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Copyright copy 2012 American Scientific PublishersAll rights reservedPrinted in the United States of America

Journal ofColloid Science and Biotechnology

Vol 1 16ndash25 2012

Encapsulation and Release Behavior fromLipid Nanoparticles Model Study with

Nile Red Fluorophore

Thomas Delmas12 Amandine Fraichard1 Pierre-Alain Bayle3Isabelle Texier1lowast Michel Bardet3 Jean Baudry2Jeacuterocircme Bibette2lowast and Anne-Claude Couffin1lowast

1CEA-LETI Campus MINATEC Deacutepartement des Technologies pour la Biologie et la Santeacute17 rue des Martyrs F-38054 Grenoble France

2Laboratoire des Colloiumldes et Mateacuteriaux diviseacutes ESPCI 10 rue Vauquelin 75231 Paris cedex 5 France3Laboratoire de Reacutesonances Magneacutetiques SCIB UMR-E CEAUJF-Grenoble 1 INAC

17 rue des Martyrs F-38054 Grenoble France

Lipid nanoparticles based on nanoemulsion templates are interesting candidates for contrast agentand active ingredient transport and delivery due to their small size biodegradable nature and highversatility An optimized nanosystem for drug delivery should present an important encapsulationratio as well as controlled release kinetics In this context the influence of the lipid nanoparticlecore composition on the encapsulation and release behavior of the Nile Red fluorophore used asa model molecule of intermediate lipophilicity is explored For that purpose the lipid nanoparticlephysical state the encapsulated molecule localization and the encapsulationrelease behavior arestudied Careful characterization of lipid nanoparticles is performed by DSC and 1H NMR analysisNile Red localization is evaluated by complementary fluorescence spectroscopy and 1H NMR tech-niques The encapsulation of Nile Red is governed and limited by its solubility in the lipids A doublelocalization of the fluorophore at the membrane and in the particle core is observed resulting ina two-phase release kinetics a quick burst release followed by a slow prolonged release Addingwax to the formulation increases the lipid nanoparticle internal viscosity while favoring Nile Redlocalization in the core which results in slower release kinetics

Keywords Lipid Nanoparticles Amorphous Physical State Localization Prolonged ReleaseNile Red

1 INTRODUCTION

A major challenge in nanomedicine is to engineer nano-structures and materials that can efficiently encapsulatedrugs at high concentration cross the cell membrane andcontrollably release the cargo content at the target siteover a prescribed period of time12 Carriers of nano-metric size are promising tools to (1) protect (2) targetand (3) improve intrinsic properties of the encapsulatedmolecules Nanoencapsulation prevents the encapsulatedspecies from direct biological interactions thus drug orcontrast agent degradation by enzymes or acid envi-ronment and helps in reducing their potential systemictoxicity Moreover encapsulating active pharmaceutical

lowastAuthors to whom correspondence should be addressed

ingredients (API) can allow improving their therapeuticefficiency by controlling their biodistribution and kineticsof releaseAmong a wide variety of nanocarriers lipid-based sys-

tems present numerous advantages over other formula-tions These nanocarriers are biocompatible biodegradableand can easily be produced by versatile and up-scalableprocesses3ndash5 Such nanoparticles have been employed forencapsulating a wide range of active pharmaceutical ingre-dients and contrast agent5 Nonetheless it is still diffi-cult to prepare lipid nanocarriers with high encapsulationratios of actives while controlling their kinetics of releaseThe internal physical state of lipid core nanoparticles hasbeen shown to dramatically affect the encapsulation andrelease properties Nanoemulsions were the first lipid core

16 J Colloid Sci Biotechnol 2012 Vol 1 No 1 2164-963420121016010 doi101166jcsb20121010


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Delmas et al Encapsulation and Release Behavior from Lipid Nanoparticles Model Study with Nile Red Fluorophore

nanocarriers to be introduced decades ago but few of thesesystems finally came to market due to formulation issuesIndeed these systems suffer from low colloidal stabilityand sustained release is difficult to achieve due to thelow viscosity of the dispersed phase6 This generally leadsto the rapid diffusion of the drugs out of the dropletsSolid lipid nanoparticles (SLN) have thus been proposedto overcome these limitations thanks to their solid coreHowever despite high expectations of such systems forprolonged release of hydrophobic molecules SLN haveshown limited controllability Crystallization of the lipidphase generally leads to druglipid phase separation andsubsequent drug expulsion providing high burst release78

Nanostructured lipid carriers (NLC) were introduced as acompromise Composed of a mixture of liquid and solidlipids the NLC core presents an imperfect crystallizationwhich favors better encapsulation ratio thanks to lowercrystallinity while allowing control over release kineticsthrough the solid character of the lipid phase9 Three dif-ferent types of NLC have been developed(1) the imperfect type whose crystallinity is lowered bycreating imperfections in the crystal lattices(2) the structureless type which is solid but amorphousand(3) the multiple OilFatWater type in which smalldroplets of liquid lipids are phase separated in the solidmatrix10

The first type has been widely reported and used to effi-ciently increase the encapsulation ratios while maintain-ing significant prolonged release11 Nonetheless whereasthe other types should theoretically allow better encapsula-tionrelease properties there is a discrepancy in the conclu-sions concerning their practical achievement For instancethe structureless type could allow high encapsulation ratioby avoiding lipid organization while favoring prolongedrelease thanks to its solid nature However some authorsaccount that their lipid core is in a super cooled melt staterather than an amorphous solid912

