International Journal of Molecular Sciences

Article Biosynthesized Multivalent Peptides Stimulate Exosome Production in Human Corneal

Changrim Lee 1, Maria C. Edman 2 , Gordon W. Laurie 3 , Sarah F. Hamm-Alvarez 1,2,* and J. Andrew MacKay 1,2,4,* 1 Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA; [email protected] 2 Department of Ophthalmology, USC Roski Eye Institute and Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; [email protected] 3 Department of Cell Biology, School of Medicine, University of Virginia, Charlottesville, VA 22908, USA; [email protected] 4 Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089, USA * Correspondence: [email protected] (S.F.H.-A.); [email protected] (J.A.M.)

 Received: 30 July 2020; Accepted: 24 August 2020; Published: 26 August 2020 

Abstract: Lacripep is a therapeutic peptide derived from the human tear , Lacritin. Lacripep interacts with -1 and induces mitogenesis upon the removal of heparan sulfates (HS) that are attached at the extracellular domain of syndecan-1. The presence of HS is a prerequisite for the syndecan-1 clustering that stimulates exosome biogenesis and release. Therefore, syndecan-1- mediated mitogenesis versus HS-mediated exosome biogenesis are assumed to be mutually exclusive. This study introduces a biosynthesized fusion between Lacripep and an elastin-like polypeptide named LP-A96, and evaluates its activity on cell motility enhancement versus exosome biogenesis. LP-A96 activates both downstream pathways in a dose-dependent manner. HCE-T cells at high confluence treated with 1 µM LP-A96 enhanced cell motility equipotent to Lacripep. However, cells at low density treated with 1 µM LP-A96 generated a 210-fold higher number of exosomes compared to those treated at low density with Lacripep. As monovalent Lacripep is capable of enhancing cell motility but not exosome biogenesis, activation of exosome biogenesis by LP-A96 not only suggests its utility as a novel molecular tool to study the Lacritin biology in the corneal epithelium but also implies activity as a potential therapeutic peptide that can further improve ocular surface health through the induction of exosomes.

Keywords: lacritin; peptide biosynthesis; corneal epithelium; exosome; syndecan-1; elastin-like polypeptides

1. Introduction The lacrimal gland-corneal axis plays a critical role in maintaining ocular health. The lacrimal gland is the major organ responsible for the of essential and electrolytes into the tear film that overlays and protects the and [1]. One of these essential tear proteins that confer anti-microbial and anti-inflammatory defense at the ocular surface is Lacritin [2]. A 12.3 kDa secreted found in human and non-human primates, Lacritin exhibits prosecretory activity in lacrimal gland acinar cells (LGAC) and mitogenic activity in corneal epithelial cells [3]. Originally discovered by the Laurie laboratory [3], several studies have revealed that the active monomeric form of Lacritin is significantly downregulated in patients suffering from chronic [4],

Int. J. Mol. Sci. 2020, 21, 6157; doi:10.3390/ijms21176157 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 6157 2 of 22 aqueous-deficient dry eye [5], contact-lens related dry eye [6], and dry eyes associated with primary Sjögren’s syndrome (SS) [7]. It remains unclear if Lacritin monomer down-regulation (in part through -dependent cross-linking at the syndecan-1 binding domain [8]) is a symptom or a direct cause of these ocular surface diseases; however, Lacritin has shown potential as a therapeutic molecule. Its supplementation enhanced tear secretion from rat LGAC [3] and monkey LGAC [9], increased basal tear secretion in rabbit LGAC [10] and dry eye mouse eyes [11], and stimulated human corneal epithelial (HCE-T) cell proliferation [12]. This composite of preclinical evidences supports the continued development of Lacritin as an ocular therapeutic. Currently, its peptide derivative, LacripepTM, is under clinical evaluation for protein replacement therapy for dry (DED) and SS-associated DED (NCT03226444). Syndecan-1 present in corneal epithelium is a known receptor for Lacritin. The cleavage of (HS) chains at the extracellular domain of Syndecan-1 by an enzyme called enables Lacritin binding to syndecan-1 [13]. Heparanase exists in inactive (proheparanase) and active (heparanase) forms. Each of these forms has been shown to engage with syndecan-1 and to mediate distinct downstream signaling events. Heparanase cleaves HS and promotes association of Lacritin and syndecan-1 [14]. This interaction promotes cell proliferation and increases the motility of corneal epithelial cells by activating PKCα and NFAT-related pathways [12]. Proheparanase can cluster syndecan-1 by binding to intact HS chains attached to syndecan-1 [15–17]. The consequent clustering and internalization of syndecan-1 is known to activate the exosome biogenesis pathway by recruiting related molecules, including ALG-2-interacting protein X (ALIX), syntenin, and endosomal-sorting complex required for transport (ESCRTs). Therefore, HS-mediated activation of mitogenic signaling (requiring removal of HS from syndecan-1) and exosome biogenesis (requiring attachment of HS to syndecan-1) in the corneal epithelium are assumed to be mutually exclusive. This study explores a newly generated self-assembled multivalent Lacritin peptide nanoparticle named LP-A96, that may activate both pathways in corneal epithelial cells. Specifically, 1 µM LP-A96 enhanced cell motility at a high cell density, whereas its ability to promote exosome biogenesis was more prominent at a low cell density. The enhancement of cell motility by LP-A96 at 1 µM was equipotent to that of the monovalent Lacripep but its ability to induce exosome biogenesis was markedly higher, enabling the production of 210-fold higher number of exosomes in a given period of time compared to 1 µM Lacripep and 58-fold higher number of exosomes compared to the complete medium. Both actions of LP-A96 occurred in parallel with mobilization of intracellular Ca2+ and syndecan-1 internalization. With increasing interest in Lacritin and in exosome biology at the ocular surface, this study serves as a foundation for understanding ocular surface homeostasis, pathophysiology, and possible therapeutic interventions.

2. Results

2.1. Biosynthesized LP-A96 Fusion Proteins Self-Assemble into Multivalent Nanoparticles For clarity, ‘LP’ refers to the lacritin-derived peptide used to generate LP-A96, and ‘Lacripep’ refers to the peptide equivalent to the one currently in clinical trials (NCT03226444). Both the LP and Lacripep used in this study are derived from the active fragment at the C-terminus of the human Lacritin: GKQFIENGSEFAQKLLKKFSLLKPWA. While both LP and Lacripep share an identical 19 sequence as a core (Table1), LP contains five additional amino acids: the N-terminal methionine encoded by the start codon followed by a glycine (MG), which is found in human lacritin. LP also contains at the C-terminus, a leucine-tryptophan-alanine (LWA). During the optimization of expression, three amino acids, leucine-lysine-proline (LKP), were removed because this region was prone to autocleavage. This cleavage was reported previously [18] and reconfirmed by an independent mass analysis (data not shown). The WA, which is also found at the next two amino acids of the endogenous Lacritin protein, was included at the C-terminus of the 19-mer core to aid in the spectroscopic estimation of concentration due to the high absorbance of the included tryptophan. Although the LP contains Int. J. Mol. Sci. 2020, 21, 6157 3 of 22 several additional amino acids compared to Lacripep, the 19-mer core that exhibits activity remained unaltered. The regions that contain those five additional amino acids were confirmed to have no activity related to syndecan-1 binding and mitogenic activity exhibited by the 19-mer core [12].

Table 1. Molecular information of LP-A96, A96, and Lacripep.

MW (kDa) Proteins Amino Acid Sequence Expected a Measured b

LP-A96 MGKQFIENGSEFAQKLLKKFSLWAG(VPGAG)96Y 39.6 39.5 A96 MG(VPGAG)96Y 37.0 37.0 Lacripep KQFIENGSEFAQKLLKKFS 2.2 2.1 a Calculated based on the indicated amino acid composition. b Exact MW of LP-A96, A96, and Lacripep were determined by MALDI-TOF-MS (AppendixA Figure A1D), reported previously [ 19], and provided by the Laurie Laboratory, respectively.

The 23-mer LP was genetically fused to the N-terminus of an elastin-like polypeptide (ELP) termed A96 (Table1). Emerging as an attractive recombinant protein–polymer choice for diverse applications, ELPs are under exploration as a drug delivery platform since their biosynthesis produces pharmacologically relevant, monodisperse, biodegradable, and biocompatible entities [20–22]. The sequence confirmed cDNA encoding the LP-A96 fusion protein was subjected to heterologous expression via bacterial fermentation. The yield after the purification was ~30 mg/L with >95% purity, as verified by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (Figure1A,B). To determine the hydrodynamic radius (Rh) and colloidal stability, purified LP-A96 was analyzed using dynamic light scattering (DLS) at days 0, 2, 4, and 7 (Figure1C,D). Unlike A96 alone, the 23-mer LP induces assembly of LP-A96 into a colloidally stable nanoparticle, which persists even below its Tt. Purified LP-A96 remained stable for at least 7 days at 37 C with average R of 90.1 nm ( 7.2 nm, ◦ h ± SD). Size-exclusion chromatography followed by multi-angle light scattering (SEC-MALS) showed that all LP-A96 monomers self-assembled to nanoparticles (AppendixA Figure A1A). The radius of gyration (Rg) was 79.3 nm ( 0.6%, SEM). The estimated shape factor (ratio between Rg and R ) of 0.88 ± h (spheres 0.8 < Rg/R < 2.4 rods) suggests that the LP-A96 particles are spherical [23,24]. ≈ h ≈ Given the thermo-responsive nature of ELPs, the optical density of an LP-A96 solution was scanned at 350 nm (OD 350) over a range of temperatures (AppendixA Figure A1B) to determine its phase transition temperature (Tt). ELPs or ELP nanoparticles become insoluble microparticles (in a process called phase separation or coacervation) that can be measured by an increase in the OD 350 [27]. Based on the generated phase diagram (AppendixA Figure A1C), LP-A96 nanoparticles are expected to remain stable colloids at physiological temperatures and not phase-separate at the concentrations used in this study. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 21 confirmed to have no activity related to syndecan-1 binding and mitogenic activity exhibited by the 19-mer core [12].

Table 1. Molecular information of LP-A96, A96, and Lacripep.

