Drug-Loaded Hydrogels for Intraocular Lenses with Prophylactic Action against Pseudophakic Cystoid Macular Edema

pha maceu ics A icle D ug-Loaded Hyd ogels o In aocula Lenses wi h P ophylac ic Ac ion agains Pseudophakic Cys oid Macula Edema Nadia To ole o 1,* , Madalena Salema-Oom 2, Soledad Anguiano Igea 3, Ca men Al a ez-Lo enzo 4, Benilde Sa amago 1and Ana Paula Se o 1,2   Ci a ion: To ole o, N.; Salema-Oom, M.; Anguiano Igea, S.; Al a ez-Lo enzo, C.; Sa amago, B.; Se o, A.P. D ug-Loaded Hyd ogels o In aocula Lenses wi h P ophylac ic Ac ion agains Pseudophakic Cys oid Macula Edema. Pha maceu ics 2021, 13, 976. h ps://doi.o g/10.3390/ pha maceu ics13070976 Academic Edi o s: Lau ence Fi zhen y and Rocio He e o-Van ell Recei ed: 7 June 2021 Accep ed: 24 June 2021 Published: 28 June 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Cen o de Química Es u u al, Ins i u o Supe io Técnico, Uni e si y o Lisbon, A enue Ro isco Pais, 1049-001 Lisbon, Po ugal; b.sa amago@ ecnico.ulisboa.p (B.S.); anapaula.se o@ ecnico.ulisboa.p (A.P.S.) 2Cen o de In es igação In e disciplina Egas Moniz, Ins i u o Uni e si á io Egas Moniz, Quin a da G anja, Mon e de Capa ica, 2829-511 Capa ica, Po ugal; [email p o ec ed] 3HGBeyond Ma e ials Science S.L., Edi icio EMPRENDIA, 15782 San iago de Compos ela, Spain; esea [email p o ec ed] 4Depa amen o de Fa macología, Fa macia y Tecnología Fa macéu ica, I + D Fa ma (GI-1645), Facul ad de Fa macia and Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS), Uni e sidade de San iago de Compos ela, 15782 San iago de Compos ela, Spain; ca men.al a ez.lo [email p o ec ed] *Co espondence: nadia. o [email p o ec ed] Abs ac : Pseudophakic cys oid macula edema (PCME), caused by ch onic in lamma ion, is he mos common cause o isual impai men in he medium- e m a e ca a ac su ge y. The e o e, he p ophylac ic opical adminis a ion o combined s e oidal and non-s e oidal an i-in lamma o y d ugs is commonly done. D ug-elu ing in aocula lenses (IOLs) gained in e es as an e icien way o o e come he compliance issues ela ed o he use o ocula d ops wi hou he need o addi ional su gical s eps. The inco po a ion o unc ional monome s and molecula imp in ing we e he ein applied o design hyd ogels sui able as IOLs and able o co-deli e s e oidal (dexame hasone sodium phospha e) and non-s e oidal (b om enac sodium) d ugs. The inco po a ion o N-(2-aminop opyl) me hac ylamide (APMA) inc eased he d ug up ake and imp o ed he in i o elease kine ics. Im- p in ing wi h b om enac esul ed in a dec eased d ug elease due o pe manen d ug bonding, while imp in ing wi h dexame hasone inc eased he amoun o dexame hasone eleased a e dual-d ug loading. The applica ion o a ma hema ical model o p edic he in i o d ug elease beha io sug- ges s he easibili y o achie ing he apeu ic d ug concen a ions o b om enac and dexame hasone in he aqueous humo o abou 2 and 8 weeks, espec i ely, which is compa ible wi h he cu en opical p ophylaxis a e ca a ac su ge y. Keywo ds: he apeu ic oph halmic lenses; pos e io segmen diseases; d ug elease; an i-in lamma o y d ug; molecula imp in ing; unc ionalized hyd ogels 1. In oduc ion Ca a ac s a e he leading cause o ision loss in he elde ly popula ion wo ldwide [ 1 , 2 ]. The pa hology consis s in he opaci ica ion o he na u al c ys alline lens and is commonly ea ed by su gical eplacemen o he damaged c ys alline wi h an a i icial in aocula lens (IOL). Al hough ca a ac su ge y is cu en ly one o he mos cos -e ec i e p ocedu es in heal hca e [ 3 ], some pos -su gical complica ions may occu . The mos common cause o isual impai men in he medium- e m a e IOL implan is pseudophakic cys oid macula edema (PCME), also called I ine-Gass synd ome [ 4 , 5 ]. PCME is caused by he p esence o ch onic in lamma ion, and i a ec s up o 3.6% o ca a ac pa ien s in i s symp oma ic acu e o m [ 6 ]. I s incidence inc eases wi h he concomi ance o p e- exis ing isk ac o s, such as con ala e al PCME, diabe es melli us, u ei is, his o y o e inal ein occlusion and e inal degene a ion, macula degene a ion, e inopa hy, epi e inal Pha maceu ics 2021,13, 976. h ps://doi.o g/10.3390/pha maceu ics13070976 h ps://www.mdpi.com/jou nal/pha maceu ics Pha maceu ics 2021,13, 976 2 o 20 memb anes, and he use o p os aglandins [ 6 ]. In mos cases, PCME can be sol ed wi h adequa e pha macological ea men . Howe e , in non- esponding pa ien s, i may become a se ious issue and lead o pe manen ision loss [ 4 , 5 , 7 ]. Fo his eason, he p ophylac ic opical adminis a ion o an i-in lamma o y d ugs is commonly done a e su ge y [ 8 ]. As mos ca a ac pa ien s a e elde ly people, p esc ip ion o ocula d ops may aise compliance issues. Mo eo e , due o he low pene a ion o d ugs h ough he co neal epi helium and he impo an d ug loss by lac ima ion, o he me hods a e needed o he in