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International Journal of Pharmaceutics 472 (2014) 282287

Contents lists available at ScienceDirect

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(0.3 mg/ml). HC displayed AL-type phase-solubility diagrams in aqueous solutions containing eitherSBEbCD or CS, although CS had negligible solubilizing effect. The ability of the NPs to encapsulate HCdecreased with increasing CS concentration during preparation of the NPs even though the SBEbCDcontent of the NPs increased with increasing CS concentration. This decrease in HC encapsulation wasrelated to the concentration; the ionic crosslinking provides better encapsulation at low initial SBEbCDand CS concentrations.

2014 Elsevier B.V. All rights reserved.

1. Introduction

Cyclodextrins (CDs) are cyclic oligosaccharides that typicallyconsist of 68 a-glucopyranose units. CDs have toroid structurewith hydrophobic outer surface and relatively lipophilic centralcavity. In the past few decades they have become importantpharmaceutical excipients, especially due to their ability toincrease aqueous solubility of various poorly soluble lipophilicdrugs (Brewster and Loftsson, 2007; Frmming and Szejtli, 1994;Loftsson and Duchene, 2007). CDs tend to self-associate to formnano- and micro-sized aggregates (diameter ranging from 1 to1000 nm) (Gonzlez-Gaitano et al., 2002; Pusks et al., 2013;Szente et al., 1998). The CD self-association is enhanced byformation of inclusion complexes with hydrophobic drug mole-cules (Jansook et al., 2010a; Loftsson et al., 2002; Messner et al.,2010). Although these self-associates are generally not stable anddissociate upon dilution (Flp et al., 2013; Messner et al., 2011b),

they can be utilized as nano-micro drug vehicles where mediadilution is negligible, like in topical drug administration to the eyeand skin (Jansook et al., 2010b; Kurkov and Loftsson, 2013; Loftssonand Brewster, 1996, 2010). Stable self-assembled CD nanoparticlescan also be formed, and various stabilizing methods do exist (Flpet al., 2012).

Chitosan (CS) is a linear polysaccharide consisting of randomlydistributed b-(1-4)-linked D-glucosamine and its N-acetylatedderivative. CS is produced by deacetylation of chitin, the maincomponent of arthropod exoskeletons such as that of shrimps andinsects. CS has for a long time been used for various medicalpurposes, including drug delivery as CS micro- and nanoparticles(Felt et al., 1998; Rampino et al., 2013). CS nanoparticles can beprepared by a number of techniques but one of the most promisingone is ionotropic gelation which is based on ionic interactionsbetween the polycationic chitosan and a polyanionic polymer,usually sodium tripolyphosphate (TPP) (Calvo et al., 1997). CSnanoparticles can also be formed by ionotropic gelation throughincorporation of polyanionic sources other than TPP. These anionsources can be different types of CDs, for example sulfobutyletherb-cyclodextrin (SBEbCD) or carboxymethyl-b-cyclodextrin(CMbCD) (Ammar et al., 2011; Krauland and Alonso, 2007;Mahmoud et al., 2011; Teijeiro-Osorio et al., 2009a,b; Trapaniet al., 2008, 2010). In some cases nonionic CDs are used togetherwith TPP to form CS nanoparticles (Jingou et al., 2011).

Abbreviations: CD, cyclodextrin; CS, chitosan; HC, hydrocortisone; SBEbCD,sulphobutylether-b-cyclodextrin sodium salt; HC/SBEbCD, HCSBEbCD inclusioncomplex; NP, CSSBEbCD nano- and microparticle.* Corresponding author. Tel.: +354 525 4464; fax: +354 525 4071.E-mail addresses: zof1@hi.is (Z. Flp), phs3@hi.is (P. Saokham), thorstlo@hi.is

(T. Loftsson).

http://dx.doi.org/10.1016/j.ijpharm.2014.06.0390378-5173/ 2014 Elsevier B.V. All rights reserved.Sulfobutylether-b-cyclodextrin/chitosantheir physicochemical characteristics

Zoltn Flp, Phennapha Saokham, Thorsteinn LoftFaculty of Pharmaceutical Sciences, University of Iceland, Hofsvallagata 53, Reykjavk 107, I

A R T I C L E I N F O

Article history:Received 27 March 2014Received in revised form 20 June 2014Accepted 21 June 2014Available online 24 June 2014

