21 effect of encapsulating arginine containing molecules on plga

Effect of Encapsulating Arginine Containing Molecules on PLGA:A Solid-State NMR Study

JEAN-BAPTISTE GUILBAUD,1 HELEN BAKER,2 BRIAN C. CLARK,3 ELISABETH MEEHAN,3 YAROSLAV Z. KHIMYAK1

1Department of Chemistry, The University of Liverpool, Crown St, Liverpool L69 7ZD, UK

2School of Chemical Engineering and Analytical Science, The University of Manchester, PO Box 88, Manchester M60 1QD, UK

3AstraZeneca, Macclesfield, Cheshire SK10 2NA, UK

Received 7 July 2009; revised 8 October 2009; accepted 19 October 2009

Published online 14 December 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.22019

Additional Sversion of this a

Corresponde1517943535; Fa

Journal of Pharm

� 2009 Wiley-Liss

ABSTRACT: Design of polymer–drug composites based on the lactide/glycolic acid often rely onthe chemical complementarity between the polymer and functional groups in a pharmaceuticalguest. We previously characterised decapeptide (AZD)/poly(D,L-lactide-co-glycolide) (PLGA) filmformulations aiming at localising the interacting groups responsible for the changes in the bulkproperties of the polymer matrix and understanding the mechanism of stabilisation of the druginto the polymer matrix. The results suggested interactions to occur between the arginineresidue in the peptide and the carbonyl end group of the polymer chains. In order to clarify therole of arginine in directing the drug–polymer interactions, arginine and hexapeptide containingarginine were encapsulated in a PLGA 50/50 polymer. Variable temperature TH

1r measurementsand WISE experiments indicated significant changes in the local dynamics of the polymerchains. These effects were enhanced near and above Tg suggesting the presence of guestspromote the appearance of backbone motion of the polymer chains. The localisation ofthe interactions on the carbonyl groups of the polymer was further confirmed by the WISEexperiments. � 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:2697–

2710, 2010

Keywords: drug interactions; solid-state
NMR; polymeric drug carrier; polymeric drugdelivery systems; poly(lactic/glycolic) acid (PLGA or PLA)
INTRODUCTION

Biomaterials have enormous impact on human healthcare and have been extensively studied for severalapplications such as tissue engineering, drug release,resorbable implants, dental surgery, etc. A widerange of polymers (polyacrylic acids, polyanhydrides,polyorthoesters, polyurethanes, polyaminoacids,polyphosphasenes, etc.) has been used for biomedicalapplications. Because of the tuneable mechanical andthermal properties, biocompatibility and biodegrad-ability, homopolymers and copolymers of lactic andglycolic acids (LA and GA respectively) have beenused extensively in drug delivery devices.1–12 Thesecarriers have several advantages such as enhancingthe therapeutic effect, prolonging the biological

upporting Information may be found in the onlinerticle.nce to: Yaroslav Z. Khimyak (Telephone: 44-x: 44-1517943588; E-mail: [email protected])

aceutical Sciences, Vol. 99, 2697–2710 (2010)

, Inc. and the American Pharmacists Association

JOURN

activity, controlling the drug release rate anddecreasing the administration frequency.13–16 A largevariety of organic molecules ranging from lowmolecular weight synthetic drugs to peptides andproteins4–6,9,17,18 have been encapsulated in PLGAhosts.

The presence of an encapsulated drug guest canhave a significant effect on the dynamic and physicalproperties of the polymer host. Several analyticaltechniques such as Raman spectroscopy,19 FTIRspectroscopy,9 MALDI-TOF,3,20,21 XPS,20 thermalanalysis,6,7 fluorescence spectroscopy22 and electro-phoresis4 have been used for the characterisation ofpolymer-loaded systems. Although these techniquescan detect the changes due to the presence of the drugand quantify the drug content, they, in general, fail toprovide information on the local organisation and theintermolecular interactions in such systems. More-over analysis of the drug is often hampered by the lowloading of the guest in the formulation. Solid-stateNMR is ideal for studies of pharmaceutical compo-sites.23 During the development of pharmaceuticalcompounds (both drugs and their formulations) it is

AL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 6, JUNE 2010 2697

2698 GUILBAUD ET AL.

necessary to characterise the material in its dis-pensed, that is mainly solid form. The chemical shiftselectivity, multinuclear ability and the relativelyhigh sensitivity of NMR to local ordering allow thecharacterisation of pharmaceutical drugs both in bulkand in dosage forms making it a technique of choicefor the study of pharmaceutical composites.24–33 Thismethod does not require any pretreatment whichmight affect the properties of the compounds. More-over, contrary to X-ray diffraction which is currentlymost suitable for crystalline materials, solid-stateNMR is also applicable to amorphous materials aswell as homogeneous or heterogeneous samples. Inthis respect, designed pulse sequences have beendeveloped to allow the selection of specific nuclearinteractions providing information on the localorganisation in the system. H-bonding can be probedvia the change in chemical shift34 or 2D correlationspectroscopy.35–39 For amorphous materials andmacromolecules, the continuous distribution of orien-tations makes the application of such techniquesadditionally challenging.

We studied the effect of incorporating an amor-phous pseudo-decapeptide, AZD (Fig. ElectronicSupplementary Information (ESI)-1), containingarginine on PLGA polymer. In this formulation ionicinteractions between the guanidine pendant groupof arginine and the free carboxylic acid chain endof PLGA have been postulated. To evaluate the role ofarginine in directing these interactions, the effect ofincorporating L-arginine into a PLGA network wasstudied. To take into account the structure of AZDwhich can result in a more complex interactionpattern due to the presence of hydrophobic group, ahexapeptide containing arginine, alanine and hydro-phobic phenylalanine (ARARAF) was also encapsu-lated in PLGA. The effect of the guests on the bulkproperties and more specifically on the cooperativemotions of the chains in PLGA was studied via theevolution of the glass transition temperature (Tg)as a function of the loading level of the guests. Dueto the amorphous nature of the polymer, diffractiontechniques are limited in characterising its localorganisation and the interactions between the guestand the polymer. Ultimately we aimed (1) to obtainstructural and localised information for these solidswith limited ordering, (2) to control the miscibility ofthe components and the homogeneity of the compo-sites and (3) to understand the relationship betweenorganisation and dynamics. Therefore, solid-stateNMR was implemented to correlate the changesobserved on a macromolecular level to the changesoccurring on a molecular level, assess the localorganisation and the probe the intermolecular inter-actions present in these systems, and evaluate theeffect of the drug loading level on the polymerdynamics.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 6, JUNE 2010

EXPERIMENTAL PART

Materials

Biodegradable linear PLGA copolymer with a LA/GAratio of 50:50 and free terminal carboxylic acid endgroups was supplied by AstraZeneca (Macclesfield,UK) and is referred to as 5050_1 (Mw¼ 12 kDa fromGPC with respect to a linear poly(styrene) standard(PolymerLab Shropshire, UK), polydispersity (PD) of1.7, Tg¼ 43.2� 0.28C). L-arginine was obtained fromSigma Aldrich Company LTD, Dorset, UK and usedwithout further purification. The hexapeptide, ala-nine–arginine–alanine–arginine– alanine–pheny-lalanine (ARARAF) was synthesised via solid phasesynthesis procedure using WANG resin and Fmoc(9-fluorenylmethyloxycarbonyl) protected aminoacids.40,41 Polymer films and their formulations wereprepared from these materials using a solvent cast-evaporation method.42 The initial PLGA copolymerand the guest were blended to obtain formulationswith a desired arginine content and dissolved in2.0 mL of dichloromethane to obtain a soft gel. Afterdissolution of the guest, the gel was cast onto a glassslide and the solvent was slowly evaporated at roomtemperature allowing the film to harden for 3 h.Thereafter the film was dried under vacuum to ensurecomplete removal of solvent.

