Fractional Laser Ablation for the Cutaneous Delivery ofTriamcinolone Acetonide from Cryomilled Polymeric Microparticles:Creating Intraepidermal Drug DepotsMayank Singhal, Sergio del Río-Sancho, Kiran Sonaje, and Yogeshvar N. Kalia*

School of Pharmaceutical Sciences, University of Geneva & University of Lausanne, 30 Quai Ernest Ansermet, 1211 Geneva,Switzerland

*S Supporting Information

ABSTRACT: The efficacy of some dermatological therapiesmight be improved by the use of “high dose” intraepidermaldrug reservoir systems that enable sustained and targeted localdrug delivery, e.g., in the treatment of keloids and hypertrophicscars. Here, a fractionally ablative erbium:YAG laser was usedto enable “needle-less” cutaneous deposition of polymericmicroparticles containing triamcinolone acetonide (TA). Themicroparticles were prepared using a freeze−fracture techni-que employing cryomilling that resulted in drug loadingefficiencies of ∼100%. They were characterized by severaldifferent techniques, including scanning electron microscopy,powder X-ray diffraction and differential scanning calorimetry. TA was quantified by validated HPLC−UV and UHPLC−MS/MS analytical methods. In vitro release studies demonstrated the effect of polymer properties on TA release kinetics. Confocallaser scanning microscopy enabled visualization of cryomilled microparticles containing fluorescein and Nile Red in thecutaneous micropores and the subsequent release of fluorescein into the micropores and its diffusion throughout the epidermisand upper dermis. The biodistribution of TA, i.e. the amount of drug as a function of depth in skin, following microparticleapplication was much more uniform than with a TA suspension and delivery was selective for deposition with less transdermalpermeation. These findings suggest that this approach may provide an effective, targeted and minimally invasive alternative topainful intralesional injections for the treatment of keloid scars.

KEYWORDS: fractional laser ablation, microparticle, skin, keloid, cryomilling

■ INTRODUCTION

Transport across the stratum corneum constitutes the rate-limiting step for the cutaneous delivery of most molecules. Itsstructure imposes a high degree of tortuosity that increases thediffusion path length and the lipid-rich character of theintercellular matrix, the principal diffusion pathway, compoundsthe challenge faced by hydrophilic molecules.1 It follows thatnumerous strategies have been explored to compromisestratum corneum integrity and to create transport conduitsthat facilitate diffusion. These include the use of microneedlesand assorted fractional ablation techniques, e.g. radio frequencyand the use of CO2 and Er:YAG lasers.2 These ablation-basedapproaches have been used to enhance drug delivery for bothlocal and systemic applications. The P.L.E.A.S.E. (Precise LaserEpidermal System; Pantec Biosolutions AG) is a fractionallyablative Er:YAG laser that emits μs pulses at a wavelength of2936 nm. These excite water molecules in the epidermis anddermis and their explosive evaporation results in the formationof micropores. Modulation of the device parameters enablesprecise control of pore density (i.e., number of pores per cm2)and pore depth. Work to-date has demonstrated that thetechnique can increase the rate and the extent of the delivery of

small molecules,3−6 peptides and proteins7 into and across theskin and that it can also be used for the intraepidermal deliveryof vaccines.8,9 Furthermore, it has also been used for the“needle-free” cutaneous delivery of biologically active antibod-ies.10

A patient treatment paradigm based on this strategy wouldinvolve a two-step approach with laser microporation beingfollowed by application of the drug formulation.11 In principle,formulation application could be repeated at the samemicroporation site as long as the pores remained open−aconservative estimate would be for 48−72 h−beyond that timeinterval, a new micropore array must be created. However, froma clinical perspective, it would be of considerable interest to beable to introduce an intraepidermal drug depot where thenatural healing process after microporation would close the skinaround it. The depot would have different drug release kineticsto a formulation applied topically to the skin surface and would

Received: September 16, 2015Revised: November 27, 2015Accepted: January 5, 2016Published: January 5, 2016

Article

pubs.acs.org/molecularpharmaceutics

© 2016 American Chemical Society 500 DOI: 10.1021/acs.molpharmaceut.5b00711Mol. Pharmaceutics 2016, 13, 500−511

enable sustained drug release over a more prolonged timeframe. This intraepidermal drug depot is clearly analogous tothe well-known subcutaneous and intramuscular depots that arefrequently used in therapy to enable prolonged systemic releaseof peptide therapeutics, e.g. gonadotropin-releasing hormoneagonists and antagonists.12

This approach would be of particular interest in thetreatment of several dermatological conditions, includingkeloids and hypertrophic scars, which are caused by anoverproliferation of dense fibrous tissue and develop as a resultof anomalies during normal physiological wound healingprocesses. These conditions affect approximately 100 millionpatients each year following elective or trauma-inducedoperations.13 Both keloid and hypertrophic scars protrudeabove the skin surface. However, keloids can extend beyond theboundaries of the original wound, grow for years, and canreappear after excision; hypertrophic scars, which are morecommon, are delimited by the original wound borders and mayregress over time.14,15 Scars affect patient quality of life bothphysically and psychologically; they are cosmetically unaccept-able and can cause pruritus, pain and contractures.16 Availabletreatment options include pressure treatment, cryotherapy,surgical removal, silicone sheeting, pulsed dye laser, andpharmacotherapy involving topical application or intralesionalinjection of corticosteroids.17,18

The corticosteroid injections, usually single or multiple 0.1−1 mL volumes of a high dose triamcinolone acetonide (TA),suspension, 10−40 mg mL−1, often coadministered withlidocaine, are the first-line treatment for keloids.19 TA haspotent glucocorticoid activity and suppresses the inflammatoryresponse during the wound healing process; it can also decreaselevels of collagenase inhibitors (alpha-1-antitrypsin and alpha-2-macroglobulin) in keloids, thereby promoting collagendegradation.20,21 Therapy can be continued for six months oreven longer, injections are given weekly or monthly dependingon the severity of the condition.22,23 Common local adverseeffects associated with the intralesional TA injection are skinand subcutaneous fat atrophy, telangiectasia and significant painat the injection site. Long-term systemic exposure of TA causesCushing’s syndrome and adrenal insufficiency, especially inchildren.24−27 Noninvasive treatment by topical administrationof corticosteroids to the keloid surface shows poor efficacy dueto poor cutaneous bioavailability, i.e. suboptimal levels of drugin the skin. Intralesional injections obviously ensure greaterdrug bioavailability but, as mentioned above, increase the risk ofsystemic exposure in addition to local site reactions and pain.28

Therefore, a delivery system providing sustained local delivery

of TA within the scar tissue while avoiding systemic exposurewould be of considerable benefit.Here, we report a new method, the freeze−fracture

technique (FFT), to prepare TA-loaded microparticles (TA-MP) that can be deposited in cutaneous micropores followingfractional laser ablation. In contrast to conventional techniques,which often display poor drug loading, the FFT enablesextremely high loading efficiencies to be achieved. Thistechnique involves grinding a solid solution of the drugpolymer mixture in a cryogenic grinder at approximately −200°C until micrometer-sized particles are obtained. The workinghypothesis was that TA-MP with high drug loading depositedin scars following fractional laser ablation would enablesustained release of TA with different release kinetics to atopically applied solution/suspension.The specific objectives of this study were (i) to develop and

to optimize a new technique to prepare stable TA-MP withhigh drug loading, (ii) to develop and to characterize TA-MPformulations using 50/50 poly(D,L-lactide-co-glycolide) andpoly(D,L-lactide) and to compare their release profiles, (iii) tovisualize MP loaded with fluorescein and Nile Red inmicroporated skin using confocal laser scanning microscopy(CLSM) and to monitor release of fluorescein from the MPdeposited in the cutaneous micropores, and (iv) to compareand contrast the TA biodistribution (the amounts of TA as afunction of position) in the skin following release from thedeposited TA-MP and after application of a TA suspension.

