Research Article Permeation of Micronized and Nanonized … · 2019. 5. 13. · and composing cells...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 787283 8 pageshttpdxdoiorg1011552013787283

Research ArticleIn Vitro Permeation of Micronized and NanonizedAlaptide from Semisolid Formulations

Radka Opatrilova Aneta Cernikova Lenka Coufalova Jiri Dohnal and Josef Jampilek

Department of Chemical Drugs Faculty of Pharmacy University of Veterinary and Pharmaceutical Sciences Palackeho 1361242 Brno Czech Republic

Correspondence should be addressed to Josef Jampilek josefjampilekgmailcom

Received 1 September 2013 Accepted 1 December 2013

Academic Editors A Concheiro J Hamman and M Ozyazici

Copyright copy 2013 Radka Opatrilova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

This study is focused on in vitro permeation of the original Czech compound a skinmucosa tissue regeneration promoter knownunder the international nonproprietary name ldquoalaptiderdquo in micronized and nanonized forms Alaptide showed a great potential forlocal applications for treatment andor regeneration of the injured skinThe abovementioned technologicalmodifications influencethe permeation of alaptide through artificial or biological membranes such as PAMPA or skin The permeation of micronized andnanonized form of alaptide formulated to various semisolid pharmaceutical compositions through full-thickness pig ear skin usinga Franz cell has been investigated in detail In general it can be concluded that the nanonized alaptide permeated through the skinless than themicronized form different observations weremade for permeation through the PAMPA system where themicronizedform showed lower permeation than the nanonized alaptide

1 Introduction

It is known that damage or deficit of skin and mucosa suchas injury wound morsus scald burn congelation radiationinjury ultraviolet irradiation electric injury traumatic injuryskin ulcer bedsore and bullous skin diseases causes degen-erative exfoliation necrosis apoptosis or apoptosis-like celldeath of skin tissue-composing cells or mucosal tissue-composing cells As for effective methods for prevention ortherapy of diseases caused by mechanical or physical damageor defect of the skin tissue or the mucosal tissues applicationof drugs which can rapidly regenerate andor reconstructthe degenerated and defected skin tissues mucosal tissuesand composing cells thereof and promote wound healing isconsidered [1ndash3]

(S)-8-Methyl-69-diazaspiro[45]decan-710-dione knownunder the international nonproprietary name (INN) ldquoalap-tiderdquo (see Figure 1) can be also considered as skinmucosatissue regeneration promoters It was designed as an analogueof melanocyte-stimulating hormone release-inhibiting factor(MIF) and synthesized by Kasafirek et al at the ResearchInstitute for Pharmacy and Biochemistry in the 80s of the

20th century [4ndash7] Alaptide is a white crystalline compoundgenerally poorly soluble stable in the sunlight and storableat ambient temperature [8 9] Since it is an optically activemolecule it is necessary to inspect the entire syntheticprocess with the intention to determine unambiguously theabsolute configuration of the final product A liquid chro-matographic method for control of alaptide enantiopuritywas developed [10] It was found out that when optically purestarting compounds are used no change of configuration(racemization) occurs and the chirally pure final productis obtained Except for the recently published X-ray powderdiffraction data revealing the unit cell parameters and thespace group of alaptide crystals [11] absolute configurationof the molecule was also determined by electronic circulardichroism spectroscopy [12]

The influence of alaptide on epidermal regenerationwas investigated in a number of tests In vivo experimentswere performed using pigs to which alaptide was appliedon experimental injury and faster skin regeneration wasobserved after the application of alaptide Similarly alaptideaccelerated curing experimental skin injuries on rats [8]Alaptide probably negatively affects the inhibition of release

2 The Scientific World Journal

O

OH3C

H

H

N

Nlowast

Figure 1 Structure of (S)-alaptide

of melanocyte-stimulating hormone and thus it increasesconcentration of melanocytes in epidermis Melanocytessignificantly influence creation and function of keratinocytesby means of melanosomas [13 14] Keratinocytes migratefrom stratum basale to stratum spinosum and stratum gran-ulosum to stratum corneum where they support epidermisregeneration [15] It is excreted unchanged mostly via urinea similar metabolic profile was also found in man [8 16 17]No toxic effects of alaptide (including skin irritation) wereobserved Induction of biotransformation enzymes CYP1A1CYP1A2 and CYP1B1 in hepatocytes did not occur [8 9 1618]

Based on the above mentioned facts it can be sum-marized that alaptide showed a great potential for localapplications for treatment andor regeneration of injuredskin or mucosa [8 19] The aim of this paper is to eval-uate the rate of permeation of alaptide through biologi-cal membranes As penetrationpermeation of compoundstothrough the membranes is important to know especiallyat local applications and micro- and nanonized substancesfeature different behavior related to penetrationpermeation[20ndash24] micronized and nanonized forms of (S)-alaptidewere prepared and permeation of both forms of alaptidethrough the membrane was also evaluated using the PAMPA(parallel artificial membrane permeability assay) technique[25ndash27] With respect to external application of both formsin vitro ability to permeate through full-thickness pig earskin was also evaluated using a Franz cell [28 29] includingpermeation from various semisolid formulations

2 Material and Methods

21 Preparation of Micronized Alaptide Alaptide was syn-thesized by the standard process as described above [4 58] Then it was micronized The particle size of the usedmicronized alaptide was measured by a microscope NIKONOptiphot 2 with a digital camera VDS CCD-1300F Theparticle size distribution (x

90) of microcrystalline alaptide

was 40 120583m

22 Preparation of Nanonized Alaptide The suspension ofmicronized alaptide (30 g) polyvinylpyrrolidone (30 g) andpurified water (240mL during milling was diluted by addi-tion of additional 150mL) was initially mixed for 12 h atambient temperature and then filtered through a mill sieveThemilling procedure was performed using a nanomill NET-ZSCH (Germany) with glass beads (03mm) the rotor speedwas 986 rpm the pump speed was 30 rpm the temperaturein the grinding chamber was within 17ndash20∘C The rotor

speed was increased to 1500 rpm after 6 h of milling Thetotal time of milling was 575 h The content of alaptide inthe suspension was 3876 gL (determined by RP-HPLC seebelow) The particle size of the prepared nanonized alaptidewas measured by Sympatec NANOPHOX 0138 P (Germany)and the particle size x