Characterisation

Differential Scanning Calorimetry (DSC)

DSC data were collected on a Perkin Diamondinstrument. Indium and zinc were used as standardsfor the calibration. A 50mL pan with hole containing10–15 mg of material was used as sample holder. Slowcooling did not induce any recrystallisation, thereforeall measurements were performed using the followingtemperature program: (1) heating from 0 to 1008C at208C/min; (2) 10 min isotherm at 1008C; (3) coolingfrom 100 to 08C at 1008C/min; (4) 2 min isotherm at08C. This sequence was repeated four times. The firstheating step was not taken in account for the calcula-tions of the glass transition temperature. Indeed, theTg observed was higher for the first heating step thanfor the other due to thermal history of the material.

Powder X-Ray Diffraction

X-ray powder diffraction patterns of L-arginine andits PLGA composites were recorded on a PanalyticalX’pert Pro Multi-Purpose X-ray diffractometer(CoKa1 radiation l¼ 1.78901 A). Data were collectedover a 2u range of 2–708.

Solid-State NMR

Solid-state NMR spectra were measured on aBruker Avance DSX-400 spectrometer operating at

DOI 10.1002/jps

EFFECT OF ENCAPSULATING ARGININE CONTAINING MOLECULES ON PLGA 2699

100.61 MHz for 13C and 400.13 MHz for 1H. Thevalues of 1H and 13C chemical shifts are referencedto TMS. 1H! 13C Cross Polarisation Magic AngleSpinning (CP/MAS) NMR experiments were carriedout an MAS rate of 7.0 kHz using zirconia rotors of4 mm in diameter. The 1H p/2 pulse was 3.0ms. TheTPPM decoupling43,44 with a decoupling field of ca.80 kHz was used during the acquisition. The Hart-mann–Hahn condition was set using glycine. RAMP-amplitude CP (starting 1H r.f. field 70 Hz, 50% slope)was implemented to recover a broad Hartmann–Hahn matching condition. The recycle delay was15.0 s for PLGA 50/50 and 8.0 s for the guestmolecules, the contact times were varied from 0.01to 16.0 ms. The recycle delays of 8 and 15 s weresufficient as no increase in intensity was observed forlonger recycle times and TH

1 of less than 1 ms weremeasured by inversion-recovery pulse sequence forthe pure guests, the pure PLGA polymers and theirformulations (data not shown). Prior to the experi-ments the samples were spun for ca. 20 min to allowthermal equilibrium to be reached.

The TH1r times were measured using separate

experiments.31 Prior to CP, the 1H magnetisationwas locked along the y axis for a variable time t. Thespin–lock field of ca. 80 kHz, a contact time of 1.0 mswith RAMP-amplitude CP (starting 1H r.f. field 70 Hz,50% slope) and an MAS rate of 7.0 kHz were used.Similarly, 13C relaxation time in the rotating frame,TC

1r, was measured at a spinning rate of 7.0 kHz.31

After CP, the 13C magnetisation is locked along the yaxis for a variable time t ranging from 0.05 to 15.0 msat a spin–lock field of ca. 60 kHz. The 13C magnetisa-tion is allowed to relax in the rotating frame duringthe time t. The intensity of the 13C peaks dependson the amount of magnetisation which has relaxedduring t. The contact time during CP was fixed to1.0 ms.

1H–13C HETeronuclear CORrelation (HETCOR)NMR was used for 1H–13C spectral editing.45 The 2DHETCOR experiments implemented here use Fre-quency-Switched Lee-Goldburg (FSLG)46,47 homo-nuclear decoupling with an on-resonance 1H r.f.field of ca. 85.6 kHz in t1 and ramp-amplitude 1H–13Ccross polarisation to ensure the broad Hartmann–Hahn matching. The Hartmann–Hahn matchingcondition was set using glycine. TPPM was used inthe direct dimension at a decoupling strength of ca.85.6 kHz. The sample volume was restricted to themiddle of the rotor to improve the r.f. homogeneity.The spinning speed was set to 10.0 kHz. States-TPPIwas employed for phase sensitive detection. Therecycle delay was 3.0 s. Two hundred microsecondscontact time was used to promote short range cross-polarisation and limit the spin–diffusion. 256 incre-ments were recorded in t1 to cover the full 1H spectralwidth.

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1H–13C WIdeline SEparation (WISE)48 was imple-mented to evaluate the mobility in the formulation.The MAS rate was set to 7.0 kHz. The 1H p/2 pulsewas 3.05ms and the TPPM decoupling43,44 with anr.f. field strength of 82.0 kHz was used during theacquisition in t2. A contact time of 200ms was used.States-TPPI was implemented to recover pureabsorption lineshape. The recycle time was 3.0 s.128 to 256 increments were recorded in t1 with anumber of transients ranging from 240 for the purepolymer to 864 for the guest–polymer composites.

Data Analysis

The NMR spectra were acquired using xwinnmr 3.5and processed with xwinnmr and TopSpin 1.3.

Nonlinear functions and nonlinear least squaredfit were used to evaluate the CP dynamics. Twofunctions, referred as the I–S (1) and I–I�–S (2)respectively, were used to study the CP kinetics:49

IðtÞ ¼ I0 1 � TIS

TI1r

!�1

exp � t

TI1r

!� exp � t

TIS

� �" #

(1)

where I0 is the absolute amplitude; T1r the relaxationtime in the rotating frame; TIS the CP time constant.

IðtÞ ¼ I0exp � t

TI1r

!1 � l exp � t

Tdf

� ��

�ð1 � lÞ exp � 3

2

t

Tdf

� �exp � 1

2

t

T22

� �� (2)

where TI1r is the I spin–lattice relaxation time in

the rotating frame; Tdf the 1H spin-diffusion timeconstant describing the strength of the homonucleardipolar interactions and the homogeneity of the I spinpool; l is defined by the number n of I spins attachedto the S spin under study (l¼ 1/(nþ 1)); T2 is the spin–spin relaxation time. Although the parameter T2

needs to be fitted, due to its low value it has very littleimpact on the quality of the fitting.49 A more detaileddescription of the different models of CP kinetics isgiven in the ESI.

The TH1r curves where fitted using a single

exponential function.

RESULTS

Choice of the Formulation and Bulk Properties

We previously studied the effect of encapsulating apseudo-decapeptide, AZD (Fig. ESI-1), containingalanine, arginine and aromatic residues in a PLGApolymer matrix on the dynamic properties of the



host.50 In this system, ionic interactions between thefree carboxylic chain ends of the polymer and the ionicarginine site of the guest have been postulated. Thus,the optimum loading level of the peptide depends onthe stoichiometry between the interacting site in theguest (guanidine group of arginine residue in thepeptide) and free carboxylic acid polymer chain ends.In the PLGA/AZD system, formulations with theloading level of approximately 10% show the bestresults from the point of view of drug dissolution,physical properties and drug release.

We found that (1) PLGA/peptide formulationspresented miscibility on both a macroscopic andmolecular level indicating the drug to be evenlydistributed in the polymer network; (2) the host localmobility was reduced due to presence of guest–polymer interactions; (3) these interactions werelocalised on the carbonyl group of PLGA.

In order to clarify the effect of the arginine residueon the interactions, arginine/PLGA formulationswith loading levels of 2.5 and 5.0 wt.% in a PLGA50/50 polymer (referred as F-Arg2.5 and F-Arg5respectively) with arginine/COhost ratios similar tothe AZD formulations were investigated. Based onthe chemical structure of the decapeptide guest othereffects including hydrogen bonding between the drugamino protons and the carbonyl groups of the polymerand/or hydrophobic interactions due to the presenceof aromatic groups should be considered. In thisrespect, a more complex amino-acid sequence (ARA-RAF) in comparison with the single L-arginine,containing arginine and aromatic fragment wasstudied in PLGA 50/50 formulations with loadinglevel of 6.8 and 13.5 wt.% (referred as F-ARARAF6.8and F-ARARAF13.5 respectively).

