Monitoring the penetration process of single microneedles withvarying tip diametersCitation for published version (APA):Römgens, A. M., Bader, D. L., Bouwstra, J. A., Baaijens, F. P. T., & Oomens, C. W. J. (2014). Monitoring thepenetration process of single microneedles with varying tip diameters. Journal of the Mechanical Behavior ofBiomedical Materials, 40, 397-405. https://doi.org/10.1016/j.jmbbm.2014.09.015

DOI:10.1016/j.jmbbm.2014.09.015

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http://dx.doi.org/101751-6161/& 2014 T(http://creativecomm

nCorresponding autE-mail address: a

Research Paper

Monitoring the penetration process of singlemicroneedles with varying tip diameters

A.M. Romgensa,n, D.L. Badera,b, J.A. Bouwstrac, F.P.T. Baaijensa,C.W.J. Oomensa

aSoft Tissue Biomechanics and Engineering, Department of Biomedical Engineering, Eindhoven University of Technology,PO Box 513, 5600 MB Eindhoven, The NetherlandsbFaculty of Health Sciences, University of Southampton, Southampton, UKcDivision of Drug Delivery Technology, Leiden Academic Centre for Drug Research, Leiden University, Leiden,The Netherlands

a r t i c l e i n f o

Article history:

Received 17 May 2014

Received in revised form

9 September 2014

Accepted 15 September 2014

Keywords:

Skin mechanics

Microneedle

Penetration force

Penetration depth

.1016/j.jmbbm.2014.09.015he Authors. Published byons.org/licenses/by-nc-

hor. Tel.: þ31 402475415..m.romgens@tue.nl (A.M

a b s t r a c t

Microneedles represent promising tools for delivery of drugs to the skin. However, before

these microneedles can be used in clinical practice, it is essential to understand the

process of skin penetration by these microneedles. The present study was designed to

monitor both penetration depth and force of single solid microneedles with various tip

diameters ranging from 5 to 37 mm to provide insight into the penetration process into the

skin of these sharp microneedles. To determine the microneedle penetration depth, single

microneedles were inserted in human ex vivo skin while monitoring the surface of the

skin. Simultaneously, the force on the microneedles was measured. The average penetra-

tion depth at 1.5 mm displacement was similar for all tip diameters. However, the process

of penetration depth was significantly different for the various microneedles. Microneedles

with a tip diameter of 5 mm were smoothly inserted into the skin, while the penetration

depth of microneedles with a larger tip diameter suddenly increased after initial superficial

penetration. In addition, the force at insertion (defined as the force at a sudden decrease in

measured force) linearly increased with tip diameter ranging from 20 to 167 mN. The force

drop at insertion was associated with a measured penetration depth of approximately

160 μm for all tip diameters, suggesting that the drop in force was due to the penetration of

a deeper skin layer. This study showed that sharp microneedles are essential to insert

microneedles in a well-controlled way to a desired depth.

& 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC

BY-NC-SA license (http://creativecommons.org/licenses/by-nc-sa/3.0/).

Elsevier Ltd. This is an open access article under the CC BY-NC-SA licensesa/3.0/).

. Römgens).

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1. Introduction

The skin provides an important physical barrier to theenvironment. It is rich in immune-responsive cells, such asLangerhans cells and dermal dendritic cells. Consequently, itprovides a promising location for vaccination. By adminis-trating the vaccine directly to the epidermal and dermallayers of the skin, a lower dose of antigens may be requiredto achieve a comparable immune response to subcutaneousor intramuscular vaccination with a hypodermic needle(Chen et al., 2010, 2011; Fernando et al., 2010; Gelinck et al.,2009; Quan et al., 2010). Vaccination with microneedlesrepresents a promising method to deliver antigens into theskin. These projections with a length of less than a millimetrecan be fabricated from various materials in a wide range ofgeometries (Donnelly et al., 2010a; Kim et al., 2012; van derMaaden et al., 2012). Microneedles are used in multiple ways.For example, hollow microneedles can be used to inject fluidsinto the skin. By contrast, solid microneedles can eithercreate micropores in the stratum corneum after which drugscan be topically applied (poke and patch) or they can releasethe vaccine into the skin after their insertion. Using this lastapproach two methods are in development, coated micro-needles and dissolvable microneedles.

