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Journal of Pharmaceutical and Biomedical Analysis 145 (2017) 682–691

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

j o ur nal ho me page: www.elsev ier .com/ lo cate / jpba

hase separation of in situ forming poly (lactide-co-glycolide acid)mplants investigated using a hydrogel-based subcutaneous tissueurrogate and UV–vis imaging

u Sun, Henrik Jensen, Nickolaj J. Petersen, Susan W. Larsen, Jesper Østergaard ∗

epartment of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark

r t i c l e i n f o

rticle history:eceived 10 June 2017eceived in revised form 30 July 2017ccepted 31 July 2017vailable online 5 August 2017

eywords:ydrogel

n situ forming implantshase separation

a b s t r a c t

Phase separation of in situ forming poly (lactide-co-glycolide acid) (PLGA) implants with agarose hydro-gels as the provider of nonsolvent (water) mimicking subcutaneous tissue was investigated using a novelUV–vis imaging-based analytical platform. In situ forming implants of PLGA-1-methyl-2-pyrrolidinoneand PLGA-triacetin representing fast and slow phase separating systems, respectively, were evaluatedusing this platform. Upon contact with the agarose hydrogel, the phase separation of the systems wasfollowed by the study of changes in light transmission and absorbance as a function of time and position.For the PLGA-1-methyl-2-pyrrolidinone system, the rate of spatial phase separation was determined andfound to decrease with increasing the PLGA concentration from 20% to 40% (w/w). Hydrogels with dif-ferent agarose concentrations (1% and 10% (w/v)) were prepared for providing the nonsolvent, water,

oly (lactide-co-glycolide acid) (PLGA)V–vis imaging

to the in situ forming PLGA implants simulating the injection site environment. The resulting implantmorphology depended on the stiffness of hydrogel matrix, indicating that the matrix in which implantsare formed is of importance. Overall, the work showed that the UV–vis imaging-based platform withan agarose hydrogel mimicking the subcutaneous tissue holds potential in providing bio-relevant andmechanistic information on the phase separation processes of in situ forming implants.

© 2017 Elsevier B.V. All rights reserved.

. Introduction

Nonsolvent induced phase separation processes findidespread application in, for instance, tissue engineering

1,2], purification [3,4] and in drug delivery research [5,6]. Drugelivery systems, known as in situ forming implants (ISFIs), haveecently attracted much attention since they may provide a lessnvasive and less painful means to insert a controlled release depotnto the target site as compared to pre-formed implants [5,6].

hese systems can be injected in form of a liquid, which transformso a solid or semi-solid depot in situ via phase separation. Theommercially available products, Atridox

®[7] and Eligard

®[8],

Abbreviations: EPR, Electron paramagnetic resonance; ISFIs, In situ formingmplants; NMP, 1-methyl-2-pyrrolidinone; PLGA, Poly (lactide-co-glycolide acid);EM, Scanning electron microscopy; TA, Triacetin; UV, Ultraviolet; Vis, Visible.∗ Corresponding author.

E-mail addresses: yu.sun@sund.ku.dk (Y. Sun), henrik.jensen@sund.ku.dkH. Jensen), nickolaj.petersen@sund.ku.dk (N.J. Petersen), susan.larsen@sund.ku.dkS.W. Larsen), jesper.ostergaard@sund.ku.dk (J. Østergaard).

ttp://dx.doi.org/10.1016/j.jpba.2017.07.056731-7085/© 2017 Elsevier B.V. All rights reserved.

employ PLGA or poly (lactide acid) (PLA) as the biodegradablepolymer dissolved in the biocompatible water miscible solvent1-methyl-2-pyrrolidinone (NMP). Upon exposure of the polymer-solvent-drug mixture to a nonsolvent, such as water or tissuefluid, the organic solvent diffuses into the surrounding aqueousphase and water penetrates into the ISFIs. This leads to phaseseparation and precipitation of the hydrophobic polymer in situ.Herein, phase separation refers to the process where the polymerseparates from the solution phase, which eventually may lead tothe formation of a solid implant at the injection site capable ofsustaining drug release. Hence, a thorough understanding of thephase separation process is paramount since it determines thedrug release characteristics and the therapeutic effects.

So far, several techniques have been applied to characterizethe phase separation process. Polymer-solvent-nonsolvent ternaryphase diagrams serve as a basic tool to describe the thermodynam-ics of nonsolvent induced phase separation [9]. Scanning electron

microscopy (SEM) [10,11], cryo-SEM [12,13], light microscopy[14,15], light transmission [16–18] or turbidity measurements[19,20] have all been applied to visualize membrane structure

Y. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 145 (2017) 682–691 683

Fig. 1. Schematic representation of the UV–vis imaging setup used at 550 nm with an agarose hydrogel matrix. Internal dimensions of the quartz cell: 8.0 × 38.0 × 1.0 mm3

(W × L × H). Light path: 1.0 mm. The width of the formulation reservoir was 2.0 mm.

Fig. 2. Representative absorbance maps obtained by imaging at 550 nm in 1% (w/v) agarose hydrogel upon addition of (A) 20% (w/w) PLGA in NMP, (B) 40% (w/w) PLGAin NMP and (C) 20% (w/w) PLGA in TA solution. The contour offset and intervals were 100 mAU. The location of the interfaces is indicated by arrows. Absorbance–positionp ntal bw d by ts re lege

onmmrpp

rofiles at selected time points shown in Fig. 3 were obtained from the black horizoere obtained as the mean intensity value of a 0.02 mm × 2 mm area, as examplifie

eparation kinetics study. (For interpretation of the references to colour in this figu

r measure the time of onset of phase separation. These tech-iques are invaluable in revealing the underlying phase separationechanisms, thus, guiding the preparation of suitable polymerembranes. For drug delivery vehicles, the water penetration

ate and phase separation rate are considered as fundamentalarameters since they govern the arrangement of water-rich andolymer-rich domains in the polymer matrix which can influence

ar shown in (A). Intensity–time profiles at specific distances from the left interfacehe red vertical bars at x = 0.4 or 0.8 mm from the left interface in (A) for the phasend, the reader is referred to the web version of this article.)

the drug release rate [5,6]. However, the above mentioned tech-niques can not provide such detailed information. In this regard,McHugh and co-workers developed an in vitro dark ground imagingmethod which allowed real-time imaging of water penetration into