Despite poor miscibility of L-arginine and PLGA,no observable crystalline domains of L-argininewere detected in the formulations by powder X-raydiffraction (Fig. ESI-2). At low loading level (up to3.0 wt.%) arginine has a plasticiser effect as lower Tg

temperatures have been measured in comparisonto the pure polymer source (Fig. ESI-3). Increasedloading level of arginine (4.0 and 5.0 wt.%) resultsin higher values of Tg suggesting hampering of thecooperative motions of the chains by the guest. In

Table 1. CP Kinetics Parameters and TH1r–VSL Times of the P

CP Dynamics

ppm I 10�7/a.u. l TH1r/ms TIS/m

C––O Lac. 170.2 1.27 — 26.6�1.2 1.02�C––O Gly. 168.1 1.13 — 28.0�1.3 0.87�CH 69.9 1.25 0.44� 0.02 22.6�2.0 —CH2 61.5 1.00 0.27� 0.02 23.0�1.9 —CH3 17.1 1.45 0.45� 0.05 29.2�2.0 —


contrast, ARARAF acts as a plasticiser over the wholerange of loading levels studied as both ARARAFcomposites show lower Tg’s compared to the polymersource.

Solid-State NMR Study of the PLGA Host

The knowledge of the local dynamics of PLGApolymers is a key factor in designing novel formula-tions and monitoring the effects of a guest. Variablecontact time cross-polarisation (CP–VCT) and vari-able spin lock TH

1r (TH1r–VSL) measurements were

used to investigate the local mobility of the PLGA50/50 films (Tab. 1, Fig. 1). The I–S model wasappropriate to fit the CP kinetics curves of thecarbonyls, while for the aliphatic resonances theI–I�–S model provided better fittings.49 Shorter TH

1r

times are observed for the CH and CH2 peaks, longerTH

1r is noted for the CH3 lactide and intermediatevalues of TH

1r are found for both carbonyl resonances.TIS times of the order of 1.0 ms were evaluated forthe carbonyls, shorter value is noted for the glycolideC––O.

Although CP kinetics of the lactide and glycolidecarbonyls are similar, the aliphatic resonancesexhibit noticeable difference in their CP kinetics.The build up of CP, mainly defined by the parametersl, is related to the number of 1H’s attached to thechemical group under study and Tdf in the I–I�–Smodel varies from a site to another. The observedvalues of l fit well to the expected ones (i.e. 0.50� 0.10for CH, 0.33� 0.10 for CH2 groups and 0.40–0.60 forthe methyl carbon). The 1H–1H spin–diffusion is moreefficient for the glycolide CH2 than for the CH lactide.This can be related to the presence of the mobile CH3

in the lactide units, which reduces the efficiency ofspin–diffusion. Unusually short Tdf observed for CH3

are indicative of its restricted mobility in the polymerfilms. Based on the CP kinetics analysis, slightlydifferent motional regimes can be assumed for theglycolide and lactide units. In line with the study ofPLGA with different LA/GA ratios, lactide units aremore mobile than the glycolide ones as suggestedby the faster kinetics observed for the glycolideresonances.

LGA 5050_1 Film

TH1r–VSL

s T2/ms Tdf/ms R2 TH1r/ms R2

0.03 — — 0.998 27.3� 1.1 0.9870.02 — — 0.998 27.5� 1.5 0.975

0.014�0.001 1.73�0.26 0.990 32.8� 0.8 0.9950.012�0.000 1.23�0.30 0.983 33.5� 1.2 0.9890.063�0.005 0.49�0.09 0.994 30.4� 0.3 0.999

DOI 10.1002/jps

Figure 1. CP kinetics curves of PLGA 5050_1 film, (a) (-&-) corresponds to the CP kineticscurve of the lactide carbonyl, (-*-) to the glycolide carbonyl; (b) (-^-) to the CH lactide, (-!-) tothe CH2 glycolide and (-~-) to the CH3 lactide.


The mono-exponential character of the TH1r relaxa-

tion decays suggests the cast films do not presentspatial heterogeneity in the nm range.31,49

Solid-State NMR Study of the Guests

Arginine

A typical 1H–13C CP/MAS NMR spectrum ofL-arginine shows sharp resonances, characteristicof a crystalline compound (Fig. 2b). The moleculararrangement of crystalline L-arginine was derivedusing powder X-ray diffraction. The powder XRD

Figure 2. (a) Stereoscopic view of the unit cell of L-argi-nine, and (b) 1H–13C CP/MAS NMR spectrum of L-arginine,an expansion of the Ca peak shows the splitting into anapproximately 2:1 doublet.

DOI 10.1002/jps

pattern was refined using the DASH software(Fig. ESI-4). L-arginine crystallises in a monocliniccrystal system (Fig. 2a). The space group is P21/a,with Z¼ 4 molecules per unit cell and two indepen-dent molecules in the asymmetric unit. The cellparameters are a¼ 11.588 A, b¼ 16.095 A and c¼5.607 A and b¼ 113.558. Hydrogen bonds occurbetween the carboxylic acid and the guanidine siteof arginine, and between the amino group and theguanidine site. The 1H–13C CP/MAS NMR spectra ofL-arginine (Fig. 2a) show splitting of the carbonyland the two low frequency aliphatic resonances, inagreement with the existence of nonequivalentarginine molecules in the unit cell. Such splittingis not visible for the peaks at 56.3 and 43.1 ppm asthe chemical environment around these sites in thenonequivalent molecules is probably very similar.The broadening of resonance at 43.1 ppm in compar-ison with the low frequency aliphatic doublet can beexplained by the residual 13C–14N dipolar couplingdue to the presence of a bound 14N atom.51–55

Therefore this resonance was assigned to the CHd2

site of the guanidine tail. Splitting in a 1:2 doubletdue to the residual 13C–14N dipolar coupling51–55 isobserved for the resonance at 56.3 ppm which wasascribed to the CHa. This assignment is corroboratedby 1H–13C HETCOR spectrum (Fig. ESI-5).

The CP kinetics (Tab. 2) for the aliphatic reso-nances of L-arginine are best described by the I–I�–Smodel. The quaternary carbons follow the I–S modeldue to the absence of directly attached protons.Efficient transfer of magnetisation and homogeneous1H–1H dipolar network are suggested by the short TIS

times observed for the C––O and Cguanidine and theshort values of Tdf times for the aliphatic resonances.A much shorter TIS time for the Cguanidine than forcarbonyls might be due to H-bonding to carboxylicacid group as indicated by the crystalline structure.Concerning the aliphatic resonances the 1H–1H spin–diffusion is enhanced along the arginine pendantgroup as shorter Tdf are observed for the CHl;b;d

2 incomparison to the CHa. The results derived from the


Table 2. CP Kinetics Parameters and TH1r–VSL Times of L-Arginine

CP Dynamics TH1r–VSL

ppm I10�7/a.u. l TH1r/ms TIS/ms T2/ms Tdf/ms R2 TH

1r/ms R2

C––O 180.7 1.30 — 20.0�0.7 0.70�0.02 — — 0.999 23.6�1.9 0.933179.5 1.28 — 19.1�0.9 0.72�0.02 — — 0.997 23.5�1.6 0.947

C guan. 158.7 0.95 — 16.8�0.7 0.30�0.01 — — 0.996 19.7�0.9 0.978CHa 56.3 2.07 0.44� 0.02 16.4�0.6 — 0.013�0.001 0.38�0.04 0.995 22.3�1.1 0.976

CHd2

43.1 2.22 0.33� 0.02 15.3�0.4 — 0.013�0.000 0.28�0.03 0.996 22.2�0.9 0.983

CHb

232.5 1.60 0.41� 0.03 16.4�0.8 — 0.010�0.001 0.21�0.03 0.989 23.3�0.9 0.982

31.8 1.55 0.30� 0.04 15.1�0.5 — 0.013�0.000 0.28�0.04 0.995 19.3�0.8 0.983

CHg2 24.8 1.95 0.32� 0.02 16.0�0.5 — 0.014�0.000 0.27�0.03 0.997 22.6�0.9 0.981

24.4 1.86 0.30� 0.02 15.2�0.5 — 0.013�0.000 0.29�0.04 0.996 20.1�0.8 0.984


CP kinetics and the assumption of efficient spin–diffusion are further confirmed by the variablespin–lock TH

1r measurements (Tab. 2), where ahomogeneous distribution of TH

1r times betweendifferent sites is observed.