90was 770 nm

23 Investigated Semisolid Formulations Directly before theapplication nanonized alaptide was dispersed by ultrasonicsto formulations to avoid possible reagglomeration The com-position of gel with alaptide 1 (ww) was the following alap-tide 1 g carboxymethylcellulose ointment (carboxymethyl-cellulose sodium 5 g Macrogol 300 10 g propylene glycol25 g methylparaben 02 g propylparaben 02 g and purifiedwater 873 g) up to 100 g The composition of cream withalaptide 1 (ww) was the following alaptide 1 g cremorneoaquasorb 95 g and propylene glycol up to 100 g Thecomposition of ointment with alaptide 1 (ww) was asfollows alaptide 1 g cera lanae hydrosa 75 g yellow vaseline20 g and liquid paraffin up to 100 g

24 Biology

241 In Vitro PAMPA Experiments The permeability ofmicronized and nanonized alaptide was evaluated in vitrousing a vertical PAMPA (parallel artificial membrane per-meability assay) system (BD gentest precoated PAMPA platesystem 96 wells)The PAMPA system constitutes a lipophilicmembrane which surface is coated by phospholipids thatsimulate intestinal wall Micronized alaptide (10mg) orthe amount of nanosuspension corresponding to 10mg ofalaptide was weighed The donor samples were prepared bydissolving the tested samples in 40mL of 001M HCl andafter 15min pH was adjusted to pH 6 using bicarbonatebuffer A carbonate buffer saline (physiological solution) withpH 74 was used as a receptor phase About 05 h beforethe experiment the PAMPA system was taken out from thefreezer and warmed up to the ambient temperature Thereceptor phase (200120583Lwell) was pipetted into the upperwells The donor phase was pipetted into the lower wells(300 120583Lwell) After the incubation time (5 h) 10 120583L of theacceptor phase was taken from each well and mixed withphysiological solution (990120583L) At least five determinationswere performed

Analysis of samples for alaptide content was performedusing an Agilent 1200 series HPLC system equipped witha diode array detection (DAD) system a quaternary modelpump and an automatic injector (Agilent Technologies Ger-many) Data acquisition was performed using ChemStationchromatography software AWaters Symmetry C

85 120583m 46times

250mm(Waters CorpMilfordMAUSA) chromatographiccolumn was used A mixture of MeCN (HPLC grade 500)and H

2O (HPLCmdashMili-Q Grade 500) was used as a

mobile phase The total flow of the column was 05mLmininjection 10 120583L column temperature 24∘C and sample tem-perature 10∘C The detection wavelength of 204 nm waschosen the time of analysis was 10min The retention time(119905119877) of alaptide was 31 plusmn 005min the limit of detection

The Scientific World Journal 3

(LOD) was 68 ngmL and the limit of quantification (LOQ)was 230 ngmL

242 In Vitro Transdermal Permeation Experiments Per-formed Using Franz Diffusion Cell Skin samples wereobtained from porcine ear Full-thickness dorsal skin wascut in fragments and stored at minus20∘C until utilized Skinsamples were slowly thawed (at 4∘C overnight and thenat ambient temperature) prior to each experiment Thepermeation through the skin of themicronized alaptide alone(1mL of suspension with the alaptide concentration of 1)nanonized alaptide alone (in the amount corresponding to1 concentration of micronized alaptide) and both formsof alaptide incorporated into ointment cream or gel wasevaluated in vitro using a vertical Franz diffusion cell (SES-Analytical Systems GmbH Germany) with a donor surfacearea of 63585mm2 and a receptor volume of 52mLThe skinwas mounted between the donor and receptor compartmentsof the Franz diffusion cell with the epidermal side up To thedonor part of the Franz diffusion cell with the volume of 1mLan investigated sample was applied in the form of solutionsuspension emulsion gel cream or ointment and then thedonor compartment of the cell was covered by Parafilm Thereceptor compartment was filled with phosphate bufferedsaline (pH 74) and was maintained at 34 plusmn 05∘C while usingcirculating water bath The receptor compartment contentwas continuously stirred using a magnetic stirring bar Theskin was kept in contact with the receptor phase for 05 hat 34 plusmn 05∘C prior to the experiment Samples (05mL) ofthe receptor phase were withdrawn at six predetermined timeintervals over 24 h (05 1 2 4 6 and 24 h) and the cell wasrefilled with an equivalent amount of fresh buffer solution Aminimum of five determinations were performed using skinfragments from aminimum of 2 animals for each compoundThe sampleswere immediately analyzed by theHPLCmethodusing the same conditions as described previouslyThe resultsof alaptide permeation are summarized in Table 1

243 Statistical Evaluation All experiments were carried out5-fold Data were expressed as means plusmn SD Differences wereevaluated by one-way analysis of the variance (ANOVA) testcompleted by Bonferronirsquos multicomparison test (ORIGINPRO7) The differences were considered significant at 119875 =005 The independent variables and responses (flux and lagtime) of all model samples were analyzed using ORIGINPRO7

3 Results and Discussion

31 In Vitro PAMPA Experiments The preliminary per-meability screening of micronized and nanonized alaptidewhich was obtained by milling process with glass beadswas performed using polyvinylidene fluoride (PVDF) thatis using PAMPA that has become a very useful and quitecheap tool for predicting intestinal permeability and is wellsuited as a ranking tool for the assessment of the compoundswith passive intestinal transport mechanisms [25ndash27] Thepermeability of nanoalaptide through PAMPA after 5 h

(06mg plusmn 001) was 2-fold higher than that of micronizedalaptide (03mg plusmn 001) Both values of permeability areexpressed as mean plusmn SD (119899 = 5 experiments)