PLGA solutions and liquid-liquid demixing [21,22]. Dark groundimaging is instrumental in establishing a fundamental knowledgeon the relationship between phase separation and drug release;

6 and Biomedical Analysis 145 (2017) 682–691

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icttwarnIhcUmTaaapihtppodaIfwTsomtt1mp

Fig. 3. The absorbance-position profiles generated along the horizontal rectangular

84 Y. Sun et al. / Journal of Pharmaceutical

owever, the technique is limited to short time scales (up to a cou-le of hours) as compared to the time span of the ISFIs, which mayelease drug over weeks to months [6]. Electron paramagnetic reso-ance (EPR) and ultrasound imaging are applicable both in vitro and

n vivo. EPR quantifies the exchange rate of solvent and nonsolvent23,24]. Ultrasound imaging visualizes the implant performancever the lifespan of ISFIs and allows quantification of phase separa-ion kinetics [25–27]. A critical point in formulation development ofSFIs is the limited availability of suitable in vitro models capable ofccurately predicting their bioperformance. Such methods are cur-ently lacking for parenteral depots. The in vitro setups utilized byhe aforementioned imaging methods rely on different geometricnd hydrodynamic conditions and certain techniques, such as EPR,equire specific sample preparation methods. This makes compar-son of the obtained results challenging. Moreover, the disparityetween the in vitro and in vivo behavior of ISFIs was reported inecent studies [28–30]. An increase in phase separation and initialrug release rates was found after subcutaneous injection of theLGA-based ISFIs as compared to that observed in buffer solution29]. The implant size, shape and the rate of polymer precipitationlso varied for PLGA-NMP systems exposed to various tissue envi-onments (e.g. necrotic, non-necrotic, or ablated tumor tissue) [28].hese findings emphasize the importance of the external environ-ent in determining the implant formation process of ISFIs.We hypothesize that essential bio-relevant, mechanistic

nformation on phase separation may be obtained using a well-ontrolled and reproducible hydrogel matrix as a model of softissue. Agarose hydrogels have been proposed as a subcutaneousissue surrogate due to their resemblance with respect to highater content and porous network [31] and has also been used

s tissue phantom in ultrasound imaging [25–27]. However, theole of agarose gel as a tissue surrogate to simulate tissue stiff-ess at the injection site for ISFIs remains to be fully explored.

t is known that the pore size and the stiffness of the agaroseydrogel matrix can be modified through alteration of the agaroseoncentration [32]. UV–vis imaging uses the native characteristicV absorbance to generate spatially and temporally resolved infor-ation on drug transport in a UV transparent medium [33,34].

he key feature of UV–vis imaging is the real-time monitoring ofbsorbance and, hence, concentration changes in the vincinity of

formulation, which makes it potentially suited for both evalu-ting the initial phase separation of ISFIs and drug release. Ourrevious work has demonstrated the potential of UV–vis imag-

ng in characterization of drug diffusion, dissolution and release inydrogel matrixes [35–38]. Motivation for the present study waso explore whether UV–vis imaging could be used to characterizehase separation of in situ forming PLGA implants and to assessotential advantages and limitations of the technique relative tother characterization methods. The aim of the research was toevelop a UV–vis imaging-based in vitro platform capable of char-cterizing and predicting the bioperformance of polymer-basedSFIs in a hydrogel matrix mimicking the subcutaneous tissue. Toocus only on the phase separation process, the drug substanceas excluded from the delivery vehicle in these initial studies.

he specific aims of the present work were i) to investigate theuitability of using UV–vis imaging to monitor phase separationf PLGA-based ISFIs with an emphasis on the solvent properties (1-ethyl-2-pyrrolidinone (NMP) or triacetin (TA)), and ii) to examine

he significance of using an agarose hydrogel as the provider ofhe nonsolvent including the effect of agarose concentration (using% or 10% (w/v) agarose hydrogel) on implant formation. Lighticrosopy and scanning electron microscopy (SEM) were used to

rovide necessary supporting information.

black bar indicated in Fig. 2 (A) at selected time points: (A) 20% (w/w) PLGA in NMP,(B) 40% (w/w) PLGA in NMP and (C) 20% (w/w) PLGA in TA.

2. Materials and methods

2.1. Materials

Agarose (type I, gel point (1.5% (w/v) agarose in water):34.5–37.5 ◦C; sulfate content: ≤ 0.15%), 1-methyl-2-pyrrolidinone(NMP) and triacetin (TA) were obtained from Sigma–Aldrich (St.Louis, MO, USA). PLGA (poly (lactide-co-glycolide acid) with lac-tide: glycolide molar ratio of 75:25, molecular weight: 20 kDa)

was purchased from Wako Pure Chemical Industries, Ltd. (Neuss,Germany). Sodium hydroxide and sodium dihydrogen phosphatemonohydrate were obtained from Merck (Darmstadt, Germany).

Y. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 145 (2017) 682–691 685

F (B) 40h ydrog

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ig. 4. Light microscope images of pre-formulations of (A) 20% (w/w) PLGA in NMP,

ydrogel and of (D) 20% (w/w) PLGA in NMP upon contact with 10% (w/v) agarose h

ll chemicals were used as received. Milli-Q water (Millipore, Bed-ord, MA) was used for preparing the solutions.

.2. Preparation of pre-formulations for in situ forming PLGAmplants

The blank pre-formulations for in situ forming PLGA implantsere prepared by adding PLGA to NMP or TA in a glass vial, followed

y mixing overnight at room temperature until the PLGA was com-letely dissolved. The resulting PLGA solutions were stored at 4 ◦C.he compositions of the pre-formulations of PLGA implants usedn the present work were 20% (w/w) and 40% (w/w) PLGA in NMPnd 20% (w/w) PLGA in TA.

.3. Preparation of agarose hydrogels

Agarose hydrogels were prepared by suspending a weighedmount of agarose powder, corresponding to 1% or 10% (w/v),n phosphate buffer solution (67 mM, pH 7.4) followed by heat-ng of the agarose suspension to 98 ◦C for approximately 30 min

o achieve complete dissolution of the agarose. Approximately00 �l of the agarose solutions were transferred to the quartzells (8.0 mm × 1.0 mm × 38 mm (W × H × L)) (Starna Scientific Ltd.,ainault, Essex, UK), and the lid of the quartz cells were placed on

% (w/w) PLGA in NMP (C) 20% (w/w) PLGA in TA upon contact with 1% (w/v) agaroseel using the quartz cell with 0.2 mm path length (100 × magnification).

top of the agarose solutions. The quartz cells containing the pre-gelswere left at room temperature for at least 1 h to ensure completegelation.

