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Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 83– 87

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

jou rn al hom e page: www.elsev ier .com/ locate / jpba

rug release profiles and microstructural characterization of cast andreeze dried vitamin B12 buccal films by positron annihilation lifetimepectroscopy

arnabás Szabóa,b, Nikolett Kállai c, Gergo Tóthd, Gergely Hetényia, Romána Zelkóa,∗

University Pharmacy Department of Pharmacy Administration, Semmelweis University, Hogyes Endre Street 7-9, H-1092 Budapest, HungaryGedeon Richter Plc., Formulation R&D, Gyömroi Street 19-21, H-1103 Budapest, HungaryDepartment of Pharmaceutics, Semmelweis University, Hogyes E. Street 9, H-1092 Budapest, HungaryDepartment of Pharmaceutical Chemistry, Semmelweis University, Hogyes E. Street 7, H-1092 Budapest, Hungary

r t i c l e i n f o

rticle history:eceived 5 August 2013eceived in revised form 16 October 2013ccepted 20 October 2013vailable online 4 November 2013

a b s t r a c t

Solvent cast and freeze dried films, containing the water-soluble vitamin B12 as model drug were pre-pared from two polymers, sodium alginate (SA), and Carbopol 71G (CP). The proportion of the CP waschanged in the films. The microstructural characterization of various samples was carried out by positronannihilation lifetime spectroscopy (PALS). The drug release kinetics of untreated and stored samples wasevaluated by the conventionally applied semi-empirical power law.

Correlation was found between the changes of the characteristic parameters of the drug release and

eywords:itamin B12

ast filmreeze-dryingissolutionositron annihilation lifetime spectroscopy

the ortho-positronium (o-Ps) lifetime values of polymer samples. The results indicated that the increaseof CP concentration, the freeze-drying process and the storage at 75% R.H. decreased the rate of drugrelease.

The PALS method enabled the distinction between the micro- and macrostructural factors influencingthe drug release profile of polymer films.

PALS)

. Introduction

The buccal mucosa offers several advantages for local drugherapy and systemic delivery of drugs that are subjected torst-pass metabolism or are unstable within the rest of the gas-rointestinal tract. With the right formulation of buccal dosageorms, the permeability and the local environment of the mucosaan be controlled in order to achieve the required drug perme-tion [1]. The rate of absorption of hydrophilic compounds is aunction of the molecular size. Smaller molecules (75–100 Da) gen-rally exhibit rapid transport across the mucosa, with permeabilityecreasing as molecular size increases. For hydrophilic macro-olecules absorption enhancers have been used to successfully

lter the permeability of the buccal epithelium, causing this routeo be more suitable for the delivery of larger molecules [2].

Buccal films are preferable over adhesive tablets in terms of flex-bility and thinness thus being less obtrusive and more acceptable

o the patient. Mucoadhesive materials are hydrophilic macro-

olecules containing numerous hydrogen-bond-forming groups.hey have been called “wet” adhesives in that they require moisture

∗ Corresponding author. Tel.: +36 1 2170927; fax: +36 1 2170927.E-mail address: zelko.romana@pharma.semmelweis-univ.hu (R. Zelkó).

731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jpba.2013.10.031

© 2013 Elsevier B.V. All rights reserved.

to become adhesive and this may be supplied by the saliva; thelatter may also act as the dissolution medium [3].

Drug release from mucoadhesive matrices is known to be acomplex interaction between diffusion, swelling and erosion mech-anisms [4,5]. The mathematical description of the entire drugrelease process is rather difficult because of the number of physicalcharacteristics that must be taken into consideration. These includethe diffusion of water into the matrix, polymer swelling and drugdiffusion out of the device, polymer dissolution, axial and radialtransport in a 3-dimensional system. Several mathematical modelshave been constructed to describe the drug release profile [6]. Eachmodel makes certain assumptions e.g. restriction of the transportphenomena to one dimension; neglect of polymer swelling or dis-solution [7], and due to these assumptions, the applicability of therespective models is restricted to certain drug–polymer systems.

Positron annihilation lifetime spectroscopy (PALS) is a pow-erful technique for the characterization of free volume fractionsand free volume size distributions in solids. In polymers, the longlifetime component, connected to the ortho-positronium (o-Ps) isexpected to give information on free volume characteristics. Deter-

mination of free volume parameters by the PALS technique givesimportant information about the open structure in solid polymers.Free volume properties are related to molecular dynamics, volumerelaxation and physical ageing, and govern transport properties

84 B. Szabó et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 83– 87

Table 1Polymeric composition of the samples.