In this context our research group has proposed a newtype of very stable amorphous lipid core nanoparticles(LNP)13 Such a nanoemulsion system can efficiently beproduced by ultrasonication and possesses a long-term sta-bility through entropic stabilization14 In order to use theseLNP as nanocarriers for drug andor contrast agent encap-sulation and delivery their drug encapsulation and releasebehaviors have to be characterized Highly hydrophobiccontrast agents have already successfully been encapsu-lated in LNP for imaging purposes1516 In this study theNile Red (NR) fluorophore is used as a model moleculeof intermediate lipophilicity (logP sim 3ndash5) such as thepotent chemotherapeutic Paclitaxel to study the influenceof the lipid particle core composition on the LNP encap-sulation and release behaviors This low cost moleculecompared to active pharmaceutical ingredients possessesmicroenvironment-sensitive fluorescence properties rele-vant for localization purposes17ndash19 This molecule is of

interest to cost-effectively get insight into the LNP encap-sulation localization and release behaviors before prop-erly optimizing the system with the desired API Thepresent paper aims to understand how internal compositioninfluences NR encapsulation and release profile from thelipid nanoparticles For that purpose we first investigatethe influence of core composition on the LNP physico-chemical properties and the core physical state We thenstudy how it affects the encapsulation and localizationof NR molecules and finally how NR release kinetics ismodified

2 MATERIALS AND METHODS

21 Materials

The wax Suppocire NBtrade (mixture of mono- di- and tri-glycerides of alkyl chain length varying from C12 to C18is a kind gift from Gattefosseacute (Saint-Priest France) Myrjs40trade (polyethylene glycol 40 stearate) and Super-refinedSoybean oil are kindly donated by Croda (ChocquesFrance) The phospholipids Lipoid s75trade (composed ofgt75 phosphatidylcholine) are purchased from Lipoid(Ludwigshafen Germany) Other chemical products arepurchased from Sigma Aldrich (Saint Quentin FallavierFrance)

22 LNP Processing

LNP are prepared by emulsion templating through ultra-sonication Both aqueous and lipid phases are separatelyprepared before mixing The lipid phase contains a blendof solid (Suppocire NBtrade) and liquid (Super refined Soy-bean oil) glycerides with phospholipids (Lipoid s75trade)while the aqueous phase is composed of a PEG surfactant(Myrj s40trade) dissolved in PBS aqueous buffer (1times Phos-phate Buffer Saline 10 mM phosphate 154 mM NaClpH 74) Standard LNP formulations are prepared at 10weight fraction with 1800 L PBS 92 mg Myrj s40trade90 mg oilwax mixture and 18 mg Lipoid s75trade Afterhomogenization at 45 C both phases are crudely mixedThen sonication cycles are performed at 45 C during a5 min period (VCX750 Ultrasonic processor 3 mm probeSonics France sonication power 25) Non encapsulatedcomponents are separated from LNP dispersions by gentledialysis overnight in 1X PBS against 1000 times their vol-ume (MWCO 12000 Da ZelluTrans) Before use LNPdispersions are filtered through 022 m cellulosic mem-brane (Millipore)In the case of LNP loaded with 01 ww Nile Red a

solution of 120 L Nile Red 10 mM in CH2Cl2 is addedto the oily phase and the solvent is evaporated Then thisdoped oily phase is used as aforementioned to produceLNP encapsulating Nile Red at a local concentration of23 nmolmg dispersed phase

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Encapsulation and Release Behavior from Lipid Nanoparticles Model Study with Nile Red Fluorophore Delmas et al

Different waxoil ratios (ww) in the core have beenused For simplification LNP samples are quoted NCXXwhere XX defines the weight fraction of wax in the LNPcore ( ww)

23 Characterization

231 Dynamic Light Scattering (DLS)

The particle size and zeta potential of the lipid nanopar-ticles are measured using a Malvern Zeta Sizer Nanoinstrument (NanoZS Malvern UK) The nanoparticle sus-pensions are diluted 100 folds with 01times PBS buffer Atleast three different LNP batches are used per conditionResults are expressed in terms of mean and standard devi-ation of all the samples for each condition each sampleresult being the mean of three independent measurementsperformed at 25 C

232 Differential Scanning Calorimetry (DSC)

DSC analysis is conducted using a TA Q200 system (TAinstrument France) Samples are weighed (2 to 5 mgfor bulk material and 10 to 20 mg LNP dispersion) intostandard aluminum sample pans which are then sealedwith a pinhole-pierced cover and an empty pan is used asreference Heating curves are recorded at a scan rate of10 Kmin from 20 C to 70 C

233 Viscosity of Lipid Core Mixtures at 40 C

The rheological properties of NC0 NC50 and NC100 bulksamples are determined using an AR 2000 rheometer (TAinstrument France) with a cone and plate fixture (diameter10 mm angle 1) Measurements are performed at 40 C inorder to avoid macroscopic issues of sample crystallinityShear rates ranging from 01 to 300 sminus1 are applied andthe viscosity value is extracted from the Newtonian flow

234 NMR Spectroscopy

1H NMR spectra are recorded using an Avance DPX500 spectrometer (Bruker Wissembourg France) operat-ing at 500 MHz They are processed and analyzed withthe MestRe-C v30 software The assignment of the 1Hand 13C NMR resonances of PEG-40-stearate (Myrj s40trade)lecithin and triglycerides mixtures (soybean oil and sup-pocire NCtrade is achieved by standard methods with theacquisition of 1H 13C and 1Hndash1H homonuclear correlation(COSY) spectra14 An accurate quantity of 01 ww ofsodium 22-dimethylsilapentane-5-sulphonate (DSS) (foraqueous samples) or tetramethylsilane (TMS) (for chloro-form dissolved samples) is added as a 0 ppm and resolu-tion reference LNP spectra are recorded at temperaturesranging from 10 to 60 C

For localization of Nile Red molecules in the LNP struc-ture the 7ndash9 ppm spectral window is particularly exploredIn order to amplify the NR signals and then selectivelyrecord them a selective 90 eBurp proton pulse coveringa spectral range of about 2000 Hz is appliedIn order to localise at a molecular level the NR within

the LNP homonuclear protonndashproton dipolar interactionsare looked for For that purpose 1H NOE (Nuclear Over-hauser Effect) measurements were carried out by applyinga pre-saturation time of 5 s 1H NOE difference spec-tra were obtained by alternatively irradiating the peak ofinterest and a control point in the spectrum followed bydata acquisition For each spectrum 2048 transients wereacquired Selective pulse was also used to favour the detec-tion of NR compound Data processing was performedin the frequency domain by subtracting the on-resonanceand off-resonance spectra to obtain the so-called NOED-IFF (Nuclear Overhauser Effect Difference Spectroscopy)spectra The interactions of NR with other componentscan be directly visualized on the resulting spectrum by theappearance of NOE signals

