ORIGINAL CONTRIBUTION

Synthesis and multidisciplinary characterization of polyelectrolytemultilayer-coated nanogold with improved stabilitytoward aggregation

Subhra Mandal & Alois Bonifacio & Francesco Zanuttin &

Valter Sergo & Silke Krol

Received: 6 October 2010 /Revised: 12 November 2010 /Accepted: 15 November 2010 /Published online: 7 December 2010# Springer-Verlag 2010

Abstract An interesting nanodrug delivery system ispolyelectrolyte multilayer-coated nanogold. For betterunderstanding of the binding of polycations or thecounter-indicative deposition of polyanions on the citrate-stabilized gold nanoparticles, we used a surface-enhancedRaman spectroscopy to characterize the orientation of thepolyions towards the gold surface. It was found that poly-allylamine replaces citrate molecules while the polyanion,poly-styrene sulfonate, intercalates in the citrate shell. Oneof the major obstacles for polyelectrolyte-coated nanogoldis its tendency to agglomerate in the presence of high ionconcentration as present, e.g., in blood. A novel encapsu-

lation protocol for polyelectrolyte multilayer coating ofgold nanoparticles was developed to successfully overcomethis drawback. Moreover, electrostatic functionalization ofthe polyelectrolyte shell with a model target molecule forcancer, folic acid, induced a significant increase in theparticle uptake in folate-receptor over-expressing breastcancer cell lines, VP 229 and MDA MB 231, compared tonon-targeted particles or cells (non-activated macrophages)not expressing the folate receptor.

Keywords Gold nanoparticles . Layer-by-layer .

Polyelectrolyte . SERS . Targeted delivery . Cancer

Introduction

Nanotechnology in medicine promises a completely newand revolutionary way for diagnosing and treating diseases.However, this has led to an increasing interest in multi-functional nanoparticles.

Colloidal gold nanoparticles (NG) and gold salts have along tradition in medical treatment of several diseases suchas rheumatoid arthritis [1–4]. Their unique properties, suchas being non-toxic, being inert against chemical modifica-tions, and emitting luminescence, make them a veryattractive tool for “theranostics,” the combination of drugor drug delivery with diagnostic features. Gold nano-particles can be used as enhancer for microwave-inducedcancer lesion treatment by radiotherapy and thermotherapy[3, 4]. Other examples of diagnostic or therapeuticapplications are the gold nanorods for fluorescence micros-copy and hyperthermia in cancer [5, 6]. While citrate-capped nanogold is considered non-toxic in other nanogoldpreparations, the capping agents show a certain toxicity [7,8] e.g., CTAB (cetyl trimethylammoniumbromide) [9], but

Electronic supplementary material The online version of this article(doi:10.1007/s00396-010-2343-2) contains supplementary material,which is available to authorized users.

S. MandalSISSA—International School for Advanced Studies,via Bonomea 265,34136, Trieste, Italy

A. Bonifacio :V. SergoCentre of Excellence for Nanostructured Materials,University of Trieste,via Valerio 2,34127, Trieste, Italy

F. Zanuttin : S. Krol (*)NanoBioMed lab @ LANADA,CBM S.c.r.l—Cluster in Biomedicine,Area Science Park, Basovizza - SS 14, Km 163.5,34149, Trieste, Italye-mail: silke.krol@ifom-ieo-campus.it

S. KrolEuropean Centre for Nanomedicine,Fondazione I.R.C.C.S. Istituto Neurologico “Carlo Besta”,IFOM-IEO-campus, via Adamello 16,20139, Milan, Italy

Colloid Polym Sci (2011) 289:269–280DOI 10.1007/s00396-010-2343-2

even here, the data are somehow contradictory [10]. But themain obstacle for using NG for medical preparation is thefact that functionalized NG tends to aggregate, as in thecase of thiol-stabilized NG [11–13]. Especially, the work ofDecher and Schneider [14] indicates that polyelectrolytemultilayer-coated NG (PNG) are aggregating in thepresence of small ions such as sodium chloride, which rendersthem useless in ionic solutions such as blood. On the otherhand, polyelectrolyte multilayer coating is an appealingmethod to functionalize gold nanoparticles for drug deliveryor theranostics via electrostatic forces [15–19].