ARARAF

ARARAF was synthesised via a solid phase process.56

Mass spectrometry indicated that all the protectinggroups were cleaved. Although their exact content inthe final product could not be determined unambigu-ously, impurities corresponding to these groups(aromatic resonances at 115–125 ppm) were detectedon the 1H–13C CP/MAS NMR spectrum (Fig. 3). Thecompound was proven to be amorphous by PXRD. Incontrast to the pure arginine, two resonances areobserved in the guanidine region (152–168 ppm). Ingeneral, carbons next to protonated amine groupsare shifted to lower frequency;57–59 thus the highfrequency resonance is ascribed to a deprotonated

Figure 3. 1H–13C CP/MAS and dipolar dephased NMRspectra of ARARAF recorded with 1.0 ms contact time and7 kHz MAS rate, � denote the spinning sidebands.


guanidine group (1H–15N CP/MAS NMR spectrum,Fig. ESI-6). The peaks attributable to the quaternaryand methyl carbons were assigned by the 1H–13Cdipolar dephasing experiments (Fig. 3).

The 1H dimension of the HETCOR spectrumrecorded with a contact time of 200ms (Fig. 4) showsoverlapping resonances of the amide and aromatic1H’s. Expected cross-peaks between the aliphatic1H (CH, CH2, CH3), the amide 1H and the C––O areobserved. Similarly to L-arginine, a contribution of theNH protons to the CH (ca. 50.0 ppm) is found. With200ms contact time only the protonated guanidinecarbon resonance (157.3 ppm) is observed due to itsfaster CP kinetics (discussed below). The deproto-nated guanidine site would become visible only atmuch longer contact times. Cross-peaks between thearomatic and NH 1H’s are detected for the guanidinecarbon suggesting spatial proximity of the guanidinegroup and the aromatic group of phenylalanine. Thisis confirmed further by the cross-peaks observedbetween the guanidine CH2 resonances (ca. 28 and40 ppm) and the aromatic protons.

Similarly to L-arginine, the CP kinetics for thecarbonyl and guanidine carbons comply with the I–Smodel (Tab. 3). Short TIS and TH

1r times are observedfor the carbonyl and the C-guanidine (157.3 ppm).The values of TIS times are similar to the onesobserved for the crystalline L-arginine. Much longerTIS and TH

1r times are observed for the deprotonat-ed guanidine carbon (162.3 ppm) suggesting anincreased mobility for this group. This was confirmedby the longer TC

1r time measured for this site (TableESI-2).

The CP kinetics of the aromatic carbons (quatern-ary at 135.1 and CH at 129.1 ppm) were fitted usingthe I–I�–S model (Tab. 3). The validity of the modelwas verified further by the values of l in agreementwith the predicted ones (0.4–0.6). The TH

1r times aresimilar to the ones for the C––O and C-guanidine(6.12–7.09 ms). Significantly different values of Tdf

are found for the quaternary (135.1 ppm) and theCH (129.1 ppm) aromatic carbons (0.76 and 0.15 ms

DOI 10.1002/jps

Figure 4. 1H–13C HETCOR spectrum of ARARAF, 200ms contact time and an MAS rate of10 kHz were used.


respectively) indicating the spin–diffusion to be moreefficient for the nonquaternary carbon.

The CP kinetics for the aliphatic resonances complywith the I–I�–S model.49 Short TH

1r times are observedfor all the resonances. Similarly to L-arginine, the rateof spin–diffusion is enhanced for CH2 in comparisonto the CH. For the methyl carbon, when the I–I�–Smodel gave unsatisfactory fitting. The I–S model ismore suitable, in spite of extremely short TIS timesobserved, suggesting that spin–diffusion and thetransfer of magnetisation from the directly attachedprotons to occur at similar rates. The results of the CPkinetics analysis are confirmed by independent TH

1r

measurements (Tab. 3).

Table 3. CP Kinetics Parameters and TH1r–VSL Times of ARA

CP Dynami

ppm I 10�7/a.u. l TH1r/ms TIS

C––O 174.6 1.39 — 6.4� 0.2 0.58�173.3 1.39 — 6.3� 0.2 0.58�

C guan. 162.3 0.81 — 8.6� 0.6 1.12�C guan. 157.3 0.93 — 5.9� 0.2 0.35�C aromatic 135.1 0.40 0.51�0.08 7.0� 1.1 —CH aromatic 129.1 1.03 0.44�0.06 6.1� 0.3 —CH 54.0 1.44 0.45�0.02 5.3� 0.3 —CH 50.2 1.63 0.43�0.02 5.4� 0.2 —CH2 41.9 0.98 0.34�0.04 4.6� 0.2 —CH2 38.2 0.65 0.46�0.04 4.7� 0.1 —CH2 27.4 0.91 0.35�0.03 5.3� 0.2 —CH3 17.2 1.16 — 7.3� 0.3 0.13�

1.21 0.83�0.07 7.1� 0.3 —

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Solid-State NMR Studies of the Formulations

Evolution of the Dynamic Parameters of theHost in Formulations

The presence of the guests has a significant effect onthe 1H–13C CP/MAS dynamics of the polymer (Tab. 4,Figs. ESI-7 and ESI-8). Measurements of TH

1r timesconfirmed changes in the mobility in the formula-tions. TH

1r is influenced by motions in the medium kHzrange (correlation times of 10�4–10�5 s) correspond-ing to segmental reorientations and backbone rota-tions. Shorter TH

1r times are observed in theformulations, especially at low loading level due tothe plasticiser effect of arginine at low concentration

RAF

cs TH1r–VSL

/ms T2/ms Tdf/ms R2 TH1r/ms R2

0.02 — — 0.998 6.0�0.2 0.9960.02 — — 0.998 5.9�0.3 0.9860.07 — — 0.991 5.9�0.1 0.9970.01 — — 0.996 6.1�0.6 0.958

0.052� 0.010 1.00� 0.61 0.968 6.1�0.2 0.9930.017� 0.002 0.15� 0.04 0.990 6.0�0.2 0.9960.016� 0.001 0.53� 0.10 0.993 6.2�0.1 0.9990.015� 0.001 0.58� 0.10 0.995 6.5�0.2 0.9970.013� 0.001 0.20� 0.05 0.994 6.2�0.1 0.9980.011� 0.001 0.12� 0.02 0.996 6.4�0.3 0.9850.013� 0.001 0.24� 0.05 0.994 6.3�0.1 0.998

0.01 — — 0.990 6.3�0.2 0.9950.027� 0.011 0.18� 0.02 0.993


Table 4. TH1r Times in ms of the PLGA 5050_1 Film and Its Guest Composites, T¼ 293 K

5050_1 F-Arg2.5 F-Arg5 F-ARARAF6.8 F-ARARAF13.5

C––O Lac. 27.3� 1.1 8.8� 0.1 14.9�0.2 16.9�1.3 25.2�1.9C––O Gly. 27.5� 1.5 8.5� 0.1 14.5�0.2 16.8�1.4 25.6�2.5CH 32.8� 0.8 8.2� 0.1 13.6�0.2 16.9�0.4 24.0�1.9CH2 33.5� 1.2 8.2� 0.2 14.1�0.3 15.9�0.5 21.4�0.7CH3 30.4� 0.3 8.2� 0.1 13.4�0.1 15.2�0.2 21.6�1.0

Figure 5. Variable temperature 1H–13C CP/MAS NMRspectra of PLGA 5050_1 film (Tg¼ 316 K), the values of TH

1r

in ms and the FWHH in Hz derived from the Gaussiandeconvoluted spectra are quoted along the peak for eachresonance.