aocula deli e y o less pe mea i e d ugs [ 9 ]. In his con ex , he de elopmen o d ug-loaded IOLs would cons i u e an e icien way o adminis e d ugs wi hou he need o addi ional s eps du ing su ge y o du ing pa ien eco e y. These de ices a e no ye p esen on he ma ke , al hough hei de elopmen has been pu sued by se e al esea che s [ 10 ]: he apeu ic IOLs ha e been designed o he sus ained elease o an ibio ics [ 11 – 15 ], an i- in lamma o y d ugs [ 13 , 14 , 16 , 17 ], immunosupp essan s [ 18 ] and an i-p oli e a i e d ugs o he p e en ion o he pos e io capsule opaci ica ion (PCO) [19–23]. As PCME p esen s a peak o incidence a 4–6 weeks a e su ge y, he adminis a ion o an i-in lamma o y d ugs is usually p esc ibed o one mon h [ 6 , 8 ]. Topical s e oids we e conside ed he classic p ophylaxis o PCME in ca a ac pa ien s [ 24 ]. Howe e , highe e icacy has been epo ed o he combined use wi h opical non-s e oidal an i- in lamma o y d ugs (NSAIDs) [ 4 ]. Indeed, co-adminis a ion o NSAIDs (e.g., nepa enac o b om enac) and co icos e oids (e.g., dexame hasone sodium phospha e) has been shown o lowe he incidence o PCME [ 24 ]. In he ESCRS PREMED s udy, an in e na ional andomized con olled clinical ial [ 11 ], he use o opical b om enac, dexame hasone sodium phospha e o a combined he apy was in es iga ed, and again he incidence o PCME was lowe in he case o he combined ea men . Se e al s a egies ha e been sugges ed in he las decade o une d ug elease om ocula de ices. Molecula imp in ing is an inno a i e echnique ha has been demon- s a ed o p olong d ug elease om se e al he apeu ic oph halmic lenses [ 25 – 28 ]. I consis s o he non-co alen associa ion be ween a d ug, ac ing as a empla e du ing he polyme iza ion s ep and unc ional monome s inco po a ed in o he polyme ic ma ix. A e polyme iza ion and d ug emo al, ailo ed ac i e si es emain imp in ed in he polyme , gene a ing an o ien ed s uc u e wi h a high a ini y o he empla e d ug. The d ug can be e-loaded in o he polyme by soaking, and, due o he in e ac ion wi h he o ien ed unc ional monome s, a highe d ug loading and a slowe elease p o ile can be achie ed. The ype and concen a ion o he unc ional monome s une he d ug a ini y o he polyme and he elease kine ics. The e o e, op imiza ion is necessa y o each combina ion o d ug and monome [29,30]. The aim o he p esen wo k is o design unc ionalized and/o imp in ed d ug-loaded hyd ogels o he p e en ion o PCME. To ou knowledge, he de elopmen o dual-loaded IOLs, able o simul aneously deli e s e oidal and non-s e oidal an i-in lamma o y d ugs, is s ill an unme medical need. Two hyd ophilic d ugs we e selec ed o hei p o en e icacy in he p e en ion o PCME: b om enac sodium, an NSAID, and dexame hasone sodium phospha e, a co icos e oid [31,32]. Two hyd ophilic monome s commonly ound in comme cial IOLs we e selec ed o p oduce he hyd ogel: HEMA (2-hyd oxye hyl me hac yla e), a componen o ac ylic IOLs, and BEM (2-bu oxye hyl me hac yla e), which is known o inc ease he ee- olume o he copolyme and he IOL lexibili y, hus equi es a smalle incision du ing implan su ge y [ 33 ]. Func ionaliza ion was done wi h wo monome s: APMA (N-(2-aminop opyl) me hac ylamide) and AAm (ac ylamide), conside ed as po en ially sui able due o he in e ac ion be ween he posi i e cha ge ha hey acqui e in aqueous solu ion and he nega i e cha ge o he selec ed d ugs in solu ion, a pH 7.4. The molecula s uc u es o he d ugs and he monome s a e p esen ed in Figu e 1. Pha maceu ics 2021,13, 976 3 o 20 Figu e 1. Molecula s uc u e o he wo selec ed d ugs (b om enac sodium and dexame hasone sodium), he hyd ogel backbone monome s (HEMA and BEM) and he unc ional monome s (APMA and AAm). 2. Ma e ials and Me hods 2.1. Ma e ials Dexame hasone sodium phospha e (CAS 2392-39-4) and b om enac sodium (CAS 91714-93-1) we e pu chased om Ca bosyn h (Comp on, UK). EGDMA (CAS 97-90-5), AIBN (CAS 78-67-1), HEMA (CAS 868-77-9) and AAm (CAS 79-06-1) we e pu chased om Sigma-Ald ich (S einheim, Ge many). BEM (CAS 13532-94-0) was pu chased om abc (Ka ls uhe, Ge many) and APMA om PolySciences (Eppelheim, Ge many). Dis illed and deionized wa e (18 M Ω cm, pH7.7) was ob ained om a Millipo e sys em. Me hanol HPLC g ade (CAS 67-56-1) was pu chased om ChemLab (Zedelgem, Belgium) and ace oni ile HPLC g ade (CAS 75-05-8) om Ca lo E ba Reagen s (Val-de-Reuil, F ance). Phospha e bu e saline (PBS), pH 7.4, was pu chased om Sigma–Ald ich (Da ms ad , Ge many). Phospha e bu e (pH 6) was p epa ed wi h he ollowing composi ion: NaOH 1.15 mM (VWR—Leu en, Belgium) and KH 2 PO 4 10 mM (ITW Reagen s—Ba celona, Spain). Po cine eyes, p o ided by a local slaugh e house (Compos elana de Ca nes S.L.