Dexamethasone Loaded Liposomes by Thin-Film Hydration and Microﬂuidic Procedures: Formulation Challenges

International Journal of Molecular Sciences

Article Dexamethasone Loaded Liposomes by Thin-Film Hydration and Microﬂuidic Procedures: Formulation Challenges

MD Al-Amin, Federica Bellato, Francesca Mastrotto, Mariangela Garofalo, Alessio Malfanti , Stefano Salmaso * and Paolo Caliceti Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Italy; [email protected] (M.A.-A.); [email protected] (F.B.); [email protected] (F.M.); [email protected] (M.G.); [email protected] (A.M.); [email protected] (P.C.) * Correspondence: [email protected]; Tel.: +39-04-9827-1602

Received: 4 February 2020; Accepted: 23 February 2020; Published: 26 February 2020

Abstract: Liposomes have been one of the most exploited drug delivery systems in recent decades. However, their large-scale production with low batch-to-batch differences is a challenge for industry, which ultimately delays the clinical translation of new products. We have investigated the effects of formulation parameters on the colloidal and biopharmaceutical properties of liposomes generated with a thin-film hydration approach and microfluidic procedure. Dexamethasone hemisuccinate was remotely loaded into liposomes using a calcium acetate gradient. The liposomes produced by microfluidic techniques showed a unilamellar structure, while the liposomes produced by thin-film hydration were multilamellar. Under the same remote loading conditions, a higher loading capacity and efficiency were observed for the liposomes obtained by microfluidics, with low batch-to-batch differences. Both formulations released the drug for almost one month with the liposomes prepared by microfluidics showing a slightly higher drug release in the first two days. This behavior was ascribed to the different structure of the two liposome formulations. In vitro studies showed that both formulations are non-toxic, associate to human Adult Retinal Pigment Epithelial cell line-19 (ARPE-19) cells, and efficiently reduce inflammation, with the liposomes obtained by the microfluidic technique slightly outperforming. The results demonstrated that the microfluidic technique offers advantages to generate liposomal formulations for drug-controlled release with an enhanced biopharmaceutical profile and with scalability.

Keywords: liposome formulation; microﬂuidic technique; dexamethasone loaded liposomes; controlled release

1. Introduction Almost 60 years ago, Alec Bangham described liposomes as swollen phospholipid systems [1,2]. The era of drug delivery using lipidic vesicles had begun. Liposomes represent a versatile drug delivery system that allows for the encapsulation of a variety of active compounds either in their lipid bilayer or in the aqueous core [3]. Doxil® developed by Barenholz and Gabizon [4] for the treatment of Acquired Immune Deficiency Syndrome (AIDS) related Kaposi sarcoma has been the first liposomal formulation approved by the Food and Drug Administration (FDA). Despite extensive research in this area, to date only 15 liposomal formulations are in clinical use [5], along with the first generic version of liposomal doxorubicin hydrochloride (Lipodox®). The approval of this generic formulation by the FDA was promoted by the shortage of Doxil® in late 2011 due to manufacturing and regulatory hurdles, which speeded up the approval of Lipodox® produced by Sun Pharma. On the other hand, the Doxil®

Int. J. Mol. Sci. 2020, 21, 1611; doi:10.3390/ijms21051611 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 1611 2 of 20 shortage raised also awareness about the issues of pharmaceutical manufacturing of liposomes and the need of novel procedures for large scale production of products with a high standard of pharmaceutical quality. Indeed, the manufacturing of liposomes presents certain major issues to be addressed: i) batch to batch variability, ii) control over the size of the liposomes, iii) the number of steps required for drug loading, iv) time-consumption and the complexity of production and v) the scalability of the process. Over the past few decades, different approaches have been proposed to manufacture liposomes. These include lipid film hydration, reverse phase evaporation, ethanol injection, ether injection, detergent removal, etc. [6–10]. Most of the methods used to formulate liposomes require additional post processing steps to increase the liposome homogeneity in term of lamellarity and size, such as freeze-and-thaw cycles, extrusion, and sonication [11,12]. Liposome size and size distribution are key features for in vivo applications, being among the main parameters dictating the biopharmaceutical behaviour of a colloidal delivery system, including the pharmacokinetic profile of the loaded drugs, access to target tissues, and the overall body biodistribution, cell uptake, and clearance by Reticuloendothelial System (RES) [13]. As the batch-to-batch variability of liposome formulations yields an unpredictable fate of the carrier upon in vivo administration, approaches for scalable formulation that limit the batch variability are needed. Formulation methods that exploit the fluid control and lipid mixing can facilitate liposome preparation in a one step process. For example, ethanol injection into an aqueous buffer [8] allows for phase separation of the lipids that spontaneously assemble into liposomes. This process, however, is usually run in a bulk reactor where the mixing of solutions is not finely controlled. More recently, microfluidic techniques where fluid mixing is performed in a geometrically constrained microenvironment, typically defined by sub-micrometre length scale and low Reynolds number, have been developed [14,15]. The rapid microfluidic mixing at the nano-litre scale allows for the nano-assembling of lipids into liposomes [16]. Indeed, the microfluidic technology efficiently controls the parameters affecting the assembly and yields highly reproducible formulations. The laminar flow of aqueous and organic solvent streams and predictable flow patterns in the microfluidic mixing result in a uniform particle size distribution. This technique allows also for controlling the size of the liposomes by setting-up proper operative conditions, namely total flow rate (TFR), flow rate ratio (FRR) of the solutions mixed, total volume (TV) processed, and solvent selection [17]. The identification of the parameters that affect the liposome features is crucial when a formulation is generated with a new procedure, namely the microfluidic, with the aim to replace an existing one. In the present study, we investigated the effect of the formulation parameters on the biopharmaceutical properties of dexamethasone hemisuccinate loaded liposomes prepared by thin-film hydration and microfluidic techniques using a calcium acetate mediated remote loading process.

2. Results

2.1. Liposome Characterization Dexamethasone free liposomes (plain liposomes), generated by thin-film hydration with different calcium acetate concentrations, were extruded through a 100 nm cut-off membrane to reduce the size and polydispersity. After extrusion, the liposomal diameter of the preparations was in the range of 144.1 13.8–162.7 17.0 nm and the poly dispersity index (PDI) was in the range of 0.08–0.39. The ± ± higher liposome size with respect to the filter pore size is due to the reversible elastic deformation of the vesicles as reported in the literature [18]. In the case of the microfluidic process, a pre-formulation study was carried out to set-up the operative conditions to generate liposomes of approximately 100 nm diameter. The size of dexamethasone free liposomes (plain liposomes) generated with different calcium acetate concentrations was in the range of 105.7 12.5–118.4 21.5 and PDI in the range of 0.17–0.28. Flow rate ratios (FRR) ± ± Int. J. Mol. Sci. 2020, 21, 1611 3 of 19 rate ratios (FRR) higher than of 1.5:1 aqueous/methanol, and a total flow rate (TFR) lower than 3 mL/min generated liposomes with a size above 200 nm, a high PDI, and low calcium acetate loading. The calcium acetate concentration used for the liposome preparation did not affect the liposome size obtained by the two techniques.

Int.2.2. J.Loading Mol. Sci. E2020fficiency, 21, 1611 and Capacity 3 of 20 The effect of calcium acetate concentration used to generate plain liposomes (from 0.1 to 1.0 M) higheron drug than loading of 1.5:1 capacit aqueousy and/methanol, efficiency andwas afirst total assessed. flow rate Both (TFR) formulations lower than obtained 3 mL/min by generated thin-film liposomeshydration withand microfluidics a size above 200 showed nm, a highthat PDI,the loading and low capacity calcium acetateand efficiency loading. of dexamethasone increasedThe calcium as the calcium acetate concentrationacetate concentration used for used the liposome to assemble preparation liposomes did increased not affect fr theom liposome0.1 M to size0.2 M, obtained which was by the expected two techniques. as a higher amount of calcium ions in the liposome core will entrap more dexamethasone in the liposomes during remote loading. On the contrary, at concentrations above 0.2 2.2.M calcium Loading acetate, Efficiency the and loading Capacity capacity and efficiency decreased (Figure 1A,B). In this case, the loadingThe capacity effect of and calcium efficiency acetate have concentration the same values used to because generate the plain study liposomes was performed (from 0.1 using to 1.0 the M) onsame drug lipid loading and dexamethasone capacity and e ffihemisuccinateciency was first concentra assessed.tion Both (2.5 mg/mL). formulations obtained by thin-film hydrationAt all andcalcium microfluidics acetate showedconcentrations that the used loading for capacitythe preparation and effi ciencyof plain of dexamethasoneliposomes, the increaseddexamethasone as the loading calcium in acetate liposomes concentration obtained by used the tomicrofluidic assemble liposomes procedure increased was found from to be 0.1 higher M to 0.2than M, in which the case was ofexpected liposomes as produced a higher amount by the thin of calcium-film hydration ions in the technique. liposome core will entrap more dexamethasoneUnder the indrug the liposomesloading conditions, during remote liposomes loading. produced On the contrary,with 0.2 atM concentrations calcium acetate above by 0.2microfluidic M calcium and acetate, thin-film the loadinghydration capacity procedures and eyieldedfficiency the decreased maximal (Figure 11.7 and1A,B). 7.0 drug/lipid In this case, w/w the% loading capacitycapacities and, respectively efficiency have, corresponding the same values to because67% higher the study loading was in performed the case using of liposomes the same obtainedlipid and by dexamethasone microfluidics. hemisuccinate Based on these concentration results, 0.2 M (2.5 calcium mg/mL). acetate was selected for preparing liposomes for the further investigations.

