Fractional Laser Ablation for the Cutaneous Delivery of Triamcinolone

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Fractional Laser Ablation for the Cutaneous Delivery of Triamcinolone Acetonide from Cryomilled Polymeric Microparticles: Creating Intra-epidermal Drug Depots Mayank Singhal, Sergio Del Rio-Sancho, Kiran Sonaje, and Yogeshvar N. Kalia Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00711 • Publication Date (Web): 05 Jan 2016 Downloaded from http://pubs.acs.org on January 14, 2016

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Molecular Pharmaceutics

Fractional Laser Ablation for the Cutaneous Delivery of Triamcinolone Acetonide from Cryomilled Polymeric Microparticles: Creating Intra-epidermal Drug Depots Mayank Singhal, Sergio del Río-Sancho, Kiran Sonaje, and Yogeshvar N. Kalia* School of Pharmaceutical Sciences, University of Geneva, 30 Quai Ernest Ansermet, 1211 Geneva, Switzerland

∗ Corresponding author: Yogeshvar N. Kalia School of Pharmaceutical Sciences University of Geneva 30 Quai Ernest Ansermet 1211 Geneva, Switzerland Tel.: +41 22 379 3355 Fax: +41 22 379 3360 Email: [email protected]

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ABSTRACT The efficacy of some dermatological therapies might be improved by the use of “high dose” intra-epidermal drug reservoir systems that enable sustained and targeted local drug delivery – e.g. in the treatment of keloids and hypertrophic scars. Here, a fractionally ablative Erbium:YAG laser was used to enable “needle-less” cutaneous deposition of polymeric microparticles containing triamcinolone acetonide (TA). The microparticles were prepared using a freezefracture technique employing cryomilling that resulted in drug loading efficiencies of ~100 %. They were characterized by several different 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. Confocal laser scanning microscopy enabled visualization of cryomilled microparticles containing fluorescein and Nile Red in the cutaneous micropores and the subsequent release of fluorescein into the micropores and its diffusion throughout the epidermis and upper dermis. The biodistribution of TA, i.e. the amount of drug as a function of depth in skin, following microparticle application was much more uniform than with a TA suspension and delivery was selective for deposition with less transdermal permeation. These findings suggest that this approach may provide an effective, targeted and minimally-invasive alternative to painful intra-lesional injections for the treatment of keloid scars.

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


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GRAPHICAL ABSTRACT


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INTRODUCTION Transport across the stratum corneum constitutes the rate-limiting step for the cutaneous delivery of most molecules. Its structure imposes a high degree of tortuosity that increases the diffusion pathlength and the lipid-rich character of the intercellular matrix, the principal diffusion pathway, compounds the challenge faced by hydrophilic molecules.1 It follows that numerous strategies have been explored to compromise stratum corneum integrity and to create transport conduits that facilitate diffusion. These include the use of microneedles and assorted fractional ablation techniques, e.g. radio-frequency and the use of CO2 and Er:YAG lasers.2 These ablation-based approaches have been used to enhance drug delivery for both local and systemic applications. The P.L.E.A.S.E.® (Precise Laser Epidermal System; Pantec Biosolutions AG) is a fractionally ablative Er:YAG laser that emits µs pulses at a wavelength of 2936 nm. These excite water molecules in the epidermis and dermis and their explosive evaporation results in the formation of micropores. Modulation of the device parameters enables precise control of pore density (i.e. number of pores per cm2) and pore depth. Work to-date has demonstrated that the technique can increase the rate and the extent of the delivery of small molecules,3-6 peptides and proteins7 into and across the skin and that it can also be used for the intra-epidermal delivery of vaccines.8,9 Furthermore, it has also been used for the “needle-free” cutaneous delivery of biologically active antibodies.10 A patient treatment paradigm based on this strategy would involve a two-step approach with laser microporation being followed by application of the drug formulation.11 In principle, formulation application could be repeated at the same microporation site as long as the pores remained open – a conservative estimate would be for 48-72 h – beyond that time interval, a new micropore array must be created. However, from a clinical perspective, it would be of


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considerable interest to be able to introduce an intra-epidermal drug depot where the natural healing process after microporation would close the skin around it. The depot would have different drug release kinetics to a formulation applied topically to the skin surface and would enable sustained drug release over a more prolonged time-frame. This intra-epidermal drug depot is clearly analogous to the well-known subcutaneous and intramuscular depots that are frequently used in therapy to enable prolonged systemic release of peptide therapeutics, e.g. gonadotropinreleasing hormone agonists and antagonists.12 This approach would be of particular interest in the treatment of several dermatological conditions, including keloids and hypertrophic scars, which are caused by an over-proliferation of dense fibrous tissue and develop as a result of anomalies during normal physiological wound healing processes. These conditions affect approximately 100 million patients each year following elective or trauma-induced operations.13 Both keloid and hypertrophic scars protrude above the skin surface. However, keloids can extend beyond the boundaries of the original wound, grow for years, and can reappear after excision; hypertrophic scars, which are more common, are delimited by the original wound borders and may regress over time.14,15 Scars affect patient quality of life both physically and psychologically; they are cosmetically unacceptable and can cause pruritus, pain and contractures.16 Available treatment options include pressure treatment, cryotherapy, surgical removal, silicone sheeting, pulsed dye laser, and pharmacotherapy involving topical application or intra-lesional injection 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 co-administered with lidocaine, are the first-line treatment for keloids.19 TA has potent glucocorticoid activity and suppresses the inflammatory response during the wound healing process; it can also decrease levels of