To study the formation of the PLGA implants in the agarosehydrogel, a cut of 2 mm in width was made in the middle ofthe hydrogel to act as a formulation reservoir. The experimentswere initiated by slowly pipetting the pre-formulations for in situPLGA implants formation into the formulation reservoir followedby placement of the lid on the cells while avoiding the entrapmentof air bubbles.

2.4. UV–vis imaging of PLGA implant formation process and dataanalysis

The UV–vis imaging measurements were performed using anActiPix D100 UV Area Imaging System (Paraytec Ltd, York, UK)(Fig. 1). The total imaging area of the UV–vis imaging system is9 mm × 7 mm consisting of 1280 × 1024 pixels (7 × 7 �m2 pixelsize); the pixels were binned 4 × 4 for increased sensitivity. Thelight source was a pulsed Xenon lamp and imaging was performed

at a wavelength of 550 nm using an integration time of 10 msand a recording rate of 5.4 images per min. All experiments wereconducted at ambient temperature (20–23 ◦C) and performed intriplicate.

686 Y. Sun et al. / Journal of Pharmaceutical and Bi

Fig. 5. (A) Representative profiles of light intensity changes as a function of time forthe pre-formulations containing 20% (w/w) PLGA in NMP and 20% (w/w) PLGA in TAat specific distances from the left interface. Data were obtained as the mean intensityvalue of a 0.02 × 2 mm2 area as exemplified by the red vertical bars at the distancesof 0.4 and 0.8 mm from the left interface highlighted in Fig. 2 (A). The arrows andtimes marked in Fig. 5 (A) indicate the critical time where the light intensity startedto decrease. (B) A plot of the square of distance (mm2) versus the critical time (min)obtained from light intensity-time profiles for the pre-formulations containing 20%air

t5htgtr(s[rc

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nd 40% (w/w) PLGA in NMP as shown in Fig. 5(A). Experiments were performedn triplicate. (For interpretation of the references to colour in this figure legend, theeader is referred to the web version of this article.)

UV–vis imaging was performed as follows: dark images (lampurned off: 10 s) and reference images with the light source of50 nm (10 s) were recorded with the quartz cell filled with agaroseydrogel and set as transmission = 100%. After 60 s of data collec-ion, UV–vis imaging was paused. The cut was made in the agaroseel followed by addition of the pre-formulation into the formula-ion reservoir (as described above), after which data collection wasesumed. The images were recorded using Actipix D100 softwareversion 1.4, Paraytec Ltd). The imaging data were read in inten-ity mode or converted to absorbance using the Actipix software39]. Pipetting of the pure solvents NMP or TA into the formulationeservoir was also performed and subjected to UV–vis imaging asontrol experiments.

.5. Light microscopy

Light microscope images were obtained at different time points

fter adding the pre-formulations to the quartz cell (0.2 mm or.0 mm light path, Starna Scientific Ltd., Hainault, Essex, UK)sing an Axiolab microcope (Carl Zeiss Microscopy GmbH, Jena,ermany) with a digital camera attached (Moticam 10, Motic

®, Xia-

omedical Analysis 145 (2017) 682–691

men, China) at 50 x or 100 x magnification. The moving distance ofthe precipitation front was determined using the Motic Image Plus2.0 software to quantify phase separation time.

2.6. Scanning electron microscopy (SEM)

The morphologies of the PLGA implants resulting from the pre-formulation containing 20% (w/w) PLGA in NMP were visualizedusing SEM (HITACHI, TM3030, Tabletop Microscope). The obtainedimplants were collected from the formulation reservoir 7 days aftersolidification to ensure the complete removal of NMP followed byvacuum drying overnight. In order to visualize the inner structureof the implant, the dried implants were cut manually to obtain alongitudinal cross-section of the samples. The samples were placedonto sticky carbon tapes and mounted on metallic stubs, followedby sputter coating with gold. The specimens were then imagedoperating the SEM at an accelerating voltage of 15 kV.

3. Results and discussion

Two types of phase separating systems have been recognizedbased on their rate of phase separation [5]. Fast phase separatingsystems, such as the PLGA-NMP-water system, result in formationof polymer membranes with a thin top layer followed by a poroussublayer. Slow phase separating systems, including the PLGA-TA-water system, lead to a homogenous dense membrane with limitedpore formation. In the present study, an agarose hydrogel-basedsetup (shown in Fig. 1) is applied to simulate the in vivo envi-ronment after subcutaneous administration of fast and slow phaseseparating PLGA-based in situ forming implants with the agarosehydrogel serving as the provider of the nonsolvent (water). In vivoISFIs are injected into the tissue using a syringe. Ideally, this shouldbe reflected in an in vitro setup. However, initial studies showedthat injection of the PLGA solutions into the hydrogel led to theformation of an irregularly shaped polymer matrix. The currentsetup containing a 2 × 8 mm2 rectangular formulation reservoir inthe hydrogel matrix serves to minimize variation caused by implantshape facilitating the subsequent data analysis and interpretation,albeit at the expense of sacrificing some degrees of biorelevance.

3.1. Monitoring of phase separation by UV–vis imaging and lightmicroscopy

Phase separation of the two polymer-solvent systems, PLGA-NMP and PLGA-TA, in the hydrogel matrixes was studied usingUV–vis imaging, light microscopy and SEM. Similar to previousmeasurements of light transmission or turbidity for characteriza-tion of phase separation [17–20,40], UV–vis imaging performedat 550 nm measured changes in light intensity during the initialtransformation of pre-formulation from a transparent liquid stateto a semi-solid or solid depot as a function of time and position.The recorded absorbance maps (Fig. 2) were processed to provideabsorbance-position profiles as shown in Fig. 3 for monitoring andquantifying phase separation of the 20% (w/w) and 40% (w/w) PLGAin NMP and 20% (w/w) PLGA in TA model systems. Light microscopywas used as a supporting technique to observe the phase separationprocess. For these experiments, a quartz cell with a 0.2 mm lightpath was used since the shorter light path allowed an improvedvisualization of the evolving microstructure (Fig. 4). In addition, toidentify the role of agarose hydrogel as a promising matrix systemfor the non-solvent for the study of ISFIs, agarose hydrogels at con-

centrations of 1% (w/v) and 10% (w/v) in phosphate buffer wereused. The corresponding final implant morphologies were com-pared to those formed in pure buffer solution as well as resultsfrom the literature.