An important challenge in using microneedles to delivervaccines into the skin in a reproducible manner is to effec-tively and reproducibly penetrate the skin. Therefore, micro-needle penetration of the skin has to be fully characterised,including the inter-subject variation in skin mechanicalproperties and the various geometries of the microneedles.It is of paramount importance to obtain sufficient control onthe depth of penetration of the microneedle into the skinsince only the part of the microneedle within the skin willdeliver the drug. In addition, the penetration depth may beimportant to specifically target Langerhans cells or dermaldendritic cells residing in the epidermal and dermal layer ofthe skin, respectively. Activation of these different cells mayresult in a different immune response (Banchereau et al.,2009; Romani et al., 2010).

During microneedle application, the skin is first indentedbefore penetration occurs. As a result, many studies revealthat the insertion depth of microneedle arrays range widelyfrom 10 to 80% of the microneedle length (Chu and Prausnitz,2011; Coulman et al., 2010; Crichton et al., 2010; Donnellyet al., 2010b; Kalluri et al., 2011; Lee et al., 2008; Li et al., 2009;Matriano et al., 2002; Roxhed et al., 2007). Various factorsaffecting microneedle penetration depth have been exam-ined. The penetration depth of a microneedle array increaseswith application velocity (Crichton et al., 2010) and applica-tion force (Donnelly et al., 2010b). Microneedle length alsoinfluenced the penetration depth, in contrast to microneedleinterspacing. Accordingly, most studies have employedmicroneedles in an array, with a high velocity or large forceto ensure effective penetration of the skin. However, thepenetration mechanism of a single microneedle is still yet tobe fully clarified.

Clearly the application force is critical. A previous studyreported that insertion force of relative blunt microneedles, withtip diameters ranging from 60 to 160 mm, ranged from 0.08 to

3.04 N and was linearly dependent on tip frontal area (Daviset al., 2004). These relatively high insertion forces motivated thedesign of sharper microneedles, with a resultant reduction ofinsertion force measured for microneedle arrays (Khanna et al.,2010; O’Mahony, 2014; Roxhed et al., 2007).

Evidently, penetration depth as well as penetration force areimportant factors in controlled application of microneedles.However, the mechanism of microneedle penetration and therelation between penetration force and depth are still not wellunderstood. Therefore, the present study was designed tomonitor both penetration force and depth of single micronee-dles, manufactured to a range of tip diameters. In particular, itwas designed to examine the influence of tip diameters on thenature of the penetration mechanisms and the absolute valuesof the penetration parameters.

2. Materials and methods

2.1. Microneedle fabrication

Single solid microneedles were fabricated in a controlledmanner by pulling glass rods with a diameter of 1 mm (WorldPrecision Instruments, Inc, Sarasota, FL, USA) with a micro-pipette puller (P-97, Sutter Instruments Company, Novato,CA, USA) (Martanto et al., 2006a). Microneedles varyingsystematically in tip diameter were produced by tuning theinput parameters of the micropipette puller. In addition, insome cases the microneedle was cut to the desired tipdiameter using a microforge (MF-830, Narishige, Japan). Foursizes of microneedles were produced with tip diameters of 5,15, 24, and 37 mm with a resolution of 1 μm. For each micro-needle, an image was made with a digital microscope todetermine its shape (Fig. 1). The tip angle between differentlysized microneedles varied from 11 to 21 degrees. The tiplength of the microneedles was approximately 3.3 mm.

2.2. Preparation of human skin samples

Abdominal human skin was obtained from 8 female patients,aged between 35 and 55 years, from the Catharina Hospital,Eindhoven, the Netherlands, according to Dutch guidelines ofsecondary used materials. The skin was transferred to thelaboratory and processed within 4 h of excision. For proces-sing, the skin was stretched on a stainless steel plate, usingmultiple forceps, and cleaned with ethanol (Geerligs et al.,2011a; Lamers et al., 2013). Subsequently, skin slices of1.2070.23 mm thickness (mean7standard deviation), whichcontained the stratum corneum, viable epidermis, and der-mis, were obtained using a dermatome (D42, Humeca,Enschede, the Netherlands). These slices were cut intosquares of approximately 3 by 3 cm. Thereafter, speckles ofGraphit 33 were sprayed on the skin samples to increasecontrast of features during imaging.