MW (kDa) Proteins Amino Acid Sequence Expected a Measured b

LP-A96 MGKQFIENGSEFAQKLLKKFSLWAG(VPGAG)96Y 39.6 39.5 A96 MG(VPGAG)96Y 37.0 37.0 Lacripep KQFIENGSEFAQKLLKKFS 2.2 2.1 a Calculated based on the indicated amino acid composition. b Exact MW of LP-A96, A96, and Lacripep were determined by MALDI-TOF-MS (Appendix A Figure A1D), reported previously [19], and provided by the Laurie Laboratory, respectively. The 23-mer LP was genetically fused to the N-terminus of an elastin-like polypeptide (ELP) termed A96 (Table 1). Emerging as an attractive recombinant protein–polymer choice for diverse applications, ELPs are under exploration as a drug delivery platform since their biosynthesis produces pharmacologically relevant, monodisperse, biodegradable, and biocompatible entities [20– 22]. The sequence confirmed cDNA encoding the LP-A96 fusion protein was subjected to heterologous expression via bacterial fermentation. The yield after the purification was ~30 mg/L with >95% purity, as verified by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (Figure 1A,B). To determine the hydrodynamic radius (Rh) and colloidal stability, purified LP-A96 was analyzed using dynamic light scattering (DLS) at days 0, 2, 4, and 7 (Figure 1C,D). Unlike A96 alone, the 23-mer LP induces assembly of LP-A96 into a colloidally stable nanoparticle, which persists even below its Tt. Purified LP-A96 remained stable for at least 7 days at 37 °C with average Rh of 90.1 nm (±7.2 nm, SD). Size-exclusion chromatography followed by multi- angle light scattering (SEC-MALS) showed that all LP-A96 monomers self-assembled to nanoparticles (Appendix A Figure A1A). The radius of gyration (Rg) was 79.3 nm (± 0.6%, SEM). The estimatedInt. J. Mol. Sci. shape2020, factor21, 6157 (ratio between Rg and Rh) of 0.88 (spheres ≈ 0.8 < Rg/Rh < 2.4 ≈ rods) suggests4 of 22 that the LP-A96 particles are spherical [23,24]. AB

C 70 D 140 25 °C 60 A96 37 °C 120 50 LP-A96 100 40 80 30 60 20 40 Percentage (%) Percentage 10 20 Hydrodynamic radius(nm) 0 0 1 10 100 1000 01234567 Hydrodynamic radius (nm) Days

FigureFigure 1. 1. LacripepLacripep promotes promotes assembly assembly of LP-A96 of LP-A96 into into stable, stable, multivalent multivalent spherical spherical nanoparticles. nanoparticles. (A) Design(A) Design of LP-A96 of LP-A96 fusion. fusion. Through Through heterologous heterologous expression expression in Escherichia in Escherichia coli, the coli LP, thewas LP fused was to fused the N-terminusto the N-terminus of the elastin-like of the elastin-like polypeptide polypeptide (ELP), A96. (ELP), (B) A96. The identity (B) The identityand purity and of purity A96 and of A96LP-A96 and LP-A96 were analyzed by Coomassie blue staining of samples resolved by SDS-PAGE (sodium dodecyl

sulfate-polyacrylamide gel electrophoresis). Arrows indicate protein bands. A 20–30% upward shift in MW (molecular weight) with respect to the expected MW for ELPs and ELP fusions in SDS-PAGE has been observed by us and others [25,26]. The exact MW of LP-A96 was measured using MALDI-TOF-MS (AppendixA Figure A1D). ( C) Comparison of the hydrodynamic radius of monomeric A96 and self-assembled LP-A96 nanoparticles at room and physiological temperatures. (D) The hydrodynamic radius of LP-A96 remains stable at 37 C for least 7 days in DPBS (10 µM, n = 3, mean SD). ◦ ± 2.2. LP-A96 Induces Ca2+ Mobilization, Enhances Cell Motility, and Stimulates Exosome Biogenesis in Corneal Epithelial Cells The central hypothesis of this study is that, because of its multivalent nanoparticulate nature relative to Lacripep, LP-A96 may have the ability to elicit pathways that enhance cell motility and stimulates exosome biogenesis in corneal epithelial cells. To test this, we started by exploring the consequences of LP-A96 treatment in HCE-T cells on cell motility and Ca2+ mobilization. Both LP-A96 and Lacripep enhanced cell motility at a dose of 1 uM in a cell monolayer scratch assay (Figure2A,B). Lacritin-mediated cell motility enhancement in corneal epithelial cells has been associated with Ca2+ mobilization [12]. It has been shown that extracellular Ca2+ translocates into the cytoplasm (influx), where it interacts with and subsequently activates downstream signaling related to cell motility. To measure Ca2+ influx, fluorescence intensity changes in individual cells incubated with Fluo-4AM were monitored by fluorescence microscopy in real-time upon addition of LP-A96 to culture media. LP-A96 induced a strong Ca2+ influx immediately after the addition (Figure2C,F). As there was no increase in fluorescence intensity upon addition of A96 (Figure2D), it was apparent that the LP component of LP-A96 was essential for inducing Ca2+ influx. However, under identical conditions, Lacripep did not induce Ca2+ influx (Figure2E). Int. J. Mol. Sci. 2020, 21, 6157 5 of 22 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 21

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FigureFigure 2. 2. LP-A96LP-A96 promotes promotes scratch scratch closure closure and and induces induces Ca2+ Cainflux2+ influx in corneal in corneal epithelial epithelial cells. (A cells.) A scratch(A) A scratch (gap) (gap)assay assaywas used was usedto observe to observe the theenhancement enhancement of ofcell cell motility. motility. The The velocity velocity of of cell cell monolayermonolayer towards thethe remainingremaining gap gap area area (devoid (devoid of cells)of cells) was was calculated calculated as outlined as outlined in (B ).in (B(B)). Gaps (B) Gapswere imagedwere imaged before before the treatment the treatment and at and 12 h at post-treatment, 12 h post-treatment, and used and to used construct to construct (A). White (A). dottedWhite dottedlines indicate lines indicate the outer the edges outer ofedges the monolayerof the monolayer adjacent adjacent to the wound to the gap.wound (C~E gap.) Human (C~E) Human corneal cornealepithelial epithelial (HCE-T) (HCE-T) cells pre-incubated cells pre-incubated with Fluo-4 with AM Fluo-4 were AM incubated were incubated with either with LP-A96, either Lacripep,LP-A96, Lacripep,or A96 (all or at A96 1 µ M).(all at The 1 μ dottedM). The line dotted at the line 5th at minute the 5th indicates minute whenindicates the when treatment the treatment was added was to addedthe culture to the medium. culture medium. The fluorescence The fluorescence intensity inte of Fluo-4nsity of AM Fluo-4 was AM monitored was monitored as a marker as a formarker Ca2+ forinflux Ca2+ (n influx= 27~33 (n cells= 27~33/treatment cells/treatment per experiment). per experiment). A representative A representative data set from data the set three from independent the three independentsets of the experiment sets of the is experiment shown. (F) Theis shown. area under (F) The the area curve under of each the fluorescence curve of each intensity fluorescence profile intensityshown in profile (C~E)showed shown thatin (C only~E) LP-A96showed extensively that only LP-A96 mobilized extensively Ca2+. A one-way mobilized ANOVA Ca2+. followedA one-way by ANOVAmultiple comparisonsfollowed by multiple was used comparisons for statistical was comparison. used for ****statisticalp < 0.0001, comparison. * p < 0.05, **** ns: p non-significant. < 0.0001, * p < 0.05,Mean ns: non-significant.SD. Mean ± SD. ± 2+ ToAlthough confirm Ca that influxthis apparent has previously increase been in exosome linked to amount lacritin-mediated was not due cell to motility, residual the LP-A96 degree co- of 2+ purifiedCa influx during required the purifi for cellcation, motility purified alone exosomes may be below were thesubjected limits ofto detectionzeta potential in this analysis. assay. IfFirstly, this is 2+ thethe case,zeta potential the superior of the Ca naturalinflux inducedexosomes by collected LP-A96 mayfrom represent regular cell evidence cultures of activation (blue bar of graph another in Figurepathway 3E) that and Lacripep the preparation does not of activate. pure LP-A96 One possibility (red bar graph in the in context Figure of 3E) syndecan-1 were compared, biology wouldwhich 2+ showedbe an activation marked ofdifferences. the exosome Secondly, biogenesis assuming pathway, that which the residual also requires LP-A96 Ca in theinflux culture [15 ,media28]. could be co-purified,To observe two whether mixtures LP-A96 were promotes prepared exosome with different biogenesis, ratios: extracellular exosome:LP-A96 vesicles at (EVs)1:1 and secreted 1:100. Theseinto the mixtures culture mediawere incubated over a three-day at 37 °C period for three were collecteddays in basal and analyzed.media, subjected Exosome to biogenesis the identical was column-basednot prominent purification when cells wereprocess at and a high then confluence analyzed (Figurefor zeta3 A).potential. However, For the at subconfluence, 1:1 mixture, instead cells ofincubated a clear separation with LP-A96 of two spawned peaks, a significantlythe profile was higher broadly number distributed of EVs (Figurebetween3B) −60~0 that weremV, exactly highly overlappingenriched with the exosome profiles markers of pure [exosome29] (Figure and3C). LP-A96. The number Two zeta of EVs potential collected values from were the culture reported media for thisfrom mixture: cells after −46.4 LP-A96 mV and treatment −24.5 mV was (black 210-fold bar and graph 58-fold in Figure higher 3E). compared For the to1:100 the mixture, amount recovered the highest in peakbasal was media observed supplemented at around with −15 Lacripep mV and and the in pro completefile was media, narrowly respectively. distributed The between mean diameter −30~0 mV, and whichthe size highly distribution resembled of the that purified of the EVspure were LP-A96. similar Lastly, among the groupszeta potential (Figure of3D). exosomes To observe purified the rate after of LP-A96exosome treatment biogenesis was/release, compared exosomes with were the collectedabove four at 36 profiles. and 72 hThe post-LP-A96 mean value treatment. and the The overall total distributionexosome number profile and of purified exosome exosomes biogenesis after/release LP-A96 rate treatment during 36 (m hagenta were found bar graph to be in significantly Figure 3E) highlydifferent resembled from those those during of the 72 natural h. This suggestsexosomes the (blue rate bar of exosomegraph in uptakeFigure/ 3E).turnover Its mean exceeds zeta thepotential rate of wasexosome −55 mV biogenesis and the/ releasemajor portion during of the the 36~72 profi hle period was narrowly (Appendix distributedA Figure betweenA4). −90~−30 mV (−48 mV and −80~−20 mV, respectively, for natural exosomes). Therefore, it was apparent that the purified EVs after LP-A96 treatment are dominated by exosomes, and not LP-A96 particles.