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collagenase inhibitors (alpha-1-antitrypsin and alpha-2-macroglobulin) in keloids, thereby promoting collagen degradation.20,21 Therapy can be continued for six months or even longer, injections are given weekly or monthly depending on the severity of the condition.22,23 Common local adverse effects associated with the intra-lesional TA injection are skin and subcutaneous fat atrophy, telangiectasia and significant pain at the injection site. Long-term systemic exposure of TA causes Cushing's syndrome and adrenal insufficiency, especially in children.24-27 Noninvasive treatment by topical administration of corticosteroids to the keloid surface shows poor efficacy due to poor cutaneous bioavailability, i.e. suboptimal levels of drug in the skin. Intralesional injections obviously ensure greater drug bioavailability but, as mentioned above, increase the risk of systemic 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 exposure would 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 following fractional laser ablation. In contrast to conventional techniques, which often display poor drug loading, the FFT enables extremely high loading efficiencies to be achieved. This technique involves grinding a solid solution of the drug polymer mixture in a cryogenic grinder at approximately -200 °C until micron-sized particles are obtained. The working hypothesis was that TA-MP with high drug loading deposited in scars following fractional laser ablation would enable sustained release of TA with different release kinetics to a topically applied solution/suspension. The specific objectives of this study were (i) to develop and to optimize a new technique to prepare stable TA-MP with high drug loading, (ii) to develop and to characterize TA-MP formulations using 50/50 poly(D, L-lactide-coglycolide) and poly(D, L-lactide) and to compare


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their release profiles, (iii) to visualize MP loaded with fluorescein and Nile Red in microporated skin using confocal laser scanning microscopy (CLSM) and to monitor release of fluorescein from the MP deposited in the cutaneous micropores, and (iv) to compare and contrast the TA biodistribution (the amounts of TA as a function of position) in the skin following release from the deposited TA-MP and after application of a TA suspension. EXPERIMENTAL SECTION Materials. TA was purchased from Hänseler AG (Herisau, Switzerland). Resomer® RG 503H (RG 503H; 50/50 poly(D, L-lactide-coglycolide), MW 24-38 kDa, intrinsic viscosity 0.32-0.44 dL g-1), Resomer® R207 (R207; poly(D, L-lactide), MW 199.8 kDa, intrinsic viscosity 1.3-1.7 dL g-1), fluorescein, and dichloromethane were purchased from Sigma-Aldrich Chemie (Buchs, Switzerland). Nile Red was purchased from TCI Europe N.V. (Belgium). Polyvinyl 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 by either the conventional oil in water (o/w) emulsion technique – OW1-TA10 (with RG 503H) and OW2-TA10 (with R207) or the newly developed FFT (FFT1-TA10 and FFT2-TA20). The composition of each MP batch and the preparation method are given in Table 1. Preparation of MP with the o/w emulsion technique involved initial dissolution of the polymer (90 mg) and TA (10 mg) in dichloromethane (5 mL) at room temperature and subsequent slow addition to 25 mL of 1% (w/v) aqueous PVA solution under homogenization at 7,500 rpm for 1 min using a Polytron® PT 2500 E Homogenizer (Kinematica AG; Luzern, Switzerland). The o/w emulsion was then transferred to 100 mL of 1% (w/v) aqueous PVA solution under homogenization at 5,000 rpm for 2 min. The final emulsion


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was left overnight at room temperature under continuous stirring to evaporate the organic solvent and to harden the MP. The resulting MP were centrifuged at 1620 × g for 5 min and the pelleted MP were washed twice with Milli-Q® water and ethanol/water (25/75) mixture to remove unloaded TA. The final microparticle pellet was redispersed in 1.0 mL Milli-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 each polymer for FFT2-TA20) and TA (50 mg and 150 mg for FFT1-TA10 and FFT2-TA20, respectively) in dichloromethane (17 mL) at 40°C and then removing the organic solvent using a Rotavapor® R-124 (Buchi Labortechnik AG; Switzerland) to obtain a solid solution of TA and polymer. The TA-polymer solid solution was freeze-fractured using a cryogenic mill (6770 Freezer/Mill®, SPEX SamplePrep; Stanmore, UK). The mill was set for 10 cycles operating at an impaction rate of 15 cps with a run time of 1.5 min and a cool time of 2 min between each cycle. Liquid nitrogen, a cryogen, was added and grinding initiated. The fractured particles were then passed through a 90 µm sieve (#170 U.S. mesh size). Particles larger than 90 µm were processed again for 10 cycles. Particles were collected and stored for one night under vacuum at room temperature to remove any residual moisture. Preparation of Fluorescein/Nile Red Loaded MP. Fluorescent dye loaded MP (FFT3-FL/NR) containing fluorescein (2 % w/w) and Nile Red (0.2 % w/w) were prepared using the same method in order to visualize the deposition and localization of MP in microporated skin and the release of their “cargo” – fluorescein – into the surrounding tissue by CLSM. Analytical Methods. A previously reported reversed-phase HPLC method was adapted and validated for quantification of TA in the in vitro release study.29 The HPLC apparatus comprised a P680A LPG-4 pump in line with an ASI-100 autosampler, a TCC-100 thermostatted column


