molecular interactions, internal structure and drug release kinetics of rationally developed...

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e 128 (2008) 60–70www.elsevier.com/locate/jconrel

Journal of Controlled Releas

Molecular interactions, internal structure and drug release kinetics ofrationally developed polymer–lipid hybrid nanoparticles

Yongqiang Li a, Ho Lun Wong a, Adam J. Shuhendler a, Andrew M. Rauth b, Xiao Yu Wu a,⁎

a Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada M5S 3M2b Experimental Therapeutics, Ontario Cancer Institute, Toronto, ON, Canada M5G 2M9

Received 9 September 2007; accepted 18 February 2008Available online 5 March 2008

Abstract

This paper presents the first study of molecular interactions of ingredients and internal nanostructure in relation to drug loading and releasemechanisms/kinetics of rationally designed solid polymer–lipid hybrid nanoparticles (PLN). The PLN were prepared by using a rationally selectedcomposition that was found in our previous work to provide optimized interactions of verapamil hydrochloride (VRP) with dextran sulfate sodium (DS)and then the VRP–DS complex with dodecanoic acid (DA). The solid-state properties of the components, their molecular interactions and themorphology, particle size and internal structure of PLN were determined by use of differential scanning calorimetry, powder X-ray diffraction, 13Cnuclear magnetic resonance, Fourier transform infrared spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering. Thedistribution of VRP in PLN was examined by TEM imaging using a cationic gold tracer. Drug release studies were conducted in various media. Drugloading as high as 36% and loading efficiencies up to 99% were achieved in the rationally formulated PLN. Hydrogen bonding between drug, polymerand lipid and a uniform distribution of amorphous VRP within the solid lipid matrix were evident. Sustained drug release from the PLN was mainlycontrolled by ion exchange and diffusion processes. The results demonstrated that strong molecular interactions among the drug, the polymer and thelipid in the optimized formulation were responsible for the improved drug loading and release performance of the PLN.© 2008 Elsevier B.V. All rights reserved.

Keywords: Polymer–lipid hybrid nanoparticles; Drug–polymer–lipid interactions; Internal nanostructure; Solid-state properties; Verapamil hydrochloride; Releasemechanism and kinetics

1. Introduction

Solid polymer–lipid hybrid nanoparticles (PLN) have beeninvestigated in the past few years by our group for enhanceddelivery of ionic, water-soluble anticancer drugs and chemo-sensitizers [1–6]. A variety of ionic and non-ionic drugs havebeen successfully encapsulated in PLN and their efficacyagainst multidrug resistant cancer cells and solid tumors hasbeen demonstrated [2–5]. Differing from solid lipid nanopar-ticles (SLN) [7–12], PLN comprises an ionic polymer thatforms complexes with ionic drugs, thus enabling sufficient drugloading and high loading efficiency [1–5]. Although SLN havea number of advantages including superior biocompatibility,sustained drug release, prospective targeting capabilities and

⁎ Corresponding author. Tel.: +1 416 978 5272; fax: +1 416 978 8511.E-mail address: [email protected] (X.Y. Wu).

0168-3659/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.jconrel.2008.02.014

feasibility of mass production, their application is limited topoorly water-soluble drugs [13–18] due to poor miscibility ofionic drugs with hydrophobic lipids. By introducing polymericcounter ions and utilizing ion-exchange mechanism [19–21], aversatile drug delivery system, PLN, has been developed [1–6].

Unlike SLN, PLN involve three components with distinctproperties, i.e., lipid, polymer, and drug. The interactions amongthese components play an important role in the successfulfabrication and performance of PLN. An in-depth investigation ofthese interactions was conducted to allow the rational design ofthis complex nanoparticle system [22]. Physicochemical char-acterization and preformulation studies were carried out usingisothermal titration calorimetry (ITC), powder X-ray diffraction(PXRD), differential scanning calorimetry (DSC) and molecularstructure-based computation of interaction parameters. Based onmaximal miscibility, an optimal lipid for a given drug–polymerpair was selected for making PLN, e.g. verapamil hydrochloride

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http://dx.doi.org/10.1016/j.jconrel.2008.02.014

61Y. Li et al. / Journal of Controlled Release 128 (2008) 60–70

(VRP) and dextran sulfate sodium salt (DS). This pioneeringwork on the interactions of the components and their effect on thesolid-state properties of the lipid–polymer–drug system has laid afoundation for the rational design of PLN systems.

However, the findings obtained in this previous work usingbulk mixtures of the tertiary system may not be translatable toPLN directly, since the formulation and fabrication of acolloidal system may be more complex than a bulk system.The main constituents of PLN may undergo changes in theirphysicochemical properties, which affect the stability, solubilityand release rate of the incorporated drug. To develop robustPLN with desired release profiles, it is essential to understandmolecular interactions of drug–polymer–lipid in nanoparticles,the physical form of drug, the internal structure of thenanoparticles, and drug loading and release mechanisms.