(ca. 15.0–17.0 ms for F-Arg5 and ca. 8.0–9.0 ms forF-Arg2.5, Tab. 4). This is in good agreement withthe results derived from the CP kinetic analysis(Tables ESI-3 and ESI-4). Similarly, a decrease of thepolymer TH

1r is observed for the ARARAF compositesespecially for the 6.8% formulation (Tab. 4) showingenhanced plasticiser effect. Clearly, the strength ofthe 1H–1H dipolar coupling network is affected in thepresence of a guest. Nevertheless this decrease of TH

1r

is difficult to relate to a specific motional change andcan be due to the increased local mobility of thematerial as well as the enhanced cooperative motionsof the chain evaluated by Tg.

Variable temperature TH1r measurements (VT–TH

1r)enabled us to interpret these data in terms ofmolecular mobility. Generally, for temperatureslower than Tg, a polymer is considered to be a rigidsolid, an increase of temperature coincides with adecrease of TH

1r. For temperatures of the order of Tg, a‘soft solid’ state is obtained and a minimum of TH

1r isreached. Above the Tg an increase of temperatureleads to an increase of TH

1r.31,60 The molecular

correlation time tc which describe the rate ofmolecular reorientation is related to the relaxationtimes.

A gradual decrease of TH1r is observed while the

temperature is increased from 293 to 333 K for thePLGA and its formulations (Figs. 5–7, Tables ESI 6–10 and Figs. ESI 9 and 10) confirming they are on thelow temperature side of the TH

1r minimum. At alltemperatures, shorter TH

1r times are observed in theformulations with respect to the initial polymer and asimilar trend to the one derived from the CP kineticsanalysis is observed—that is TH

1r(polymer)>TH1r

(F-Agr5)>TH1r(F-Arg2.5) (Tab. 4 and Tables ESI 6–8).

In line with observations of Dastbaz et al. for purePLGA61–63 this can be interpreted as an indicationof enhanced mobility (decrease of the molecular tc).Such decrease in TH

1r times is even more pronouncedfor the ARARAF composites (Fig. 7, Fig. ESI 10 andTables ESI 9 and 10). In the ARARAF composites,above Tg the efficiency of CP is significantly reduceddue to a combination of a very short TH

1r value forneighbouring protons and a partial motional aver-aging of the 1H–13C dipole–dipole interaction respon-sible for cross-polarisation.

This change in the mobility has a direct effect ofthe 1H–13C CP/MAS spectra. For the pure PLGA, the


relative intensity as well as the linewidths areaffected upon the increase of temperature. Uponpassing through Tg, the ester carbonyls, the lactideCH and in particular the CH2 glycolide resonancesbroaden whereas the lactide CH3 resonance sharpens(Fig. 5). The broadening of the carbonyl and aliphaticresonances can be related to a combination of reduceddecoupling efficiency and molecular motions. Thisreduced decoupling efficiency can be explained by theappearance of significant backbone motions at asimilar frequency as the 1H decoupling field (about80 kHz), which partially uncouples the proton fromthe decoupling field.61–65 Similar trends are observedfor the arginine and ARARAF formulations (Fig. 6and Fig. ESI-9 for the arginine formulations andFig. 7 and Fig. ESI-10 for the ARARAF composites).The broadening of the C––O, CH and CH2 peaks andthe narrowing of the CH3 line are more pronouncedthan for the pure polymer, especially for the arginine2.5% formulation and for both ARARAF composites.The broadening of the resonances occurs at lowertemperature in the formulations suggesting thepresence of guest in the polymer matrix promotesof backbone motions of the polymer chains.

TH1 relaxation time probes faster motions in the

MHz range (correlation time tc of 10�8–10�9 s), that is

DOI 10.1002/jps

Figure 6. Variable temperature 1H–13C CP/MAS NMRspectra of F-Arg2.5 (Tg¼ 315 K), the values of TH

1r in ms andthe FWHH in Hz derived from the Gaussian deconvolutedspectra are quoted along the peak for each resonance.


methyl rotation. Similar TH1 times have been observed

for the unloaded PLGA film and the arginine formu-lation (data not shown) suggesting the presence ofthis guest does not affect the fast motion regimeevaluated by TH

1 .

WISE Experiments

We used 2D WISE experiments to separate andcorrelate the wide-line (static) proton spectrum withits spectroscopically resolved 1H–13C CP/MAS spec-trum.48 The 1H lineshape extracted at each site

Figure 7. Variable temperature 1H–13C CP/MAS NMRspectra of F-ARARAF6.8 composite (Tg¼ 313 K), the valuesof TH

1r in ms and the FWHH in Hz derived from the Gaussiandeconvoluted spectra are quoted along the peak for eachresonance.

DOI 10.1002/jps

contains valuable information on the local mobility.The greater the mobility of a 1H site, the narrower itscorresponding 1H NMR peak.

Figures 8A and 9A display the 1H–13C WISEspectra of the arginine and ARARAF formulationsrespectively as well as that of the correspondingPLGA film. Although the spectra are similar, someimportant features can be pointed out. The CH lactideand CH2 glycolide resonances exhibit very broad linesconfirming the existence of a strong 1H–1H dipolarcoupling network in the PLGA 5050_1 and thecomposites. The presence of arginine and ARARAFdoes not affect the lineshape for these resonancessignificantly indicating similar mobility of PLGAsindependently of the composition. The relativeintensity of the methyl is enhanced in the presenceof a guest, which was previously observed onthe 1H–13C CP/MAS NMR spectra (Figs. ESI-7 andESI-8).

Broad and complex lineshapes are observed for1H lines corresponding to the carbonyl glycolide, andthe aliphatic CH and CH2 groups. The 1H widelinesare broader for the glycolide resonances than for thelactide (refer to C––O and CH, CH2, Tab. 5) which isrelated to the difference in the surrounding protonpopulations participating to the CP (CH2 for theglycolide carbonyl; CH and the mobile CH3 for thelactide carbonyl).

The presence of arginine and ARARAF affects theglycolide and the lactide units of the host in a differentway (Figs. 8B and 9B). The presence of arginineresults in a sharpening of the proton widelines forthe glycolide resonances (Tab. 5). This effect is morepronounced in the 2.5% composite especially forthe carbonyl glycolide. Such changes suggest that theglycolide units are more likely to be more accessible tointeraction with the guest. Similarly to the argininecomposites, broad and complex lineshapes areobserved for the carbonyl glycolide, and the aliphaticCH and CH2 in the ARARAF formulations. In thesecomposites, broader 1H widelines are also noted forthe glycolide resonances with respect to the lactideones. A substantial narrowing of the carbonylglycolide peak is observed in the ARARAF formula-tions especially at high loading level; no significantchanges are noted for the C––O lactide resonance andfor the aliphatic 1H widelines.

In all the formulations, the changes observed forthe glycolide rather than the lactide resonancessuggest that the former are more likely to be affectedby the guest. We also note that differences indynamics between PLGA and different formulationsindicated by the measurements of TH

1r relaxationtimes are significantly less pronounced in therespective WISE spectra. This clearly indicates thatthese measurements correspond to different motionalregimes of the polymeric matrix.


Figure 8. (A) 1H–13C WISE spectra of (a) PLGA 5050_1 film, (b) 550_1/Arg2.5% and(c) 5050_1/Arg5%, MAS¼ 7 kHz, contact time¼ 200ms. (B) Carbonyl 1H projections of the1H–13C WISE spectra of 5050_1 (black), F-Arg2.5 (red) and F-Arg5 (green).