Y. Sun et al. / Journal of Pharmaceutical and Bi

Ff

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liquid-liquid demixing was delayed since influx of water and efflux

ig. 6. SEM images of (A) top surface layer and (B) cross sections of the implantsormed from 20% (w/w) PLGA in NMP in phosphate buffer.

As seen in Fig. 2 and Fig. 3, the highest apparent absorbanceoptical density) was found at the interfaces between the pre-ormulation and agarose hydrogel. The absorbance in the interiorf the formulation reservoir increased over time. The overallbsorbance increases in the PLGA-TA system were small as com-ared to those observed for the PLGA-NMP system. At t = 0 min,axima with similar absorbance appeared at the interfaces for

tudies of the pre-formulations as well as the pure solvents (Fig. S1nd S2). However, this initial absorbance was small as comparedo the absorbance change induced by phase separation (Fig. 3). Thepparent absorbance in the interior of the formulation reservioras below zero, because the transparent pre-formulation solutions

llowed more light to pass through the quartz cell than the 1% (w/v)garose hydrogel used as imaging reference.

From the absorbance-position profiles of 20% (w/w) PLGA inMP (Fig. 3A), a sharp increase in absorbance around the inter-

aces occurred within 1 h, which may be explained by diffusionf NMP into the agarose hydrogel matrix and water penentrationnto the polymer solution triggering the rapid PLGA precipitationt the interfaces. This observation is in accordance with previoustudies showing that the use of NMP, a co-solvent with high mis-ibility with water [6], resulted in formation of solidified polymerayers at the interface where the exchange of NMP and water ishe most rapid [11]. To this end, a solidification region exists in theLGA-water-NMP ternary phase diagram at high PLGA concentra-

ion and low NMP and water concentrations [22]. This solidifiedLGA layer at the interface restrains subsequent NMP efflux andater influx through the polymer solution. Thus, in the sublay-

omedical Analysis 145 (2017) 682–691 687

ers, PLGA precipitate relatively slowly as compared to the rate andextent occurring. From the absorbance-position profiles (Fig. 3A), acorresponding gradual increase in absorbance in the interior of for-mulation reservoir until 9 h was observed, which can be ascribedto the penetration of water into the PLGA solution inducing phaseseparation leading to optically more dense structures. Addition of asmall amount of water to a PLGA solution leads to two co-existingphases, a water-rich polymer-lean phase and a polymer-rich phase[22]. It was also noticed that after 9 h, the absorbance within theformulation reservoir had approached the maximum value withlimited changes occurring hereafter. This indicates that the poly-mer precipitation process throughout the pre-formulation may beclose to completion 9 h after exposure of the pre-formulation to theagarose hydrogel. The observations from the UV–vis imaging weresupported by results obtained using light microscopy (Fig. 4A).The thin dark layer that appeared at the interface 5 min afteraddition of the pre-formulation of 20% (w/w) PLGA in NMP cor-responds to the formation of solid PLGA. Next to the interface, thenascent void structures (water-rich phase) were also observed andgrew over time in the direction from the pre-formulation-agarosehydrogel interface toward the center of the formulation reservoir.The growing dark layer in the interior of formulation reservoirobserved using light microscopy (Fig. 4A) as well as the increaseof absorbance at the corresponding position in the absorbance-position profiles obtained from imaging at 550 nm (Figs. 2 A and3 A) indicate PLGA precipitation over time in the direction from theinterface to the interior.

The 40% (w/w) PLGA in NMP pre-formulation was characterizedby a rapid increase in absorbance within 1 h as compared to the 20%(w/w) PLGA in NMP pre-formulation (Fig. 3B) and remained fairlyconstant hereafter. It is suggested that the 40% (w/w) PLGA in NMPpre-formulation results in a faster formation of solidified polymerlayer than for the 20% (w/w) PLGA in NMP pre-formulation. Fur-thermore, the interior of formulation reservoir exhibited a similartrend in absorbance changes as the pre-formulation of 20% (w/w)PLGA in NMP regarding a gradual increase in absorbace within thefirst 9 h followed by small absorbance changes from 9 h to 24 h.However, the increase in absorbance in the reservoir interior ofthe 40% (w/w) PLGA-NMP pre-formulation was slower than thatof the 20% (w/w) PLGA-NMP pre-formulation. This may be due tothe fast formation of the solidified polymer layer and the higherviscosity of 40% (w/w) PLGA in NMP pre-formulation, hinderingthe diffusional exchange of NMP and water. Light microscopy pic-tures (Fig. 4B) also supported this interpretation. Compared to thepre-formulation with 20% (w/w) PLGA in NMP, a thicker ‘skin’ (darklayer) was found at the interfaces at 5 min for the 40% (w/w) PLGA inNMP pre-formulation. The solidified ‘skin’ formed a barrier for theexchange of NMP and water. Thereby, phase separation occurredrelatively slowly next to the ‘skin’. The immediate precipitation ofthe PLGA at the interface as well as a gradual phase separation in theinner pre-formulation leads to the formation of a PLGA membranecharacterized by a dense shell and porous inner structure [21].

The pre-formulation containing TA showed a distinctly differentbehavior (Fig. 3C) as compared to the pre-formulations containingNMP. It was observed that the absorbance at the interfaces onlyincreased slightly and the absorbance in the interior of formulationreservoir remained constant during the first hour. The difference inabsorbance between the interfaces and the interior of the reservoirwas small compared to that of the PLGA-NMP pre-formulations.Light microscopy pictures did not show formation of macrovoidstructures and phase separation was slower (Fig. 4C). These obser-vations are consistent with previous findings [22] showing that

of TA was restrained due to the low miscibility of TA in water(water solubility of TA is 61.2 g/L at 20 ◦C [6]). Thus, a dense layer ofPLGA precipitating at the interface was not seen. Instead of form-

688 Y. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 145 (2017) 682–691

F A in N( oomed

itpt

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ig. 7. SEM images of the cross sections of the implants formed from 20% (w/w) PLGB), middle layer (C) to the interior (D) and in 10% (w/v) agarose hydrogel (E) and z

ng a solid depot as for the PLGA-NMP system, the PLGA-TA systemransformed into a semi-fluid liquid polymer matrix as describedreviously [22]. This may explain the small absorbance change forhe PLGA-TA system throughout the 24 h (Fig. 2C and Fig. 3C).

