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International Journal of Pharmaceutics 458 (2013) 99– 103

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

j o ur nal ho me page: www.elsev ier .com/ locate / i jpharm

olymer structure and antimicrobial activity ofolyvinylpyrrolidone-based iodine nanofibers prepared withigh-speed rotary spinning technique

stván Sebea, Barnabás Szabóa,b, Zsombor K. Nagyc, Dóra Szabód,ászló Zsidaie, Béla Kocsisd, Romána Zelkóa,∗

University Pharmacy Department of Pharmacy Administration, Semmelweis University, Hogyes Endre Street 7–9, Budapest H-1092, HungaryFormulation R&D, Gedeon Richter Plc., Gyömroi Street 19–21, Budapest H-1103, HungaryDepartment of Organic Chemistry and Technology, Budapest University of Technology and Economics, Muegyetem rkp. 3, Budapest H-1111, HungaryInstitute of Medical Microbiology, Semmelweis University, Nagyvárad Square 4, Budapest H-1089, HungaryFaculty of Mechanical Engineering, Szent István University, Páter Károly Street 1, Gödöllo H-2100, Hungary

r t i c l e i n f o

rticle history:eceived 7 September 2013ccepted 8 October 2013vailable online 18 October 2013

eywords:

a b s t r a c t

Poly(vinylpyrrolidone)/poly(vinylpyrrolidone-vinylacetate)/iodine nanofibers of different polymerratios were successfully prepared by a high-speed rotary spinning technique. The obtained fiber matswere subjected to detailed morphological analysis using an optical and scanning electron microscope(SEM), while the supramolecular structure of the samples was analyzed by positron annihilation life-time spectroscopy (PALS). The maximum dissolved iodine of the fiber samples was determined, and

igh-speed rotary spinningVP-iodineiber compositeositron annihilation lifetime spectroscopyPALS)

microbiological assay was carried out to test their effect on the bacterial growth. SEM images showedthat the polymer fibers were linear, homogenous, and contained no beads. The PALS results, both theo-positronium (o-Ps) lifetime values and distributions, revealed the changes of the free volume holesof fibers as a function of their composition and the presence of iodine. The micro- and macrostructuralcharacterisation of polymer fiber mats enabled the selection of the required composition from the point

woun

ntimicrobial activity of their applicability as a

. Introduction

During the last decade, the formulation of nanofibrous materi-ls loaded with different drugs for biomedical applications suchs dressing materials for wound treatment and for local cancerreatment has evoked considerable interest. To decontaminate theacteria invasion, biocides, silver (Chen and Chiang, 2010) iodineomplex (Ignatova et al., 2007) have been added to the electrospunanofibers. Antibacterial and anticancer agents can be easily incor-orated in fibers and their release profile can be controlled throughhanges in the fibers morphology, porosity, and composition. Thearge specific surface area of the materials, the possibility for grad-al release, and site-specific local delivery of the active compounds

ead to cytotoxicity decrease and enhancement of the therapeuticffect of the drugs (Ignatova et al., 2013).

Several methods have been developed to produce nanofibers,

uch as phase separation (Ma and Zhang, 1999), template (Ikegamet al., 2003), self-assembly (Hong et al., 2003), meltblowing (Ellisont al., 2007), electrospinning (Doshi and Reneker, 1995; Reneker

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

378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijpharm.2013.10.011

d dressing.© 2013 Elsevier B.V. All rights reserved.

and Yarin, 2008), and forcespinning (Lozano and Sarkar, 2009;Sarkar et al., 2010; McEachin and Lozano, 2012). From the availablemethods, electrospinning is the most widely used and promisingmethod of producing fine fibers. Although a simple and laboratoryefficient method for fiber production, the electrospinning methodposes many disadvantages including the use of a high-voltagepower source (>10 kV), limited to solvents within a certain rangeof dielectric constant, low fiber yield, and wide diameter distribu-tion (McEachin and Lozano, 2012; Valipouri et al., 2013). In orderto increase the yield Wang et al. (2008) developed a self-bundlingelectrospinning method for generating continuous aligned electro-spun fiber yarn.

Sarkar et al. (2010) developed a new method calledForcespinningTM to increase nanofiber production rate whilealso providing a broader opportunity of the type of materialsthat could be spun into nanofibers. The electrostatic force neededto create nanofibers in the electrospinning process is replacedby centrifugal forces (Sarkar et al., 2010). This method enablesthe application of polymer solutions as well as solid materials

through melt spinning to create fibers. The conductivity and/orelectrostatic charges are not relevant parameters for the solutionproperties to create fibers; therefore, the spectrum of materialsto be spun when compared with electrospinning is considerably

100 I. Sebe et al. / International Journal of Pharmaceutics 458 (2013) 99– 103

Table 1The concentration and ratio of different types of PVP hydrogels.

