RESEARCH ARTICLE

Microneedle-assisted transdermal delivery of Zolmitriptan: effect of microneedlegeometry, in vitro permeation experiments, scaling analyses and numericalsimulations

Chandra Teja Uppuluria, Jyothirmayee Devinenia, Tao Hanb, Atul Nayakb, Kartik J. Nairc, Benjamin R. Whitesidec,Diganta B. Dasb and Buchi N. Nalluria

aDepartment of Pharmaceutics, KVSR Siddahrtha College of Pharmaceutical Sciences, Vijayawada, India; bDepartment of Chemical Engineering,Loughborough University, Loughborough, UK; cDepartment of Engineering and Informatics, University of Bradford, Bradford, UK

ABSTRACTObjective: The present study was aimed to investigate the effect of salient microneedle (MN) geometryparameters like length, density, shape and type on transdermal permeation enhancement of Zolmitriptan(ZMT).Methods: Two types of MN devices viz. AdminPatchVR arrays (ADM) (0.6, 0.9, 1.2 and 1.5mm lengths) andlaboratory fabricated polymeric MNs (PM) of 0.6mm length were employed. In the case of PMs,arrays were applied thrice at different places within a 1.77 cm2 skin area (PM-3) to maintain the MN densitycloser to 0.6mm ADM. Scaling analyses was done using dimensionless parameters like concentration ofZMT (Ct/Cs), thickness (h/L) and surface area of the skin (Sa/L

2).Results: Micro-injection molding technique was employed to fabricate PM. Histological studies revealedthat the PM, owing to their geometry/design, formed wider and deeper microconduits when compared toADM of similar length. Approximately 3.17- and 3.65-fold increase in ZMT flux values were observed with1.5mm ADM and PM-3 applications when compared to the passive studies. Good correlations wereobserved between different dimensionless parameters with scaling analyses. Numerical simulations, usingMATLAB and COMSOL software, based on experimental data and histological images provided informationregarding the ZMT skin distribution after MN application.Discussion: Both from experimental studies and simulations, it was inferred that PM were more effectivein enhancing the transdermal delivery of ZMT when compared to ADM.Conclusions: The study suggests that MN application enhances the ZMT transdermal permeation and thegeometrical parameters of MNs play an important role in the degree of such enhancement.

ARTICLE HISTORYReceived 19 December 2016Revised 1 March 2017Accepted 23 March 2017

KEYWORDSHistological sections;microneedles; microneedlegeometry; numericalsimulations; scalinganalyses; Zolmitriptan

Introduction

Migraine is a chronic, debilitating headache affecting �19% of thepopulation globally, with 2–3 times more incidence in womenthan in men. It is a neurovascular disorder whose pathophysiologyis still not clearly understood. An attack/episode of migraine maylast from 2 to 72 h, associated with characteristic symptoms like apulsating and throbbing headache generally on one side of thehead, nausea, vomiting, sensitivity to light, etc. that generally wor-sens with physical activity. Triptans are 5HT or serotonin receptoragonists that act specifically on migraine. These cause constrictionof the dilated cranial blood vessels, which is thought to be thecause of migraine1,2.

The first among this class of drugs is Sumatriptan, followed bynewer ones viz. Zolmitriptan, Naratriptan, Rizatriptan, Eletriptan,Almotriptan, Avitriptan and Frovatriptan. Zolmitriptan (ZMT) wasthe second triptan available in USA and many countries, and wasdeveloped with the goal of creating a more bioavailable, longerhalf-life, more rapidly absorbed and centrally active oral tabletthan Sumatriptan2. ZMT is available as oral tablet, nasal spray andoral dispersible tablet formulations, with an optimum dose being2.5mg orally, with a maximum daily dose of 10mg/day3. With all

three formulations, the bioavailability is between 40 and 48%.Nasal sprays of ZMT do offer a pharmacokinetic advantage of arapid absorption across nasal mucosa, which results in amuch faster onset of action. However, the overall therapeutic gainor efficacy is not improved significantly when compared to oralformulations. All these formulations of ZMT were reported to havea good chance of therapeutic failure with the first dose, requiringa second dose within 2 h to avoid a recurrence of the attackwithin 24 h3,4.

An ideal delivery system for ZMT must improve the bioavail-ability and ensure a good therapeutic gain within 2 h, i.e. a fasteronset of action and a low 24 h recurrence rate when compared toexisting options, without compromising patient compliance andease of handling. Transdermal delivery has gained a lot of interestas an alternative to oral and parenteral routes, and research isgoing on extensively to open this route for systemic administra-tion of a vast array of drug molecules including large moleculeslike insulin, growth hormone, etc5.

ZMT is a relatively hydrophilic molecule with a log p values of1.6 and does not have ideal properties to permeate through skinand hence a suitable permeation enhancement technique should beemployed to achieve clinically significant rates of drug permeation.