—San iago de Compos ela, Spain), we e imme sed in PBS solu ion, anspo ed in an ice ba h and used wi hin h ee hou s a e collec ion. Human lens epi helial cells (ATCC-CRL-11421 1 B-3) we e pu chased om LCG S anda ds (Ba celona, Spain). Dulbecco’s Modi ied Eagle’s Medium (DMEM), e al bo ine se um, penicillin, s ep omycin, yellow e azolium (3-(4,5- dime hyl hiazolyl-2)-2,5-diphenyl e azolium b omide) (MTT), dime hyl sul oxide (DMSO, ≥ 99%), isop opanol and hyd ochlo ic acid we e pu chased om Sigma-Ald ich (S einheim, Ge many). IGEPAL®was pu chased om Me ck (Da ms ad , Ge many). 2.2. Molecula In e ac ion Analysis The modeling o he d ug-monome in e ac ion was pe o med o selec componen s po en ially sui able o he d ug- eleasing hyd ogels, using he Au oDock Tools 1.5.6 so - wa e (MGL Tools, Sc ipps Resea ch, La Jolla, CA, USA). The 3D model o he molecules was ob ained om he PubChem da abase [ 34 ] and iles we e con e ed wi h OpenBabel GUI Pha maceu ics 2021,13, 976 4 o 20 so wa e (OpenEye Scien i ic, San a Fe, NM, USA). Then, each monome -d ug couple was analyzed. B ie ly, he d ug molecule was selec ed as ‘mac omolecule’, and he monome as ‘ligand’. The g id was gene a ed wi h de aul se ings. Molecula docking was pe o med h ough he Lama ckian gene ic algo i hm. The ob ained con o ma ions we e anked by ene gy, and he lowes ene gy con o ma ion was chosen as a esul . All o sions we e allowed o o a e du ing docking. The ee ene gy o in e ac ion, E, and he dissocia ion cons an , Ki, we e ob ained as ou pu [35]. 2.3. Hyd ogels Syn hesis Nine di e en hyd ogels we e p epa ed, as summa ized in Table 1. G oup A co - esponds o non-imp in ed hyd ogels, g oup B o b om enac-imp in ed hyd ogels and g oup C o dexame hasone-imp in ed hyd ogels. Fo each g oup, h ee di e en monome composi ions we e es ed o in es iga e he in luence o APMA and AAm as unc ional monome s. Table 1. Composi ion o he nine o mula ions es ed as po en ial IOL ma e ials. A1, A2 and A3 a e non-imp in ed hyd ogels. B1, B2, B3 a e b om enac-imp in ed hyd ogels. C1, C2 and C3 a e dexame hasone-imp in ed hyd ogels. Code HEMA BEM APMA AAm B om enac Sodium Dexame hasone Sodium AIBN EGDMA A1 2.4 mL 600 µL 10 mM 80 mM A2 2.4 mL 600 µL 100 mM 10 mM 80 mM A3 2.4 mL 600 µL 100 mM 10 mM 80 mM B1 2.4 mL 600 µL 25 mM 10 mM 80 mM B2 2.4 mL 600 µL 100 mM 25 mM 10 mM 80 mM B3 2.4 mL 600 µL 100 mM 25mM 10 mM 80 mM C1 2.4 mL 600 µL 12.5 mM 10 mM 80 mM C2 2.4 mL 600 µL 100 mM 12.5 mM 10 mM 80 mM C3 2.4 mL 600 µL 100 mM 12.5 MM 10 mM 80 mM The monome mix u es (3 mL) we e s i ed o 24 h a oom empe a u e. Then, he solu ions we e injec ed in o molds made o silanized glass wi h a 0.5 mm silicone ubbe sepa a o . The mal polyme iza ion was pe o med h ough a i s s ep incuba ion a 50 ◦ C o 12 h, ollowed by a second s ep a 70 ◦ C o 24 h. A e polyme iza ion, he hyd ogels we e boiled o 15 min in 900 mL o dis illed wa e . Then, he ob ained polyme shee s we e cu in o 6 mm discs using a biopsy punche o esemble he size o comme cial IOLs and subjec ed o an addi ional washing s ep o emo e he un eac ed monome s and he d ug molecules used as a empla e in he imp in ed ne wo ks. The washing p ocedu e was pe o med by soaking he discs unde s i ing in dis illed wa e (14 days, enewed wice a day), ollowed by soaking in e hanol (48 h), NaCl 0.9% (12 h) and dis illed wa e (48 h). Du ing he las s ep, no peaks associa ed wi h he p esence o esidual monome s o he d ug could be de ec ed by UV- is spec ome ic analysis o he washing solu ion. The ob ained discs we e hen d ied a 35 ◦ C o 72 h p io o s o age. D y discs weighed app oxima ely 13 mg. 2.4. D ug Loading and Release Single d ug loading was pe o med o e alua e he in e ac ion be ween he hyd ogels and each d ug, he in luence o he unc ional monome s and he e ec o molecula imp in ing on he elease p o iles. Non-imp in ed hyd ogels (A1, A2, A3) and b om enac- imp in ed hyd ogels (B1, B2, B3) we e loaded by soaking each d y disc (n= 3) in o 1 mL o b om enac sodium solu ion (1 mg/mL in PBS). Non-imp in ed hyd ogels (A1, A2, A3) and dexame hasone-imp in ed hyd ogels (C1, C2, C3) we e loaded by soaking each d y disc (n= 3) in o 1 mL o dexame hasone sodium solu ion (5 mg/mL in PBS). A highe concen a ion was used o dexame hasone since p elimina y s udies (da a no shown) indica ed ha he amoun o dexame hasone loaded and eleased om he hyd ogels was Pha maceu ics 2021,13, 976 5 o 20 much lowe han ha o b om enac. Soaking was pe o med a 36 ◦ C o 1 week unde agi a ion a 180 pm. A e he e alua ion o he single-d ug elease p o ile, dual d ug loading o he bes hyd ogels was pe o med by soaking each d y disc (n= 3) in o 1 mL o dual d ug solu ion (i.e., 1 mg/mL b om enac sodium + 5 mg/mL dexame hasone sodium in PBS). Soaking was done a di e en empe a u es (4 ◦ C, 36 ◦ C o 60 ◦ C) and soaking imes (1, 2 o 4 weeks) o iden i y he op imal loading condi ions. The elease p o iles o bo h single- and dual-loaded hyd ogels we e e alua ed in i o