Physicochemical Characterization of α-, β-, and γ-Cyclodextrins Bioesterified with Decanoate...

Published: June 28, 2011

r 2011 American Chemical Society 3031 dx.doi.org/10.1021/bm2006664 | Biomacromolecules 2011, 12, 3031–3038

ARTICLE

pubs.acs.org/Biomac

Physicochemical Characterization of r-, β-, and γ-CyclodextrinsBioesterified with Decanoate Chains Used As Building Blocks ofColloidal NanoparticlesLuc Choisnard,*,† Annabelle G�eze,† C�ecile Vanhaverbeke,† Josias B. G. Yam�eogo,†,‡ Jean-Luc Putaux,§

Bernard Brasme,|| Laure Jullien,^ St�ephanie Boullanger,§ Claire Elfakir,# and Denis Wouessidjewe†

†DPM, UMR CNRS 5063, ICMG FR 2607, UFR of Pharmacy, University of Grenoble, France‡UFR/SDS, University of Ouagadougou, Burkina Faso§CERMAV, UPR CNRS 5301, ICMG FR 2607, Grenoble, France

)ICMG FR2607, UFR Chemistry, University of Grenoble, France^DCM, UMR 5250, ICMG FR 2607, UFR Chemistry, University of Grenoble, France#ICOA, UMR CNRS 6005, FR 2708, UFR Sciences, University of Orl�eans, France

bS Supporting Information

’ INTRODUCTION

R-, β-, and γ-Cyclodextrins (CDs) are cyclic oligosaccharidesconsisting of six, seven, and eight glucopyranose units, respec-tively, linked by R-(1,4) bonds.1 These molecules exhibit inter-esting properties in terms of size of cavity, water solubility, andcomplexation ability. They are thus widely used as pharmaceu-tical excipients to formulate drugs with poor bioavailability.2

Cyclodextrins have been chemically modified in various ways.During the past two decades, numerous amphiphilic CDs havebeen obtained by grafting different substituents on the primary,secondary, or both faces of natural CDs.3�6 Some of these amphi-philic CD derivatives were shown to self-assemble into nanopar-ticles that can be used as potential drug carriers.7,8 Recently, anenzymatic pathway synthesis consisting of a one-step thermolysintransesterification of the CDs with various vinyl ester donorswas successfully carried out.9,10 Typically, decanoate β-CD ester(β-CD-C10) was obtained as described in Scheme 1:

One of the advantages of this enzymatic procedure is thepossibility of producing various CD derivatives easily and in largeamounts. The amphiphilic CD derivatives obtained by selectiveenzymatic substitution of aliphatic chains of different lengths(C6 to C16) offer a wide range of particle morphologies oncenanoprecipitated.11 Some of the nanoparticles formulated withβ-CD-C10 exhibit a pellicular onion-like structure and may be

used as original pharmaceutical drug carriers.9 A good knowledgeof the amphiphilic CDmixture composition used as raw materialis a prerequisite for a better understanding of the ultrastructure ofthe self-assembled nanosystems as well as a key point for theirindustrial development. In the pharmaceutical domain, thechemically modified CDs used at the industrial scale are usuallycharacterized by the degree of substitution (DS) of the batch.

As reported in the literature, it is well-established that theregioselectivity of thermolysin against the hydroxyl in position 2of the glucopyranose unit remains only partial, meaning thatpositions 6 and 3 could also be concerned by the enzymatic reactionto some extent.12 In addition, the large number of potential hydroxylgroups of the parent CD (i.e., 7 hydroxyl groups in position 2 forthe β-CD) leads to a complex polydisperse mixture of amphi-philic derivatives with different DSs. Moreover, position isomersin a given acylated species presenting the same DS are expected.Some past efforts have been made to improve the global knowl-edge of composition of CD derivatives composition, even if theirprecise characterization remains very difficult. Indeed, Kiekenet al. have investigated the direct separation of amphiphilic CD

Received: May 16, 2011Revised: June 28, 2011

ABSTRACT: Nanoparticles of amphiphilic R-, β-, and γ-cyclodextrins were obtained by formulation of cyclodextrinsenzymatically transesterified with vinyl decanoate. The productof this synthesis is a mixture of bioesterified cyclodextrins withvarious degrees of substitution (DS) presenting for a same DSdifferent regio-isomers. In a first step, the efficiency of aMALDI-TOF procedure to characterize the average molecularweight of the derivative bulk mixture was demonstrated by comparing the results with those obtained from complementary NMRand HPLC techniques. In a second step, the ultrastructure of nanoparticles prepared from three different batches of synthesis wasinvestigated and correlated with the average molecular weight and DS of the parent derivative.