Natural pretension in the skin leads to shrinkage duringthe cutting process. As the penetration can be influenced bythe tensions within the skin samples (Butz et al., 2012), thein vivo state should be mimicked as closely as possible. Thiswas achieved by attaching a metal ring of 7.5 g weight withan inner diameter of 15 mm on the unstretched skin samples

Fig. 1 – Solid microneedles with a tip diameter of 5, 15, 24,and 37 lm. The microneedles were fabricated using amicropipette puller. Bar indicates 50 lm.

55

Skin

PDMS

Micr

osco

pe

DepthWidth

Force

Fig. 2 – The surface of the skin was recorded to determinethe penetration depth of the microneedle. (A) Video frameused to measure the width of the microneedle at thelocation where the surface of the skin was penetrated (whitebar). (B) Schematic overview of the setup. From themeasured width, the penetration depth was deduced,knowing the shape of the microneedle.

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with super glue (Ultra gel, Pattex). Subsequently, the skinsample was stretched by the weight when it was placed on arod with a smaller diameter than the ring. A second metalring with an inner diameter of 4.4 mm was then glued on topof the stretched skin. Using digital image correlation, pilottests indicated that the skin sample is stretched between 15and 25%, considered to represent the skin strains encoun-tered in vivo. During and after sample preparation, the skinsamples were stored in Hank's Balanced Salt Solution (HBSS,Lonza Biowhittaker, Switzerland), supplemented with 1%penicillin/streptavidin and 0.2% Fungin at 4 1C until furtheruse. All experiments were performed within 48 h of surgery.

2.3. Microneedle penetration of the human skin

The microneedles were attached to a custom designed micro-indentation device (Vaenkatesan et al., 2006). With this device,displacement of the microneedle can be applied. Under optimalconditions, the displacement and force can be measured with aresolution of 15 nm and 15 mN, respectively.

Skin samples were allowed to warm up to room tempera-ture. Subsequently, the samples were taken out of the HBSS.The larger metal ring and excessive skin around the smallerring were removed. Any excess fluid was removed from itstop surface, and the sample, with the small metal ring stillattached, was placed on a substrate of polydimethylsiloxane(PDMS). The sample was positioned in such a way that themicroneedle was located above the centre of the smallermetal ring attached to the skin surface.

Microneedles were indented to a predetermined maximaldisplacement at a velocity of 0.05 mm/s. This velocity assured

quasi-static conditions and reliable force data. When thepredetermined maximal displacement was reached, themicroneedles were immediately retracted at the same rateuntil the tip was positioned 500 mm above the skin surface.During microneedle indentation, the surface of the skin wascontinuously imaged from an angle of approximately 55degrees with a monochromatic CCD camera (DMK 41BU02.H,The Imaging Source, Germany), which was attached to a longdistance microscope (QM100, Questar, New Hope, PA, USA)(Fig. 2). This resulted in a series of video frames and forcedata for each experiment.

Several series of experiments were conducted. One seriesof experiments focussed on the influence of the skin donoron the penetration depth. These experiments were conductedwith microneedles with a tip diameter of 5 mm using samplesof 5 different skin donors. A second series of experimentsfocussed on the effect of the tip diameter on the resultingpenetration depth and force. Tip diameters varied between 5and 37 mm. Experiments of both series were also used toevaluate the penetration depth measurements. A summaryof the different experiments is provided in Table 1.

Table 1 – Summary of the experiments performed to determine the influence of the used skin donor and the tip diameter.Experiments of both series were also used to evaluate the protocol of the penetration depth measurements.

Type of test Microneedle tipdiameter [mm]

Maximal displacement ofmicroneedle [mm]

Number ofsamples

Number of skindonors

Evaluation of penetration depthmeasurements

5 0.25, 0.50, 0.75, 1.00, 1.25, and1.50

44 815 924 937 9

Influence of skin donor on penetrationdepth

5 0.25, 0.50, 0.75, 1.00, 1.25, and1.50

33 5

Influence of tip diameter on penetrationforce and depth

5 1.50 12 415 624 6

737

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2.4. Data and statistical analysis

2.4.1. Penetration depthThe width of the microneedle at the location where itpenetrated the top surface of the skin was determined byanalysing individual video frames (Fig. 2). From this widthand the image of the microneedle shape (Fig. 1), the depth ofthe microneedle in the skin could be calculated. This wasperformed for multiple frames of each sample, each sepa-rated by approximately 0.1 mm displacement, using a customwritten program in Matlab (R2013a, The MathWorks, Inc.,Natick, MA, USA) and manually selecting the width of themicroneedles in the images. The estimated depth of themicroneedle was associated with corresponding values forpenetration force and displacement.