Int.Int. J. J.Mol. Mol. Sci. Sci. 20202020, 21, 21, x, 6157FOR PEER REVIEW 6 of of 22 21

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Figure 3. LP-A96 activates exosome biogenesis in corneal epithelial cells. (A) Nanoparticle tracking Figure 3. LP-A96 activates exosome biogenesis in corneal epithelial cells. (A) Nanoparticle tracking analysis was used to quantify extracellular vesicles (EVs) in the collected HCE-T cell culture media analysisunder each was treatment used to quantify at near maximum extracellular confluency. vesicles Cells(EVs) cultured in the collected to 80~90% HCE-T confluency cell culture were starved media underfor another each treatment 24 h using at near basal maximum media prior confluency. to the treatment. Cells cultured EVs in to the80~90% culture confluency media were were purified starved forafter another 3 days 24 of h treatment.using basal ( Bmedia) EVs prior in the to collected the treatm HCE-Tent. EVs cell in culture the culture media media under were each purified treatment after at 3subconfluency days of treatment. (30%). ( CellsB) EVs were in supplementedthe collected withHCE-T diff erentcell culture agents uponmedia seeding under (0.5 each 10treatment5 cells/well at × subconfluencyin 12-well plate). (30%). EVs Ce inlls the were culture supplemented media were purifiedwith differen after 3t agents days of upon seeding. seeding The raw(0.5 × increase 105 cells/well in EV inamount 12-well upon plate). LP-A96 EVs in treatment the culture was media corrected were based purifi oned the after Western 3 days blot-based of seeding. analysis The raw (Appendix increaseA in EVFigure amount A2) beforeupon beingLP-A96 analyzed treatment for foldwas change.corrected For based (A,B), on all treatmentsthe Western were blot-based at 1 µM. (analysisC) EVs (Appendixcollected after A Figure LP-A96 A2) treatment before being were analyzed enriched for inthe fold exosome change. markers For (A,B CD9), all and treatments TSG101. were (D) Barat 1 μgraphsM. (C) showEVs thecollected size distribution after LP-A96 of purified treatment exosomes. were enriched The three in parallel the exosome gray lines markers indicate CD9 50, 150,and TSG101.and 250 ( nm,D) Bar respectively. graphs show Representative the size distribution data from of three purified independent exosomes. sets The of the three experiment parallel gray is shown. lines indicateInserted 50, numbers 150, and indicate 250 nm, mean respectively. hydrodynamic Represen diametertative ofdata purified from three exosomes. independent (E) Zeta sets potential of the experimentanalyses of is exosomes, shown. Inserted LP-A96, numbers and combinations indicate me of bothan hydrodynamic at different ratios. diameter The four of purified parallel exosomes. gray lines (Eindicate) Zeta potential90, 60, analyses30, and of 0 mV,exosomes, respectively. LP-A96, Inset an valuesd combinations are the mean of both zeta at potentials different reported ratios. The by − − − fourthe built-inparallel software.gray lines A indicate one-way −90, ANOVA −60, −30, followed and 0 mV, by multiple respectively. comparisons Inset values was used are the for mean statistical zeta comparison. * p < 0.05. Mean SD. potentials reported by the built-in± software. A one-way ANOVA followed by multiple comparisons was used for statistical comparison. * p < 0.05. Mean ± SD.

To identify the presence of any residual LP-A96 particles that were not detected during the zeta potential analysis, collected exosomes (used to construct Figure 3A and magenta bar graph in Figure 3E) were analyzed by Western blotting to detect ELPs using an anti-ELP antibody [30] along with a pre-determined amount of LP-A96 (Appendix A Figure A2). LP-A96 nanoparticles co-purified with the increased exosome yield were detected at low abundance as estimated by Western blotting,

Int. J. Mol. Sci. 2020, 21, 6157 7 of 22

To confirm that this apparent increase in exosome amount was not due to residual LP-A96 co-purified during the purification, purified exosomes were subjected to zeta potential analysis. Firstly, the zeta potential of the natural exosomes collected from regular cell cultures (blue bar graph in Figure3E) and the preparation of pure LP-A96 (red bar graph in Figure3E) were compared, which showed marked differences. Secondly, assuming that the residual LP-A96 in the culture media could be co-purified, two mixtures were prepared with different ratios: exosome:LP-A96 at 1:1 and 1:100. These mixtures were incubated at 37 ◦C for three days in basal media, subjected to the identical column-based purification process and then analyzed for zeta potential. For the 1:1 mixture, instead of a clear separation of two peaks, the profile was broadly distributed between 60~0 mV, exactly − overlapping the profiles of pure exosome and LP-A96. Two zeta potential values were reported for this mixture: 46.4 mV and 24.5 mV (black bar graph in Figure3E). For the 1:100 mixture, the highest peak − − was observed at around 15 mV and the profile was narrowly distributed between 30~0 mV, which − − highly resembled that of the pure LP-A96. Lastly, the zeta potential of exosomes purified after LP-A96 treatment was compared with the above four profiles. The mean value and the overall distribution profile of purified exosomes after LP-A96 treatment (magenta bar graph in Figure3E) highly resembled those of the natural exosomes (blue bar graph in Figure3E). Its mean zeta potential was 55 mV − and the major portion of the profile was narrowly distributed between 90~ 30 mV ( 48 mV and − − − 80~ 20 mV, respectively, for natural exosomes). Therefore, it was apparent that the purified EVs after − − LP-A96 treatment are dominated by exosomes, and not LP-A96 particles. To identify the presence of any residual LP-A96 particles that were not detected during the zeta potential analysis, collected exosomes (used to construct Figure3A and magenta bar graph in Figure3E) were analyzed by Western blotting to detect ELPs using an anti-ELP antibody [ 30] along with a pre-determined amount of LP-A96 (AppendixA Figure A2). LP-A96 nanoparticles co-purified with the increased exosome yield were detected at low abundance as estimated by Western blotting, comprising about 10% of the total particles purified. Based on this densitometric analysis, the pure increase in exosome yield (210-fold and 58-fold increase compared to Lacripep and complete media treatment, respectively) associated with LP-A96 treatment was corrected (Figure3A). Before correction, it was about 235-fold and 68-fold, respectively. Exosomes mediate intercellular signaling. Their cargos, especially ribonucleic acids (RNAs), are important mediators for intercellular communication [31]. As LP-A96 stimulates exosome production, it was of interest to determine whether this important exosomal cargo was increased proportionally to the exosome number increase. During a 36 h incubation, cells treated with LP-A96 produced about 12-fold higher numbers of exosomes compared to cells exposed to complete media (Figure4A). This increase in exosome abundance was correlated with a commensurate increase in both the total yield of non-coding small RNAs (9-fold) and miRNAs (17-fold) (Figure4B,C). miRNAs are a subset of non-coding small RNAs often enriched in exosomes. When content was normalized to particle number, the amount of exosomal non-coding small RNAs and miRNAs per exosome particle was similar between groups (Figure4D,E). About 60% of exosomal small RNAs were miRNAs in exosomes stimulated by LP-A96, which was slightly greater than that of exosomes stimulated by complete media, but there was no statistically significant difference (Figure4F). This indicates that LP-A96 treatment produces exosomes that are likely functional and loaded with the proper amount of cargo RNA, similar to those produced under physiological growth condition, and not ‘blank’ exosomes. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 21 comprising about 10% of the total particles purified. Based on this densitometric analysis, the pure increase in exosome yield (210-fold and 58-fold increase compared to Lacripep and complete media treatment, respectively) associated with LP-A96 treatment was corrected (Figure 3A). Before correction, it was about 235-fold and 68-fold, respectively. Exosomes mediate intercellular signaling. Their cargos, especially ribonucleic acids (RNAs), are important mediators for intercellular communication [31]. As LP-A96 stimulates exosome production, it was of interest to determine whether this important exosomal cargo was increased proportionally to the exosome number increase. During a 36 h incubation, cells treated with LP-A96 produced about 12-fold higher numbers of exosomes compared to cells exposed to complete media (Figure 4A). This increase in exosome abundance was correlated with a commensurate increase in both the total yield of non-coding small RNAs (9-fold) and miRNAs (17-fold) (Figure 4B,C). miRNAs are a subset of non-coding small RNAs often enriched in exosomes. When content was normalized to particle number, the amount of exosomal non-coding small RNAs and miRNAs per exosome particle was similar between groups (Figure 4D,E). About 60% of exosomal small RNAs were miRNAs in exosomes stimulated by LP-A96, which was slightly greater than that of exosomes stimulated by complete media, but there was no statistically significant difference (Figure 4F). This indicates that LP-A96 treatment produces exosomes that are likely functional and loaded with the proper amount of cargo RNA, similar to those produced under physiological growth condition, and Int.not J. ‘blank’ Mol. Sci. exosomes.2020, 21, 6157 8 of 22

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D 8 E 3 F 100 80 6 2 60 4 exosomes) exosomes)

5 5 40 1

2 amount miRNA per exosome (%) 20 (pg/10 (pg/10 Small RNA amount RNA Small miRNA ratio/small miRNA RNA 0 0 0 96 ia 96 ia 96 ia -A ed -A ed -A ed LP e M LP e M LP e M et et et pl pl pl m m m Co Co Co Figure 4. LP-A96 induced exosomes are properly loaded with RNAs. (A) Exosomes purified Figure 4. LP-A96 induced exosomes are properly loaded with RNAs. (A) Exosomes purified from from subconfluent HCE-T cells after 36 h of incubation with either 1 µM LP-A96 in basal media subconfluent HCE-T cells after 36 h of incubation with either 1 μM LP-A96 in basal media or complete or complete media shows stimulated exosome biogenesis and release by LP-A96 treatment. These media shows stimulated exosome biogenesis and release by LP-A96 treatment. These exosomes were exosomes were subjected to RNA isolation. (B,C) Elevated small RNA (0~270 nucleotides) and miRNA subjected to RNA isolation. (B,C) Elevated small RNA (0~270 nucleotides) and miRNA (10~40 (10~40 nucleotides) amount reflects an increase in total exosome amount shown in (A). (D) Small RNAs, nucleotides) amount reflects an increase in total exosome amount shown in (A). (D) Small RNAs, (E) (E) miRNAs, and (F) miRNA-to-small RNA ratio per exosome particle under two different treatment miRNAs, and (F) miRNA-to-small RNA ratio per exosome particle under two different treatment conditions are similar. n = 3. Mean SD. * p < 0.05, ns: non-significant. conditions are similar. n = 3. Mean ± SD. * p < 0.05, ns: non-significant. The appearance of cells treated with LP-A96 was also notably different from the cells treated with other agents (Figure5A) as characterized by the existence of perinuclear refractive organelles (arrows). To confirm that the increase in exosome amount upon LP-A96 treatment was not a result of secreted intracellular vesicles upon cell death, cells were treated with LP-A96 to see if it exhibited any cytotoxicity. During the course of three days, LP-A96 did not appear to exhibit any cytotoxicity (Figure5B). The proliferation profile of LP-A96 treated cells was similar to that of the cells treated with basal media, Lacripep, or A96. The rate of proliferation was less than that achieved in complete media. At 24 h, the proliferation rate with LP-A96 treatment compared to that with A96 treatment was statistically significant, suggesting a mitogenic effect during the initial 24 h period. However, a mitogenic effect of LP-A96 was not prominent after this period. The difference in cell appearance may be an indirect indication of a change in the intracellular membrane machinery associated with exosome biogenesis and increased vesicular accumulation in multivesicular bodies or other membrane compartments prior to secretion. Based on the above analyses, we concluded that: (i) the cellular response to exosome biogenesis is more prominent when cell density is low; (ii) purified EVs are exosomes and their increase is dominated by exosomes; and (iii) this increase is not related to the release of membranes upon cell death. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 21