CONTACT Buchi N. Nalluri buchinalluri@yahoo.com Department of Pharmaceutics, KVSR Siddahrtha College of Pharmaceutical Sciences, Vijayawada 520010,Andhra Pradesh, India� 2017 Informa UK Limited, trading as Taylor & Francis Group

DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY, 2017http://dx.doi.org/10.1080/03639045.2017.1313862

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Only a few studies have reported on the transdermal delivery ofZMT, using chemical enhancers and iontophoresis as permeationenhancement techniques6–8. Subedi et al., 20117 studied the effectof different formulation variables like the specific types of pressuresensitive adhesives (PSA), solvent system, permeation enhancers,crystallization inhibitors, etc. on transdermal flux of ZMT andreported that permeation enhancers (polyoxyethylene alkyl ethers)resulted in a considerable increase in transdermal flux of ZMT, butinduced ZMT crystallization7. Lui and Fang, 20158, also developedan adhesive transdermal patch containing ZMT using DURO-TAKVR

87–4098 (PSA) and 10% Azone as permeation enhancer at a drugload of 4% ZMT and reported that 88 ± 51 ng/mL ZMT wasdetected in systemic circulation within 15min of administration ofthe patches in vivo using rabbit as an animal model, and con-cluded that further optimization can enable effective delivery ofZMT via a transdermal route8.

Extensive work in the literature emphasizes that microneedles(MN), which are minimally invasive systems that can overcome thebarrier functions of skin and improve the permeation of drug mol-ecules including macromolecules, could be used to effectivelyenhance the permeation of ZMT via a transdermal route9–14. Itwas also reported that such enhancement is dependent on manyaspects of MN design, such as type, shape of MNs, density, materi-als, etc. There is also an interplay of the effect of these variablesand, as such, their effects on the permeation of a drug moleculeare generally non-intuitive. As a result, their effects are beststudied for specific case15,16.

Consistent with this idea, the present study was designed usinga systematic approach to investigate the efficiency of MNs inenhancing the transdermal permeation of ZMT and to explore theeffect of MN geometrical parameters viz. type, MN length, design/shape and density on the extent of ZMT transdermal permeationenhancement. Two types of MN devices viz. commercially availableAdminPatchVR arrays (ADM) (0.6, 0.9, 1.2 and 1.5mm length) andlab fabricated polymeric MN arrays (PM) (0.6mm length) of differ-ent MN densities were employed for the same purpose (Figure 1).A number of different techniques such as etching, lithography andmolding were discussed widely in the literature for the manufactureof MNs of different dimensions (100–1800mm in length), shapes withdifferent materials (plastics, silicon, ceramics, metals, etc.). Injectionmolding offers several advantages in terms of ease of scalability forbulk manufacture, range of materials and good reproducibility17.

In this study, micro-injection molding was employed for fabrica-tion of the PM using PEEK LT-3 (polyether ether ketone) asreported in Nair et al18.

Materials and methods

Materials

AdminPatchVR MN arrays were purchased from AdminMed,Sunnyvale, CA. ZMT was obtained from Mylan LaboratoriesLimited, Hyderabad, India; sodium chloride, isopropyl alcohol, pro-pylene glycol (PG) and 1% w/v Safranin solution from LobaChemie, Mumbai, India; formic acid, acetonitrile, methanol andHPLC water from Merck Specialities Pvt. Ltd, Mumbai, India;haematoxylin and eosin stain from Sigma-Aldrich, Bengaluru, India.All the reagents and chemicals used in the study were of HPLCgrade. Pig ear skin was obtained from local abattoirs.

Fabrication of PM

MN array components consisted of 25 conical needles, each0.6mm in length with a base diameter of 0.3mm, in a 5� 5 arrayover a 0.5� 0.5 cm2 area, supported by a circular substrate ofdiameter 17.5mm and thickness 0.5mm. Moulding of these com-ponents was performed using a Wittmann–Battenfeld Micro-Power15 micro-injection molding machine as shown in Figure 2(A). Thismachine offers a high repeatability, accurate dosing, clean roomfacility and robotic component handling which is ideal for thebulk manufacture of MNs. The MN mold insert (Figure 2(B)) wasmade out of Stavax ESR (Bohler-Uddeholm Corporation, Elgin, IL).Sink electrical discharge machining (EDM) was used to createnegative MN features into the insert and was outsourced toIsometric Tool & Design, Inc. (New Richmond, WI).

The polymer used for this study is PEEK (polyetheretherketone)LT-3 grade material, which is an FDA-approved semi-crystallinebiomaterial manufactured by Invibio Inc., Lancashire, UK and iswidely employed for medical use. The attractive characteristics ofPEEK LT-3 for this application include its excellent processability,dimensional stability, mechanical strength, chemical resistance andresistance to gamma and ethylene oxide sterilization methods. Toensure the moisture content of the material was suitable for

Figure 1. Different MNs used in the study.

2 C. T. UPPULURI ET AL.

processing, the material was dried at a temperature of 150 �C for4 h prior to molding.