3032 dx.doi.org/10.1021/bm2006664 |Biomacromolecules 2011, 12, 3031–3038

Biomacromolecules ARTICLE

derivatives by reverse-phase high-performance liquid chroma-tography (RP-HPLC) coupled to evaporative light scatteringdetectors (ELSDs) or mass spectrometer (MS).13 However,despite a significant improvement, the complexity of the mixtureand the difficulty to isolate pure fractions prohibit any simple andextensive quantitative determination.

As a complement to this first approach, it could be moreinteresting and relevant to improve the knowledge of the mixturecomposition without any species separation. To achieve thisobjective, we referred to matrix-assisted laser desorption ioniza-tion time-of-flight (MALDI-TOF) mass spectral analysis, whichwas used to characterize the relativemolecular weight distribution oflow-molecular-weight polymers presenting a low polydispersity.14

For a given polymer mixture, the number-average molecular weight(Mn) corresponds to the arithmetic average of the molecularweights of the individual oligomer and the weight-averagemolecular weight (Mw) emphasizes the mass of the polymerso that the heaviest molecules are more important. The poly-dispersity index (PDI = Mw/Mn) measures the distribution ofindividual molecular masses in the polymer batch. Mass spectro-metry analysis was also successfully used to determine theaverage DS of CD derivatives.15

For the sake of efficiency, this MALDI-TOF analysis implicitlyassumes that for all possible CD derivatives with a given DS, theanalysis is carried out in the range of molecular weights wherethere is a linear relationship between the signal intensity (Ii) andthe number of molecules (ni)

Ii ¼ k 3 ni ð1Þwhere k is a constant independent of the DS of a particular CDderivative

∑Ii ¼ k 3∑ni ð2ÞUnder this condition, one of the most important characteristicsof the MALDI-TOF procedure is the possibility to access to therelative proportion of CD species that present a specific DS in theCD mixture. Obviously, this assumption is valid if there is nomass discrimination (i.e., fragmentation) against any fraction ofCDmixture during theMALDI procedure; otherwise, MALDI-TOFwould fail in providing a correct molecular weight distribution.16,17

To validate the MALDI-TOF assumption in the lack of any pureamphiphilic CD standard available, we have to confirm theaverage molecular weight obtained by MALDI-TOF analysiswith an independent analytical method. HPLC titration of thevinyl decanoate (VD) at the end point of the transesterificationreaction appears to be a rapid procedure that can be applied toindirectly determine Mn and the total degree of substitution(TDS) of CD derivatives. However, this titration is an indirectprocedure, and to get an accurate result, it has to be assumed thatafter stopping the reaction the amount of VD disappeared fromthe initial reaction medium is totally grafted onto the CD

backbone without any significant formation of undesirable by-product and VD hydrolysis. In complement, nuclear magneticresonance (NMR) spectroscopy is widely used in pharmaceuticalinvestigation to characterize the composition of oligo- andpolymers. Indeed, the compendial European Pharmacopoeiadescribes in the monograph of β-cyclodextrin-2-hydroxypropylether the 1H NMR procedure for the determination of molarsubstitution defined as the ratio of the number of hydroxypropylgroups per anhydroglucose unit.18 Mn can be easily calculatedfrom this standardprocedure ofmolecular substitutiondetermination.

The aim of this work was, first, to validate the accuracy of theMALDI-TOF procedure to determine the molecular weight ofvarious R-, β-, or γ-CD-C10 batches. This study was carried outby comparing the agreement of Mn values determined by threeanalytic techniques, namely, HPLC, MALDI-TOF, and NMR.In a second step, a correlation was established between themorphology and structure of colloidal nanoparticles preparedfrom selected fractions of β-CD-C10 and their physicochemicalcharacteristics, as previously determined by the MALDI-TOFprocedure.

