**5. Conclusions**

A novel HA–lipid conjugate was successfully synthesized for the preparation of light-activated indocyanine green liposomes. HA-coated liposomes were functional in light-triggered drug release in vitreous and plasma samples. The HA-coated liposomes showed adequate stability and mobility in the vitreous humor. Protein corona formation on the HA-coated liposomes was characterized in the vitreous and plasma samples, and its properties were compared to PEG-coated and uncoated liposomes. Suitable stability and pharmacokinetic properties are vital for triggered release delivery systems that rely on controlled phase changes. The results showed the significance of nanoparticle coating materials on the stability, as well as on the interactions with vitreal and plasma components. These in turn may affect the immunological reactions to the drug delivery system, the implications of which are essential for intravitreal therapy due to the immunoprivilege of the eye. Future studies should further elucidate the mechanisms and consequences of corona formation in vitreous humor, taking into consideration the molecular crowding activity of structural HA. In conclusion, the HA-coated light-activated liposomes are a promising drug delivery system for intravenous and ocular applications.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/12/8/763/s1: Figure S1. <sup>1</sup>H-NMR and FT-IR spectra of the synthetized DSPE–HA conjugate; Figure S2. Size, T<sup>m</sup> profiles, heat- and light-triggered calcein releases, and ICG absorbances; Figure S3. Size, T<sup>m</sup> profiles, heat-triggered calcein releases, and ICG absorbances; Figure S4. Dynamic light scattering and large-angle light scattering; Figure S5. Heatmap of the 178 proteins identified in human plasma; Figure S6. Heatmap of the 728 proteins identified in porcine vitreous humor; Figure S7. Properties of the top 20 enriched proteins; Figure S8. Enriched functions and gene interaction network of HC proteins only enriched on HA-coated liposomes in plasma; Figure S9. Enriched biological functions and gene interaction network based on top 20 most abundant HC proteins in plasma; Figure S10. Enriched biological functions and gene interaction network based on top 20 most abundant HC proteins in vitreous humor; Table S1. Venn diagram of the protein-encoding genes in the plasma HC intersection; Table S2. Venn diagram of the protein-encoding genes in the vitreous HC union; Table S3. Enriched biological functions in the HC-enriched outlier sets per liposome group in plasma; Table S4. Top 100 enriched biological functions of the top 20 most abundant HC proteins in plasma; Table S5. Enriched biological functions based on the top 20 most abundant HC proteins in vitreous humor; Supplementary File 1. Plasma matrix and enriched (MS Excel); Supplementary File 2. Vitreous full matrix and enriched (MS Excel); Supplementary File 3. Top 20 plasma (PDF); Supplementary File 4. Top 20 vitreous (PDF); Supplementary File 5. Top 20 plasma vs. vitreous (PDF); Supplementary File 6. Common gene subsets plasma and vitreous (PDF).

**Author Contributions:** Conceptualization, O.K.K., A.U., and T.L.; methodology, O.K.K., S.T., P.P., N.G.J., T.V., and T.L.; software, P.P., J.N., and H.A.; formal analysis, O.K.K., S.T., S.B., and T.L.; investigation, O.K.K., S.T., S.B., R.S., T.R., N.G.J., J.N., and T.L.; resources, H.A., T.V., A.U., and T.L.; data curation, O.K.K. and T.L.; writing—original draft preparation, O.K.K., R.S., and T.L.; writing—review and editing, O.K.K., S.T., J.N., H.A., T.V., A.U., and T.L.; visualization, O.K.K., S.T., and T.L.; supervision, A.U. and T.L.; project administration, T.L.; funding acquisition, O.K.K., A.U., and T.L. All authors read and agree to the published version of the manuscript.

**Funding:** This research was funded by the Academy of Finland (#311122, #317336), Business Finland (#4208/31/2015), EU Horizon 2020 Marie Skłodowska-Curie Innovative Training Networks NANOMED project (#676137), Orion Research Foundation (#9-8214-9), Phospholipid Research Center, Emil Aaltonen Foundation, Instrumentarium Science Foundation, Mary and Georg C. Ehrnrooth Foundation, Evald and Hilda Nissi Foundation, Inkeri and Mauri Vänskä Foundation, Paulo Foundation, and Päivikki and Sakari Sohlberg Foundation. Open access funding provided by University of Helsinki.

**Acknowledgments:** Marja Hagström is acknowledged for her assistance with sample preparation for proteomics analysis. The University of Helsinki EV core is acknowledged for the flow cytometry support.

**Conflicts of Interest:** The authors declare no conflicts of interest.