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compartment, and a UV170D detector (Dionex; Voisins LeBretonneux, France). Briefly, the samples were analyzed using a LiChrospher® 100, RP-18 (4 x 250 mm and 5 µm particle size) column (BGB Analytik AG; Boeckten, Switzerland) with acetonitrile:water (60:40, v/v) as mobile phase (flow rate: 0.6 mL min-1). TA was quantified using a detection wavelength of 266 nm; the retention time was 6.35 ± 0.1 min and the assay was linear (r2 = 0.999) in the concentration range of 0.5 – 50 µg mL-1. The specificity of the analytical method was tested in the presence of skin matrix compounds (Supporting Information: Figure S1, Table S1). An isocratic UHPLC-MS/MS method was developed and validated to determine the biodistribution of TA, that is, the amounts of TA deposited as a function of position in the different skin layers during skin transport studies (Supporting Information: Figure S2, Table S2). A Waters Acquity® UPLC® core system (Baden-Dättwil, Switzerland) comprising a binary solvent pump and sample manager with a Waters XEVO® TQ-MS tandem quadrupole detector (Baden-Dättwil, Switzerland) equipped with electron spray ion source was used for the UHPLC-MS/MS analysis together with a Waters XBridge™ BEH C18 column (2.1 x 50 mm and 2.5 µm particle size). The mobile phase comprised 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-MS detector was operated in positive ion mode using multiple reaction monitoring. The mass transition ion-pair was m/z 435.277 →415.154 (removal of hydrogen fluoride). MS source parameters were as follows: ion spray capillary voltage, 1.8 kV; cone voltage, 19 V; desolvation gas temperature, 500 °C; cone gas flow rate, 1000 L h-1; and collision energy, 2 V. Data acquisition was carried out using MassLynx V4.1 software. Characterization of MP. TA content in the MP was determined by dissolving approximately 5 mg of MP in 1 mL of dichloromethane and diluting 25 times with methanol before HPLC


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analysis. The drug loading and encapsulation efficiencies of MP were calculated using eqs 1 and 2, respectively.

Drug loading % =

Encapsulation efficiency % =

x 100

+ ,

(1) x 100

(2)

To measure MP size and size distribution, approximately 15 mg of MP were suspended in 7 mL of 1% aqueous PVA solution and analyzed using a Mastersizer S (Malvern Instruments Ltd; Malvern, UK). Surface morphology of the MP was studied using scanning electron microscopy (SEM) (JEOL JSM-7001FA, JEOL USA, Inc.; CA) after mounting the MP on a conductive carbon surface and coating with a thin layer of gold (~21.1 nm) using a high vacuum sputter coater (Leica EM SCD500, Anatech USA; CA) prior to microscopy. Powder X-ray diffraction (PXRD) patterns were measured using an Agilent Supernova Diffractometer (CA) and Cu radiation by loading the samples in a 100 µm MiTeGen cryoloop using fomblin oil. A rotation of 180° was made during 6 min. The pattern was then integrated using the CrysAlisPro Agilent software. MP phase transitions were analyzed by differential scanning calorimetry (DSC) (SSC/5200, Seiko Instruments; UK). Approximately 5 mg of each MP was weighed in an aluminum pan and the thermograph was recorded over a heating range of 30°C to 320°C at 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 enhancer was selected as the dissolution medium for the in vitro release study (Supporting Information: Figure S3). A TA suspension, with a composition similar to the Kenalog® -40 injection (Bristol-Myers Squibb), was prepared as a control formulation. FFT1TA10 and FFT2-TA20 (5 mg; – containing 0.5 and 1 mg TA, respectively) and 12.5 µL of TA


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suspension (TA, 0.5 mg) were dispersed in 50 mL of dissolution medium. All samples were kept in a shaker bath maintained at 34.5 ± 1 °C and 300 rpm. Aliquots (1 mL) were withdrawn at predetermined time intervals and centrifuged (Eppendorf Centrifuge 5804; Schönenbuch, Switzerland) at 10621 x g for 5 min. An aliquot (0.5 mL) of supernatant was taken and analyzed for TA content using HPLC while the remaining 0.5 mL was diluted with a fresh 0.5 mL of dissolution media and vortexed to resuspend the MP and transferred back to the dissolution container. All samples were analyzed in triplicate. The changes in the physical properties of the MP after the in vitro release study were also monitored. For this, the MP incubated in release medium for one week were collected by centrifugation (1620 x g, 5 min) and dried under vacuum 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 local abattoir (CARRE; Rolle, Switzerland). After cleaning with running cold water, the skin from the outer region of ears was carefully excised from the underlying cartilage using a scalpel. The excised full thickness skin samples (2 mm) were then punched into 32 mm circular discs and then stored at -20 °C for no more than 1 month before use. Frozen skin samples were thawed and equilibrated in 0.9 % NaCl solution for 30 min before microporation using an Er:YAG laser (P.L.E.A.S.E.® , Pantec Biosolutions AG; Ruggell, Liechtenstein). Skin surface moisture was removed and then the samples were mounted on a custom designed assembly. Microporation parameters were set to provide 300 pores cm-2 (15% pore density) at a fluence of 90 J cm-2. MP Deposition in P.L.E.A.S.E.® Porated Skin and Visualization by CLSM. The P.L.E.A.S.E.® porated skin samples were mounted in Franz diffusion cells (area 2.0 ± 0.1 cm2)