Sample ID SA (w/w%) CP (w/w%)

1 3.0 –2 3.0 0.153 3.0 0.254 4.5 –5 4.5 0.156 4.5 0.257 6.0 –8 6.0 0.159 6.0 0.25

S

stoi

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2

2

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2

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2

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carrier-free 22NaCl was used. Its activity was around 2 × 105 Bq and

A: sodium alginate, CP: Carbopol 71 G.

uch as gas permeation, gas separation and drug release. Besideshe size of the drug molecules, the drug release rate depends alson the radius of the fractional free volume and, naturally, on thenterconnectivity of the holes [8–10].

The main objective of this work was to find a correlationetween the drug release profiles and the supramolecular structure11] of buccal films and wafers as a function of their compositionnd the way of their preparation.

. Materials and methods

.1. Materials

Sodium alginate (SA) from brown algae (Mw = 12–40 kDa; CAS005-38-3) was obtained from Sigma (St. Louis, MO, USA) andarbopol® 71 G NF (CP; acrylic acid cross-linked with polyalkenylthers or divinyl glycol; Mw = 237 kDa) was provided by NoveonCleveland, OH, USA), vitamin B12 was received from Gedeonichter Plc. (Budapest, Hungary).

.2. Sample preparation

Samples were prepared by dissolving the necessary amount ofolymeric excipients (Table 1) in distilled water and 2 mg vita-in B12/10 g hydrogel under stirring at room temperature for 48 h.lthough the gels were slightly acidic (pH = 4.5–6.5), in order tobtain transparent gels and consequently homogeneous films, theA-CP ratios of the compositions were determined based on theuthors’ previous study [12]. Cast films were made by solventvaporation of 1.6 g hydrogel in metal plates of 3.5 cm diameternd dried at 22 ± 2 ◦C temperature and 50 ± 5% relative humidityor 24 h. Freeze dried samples (wafers) were produced from theame amount of hydrogels in the same plates by fast freeze-dryingith the following parameters: freezing temperature and period:20 ◦C, 12 h; tray temperature and period: 6 ◦C and 24 h; sample

emperature: (−2)–(−3) ◦C. The prepared samples were stored at0 ± 2 ◦C and 75 ± 5% relative humidity for 4 weeks.

.3. In vitro dissolution test

In vitro dissolution test was performed in a multibath (n = 8) dis-olution test system Hanson SR8-Plus (Hanson Research, USA). Thepparatus was used with paddles at stirring speed of 50 rpm. Eachlm and wafer was dissolved in 300 ml of pH = 6.8 phosphate bufferPh. Eur. 7.). The number of parallels was 3. Dissolution mediumas temperature controlled at 37.0 ± 0.5 ◦C. 500 �l of samples were

ithdrawn at 15 min regular intervals without replacing by freshedium. The samples were filtered through 10 �m sample filter

Hanson Research, USA) before further analysis.

Fig. 1. The supposed structure of product ion.

2.4. HPLC-MS analysis

The B12 concentration was determined by HPLC MS/MS in MRM(multiple reaction monitoring) mode based on previous literaturedata after the in vitro dissolution test [13]. HPLC analysis was per-formed by an Agilent 1260 Infinity LC system in conjunction withan Agilent 6460 triple-quadrupole mass spectrometer. The columnwas a Waters Sunfire C18 (50 mm × 2.1 mm × 3.5 �m) maintainedat 25 ◦C. The mobile phase consisted of 10% acetonitrile in watercontaining 0.1% formic acid, the flow rate was set to 0.5 ml/min. Themass spectrometer was operated in conjunction with a Jet Streamelectrospray ion source in positive ion mode. In the MRM mode theprecursor and the product ions were 678.5 ([M+2H]2+) and 146.9(Fig. 1), respectively. Other optimized parameters were the follow-ing: fragmentor voltage: 135 V, collision energy 50 V, dwell time:200 ms, delta EMV: 30 V. Flow and temperature of the drying gas(N2) in the ion source: 7 l/min and 300 ◦C, pressure of the nebulizergas (N2): 45 psi, capillary voltage: 3500 V, sheath gas flow and tem-perature: 11 l/min and 300 ◦C. Mass spectra were processed usingAgilent MassHunter B.02.00 software.

The HPLC-MS method was validated according to the ICHguideline Q2 (R1) [14]. Results of the validation studies were asfollows; calibration curves of B12 were linear in the concentra-tion range of 5–300 ng/ml (using 7 different concentration points,y = 410.23x + 13.2R2 = 0.9998). The samples were diluted in appro-priate manner if it was necessary. Limit of quantitation was2.5 ng/ml. Recovery was greater than 96%. Intra- and inter-dayrelative standard deviation (low, mid and high concentrationsof the standards in three parallel runs on the same day and onthree successive days, respectively) was less than 4.65 and 7.65%,respectively. Therefore HPLC method for B12 was determined to bereliable, linear, precise, accurate and selective.