o 25 days. The d ug-loaded hyd ogels we e emo ed om he soaking solu ion, insed in dis illed wa e , and gen ly blo ed wi h abso ben pape . Then, each disc was comple ely imme sed in 3 mL o PBS and placed in a shake a 36 ◦ C and 180 pm. A each ime poin , aliquo s o 0.3 mL we e collec ed o be analyzed and we e eplaced by he same olume o esh PBS. 2.5. D ug Quan i ica ion D ug quan i ica ion was pe o med by UV-Vis spec oscopy (Mul iscanGO, The mo- Scien i ic, Po o Sal o, Po ugal) in all es s, excep when explici ly s a ed o he wise. The abso bance o b om enac sodium and dexame hasone sodium in wa e o PBS was de ec ed espec i ely a he wa eleng hs o 268 nm and 242 nm. In u n, in me hanol, d ugs we e de ec ed a 260 nm and 238 nm, espec i ely. A calib a ion cu e was ob ained o each d ug, in he a ious liquid media. To quan i y each d ug in dual-d ug solu ions, he en i e spec um was decon olu ed acco ding o he me hod p e iously de eloped by Kim and Chauhan [ 36 ], which conside ed he dual-d ug spec um as a linea combina ion o he single-d ug spec a. A leas -squa e i be ween he dual-d ug and single-d ug spec a we e applied o de e mine he concen a ion o each d ug. Due o he di icul ies encoun e ed in he applica ion o he decon olu ion me hod o low d ug concen a ions, ch oma og aphic analysis was used o quan i ica ion in he i s 48 h o dual d ug elease in i o (Sec ion 2.4) and du ing he ex- i o es ing (Sec ion 2.8). A Wa e s HPLC appa a us (Mil o d, MA, USA) wi h a UV de ec o was used. A C18 column ( 4.6 ×150 mm , ACE ® , Abe deen, UK) wi h 5 µ m po es was selec ed. The analysis was pe - o med a oom empe a u e, using as mobile phase a mix u e o 0.01 M phospha e bu e (pH 6.0) and ace oni ile (72:28 / ), wi h a low a e o 1 mL/min. Fo he in i o elease and ex- i o es ing, he selec ed injec ion olumes we e 20 µ L and 80 µ L, espec i ely. Dexame hasone sodium and b om enac sodium we e quan i ied a 242 nm and 265 nm, espec i ely, wi h a e en ion ime o 4.20 min and 14.65 min. HPLC calib a ion cu es we e alida ed o linea i y, accu acy and p ecision. All quan i ica ion measu emen s we e pe o med a leas in iplica e. 2.6. D ug Amoun Loaded To e alua e he o al amoun o d ug loaded in he bes ma e ials, hyd ogel discs (n= 3) we e insed a e loading in a dual-d ug solu ion, gen ly blo ed and placed in glass ials wi h 3 mL me hanol. Each 24 h, he ex ac ion medium was analyzed by UV- is spec oscopy and eplaced by esh me hanol. The amoun o d ug loaded was quan i ied using he p e iously desc ibed decon olu ion me hod [ 36 ] wi h calib a ion cu es o each d ug in me hanol. The analysis was epea ed un il no d ug could be de ec ed in he ex ac ion medium. The o al amoun o d ug ex ac ed co esponded o he sum o he d ug amoun s quan i ied a each measu emen . 2.7. Physical and Mechanical Cha ac e iza ion To e alua e he liquid up ake o he hyd ogels, ba e hyd ogel discs (n= 3) we e weighed a e he washing and d ying p ocedu e. Then, each disc was imme sed in 3 mL o PBS a 36 ◦ C unde agi a ion (100 pm). The weigh a ia ion was moni o ed a = 0.5, 1, 2.5, 5, 8 h and e e y 24 h un il a pla eau alue was eached. Liquid up ake was calcula ed Pha maceu ics 2021,13, 976 6 o 20 by Equa ion (1), whe e w 0 and w ep esen he weigh o he d y hyd ogel and o he hyd ogel a ime , espec i ely. Liquid up ake (%) = [(w −w0)/w0]×100 (1) The same equa ion was used o e alua e he swelling o he hyd ogels a e dual d ug loading. In his case, w o samples (n= 3) was measu ed a = 1 week o soaking in 1 mL o dual d ug solu ion (i.e., 5 mg/mL dexame hasone sodium + 1 mg/mL b om enac sodium in PBS, 180 pm, T = 36 ◦C). Hyd ogels (n ≥ 3) we e es ed o ansmi ance a e hyd a ion in PBS and a e dual d ug loading unde he p e iously de ined condi ions. T ansmi ance measu emen s we e pe o med using a UV- is spec opho ome e (Mul iscanGO, The moScien i ic, Po o Sal o, Po ugal). The wa eleng h in e al o 200–800 nm was scanned wi h 1 nm in e als. The ensile modulus o he hyd ogels was e alua ed wi h a TA.XT Exp ess Tex u e Analyze (S able Mic o Sys ems, Godalming, UK). Dog-bone shaped samples (n ≥ 5, hickness ≈ 0.5 mm, wid h ≈ 2.5 mm) we e es ed a e hyd a ion in PBS and a e dual d ug loading unde he p e iously de ined condi ions. The es was pe o med a 0.3 mm/s up o ailu e, wi h a igge o ce o 0.005 N [ 28 ]. Young’s modulus was de e mined om he ini ial slope (ε= 0–5%) o he ob ained s ess-s ain cu e. The a e age mesh size ξ o he hyd ogels was calcula ed by Equa ion (2), whe e l c−c is he leng h o he ca bon-ca bon bond in he hyd ogel backbone, C F is he Flo y cha ac e is ic a io, ρ is he d y hyd ogel densi y, M is he molecula weigh o he epea ing uni , G 0 (0) is he ze o- equency shea s o age modulus (de ined as G 0 (0) = Young’s modulus × 1/3, assuming a Poisson a io o 0.5) and ϕ is he olume ac ion o he polyme in he swollen