’EXPERIMENTAL SECTION

Materials. Thermolysin (EC 3.4.24.27), a protease type X isolatedfrom Bacillus thermoproteolyticus rokko, anhydrous DMSO (99%), VD(95%), and Celite were obtained from Sigma Aldrich (Sigma Aldrich,L’Isle D’Abeau Chesnes, France). R-, β-, and γ-CD (kleptose) sampleswere provided by Roquette (Roquette, Lestrem, France). Anhydrousacetone was purchased from VWR (VWR International, Lyon, France).Water was freshly deionized in our laboratory.Amphiphilic Cyclodextrin Synthesis. Thermolysin (50 mg)

was immobilized on Celite (0.5 g) using a deposition techniqueaccording to the previously reported procedure.9 Before use, the watercontent of CDs was measured in triplicate using an Ohaus MB45moisture analyzer apparatus. The values of CD relative humidity were10.4, 13.1, and 9.2% for R-, β-, and γ-CDs, respectively. We mixed 0.5 gof R-, β-, or γ-CD and various amounts of VD ester in 5 mL of DMSOand placed it in a capped glass vial containing the immobilized thermo-lysin. The amounts of VD added to the medium were chosen to obtainafter the reaction various batches of CD derivatives that present TDSvalues ranging at about 1 to 7, 1 to 8, and 1 to 10 substitutions for R-, β-,and γ-CD-C10, respectively. The heterogeneous mediumwas vigorouslystirred (350 rpm), and the reaction was carried out at 45 �C for 72 h or atleast until the vinyl fatty acid upper-phase completely disappeared. Atthe end of the reaction, the immobilized thermolysin was removed fromthe liquid phase by centrifugation (1500 rpm; 15 min), and the upperphase was filtered on filter paper MN 615 1/4 L 110 mm (Macherey-Nagel, D€uren, Germany). A sample of ∼100 μL was taken for HPLCtitration of residual VD ([VD]f). The remainder filtrate was diluted in∼10 mL of deionized water, the medium was frozen at�80 �C, and theDMSO/water mixture was removed from the amphiphilic CD derivativesby sublimation under reduced pressure (20 �C, 2 � 10�3 mbar; 48 h).

Scheme 1. Schematic Route of β-CD-C10 Synthesis with Thermolysin



HPLCAnalysis andValidation.AShimadzuHPLCsystem(Shimadzu,Kyoto, Japan) was used equipped with an SCL-10AVP system controller, LC-10ADVPpump,DGU-14Adegasser, SIL-10ADVP autosampler, and an SPD-M10AVP photodiode array (PDA) detector. The detector was set at λmax =203 nm, and peak areas were integrated automatically by computer using aShimadzu LC-Solution v1.25 software program. The experiments were carriedoutona reversedphase Interchim(Montluc)on,France)NucleosilC18column(150mm� 4.6mmI.D., with a particle size of 5μmandpore size of 100Å). Asecurity guard cartridgeC18 (10.0mm� 4.0mm I.D.) was used to protect theanalytical column. TheHPLC systemwas operated isocratically using amobilephase of water�methanol (1/9 v/v), and the column was operated at 30 �C.Themobile phase ofwater�methanol (1/9V/V) was pumped at a flow rate of0.7 mL/min. Before use, the mobile phase was filtered through a 0.45 μmmembrane filter (Millipore).The injection volumewas10μL for both standardand samples, and all the determinations were carried out in triplicate. The voidtime was measured using a sodium nitrite solution in the mobile phase (t0 =2.889 min). The retention time for VD peaks appears at tR = 12.516 min(Figure S1 reported in Supporting Information). Then, the retention factor ofVD for this HPLC procedure is found to be k = 0.769. Quantitativedetermination of VD was performed by diluting the standard product invarious volumes of themobile phase (water�methanol 1/9V/V) and injectinga 10 μL aliquot. The total analysis time was 16 min.

A statistical approach based on the total error profiles was carriedout to validate the HPLCmethod.19�23 To determine the coefficients ofthe linear regression model, six calibration standards of VD (0.085 to1.015 mg 3mL

�1) were prepared and analyzed in triplicate over 3 days.Independent validation standards were realized with the same six levelsof VD spiked into medium (e.g., DMSO, Celite, cyclodextrin), whichwas as similar as possible to thematrix used for enzymatic routine samplesynthesis. Each validation standard was analyzed with three replicationson 3 days. No changes in retention time or peak symmetry of VD wereobserved between the chromatogram profile of validation and calibra-tion standard. For accuracy profile validation, the acceptability limitcriterion was fixed at λ = 25%, and the probability to have ameasurementwithin the acceptability limits was fixed with a β-tolerance limit of 80%with regards to the goals of the analytical procedure without taking intoaccount any regulatory requirements. The calibration curve was ob-tained from results of absorbance measured on each calibration standardusing a least-squares linear regression (slope = 15 075 113.9 ; intercept =672 894.2). The linearity was confirmed with an R2 value of 0.9965. Thetotal error of theHPLCmethod was evaluated from the accuracy profiles(Figure 1), which show the trueness (bias) of the method, which isrepresented by the recovery (%) and the precision symbolized by

unilateral inferior and superior confidence limits. The lower and upperlimits of quantification (LOQ) were determined by computing thesmallest and highest concentration levels, beyond which confidencelimits go outside the acceptance limit. The dosing range for the methodis located between the lower and upper LOQ. As shown in Figure 1 forthe HPLC titration of VD, the dosing range is included within theacceptance limits for concentration levels in between 0.170 (lowerLOQ) and 1.016 mg 3mL�1.