12 12

10 10

8 8

6 6 (w/w%) (w/w%) 4 4 loading capacity

2 efficiency loading 2

0 0 0.1 0.2 0.5 1 0.1 0.2 0.5 1 calcium acetate (M) calcium acetate (M) (A) (B)

Figure 1. The loadingloading capacity (A) and eefficiencyfficiency (B)) ofof dexamethasonedexamethasone hemisuccinatehemisuccinate in liposomes assembled at increasing concentrationsconcentrations of calcium acetate. The l liposomesiposomes were obtained by thin thin-film-film hydration (■) and microfluidic microfluidic (■) techniques. Calcium Calcium acetate acetate loaded loaded liposomes liposomes and dexamethasone dexamethasone hemisuccinathemisuccinatee werewere incubated incubated at at 2.5 2.5 mg mg/mL,/mL, respectively. respectively. The loadingThe loading capacity capacity was expressed was expressed as loaded as loadeddrug/lipid drug/lipidw/w% ratio; w/w% the ratio; loading the loading efficiency efficiency was expressed was expressed as (loaded as drug(loaded/fed drug/fed drug) w/ wdrug% ratio.) w/w% ratio. At all calcium acetate concentrations used for the preparation of plain liposomes, the dexamethasoneA drug loading loading time in liposomescourse study obtained showed by thethat microfluidic the dexamethasone procedure loadi was foundng efficiency to be higher and thancapacity in the increased case of liposomeswith the incubation produced time by the until thin-film 60 min hydration, either in technique.the case of liposomes prepared by thin-filmUnder hydration the drug loadingor microfluidic conditions,s (Figure liposomes 2), producedwhich corresponds with 0.2 M calciumto the maximal acetate by 11.7 microfluidic and 7.0 drug/lipidand thin-film w/w hydration% loading procedurescapacity for yielded the liposomes the maximal obtained 11.7 by and microfluidics 7.0 drug/lipid andw /byw% thin loading-film capacities,hydration, respectively,respectively, correspondingas discussed for to Figure 67% higher 1. Once loading again, in the the microfluidic case of liposomes procedure obtained allowed by formicrofluidics. a higher loading Based capacity on these and results, efficiency 0.2 M calciumat each timepoint acetate was with selected respect for to preparing the thin-film liposomes hydration for gtheenerated further liposomes. investigations. To note that similarly to what was reported for Figure 1, the loading capacity and efficiencyA drug loading have timethe same course values study showedbecause thatthe thestudy dexamethasone was performed loading using effi theciency same and lipid capacity and dexamethasoneincreased with the hemisuccinate incubation time concentration until 60 min, (2.5 either mg/mL). in the case of liposomes prepared by thin-film hydration or microfluidics (Figure 2), which corresponds to the maximal 11.7 and 7.0 drug /lipid w/w% loading capacity for the liposomes obtained by microfluidics and by thin-film hydration, respectively, as discussed for Figure1. Once again, the microfluidic procedure allowed for a higher loading capacity and efficiency at each timepoint with respect to the thin-film hydration generated liposomes. To note that similarly to what was reported for Figure1, the loading capacity and e fficiency have the same Int. J. Mol. Sci. 2020, 21, 1611 4 of 20 values because the study was performed using the same lipid and dexamethasone hemisuccinate concentrationInt. J. Mol. Sci. 2020 (2.5, 21,mg 1611/mL). 4 of 19

12 12 10 10 8 8 6

(w/w %)(w/w 6 (w/w) %) 4

loading capacity 4 loading efficiency efficiency loading 2 2 0 0 20 40 60 120 incubation time (min) 20 40 60 120 incubation time (min) (A) (B)

Figure 2. The l loadingoading capacity ( A)) and eefficiencyfficiency (B) ofof dexamethasonedexamethasone hemisuccinate in calcium acetate pre-loadedpre-loaded liposomes liposomes at increasingat increasing incubation incubation times oftime thes liposomesof the liposomes with the drug.with Liposomesthe drug. wereLiposomes obtained were by thin-filmobtained hydrationby thin-film () hydration and microfluidic (■) and ( )microf techniques.luidic Calcium(■) techniques. acetated Calcium loaded acetatedliposomes loaded and dexamethasone liposomes and hemisuccinate dexamethasone were incubatedhemisuccinate at 2.5 mgwere/mL, incubated respectively. at 2.5 The mg/mL, loading respectively.capacity was expressedThe loading as loaded capacity drug was/lipid expressedw/w% ratio; as theloaded loading drug/lipid efficiency w/w was% expressed ratio; the as loading (loaded efficiencydrug/fed drug) was expressedw/w% ratio. as (loaded drug/fed drug) w/w% ratio.

The eeffectffect of the lipidlipid/dexamethasone/dexamethasone hemisuccinate fed ratio on the loading process was evaluated by either maintaining a constant lipid concentration and increasing the drug concentration or maintaining a constant drug concentration and increasing the lipid concentration (Figure3 3).). UnderUnder a constant lipid concentration, the loading capacity (Figure 33A)A) increasesincreases asas thethe drugdrug concentrationconcentration increases toto reachreach the the maximal maximal values values at at 1.25 1.25 mg mg/mL/mL drug drug concentration, concentration yielding, yielding the maximalthe maximal 12.6 12.6 and and4.5 drug 4.5 /drug/lipidlipid w/w% w/w loading% loading capacity capacity for liposomes for liposomes obtained obtained with microfluidic with microfluidic and thin-film and hydration, thin-film hydrationrespectively,, respectively, which corresponds which corresponds to a 179% higher to a 179% loading higher for the loading liposomes for the obtained liposomes by microfluidics. obtained by Themicrofluidics drug loading. The e ffidrciencyug loading (Figure ef3ficiencyB) agreed (Figure with the3B) capacity agreed with profiles. the Indeed,capacity the profiles. loading Indeed, e fficiency the loadingincreased efficiency up to the increased plateau and up to then the decreased. plateau and then decreased. When the lipid concentration is increased, more vesicles are present in the medium at a constant drug concentration,concentration, whichwhich results results in in a higha high total total drug drug loading loading and and a constant a constant capacity capacity (lipid (lipid/dr/drug ratio)ug ratio)is achieved is achieved (Figure (Figure3C). The 3C). higher The thehigher number the number of vesicles of obtainedvesicles obtained by the lipid by the concentration lipid concentration increase, increase,the higher the the higher amount the ofamount drug loadedof drug inloaded the vesicles, in the vesicles, which resultswhich results in an increased in an increased efficiency efficiency of the processof the process (Figure (Figure3D). Microfluidic 3D). Microfluidic and thin-film and thin hydration-film hydration procedures procedures yielded ayielded maximal a maximal 11.7 and 11.7 7.0 anddrug 7.0/lipid drug/lipidw/w% loading w/w% loading capacity, capacity, respectively, respectively, which corresponds which corresponds to a 67% to higher a 67% loadinghigher loading for the forliposomes the liposomes obtained obtained by microfluidics by microfluidics than those than generated those generated by thin-film by thin hydration.-film hydration. Overall, thisthis study study shows shows that that drug drug loading loading can be can optimised be optimised by the selectionby the ofselection proper conditions.of proper Theconditions. liposomes The obtained liposomes by obtained microfluidics by microfluidics yielded a higher yielded drug a loadinghigher drug capacity loading with capacity respect to with the respectformulation to the obtained formulation by thin-film obtained hydration. by thin-film Based hydration. on these Based results, on thethese 10 results, mg/mL the lipid 10 formulationsmg/mL lipid formulationsobtained with obtained 0.2 M calcium with 0. acetate2 M calcium and loaded acetate by and 60 min loaded incubation by 60 min with incubation 2.5 mg/mL with dexamethasone 2.5 mg/mL dexamethasonehemisuccinate were hemisuccinate selected to were obtain selected the highest to obtain dexamethasone the highest concentration:dexamethasone 0.81 concentration: and 0.98 mg 0.81/mL andfor liposomes 0.98 mg/mL obtained for liposomes by thin-film obtained hydration by thin and-film microfluidics, hydration and respectively. microfluidics, respectively.