■ EXPERIMENTAL SECTION

Materials. TA was purchased from Hanseler AG (Herisau,Switzerland). Resomer RG 503H (RG 503H; 50/50 poly(D,L-lactide-co-glycolide), MW 24−38 kDa, intrinsic viscosity 0.32−0.44 dL g−1), Resomer R207 (R207; poly(D,L-lactide), MW199.8 kDa, intrinsic viscosity 1.3−1.7 dL g−1), fluorescein, anddichloromethane were purchased from Sigma-Aldrich Chemie(Buchs, Switzerland). Nile Red was purchased from TCIEurope N.V. (Belgium). Poly(vinyl alcohol) (PVA; MW 72kDa) was purchased from Axon lab AG (Baden, Switzerland).All other chemicals and solvents were of analytical grade.

Preparation of TA Loaded MP. These were prepared byeither the conventional oil in water (o/w) emulsion technique,OW1-TA10 (with RG 503H) and OW2-TA10 (with R207), orthe newly developed FFT (FFT1-TA10 and FFT2-TA20). Thecomposition of each MP batch and the preparation method aregiven in Table 1. Preparation of MP with the o/w emulsiontechnique involved initial dissolution of the polymer (90 mg)and TA (10 mg) in dichloromethane (5 mL) at roomtemperature and subsequent slow addition to 25 mL of 1%

Table 1. Composition and Characterization of the Different Microparticle Formulations

methoda batchTAb

(%)RG 503H

(%)R 207(%)

FL/NRc

(%)mean size (d[4,3])

(μm)size distribn (d10, d50,

d90) (μm) EEd (%) DLe (%)

o/w emulsion technique OW1-placebo

100.0 11.39 ± 3.0 1.5, 8.16, 15.28 NA NA

OW1-TA10 10.0 90.0 8.17 ± 2.1 1.12, 6.57, 13.55 5.4 ± 0.3 0.54 ± 0.03OW2-placebo

100.0 9.84 ± 2.4 1.53, 8.99, 15.31 NA NA

OW2-TA10 10.0 90.0 14.84 ± 4.2 1.51, 13.63, 24.12 6.8 ± 0.2 0.68 ± 0.02freeze−fracture technique(FFT)

FFT1-TA10 10.0 90.0 89.23 ± 5.7 35.37, 81.97, 141.85 99.9 ± 1.7 9.8 ± 0.2FFT2-TA20 20.0 40.0 40.0 81.64 ± 4.4 29.26, 87.79, 139.44 101.6 ± 2.1 20.2 ± 0.2FFT3-FL/NR

97.8 2.0/0.2 86.09 ± 3.2 31.41, 78.80, 127.55 NA NA

aMethod of preparation. bTriamcinolone acetonide. cFluorescein/Nile Red. dEncapsulation efficiency. eDrug loading.

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(w/v) aqueous PVA solution under homogenization at 7,500rpm for 1 min using a Polytron PT 2500 E homogenizer(Kinematica AG; Luzern, Switzerland). The o/w emulsion wasthen transferred to 100 mL of 1% (w/v) aqueous PVA solutionunder homogenization at 5,000 rpm for 2 min. The finalemulsion was left overnight at room temperature undercontinuous stirring to evaporate the organic solvent and toharden the MP. The resulting MP were centrifuged at 1620g for5 min, and the pelleted MP were washed twice with Milli-Qwater and ethanol/water (25/75) mixture to remove unloadedTA. The final microparticle pellet was redispersed in 1.0 mL ofMilli-Q water and dried at room temperature under vacuum.For the FFT, MP were prepared by first dissolving the polymer(450 mg of RG 503H for FFT1-TA10 and 300 mg of eachpolymer for FFT2-TA20) and TA (50 mg and 150 mg forFFT1-TA10 and FFT2-TA20, respectively) in dichloromethane(17 mL) at 40 °C and then removing the organic solvent usinga Rotavapor R-124 (Buchi Labortechnik AG; Switzerland) toobtain a solid solution of TA and polymer. The TA−polymersolid solution was freeze−fractured using a cryogenic mill(6770 freezer/mill, SPEX SamplePrep; Stanmore, U.K.). Themill was set for 10 cycles operating at an impaction rate of 15cps with a run time of 1.5 min and a cool time of 2 minbetween each cycle. Liquid nitrogen, a cryogen, was added andgrinding initiated. The fractured particles were then passedthrough a 90 μm sieve (#170 U.S. mesh size). Particles largerthan 90 μm were processed again for 10 cycles. Particles werecollected and stored for one night under vacuum at roomtemperature to remove any residual moisture.Preparation of Fluorescein/Nile Red Loaded MP.

Fluorescent dye loaded MP (FFT3-FL/NR) containingfluorescein (2% w/w) and Nile Red (0.2% w/w) were preparedusing the same method in order to visualize the deposition andlocalization of MP in microporated skin and the release of their“cargo”fluoresceininto the surrounding tissue by CLSM.Analytical Methods. A previously reported reversed-phase

HPLC method was adapted and validated for quantification ofTA in the in vitro release study.29 The HPLC apparatuscomprised a P680A LPG-4 pump in line with an ASI-100autosampler, a TCC-100 thermostated column compartment,and a UV170D detector (Dionex; Voisins LeBretonneux,France). Briefly, the samples were analyzed using aLiChrospher 100, RP-18 (4 × 250 mm and 5 μm particlesize) column (BGB Analytik AG; Boeckten, Switzerland) withacetonitrile:water (60:40, v/v) as mobile phase (flow rate: 0.6mL min−1). TA was quantified using a detection wavelength of266 nm; the retention time was 6.35 ± 0.1 min, and the assaywas linear (r2 = 0.999) in the concentration range of 0.5−50 μgmL−1. The specificity of the analytical method was tested in thepresence of skin matrix compounds (Figure S1, Table S1). Anisocratic UHPLC−MS/MS method was developed andvalidated to determine the biodistribution of TA, that is, theamounts of TA deposited as a function of position in thedifferent skin layers during skin transport studies (Figure S2,Table S2). A Waters Acquity UPLC core system (Baden-Dattwil, Switzerland) comprising a binary solvent pump andsample manager with a Waters XEVO TQ-MS tandemquadrupole detector (Baden-Dattwil, Switzerland) equippedwith electron spray ion source was used for the UHPLC−MS/MS analysis together with a Waters XBridge BEH C18 column(2.1 × 50 mm and 2.5 μm particle size). The mobile phasecomprised Milli-Q water (0.1% formic acid):acetonitrile (55:45,v/v) maintained at 30 °C with a flow rate of 0.3 mL min−1. The

injection volume was 5 μL. The Waters XEVO TQ-MSdetector was operated in positive ion mode using multiplereaction monitoring. The mass transition ion pair was m/z435.277 → 415.154 (removal of hydrogen fluoride). MS sourceparameters were as follows: ion spray capillary voltage, 1.8 kV;cone voltage, 19 V; desolvation gas temperature, 500 °C; conegas flow rate, 1000 L h−1; and collision energy, 2 V. Dataacquisition was carried out using MassLynx V4.1 software.

