, differential enrichment of 76 vitreous proteins out of 504 was observed after filtering out proteins not present in at least three samples. Two identified proteins were not listed in the human or animal vitreous proteomes listed above: gamma-synuclein (SNCG) and mitochondrial ATP synthase subunit alpha (ATP5F1A). As these were found enriched in the HC, only 13 out of the 15 HC enriched proteins have been previously reported constitutive to the vitreous.
Adipogenesis regulatory factor (ADIRF) has not been previously reported in the vitreous. beta and gamma that enriched in the HC, is also a GADPH interaction partner along with enolase 1 (ENO1). The primary enriched function was glucose metabolism, same as the primary function of the overlapping genes between our study and the other human vitreous proteomes. As discussed above, it is possible that protein contaminants influence corona formation through physical interactions with constitutive proteins or limit binding sites on the NPs. Clusterin (CLU), which has been proposed to mediate the stealth activity of polyethylene glycol towards MPS, [8] was enriched in the source and slightly more enriched on the non-pegylated AL2. Although complement components were present in the vitreous source and are known to bind nanocarriers or corona proteins in serum and plasma, [9] complement components C3 and C4A, the latter of which was the 9th most enriched protein on AuNPs and SiNPs, [5] were relatively enriched in the liposome SC but depleted in the HC.
Both C3 and clusterin are components of drusen, which may increase in concentration with age and in AMD. [10] The liposome-associated proteins were compared with earlier studies on protein localization in vitreous substructures, [3] with 15 genes associated with the vitreous cortex and five with core, but most did not localize to any substructure (Table S4). Figure 3 and Table 2 include three keratins that showed uncharacteristic peaks for contaminants and were also contained in one report on the human vitreal proteome. [2] However, these were excluded from further data analysis as probable contaminants from the liposome preparation or analysis steps.
The comparisons with previously published proteomes suggest that the liposomal HC and SC enriched proteins are probable corona constituents in human patients following liposome intravitreal injections. Earlier studies have noted that most vitreal proteins are intracellular but probably carry out specific functions, since they organize into pathways based on biological functions. [3,7] For instance, the enrichment of glucose metabolism pathways confirms that these proteins are highly abundant in all parts of the vitreous and energy metabolism is one of its key biological functions. [3] The overlap between our study and the other proteomes showed a strong association with glucose metabolism. These metabolic proteins are carried by microvesicles, [3] which might contribute to their enrichment on liposomes.