In the present work, we address several challenges inrelation to the layer-wise deposition of polyelectrolytes onnanogold. One open question was the counter-indicativeself-assembly of the polyanion on the negatively chargedcitrate-stabilized gold surface, which we reported earlier[17]. So, we studied the orientation and the bindingmechanism of both poly-allylamine hydrochloride (PAH)and poly-styrene sulfonate toward the NG surface withsurface-enhanced Raman spectroscopy (SERS).

Next, we improved the stability of the polyelectrolytemultilayer-coated nanogold against aggregation in the presenceof ions by introducing a new protocol. As working hypothesis,we assume that the low stability against agglomeration reportedby Decher and Schneider [14] may be due to the excessivelength of the polyelectrolytes (PEs) with respect to the particlesize leading to incomplete wrapping and in turn to bridgingflocculation. In the literature, it was reported that weakpolyelectrolytes change their conformation from a chain to arandom coil structure in dependence of ionic strength due tothe shielding of the intramolecular repulsive forces [20].Therefore, we decided to collapse the PE by pre-incubation insodium chloride solution and deposit randomly coiledpolyelectrolytes. The resulting nanoparticles were found tobe stable in high ionic strength (0.5 M NaCl) or even in themultivalent ion-containing Ringer’s solution, a well-knownmedicinal solution.

Finally, we tested electrostatically bound targetingmoieties against cancer cells. Due to the over-expressionof folate receptors [21–25] in most cancers and because fastdividing cancer cells require high amounts of folic acid forrapid nucleic acid biosynthesis [26, 27], folic acid waschosen as model for cancer-targeted receptor-mediated up-take of the functionalized PNG. Many reports on targetednanoparticle delivery to cancer cells describe the function-alization by covalent binding of folic acid to polymers ordrug molecules [24, 25, 27–30]. But covalent binding canproduce toxic by-products, reduce drug efficiency, or hinderrecognition by the receptor.

The efficiency in introducing receptor-mediated up-takeby the folic acid labeling of the polyelectrolyte multilayer-coated nanogold was studied in two breast cancer cell lines(VP229, MDA MB 231) known to over-express the folate

receptor, and the specificity of this up-take was supportedby a competition assay with free folic acid and incomparison to non-activated macrophages that (1) do notexpress the folate receptor and (2) are part of the immuneresponse. The electrostatic labeling of the polyelectrolyte-coated nanogold with folic acid leads to a significantlyimproved particle uptake.

In summary with the present work, we gained insight inthe molecular binding of the first polyelectrolyte layer andpossible pitfalls for toxicity coming from an incompletereplacement of capping molecules. We stabilized andfunctionalized the multilayer-coated nanogold particles sothat now they can become useful for future medicalapplications.

Experimental

Materials The polycations, PAH (MW 15 kDa) and poly-fluorescein allylamine hydrochloride (FITC-PAH; MW15 kDa), as well as the polyanion poly-styrene-4-sulfonatesodium (PSS; MW 4.3 kDa), folic acid, sodiumtetrachloroaurate-(III) dihydrate (NaAuCl4·2H2O), sodiumcitrate tribasic dihydrate, sodium chloride, potassiumchloride, and calcium chloride were purchased from Sigma(Milan, Italy) and used without further purification.MEGM® SingleQuots medium, fetal bovine serum (FBS),fetal calf serum (FCS), and DMEM medium were pur-chased from Lonza (USA). For all experiments andwashing steps, Milli-Q grade (MQ) water with a resistanceof 18.2 MΩ/cm2 was used.

Gold nanoparticle preparation, coating, and functionaliza-tion Gold nanoparticles (NG) were prepared according toTurkevich [31]. The concentration of the resulting colloidalgold solution was calculated from the absorption deter-mined at λ=518 nm with the UV–vis spectrophotometerDU®730 (BeckmanCoulter, Italy) by the Lambert–Beerequation using the values for ε from Liu et al. [32] to be 35±0.5 nM. This solution was stable for at least 3 monthswithout a visually significant agglomeration. The particlesize was determined with a transmission electron microscope(TEM), JEM-2100F (JEOL, UK), while the polydispersityindex (PDI), hydrodynamic diameter, and surface chargewere measured by dynamic light scattering (DLS) and ζ-potential analysis on Zeta-sizer (Nano-ZS, Malvern, UK). Ingeneral, DLS serves as a quality control of the preparation.Gold nanoparticle preparations with one single peak in DLSand a PDI below 0.2 were used for further experiments.TEM measurements showed NG with a diameter of 15±1 nm, while the DLS analysis determines the particlediameter with 21±2 nm. The size difference between TEMand DLS can be explained by the citrate/structured watershell around the particles which stabilizes the NG in solution

270 Colloid Polym Sci (2011) 289:269–280

and influences the measurement with DLS but is not visiblein TEM.