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40 14

12 30 10

8 20

6 (w/w %) (w/w%)

4 efficiency loading

loading capacity capacity loading 10 2

0 0 2.5 ; 0.25 2.5 ; 0.625 2.5 ; 1.25 2.5 ; 2.5 2.5 ; 0.25 2.5 ; 0.625 2.5 ; 1.25 2.5 ; 2.5 lipid and dexamethasone (mg/mL) lipid and dexamethasone (mg/mL) (A) (B) 14

12 40 10 30 8

6 (w/w %) 20 (w/w%)

loading capacity capacity loading 4

loading efficiency efficiency loading 10 2

0 0 2.5 ; 2.5 5 ; 2.5 10 ; 2.5 lipid and dexamethasone (mg/mL) 2.5 ; 2.5 5 ; 2.5 10 ; 2.5 lipid and dexamethasone (mg/mL) (C) (D)

FigureFigure 3. 3. TheThe l loadingoading capacitycapacity ((AA,C and) and C) e ffiandciency efficiency (B,D) of(B dexamethasone and D) of dexamethasone hemisuccinate hemisuccinate in liposomes inat aliposomes fixed lipid at concentration a fixed lipid of 2.5concentration mg/mL (A,B of) and 2.5 a mg/mL fixed dexamethasone (A and B) and hemisuccinate a fixed dexamethasone concentration hemisuccinateof 2.5 mg/mL( Cconcentration,D) while varying of 2.5 their mg/mL weight (C ratio.and D Liposomes) while varying were obtainedtheir weight by thin-film ratio. Liposomes hydration were() and obtained microfluidic by thin (-film) techniques. hydration The (■) loadingand microfluidic capacity was(■) techniques. expressed asThe loaded loading drug capacity/lipid w was/w% expressedratio; loading as loaded efficiency drug/lipid was expressed w/w% ratio; as (loaded loading drug efficiency/fed drug) wasw/w expressed% ratio. as (loaded drug/fed drug) w/w% ratio. 2.3. Size, Morphology, and Colloidal Stability of Dexamethasone Loaded Liposomes 2.3. Size,The M incubationorphology, of and liposomes Colloidal with Stability dexamethasone of Dexamethasone hemisuccinate Loaded Liposomes during the remote loading process did notThe induce incubation a size increaseof liposomes of liposomes with dexamethasone (Figure4A). Furthermore, hemisuccinate neither during the incubation the remote time, loading nor the processlipid/drug did ratio not induce affected a thesize vesicle increase size of and liposomes polydispersity (Figure (Figure4A). Furthermore,4B–D). In general, neither the the formulation incubation time,obtained nor bythe thin-film lipid/drug hydration ratio affected procedure the vesicle provided size larger and batch-to-batchpolydispersity di(Figurefferences, 4B– whichD). In reflectedgeneral, theinthe formulation larger standard obtained deviation by withthin- respectfilm hydration to that obtained procedure by microfluidic provided liposomelarger batch preparation.-to-batch differences,Formulations which obtainedreflectedby in thin-film the larger hydration standard and deviation microfluidic with procedures respect to under that theobtained optimized by microfluidicconditions reported liposome above, preparation. with size of 140.9 19.5 nm (PDI 0.07) and 103.7 3.5 nm (PDI 0.13) ± ± (Figure S1), respectively, were characterized.

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200 1 200 1.0

0.8 0.8 150 150 0.6 0.6 100 100 PDI

0.4 PDI 0.4 size (nm) size (nm) 50 50 0.2 0.2

0 0 0 0.0 0.1 M 0.2 M 0.5 M 1 M 20 40 60 120 incubation time (min) calcium acetate (M) (A) (B) 200 1.0 200 1.0

0.8 0.8 150 150 0.6 0.6 100 100 PDI 0.4 0.4 PDI size (nm) size (nm) 50 50 0.2 0.2

0 0.0 0 0.0 2.5 ; 0.25 2.5 ; 0.625 2.5 ; 1.25 2.5 ; 2.5 2.5 ; 2.5 5 ; 2.5 10 ; 2.5 lipid and dexamethasone (mg/mL) lipid and dexamethasone (mg/mL) (C) (D)

FigureFigure 4. 4. TheThe size size of of dexamethasone dexamethasone hemisuccinate hemisuccinate loaded loaded liposomes liposomes obtained obtained by by thin thin-film-film hydration hydration (■() )and and microfluidic microfluidic (■ () )techniques techniques and and the the corresponding corresponding PDI PDI ( (■, ,and and ■, ,respectively). respectively). (A (A) )liposomes liposomes assembledassembled at at increasing increasing calcium acetateacetate concentrationconcentration and and loaded loaded by by contact contact of of 2.5 2.5 mg mg/mL/mL of lipidsof lipids and anddexamethasone dexamethasone hemisuccinate; hemisuccinate; (B) liposomes (B) liposomes loaded atloaded increasing at contactincreasing time withcontact dexamethasone time with dexamethasonehemisuccinate byhemisuccinate contact of 2.5 by mg contact/mL of of lipids 2.5 mg/mL and dexamethasone of lipids and dexamethasone hemisuccinate,; hemisuccinate,; (C): liposomes (Cloaded): liposomes at increasing loaded dexamethasone at increasing concentration dexamethasone and fixedconcentration lipid concentration and fixed (mg lipid/mL); concentration (D) liposomes (mg/mL);loaded at increasing(D) liposomes lipid concentrationloaded at incr andeasing fixed dexamethasonelipid concentration concentration and fixed (mg dexamethasone/mL). concentration (mg/mL). In all cases, the liposome diameters observed by cryogenic transmission electron microscopy (Cryo-TEM)Formulations well correlatedobtained by with thin the-film Dynamic hydration Light and Scattering microfluidic (DLS) procedures measurements. under Thethe optimized Cryo-TEM conditionsimages of reported liposomes above, obtained with withsize of the 140.9 two ± procedures19.5 nm (PDI showed 0.07) and vesicular 103.7 ± 3.5 structures nm (PDI with 0.13) a(Figure darker Sliposome1), respectively, membrane were surroundingcharacterized. the inner aqueous compartment containing darker rod-shaped structuresIn all cases, corresponding the liposome to thediameter nanocrystallines observed calcium-dexamethasoneby cryogenic transmission hemisuccinate electron microscopy complex (Cryo(Figure-TEM)5 and well Figure correlated S2). This with was the in Dynamic agreement Light with Scattering the observations (DLS) measurements. reported in the The literature Cryo-TEM [ 19]. imagesThe liposomes of liposomes obtained obtained by thin-film with the hydration two procedures and hand showed extrusion vesicular showed structures a non-homogeneous with a darker liposomemultilamellar membrane structure surrounding (Figure5A,C) the while inner the aqueous liposomes compartment obtained by microfluidiccontaining proceduredarker rod appeared-shaped structuresas homogeneous corresponding unilamellar to the vesicles nanocrystalline (Figure5B,D). calcium-dexamethasone hemisuccinate complex (FigureThe 5 and liposome S2). This formulations was in agreement were fairly with stable the observation over two weekss reported incubation in the at literature room temperature [19]. The liposomes(Figure6). obtained by thin-film hydration and hand extrusion showed a non-homogeneous multilamellar structure (Figure 5A,C) while the liposomes obtained by microfluidic procedure appeared as homogeneous unilamellar vesicles (Figure 5B,D).