235 Fluorescence Spectroscopy

NR localization study is performed using a TECAN Infi-nite 1000 spectrometer (TECAN Lyon France) First NileRed fluorescence spectra Xiexc = 505 nm) are recordedseparately in the different LNP components i pure oil(01 ww dye) pure wax (01 ww dye) PEG surfac-tant aqueous solution (with concentrationgt critical micelleconcentration 01 ww dye) phospholipid aqueous solu-tion (01 ww dye) Each fluorescence spectrum Xi isthen normalised by the fluorescence intensity integral ofthe NR fluorescence spectrum in the component i to givenormalized fluorescence spectrum xi

Fluorescence spectra Y (exc= 505 nm) are thenrecorded for the different LNP samples with differentwaxoil core ratios (01 ww dye loading) The fluores-cence spectrum of each NR-loaded LNP (as displayed inFig 3(a)) is then normalised by the overall fluorescenceintensity under the curve to obtain ldquonormalizedrdquo fluores-cence spectra y of Nile Red dispersed in LNP as displayedin Figure 3(a) insert In order to get qualitative informationabout Nile Red localization each normalized spectrum yof LNP NCXX is then deconvoluted over the normalizedNR spectra xi obtained previously in the different LNPcomponents using the Excel software For this purposethe proper i coefficients allowing minimizing the differ-ence between the experimental LNP spectrum and the fitspectrum (yminus ixi with i = 1 are determined i

represents the of Nile Red localized in the i compart-ment Due to the significant spectral overlap between thefluorescence spectra of Nile Red in oil and wax on oneside which represent core compartimentation and PEGsurfactants and phospholipids on the other side which rep-resent shell location qualitative NR localization is then

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expressed as of NR localized in the two main LNPstructural compartments the core (wax and oil) and theshell (PEG surfactants and phospholipids)

24 NR Encapsulation Efficiency

Previous to quantification of Nile Red payload in nanopar-ticles the fluorophore is extracted after destruction of thenanoparticles according to the following protocol adaptedfrom the literature20 Briefly extraction is performed bydissolving 200 L LNP dispersion (10 weight fraction)in tetrahydrofuran (ratio 13 vv) at room temperature fol-lowed by a selective lipid precipitation in methanol (ratio12 vv) at 4 C After centrifugation the supernatant is fil-tered on a Minisartreg 02 m filter (Sartorius GoettingenGermany) and an aliquot of 20 L of filtrate is analyzed byHPLC-UV method The efficiency of this extraction proto-col is gt95 Chromatographic separation is carried out ona Supelcosil C18 column (250times46 mm 5 m) (SupelcoUSA) using a Waters 2495 separation module with aWaters 2487 dual absorbance spectrometer (Waters Saint-Quentin-en-Yvelines France) at detection wavelengths of227 nm and 552 nm The elution is performed with an iso-cratic mixture of MethanolWater (9010 vv) with a flowrate of 1 mLmin With this protocol NR retention time is72 min Quantification is achieved by comparison betweenthe Nile Red observed peak area for n = 3 samples performulation with a calibration curve obtained under thesame conditionsThe encapsulation efficiency is calculated by dividing

the amount of Nile Red still encapsulated in the LNP afterpurification by dialysis by the amount of Nile Red insideLNP before purification (Eq (1)) This is performed byNR extraction and quantification over the purified samples(right after dialysis) by the previously described HPLCprotocol The maximum loading ratio is then given by themaximum payload achievable without significant loss dur-ing the purification step (encapsulation efficiency gt80)

Encapsulation efficiency

= amount of NR after dialysisamount of NR before dialysis

times100 (1)

25 In Vitro NR Release Kinetics

The release study is carried out using a dialysis pro-tocol In brief after preparation NR loaded LNP sam-ples (300 L of 10 dispersed phase weight fraction)are placed in 500 L Quix Sep micro-dialysis capsules(Fisher Bioblock Illkirch France) and subjected to inten-sive dialysis in 1000 times their volume (MWCO 12000Da MW ZelluTrans) The amount of molecules still encap-sulated is determined by dividing the payload of NR inLNP after different periods of time to the original payloadafter LNP preparationpurification following the extrac-tionHPLC quantification protocol previously described

The release rate is then given by Eq (2) Temperature-dependent release kinetics is performed in 1times PBS bufferat three temperatures (4 C room temperature (25 C) and40 C) over 8-day periods

release=(1minus amount of NR after dialysis

amount of NR before dialysis

)times100

(2)

3 RESULTS

31 LNP Formulation

The nanocarrier system described herein is based on anoil-in-water emulsion The lipid nanoparticles (LNP) arecomposed of an oily core (mixture of solid and liquidlipids) which is stabilized by a surfactant shell combi-nation of hydrophilic (PEG surfactant) and lipophilic sur-factants (phospholipids) The oily phase contains soybeanoil (Super Refined Oiltrade) semi-synthetic wax (SuppocireNBtrade or a mixture of both in which saturated and unsat-urated long-chain triglycerides (saturated C12ndashC18 for Sup-pocire NBtrade and unsaturated C16ndashC18 for soybean oil)are present Due to their high lipophilic character thesespecies have very low solubility in the continuous phasethen restraining possible destabilization of the colloidaldispersion through Ostwald ripening13 Moreover soy-bean oil is acknowledged for its biocompatibility andis FDA-approved for parenteral injection Such compo-sition allows obtaining long term stable formulations asaqueous dispersions1314 It was previously demonstratedthat adding wax to the lipid core increases LNP coreviscosity13 This study is focused on 50 nm-diameter par-ticles obtained by optimization through an experimen-tal design14 This formulation has been selected for itssize and neutral surface charge ensuring low polydis-persity favoring physical stability by Ostwald Ripeningprevention13 and passive accumulation in tumors1521