Characterization of the state of VRP within PLN is achallenge due to the transformation of VRP from crystallinestate to amorphous state following complexation with DS andthe submicron size of the particles. In the present work solid-state 13C NMR with cross polarization (CP) and magic-anglespinning (MAS) was used in combination with DSC, PXRDand Fourier transform infrared spectroscopy (FT-IR) to examinethe molecular dynamics of components and the internalstructure of PLN. The CP–MAS 13C NMR detected differencesin chemical shifts of drug and excipient in the solid phase. NMRis a useful technique for simultaneously investigating the stateof drug, excipients and their environment based on their specificchemical structures. 1H NMR has been applied to characterizethe composition and structure of nanostructured lipid carriers(NLC) [23–26]. Solid-state 13C NMR has been used effectivelyto identify the form of drug within solid matrix, molecularmobility and molecular interactions between drug and excipi-ents in a variety of dosage forms including SLN [27–30].

The internal structure of nanoparticles, in particular thedistribution of a drug within the solid matrix, is a key contributorto the drug release profile. Nanoparticles with drug concentratedon the surface normally exhibit a large initial burst release,whereas those with uniformly loaded drug tend to provide a moresustained release with a small burst effect. To determine thedistribution of VRP within the PLN, ultra-fine cationic goldnanoparticles (0.8 nm) were utilized. The rationale for thisapproach is that cationic gold nanoparticles, like VRP, form acomplex with anionic polymers and a very small quantity ofpolymer–gold complex will not influence the overall formationand lipid partition of the drug–polymer complex. Informationabout the morphology, solid-state properties and internal structureof PLNwill help elucidate the underlyingmechanisms of the drugloading and release behavior of PLN.

2. Materials and methods

2.1. Materials

VRP, DA (MP: 44–46 °C), DS (MW: 5000 Da), Tween 80,and other chemicals unless otherwise specified, were purchasedfrom Sigma-Aldrich Canada (Oakville, ON, Canada). PluronicF68 was a gift from BASF Canada Inc. (Mississauga, ON,

Canada). Spectra/Por® dialysis tubes (MWCO: 10,000 Da) andMillipore filters (MWCO: 30,000 Da) were supplied bySpectrum Laboratories Inc. (Rancho Dominguez, CA, USA)and Millipore Inc. (Toronto, ON, Canada), respectively.Cationic gold tracer (ultrasmall, EM Grade) with a concentra-tion of 5×1015 particles/ml and a mean diameter of 0.8 nm wasbought from Electron Microscopy Sciences Inc. (Washington,PA, USA). Distilled and deionized (DDI) water was preparedwith a Milli-Q water purifier (Milli-Pore Inc.). Calciumchloride, potassium dihydrogen phosphate and disodiumhydrogen orthophosphate anhydrous (dibasic) were purchasedfrom Fisher Chemicals (Pittsburgh, PA, USA).

2.2. Preparation of PLN, blank SLN and gold/VRP-PLN

PLN were prepared by a modified emulsion methodfollowed by ultrasonication [31]. Briefly, the lipid, DA, wasmelted at 50 °C, about 5 °C above its melting temperature. Aknown amount of VRP was added into the molten lipid,followed by the addition of an aqueous solution of Pluronic F68and Tween 80 at the same temperature. Known amounts of DSwere then added slowly to the mixture during stirring with amagnetic stirrer (Corning stirring plate) at 700 rpm until anequivalent charge ratio of DS to VRP was obtained, which waspre-calculated based on the moles of VRP in the solution andthe nominal number of SO3

− groups available on each repeatingunit of DS, i.e., 2.3, according to the monograph of DS from thesupplier. The prepared coarse oil-in-water (o/w) emulsion wascontinuously stirred for 20 min and then sonicated (VWR,Model 75HT) for another 5 min. Nanoparticles were obtainedby dispersing the final emulsion into cold water at 2–4 °C undermagnetic stirring at an emulsion to water volume ratio of 1:15.The final PLN suspensions were centrifuged, washed and thenlyophilized for 24 h for DSC and PXRD analysis. Blank SLNwere prepared similarly without addition of the drug and thepolymer. Briefly an aqueous solution of Pluronic F68 andTween 80 at 50 °C was introduced to molten DA. An o/wemulsion was produced under vigorous stirring and thensonicated for 5 min. PLN were composed of DA, VRP, DS,Pluronic F68 and Tween 80, while the blank SLN consisted ofDA, Pluronic F68 and Tween 80.

The gold/VRP-PLN were produced using the methoddescribed above with minor modification. To avoid significantchanges in the partition behavior of the gold/VRP–polymerhybrid complex molecules, only one one-thousandth of theVRP mole charges was replaced by the cationic gold tracer.Aliquot amounts of DS were added to a hot mixture of meltlipid, VRP and surfactants to allow the formation of VRP–DScomplexation, followed by introduction of aliquots of cationicgold particles which then bind the residual unoccupied bindingsites on DS. The gold/VRP-PLN included the ingredients, DA,VRP, gold particles, DS, Pluronic F68 and Tween 80.