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Int. J. Mol. Sci. 2020, 21, 1611 A B 7 of 19

A B

C D

Figure 5. Cryogenic transmission electron microscopy (Cryo-TEM) image of liposomes obtained by

thin-film hydration (A and C) and microfluidic (B and D) techniques. A and B: liposomes loaded with dexamethasone hemisuccinate. C and D: dexamethasone free liposomes (plain liposomes). Red Figure 5. Cryogenic transmission electron microscopy (Cryo-TEM) image of liposomes obtained by Figurearrows 5.indicateCryogenic lamellae transmission in multilamellar electron microscopy vesicles, (Cryo-TEM)and yellow imageharrows of liposomesindicate obtainedcalcium- thin-film hydration (A and C) and microfluidic (B and D) techniques. A and B: liposomes loaded with bydexamethasone thin-ﬁlm hydration hemisuccinate (A,C) and nanocrystalline microﬂuidic (complexes.B,D) techniques. Liposomes A and were B: liposomesassembled loaded with 0.2 with M dexamethasone hemisuccinate. C and D: dexamethasone free liposomes (plain liposomes). Red dexamethasonecalcium acetate hemisuccinate.and loaded by Cincubation and D: dexamethasone for one hour freewith liposomes dexamethasone (plain liposomes). hemisuccinate Red arrowsat a 10 arrows indicate lamellae in multilamellar vesicles, and yellow harrows indicate calcium- indicateand 2.5 mg/mL lamellae concentration in multilamellar of lipid vesicles, and drug, and respectively. yellow harrows Size bar: indicate 200 nm. calcium-dexamethasone hemisuccinatedexamethasone nanocrystalline hemisuccinate complexes.nanocrystalline Liposomes complexes. were Liposomes assembled were with assembled 0.2 M calcium with acetate 0.2 M andThecalcium loadedliposome acetate by incubation formulaand loadedtions for by onewere incubation hour fairly with forstable dexamethasone one overhour twowith weeks hemisuccinatedexamethasone incubation at hemisuccinate a 10at androom 2.5 temperature mgat /amL 10 (Figureconcentrationand 6) 2.5. mg/mL of concentration lipid and drug, of respectively.lipid and drug, Size respectively. bar: 200 nm. Size bar: 200 nm.

The liposome formulations200 were fairly stable over two weeks incubation1.0 at room temperature (Figure 6). 0.8 150200 1.0

0.6 0.8 100150

0.4 PDI size (nm) 0.6 10050 0.2 0.4 PDI size (nm)

500 0.0 0.2 0 3 6 24 48 120 168 336 time (hours) 0 0.0 Figure 6. TheThe stability stability profile profile of of liposomes0 liposomes3 6obtained obtained24 48 by by thin120 thin-film-film168 hydration336 hydration (■) (and) and microfluidic microfluidic (■) (procedures) procedures and and the the corresponding corresponding PDI PDI (■ (timeandand ■(hours), respectively)., respectively). The The size size was was measured measured by by DLS. DLS. Liposomes were assembled with 0.2 M calcium acetate and loaded by incubation for one hour with Figure 6. The stability profile of liposomes obtained by thin-film hydration (■) and microfluidic (■) dexamethasone at a 10 and 2.52.5 mgmg/mL/mL concentrationconcentration ofof lipidlipid andand drug,drug, respectively.respectively. The datadata are procedures and the corresponding PDI (■ and ■, respectively). The size was measured by DLS. presented asas meanmean ± SD ( n = 3).3). Liposomes were assembled± with 0.2 M calcium acetate and loaded by incubation for one hour with dexamethasone at a 10 and 2.5 mg/mL concentration of lipid and drug, respectively. The data are presented as mean ± SD (n = 3).

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2.4.2.4. InIn vitrovitro ReleaseRelease TheThe drugdrug release release studies studies carried carried out out under under physiological physiological pH pH and and osmolarity osmolarity showed showed that thethat two the formulationstwo formulations have similarhave similar release release profiles profiles with an with initial an burstinitial in burst the first in the 24 first h followed 24 h followed by a slow by release a slow (Figurerelease7 ).(Figure However, 7). However, the liposomes the liposomes produced byproduced microfluidic by microfluidic techniques technique showed as higher showed burst a higher than theburst ones than produced the ones by produced thin-film by hydration. thin-film In hydration. both cases In the both remaining cases the drug remaining was released drug inwas about released four weeks,in about which four isweeks, consistent which with is consistent the slow dissolution with the slow of the dissolution nanocrystalline of the calcium-dexamethasonenanocrystalline calcium- hemisuccinatedexamethasone complexes hemisuccinate into the complexes vesicles. into the vesicles.

100

cumulative (%) release cumulative 20

0 0 100 200 300 400 500 600 time (hours) FigureFigure 7.7. Dexamethasone releaserelease profilesprofiles ofof liposomesliposomes obtainedobtained byby thin-filmthin-film hydrationhydration ((■)) andand microfluidicmicrofluidic ( (■)) techniques techniques in in 20 20 mM mM HEPES, HEPES, 150 150 mM mM NaCl, NaCl, pH pH 7.4. 7.4. The The data data are are presented presented as as mean mean ± SD± SD (n (=n 3).= 3).

2.5.2.5. InIn vitrovitro BiologicalBiological StudiesStudies TheThein in vitro vitrocytotoxicity cytotoxicity of of dexamethasone dexamethasone loaded loaded liposomes liposomes was was studied studied on ARPE-19 on ARPE retina-19 retina cells, whichcells, which represents represent a consolidateds a consolidated cell model cell formodel dexamethasone for dexamethasone activity assay.activity The assay. retinal The pigment retinal epitheliumpigment epithelium (RPE) undergoes (RPE) undergoes oxidative stress, oxidative which stress induces, which local induces inflammation local inflammation in the eye correlated in the eye to Age-relatedcorrelated to Macular Age-related Degeneration Macular (AMD). Degeneration This is responsible(AMD). This of is severe responsible vision lossof severe in aged vision individuals loss in inaged developed individuals countries in developed [20]. Thus, countries the exploitation [20]. Thus, of the novel exploitation delivery systems,of novel suchdelivery as liposomes, systems, such for theas liposomes, local administration for the local of anti-inflammatory administration of corticosteroidsanti-inflammatory can becorticosteroids beneficial for can the improvementbe beneficial for of existingthe improvement treatments of and existing patient treatments compliance. and patient compliance. TheThe cellcell cultureculture studiesstudies showedshowed thatthat bothboth dexamethasonedexamethasone freefree liposomesliposomes andand dexamethasonedexamethasone loadedloaded liposomesliposomes obtainedobtained byby thin-filmthin-film hydration hydration and and microfluidics microfluidics were were not not cytotoxic. cytotoxic. Indeed,Indeed, 2424 hh ofof cellcell incubation, incubation, which which corresponds corresponds to to the the burst burst release release time, time, did did not elicitnot elicit cytotoxicity cytotoxicity even even at a high at a drughigh concentrationdrug concentration (Figure (Figure8). According 8). According to the literature, to the literature, in the case in the of control case of cells control incubated cells incubated with an equivalentwith an equivalent concentration concentration of free dexamethasone of free dexamethasone hemisuccinate, hemisuccinate, cytotoxicity cytotoxicity was observed was onlyobserved at a highonly drugat a high concentration drug concentration (100 µM) [(10021,22 µM)]. [21,22].

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100

40 Cell vability (%) 20

0 NC H HE M ME DMS Figure 8. The in vitro cytotoxicity profile of dexamethasone hemisuccinate loaded liposomes obtained Figure 8. The in vitro cytotoxicity profile of dexamethasone hemisuccinate loaded liposomes obtained by thin thin-film-film hydration and microfluidic microfluidic procedures. The c concentrationoncentration refers to the dexamethasone hemisuccinate or or equimolar equimolar lipid lipid concentration concentration in inthe the case case of empty of empty liposomes liposomes with withrespect respect to drug to loadeddrug loaded liposomes: liposomes: 1 µM (■ 1),µ 10M( µM), (■ 10), µandM( 100), µM and (■ 100). SampleµM( ).identity: Sample NC, identity: negative NC, control negative-non treatedcontrol-non cells;treated H, dexamethasone cells; H, dexamethasone hemisuccinate hemisuccinate loaded liposomes loaded obtained liposomes by obtained thin-film by hydration; thin-film HE,hydration; dexamethasone HE, dexamethasone free liposomes free liposomes obtained obtained by thin by thin-film-film hydration; hydration; M, M, dexamethasone hemisuccinate loadedloaded liposomes liposomes obtained obtained by microfluidics;by microfluidics; ME, dexamethasoneME, dexamethasone free liposomes free liposomes obtained obtainedby microfluidics; by microfluidics; DMS, dexamethasone DMS, dexamethasone hemisuccinate hemisuccinate in solution. in Thesolution. data areThe presented data are aspresented mean ± asSD mean (n = 3).± SD (n = 3).