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and silicone grease was applied at the edges to ensure a water tight seal. The receiver compartments were filled with release medium (10 mL PBS at pH 7.4 with 1% Tween 80) and skin was equilibrated for 30 min. For the confocal microscopy experiments, 2.5 mg of FFT3FL/NR was suspended in 200 µL of Milli-Q® water and applied to the external surface of the microporated skin samples. The receiver phase was maintained at 34.5 ± 1 °C. The experiments to study MP deposition and release of fluorescein in the skin were performed for two formulation application times of 30 min and 48 h. After completion of the experiments, the diffusion cells were disassembled and the skin samples were gently dried with a paper towel. The microporated area of the skin was dissected and snap-frozen in 2-methylbutane cooled with liquid nitrogen at 160 °C. Then the skin was sliced into 40 µm thick sections using a cryotome (Microm HM 560 Cryostat, Walldorf, Germany). The sections were then fixed in 4% paraformaldehyde and counterstained with Hoechst blue 33258 to visualize nuclei. Finally, the stained tissue sections were visualized under a confocal laser scanning microscope (LSM 700, Zeiss; Germany); Nile Red enabled localization of the MP and the release and diffusion of fluorescein from the MP was monitored using its characteristic green fluorescence. The confocal images were analyzed using Zen software (Carl Zeiss, Germany) and processed using Image J 1.45s software. The microporated area was also visualized under an optical microscope to examine MP deposition. Biodistribution of TA in the P.L.E.A.S.E.® Porated Skin. The same experimental set-up was also used to study the biodistribution of TA. A solution containing 2.5 mg of FFT1-TA10 (corresponding to 0.25 mg of TA) suspended in 200 µL of Milli-Q® water was applied to the microporated skin for 48 h. The receiver phase was maintained at 34.5 ± 1 °C. Cutaneous delivery was compared with that from a TA suspension (200 µL, 0.25 mg of TA). After completion of the study, skin biodistribution of TA released from MP was investigated as a


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function of depth by quantifying the amount of TA present in five lamellae each with a thickness of 100 µm going from the skin surface to a nominal depth of 500 µm. The lamellae were obtained after snap-freezing the skin samples and cryotoming (as described above). TA was also extracted from the remaining dermis. TA from each cryotomed skin lamella was extracted using 4 mL methanol:water (1:1) mixture. The amount of TA diffused from the skin into the receiver compartment after 48 h was also determined. TA biodistribution study samples were analyzed by the UHPLC-MS/MS method described above. 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) or Student t-test. The level of significance was fixed at α=0.05. RESULTS AND DISCUSSION Characterization of MP. The size distribution and drug encapsulation efficiencies of MP are shown in Table 1. The polymers were selected on the basis of their degradation behavior and their ability to control drug release. PLGA polymers 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 is capped with an ester function as in R207 which can result in extended release for periods of up to several months. Therefore, it was decided to also investigate a mixture of R207 and RG 503H (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, stabilizer molecular weight,31


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stabilizer concentration, and solvent evaporation rate.29 In contrast, controlling the size of MP prepared by the FFT is more straightforward. The drug-polymer matrix is processed in a cryomill until the desired MP size is achieved and only the number of milling cycles needs to be optimized at a given set of operating parameters such as frequency and impact duration along with the cool time to harden the polymer-drug mixture between each cycle. Furthermore, TA-MP prepared by the FFT, FFT-TA10 and FFT-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 (with RG 503H) and OW2-TA10 (with R 207), showed much lower encapsulation 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 irregularly shaped (Figure 1). In the case of the o/w emulsion technique, TA, which is practically insoluble in water, precipitated in the aqueous phase and crystals were observed together with the TA-MP in the SEM images (Figure 1A,b and 1B,b). This effect was probably due to transient diffusion of dichloromethane into the aqueous phase, which caused a temporary increase in TA solubility. As the dichloromethane slowly left the aqueous phase, 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 the precipitated TA. In contrast to the o/w emulsion technique, the FFT involves preparation of a drug-polymer solid solution where drug and polymer(s) are co-dissolved in an organic solvent, which is subsequently 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 polymer matrix under these conditions results in a decrease in the fracture resistance of the polymers.33 Therefore, when impaction forces are