Moulding trials were performed to optimize the process param-eters based on measurement of the needle dimensions and sub-strate flatness. The optimized parameters for the molding of PMare shown Table 1. Once the process was stabilized, samples werecollected for the subsequent experimental work.

MN dimensional evaluation

Scanning electron microscopeA Hitachi TM-3000 table-top Scanning Electron Microscope (Tokyo,Japan) was used for analysis of the MN insert and needle geom-etry. The Hitachi TM-300 has a magnification from 15–30,000�,sample chamber of 700mm diameter and 50mm thickness. It fea-tures 5 kV, 15 kV and analysis observation modes. Because of thehigh aspect ratio and low contrast of the MNs, analysis mode wasused for imaging. The major advantage of using TM-3000 SEMwas it works under low vacuum and does not need specimencoating.

Confocal laser microscopeIt is very important to measure the tip radius and height of theMNs and the most common methods used are optical or electronmicroscopy. Because of its steep and complex structures, a 3Dimage analysis gives a better measurement of the needle geom-etry and quality control. In this study, an Olympus vertical

scanning laser confocal microscope LEXT OLS 4000 was used toaccurately measure the tip radius and height of MN arrays.

The device offers a broad magnification range from 108 to17,280� and the exact 3D reconstruction of the MNs. The confocallaser microscope LEXT scans the surfaces with a laser beam withthe wavelength of 405 nm thus allowing submicron visualizationof material and component surfaces with the resolution of downto 0.10 mm. Measurements were taken with the 20� lens using thewide range stitching feature with 20% overlap to produce a meas-urement area of 5� 5mm. A five level brightness switch (Table 2)was enabled to accurately illuminate the specimen.

Atomic force microscopeThe most important feature of the MN is that the tip has to be sharpin order to effectively pierce the skin. So as to confirm the measure-ments made from confocal laser microscope, AFM (MFP-3D AsylumResearch, Santa Barbara, CA) was used to measure the tip radius ofthe MNs. All AFM scans were made using an MFP-3D scanner fromAsylum Research. Silicon nitride cantilever tips (Applied Nano, SantaClara, CA) with a tip radius of 15 nm and spring constant of 0.3 Nmwere used. Images were interpreted using integrated MFP-3DTM Igorsoftware (Asylum Research). Errors in the piezo-linearity were cor-rected for by using zero- and first-order flattening.

High-performance liquid chromatography method

Rapid-high high-performance liquid chromatography photodiodearray (RP-HPLC-PDA) method was developed specifically for the

Figure 2. (A) Battenfeld micro-power 15 molding machine and (B) 3D drawing of the microneedle insert.

Table 1. Microinjection molding parameters.

Parameter Value

Melt temperature (�C) 400Maximum injection velocity (mm/s) 750Clamping force (kN) 50Holding pressure (bars) 600Mould temperature (�C) 210

Table 2. Brightness switch settings usedfor the confocal measurements.

Position (lm) Brightness

600 49550 47.1520 49.120 75.50 48.4

DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 3

analysis of ZMT in the transdermal permeation samples.A Shimadzu Prominence HPLC system provided with DGU-20A3degasser, LC-20AD binary pumps, SIL-20AHT auto sampler andSPD-M20A PDA detector was used for analysis of the samples.Data acquisition was carried out using LC solutions software.Separation was achieved on an Agilent Eclipse column(150� 4.6mm; 5 mm). The mobile phase comprised of 0.02% v/vformic acid: methanol 75:25 (v/v) at a flow rate of 1ml/min withan injection volume of 20 mL and the eluents were monitored at221 nm. The developed method was validated as per theInternational Council for Harmonisation of Technical Requirementsfor Pharmaceuticals for Human Use (ICH) guidelines.

Solubility studies

The solubility of ZMT was studied in different vehicle combina-tions of PG, polyethylene glycol-400 (PEG) and saline (S) at 70:30,80:20 and 90:10% v/v ratios. To each vehicle system, an excessamount of ZMT was added and vortexed for 1min in order toobtain a saturated solution and the solutions were equilibrated at37 �C in an orbital shaker for 24 h. After equilibration, the sampleswere centrifuged at 3000 rpm for 10min and filtered through anylon syringe filter (0.45mm) and all the samples were appropri-ately diluted and analyzed by HPLC method.

Skin preparation

Pig ears were collected from the local abattoirs immediately afteranimals were killed by electric current. The ears were transported tothe laboratory in a cooling box without previous treatment. In thelaboratory, the pig ears were washed carefully with distilled waterand the hair was removed from the external part of pig ear usingan electrical hair clipper. Carefully the full-thickness skin from theexternal part of the pig ear was separated from underlying cartilageusing a scalpel and excess fat underlying the skin was removed to athickness of 1.2mm was employed for the in vitro transdermal per-meation studies. The dermis side was wiped with isopropyl alcoholto remove the residual adhering fat. Processed skin samples wereindividually wrapped in plastic bags without air entrapment andstored in a deep freezer at �20 �C until further use.