Keywords:Cyclodextrin

A B S T R A C T

Metastable polymer/cyclodweight chitosan (CS), Mw apolysaccharide containing b-cyclodextrin derivative wmolecule. Ionotropic gelatiolow molecular weight catiodiameter of the NPs rangedratio during NP preparationano- and microparticles and

on *and

trin nano- and microparticles (NPs) were prepared from low molecularut 10 kDa, and sulfobutylether b-cyclodextrin (SBEbCD). CS is a cationicumerous protonated amino groups (pKa about 6.5). SBEbCD is ah six to seven negatively charged sulfobutyl ether groups per cyclodextrin

technique was used to prepare the NPs. The NP matrix was composed ofic CS polysaccharide cross-linked with polyvalent anions (SBEbCD). Theom 200 to almost 1000 nm and was controlled by the CS:SBEbCD molarHydrocortisone (HC) is a lipophilic drug with limited aqueous solubility

l of Pharmaceutics

evier .com/ loca te / i jpharm

K1:1 slopeS01 slope (1)

CE slope1 slope

D=CD complexCD

K1:1 S0 (2)

where S0 is the intrinsic solubility of the drug.

2.4. Preparation of the nanoparticles

SBEbCD/CS nano- and microparticles (NPs) were preparedaccording to the ionotropic gelation method (Mahmoud et al.,2011), by mixing SBEbCD and CS solutions of equal weightconcentrations (0.25, 0.5, 1.0, 2.0 w/v%). SBEbCD was dissolved inwater and CS in pH 4 acetic acid. The volume ratio of the twosolutions was determined by titration. Thus, SBEbCD solution in a

Chitosan concentration CS/SBE-b-CD volume ratio

0.15 1.5/10.33 2/10.69 2.25/11.47 2.75/1

Z. Flp et al. / International Journal of Pharmaceutics 472 (2014) 282287 2832. Materials and methods

2.1. Materials

Sulfobutylether b-cyclodextrin sodium salt (SBEbCD) withmolar substitution of 0.9, molecular weight (Mw) 2163 Da, wasobtained from CyDex Pharmaceuticals (Lawrence, KS, USA),chitosan mesylate salt (CS, 95% deacetylation degree, Mw 10kDa) was prepared as described in the following references:(Rnarsson et al., 2008; Song et al., 2010). Hydrocortisone (HC)was purchased from Fagron (Nieuwerkerk aan den Ussel, TheNetherlands). Methanol and tetrahydrofuran were purchased fromSigmaAldrich (St. Louis, MO, USA). All the solutions and themobile phase for HPLC measurement were prepared using Milli-Qwater (Millipore, Billerica, MA, USA).

2.2. Quantitative determination of hydrocortisone concentration

Quantitative determination of hydrocortisone was performedon a reversed-phase high performance liquid chromatographic(HPLC) system from Dionex Softron GmbH (Germany) Ultimate3000 series consisting of a LPG-3400A pump with a built-indegasser, a WPS-3000-TSL autosampler column compartment, aVWD-3100 variable wavelength UVvis detector and PhenomenexLuna C18 150 mm 4.60 mm, 5 micron column (Phenomenex, UK)with a matching HPLC KrudKatcher Ultra Column in-line lter(Phenomenex, UK). The mobile phase consisted of methanol, waterand tetrahydrofuran 70:29:1 (volume ratios). The ow rate was1.0 ml/min, and the retention time was 3.1 min.

2.3. Phase-solubility

Saturated solutions of HC were prepared in water, as well as, inaqueous SBEbCD solutions and the solubility was determined bythe heating method (Loftsson and Hreinsdttir, 2006). Excessamount of hydrocortisone was added to aqueous solutioncontaining from 0 to 10% (w/v) SBEbCD and the suspension wassonicated at 55 5 C for 60 min in sealed glass vials. Thesuspension was allowed to equilibrate at room temperature for7 days under constant agitation (KS 15 A shaker, EB Edmund BhlerGMbH, Germany). Preliminary experiments showed that 7 days aremore than enough time to reach solubility equilibrium. Afterequilibrium, the suspension was ltered through a 0.45 mm RCmembrane lter (Whatman, Germany), the ltrate diluted with

Table 1Final concentrations of SBEbCD and CS solution and ratios.

