encapsulation of drug nano particles in self-assembled

7/31/2019 Encapsulation of Drug Nano Particles in Self-Assembled

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Encapsulation of Drug Nanoparticles in Self-AssembledMacromolecular Nanoshells

Alisar S. Zahr, Melgardt de Villiers, and Michael V. Pishko*,

Departments of Chemical Engineering, Chemistry, and Materials Science & Engineering,The Pennsylvania State University, University Park, Pennsylvania 16802, and

Department of Basic Pharmaceutical Sciences, School of Pharmacy,

University of Louisiana at Monroe, Monroe Louisiana 71209

Received August 27, 2004. In Final Form: October 15, 2004

Layer-by-Layer (LbL) stepwise self-assembly of the polyelectrolytes poly(allylamine hydrochloride) andpoly(styrenesulfonate) was used to create a macromolecular nanoshell around drug nanoparticles(approximately150 nmin diameter). Dexamethasone, a steroidoften used in conjugation withchemotherapy,was chosen as a model drug and was formulated into nanoparticles using a modified solvent-evaporationemulsification method. Measurementof the zeta potential (-potential) aftereach polyelectrolyte layerwaselectrostatically added confirmed the successful addition of each layer. Additionally, data acquired fromX-ray photon spectroscopy (XPS) indicated the presence of peaks representative of each physisorbedpolyelectrolyte layer. Surface modification of the nanoshell was performed by covalently attaching poly-(ethyleneglycol) (PEG) with a molecular weightof 2000 to theoutersurface of thenanoshell. Zeta potentialmeasurements andXPS indicatedthe presence ofPEG chainsat thesurfaceof thenanoshell.The polymericnanoshell on the surface of the drug nanoparticle provides a template upon which surface modifications

can be made to create a stealth or targeted drug delivery system.

Introduction

Polymer drug delivery systems have been and willcontinueto be importantin thetreatmentof cancer,geneticdiseases, and other ailments.1,2 The development of thesedelivery systems has been impacted by nanotechnology,3

leading to the use of nanoparticles for drug delivery andgene delivery. Nanoparticles for drug delivery may bedefined as submicrometer colloidal particles (


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of polyelectrolytes, a technique introduced by Decher andcolleagues in the early 1990s.21 The LbL method exploitselectrostatic interactions between oppositely chargedpolyelectrolytes to create thin multilayer polymer filmsof alternating positive and negative charges with na-nometer scale thickness.22-25

In previous studies using LbL assembly, researchershave focused on using dissolvable core particles such aspolystyreneand melamine formaldehyde(MF) to fabricatehollow-shell microcapsules.20,25,26 Here we describe the use

of LbL self-assembly to fabricate a nanoshell composed ofpolycationic poly(allylamine hydrochloride), PAH, andpolyanionicpoly(styrene-4-sulfonate), PSS,polymer layersthat encapsulate drug nanoparticles, thus creating ananoparticle with a solid drugcore that hasa surface thatmay be readily modified to improve biocompatibility orfacilitate targeting. The key concept is that the assemblytakes place directly onto the drug nanoparticles. Here,nanoparticles of dexamethasone were fabricated by amodifiedsolventevaporationmethod.17,27 Dexamethasoneisa syntheticglucocorticoid that canbe administeredorallytotreat inflammatory conditionssuch as arthritis,asthma,and allergic conditions of the nose and eyes.28,29 It is alsoroutinelygiven to patients beforethey areaboutto receivechemotherapy because it can help alleviate and prevent

nausea and other side effects associated with chemo-therapy.29 The polyelectrolyte (PE) pair PAH/PSS is usedto illustrate that the assembly can indeed be achieved.With the LbL assembly a controlled drug deliverymay beaccomplished. One factor that controls the diffusion ofthe drug out of the encapsulation is the thickness of eachpolymer layer. The polymer layers therefore help controldrug release, which is desirable for any drug deliverysystem. In addition to creating an encapsulated drugparticle, the nanoshell of the encapsulation is surfacemodified with poly(ethylene glycol), PEG, to render abiocompatible surface. In this paper, we present resultsforthe LbLencapsulation of dexamethasonedrug particlesand for the surface modification of the nanoshell by thecovalent attachment of PEG.

Experimental Section

Materials. Poly(allylamine) hydrochloride (PAH, MW60 000)was purchased from Polysciences, Inc, USA. Poly(styrene-sulfonate) (PSS, MW 70 000) was purchased from AldrichChemicals, USA. USP grade dexamethasone was obtained fromSigma Chemicals, USA. Succinimidyl ester of polyeethyleneglycol) propionic acid (mPEG-SPA, MW 2000) was purchasedfromNektar, USA. Acetone and n-heptane were purchased fromSigma Chemicals,USA. Ultrapure water used forall experimentsand cleaning steps was obtained from a Barnstead NanopureDiamond RO systemhaving a specific resistance of greater than18 M/cm.

