3.4.3. Properties of the Corona-Enriched Proteins

The HC and SC subsections in plasma were enriched with 17–47 proteins per formulation, while some vitreous corona samples had up to 620 enriched proteins per formulation (log<sup>2</sup> fold-change ≥ 1.00). Notably, the HA-coated formulation with ICG (F4) and the 50 nm PEG-liposome (F7) were enriched with only 17 proteins in plasma. The HA liposome with ICG also had the smallest number of enriched proteins in its vitreous HC (102). The smaller dynamic concentration range of proteins in the vitreous sample is reflected in the narrower range of fold-changes, suggesting that the abundances of adsorbed proteins are also lower in vitreous than in plasma

samples (Supplementary File 2). To ensure comparability between samples and biological matrices, the top 20 most abundant proteins after median normalization were selected for the analysis of protein physicochemical properties (Supplementary Files 3 and 4). Their protein sequences were used to calculate the grand average of hydropathy (GRAVY), the theoretical isoelectric point (pI), the molecular weight (MW), and the number of the aromatic residues phenylalanine, tyrosine, tryptophan, and histidine (Ar (+His)) for the top 20 corona proteins in the corona subsections and the plasma or vitreous sources. The results for HC are displayed in Figure 7, while the corresponding figure for SC is shown in Figure S7 (Supplementary Materials). Summary data for both corona subsections in plasma and vitreous samples are provided as Supplementary File 5. abundances of adsorbed proteins are also lower in vitreous than in plasma samples (Supplementary File 2). To ensure comparability between samples and biological matrices, the top 20 most abundant proteins after median normalization were selected for the analysis of protein physicochemical properties (Supplementary Files 3–4). Their protein sequences were used to calculate the grand average of hydropathy (GRAVY), the theoretical isoelectric point (pI), the molecular weight (MW), and the number of the aromatic residues phenylalanine, tyrosine, tryptophan, and histidine (Ar (+His)) for the top 20 corona proteins in the corona subsections and the plasma or vitreous sources. The results for HC are displayed in Figure 7, while the corresponding figure for SC is shown in Figure S7 (Supplementary Materials). Summary data for both corona subsections in plasma and vitreous samples are provided as Supplementary File 5.

the vitreous sample is reflected in the narrower range of fold-changes, suggesting that the

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clusterin (CLU) on the 50 nm anionic and 100 nm PEG-coated liposomes, which were depleted or less enriched on the HA-coated liposome (dashed box **C**). Interestingly, variants of glyceraldehyde phosphate dehydrogenase (GAPDHS) and tubulin (TUBA1A) were enriched on the HA-coated liposomes (**d**). One replicate of the PEG-coated 100 nm liposome showed high enrichment of these variants along with immunoglobulin family proteins (boxes **b**). An obsolete immunoglobulin subtype was enriched on the HA-coated liposome (**e**). The range for relative enrichment (*red*) and depletion

The HC and SC subsections in plasma were enriched with 17–47 proteins per formulation, while some vitreous corona samples had up to 620 enriched proteins per formulation (log<sup>2</sup> fold-change ≥ 1.00). Notably, the HA-coated formulation with ICG (F4) and the 50 nm PEG-liposome (F7) were

(*blue*) is two standard deviations from the mean on the log<sup>2</sup> scale.

3.4.3. Properties of the Corona-Enriched Proteins

**Figure 7.** Properties of the top 20 enriched proteins in the hard coronas (HC) and the plasma or vitreous sources (P or V) with the max–min range, first and second quartiles, median, and mean (+). The color-coded groups are neutral 100 nm (*blue*), anionic 100 nm HA-coated (*green*), and small 50 nm anionic (*orange*) liposomes. GRAVY: grand average of hydropathy (positive score indicates hydrophobicity); pI: theoretical isoelectric point (pI under pH indicates net negative charge); MW: molecular weight; Ar (+His): aromatic residues phenylalanine, tyrosine, tryptophan, and histidine. Note the different *y*-axis ranges. **Figure 7.** Properties of the top 20 enriched proteins in the hard coronas (HC) and the plasma or vitreous sources (P or V) with the max–min range, first and second quartiles, median, and mean (+). The color-coded groups are neutral 100 nm (*blue*), anionic 100 nm HA-coated (*green*), and small 50 nm anionic (*orange*) liposomes. GRAVY: grand average of hydropathy (positive score indicates hydrophobicity); pI: theoretical isoelectric point (pI under pH indicates net negative charge); MW: molecular weight; Ar (+His): aromatic residues phenylalanine, tyrosine, tryptophan, and histidine. Note the different *y*-axis ranges.

There are no statistically significant correlations between liposome size or surface charge and the protein physicochemical properties. However, the anionic formulations had individual hydrophobic proteins in their HCs in plasma. The contributors of these positive hydropathicity values in plasma are immunoglobulin heavy chain V-III regions BRO and TIL. The median molecular weight of HC proteins is ~50 kDa in both plasma and vitreous samples, while the corresponding means are ~87 kDa and ~54 kDa, respectively, with higher variance in plasma. The anionic 50 nm liposomes (F6–F7) did not bind proteins with molecular weights above 200 kDa in their plasma HCs. The trend with aromatic residues mirrors that of the molecular weight in plasma. In vitreous humor, the second quartile of the pI is above the pH, indicating a higher abundance of proteins with a positive charge, while the majority of proteins in plasma and vitreous samples hold a negative charge at physiological pH. The top-range high-molecular-weight protein on the HA-coated liposome with ICG (F4) in vitreous is fibronectin (FN1). The protein properties for the top 20 proteins in SCs largely mirror the plasma and vitreous sources (Figure S7, Supplementary Materials).