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applied to the polymer it is less able to undergo plastic deformation and low intensity forces are sufficient to induce fracturing. The SPEX® 6770 Freezer/Mill® used in the present study for MP preparation is a small cryogenic mill that is specifically designed for cryogenic grinding 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 and FFT2-TA20 were assessed by PXRD (Figure 2A). Crystal diffraction peaks were clearly visible in the diffraction pattern of pure TA (Figure 2A,a) and they were also observed in the physical mixtures of TA-RG 503H and TA-R207/RG 503H (Figure 2A,d and 2A,e, respectively). However, they were absent in the FFT1-TA10 and FFT2-TA20 samples (Figure 2A,f and 2A,g, respectively) confirming that TA had been completely transformed into an amorphous form in these TA-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, and FFT1-TA10 and FFT2-TA20 in order to assess the physical state of TA in the TA-MP. As shown in Figure 2B, the endothermic peak of pure TA was at 271.5 °C which corresponds to its melting point. The glass transition points for RG 503H and the R207/RG 503H mixture were 53.2 °C and 60.2 °C, respectively. The DSC thermograms of the physical mixtures of TA with RG 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 the polymer upon heating.34 The melting endotherm of TA was shifted to a lower temperature in FFT2-TA20 (241 °C) but no TA melting endotherm was seen in FFT1-TA10. The absence of a TA melting transition (Tm) in FFT1-TA10 was due to the complete dissolution of the 10% TA load inside the MP formulation. The TA content in FFT2-TA20 was twice as high (i.e. 20 %) and given that TA has a limited


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solubility in R207 and that the polymer also has a higher molecular weight and is more viscous (MW 199.8 kDa and intrinsic viscosity 1.3-1.7 dL g-1, respectively), some drug was precipitated from the polymer and underwent a melting transition. In Vitro Drug Release. Complete release of TA from the suspension formulation, which was similar in composition to Kenalog® -40 injection, was achieved in 90 min (Figure 3). MP formulations prepared by FFT displayed sustained release profiles; for FFT1-TA10, 91.83 ± 1.72 % was released after 7 days, and in the case of FFT2-TA20, 50.33 ± 2.24 % was released after 14 days (Figure 3). The differences between the two polymers were due to differences in the inherent viscosity, molecular weight and the nature of the end-groups. Polymer matrices with free carboxyl groups such as PLGA (RG 503H) undergo faster water absorption, hydrolysis and erosion than endcapped polymers with an ester termination (e.g. PLA (R207)).35 Therefore, for FFT1-TA10 a triphasic release profile was observed. First, hydrophobic TA diffused through the external surface of the polymeric MP immediately after coming in contact with the release media; this gave an initial release of 8.24 ± 0.62 % within the first 2 h. During the second phase of the FFT1-TA10 tri-phasic release profile, constant release was observed between days 1 to 4. This second phase involved formation of small water-filled pores and hydrolytic degradation that led to the development of a porous connected network inside the MP matrix and ensured rapid TA release. The third phase corresponded to TA release due to surface erosion of the MP and diffusion of TA from the MP core. Since water absorption was slower in FFT2-TA20 because of its more hydrophobic polymer mixture, only 2.65 ± 0.14 % TA diffused through the exposed external region of MP in 2 h followed by constant, slow release up to the end of the study period of 14 days.


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MP degradation was visualized by SEM analysis of MP recovered upon completion of the release study (Figure 1C,c and 1D,c). For FFT1-TA10, the integrity was markedly impaired as several “pores” were observed on the surface possibly contributing to the observed faster degradation of polymer and release of the drug (Figure 1C,c). In contrast, the integrity of FFT2TA20 was intact and TA crystals were found in large numbers on their surface because of slow diffusion and the high TA content in the particles (Figure 1D,c). The slower degradation of the FFT2-TA20 formulation was correlated to the higher molecular weight and lower porosity of the polymer matrix due to R207.36 An inverse relation between drug release and polymer molecular weight or viscosity has also been reported.37 Increasing viscosity decreases the permeability of the polymer to release media which in turn slows the drug release process.30,38 The in vitro drug release studies showed that the TA-MP provided sustained release under sink conditions but these obviously 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 scars and of course healthy skin.39,40 It was reported that keloid scars lack microvascular connections and suffer from inadequate blood supply because of excessive collagen growth. Deficient vascularization might prove to be an advantage for sustained 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 release study crystallized on the surface of the particles (Figure 2A,h and 2A,i). This was consistent with the SEM images (Figure 1C,c and 1D,c). By the end of the release study, TA precipitated on the surface of both MP formulations and gave rise to small Tm peaks at ~231.0 °C together with the


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pure TA Tm at 271.5 °C (Figure 2B). During the release study TA was released into the dissolution media and the ratio of TA to polymer 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 lower temperature after the release study.41 These observations were in agreement with a published report where the miscibility of lumefantrine in hydroxypropyl methylcellulose, selected as a dispersing agent, decreased the Tm of wet-milled lumefantrine.42 Using CLSM to Visualize MP Deposition and Fluorescein Release in Laser Porated Skin. Optical microscopy 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 skin surface (Figure 4); equivalent 2D images with human skin are given in the Supporting Information (Figure S4). CLSM enabled visualization and a 3D reconstruction of the micropores (Figure 5), delimited by Hoechst Blue staining of neighboring epidermal cell nuclei (Figure 5A and 5B), containing FFT3-FL/NR that were readily localized due to the strong NR fluorescence (Figure 5C and 5D). The depth of the micropores was ~140 µm with a diameter of ~220 µm. P.L.E.A.S.E.® technology enables a precise control on delivery kinetics by modulation of pore density (number of pores per cm2) and fluence (laser energy per cm2).5-7,43 The fluence can be increased to obtain deeper micropores that penetrate further into skin. As keloids and hypertrophic scars are thick collagen tissues, higher fluences may be of interest; deeper pores would also be capable of accommodating more MP. The release behavior of fluorescein from the FFT3-FL/NR trapped in the micropores was visualized by monitoring the presence of its characteristic green fluorescence in the epidermis as it diffused out of the MP (Figure 6). Images were recorded after two application times (i) 30 min (Figure 6A-C) and (ii) 48 h (Figure 6D-F). After 30 min, the green fluorescence was already