2.3. Transmission electron microscopy

The morphology of VRP-PLN and the gold/VRP-PLN wasexamined by transmission electron microscopy (TEM) (Hitachi

62 Y. Li et al. / Journal of Controlled Release 128 (2008) 60–70

7000, Tokyo, Japan). For VRP-PLN, the samples were stainedwith 2% (w/v) negatively charged phosphotungstic acid (PTA)for 2 min before TEM imaging. For the gold/VRP-PLN, theimaging was conducted without application of the PTA stainingto avoid interference. Due to the extremely small size of thegold tracer particles, a high magnification of 200,000 wasapplied for the imaging.

2.4. Measurements of particle size and zeta potential

The hydrodynamic diameters and zeta potentials of the PLNwere measured using a dynamic laser scattering (DLS) zetasizer(Nicomp, Model 380 ZLS, Particle Sizing Systems Inc., SantaBarbara, CA, USA) with a He/Ne laser source of 632.8 nm. Thesamples were diluted with DDI water and measured at a fixedangle of 90° at 25 °C. Zeta potential was determined at 25 °C inan electric field about 24.5 V/cm. All measurements wererepeated three times.

2.5. Determination of drug loading

Following the preparation, 3 ml of undiluted PLN samplewas centrifuged through a membrane filter unit (Millipore Inc.MWCO: 30,000 Da) using a Centra-CL3 ventilated centrifuge(Thermo IEC Inc., Needham, Heights, MA, USA) at 4000 rpmfor 45 min. An aliquot of 1 ml of filtrate was sampled from therecovery chamber and incubated with an equal volume of0.3 M CaCl2 solution for 24 h at room temperature to allowcomplete release of the bound VRP from the PLN. The VRPcontent in the aqueous filtrate was analyzed with an UV–visspectrophotometer (Hewlett-Packard 8452A, Palo Alto, CA.USA). Drug loading efficiency (DLE) and drug loadingcapacity (DLC) were calculated using the following equa-tions:

DLE kð Þ ¼ Winitial drug added �Wfree drug

Winitial drug added� 100: ð1Þ

DLC kð Þ ¼ Wdrug in PLN

Wlipid� 100: ð2Þ

2.6. Differential scanning calorimetry (DSC)

Thermal analysis was conducted using DSC 2010 differentialscanning calorimeter (TA Instruments, New Castle, DE, U.S.A.).The temperature and heat flow calibrations were performed at aheating rate of 5 °C/min from −20 to 250 °C with indium as astandard substance. Powder samples, 5–10 mg each, wereanalyzed at the same settings under a purge of nitrogen (50 ml/min). Each analysis was performed in triplicate.

2.7. Powder X-ray diffraction (PXRD)

Powder X-ray diffraction patterns of various dry samples,200 mg each, were measured at room temperature with aSiemens D5000 θ/2θ diffractometer (Karlsruhe, Germany). Thecommon Cu-Kα radiation source was operated at a voltage of50 kV and a current of 35 mA, and the secondary beam was

monochromatized by a Kevex solid-state detector. The sampleswere scanned from 2° to 35° (2θ) with a step size of 0.02° and astep interval of 1.2 s. The data were processed by DiffracPlus™software accompanied with the instrument.

2.8. 13C nuclear magnetic resonance (NMR) spectroscopy

In order to eliminate the influence of water, PLN suspensionwas centrifuged and dried in a dessicator. Approximately200 mg of the dried PLN samples was then introduced into theprobe rotor. Solid-state 13C NMR spectra of VRP, DS, VRP–DScomplex, DA, blank SLN and PLN loaded with 12.4% of VRPwere obtained at 75.46 MHz using a Bruker Avance™ DSX-400 high resolution solid-state spectrometer (Bruker BioSpinGmbH, Rheinstetten, Germany) at ambient temperature. Thespectra were recorded using cross polarization–magic-anglespinning (CP–MAS) with a 90° pulse width of 5 ms, a delaytime of 4 s, a contact time for cross polarization of 1 ms and aspinning frequency rate of 5 kHz. Chemical shifts werecalibrated to an external standard of adamantane with achemical shift of 38.56 ppm.

2.9. Fourier transform infrared (FT-IR) spectroscopy

Transmission infrared spectra of VRP alone, VRP–DSphysical mixture, VRP–DS complex and VRP-loaded PLN(12.4%, w/w) were recorded on a Fourier transform infraredspectrophotometer (Perkin-Elmer Spectrum BX, Perkin-ElmerInc., Norwalk, CT, USA) using KBr discs, each containing 2 mgof a sample. The FT-IR spectra were obtained by averaging 32scans at a resolution of 2 cm−1.