The polyelectrolyte (PE) solutions were prepared in a phos-phate-buffered saline solution (PBS, pH 7.4) consisting of 1.1

mM potassium phosphate monobasic, 3 mM sodium phosphatedibasic heptahydrate, and 0.15 M NaCl. For the covalentattachment of PEGto thenanoshell,a 0.1M sodium bicarbonatebuffer solution at pH 8.34 was used.

Centrifuge filters of sizes 0.1and 0.2m were purchased fromMillipore, USAand Nalgene syringe filters werepurchased fromNalge Nunc International, USA, respectively. An EppendorfCentrifuge 5810 was used for the centrifugation procedures.

Preparation of Dexamethasone Nanoparticles. Nano-particlesof dexamethasone werepreparedby a modifiedsolvent-evaporation emulsification method as previously reported in the

literature.17,27 Briefly, a 30 mg/mL solution of USP gradedexamethasone in acetone (1.0% w/v) wasemulsified with twicethe volume of n-heptane for 30 min at room temperature. Thedrug particles were collected by centrifugation at 5000 rpm for10 min andresuspendedin PBS.A second evaporation procedurewas performed to ensure that n-heptane was removed from thedrug particles. The drug particles were suspended in the buffersolution and constantly stirred under low heat for 1 h. The drugparticles again were collected, centrifuged, and redispersed inPBS-buffered solution. Finally, they were centrifuge filteredthrough a 0.2 and 0.1 m pore size filter as to achieve a morehomogeneous sizedistribution and to eliminateaggregated drugparticles. Each fraction was resuspended in PBS solution andstored in a refrigerator for future use.

Layer-by-Layer Self-Assembly. ThepH ofthe PEsolutionswere maintained at 7.4 for both polycationic and polyanionic

adsorption. The assembly procedure was as follows: 1.0 mL ofa 20 mg/mL PE solution was mixed with a solution of 30 mg/mLof drug particles and sonicated for 5 min followed by 20 minincubation under gentle shaking. After each layer was added,three cycles of centrifugation, removal of supernatant, andresuspension in 1.5 mL of buffer solution were performed toensure theremoval of unbound PE. The centrifugation step wasperformed at 5000 rpm for 5-10 min. The process continued byalternating the polycationic and polyanionic layers until thedesired number of layers was added.

Surface Modification of the Nanoshell. The surfacemodification of the nanoshell with PEG required that the lastlayer of thenanoshell contain a reactive amine group. ThePEG-conjugationprocedure hasbeen modifiedfrompreviouslyreportedprotocols for peptides and proteins.30,31 The reaction requiresthe reactive amine group concentration to be between 5 and 20mg/mL.The solution ofdrug particlestherefore hadto be diluted1.5 times and suspended in a 0.1 M sodium bicarbonate bufferatpH 8.34. Next, 100Lofa2mg/100L solution of mPEG-SPAwasadded to thedrug particles. Thereactiontook place over theperiod of 1 h under gentle shaking at room temperature. Afteran hour, the suspension was centrifuged and the pellet wasresuspended in a 0.1 M PBS-buffered solution. This cleaningprocess was repeated two more times.

Characterization. -PotentialMeasurements.The-potentialof each adsorbing layer was determined with a BrookhavenZetaPALSinstrument. Aqueous electrodes, AQ 422 and AQ 319,were used for these measurements. Each sample was run twice,with each run consisting of 10 or more data points.

X-ray Photoelectron Spectroscopy. X-ray photoelectron spec-troscopy(XPS)analyseswere performedusing a KratosAnalytical

Axis Ultra instrument. The X-ray source was a monochromaticaluminum (1486.6 eV) and powered at 280 W. Survey and high-

resolution spectra were collected at a takeoff angle of 90 withrespectto thesample plane. Theanalysis of thesepowdersamplesrequired thechargeneutralizationto be turnedon at lowenergy.

All spectra were referenced for C-C inthe carbon1s peakat 285eV. Survey spectra were collected from 0 to 1200 eV with passenergy of 160 eV, high sensitivity scans for the PSS layer werecollected at a pass energy of 80 eV, and high-resolution spectrawere collected for each detected element (C, O, N, and F) withpass energy of 20 eV. Quantification was performed by derivingrelative sensitivity factors from the given polymer standards:poly(ethyleneimine), PEI,poly(ethylene terephthalate), PET,andpoly(tetrafluoroethylene), PTFE.

(20) Moya, S.; Sukhorukov, G. B.; Auch, M.; Donath, E.; Mohwald,H. J. Colloid Interface Sci. 1999, 216, 297-302.