The results of PAMPA experiments and testing in Franzcells (see below) differed completely It seems that thepermeation of micronized and nanonized alaptide is mostsignificantly influenced by the use of the real skin as a barrierin Franz cells and artificial PVDF membrane in PAMPAThe major drawback of the PAMPA technique is that it canonly predict passive diffusion and is therefore unable togenerate the full description of the permeability process atthe real skin The skin is a complex organ that can influenceand change pharmacokinetics of the administered drugs bya number of various interactions [29 30] On the otherhand PAMPA is an excellent tool to rapidly predict passivepermeability through the gastrointestinal tract with highthroughput efficiency asmentioned above Other parametersthat may influence the results are solubility supramolec-ular superassembly properties of nanonized alaptide andnanoparticle stabilizer properties but the explanation ofthese parameters is not the aim of this study

32 In Vitro Transdermal Permeation Experiments PerformedUsing Franz Diffusion Cell The main focus was the inves-tigation of nanonized and micronized alaptide permeationthrough porcine ear skin Firstly both forms were evalu-ated using a phosphate buffer (pH 74) as a control donorinert vehicle that did not support permeation consequentlyboth forms were incorporated into semisolid formulationsPharmaceutical compositions were prepared in the form ofointment cream and gel with the content of alaptide inconcentration 1 (ww) Alaptide was used in microcrys-talline form or in the form of nanoparticles Porcine earskin was selected for initial evaluation of permeation as thistissue is a suitable in vitro model of human skin [31 32]Porcine skin proved to be histologically and biochemicallysimilar to human skin therefore full-thickness pig ear skinhas been used in numerous percutaneous absorption studies[33]The skin permeation experiments were performed usingstatic Franz diffusion cells [28] As it could be expectedthe presented transdermal permeation results of micronizedand nanonized alaptide and the data obtained using PAMPAmethod are different The principal difference is due to dif-ferent properties of the transport ldquomembranerdquo the PAMPAdiaphragm is an artificial model of the cell lipid bilayerldquosimulatingrdquo the intestinal wall whereas the ear porcine skinis a much more complex naturally constructed barrier asdiscussed above

In transdermal experiments as a result of the sampling oflarge volumes from the receptormedium (and replenishmentwith equal volumes of the fresh medium) the receptormedium was constantly being diluted Consequently thereceptor compartment concentration of alaptide was cor-rected for sample removal and replenishment using thefollowing

1198621015840

119899= 119862119899(119881119905

119881119905minus 119881119904

)(1198621015840

119899minus1

119862119899minus1

) (1)


Table 1 Cumulative permeated amounts119876119905per unit area (120583gcm2) ofmicronizednanonized alaptide from buffer and from various semisolid

formulations achieved in in vitro transdermal permeation experiments performed using Franz diffusion cell Cumulative permeations areexpressed as mean plusmn SD (119899 = 5 experiments)

Time (h) Alaptide Cumulative permeated amounts 119876119905(120583gcm2)

Buffer Gel Cream Ointment

05 Micro 126 plusmn 19a 2205 plusmn 111abc 899 plusmn 40a 753 plusmn 54cd

Nano 69 plusmn 20a 1057 plusmn 73a 2467 plusmn 215a 589 plusmn 74a

1 Micro 401 plusmn 80a 4016 plusmn 309c 2067 plusmn 89a 848 plusmn 73de

Nano 185 plusmn 76a 1656 plusmn 70ab 4881 plusmn 142b 650 plusmn 82ab

2 Micro 1508 plusmn 72a 6264 plusmn 2111d 5687 plusmn 72b 967 plusmn 59fg

Nano 1473 plusmn 92a 3427 plusmn 799bc 67607 plusmn 219b 729 plusmn 67bc

4 Micro 4183 plusmn 246b 14745 plusmn 3069f 14145 plusmn 213d 1124 plusmn 73h

Nano 3459 plusmn 218b 10275 plusmn 280e 9175 plusmn 135c 812 plusmn 72cd

6 Micro 11890 plusmn 1059d 19276 plusmn 2412g 25337 plusmn 92f 1264 plusmn 37i

Nano 10293 plusmn 2126c 18509 plusmn 277g 18638 plusmn 241e 909 plusmn 76ef

24 Micro 38971 plusmn 2956f 137668 plusmn 928i 106026 plusmn 182h 1519 plusmn 95j

Nano 21780 plusmn 2957e 97741 plusmn 3903h 84994 plusmn 4610g 1030 plusmn 103gh

The means followed by different letters are significantly different at 119875 = 005 The analysis was performed for both micro- and nanoforms of alaptide withinindividual studied system (buffer as well as 3 semisolid formulations)

where 1198621015840119899is corrected drug concentration in the 119899th sample

119862119899is measured drug concentration in the 119899th sample 1198621015840

119899minus1

is corrected drug concentration in the (119899 minus 1)th sample 119862119899minus1

is measured drug concentration in the (119899 minus 1)th sample 119881119905is

total volume of receptor solution119881119904is volume of the sample

and 11986210158401is 1198621[34]

The corrected data were expressed as the cumulativedrug permeation (119876

119905) per unit of skin surface area using the

following

119876119905=1198621015840

119899

119860 (2)

where 119860 = 06359 cm2 in our experimentIf the cumulative amount of drug per unit area (119876

119905[120583g])

in the receiver chamber is plotted as a function of time (119879 [h])steady state permeation flux (119869 [120583ghcm2]) can be calculatedfrom the slope of the linear portion of the curve [35] Ittakes some time for achieving the steady state of maximumpermeation and this timemay be considered to be the lag time(119879lag [h])The lag time can be determined by extrapolating thelinear portion of cumulative amount of permeation per unitarea (119876

119905) versus time (119879 [h]) curve to the abscissa [36]