2.10. In vitro release study

A dialysis bag technique, widely used for studying drugrelease kinetics of nanoparticulate carrier systems [32–35], wasmodified in this work to maintain a sink condition and achievesatisfactory reproducibility. Briefly, 6 ml of an undiluted PLNsuspension was sealed in a dialysis tubing (MWCO: 10,000 Da,SpectraPor, CA, USA) and immersed in 250 ml of preheatedrelease medium of various compositions: DDI water, pH 6.5;NaCl solutions of various concentrations; 0.15 M KCl solution,pH=5.3; 0.05 M CaCl2 solution, pH=5.5; or phosphate buffer,pH=7.4, µ=0.15 M. The release study was conducted at 37±0.5 °C using a USP dissolution apparatus 2 (paddle method)(Kalish dissolution tester, Model 72RL, Toronto, Canada) witha stirring rate at 50 rpm. The release medium was continuouslypumped to a flow cell and assayed with a UV–vis spectro-photometer (Hewlett-Packard 8452A, Palo Alto, CA, USA) at278 nm. All experiments were performed in triplicate.

2.11. Statistical analysis

Two-sided, unpaired student t-test was applied to compare twoindependent means. A value of pb0.025 was considered to bestatistically significant. All values were expressed as the meanvalue±standard deviation (S.D.) of three independent experiments.

Table 1Loading efficiency of verapamil hydrochloride (VRP) into the PLN as a functionof the VRP to lipid matrix ratio (w/w), (n=3)

Verapamil HCl/lipid ratio(w/w)

Drug loading efficiency(%) a

Drug loading capacity(%) a

1:20 99.2±0.6 5.0±0.031:8 97.7±0.4 12.2±0.11:4.4 92.6±0.4 21.0±0.11:2.5 90.3±1.4 36.1±0.6

The DS:VRP charge ratio was fixed at 1:1.a Mean±S.D.


3. Results

3.1. Morphology, particle size and size distribution and surfacecharge

Fig. 1a and b presents TEM photographs of VRP-PLN andgold/VRP-PLN, respectively. The PLN have a nearly sphericalshape with no obvious particle aggregation and a mean particlediameter of 342.5 nm with a standard deviation of 54.8 nm whenfit with a Gaussian distribution function. The gold tracer particlesare evenly distributed within PLN (Fig. 1b). The particle size of

Fig. 1. Morphological examination and particle size distribution of PLN (12.4%drug loading (w/w), with respect to the lipid): a) transmission electronmicroscopy (TEM) images of PLN; and b) TEM image of VRP/gold-PLN.

gold/VRP-PLN and blank SLN were not significantly differentfrom that ofVRP-PLN (data not shown). The zeta potentials (ζ) ofblank SLN, VRP-PLN and gold/VRP-PLN are all negative:−20.41±0.43 mV (n=3) for blank SLN, −20.14±0.52 mV(n=3) for PLN containing 12.4% VRP, and −29.44±0.62 mV(n=3) for gold/VRP-PLN.

Fig. 2. Solid-state property characterization of PLN. (a) Differential scanningcalorimetry thermograms of (i) blank SLN and (ii) PLN containing12.4% drug;and (b) overlaid powder X-ray diffraction crystallographs of (i) blank SLN and(ii) PLN containing 12.4% drug.

Fig. 3. 13C CP–MAS solid-state NMR spectra of a) verapamil hydrochloride (VRP), dextran sulfate sodium salt (DS) and VRP–DS complex; b) dodecanoic acid (DA),blank SLN and VRP-PLN.


3.2. Drug loading efficiency (DLE) and drug loading capacity(DLC)

Table 1 shows the dependence of DLE and DLC on theweight ratio (w/w) of VRP to lipid (VRP/LIP). Although theDLE generally declines with increasing VRP/LIP, it remains atca. 90% even when the ratio is as high as 1:2.5. In an oppositetrend, the DLC increases from 5.0% to 36.1% as the VRP/LIPweight ratio is increased from 1:20 to 1:2.5.

3.3. Solid-state properties

Solid-state properties such as melting point (M.P.) andcrystallinity of PLN components were characterized by DSCand PXRD as previously reported for bulk samples [22]. Fig. 2acompares the DSC thermograms of blank SLN (curve i) andPLN containing 12.4% VRP (curve ii). The onset of VRP-PLNmelting occurs at 30.9 °C, lower than blank SLN at 33.1 °C. TheM.P. of DA (43.7 °C) in VRP-PLN is slightly lower than that in

Table 2Comparison of 13C CP/MAS NMR chemical shifts at 75.46 MHz of typicalgroups of VRP, DA, VRP–DS complex, blank SLN and PLN containing VRP–DS

a)

Carbons 13C NMR chemical shift, δ (ppm)

VRP VRP–DS complex PLN

C21 107.0 – –C2 110.3 112.3 114.4C5, C24 111.9 – –C6, C25 122.4 120.9 –C20 128 – 130.2C1 130.6 129.9 132.5C23 149.1 – 149.4C4 150.2 149.1 150.7C3, C22 155.1 – 151.3C7 55.9 56.2 56.4C8 55.1 56.2 55.3C26 53.8 56.2 53.9C27 53.1 56.2 53.9

b)

Carbons 13C NMR chemical shift, δ (ppm)