The cyt cytofluorimetricofluorimetric study showed that the liposomes obtained either by by thin thin-film-film hydration hydration or by microfluidicmicrofluidic techniquestechniques undergo undergo time time dependent dependent association association to ARPE-19 to ARPE cells-19 (Figurecells (Figure9A,B), which9A,B), whichis in agreement is in agreement with the with literature the literature [23,24]. No[23,24] significant. No significant difference difference in the association in the association of the vesicles of the to vesiclesthe cells to was the detected cells was for thedetected two formulations, for the two indicatingformulations that, indicating the size and that structural the size di ffanderences structural of the differencestwo products of dothe not two a ffproductsect the interaction do not affect with the cells. interaction Confocal with microscopy cells. Confocal showed microscopy that the fluorescein showed thatlabelled the liposomesfluorescein associate labelled to liposomes the cells and associate are mostly to detectedthe cells as and green are fluorescent mostly detected spots either as green in the fluorescentproximity of spots the celleither membrane in the proximity or in the of cytosol the cell (Figure membrane9C,D). o Ther in similarthe cytosol density (Figure in cells 9C,D). treated The similarwith liposomes density obtainedin cells treated by thin-layer with liposomes hydration obtained and microfluidic by thin-layer techniques hydrat agreesion and with microfluidic the results techniquesobtained by agrees the cytofluorimetric with the results analysis. obtained by the cytofluorimetric analysis. The anti-inflammatory activity of the formulations was assessed by assaying the inhibition of pro-inflammatory cytokine, namely Interleukin 6 (IL-6) released by lipopolysaccharide (LPS) stimulation of ARPE-19 cell, induced by 0, 1, and 10 µM dexamethasone loaded liposomes [25]. Empty liposomes produced with the two techniques did not present anti-inflammatory activity (Figure 10) while the dexamethasone-loaded liposomes obtained by thin-film hydration and microfluidic techniques significantly reduced the pro-inflammatory activity induced by LPS. At 1 µM dexamethasone concentration, the liposomes obtained by thin-film hydration procedure and the liposomes obtained by microfluidics were 2.3-times and three-times more efficient than free dexamethasone, respectively. Control plain liposomes (drug free) did not display pro-inflammatory activity on non-stimulated cells (no LPS treatment; Figure S4).

Int. J. Mol. Sci. 2020, 21, 1611 10 of 20 Int. J. Mol. Sci. 2020, 21, 1611 10 of 19

FigureFigure 9. 9.( A(A)) TheThe cytofluorimetriccytofluorimetric profiles profiles of of ARPE ARPE-19-19 cells cells incubated incubated with with fluorescently fluorescently labelled labelled dexamethasonedexamethasone hemisuccinate hemisuccinate loadedloaded liposomes obtained obtained by by thin thin-film-film hydration hydration (A2 (A2 and and A3) A3) and and by by microfluidicmicrofluidic techniques techniques (A5 (A5 andand A6)A6) incubated for for 2 2 (A2 (A2 and and A5) A5) and and 6 6(A3 (A3 and and A6) A6) hours. hours. Untreated Untreated cellscells were were incubated incubated for for 2 (A1) 2 (A1) and and 6 (A4) 6 (A4) hours hours with with culture culture medium. medium. (B) Mean (B) Mean Fluorescence Fluoresce Intensitynce (MFI)Intensity of ARPE-19 (MFI) of cellsARPE incubated-19 cells incubated with fluorescently with fluorescently labelled labelled dexamethasone dexamethasone hemisuccinate hemisuccinate loaded liposomesloaded liposomes obtained obtained by thin-film by thin hydration-film hydration () and microfluidic(■) and microfluidic () techniques (■) techniques and control and control untreated cellsuntreated ()(n =cells3). ( Confocal■) (n = 3). images Confocal of ARPE-19 images cellsof ARPE incubated-19 cells with incubated dexamethasone with dexamethasone hemisuccinate hemisuccinate loaded liposomes obtained by thin-film hydration (C) and microfluidics (D); liposomes loaded liposomes obtained by thin-film hydration (C) and microfluidics (D); liposomes were labelled were labelled with fluorescein-DHPE (green), cell membrane with wheat germ agglutinin-Alexa with fluorescein-DHPE (green), cell membrane with wheat germ agglutinin-Alexa fluor-633 (red), fluor-633 (red), nuclei with DAPI (blue). Bright field is reported in Figure S3. nuclei with DAPI (blue). Bright field is reported in Figure S3. The anti-inflammatory activity of the formulations was assessed by assaying the inhibition of pro-inflammatory cytokine, namely Interleukin 6 (IL-6) released by lipopolysaccharide (LPS) stimulation of ARPE-19 cell, induced by 0, 1, and 10 μM dexamethasone loaded liposomes [25]. Empty liposomes produced with the two techniques did not present anti-inflammatory activity (Figure 10) while the dexamethasone-loaded liposomes obtained by thin-film hydration and microfluidic techniques significantly reduced the pro-inflammatory activity induced by LPS. At 1 μM dexamethasone concentration, the liposomes obtained by thin-film hydration procedure and the liposomes obtained by microfluidics were 2.3-times and three-times more efficient than free dexamethasone, respectively. Control plain liposomes (drug free) did not display pro-inflammatory activity on non-stimulated cells (no LPS treatment; Figure S4).

Int. J. Mol. Sci. 2020, 21, 1611 11 of 20 Int. J. Mol. Sci. 2020, 21, 1611 11 of 19

Figure 10. TheThe inin vitr vitroo ILIL-6-6 release release by byARPE ARPE-19-19 cells cells after after stimulation stimulation with withlipopolysaccharide lipopolysaccharide (LPS) (LPS)and treatment and treatment with withliposomes. liposomes. Cells Cells were were treated treated with with dexamethasone dexamethasone hemisuccinate loadedloaded liposomesliposomes at 11 µµMM( (■)) andand 1010 µµMM( (■)) drugdrug concentrationsconcentrations oror anan equimolarequimolar concentrationconcentration of emptyempty liposomes.liposomes. Abbreviations: NC, negative control (non (non-treated-treated cells); PC, positive control (cells(cells treatedtreated only with LPS); H, pre-stimulatedpre-stimulated cells treated with dexamethasone hemisuccinate loaded liposomes obtained by thin thin-film-film hydration; hydration; HE, HE, pre pre-stimulated-stimulated cells cells treated treated with with empty empty liposomes liposomes obtained obtained by bythin thin-film-film hydration; hydration; M, M, pre pre-stimulated-stimulated cells cells treated treated with with dexamethasone hemisuccinate loadedloaded liposomesliposomes obtainedobtained byby microfluidics;microfluidics; ME,ME, pre-stimulatedpre-stimulated cellscells treatedtreated withwith empty liposomes obtained by microfluidics;microfluidics; and DMS,DMS, pre-stimulatedpre-stimulated cellscells treatedtreated withwith freefree dexamethasonedexamethasone hemisuccinatehemisuccinate in solution.solution. The The data data are are presented presented as m asean mean ± SD (nSD = 3). (n P= >3). 0.0.5,P ns;> 0.0.5, P ≤ 0.05, ns; *;P P ≤ 0.05,0.01, **; *; P ≤ 0.001,0.01, *** **; ± ≤ ≤ Pwith0.001 respect, *** withto respectthe positive to the positivecontrol control(PC). Additional (PC). Additional statistical statistical analysis analysis was was performed performed on ≤ dexamethasone loaded liposomes vs.vs. free dexamethasone hemisuccinate and plain liposomes (drug free)free) obtainedobtained byby thin-filmthin-film hydrationhydration vs.vs. plain liposomes obtained by microfluidics microfluidics (See Table S1S1 inin Supplementary Materials).Materials).