Permeability coefficient (119870119901[cmh]) can be calculated

according to the following

119870119901=119869

119862119889

(3)

where 119862119889is drug concentration in the donor compartment

(in our experiment 10 ww ie 10 times 104 120583gmL) It isassumed that under sink conditions drug concentration inthe receptor compartment is negligible compared to that inthe donor compartment [37 38]

Cumulative permeated amounts per unit area were cal-culated from individual experimental data see Table 1 Thedependences of the cumulative amounts per unit area of

the micronizednanonized alaptide permeated through theporcine ear skin on the time [h] (the permeation profiles ofmicronized and nanonized forms of alaptide from variousmedia) are illustrated in Figures 2(a)ndash2(d)

The general results of alaptide alone (only from bufferwithout any formulation) showed that micronized alaptidepermeated significantly more than nanonized alaptide nev-ertheless permeation of micronized alaptide was really lowThe amount of nanonized alaptide that permeated throughthe skinwas approximately 2-fold less than the amount of per-meated micronized alaptide When alaptide was formulatedin semisolid formulations the observed in vitro transdermalpermeation was higher for gel and cream than for bufferIn general it can be stated that more hydrophilic semisolidformulation means higher permeation which is connectedwith the moisturizing effect on stratum corneum [29] andthe presence of propylene glycol compared with the ointmentcomposition The permeation of both forms of alaptidethrough the skin from ointment was minimal neverthelessmore alaptide permeated from ointment within 1 h than frombuffer In fact it is well known that viscous formulationsreduce the diffusion coefficient of themolecule in the vehiclethus retarding or avoiding its skin partitioning and henceabsorption In addition extremely lipophilic formulationscompete with stratum corneum lipophilicity hamperingthe stratum corneum partitioning of the penetrating agentIn contrast occlusive formulations may favor a moderateincrease in absorption as previously mentioned [21 29 39]

The field of nanoparticle drug delivery to the skin hasprogressed over the last dozen years to a point wherethere are well-characterized tools that have the capacity forcustomized pharmacokinetics Although in general transportmechanisms support nanomaterial penetration [40ndash43] thepromise of nanoparticle-mediated drug delivery into theepidermis and dermis without barrier modification has metwith little success It can be stated that the penetration of


0500

1000150020002500300035004000

0 4 8 12 16 20 24Time (h)

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(a)

Time (h)

02000400060008000

100001200014000

0 4 8 12 16 20 24

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(b)

Time (h)

MicroNano

0

2000

4000

6000

8000

10000

12000

0 4 8 12 16 20 24

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(c)

Time (h)

MicroNano

020406080

100120140160

0 4 8 12 16 20 24Cu

mul

ativ

e am

ount

(120583g

cm2)

(d)

Figure 2 In vitro penetration-time profile of micronizednanonized alaptide through skin from buffer (a) and from the followingpharmaceutical compositions gel (b) cream (c) and ointment (d) Points with error bars represent mean values of cumulative permeations(mean plusmn SD) determined in five experiments

particulate materials into the skin is a very complex issueTherefore the interaction of nanoparticles with the skin andespecially skin models is an intriguing field However thedata obtained do not show a clear image of the effect ofnanocarriers In particular the penetration of such particlesis an open and controversially discussed issue The litera-ture reports different results mainly on pig or murine skinshowing strong penetration (pig and mouse) or the oppositeLooking only at the sizes of the particles no conclusivepicture can be obtained either The fact that nanoparticles donot penetratepermeate or they expressed decreased penetra-tionpermeation was described and discussed in some recentpapers [20ndash24 44]

Due to the small number of time intervals in whichpermeation of alaptide was determined as well as the factthat in the time interval 6ndash12 h samples were not drawnit is not possible to evaluate steady-state permeation fluxesTherefore apparent fluxes the corresponding (119879lag) andpermeability coefficients (119870

119901) were calculated from lin-

earized dependences of six determined cumulative perme-ated amounts per unit area on time for buffer gel andcream formulations (they showed correlation coefficients (119903)ranging from 0980 to 0999) Only the apparent flux ofnanoparticles of alaptide in buffer was evaluated from theinterval 05ndash4 h The following permeation parameters arepresented in Table 2 nonsteady-state flux (119869) corresponding

0

100

200

300

400

500

600

700

Micro Nano Micro Nano Micro NanoBuffer Gel Cream

Flux

(120583g

cm2h

)

Figure 3 Range of nonsteady permeation flux (mean of fiveexperiments plusmn SD) of micronized and nanonized alaptide fromvarious media

lag time (119879lag) and apparent permeability coefficient (119870119901)

Although these values are only informative they can servefor comparison of permeation effectiveness of alaptide frombuffer and hydrophilic semisolid formulations The values ofnonsteady-state permeation fluxes are illustrated in Figure 3

It was not possible to calculate the above mentionedparameters for ointment composition with both forms ofalaptide In this case cumulative permeated amounts ofalaptide per unit area were more than one order lower


Table 2 Selective permeation parameters of micronized and nanonized alaptide through full-thickness pig ear skin from various medianonsteady-state permeation flux (119869) corresponding lag time (119879lag) and apparent permeability coefficient (119870

119901)

Medium Alaptide J (120583gcm2h) 119879lag (h) 119870119901times 10minus3 (cmh)

Buffer Micro 1684 plusmn 87 069 plusmn 038 1684Nano 1254 plusmn 60lowast 076 plusmn 001lowast 1254

Gel Micro 5880 plusmn 48 103 plusmn 023 5880Nano 4206 plusmn 175 099 plusmn 009 4206

Cream Micro 4522 plusmn 08 057 plusmn 001 4522Nano 3544 plusmn 197 027 plusmn 003 3544

lowast05ndash4 h

compared with other investigated systems and it can bestated that ointment medium due to its constitution andsmall content of water did not supportinhibited permeationof alaptide from the composition Thus alaptide with min-imal permeation through the skin without achievement ofsystemic concentration can curatively act only in the upperskin layer which is desirable from the point of view of variousskin defects Nevertheless also in case of this medium theobvious trend discussed above is evident namely the factthat the permeation of microparticles was higher than thatof nanoparticles