(21) Decher, F. Science 1997, 277, 1232-1237.(22) Ai, H.; Jones, S. A.; Lvov, Y. M. Cell Biochem. Biophys. 2003,

39, 23-43.(23) Ai, H.; Jones, S. A.; de Villiers, M. M.; Lvov, Y. M. J. Controlled

Release 2003, 86, 59-68.(24) Clark,S. L.;Hammond, P. T.Adv. Mater. 1998, 10, 1515-1519.(25) Donath, E. S.; Caruso, F.; Davis, S. A.; Mohwald, H. Angew.

Chem., Int. Ed. 1998, 37, 2201-2205.(26) Tiourina, O. P.; Antipov, A. A.; Sukhoarukov, G. B.; Larionova,

N. I.; Lvov, Y.; Mohwald, H. Macromol. Biosci. 2001, 1, 209-214.(27) ODonnell, P. B.; McGinity,J. W.Adv. Drug Delivery Rev. 1997,

28, 25-42.( 2 8 ) M e d i c i n e N e t . co m . h t t p : / / w w w . me d i c i n e n e t . c o m/

dexamethasone_oral/article.htm (accessed Jun 2004).(29) Hickey, T.;Kreutzer, D.;Burgess, D. J.;Moussy, F.Biomaterials

2002, 23, 1649-1656.

(30) Veronese, F. M. Biomaterials 2001, 22, 405-417.(31) Roberts, M. J.;Bentley,M. D.;Harris, J. M.Adv. Drug Delivery

Rev. 2002, 54, 459-476.

404 Langmuir, Vol. 21, No. 1, 2005 Zahr et al.


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Sample preparation was as follows: Suspended drugparticleswere centrifuged and the pellet was dried in a vacuum ovenovernight at room temperature. The powdered sample wasmounted on silicon wafersusingdouble-sided carbontape. Eachsilicon wafer was then placed on a sample holder and mountedinto the transfer arm of the instrument.

Scanning Electron Microscopy (SEM). Images of the drugparticles encapsulated within the polymeric nanoshell wereobtained from a Hitachi S-3000H unit. Sample preparationincluded pipetting 200 L of the suspended drug particles indeionizedwaterontoa gold-coated wafer.The samplewas allowed

to dryunderambient conditions fora daypriorto imaging. Next,the sample was gold sputtered for 30 s to minimize charging.

Additionally, SEM with energy-dispersive X-ray spectroscopy,EDS, was used to determine the atomic composition of the drugparticles. This procedure did not include sputter-coating thesample.

Transmission ElectronMicroscopy (TEM).A JEOL JEM 1200EXII TEM was used to image the encapsulated drug particles.Drug nanoparticles (in solution of deionized water) were placedon copper grids for imaging. A Tietz camera was used to takedigital images. All images were acquired at a tension of 80 kVand current density of 20 pA/cm2.

Particle Size Distribution. Theparticlesize distributionof thecentrifuge filtered sample fractions was analyzed by a ZetaSizerNano ZS. Material indexes of refraction for both PEs were setto 1.46, and PBS solution was set to 1.33. A volume of 1.5 mL

ofthe drug particle solutionwas pipetted into a disposable cuvette.Data were collected at room temperature.

Results and Discussion

Preparation of Dexamethasone Nanoparticles.Dexamethasone nanoparticles using a modified solvent-evaporation oil-in-water emulsification protocol.17,27 Thismethodhasbeen usedto fabricate polymeric drugparticleswhen the drug is hydrophobic. Here, an aqueous phasecontaining a surfactant was mixed with an organic phasecontaining the drug. Next, the organic solvent wasevaporated by either heating or high-speed homogeniza-tion under ambient conditions. As the organic phasepartitioned into the aqueous phase and evaporated at theliquid/air interface, spherical drug nanoparticles wereformed.27 While this process indeed producednanometer-sized drug nanoparticles, the size distribution can beheterogenous.2,17,27 However, this size distribution can becontrolled by using high-speedhomogenizationand highersurfactant concentrations.17

In the procedure described here, USP grade dexa-methasone powder was dissolved in acetone and thissolution was emulsified with a second organic phase ofn-heptane. The organic solvent pair was chosen based ontheir vapor pressures. Thefirst organicsolution must havea higher vapor pressure than the second, and mostimportantly, the drug must be insoluble in the secondorganic phase. As the emulsion proceeds, acetone evapo-rates, leaving the dexamethasone drug particles sus-

pended in the second organic phase. The drug particleswere then collected by centrifugation and resuspended ina 0.1 M PBS solution. To ensure that there were few ornotraces ofn-heptane on thesurface of thedrug particles,the drug particle solution was allowed to mix under highagitation rate and low heat to allow further n-heptanemoleculesto evaporate.The drug particleswere collected,centrifuged, and resuspended in buffer solution, and thisprocedure was performed three times.