Application of MNs on skin samples

Prior to the in vitro skin permeation experiments, the skin sampleswere allowed to be brought to room temperature and then theskin surface was carefully washed with saline. Two types of MNdevices viz. ADM (0.6, 0.9, 1.2 and 1.5mm) and PM (0.6mm) wereused to poke the skin surface under thumb pressure. In the caseof PM, both single (PM-1) and triple (PM-3) insertions at differentplaces within a 1.77 cm2 skin area (PM-3) were made in order tomaintain the MN density closer to ADM of 0.6mm length asshown in Figure 1. The MNs were periodically checked during thisprocess for potential damage using a stereomicroscope.

Surface visualization and histological examination of the skinsamples

To visually confirm the disruption of skin layers by the MNs, thearrays were pressed over the pig ear skin under thumb pressureand held for 1min. Then, the skin was stained with safranin dye(1% w/v in water) and wiped with isopropanol cotton swabs forthe identification of the microconduits formed. In the case of the

histological studies, the skin section samples with and without MNtreatments were prepared after staining with hematoxylin andeosin for visualization of skin layers and to display a clear indenta-tion by MN penetration. The sections were observed under amicroscope (Olympus; Noida, India). The width and depth ofmicroconduits formed were also calculated in triplicate (n¼ 3 skinsamples for each MN) with the help of Toup View 3.2 Software(Irwin, CA). For control, skin samples without MN treatment werealso prepared.

In vitro skin permeation studies

The in vitro transdermal permeation studies were performed usinga vertical type Franz diffusion cell apparatus fitted with a watercirculation system, a water heater and an eight-stage magneticstirrer (Orchid Scientifics, Nasik, India). Franz diffusion cells with aneffective diffusion area of 1.77 cm2 and a receptor volume of�14ml were used. Saline was used as the receptor fluid. Pig earskin was mounted between the donor and receptor cells with thestratum corneum (SC) facing towards the donor cell. The receptormedium was stirred for uniform drug distribution at a speed of600 rpm throughout the experiment. Care was taken to preventthe entrapment of air bubbles at the interface of the skin (dermis)and the receptor solution. The surface of the skin was maintainedat 32 �C using a circulating water bath. After equilibration, 500 mLof donor solutions containing excess amount of ZMT were appliedon to the skin. Samples (500mL) were withdrawn from the recep-tor fluid at 6-h increments up to 48 h and replaced with the freshsaline to maintain a constant volume. All the samples were storedat 4 �C, prior to the HPLC analysis.

The cumulative permeation profiles were plotted for the cumu-lative amount of drug permeated (nmoL/cm2) as a function oftime, for untreated and microneedle-treated skin. The flux valuesand the respective lag times were obtained from the slope andthe X-intercept of the steady-state portion of the cumulative per-meation profiles. Apparent permeability and diffusion coefficientvalues were computed from Fick’s first law of diffusion:

1A

dMdt

� �¼ Js ¼ KpDC

Js is the steady-state flux (nmoL/cm2/h), M is the cumulative

amount of drug permeating the skin (nmoL/cm2), A is the area ofthe skin (1.77 cm2), Kp is the apparent permeability coefficient (cm/h) and DC is the difference in concentrations of ZMT in the donorand receiver. Sink conditions were maintained in the receiverthroughout the experiment and hence DC was approximated tobe the drug concentration in the donor compartment.

Enhancement ratios were also computed to evaluate the rela-tive efficiency of different MNs on the ZMT skin permeationenhancement. The enhancement ratios were calculated as follows:

Enhancement ratio ¼Cumulative amount or fFlux obtained after MN applicationCumulative amount or fFlux obtained from passive studies

ZMT content in skin

After the completion of the permeation studies, skin samples werestudied for drug disposition. The skin tissue exposed to the donorsolution was cut with a scalpel and washed with filtered water

4 C. T. UPPULURI ET AL.

and blotted with a paper towel in order to remove the drug whichadhered to the surface. Then skin was minced with a scalpel, andplaced in a pre-weighed vial. The drug was extracted from theskin by equilibrating with 5ml of acetonitrile at 32 �C in an orbitalshaker. The solutions were then analyzed by HPLC to determinethe ZMT content.

Scaling analyses

Dimensionless correlations between the amount of ZMT perme-ated (Ct/Cs) and other variables such as the thickness (h/L) and thesurface area (Sa/L2) of the skin were made in order to gaininsights into the overall phenomenon of ZMT transdermal perme-ation enhancement by MN application. The scaling analyses werecarried out based on the principles of the Buckingham p theoremwhere it is defined that the dimensionless concentration of adrug, which permeates through skin, can be defined in terms ofkey non-dimensional parameters (e.g. MN lengths) using the pro-cedures described in previous articles13,19.

Equation 1 describes the relationship of all the model parame-ters used for such analyses (Table 3).