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clearly visible and mostly situated in and around the micropores (Figure 6B and 6C) whereas after 48 h, it was distributed throughout the epidermis and had also reached the dermis (Figure 6E and 6F). Biodistribution of TA in the Epidermis and Upper Dermis. The TA biodistribution profile, that is the amount of TA present as a function of depth within the epidermis and dermis, was determined after application of FFT1-TA10 and TA suspension for 48 h. The amount of TA present in five lamellae each with a thickness of 100 µm going from the skin surface to a nominal depth of 500 µm was quantified; it was also measured in the remaining dermal tissue. Comparison of the TA biodistribution profiles in skin following application of FFT1-TA10 and the TA suspension showed significant differences. The biodistribution observed with FFT1TA10 was appreciably more uniform (Figure 7A). Significantly greater amounts of TA were present in the upper lamellae following application of the TA suspension. Indeed, the amount of TA deposited in the first two lamellae was 11 times higher from TA suspension (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 250 µg of TA, this ensured that the TA solution was at saturation and that the thermodynamic activity of TA was at a maximum throughout the duration of the experiment. This facilitated partitioning from the solution into the micropore wall and the resulting high concentration gradient drove TA diffusion into the epidermis and resulted in the increased amounts in the first and second skin lamellae. The first two lamellae nominally descend from the skin surface to a depth of ~200 µm, which is the approximate depth of the micropores. The presence of much greater amounts of TA in the upper lamellae was responsible for the 6-fold higher total TA deposition in the skin from the TA suspension as compared to FFT1-TA10 (14.32


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± 5.21 and 2.48 ± 0.54 µg cm-2, respectively) since the amounts of TA in subsequent lamellae were more similar. Cumulative permeation of TA was almost eight-fold higher for the TA suspension 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 TA suspension for 48 h resulted in cumulative permeation across the skin of 27.8 ± 4.4 % of the applied TA dose whereas the corresponding amount for FFT1-TA10 was only 2.92 ± 0.3 %, the risk of systemic exposure in vivo can be significantly attenuated (Figure 7C). Thus, very different release kinetics and distribution profiles were obtained with FFT1-TA10. It was clearly able to control the release of TA into the micropore and the surrounding tissue, which was much more uniform. The estimated concentration of TA in the uppermost lamella following application of FFT1-TA10 was 80.9 ± 42.0 µg mL-1 whereas the concentration of TA in the fifth lamella was estimated to be 19.6 ± 5.7 µg mL-1. Although there are no reports on the TA concentrations achieved after intra-lesional injection, oral administration of TA (5 mg) results in mean Cmax values of 10.5 ± 6.2 ng mL-1.46 Given that these plasma concentrations are sufficient to produce pharmacological effects in target tissue but are much lower than the concentrations observed with FFT1-TA10 in the skin, we believe that the TA levels following controlled release of TA from the deposited MP are more than sufficient to be of pharmacological relevance – despite being much lower than those achieved following application of the TA suspension – and that this is a promising approach for the successful treatment of keloids. It is far more selective for localized skin delivery and limits systemic exposure. Moreover, if a higher dose were to be required, deposition of a larger volume of MP with higher TA loading could be used to further enhance TA delivery; alternatively, the pore density could also be increased.


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Translation from the Bench to Clinical Practice. A hypothetical treatment protocol for keloid scars using a combined laser microporation and TA-MP is shown in Figure 8. The TA-MP would most likely be applied as a semi-solid formulation since this could be spread over the entire microporated area on the scar surface. The semi-solid formulation would also provide flexibility with respect to the application area given that the shape and area of keloids can vary considerably. Furthermore, the act of spreading and massaging the formulation across the microporated area would also facilitate entry of the TA-MP into the micropores; it has previously been demonstrated that massaging favored entry of nanoparticulate carriers into hair follicles.47,48 The formulation could be covered with an occlusive dressing which, in addition to hydrating the skin, would also reduce the risk of infection. Treatment periodicity would obviously have to be determined in clinical trials but it was recently reported that TiO2 microparticles were retained in the skin for up to 30 days following application after laser microporation.49 CONCLUSIONS The aim of the project was to develop a method to enable the intra-epidermal delivery of drug reservoir systems, using microparticle-based formulations, with a view to provide an alternative strategy to treat keloids or hypertrophic scars. P.L.E.A.S.E.® microporation enabled delivery and deposition of TA-MP which could serve as local depots in vivo for sustained release of TA even after micropore closure. The MP present in the micropores might interact with the epidermal/dermal tissue and “sticky” interstitial fluid in vivo; this might increase adhesion and facilitate retention. It has been reported that keloid scars lack microvascular connections and suffer from inadequate blood supply because of excessive collagen growth – moreover, according to some studies, vascular density in keloid scars is less than that in hypertrophic scars.39,40 Deficient vascularization might prove to be an advantage for sustained drug release