To use the LbL assembly technique to fabricate thinpolymer shells on drug nanoparticles, it was necessarythat the drug particles must be charged. To determinethe charge on the drug nanoparticles, the -potential ofthe suspended solution was measured. The average-potential for dexamethasone nanoparticles was deter-

minedtobe-22.01mV((2.98). This was anticipatedsincedexamethasone possesses a hydroxyl two carbons awayfrom a fluorine, thus lowering the pKa of this OH moiety.It wasimportant that thedrug particlesbe highlychargedin order to prevent partial desorption of the first layer

which canresultupon theadsorptiona stronger PE layer.32

The sample was separated into two fractions. The twofractions were centrifuged filtered through 0.1 and 0.2m filters, respectively. Size distribution data of the twofractionswere determined. Beforethe datawerecollected,thefractionswere sonicated for10 minto break aggregateddrug particles. Figure 1 showsthe sizedistribution resultsfrom each fraction of a given sample. The average size ofdrug particles filtered through the 0.1 and 0.2 m poresize filters were 47.66 nm ((8.0 nm) and 146.54nm ((8.0nm), respectively.

The results suggest that, aftercentrifugationto removelargeagglomerates,a low polydispersecolloidalsuspensionis attainable. The size of the drug delivery system playsan important role in the release of the therapeutic agent

that is encapsulated within the polymeric nanoshell.Factors that affect the particle size include homogenizerspeed, surfactant concentration, concentration of thewater-soluble solvent, and organic phase solvent viscos-ity.2,17

Layer-by-Layer Self-Assembly. The -potential, mea-sured at the surface of the hydrodynamic shear, alsoconfirms the presence of alternating layers.33 We chosethe PAH/PSS polyelectrolyte pair because this is a well-studied combination and the process variables including

(32) Kato,N.; Schuetz,P.; Fery,A.; Caruso, F.Macromolecules 2002,35, 9780-9787.

(33) Coombes,A. G. A.;Tasker, S.;Lindblad,M.; Holmgren, J.;Hoste,K.; Toncheva, V.; Schacht, E.; Davies, M. C.; Illum, L.; Davis, S. S.

Biomaterials 1997, 18, 1153-1161.

Figure 1. Size distribution of dexamethasone drug particles:(a) the first fraction was centrifuged through a 0.2 m filter;(b) the second fraction of drug particles that was centrifugedthrough a 0.1 m filter.

Encapsulation of Drug Nanoparticles Langmuir, Vol. 21, No. 1, 2005 405


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salt concentration, pH, incubation times, and washingprocess have been established.32,34-36

The LbL adsorption procedure involved the alternatingaddition of 20 mg/mL of each PE solution onto the coredrug particles.The salt concentrations forthe PE,washing,and resuspension solutions were all 0.1 M PBS solution.The salt concentrations were maintained constant todemonstrate that the PE layers can be successfullyadsorbed to the drug nanoparticles. The thickness of theadsorbing layers may be increased by adjusting the saltconcentration in the buffer solution.32,36

The -potential results obtained afterthree layers havebeen added to the negatively charged nanodrug particles

verify the successful LbL assembly required for drugencapsulation (Table 1). The -potential is not a directmeasure of the surface potential but an experimentallymeasured potential that is responsible for interparticleforces.37,38 The larger -potential, -38.06 ((1.65), of thePSS layer accounts for the bulky and rigid styrene-sulfonate group that is able to extend further toward thesolution, thus contributing more to the measured -po-tential.32,39 The results collected from Mohwald and co-workerssuggest that weakPEs such as PAHadsorblooselyto the core particle and this interaction can allow PSS tocompete with PAH for binding sites on the core particle.40

This observation can also be further explained as follows:Although the overall charge of the drug particles isnegative, there can still be patches of neutral or positivecharges on the particle surface,41 i.e., there is not a

homogeneousdistributionof chargeon the particle surface.The interaction between two polyelectrolytes is alsostronger than that between PAH and the drug particlesbecause multivalent interactions characteristic of ionicpolymersproduce strongirreversible forces.40 The findingsfrom the literature show that the -potential of the firstlayer, PAH, on polystyrene latex spheres was approxi-mately 25 mV less than that of the third PAH layer. 32 Inour results this difference was not observed. The -potentials for the first and third layer were comparable,differing only by 1.34 mV.

The results from the electrophoretic mobility studiesindicated the success of LbL assembly depends on thechargeof theadsorbingPE layer.Subsequently,the chargeis dependent upon the pH of the solution. For example,

at pH 7.4 the amine group is protonated and carries apositive charge (pKa PAH ) 8.5). Kato and colleaguesstudied the influence of pH on the -potential of the PAH

layer32 and were able to measure the -potential as afunction of pH ranging between 2 and 12. From theirresults it was clear that the optimal pH that producesthehighest electrophoretic mobility is 7.0.32 At pH 8.0 the-potential was0 mV andbelow this pH thevalues plateauto -45 mV. The pH of the media containing the drugnanoparticles described here was approximately 7.4 tomimic physiological conditions. At pH 7.4 the -potentialof dexamethasone nanoparticles encapsulated with PAHwas in agreement with theresults of Kato and colleagues.