The permeability of alaptide from the evaluated mediawas significantly influenced by the composition (see Fig-ure 3) especially by hydrophilicity of the whole formulationand proportions of possible surfactantcosurfactant in themixture

The apparent permeability coefficient 119870119901representing

absorption rate showed a wide range from 5880 times 10minus3 cmh(micro gel) to 1254 times 10minus3 cmh (nano buffer) that islog119870119901

ranging from minus123 to minus190 It is evident againthat nanonized alaptide from all the media showed lowerpermeation and the penetration effectiveness of alaptide isinfluenced by formulation According to the values of thelogarithm of the permeability coefficient it is possible to statethat the permeation of micronized alaptide from buffer isclose to nicotine (log119870

119901= minus177) [45] while permeation

of the nanonized alaptide from buffer is similar to sufentanil(log119870

119901= minus192) [45] It can be stated that penetration ability

of alaptide is not small Permeation through the skin frombuffer of both forms of alaptide met quite good correlationwith the result of regression analysis (4) of QSAR approachfor skin permeability that was published by Barratt [45]

log119875119888= minus000734MV + 0769 log119875 minus 27713 (4)

where 119875119888is permeability coefficient MV is molecular volume

(predicted by CS ChemOffice Ultra ver 100 (CambridgeSoftCambridge MA USA) expressed as a molar refractivityMR) and log119875 is logarithm of octanol-water partitioncoefficient 119875ow (for alaptide MR = 4727 cm3mol andexperimental log119875ow = 139 [8 9]) The calculated valuelog119875119888= minus205 quite corresponds to experimentally found

logarithmvalues of the permeability coefficient ofmicronized(log119870

119901= minus177) and nanonized (log119870

119901= minus190) alaptide

from buffer that is without any modifier of permeationConsequently it can be stated that apparent permeability

coefficients calculated from linearized dependences of cumu-lative permeated amounts on time (05ndash6 h for nanobufferand 05ndash24 h for other investigated systems) can be used forcomparison of permeation of both forms of alaptide fromvarious mediasystems

Based on the above mentioned facts it can be assumedthat the evaluated pharmaceutical compositions can be usedwith success in combination with anti-inflammatory drugsanalgesics antimicrobial chemotherapeutics andor antihe-morrhagics As was mentioned in the introduction alaptideadministered topicallylocally supportsinduces much fasterregeneration and healing of the skin than in the case whenonly drugs alone from the above mentioned therapeuticgroups are present in the formulation The selection of themicronized or nanonized form of alaptide and the type of for-mulation can influence the depth and the rate of permeationto the skin that is the curative effect Such pharmaceuticalcompositions containing alaptide can be used for formulationof alaptide alone or for combination of alaptide with otherdrugs and can be applied for treatment of skin and mucosallesions for example burns raws decubitus and venousulcerations The therapy of such skin lesions is very difficultlong-standing and not always quite successful thereforeit is suitable to have available as broad variety of effectivetherapeutics as possible [9]

4 Conclusion

In general it can be concluded that nanonized alaptidepermeated through PAMPAmore than the micronized formDifferent observationswere obtained for permeation throughthe skin where micronized alaptide permeated ca 2-foldmore from the buffer and other semisolid pharmaceuti-cal compositions than the nanonized form neverthelesshydrophilic and hydrophobic media significantly influencedthe permeation of alaptide Both forms of alaptide permeatedmore effectively from hydrophilic gel and cream wherealso the transdermal enhancer propylene glycol was presentthan from buffer The permeation of both forms of alaptidethrough the skin from ointment was minimal The constitu-tion of ointment did not support permeation of alaptide thusalaptide with minimal permeation through the skin withoutachievement of systemic effect can act only in the upper skinlayer which is desirable from the point of view of various skindefects


Conflict of Interests

The authors declare no conflict of interests

Acknowledgment

This study was supported by the Grant Agency of the CzechRepublic (Czech Science Foundation) Project no GACRP304112246

References

[1] E M De Souza Russo and V F T Russo Inventors CristaliaProdutos Quimicos e Farmaceuticos Ltd assignee ldquoPharma-ceutical compositions of topic use applied in treatment of skinandor mucous injuries use of compositions in treatment ofskin andormucous injuries and use of compounds in treatmentof skin andor mucous injuriesrdquoWO 2002083086 A1 2002

[2] K Hashimoto K Nakata M Sakanaka and J Tanaka Inven-tors Japan Science and Technology Corporation assigneeldquoSkin tissue regeneration promoters comprising ginsenosideRb1rdquo EP1295893 A1 2003

[3] S G Choi E J Baek E Davaa et al ldquoTopical treatment of thebuccal mucosa and wounded skin in rats with a triamcinoloneacetonide-loaded hydrogel prepared using an electron beamrdquoInternational Journal of Pharmaceutics vol 447 pp 102ndash1082013

[4] E Kasafirek A Sturc and A Roubalova ldquoLinear tri- andtetrapeptides acting as prodrugsrdquo Collection of CzechoslovakChemical Communications vol 57 pp 179ndash187 1992

[5] E Kasafirek M Rybak I Krejci A Sturc E Krepela andA Sedo ldquoTwo-step generation of spirocyclic dipeptides fromlinear peptide ethyl ester precursorsrdquo Life Sciences vol 50 no3 pp 187ndash193 1992

[6] M E Celis S Taleisnik andRWalter ldquoRegulation of formationand proposed structure of the factor inhibiting the release ofmelanocyte-stimulating hormonerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 68 no7 pp 1428ndash1433 1971

[7] M Petersson and K Uvnas-Moberg ldquoProlyl-leucyl-glycinam-ide shares some effects with oxytocin but decreases oxytocinlevelsrdquoPhysiology andBehavior vol 83 no 3 pp 475ndash481 2004

[8] S Radl E Kasafirek and I Krejci ldquoAlaptiderdquo Drugs of theFuture vol 15 pp 445ndash447 1990