Characterization of MP. TA content in the MP wasdetermined by dissolving approximately 5 mg of MP in 1 mL ofdichloromethane and diluting 25 times with methanol beforeHPLC analysis. The drug loading and encapsulation efficienciesof MP were calculated using eqs 1 and 2, respectively.

= ×drug loading (%)mass of drug in microparticles

mass of microparticles100

(1)

= ×

encapsulation efficiency (%)experimental drug loading

theoretical drug loading100

(2)

To measure MP size and size distribution, approximately 15mg of MP was suspended in 7 mL of 1% aqueous PVA solutionand analyzed using a Mastersizer S (Malvern Instruments Ltd.;Malvern, U.K.). Surface morphology of the MP was studiedusing scanning electron microscopy (SEM) (JEOL JSM-7001FA, JEOL USA, Inc.) after mounting the MP on aconductive carbon surface and coating with a thin layer of gold(∼21.1 nm) using a high vacuum sputter coater (Leica EMSCD500, Anatech USA) prior to microscopy. Powder X-raydiffraction (PXRD) patterns were measured using an AgilentSupernova Diffractometer and Cu radiation by loading thesamples in a 100 μm MiTeGen cryoloop using fomblin oil. Arotation of 180° was made during 6 min. The pattern was thenintegrated using the CrysAlisPro Agilent software. MP phasetransitions were analyzed by differential scanning calorimetry(DSC) (SSC/5200, Seiko Instruments; U.K.). Approximately 5mg of each MP was weighed in an aluminum pan, and thethermograph was recorded over a heating range of 30 to 320 °Cat a heating rate of 10 °C min−1.

In Vitro Drug Release Study. Phosphate buffered saline(PBS; pH 7.4) containing 1% Tween 80 as solubility enhancerwas selected as the dissolution medium for the in vitro releasestudy (Figure S3). A TA suspension, with a composition similarto that of the Kenalog-40 injection (Bristol-Myers Squibb), wasprepared as a control formulation. FFT1-TA10 and FFT2-TA20(5 mg; containing 0.5 and 1 mg of TA, respectively) and 12.5μL of TA suspension (TA, 0.5 mg) were dispersed in 50 mL ofdissolution medium. All samples were kept in a shaker bathmaintained at 34.5 ± 1 °C and 300 rpm. Aliquots (1 mL) werewithdrawn at predetermined time intervals and centrifuged(Eppendorf Centrifuge 5804; Schonenbuch, Switzerland) at10621g for 5 min. An aliquot (0.5 mL) of supernatant wastaken and analyzed for TA content using HPLC while theremaining 0.5 mL was diluted with a fresh 0.5 mL of dissolutionmedia and vortexed to resuspend the MP and transferred backto the dissolution container. All samples were analyzed intriplicate. The changes in the physical properties of the MPafter the in vitro release study were also monitored. For this,the MP incubated in release medium for 1 week were collectedby centrifugation (1620g, 5 min) and dried under vacuum

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overnight. These MP were then analyzed using PXRD, DSC,and SEM as described above.Preparation of Skin and P.L.E.A.S.E. Microporation.

Porcine ears were obtained shortly after sacrifice from a localabattoir (CARRE; Rolle, Switzerland). After cleaning withrunning cold water, the skin from the outer region of ears wascarefully excised from the underlying cartilage using a scalpel.The excised full thickness skin samples (2 mm) were thenpunched into 32 mm circular disks and then stored at −20 °Cfor no more than 1 month before use. Frozen skin sampleswere thawed and equilibrated in 0.9% NaCl solution for 30 minbefore microporation using an Er:YAG laser (P.L.E.A.S.E.,

Pantec Biosolutions AG; Ruggell, Liechtenstein). Skin surfacemoisture was removed, and then the samples were mounted ona custom designed assembly. Microporation parameters wereset to provide 300 pores cm−2 (15% pore density) at a fluenceof 90 J cm−2.

MP Deposition in P.L.E.A.S.E. Porated Skin andVisualization by CLSM. The P.L.E.A.S.E. porated skinsamples were mounted in Franz diffusion cells (area 2.0 ±0.1 cm2), and silicone grease was applied at the edges to ensurea watertight seal. The receiver compartments were filled withrelease medium (10 mL of PBS at pH 7.4 with 1% Tween 80),and skin was equilibrated for 30 min. For the confocal

Figure 1. SEM images. (A) PLGA (RG 503H) microparticles prepared by the oil in water (o/w) emulsion technique: (a) OW1-placebo; (b) OW1-TA10, before washing with ethanol/water (25/75); and (c) OW1-TA10, after washing with ethanol/water (25/75). (B) PLA (R 207) microparticlesprepared by the o/w emulsion technique: (a) OW2-placebo; (b) OW2-TA10, before washing with ethanol/water (25/75); and (c) OW2-TA10, afterwashing with ethanol/water (25/75). (C) PLGA microparticles prepared by the freeze−fracture technique: (a) FFT1-TA10; (b) FFT1-TA10,individual microparticle before release study; and (c) FFT1-TA10, individual microparticle after release study. (D) PLGA/PLA microparticlesprepared by the freeze−fracture technique: (a) FFT2-TA20; (b) FFT2-TA20, individual microparticle before release study; and (c) FFT2-TA20,individual microparticle after release study.

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microscopy experiments, 2.5 mg of FFT3-FL/NR wassuspended in 200 μL of Milli-Q water and applied to theexternal surface of the microporated skin samples. The receiverphase was maintained at 34.5 ± 1 °C. The experiments to studyMP deposition and release of fluorescein in the skin wereperformed for two formulation application times of 30 min and48 h. After completion of the experiments, the diffusion cellswere disassembled and the skin samples were gently dried witha paper towel. The microporated area of the skin was dissectedand snap-frozen in 2-methylbutane cooled with liquid nitrogenat −160 °C. Then the skin was sliced into 40 μm thick sectionsusing a cryotome (Microm HM 560 Cryostat, Walldorf,Germany). The sections were then fixed in 4% paraformalde-hyde and counterstained with Hoechst blue 33258 to visualizenuclei. Finally, the stained tissue sections were visualized undera confocal laser scanning microscope (LSM 700, Zeiss;Germany); Nile Red enabled localization of the MP, and therelease and diffusion of fluorescein from the MP was monitoredusing its characteristic green fluorescence. The confocal imageswere analyzed using Zen software (Carl Zeiss, Germany) andprocessed using ImageJ 1.45s software. The microporated areawas also visualized under an optical microscope to examine MPdeposition.Biodistribution of TA in the P.L.E.A.S.E. Porated Skin.

The same experimental setup was also used to study thebiodistribution of TA. A solution containing 2.5 mg of FFT1-TA10 (corresponding to 0.25 mg of TA) suspended in 200 μLof Milli-Q water was applied to the microporated skin for 48 h.The receiver phase was maintained at 34.5 ± 1 °C. Cutaneousdelivery was compared with that from a TA suspension (200μL, 0.25 mg of TA). After completion of the study, skinbiodistribution of TA released from MP was investigated as afunction of depth by quantifying the amount of TA present infive lamellae each with a thickness of 100 μm going from theskin surface to a nominal depth of 500 μm. The lamellae wereobtained after snap-freezing the skin samples and cryotoming(as described above). TA was also extracted from the remainingdermis. TA from each cryotomed skin lamella was extractedusing 4 mL of methanol:water (1:1) mixture. The amount ofTA diffused from the skin into the receiver compartment after48 h was also determined. TA biodistribution study sampleswere analyzed by the UHPLC−MS/MS method describedabove.Statistical Analysis. Data were expressed as mean ± SD.