ID Concentration (% w/w) Ratio of PVP25:PVP 64

Total concentrationof PVP (% w/w)

Concentration of PVP–iodinecomplex (% w/w)

PVP 25 PVP 64

I 0 57 0:1 57 2.9II 18 36 1:2 54 3.1

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IV 38 19 2:1

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roadened. Controllable parameters include the rotational speedf the spinneret, the design of the collection system, and the shapend size of the needles or nozzles. Several materials have beenuccessfully forcespun in the nanometer range such as polyamide,olyethylene oxide, polytetrafluoroethylene, polylactic acid, poly-aprolactone, indium tin oxide and titanium dioxide nanowiresVazquez et al., 2012).

Poly(vinylpyrrolidone) (PVP) is a synthetic polymer with goodiocompatibility. PVP bound iodine (PVP-I) was developed by NASAor the Apollo program. PVP-I is beneficial on burn wounds due tots effect on the reduction of bacterial colony (Uslu and Aytimur,012). It was reported that PVP–I complex gradually releasedctive iodine. Because of the broad-spectrum microbicidal activityf iodine, electrospun PVP-I nanofibers had external antibacte-ial, antimycotic, and antiviral applications (Fang et al., 2011). Theighly porous mat structure and well interconnected pores are par-icularly important for exuding fluid from the wound; the smallores and very high specific surface area not only inhibit the exoge-ous microorganism invasions, but also assist the control of fluidrainage.

The purpose of the present study was to prepare PVP 25/PVP4/iodine fiber mat by high- speed rotary spinning technique ando characterize the micro- and macrostructural properties of therepared fiber mat. Another aim was to find a correlation betweenheir supramolecular structure and antimicrobial activity.

. Experimental

.1. Materials

Commercial product Braunol (aqueous solution of PVP–iodineomplex as the active substance, B. Braun Medical AG,witzerland) was used. Fig. 1 illustrates the chemical struc-ure of povidone–iodine complex. Excipients were PVP 25poly(vinylpyrrolidone), Kollidon 25, BASF, Germany), PVP 64poly(vinylpyrrolidone-vinylacetate), Kollidon 64 VA, BASF,ermany), Ph.Eur. (96% ethanol). The Kollidon’s were kind gifts

rom BASF Ltd.

.2. Sample preparation

Hydrogels were prepared with polyvinyl-pyrrolidones (PVP) ofifferent molecular weights. Samples were regularly stirred untilhe complete homogenization at room temperature. The ratio andoncentrations of the applied PVPs are listed in Table 1. The aqueous

Fig. 1. Chemical structure of povidon–iodine complex.

56 2.957 2.960 2.7

medium of the hydrogels was Braunol which contains surfactant (inamount up to 6.8%), NaH2PO4, NaIO3, and NaOH in addition to thePVP–iodine complex (7.5%).

2.3. Viscosity measurement

The viscosity of the hydrogels was measured by Kinexus ProRheometer (Malvern, Worchestershire, UK) in parallel plate config-uration. The upper moved and rotated disc was a 40-mm diameterstainless steel plate. The lower portion was a stainless steel Peltierplate and the applied gap between the plates was 0.15 mm. The pro-cess was carried out at 25 ± 0.1 ◦C and this temperature was kept byPeltier thermoelement. The results are the average of 10 parallels.

2.4. High speed rotary spinning technique

The fiber formation was carried out with a WSE 602M (AEG,Germany) motor based on a high-speed rotary spinning technique.The rotating reservoir was a polyamide-copper spinneret with80 ml internal volume. The various fibers made from the PVP hydro-gels at 3500 RPM with about 1 g/min production rate. The rotatingspeed was controlled with a toroidal transformator and measuredwith laser revolution counter (DT-10L, Voltcraft, Germany). Theinternal diameter of the stainless steel nozzles was 0.5 mm.

2.5. Modeling of the mechanical load

The mechanical stress of the designed spinneret was analyzedby Ansys v12.1 finite element modeling software. In the case ofthe stainless steel nozzles and the copper cover used the appliedmaterials for the analysis, in the case of polyamide the fragilepolyethylene was chosen from the database of the software. Forthe analysis the spinneret was composed by 9946 tetrahedron ele-ments and 19,520 nodes. The highest equivalent stress values atthe maximal rotation speed (11,000 RPM, 1152 rad/s with 0.2 mmeccentricity) and the yield strength of the materials are in Table 2.