2.5. Drug release profiles

To examine the release mechanism of active ingredient from theprepared cast films and wafers, the results were analyzed accordingto the following equation:

Mt

M∞= k · tn (1)

where Mt and M∞ are the cumulative amount of the released drugat time t and infinite time, k is a kinetic constant dependant onthe structural geometries of the drug–polymer system, n is thediffusional coefficient related to release mechanism [5,15,16]. Fornon-Fickian release, the n value falls between 0.5 and 1.0, whereasin the case of Fickian diffusion, n = 0.5; for zero-order release (caseII transport), n = 1, and for super case II transport, n > 1 [17]. Thekinetic parameters were determined using the least residual squaremethod and Microsoft Excel 2007 software.

2.6. Positron annihilation lifetime spectrometry (PALS)

For positron lifetime measurements a positron source made of

the active material was sealed between two Kapton® (polyimide)foils. The thin films were cut into 8 peaces and 4-4 layers wereplaced to both sides of the sealed positron source and covered with

B. Szabó et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 83– 87 85

aptceaetctdtpimatpowc

fEwCdb

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2

dDEp

Fig. 3. The k and the n parameters of the cast films. Black lines: without storage,red lines: after 4 weeks of storage. (For interpretation of the references to color infigure legend, the reader is referred to the web version of the article.)

Fig. 4. The k and the n parameters of the wafers. Blue lines: without storage, greenlines: after 4 weeks of storage. (For interpretation of the references to color in figure

Fig. 2. The scheme of the PALS measuring cycles.

luminum foil to protect the sample. The thicker wafers were pre-ared in the same way but only one layer was placed each side ofhe source. Lifetime spectra were measured with a fast-fast coin-idence system [18] based on BaF2/XP2020Q detectors and Ortec®

lectronics. Each spectrum was recorded in 4096 channels of annalyser card for 3600 s. Spectra contained about 106 coincidencevents in the case of cast films and a little less, about 8 × 105 inhe case of wafers. Fig. 2 illustrates the scheme of the measuringycles. The 22Na positron source decomposes to a positron, a neu-rino and a short-life 22Ne atom, which relax from its excited stateuring a high-energy photon emission. This high energy photon ishe start sign. The emitted positron annihilates with an electron androduces two photons with 512 keV energy. One of these photons

s the stop sign. If no stop sign in 4000 ps after the start sign, theeasuring cycle starts over. Three parallel spectra were measured

t each composition to increase the reliability. After summarizinghe parallels, spectra were evaluated by the RESOLUTION com-uter code [19], the indicated errors are the standard deviationsf the lifetime parameters obtained. Three lifetime componentsere found in all the samples and the longest lifetime component

onsidered as ortho-positronium (o-Ps) lifetime.A successful quantum mechanical model for the calculation of

ree volume hole sizes (Eq. (2)) has been developed by Tao andldrup [20,21]. The model postulates an electron layer at the poreall, with which the ortho-positronium can interact and decay.alculation of the overlap integral of the positronium probabilityensity function with this electron layer yields a direct relationetween positronium lifetime and the hole radius:

o-Ps = 1�

·[

1 − R

R + R0+ 1

2�· sin

(2�R

R + R0

)]−1(2)

here � is the annihilation rate of the Ps in the electron layer� = 2 ns−1), the R0 constant refers to the width of the electron layern the hole (R0 = 0.166 nm), the �o-Ps is the measured o-Ps lifetime,

is the calculated radius of the free hole.

.7. Microscopic visualization of the dissolution behaviour

The physical changes and disintegration of samples of 4.0 mm

iameter in 5 ml dissolution medium were investigated by USBigital Microscope (Digimicro Scale, Drahtlose Nachrichtentechnikntwicklungs- und Vertriebs GmbH; Dietzenbach, Germany). Thehotomicrographs were taken at predetermined time intervals. A

legend, the reader is referred to the web version of the article.)

standard micrometer scale was used for the calibration with themagnification set in a way that 1 pixel corresponded to 7.4 �m.

3. Results and discussion

The drug release exponents (n values) of different samples werebetween 0 and 0.7 regardless of the composition and the way ofpreparation. The smaller values of release exponent (n < 0.5) maybe due to drug diffusion partially through a swollen matrix andwater filled pores in the formulations. Drug release decreased withincreasing the concentration of CP. Along with the increase of CP therate of drug release (k) decreased as it can be seen in Figs. 3 and 4.The release rates were characterized based on the Korsmeyer &Peppas model by the time needed to 60% drug release (Fig. 5).Since CP is insoluble in the dissolution medium and its swellingbehaviour is attributed to the unchanged COOH group that gethydrated by forming hydrogen bonds in imbibing with water andtherefore extending polymer chains. The increased o-Ps lifetime

values and the consequent bigger free volume holes indicate theextended polymer chains after one month’s storage at elevatedconditions of relative humidity (Fig. 6).