A series of formulations with similar surfactant shellcomposition and constant lipid core weight are first pre-pared The lipid core composition ranges from NC0 (0wax100 oil) to NC100 (100 wax0 oil) while NRloading is varied from 001 to 01 ww of the dispersedphase Changing core composition does not affect the par-ticle surface charge (zeta potential) while great amountof oil in lipid core tends to increase the LNP diameter(Table I) Indeed a 10 increase in hydrodynamic diam-eter is observed when increasing the amount of oil in thecore from pure wax to pure oil core nanoparticles Aspreviously detailed such increase in LNP diameter whenvarying the oilwax ratio has to be related to the differencesof viscosity between the wax and oil dispersed phases dur-ing the sonication process13 Meanwhile the encapsulationof Nile Red does not significantly affect the surface chargeand particle size properties The zeta potential thus remainsnearly neutral with values close to minus6 mV



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Table I LNP physicochemical properties as a function of core composition and Nile Red loading particle hydrodynamic diameter (d) and zetapotential ( (average and standard deviation over n= 3 samples)

Core composition

NC0 NC50 NC100NR concentration( ww dispersed phase) d (nm) (mV) d (nm) (mV) d (nm) (mV)

0 652plusmn13 minus59plusmn21 589plusmn09 minus60plusmn11 502plusmn25 minus43plusmn08001 645plusmn41 minus72plusmn21 614plusmn29 minus65plusmn23 546plusmn19 minus52plusmn19005 667plusmn42 minus69plusmn26 621plusmn32 minus59plusmn31 563plusmn18 minus59plusmn2401 684plusmn38 minus65plusmn22 644plusmn31 minus68plusmn29 557plusmn21 minus60plusmn31

32 Influence of Core Composition on InternalPhysical State

The LNP internal physical state is investigated by Dif-ferential Scanning Calorimetry (DSC) and 1H NMRtechniquesDSC analysis allows investigating whether the lipids are

crystallized As compared to bulk lipid mix DSC thermo-grams of 50 nm-diameter particles do not display any ther-mal event along heating from room temperature to 60 CIt therefore indicates that lipids are under amorphous statewhen nanoformulated as LNP whereas they are in a crys-talline state as a bulk This was observed to be true what-ever the LNP internal composition from NC0 to NC100(0 wax to 100 wax) over 8-month storage at both 4 Cand room temperatureThe LNP internal viscosity was monitored by NMR

spectroscopy by following the evolution of LNP 1H NMRspectra as function of core composition and temperatureIndeed peak resolution is related to molecular mobilitywhen increasing the viscosity of a sample peak resolutionis seen to decrease in liquid-state 1H NMR through peakbroadening In extreme limit for a completely solid matrixno signal is expected to appear in spectra recorded underliquid-state NMR conditions As previously described inthe LNP 1H NMR spectrum it is possible to assign specificsignals representative of particle locations14 Two signals(labeled by square and triangle symbols) are located at13 ppm and 37 ppm respectively attributed to the methylprotons of alkyl chains and the methylene protons ofPEG surfactant (Fig 1(a)) Whatever core composition thewidth related to PEG chain peaks does not change whiletemperature decreases from 35 C to 10 C as depicted bypeak width ratio close to 09ndash10 in Figure 1 These chainsbelonging to the external membrane and being exposed towater are not significantly affected by changes in the lipidcore internal composition On the contrary when lookingat a peak specific to the internal lipids (13 ppm) sig-nificant differences can be observed Indeed whereas thepeak width ratio is close to 095 and thus the peak reso-lution only slightly decreases when going from 35 C to10 C in the case of pure oil core (NC0) loss of resolu-tion and peak broadening are more significant with a corecomposed of 50 oil50 wax (NC50) and even more

Fig 1 Particle internal viscosity assessed by 1H NMR (a) 1H-NMRspectrum of LNP NC75 at room temperature (b) Peak width ratio attwo different temperatures (10 C and 35 C) as a function of corecomposition (NC0 NC50 and NC100)

when looking at a pure wax core LNP (NC100) these twocompositions displaying peak width ratios of respectively045 and almost 025 (Fig 1(b)) These differences in peakresolution account for significant differences in viscositiesdepending on core composition and temperature The morewax in the core and the colder the more viscous

33 Nile Red Encapsulation Efficiency

The influence of core composition on the maximum NRpayload is investigated by formulating LNP of differentwaxoil core ratio with increasing amounts of Nile RedThe encapsulation efficiency is determined following NRextraction from nanoparticles by quantification using theHPLC protocol described in the Materials and Methodssection (Fig 2) The maximum accessible loading ratio isgiven by the maximum payload achievable without sig-nificant loss during the purification process The limit of



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Enc

apsu

latio

n ef

fici

ency

(

ww

)

NR concentration ( ww)

20

40

60

80

100

0001 001 01 1

NC 0

NC 50

NC 100

0

20

40

60

80

100

0001 001 01 1

NC 0

NC 50

NC 100

Fig 2 Nile Red encapsulation determination of the maximum payload(mean and standard deviation over n= 3 samples per condition) for dif-ferent core compositions (NC0 diamond symbol NC50 square symbolNC100 triangle symbol) A picture of LNP encapsulating Nile Red atincreasing concentrations is given at a glance

significant loss is fixed at 20 since the LNP process-ing efficiency is close to 80ndash85 due to the low volumesused which leads to some material loss on the glass vialsAll core compositions behave similarly giving a maximumpayload of around 01 ww (Fig 2) Modulating the corecomposition by varying the waxoil core ratio thereforedoes not allow modifying the NR loading ratio