Outliers determined using the Grubbs test were discarded.Results were evaluated statistically using analysis of variance(ANOVA followed by Student−Newman−Keuls test) orStudent t test. The level of significance was fixed at α = 0.05.

■ RESULTS AND DISCUSSIONCharacterization of MP. The size distribution and drug

encapsulation efficiencies of MP are shown in Table 1. Thepolymers were selected on the basis of their degradationbehavior and their ability to control drug release. PLGApolymers with a carboxylic acid end group, e.g., RG 503H,generally provide faster release in comparison to PLA polymers,e.g., R207. This can be further prolonged when the end group iscapped with an ester function as in R207 which can result inextended release for periods of up to several months. Therefore,it was decided to also investigate a mixture of R207 and RG503H (1:1) in order to achieve an intermediate release profile.It is known that the size of MP prepared by the o/w

emulsion technique is a function of several variables including

polymer concentration,30 solvent, drug content, stabilizermolecular weight,31 stabilizer concentration, and solventevaporation rate.29 In contrast, controlling the size of MPprepared by the FFT is more straightforward. The drug−polymer matrix is processed in a cryomill until the desired MPsize is achieved, and only the number of milling cycles needs tobe optimized at a given set of operating parameters such asfrequency and impact duration along with the cool time toharden the polymer−drug mixture between each cycle.Furthermore, TA-MP prepared by the FFT, FFT-TA10 andFFT-TA20, had encapsulation efficiencies of effectively 100%(99.9 ± 1.7% and 101.6 ± 2.1%, respectively). In contrast, TA-MP prepared by the o/w emulsion technique, OW1-TA10 (withRG 503H) and OW2-TA10 (with R 207), showed much lowerencapsulation efficiencies of 5.4 ± 0.3% and 6.8 ± 0.2%,respectively.SEM images showed that OW1-TA10 and OW2-TA10 were

spherical, whereas FFT-TA10 and FFT-TA20 were irregularlyshaped (Figure 1). In the case of the o/w emulsion technique,TA, which is practically insoluble in water, precipitated in theaqueous phase, and crystals were observed together with theTA-MP in the SEM images (Figure 1A,b and 1B,b). This effectwas probably due to transient diffusion of dichloromethane intothe aqueous phase, which caused a temporary increase in TAsolubility. As the dichloromethane slowly left the aqueousphase, TA precipitated out as its solubility decreased.32

Therefore, the final two washings were done with ethanol/water mixture (25/75) to dissolve and so remove theprecipitated TA.In contrast to the o/w emulsion technique, the FFT involves

preparation of a drug−polymer solid solution where drug andpolymer(s) are codissolved in an organic solvent, which issubsequently removed to form a drug−polymer solid matrix.The FFT is based on the concept of “cryogenic hardening”,which involves cooling a substance to cryogenic temperatures(CT, <−150 °C). Decreased molecular mobility of the polymermatrix under these conditions results in a decrease in thefracture resistance of the polymers.33 Therefore, whenimpaction forces are applied to the polymer, it is less able toundergo plastic deformation and low intensity forces aresufficient to induce fracturing. The SPEX 6770 freezer/millused in the present study for MP preparation is a smallcryogenic mill that is specifically designed for cryogenicgrinding of tough and/or temperature sensitive materials.The crystalline states of TA, RG 503H, R207/RG 503H

mixture, TA−polymer physical mixtures, and FFT1-TA10 andFFT2-TA20 were assessed by PXRD (Figure 2A). Crystaldiffraction peaks were clearly visible in the diffraction pattern ofpure TA (Figure 2A,a) and they were also observed in thephysical mixtures of TA-RG 503H and TA-R207/RG 503H(Figures 2A,d and 2A,e, respectively). However, they wereabsent in the FFT1-TA10 and FFT2-TA20 samples (Figures 2A,fand 2A,g, respectively), confirming that TA had beencompletely transformed into an amorphous form in theseTA-MP and that no drug was present on the MP surface.DSC thermograms were also recorded for TA, RG 503H,

R207/RG 503H mixture, TA−polymer physical mixtures, andFFT1-TA10 and FFT2-TA20 in order to assess the physical stateof TA in the TA-MP. As shown in Figure 2B, the endothermicpeak of pure TA was at 271.5 °C, which corresponds to itsmelting point. The glass transition points for RG 503H and theR207/RG 503H mixture were 53.2 and 60.2 °C, respectively.The DSC thermograms of the physical mixtures of TA with RG

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503H and R207/RG 503H also exhibited the TA melting peak,but instead of a sharp endotherm, a broad peak was observed.These broad peaks were found in the range from 260 °C to>300 °C due to solid state interactions between TA and thepolymer upon heating.34 The melting endotherm of TA wasshifted to a lower temperature in FFT2-TA20 (241 °C), but no

TA melting endotherm was seen in FFT1-TA10. The absence ofa TA melting transition (Tm) in FFT1-TA10 was due to thecomplete dissolution of the 10% TA load inside the MPformulation. The TA content in FFT2-TA20 was twice as high(i.e., 20%), and, given that TA has a limited solubility in R207and that the polymer also has a higher molecular weight and ismore viscous (MW 199.8 kDa and intrinsic viscosity 1.3−1.7dL g−1, respectively), some drug was precipitated from thepolymer and underwent a melting transition.

In Vitro Drug Release. Complete release of TA from thesuspension formulation, which was similar in composition toKenalog-40 injection, was achieved in 90 min (Figure 3). MPformulations prepared by FFT displayed sustained releaseprofiles; for FFT1-TA10, 91.83 ± 1.72% was released after 7days, and in the case of FFT2-TA20, 50.33 ± 2.24% wasreleased after 14 days (Figure 3).The differences between the two polymers were due to

differences in the inherent viscosity, molecular weight, and thenature of the end groups. Polymer matrices with free carboxylgroups such as PLGA (RG 503H) undergo faster waterabsorption, hydrolysis, and erosion than end-capped polymerswith an ester termination (e.g., PLA (R207)).35 Therefore, forFFT1-TA10 a triphasic release profile was observed. First,hydrophobic TA diffused through the external surface of thepolymeric MP immediately after coming in contact with therelease media; this gave an initial release of 8.24 ± 0.62% withinthe first 2 h. During the second phase of the FFT1-TA10triphasic release profile, constant release was observed betweendays 1 and 4. This second phase involved formation of smallwater-filled pores and hydrolytic degradation that led to thedevelopment of a porous connected network inside the MPmatrix and ensured rapid TA release. The third phasecorresponded to TA release due to surface erosion of the MPand diffusion of TA from the MP core. Since water absorptionwas slower in FFT2-TA20 because of its more hydrophobicpolymer mixture, only 2.65 ± 0.14% TA diffused through theexposed external region of MP in 2 h followed by constant,slow release up to the end of the study period of 14 days.MP degradation was visualized by SEM analysis of MP

recovered upon completion of the release study (Figures 1C,cand 1D,c). For FFT1-TA10, the integrity was markedly impairedas several “pores” were observed on the surface possiblycontributing to the observed faster degradation of polymer andrelease of the drug (Figure 1C,c). In contrast, the integrity ofFFT2-TA20 was intact and TA crystals were found in largenumbers on their surface because of slow diffusion and the highTA content in the particles (Figure 1D,c). The slowerdegradation of the FFT2-TA20 formulation was correlated tothe higher molecular weight and lower porosity of the polymermatrix due to R207.36 An inverse relation between drug releaseand polymer molecular weight or viscosity has also beenreported.37 Increasing viscosity decreases the permeability ofthe polymer to release media, which in turn slows the drugrelease process.30,38