[9] J Jampılek R Opatrilova A Rezacova Z Oktabec and JDohnal Inventors Faculty of Pharmacy University of Veteri-nary and Pharmaceutical Sciences Brno assignee ldquoAlaptideMethods of effecting its solubility membrane permeation andpharmaceutical compositions for human and veterinary appli-cationsrdquo PTCCZ2012000074 2012

[10] M Dousa and K Lemr ldquoLiquid chromatographic method forenantiopurity control of alaptide using polysaccharide station-ary phasesrdquo Journal of Separation Science vol 34 no 12 pp1402ndash1406 2011

[11] J Maixner J Rohlıcek B Kratochvıl and A Sturc ldquoX-ray powder diffraction data for alaptide 8(S)-methyl-69-diazaspiro45decane-710-dione or cyclo(l-alanyl-1-amino-1-cyclopentan carbonyl) cyclo(l-Ala-Acp)rdquo Powder Diffractionvol 24 pp 32ndash34 2009

[12] O Julinek V Setnicka A Rezacova J Dohnal V VosatkaandMUrbanova ldquoProduct of alaptide synthesis determination

of the absolute configurationrdquo Journal of Pharmaceutical andBiomedical Analysis vol 53 pp 958ndash961 2010

[13] T Burns S Breathnach N Cox and C Griffiths RookrsquosTextbook of Dermatology Blackwell Publishing Oxford UK7th edition 2004

[14] W James T Berger and D Elston Andrewsrsquo Diseases of theSkin Clinical Dermatology Saunders Philadelphia PA USA10th edition 2006

[15] F M Watt ldquoThe epidermal keratinocyterdquo BioEssays vol 8 no5 pp 163ndash167 1988

[16] J Vanzura K Kosar and E Kasafirek ldquoInhibition of prolifer-ative activity by cyclic dipeptides spirocyclic derivatives of 1-aminocyclopentanecarboxylic acidrdquo Toxicology Letters vol 31no 3 pp 189ndash193 1986

[17] R Lapka ldquoPharmacokinetics and brain entry of alaptide a novelnootropic agent in mice rats and rabbitsrdquo Journal of Pharmacyand Pharmacology vol 43 no 12 pp 874ndash876 1991

[18] K Kosar and J Vanzura ldquoEmbryotoxicity of L-prolyl-L-leucyl-glycinamide cyclo(1-amino-cyclopentanecarbonyl-alanyl) andcyclo(glycyl-leucyl) new potential neuropeptides in chickembryosrdquo Pharmazie vol 43 no 10 pp 715ndash716 1988

[19] Bioveta-Alaptid veterinary ointmentcz httpwwwbiovetaczenveterinary-divisionproductsnew-products-for-dogs-and-catsalaptid-veterinary-ointmenthtml

[20] M Schneider F Stracke S Hansen and U F SchaeferldquoNanoparticles and their interactions with the dermal barrierrdquoDermatoendocrinol vol 1 pp 197ndash206 2009

[21] B Baroli ldquoPenetration of nanoparticles and nanomaterials inthe skin fiction or realityrdquo Journal of Pharmaceutical Sciencesvol 99 no 1 pp 21ndash50 2010

[22] T W Prow J E Grice L L Lin et al ldquoNanoparticles andmicroparticles for skin drug deliveryrdquo Advanced Drug DeliveryReviews vol 63 no 6 pp 470ndash491 2011

[23] C S J Campbell L R Contreras-Rojas M B Delgado-Charro and R H Guy ldquoObjective assessment of nanoparticledisposition in mammalian skin after topical exposurerdquo Journalof Controlled Release vol 162 pp 201ndash207 2012

[24] E Kimura Y Kawano H Todo Y Ikarashi and K SugibayashildquoMeasurement of skin permeationpenetration of nanoparticlesfor their safety evaluationrdquo Biological amp Pharmaceutical Bul-letin vol 35 pp 1476ndash1486 2012

[25] M Kansy F Senner and K Gubernator ldquoPhysicochemical highthroughput screening parallel artificial membrane permeationassay in the description of passive absorption processesrdquo Journalof Medicinal Chemistry vol 41 no 7 pp 1007ndash1010 1998

[26] A Avdeef and O Tsinman ldquoPAMPAmdasha drug absorptionin vitro model 13 Chemical selectivity due to membranehydrogen bonding in combo comparisons of HDM- DOPC- and DS-PAMPA modelsrdquo European Journal of PharmaceuticalSciences vol 28 no 1-2 pp 43ndash50 2006

[27] K Y Tam M Velicky and R A W Dryfe ldquoThe importanceof and different approaches to permeability determinationrdquo inPhysico-Chemical Methods in Drug Discovery and DevelopmentZ Mandic Ed pp 121ndash164 IAPC Zagreb Croatia 2012

[28] T J Franz ldquoPercutaneous absorption On the relevance of invitro datardquo Journal of Investigative Dermatology vol 64 no 3pp 190ndash195 1975

[29] J Jampilek and K Brychtova ldquoAzone analogues classifica-tion design and transdermal penetration principlesrdquo MedicalResearch Review vol 32 pp 907ndash947 2012


[30] J Jampilek ldquoTransdermal application of drugs and techniquesaffecting skin barrierrdquo Journal of Bioequivalence amp Bioavailabil-ity vol 5 pp 233ndash235 2013

[31] U Jacobi M Kaiser R Toll et al ldquoPorcine ear skin an in vitromodel for human skinrdquo Skin Research and Technology vol 13no 1 pp 19ndash24 2007

[32] C Herkenne A Naik Y N Kalia J Hadgraft and R H GuyldquoPig ear skin ex vivo as a model for in vivo dermatopharma-cokinetic studies in manrdquo Pharmaceutical Research vol 23 no8 pp 1850ndash1856 2006

[33] W Meyer R Schwarz and K Neurand ldquoThe skin of domesticmammals as a model for the human skin with special referenceto the domestic pigrdquo Current Problems in Dermatology vol 7pp 39ndash52 1978

