3.4.2. Hard and Soft Corona Composition

The HC and SC corona fractions from Figure 4C,D were eluted for high-resolution proteomics analysis with nanoliquid chromatography tandem mass spectrometry. Sufficient chromatographic separation was obtained with a one-hour linear gradient for plasma, while the vitreous samples required three-hour gradients. Figure 5 shows the relative enrichment and hierarchical clustering of the top 60 most abundant proteins in human plasma after median normalization. A list and a heatmap of the 178 proteins identified in at least three samples are provided in Figure S5 (Supplementary Materials). *Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 12 of 25

**Figure 5.** Heatmap of the top 60 most abundant proteins in at least three samples in human plasma after median normalization (178 proteins). Distinct clusters are observed for the HC, SC, and plasma source (including 1× and 7× diluted, surface plasmon resonance (SPR) instrument flow-through, and untreated). The HC subsections of the tested formulations cluster based on their anionic (*light-red box*) or neutral (*light-blue box*) surface charge. The corresponding significantly enriched proteins are highlighted with black boxes (**A**–**C**). The *dashed boxes* indicate the enrichment of hyaluronic acidbinding inter-alpha-trypsin inhibitor heavy chain proteins (ITIH2, ITIH4) and fibronectin (FN1). The range for relative enrichment (*red*) and depletion (*blue*) is two standard deviations from the mean on the log<sup>2</sup> scale. **Figure 5.** Heatmap of the top 60 most abundant proteins in at least three samples in human plasma after median normalization (178 proteins). Distinct clusters are observed for the HC, SC, and plasma source (including 1× and 7× diluted, surface plasmon resonance (SPR) instrument flow-through, and untreated). The HC subsections of the tested formulations cluster based on their anionic (*light-red box*) or neutral (*light-blue box*) surface charge. The corresponding significantly enriched proteins are highlighted with black boxes (**A**–**C**). The *dashed boxes* indicate the enrichment of hyaluronic acid-binding inter-alpha-trypsin inhibitor heavy chain proteins (ITIH2, ITIH4) and fibronectin (FN1). The range for relative enrichment (*red*) and depletion (*blue*) is two standard deviations from the mean on the log<sup>2</sup> scale.

B (APOB) and E (APOE), as well as clusterin (CLU) (Figure 5B). The HA-coated liposomes were also enriched with the extracellular matrix component fibronectin (FN1) and the hyaluronic acid-binding proteins inter alpha trypsin inhibitors (ITIH2 and ITIH4) (dashed boxes in Figure 5). In addition, the HA-coated liposome with ICG (F4) was enriched with additional immunoglobulin light-chain

The vitreous corona samples had 728 proteins that were identified in at least three samples (Figure S6; Supplementary Materials). The relative enrichment and hierarchical clustering of the top 60 most abundant proteins after median normalization are shown in Figure 6. In the vitreous sample, the corona subsections do not cluster based on charge or formulation. The relatively enriched HC proteins on the 50 nm anionic liposomes (F6–F7) and to a lesser extent on the 100 nm PEG-coated liposome (F2) include the cytoskeletal protein tubulin alpha (TUBA1B) and the glucose metabolismassociated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Figure 6A). Enrichment of tubulin beta chain variants (P02554; A0A287A275) was more pronounced in the PEG-coated liposome HCs (Figure 6B). These formulations were also enriched with clusterin (CLU) and hemoglobin

regions, which was not observed in the absence of ICG (F5; Figure 5C).

The HC, SC, and source plasma cluster into distinct groups (Figure 5). In addition, the anionic and neutral liposome groups cluster regardless of liposome size. The notably enriched proteins in the

The HC, SC, and source plasma cluster into distinct groups (Figure 5). In addition, the anionic and neutral liposome groups cluster regardless of liposome size. The notably enriched proteins in the *anionic* HC intersection are fibrinogen alpha and beta (FGA and FGB), complement component C3, thrombin (F2), and immunoglobulin variable regions (Figure 5A). *Neutral* liposomes were enriched with fibrinogen gamma (FGG), complement component C4, fibronectin (FN1), and apolipoproteins B (APOB) and E (APOE), as well as clusterin (CLU) (Figure 5B). The HA-coated liposomes were also enriched with the extracellular matrix component fibronectin (FN1) and the hyaluronic acid-binding proteins inter alpha trypsin inhibitors (ITIH2 and ITIH4) (dashed boxes in Figure 5). In addition, the HA-coated liposome with ICG (F4) was enriched with additional immunoglobulin light-chain regions, which was not observed in the absence of ICG (F5; Figure 5C).