DA Blank SLN Difference (Hz) a

C12 15.1 15.2 7.6C5–C11 25.2 25.3 7.6C4 33.2 33.3 7.6C3 33.9 34.0 7.6C2 34.8 34.9 7.6C1 181.8 181.9 7.6a Calculated as: Difference (Hz)=(δBlank SLN−δDA)×Radio frequency of the

instrument in MHz.


blank SLN, 44.2 °C. A previous study showed that the M.P. ofVRP in a binary physical blend of VRP and DS at an equalcharge ratio decreased from 145.5 °C to 114.05 °C [22].However, the melting peak of VRP is absent in the VRP-PLN,suggesting that it is in an amorphous state after complexationwith DS. These results agree with our previous findings in thebulk VRP–DS–lipid system, suggesting negligible effect of thesurfactants used in the preparation of the PLN on the solid-stateproperties VRP.

The overlaid PXRD diffraction patterns of blank SLN andVRP-PLN are consistent with the results of DSC thermalanalysis. Fig. 2b depicts that DA remains in a crystalline state inboth blank SLN and VRP-PLN, as evidenced by the presence ofits characteristic diffraction peaks, e.g. at 3.00°, 6.40°, 9.66°,21.51° and 23.88°. The PXRD diffraction pattern of VRP-PLN(curve ii) exhibited no peaks associated with VRP, indicatingthat VRP is essentially amorphous following the complexationwith DS and subsequent incorporation into the lipid matrix. It iseither molecularly dissolved in the solid lipid matrix ordispersed as an amorphous material embedded within thesolid lipid matrix.

3.4. Internal structure of PLN

CP–MAS solid-state 13C NMR spectroscopy is a powerfulnon-destructive technique for investigating the microenviron-ment of pharmaceutical materials in solid dosage forms [36–38], and excipient interactions, including H-bonding, in variousformulations [26,28,39,40]. The 13C CP–MAS NMR spectra ofVRP, DS, VRP–DS complex, DA, blank SLN and VRP-PLNare compared in Fig. 3. The aromatic carbons of VRP are ofparticular interest since these characteristic carbons are sensitiveto 13C shielding pertinent to changes in the microenvironmentand other compounds in this study display no resonance activityin this region [41–43]. The breadth and shape of the peaks (Fig.3a) indicate that non-encapsulated VRP is crystalline, consistentwith the DSC and PXRD patterns.

The characteristic region for DS ranges from 60 to 100 ppm,where the resonances of 13C occur on the repeating unit ofglucose [44,45]. The signals of C1 are broad multiplets andabsorb at 97.20 ppm. The resonance of C2 occurs at 75.36 ppmwhile the C3 signals are split into two peaks: one broad peakwith a maximum resonance at 75.90 ppm, and the other at80.17 ppm. Since the reactivity of carbon atoms in a glucosering is in the order: C3NC2NC4, the absorption of non-sulfatedC4 is affected by the sulfated C3 and C2 and thus exhibitresonances at 69.13 and 67.93 ppm. C5 absorbs at 70.27 ppmand the broad multiplets of C6 occur at 65.99 ppm. Broad peaksin the spectrum of DS indicate an amorphous state of DS(Fig. 3a).

The differences between the 13C chemical shifts of thefunctional groups in VRP alone and in the VRP–DS complex ofan equal charge ratio are illustrated in Fig. 3a and summarizedin Table 2a. The significant changes in the chemical shifts andthe shape of peaks in the spectrum of VRP–DS complex revealstrong molecular interactions between VRP and DS. Thedisappearance of some peaks suggests the formation of a new

species, i.e., the VRP–DS complex, and the alteration in theVRP solid form upon complexation. The broad and Gaussianpeaks illustrated in Fig. 3c indicate that VRP–DS complex isamorphous.

The peak at 180 ppm for carbonyl group is characteristic forDA (Fig. 3b). The sharp peaks suggest that DA is in a compactand crystalline form. As shown in the figures and Table 2b,there is no significant difference between the spectra and the13C chemical shifts between DA and blank SLN. As neitherVRP nor DS is present in the preparation of blank SLN, the lackof change in chemical shifts is indicative of an insignificanteffect of the surfactants used for the preparation of PLN on themolecular environment of DA.

The spectrum of VRP-PLN (Fig. 3b) is significantly differentfrom that of blank SLN. The characteristic broad peaks in theregion from 50 to 155 ppm confirm the presence of amorphousVRP–DS complex molecules of restricted mobility within thesolid lipid matrix of PLN. Nonetheless, marked difference existsin the peak shape of VRP–DS complex within PLN (Fig. 3b)and those of VRP–DS complex alone (Fig. 3a). The multipletswith broad line widths and weak amplitudes indicate a changeof microenvironment of the VRP–DS complex and interactionswith the solid lipid matrix. The chemical shifts of 13C NMRspectrum of VRP–DS complex and VRP-PLN are alsocompared in Table 2a.