34 Nile Red Localization

Nile Red localization is investigated by fluorescence and1H NMR studies As Nile Red fluorescence is known tobe very sensitive to its microenvironment1722 this featureis used to evaluate the dye localization once encapsulatedin LNP of different core compositions The NR fluores-cence spectra while dispersed at 01 ww in pure oilpure wax or in PEG or phospholipid aqueous solutionsare recorded (Fig 3(a) insert) As already described inthe literature fluorescence intensity and peak maximumshift are observed1718 Apolar environment (oil and wax)lead to emission maxima of asymp580ndash610 nm whereas morepolar environment (phospholipids and PEG surfactants)lead to an emission maximum of around asymp630ndash640 nmFluorescence spectra recorded for the different NR loadedLNP compositions are reported in Figure 3(a) highlightingsignificant differences depending on particle core compo-sition It is also noticed that these fluorescence profileshave a very large bandwidth in comparison to the emis-sion bands observed for NR in the presence of each ingre-dient taken separately (Fig 3(a)) Therefore it appearsthat NR loaded LNP emission results from the differentcomponents Each LNP fluorescence spectrum is there-fore deconvoluted over the four different components toachieve a qualitative analysis of NR localization (Materialsand Methods section) Nevertheless the significant over-lapping of NR fluorescence within LNP membrane com-ponents (phospholipids and PEG surfactant) and LNP core

Fig 3 Nile Red localization assessed by fluorescence analysis(a) Fluorescence spectra of Nile Red-loaded LNP with different corecompositions insert deconvolution of the fluorescence spectrum of NileRed-loaded LNP NC50 using Nile red fluorescence spectra in the dif-ferent components of LNP (soybean oil Suppocire NBtrade phospho-lipids Myrj 53trade) (b) Nile Red localization as a function of LNP corecomposition

components (wax and oil) lead us to consider two-partitionlocalization for NR molecules (core and shell compart-ments) This analysis reveals that Nile Red is located bothin the membrane and in the lipid core with various per-centages (Fig 3(c)) Interestingly the relative proportionof Nile Red at the particle shell or into the core dependson the lipid core composition Indeed the more wax in thecore the more NR is located into the coreTo gain further insight into this NR double localiza-

tion NR loaded LNP of NC75 core composition (75wax25 oil) are studied by classic 1H NMR spectroscopyand NOEDIFF (Nuclear Overhaser Effect Difference Spec-troscopy) experiment First a temperature study is per-formed recording the evolution of peak resolution andwidth associated to NR molecules as a function of temper-ature As can be seen in Figure 4(a) the resolution of NileRed peaks dramatically decreases when cooling the systemfrom 35 C to 10 C (focus in the 70 to 90 ppm range forwhich only Nile Red peaks are observed) This decreasein peak resolution is related to a decrease in molecular



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Fig 4 Nile Red localization assessed by NMR study (a) Nile Red chemical structure and selective pulse effect of the temperature (b) NOEDIFFspectra at 25 C by irradiation of respectively 37 33 and 13 ppm signals (Nile Red specific peaks are marked by an arrow) Formulation used NC75encapsulating 01 ww NR

mobility due to a microenvironment viscosity increaseas explained above for core components Nonethelesseven at 10 C it is still possible to observe peaks eventhough they are dramatically broaden This qualitativelysupports the fact that most NR molecules are localizedin the particle core but also suggests that a part of themremains located at the surface when considering a NC75core composition To finally conclude about NR localiza-tion complementary quantitative NOEDIFF experimentsare performed The principle is to use NOE transfer tolocalize NR molecules in the LNP structure by evaluatingthe efficiency of magnetization transfer between LNP com-ponents and NR molecules The characteristic NMR peaksassociated to PEG chains (37 ppm) choline heads ofthe lecithin (33 ppm) and hydrocarbon part (13 ppm) ofthe core components are thus excited individually and theeffect on Nile Red signals is assessed (Fig 4(b)) For sim-ilar excitation conditions larger signals are obtained whenexciting core components (hydrophobic chains) comparedto PEG chains or lecithin Yet here again some signalsare still observed with membrane components (lecithinand PEG) These NOEDIFF results are consistent witha double localization already suggested by fluorescencespectroscopy These findings confirm that most of NR

molecules localizes in the core while a small amount alsolocalizes at the membrane of LNP

35 Nile Red In Vitro Release

The influence of core composition and temperature on NileRed release kinetics is then investigated For that purposeLNP formulations with 3 different waxoil ratios (NCONC50 NC100) at the maximum payload (01 ww NR)are prepared and purified before undergoing in vitrorelease under dialysis at different temperatures 4 C25 C and 40 C The results are displayed in Figure 5Each release curve presents two noticeable parts (1) aquick burst release occurring during the first 5 hoursand (2) a slow prolonged release for times gt5 hoursWhen considering the burst release no significant temper-ature effect is observed and 20ndash30 of the encapsulatedmolecules escape from LNP during the first 5 hours of theexperiments Temperature has a more pronounced influ-ence on the prolonged release Indeed whereas no sig-nificant prolonged release is observed over 150 hours at4 C and 25 C 70ndash80 NR molecules are released at40 C after the same time More interestingly whereasno influence of core composition is observed at 4 C and



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Fig 5 Nile Red in vitro release at different temperatures (a) 4 C(b) 25 C and (c) 40 C LNP of 3 different core compositions loadedwith 01 NR ww are compared NC0 (diamond symbol) NC50 (squaresymbol) and NC100 (triangle symbol)

25 C on the burst and prolonged release significant dif-ferences occur at 40 C Increasing the amount of waxin the core thus leads to a reduced burst release (up to40 for NC0 35 for NC50 and less than 25 forNC100) (Fig 5(c)) Similarly increasing the amount ofwax in the core leads to slower prolonged release kineticsFor NC0 LNP NR release levels off at 80 of total NRmolecules after 75 hours In parallel such level of releaseis reached after 150 hours for NC50 LNP and is still notachieved after 150 hours for NC100 LNP samples