The results are in line with the accepted mechanisms forast phase inverting systems and slow phase inverting systems5,6,11,22]. Light microscopy can provide important informationegarding the microstructure evolving upon contact with thequeous environment. However, one of the drawbacks of light

icroscopy is that it can only follow the early events of the phase

eparation process. UV–vis imaging performed at 550 nm measuredhe absorbance change from the onset of phase separation to theormation of a final polymer membrane as a function of time (until

MP in 1% (w/v) agarose hydrogel, (A) showing three-layer structure from interface picture (F) showing the interior structure.

24 h) and position facilitating, potentially, a more thorough under-standing of the initial phase separation process of ISFIs.

3.2. Phase separation kinetics

During phase separation, the polymer precipitates as the non-solvent diffuses into the polymer solution and the successive layerschange from a fluid state to a gel state. It is often assumed that therate of the precipitation front propagation can be used as a measure

of the phase separation rate of the investigated solvent, nonsolventand polymer systems [41,42].

The apparent absorbance changes observed at 550 nm duringphase separation may be ascribed to absorbance, light reflectance

and Biomedical Analysis 145 (2017) 682–691 689

aasoriaswTpsttsicloisos[c2eforftsmdp0(pcbaimcuodtf

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Fig. 8. Formation of a surface liquid layer of 20% (w/w) PLGA in NMP upon contact

Y. Sun et al. / Journal of Pharmaceutical

nd light scattering, which makes the assessment of the phase sep-ration kinetics using the absorbance data challenging. In order toimplify the data interpretation, as well as to facilitate alignmentf the obtained results with the literature, transmittance data asead from the pixel intensities were utilized. Liquid-liquid demix-ng or polymer precipitation would lead to optical inhomogeneitynd thus a decrease in transmission of light through the polymerolution. The critical time where the light intensity began to dropas taken as a measure of the time of onset of phase separation.

he intensity-time profiles at selected distances from the hydrogel-re-formulation interface within the pre-formulation (Fig. 2A) arehown in Fig. 5A. The profiles in Fig. 5A were consistent withhe two general mechanisms of liquid-liquid demixing based onhe solvent properties [16] describing that systems with the lowolvent/water miscibility (PLGA-TA) underwent a delayed phasenversion in comparison to systems with high solvent/water mis-ibility (PLGA-NMP) which showed a faster and sharper decline inight intensity. For the PLGA-NMP system, at the distances of 0.4, 0.6r 0.8 mm from the interface, phase separation occurred at approx-mately 6.9, 14.4 or 23.2 min, respectively (Fig. 5A). In the plot ofquare distance (mm2) versus critical time (min) (Fig. 5B), the slopebtained from linear regression is related to the rate of spatial phaseeparation (i.e. implant formation) for the selected ternary systems41,42]. The rate of spatial phase separation (mm2/min) after cal-ulation was given as 0.030 ± 0.002 and 0.021 ± 0.001 mm2/min for0% (w/w) and 40% (w/w) PLGA in NMP. The same approach, how-ver, could not be applied to the PLGA-TA system. The PLGA implantormed from the PLGA-TA system was in a fluid state leading tonly small changes in intensity over time (Figs. 3C and 5A), whichesulted in a large uncertainty when determining the critical timerom the intensity-time profiles compared to the PLGA-NMP sys-ems as seen in Fig. S3. The phase separation rate for the PLGA-NMPystem was also determined using the light microscopy pictures byeasuring the moving distance (mm) of the precipitation front at

ifferent times (min). Using the same approach, the rate of spatialhase separation (mm2/min) measured by light microscopy was.051 ± 0.007 and 0.030 ± 0.001 mm2/min for 20% (w/w) and 40%w/w) PLGA in NMP, respectively. For the PLGA-NMP system, thehase separation rate decreased by a factor of ∼0.6 when the PLGAoncentration in NMP increased from 20% (w/w) to 40% (w/w) foroth the imaging and the light microscopy methods although thebsolute values differed. For the pre-formulation of 40% (w/w) PLGAn NMP, the liquid–liquid phase separation rate has been deter-

ined to be 0.0127 mm2/min by dark ground imaging [21] which isomparable to the current results. The differences between the val-es may be related to different measuring principles and geometryf the setup used in the dark ground imaging system. The averageiffusion coefficient (De) of the nonsolvent (water) during the ini-ial phase separation for the PLGA-NMP systems can be estimatedrom the slope (Fig. 5B) and Eq. (1) [10,43]:

2 = 4Det (1)

here d is the moving distance of the precipitation front (mm) and is the critical time. The calculated De values in the phase invertingayer were 1.3 × 10−10 m2/s and 0.9 × 10−10 m2/s for 20% and 40%w/w) PLGA in NMP, respectively, which are almost one order of

agniture lower than the mutual diffusion coefficient of NMP andater (8.29 × 10−10 m2/s [44]) when the mole fraction of NMP is

.95. This indicates that transport by diffusion may account for aignificant component of the transport occurring during phase sep-ration and that the formation of the dense PLGA layer limits theater influx rate.

with 1% (w/v) agarose hydrogel observed under light microscopy (magnification50 × ) using 1 mm light path quartz cell: (A) before phase separation, (B) and (C) theoccurrence of the liquid layer 1 h after addition.