The results attained by Kato and co-workers furtherconfirm that the LbL assembly on the dexamethasonedrug particles was successful.

To further characterize the LBL encapsulation ofdexamethasone drug particles, XPS survey scans, highsensitivity scans, and high resolution scans werecollected.

XPS survey scans and high sensitivity scans (Figure 2)illustrated the distinguishing characteristic peaks ofdexamethasone and the added polyelectrolyte layers. Theshort dwell time for the survey scan was unable to pickup the sulfur 2p peak; consequently a pass energy of 80eV was used to collect spectra for this PE addition. TheF 1s peak at 700 eV, N 1s peak at 400 eV, and S 2p peaksat 166 eV confirmed the presence of fluorine, PAA, and

PSS, respectively. The F 1s peak is the distinguishingcharacteristic atom present in the steroid chemicalstructure. As the layers were added, the fluorine peakbegan to decrease in intensity, confirming the presenceof added polyionic layers.

Relative atomic compositions were calculated for eachcharacteristic atom of the adsorbed PE layer, Table 2.The calculated relative atomic compositions were normal-ized, and residual contaminants, silicon and phosphate,were subtracted from each atomic composition. Usingthese data, comparisonswere madebetween theadsorbingPE layers and their standards. When PAH was added,the atom percent of fluorine, 2.9%, decreased whencompared to the standard dexamethasone powder, 3.6%.

As the second layer was added, the fluorine atom percent

continued to decrease to 1.6%. This observation clearlydemonstrates the addition of the layers. The amounts ofnitrogen and sulfur at the surface of the drug particleswere small when compared to their standards. It wasexpected that the percentatomic compositions of nitrogenand sulfur would be small because the thickness ofadsorbing PE layers was very thin. Although the con-centrations for nitrogen and sulfur are minimal, the factthat the spectra appeared at the designated bindingenergies affirmed that the LbL assembly was successful.

A high-resolution scan of the N 1s peak of the PAHlayer, Figure 3, was captured to determine the surfacefunctionality of the nitrogen atoms present in the ad-sorbing PE layer. This high-resolution scan contained aconsiderable amount of noise which may be attributed tothe protonated state of the nitrogen. The two peaks werecurve fit, and the resulting peaks indicated differentprotonation states of nitrogen. The protonated amine(-NH3+) peak at 401.2 eV corresponded to similar valuesof 401.6 eV found in the literature.42 This result was inagreement with results from the -potential measure-ments that indicated the amine was protonated. Sincetherelativepeak amounts of protonated and deprotonatednitrogen would change after the chemisorption of PEG,it was important that the N 1s peak was characterizedunder high resolution. Changes in protonation state ofthe N 1s peak would further confirm the conjugation of

(34) Shenoy, D. B.; Antipov, A. A.; Sukhorukov, G. B.; Mohwald, H.Biomacromolecules 2003, 4, 265-272.

(35) Goldenberg, L. M.; Jung,B.-D.; Wagner, J.; Stumpe, J.; Paulke,B.-R.; Gornitz, E. Langmuir 2003, 19, 205-207.

(36) Lvov, Y.;Decher, G.;Mohwald, H.Langmuir 1993, 9, 481-486.(37) Sennet, P.; Oliver, J. P. Chemistry and Physics of Interfaces;

American Chemical Society: Washington, DC, 1965; pp 33-50.(38) OBrien, R. W.;White, L. R.J. Chem. Soc 1978, 74, 1607-1626.(39) Caruso, F.; Mohwald, H. J. Am. Chem. Soc. 1999, 121, 6039-

6046.(40) Sukhorukov, G. B.; Donath, E.; Lichtenfeld, H.; Knippel, E.;

Knippel, M.; Budde,A.; Mohwald, H. ColloidsSurf., A 1998, 137,253-266.

(41) Feick, J. D.; Velegol, D. Langmuir 2002, 18, 3454-3458. (42) Alexander, M. R.; Jones, F. R. Carbon 1996, 34, 1093-1102.

Table 1. The -Potential (mV) for Core Particles andEach Adsorbing Layer

layer no. layer descriptionmean

potential (mV) std deva

0 dexamethasone particles -22.01 2.981 PAH 27.25 1.702 PSS -36.89 2.543 PAH 25.91 1.65

a The standard deviation is based upon n > 10.



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PEG to the PAH layer, as this reaction would change theprimary amine to a secondary amine.