The appearance of cells treated with LP-A96 was also notably different from the cells treated with other agents (Figure 5A) as characterized by the existence of perinuclear refractive organelles (arrows). To confirm that the increase in exosome amount upon LP-A96 treatment was not a result of secreted intracellular vesicles upon cell death, cells were treated with LP-A96 to see if it exhibited any cytotoxicity. During the course of three days, LP-A96 did not appear to exhibit any cytotoxicity (Figure 5B). The proliferation profile of LP-A96 treated cells was similar to that of the cells treated with basal media, Lacripep, or A96. The rate of proliferation was less than that achieved in complete media. At 24 h, the proliferation rate with LP-A96 treatment compared to that with A96 treatment was statistically significant, suggesting a mitogenic effect during the initial 24 h period. However, a mitogenic effect of LP-A96 was not prominent after this period. The difference in cell appearance may be an indirect indication of a change in the intracellular membrane machinery associated with exosome biogenesis and increased vesicular accumulation in multivesicular bodies or other membrane compartments prior to secretion. Based on the above analyses, we concluded that: (i) the cellular response to exosome biogenesis is more prominent when cell density is low; (ii) purified EVs are exosomes and their increase is dominated by exosomes; and (iii) this increase is not related to the release of membranes upon cell death.Int. J. Mol. Sci. 2020, 21, 6157 9 of 22

ABComplete media Lacripep 2.0 Complete media LP-A96 1.5 Lacripep dPBS A96 1.0 Digitonin A96 LP-A96

0.5

Absorbance at450 nm *

0.0 0244872 Hours

Figure 5. LP-A96-mediated exosome biogenesis does not affect cell viability in corneal epithelial Figure 5. LP-A96-mediated exosome biogenesis does not affect cell viability in corneal epithelial cells. cells. (A) Brightfield images of HCE-T cells at 48 h post-treatment. The appearance of LP-A96 (A) Brightfield images of HCE-T cells at 48 h post-treatment. The appearance of LP-A96 treated HCE- treated HCE-T cells at 48 h was typical of that observed throughout the 3-day incubation period, T cellswhile at all48 otherh was treatments typical of did that not observed affect the appearancethroughout of the the 3-day cells. Yellowincubation arrows period, indicate while possible all other treatmentsperinuclear did compartments.not affect the (Bappearance) Cell viability of uponthe ce 1 lls.µM Yellow LP-A96 treatmentarrows indicate was quantified possible alongside perinuclear compartments.other treatments. (B) Cell 50 µ Mviability Digitonin upon was used1 μM as aLP-A96 cytotoxic treatment agent. All was experiments quantified were alongside performed other treatments.when cells 50 were μM subconfluentDigitonin was (30%). used At as 24 a h, cytotoxic LP-A96 vs. agent. A96 treatment: All experimentsp = 0.012*; were LP-A96 performed vs. dPBS: when cellsp were= 0.052. subconfluent A one-way ANOVA(30%). At followed 24 h, LP-A96 by multiple vs. comparisonsA96 treatment: was usedp = 0.012*; for statistical LP-A96 comparison. vs. dPBS: p = * p < 0.05. Mean SD. 0.052. A one-way ANOVA± followed by multiple comparisons was used for statistical comparison. * p 2.3.< 0.05. LP-A96 Mean Is ± Internalized SD. via Dynamin-Mediated Endocytosis and Colocalizes with Syndecan-1 in Corneal Epithelial Cells 2.3. LP-A96 is Internalized Via Dynamin-Mediated Endocytosis and Colocalizes with Syndecan-1 in Corneal Syndecan-1 is known to be internalized upon clustering at the cell surface [32]. As LP-A96 is Epithelialexpected Cells to cluster syndecan-1 on the cell surface and induce its endocytosis, internalization and subcellularSyndecan-1 colocalization is known withto be syndecan-1 internalized would upon be expected. clustering First, at thethe internalizationcell surface [32]. of LP-A96 As LP-A96 was is expectedexplored. to cluster Cells were syndecan-1 treated with on eitherthe cell amiloride surfac ore and dynasore induce before its endocytosis, the addition of internalization LP-A96, which and subcellularrespectively colocalization inhibit pinocytosis with syndecan-1 or receptor-mediated would be endocytosisexpected. First, [33]. Afterthe internalization 15 min of incubation, of LP-A96 a significant fraction of LP-A96 is internalized (Figure6A), partly in an amiloride (Figure6B) and was explored. Cells were treated with either amiloride or dynasore before the addition of LP-A96, completely in a dynasore-dependent manner (Figure6C,D). Dynasore treatment did not a ffect cell whichviability respectively and inhibition inhibit of LP-A96pinocytosis endocytosis or receptor-mediated was restored after dynasore endocytosis was removed [33]. After (Appendix 15 minA of incubation,Figure A3 a significant). Further confirmationfraction of LP-A96 was sought is intern to observealized (Figure if internalized 6A), partly LP-A96 in an colocalize amiloride with (Figure syndecan-1. After 15 min of incubation, LP-A96 colocalized intracellularly with syndecan-1 in the

vicinity of the nucleus (Figure6E). Therefore, it is highly likely that LP-A96 is internalized through a receptor-mediated pathway associated with syndecan-1. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 21

6B) and completely in a dynasore-dependent manner (Figure 6C,D). Dynasore treatment did not affect cell viability and inhibition of LP-A96 endocytosis was restored after dynasore was removed (Appendix A Figure A3). Further confirmation was sought to observe if internalized LP-A96 colocalize with syndecan-1. After 15 min of incubation, LP-A96 colocalized intracellularly with syndecan-1 in the vicinity of the nucleus (Figure 6E). Therefore, it is highly likely that LP-A96 is Int.internalized J. Mol. Sci. 2020 through, 21, 6157 a receptor-mediated pathway associated with syndecan-1. 10 of 22

Non-Treat (DMSO) + Amiloride (1 mM) + Dynasore (80 µM) 2.5 A B C D *** **** 2.0 ****

1.5

1.0 (fold-change) 0.5 Fluorescence Intensity Fluorescence

0.0 at e re re rid o -T ilo as n m yn No A D

E LP-A96 Syndecan-1 Nucleus Merge + 2° AF488 + anti-Syndecan-1 + 2° AF488 - anti-Syndecan-1

FigureFigure 6. LP-A96 is internalized via dynamin-dependent endocytosisendocytosis and and colocalized colocalized with with syndecan-1 syndecan- in corneal epithelial cells. (A–D) HCE-T cells were treated with either DMSO (vehicle control), amiloride 1 in corneal epithelial cells. (A–D) HCE-T cells were treated with either DMSO (vehicle control), (1 mM), or dynasore (80 µM) prior to addition of 1 µM rhodamine-labeled LP-A96. (D) Quantified amiloride (1 mM), or dynasore (80 μM) prior to addition of 1 μM rhodamine-labeled LP-A96. (D) fluorescence intensity per cell under each treatment. n = 39 for DMSO, n = 49 for amiloride, n = 48 Quantified fluorescence intensity per cell under each treatment. n = 39 for DMSO, n = 49 for amiloride, for dynasore. Representative images are shown from the three independent experiments. (E) LP-A96 n = 48 for dynasore. Representative images are shown from the three independent experiments. (E) was colocalized with syndecan-1 in HCE-T cells. (Pearson’s correlation = 0.8, n = 32). 1 µM LP-A96 was colocalized with syndecan-1 in HCE-T cells. (Pearson’s correlation = 0.8, n = 32). 1 μM rhodamine-labeled LP-A96 were incubated with the cells for 15 min at 37 C prior to fixation. Secondary rhodamine-labeled LP-A96 were incubated with the cells for 15 min◦ at 37 °C prior to fixation. immunofluorescence revealed apparent colocalization of rhodamine-labeled LP-A96 and Syndecan-1. Secondary immunofluorescence revealed apparent colocalization of rhodamine-labeled LP-A96 and Anti-syndecan-1 antibody was further detected by Alexa Fluor 488 conjugated secondary antibody. Syndecan-1. Anti-syndecan-1 antibody was further detected by Alexa Fluor 488 conjugated secondary Nuclei were stained with DAPI (4 ,6-diamidino-2-phenylindole). A one-way ANOVA followed by antibody. Nuclei were stained with0 DAPI (4′,6-diamidino-2-phenylindole). A one-way ANOVA multiple comparisons was used for statistical comparison. **** p < 0.0001, *** p < 0.001. Mean SD. followed by multiple comparisons was used for statistical comparison. **** p < 0.0001, *** p <± 0.001. 2.4. LP-A96Mean ± DeliveredSD. by Contact Lens Enhances Cell Motility in Corneal Epithelial Cells

2.4. LP-A96Eyedrops Delivered are the by most Cont convenientact Lens Enhances and frequently Cell Motility used in route Corneal of administration Epithelial Cells for ocular surface drug delivery. Although it is convenient and effective, a frequent need for topical administration is associatedEyedrops with are lower the patientmost convenient compliance. and Thus, frequently strategies used to route enable of sustained administration release for of ocular drugs thatsurface are edrugffective delivery. when givenAlthough topically it is areconvenient of great interest.and effective, Such strategiesa frequent may need maintain for topical an active administration drug on the is ocularassociated surface with for lower a longer patient period compliance. of time. OneThus, such strategies method to is enable to use sustained contact lenses release as aof drug drugs depot. that Contactare effective lenses when have given been studied topically as are a therapeutic of great intere platformst. Such to manage strategies ocular may anterior maintain segment an active disorders drug beyondon the ocular their primary surface functionfor a longer of providing period of millions time. One of people such method with glasses-free is to use contact vision correction lenses as [a34 drug,35]. Baseddepot. on Contact the previous lenses demonstrationhave been studied of adsorption as a therapeutic of ELPs platform to and their to manage delivery ocular to the cornealanterior epithelial segment cellsdisorders upon beyond release fromtheir theprimary commercially function availableof providin contactg millions lenses of [ 18people], the abilitywith glasses-free of contact lensesvision tocorrection deliver functional[34,35]. Based LP-A96 on the to previous corneal epithelialdemonstration cells wasof adsorption tested. To of begin ELPs with,to and the their adsorption delivery and release kinetics of LP-A96 in contact lenses was determined using a fluorescence-based method. Fluorescein-labeled LP-A96 was incubated with contact lenses to analyze the concentration-dependent adsorption (Figure7A), time-dependent adsorption (Figure7B), and time-dependent release kinetics (Figure7C). Parameters estimated from the fit are reported in Table2. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 21

to the corneal epithelial cells upon release from the commercially available contact lenses [18], the ability of contact lenses to deliver functional LP-A96 to corneal epithelial cells was tested. To begin with, the adsorption and release kinetics of LP-A96 in contact lenses was determined using a fluorescence-based method. Fluorescein-labeled LP-A96 was incubated with contact lenses to analyze the concentration-dependent adsorption (Figure 7A), time-dependent adsorption (Figure 7B), and Int.time-dependent J. Mol. Sci. 2020, 21 release, 6157 kinetics (Figure 7C). Parameters estimated from the fit are reported in11 Table of 22 2.