Fig. 4. Transmission infrared spectra for a) VRP alone, b) VRP–DS physicalmixture, c) VRP–DS complex, and d) VRP-PLN.


3.5. Molecular interactions determined by FT-IR

The transmission infrared spectra of VRP alone (a), VRP–DS physical mixture (b), VRP–DS complex (c), and PLNloaded with 12.4% VRP (d) are compared in Fig. 4, and thecharacteristic IR absorption frequencies of VRP in varioussamples are summarized in Table 3. The drug–polymer–lipidinteractions are demonstrated by the absence and shift ofstretching peaks of the characteristic functional groups in VRP[41], as listed in Table 3. Comparison of Fig. 4a (VRP) and b(VRP and DS physical mixture) reveals some change in theintensity and shape of the peaks of the N–H stretchingvibrations of the protonated amine of VRP in the region of

Table 3Comparison of characteristic bands of VRP, VRP–DS physical mixture, VRP–DS complex, and PLN in FT-IR spectra

Characteristicbands

Wavenumber, cm−1

VRP VRP–DSphysicalmixture

VRP–DScomplex

PLN

C–H stretchingvibrations of themethoxy groups

2840 2840 2838 2850

N–H stretchingvibrations of theprotonated amine

2800–2300(broad, complex)

2800–2300(broad, complex)

– –

C`N stretchingvibrations

2237 2237 2234 2208

Skeletal stretchingvibrations ofthe benzene ring

1609 1609 1606 16081592 1592 1592 15901517 1518 1518 1517

C–O stretchingvibrations of thearomatic ethers

1260 1260 1262 1250

Fig. 5. Dependence of in vitro drug release profile of PLN on a) the ionicstrength of the release medium; b) the type of counter ions in the medium; and c)the drug to lipid ratio (w/w) of the PLN. The PLN contained 12.4%w/w VRP,with respect to the lipid. The release experiments were conducted at 37 °C in anelectrolyte aqueous solution or DDI water (a and b) or in a pH=7.4 phosphatebuffer solution of μ=0.15 M (c). (n=3).

2800 cm−1 to 2300 cm−1, suggesting molecular interactionsbetween VRP and DS. More strikingly, the N–H stretchingvibrations peak is absent in VRP–DS complex (Fig. 4c) andPLN (Fig. 4d), which confirms a significant microenvironmentchange of protonated amine group in VRP, due to the formationof R3NH

+(SO3−) ion pair. As for the skeletal stretching vibration

of the benzene ring at 1609 cm−1, 1592 cm−1 and 1517 cm−1,


no significant changes are seen among these four samples.Nonetheless, there are significant changes in the peak shape(Fig. 4) and the wavenumber (Table 3) for the stretchingvibrations of the –OCH3 group, –C`N group, and the C–Ogroup of the aromatic ethers in VRP in these different samples.These changes are indicative of strong hydrogen bondingamong VRP, DS and DA.

3.6. Factors influencing release kinetics of VRP from PLN

The kinetics of VRP release from PLN with various initialloading in release media of different pH and/or ionic strengthswas studied and the release profiles are presented in Fig. 5.

3.6.1. Effect of ionic strength and type of counter ionsThe effect of the ionic strength (µ) of the release medium on

the release profile of PLN is shown in Fig. 5a. Using DDI wateras release medium, where no ion exchange occurs, the release ofVRP from PLN containing 12.4% drug is slow with only 27%(w/w) of the incorporated drug being released in 24 h. As theionic strength of the release medium is increased from 0 to0.15 M NaCl, the release rate increases significantly. Thefraction of drug released in 24 h is increased to 61% in 0.5 mM(0.0005 M) NaCl and to 77% in 0.015 M NaCl. However, as thesalt concentration is increased from 0.015 M NaCl (pH 5.4) to0.15 M NaCl (pH 5.3), the fraction of drug release is onlyincreased to 80% (w/w), statistically insignificant (pN0.10).

In an ion-exchange process, the species of counter ions in therelease medium affects drug release kinetics of PLN, dependingon the affinity or apparent association constant between thecounter ions and the sulfate group (–SO3

−) on DS. Fig. 5bcompares the release curves of PLN containing 12.4% drug inmedia with the same ionic strength of 0.15 M (i.e., 0.05 MCaCl2, 0.15 M NaCl, and 0.15 M KCl). A trend in release rate isobserved for PLN in a medium containing different counterions: Ca2+NNa+NK+, although only the release curves for PLNin 0.05 M CaCl2 and 0.15 M KCl are significantly differentbased on the value of similarity factor (f2) [19]. This finding isconsistent with the rank of binding affinity between DS andmetal ions, i.e., Ca2+NNa+NK+, reported in literature [46,47].