Characterization by Electron Microscopy. To visuallydetermine whether LbL assembly successfully encapsu-lated the drug particles, SEM and TEM were used toexamine the surface characteristics and size of the drug

delivery nanoparticles. High-resolution microscopy canbe used to view these nanoparticles at a resolution of 1nm.2A SEM image of the drug nanoparticles encapsulatedwith two PE layers is shown in Figure 4. The particleswere passed through a 0.2 m centrifuge filter to removelarge agglomerates. The size distribution of the particlescaptured in this dry state illustrates a relatively homo-geneous distribution in good agreement with the laserlight scattering results shown in Figure 1. The drugparticles were spherical and nanometer sized, approxi-mately 150 nm in diameter. SEM-EDS was performed onthese nanoparticles and indicated that fluorine waspresent in the drug nanoparticles. This was expectedbecause the structure of dexamethasone possesses afluorine atom.

To visualizethesenanoshell-encapsulated nanoparticlesin more detail, TEM was used. A TEM image of anencapsulated drug particle with two PE layers is shownin Figure 5a. The diameter of this encapsulated drugparticle was approximately 130 nm. At this magnificationof 300 000, twoPE layers were evident. Also, the thickness

of the two PE layers was less than 10 nm as shown inFigure 5b. This verified that LbL assembly may be usedto fabricate a nanometer scale nanoshell to encapsulatedrugnanoparticles. Thedata acquired fromSEM andTEMare in agreement with each other and with the sizedistribution data collected by laser light scattering.

Modification of the Nanoshell. After the drugparticles were encapsulated, the surface was modifiedbecause chemically tailoring the exterior surface of thenanoshell may permit the nanoparticles to be targeted orbecome stealthy in vivo. The outer PAH layer providedthe appropriate chemistry for the covalent attachment ofPEG.30,31 Surface modification of the nanoshell wasaccomplished by the covalent attachment of mPEG-SPAto the PAH layer of the encapsulated drug particles via

Figure 2. XPS survey scans with pass energy 160 eV: (A) spectrum of dexamethasone; (B) dexamethasone encapsulated withPAH; (C) spectrum of dexamethasone encapsulated with PAH and PSS; (D) spectrum of PEG surface modified dexamethasone.

Table 2. XPS Elemental Analysis of Encapsulated and PEG-Modified Dexamethasone Drug Particles

rel atomic composition (%)a

surface condition O (1s) C (1s) N (1s) F (1s) S (2p) Na (1s, 2s)dexamethasone+ PAH 13.5 80.1 3.4 2.9 0 0dexamethasone+ PAH/PSS 15.1 82 1.1 1.6 0.11 0PEG-modified dexamethasone 15.9 79.5 2.4 2.1 0 0PAH standard 0 75 25 0 0 0PSS standard 25.3 46.8 0 0 7 7.4, 6.8PEG standard 30.1 69.6 0.3 0 0 0

a Relative atomic percents represent normalized atomic compositions.



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the reaction between the hydroxysuccinimidyl (NHS)moiety of mPEG-SPA with an amine on PAH. The

requirement for this reaction to take place is for the pH

of the buffered solution to be between 8.2 and 8.5. At thisbasic pH, the protonated amine of the PAH layer becomesdeprotonated and PEG can be covalently attached to thissecondary amine.Consequently,thechemisorptionof PEGto thenanoshell of theencapsulated drug particle createda neutralization in charge of the nanoparticle.

PEGcoatings create a cloud of solvated hydrophilic andneutral chains at the particle surface which can repelapproachingplasmaandblood proteins.15,16 PEGpolymerswith molecular weights between 2000 and 5000 haveshown to increase half-life circulation times for liposomedrug delivery vehicles.15,43 In thismolecular weight range,PEG chains are stretchedin solutionas elongated, flexiblecoils, which allow water molecules to hydrate theirchains.15 Furthermore, this hydration serves to minimizeprotein adsorption and opsonization by the MPS.15,16,43

The fluctuation of-potential between small positiveand negative values was observed after PEG was chemi-sorbed. This fluctuation was indicative of a -potentialclose to the isoelectric point of the drug particles. TheZetaPALS instrument will not show a -potential of 0 mVbut instead provide results within a range close to 0 mV.The-potential of thenanoparticles after PEGconjugationwas found to be 0.0275 ((0.138, n) 5). When comparingthese results with the -potential after one layer of PAHwas added, one can indeed notice the difference. The

(43) Gref, R.; Luck, M.; Quellec, P.; Marchand, M.; Dellacherie, E.;Harnish, S.; Blunck, T.; Muller, R. H. Colloids Surf., B 2000, 18, 301-313.