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from MP since drug elimination would be reduced and drug levels maintained much longer resulting in a prolonged, localized action. The preliminary results obtained in this study are extremely promising; however, there are caveats: (i) healthy porcine skin was used to estimate drug delivery into “diseased” human skin – more specifically, the skin does not have the same structure as an actual keloid scar, (ii) the MP were applied to fractionally ablated skin using aqueous solutions – it is clear that for clinical use, the TA-MP will need to be administered from semi-solid formulations that will most likely be massaged into the porated skin, this should improve uptake and (iii) the effect of occlusive / non-occlusive dressings on delivery kinetics would also need to be explored – this is also likely to impact upon the rate of pore closure. The combination of laser microporation with MP based delivery systems and the creation of intraepidermal/dermal drug depots should certainly be of interest for the sustained and/or targeted local therapy of other dermatological conditions: (i) those that require maintenance or prolonged therapy since it would reduce the frequency of administration, (ii) where topical application of conventional formulations, e.g. semi-solids to intact skin is ineffective due to insufficient drug penetration and hence poor cutaneous bioavailability, (iii) where oral therapy results in low cutaneous bioavailability and poor efficacy, (iv) for drugs where systemic exposure results in the risk of unacceptable adverse effects, and (v) where cross-contamination might also be a risk factor. Therefore, in addition to enabling sustained local drug concentrations and modifying release kinetics, this approach might also improve patient compliance. For example, patients suffering from chronic skin conditions, e.g. with recalcitrant psoriatic plaques, require long-term use of medications and lack of adherence to therapy can be an issue. However, it is clear that each potential application will have to be evaluated individually and some of these will be explored in future preclinical and clinical studies.


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ASSOCIATED CONTENT Supporting Information Additional data concerning the validation of analytical method using HPLC-UV and UHPLC MS/MS, selection of dissolution study media, and MP deposition in laser porated human skin. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Tel: +41 22 379 3355. Fax: +41 22 379 3360. Email: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS MS is grateful to the Swiss Federal Commission for Scholarships for Foreign Students and the University of Geneva for their financial support. KS and SdR thank the University of Geneva and the Swiss Commission for Technology Innovation for financial support (CTI 13933.2). We thank Dr. Céline Besnard, Laboratoire de Cristallographie, UNIGE for PXRD characterization. We also acknowledge the UNIGE Bioimaging platform for access to the confocal imaging facilities. ABBREVIATIONS TA, triamcinolone acetonide; MP, microparticles; TA-MP, triamcinolone acetonide loaded microparticles; HPLC-UV, high-performance liquid chromatography with ultraviolet detection; UHPLC-MS/MS, ultra high-performance liquid chromatography with tandem mass spectrometry


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detection; LOD, limit of detection; LOQ, limit of quantification; Er:YAG, erbium-doped yttrium aluminium garnet; PBS, phosphate buffered saline; o/w, oil in water; PVA, polyvinyl alcohol; FFT, freeze-fracture technique; PLGA, poly(D, L-lactide-coglycolide); PLA, poly(D, L-lactide); EE, encapsulation efficiency; DL, drug loading; SEM, scanning electron microscopy; PXRD, powder X-ray diffraction; DSC, differential scanning calorimetry; CLSM, confocal laser scanning microscopy; FL, fluorescein; NR, nile red.


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acetonide after intravenous, oral, and inhaled administration. J. Clin. Pharmacol. 1995, 35, 11871193. (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 delivery into the hair follicles. Eur. J. Pharm. Biopharm. 2007, 66, 159-164. (48) Mak, W. C.; Patzelt, A.; Richter, H.; Renneberg, R.; Lai, K. K.; Rühl, E.; Sterry, W.; Lademann, J. Triggering of drug release of particles in hair follicles. J. Control. 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 laser microporation. J. Biomed. Opt. 2013, 18, 111406. (50) Carantino, I.; Florescu, I. P.; Carantino, A. Overview about the keloid scars and the elaboration of a non-invasive, unconventional treatment. J. Med. Life 2010, 3, 122-127.


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(51) Hunasgi, S.; Koneru, A.; Vanishree, M.; Shamala, R. Keloid: A case report and review of pathophysiology and differences between keloid and hypertrophic scars. J. Oral Maxillofac. Pathol. 2013, 17, 116-120. (52) Pantec Biosolutions, Laser microporation technology P.L.E.A.S.E.®

Platform,

http://www.pantec-biosolutions.com/en/technology/laser-microporation, accessed: July, 2015.


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TABLES

Table 1. Composition and characterization of the different microparticle formulations. Methoda Batch

TAb RG R (%) 503H 207 (%) (%)

FL/NRc Mean size (%) (d [4,3])

Size distribution

EEd

DLe

(%)

(%)

(d10, d50, d90) (µm)

(µm) o/w emulsion technique

Freezefracture technique (FFT)