[34] H Wu C Ramachandran N D Weiner and B J RoesslerldquoTopical transport of hydrophilic compounds using water-in-oil nanoemulsionsrdquo International Journal of Pharmaceutics vol220 no 1-2 pp 63ndash75 2001

[35] N Akhtar M U Rehman H M S Khan F Rasool T Saeedand G Murtaza ldquoPenetration enhancing effect of polysorbate20 and 80 on the in vitro percutaneous absorption of L-ascorbicacidrdquo Tropical Journal of Pharmaceutical Research vol 10 no 3pp 281ndash288 2011

[36] L Panigrahi S Pattnaik and S K Ghosal ldquoThe effect of pHand organic ester penetration enhancers on skin permeationkinetics of terbutaline sulfate from pseudolatex-type transder-mal delivery systems throughmouse and human cadaver skinsrdquoAAPS PharmSciTech vol 6 no 2 pp E167ndashE173 2005

[37] C THuangM J Tsai YH Lin et al ldquoEffect ofmicroemulsionson transdermal delivery of citalopram optimization studiesusing mixture design and response surface methodologyrdquoInternational Journal of Nanomedicine vol 2013 pp 2295ndash23042013

[38] Y-S Rhee J-G Choi E-S Park and S-C Chi ldquoTransdermaldelivery of ketoprofen using microemulsionsrdquo InternationalJournal of Pharmaceutics vol 228 no 1-2 pp 161ndash170 2001

[39] L Kennish and B Reidenberg ldquoA review of the effect ofocclusive dressings on lamellar bodies in the stratum corneumand relevance to transdermal absorptionrdquo Dermatology OnlineJournal vol 11 no 3 p 7 2005

[40] B Bhushan Handbook of Nanotechnology Springer BerlinGermany 2004

[41] C Buzea I I Pacheco and K Robbie ldquoNanomaterials andnanoparticles sources and toxicityrdquo Biointerphases vol 2 ppMR17ndashMR71 2007

[42] E Corredor P S Testillano M-J Coronado et al ldquoNanoparti-cle penetration and transport in living pumpkin plants in situsubcellular identificationrdquo BMC Plant Biology vol 9 article 452009

[43] A Verma and F Stellacci ldquoEffect of surface properties onnanoparticle-cell interactionsrdquo Small vol 6 no 1 pp 12ndash212010

[44] A C Watkinson A L Bunge J Hadgraft and M E LaneldquoNanoparticles do not penetrate human skin-a theoreticalperspectiverdquo Pharmaceutical Research vol 30 pp 1943ndash19462013

[45] M D Barratt ldquoQuantitative structure-activity relationships forskin permeabilityrdquo Toxicology in Vitro vol 9 no 1 pp 27ndash371995

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


O

OH3C

H

H

N

Nlowast

Figure 1 Structure of (S)-alaptide

of melanocyte-stimulating hormone and thus it increasesconcentration of melanocytes in epidermis Melanocytessignificantly influence creation and function of keratinocytesby means of melanosomas [13 14] Keratinocytes migratefrom stratum basale to stratum spinosum and stratum gran-ulosum to stratum corneum where they support epidermisregeneration [15] It is excreted unchanged mostly via urinea similar metabolic profile was also found in man [8 16 17]No toxic effects of alaptide (including skin irritation) wereobserved Induction of biotransformation enzymes CYP1A1CYP1A2 and CYP1B1 in hepatocytes did not occur [8 9 1618]

Based on the above mentioned facts it can be sum-marized that alaptide showed a great potential for localapplications for treatment andor regeneration of injuredskin or mucosa [8 19] The aim of this paper is to eval-uate the rate of permeation of alaptide through biologi-cal membranes As penetrationpermeation of compoundstothrough the membranes is important to know especiallyat local applications and micro- and nanonized substancesfeature different behavior related to penetrationpermeation[20ndash24] micronized and nanonized forms of (S)-alaptidewere prepared and permeation of both forms of alaptidethrough the membrane was also evaluated using the PAMPA(parallel artificial membrane permeability assay) technique[25ndash27] With respect to external application of both formsin vitro ability to permeate through full-thickness pig earskin was also evaluated using a Franz cell [28 29] includingpermeation from various semisolid formulations

2 Material and Methods

21 Preparation of Micronized Alaptide Alaptide was syn-thesized by the standard process as described above [4 58] Then it was micronized The particle size of the usedmicronized alaptide was measured by a microscope NIKONOptiphot 2 with a digital camera VDS CCD-1300F Theparticle size distribution (x

90) of microcrystalline alaptide

was 40 120583m

22 Preparation of Nanonized Alaptide The suspension ofmicronized alaptide (30 g) polyvinylpyrrolidone (30 g) andpurified water (240mL during milling was diluted by addi-tion of additional 150mL) was initially mixed for 12 h atambient temperature and then filtered through a mill sieveThemilling procedure was performed using a nanomill NET-ZSCH (Germany) with glass beads (03mm) the rotor speedwas 986 rpm the pump speed was 30 rpm the temperaturein the grinding chamber was within 17ndash20∘C The rotor

speed was increased to 1500 rpm after 6 h of milling Thetotal time of milling was 575 h The content of alaptide inthe suspension was 3876 gL (determined by RP-HPLC seebelow) The particle size of the prepared nanonized alaptidewas measured by Sympatec NANOPHOX 0138 P (Germany)and the particle size x

90was 770 nm

23 Investigated Semisolid Formulations Directly before theapplication nanonized alaptide was dispersed by ultrasonicsto formulations to avoid possible reagglomeration The com-position of gel with alaptide 1 (ww) was the following alap-tide 1 g carboxymethylcellulose ointment (carboxymethyl-cellulose sodium 5 g Macrogol 300 10 g propylene glycol25 g methylparaben 02 g propylparaben 02 g and purifiedwater 873 g) up to 100 g The composition of cream withalaptide 1 (ww) was the following alaptide 1 g cremorneoaquasorb 95 g and propylene glycol up to 100 g Thecomposition of ointment with alaptide 1 (ww) was asfollows alaptide 1 g cera lanae hydrosa 75 g yellow vaseline20 g and liquid paraffin up to 100 g