CtCs

¼ K SaL4Ke

VdhD

� �n

(1)

Where, “K” is a dimensionless constant and “n” is an unknownpower; Ct and Cs are the amount of ZMT permeated at a giventime “t” (48 h) and the amount of drug loaded in the donor com-partment for diffusion (surface concentration on skin); Sa is thesurface area of skin available for diffusion; L is the length of micro-needles; Ke and Vd are the first-order elimination constant and thevolume of receptor fluid; “h” is the thickness of skin and D is thediffusion coefficient of ZMT in skin.

Using Equation 1, the correlations between the dimensionlessZMT concentration (Ct/Cs) against the dimensionless parameters ofthe study (h/L and Sa/L

2) have been established considering thatall other variables remain unchanged.

Numerical simulations of experimental studies

A set of well-defined numerical simulations were carried out inorder to gain insights into the specific effects of MN shape, dimen-sions, force of insertion of MN, etc. on the overall permeationenhancement of ZMT, and also to obtain the information aboutZMT distribution within the skin layers during permeation, whichare otherwise difficult to obtain directly from the experimentaldata. The simulations were carried out using MATLAB (MathWorks,Natick, MA) and COMSOL Multiphysics (COMSOL Multiphysics Pvt.

Ltd., Stockholm, Sweden) programs. The MATLAB program wasuse to process the histological sections images of skin treatedwith MNs and these processed images were further coupled withthe experimental permeation parameter, e.g. passive diffusioncoefficient and imported into COMSOL simulator software13,20.

Statistical analysis of the data

Results of the experimental data were subjected to statistical ana-lysis by one-way analysis of variance (ANOVA) [using Fisher’s leastsignificance difference (LSD) post hoc test] using SYSTAT 13 soft-ware (Systat Software Inc., San Jose, CA). Results with a p values of

Figure 3. (A) 3D confocal image of (a) front view of the PEEK MN array, (b) single MN of PEEK and (c) line measurement of the PM array; (B) SEM image of (a) MNarray of PEEK, (b) top view of the needle and (c) single MN of PEEK; (C) AFM image of the PEEK MN tip and (b) line graph showing the spherical cap radius of the nee-dle; and (D) 3D confocal image of 0.6mm ADM showing its design parameters.

6 C. T. UPPULURI ET AL.

microconduits across skin layers was clearly evident. The averagepenetration depth (vertical) (n¼ 3) of the MNs was found to be180.26 ± 30.39, 302.22 ± 22.28, 400.85 ± 20.83, 478.48 ± 67.72 and338.20 ± 22.66 mm for ADM (0.6, 0.9, 1.2 and 1.5mm) and PM,respectively.

In vitro permeation studies

The comparative in vitro pig ear skin permeation profiles of ZMTwithout and with MN application were shown in Figure 8. VariousZMT permeation parameters viz. cumulative amount permeated atthe end of 48 h, steady-state flux, lag time, permeability and diffu-sion coefficients without and with microneedle treatment weregiven in Table 5.

Scaling analyses

Using scaling analyses, correlations were developed between thedimensionless parameters, namely, the amount of ZMT permeated(Ct/Cs) and skin thickness (h/L) and surface area of skin (Sa/L

2) inorder to derive a better understanding on the effect of specificgeometry parameters of MN, like length (L), on overall permeationenhancement by MNs of same design (ADM) (Figure 9(A,B),respectively)13,19. Equations 2 and 3 describe such relationshipsamong the considered parameters within the given range.

Table 4. Different geometry parameters of MNs used in the study.

Parameter

ADMa

PM0.6 0.9 1.2 1.5

Length (lm) 600 900 1200 1500 560 ± 30Number of MNs (1.77 cm2) 187 85 41 31 25Shape Flat (2D) Conical (3D)Thickness of each MN (lm) 78 (uniform till tip)b 300 (�30 at tip)Thickness of array base (lm) 100–200b 300Material Medical grade stainless steel (SS 316L) PEEK LT-3 (polyether ether ketone)aValues are from manufacturer21.bThe width and thickness of the MN and the base varies with length.

Figure 4. (A) RIZ chromatogram; (B) UV spectrum; (C) Peak purity index.

Figure 5. Solubility data of ZMT in PG:S and PEG:S solvent systems.

DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 7

These correlations were determined for given thickness and surfacearea of skin while the MN length varies (0.6, 0.9, 1.2 and 1.5mm).

CtCs

¼ 5:52 � x10�3 hL

� ��0:624for 0:8 � h

L� 2

(2)

CtCs

¼ 2:47 � x10�2 SaL2

� ��0:312for 78:5 � Sa

L2� 491:5

(3)

Good correlation was observed (R2> 0.83) between the dimen-sionless parameters of the study (Figure 9).

Numerical simulations of experimental studies

The numerical simulations were carried out using the proceduresdiscussed in previous articles based on in vitro passive diffusioncoefficient values, coupled with histological section images (of cor-responding MNs)13,20. Different stages in processing the histo-logical section images by MATLAB and COMSOL programs forsimulations are shown in Figure 10. The numerical simulationswere able to provide information regarding the skin distributionof ZMT after applying the MNs at any time point and depth(Figure 10(D)).