From a one-way ANOVA computation, the results generated fromboth intermediate precision and trueness study for each concentrationlevel are reported in Table 1. Trueness expressed in terms of relative bias(%) refers to the closeness of agreement between the obtained valuesand known exact concentration of the spiked samples. Nomatrix effect isdetected within the dosing range of concentrations because the relativebias never exceeded 3.5%.

Even if no specific study of robustness was carried out, the stability ofHPLC column has been regularly checked over the period of this study.No changes in retention time or peak symmetry were seen, and thepressure drop across the columns remained constant during this period,indicating that the column remained undamaged by the procedures.MALDI-TOF Measurements. CD-C10 derivatives were solubi-

lized in DMSO (200 pmol/μL). The matrix solution was preparedby solubilizing DHB (2,5-dihydroxybenzoic acid, Sigma) in DMSO(50 mg/mL). For analysis, a mixture of 0.5 μL of CD-C10 derivativessolution and 0.5 μL of DHB solution was deposited on the sampleholder and dried overnight at atmospheric pressure. Mass spectra wereacquired using a time-of-flight mass spectrometer (Autoflex S�erie 1,Bruker Daltonics) in reflectron positive ion mode (260 cm effectiveflight path; 20 kV). Voltages were IS1 19 kV and IS2 16 kV, and the PIEdelay was 90 ns. A nitrogen laser was used for desorption/ionization (λ =337 nm, 0.5 ns, pulse energy of 100 μJ). Time-of-flight spectra weregenerated by signal averaging of 10 deposits on sample holder with 240laser shots (diameter < 150 μm) by deposit.NMR Spectroscopy. NMR spectra were recorded at 25 �C on a

400 MHz Bruker Avance spectrometer using Bruker Topspin software.Each derivative (∼6 nmol) was dissolved in 700 μL of DMSO-d6. Eachexperiment was performed with a relaxation time (D1) of 5 s. Baselinecorrection was applied to each spectrum after Fourier transformationand phase correction, without multiplication of the FID. Signal inten-sities were measured in two regions of the 1H spectrum, correspondingto terminal CH3 of decanoate chains (0.72 to 0.93 ppm) and H1 signals(4.70 to 5.02 ppm for R-CD-C10 ; 4.75 to 5.22 ppm for β-CD-C10, and4.79 to 5.38 for γ-CD-C10).Preparation of Colloidal Suspensions. β-CD-C10 nanoparti-

cles were prepared using the solvent displacement method.11 In brief,10 mg of CD-C10 derivatives was dissolved in anhydrous acetone (10 mL)to reach a concentration of 1 mg 3mL�1. This solution was thermostated(25 �C) in double envelope equipment and poured in an equal volume

Figure 1. Accuracy profiles of vinyl decanoate quantification by HPLCmethod.

Table 1. Results of Intermediate Precision and TruenessStudy for Each Concentration Levela

standard validation

concentration

(mg 3mL�1)

intermediate

precision SD (sIP)

trueness

relative bias (%)

recovery

(%)

0.171 0.019 �3.00 97.00

0.255 0.016 �3.43 96.57

0.513 0.049 �2.44 97.56

0.773 0.075 1.52 101.52

1.005 0.098 �2.54 97.46a Intermediate precision standard deviation sIP is estimated from thebetween-days variance (sB

2) and repeatability variance (sr2) according to

the following relation: sIP = (sB2 + sr

2)1/2.