100 70 100 AB60 C 80 80 50 60 40 60

30 40 40 Qe (mg/g)Qe Qe (mg/g)

20 Retention (%) 20 20 10

0 0 0 0246810 0 10203040506070 0 24487296 Ce (mg/mL) Time (h) Time (h) D 40 12 EFns ** ns 30 8

20

g protein (fold-change) protein g 4 μ 10 % Gap remaining after 24hr after remaining % Gap

0 Particles/ 0 dPBS A96 LP-A96 CM dPBS A96 only LP-A96 CM Agent delivered by contact lens Agent delivered by contact lens

FigureFigure 7. 7. ContactContact lens-delivered lens-delivered LP-A96 LP-A96 maintains maintains its its activity activity in incorneal corneal epithelial epithelial cells. cells. (A) (AConcentration-dependent) Concentration-dependent adsorption adsorption of LP-A96 of LP-A96 to contact to contact lenses ov lenseser a 24 over hr aperiod. 24 h (B period.) Time- (Bdependent) Time-dependent adsorption adsorption of 2 mg/mL of 2 LP-A96 mg/mL LP-A96to contact to lenses. contact (C lenses.) Time-dependent (C) Time-dependent release of release LP-A96 offrom LP-A96 contact from lenses. contact (D lenses.) Study (D design) Study of design the contact of the lens-med contact lens-mediatediated delivery deliveryof LP-A96 of LP-A96to a scratch to a(gap) scratch generated (gap) generated corneal cornealepithelial epithelial cell monolayer. cell monolayer. (E) Gap closure (E) Gap activity closure of activity HCE-T of cells HCE-T incubated cells incubatedwith contact with contactlenses lensesloaded loaded with withdifferent different agents. agents. (F) (FAfter) After gap gap closure closure activityactivity measurement,measurement, exosomesexosomes were were purified purified fromfrom cultureculture media and quantified quantified using using na nanoparticlenoparticle tracking tracking analyzer. analyzer. (F) (FExosome) Exosome abundance abundance in the in theculture culture media media after after 24 hr 24period. h period. A one-way A one-way ANOVA ANOVA followed followed by multiple by multiplecomparisons comparisons was used was for used statistical for statistical comp comparison.arison. CM indicates CM indicates complete complete media. media. * p <* 0.05,p < 0.05, ns: non- ns: non-significant. Mean SD. significant. Mean ± SD.± Table 2. Adsorption and release kinetics of LP-A96 in Proclear CompatiblesTM contact lenses. Table 2. Adsorption and release kinetics of LP-A96 in Proclear CompatiblesTM contact lenses. Concentration- Time-Dependent Dependent Time-Dependent Time-DependentReleaseTime-Dependent Parameters Concentration- DependentParameters Adsorption (Mean Parameters Parameters Adsorption (Mean Parameters Adsorption (Mean (95% Parameters(Mean (95%Release CI)) (Mean Adsorption (Mean (95% CI)) (95% CI)) (95% CI)) CI)) (95% CI)) Plateau Fast Release Fast Release QQmm (mg/g)(mg/g) 78.6 (68.5–88.8)78.6 (68.5–88.8) Plateau (mg/g) 55.8 (46.1–65.5)55.8 (46.1–65.5) 73.3 (65.8–80.9)73.3 (65.8–80.9) (mg/g) (%) (%) 1 Kd (mg/L) 0.78 (0.35–1.21) Fast binding (%) 25.6 (15.8–35.4) kfast (h− ) 0.068 (0.055–0.082) 2 1Fast binding 1 0.068 (0.055– Kd (mg/L)R 0.780.84 (0.35–1.21) kfast (h− ) 2.36 (0.0–4.86)25.6 (15.8–35.4)kslow (h− ) kfast 0.0011(h−1) (0.0–0.0044) 1 (%) 0.082) kslow (h− ) 0.041 (0.0–0.069) t1/2, fast (h) 10.1 (8.4–12.7) t (h) 0.29 (0.14-Inf.) t (h) 645.5 (157.3-Inf.)0.0011 (0.0– R2 0.84 1/2, fast kfast (h−1) 2.36 (0.0–4.86)1/2,slow kslow (h−1) 2 t1/2, slow (h) 16.9 (10.1–51.1) R 0.99 0.0044) 2 R kslow (h−1) 0.95 0.041 (0.0–0.069) t1/2, fast (h) 10.1 (8.4–12.7)

t1/2, fast (h) 0.29 (0.14-Inf.) t1/2, slow (h) 645.5 (157.3-Inf.) 2 Based on these parameters, contact lensest1/2, slow were (h) incubated16.9 with(10.1–51.1) 0.4 mg of LP-A96R for 24 h;0.99 release 2 from lenses adsorbed under this condition wasR expected to increase0.95 LP-A96 concentration in culture media up to 1~2 µM over 24 h. After in solution adsorption, LP-A96 loaded contact lenses were transferredBased to on HCE-T these cellparameters, cultures andcontact incubated lenses forwere 24 incu h. Justbated before with the 0.4 contact mg of LP-A96 lens transfer, for 24 a h; scratch release wasfrom generated lenses adsorbed on HCE-T under monolayers this condition (Figure was7D). expe Thected cells to incubatedincrease LP-A96 with LP-A96 concentration loaded in contact culture lensesmedia showed up to equipotent1~2 μM over cell 24 motility h. After enhancement in solution toadsorption, the cells incubated LP-A96 loaded with an contact intact contact lenses lenswere intransferred complete media to HCE-T (Figure cell7 E),cultures which and was incubated significantly for higher24 h. Just than before that observed the contact with lens A96 transfer, loaded a contact lens treatment. However, exosome production during this 24 h period was not significantly di fferent (Figure7F). This may be because LP-A96-mediated exosome biogenesis is not prominent under conditions of maximum HCE-T cell confluency (Figure3A) and the duration of incubation was only 24 h. Nevertheless, the data clearly show that the contact lenses can deliver functional LP-A96 to corneal epithelial cells. Int. J. Mol. Sci. 2020, 21, 6157 12 of 22

3. Discussion This study describes the construction of a multivalent Lacritin-derived peptide nanoparticle named LP-A96 and its biological effects in corneal epithelial cells. Under different conditions in corneal epithelial cells, LP-A96 enhanced cell motility and stimulated exosome biogenesis in parallel with intracellular Ca2+ mobilization. Of these functions, only the ability to enhance cell motility is shared with the monovalent Lacripep, while the stimulation of exosome biogenesis was unique to the multivalent nanoparticle. When cells are sparse and not in contact with each other, LP-A96 appears to be able to direct the intracellular machinery to evoke exosome biogenesis instead of cell motility. On the other hand, LP-A96 did not activate exosome biogenesis when the cells were confluent but were capable of enhancing cell motility. This cell density-dependent differential activation can be explained by the activation of different cellular mechanism that ensures cell-to-cell communication. When cells are confluent, there is no need for cells to initiate another intercellular communication mechanism, i.e., stimulate production and release of extracellular vesicles for their communication, because cells are able to directly communicate through physical contacts. However, when cells are sparse, the most effective way to communicate with each other would be to stimulate production and release of extracellular vesicles including exosomes containing signaling mediators which can be internalized. This study only tested these two opposing conditions, near-maximal confluency and low cell density. Future studies identifying the conditions under which cells become unresponsive to signaling to evoke exosome biogenesis would allow a better understanding of the syndecan-1 biology in the corneal epithelium. With this, several aspects should also be explored regarding ocular surface physiology. First, LP-A96 treatment stimulated exosome biogenesis/release during the three-day period (Figure3B). Within a three-day period, the amount of total exosome and the rate of exosome biogenesis/release was higher during the 0~36 h period compared to those of the 0~72 h period (AppendixA Figure A4). This indicates that the exosome uptake/turnover rate may exceed the exosome biogenesis/release rate during the 36~72 h period. As this study focuses on the discovery of the enhancement of exosome biogenesis upon LP-A96 treatment, biogenesis-release-uptake and other biological aspects of ocular exosomes in corneal epithelial cells will be explored in future studies. Second, since LP-A96 stimulates exosome biogenesis, it is possible that the expression and activity profiles of heparinase and proheparanase may change upon addition of LP-A96. As this enzyme is known to be heavily involved in homeostasis [16], a more comprehensive examination of its expression and activity upon LP-A96 treatment in corneal epithelium will allow better insights regarding the biological influence of LP-A96. Third, the involvement of ocular exosomes in corneal wound healing is well documented [36,37]. As a significantly higher number of exosomes that carry RNAs are produced and secreted upon LP-A96 treatment, deep sequencing of miRNAs might shed insights regarding the molecular mechanisms targeted on the ocular surface and possibly the draining lymph nodes by LP-A96-evoked exosomal shedding. Fourth, LP-A96 was colocalized with syndecan-1 in corneal epithelial cells, possibly via association of LP-A96 with syndecan-1 [14]. Further studies addressing direct binding of LP-A96 to syndecan-1 and to other proposed co-receptors, such as G-protein coupled receptors (GPCRs) [12], and their internalization/activation in corneal epithelial cells will be of great importance to understand Lacritin biology at the ocular surface. The optimal concentration for Lacritin’s mitogenic activity is reported to be 1~10 nM in human salivary gland ductal cells [12]. At this concentration, however, neither Lacripep nor LP-A96 enhanced cell motility in corneal epithelial cells. The chosen concentration for this study was 1 µM, for both Lacripep and LP-A96, based on a dose-dependent scratch closure assay (Figure2E). As similar concentrations were reported for corneal-lacrimal gland axis in rabbit (0.8~8 µM) [10], mouse (4 µM) [11], and monkey (0.1~1 µM) [9] models, corneal epithelium seems to require a higher dose of Lacritin or its derivative Lacripep compared to salivary gland ductal cells. At 1 µM, only LP-A96 induced Ca2+ mobilization but Lacripep did not. Although Lacritin (3.2 nM) was able to evoke Ca2+ influx in HCE-T cells [3], it is possible that the structural differences between the full-length versus derivatized Int. J. Mol. Sci. 2020, 21, 6157 13 of 22 peptide fragment affect the binding affinity as well as the degree of Ca2+ mobilization at this lower dose. Thus Lacripep-mediated Ca2+ mobilization may be transient or under the limits of detection. Another possibility would be that the chosen dose (1 µM) is sub-optimal to detect significant changes based on the ‘bell-shaped’ dose-response curve observed in the parent molecule. Lacritin. Since the current study was conducted with a single dose (1 µM) and is focused on highlighting differences between the free peptide and its multimeric form in eliciting exosome biogenesis, further studies will be needed to define the dose-dependency between Lacripep and Lacritin with respect to both cell motility and calcium signaling. In addition to actions on the cornea which have been described, Lacritin is a known tear secretagogue. Upon expression and secretion from LGAC (lacrimal gland acinar cells), Lacritin promotes tear secretion from lacrimal glands to sustain the tear film and maintain ocular surface homeostasis [10]. Upon the development of the LP-A96, its secretagogue activity was tested in primary rabbit LGAC. However, LP-A96 did not show any meaningful secretagogue activity. For this reason, our research efforts were focused on elucidating biological effects in corneal epithelial cells rather than in LGAC. Despite several observational reports of Lacritin’s secretagogue activity, the intracellular mechanisms that promote increased tear secretion are not well understood. Comparison of Lacritin’s secretagogue activity to the well-characterized cholinergic agonist, carbachol, in primary monkey LGACs by Fujii et al. showed that Lacritin stimulated tear secretion in a Ca2+ and PKCα independent manner while carbachol required both for its activity [9]. Based on the fact that Ca2+ and PKCα are essential molecules for Lacritin-mediated signaling in other epithelial cells, Lacritin’s receptor and intracellular signaling pathway in LGACs seem to be different from what is generally accepted in corneal epithelial cells. Identification of its receptor(s) and its respective intracellular signaling as well as how LP-A960s multivalency may impact tear secretion, the spectrum of proteins secreted, and exosome production in LGAC will provide more information on how LP-A96 may be used therapeutically in lacrimal glands. Multivalency was achieved by genetically fusing LP to ELP recombinant polypeptides. Compared to unimeric ELP A96, the hydrodynamic radius of LP-A96 indicate that the amphipathic peptide, LP, drives nano-assembly of LP-A96. Similar assembly by another amphipathic peptide-ELP fusion named L4F-A192 was reported previously [38]. Cryo-TEM imaging revealed that L4F-A192 forms a vesicular structure with a wall thickness of 8.4 nm. Based on the similar amphipathic nature between LP and L4F peptides, it is possible that LP-A96 nano-assembly may form vesicles. Investigating the nanostructures formed by LP-A96 as well as other amphipathic peptide-ELP fusions will allow a better understanding of polymer-mediated nano-assembly. Drugs that are currently prescribed for the treatment of various ocular surface and anterior chamber disorders have been investigated for contact lens-based delivery to enhance their therapeutic performance. These include drugs for ocular infection (Ciprofloxacin), corneal injury (EGF), allergic conjunctivitis (Ketotifen fumarate), dry eye (Re-wetting agents/hyaluronic acid, Cyclosporin A) and glaucoma (Acetozolamide, Timolol) [39]. Despite these advances, it would be desirable to provide a contact lens drug delivery device which is relatively simple in design; which does not require complicated and expensive manufacturing processes; which does not significantly impair or interfere with the patient’s vision; and which would not require a substantial change in the practice patterns of eye physicians and surgeons. As LP-A96 released from the contact lenses was functional, delivery of LP-A96 through contact lenses could serve as an alternative route of administration to improved ocular surface health. To conclude, this study demonstrates a simple yet effective peptide modality that is capable of stimulating both cell motility and exosome biogenesis in corneal epithelium, mediated through Lacritin biology. As exosomes mediate cellular communication, signaling, and immune modulation, the biological and therapeutic effects of both exosomes and LP-A96 in the context of dry eye diseases (DED) [40,41] will greatly advance our knowledge towards the pathophysiology of DED and aid in providing a better way to improve daily lives of DED patients. Int. J. Mol. Sci. 2020, 21, 6157 14 of 22