Discussion

The dimensions of the PM were found to be consistent andrepeatable with good tip shape, confirming the complete filling ofthe PEEK into the MN insert cavity under the maintained process-ing conditions (Table 1) and the technique used is reliable for thebulk manufacture of PMs. Compression tests of PM on a steelplate using Bose Electroforce 3100 instrument with a 225 N loadcell and WintestVR software (Bose, Eden Prairie, MN) revealed thatthe PMs were able to withstand compression forces of up to8 N18.

A rapid and sensitive HPLC-PDA method was developed for theselective quantification of ZMT in transdermal permeation studies.The method complied with all the requirements of ICH guidelines

and was successfully employed for the quantitative estimation ofZMT in various samples throughout the study.

Solubility of ZMT was relatively higher in PG:S when comparedwith PEG:S combinations (p> .05) (Figure 5). However, a significantdifference was not observed among PG:S vehicle combinations(p> .05). PG:S at 70:30% v/v was selected for the ZMT in vitro skinpermeation studies.

Surface visualization of MN-treated skin showed clear distinc-tions in the number of the microconduits formed due to the dif-ferences in length and density of MNs in the ADM and PM devices(Figure 6). Skin layer disruption and the formation of microconduitswas clearly evident from histological section images (Figure 7).The penetration depth �25–35% of the original needle length forADM and 55–60% for the PM MN. Even though the length of theMNs differs in ADM, the percentage of MN penetration is almostsame, which is an indication of uniformity in thumb pressure underwhich MNs were applied at different times.

With the ADM devices, as the length of the MNs increased thepenetration depth also increased. However, the microconduitswere found to be wider and deeper with PM when compared toADM of similar lengths i.e. 0.6mm. These differences in the effi-ciency of creating microconduits in skin layers between the twotypes of MN devices (ADM and PM) may be attributed to the dif-ferences in geometry parameters like shape, design and type offabricating material. Regarding the shape/design, the PMs wereconical (3D) in shape, and the microconduits formed by PM werewider, while the microconduits formed using ADM looked merelylike a cut on the skin (2D) as the arrays of ADM are low in thick-ness (2D) (Figure 3). Furthermore, owing to the sturdy and thickbase (when compared to ADM) that supports the solid PM (Figure 1),the effective resistance (because of the viscoelastic nature) of theskin during application of arrays may be less for PM, while theADM might have experienced greater resistance which is becauseof their thin base and array design21, resulting in a relatively shal-low penetration with similar MN lengths (0.6 and 0.9mm ADM).

Significant enhancement in ZMT permeation was observedafter the application of MNs into the skin (p< .05) when comparedto passive permeation studies (Figure 8). A 1.83, 1.91, 2.35 and3.17-fold increase in the cumulative amount (48 h) of ZMT

Figure 6. Surface images of stained skin without and with MN treatment.

8 C. T. UPPULURI ET AL.

permeated was observed with 0.6, 0.9, 1.2 and 1.5mm ADM treat-ments, respectively, when compared to the passive permeationamount. A similar trend was observed with other permeationparameters like permeability and diffusion coefficient values(Table 5). Significant reduction in the lag time was observed afterthe application of the 1.5mm ADM when compared to that withthe passive data and other ADM lengths (p< .05). However, thereis no statistically significant difference between the 0.9 and1.2mm ADM treatments in terms of lag time (p> .05).

The ZMT flux values were found to be in the order of1.5> 1.2> 0.9� 0.6mm>passive with ADM treatments. A 2.26-fold increase in ZMT flux was observed with the 1.5mm whencompared to passive studies. However, there is no statistically sig-nificant difference between the 0.6 and 0.9mm treatments(p> .05). It was observed that the enhancement of ZMT perme-ation was only marginal with the application of the 0.6 and0.9mm ADM when compared to passive studies (Figure 8). Thismay be due to the fact that the skin disruption/penetrationcaused by 0.6 and 0.9mm ADM was less, as supported by thehistological section images (Figure 7). Moreover, the ZMT perme-ation was high with the 1.5mm ADM when compared to 0.6, 0.9and 1.2mm even though the density of the MNs is low, which ispossibly due to longer needle lengths which, in turn, resulted indeeper skin penetration.

With the PM application, �1.37- and 3.65-fold increase in thecumulative amount (48 h) of ZMT permeated was observed withthe PM-1 and PM-3 treatments, respectively, when compared topassive permeation amounts. The lag times were found to be

significantly lower for PM-3 when compared to passive and PM-1treatments (p< .05). The ZMT flux values were found to be in theorder of PM-3> PM-1> passive treatments. A 1.25- and 2.96-foldincrease in ZMT flux was observed with the PM-1 and PM-3,respectively, when compared to passive studies. A similar trendwas observed with other ZMT permeation parameters like perme-ability and diffusion coefficient values (Table 5) with both the PMtreatments.