of distilled water under magnetic stirring (500 rpm). The nanoparticlesspontaneously formed, and the organic solvent was then removed underreduced pressure. The suspension was concentrated at 40 �C undervacuum until a final aqueous volume equal to 70% of initial distilledwater volume. The aqueous suspension was then filtered through 0.8 μmfilters.Transmission Electron Microscopy. Droplets of 0.01% (w/v)

suspensions were deposited onto glow-discharged carbon-coated micro-scopy grids. The excess liquid was rapidly blotted, and prior to completedrying, a droplet of 2% (w/v) uranyl acetate negative stain was added.After 1 min, the stain in excess was blotted, and the remaining liquid filmwas allowed to dry. The samples were observed using a Philips CM200microscope operating at 80 kV. The images were recorded on KodakSO163 films.Small-Angle X-ray Scattering. SAXS experiments were carried

out on the BM02 (D2AM) beamline at European Synchrotron Radia-tion Facility (Grenoble, France). A series of glass capillaries (1.5 mmouter diameter) were filled with concentrated (1% w/v) nanoparticlesuspensions and attached to an automatic sampler changer. Scatteringpatterns were recorded at an energy of 16 keV (λ = 0.7768 Å) with aCCD detector placed at a distance of ∼220 mm. The scattered signalwas recorded during 200 s exposures. The diffraction patterns werecalibrated using a silver behenate standard.

’RESULTS AND DISCUSSION

Cross Validation of Quantification by HPLC, NMR, andMS.TheMALDI-TOF procedure applicability for molecular weightdetermination of various R-, β-, or γ-CD-C10 batches wasfirst investigated. This objective was achieved by probing thatthe Mn samples values determined by HPLC, MALDI-TOF,and NMR techniques were all in agreement. To reach thisobjective, we added the thermolysin to different synthesisbatches through different initial CD/VD ratios to produceseveral batches of amphiphilic cyclodextrin characterized by alarge range of TDS.First, an HPLC procedure for quantitative measurement of

VD was developed and statistically validated. Then, knowing theconcentration of VD and parent CD initially introduced in thereaction medium ([VD]0 and [CD]0, respectively), the unreactedVD remaining in themedium ([VD]f) at the end of the reaction hasbeen titrated to determine TDSHPLC of the amphiphilic CDderivatives (eq 3). It was assumed that the residual VD disappearedfrom the initial reaction medium was integrally grafted on the CDbackbone without formation of byproduct.24

TDSHPLC ¼ ½VD�0 � ½VD�f½CD�0

ðmol of aliphatic chain=mol of parent CDÞ

ð3Þ

The average molar weight in number of CD-C10 mixture(MnHPLC

CD-C10) was calculated knowing the TDSHPLC

MCD � C10nHPLC ¼ MCD þ TDSHPLC � ðMCH3 � ðCH2Þ8 � CO� �MHÞ

¼ MCD þ 154� TDSHPLC ðg 3mol�1Þ ð4Þwhere MCD is the molar weight of the R-, β-, or γ- parent CD,MCH3�(CH2)8�CO� is the molar weight of ester residue, and MH isthe molar mass of the hydrogen atom removed from the CDbackbone during the transesterification reaction.All of the batches of synthesis were also analyzed by 1H NMR

spectrometry. The signal area integrals from the hydrogen atoms

corresponding to the aliphatic terminal methyl group (Amethyl protons)and from the anomeric proton of the CD (Aglycosidic H1 protons)were measured. The 1H NMR signal of a methyl group wouldhave an integrated intensity corresponding to three times that ofthe decanoate chain. In addition, anomeric proton would have anintegrated intensity corresponding to 6, 7, or 8 times the CDbackbone for R-, β-, or γ-CD, respectively. The total degree ofsubstitution of parent cyclodextrin (TDSNMR) can be calculatedusing following equation

TDSNMR ¼ n� Amethylprotons

3� AglycosidicH1protonðmol 3mol�1Þ ð5Þ

where n = 6, 7, or 8 for R-, β-, or γ-CD, respectively.By analogy to eq 4, the average molar mass in number of

CD-C10 mixture (MnNMRCD�C10) was deduced from TDSNMR

MCD � C10nNMR ¼ MCD þ 154� TDSNMR ðg 3mol�1Þ ð6Þ

The amphiphilic derivative was also directly analyzed byMALDI-TOF spectrometry. A typical spectrum of a γ-CD-C10 mixtureis shown in Figure 2. Peaks spacing of ∼154.13 Da reflect thedecanoate chain (�CO�(CH2)8�CH3) repeated unit graftedon the cyclodextrin backbone. It should be pointed out thatthe γ-CD-C10 mixture contains a small fraction of overacylatedmaterial (peaks 2465 and 2466 with a relative intensity to2463 = 12.4 and 0.8%, respectively). This result evidences thepossible attack of the hydroxyl groups in positions 3 and 6 bythermolysin.