ne wo k [ 37 ]. The alues o hose pa ame e s a e gi en in Supplemen a y Ma e ials Table S1. ξ=lc−cs2 CFρRT M G0(0)ϕ−1/6 (2) 2.8. P edic ion o In Vi o E icacy A p e iously de eloped ma hema ical model was applied o es ima e he d ug con- cen a ion in he aqueous humo o e ime, a e he hyd ogel implan a ion [ 38 ]. The inpu da a included he hickness ( ≈ 0.45 mm) and diame e ( ≈ 6 mm) o he hyd ogel discs and he loading condi ions, namely, olume (1 mL), he concen a ion o he loading solu ion (1 mg/mL b om enac sodium and 5 mg/mL dexame hasone sodium), and soaking ime ( = 1 week)). The pa i ion coe icien K be ween he hyd ogel and he elease medium, he e ec i e di usi i y D o he d ugs h ough he hyd ogel ne wo k, and he d ug loss h ough he co nea we e also equi ed o he es ima ion o he beha io o he hyd ogels in i o. K was calcula ed as he a io be ween he d ug concen a ion in he hyd ogel disc (C disc ) and in he elease medium (C medium ) a equilib ium (Equa ion (3)) [ 39 ]. Wi h his pu pose, dual-loaded A1, A2, B2 and C2 hyd ogels we e placed in 10 mL PBS and C medium was analyzed weekly un il he equilib ium was eached. Then, he d ug amoun in he hyd ogels was ex ac ed wi h me hanol as desc ibed abo e and no malized by he disc olume o ob ain Cdisc. K=Cdisc/Cmedium (3) The e ec i e di usi i y D was ob ained as p e iously desc ibed by Pimen a e al. [ 38 ]. B ie ly, conside ing he hyd ogel as a uni o m hickness ilm and applying Fick’s second law (Equa ion (4)), i is possible o ob ain a one-dimensional di usion equa ion (Equa ion (5)) which can be i ed o he in i o elease da a. C and A ep esen , espec i ely, he d ug concen a ion in he hyd ogel and i s su ace a ea, while C and V e e o he concen a ion and olume o he elease medium, espec i ely. ∂C/∂ =D(∂2C/∂y2) (4) Pha maceu ics 2021,13, 976 7 o 20 −2DA(∂C/∂y)y=h = V (∂C /∂ ) (5) The d ug pe meabili y h ough he co nea, P co nea , was equi ed o es ima e he d ug loss h ough he issue. The pe meabili y alues o b om enac sodium and dexame hasone sodium we e ob ained wi h ex i o es s which in ol ed F anz di usion cells and po cine co neas, as desc ibed in a p e ious publica ion [ 40 ]. B ie ly, co neas we e excised wi h 2–3 mm o su ounding scle a o suppo and hen placed wi h he endo helium acing he dono chambe o he F anz cell o simula e he in i o condi ions o he issue when exposed o in aocula d ug sou ces. A2 and C2 hyd ogel discs (n= 3), loaded wi h bo h b om enac and dexame hasone, we e insed om he loading solu ion, placed in each dono chambe , and co e ed wi h 1 mL PBS. The ecep o chambe was illed wi h 6 mL PBS. F anz cells we e placed in a ba h a 37 ◦ C wi h magne ic s i ing o he ecep o chambe . A each ime poin (0.5, 1, 2, 3, 4, 5, 6 h), 1 mL o PBS was emo ed om he ecep o chambe and subs i u ed wi h esh PBS. A e he las ime poin , he olume o he dono chambe was collec ed o analysis. D ug quan i ica ion was pe o med as explained abo e (Sec ion 2.5). All samples we e il e ed (0.22 µ m PTFE hyd ophilic sy inge il e s, Scha lau ® ) be o e es ing. The cumula i e amoun s o pe mea ed d ug we e i ed o a linea leas squa es eg ession (Equa ion (6)), and he slope o he eg ession was he s eady-s a e lux J. The P co nea o each d ug was ob ained as he a io be ween J and he d ug concen a ion in he dono chambe a = 6 h (Equa ion (7)) [41,42]. Cumula i e mass pe mea ed/Su ace a ea = J + q (6) Pco nea = J/[Dono ] =6h (7) The d ug concen a ion in he aqueous humo o e ime (C aqueous ) was hen ob ained h ough Equa ion (8) [ 38 ], conside ing he olume o he an e io chambe ( Vaqueous = 0.250 cm3 ), he d ug accumula ion in he aqueous humo due o he elease om he hyd ogel ( ∂ C/ ∂ y) and he d ug loss ela ed o he aqueous humo eno a ion a e ( Φ = 0.0025 mL/min) and h ough he co nea (P co nea A co nea ), wi h A co nea = 1.3 cm 2 . The bounda y condi ions s a e he symme y a he cen e o he hyd ogel ( ∂ C/ ∂ y = 0 a y = 0), he assump ion o sink condi ion a he i eous-IOL bounda y (C = 0 a y = − h, i.e., on he pos e io lens su ace), and he equilib ium be ween he concen a ion on he an e io su ace o he lens and he aqueous humo ( C = KCaqueous a y = h ). The ini ial condi ion is he d ug concen a ion in he lens a equilib ium (C = Ci ∀y, a = 0). Vaqueous (∂Caqueous/∂ )=DA(∂C/∂y)y=h −(Pco neaAco nea +Φ) Caqueous (8) 2.9. Cy o oxici y A2 and C2 hyd ogel discs (n= 4) we e loaded wi h bo h d ugs as desc ibed abo e hen insed, blo ed and d ied unde acuum o 24 h a 40 ◦ C. Due o he he mosensi i i y o co icos e oids [ 43 ], s e iliza ion was hen pe o med on d y discs by γ - adia ion wi h a dose a e o 2.5 kGy/h a oom empe a u e un il a o al dose o 25 kGy was eached [ 13 ]. Unloaded A2 and C2 hyd ogel discs (n= 4) we e also s e ilized and es ed. Cy o oxici y was s udied by indi ec con ac [ 28 , 44 ], placing he hyd ogels in cell cul- u e inse s (T