3.6.2. Effect of drug loadingUsing a pH 7.4 phosphate buffer (PBS, Na2HPO4, KH2PO4,

µ=0.15 M) as the release medium, the effect of drug loading onthe release profiles of PLN was investigated. Fig. 5c presentsthe release curves of PLN containing 5%, 12.4%, or 22.5% (w/w) VRP initially together with that for a VRP solution as acontrol which contains the same amount of VRP and surfactantsas the 12.4% VRP-loaded PLN. It is seen that the in vitro drugrelease rate decreases with increasing drug loading capacity. Forall VRP-PLN samples, a lag time is present with 10% or lessVRP being released in the first 5 h. This sigmoidal shape of therelease curves differs significantly from those in salt solutions ofpH 5.3–6.5 (Fig. 5a and b) and that of a VRP solution whichreaches 86% release through the membrane in 5 h (top curve inFig. 5c). This result suggests a determinant role of VRPdissolution in pH 7.4 PBS as discussed below.

4. Discussion

Based on optimized drug–polymer–lipid interactions, PLNwere prepared in the presence of non-ionic surfactants. ThesePLN exhibited distinct features in drug loading, physicochem-ical properties, drug distribution within the lipid matrix andtheir drug release behavior.

4.1. High drug loading capacity of PLN

Formation of ionic complexes between VRP and DS isessential for the preparation of VRP-PLN. The complexation ofVRP with DS could be affected by the degree of substitution,chain length and branching density of the anionic polymer. Theclinically applicable low-molecular-weight DS has a highdegree of sulfate substitution (2.3 nominal number of sulfategroups on each repeating unit of DS) [48–50]. The high chargedensity of DS results in a high binding capacity of DS withcationic drugs, which in turn imparts a potentially high loadingcapacity to the PLN. Furthermore, the low-molecular weightand low branching density (b5%) of DS result in high watersolubility and a more stretched chain configuration. As a result,the complexation between VRP and DS becomes morecomplete and reproducible.

The solid-state compatibility or miscibility of DA in VRP–DS complexes of equal charge ratio contributes to the high drugloading capacity of PLN. Stoichiometric complexation of VRPand DS in the VRP–DS complex results in a high degree ofhydrophobicity due to the complete neutralization of thenegative charges on DS [22]. This is further supported by thecomparable zeta potential values of blank SLN and VRP-PLN,i.e., −20.41±0.43 mV and −20.14±0.52 mV, respectively.

4.2. Thermal properties and crystallinity

The different shapes of the endothermic peaks of blank SLNand VRP-PLN indicate the different structures of the nanoparti-culates. The DA within VRP-PLN exhibited a broader meltingpeak and lower M.P. than the blank SLN (Fig. 2a), suggesting aless stable DA solid form [51] in the presence of VRP–DScomplex. Nonetheless, the crystalline structure of DA remainedunchanged in the blank SLN and VRP-PLN, as indicated by thevirtually identical diffractograms of blank SLN and VRP-PLN(Fig. 2b). That VRP in PLN exists in amorphous state may beattributable to its strong complexation with amorphous DS in thelipid phase hindering crystallization. The lack of stereo-regularityand the rigidity of glucose ring of DS may be unfavorable to thecrystallization of VRP within PLN thermodynamically.

4.3. Form and distribution of VRP within PLN

A uniform molecular distribution of VRP within PLN issupported by the experimental results of high miscibilitybetween the VRP–DS complex and DA, the amorphous stateof VRP in PLN, the H-bonding between VRP–DS complex andDA and the sustained release of PLN in the release media ofvarious compositions.


No significant difference between the 13C NMR spectra of DAand blank SLN suggests that the microenvironment for DAremains relatively unchanged (Fig. 3b). However, molecularinteractions between VRP–DS complex and DA do exist,otherwise the 13C NMR for the VRP-PLN would be the simplesuperimposition of the individual spectra of VRP–DS complexand DA. The changes in the chemical shifts and peak shape in thesolid-state NMR spectrum of PLN as compared to VRP–DScomplex alone (also see Table 2a) evidence the formation of H-bonding due to the delocalization of electrons [52]. In this study,the high field shift of the aromatic carbons on the VRP–DScomplex in the PLN implies a change in the 13C shieldingcomponent. This could be attributed to the intermolecular H-bonding between the –OH group on DA and the CH3O– groups(C7, C8, C26, C27), –C`N group (C15) on VRP, and the –O–groups and O atoms on DS. Supporting this hypothesis are theremarkable differences in the chemical shifts and the peak shapesof the characteristic carbon atoms onVRP (C7, C8, C26, C27) andthose in the VRP–DS complex and in the VRP-PLN (Table 2b).

In addition to the 13C NMR results, the shifts of functionalgroups and the changes in stretching peaks in the FT-IR spectrafurther indicate the existence of intermolecular H-bondingbetween the VRP–DS complex and the lipid matrix of PLN(Table 3). The intermolecular H-bonding contributes to the highmiscibility between VRP–DS complex and lipid matrix, assuggested by findings in other drug systems [27], which leads tothe molecular distribution of VRP within the solid lipid matrixof PLN.