24 Biology

241 In Vitro PAMPA Experiments The permeability ofmicronized and nanonized alaptide was evaluated in vitrousing a vertical PAMPA (parallel artificial membrane per-meability assay) system (BD gentest precoated PAMPA platesystem 96 wells)The PAMPA system constitutes a lipophilicmembrane which surface is coated by phospholipids thatsimulate intestinal wall Micronized alaptide (10mg) orthe amount of nanosuspension corresponding to 10mg ofalaptide was weighed The donor samples were prepared bydissolving the tested samples in 40mL of 001M HCl andafter 15min pH was adjusted to pH 6 using bicarbonatebuffer A carbonate buffer saline (physiological solution) withpH 74 was used as a receptor phase About 05 h beforethe experiment the PAMPA system was taken out from thefreezer and warmed up to the ambient temperature Thereceptor phase (200120583Lwell) was pipetted into the upperwells The donor phase was pipetted into the lower wells(300 120583Lwell) After the incubation time (5 h) 10 120583L of theacceptor phase was taken from each well and mixed withphysiological solution (990120583L) At least five determinationswere performed

Analysis of samples for alaptide content was performedusing an Agilent 1200 series HPLC system equipped witha diode array detection (DAD) system a quaternary modelpump and an automatic injector (Agilent Technologies Ger-many) Data acquisition was performed using ChemStationchromatography software AWaters Symmetry C

85 120583m 46times

250mm(Waters CorpMilfordMAUSA) chromatographiccolumn was used A mixture of MeCN (HPLC grade 500)and H

2O (HPLCmdashMili-Q Grade 500) was used as a

mobile phase The total flow of the column was 05mLmininjection 10 120583L column temperature 24∘C and sample tem-perature 10∘C The detection wavelength of 204 nm waschosen the time of analysis was 10min The retention time(119905119877) of alaptide was 31 plusmn 005min the limit of detection











1198621015840

119899= 119862119899(119881119905

119881119905minus 119881119904

)(1198621015840

119899minus1

119862119899minus1

) (1)





















119899minus1




and 11986210158401is 1198621[34]



following

119876119905=1198621015840

119899

119860 (2)


119905[120583g])





119870119901=119869

119862119889

(3)








0500

1000150020002500300035004000

0 4 8 12 16 20 24Time (h)

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(a)

Time (h)

02000400060008000

100001200014000

0 4 8 12 16 20 24

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(b)

Time (h)

MicroNano

0

2000

4000

6000

8000

10000

12000

0 4 8 12 16 20 24

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(c)

Time (h)

MicroNano

020406080

100120140160

0 4 8 12 16 20 24Cu

mul

ativ

e am

ount

(120583g

cm2)

(d)






0

100

200

300

400

500

600

700


Flux

(120583g

cm2h

)







119901)





lowast05ndash4 h



















4 Conclusion





Acknowledgment


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of











1198621015840

119899= 119862119899(119881119905

119881119905minus 119881119904

)(1198621015840

119899minus1

119862119899minus1

) (1)





















119899minus1




and 11986210158401is 1198621[34]



following

119876119905=1198621015840

119899

119860 (2)


119905[120583g])





119870119901=119869

119862119889

(3)








0500

1000150020002500300035004000

0 4 8 12 16 20 24Time (h)

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(a)

Time (h)

02000400060008000

100001200014000

0 4 8 12 16 20 24

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(b)

Time (h)

MicroNano

0

2000

4000

6000

8000

10000

12000

0 4 8 12 16 20 24

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(c)

Time (h)

MicroNano

020406080

100120140160

0 4 8 12 16 20 24Cu

mul

ativ

e am

ount

(120583g

cm2)

(d)






0

100

200

300

400

500

600

700


Flux

(120583g

cm2h

)







119901)





lowast05ndash4 h



















4 Conclusion





Acknowledgment


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





















119899minus1




and 11986210158401is 1198621[34]



following

119876119905=1198621015840

119899

119860 (2)


119905[120583g])





119870119901=119869

119862119889

(3)








0500

1000150020002500300035004000

0 4 8 12 16 20 24Time (h)

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(a)

Time (h)

02000400060008000

100001200014000

0 4 8 12 16 20 24

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(b)

Time (h)

MicroNano

0

2000

4000

6000

8000

10000

12000

0 4 8 12 16 20 24

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(c)

Time (h)

MicroNano

020406080

100120140160

0 4 8 12 16 20 24Cu

mul

ativ

e am

ount

(120583g

cm2)

(d)






0

100

200

300

400

500

600

700


Flux

(120583g

cm2h

)







119901)





lowast05ndash4 h



















4 Conclusion





Acknowledgment


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0500

1000150020002500300035004000

0 4 8 12 16 20 24Time (h)

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(a)

Time (h)

02000400060008000

100001200014000

0 4 8 12 16 20 24

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(b)

Time (h)

MicroNano

0

2000

4000

6000

8000

10000

12000

0 4 8 12 16 20 24

Cum

ulat

ive a

mou

nt (120583

gcm

2)

(c)

Time (h)

MicroNano

020406080

100120140160

0 4 8 12 16 20 24Cu

mul

ativ

e am

ount

(120583g

cm2)

(d)






0

100

200

300

400

500

600

700


Flux

(120583g

cm2h

)







119901)





lowast05ndash4 h



















4 Conclusion





Acknowledgment


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



119901)





lowast05ndash4 h



















4 Conclusion





Acknowledgment


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




Acknowledgment


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Permeation of Micronized and Nanonized … · 2019. 5. 13. · and composing cells...

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Transcript of Research Article Permeation of Micronized and Nanonized … · 2019. 5. 13. · and composing cells...