4. Materials and Methods

4.1. Synthesis, Expression, and Purification of LP-A96 and A96 The pET-25b(+) vector (#69753, Millipore-Sigma, Burlington, MA, USA) was purchased and further modified for ELP fusion cloning [19]. A chemically synthesized oligonucleotide cassette encoding the amino acids MGKQFIENGSEFAQKLLKKFSLWA was ligated to the N-terminus of ELP A96 to generate LP-A96. The resulting fusion plasmids were sequenced, transformed into, and expressed in ClearColi® BL21(DE3) Electrocompetent Cells (#60810, Lucigen, Middleton, WI, USA). Cells were fermented in terrific broth media supplemented with 1 mM NaCl for 24 h at 37 ◦C without IPTG induction. After centrifugation, 1 g of biomass (cell pellets) was resuspended in a 4 mL of 1:1 mixture of 1-butanol and ethanol (i.e., mixture of 8 mL 1-butanol and 8 mL ethanol was directly added to 4 g of cell pellet) [42]. Cell pellets were resuspended thoroughly by vortexing (10 s) and left under constant agitation at room temperature for 15 min. After transferring to 50 mL conical tubes, the suspension was centrifuged at 4000 rpm for 10 min using a Sorvall RC-3C Plus Centrifuge. Only the organic phase (upper phase) that contains LP-A96 was collected and transferred to a clean 50 mL conical tube with 5 mL Dulbecco’s phosphate-buffered saline (dPBS, without Ca2+ and Mg2+). The whole solution was placed under a mild focused air stream with constant stirring, and left overnight to passively evaporate organic solvents. Collected samples were then dialyzed against dPBS for 24 h under sink condition to remove residual organic solvents. Purified proteins were sterile filtered (200 nm pore, #PN 4612, Pall Corp., NY, USA) after dialysis and used for subsequent assays. Lacripep was provided by the Laurie Laboratory.

4.2. Biophysical Characterization of LP-A96 and A96 The purity of A96 and LP-A96 fusion proteins was analyzed using SDS-PAGE. The molar extinction coefficients (ε) of A96 and LP-A96 were calculated at 1285 and 6970 M 1 cm 1 [43]. Serial dilutions − · − in Edelhoc buffer were prepared, measured and averaged to acquire the best estimate of protein concentration in dPBS using Equation (1) [43,44].

A280 (A330 2) C = − × . (1) protein ε

The hydrodynamic radius (Rh) at 25 ◦C and 37 ◦C was determined using dynamic light scattering (DLS). Proteins in dPBS (50 µL at 10 µM) were loaded onto a 384-well plate followed by layering with two drops of mineral oil to prevent evaporation, and the whole plate was centrifuged for 1 g min × at 1000 rcf to remove any remaining air bubbles. Triplicate samples were analyzed using a Wyatt Dynapro plate reader and by built-in software DYNAMICS V7 (Wyatt Tech. Co., CA, USA). Rh was measured first at 25 ◦C and then the temperature was immediately increased to 37 ◦C, where the second measurement was made. The plate was subsequently incubated at 37 ◦C for 7 days, while Rh was measured at days 2, 4, and 7 to observe the stability. Size exclusion chromatography followed by multiangle light scattering (SEC-MALS) was used to determine the radius of gyration (Rg), absolute molecular weight, and oligomeric state of the sample. 10 µM sample in 100 µL dPBS was injected onto a Shodex size exclusion column (KW-803, Showa Denko K.K, Tokyo, Japan) at 0.5 mL/min. The eluents were analyzed on a Helios system (Wyatt Tech. Co., CA, USA) maintained at 25 ◦C and the data were fit to a Debye model, which best explained the data to determine the Rg. The transition temperature (Tt) of proteins were obtained using an optical density. The absorbance at 350 nm, A, was measured in a DU800 UV-Vis spectrophotometer (Beckman Coulter, CA, United States) under a temperature gradient of 0.5 ◦C/min. The Tt at each concentration was defined as the temperature at which the maximum first derivative, dA/dT, was achieved using Equation (2). The Ai is Int. J. Mol. Sci. 2020, 21, 6157 15 of 22

defined as the absorbance recorded at Ti temperature. The Tt from each concentration was used to plot the phase diagram and fit with Equation (3) to obtain slope, m, and intercept b (Table A1).

dA Ai+1 Ai = − (2) dT T + T i i 1 − i h i Tt = b m log C (3) − 10 protein 4.3. Cell Culture, Scratch Assay, and Cytotoxicity Human corneal epithelial SV40-transformed cells (HCE-T, Riken Cell Bank, Japan) were cultured in KSFM media (#17005042, Life Technologies, Rockville, MD, USA) supplemented with bovine pituitary extract (BPE) and epidermal growth factor (EGF) according to the manufacturer’s recommendation (‘cells’ hereafter). Complete media refers to KSFM supplemented with EFG and BPE and basal media refers to KSFM media alone without EGF and BPE. Cells in passage number 4~7 were used for all cell-based assays. For imaging, a Zeiss LSM880 Confocal Microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with Airyscan, was used (‘confocal microscope’ hereafter). For image analysis, ZEN2 Blue Edition software (Carl Zeiss AG, Oberkochen, Germany) was used (‘ZEN20 hereafter). For evaluation of cell motility, cells were cultured in 24 well plates. At 80% confluency, cells were cultured with basal media for another 24 h. A scratch was generated on the cell monolayer using a 200 µL pipette tip. After washing twice with dPBS, cells were cultured with 2 mL of fresh basal media supplemented with 1 µM Lacripep, A96, or LP-A96. These doses were chosen from preliminary studies evaluating optimal dosages of Lacripep and were kept constant in subsequent functional assays. The area that was devoid of cells was imaged under the confocal microscope at 0 and 13 h post-treatment and analyzed using ZEN2. Cells treated with basal media and complete media served as negative and positive controls, respectively. Identical sets of HCE-T cells were also prepared to measure cell motility upon contact lens-mediated delivery of LP-A96. Contact lenses incubated with 400 µg of either LP-A96 or A96 in dPBS (or 10 µM in 1 mL) for 24 h at room temperature under constant agitation were briefly washed in dPBS and transferred to cultures. After 24 h incubation at 37 ◦C, the cell monolayer was imaged to measure cell motility and cell culture media was collected and processed for exosome purification. For cell cytotoxicity/proliferation upon LP-A96 treatment, cells were seeded with basal media at 0.1 x 104 cells/well in 96-well plate one day prior to the experiment. On the next day, cells were washed with dPBS and incubated with 1 µM Lacripep, A96, or LP-A96 in basal media. 50 µM Digitonin (#D141, Sigma-Aldrich, St. Louis, MO, USA) was used as a cytotoxic agent. At 24, 48, and 72 h post-treatment, cell proliferation was measured using WST-1 reagent (#5015944001, Sigma-Aldrich, St. Louis, MO, USA) per manufacturer’s protocol.

4.4. Confocal Fluoresence Imaging For calcium signaling, cells at 50% confluency in 35 mm glass-bottom culture dishes were further cultured with basal media for 24 h. Cells were rinsed with dPBS and incubated at room temperature for 20 min with fresh basal media supplemented with 2.5 µM Fluo-4 AM (#F14201, Invitrogen, Carlsbad, CA, USA). After this period, the NaCl Ringer buffer (145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM KH2PO4, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES, osmolarity 300, pH 7.4) was used to rinse and incubate cells at room temperature for another 30 min. After this period, NaCl Ringer buffer was changed to Ca2+ deprived NaCl Ringer buffer (1 mM Ca2+ was replaced with 0.5 mM EGTA) and incubated for 10 min. After this period, cells were excited at 488 nm and their emission was recorded in real-time at 510 nm under the confocal microscope. The fluorescent intensity profile was recorded upon addition of 1 µM Lacripep, A96, or LP-A96. The fluorescence profile from each cell was converted Int. J. Mol. Sci. 2020, 21, 6157 16 of 22

to fold-change using Equation (2). F0 is the average fluorescence intensity measured during the first 5 min (before the addition of the treatment) and Ft is the measured fluorescence intensity at every sec.