Figure 2. MALDI-TOF spectrum of a γ-CD-C10 mixture obtaineddirectly after freeze-drying of the reaction medium. 1, m/z zone ofisotope of γ-CD-C10 ester with four aliphatic substitutions and cationadduct (most abundant: m/z (M+Na+) = 1935.9); 2, γ-CD-C10 esterwith five aliphatic substitutions (most abundant: m/z (M+Na+) =2090.0); 3, γ-CD-C10 ester with six aliphatic substitutions (mostabundant: m/z (M+Na+) = 2244.2); 4, γ-CD-C10 ester with sevenaliphatic substitutions (most abundant: m/z (M+Na+) = 2398.3; m/z(M+K+) = 2414.3);5,γ-CD-C10 ester with eight aliphatic substitutions(most abundant: m/z (M+Na+) = 2552.4); 6, γ-CD-C10 ester withnine aliphatic substitutions (most abundant: m/z (M+Na+) = 2706.6);7, γ-CD-C10 ester with ten aliphatic substitutions (most abundant:m/z (M+Na+) = 2860.7). Peaks with star (*) are M+K+ adduct.



From the peak area of each amphiphilic species in the chromato-gram, the number-average relative molar mass of amphiphilic CDmixture (MnMALDI

CD�C10) was determined using the following equation

MCD � C10nMALDI ¼ ∑ðSi �MiÞ

∑Siðg 3mol�1Þ ð7Þ

where Si is the integrated area under each amphiphilic CDderivative (i = 1 to p) peak, and Mi is the chemical mass of thecorresponding amphiphilic CD derivative. The molar mass wascomputed using isotope-averaged molar mass corrected for bothsodium and potassium adducts.25

The number-average molar mass of each resulting batch ofsynthesis was determined both by indirect HPLC procedurebased on VD titration (MnHPLC

CD-C10), direct 1H NMR quantification(MnNMR

CD�C10), and MALDI-TOF analysis (MnMALDICD�C10). To evaluate

the agreement of molar mass determinations between theMALDI-TOF procedure and the two other approaches, weplotted the variation of MnHPLC

CD-C10 versus MnMALDICD�C10 and MnNMR

CD�C10

versusMnMALDICD�C10 (Figure 3). A linear model relationshipMnMALDI

CD�C10 =a�MnHPLC

CD-C10 + b andMnMALDICD�C10 = a�MnNMR

CD�C10 + b was computedfor all comparisons:As shown in Table S1 reported in the Supporting Information,

the linearity of the relationship betweenMnHPLCCD-C10 orMnNMR

CD�C10 andMnMALDI

CD�C10 was confirmed for the R-, β-, and γ-CD-C10 series(R2 > 0.93). Ideally, no systematic bias was noticeable betweenthe two analytical methods, and the results can be considered tobe identical if the intercept of model b = 0 and the slope a = 1.From the results of the Student t test reported in Table S1 that is

incorporated in the Supporting Information, it can be concludedthat whatever the nature of R-, β-, and γ-CD-C10 derivatives, thethree methods of Mn

CD�C10 estimation provide an equivalentresult (probability {b = 0} > 5% and probability {a = 1} > 5%).The discrepancies between Mn

CD�C10 values from either HPLCtitration or 1HNMR approach andMALDI-TOF data are always<8%, which is believed to be a more than acceptable result.As a consequence of this cross-validation of results, it can be

concluded that the initial hypothesis concerning the HPLC andMALDI-TOFmethodologies are confirmed. First, even if it is notpossible to support that any part of VD was hydrolyzed andbyproduct formed during the step of synthesis, the amount of VDconcerned by these phenomena is marginal in regard to the VDamount effectively consumed for the synthesis of CD ester. Second,the MALDI-TOF procedure did not provide mass discrimina-tions between the different species present in the batch mixtureof the amphiphilic derivatives. Furthermore, there is the samelinear relationship between the signal intensity of MALDI-TOFmeasurement and the number of molecules of CD derivativesand thus whatever the degree of aliphatic chain substitution. Inother words, for a given CD-C10 mixture enzymatically synthe-sized, the MALDI-TOF spectra give access to a good overallestimation of the relative proportion of species in presence.These results are in accordance with those reported by Mercieret al. when characterizing the commercial randomly methylatedβ-CD using MALDI-TOF, ionspray (IS), and atmosphericpressure chemical ionization (APCI).15