3.3. Morphologies of the PLGA implants

The biological environment that ISFIs are exposed to is com-plex and may affect performance of the ISFIs. Patel et al. [28] andHernandez et al. [30] found that drug release rates and implant mor-phologies of ISFIs varied with the injection site stiffness. For agarosehydrogels, the pore size and stiffness of the resulting matrix can bemodified by varying the agarose concentration [32]. This promptedus to explore whether agarose hydrogels of different concentra-tions would lead to differences with respect to implant formation.For this purpose, 20 �l of the pre-formulation contaning 20% (w/w)PLGA in NMP was added to different aqueous environments, whichwere 100 ml of stagnant 67 mM phosphate buffer solution (pH 7.4)in a beaker, 1% (w/v) and 10% (w/v) agarose gel prepared in thequartz cell and 1% agarose hydrogel prepared in a petri dish accord-ing to [45]. The morphologies of the resulting implants as detectedby SEM are shown in Fig. 6 and Fig. 7. The PLGA implants formed inthe phosphate buffer (67 mM, pH 7.4) presented a typical structurefor a fast phase separating system [21]: a thin top layer (Fig. 6A)followed by the interconnected porous network and finger likecavities (Fig. 6B). The PLGA implants formed in 1% (w/v) agarosehydrogel (both in the quartz cell and in bulk agarose) also con-tained the typical structure formed through a fast phase separatingprocess (Fig. 7A). However, on the top of the solid PLGA layer, athin polymer beads-like structure was observed (Fig. 7B and C)

which was not found on the PLGA implants formed in the phos-phate buffer. It is also worth to mention that in the light microscopyphotographs, an extra transparent layer was observed in the hydro-gel side next to the interface (Fig. 8). It appeared after initiation of

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he phase separation and grew overtime. This could be the result ofhe accumulation of water and NMP at the interface, which destabi-ized the surface layer and induced an additional phase separationia nucleation and growth of a polymer rich phase. Indeed, the for-ation of an additional gel layer on the surface of polymer solution

as been reported in some previous studies [15,46]. Guillen et al.bserved that manually introducing a small amount of water onhe edge of polymer solution prior to the exposure to the bulkater, the formation of the liquid layer following the nucleation

f a polymer-rich phase was seen at the interface [15]. In our case,he nonsolvent water was gradually ‘supplied’ through the agaroseydrogel, which could assist the buildup of this additional layer.

At an agarose concentration of 10% (w/v), the PLGA implantshowed a homogenous porous structure (Fig. 7E) and a reducedorosity as compared to that observed in the 1% (w/v) agaroseydrogel. The corresponding light microscopy images obtained in0% (w/v) agarose are shown in Fig. 4D. The rate of phase separationas determined as 0.030 ± 0.002 mm2/min; lower than that found

n 1% (w/v) agarose gel (0.051 ± 0.007 mm2/min). The formationnd growth of the finger like cavities were inhibited using 10% (w/v)garose as the nonsolvent (Fig. 4D). The 10% (w/v) agarose hydrogelresents a more rigid texture possibly reducing the mass transportate of water in the hydrogel, which in turn suppresses the growthf a nascent pore to a finger like macrovoid structure (Fig. 4D).igher magnification shows regular loosely packed polymer par-

icles in the interior of the implants (Fig. 7F). This may be related to phase separation through nucleation of the polymer-rich phase athe location where the solvent concentration is highest due to theonger water penetration distance to the surrounding hydrogels.his additional phase separation process have also been observedor other polymer-solvent systems under the condition of a delayednset of liquid-liquid demixing [47,48].

Patel et al. [28] and Solorio et al. [29] found significant dif-erences in implant morphology in terms of shape and porosityepending on whether the implants were formed in vitro or inivo. A recent study showed that the implant morphology ofLGA-based ISFIs formed in tissue-mimicking phantoms resem-led the morphology found in vivo [30]. Here, we have foundhat agarose hydrogels of various concentrations affect the finalmplant microstructure attained, indicating that the matrix in

hich implants are formed is of importance and should be inves-igated further in the pursue of in vivo predictive in vitro testing

ethods.

. Conclusions

In the present study, the effect of the solvent properties as wells the external environment on the phase separation behavior ofLGA-based in situ forming implants and implant morphology wasvaluated using a novel agarose hydrogel-based setup. In contrasto the traditional use of water or buffer solution as the nonsolvent,he agarose hydrogel serving as a simple model of biological softissue was utilized to provide the nonsolvent, water, for the ISFIs.pon contact with the hydrogel matrix, polymer-solvent systemsf PLGA-NMP and PLGA-TA underwent distinctive changes in lightransmission and absorbance as a function of time and position. Thenterpretation of the absorbance and intensity profiles supportedhe two general classes of non-solvent induced phase separation,epending on the miscibility of solvent and nonsolvent, which cane monitored and quantified by UV–vis imaging. We observed a dif-erent microstructure for the PLGA implants formed in the hydrogel

atrix as compared to buffer solution. However, additional inves-igations are needed to explore the role of agarose hydrogel as aromising tissue surrogate for in vitro testing of ISFIs. The ability toonitor time-resolved events in the immediate vicinity (within �m

[

omedical Analysis 145 (2017) 682–691

to mm range) of the formulation makes UV–vis imaging attractivefor investigation of in situ forming drug delivery systems. More-over, the developed in vitro platform combining the use of UV–visimaging, light microscopy and scanning electron microscopy holdspromise for simultaneous monitoring of phase separation processand drug release. Studies along these lines are currently pursured.

Acknowledgements

This research did not receive any specific grant from fundingagencies in the public, commercial, or not-for-profit sectors. Theauthors alone are responsible for the content and writing of thispaper. Jesper Østergaard is member of the Scientific Advisory Boardof Paraytec Ltd. (York, UK).

References

[1] X. Liu, A.L. Miller II, B.E. Waletzki, M.J. Yaszemski, L. Lu, Novel biodegradablepoly (propylene fumarate)-co-poly (l-lactic acid) porous scaffolds fabricatedby phase separation for tissue engineering applications, RSC Adv. 5 (2015)21301–21309.

[2] N. Kasoju, N. Hawkins, O. Pop-Georgievski, D. Kubies, F. Vollrath, Silk fibroingelation via non-solvent induced phase separation, Biomater. Sci. 4 (2016)460–473, http://dx.doi.org/10.1039/c5bm00471c.

[3] X. Lin, K. Wang, Y. Feng, J.Z. Liu, X. Fang, T. Xu, H. Wang, Compositeultrafiltration membranes from polymer and its quaternaryphosphonium-functionalized derivative with enhanced water flux, J. Membr.Sci. 482 (2015) 67–75.

[4] T.A. Tweddle, O. Kutowy, W.L. Thayer, S. Sourirajan, Polysulfone ultrafiltrationmembranes, Ind. Eng. Chem. Prod. Res. Dev. 22 (1983) 320–326.

[5] M. Parent, C. Nouvel, M. Koerber, A. Sapin, P. Maincent, A. Boudier, PLGAin situ implants formed by phase inversion: critical physicochemicalparameters to modulate drug release, J. Control. Release. 172 (2013) 292–304,http://dx.doi.org/10.1016/j.jconrel.2013.08.024.