DISCUSSION

Structure and Miscibility

No observable crystalline arginine domains aredetected in the arginine formulation by powderX-ray diffraction. In the case of the amorphousARARAF, a single glass transition is observed forthe composites indicating a good miscibility of thedrug and polymer over the range of concentrationstudied (13.5 wt.% for ARARAF). In contrast to theAZD composites, plasticiser effects are observed forF-Arg2.5 and the ARARAF composites suggesting theenhanced cooperative motions of the polymer chains.

The miscibility of the drug and polymer on amolecular level is confirmed further by the solid-stateNMR measurements. The relaxation times in therotating frame for the polymer are significantlydifferent in the formulation than those of the purepolymer. In the case of a biphasic system, similarrelaxation times to the ones of the pure compoundswould be expected.66–68 The fact that only onerelaxation component can be detected reflectsthe structural homogeneity (a few nm scale) in theformulation that can be averaged out by spin–diffusion on the experimental time scale (1–10 ms).69


Similarly to the AZD formulations, based on X-raydiffraction, thermal analysis and solid-state NMR,one can assume guest–polymer solid solutions to bepresent. The possibility of the existence of guestclusters with sizes below the detection limit of NMR(ca. 2 nm) embedded in the guest–polymer solidsolution should not be excluded.

Evolution of PLGA Dynamics in Formulation

At natural abundance, only a weak signal correspond-ing to the guest is observed for the composites,making a reliable analysis of their relaxationbehaviour challenging. Thus the changes in therelaxation of the PLGA polymer, the main componentof the systems, are considered.

The variable temperature TH1r measurements indi-

cate an enhanced local mobility of the polymer in thepresence of a guest. A narrowing of the CH3 peaksand a broadening of the aliphatic CH and CH2

are observed upon the increase of the temperatureexplained by the presence of backbone motions with afrequency similar to that of the decoupling field (ca.80 kHz).61–64 These effects depend on the loading levelof the guests and are enhanced when the guest hasa plasticiser effect (F-ARG2.5, F-ARARAF6.8 andF-ARARAF13.5). In contrast, in the AZD composites

DOI 10.1002/jps

Figure 9. (A) 1H–13C WISE spectra of (a) PLGA 5050_1 film, (b) 550_1/ARARAF6.8% and(c) 5050_1/ARARAF13.5%, MAS¼ 7 kHz, contact time¼ 200ms. (B) Carbonyl 1H projections ofthe 1H–13C WISE spectra of 5050_1 (black), F-ARARAF6.8 (red) and F-ARARAF13.5 (green).


these changes (decrease of TH1r and changes in the

linewidths of the 1H–13C CP/MAS spectra) were morepronounced at the higher loading level (20 wt.%). InF-AZD10, TH

1r times similar to those for the polymersource were found, suggesting the drug at low loadinglevel did not disturb the polymer matrix significantlyand if anything rigidified the system. For ARARAFcomposites, a broadening of the lactide CH3 isobserved above the Tg.

The dynamics of carbonyl groups depend on theloading level and the nature of the guest. For AZDcomposites, a broadening of the PLGA carbonyl linefor the 20% composite was observed whereas the

Table 5. 1H Full Width at Half Height (FWHH) in kHz ofthe Pure PLGA 5050_1 and Its Composites, 1H Wide LinesExtracted from the WISE Spectra

5050_1 F-Arg2.5 F-Arg5

F-ARARAF

6.8 13.5

C––O Lac. 19.1 17.5 19.0 19.5 19.6C––O Gly. 35.1 23.2 33.4 31.4 22.0CH 33.9 32.7 33.6 35.7 34.9CH2 48.5 44.3 47.3 51.2 53.4CH3 18.3 16.1 17.2 18.8 18.8

DOI 10.1002/jps

FWHH of the carbonyl in the 10% formulationremained almost unchanged upon an increase oftemperature. In contrast to these observations, agradual broadening of the carbonyl resonance, muchmore pronounced around Tg than for the purepolymer source, is observed in the case of the arginineand especially for the ARARAF composites. Incontrast to F-AZD10, arginine and ARARAF donot rigidify the polymer network and, similarly toF-AZD20, promote backbone motions of the polymerchains.

Localisation of the Interactions

The changes in the relaxation behaviours of thepolymer in the presence of a guest can be attributed tointermolecular interactions between the guest andthe polymer. Based on the variable temperature TH

1r

measurements, the localisation of these interactionsto a specific functional group could not be addressedunambiguously. The presence of the guests affects thestrength of the 1H–1H dipolar coupling as suggestedby the changes in the linewidth and lineshape of thealiphatic 1H wide lines in the WISE spectra of thecomposites. These effects depend of the loading leveland the nature of the guest involved. In contrast tothe AZD composites, little modifications in the



1H linewidths are observed in the arginine andARARAF formulations and therefore it is difficult torelate these variations to changes in local mobility.These results are unexpected considering the sig-nificant decrease of TH

1r observed for the aliphaticresonances in the composites with respect to theirpolymer sources. This suggests that different typesof motions are revealed by the lineshape analysis ofWISE spectra for these resonances.

The 1H wide lines corresponding to the carbonylsites vary significantly in the presence of a peptideguest. Since these 13C sites do not have directlyattached protons, the corresponding 1H widelines areindicative of the overall motions in the system. It isthe presence of the guest that affects the carbonylgroups preferentially. In the pure polymer host, thecarbonyl groups only cross-polarise from the lactideand glycolide aliphatic protons. In the formulations,the proton source is extended to the guest molecules.The mobility of the carbonyl groups and theirsurrounding is decreased in the 10% composite andenhanced when the loading level of AZD is 20%. Theresults from the WISE studies unambiguouslyindicated the presence of drug–polymer interactionslocalised on the polymer carbonyls.

Opposite effects are observed for the arginine andARARAF formulations. A decrease of the linewidthof the proton peak of glycolide carbonyl is observedwhereas the lactide carbonyl is essentially un-changed. Taking into account the results of VT–TH

1r

measurements one would expect the changes in thelinewidth to be more significant (also consideringthe effects of temperature are more pronounced inthese composites than for the AZD formulations).These results confirm the presence of arginine andARARAF enhances the local mobility of the polymer.More importantly, they also suggest that arginine isnot the only functional group directing the interac-tions between the polymer and the guests.

The WISE experiments also indicate that changesin the linewidth of the carbonyl resonances are lesspronounced for the lactide units. This highlights adifference in the affinity of the monomer units to theguests. The glycolide units are preferentially affectedby the presence of a guest. One can assume a higheraffinity of the glycolide unit for interactions than thelactide unit. This can be related to the presence of themethyl carbon in the lactide unit which creates sterichindrance and makes the interactions less probable.

CONCLUSIONS

The effects of incorporating arginine and smallpeptide guest into a PLGA polymer were investigated.A good miscibility of the guests in the composites isconfirmed by thermal analysis and X-ray diffraction


and changes in the TH1r of the polymer. The presence of

guest–polymer interactions in the formulations isfurther confirmed by the changes observed for VT–TH

1r

of the polymer with respect to the initial polymer.From the VT–TH

1r experiments, changes in linewidthare observed upon increasing temperature. Thesechanges in the linewidth are related to the appear-ance of segmental reorientations and backbonemotions which are enhanced near and above Tg. Inthe presence of both guests, the increase in linewidthof the carbonyl resonances is also more pronouncedthan that of the aliphatic resonances and becomessignificant above Tg indicating the carbonyl reso-nances are preferentially affected. The localisation ofthe interactions on the carbonyl is further confirmedby the WISE experiments. In all the formulations, anarrowing of the 1H widelines is found for thecarbonyls and it is more pronounced for the linescorrelating to the glycolide carbonyl resonances.

All these results confirm the presence of guest–polymer interactions involving the polymer carbonylgroups in the composite. The strength and extent ofthe interactions depend on the nature of the guest andits loading level. The effect of AZD on the polymermatrix cannot be exclusively restricted to interactionbetween the carboxylic polymer chain end and theguanidine groups of AZD as the rigidification ofthe polymer matrix is not observed in arginineand ARARAF composites and was only found inthe F-AZD10. This confirms the complexity of themechanism of interactions which can include H-bonding, ionic interactions and also the hydrophobic/hydrophilic effect.