2.6. Microscopic visualization by scanning electron microscopy(SEM) and optical microscope with digital enlargement

Morphology and average diameter of the samples were investi-gated by a JEOL 6380LVa (JEOL, Tokyo, Japan) type scanning electronmicroscope. Parts of samples were fixed by conductive carbon

adhesive tape. The adjusted accelerating high voltage and workingdistance were 15 and 20 kV and 10 mm and 12 mm, respectively.The fiber mats were the subjects of morphological examination inorder to select the individual filaments from the intact contiguous

Table 2The equivalent stress values and the yield strength of the applied materials.

Part of the spinneret Max. stress (MPa) Yield strength (MPa)

Stainless steel nozzles 47.5 240Copper cover 77.7 130Polymer base 5.6 26–33

I. Sebe et al. / International Journal of Pharmaceutics 458 (2013) 99– 103 101

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ig. 2. Morphology of the forcespun fibers containing iodine (left: SEM photo, mahoto, magnification: 50×).

at domains. The average diameters of fibers were determinedrom 10 different randomly selected filaments. The visual morphol-gy of fiber tissues was also investigated by USB Digital MicroscopeDigimicro Scale, Drahtlose Nachrichtentechnik Entwicklung- undertriebs GmbH; Germany).

.7. Positron annihilation lifetime spectroscopy (PALS)

For positron lifetime measurements, a positron source made ofarrier-free 22NaCl was used. Its activity was around 105 Bq, and thective material was sealed between two very thin Ti foils. Lifetimepectra were measured with a fast-fast coincidence system basedn BaF2/XP2020Q detectors and Ortec® electronics. Every spectrumas recorded in 4096 channels of an analyzer card for 3600 s and

ach contained about 1.5 × 106 coincidence events. Three parallelpectra were measured at each concentration to increase reliabil-ty. After summarizing the parallels, spectra were evaluated by theESOLUTION computer code (Kirkegaard et al., 1981); the indi-ated errors are the deviations of the lifetime parameters obtained.hree lifetime components were found in all the samples. The MELTode (Shukla et al., 1993) was used to extract lifetime distributionsrom the spectra. These latter evaluations were used to characterizehe size distribution of free volume holes in the samples throughrtho-positronium (o-Ps) lifetime.

.8. Determination of iodine content

The measurements were carried out in a quartz cuvetteHELLMA J100-Q, Jena, Germany) in 96% w/w ethanol. The alcoholic

edium was selected to increase the stability of the iodine com-lex in the course of the measurement. Miniature indifferent wireas placed into the cuvette in order to stir the solution by a mag-etic stirrer (Yellow Line MSH Basic, IKA, USA) using the highest

evel of stirring at 25 ◦C. The samples were investigated from differ-nt compositions of PVP-fibers with model drug. The drug contentsere analyzed by Agilent 8453 UV–VIS (Agilent, USA) spectropho-

ometer at a 291-nm wavelength. The measuring time was 4 minn average until the absorbance values leveled out to a constantalue. The latter was considered as a maximum absorbance whichas applied for the determination of the maximum iodine concen-

ration of the samples.

.9. Tensile strength measurements of fiber mats

The tensile strength tests of the randomly oriented fiber matsere executed on a universal material testing instrument type

wick Z005 (Zwick Roell GmbH, Germany) at 5 mm/min speed and5 mm clamping length using 15 N upper force limits at room tem-erature. The following equation was applied for the determinationf the tensile strength values

(Pa; N/m2) = Fmax(N) · � (kg/m3)TEX (kg/m)

(1)

ation: 55×; center: SEM photo, magnification: 2000×; right: Optical microscopic

where Fmax is the maximum force, � is the average density of thefibers, TEX is the linear density which equals to W/L, where W is themass of the fibers, and L is the length of fiber (Andrady, 2008).

2.10. Antimicrobial susceptibility testing

For the antimicrobial susceptibility tests the following bacterialstrains were used: Bacillus subtilis ATCC 6633, Staphylococcus aureusATCC 29923, Streptococcus pyogenes ATCC 30013, Escherichia coliATCC 25922, Pseudomonas aeruginosa ATCC 27853. Long-term stor-age of bacterial strains was performed at −70 ◦C in a brain–heartinfusion (BHI) broth with 20% glycerol.