OW1Placebo

-

100.0

-

-

11.39 ± 3.0

1.5, 8.16, 15.28

NA

NA

OW1TA10

10.0

90.0

-

-

8.17 ± 2.1

1.12, 6.57, 13.55

5.4 ± 0.3

0.54 0.03

OW2Placebo

-

-

100.0

-

9.84 ± 2.4

1.53, 8.99, 15.31

NA

NA

OW2TA10

10.0

-

90.0

-

14.84 ± 4.2

1.51, 13.63, 24.12

6.8 ± 0.2

0.68 0.02

FFT1TA10

10.0

90.0

-

-

89.23 ± 5.7

35.37, 141.85

81.97,

99.9 1.7

9.8 ± 0.2

FFT2TA20

20.0

40.0

40.0

-

81.64 ± 4.4

29.26, 139.44

87.79,

101.6 ± 2.1

20.2 0.2

FFT3FL/NR

-

97.8

-

2.0/0.2

86.09 ± 3.2

31.41, 127.55

78.80,

NA

NA

a

Method of preparation

b

Triamcinolone acetonide

c

±

±

Fluorescein/Nile Red

d e

±

±

Encapsulation efficiency

Drug loading


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FIGURE LEGENDS Figure 1. SEM images of (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), (c) OW1-TA10, after washing with ethanol/water (25/75). (B) PLA (R 207) microparticles prepared by the o/w emulsion technique: (a) OW2-Placebo, (b) OW2-TA10, before washing with ethanol/water (25/75), and (c) OW2-TA10, after washing 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, (c) FFT1-TA10, individual microparticle after release study. (D) PLGA/PLA microparticles prepared 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. Figure 2. Physical characterization of MP. (A) Powder X-ray diffraction patterns of (a) triamcinolone acetonide (TA), (b) RG 503H polymer, (c) R207/RG 503H polymer mixture (50:50), (d) TA-RG 503H physical mixture, (e) TA-R207/RG 503H physical mixture, (f) FFT1TA10, individual microparticle before the release study, (g) FFT2-TA20, individual microparticle before the release study, (h) FFT1-TA10, individual microparticle after the release study, (i) FFT2-TA20, individual microparticle after the release study. (B) Differential scanning calorimetry thermograms of (a) triamcinolone acetonide (TA), RG 503H polymer, TA-RG 503H physical mixture, FFT1-TA10 before the release study, and FFT1-TA10 after the release study; (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 release study.


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Figure 3. Release profiles of triamcinolone acetonide (TA) from the microparticle formulations, FFT1-TA10, FFT2-TA20 and a drug suspension during (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 4X magnification of individual micropores; (C) deposited microparticles (FFT3-FL/NR) in the micropore array; and (D) view at 4X magnification showing close-ups of the microparticles deposited in individual micropores. Figure 5. 3D Confocal laser scanning microscopy images of microporated skin following P.L.E.A.S.E.® poration; (A) micropore array (Hoechst Blue stained the exposed nuclei in the viable epidermis under the stratum corneum) after microporation, (B) single micropore, (C) micropore array with deposited microparticles (FFT3-FL/NR), which are identified by the strong Nile Red fluorescence, 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) 3D reconstruction, (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 surrounding epidermis and dermis. The signal is present throughout the epidermis after 48 h. Figure 7. (A) Comparison of TA biodistribution in skin to a total depth of 500 µm at a resolution of 100 µm after application of the TA suspension and FFT1-TA10 formulations for 48 h. (B) Comparison of TA permeation after 48 h application of TA suspension formulation and FFT1-


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TA10 – suggests that the former would significantly increase the risk of systemic exposure in vivo. (C) Comparison of total TA release from TA suspension formulation and FFT1-TA10 after application for 48 h in terms of the proportions deposited in and permeated across the skin (expressed as a percentage of the amount applied). (Mean ± SD; n ≥ 5). 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 acetonide loaded microparticles (TA-MP). (A) Image of keloid scar showing the surface morphology.50 (B) Histology image of the keloid scar displaying epidermis and dermis where densely packed hypocellular collagen fibers are clearly visible.51 (C) A schematic representation of a keloid scar showing epidermis, 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 and decreases the levels of collagenase inhibitors which results in the breakdown of the collagen fibril network in the dermis and a reduction in the thickness of keloid scar. (H) Histology of healthy skin. Image of P.L.E.A.S.E.® professional laser device is displayed in the centre.52


36

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Figure 1


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Figure 2


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Figure 3

100

TA release (%)

A

80 60 40 20 0 0

2

4

6

8

10

12

12

14

Time (h)

B

100

TA Release (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60 40 20 0 0

2

4

6

8

10

Time (days) TA Suspension

FFT1-TA10

FFT2-TA20


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Figure 4


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Figure 5


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Figure 6


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Figure 7


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Figure 8


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FOR TABLE OF CONTENTS ONLY

Cutaneous micropores created by fractional laser ablation enable intra-epidermal delivery of triamcinolone acetonide microparticles. Optical microcopy reveals the micropore array. Confocal laser scanning microscopy confirms the presence of Nile Red-containing microparticles in the micropores and also demonstrates the release and distribution of fluorescein (green) in the epidermis and dermis.


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Table of contents entry


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Figure 1 170x177mm (300 x 300 DPI)



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Figure 2 147x271mm (600 x 600 DPI)


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Figure 3 1448x602mm (96 x 96 DPI)



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Figure 4 97x56mm (300 x 300 DPI)


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Figure 6 114x76mm (300 x 300 DPI)


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Figure 8 117x86mm (300 x 300 DPI)


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Fractional Laser Ablation for the Cutaneous Delivery of Triamcinolone

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