REFERENCES

1. Geze A, Chourpa I, Boury F, Benoit JP, Dubois P. 1999. Directqualitative and quantitative characterization of a radiosensi-tizer, 5-iodo-20-deoxyuridine within biodegradable polymericmicrospheres by FT-Raman spectroscopy. Analyst 124:37–42.

2. Hyde TM, Gladden LF, Payne R. 1995. A nuclear-magnetic-resonance imaging study of the effect of incorporating a macro-molecular drug in poly(glycolic acid-co-dl-lactic acid). J ControlRelease 36:261–275.

3. Lee JW, Gardella JA. 2003. Simultaneous time-of-flight sec-ondary ion MS quantitative analysis of drug surface concen-tration and polymer degradation kinetics in biodegradablepoly(L-lactic acid) blends. Anal Chem 75:2950–2958.

4. Progent F, Taverna M, Le Potier I, Gopee F, Ferrier D. 2002.A study of the binding between polymers and peptides, usingaffinity capillary electrophoresis, applied to polymeric drugdelivery systems. Electrophoresis 23:938–944.

5. BlancoPrieto MJ, Fattal E, Gulik A, Dedieu JC, Roques BP,Couvreur P. 1997. Characterization and morphological analy-sis of a cholecystokinin derivative peptide-loaded poly(lactide-co-glycolide) microspheres prepared by a water-in-oil-in-wateremulsion solvent evaporation method. J Control Release 43:81–87.

DOI 10.1002/jps


6. Passerini N, Craig DQM. 2002. Characterization of ciclosporinA loaded poly (D,L lactide-co-glycolide) microspheres usingmodulated temperature differential scanning calorimetry.J Pharm Pharmacol 54:913–919.

7. Hill VL, Passerini N, Craig DQM, Vickers M, Anwar J, FeelyLC. 1998. Investigation of progesterone loaded poly(D,L-lac-tide) microspheres using TMDSC, SEM and PXRD. J ThermAnal 54:673–685.

8. Fu YJ, Shyu SS, Su FH, Yu PC. 2002. Development of biode-gradable co-poly(D,L-lactic/glycolic acid) microspheres for thecontrolled release of 5-FU by the spray drying method. ColloidSurf B Biointerfaces 25:269–279.

9. Fu K, Griebenow K, Hsieh L, Klibanov AM, Langer R. 1999.FTIR characterization of the secondary structure of proteinsencapsulated within PLGA microspheres’. J Control Release58:357–366.

10. Faisant N, Siepmann J, Benoit JP. 2002. PLGA-based micro-particles: Elucidation of mechanisms and a new, simple math-ematical model quantifying drug release. Eur J Pharm Sci 15:355–366.

11. Govender T, Stolnik S, Garnett MC, Illum L, Davis SS. 1999.PLGA nanoparticles prepared by nanoprecipitation: Drug load-ing and release studies of a water soluble drug. J ControlRelease 57:171–185.

12. Kiss E, Vargha-Butler EI. 1999. Novel method to characterizethe hydrolytic decomposition of biopolymer surfaces. ColloidSurf B Biointerfaces 15:181–193.

13. Langer R. 1995. Biomaterials and Biomedical Engineering.Chem Eng Sci 50:4109–4121.

14. Langer R. 2000. Biomaterials in drug delivery and tissueengineering: One laboratory’s experience. Acc Chem Res 33:94–101.

15. Uhrich KE, Cannizzaro SM, Langer RS, Shakesheff KM. 1999.Polymeric systems for controlled drug release. Chem Rev 99:3181–3198.

16. Albertsson A-C, Varma IK. 2003. Recent developments in ringopening polymerization of lactones for biomedical applications.Biomacromolecules 4:1466–1486.

17. Miyajima M, Koshika A, Okada J, Ikeda M. 1999. Effect ofpolymer/basic drug interactions on the two-stage diffusion-controlled release from a poly(L-lactic acid) matrix. J ControlRelease 61:295–304.

18. Tsai TM, Mehta RC, DeLuca PP. 1996. Adsorption of peptidesto poly(D,L-lactide-co-glycolide). 1. Effect of physical factors onthe adsorption. Int J Pharm 127:31–42.

19. Murza A, Alvarez-Mendez S, Sanchez-Cortes S, Garcia-RamosJV. 2003. Interaction of antitumoral 9-aminoacridine drug withDNA and dextran sulfate studied by fluorescence and surface-enhanced Raman spectroscopy. Biopolymers 72:174–184.

20. Walker AK, Qiu HB, Wu YL, Timmons RB, Kinsel GR. 1999.Studies of peptide binding to allyl amine and vinyl acetic acid-modified polymers using matrix-assisted laser desorption ioni-zation mass spectrometry. Anal Biochem 271:123–130.

21. Zhang J, Kinsel GR. 2002. Quantification of protein-polymerinteractions by matrix-assisted laser desorption/ionizationmass spectrometry. Langmuir 18:4444–4448.

22. Vila A, Sanchez A, Perez C, Alonso MJ. 2002. PLA-PEG nano-spheres: New carriers for transmucosal delivery of proteins andplasmid DNA. Polym Adv Technol 13:851–858.

23. Tishmack PA, Bugay DE, Byrn SR. 2003. Solid-state nuclearmagnetic resonance spectroscopy—Pharmaceutical applica-tions. J Pharm Sci 92:441–474.

24. Lubach JW, Padden BE, Winslow SL, Salsbury JS, MastersDB, Topp EM, Munson EJ. 2004. Solid-state NMR studies ofpharmaceutical solids in polymer matrices. Anal Bioanal Chem378:1504–1510.

25. Saindon PJ, Cauchon NS, Sutton PA, Chang CJ, Peck GE,Byrn SR. 1993. Solid-state nuclear-magnetic-resonance (NMR)

DOI 10.1002/jps

spectra of pharmaceutical dosage forms. Pharm Res 10:197–203.

26. Wulff M, Alden M, Tegenfeldt J. 2002. Solid-state NMR inves-tigation of indomethacin-cyclodextrin complexes in PEG 6000carrier. Bioconjug Chem 13:240–248.

27. Yoshioka S, Aso Y, Kojima S, Sakurai S, Fujiwara T, Akutsu H.1999. Molecular mobility of protein in lyophilized formulationslinked to the molecular mobility of polymer excipients, asdetermined by high resolution C-13 solid-state NMR. PharmRes 16:1621–1625.

28. Watanabe T, Hasegawa S, Wakiyama N, Kusai A, Senna M.2003. Comparison between polyvinylpyrrolidone and silicananoparticles as carriers for indomethacin in a solid statedispersion. Int J Pharm 250:283–286.

29. Spiess HW. 2004. Advanced solid-state nuclear magnetic reso-nance for polymer science. J Polym Sci A Polym Chem 42:5031–5044.

30. Spiess HW. 2001. Multidimensional solid-state NMR of struc-ture and dynamics of polymers. Macromol Symp 174:111–119.

31. Voelkel R. 1988. High-resolution solid-state 13C-NMR spectro-scopy of polymers. Angew Chem Int Ed Engl 27:1468–1483.

32. Blumich B, Spiess HW. 1988. Two-dimensional solid-stateNMR-spectroscopy—New possibilities for the investigation ofthe structure and dynamics of solid polymers. Angew Chem IntEd Engl 27:1655–1672.

33. Brown SP, Spiess HW. 2001. Advanced solid-state NMR meth-ods for the elucidation of structure and dynamics of molecular,macromolecular, and supramolecular systems. Chem Rev 101:4125–4155.

34. Harris RK. 2004. NMR crystallography: The use of chemicalshifts. Solid State Sci 6:1025–1037.

35. Brus J, Dybal J. 2002. Hydrogen-bond interactions in organi-cally-modified polysiloxane networks studied by 1D and 2DCRAMPS and double-quantum 1H MAS NMR. Macromolecules35:10038–10047.