(Ft F0) Fold_change = − . (4) F0

To observe cellular uptake of LP-A96, cells were cultured in 35 mm glass-bottom culture dishes (#P35G-0-10-C, MatTek Corp. Ashland, MA, USA) in 1.2 mL of basal media supplemented with 10 µL of either DMSO, 1 mM amiloride (#A7410, Sigma-Aldrich, St. Louis, MO, USA), or 80 µM dynasore (#D7693, Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 ◦C. Cells were washed with dPBS twice and then incubated at 37 ◦C for 10 min with 50 µL of solution that was comprised of 1 µL of NucBlueTM Live Cell Stain ReadyProbesTM reagent (#R37605, Molecular Probes, Eugene, OR, USA), 0.5 µL of rhodamine-labeled LP-A96 (1 µM final concentration), and 48.5 µL of basal media. After a 10 min incubation period, cells were washed with dPBS twice and incubated with Live Cell Imaging Solution (#A14291DJ, Molecular Probes, Eugene, OR, USA). The fluorescence was imaged under the confocal microscope and the intensity was analyzed using ZEN2. To assay for cell viability upon dynasore treatment, cells cultured in 35 mm glass-bottom culture dishes with either complete medium, 80 µM dynasore, or 50 µM digitonin for 1 h were incubated with annexin V-FITC and propidium iodide (#V13242, Thermo Fisher, Waltham, MA, USA) and imaged per manufacturer’s recommendation. To colocalize LP-A96 and syndecan-1, 1 µM rhodamine-labeled LP-A96 in 400 µL basal media was incubated with cells at 37 ◦C for 20 min and fixed/permeabilized with ice-cold methanol:acetone (1:1) mixture for 10 min at 20 C, and then processed for blocking (1% BSA), anti-Syndecan-1 antibody − ◦ incubation (1:30 dilution, #SAB1305542, Sigma-Aldrich, St. Louis, MO, USA), and secondary antibody incubation (1:200 dilution, #A21202, Invitrogen, NY, USA). Nuclei were stained with DAPI solution (1:500 dilution, #62248, Thermo Fisher, Waltham, MA, USA) during secondary antibody incubation. Cells were imaged using a confocal microscope and analyzed with ZEN2.

4.5. Exosome Purification and Analysis Cells were seeded into 12-well plates at a density of 0.5 105 cells/well. 1 µM of either Lacripep, × A96, or LP-A96 were added during seeding. Cells seeded with dPBS or complete medium served as negative and positive controls, respectively. Cells were then incubated at 37 ◦C for 72 h, undisturbed. After this period, culture media were collected and subjected to exosome purification. After removal of culture media, cells were immediately collected in 50 µL radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail (#78430, Thermo Fisher, Waltham, MA, USA) to measure total protein concentration. To purify exosomes, collected culture media under each condition was cleared of cell debris and microparticles. To do this, the media was spun down at 300 g for 5 min. Collected supernatant after this centrifugation was then spun down at 2000 g for 10 min. Collected supernatant after this × centrifugation was then spun down at 10,000 g for 30 min. Collected supernatants after these three × centrifugations were concentrated and subjected to column purification (#qEVoriginal/70 nm, iZon Sciences, Medford, MA, USA) that is optimized for exosome purification. The purified exosomes were analyzed for the amount and the size using ZetaView® nanoparticle tracking analyzer (PMX-120, Particle Metrix GmbH, Meerbusch, Germany). The number of exosomes was normalized to the protein concentration in cell lysates measured using the Micro BCA™ Protein Assay Kit (#23235, Thermo Fisher, Waltham, MA, USA). Exosomes in 0.1X dPBS were used for zeta potential analysis in ZetaView®. Exosomes were resolved by SDS-PAGE and blotted with antibodies to CD9 (1:250 dilution, #MA1-80307, Invitrogen, NY, USA), TSG101 (1:500 dilution, #ab3071, Abcam, Cambridge, MA, USA) and Alix (1:500 dilution, #2171, Cell Signaling Technology, Danvers, MA, USA). Primary antibodies were incubated overnight at 4 ◦C. Donkey anti-mouse (#925-68072, 1:5000 dilution) and goat anti-rabbit Int. J. Mol. Sci. 2020, 21, 6157 17 of 22

(#925-32211, 1:5000 dilution) secondary antibodies were purchased from LI-COR (Lincoln, NE, USA) for fluorescence imaging. Exosomal RNAs were isolated using miRNeasy Serum/Plasma Kit (#217184, Qiagen, Hilden, Germany) and analyzed with 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA).

4.6. Adsorption and Release Kinetics of LP-A96 from Contact Lenses Commercially available ProclearTM 1 Day disposable contact lenses (CooperVision, Inc., Lake Forest, CA, USA) were washed three times with dPBS prior to any studies. To measure the concentration-dependent adsorption of LP-A96 to the contact lenses, excised contact lens pieces (5 mm 5 mm, about 1~2 mg in weight) were incubated with 100 µL of fluorescein-labeled LP-A96 × (#46410, Thermo Fisher, Waltham, MA, USA) for 24 h. After the incubation, contact lens pieces were gently and briefly washed three times in dPBS to remove any unbound material and the fluorescence intensity was measured in 96-well plate using Synergy H1 Hybrid Multi-Mode Reader (BioTek Instruments, Inc., VT, USA). The data were fit with the Langmuir isotherm model (Equation (3)). The Qe (mg/g) and Ce (mg/mL) are the amount of adsorbed protein per gram of contact lens and protein concentration at equilibrium, respectively. The Qm (mg/g) is the maximum amount of protein adsorbed per gram of contact lens. The Kd (mg/L) is an equilibrium binding constant.

Ce Qm Qe = × . (5) Ce + Kd

To measure the time-dependent adsorption of LP-A96 to the contact lens, the fluorescence intensity of excised contact lens pieces that were incubated in 100 µL LP-A96 (2 mg/mL) was retrieved at pre-determined time points, gently and briefly washed three times in dPBS to remove any unbound material, and then measured in 96-well plate using Synergy H1 Hybrid Multi-Mode Reader. The acquired data were fit with the two-phase association model. To determine the release profile of LP-A96 from the contact lenses, intact contact lenses were incubated with 1 mL of fluorescein-labeled LP-A96 (50 µM) overnight at room temperature in a 24-well plate. On the next day, the contact lens was gently and briefly washed three times in dPBS to remove any unbound material and then placed in 4 mL dPBS (pH 7.4) inside a 50 mL conical tube under constant agitation (70 rpm, 37 ◦C). During the first 24 h, 100 µL dPBS was sampled at predetermined intervals. The dPBS was replaced completely with fresh dPBS after 24 h and for every 24 h thereafter. Collected samples were stored at 4 ◦C until further analysis. The amount of fluorescein released into the dPBS and the remaining fluorescence intensity on the contact lens after 96 h were measured using a Synergy H1 Hybrid Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA; Ex: 485 nm/Em: 528 nm). The acquired data were fit the two-phase dissociation model.

Author Contributions: Conceptualization, J.A.M. and S.F.H.-A.; methodology, J.A.M. and S.F.H.-A.; software, C.L.; validation, C.L., M.C.E., J.A.M., and S.F.H.-A.; formal analysis, C.L., M.C.E.; investigation, J.A.M. and S.F.H.-A.; resources, G.W.L., J.A.M. and S.F.H.-A.; data curation, C.L., M.C.E.; writing—original draft preparation, C.L.; writing—review and editing, M.C.E., G.W.L., J.A.M. and S.F.H.-A.; visualization, C.L.; supervision, M.C.E., J.A.M. and S.F.H.-A.; project administration, S.F.H.-A.; funding acquisition, G.W.L., J.A.M. and S.F.H.-A. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by R01 EY026635 to SHA and JAM, EY 011386 to SHA, GM 114839 to JAM, P30 EY029220 to the USC Department of Ophthalmology, CA014089 to the USC Norris Comprehensive Cancer Center, Gavin S. Herbert Endowed Chair of Pharmaceutical Sciences, and an unrestricted grant from Research to Prevent Blindness (RPB) to the USC Department of Ophthalmology. Acknowledgments: The authors would like to thank Junji Watanabe at the University of Southern California (USC) School of Pharmacy Translational Research Core, Shuxing Li at the Center for Excellence in NanoBiophysics in the USC Dornsife College of Letter, Arts and Sciences, and Alan Epstein at the USC Keck School of Medicine for providing the anti-ELP antibody, AK1. The authors also thank Jeff Romano at the University of Virginia in the Laurie Laboratory for the assistance. Conflicts of Interest: G.W.L. holds patents or patents pending on the use of lacritin for treating dry eye, and is cofounder of TearSolutions, Inc., that is currently testing the efficacy of a lacritin synthetic peptide for Sjogren’s Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 17 of 21 Int. J. Mol. Sci. 2020, 21, 6157 18 of 22 Conflicts of Interest: G.W.L. holds patents or patents pending on the use of lacritin for treating dry eye, and is cofounder of TearSolutions, Inc., that is currently testing the efficacy of a lacritin synthetic peptide for Sjogren’s syndromesyndrome drydry eyeeye inin aa phasephase 22 clinicalclinical trial.trial. J.A.M. is an inventor on a patent related to lacritin-ELP technology. TheThe fundersfunders hadhad nono rolerole inin thethe designdesign ofof thethe study;study; inin thethe collection,collection, analyses,analyses, oror interpretationinterpretation ofof data;data; inin thethe writing of the manuscript, or in the decision to publish the results. writing of the manuscript, or in the decision to publish the results. Appendix A Appendix A

10 3 100 2 10 LP-A96 A96

A Molar (g/mol) Mass B C 5 µM 25 µM 90 108 10 µM 50 µM 20 µM 100 µM 80 A96 2 40 µM 200 µM 6 10 70 LP-A96 1 104 60 1 50 102 40 Relative UV (280 nm) scale (280 nm) UV Relative Optical density at 350nm 0 100 0 Transition temperature (°C) 04812 30 25 50 75 100 1 10 100 1000 Volume (mL) Temperature (oC) Concentration (µM) D 1