CS and SBEbCD initialconcentration (w/v%)

SBEbCD concentration

(mM)

0.25 0.46 0.5 0.77 1.0 1.42 2.0 2.47 mobile phase and analyzed by HPLC.To determine phase-solubility of HC in aqueous SBEbCS

solution, HC saturated 010% (w/v) CS solutions were preparedas described above and the amount of dissolved HC wasdetermined by HPLC. Phase-solubility proles were determinedaccording to the method of Higuchi and Connors (Higuchi andConnors, 1965). The apparent stability constant (K1:1) and thecomplexation efciency (CE) were determined from the slope ofthe linear phase-solubility diagrams (the total drug solubilityversus total SBEbCD or chitosan concentration in millimoles unit)(Loftsson et al., 2005):syringe was gradually added to a stirred (250 rpm) CS solution atroom temperature until the clear solution turned opalescent. Thedetermined volume ratios and concentrations are shown in Table 1.During following preparations of identical composition the twosolutions were mixed in the determine volume ratios and thenstirred for 60 min. Thus, prepared SBEbCD/CS NPs are depicted inFig. 1.

The HC loaded NPs were obtained by rst preparing the HC/SBEbCD inclusion complexes (as described in Section 2.2) and thenmixing the aqueous complex solution with a CS solution under thesame conditions as previously described for preparation of theunloaded particles. Every sample was prepared in a triplicate.Fig. 1. Schematic depiction of the ionic interactions between SBEbCD and CS in thenano- and microparticles.

rst or higher order with respect to HC but rst-order with respectto SBEbCD and CS (Higuchi and Connors,1965). The proles (Fig. 2)show that SBEbCD solubilized HC in aqueous solutions, while CS hasinsignicant effect on the HC solubility in the observed concentra-tion range (05 mM). The intrinsic solubility (S0), the apparentstability constants (K1:1) and the observed complexation efcien-cies (CE) are displayed in Table 2. The CE of the HC/SBEbCD complexindicates that the HC:SBEbCD molar ratio is 2:3 in aqueous SBEbCDsolutions saturated with HC, i.e. that two out of every three SBEbCDmolecules are forming a complex with HC. In general the CE of CDs isbelow unity and, thus, the observed CE of SBEbCD is very high

Table 2The constants obtained from the HC phase-solubility study in aqueous SBEbCD andchitosan solutions.

Parameter HC/SBEbCD HC/chitosan

Intrinsic solubility (S0)(mM) 0.71Stability constant (K1:1)(M1) 3100 49Complexation efciency (CE) 2.2 0.035

284 Z. Flp et al. / International Journal of Pharmaceutics 472 (2014) 2822872.5. Permeation studies

Drug permeation studies from solutions containing saturatedHC/SBEbCD complexes (donor phase, 2 ml), or HC saturated HC/SBEbCD/CS particles, were carried out in unjacketed Franzdiffusion cells with diffusion area of 1.77 cm2 (SES GmbH Analysesysteme, Germany). The receptor phase (12 ml) consistedof pH 7.4 phosphate buffered saline (PBS) containing 2% (w/v) gCD.The donor and receptor compartments were separated by a singlelayer semi-permeable cellulose ester membrane (Biotech CE,Spectrum Europe, Breda, NL), with molecular-weight-cutoff(MWCO) of 810 kDa, which had been soaked in the receptorphase overnight. The MWCO of the membrane was chosen to belarger than the Mw of the HC/SBEbCD complex (2.5 kDa) and, thus,the free complex was able to permeate the membrane. The studywas carried out at room temperature under constant stirring(300 rpm). Samples were taken at 2.0, 2.5, 3.0, 4.0 and 4.5 h, andreplaced immediately with equal amount of pure receptor phase.The ux (J) was calculated from the linear part of the permeationprole and the apparent permeation coefcient (Papp) wascalculated by the following equation:

J dq=dtA

Papp Cd (3)

where dq/dt is the slope (in mg/h) of HC permeated through themembrane over time, A is the diffusion area (1.77 cm2) and Cd is thetotal drug concentration in the donor phase.

The initial HC ux from pure HC-saturated water (J0) wassubtracted from the determined HC ux values of the saturatedHC/SBEbCD complexes. In the case of the NPs, which are non-saturated HC solutions, the ux values were likewise adjusted bysubtracting J0 from the determined values accounting for the drugdilution according to Ficks rst law of diffusion.

2.6. Encapsulation efciency

The encapsulation efciency (EE) of HC in the NPs wascalculated by an indirect method. Nanoparticles were isolatedby centrifugation (10,000 min1, 90 min) and dissolved HC in thesupernatant quantied by HPLC. The EE in % was calculated asfollows:

EE Total amount of drug Amount of unbound drugTotal amount of drug

100 (4)

2.7. Particle size measurement

The mean hydrodynamic diameter of the nanoparticles andpolydispersity index was determined by dynamic light scattering(DLS) using Nanotrac Wave from Microtrac, USA. Each sample weremeasured 3 times for 60 s at 25 C and the reported values are themean values standard deviation (SD).