Even though, the skin penetration by PM device (with 0.6mmarray) was significantly greater when compared to 0.6 and 0.9mmADM (Figure 7), the overall permeation enhancement in terms offlux, cumulative amount permeated etc. achieved with PM-1 was

Figure 7. Penetration depth calculation of ADM and PM in skin (ToupViewVR

screenshot).

0

250

500

750

1000

1250

0 6 12 18 24 30 36 42 48

Cum

ulat

ive

amou

nt p

erm

eate

d(n

moL

/cm

2 )

Time (h)

Passive 0.6 ADM0.9 ADM 1.2 ADM1.5 ADM PM-1PM-3

Figure 8. Comparative in vitro skin permeation profiles of ZMT from passive andMN treatments.

DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 9

found to be closer to those of 0.6 and 0.9mm ADM (p> .05). Thismay be because the needle density of 0.6 and 0.9mm ADM wasgreater when compared to PM-1 (Table 4), which may have com-pensated the variation in the extent of skin penetration amongthese MNs, and thus no significant variation in overall permeationenhancement was observed among these three MNs (p> .05).

In the case of the PM, a single application of array didn’t coverthe 1.77 cm2 surface area of the skin and hence in order to main-tain the MN density closer to the ADM of 0.6mm length, thearrays were applied three times at different places within a1.77 cm2 skin area (PM-3) as shown in Figure 1. As the needledensity was increased with PM-3, the ZMT transdermal permeationincreased markedly and was similar to that obtained with 1.5mmADM (p> .05).

Moreover, it is intriguing to note although the needle densityof PM-3 (75 MNs) was significantly greater when compared to the1.5mm ADM (31 MNs), the lag times were found to be signifi-cantly shorter with 1.5mm ADM (p< .05), which may be becauseof deeper (vertical) penetration into skin. Even though the depthof the penetration was significantly lower with PM when com-pared to 1.5mm ADM, the comparable permeation enhancementby PM-3 is may be because of higher needle density as stated ear-lier and wider microconduits formed as evidenced by histologicalsection images (Figure 7).

The enhancement in ZMT permeation was found to be in theorder of 1.5mm ADM� PM-3> 1.2mm ADM >0.9mm ADM�0.6mm ADM� PM-1> passive. Even though no correlation ofZMT skin content was observed with different MN treatments, sig-nificantly higher amounts of ZMT were found to be distributed inskin layers at the end of 48 h with MN-treated studies and is anindication of potential ZMT skin deposition.

Patel et al. (2009)6, investigated the effect of iontophoresison transdermal permeation of ZMT both in vitro and in vivo.

The cumulative amount of ZMT permeated was found to be�263.7 ± 92.7 mg/cm2, in vitro, at the end of 6 h with continuousapplication of current (0.25mA/cm2). However, the preliminarystudies carried out in the present investigation showed a similarresult (cumulative amount permeated) at the end of 48 h with1.5mm ADM and PM-3 applications (251.08 ± 17.07 and289.85 ± 64.58mg/cm2, respectively).

Moreover, iontophoresis was reported to be associated withitching, redness and skin irritation and tingling sensation, makingiontophoresis less patient compliant22. The MN application, espe-cially the poke and patch technique discussed in this investigation,is painless and easy to administer when compared to iontophor-esis, thus alleviating any issues regarding patient compliance.

Overall, further enhancement in the cumulative amount perme-ated and other permeation parameters like steady-state flux ofZMT may be achieved by further optimizing the MN geometry,especially of the PM in terms of design, MN length and density,and other parameters of the study like drug loading, donorvehicle, formulation, etc. thus resulting in more rapid and effectiveZMT delivery.

Dimensionless correlations were developed for ADM MNs(same type but differing in length) using scaling analyses. Goodcorrelations were observed between the dimensionless parameters(Ct/Cs versus h/L and Ct/Cs versus Sa/L

2) (Figure 9). These correla-tions can be used to predict the amount of ZMT permeated withhigh accuracy for other MN lengths in the range of 0.6–1.5mmwith similar design as ADM.

From the results of numerical simulations, the flux was repre-sented by white arrows in Figure 10(D) showing obvious incre-ment at the upper layers of the skin which is caused by MNpenetration. This increment may be extrapolated to a higher drugaccumulation rate at the blood stream as well as shorter lag timefor the diffusion to reach its steady state after the application of

Table 5. Permeation parameters of ZMT without and with MN treatments.