2 3

FigureFigure A1.A1.Biophysical Biophysical characteristics characteristics of LP-A96. of LP-A96. (A) LP-A96 (A elutes) LP-A96 from sizeelutes exclusion from chromatographysize exclusion followedchromatography by multiangle followed light by scattering multiangle (SEC-MALS) light scattering as a single (SEC-MALS) peak (solid as line a accordingsingle peak to left(solidY-axis) line consistentaccording withto left a Y nanoparticle-axis) consistent with with a radius a nanoparticle of gyration with of 79.3a radius nm (redof gyration dotted of line 79.3 according nm (red dotted to the rightline accordingY-axis). (B to) Thethe right ELP-mediated Y-axis). (B phase) The ELP-mediated separation is monitored phase separation using optical is monitored density using at 350 optical nm as adensity function at of350 temperature nm as a function using UV–Visof temper spectrophotometryature using UV–Vis (10 spectrophotometryµM in dPBS). ELP phase(10 μM separation in dPBS). increasesELP phase the separation optical density. increases Transition the optical temperatures density. were Transition identified temperatures using Equation were (4). identified (C) A log-linear using relationshipEquation (4). between (C) A log-linear transition relationship temperature between and concentration transition temperature was fit using and Equation concentration (3). Parameters was fit ofusing the fitEquation are reported (3). Parameters in Table A1of .the Dotted fit are lines reported indicate in Table 95% A1. CI. (DottedD) MALDI-TOF-MS lines indicate 95% analysis CI. (D of) LP-A96.MALDI-TOF-MS The molecular analysis weight of LP-A96. of 39,521 The Damolecular (black arrowweight 1) of is 39,521 consistent Da (black with arrow expected 1) is molecularconsistent weightwith expected reported molecular in Table1. Minorweight species reported identified in Table at 79,1871. Minor Da species (black arrowidentified 2) and at 118,797 79,187 DaDa (black(black arrowarrow 3)2) areand consistent 118,797 Da with (black the arrow masses 3) expected are consiste fornt transglutaminase-mediated with the masses expected for dimers transglutaminase- and timers of LP-A96mediated [8]. dimers and timers of LP-A96 [8]. Table A1. Parameters of LP-A96 and A96 phase separation. Table A1. Parameters of LP-A96 and A96 phase separation. Phase Diagram a Proteins Phase Diagram a Slope, m [◦C/log10(µM)] y-intercept, b [◦C] Proteins Slope, m [°C/log10(μM)] y-intercept, b [°C] (Mean [95% CI]) (Mean [95% CI]) (Mean [95% CI]) (Mean [95% CI]) 8.2 58.3 LP-A96 −8.2 − 58.3 [ 10.7– 5.7] [55.3–61.3] LP-A96 − − [−10.7–−5.7] 21.7 [55.3119.5–61.3] A96 − −21.7[ 29.0– 14.4] [105.9–133.2]119.5 A96 − − [−29.0–a −Fit14.4] according to Equation (3). [105.9–133.2] a Fit according to Equation (3).

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Figure A2. Western blot-based densitometric analysis for residual LP-A96 in purified EV samples. FigurePurifiedFigure A2.A2. EVs WesternWestern from three blot-basedblot-based independ densitometricdensitometricent experiments analysisanalysis (Experiments forfor residualresidual #1~3, LP-A96LP-A96 lane 1~3) inin were purifiedpurified blotted EVEV samples.forsamples. ELPs PurifiedusingPurified anti-ELP EVs EVs from from antibody three three independent independAK1 [30] toent detect experiments experiments and qu (Experimentsantify (Experiments residual #1~3, #1~3, LP-A96 lane lane co 1~3) 1~3)-purified werewereblotted blottedwith exosomes.for forELPs ELPs usingTousing determine anti-ELPanti-ELP this, antibodyantibody firstly, AK1particleAK1 [[30]30 ]concentration toto detectdetect andand of quantifyqu pureantify LP-A96 residualresidual was LP-A96LP-A96 determined co-purifiedco-purified (particle withwith number/mL) exosomes.exosomes. TousingTo determine determine nanoparticle this, this, firstly, firstly,tracking particle particle analyz concentration concentrationer. Secondly, of ofserial pure pure LP-A96dilutions LP-A96 was was of determinedLP-A96 determined were (particle (particle prepared number number/mL) and their/mL) usingparticleusing nanoparticlenanoparticle concentration trackingtracking was correlated analyzer.analyzer. to Secondly,Secondly, the band serial serialintensity dilutionsdilutions in Western ofof LP-A96LP-A96 blot (ELP werewere Standard preparedprepared #1~3, andand theirlanetheir particle4~6).particle Lastly, concentrationconcentration the standard waswas curve correlatedcorrelated generated toto thethe based bandband on intensityin tensitythis correlationin inWestern Western was blot blot used (ELP (ELP toStandard extrapolateStandard #1~3, #1~3, thelane laneLP- 4~6).A964~6). particle Lastly,Lastly, the amountthe standard standard co-purified curve curve generated generatedin EV samples based based on after on this this LP-A96 correlation correlation treatment was was used (laneused to extrapolate 1~3).to extrapolate This thecalculation LP-A96the LP- particlewasA96 used particle amount to reflect amount co-purified the co-purified raw increase in EV in samples in EV exosomes samples after in LP-A96after Figure LP-A96 treatment 3B. treatment (lane 1~3).(lane This1~3). calculationThis calculation was usedwas used to reflect to reflect the raw the increase raw increase in exosomes in exosomes in Figure in Figure3B. 3B. ABCComplete media Dynasore (80 µM) Digitonin (50 µM) ABCComplete media Dynasore (80 µM) Digitonin (50 µM) Annexin V-FITC Annexin Propidium iodide Propidium Annexin V-FITC Annexin Propidium iodide Propidium

D E F 4 G ns D E F 4 G ns 3 **** **** 3 **** **** 2

2 (fold-change) 1 (fold-change) Non-Treat (DMSO) Dynasore (80 µM) Dynasore (80 µM) 1 ↓ cell per Intensity Fluorescence Non-Treat (DMSO) Dynasore (80 µM) Dynasore (80 µM) 0 O e ➝ Remove dynasore and cell per Intensity Fluorescence S or e ↓ 0DM as or ut incubate in blank yn as o O D e yn sh ➝ Remove dynasore and S or D wa e basal media for 4 hr DM as r or ut yn h as o incubate in blank D 4 n sh Dy a basal media for 4 hr r w 4 h Figure A3. Dynasore inhibits endocytosis of LP-A96 without cytotoxicity. ( A–CA–C) Cell viability under eachFigure treatmenttreatment A3. Dynasore waswas visualized visualized inhibits usingendocytosis using annexin annexin of V LP-A96 andV an propidiumd propidium without iodide. cytotoxicity. iodide. Cells Cells were (A–C were incubated) Cell incubated viability for 1 for h under with 1 h µ µ completewitheach completetreatment media, media,was 80 visualizedM 80 dynasore, μM dynasore, using or 50 annexin Mor digitonin. 50 V μ anMd digitonin. propidium Cells treated Cells iodide. with treated dynasoreCells with were diddynasore incubated not experience did for not 1 h cellexperiencewith death. complete (cellD–F death. )media, Endocytosis (D–F 80 )μ EndocytosisM of dynasore, LP-A96 is of restoredor LP-A96 50 μM afteris restoreddigitonin. dynasore after Cells has dynasore beentreated removed. has with been dynasore Cells removed. pre-treated did Cells not µ withpre-treatedexperience (D) DMSO withcell death. ( orD) ( DMSOE )(D–F dynasore )or Endocytosis (E) weredynasore further of wereLP-A96 incubated further is restored incubated with after 1 M with dynasore rhodamine-labeled 1 μM hasrhodamine-labeled been removed. LP-A96 Cells LP- for F 15A96pre-treated min for and 15 min imaged. with and (D )imaged. ( DMSO) Cells or( incubatedF )( ECells) dynasore incubated for additional were for further additional 4 h incubated in basal 4 h media in with basal after1 μmediaM dynasore rhodamine-labeled after dynasore was removed was LP- were further incubated with 1 µM rhodamine-labeled LP-A96 for 15 min and imaged. (G) Fluorescence removedA96 for 15 were min further and imaged. incubated (F) Cells with incubated1 μM rhodamine-labeled for additional 4 LP-A96h in basal for media 15 min after and dynasore imaged. (wasG) intensity changes show restoration of LP-A96 endocytosis after 4 h. **** p < 0.0001, ns: non-significant. Fluorescenceremoved were intensity further changesincubated show with restorat 1 μM ionrhodamine-labeled of LP-A96 endocytosis LP-A96 afterfor 15 4 h.min **** and p

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AB12 4 ** 10 *** 3 8 )/µg protein 8 )/µg protein/hr

6 7 2

4 1 2 Particles (x10

0 (x10 Particles 0 0~36 0~72 0~36 0~72 Incubation hour Incubation hour

Figure A4. Time-dependent differential release rate of exosomes from HCE-T cells. (A) Exosome amountsFigure A4. in cultureTime-dependent media were differential quantified release and ( Brate) the of rate exosomes of release from was HCE-T calculated cells. at (A 36) Exosome and 72 h post-treatment.amounts in culture HCE-T media cells were were quantified treated with and 1 (µBM) the LP-A96. rate of The release total was amount calculated of exosomes at 36 and and 72 the h ratespost-treatment. of exosome HCE-T biogenesis cells/ releasewere treated were higher with 1 during μM LP-A96. the 0~36 The h periodtotal amount compared of exosomes to 0~72 h and period. the Thisrates suggestsof exosome the biogenesis/release rate of exosome uptakewere higher/turnover during exceeds the 0~36 the h rate period of exosome compared biogenesis to 0~72 h/ releaseperiod. duringThis suggests 36~72 hthe period rate asof cellsexosome reach uptake/turnover confluence. *** p exceeds< 0.001, **thep rate< 0.01. of MeanexosomeSD. biogenesis/release during 36~72 h period as cells reach confluence. *** p < 0.001, ** p < 0.01. Mean ±± SD. References References 1. Dartt, D.A. Neural regulation of lacrimal gland secretory processes: Relevance in dry eye diseases. Prog. Retin. 1. EyeDartt, Res. D.A.2009 Neural, 28, 155–177. regulation [CrossRef of lacrimal][PubMed gland] secretory processes: relevance in dry eye diseases. Prog 2. Laurie,Retin Eye G.W.; Res. Olsakovsky,2009, 28, 155–177, L.A.; Conway, doi:10.1016/j.preteyeres.2009.04.003. B.P.; McKown, R.L.; Kitagawa, K.; Nichols, J.J. Dry eye and designer 2. ophthalmics.Laurie, G.W.;Optom. Olsakovsky, Vis. Sci. L.A.;2008 Conway,, 85, 643–652. B.P.; [CrossRefMcKown,][ R.L.;PubMed Kitagawa,] K.; Nichols, J.J. Dry eye and 3. Sanghi,designer S.; ophthalmics. Kumar, R.; Optom. Lumsden, Vis. A.;Sci. Dickinson,2008, 85, 643–652, D.; Klepeis, doi:10.1097/OPX.0b013e318181ae73. V.; Trinkaus-Randall, V.; Frierson, H.F., Jr.; 3. 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