2.8. Determination of SBEbCD incorporation in the particles

SBEbCD was quantied by spectrophotometric analysis offading phenolphthalein alkaline solution (Monza da Silveira et al.,1998). Briey, sample and standard SBEbCD solution were dilutedwith aqueous pH 10.0 borate buffer solution containing 40 mMphenolphthalein. The absorbance at 550 nm was measured byPerkinElmer Lambda 35 UV/vis spectrometer, USA. The standardcurve was linear for SBEbCD concentrations ranging from 1 to750 mg/ml. Nano- and microparticles were isolated by centrifuga-tion (10,000 min1, 90 min) and free SBEbCD in the supernatantwas quantied. The incorporation efciency (IE) of SBEbCD in %was calculated as follows:

IE Total amount of CD Amount of unbound CDTotal amount of CD

100 (5)

2.9. In-vitro release studies

In-vitro release studies were performed as described inSection 2.5 using Franz diffusion cells and 810 kDa MWCOmembrane. Samples were collected at 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 10and 12 h. The results are represented as the mass percentage of HCthat had permeated at given time point assuming 100% permeationat innite time (t = 1), i.e. at t = 1 the HC concentration in thedonor and receptor phases are identical.

The release kinetics of HC were described via mathematicalmodels such as the zero-order, rst-order, Higuchi, and Kors-meyer-Peppas models using regression analysis (Dash et al., 2010).

3. Results and discussions

3.1. Phase-solubility

The phase-solubility studies were performed to investigate theinteraction of HC with both SBEbCD and CS. As expected, the phase-solubility proles were of AL-type indicating that the complex was

Fig. 2. Phase-solubility of HC with SBEbCD (&) and chitosan (^).

(Loftsson and Brewster, 2012). On the other hand, the HC:CS molarratio is 1:30 in aqueous CS solutions saturated with HC and, thus,only one out of every 30 CS molecules is forming a complex with HC.Furthermore, the Mw of CS is about 5 times greater than that ofSBEbCD showing that on a weight basis CS has even much less effecton the HC solubility than SBEbCD. Thus, the direct CS solubilizationof HC in aqueous solutions containing both SBEbCD and CS can forall practical purposes be ignored.

3.2. Permeability studies

The resultof permeabilitystudies(Fig.3)shows that theuxofHCfrom the SBEbCD complexes is higher than the HC ux from theloaded NPs at identical SBEbCD comcertration. Although SBEbCDhas lower tendency to self-aggregate than other bCD derivatives itdoes form aggregates that are unable to permeate the semi-permeable membranes with MWCO 810 kDa. According to Eq. (3)there should be a linear relationship between J and the SBEbCDconcentration and the negative deviation from linearity has beenshown to be due to self-association of the complexes to formnanoparticles (Messner et al., 2011a). However, inpresence of CStheNP formation is enhanched due to ionpair formation between theanionic C/SBEbCD complexes and cationi CS polymer resulting information of relatively stable NP that are unable to permeate the

membrane (Fig. 1). HC/SBEbCD complexes form metastable nano-particles in aqueous solution that dissociate upon media dilution.

3.3. Hydrocortisone encapsulation efciency

Fig. 4a shows decreasing HC encapsulation, i.e. HC loading, withincreasing CS concentration. This is most probably due to changesin the size and composition of the NPs. Although the SBEbCDcontent of the NP increases with increasing CS concentration theirsizes become larger making SBEbCD less accessible especially atthe core of the NPs. The larger NPs release the drug more easilythan the smaller ones and the smaller NPs tend to retain their drugcontent better.

3.4. Particle size and polydispersity index

As shown in Fig. 4b, particle size of the loaded and unloadedNPs are in all cases less than 1000 nm (1 mm). NP solutions of all

Z. Flp et al. / International Journal of Pharmaceutics 472 (2014) 282287 285Fig. 3. Permeation proles of HC/SBEbCD (&) and HC loaded NPs (^); (a) aqueoussolutions containing either HC/SBEbCD complexes or the HC loaded NPs; (b)normalized ux diagram, the ux of free HC molecules through the membrane wassubtracted.Fig. 4. NP properties at different concentrations (a) encapsulation of HC in the NPs;(b) particle size of the unloaded (black) and loaded (grey) NPs; (c) incorporationefciency of SBEbCD in the NPs; CS and SBEbCD volume ratios were 1.5/1, 2/1, 2.25/1, 2.75/1, respectively.

mixing ratios were monodisperse and the polydispersity index(PDI, calculated by the DLS software from Microtrac, USA) waslower than 1 indicating the uniformity of particle size. Increasingthe concentration of the components leads to particle size increase.At low CS and SBEbCD concentrations, the particle size of the HCloaded NPs was signicantly greater than that of the unloaded NPs.Presence of HC affects the NP size in this concentration range. Theparticle size did increase with increasing CS concentration but thesize difference between unloaded and loaded NPs decreased withincreasing CS concentration. This correlates with the observationthat HC encapsulation decreases with increasing CS concentration(see Section 3.3). Thus, NPs containing relatively large amount ofHC are smaller than unloaded NPs or NPs containing relatively lowamounts of HC.