4 DISCUSSION

41 Influence of Dye Solubility and Localization onEncapsulation

LNP core composition does not significantly change theirNR loading capabilities and different core compositionslead to similar maximum loading of about 01 ww This

has to be related to the NR solubility in the melted macro-scopic mixtures of lipid compounds (Table II) IndeedNR is slightly more soluble in wax containing mixturesafter 3 weeks storage at room temperature NonethelessNR crystallizes in all lipid mixtures when at concentra-tions higher than 01 ww Both liquid and solid lipidsare triglycerides with similar chemical entities Soybeanoil is composed of fatty acids with C16ndashC18 alkyl chainlength while Suppocire NBtrade is a more complex mix-ture of mono- di- and triglycerides with various lengthsof alkyl chain ranging from C12 to C18 with a hydroxylvalue of 20ndash30 Therefore the presence of partial di-and monoglycerides and the variation in chain lengthcould improve the NR solubility in the presence of thewax However all tested lipid mixtures exhibit a solu-bility limit close to 01 ww in lipid bulks As NRhas a double localization once nanoformulated (in thecore and in the membrane) this slight variation of coresolubility (lipid solubility) could be compensated by amembrane re-localization making all LNP samples dis-play a similar 01 ww maximum loading capacity Thislow loading can be explained by the low lipophilicityof the NR molecule (logP = 35) and is emphasized bythe huge amount of hydrophilic surfactants correspondingto almost 50 ww of the dispersed phase This load-ing ratio is well below the classical 10 API contentas encountered in well-established pharmaceutical formu-lations which should be sufficient to allow the requiredtherapeutic dose of API to be administrated In compari-son loading ratios up to 6ndash10 ww in LNP have beensuccessfully achieved for molecules with higher lipophiliccharacter such as the DiD fluorophore16 or photosensi-tizers used in photodynamic therapy Although chemicalmodifications of the desired API may be performed inorder to increase its lipophilicity by alkyl chains graft-ing the LNP system offers a promising suitability as highlipophilicity API delivery systemsYet whereas not significantly impacting the maximum

loading achievable changing core composition notablyaffects NR localization as demonstrated by fluorescenceand NMR studies (Figs 3 and 4) Adding wax to the parti-cle core promotes localization of NR molecules inside thelipid core instead of at the membrane compartment This

Table II Nile Red solubility in bulk lipid mixtures just after preparation(t0 and after 3-week storage at 25 C soluble (sol) insoluble (times) andappearance of a few crystals (asymp

NR concentration ( ww dispersed phase)

005 01 025Bulk lipidmixtures t0 t0 +3 weeks t0 t0 +3 weeks t0 t0 +3 weeks

NC0 Sol Sol Sol times times timesNC50 Sol Sol Sol asymp times timesNC100 Sol Sol Sol Sol times times



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feature is consistent with the preferential solubility of NRdye in wax compared to soybean oil

42 Release Profiles

As evidenced in Figure 5 the NR release profile fromLNP presents a two-step process (1) a burst release and(2) a prolonged release It has first to be noted that LNPsize remains nearly constant along the release experimentstherefore suggesting that NR release does not rely on parti-cle destabilization The burst release frees around 20ndash30of total NR molecules during the first 5 hours of experi-ments At 4 and 25 C the amount of released moleculesis not significantly affected by core composition How-ever at 40 C increasing the amount of wax in the coreleads to decreased burst release Interestingly the amountof NR molecules released during the burst phase increaseswith the amount of NR localized at the membrane asquantified by the fluorescence measurements (Fig 6(a))These NR molecules belonging to the shell in contact withthe continuous aqueous phase could easily be releaseddue to osmotic pressure exerted on the PEG surfactant byhigh dilution and intensive dialysis in micro dialysis cap-sules This occurrence can lead to surfactant desorption

Fig 6 Nile Red burst release (a) Correlation between Nile Red mem-brane localization (assessed by fluorescence spectroscopy) and Nile Redburst release for 3 different core compositions (NC0 NC50 NC100) at25 C (b) Nile Red release from NC100 LNP loaded with 01 wwfluorophore release at 40C in PBS versus release at 40 C in PBSsaturated with PEG surfactants (Mirj s40trade above their critical micelleconcentration)

and could account for the observed burst release To fur-ther support this hypothesis a complementary NR releaseexperiment is performed lowering the osmotic pressureon the surfactants by saturating the release medium withsurfactants (with a surfactant PEG concentrationgt criticalmicelle concentration) As shown in Figure 6(b) burstrelease occurs during the first 15 minutes and is signifi-cantly reduced under saturated medium conditions After2 hours both releases become similar probably due to thesolubilization of NR in the presence of PEG surfactantsmicellesMeanwhile the Nile Red prolonged release is notably

affected by temperature and core composition (Figs 5 7)Indeed whereas no prolonged release is observed at 4 Cand 25 C a significant release is obtained at 40 Cand the kinetics of release is dramatically slowed downwhen adding wax to the LNP core (Fig 7(a)) As noconvection process can exist on such nano-compartmentNile Red release should be related to the diffusion of theNR molecules through the LNP structure in particularthrough the lipid matrix as already described for otherlipid nanocarriers2324 Since the nanoscale does not allowus to assess the micro-viscosity of the particle lipid corewe performed viscosity measurements on the lipid bulksat 40 C (temperature for which all lipid blends are under

Fig 7 Nile Red prolonged release (a) Effect of temperature and com-position on Nile Red prolonged release (b) Correlation between the vis-cosity of the bulk lipid mixtures (NC0 NC50 and NC100) and prolongedLNP release kinetics at 40 C



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liquid form) As shown in Figures 7(b) at 40 C the pro-longed release kinetics scales as the inverse of the lipidmixture viscosity as expected for a diffusion-driven pro-cess according to the Einstein-Stokes law It is indicativethat the prolonged release kinetics mainly depends on theintrinsic micro-viscosity of the LNP core Diffusion ofmolecules is a temperature-activated process25 From thisstudy it can be stated that it is possible to modify therelease kinetics by varying the core composition and thetemperature release conditions which affect the microvis-cosity and the associated local diffusion coefficient of theencapsulated molecules