answell ® , Co ning, Glendale, AZ, USA; 12 mm diame e ) and using human lens epi helial cells acco ding o he ISO 10993-5: 2009 s anda d. B ie ly, cells we e cul u ed in a T75 lask wi h DMEM supplemen ed wi h 20% e al bo ine se um, 1% an ibio ics (penicillin-s ep omycin solu ion: 10,000 U/mL penicillin, 10 mg/mL s ep omycin) and incuba ed a 37 ◦ C in a humidi ied 5% CO 2 incuba o . Cell suspension, om passages 3 o 6, was seeded in each well o a 12-wells cul u e pla e o ob ain 1 × 10 5 cells/well. Pla es we e incuba ed o 24 h a 37 ◦ C unde he same g ow h condi ions. Meanwhile, d ug- loaded and unloaded hyd ogels we e hyd a ed o e nigh in 1 mL o d ug loading solu ion (s e ile il e ed 0.22 µ m) o PBS, espec i ely. A e p oli e a ion, he cul u e medium was eno a ed, hyd ogels we e ans e ed in o he inse s and hese we e placed in he wells. An addi ional 200 µ L o esh medium was added o ensu e a comple e imme sion o he Pha maceu ics 2021,13, 976 8 o 20 hyd ogels. Nega i e (DMEM) and posi i e (DMEM wi h 10% DMSO) con ol wells ( n= 4 ) we e also p epa ed. A e incuba ion o 24 h, inse s we e emo ed, cells we e insed wi h esh PBS and incuba ed wi h MTT solu ion (MTT dissol ed in se um- ee DMEM a a concen a ion o 0.5 mg/mL, om a s ock solu ion o 5 mg/mL MTT in PBS) o 3 u he hou s. Then, a o mazan-dissol ing solu ion (0.1% IGEPAL in isop opanol wi h hyd ochlo ic acid 4 mM) was added o each well. Pla es we e shaken in a da k en i onmen a oom empe a u e o 1 h. The abso bance was measu ed a 595 nm in a mic opla e eade (Pla os R 496, AMP Diagnos ic, G az, Aus ia). The ela i e quan i ica ion o cell iabili y was no malized o he nega i e con ol. 2.10. S a is ical Analysis Quan i a i e da a a e p esen ed as he mean ± s anda d de ia ion. S a is ical analysis was pe o med on P ism 8.0.1 so wa e (G aphPad, San Diego, CA, USA) by - es o compa e wo g oups o samples and by one-way ANOVA o compa e mul iple g oups wi h Tukey’s mul iple compa isons pos -hoc es . The no mali y o all a iables was assessed by he Shapi o–Wilk es . The signi icance le el was se a p< 0.05. The sample size was es ima ed o ob ain a e age alues wi h a maximum e o o 7% and a con idence le el o 95%. Fo mechanical es s, due o echnical cons ain s, a maximum e o o 14% wi h a con idence le el o 90% was admi ed. 3. Resul s and Discussion 3.1. Molecula In e ac ion Analysis Hyd ogels wi h a HEMA-BEM backbone we e he ein designed as IOL ma e ials able o be simul aneously loaded wi h b om enac sodium and dexame hasone sodium. APMA and AAm we e selec ed as unc ional monome s a e molecula docking analysis (Table 2), as hey exhibi ed much s onge in e ac ion pa ame e s (i.e., highe ee ene gy, E and lowe dissocia ion cons an , Ki) wi h he d ug molecules i compa ed o he ma e ial backbone. Table 2. F ee ene gy o in e ac ion (E) and dissocia ion cons an (Ki) o b om enac sodium and dexame hasone sodium wi h HEMA, BEM, APMA and AAm monome s. DRUG HEMA BEM APMA AAm E [Kcal/mol] Ki [mM] E [Kcal/mol] Ki [mM] E [Kcal/mol] Ki [mM] E [Kcal/mol] Ki [mM] Dexame hasone sodium −1.47 83.32 −1.10 156.55 −2.77 9.30 −2.06 30.74 B om enac sodium −1.54 74.48 −1.27 117.06 −3.45 2.95 −2.20 24.43 The ee ene gy alue desc ibes he in e ac ion be ween wo molecules, namely he d ug and he ligand (monome ). A mo e nega i e alue o E indica es a highe in e ac- ion ene gy be ween he pai . The selec ed unc ional monome s APMA and AAm exhibi highe in e ac ion wi h he d ugs i compa ed o he backbone componen s o he hyd ogels (HEMA and BEM). The e o e, he p esence o APMA and AAm could esul in he o ma- ion o sui able binding poin s o he d ugs in he hyd ogels. In pa icula , he in e ac ion be ween he d ugs and APMA p esen s mo e nega i e alues, o bo h dexame hasone and b om enac, and he e o e highe a ini y can be expec ed. Molecula docking analysis was p e iously used in a ious d ug- elease applica ions, wi h epo ed E alues be ween − 3.5 and − 9.7 Kcal/mol o s ong bu s ill e e sible d ug-ca ie in e ac ions [ 45 , 46 ]. These alues a e compa able o he ee ene gy, E, be ween APMA and he selec ed d ugs. The dissocia ion cons an (Ki) p o ides in o ma ion abou he d ug elease om he ma e ial. I desc ibes he in e ac ion be ween he monome (M) and he d ug (D), by indica ing he endency owa ds he o ma ion o wo sepa a e species (M + D) as opposed o he MD complex. A low Ki alue indica es ha he MD complex p edomina es o e he Pha maceu ics 2021,13, 976 9 o 20 species in sepa a e (Equa ion (9)) [ 47 ]. Lowe Ki alues we e ob ained o he unc ional monome s APMA and AAm i compa ed o he backbone monome s. Ki = [M][D]/[MD] (9) Less nega i e E alues and high Ki alues, and he e o e low molecula a ini y, we e ob ained by he in e ac ion analysis o BEM wi h bo h d ugs. The balance be ween he p esence o BEM in he hyd ogel backbone and he p esence o he unc ional