The in vitro drug release studies showed that the TA-MPprovided sustained release under sink conditions, but theseobviously do not reflect the microenvironment of the skin and,more specifically, diseased skin. According to some studies,vascular density in keloid scars is less than in hypertrophic scarsand of course healthy skin.39,40 It was reported that keloid scarslack microvascular connections and suffer from inadequateblood supply because of excessive collagen growth. Deficientvascularization might prove to be an advantage for sustained

Figure 2. Physical characterization of MP. (A) Powder X-raydiffraction patterns of (a) triamcinolone acetonide (TA), (b) RG503H polymer, (c) R207/RG 503H polymer mixture (50:50), (d)TA−RG 503H physical mixture, (e) TA−R207/RG 503H physicalmixture, (f) FFT1-TA10, individual microparticle before the releasestudy, (g) FFT2-TA20, individual microparticle before the releasestudy, (h) FFT1-TA10, individual microparticle after the release study,and (i) FFT2-TA20, individual microparticle after the release study.(B) Differential scanning calorimetry thermograms of (a) triamcino-lone acetonide (TA), RG 503H polymer, TA−RG 503H physicalmixture, FFT1-TA10 before the release study, and FFT1-TA10 after therelease study; and (b) triamcinolone acetonide (TA), R207/RG 503H(50:50) polymer mixture, TA−-R207/RG 503H physical mixture,FFT2-TA20 before the release study, and FFT2-TA20 after the releasestudy.

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drug release from MP since, in the absence of blood capillaries,drug levels can be maintained over a longer time period,resulting in a more prolonged local action.The PXRD pattern of MP recovered after the release study

showed a crystalline peak due to TA in both FFT formulations,suggesting that TA released from the TA-MP during the releasestudy crystallized on the surface of the particles (Figure 2A,hand 2A,i). This was consistent with the SEM images (Figures1C,c and 1D,c). By the end of the release study, TAprecipitated on the surface of both MP formulations and gaverise to small Tm peaks at ∼231.0 °C together with the pure TATm at 271.5 °C (Figure 2B). During the release study TA wasreleased into the dissolution medium, and the ratio of TA topolymer in the formulation was reduced. The decrease in Tm

depends on the ratio of TA to the external phase present (i.e.,the polymer matrix), thus the Tm is reduced to even lowertemperature after the release study.41 These observations were

in agreement with a published report where the miscibility oflumefantrine in hydroxypropyl methylcellulose, selected as adispersing agent, decreased the Tm of wet-milled lumefan-trine.42

Using CLSM To Visualize MP Deposition andFluorescein Release in Laser Porated Skin. Opticalmicroscopy images of porcine skin samples (in 2D and 3D)following P.L.E.A.S.E. poration and application of FFT3-FL/NR clearly showed the presence of micropore array on the skinsurface (Figure 4); equivalent 2D images with human skin aregiven in Figure S4. CLSM enabled visualization and a 3Dreconstruction of the micropores (Figure 5), delimited byHoechst Blue staining of neighboring epidermal cell nuclei(Figures 5A and 5B), containing FFT3-FL/NR that werereadily localized due to the strong NR fluorescence (Figures 5Cand 5D). The depth of the micropores was ∼140 μm with adiameter of ∼220 μm. P.L.E.A.S.E. technology enables a precise

Figure 3. Release profiles of triamcinolone acetonide (TA) from the microparticle formulations, FFT1-TA10, FFT2-TA20, and a drug suspensionduring (A) 12 h and (B) 14 days. Data represent mean values of three replicates ± standard deviation.

Figure 4. Optical microscopy images of laser-porated skin showing (A) micropore array; (B) view at 4× magnification of individual micropores; (C)deposited microparticles (FFT3-FL/NR) in the micropore array; and (D) view at 4× magnification showing close-ups of the microparticlesdeposited in individual micropores.

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control on delivery kinetics by modulation of pore density(number of pores per cm2) and fluence (laser energy percm2).5−7,43 The fluence can be increased to obtain deepermicropores that penetrate further into skin. As keloids andhypertrophic scars are thick collagen tissues, higher fluencesmay be of interest; deeper pores would also be capable ofaccommodating more MP.The release behavior of fluorescein from the FFT3-FL/NR

trapped in the micropores was visualized by monitoring thepresence of its characteristic green fluorescence in theepidermis as it diffused out of the MP (Figure 6). Imageswere recorded after two application times: (i) 30 min (Figure6A−C) and (ii) 48 h (Figure 6D−F). After 30 min, the greenfluorescence was already clearly visible and mostly situated inand around the micropores (Figures 6B and 6C), whereas, after48 h, it was distributed throughout the epidermis and had alsoreached the dermis (Figures 6E and 6F).

Biodistribution of TA in the Epidermis and UpperDermis. The TA biodistribution profile, that is, the amount ofTA present as a function of depth within the epidermis anddermis, was determined after application of FFT1-TA10 and TAsuspension for 48 h. The amount of TA present in five lamellaeeach with a thickness of 100 μm going from the skin surface toa nominal depth of 500 μm was quantified; it was alsomeasured in the remaining dermal tissue. Comparison of theTA biodistribution profiles in skin following application ofFFT1-TA10 and the TA suspension showed significant

Figure 5. 3D confocal laser scanning microscopy images ofmicroporated skin following P.L.E.A.S.E. poration: (A) microporearray (Hoechst Blue stained the exposed nuclei in the viable epidermisunder the stratum corneum) after microporation; (B) singlemicropore; (C) micropore array with deposited microparticles(FFT3-FL/NR), which are identified by the strong Nile Redfluorescence; and (D) single micropore with deposited microparticles.

Figure 6. Confocal laser scanning microscopy images showing the FFT3-FL/NR microparticles (containing Nile Red and fluorescein (green))deposited in micropores present in porcine skin following P.L.E.A.S.E. poration after formulation application for 30 min [(A) XY plane, (B) 3Dreconstruction, and (C) XZ plane] and after formulation application for 48 h [(D) XY plane, (E) 3D reconstruction, and (F) XZ plane]. The FFT3-FL/NR microparticles remain localized within the pores as evidenced by the Nile Red signal, whereas fluorescein diffuses into the surroundingepidermis and dermis. The signal is present throughout the epidermis after 48 h.

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differences. The biodistribution observed with FFT1-TA10 wasappreciably more uniform (Figure 7A). Significantly greater

amounts of TA were present in the upper lamellae followingapplication of the TA suspension. Indeed, the amount of TAdeposited in the first two lamellae was 11 times higher from TAsuspension (12.28 ± 4.47 μg cm−2) than the TA-MP (1.13 ±0.38 μg cm−2).TA has very poor aqueous solubility, estimated at 15−20 μg

mL−1;44,45 therefore, given that the suspension contained 250μg of TA, this ensured that the TA solution was at saturationand that the thermodynamic activity of TA was at a maximumthroughout the duration of the experiment. This facilitatedpartitioning from the solution into the micropore wall, and theresulting high concentration gradient drove TA diffusion intothe epidermis and resulted in the increased amounts in the firstand second skin lamellae. The first two lamellae nominallydescend from the skin surface to a depth of ∼200 μm, which isthe approximate depth of the micropores. The presence ofmuch greater amounts of TA in the upper lamellae wasresponsible for the 6-fold higher total TA deposition in the skinfrom the TA suspension as compared to FFT1-TA10 (14.32 ±5.21 and 2.48 ± 0.54 μg cm−2, respectively) since the amountsof TA in subsequent lamellae were more similar. Cumulativepermeation of TA was almost 8-fold higher for the TAsuspension in comparison to FFT1-TA10: 29.48 ± 9.32 μg cm−2