Correlation of Nanoparticle Structure to β-CD-C10 Physi-cochemical Characteristics. In this part of our study, theMALDI-TOF procedure was used to determine the physicochemicalcharacteristics of three different β-CD-C10 batches synthesized toobtain a low, medium, and high TDS. The ultrastructure of thecorresponding formulated nanoparticles was investigated by SAXS.It can be expected that depending on the mixture of the CD-C10,under similar conditions of nanoprecipitation, nanosystems withdifferent types of morphologies and ultrastructures may be gener-ated. Then, a correlation between nanoparticle ultrastructures andthe TDS can be expected. In complement to TDS, the MnMALDI

CD�C10

determination was computed, and the averagemolar mass in weightof amphiphilic CDmixture (MwMALDI

CD�C10 ) was calculated by analogy tothe small polymerMALDI-TOF analysis according to the followingequation14

MCD � C10wMALDI ¼

∑p

i¼ 1ðSi �M2

i Þ

∑p

i¼ 1Si �Mi

ðg 3mol�1Þ ð8Þ

Knowing the MnMALDICD�C10 value, the average TDSMALDI can also be

calculated

TDSMALDI ¼ ðMCD � C10nMALDI �MCDÞ

ðMCH3 � ðCH2Þ8 � CO� �MHÞ

¼ ðMCD � C10nMALDI �MCDÞ

154ð9Þ

By analogy to Griffin’s equation proposed for nonionic surfactants,the ratio between the hydrophilic and the hydrophobic character ofCD derivatives is determined by calculating the theoretical value ofhydrophilic�lipophilic balance (HLBMALDI).

26�29 The empiricHLB scale, still simple and valid to compare substances in a same

Figure 3. Scatter diagrams of the number-average molar mass (g 3mol�1)

of R-, β-, and γ-CD-C10 derivatives determined from HPLC (MnHPLCCD-C10)

versus MALDI-TOF (MnMALDICD�C10) or from NMR (MnNMR

CD�C10) versusMALDI-TOF (MnMALDI

CD�C10) procedure with the regression line (solid line)and the 95% confidence interval for the regression line (dashed lines).



family of surfactants, is

HLBMALDI ¼

Mhydro

MCD � C10nMALDI

!

5� 100

¼

MCD þ TDSMALDI � ðM�CO � O� �MHÞMCD � C10

nMALDI

!

5� 100

¼ MCD þ TDSMALDI � 43

MCD � C10nMALDI

!� 20 ð10Þ

where Mhydro is the molar mass corresponding to the hydrophilicmoiety (CD ring including the ester function M�CO�O�=44 g 3mol

�1 minus MH = 1 g 3mol�1, the molar mass of the

hydrogen atom removed from the hydroxyl moiety during thereaction of esterification).If the shape of molar mass distribution of amphiphilic CD

derivatives shown in the MALDI-TOF spectrum correspondsroughly to normal distribution of mass, a standard deviation σand a mean (MnMALDI

CD�C10) of the molar mass frequency distributioncould be determined. For variations between different batches ofsynthesis, it is more convenient to use the relative standarddeviation RSD (%) = 100 � (s)/(MnMALDI

CD�C10) than the standarddeviation (s) itself.The characterization of three different batches of β-CD-C10 is

summarized in Table 2. It can be noted that the amplitude of 95%confident interval measured onMnMALDI

CD�C10 andMwMALDICD�C10 is always

below themolarmass of one aliphatic chain (154 g 3mol�1). Eachbatch was submitted to identical conditions of nanoprecipitation.The colloidal suspensions were characterized by direct observa-tion using TEM after negative staining of the preparations(Figure 4). The concentrated suspensions of the same nanosys-tems (1% w/v) were analyzed by SAXS as well (Figure 5).Figures 4 and 5 illustrate the evolution of the morphology and

ultrastructure of nanoparticles made of β-CD-C10 with a differ-ent TDS. The TEM image of batch 1 (TDS = 3.8) shows80�120 nm spheroidal particles that can slightly deform whenthey are confined (Figure 4a). The corresponding SAXS profilecontains one strong peak at q1 = 1.33 nm

�1 and one weak peak atq2 = 2.66 nm�1. The fact that the scattering vector of the weakpeak is twice that of the strong peak suggests that the structure islamellar, with a repeat distance of d = 2π/q1 = 4.71 nm. Althoughit has not been verified by recording high-resolution cryo-TEMimages of the colloidal suspension, it is likely that the sphericalnanoparticles have a multilamellar structure similar to that

previously observed by Choisnard et al. on a suspension ofβ-CD-C10 particles with TDS = 4.1.9