[6] R.R.S. Thakur, H.L. McMillan, D.S. Jones, Solvent induced phaseinversion-based in situ forming controlled release drug delivery implants, J.Control. Rel. 176 (2014) 8–23, http://dx.doi.org/10.1016/j.jconrel.2013.12.020.

[7] G. Southard, R. Dunn, S. Garrett, The drug delivery and biomaterial attributesof the ATRIGEL® technology in the treatment of periodontal disease, ExpertOpin. Investig. Drugs 7 (1998) 1483–1491, http://dx.doi.org/10.1517/13543784.7.9.1483.

[8] O. Sartor, Eligard: leuprolide acetate in a novel sustained-release deliverysystem, Urology 61 (2003) 25–31.

[9] A.J. Reuvers, Membrane Formation: Diffusion Induced Demixing Processes inTernary Polymeric Systems, University of Twente, 1987.

10] H. Strathmann, K. Kock, P. Amar, R.W. Baker, The formation mechanism ofasymmetric membranes, Desalination 16 (1975) 179–203, http://dx.doi.org/10.1016/S0011-9164(00)82092-5.

11] T.-H. Young, L.-W. Chen, Pore formation mechanism of membranes fromphase inversion process, Desalination 103 (1995) 233–247, http://dx.doi.org/10.1016/0011-9164(95)00076-3.

12] S.S. Prakash, L.F. Francis, L.E. Scriven, Microstructure evolution in dry castcellulose acetate membranes by cryo-SEM, J. Membr. Sci. 283 (2006)328–338, http://dx.doi.org/10.1016/j.memsci.2006.07.001.

13] S.S. Prakash, L.F. Francis, L.E. Scriven, Microstructure evolution in dry-wet castpolysulfone membranes by cryo-SEM: A hypothesis on macrovoid formation,J. Membr. Sci. 313 (2008) 135–157, http://dx.doi.org/10.1016/j.memsci.2008.01.006.

14] R. Matz, The structure of cellulose acetate membranes 1, Develop. PorousStruc. Anisotr. Memb. Desal. 10 (1972) 1–15, http://dx.doi.org/10.1016/S0011-9164(00)80243-X.

15] G.R. Guillen, G.Z. Ramon, H.P. Kavehpour, R.B. Kaner, E.M.V. Hoek, Directmicroscopic observation of membrane formation by nonsolvent inducedphase separation, J. Membr. Sci. 431 (2013) 212–220, http://dx.doi.org/10.1016/j.memsci.2012.12.031.

16] J.H. Hao, S. Wang, Studies on membrane formation mechanism by the lighttransmission technique. I, J. Appl. Polym. Sci. 87 (2003) 174–181.

17] C.-Y. Kuo, S.-L. Su, H.-A. Tsai, Y.-S. Su, D.-M. Wang, J.-Y. Lai, Formation andevolution of a bicontinuous structure of PMMA membrane during wetimmersion process, J. Membr. Sci. 315 (2008) 187–194, http://dx.doi.org/10.1016/j.memsci.2008.02.034.

18] H.-A. Tsai, C.-Y. Kuo, S.-L. Su, D.-M. Wang, J.-Y. Lai, The morphologicalevolution of solvent-containing PMMA membranes in various solventremoval processes, J. Membr. Sci. 345 (2009) 288–297, http://dx.doi.org/10.1016/j.memsci.2009.09.011.

19] C. Stropnik, V. Musil, M. Brumen, Polymeric membrane formation by

wet-phase separation; turbidity and shrinkage phenomena as evidence forthe elementary processes, Polymer 41 (2000) 9227–9237.

20] C. Stropnik, V. Kaiser, Polymeric membranes preparation by wet phaseseparation: mechanisms and elementary processes, Desalination 145 (2002)1–10, http://dx.doi.org/10.1016/s0011-9164(02)00322-3.

and Bi

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[polysulfone structures obtained by wet phase separation, Eur. Polym. J. 43(2007) 2515–2524, http://dx.doi.org/10.1016/j.eurpolymj.2007.03.012.

[48] T.-H. Young, D.-M. Wang, C.-C. Hsieh, L.-W. Chen, The effect of the secondphase inversion on microstructures in phase inversion EVAL membranes, J.Membr. Sci. 146 (1998) 169–178.

Y. Sun et al. / Journal of Pharmaceutical

21] P.D. Graham, K.J. Brodbeck, A.J. McHugh, Phase inversion dynamics of PLGAsolutions related to drug delivery, J. Control. Rel. 58 (1999) 233–245.

22] K.J. Brodbeck, J.R. DesNoyer, A.J. McHugh, Phase inversion dynamics of PLGAsolutions related to drug delivery: part II. The role of solutionthermodynamics and bath-side mass transfer, J. Control. Rel. 62 (1999)333–344.

23] S. Kempe, H. Metz, K. Mader, Do in situ forming PLG/NMP implants behavesimilar in vitro and in vivo? A non-invasive and quantitative EPRinvestigation on the mechanisms of the implant formation process, J. Control.Rel. 130 (2008) 220–225, http://dx.doi.org/10.1016/j.jconrel.2008.06.006.

24] S. Kempe, H. Metz, P.G.C. Pereira, K. Mäder, Non-invasive in vivo evaluation ofin situ forming PLGA implants by benchtop magnetic resonance imaging(BT-MRI) and EPR spectroscopy, Eur. J. Pharm. Biopharm. 74 (2010) 102–108,http://dx.doi.org/10.1016/j.ejpb.2009.06.008.

25] L. Solorio, A.M. Olear, H. Zhou, A.C. Beiswenger, A.A. Exner, Effect of cargoproperties on in situ forming implant behavior determined by noninvasiveultrasound imaging, Drug Deliv, Transl. Res. 2 (2012) 45–55, http://dx.doi.org/10.1007/s13346-011-0054-y.

26] L. Solorio, B.M. Babin, R.B. Patel, J. Mach, N. Azar, A.A. Exner, Noninvasivecharacterization of in situ forming implants using diagnostic ultrasound, J.Control. Rel. 143 (2010) 183–190, http://dx.doi.org/10.1016/j.jconrel.2010.01.001.