3. Discussion InIn thisthis comparativecomparative study, twotwo liposomeliposome formulationsformulations were produced by microfluidicsmicrofluidics and by conventional thin-filmthin-film hydration.hydration. The The plain plain liposomes were produced using calcium acetate,acetate, as 2+ calcium ionsions inin thethe aqueousaqueous corecore ofof liposomesliposomes establishestablish aa CaCa2+ gradient that provides the entrapment of dexamethasonedexamethasone hemisuccinate, hemisuccinate, the the drug drug model model used used in this in study, this instudy the vesicle, in the [19 vesicle,26,27]. [19,26,27] Although. onlyAlthough certain only drugs certain can bedrugs loaded can inbe liposomes loaded in byliposomes remote loading,by remote the loading, opportunity the opportunity to encapsulate to drugsencapsulate in the drugs liposome in the core liposome by complexation core by complexation with ions represents with ions the represents most efficient the most procedure efficient to ensureprocedure high to payloads. ensure high The firstpayloads. drug to The be encapsulatedfirst drug to by be this encapsulated procedure wasby Doxorubicin,this procedure whose was encapsulationDoxorubicin, whose in liposomes encapsulation was patented in liposomes back in was the patented 1990s [28 back]. Throughout in the 1990s the [28] years,. Throughout other drugs the haveyears been, other successfully drugs have encapsulated been successfully by remote encapsulated loading, by including remote loading, corticosteroids including [27]. corticosteroids According to the[27] physicochemical. According to features,the physicochemical amphipathicity, features, and low acidicamphipathic character,ity, dexamethasone and low acidic hemisuccinate character, isdexamethasone an excellent candidate hemisuccinate for remote is an loadingexcellent into candidate liposomes for [remote29]. loading into liposomes [29]. The lightlight scatteringscattering andand Cryo-TEMCryo-TEM analysis showed that thethe thin-filmthin-film hydrationhydration procedureprocedure yieldsyields largerlarger morphologicallymorphologically heterogeneousheterogeneous multilamellarmultilamellar liposomes,liposomes, whilewhile smallersmaller homogeneoushomogeneous unilamellar vesicles are obtainedobtained with thethe microfluidicmicrofluidic procedure.procedure. This proves that the microfluidicmicrofluidic approach allowsallows forfor aa moremore structure-controlledstructure-controlled formulation.formulation. Furthermore, at at the same lipidlipid concentration, multilamellar liposomes possess a higher amount of lipidlipid moleculesmolecules perper liposomeliposome vesicle with respect to unilamellar liposomes, which account for a smaller aqueous volume normalized for the lipids and, in turn, a lower drug loading capacity (expressed as drug/lipid w/w% ratio).

Int. J. Mol. Sci. 2020, 21, 1611 12 of 20 vesicle with respect to unilamellar liposomes, which account for a smaller aqueous volume normalized for the lipids and, in turn, a lower drug loading capacity (expressed as drug/lipid w/w% ratio). The drug loading was found to be affected by the calcium concentrations used for the liposome preparation. Liposomes obtained by the two preparation procedures showed that the drug loading capacity and efficiency increased as the calcium concentration increased to reach the maximal values at 0.2 M, and then decreased at higher calcium concentrations. This behaviour could be attributed to the hyperosmotic condition of the liposome core when liposomes are dialysed vs. isosmotic buffer to remove non encapsulated calcium acetate, which may alter the membrane permeability and cause partial loss of the calcium acetate of the liposome aqueous core. The time course of drug loading profiles were in agreement with the literature, which reports that the drug loading by remote control occurs in 1 h [19] while a longer incubation may decrease the loading because of slow dissipation of the ion gradient from the liposome core [30]. The incubation of liposomes with increasing concentrations of dexamethasone hemisuccinate yielded a plateau in drug loading capacity, which is achieved with liposomes prepared by using 1.25 mg/mL dexamethasone-hemisuccinate. As a consequence, in the case of liposomes prepared with a higher drug concentration, the drug loading efficiency dramatically decreases. The maximal drug loading capacity with 1.25 mg/mL dexamethasone hemisuccinate may be attributable to the intravesicle calcium consumption by drug complexation. On the other side, when the lipid concentration increases, the loading capacity expressed as loaded drug/lipid w/w% ratio does not increase. Indeed, the increase of lipid concentration results in the increase of concentration of liposome particles but not in the increase of the liposome drug payload (expressed as drug/lipid w/w% ratio). In all preparations with the same lipid concentration, the loading capacity is higher in the case of the liposomes obtained by microfluidics with respect to those obtained by thin-film hydration, which is due to the overall higher intravesical volume of the former monolayer vesicles with respect to the multilayer vesicles produced by thin-film hydration. The dexamethasone entrapment into the liposome core has been observed by Cryo-TEM imaging, which showed dark nanocrystals within the liposome aqueous compartment resulting in “coffee bean” like vesicles, which are similar to the vesicles obtained by remote loading of anionic corticosteroids, as reported in the literature [31]. The structures in the vesicle aqueous core indicate that the calcium dexamethasone hemisuccinate is in a “precipitated form”. The microfluidic procedure was found to produce liposomes with low batch-to-batch variability and higher stability during the drug loading process, resulting in a higher production reproducibility compared to the liposomes produced by thin-film hydration. This evidence confirms that the microfluidic based formulation of liposomes, by virtue of the chaotic advection mixing process into the channels of a staggered herringbone micromixer (SHM) at low shear stress provides a quick and locally controlled process of assembly of the lipids [32]. The in vitro release profiles reflected the structural properties of the liposomes. The burst release of the drug in the first 24 h is attributable to the dexamethasone in solution in the liposome core, which is in equilibrium with the “nanocrystalline form” observed by Cryo-TEM. The higher burst obtained with the liposomes produced by microfluidics with respect to the ones produced by thin-film hydration is likely due to the higher dexamethasone concentration in the liposome aqueous core. Notably, for both the liposome formulations obtained with the two techniques, while the initial fast drug release is dictated by the diffusion of a soluble drug fraction from the aqueous compartment of the vesicles, the second phase of slower release is guided by the dissolution rate of the drug from the nanocrystalline structures in the aqueous core of the liposomes. In vitro cell studies were carried out using retinal pigmented epithelium cells (ARPE-19) to obtain pharmacological information of the two liposomal formulations. The ARPE-19 cell line was selected because dexamethasone has been used to treat inflammation in a variety of ocular diseases including diabetic macular oedema, retinal vein occlusion [33,34], and in combination with anti-Vascular Endothelial Growth Factor (anti-VEGF) agents for the therapy of age related macular degeneration Int. J. Mol. Sci. 2020, 21, 1611 13 of 20

(AMD) [35]. Both formulations were found to be biocompatible under the experimental conditions, even at high drug concentrations, which agreed with the slow release of a major fraction of loaded drug. On the other hand, both formulations showed a high anti-inflammatory performance. The non-direct correlation of the inhibition of IL-6 release by cells with respect to the dexamethasone concentration in the medium is in agreement with the observations reported in the literature [25]. The higher activity of the liposomal formulations with respect to the free drug, despite the partial drug availability of the dexamethasone in the liposomes and the complete availability of free dexamethasone, can be attributed to the liposome capacity to associate to cells as observed by cytofluorimetry and confocal microscopy and intracellularly deliver the anti-inflammatory drug. The better performance of the liposomes obtained by microfluidics with respect to the ones obtained by thin-film hydration can be ascribed to a higher burst release in the first few hours, which can provide a higher intracellular concentration of dexamethasone.

4. Materials and Methods

4.1. Materials Hydrogenated phosphatidyl choline from soyabeans (HSPC, 98%, Tm 52.5 C) was ≥ ◦ received as a gift from Lipoid GmBH (Ludwigshafen, Germany). Cholesterol was purchased from Sigma-Aldrich (St Louis, MO, USA). The glucocorticoid pro-drug dexamethasone hemisuccinate (1,4-Pregnadien-9α-Fluoro-16α-Methyl-11β,17,21-Triol-3,20-Dione 21-Hemisuccinate) was purchased from Steraloids Inc (Newport, RI, USA). Dulbecco’s Modified Eagle’s Medium/nutrient mixture F-12 without glutamine was purchased from Aurogene (Rome, Italy). Penicillin-Streptomycin solution (10,000 units penicillin and 10 mg streptomycin/mL), L-glutamin (200 mM), Trypsin (10 ), Lipopolysaccharides from Escherichia coli O111:B4 × (LPS), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Fetal Bovine Serum (FBS), and Phosphate Buffered Saline (PBS) were purchased from Sigma (St Louis, MO, USA). N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Fluorescein-DHPE) was purchased from Biotium Inc. (Fremont, CA, USA). Wheat germ agglutinin (WGA) Alexa Fluor 633 conjugate was purchased from Molecualr Probes (Eugene, OR, USA). 40,6-Diamidino-2-phenylindole (DAPI) was purchased from Vector laboratories, Inc. (Burlingame, CA, USA). An IL-6 ELISA duo set kit was purchased from R&D systems (Minneapolis, MN, USA). The water used for all experiments was produced with the Millipore Milli-Q purification system (Massachusetts, MA, USA) and filtered sterilized. All chemicals used in this study were of high analytical grade.