Figure 3. XPS high-resolution scans of N 1s peaks: (A) 63.6%and 36.4% for dexamethasone encapsulated with PAH; (B)29.1% and 70.8%for surface-modified dexamethasone particleswith PEG. The curve fits provided percent concentrations foreach peak at positions 401 and 399 eV, respectively.

Figure 4. SEM image of dexamethasone drug particlesencapsulated with two PE layers. The drug particles werefiltered though a 0.2 m centrifuge filter before the image wastaken.

Figure 5. TEM image of dexamethasone drug particle: (a)drug particle encapsulated with two layers of polyelectrolytes,magnification 150k; (b) thickness of the two layers can be seenand is approximately 10 nm, magnification 300k.



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addition of PEG to the surface of the encapsulated drugparticles resulted in the neutralization of the overallcharge. This result was expected because first PEG is anonionic polymer and, second, the covalent attachmentrequires a noncharged secondary amine. Neutralizationof nanoparticlecharge is useful in preventing aggregation,sterically stabilizing the drug particles, and preventingopsonization by the MPS.15,16

Analysis of high-resolution spectra of the C 1s, O 1s,and N 1s peaks by XPS further confirmed the chemi-sorption of PEG. High resolution spectra allowed ad-ditional surface sensitivity because at the lower X-rayenergyof 20 eV, atomicchargestates can be resolved withmore detail and with a detection error of less than 1%.

Comparisonswere made between high-resolution spectraof dexamethasone standard, drug particles with PAHlayers, and surface-modified drug particles to verify thecovalent attachment of PEG to the outer portion of thenanoshell. Changes in the oxidation state of the C 1s, O1s, and N 1s spectra clearly indicated the surfacemodification of the nanoshell.

In the structure of PEG there is a repeating glycol unitof C-C-O in the backbone. This functionality wasobserved in the C 1s (atomic orbital 1s of carbon) peak asa shoulder at 286.34 eV (Figure 6). Curve fit analysis wasperformed on these high-resolution spectra, and therelative peakareas (%) for specific carbon chemical bondsaresummarizedin Table 3. Comparison between thepeakareas of two chemical bonds Cs(O,N) and CdO,CsF

present at the surface of the drug particles indicated thechemisorption of PEG. Thesetwochemical bonds or speciesrepresent the surfaces of PEG modified drug particlesand nonsurface modified drug particles, respectively.There was an apparent increase in Cs(O,N) (286.34 eV)peak area and decrease in CdO,CsF (287.64 eV) peakarea when PEG was covalentlyattached to the PAH layerof encapsulated drug particles. Also, a visual comparisoncan be made between the shoulder at 286.34 eV in Figure6a andFigure 6b to illustratethatthere wasan observableincrease in this shoulder after PEG waschemisorbed. Themore defined shoulder at 286.34 eV in Figure 6b wasamplified due to the presence of PEG. The data collectedfrom the C 1s spectra therefore verified the surfacemodification by PEG to the nanoshell.

Additionally,oxygen intensity increased after PEG was

covalently attached to the nanoshell. A comparison wasmade between high-resolution O 1s (atomic orbital 1s foroxygen) spectra for drug encapsulated with PAH andsurface-modified drug particles. This comparison wasmade by observing the change in intensity for the O 1speak andthe changein atomic compositionof oxygenafterPEGwas chemisorbed. Thepeak intensityof drugparticlesmodified with PEG was approximately 10 CPS unitshigher than that for nonmodified drug particles (Figure7).Thisis an important observation because PEG containsmany oxygen atoms. Therefore this apparent increase inintensityof the O 1s peak wasanticipated. Thecomponentanalysis also suggested the presence of PEG because theoxygen atomic relative composition increased from 13.5

% to 15.9% after PEG was chemisorbed to the nanoshell(Table 2). With these two results from analysis of the O1s spectra, it was further concluded that the nanoshellwas successfully modified by PEG.

Finally, theN 1s (atomic orbital1s for nitrogen)spectraprovided results that confirmed the presence of PEG atthe outer layer of the nanoshell. In Figure 3, two high-resolution spectra illustrate two differentelectronic statesfor nitrogen. As previously discussed the two peaks in thespectra represent a protonated and nonprotonated elec-tronic state of nitrogen. Before the addition of PEG theprotonated amine peak at 401.2 eV had a relative peakarea of 63.6%. After PEG was chemisorbed, this peakbecame a weaker shoulder with relative peak area of29.1%. Additionally, the relative peak areas for the

secondary amine at 399 eV increased as the protonatedamine peak decreased upon the chemisorption of PEG.The XPS results agreed with -potential measurementswith regards to the surface charge of the encapsulateddrug particles. With an increase in the secondary, non-protonated nitrogen peak, the charge at the surface of thedrug particles would be close to neutral. These resultswere important because they demonstrated that byobserving changes in the N 1s spectra, one could confirmwhether PEG was successfully chemisorbed to thenanoshell.