36. Potrzebowski MJ. 2003. What high-resolution solid-state NMRspectroscopy can offer to organic chemists. Eur J Org Chem2003:1367–1376.

37. Brus J, Petrickova H, Hlavata D, Strachota A. 2004. Self-organization, structure, dynamic properties, and surface mor-phology of silica/epoxy films as seen by solid-state NMR, SAXS,and AFM. Macromolecules 37:1346–1357.

38. Mao JD, Xing B, Schmidt-Rohr K. 2001. New structural infor-mation on a humic acid from two-dimensional H-1-C-13 corre-lation solid-state nuclear magnetic resonance. Environ SciTechnol 35:1928–1934.

39. Bockmann A, Juy M, Bettler E, Emsley L, Galinier A, Penin F,Lesage A. 2005. Water-protein hydrogen exchange in the micro-crystalline protein Crh as observed by solid state NMR spectro-scopy. J Biomol NMR 32:195–207.

40. Chan WC, White PD. 2000. Fmoc solid phase peptide synthesis:A practical approach, 1st ed., Oxford, UK: Oxford UniversityPress. p 288.

41. Atherton A, Sheppard RC. 1989. Solid phase peptide synthesis:A practical approach. The Practical Approach Series Paper-back, Oxford, UK: Oxford University Press. p 216.

42. Jeong JH, Lim DW, Han DK, Park TG. 2000. Synthesis,characterization and protein adsorption behaviors of PLGA/PEG di-block co-polymer blend films. Colloid Surf B Biointer-faces 18: 371–379.

42. Jeong JH, Lim DW, Han DK, Park TG. 2000. Synthesis,characterization and protein adsorption behaviors of PLGA/PEG di-block co-polymer blend films. Colloid Surf B Biointer-faces 18:371–379.

43. Bennett AE, Rienstra CM, Auger M, Lakshmi KV, Griffin RG.1995. Heteronuclear decoupling in rotating solids. J ChemPhys 103:6951.



44. Khitrin AK, Fujiwara T, Akutsu H. 2003. Phase-modulatedheteronuclear decoupling in NMR of solids. J Magn Reson162:46–4653.

45. vanRossum BJ, Forster H, deGroot HJM. 1997. High-field andhigh-speed CP-MAS 13C NMR heteronuclear dipolar-correla-tion spectroscopy of solids with frequency-switched Lee-Gold-burg homonuclear decoupling. J Magn Reson 124:516–519.

46. Bielecki A, Kolbert AC, Levitt MH. 1989. Frequency-switchedpulse sequences—Homonuclear decoupling and dilute spinNmr in solids. Chem Phys Lett 155:341–346.

47. Laws DD, Bitter HML, Jerschow A. 2002. Solid-state NMRspectroscopic methods in chemistry. Angew Chem Int Ed Engl41:3096–3129.

48. Schmidtrohr K, Clauss J, Spiess HW. 1992. Correlation ofstructure, mobility, and morphological information in hetero-geneous polymer materials by 2-dimensional wideline-separa-tion Nmr-spectroscopy. Macromolecules 25:3273–3277.

49. Kolodziejski W, Klinowski J. 2002. Kinetics of cross-polariza-tion in solid state NMR: A guide for chemists. Chem Rev102:613–628.

50. Guilbaud JB. 2007. Solid-state NMR studies of polymer-druginteractions in pharmaceutical formulations. PhD thesis.Department of Chemistry, the University of Liverpool. p 230.

51. Harris RK, Olivieri AC. 1992. Quadrupolar effects transferredto spin-1/2 magic-angle spinning spectra of solids. Prog NuclMagn Reson Spectrosc 24:435–456.

52. Orr RM, Duer MJ. 2006. Decoupling residual dipolar couplingbetween 13C and 14N spin pairs in CPMAS NMR. Solid StateNucl Magn Reson 30:130–134.

53. Eichele K, Lumsden MD, Wasylishen RE. 1993. 14N coupleddipolar-chemical shift 13C NMR-spectra of the amide frag-ment of peptides in the solid-state. J Phys Chem 97:8909–8916.

54. Hexem JG, Frey MH, Opella SJ. 1982. Molecular and struc-tural information from 14N-13C dipolar couplings manifested inhigh-resolution 13C NMR-spectra of solids. J Chem Phys 77:3847–3856.

55. Hexem JG, Frey MH, Opella SJ. 1981. Influence of 14N on13C NMR-spectra of solids. J Am Chem Soc 103:224–226.

56. Sewald N, Jakubke HD. 2002. Peptides: Chemistry and biology,1st ed., Weinheim, Germany: Wiley-VCH Verlag GmbH & Co.p 590.

57. Hague DN, Moreton AD. 1994. Protonation sequence of linearaliphatic polyamines by 13C NMR-spectroscopy. J Chem SocPerkin Trans 2:265–270.


58. Somashekar BS, Gowda GAN, Ramesha AR, Khetrapall CL.2005. Protonation of trimipramine salts of maleate, mesylateand hydrochloride observed by 1H, 13C and 15N NMR spectro-scopy. Magn Reson Chem 43:166–170.

59. Somashekar BS, Gowda GAN, Ramesha AR, Khetrapal CL.2004. Differential protonation and dynamic structure of dox-ylamine succinate in solution using H-1 and C-13 NMR. MagnReson Chem 42:636–640.

60. Xue G, Ji GD, Yan H, Guo MM. 1998. Morphology and mole-cular motion of poly(ethylene terephthalate) in polymer/oligo-mer gel. Macromolecules 31:7706–7711.

61. Dastbaz N, Middleton DA, George A, Reid DG. 1999. Moleculardynamics of poly(lactide-co-glycolide) controlled pharmaceuti-cal release polymers: Preliminary solid state NMR. Mol Simul22:51–55.

62. Muller K. 1992. Guest molecule-dynamics in thiourea inclu-sion-compounds as studied by 13C MAS NMR-spectroscopy.J Phys Chem 96:5733–5738.

63. Rothwell WP, Waugh JS. 1981. Transverse relaxation of dipo-lar coupled spin systems under RF-irradiation—Detectingmotions in solids. J Chem Phys 74:2721–2732.

64. Vanderhart DL, Earl WL, Garroway AN. 1981. Resolution in13C NMR of organic-solids using high-power proton decouplingand magic-angle sample spinning. J Magn Reson 44:361–401.

65. Asano A, Takegoshi K. 2001. Free volume study of amorphouspolymers detected by solid-state 13C NMR linewidth experi-ments. J Chem Phys 115:8665–8669.

66. Zheng SX, Guo QP, Mi YL. 1998. Examination of miscibilityat molecular level of poly(hydroxyether of bisphenol A) poly-(N-vinyl pyrrolidone) blends by cross-polarization magicangle spinning 13C nuclear magnetic resonance spectroscopy.J Polym Sci B Polym Phys 36:2291–2300.

67. Cojocariu G, Natansohn A. 2003. Solid-state NMR investiga-tion of morphology in poly(N-vinylcarbazole) complexes withpoly(ethylene glycol) monomethyl ether 3,5-dinitrobenzoate.Macromolecules 36:2404–2411.

68. Kao HM, Chao SW, Chang PC. 2006. Multinuclear solid-stateNMR, self-diffusion coefficients, differential scanning calori-metry, and ionic conductivity of solid organic-inorganic hybridelectrolytes based on PPG-PEG-PPG diamine, siloxane, andlithium perchlorate. Macromolecules 39:1029–1040.

69. Xiong JC, Maciel GE. 1997. Variable-temperature high-resolu-tion proton NMR study of laboratory-frame and rotating-framespin-lattice relaxation in coals. Energy Fuels 11:866–878.

DOI 10.1002/jps

21 effect of encapsulating arginine containing molecules on plga

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