and 3.83 ± 0.90 μg cm−2 (2.92 ± 0.3% of applied dose),respectively (Figure 7B). Given that application of TAsuspension for 48 h resulted in cumulative permeation acrossthe skin of 27.8 ± 4.4% of the applied TA dose whereas thecorresponding amount for FFT1-TA10 was only 2.92 ± 0.3%,the risk of systemic exposure in vivo can be significantlyattenuated (Figure 7C). Thus, very different release kineticsand distribution profiles were obtained with FFT1-TA10. It wasclearly able to control the release of TA into the micropore and

the surrounding tissue, which was much more uniform. Theestimated concentration of TA in the uppermost lamellafollowing application of FFT1-TA10 was 80.9 ± 42.0 μg mL−1,whereas the concentration of TA in the fifth lamella wasestimated to be 19.6 ± 5.7 μg mL−1. Although there are noreports on the TA concentrations achieved after intralesionalinjection, oral administration of TA (5 mg) results in meanCmax values of 10.5 ± 6.2 ng mL−1.46 Given that these plasmaconcentrations are sufficient to produce pharmacological effectsin target tissue but are much lower than the concentrationsobserved with FFT1-TA10 in the skin, we believe that the TAlevels following controlled release of TA from the depositedMP are more than sufficient to be of pharmacologicalrelevancedespite being much lower than those achievedfollowing application of the TA suspensionand that this is apromising approach for the successful treatment of keloids. It isfar more selective for localized skin delivery and limits systemicexposure. Moreover, if a higher dose were to be required,deposition of a larger volume of MP with higher TA loadingcould be used to further enhance TA delivery; alternatively, thepore density could also be increased.

Translation from the Bench to Clinical Practice. Ahypothetical treatment protocol for keloid scars using acombined laser microporation and TA-MP is shown in Figure8. The TA-MP would most likely be applied as a semisolidformulation since this could be spread over the entiremicroporated area on the scar surface. The semisolidformulation would also provide flexibility with respect to theapplication area given that the shape and area of keloids canvary considerably. Furthermore, the act of spreading andmassaging the formulation across the microporated area wouldalso facilitate entry of the TA-MP into the micropores; it haspreviously been demonstrated that massaging favored entry ofnanoparticulate carriers into hair follicles.47,48 The formulationcould be covered with an occlusive dressing which, in additionto hydrating the skin, would also reduce the risk of infection.Treatment periodicity would obviously have to be determinedin clinical trials, but it was recently reported that TiO2microparticles were retained in the skin for up to 30 daysfollowing application after laser microporation.49

■ CONCLUSIONSThe aim of the project was to develop a method to enable theintraepidermal delivery of drug reservoir systems, usingmicroparticle-based formulations, with a view to provide analternative strategy to treat keloids or hypertrophic scars.P.L.E.A.S.E. microporation enabled delivery and deposition ofTA-MP, which could serve as local depots in vivo for sustainedrelease of TA even after micropore closure. The MP present inthe micropores might interact with the epidermal/dermal tissueand “sticky” interstitial fluid in vivo; this might increaseadhesion and facilitate retention. It has been reported thatkeloid scars lack microvascular connections and suffer frominadequate blood supply because of excessive collagen growth;moreover, according to some studies, vascular density in keloidscars is less than that in hypertrophic scars.39,40 Deficientvascularization might prove to be an advantage for sustaineddrug release from MP since drug elimination would be reducedand drug levels maintained much longer, resulting in aprolonged, localized action. The preliminary results obtainedin this study are extremely promising; however, there arecaveats: (i) healthy porcine skin was used to estimate drugdelivery into “diseased” human skin; more specifically, the skin

Figure 7. (A) Comparison of TA biodistribution in skin to a totaldepth of 500 μm at a resolution of 100 μm after application of the TAsuspension and FFT1-TA10 formulations for 48 h. (B) Comparison ofTA permeation after 48 h application of TA suspension formulationand FFT1-TA10 suggests that the former would significantly increasethe risk of systemic exposure in vivo. (C) Comparison of total TArelease from TA suspension formulation and FFT1-TA10 afterapplication for 48 h in terms of the proportions deposited in andpermeated across the skin (expressed as a percentage of the amountapplied). (Mean ± SD; n ≥ 5.)

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does not have the same structure as an actual keloid scar; (ii)the MP were applied to fractionally ablated skin using aqueoussolutions; it is clear that, for clinical use, the TA-MP will needto be administered from semisolid formulations that will mostlikely be massaged into the porated skin; this should improveuptake; and (iii) the effect of occlusive/nonocclusive dressingson delivery kinetics would also need to be explored; this is alsolikely to impact the rate of pore closure. The combination oflaser microporation with MP based delivery systems and thecreation of intraepidermal/dermal drug depots should certainlybe of interest for the sustained and/or targeted local therapy ofother dermatological conditions: (i) those that requiremaintenance or prolonged therapy since it would reduce thefrequency of administration; (ii) where topical application ofconventional formulations, e.g., semisolids, to intact skin isineffective due to insufficient drug penetration and hence poorcutaneous bioavailability; (iii) where oral therapy results in lowcutaneous bioavailability and poor efficacy; (iv) for drugs wheresystemic exposure results in the risk of unacceptable adverseeffects; and (v) where cross-contamination might also be a riskfactor. Therefore, in addition to enabling sustained local drugconcentrations and modifying release kinetics, this approachmight also improve patient compliance. For example, patientssuffering from chronic skin conditions, e.g., with recalcitrantpsoriatic plaques, require long-term use of medications, andlack of adherence to therapy can be an issue. However, it isclear that each potential application will have to be evaluatedindividually, and some of these will be explored in futurepreclinical and clinical studies.

■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.molpharma-ceut.5b00711.

Additional data concerning validation of analyticalmethods, selection of dissolution study media, and MPdeposition in laser porated human skin (PDF)

■ AUTHOR INFORMATION

Corresponding Author*Tel.: +41 22 379 3355. Fax: +41 22 379 3360. E-mail: Yogi.Kalia@unige.ch.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

M.S. is grateful to the Swiss Federal Commission forScholarships for Foreign Students and the University of Genevafor their financial support. K.S. and S.d.R.-S. thank theUniversity of Geneva and the Swiss Commission forTechnology Innovation for financial support (CTI 13933.2).We thank Dr. Celine Besnard, Laboratoire de Crystallographie,UNIGE, for PXRD characterization. We also acknowledge theUNIGE Bioimaging platform for access to the confocal imagingfacilities.