monome s may de e mine he d ug elease kine ics om he ma e ial. 3.2. Physical and Mechanical Cha ac e iza ion o he Unloaded Hyd ogels P io o d ug-loading, hyd ogels we e subjec ed o physical and mechanical cha - ac e iza ion o p o e hei sui abili y as an IOL ma e ial. All hyd ogel discs eached an equilib ium liquid con en in he i s 6 h o soaking (Figu e 2A). Then, he liquid con en emained s able o he subsequen ime poin s. The highes liquid up ake was ob ained o hyd ogels con aining APMA (i.e., A2, B2, C2 hyd ogels, liquid up ake ≈ 32.2 %). The addi ion o AAm o he hyd ogel composi ion (i.e., A3, B3, C3 hyd ogels), on he con a y, did no cause any di e ence in he swelling beha io o he ma e ial. No s a is ical di e - ence was e idenced be ween he d ug-imp in ed and non-imp in ed hyd ogels wi h he same monome composi ion (p≥0.2). All hyd ogels, excep C1 and C3, exhibi ed a ligh ansmi ance highe han 90% a λ > 550 nm (Figu e 2B), which was conside ed he minimum op ical equi emen o he ma e ials [ 48 ]. As C1 and C3 hyd ogels a e bo h imp in ed wi h dexame hasone sodium, he lowe ligh ansmission may ha e been caused by d ug p ecipi a ion in he p epolyme mix u e, o ming d ug clus e s du ing polyme iza ion, which we e no dissol ed du ing boiling. Di e en ly, C2 hyd ogel, also imp in ed wi h dexame hasone, did no exhibi he same p oblem. The p esence o APMA in C2 (Table 2) may ha e inc eased he solubili y o dexame hasone sodium in he p epolyme mix u e, hus a oiding p ecipi a ion. As can be no iced om he spec um o A2 hyd ogel, APMA also ac ed as a weak UV ligh il e o λ< 350 nm. B1, B2 and B3 hyd ogels, imp in ed wi h b om enac sodium, also exhibi ed ligh il e p ope ies up o λ≈ 450 nm. The p esence o un emo ed d ug molecules in he polyme s a e he washing phase could jus i y he phenomenon, especially conside ing ha he UV-block e ec is mo e e iden in he B2 hyd ogel, unc ionalized wi h APMA, which has a s onge binding in e ac ion wi h he d ug (Table 2). In ac , he yellowish colo o b om enac sodium was obse ed in B2 hyd ogels e en a e epea ed washing. The ensile moduli o he hyd a ed hyd ogels a e epo ed in Figu e 2C. In gene al, all alues (1.9–4.2 MPa) lie wi hin he ange epo ed o p e iously de eloped IOL ma e ials and hyd ophilic comme cial lenses (i.e., 0.5–4 MPa [ 16 , 49 , 50 ]). Molecula imp in ing did no signi ican ly al e he mechanical p ope ies o he hyd ogels. The p esence o APMA (in A2, B2, C2 hyd ogels) lowe ed he ensile modulus o he ma e ial ( ≈ 2.1 MPa). This should also be ela ed o he inc eased swelling o he hyd ogels p epa ed wi h APMA (Figu e 2A), which inc eases he in e molecula bonding be ween he hyd ogel chains and wa e molecules, as opposed o hyd ogel-hyd ogel in e ac ions. The calcula ed alues o he a e age mesh size a e epo ed in he Supplemen a y Ma e ials, Table S1. The a e age mesh size o A1 hyd ogels was equal o 2.30 nm, which is cohe en wi h p e iously epo ed alues o HEMA-based hyd ogels [ 51 , 52 ]. The mesh sizes o AAm- unc ionalized hyd ogels we e equal o 2.93, 2.76 and 2.87 nm o A3, B3 and C3 hyd ogels, espec i ely. When unc ionalized wi h APMA, he mesh size inc eased up o 3.22, 3.31 and 3.47 nm o A2, B2 and C2 hyd ogels, espec i ely. The la ge mesh sizes o APMA- unc ionalized hyd ogels a e associa ed wi h highe liquid up ake and lowe Young’s modulus (Figu e 2A,C). Pha maceu ics 2021,13, 976 16 o 20 The ensile modulus was no signi ican ly di e en in bo h A2 (2.3 ± 0.6 s. 1.6 ±0.3 MPa ) and C2 (1.9 ±0.8 s. 1.4 ±0.3 MPa) hyd ogels be o e and a e loading, espec i ely (Figu e 5C). In i o cell iabili y es s we e pe o med using unloaded and dual-loaded discs o e alua e he biocompa ibili y o he ma e ial and he d ug eleased. Cell iabili y was 88 ± 1% and 89 ± 2% o unloaded A2 and C2 hyd ogels, and 81 ± 1% and 79 ± 1% o d ug-loaded A2 and C2 hyd ogels, espec i ely (Figu e 5D), hence conside ed non-cy o oxic acco ding o he ISO 10993-5: 2009 s anda d. Al hough a dec ease in cell iabili y was obse ed in he p esence o d ugs, i should be conside ed ha cell es s we e pe o med wi hou mimicking he con inuous eno a ion o he aqueous humo , which is expec ed o educe he ime o con ac o he d ug and cy o oxici y in i o . A e eye implan a ion o d ug-loaded hyd ogels, a peak in d ug concen a ion in he aqueous humo is expec ed in he i s day (Figu e 4), wi h maximum concen a ions o dexame hasone and b om enac equal o 12.6 µ g/mL and 71.0 µ g/mL om C2 and A2 hyd ogels, espec i ely (Table 5). These alues a e much lowe han he concen a ions o cu en ly used eye d ops (900 µ g/mL b om enac colly ium, Yellox ® [ 59 ]; 1 mg/mL dexame hasone colly ium, Maxidex ® [ 60 ]) o in aocula injec ions (up o 1.1–1.6 mg/mL o dexame hasone [ 32 , 61 ]) and a e, he e o e, expec ed o be non- oxic o cells. As d ug-loaded IOLs elease he d ug di ec ly in o he an