4.2. Assembly of Liposomes Dexamethasone hemisuccinate loaded liposomes were formulated with a two-step process: In the first step, calcium acetate loaded liposomes were assembled and then they were incubated with dexamethasone hemisuccinate for remote loading. Calcium acetate loaded liposomes were either fabricated by conventional thin-film hydration or by a microfluidic process. Thin-film hydration process. Liposomes were prepared according to the thin-film hydration process reported in the literature [1]. Briefly, a stock solution of HSPC and cholesterol in CHCl3 was prepared at a concentration of 6.67 mg/mL. A 2:1 HSPC/Cholesterol molar ratio solution was generated in a 10 mL round bottom flask by mixing two stock solutions (2405.2 µL of HSPC and 593.3 µL cholesterol resulting in 20 mg lipid). The organic solvent was removed under reduced pressure at 37 ◦C using a rotary evaporator. Then the lipid film was hydrated with 1 mL of calcium acetate solution in MQ water at different concentrations (a 0.1–1 M range). The lipidic vesicle suspension underwent 10 freeze-and-thaw cycles and extrusion after each cycle through a polycarbonate membrane with Int. J. Mol. Sci. 2020, 21, 1611 14 of 20 pores of 100 nm cut-off diameter using an Avanti mini extruder (Avanti Polar Lipids Inc. Alabaster, AL, USA). Microfluidic process. Liposomes were also prepared using a NanoAssemblr® Benchtop system (Precision Nano System, Vancouver, BC, Canada). Briefly, a 2:1 HSPC/Cholesterol molar ratio mixture in methanol was generated at a concentration of 30 mg/mL. Calcium acetate solution in milliQ water in the concentration range of 0.1–1 M was used as aqueous phase. Liposomes were assembled by processing 1 mL of the lipidic organic solution and 1.5 mL of the calcium acetate solution using a benchtop NanoAssembler system equipped with a Staggered Herringbone Mixing cartridge resulting into liposome assembly by nano-precipitation [36]. The Total volume (TV), Flow rate ratio (FRR) between the aqueous and organic stream, and total flow rate (TFR) were set to 2.5 mL, 17 mL/min, and 1.5:1, respectively, to generate calcium acetate liposomes. Methanol was removed from the formulations by rotary evaporation under reduced pressure for 1 6 min at 35 ◦C. The complete removal of methanol was confirmed by H NMR analysis with a Bruker 400 MHz NMR Avance spectrometer (Billerica, MA, USA).

4.3. Remote Loading of Dexamethasone Hemisuccinate We dialyzed 1 mL of freshly prepared calcium acetate loaded liposomes formulated by thin-film hydration or microfluidic procedures (20 mg/mL lipids) against 1 L of 20 mM HEPES, 150 mM NaCl, pH 7.4 overnight using a 100 Kda float-a-lyzer to remove non loaded calcium acetate and generate a calcium gradient. The resulting lipid concentration after dialysis was assessed by the Stewart assay [37]. Liposomes were diluted with the same buffer when needed. A 5 mg/mL dexamethasone hemisuccinate stock solution was prepared in 20 mM HEPES, 150 mM NaCl, pH 7.4. Incubation time effect on loading. A dedicated study was performed to elucidate the effect of the incubation time on remote loading. We pre-incubated 500 µL volume of 5 mg/mL dexamethasone hemisuccinate solution and 500 µL samples of 5 mg/mL calcium acetate loaded liposomes (obtained using 0.2 M calcium acetate) at 65 ◦C for 5 min and then mixed them together. The resulting liposomes/dexamethasone hemisuccinate mixture was incubated for 20, 40, 60, or 120 min at 65 ◦C and then cooled down at 4 ◦C. Non-loaded dexamethasone hemisuccincate was removed by dialysis against 1 L of 20 mM HEPES, 150 mM NaCl, pH 7.4 overnight using a 100 KDa float-a-lyzer. Lipid/dexamethasone hemisuccinate ratio effect on loading. 500 µL volume of dexamethasone hemisuccinate solutions at increasing concentrations from 0.5, to 5 mg/mL and 500 µL samples of 5 mg/mL calcium acetate loaded liposomes (obtained using 0.2 M calcium acetate) were pre-incubated at 65 ◦C for 5 min and then each of the dexamethasone hemisuccinate solutions was added and mixed to one liposome sample and mixtures were incubated for 60 min at 65 ◦C and then cooled down at 4 ◦C. Additionally, 500 µL of 5 mg/mL dexamethasone hemisuccinate solutions and calcium acetate loaded liposomes (obtained using 0.2 M calcium acetate) at increasing lipid concentrations from 5 to 20 mg/mL were pre-incubated at 65 ◦C for 5 min. Then the dexamethasone hemisuccinate solution was added and mixed to each of the liposome samples and mixtures were incubated for 60 min at 65 ◦C and then cooled down at 4 ◦C. Non-loaded dexamethasone hemisuccincate was removed by dialysis as reported above. The concentration of lipids after dialysis was assessed by a Stewart assay. A validation assay of the dialysis process was performed by dialysing a 2.5 mg/mL solution of dexamethasone hemisuccinate against 1 L of 20 mM HEPES, 150 mM NaCl, pH 7.4 with the same procedure.

4.4. Loading Efficiency and Capacity Assessment The dexamethasone hemisuccinate loaded into liposomes was quantified by spectrophotometric analysis. We diluted 20 µL of drug loaded liposomes with 800 µL of methanol and sonicated for 30 min in a water bath sonicator to disassemble the liposomes and dissolve the drug. Afterward, Int. J. Mol. Sci. 2020, 21, 1611 15 of 20 dexamethasone concentration was assessed by spectrophotometric analysis at 242 nm. All experiments were performed in triplicate. The loading capacity and loading efficiency were calculated using the following formula:

Loading Capacity (lipid/drug w/w) (%) = (1) [Dexamethasone encapsulated in liposomes (mg/mL) Lipids (mg/mL)] 100 ÷ × Loading Eﬃciency (w/w) (%) = (2) [Dexamethasone encapsulated in liposomes (mg) processed Dexamethasone (mg)] 100 ÷ × 4.5. Size Analysis and Stability The size and poly-dispersity index (PDI) of liposomal formulations were measured by photo correlation spectroscopy (PCS) using a Zetasizer NanoZS (Malvern Instrument LTD, Malvern, UK). The liposomes were diluted before analysis with 20 mM HEPES, 150 mM NaCl, pH 7.4 to a concentration of 0.5 mg/mL. The size analysis was based on the Intensity. The liposome colloidal stability was performed by incubating 10 mg/mL drug loaded liposomes in 20 mM HEPES, 150 mM NaCl, pH 7.4 at 25 ◦C for 14 days. The liposomes were diluted to 0.5 mg/mL with 20 mM HEPES, 150 mM NaCl, pH 7.4 at scheduled time points before analysis and the size and poly dispersity index (PDI) were measured as reported above.

4.6. Cryogenic Transmission Electron Microscopy (Cryo-TEM) We applied 3.5 µL of dexamethasone hemisuccinate loaded liposomes and dexamethasone hemisuccinate free liposomes at a 10 mg/mL lipid concentration on copper 300-mesh Quantifoil R2/1 holey carbon grid, previously glow discharged for 30 s at 30 mA using a GloQube system (Quorum Technologies, Laughton, UK). After 60 s, the grid was plunge-frozen in liquid ethane using a Vitrobot Mk IV (Thermo Fisher Scientific, Waltham, MA, USA) operating at 4 ◦C and 100% Relative Humidity (RH). Images of the vitrified specimens were acquired using a Talos Arctica transmission electron microscope operating at 200 kV and equipped with a CETA 16M camera (Thermo Fisher Scientific, Waltham, MA, USA). Images with applied defocus values between 2 and 4 µm were acquired with a − − total exposure time of 1 s and a total accumulated dose of 16 electrons per A2 at nominal magnifications of 22,000 , corresponding to a pixel size of 4.7 Å/pixel at the specimen level. × 4.7. Release Study We transferred 1 mL of 10 mg/mL freshly prepared dexamethasone hemisuccinate loaded liposomes in 20 mM HEPES, 150 mM NaCl, pH 7.4 into a 20 KDa MWCO Spectra/Por® Float-ALyzer® and dialyzed against 2 L of the same buffer. The buffer was thermostated at 37 ◦C throughout the study. At scheduled time intervals, 10 µL of liposomes were withdrawn and after dilution in 500 µL of methanol dexamethasone, the concentration was assessed by spectrophotometric analysis. The release study was carried out for 30 days. The released percentage of dexamethasone was plotted versus time.