5 CONCLUSION

The relationship between the core composition of lipidnanoparticles their internal physical state the localizationof encapsulated species and their encapsulationreleaseproperties was here studied using Nile Red as a modelof intermediate lipophily molecule We demonstrated theamorphous character of LNP and further underlined thatchanging the particle core composition allows its inter-nal physical state to be tuned from very fluid to highlyviscous Changing the LNP core composition has beenshown to change the Nile Red localization and dramati-cally affect NR release Whatever release condition a burstrelease which may rely on desorption of PEG surfactantswas observed in the first 5 hours Under storage conditions(4 C and 22 C) we expect no significant leakage as longas no surfactant desorption can occur Under physiologi-cal conditions the release kinetics is mainly driven by theinternal physical state of LNP nanoparticles Increasing theamount of wax in the core is shown to significantly slowthe Nile Red release kinetics Attempts will be made infurther investigations to reinforce the LNP shell by mod-ifying membrane composition and making it more rigidand therefore limiting burst release The micro-viscosityof the LNP core related to lipid composition could helpcontrolling the release kinetics This fundamental under-standing of the physicochemical basis directing the kinet-ics of release should now allow the fine tuning of suchbehavior for further applications of the LNP nanocarrieras a drug delivery system

Abbreviations

API Active Pharmaceutical Ingredient DLS DynamicLight Scattering DSC Differential Scanning CalorimetryLNP Lipid NanoParticles NOEDIFF Nuclear OverhauserEffect Difference Spectroscopy NR Nile Red PBS Phos-phate Buffer Saline

Acknowledgments We acknowledge the Molinspira-tion Property Calculation Service (wwwmolinspirationcom) for logP calculation The authors thank CatherinePicart and Thomas Boudou for help during the fluores-cence measurements and Rachel Auzely and Eric Baymafor rheology support This work was supported by theCommissariat agrave lrsquoEnergie Atomique et aux Energies Alter-natives (CEA) and the French National Research Agency(ANR) through Carnot funding

References and Notes

1 V P Torchilin Adv Drug Deliv Rev 58 1532 (2006)2 L Zhang F X Gu J M Chan A Z Wang R S Langer and

O C Farokhzad Clin Pharmacol Ther 83 761 (2008)3 M D Joshi and R H Muumlller Eur J Pharm Biopharm 71 161

(2009)4 W Mehnert and K Maumlder Adv Drug Deliv Rev 47 165 (2001)5 R H Muumlller K Maumlder and S H Gohla Eur J Pharm Biopharm

50 161 (2000)6 B Mangenheim M Y Lvy and S Benita Int J Pharm 94 115

(1993)7 H Bunjes M H J Koch and K Westesen Langmuir 16 5234

(2000)8 C Freitas and R H Muumlller Eur J Pharm Biopharm 47 125

(1999)9 H Bunjes K Westesen and M J H Koch Int J Pharm 129

(1996)10 R H Muumlller M Radtke and S A Wissing Int J Pharm 242 121

(2002)11 E B Souto and R H Muumlller Handbook of Experimental

Pharmacology edited by M Schaumlfer-Korting Heidelberg (2010)pp 115ndash141

12 K Westesen and H Bunjes Int J Pharm 115 129 (1995)13 T Delmas H Piraux A C Couffin I Texier F Vinet P Poulin

M E Cates and J Bibette Langmuir 27 1683 (2011)14 T Delmas A C Couffin P A Bayle F de Creacutecy E Neumann

F Vinet M Bardet J Bibette and I Texier J Colloid Interf Sci360 471 (2011)

15 I Texier M Goutayer A Da Silva L Guyon N DjakerV Josserand E Neumann J Bibette and J Vinet J Biomed Opt14 054005 (2009)

16 J Gravier F Navarro T Delmas F Mittler A C Couffin F Vinetand I Texier J Biomed Opt 16 096013 (2011)

17 P Greenspan and S D Fowler J Lipid Res 26 781 (1985)18 A Y Jee S Park and M Lee Chem Phys Lett 477 112 (2009)19 C Lin J Zhao and R Jiang Chem Phys Lett 464 77 (2008)20 A Lamprecht and J P Benoicirct J Chromat B 787 415 (2003)21 M Goutayer S Dufort V Josserand A Royegravere E Heinrich

F Vinet J Bibette J L Coll and I Texier Eur J PharmBiopharm 75 137 (2010)

22 V Teeranachaideekul P Boonme E B Souto R H Muumlller andV B Junyaprasert J Control Release 128 134 (2008)

23 V Teeranachaideekul R H Muumlller and V B Junyaprasert Int JPharm 340 198 (2007)

24 J J Wang K S Liu K C Sung C Y Tsai and J Y Fang Eur JPharm Sci 38 138 (2009)

25 J Guery J Baudry D A Weitz P Chaikin and J Bibette J PhysRev E 79 060402(R) (2009)

Received 15 February 2012 Accepted 6 March 2012



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nanocarriers to be introduced decades ago but few of thesesystems finally came to market due to formulation issuesIndeed these systems suffer from low colloidal stabilityand sustained release is difficult to achieve due to thelow viscosity of the dispersed phase6 This generally leadsto the rapid diffusion of the drugs out of the dropletsSolid lipid nanoparticles (SLN) have thus been proposedto overcome these limitations thanks to their solid coreHowever despite high expectations of such systems forprolonged release of hydrophobic molecules SLN haveshown limited controllability Crystallization of the lipidphase generally leads to druglipid phase separation andsubsequent drug expulsion providing high burst release78

Nanostructured lipid carriers (NLC) were introduced as acompromise Composed of a mixture of liquid and solidlipids the NLC core presents an imperfect crystallizationwhich favors better encapsulation ratio thanks to lowercrystallinity while allowing control over release kineticsthrough the solid character of the lipid phase9 Three dif-ferent types of NLC have been developed(1) the imperfect type whose crystallinity is lowered bycreating imperfections in the crystal lattices(2) the structureless type which is solid but amorphousand(3) the multiple OilFatWater type in which smalldroplets of liquid lipids are phase separated in the solidmatrix10

The first type has been widely reported and used to effi-ciently increase the encapsulation ratios while maintain-ing significant prolonged release11 Nonetheless whereasthe other types should theoretically allow better encapsula-tionrelease properties there is a discrepancy in the conclu-sions concerning their practical achievement For instancethe structureless type could allow high encapsulation ratioby avoiding lipid organization while favoring prolongedrelease thanks to its solid nature However some authorsaccount that their lipid core is in a super cooled melt staterather than an amorphous solid912