To determine the influence of the skin donor on thepenetration depth, a linear regression model was used withdata of the measured penetration depths. Subsequently, theR-squared value was determined. To determine the influenceof microneedle tip diameter, the mean penetration depth atevery subsequent 150775 mm displacement was compared. Anon-parametric Kruskal–Wallis test was performed followedby Dunn–Bonferroni post-hoc test to test for significantdifferences between microneedles per displacement range,with a significant value set at 5% (i.e. po0.05).

2.4.2. Evaluation of penetration depth measurementsTo evaluate the use of video frames to determine theprogression of penetration depth, two additional methodsto determine the maximal penetration depth were used.Before each experiment, the microneedles were dipped inmethylene blue. An image of the microneedle was recordedafter each test. From this image, the maximum penetrationdepth of the microneedle was determined by measuring thedistance from the tip until the location where no methyleneblue was visible (hereafter called ‘depth according to theimage’), similar to the method used by van der Maaden et al.(2014). These images also confirmed that none of the micro-needles failed during the experiments. In addition, thepenetration depth during retraction of the microneedle wasmeasured using video frames (hereafter called ‘depth atretraction’). This was defined as the depth determined fromthe video frame corresponding to the moment the force

during retraction became zero, because then the microneedlewas still at its maximal depth within the skin.

2.4.3. Penetration forceAs reported in previous studies, a sudden decrease in theforce can be observed when the skin is penetrated (Daviset al., 2004; Khanna et al., 2010). These drops were alsoobserved in our experiments. The initial drop in force wasdefined as the first decrease in force larger than 0.685 mN.This threshold value was determined by measuring themaximal noise of the force signal under the current condi-tions, and multiplying it by 5. For each sample, three para-meters were determined, namely, the force at the initial drop,the displacement at the initial drop, and the total decrease inforce during the initial drop.

To examine the differences in the three parameters withmicroneedle tip diameter, a one-way analysis of variances(ANOVA) with a Bonferroni post-hoc test was performed.Normality of the test data and equality of variance betweenthe groups were verified with Shapiro–Wilk and Levene'stests, respectively. The assumption of equality of variancewas violated for one parameter (po0.05). All statistical ana-lyses were performed using SPSS (IBM SPSS Statistics 22, IBMCorp., Armonk, NY, USA).

3. Results

3.1. Evaluation of penetration depth measurements

The method of using video recordings of the skin surface todetermine the penetration depth was evaluated by compar-ing the depth according to the video at maximal displace-ment with the depth at retraction and depth according to theimage. It was seen that the depth from the video at maximaldisplacement was slightly higher than both the depth atretraction and according to the image; the latter two valueswere similar. This indicates that the penetration depthmeasured with the video frames was overestimating thepenetration depth. By comparing the depth of the video atmaximal displacement (dv) with the depth at retraction (dr)using a linear regression model (dv ¼ 0:75 � dr þ 2:35 μm,R2¼0.78) it was determined that the depth was overestimated

0 0.3 0.6 0.9 1.2 1.50

100

200

300

400

Microneedle displacement [mm]

Pen

etra

tion

dept

h [µ

m]

*

* ** * *

**

5 µm tip15 µm tip24 µm tip37 µm tip

Fig. 4 – The effect of microneedle tip diameter on thepenetration depth related to the microneedle displacement.The penetration depth was significantly different betweenmicroneedles at lower displacement. Significant differencesbetween microneedles are indicated with n (po0.05).Number of data points is 2 to 21 per bar.

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by approximately 34%. Subsequent data shown was correctedby the value of the slope of the linear regression model.

3.2. Influence of skin donor

To determine the influence of skin donor on the penetrationdepth, experiments were performed on samples from 5different skin donors using microneedles with a tip diameterof 5 mm. Fig. 3 demonstrates that the penetration depthincreased linearly with microneedle displacement (R2 variedfrom 0.73 to 0.97 for the various skin donors). It is evident thatthe slopes of the linear regression models were generallywithin 12% of each other.

3.3. Influence of tip diameter

To determine the influence of microneedle tip diameter onthe penetration process, the penetration experiments wereperformed with the four different tip diameter microneedleson at least 6 skin samples per diameter. In 2 measurementsfor both the 5 and the 37 mm diameter microneedles therewas no drop in force larger than the threshold value of0.685 mN and these 4 samples were excluded from furtheranalysis.