86 B. Szabó et al. / Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 83– 87

Table 2Correlation coefficients between o-Ps lifetime and drug release time values as a function of the concentration of sodium alginate.

Sodium alginate (w/w%)

3 4.5 6.0

Film without storage 0.939 0.999 significant 0.464Film (4 weeks storage) 0.999 significant 0.998 significant −0.955Wafer without storage 0.959 0.586 0.987Wafer (4 weeks storage) −0.458 0.381 0.773

Fig. 5. Dissolution time request for 60% released drug (n = 3). Color codes: black:cst

rmoNr

Fig. 6. o-Ps lifetime values of the different samples (n = 3). Color codes: black: cast

ast film without storage, red: cast film after 4 weeks of storage, blue: wafer withouttorage, green: wafer after 4 weeks of storage. (For interpretation of the referenceso color in figure legend, the reader is referred to the web version of the article.)

It was observed that films containing CP exhibited slower drugelease indicating better matrix characteristics due to denser poly-

eric chains and improved mechanical properties. The presence

f CP both in the films and wafers ordered the SA chains [22].ot only the increase of CP content decreased the rate of drug

elease but also the freeze-drying process and the storage, as well.

Fig. 7. Microscopic photos of a freeze dried (up) and a cast unstored

film without storage, red: cast film after 4 weeks of storage, blue: wafer withoutstorage, green: wafer after 4 weeks of storage. (For interpretation of the referencesto color in figure legend, the reader is referred to the web version of the article.)

The freeze drying resulted in higher macroscopic porosity and big-ger specific surface area of the polymeric systems thus increasing

the thickness of the diffusion layer in contact with the dissolu-tion media thus forming a rate controlling barrier. Boateng et al.in 2010 [23] also observed difference between the rate of drugdissolution from the polymeric matrices of film and wafer dosage

films (down) in contact with 5 ml of the dissolution medium.

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B. Szabó et al. / Journal of Pharmaceut

orms which is correlated very well with results obtained fromwelling studies. The rates of swelling were significantly higheror wafers that can be attributed to the differences in the micro-tructures between the porous wafers and dense continuous films24]. The extent of swelling was different in the case of freeze driedamples compared to the cast films after one-month of storage.he freeze-drying process rearranged the sample structure [25].he latter resulted in less extent of swelling which is indicatedy the smaller o-Ps lifetime values and the consequent free vol-me holes. The freeze drying process resulted in lamellar structurend the formed sheets can be considered separate films. The big-er specific surface enabled increased water absorption. This effects more dominant from the point of microstructural view in thease of freeze dried samples than the concentration of Carbopol inhe examined range forming homogeneous films. The latter is con-rmed by the more similar o-Ps lifetime values of various freezeried samples (Fig. 6). Fig. 7 visualizes the behaviour of cast andreeze dried films in contact with the dissolution medium. Althoughn the case of freeze dried samples, erosion of the divided sheets cane observed in contrast to the cast films, but the n release exponentemained below 0.5 indicating that the rate-limiting mechanismf drug release is the diffusion from the individual sheets in thease of freeze dried samples, as well. The observed phenomenons in good compliance with the results of other workers [5,23] andhey stated that in this category of drug release kinetic, the domi-ant mechanism for drug transport is due to diffusion through theores of polymer and the polymer chains relax and disperse in theedia erosion occur which is known as erosion-controlled release

ystems. We have applied the Bivariate Correlation procedure toetermine the relationship between the variables. The Bivariateorrelations procedure computes Pearson’s correlation coefficientcovariate divided by the multiplication of standard deviations),ith their significance levels. The results confirmed our experimen-

al experiences and summarized in Table 2.

. Conlusions

The microstructural characterization of polymeric films basedn their free volume changes enables the design of delivery sys-ems of required drug release profile and stability. Useful predictionas obtained by PALS method concerning the effect of swellableolymer and storage on the diffusion-based drug release profile ofolymer films. Similar free volumes of the same polymer primarytructures referred to similar supramolecular structure and rate ofrug release while the macrostructural difference between filmsnd wafers modified the drug release mechanism.

unding

The work was supported by the Seventh Framework Program ofhe EU (grant number: TÁMOP-4.2.2/B-10/1-2010-0013).

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d Biomedical Analysis 89 (2014) 83– 87 87

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