Skin treatment

Permeation parameters

Steady-state flux(nmoL/cm2/h) Lag time (h)

Permeability coefficient(�10�5) (cm/h)

Diffusion coefficient(�10�10) (cm2/s)

Skin content(lg/g)

Passive 10.138 ± 0.803 20.83 ± 0.63 4.593 ± 0.364 15.309 ± 1.212 1627.67 ± 164.230.6 ADM 15.019 ± 0.164 17.66 ± 1.37 6.797 ± 0.074 22.658 ± 0.248 1772.13 ± 205.880.9 ADM 14.847 ± 0.398 14.04 ± 1.71 6.725 ± 0.180 22.418 ± 0.601 1800.12 ± 302.821.2 ADM 19.168 ± 1.311 14.52 ± 0.97 8.683 ± 0.594 28.943 ± 1.979 2630.95 ± 129.191.5 ADM 22.872 ± 1.666 13.20 ± 0.39 10.361 ± 0.755 34.536 ± 2.515 2832.67 ± 367.25PM-1 12.621 ± 2.225 20.36 ± 1.45 5.712 ± 1.007 19.041 ± 3.356 2124.60 ± 121.57PM-3 30.095 ± 4.754 16.46 ± 1.85 13.621 ± 2.152 45.403 ± 7.172 1899.31 ± 152.33

0.0035

0.0045

0.0055

0.0065

0.0075

(A) (B)

Ct/C

s

h/L

0.0035

0.0045

0.0055

0.0065

0.0075

0.7 1.05 1.4 1.75 2.150 200 350 500

Ct/C

s

Sa/L2

y = −0.3122x −1.6065

−3

−2.5

−2

−1.5

−1

log

Ct/C

s

log Sa/L2

y = −0.6244x −2.2588R2 = 0.8339

R2 = 0.8339

−3

−2.5

−2

−1.5

−1

1.5 1.8 2.1 2.4 2.7 3−0.2 0 0.2 0.4

log

Ct/C

s

log h/L

Figure 9. Scaling relationship of different dimensionless groups for permeation of RIZ: (A) effects of dimensionless length of MNs and (B) effects of dimensionless sur-face area for diffusion.

10 C. T. UPPULURI ET AL.

MNs. Based on the simulation results, the diffusion profile of ZMTshowed significant permeation increment with 1.5mm ADM andPM when compared to other MNs in the study. Furthermore, theadvantage of greater penetration by PM can be further magnifiedby increasing the MN density, as in the case of PM-3.

These simulations can be of great research value as they maybe used to predict the degree of transdermal permeationenhancement of ZMT with other (new) dimensions and designs ofMNs using the respective histological section images and the pas-sive diffusion coefficient data.

Conclusions

Two types of MN devices (ADM and PM) were employed in thisinvestigation to derive a greater understanding on the effects ofvarious geometry parameters specifically on the transdermal per-meation of ZMT by MN application. It was observed that the

application of MNs significantly enhances the transdermal perme-ation of ZMT across pig ear skin. In addition, the type/shape, dens-ity and more importantly, the length of MNs (depth of penetrationinto skin) were found to play a crucial role in the overall perme-ation enhancement of ZMT using this technique. It may beinferred that the transdermal delivery of ZMT, at clinically signifi-cant levels and in a painless and non-invasive manner, may bypossible using MN applications. This investigation can form a basisfor further studies (in vivo) and for optimization of various MNparameters to achieve successful delivery of ZMT via MN-assistedtransdermal delivery systems.

Acknowledgements

The authors are thankful to Mylan Pharmaceuticals India Ltd,Hyderabad, India, for providing a gift sample of ZMT, to Dr.Naveen, Department of Pathology, Dr. Pinnamaneni Siddhartha

Figure 10. (A) The histological section images of skin. (B,C) Images treated and processed with MATLAB program. (D) Images simulated in COMSOL.

DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 11

Institute of Medical Sciences and Research Foundation,Vijayawada, India, for providing the required facilities for takinghistological sections of skin samples and also to the SiddharthaAcademy of General and Technical Education, Vijayawada, India,for providing necessary facilities to carry out the research work.

Disclosure statement

The authors report no conflicts of interest. The authors alone areresponsible for the content and writing of this article.

Funding

The authors extend their sincere thanks to Department of Scienceand Technology, Ministry of Science and Technology, Governmentof India and the British Council, London, UK, for funding thisresearch work under the DST-UKIERI scheme (DST/INT/UK/P-60/2014).

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https://www.researchgate.net/publication/315789468

Microneedle-assisted transdermal delivery of Zolmitriptan: effect of microneedle geometry, in vitro permeation experiments, scaling analyses and numerical simulationsIntroductionMaterials and methodsMaterialsFabrication of PMMN dimensional evaluationScanning electron microscopeConfocal laser microscopeAtomic force microscope

High-performance liquid chromatography methodSolubility studiesSkin preparationApplication of MNs on skin samplesSurface visualization and histological examination of the skin samplesIn vitro skin permeation studiesZMT content in skinScaling analysesNumerical simulations of experimental studiesStatistical analysis of the data

ResultsFabrication and characterization of PMAnalytical methodSolubility studiesSurface visualization and histological examination of skin samplesIn vitro permeation studiesScaling analysesNumerical simulations of experimental studies

DiscussionConclusionsAcknowledgementsDisclosure statementReferences