3.3.1. Penetration depthAlthough the mean penetration depth at the maximal displace-ment of 1.5mm was approximately 250 mm for all tip diameters(Fig. 4), the progression to this depth varied considerably. For thesmallest tip diameter of 5 μm, the microneedle was smoothlyinserted into the skin, which was characterised by a linearincrease in depth with microneedle displacement. In contrast,

0 0.5 1 1.5

100

200

300

400

500

Microneedle displacement [mm]

Pen

etra

tion

dept

h [µ

m]

LRM 1LRM 2LRM 3LRM 4LRM 5

0

Fig. 3 – The penetration depth of a microneedle with a tipdiameter of 5 lm at a range of displacements tested ondifferent skin donors. Single measurements are shown for atotal of 33 samples from 5 skin donors with an associatedlinear regression model (LRM) (R2 is 0.79, 0.86, 0.73, 0.87, and0.97, respectively). The slopes of the linear regression modelfor the different skin donors were 221, 141, 149, 149, and133 lm/mm, respectively. The diamonds, asterisks, squares,circles, and triangles represent single measurements ofdonor 1 to 5, respectively.

for larger tip diameters the penetration depth was initiallysmaller than the 5 mm tip microneedle, as the skin was pre-dominantly indented. This was followed by a sudden insertioninto the skin, as reflected in the sudden increase in depth afterapproximately 0.75mm displacement, particularly evident withthe 37 mm tip microneedle (Fig. 4). In some samples, this suddenincrease in depth was clearly visible on the video recordings ascan be seen in Fig. 5. Subsequently, the penetration depth rapidlyincreased up to a similar magnitude to the 5 mm tip diameter. Asignificant difference between the microneedle groups wasfound for displacements from 0 to 0.9mm (po0.05).

3.3.2. Penetration forceThe measured force during penetration increased non-linearlywith displacement up to forces of approximately 320mN. Thepenetration depth at a certain force is shown in Fig. 6. Similar tothe relationship between penetration depth and microneedledisplacement (Fig. 4), the depth at a certain force was differentfor the four microneedle diameters. It was evident that thesharper microneedles penetrated the skin at lower forces thanthe more blunt microneedles.

The force and displacement at the initial drops and thetotal decrease in force during these drops were compared forthe four microneedles (Fig. 7). The force at the initial droplinearly increased with tip diameter with means rangingfrom 20 to 167 mN. A statistically significant difference inthe force at the initial drop between the largest and twosmallest diameter tip microneedles was found (po0.05),together with a difference between the 24 mm and 5 mm tipmicroneedles. The total decrease in force during the initialdrop also increased with tip diameter from 1.5 to 30.4 mN,with a significant difference between 5 and 37 mm tipdiameters. There was no significant difference in displace-ment at the initial drop, which occurred at approximately1.1 mm displacement. If the penetration depth within thedisplacement range where the initial drop in force was

}

51 μm

}

55 μm

}

67 μm

0 0.5 1 1.50

50

100

150

Microneedle displacement [mm]

Forc

e [m

N]

0 0.5 1 1.50

50

100

150

Microneedle displacement [mm]

Forc

e [m

N]

0 0.5 1 1.50

50

100

150

Microneedle displacement [mm]

Forc

e [m

N]

Fig. 5 – Video frames of the penetration of a 37 lm tip microneedle indicated a sudden increase in penetration depth.Top: from left to right three subsequent video frames at 15 frames per second. The dashed lines mark the edges of themicroneedle. The width of the microneedle at the location where it penetrated the skin is indicated; bottom: thecorresponding force curve. The dot shows the force corresponding to the video frame.

0 100 200 300 4000

100

200

300

400

Forc

e [m

N]

Penetration depth [µm]

5 µm 15 µm 24 µm

37 µm

Fig. 6 – The penetration depth related to the measured forceis shown in the figure per microneedle group.

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measured (range mean7standard deviation) was comparedfor the various tip diameters, it is worth noting that thepenetration depth was approximately 160 mm for all groups.This suggests that the microneedles had already penetratedthe upper layer of the skin and thus, at the time an initialdrop in force was measured, penetration involved one of thedeeper skin layers.