The antimicrobial susceptibility tests were performed usingconfluent growth of each bacteria (0.5 McFarland) on Mueller-Hinton (MH) agar or MH agar supplemented with 5% sheep blood(Becton Dickinson, Sparks, MD) (for S. aureus and S. pyogenes), andincubated at 37 ◦C overnight (CLSI2005).

For antimicrobial testing the following fiber discs were used: (i)discs with polymers S-I, S-II, S-III, S-IV, S-V alone; (ii) discs withpolymers containing iodine S-IB, S-IIB, S-IIIB, S-IVB, S-VB.

For antimicrobial susceptibility testing the following hydrogelswere also used: (iii) hydrogels with polymers S-I, S-II, S-III, S-IV, S-V alone; (iv) hydrogels with polymers containing iodine S-IB, S-IIB,S-IIIB, S-IVB, S-VB.

Discs (diameter =13 mm, average width = 1 mm) were preparedfrom the forcespun fibers of amorphous glassy polymers withSpecac type pneumatic press at 1000 N compression force. Glassy-to-rubbery transition can be visually observed due to the swellingof discs in contact with the agar gel.

2.10.1. Kinetic assayThe in vitro bactericidal activity of the fiber discs – S-IB and S-VB

– was determined for S. pyogenes ATCC 30013 using the time-killmethods (CLSI, 2005).

The initial concentrations of S. pyogenes ATCC 30013 was 7.602log10 colony-forming units (CFU)/ml. The concentrations of thepolymers were the following S-IB: 21.38 mg/ml S-VB: 21.5 mg/ml.

The viable bacterial counts were determined after 1, 3, 6, 12 and24 h following the incubation with polymers IB and Vb. Dilutionswere used to minimize the carryover effect of antimicrobial agent.The amount of 0.1 ml was subcultured on agar plates and incubatedat 37 ◦C for 24 h for the CFU determination.

2.11. Fiber tissue preparation

The collected and untangled fibers were laid on a stainless steelplate covered by Teflon® and they were pressed by the same plate.Thus, the evolved membrane was layered on each other across tobecome raster structure. The size of the area was 20 mm2.

3. Results and discussion

Fig. 2 illustrates the scanning electron microscopic photo ofthe fiber web and also represents the fiber mat structure of the

102 I. Sebe et al. / International Journal of Pharmaceutics 458 (2013) 99– 103

Table 3Summary of the average diameter and the maximum dissolved iodine of different fiber samples.

ID Ratio of PVP 25:PVP 64 Average diameter of the fibers (�m) Maximum I2 concentration (% w/w)

Without iodine Containing iodine (B) Theoretical Measured

S-I 0:1 2.49 ± 0.18 4.34 ± 0.11 4.8 2.7 ± 0.04S-II 1:2 6.13 ± 0.10 5.70 ± 0.20 5.4 3.5 ± 0.07S-III 1:1 6.04 ± 0.21 6.21 ± 0.08 5.0 2.2 ± 0.12S-IV 2:1 5.96 ± 0.03 7.67 ± 0.17 4.8 2.1 ± 0.14

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with their available iodine content, thus the method eliminatedthe effect of diffusion. The antimicrobial effect of fibers was in goodagreement with their available iodine contents determined by theirsupramolecular structure in case of the representative S. pyogenes.

Fig. 3. (a) The o-Ps lifetime values of the fibers (black straight line: fibers without

S-V 1:0 6.42 ± 0.14

otential wound dressing. The average diameters of the emptybers are similar in the case of S-II–S-V but the PVP 25 (S-I) showed

significantly lower value. The possible reason of this phenomenonould be that the presence of vinylacetate ordered the polymerhains via intermolecular hydrogen bonding thus decreasing thentermolecular distance within the polymer chains resulting inenser and thinner fibers. The presence of iodine increased theber diameter in the case of S-IB and S-IVB. Table 3 summa-izes the average diameter and the maximum dissolved iodinef different fiber samples. The differences between the maximumodine concentrations could be partially explained by the differenttructure-dependent phase separation in the rotating device duringhe fiber formation and the iodine sublimation from the obtainedbers. The higher the viscosity of the hydrogels, the less the iodineoncentration of the resultant fibers.

Fig. 3a illustrates the o-Ps lifetime values of the different emptyber mats and the same samples containing iodine while Fig. 3bhows the lifetime distributions of the corresponding samples.