Figure 8. Scheme showing protocol for the treatment of a keloid scar by P.L.E.A.S.E. laser microporation and deposition of triamcinolone acetonideloaded microparticles (TA-MP). (A) Image of keloid scar showing the surface morphology.50 (B) Histology image of the keloid scar displayingepidermis and dermis where densely packed hypocellular collagen fibers are clearly visible.51 (C) A schematic representation of a keloid scar showingepidermis, dermis, subcutaneous tissue, collapsed blood vessel, densely packed collagen bundles, blood vessel, and excess of fibroblasts. (D)Fractional ablation by P.L.E.A.S.E. Er:YAG laser to create micropores. (E) Deposition of TA-MP in the microporated skin. (F) Pore healing (i.e.,closure) with time and retention of MP in the pores along with sustained drug release from MP. (G) TA exerts an anti-inflammatory effect anddecreases the levels of collagenase inhibitors, which results in the breakdown of the collagen fibril network in the dermis and a reduction in thethickness of keloid scar. (H) Histology of healthy skin. Image of P.L.E.A.S.E. professional laser device is displayed in the center.52

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■ ABBREVIATIONS USED

TA, triamcinolone acetonide; MP, microparticles; TA-MP,triamcinolone acetonide loaded microparticles; HPLC−UV,high-performance liquid chromatography with ultravioletdetection; UHPLC−MS/MS, ultrahigh-performance liquidchromatography with tandem mass spectrometry detection;LOD, limit of detection; LOQ, limit of quantification; Er:YAG,erbium-doped yttrium aluminum garnet; PBS, phosphatebuffered saline; o/w, oil in water; PVA, poly(vinyl alcohol);FFT, freeze−fracture technique; PLGA, poly(D,L-lactide-co-glycolide); PLA, poly(D,L-lactide); EE, encapsulation efficiency;DL, drug loading; SEM, scanning electron microscopy; PXRD,powder X-ray diffraction; DSC, differential scanning calorim-etry; CLSM, confocal laser scanning microscopy; FL,fluorescein; NR, Nile Red

■ REFERENCES(1) Naik, A.; Kalia, Y. N.; Guy, R. H. Transdermal drug delivery:overcoming the skin’s barrier function. Pharm. Sci. Technol. Today2000, 3, 318−326.(2) Gratieri, T.; Alberti, I.; Lapteva, M.; Kalia, Y. N. Next generationintra- and transdermal therapeutic systems: Using non- and minimally-invasive technologies to increase drug delivery into and across the skin.Eur. J. Pharm. Sci. 2013, 50, 609−622.(3) Taudorf, E. H.; Lerche, C. M.; Vissing, A. C.; Philipsen, P. A.;Hannibal, J.; D’Alvise, J.; Hansen, S. H.; Janfelt, C.; Paasch, U.;Anderson, R. R.; Haedersdal, M. Topically applied methotrexate israpidly delivered into skin by fractional laser ablation. Expert Opin.Drug Delivery 2015, 12, 1−11.(4) Erlendsson, A. M.; Taudorf, E. H.; Eriksson, A. H.; Haak, C. S.;Zibert, J. R.; Paasch, U.; Anderson, R. R.; Haedersdal, M. Ablativefractional laser alters biodistribution of ingenol mebutate in the skin.Arch. Dermatol. Res. 2015, 307, 515.(5) Yu, J.; Bachhav, Y. G.; Summer, S.; Heinrich, A.; Bragagna, T.;Bohler, C.; Kalia, Y. N. Using controlled laser-microporation toincrease transdermal delivery of prednisone. J. Controlled Release 2010,148, 71−73.(6) Bachhav, Y. G.; Summer, S.; Heinrich, A.; Bragagna, T.; Bohler,C.; Kalia, Y. N. Effect of controlled laser microporation on drugtransport kinetics into and across the skin. J. Controlled Release 2010,146, 31−36.(7) Bachhav, Y. G.; Heinrich, A.; Kalia, Y. N. Controlled intra- andtransdermal protein delivery using a minimally invasive Erbium:YAGfractional laser ablation technology. Eur. J. Pharm. Biopharm. 2013, 84,355−364.(8) Weiss, R.; Hessenberger, M.; Kitzmuller, S.; Bach, D.;Weinberger, E. E.; Krautgartner, W. D.; Hauser-Kronberger, C.;Malissen, B.; Boehler, C.; Kalia, Y. N.; Thalhamer, J.; Scheiblhofer, S.Transcutaneous vaccination via laser microporation. J. ControlledRelease 2012, 162, 391−399.(9) Terhorst, D.; Fossum, E.; Baranska, A.; Tamoutounour, S.;Malosse, C.; Garbani, M.; Braun, R.; Lechat, E.; Crameri, R.; Bogen,B.; Henri, S.; Malissen, B. Laser-Assisted Intradermal Delivery ofAdjuvant-Free Vaccines Targeting XCR1+ Dendritic Cells InducesPotent Antitumoral Responses. J. Immunol. 2015, 194, 5895−5902.(10) Yu, J.; Kalaria, D. R.; Kalia, Y. N. Erbium:YAG fractional laserablation for the percutaneous delivery of intact functional therapeuticantibodies. J. Controlled Release 2011, 156, 53−59.(11) Zech, N. H.; Murtinger, M.; Uher, P. Pregnancy after ovariansuperovulation by transdermal delivery of follicle-stimulating hormone.Fertil. Steril. 2011, 95, 2784−2785.(12) Teutonico, D.; Montanari, S.; Ponchel, G. Leuprolide acetate:pharmaceutical use and delivery potentials. Expert Opin. Drug Delivery2012, 9, 343−354.(13) Gauglitz, G. G.; Korting, H. C.; Pavicic, T.; Ruzicka, T.; Jeschke,M. G. Hypertrophic scarring and keloids: pathomechanisms and

current and emerging treatment strategies. Mol. Med. 2011, 17, 113−125.(14) Peacock, E. E., Jr.; Madden, J. W.; Trier, W. C. Biologic basis forthe treatment of keloids and hypertrophic scars. South. Med. J. 1970,63, 755−760.(15) Mancini, R. E.; Quaife, J. V. Histogenesis of experimentallyproduced keloids. J. Invest. Dermatol. 1962, 38, 143−181.(16) O’Sullivan, S. T.; O’Shaughnessy, M.; O’Connor, T. P.Aetiology and management of hypertrophic scars and keloids. Ann.R. Coll. Surg. Engl. 1996, 78, 168−175.(17) Mutalik, S. Treatment of keloids and hypertrophic scars. IndianJ. Dermatol. Venereol. Leprol. 2005, 71, 3−8.(18) Juckett, G.; Hartman-Adams, H. Management of keloids andhypertrophic scars. Am. Fam. Physician 2009, 80, 253−260.(19) Niessen, F. B.; Spauwen, P. H.; Schalkwijk, J.; Kon, M. On thenature of hypertrophic scars and keloids: a review. Plast. Reconstr. Surg.1999, 104, 1435−1458.(20) Diegelmann, R. F.; Bryant, C. P.; Cohen, I. K. Tissue alpha-globulins in keloid formation. Plast. Reconstr. Surg. 1977, 59, 418−423.(21) Golladay, E. S. Treatment of keloids by single intraoperativeperilesional injection of repository steroid. South. Med. J. 1988, 81,736−738.(22) Sherris, D. A.; Larrabee, W. F., Jr.; Murakami, C. S. Managementof scar contractures, hypertrophic scars, and keloids. Otolaryngol. Clin.North Am. 1995, 28, 1057−1068.(23) Jalali, M.; Bayat, A. Current use of steroids in management ofabnormal raised skin scars. surgeon: journal of the Royal Colleges ofSurgeons of Edinburgh and Ireland 2007, 5, 175−180.(24) Kumar, S.; Singh, R. J.; Reed, A. M.; Lteif, A. N. Cushing’ssyndrome after intra-articular and intradermal administration oftriamcinolone acetonide in three pediatric patients. Pediatrics 2004,113, 1820−1824.(25) Finken, M. J.; Mul, D. Cushing’s syndrome and adrenalinsufficiency after intradermal triamcinolone acetonide for keloid scars.Eur. J. Pediatr. 2010, 169, 1147−1149.(26) Teelucksingh, S.; Balkaran, B.; Ganeshmoorthi, A.; Arthur, P.Prolonged childhood Cushing’s syndrome secondary to intralesionaltriamcinolone acetonide. Ann. Trop. Paediatr. 2002, 22, 89−91.(27) Liu, M. F.; Yencha, M. Cushing’s syndrome secondary tointralesional steroid injections of multiple keloid scars. Otolaryngol.–Head Neck Surg. 2006, 135, 960−961.(28) Mishra, S. Safe and less painful injection of triamcenoloneacetonide into a keloid–a technique. J. Plast. Reconstr. Aesthet. Surg.2010, 63, 205.(29) Sabzevari, A.; Adibkia, K.; Hashemi, H.; Hedayatfar, A.;Mohsenzadeh, N.; Atyabi, F.; Ghahremani, M. H.; Dinarvand, R.Polymeric triamcinolone acetonide nanoparticles as a new alternativein the treatment of uveitis: in vitro and in vivo studies. Eur. J. Pharm.Biopharm. 2013, 84, 63−71.(30) Birnbaum, D. T.; Kosmala, J. D.; Henthorn, D. B.; Brannon-Peppas, L. Controlled release of β-estradiol from PLAGA micro-particles:: The effect of organic phase solvent on encapsulation andrelease. J. Controlled Release 2000, 65, 375−387.(31) Jeong, Y. I.; Song, J. G.; Kang, S. S.; Ryu, H. H.; Lee, Y. H.;Choi, C.; Shin, B. A.; Kim, K. K.; Ahn, K. Y.; Jung, S. Preparation ofpoly(DL-lactide-co-glycolide) microspheres encapsulating all-transretinoic acid. Int. J. Pharm. 2003, 259, 79−91.(32) Layre, A.-M.; Gref, R.; Richard, J.; Requier, D.; Chacun, H.;Appel, M.; Domb, A. J.; Couvreur, P. Nanoencapsulation of acrystalline drug. Int. J. Pharm. 2005, 298, 323−327.(33) Chen, Z.-K.; Yang, J.-P.; Ni, Q.-Q.; Fu, S.-Y.; Huang, Y.-G.Reinforcement of epoxy resins with multi-walled carbon nanotubes forenhancing cryogenic mechanical properties. Polymer 2009, 50, 4753−4759.(34) Adibkia, K.; Shadbad, M. R. S.; Nokhodchi, A.; Javadzedeh, A.;Barzegar-Jalali, M.; Barar, J.; Mohammadi, G.; Omidi, Y. Piroxicamnanoparticles for ocular delivery: Physicochemical characterization andimplementation in endotoxin-induced uveitis. J. Drug Target. 2007, 15,407−416.