In more than 50% of tests with the three smallest tipdiameters, more than one drop in force larger than thethreshold value was recorded within a single test. By

contrast, for the 37 mm tip diameter only one drop in forcegreater than the threshold value was evident within asingle test.

4. Discussion

It is important that insertion of microneedles into the skin iscontrolled to ensure a reproducible depth of insertion whenthe technique will be adopted in the clinic. In the presentstudy, the penetration depth and insertion force of singlemicroneedles with various tip diameters, ranging from 5 to37 μm, were determined. Recordings demonstrated a moresmooth penetration of the skin by microneedles of smaller tipdiameters. This was supported both by the measurements ofthe penetration depth, which increased more linearly forsharp microneedles than for blunter microneedles, and thesudden drop in penetration force, which was larger inmagnitude for the blunter microneedles. Therefore, sharpmicroneedles with diameters less than 15 μm are essential toinsert microneedles in a well-controlled manner to adesired depth.

In the present study, no difference in penetration depthwas found between different skin donors. The measuredpenetration depths were slightly lower than reported in aprevious study using micropipette pulled microneedles witha tip opening of 22 to 48μm (Martanto et al., 2006a). Theseauthors reported a penetration depth of 300 to 350 mm at adisplacement of 1080 mm. This discrepancy can be due to thesubstrate employed, which was stainless steel in theprevious study.

5 15 24 370

50

100

150

200

250

300

Microneedle diameter [µm]

Forc

e at

initi

al d

rop

[mN

]

*

**

5 15 24 370

10

20

30

40

50

60

70

Microneedle diameter [µm]To

tal d

ecre

ase

in fo

rce

[mN

] *

5 15 24 370

0.5

1

1.5

Microneedle diameter [µm]

Dis

plac

emen

t at i

nitia

l dro

p [m

m]

Fig. 7 – The influence of microneedle tip diameter on the force and displacement at the initial drop and the total decrease inforce during this drop. Mean and standard deviation are shown. N¼10, 6, 6, and 5 for the microneedle groups, from small tolarge tip respectively. n indicates a significant difference between two groups with po0.05.

100 150 2000

1

2

3

4

Microneedle tip diameter [µm]

Forc

e at

initi

al d

rop

[N]

Davis et al., 2004Current study

500

Fig. 8 – (A) The force at the initial drop as function of themicroneedle tip diameter measured by Davis et al. (2004)and in the current study. (B) For microneedles with a tipdiameter less than approximately 80 μm, the force to stretchthe surface of the skin, which is proportional to r (indicatedby the dashed area), exceeds the force to compress the skinunderneath the microneedle, which is proportional to r2

(indicated by the dark area underneath the microneedle).(C) For tip diameters larger than 80 μm, compression of theskin volume under the skin gets predominant.

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Previous studies determined the penetration force of variousmicroneedles by measuring a sudden decrease in force exertedon the microneedle (Davis et al., 2004; Khanna et al., 2010).The authors assumed that, at the moment a drop in the forcewas measured, the stratum corneum was ruptured. This inter-pretation is questioned by the present findings, since the initialdrop in force corresponded to a measured penetration depth ofapproximately 160 μm for all tip diameters. This depth approx-imates to the depth of the transition from the papillary to thereticular dermis. The reticular dermis consists of a denselypacked collagen fibre network and these fibres are likely toresist penetration by a microneedle. The present findingsindicate that the penetration of the stratum corneum andviable epidermis was smooth in nature and therefore did notresult in a measurable drop in the force even with a verysensitive load cell. Therefore, it is conceivable that previousstudies also measured the penetration of a deeper skin layer. Inmany of the present experiments, more than one drop largerthan the threshold value was measured within a single test.This indicated that the microneedles were gradually insertedinto the skin, as previously suggested (Gittard et al., 2013). Insome cases, drops in force were of smaller magnitude than thethreshold values. These may represent penetration of thestratum corneum and/or noise in the force signal.