The results indicate that the presence of vinylacetate increasedhe o-Ps lifetime values, consequently the free volume holes ofhe fibers and ordered the PVP chains that was confirmed by theecreased width of the o-Ps lifetime distribution curves. The nar-ower the distribution curves the more uniform the hole sizes of theolymer samples. The presence of iodine significantly decreasedhe o-Ps lifetime values of the samples since the iodine filled inhe free volume holes of polymers (Ramani and Ranganathaiah,001). The viscosity changes of the samples are in good agree-ent with the results of the microstructural characterization based

n the o-Ps lifetime values. In the presence of iodine the viscosityncreased indicating the filled in free volume holes of the polymers

hich is the most noticeable in the case of S-VB. In the latter casehe iodine decreased the free volume of S-V in the largest extent.he tensile strength values followed the tendency of the viscosityhanges. The mechanical resistance of the mono-component sam-les is higher than that of the composites containing two types ofolymers in different ratios. The presence of the second componentaused the inhomogeneity of the samples thus creating a looser andess homogeneous fiber structure of lower tensile strength. Notnly the second polymer component, but the presence of iodinelso decreased the mechanical strength of the fibers. The mini-um size of the free volume holes is nearly identical to the radius

f iodine atom size (140 pm); thus, the iodine is able to fill in theree volume holes of the polymers bigger than its molecular size.his phenomenon resulted in the drastic reduction of the tensiletrength of fibers.

The size of inhibition zone was a measure of the compound’sffectiveness. The samples were showed significant growth inhi-ition for the examined bacterial strains for each fiber sample.he fiber discs hydrated on contact with the agar–agar gel thatlays an important role in controlling the rate of iodine diffusion;

hus, the concentration of iodine at the border of the hydrated rub-ery type polymeric disc front determines its further diffusion inhe agar gel. Since the change of the free iodine diffusion of theamples in the agar gel depends on its initial concentration at the

6.08 ± 0.15 4.3 0.6 ± 0.07

border of the swollen hydrogel zone, the effective iodine concen-tration primarily depends on the structure of polymeric fiber discs.The diatomic elemental form of iodine (free iodine) can be con-sidered responsible for the antimicrobial activity. The inhibitionzones were calculated as an elliptic surface and its extent corre-lated well with the maximum concentration of dissolved iodine(Fig. 4). Fig. 5 represents the results of the time kill assay of the twodifferent monocomponent fibers (S-IB, S-VB) in comparison withthe control sample. While the changes of the sizes of the inhibitionzones were strongly influenced by the diffusion characteristics ofthe fiber discs, the kinetic profile of dissolved fibers was correlated

iodine, red dotted line: fibers with iodine). (a) Positron annihilation lifetime dis-tribution curves of the fibers (colors: black: S-I, blue:S-II, cyan: S-III, green: S-IV,red: S-V; straight lines: fibers without iodine, dotted lines: fibers with iodine). (Forinterpretation of the references to color in this figure legend, the reader is referredto the web version of this article.)

I. Sebe et al. / International Journal of Ph

Fig. 4. Maximum concentration of the dissolved iodine (black) and the inhibitionzones of different fiber discs (blue) in the case of S. pyogenes ATCC 30013. (For inter-pretation of the references to color in this figure legend, the reader is referred to theweb version of this article.)

Fig. 5. Kinetic profile of S. pyogenes ATCC 30013 in the presence of S-I and S-V(it

4

acttcssa

polyvinylidene fluoride nanofibrous membranes by ForcespinningTM. Polym.Eng. Sci. 52, 2260–2265.

Wang, X., Zhang, K., Zhu, M., Yu, H., Zhou, Z., Chen, Y., Hsiao, B.S., 2008. Continu-

colors: blue: S-IB, orange: S-VB, dark cyan: control is S. pyogenes ATCC 30013). (Fornterpretation of the references to color in this figure legend, the reader is referredo the web version of this article.)

. Conclusions

The high-speed rotary spinning technique was successfullypplied for the preparation of polymer fibers containing the bio-ide iodine. Positron lifetime distributions revealed the changes ofhe free volume of fibers as a function of their composition andhe presence of iodine. The supramolecular structure of the fibers

ould be changed by modifying the ratio and the type of the con-isting polymers. This change of the fiber composition allows theelection of fiber composites containing iodine of required physicalttributes and antibacterial activity.

armaceutics 458 (2013) 99– 103 103

Acknowledgments

The authors are grateful to the BASF Ltd. (Ludwigshafen,Germany) for the gifted Kollidon samples. The work described inthis paper was supported by the Seventh Framework Program ofthe EU, TÁMOP-4.2.2/B-10/1-2010-0013.

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