The results obtained from the surface modification ofdrug particles with PEG were in agreement with valuesfound in the literature for the PEG-modified bare silica

Figure 6. High-resolution scans of the C 1s peak for (A)dexamethasone particles encapsulated with PAH and (B)dexamethasone particles encapsulated with PAH/PEG. Theshoulder at 286.34 eV represents C-O and C-N bonds. Thisshoulder indicated the covalent attachment of PEGto the PAHencapsulated dexamethasone drug particles.

Table 3. XPS Binding Energies and Peak Areas forEncapsulated Dexamethasone Drug Particles

binding energy (eV) (rel peak area %) C 1s

surface condition CsC Cs(O,N) CdO, CsF

drug particles + PAH 285.05 (62.70) 286.25 (29.1) 287.64 (8.2)PEG-modified drug

nanoparticles284.9 (64.3) 286.34 (32.0) 287.64 (3.7)



8/8

andmetal oxide surfaces.44,45 These studiesconcluded thatan increase in C-O intensity of the high-resolution C 1sspectrum, together with an overall increase in oxygencontent, indicatedthesuccessfulattachmentof PEO chainsonto the surfaces. The same observation was madein thisLbL-modified drug nanoparticle. Results attained from

XPS alone indicated the presence of PEG chains at theouter layer of the nanoshell.

The impetus for modifying the outer portion of thenanoshell with PEG was to create potential stealthnanoparticles.One hypothesis suggests that protein ad-sorption to high molecular weight PEG is disfavored bysteric repulsion.10,15 The adsorption of blood proteins tothe glycol chains restricts the conformation of the chains,

which is entropically disfavored.46 PEGs hydrophilicnature helps to minimize protein adsorption therebyreducing recognition by the MPS (hence the stealthadjective applied to many such systems). Also, theflexibility andthe mobility of PEGchains help accomplishthe task of reducing protein adsorption; this is dependenton themolecularweightof themolecule.15As the molecularweightincreases, thechainsfoldinto a hydrated coil,whichcan form a repulsive hydrated layer. This mechanism ischaracteristic of molecular weights between 1500 and

3500.15 For the drug delivery system described here, amolecular weight of 2000 was used, thus creating thepotential for a stealth drug delivery system.

Conclusion

LbLself-assembly of polyelectrolytes was demonstratedto be successful in encapsulating dexamethasone drugnanoparticles. The stepwise assembly of alternatingcharged polymer electrolyte solutions, PAHand PSS, wasalso demonstrated. -potential and XPS experimentalresults were in agreement and illustrated that theiradsorption was present at thesurface of thedrug particles.SEM and TEM images captured encapsulated drugparticles, and a magnified image of the encapsulated PE

layer affirmed the nanometer scale thickness. The nextstep in this research is to show that LbL assembly can beperformed with biomacromolecules. To further improvethe biocompatibilityof this drug delivery system, possiblepolyelectrolytes that will be investigated are polypeptidepoly-L-lysine and anticoagulant heparin sulfate.47

The surface of the nanoshell or encapsulated drug wassurface modified by thechemisorption of PEG(MW 2000).The overall charge of the drug particles was neutralafterthe covalent attachment of PEG to amine reactive sites.The results obtained by XPS provided sufficient informa-tion to conclude that PEG chains were present at thesurface. Theincreasein C-O intensity andoxygen atomicpercentages affirmed that a stealth drug delivery systemis feasible. Thesurfacedensity andcoverageof PEGchains

were not investigated in this study, but future studieswill be performed to demonstrate how surface coveragemay affect the compatibility of a nanoparticulate drugdelivery system in vivo.

Acknowledgment. This work was funded by theNational Science Foundation (NIRT BES-0210298). The

XPS instrument was funded by NSF-DMR 0114104. Theauthors appreciate Jeff Shallenberger and Bob Hengste-beck for their assistance in XPS analysis. The authorsalsoappreciate our numerousconsultations with Professor

Yuri D. Lvov.

LA0478595

(44) Wasserman, S. R.;Tao, Y.-T.; Whitesides,G. M.Langmuir1989,5, 1074-1087.

(45) Kenausis, G. L.; Voros, J.; Elbert, D. L.; Huang, N.; Hofer, R.;Ruiz-Taylor, L.;Textor, M.;Hubbell,J. A.;Spencer,N. D.J. Phys. Chem.

B 2000, 104, 3298-3309.

(46) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115,

10714-10721.(47) Angelova, N.; Hunkeler, D. Trends Biotechnol. 1999, 17, 409-

421.

Figure 7. XPS high-resolution scans of O 1s peak for (A)dexamethasone encapsulated with PAH and (B) surface-modified dexamethasone particles with PEG.


encapsulation of drug nano particles in self-assembled

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