e io chambe , d ug loss associa ed wi h eye d ops is a oided making lowe d ug concen a ions su icien o ob ain a he apeu ic e ec . Indeed, less han 5% o he dose ins illed wi h eye d ops is expec ed o each he aqueous humo [ 62 – 64 ]. The p esence o an ini ial peak in d ug concen a ion a e implan a ion o he d ug-loaded IOL could be bene icial o he in ended ea men , as he adminis a ion o an i-in lamma o y d ugs is mos equi ed in he pe iope a i e pe iod [6,8]. P io o being es ed o cell iabili y in i o , he hyd ogels we e s e ilized in a d y s a e by γ - adia ion. In he iew o a u u e comme cial applica ion o he designed hyd ogels, u he es s a e needed o e alua e he e ec o s e iliza ion on he ma e ial beha io o e ime, and he possibili y o s e ilizing he ma e ials in he loading solu ion, o example using high hyd os a ic p essu e (HPP) [65]. Ma hema ical modeling was used in his s udy and cons i u es a use ul ool o he es ima ion o he e icacy o he de ices in he ea ly s age o p oduc de elopmen . Howe e , in i o animal s udies will ha e o p ecede any comme cial applica ion o he p oposed hyd ogels o e alua e he e icacy o he d ug-elu ing IOLs in pos -su gical ocula condi ions. Despi e he signi ican achie emen s ob ained in he las decade on he design o he apeu ic IOLs, such de ices a e no cu en ly p esen on he ma ke and la ge-scale clinical ials a e missing o a signi ican ad ance in he ield [66]. 4. Conclusions Two di e en s a egies, namely he addi ion o unc ional monome s and molecula imp in ing, we e he ein in es iga ed o une he elease p o ile o wo an i-in lamma o y d ugs om HEMA-BEM hyd ogels, wi h he aim o de eloping sui able ma e ials o he ab ica ion o IOLs wi h p ophylac ic ac ion agains PCME. B om enac sodium and dexam- e hasone sodium we e simul aneously loaded in o he hyd ogels. D ug-loaded hyd ogels exhibi ed sui able physical and mechanical p ope ies o be used as oldable in aocula lenses. The non- oxici y o he hyd ogels and o he deli e ed d ugs was assessed in i o wi h human lens epi helial cells. The inco po a ion o APMA in he polyme ic ma ix imp o ed he elease kine ics o he d ugs. Molecula imp in ing wi h dexame hasone sodium inc eased he amoun o dexame hasone eleased a e dual-d ug loading. Hyd o- gels A2 (non-imp in ed con aining APMA) and C2 (dexame hasone imp in ed con aining APMA) op imized he elease o b om enac and dexame hasone, espec i ely. B om enac is expec ed o be eleased a he apeu ic concen a ions in he aqueous humo o abou 2 weeks a e A2 hyd ogel implan a ion, while C2 is expec ed o sus ain he deli e y o dexame hasone o up o 8 weeks. These du a ions a e compa ible wi h he cu en opical an i-in lamma o y p ophylaxis o PCME a e ca a ac su ge y. Dual-loaded IOLs made o he de eloped hyd ogels will be able o e icien ly subs i u e opical applica ion, sol e Pha maceu ics 2021,13, 976 17 o 20 he compliance issues associa ed wi h he use o eye d ops and add ess an unme medical need o he p e en ion o PCME. Supplemen a y Ma e ials: The ollowing a e a ailable online a h ps://www.mdpi.com/a icle/ 10.3390/pha maceu ics13070976/s1, Figu e S1: Ligh ansmi ance, measu ed a λ = 550 nm, o A1, A2, B2 and C2 hyd ogels a e dual-d ug loading wi h di e en soaking pa ame e s; Figu e S2: Cumula i e amoun o d ug eleased om A1, A2, B2 and C2 hyd ogels in i o a e dual-d ug loading a di e en empe a u es and soaking imes; Figu e S3: Linea eg ession o he cumula i e mass o b om enac sodium pe mea ed h ough he co nea; Figu e S4: De e mina ion o he e ec i e di usi i y (D) by cu e- i ing o he cumula i e amoun o d ug eleased om A1, A2, B2 and C2 hyd ogels o e ime in i o. Table S1: Pa ame e s used o he calcula ion o he a e age mesh size ξ : densi y o he d y hyd ogel ( ρ ), ze o- equency shea s o age modulus (G 0 (0)) and olume ac ion o he polyme in he swollen ne wo k (ϕ). Au ho Con ibu ions: Concep ualiza ion, N.T., B.S., A.P.S. and C.A.-L.; me hodology, N.T., B.S., A.P.S. and C.A.-L.; so wa e, N.T.; alida ion, N.T.; o mal analysis, N.T.; in es iga ion, N.T, M.S.-O. and S.A.I.; esou ces, C.A.-L. and A.P.S.; da a cu a ion, N.T.; w i ing—o iginal d a p epa a ion, N.T.; w i ing— e iew and edi ing, N.T., B.S., C.A.-L., M.S.-O. and A.P.S.; isualiza ion, N.T.; supe ision, C.A.-L., B.S. and A.P.S.; p ojec adminis a ion, A.P.S.; unding acquisi ion, A.P.S. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This p ojec has ecei ed unding om he Eu opean Union’s Ho izon 2020 esea ch and inno a ion p og amme unde he Ma ie Skłodowska-Cu ie g an ag eemen N ◦ 813440 (OR-BITAL— Ocula Resea ch by In eg a ed T aining and Lea ning) and is also suppo ed by Fundação pa a a Ciência e Tecnologia (FCT) [UID/QUI/00100/2019, UIDB/00100/2020, and UID/BIM/04585/2020]. Ins i u ional Re iew Boa d S a emen : No applicable. 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