The vitreous corona samples had 728 proteins that were identified in at least three samples (Figure S6; Supplementary Materials). The relative enrichment and hierarchical clustering of the top 60 most abundant proteins after median normalization are shown in Figure 6. In the vitreous sample, the corona subsections do not cluster based on charge or formulation. The relatively enriched HC proteins on the 50 nm anionic liposomes (F6–F7) and to a lesser extent on the 100 nm PEG-coated liposome (F2) include the cytoskeletal protein tubulin alpha (TUBA1B) and the glucose metabolism-associated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Figure 6A). Enrichment of tubulin beta chain variants (P02554; A0A287A275) was more pronounced in the PEG-coated liposome HCs (Figure 6B). These formulations were also enriched with clusterin (CLU) and hemoglobin subunit zeta (HBZ) in their HCs in addition to beta-crystallin (CRYBB1), which were mostly depleted on the HA-coated liposome (F4). The enrichment of the Dickkopf Wnt signaling pathway inhibitor 3 (A0A286ZNN6; DKK3) and an uncharacterized Ig-like protein (A0A286ZJL9) was less pronounced in these groups (Figure 6A,B), both of which were depleted on the HA-coated liposome (Figure 6C). Although GAPDH and TUBA1B were also enriched on replicates of the HA-coated liposome, their variants GADPHS and tubulin alpha chain (TUBA1A) showed higher enrichment (Figure 6d). The 50 nm anionic liposomes were also enriched with the retinol-binding protein 3 (RBP3) and albumin (ALB) in their HCs, which were mostly abundant in the SCs of the other formulations. One biological replicate of the PEG-coated liposome was an outlier, with a high abundance of immunoglobulins (Figure 6b,e,f). Both the HA-coated (F4) and PEG-coated liposomes (F2) with ICG were enriched with A0A286ZTT9, which is a 14-kDa immunoglobulin subtype deleted from the UniProt repository as obsolete in December 2019 (Figure 6f). Many of the other protein identifications that were not mapped into genes in Figure 6 are also immunoglobulins. Some of these proteins were found highly enriched in the SC of the HA-coated liposome with ICG (F4) and in some individual SC replicates (Figure 6e). The commonality between the source controls with different treatments (undiluted, diluted, SPR instrument flow-through) was 70% for plasma and 42% for vitreous samples.

subunit zeta (HBZ) in their HCs in addition to beta-crystallin (CRYBB1), which were mostly depleted on the HA-coated liposome (F4). The enrichment of the Dickkopf Wnt signaling pathway inhibitor 3 (A0A286ZNN6; DKK3) and an uncharacterized Ig-like protein (A0A286ZJL9) was less pronounced in these groups (Figure 6A–B), both of which were depleted on the HA-coated liposome (Figure 6C). Although GAPDH and TUBA1B were also enriched on replicates of the HA-coated liposome, their variants GADPHS and tubulin alpha chain (TUBA1A) showed higher enrichment (Figure 6d). The 50 nm anionic liposomes were also enriched with the retinol-binding protein 3 (RBP3) and albumin (ALB) in their HCs, which were mostly abundant in the SCs of the other formulations. One biological replicate of the PEG-coated liposome was an outlier, with a high abundance of immunoglobulins (Figure 6b,e,f). Both the HA-coated (F4) and PEG-coated liposomes (F2) with ICG were enriched with A0A286ZTT9, which is a 14-kDa immunoglobulin subtype deleted from the UniProt repository as obsolete in December 2019 (Figure 6f). Many of the other protein identifications that were not mapped into genes in Figure 6 are also immunoglobulins. Some of these proteins were found highly enriched in the SC of the HA-coated liposome with ICG (F4) and in some individual SC replicates (Figure 6e).

**Figure 6.** Heatmap of the top 60 most abundant proteins in porcine vitreous humor in at least three samples after median normalization (728 proteins). Distinct clusters are observed for the HC, SC, and plasma source. The HC subsections of the tested formulations do not cluster based on their anionic or neutral surface charge. The boxes **A**–**B** indicate the enrichment of tubulins, glyceraldehyde phosphate dehydrogenase (GAPDH), hemoglobin (HBZ), Dickkopf-related protein 3 (A0A286ZNN6), and **Figure 6.** Heatmap of the top 60 most abundant proteins in porcine vitreous humor in at least three samples after median normalization (728 proteins). Distinct clusters are observed for the HC, SC, and plasma source. The HC subsections of the tested formulations do not cluster based on their anionic or neutral surface charge. The boxes (**A**–**B**) indicate the enrichment of tubulins, glyceraldehyde phosphate dehydrogenase (GAPDH), hemoglobin (HBZ), Dickkopf-related protein 3 (A0A286ZNN6), and 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**,**e**)), which also enriched on the HA-coated liposome (**d**,**f**). An obsolete immunoglobulin subtype was enriched on the HA-coated liposome (**e**). The range for relative enrichment (*red*) and depletion (*blue*) is two standard deviations from the mean on the log<sup>2</sup> scale.