In this context our research group has proposed a newtype of very stable amorphous lipid core nanoparticles(LNP)13 Such a nanoemulsion system can efficiently beproduced by ultrasonication and possesses a long-term sta-bility through entropic stabilization14 In order to use theseLNP as nanocarriers for drug andor contrast agent encap-sulation and delivery their drug encapsulation and releasebehaviors have to be characterized Highly hydrophobiccontrast agents have already successfully been encapsu-lated in LNP for imaging purposes1516 In this study theNile Red (NR) fluorophore is used as a model moleculeof intermediate lipophilicity (logP sim 3ndash5) such as thepotent chemotherapeutic Paclitaxel to study the influenceof the lipid particle core composition on the LNP encap-sulation and release behaviors This low cost moleculecompared to active pharmaceutical ingredients possessesmicroenvironment-sensitive fluorescence properties rele-vant for localization purposes17ndash19 This molecule is of

interest to cost-effectively get insight into the LNP encap-sulation localization and release behaviors before prop-erly optimizing the system with the desired API Thepresent paper aims to understand how internal compositioninfluences NR encapsulation and release profile from thelipid nanoparticles For that purpose we first investigatethe influence of core composition on the LNP physico-chemical properties and the core physical state We thenstudy how it affects the encapsulation and localizationof NR molecules and finally how NR release kinetics ismodified

2 MATERIALS AND METHODS

21 Materials

The wax Suppocire NBtrade (mixture of mono- di- and tri-glycerides of alkyl chain length varying from C12 to C18is a kind gift from Gattefosseacute (Saint-Priest France) Myrjs40trade (polyethylene glycol 40 stearate) and Super-refinedSoybean oil are kindly donated by Croda (ChocquesFrance) The phospholipids Lipoid s75trade (composed ofgt75 phosphatidylcholine) are purchased from Lipoid(Ludwigshafen Germany) Other chemical products arepurchased from Sigma Aldrich (Saint Quentin FallavierFrance)

22 LNP Processing

LNP are prepared by emulsion templating through ultra-sonication Both aqueous and lipid phases are separatelyprepared before mixing The lipid phase contains a blendof solid (Suppocire NBtrade) and liquid (Super refined Soy-bean oil) glycerides with phospholipids (Lipoid s75trade)while the aqueous phase is composed of a PEG surfactant(Myrj s40trade) dissolved in PBS aqueous buffer (1times Phos-phate Buffer Saline 10 mM phosphate 154 mM NaClpH 74) Standard LNP formulations are prepared at 10weight fraction with 1800 L PBS 92 mg Myrj s40trade90 mg oilwax mixture and 18 mg Lipoid s75trade Afterhomogenization at 45 C both phases are crudely mixedThen sonication cycles are performed at 45 C during a5 min period (VCX750 Ultrasonic processor 3 mm probeSonics France sonication power 25) Non encapsulatedcomponents are separated from LNP dispersions by gentledialysis overnight in 1X PBS against 1000 times their vol-ume (MWCO 12000 Da ZelluTrans) Before use LNPdispersions are filtered through 022 m cellulosic mem-brane (Millipore)In the case of LNP loaded with 01 ww Nile Red a

solution of 120 L Nile Red 10 mM in CH2Cl2 is addedto the oily phase and the solvent is evaporated Then thisdoped oily phase is used as aforementioned to produceLNP encapsulating Nile Red at a local concentration of23 nmolmg dispersed phase



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23 Characterization

















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times100 (1)






)times100

(2)

3 RESULTS

31 LNP Formulation





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Core composition













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Enc

apsu

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fici

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ww

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20

40

60

80

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0001 001 01 1

NC 0

NC 50

NC 100

0

20

40

60

80

100

0001 001 01 1

NC 0

NC 50

NC 100










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4 DISCUSSION











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42 Release Profiles








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5 CONCLUSION


Abbreviations




























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23 Characterization

















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times100 (1)






)times100

(2)

3 RESULTS

31 LNP Formulation





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Core composition













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Enc

apsu

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fici

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(

ww

)


20

40

60

80

100

0001 001 01 1

NC 0

NC 50

NC 100

0

20

40

60

80

100

0001 001 01 1

NC 0

NC 50

NC 100










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4 DISCUSSION











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42 Release Profiles








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5 CONCLUSION


Abbreviations




























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times100 (1)






)times100

(2)

3 RESULTS

31 LNP Formulation





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Core composition













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Enc

apsu

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(

ww

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20

40

60

80

100

0001 001 01 1

NC 0

NC 50

NC 100

0

20

40

60

80

100

0001 001 01 1

NC 0

NC 50

NC 100










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4 DISCUSSION











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42 Release Profiles








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5 CONCLUSION


Abbreviations




























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Core composition













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Enc

apsu

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fici

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(

ww

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20

40

60

80

100

0001 001 01 1

NC 0

NC 50

NC 100

0

20

40

60

80

100

0001 001 01 1

NC 0

NC 50

NC 100










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4 DISCUSSION











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42 Release Profiles








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5 CONCLUSION


Abbreviations




























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Enc

apsu

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n ef

fici

ency

(

ww

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20

40

60

80

100

0001 001 01 1

NC 0

NC 50

NC 100

0

20

40

60

80

100

0001 001 01 1

NC 0

NC 50

NC 100










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4 DISCUSSION











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42 Release Profiles








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5 CONCLUSION


Abbreviations




























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4 DISCUSSION











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42 Release Profiles








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5 CONCLUSION


Abbreviations




























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4 DISCUSSION











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42 Release Profiles








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5 CONCLUSION


Abbreviations




























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42 Release Profiles








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5 CONCLUSION


Abbreviations




























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5 CONCLUSION


Abbreviations



























encapsulation and release behavior from lipid nanoparticles: model study … · 2017-07-21 ·...

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