In the present study, the force at the initial drop linearlyincreased with tip diameter. This seems contrary to a pre-vious study by Davis et al. (2004), who reported a linearrelationship between the force at the initial drop and the tiparea of the microneedles, with diameters ranging from 60 to160 μm. However, pooling data from the two studies revealedan interesting relationship between force at initial drop andtip diameter, as illustrated in Fig. 8a. It is apparent that theforce at the initial drop is linearly related to the radius (r) forsharp microneedles, while for tip diameters larger thanapproximately 80 μm the force at the initial drop is propor-tional to r2. This may be explained by the combination offorces the microneedle exerts on the skin (Fig. 8b and c). Themicroneedle stretches the skin surface, resulting in a high

stress in the skin in a ring around the edges of the micro-needle. The size of this ring is equivalent to the circumfer-ence of the microneedle tip, which is proportional to r. In

j o u r n a l o f t h e m e c h a n i c a l b e h a v i o r o f b i o m e d i c a l m a t e r i a l s 4 0 ( 2 0 1 4 ) 3 9 7 – 4 0 5404

addition to stretching the skin, the force will be influenced bycompression of the volume of skin underneath the micro-needle, which is proportional to r2. Results indicate that theforce to stretch the surface dominates over compressing theskin at small tip diameters (Fig. 8b), while the inverse is thecase at increasing tip radii (Fig. 8c).

The present study benefits from the use of a singlemicroneedle to penetrate the skin. Previous studies measuredthe force of insertion of a microneedle array, and thendivided this force by the number of microneedles in the array(Kochhar et al., 2013; Olatunji et al., 2013; O’Mahony, 2014;Roxhed et al., 2007). However, pilot studies performed withmicroneedle arrays (array of 4�4 microneedles of 300 mm)revealed that microneedles did not significantly penetrate theskin before the back plate of the array made contact with theskin, thus influencing the measured force. Therefore, whenusing an array, a measure of the force per microneedle willundoubtedly be influenced by the microneedle density withinthe array. By using a single microneedle without a back plate,we were able to accurately measure the force of a singlemicroneedle. Although the insertion process may be differentwhen microneedles are applied in an array, it is important tofirst understand this process for a single microneedle.

Specific aspects of the study design are worthy of discus-sion. First, the penetration depth was corrected to overcomethe overestimation when measuring the microneedle widthat its boundary with the skin. This overestimation wascaused by the skin curling around the microneedle duringthe insertion process. Therefore, the visible boundary of theskin with the microneedle was not always equal to the pointwhere the microneedle actually entered the skin. This effectwas reported by Martanto et al. (2006b), who fixated the skinwith the microneedle in place. In the histological images, thedeflection of the skin is visible. However, when the micro-needle was retracted after penetration, the deflection of theskin recovered while the microneedle was still penetratingthe skin, which made it possible to correct for the curling ofthe skin. Apart from this correction, it was estimated that themaximum error in determining the penetration depth usingthe video frames was approximately 30 mm. Second, Figs. 3and 4 depict a larger spread in penetration depth above 1 mmdisplacement compared to depths under 1 mm displacement.This may be a result of the slight variation in skin thickness:depending on the sample thickness the PDMS substrate wasindented to a larger extent. With a Young's modulus ofapproximately 0.8 MPa, PDMS is stiffer than subcutaneoustissue, which has a Young's modulus of 20–30 kPa (Gefen andHaberman, 2007). Therefore, the use of PDMS as a substrateinstead of subcutaneous tissue, may have an influence on theabsolute values found for the penetration depth in relation-ship to the microneedle displacement. However, this differ-ence in substrate stiffness is not expected to unduly influencethe major findings of the present study. Similarly, the relativehumidity of the environment may affect the penetrationprocess of the stratum corneum, since its stiffness is knownto be dependent on the relative humidity (Geerligs et al.,2011b; Yuan and Verma, 2006). Nevertheless, the overallinfluence of the stratum corneum on the penetration processseemed to be limited. In addition, the present study wasperformed with human skin tissue, which has more

physiological relevance to previous studies involving porcineor murine skin samples (Crichton et al., 2010; Donnelly et al.,2010b; Gittard et al., 2013).

To our knowledge, this is the first study that visualised themicroneedle penetration depth and skin deflection during theinsertion process of a single microneedle while measuringthe force. By performing these experiments for microneedleswith a range of tip diameters, a more complete insight intothe penetration process was obtained. It can be concludedthat microneedles with a smaller tip diameter penetrate theskin more smoothly than microneedles with a larger tipdiameter. Therefore, if we want to controllably insert micro-needles to a desired depth for the purpose of vaccine delivery,sharp microneedles (o15 mm) are necessary.

Acknowledgements

This research is supported by the Dutch Technology Founda-tion STW, which is part of the Netherlands Organisation forScientific Research (NWO), and which is partly funded by theMinistry of Economic Affairs (Project no. 11259).

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