4.8. In vitro Cytotoxicity The in vitro cytotoxicity of dexamethasone hemisuccinate loaded liposomes was performed by incubating the human retinal pigmented epithelia cell line (ARPE-19) with the formulations. ARPE-19 cells were cultured at 37 ◦C, in 5% CO2 atmosphere, using DMEM/F12 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS, 2 mM L-Glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin. The cytotoxicity study was performed by seeding ARPE-19 cells at 3 104 cells/well in a 96-well plate; the cells were grown for 24 h in a humidified 5% CO atmosphere at × 2 37 ◦C. Then the medium was discharged, the cells were washed two times with PBS and incubated with 200 µL of liposomes or free dexamethasone hemisuccinate in DMEM/F12 without FBS at equivalent drug concentrations of 1, 10, and 100 µM. Drug free liposomes at equivalent concentrations of lipids Int. J. Mol. Sci. 2020, 21, 1611 16 of 20 were used as a negative control. After 24 h, the medium was removed and the cells were washed carefully with PBS and then incubated with 200 µL/well of complete medium containing 20 µL of 5 mg/mL MTT solution in PBS for three hours at 37 ◦C. Afterward, the medium was removed and 200 µL/well of DMSO was added. After 30 min the plate was read spectrophotometrically with a microplate reader (Microplate autoreader, EL311SK, Biotek Inc, Winooski, VT, USA) equipped with a 570 nm light filter. The analysis was performed in triplicate and cell viability was expressed as percentage referring to non-treated cells.

4.9. In vitro Cell Association of Liposomes Cytofluorimetric study. ARPE-19 cells (2 105/well) were seeded on a 24-well plate in DMEM/F12 × supplemented of 10% FBS and grown for 24 h. The medium was then removed, and the cells were washed twice with PBS. We added 500 µL/well of a 0.5 mg/mL of dexamethasone loaded liposomes labelled with 0.3 mol% of fluorescein-DHPE with respect to the lipids. The cells were incubated with liposomes for 2 and 6 h. Afterward, the liposome containing medium was removed, and the cells were washed twice with PBS and then with PBS added of 10% FBS. Cells were then detached by treatment with 200 µL/well of 0.125 mg/mL trypsin for 8 min at 37 ◦C and then transferred in FACS tubes containing 200 µL PBS buffer, pH 7.4, 0.5% BSA, 5 mM EDTA, 2 mM NaN3, 4% PFA. Cells were stored at 4 ◦C in the dark prior to analysis. Samples underwent cytofluorimetric analysis using a BD FACS CANTOTM II (BD Biosciences, San Jose, CA, USA) and the fluorescence deriving from Fluoroscein-DHPE labelled liposomes was detected using a laser emitting at 496 nm. Confocal microscopy. ARPE-19 cells (8 104/well) were seeded on 13 mm2 cover slips introduced × in a 24-well plate using DMEM/F12 with 10% FBS and incubated for 24 h. The medium was then removed, and the cells were washed twice with PBS. We added 300 µL of a 0.5 mg/mL of dexamethasone loaded liposomes labelled with 0.3 mol% of fluorescein-DHPE with respect to the lipids in FBS free medium to each well, and the cells were incubated for 6 h. Afterward, the medium was removed, and the cells were washed two times with PBS and two times with PBS added of 10% FBS. The cells were then fixed by 20 min of treatment with 200 µL/well of PBS buffer, pH 7.4, containing 4% (w/v) PFA. The solution was then discharged, and the cells were washed three times with PBS. The staining of cell nuclei was performed by incubating the samples with 200 µL of a 5 µg/mL solution of DAPI in PBS for 10 min. After discharging the solution, the cell membranes were labelled by incubating the cells with 200 µL/well of a 5 µg/mL solution of Wheat germ agglutinin-Alexa fluor-633 conjugate in PBS for 10 min. The solution was then discharged, and the cells were washed three times with PBS and two times with mQ water. The coverslips were mounted on a glass slide using a mounting medium prepared with 10 w/v% of Mowiol®4-88 (Sigma-Aldrich (St Louis, MO, USA) in a 1:3.8 glycerol/0.13 M Tris-HCl buffer pH 8.5 v/v ratio mixture. The samples were maintained at 4 ◦C in the dark until microscopic examination. The cell samples were imaged using a Zeiss LSM 800 confocal microscope (Carl Zeiss, Jena, Germany) equipped with a 63 , n.a. 1.4, oil immersion objective and a ZEN 2.1, blue edition software × (Carl Zeiss, Jena, Germany). Lasers with emission wavelengths at 405, 488, and 640 nm were used to detect DAPI, fluorescein-DHPE, and WGA-Alexa Fluor-633, respectively. To avoid emission crosstalk, each emission fluorescence was recorded independently with a specific detector and optical cut-off filter over the entire emission spectrum of related chromophores. Image analyses were performed using ImageJ 1.47v (National Institutes of Health software package by Wayne Rasband; Bethesda, MD, USA).

4.10. In vitro Anti-inflammatory The anti-inflammatory activity of dexamethasone hemisuccinate loaded liposomes was investigated by assessing the inhibition of IL-6 release by LPS stimulated ARPE-19 cells. Cells were seeded at a 1.2 105 cells/well density in a 24-well plate with complete medium and grown for × 24 h in a humidified 5% CO2 atmosphere at 37 ◦C. Then, the medium was discharged, the cells were Int. J. Mol. Sci. 2020, 21, 1611 17 of 20 washed two times with PBS and incubated with 500 µL/well of 5 µg/mL LPS in DMEM/F12 medium without FBS for 12 h. The LPS containing medium was then removed and the cells were washed with PBS and 500 µL/well of dexamethasone hemisuccinate loaded liposomes or free drug at 1 and 10 µM in DMEM/F12 medium without FBS was added. Drug free liposomes at equivalent concentrations of lipids were then used as a control. The cells were incubated with the samples for 24 h. The medium of each well was collected, centrifuged at 13,000 rpm for 8 min, and analysed with human IL-6 ELISA Duo Set kit (R&D systems, Inc, Minneapolis, MN, USA) according to the procedure reported by the provider to quantify the concentration of IL-6 released by the cells upon suitable dilutions. In order to assess the potential of the carrier to induce inflammation, non-stimulated cells (no LPS treatment) were incubated with plain liposomes (no drug loaded) at lipid concentrations equivalent to those of the 1 and 10 µM dexamethasone hemisuccinate loaded liposomes for 12 h. Afterward, the liposomes containing medium was removed, the cells were washed with PBS and further incubated with medium without FBS for 24 h. The cell supernatants were then tested as reported above.

5. Conclusions The comparative study reported here attempted to fill, at least in part, the lack of information regarding the exploitation of microfluidics in liposome production in comparison to traditional techniques, in view of pharmaceutical scaling developments. This study shows that the two procedures investigated for liposome preparation, namely thin-film hydration and microfluidics, yield liposomes with different colloidal features. The drug loading and release profile of the vesicles are affected by the different lamellarity, size, and internal aqueous volume of the liposomes obtained with the two processes rather than by the drug loading conditions. With respect to the traditional techniques, microfluidics offer an opportunity to significantly improve the quality of the pharmaceutical product by providing reproducible liposome production, including reduction of the post-production processing steps (i.e., sonication, extrusion, freezing, and thawing) to generate liposomes with desirable characteristics of size and homogeneity, which conceivably results in time savings. The industrial production of pharmaceutical grade products can exploit microfluidic equipment operating under Good Manufacturing Practice (GMP) conditions and can be designed to translate small batch to high-throughput productions [38]. However, comparative and extensive cost analyses for industrial production generating nanopharmaceutics for clinical applications are not yet available.

Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/5/1611/s1. Author Contributions: M.A.-A.: investigations, experimental execution, writing—original draft; F.M.: experimental execution and data analysis; M.G.: experimental execution, and data analysis; F.B.: sample experimental execution; A.M.: sample experimental execution; P.C.: data discussion, grant management, experimental supervision, writing contributions; S.S.: conceptualization, supervision of experimental operations, draft preparation. All authors have read and agreed to the published version of the manuscript. Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement N◦ 722717. Federica Bellato was supported by the ARD-B fellowship from BIRD 2019 grant scheme of the University of Padova. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations

TFR Total Flow rate TV Total Volume FRR Flow Rate Ratio PDI Poly Dispersity Index Cryo-TEM Cryogenic Transmission Electron Microscopy AMD Age Related Macular Degeneration RPE Retinal Pigmented Epithelium Int. J. Mol. Sci. 2020, 21, 1611 18 of 20

LPS Lipopolysaccharide IL-6 Interleukin-6 ELISA Enzyme Linked Immune Sorbent Assay EPR Enhanced Permeability and Retention RES Reticuloendothelial System E. coli Escherichia Coli MWCO Molecular Weight Cut-off HSPC Hydrogenated Soybean Phosphatidyl Choline DMEM/F12 Dulbecco’s Modified Eagle’s Medium F12 FBS Fetal Bovine Serum MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide PBS Phosphate Buffer Saline HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid DMSO Dimethyl Sulfoxide PFA Paraformaldehyde

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