The nanoparticles in batch 3 (TDS = 6.6) exhibit a barrelshape, with a length varying from 100 to 300 nm and a width of80�150 nm (Figure 4c). Five peaks can be seen in the SAXSprofile at q = 1.63, 2.82, 3.26, 4.31, and 4.99 nm�1 (Figure 5).The ratios of the q value of peaks 2 to 5 to that of peak 1 are 1.73,2.00, 2.65, and 3.05, respectively. The progression (approximately 1,√3,√4,√7, and

√9) suggests a hexagonal structure with a cell

parameter a = 4π/√3q1 = 4.45 nm. Interestingly, the sample

with TDS = 5.3 (batch 2) seems to be intermediate, in terms ofboth morphology and ultrastructure. Spherical particles coexistwith more elongated ones (Figure 4b). The SAXS profile containsthree clear peaks at q = 1.61, 2.28, and 3.23 nm�1, and peak 1exhibits a shoulder at q =∼1.32 nm�1 (Figure 5). By comparingthe TEM image and SAXS profile of batch 2 with those of batches1 and 3, we can assume that the sample is a mixture of particles,the more spherical ones having a lamellar organization, whereasthe more elongated ones have a hexagonal structure. However,we cannot rule out that some particles may have a mixed structure,with a coexistence of lamellar and hexagonal regions. Complementaryhigh-resolution cryo-TEM images of unstained particles must berecorded to clarify this point.

Figure 4. TEM images of negatively stained nanoparticles preparedfrom β-CD-C10 with different total substitution degrees: (a) 3.8, (b) 5.3,and (c) 6.6. Scale bars: 100 nm.

Figure 5. SAXS profiles of suspensions of β-CD-C10 nanoparticles withdifferent total substitution degrees (indicated on the right). Arrowheadspoint the main diffraction peaks.

Table 2. Physicochemical Characterization ofThree Different Batches of β-CD-C10

a

characteristic ( t5%;9 3 s(1/n)1/2 batch 1 batch 2 batch 3

TDSMALDI ( 0.5 3.8 5.3 6.6

MnMALDICD C10 ( 73.7 1774.0 1952.8 2156.1

MwMALDICD C10 ( 72.1 1739.9 1967.4 2171.2

HLBMALDI ( 0.3 15.1 14.0 7.8

RSD (%) distribution of β-CD-C10 mixture 11.4 8.7 8.4a 95% confidence interval was calculated using the standard deviationestimated from 10 repetitions of MALDI-TOF analysis on a typicalβ-CD-C10 mixture. Batch values were determined without replication(n = 1).



’CONCLUSIONS

Significant efforts were made to demonstrate that MALDI-TOF was able to determine the molar mass, TDS, and HLB ofvarious CD-C10 mixtures by comparison with alternative HPLCand NMR technics. The correlation between the nanoparticleultrastructure and the TDS has been demonstrated in the caseof β-CD-C10 derivatives and can be extended to MnMALDI

CD�C10 orMwMALDI

CD�C10 in place of TDSMALDI. Similar analyses have to becarried out on systems prepared from R- and γ-CD-C10 deriva-tives to see if such correlation also exists. After this preliminarystudy, further investigations will be carried out to clarify thephysicochemical driving force subjacent to the ultrastructure ofthe nanosystems. The work is in progress to study the impactof the characteristics of the molar mass frequency distribution ofCD-C10 mixture (mean MnMALDI

CD�C10 and σ) on the ultrastructureof amphiphilic CD building blocks. It would also be interesting tostudy the effect of formulation of R-, β-, and γ-CD-C10 withidentical HLB parameters on the ultrastructure of the corre-sponding nanoparticles.

’ASSOCIATED CONTENT

bS Supporting Information. HPLC chromatogram of so-dium nitrite (2.889 min) and standard of VD (VD: 12.516 min)and Student t test for slope and intercept significance of linearrelationship MnMALDI

CD�C10 = a � MnHPLCCD�C10 + b and MnMALDI

CD�C10 = a �MnNMR

CD�C10 + b for the R-, β-, and γ-CD-C10 mixtures. Thenormality of the data distribution has previously been checked(p value <0.05) using a Shapiro�Wilk W test. CI is the 95%confidence interval; R2 is the reliability coefficient of the linearregression. This material is available free of charge via theInternet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*Tel: +33 476635301. Fax: +33 476635322. E-mail: [email protected].

’ACKNOWLEDGMENT

We thank the French CRG at ESRF for granting beam timeon the BM02 beamline and especially Dr. Cyrille Rochas for hiskind assistance during the experiments. We also would liketo acknowledge technical assistance of Delphine Levilly andChristine Brunet-Manquat.

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