27] L. Solorio, A.M. Olear, J.I. Hamilton, R.B. Patel, A.C. Beiswenger, J.E. Wallace, H.Zhou, A.A. Exner, Noninvasive characterization of the effect of varying PLGAmolecular weight blends on in situ forming implant behavior usingultrasound imaging, Theranostics 2 (2012) 1064–1077.

28] R.B. Patel, L. Solorio, H. Wu, T. Krupka, A.A. Exner, Effect of injection site onin situ implant formation and drug release in vivo, J. Control. Release. 147(2010) 350–358, http://dx.doi.org/10.1016/j.jconrel.2010.08.020.

29] L. Solorio, A.A. Exner, Effect of the subcutaneous environment onphase-sensitive in situ-forming implant drug release, regradation, andmicrostructure, J. Pharm. Sci. 104 (2015) 4322–4328, http://dx.doi.org/10.1002/jps.24673.

30] C. Hernandez, N. Gawlik, M. Goss, H. Zhou, S. Jeganathan, D. Gilbert, A.A.Exner, Macroporous acrylamide phantoms improve prediction of in vivoperformance of in situ forming implants, J. Control. Rel. 243 (2016) 225–231,http://dx.doi.org/10.1016/j.jconrel.2016.10.009.

31] D. Klose, N. Azaroual, F. Siepmann, G. Vermeersch, J. Siepmann, Towards morerealistic in vitro release measurement techniques for biodegradablemicroparticles, Pharm. Res. 26 (2009) 691–699, http://dx.doi.org/10.1007/s11095-008-9747-4.

32] A.P. Balgude, X. Yu, A. Szymanski, R.V. Bellamkonda, Agarose gel stiffnessdetermines rate of DRG neurite extension in 3D cultures, Neural Tissue Eng.22 (2001) 1077–1084, http://dx.doi.org/10.1016/S0142-9612(00)00350-1.

33] Y. Sun, J. Østergaard, Application of UV Imaging in formulation development,Pharm. Res. (2016), http://dx.doi.org/10.1007/s11095-016-2047-5.

34] J. Østergaard, J. Lenke, S.S. Jensen, Y. Sun, F. Ye, UV imaging for in vitrodissolution and release studies: initial experiences, Dissolut. Technol. 21

(2014) 27–38.

35] S.S. Jensen, H. Jensen, C. Cornett, E.H. Møller, J. Østergaard, Real-time UVimaging identifies the role of pH in insulin dissolution behavior inhydrogel-based subcutaneous tissue surrogate, Eur. J. Pharm. Sci. 69 (2015)26–36, http://dx.doi.org/10.1016/j.ejps.2014.12.015.

omedical Analysis 145 (2017) 682–691 691

36] S.S. Jensen, H. Jensen, E.H. Møller, C. Cornett, F. Siepmann, J. Siepmann, J.Østergaard, In vitro release studies of insulin from lipid implants in solutionand in a hydrogel matrix mimicking the subcutis, Eur. J. Pharm. Sci. 81 (2016)103–112, http://dx.doi.org/10.1016/j.ejps.2015.10.011.

37] F. Ye, A. Yaghmur, H. Jensen, S.W. Larsen, C. Larsen, J. Østergaard, Real-timeUV imaging of drug diffusion and release from Pluronic F127 hydrogels, Eur. J.Pharm. Sci. 43 (2011) 236–243, http://dx.doi.org/10.1016/j.ejps.2011.04.015.

38] F. Ye, S.W. Larsen, A. Yaghmur, H. Jensen, C. Larsen, J. Østergaard, Real-timeUV imaging of piroxicam diffusion and distribution from oil solutions intogels mimicking the subcutaneous matrix, Eur. J. Pharm. Sci. 46 (2012) 72–78,http://dx.doi.org/10.1016/j.ejps.2012.02.011.

39] S.S. Jensen, H. Jensen, D.M. Goodall, J. Østergaard, Performance characteristicsof UV imaging instrumentation for diffusion, dissolution and release testingstudies, J. Pharm. Biomed. Anal. 131 (2016) 113–123, http://dx.doi.org/10.1016/j.jpba.2016.08.018.

40] S.H. Yoo, J.H. Kim, J.Y. Jho, J. Won, Y.S. Kang, Influence of the addition of PVPon the morphology of asymmetric polyimide phase inversion membranes:effect of PVP molecular weight, J. Membr. Sci. 236 (2004) 203–207, http://dx.doi.org/10.1016/j.memsci.2004.02.017.

41] J.S. Taurozzi, C.A. Crock, V.V. Tarabara, C60-polysulfone nanocompositemembranes: entropic and enthalpic determinants of C60 aggregation and itseffects on membrane properties, Desalination 269 (2011) 111–119, http://dx.doi.org/10.1016/j.desal.2010.10.049.

42] X. Li, C. Chen, J. Li, Formation kinetics of polyethersulfone with cardomembrane via phase inversion, J. Membr. Sci. 314 (2008) 206–211, http://dx.doi.org/10.1016/j.memsci.2008.01.042.

43] S.K. Yong, J.K. Hyo, Y.K. Un, Asymmetric membrane formation via immersionprecipitation method. I. Kinetic effect, J. Membr. Sci. 60 (1991) 219–232.

44] L. Ambrosone, G. D’Errico, R. Sartorio, V. Vitagliano, Analysis of velocitycross-correlation and preferential solvation for the systemN-methylpyrrolidone-water at 20(C), J. Chem. Soc. Faraday Trans. 91 (1995)1339–1344.

45] S.S. Jensen, H. Jensen, E.H. Møller, C. Cornett, F. Siepmann, J. Siepmann, J.Østergaard, In vitro release studies of insulin from lipid implants in solutionand in a hydrogel matrix mimicking the subcutis, Eur. J. Pharm. Sci. 81 (2016)103–112, http://dx.doi.org/10.1016/j.ejps.2015.10.011.

46] P. Menut, Y.S. Su, W. Chinpa, C. Pochat-Bohatier, A. Deratani, D.M. Wang, P.Huguet, C.Y. Kuo, J.Y. Lai, C. Dupuy, A top surface liquid layer duringmembrane formation using vapor-induced phase separation (VIPS)—Evidenceand mechanism of formation, J. Membr. Sci. 310 (2008) 278–288, http://dx.doi.org/10.1016/j.memsci.2007.11.016.

47] V. Kaiser, C. Stropnik, V. Musil, M. Brumen, Morphology of solidified