Uniform distribution of VRP–DS complex in PLN was alsoevidenced by the TEM image of gold/VRP-PLN (Fig. 1b). Thecationic gold tracer, representing VRP, distributed randomlywithin the solid lipid matrix of PLN with no detectableaggregation. The cationic gold nanoparticles and VRP bind withDS to form a hydrophobic gold/VRP–DS complex which isincorporated in the gold/VRP-PLN.

4.4. Drug release characteristics of PLN

With the rational selection of lipid and drug–polymer pair,the release kinetics of PLN showed some unique features. Therewas an absence of initial burst release due to the uniformdistribution of VRP–DS in the PLN matrix rather than just onthe PLN surface. In addition, a strong dependence of releaseprofiles of the PLN on the properties of release mediumsupports the role of drug solubility and ion exchange in therelease mechanisms.

The much slower release of VRP from PLN in DDI waterthan in electrolyte solutions (Fig. 5a) suggests that ion exchangeis a dominating release mechanism of VRP-PLN. Despite thepolar structure of DS containing ether bonds and –OH groups,the strong electrostatic interaction between VRP and DS makesthe complex hydrophobic enough to stay in the lipid phase forprolonged times. In addition, the lipophilic nature and solidmatrix of PLN and immobility of the polymer chains slow waterpenetration and drug diffusion. In the absence of electrolytes,diffusion is the main release mechanism and thus the release rateis low.

As demonstrated by previous experimental findings andmathematical modeling [1,21,53], the ion exchange betweenthe counter ions and drug ions in the VRP–DS complex canbe used to explain the elevated VRP release rate when Ca2+

was used in place of Na+ and as NaCl concentration wasraised from zero (DDI water) to 0.005 M and to 0.015 M.However, this mechanism fails to explain why a furtherincrease in NaCl concentration to 0.15 M did not generate acorresponding higher release rate. It is possible that self-association or aggregation of the amphiphilic VRP occurs inaqueous salt solutions of high concentration [54]. This self-association could lead to a lower solubility of VRP in0.15 M NaCl solution, which offsets the gain in ion-exchange rate due to higher electrolyte concentration.

As mentioned above, the sigmoidal shape of the releasecurves of VRP-PLN in Fig. 5c (measured in pH 7.4 PBS) ismarkedly different from those in Fig. 5a and b (measured inelectrolyte solutions of pH 5.3–5.5), despite the same ionicstrength. This marked difference may be due to the pH-dependent solubility of VRP, which drops dramatically fromN100 mg/ml at pHb6.35 to 0.75 mg/ml at pH 7.4 [41,55]. VRPmolecules exist mainly as a free base at pH 7.4 and are mostlyprotonated at pHb6.5, which determines its pH-dependentwater solubility. Conceivably when the release mediumpenetrates into the tiny pores of the solid lipid matrix of PLNand the counter ions free VRP from VRP–DS complex throughion exchange, the dissociated VRP may precipitate in the pH 7.4PBS that fills the pores instead of diffuse out. The driving forcefor VRP molecules to diffuse, i.e., the drug concentrationgradient at pH 7.4, is much lower than that at the lower pH,resulting a “lag time” in drug release. As the ion exchange anddrug release progress, more flexible and hydrophilic polymerchains are produced. Eventually the dissociated DS moleculesare dissolved and diffuse out, leaving the solid lipid matrix moreporous and accessible for aqueous medium to diffuse in, whichaccounts for the later acceleration of release.

5. Conclusions

This work has demonstrated, using the VRP–DS and DAsystem as an example, that rationally designed PLN can bereadily prepared to possess high drug loading capacity andsustained release profiles. This study has elucidated theunderlying molecular mechanisms of drug–polymer–lipidinteractions which are essential for the successful developmentof a PLN system. The complexation between VRP and DS andstrong hydrogen bonding among various groups on the drug,polymer and lipid contribute to the high drug loading efficiencyand capacity, as well as the uniform distribution of VRP–DS inthe lipid matrix. The intermolecular interactions of drug–polymer–lipid account for the excellent loading properties andsustained drug release of the PLN. Owing to the ioniccomplexation of VRP and DS, drug release from PLN ismainly controlled by ion exchange and diffusion. However, thepH- and electrolyte-dependent solubility of VRP may also playa role in the release profile. The methodology and approachesdeveloped in this work can be extended to the rational design of


other drug delivery systems based on the understanding ofmolecular interactions of various components.

Acknowledgements

This work was partially supported by the Canadian Institutesof Health Research. The authors would like to thank Drs. S.Petrov, A. Baer, H. Zhang and Mr. B. Calvieri, University ofToronto, for their technical supports on the PXRD, 13C NMR,SEM and TEM imaging, respectively; Dr. T. V. Chalikian forvaluable inputs, Drs. L. P. Kotra and C. J. Allen for kindpermission on the use of freeze drier and FT-IR spectro-photometer, respectively. Financial support of the NaturalSciences and Engineering Research Council PostgraduateScholarship doctoral level (NSERC PGS D) to Y. Li, OntarioGraduate Scholarship to A. Shuhendler, and University ofToronto Top-Up awards to both are also gratefully acknowledged.

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