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DOI: 10.1021/acs.molpharmaceut.5b00711Mol. Pharmaceutics 2016, 13, 500−511

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(35) Tracy, M. A.; Ward, K. L.; Firouzabadian, L.; Wang, Y.; Dong,N.; Qian, R.; Zhang, Y. Factors affecting the degradation rate ofpoly(lactide-co-glycolide) microspheres in vivo and in vitro.Biomaterials 1999, 20, 1057−1062.(36) Anderson, J. M.; Shive, M. S. Biodegradation and biocompat-ibility of PLA and PLGA microspheres. Adv. Drug Delivery Rev. 1997,28, 5−24.(37) Vert, M.; Mauduit, J.; Li, S. Biodegradation of PLA/GApolymers: increasing complexity. Biomaterials 1994, 15, 1209−1213.(38) Zidan, A.; Sammour, O.; Hammad, M.; Megrab, N.; Hussain,M.; Khan, M.; Habib, M. Formulation of anastrozole microparticles asbiodegradable anticancer drug carriers. AAPS PharmSciTech 2006, 7,38−46.(39) Kurokawa, N.; Ueda, K.; Tsuji, M. Study of microvascularstructure in keloid and hypertrophic scars: Density of microvessels andthe efficacy of three-dimensional vascular imaging. J. Plast. Surg. HandSurg. 2010, 44, 272−277.(40) Kischer, C. W.; Thies, A. C.; Chvapil, M. Perivascularmyofibroblasts and microvascular occlusion in hypertrophic scarsand keloids. Hum. Pathol. 1982, 13, 819−824.(41) Chokshi, R. J.; Sandhu, H. K.; Iyer, R. M.; Shah, N. H.; Malick,A. W.; Zia, H. Characterization of physico-mechanical properties ofindomethacin and polymers to assess their suitability for hot-meltextrusion processs as a means to manufacture solid dispersion/solution. J. Pharm. Sci. 2005, 94, 2463−2474.(42) Gahoi, S.; Jain, G. K.; Tripathi, R.; Pandey, S. K.; Anwar, M.;Warsi, M. H.; Singhal, M.; Khar, R. K.; Ahmad, F. J. Enhancedantimalarial activity of lumefantrine nanopowder prepared by wet-milling DYNO MILL technique. Colloids Surf., B 2012, 95, 16−22.(43) Bachhav, Y. G.; Heinrich, A.; Kalia, Y. N. Using lasermicroporation to improve transdermal delivery of diclofenac:Increasing bioavailability and the range of therapeutic applications.Eur. J. Pharm. Biopharm. 2011, 78, 408−414.(44) Block, L. H.; Patel, R. N. Solubility and dissolution oftriamcinolone acetonide. J. Pharm. Sci. 1973, 62, 617−621.(45) Behl, C. R.; Block, L. H.; Borke, M. L. Aqueous solubility of 14c-triamcinolone acetonide. J. Pharm. Sci. 1976, 65, 429−430.(46) Rohatagi, S.; Hochhaus, G.; Mollmann, H.; Barth, J.; Galia, E.;Erdmann, M.; Sourgens, H.; Derendorf, H. Pharmacokinetic andpharmacodynamic evaluation of triamcinolone acetonide after intra-venous, oral, and inhaled administration. J. Clin. Pharmacol. 1995, 35,1187−1193.(47) Lademann, J.; Richter, H.; Teichmann, A.; Otberg, N.; Blume-Peytavi, U.; Luengo, J.; Weiß, B.; Schaefer, U. F.; Lehr, C.-M.; Wepf,R.; Sterry, W. Nanoparticles − An efficient carrier for drug deliveryinto the hair follicles. Eur. J. Pharm. Biopharm. 2007, 66, 159−164.(48) Mak, W. C.; Patzelt, A.; Richter, H.; Renneberg, R.; Lai, K. K.;Ruhl, E.; Sterry, W.; Lademann, J. Triggering of drug release ofparticles in hair follicles. J. Controlled Release 2012, 160, 509−514.(49) Genina, E. A.; Bashkatov, A. N.; Dolotov, L. E.; Maslyakova, G.N.; Kochubey, V. I.; Yaroslavsky, I. V.; Altshuler, G. B.; Tuchin, V. V.Transcutaneous delivery of micro- and nanoparticles with lasermicroporation. J. Biomed. Opt. 2013, 18, 111406.(50) Carantino, I.; Florescu, I. P.; Carantino, A. Overview about thekeloid scars and the elaboration of a non-invasive, unconventionaltreatment. J. Med. Life 2010, 3, 122−127.(51) Hunasgi, S.; Koneru, A.; Vanishree, M.; Shamala, R. Keloid: Acase report and review of pathophysiology and differences betweenkeloid and hypertrophic scars. J. Oral Maxillofac. Pathol. 2013, 17,116−120.(52) Pantec Biosolutions, Laser microporation technologyP.L.E.A.S.E.® Platform, http://www.pantec-biosolutions.com/en/technology/laser-microporation, accessed: July, 2015.

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