3.5. SBEbCD incorporation efciency

The SBEbCD incorporation efciency of the NPs is dened as theamount of SBEbCD that is bound to CS in the NPs divided by thetotal amount of SBEbCD in the solution (Eq. (5)). It was calculatedfrom the concentration of free SBEbCD in the supernatant aftercentrifugation. As expected SBEbCD incorporation increased withincreasing CS concentrations, i.e. when the CS:SBEbCD ratio

286 Z. Flp et al. / International Journal of Pharmaceutics 472 (2014) 282287increased (Fig. 4c). Increasing the CS amount in the NPs allows theNPs to bind larger number of SBEbCD molecules and the SBEbCDmolecules are bound more tightly. It appears that the NPs becomelarger and contain larger number of SBEbCD molecules withincreasing CS:SBEbCD ratio but at the same time their ability totake up HC molecules decreases.

3.6. In vitro release studies

The in vitro HC release proles of three different formulationsand a reference are shown in Fig. 5. The reference solution was purewater saturated with HC. The aim was to compare the HC releaseproles from the HC/SBEbCD complex, the HC/CS complex and NCloaded NPs. All the formulations tested and shown in Fig. 5contained 0.27 mg of HC per ml. The HC loaded NP formulationreleased HC at a slower rate than the HC/SBEbCD formulation. Thiswas to be expected since the NP formulations contains larger andmore stable NP than the HC/SBEbCD solution. However, thestability of the NPs are much better than the aggregates of the HC/

Fig. 5. HC release prole of the different solutions (C0 = 0.27 mg/ml); HC referencein water (); HC/SBEbCD complex (); HC loaded NPs (); HC in aqueous CSsolution (- -).SBEbCD inclusion complexes. All formulations tested displayedrst-order release model following the Higuchi model (Dash et al.,2010).

4. Conclusions

CS/SBEbCD nano- and microparticles were prepared frombiocompatible excipients, i.e. SBEbCD and low molecular weightCS, without addition of other excipients. The NP matrix consisted ofpolyvalent cations (CS polysaccharide) cross-linked with polyva-lent anions (SBEbCD). Due to the lipophilic nature of the CDcavities the very hydrophilic particles could be loaded with poorlywater-soluble lipophilic drugs such as HC. The size of the particles(diameter 2001000 nm) and the drug release rate could becontrolled by changing the initial concentration of SBEbCD and CSduring the NP preparation. In aqueous solutions the NPs werecharacterized as metastable whereas self-assembled cyclodextrinnanoparticles readily dissociate upon media dilution. However, thepKa of CS is about 6.5 and, thus, their stability is pH dependent andat its maximum under acidic conditions but decreasing withincreasing pH above 7. This pH dependent stability of the CS/SBEbCD NP allows for pH controlled drug release.

Acknowledgements

This work was supported by a grant from the EuropeanCommission for Research through the Marie Curie Initial TrainingNetworks (FP7-PEOPLE-ITN-2008 Project no. 237962). We wouldlike to thank Vivek Gaware for supplying chitosan for our research.

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Z. Flp et al. / International Journal of Pharmaceutics 472 (2014) 282287 287

Sulfobutylether--cyclodextrin/chitosan nano- and microparticles and their physicochemical characteristics1 Introduction2 Materials and methods2.1 Materials2.2 Quantitative determination of hydrocortisone concentration2.3 Phase-solubility2.4 Preparation of the nanoparticles2.5 Permeation studies2.6 Encapsulation efficiency2.7 Particle size measurement2.8 Determination of SBECD incorporation in the particles2.9 In-vitro release studies

3 Results and discussions3.1 Phase-solubility3.2 Permeability studies3.3 Hydrocortisone encapsulation efficiency3.4 Particle size and polydispersity index3.5 SBECD incorporation efficiency3.6 In vitro release studies

4 ConclusionsAcknowledgementsReferences