The multilayer coating was performed according to theprotocol described by Chanana et al. [17] and compared toparticles coated with a novel coating procedure. In brief, thepolyelectrolytes were dissolved in MQ grade water to aconcentration of 10 mg/mL for PSS and 2 mg/mL for PAH.The procedure is similar to that described later for the firstlayers of particles prepared with the novel procedure. Butthe novel encapsulation protocol to improve the stabilityagainst aggregation includes that the first five PE layerswere deposited from polyelectrolytes in pure MQ waterwhile all following layers were deposited from 0.5 Msodium chloride. PSS (4.3 kDa) was used in a supersatu-rated concentration of 10 mg/mL in order to guarantee afast and complete surface coverage. PAH (15 kDa) wassolved in 0.5 M NaCl solution to a concentration of 2 or3 mg/mL. All PE solutions were prepared 1 day in advance.The high ionic strength as well as the pre-incubation of1 day is necessary in order to induce the proposedconformational change. The following polyelectrolytesdissolved in 0.5 M NaCl will be tagged with the index0.5, e.g., PSS0.5, the same labeling will be used for PNGwith the last two layers from PSS0.5 or PAH0.5, and theywill be referred to as PNG0.5.

For the first layer, the NG stock solution was addeddrop-wise under continuous vortexing to either the poly-cation or the polyanion solution. Then, the mixture waskept for 20 min in the dark to allow a sufficient andhomogenous coating of the nanoparticle. The protectionfrom light is only a precaution to avoid light-inducedaggregation of the gold nanoparticles. Next, the PNG werewashed twice in MQ-water by centrifugation 18,000 × g for30 min, followed by removal of the supernatant andredispersion of the pellet in water. For the next layers, theprocedure was repeated with an oppositely charged PE.Again, the coated gold was added drop-wise to thepolyelectrolyte solution. For the normal PNG, this proce-dure was repeated until the desired number of layers wasreached. For fluorescent coatings on the gold nanoparticles(FPNG), FITC-PAH (2 mg/mL) was used instead ofunlabeled PAH.

Preliminary experiments showed that coated particlesaggregate immediately if the first five layers are depositedfrom polyelectrolytes solved in 0.5 M sodium chloride.Hence, the PNG0.5 were coated with the first five layersfrom aqueous PEs and all following layers deposited fromPEs dissolved in 0.5 M NaCl. For these layers, the particleswere washed twice by centrifugation at 12,000 × g for30 min and resuspended in fresh 0.5 M NaCl solution toremove the unbound polyelectrolyte. The PDI value forcitrate-stabilized gold nanoparticles was usually 0.192±0.017, whereas for coated particles, the value varies from

0.23 to 0.27. A detailed coating procedure is depicted in thescheme in Fig. 1.

Folic acid (Fo) functionalized NG (Fo-PNG) forreceptor-mediated up-take in cancer cells was prepared byadding drop-wise a folic acid colloidal suspension (70 μg/mL; solubility limit for folic acid is 1.6 μg/mL) to PNG0.5

coated with the layer sequence [(PAH/PSS)2/PAH/(PSS0.5/PAH0.5)] under constant vortexing, followed by 30-minincubation in the dark. The particles were used after fourwashing steps. In order to visualize the folate receptor-mediated endocytosis by confocal microscopy, fluorescentFPNG with the following sequence of layers [(FITC-PAH/PSS)2/FITC-PAH/(PSS0.5/FITC-PAH0.5)] were functional-ized with folic acid. Here, the particles were stored 2 h inthe dark at room temperature. Then, the presence of folicacid was shown by DLS, UV–vis, and SERS. Eachmeasurement was repeated for a minimum of 10 times.

Particle agglomeration by small ions The PNG and PNG0.5

stability was tested in two ionic media. The coated anduncoated gold nanoparticles were centrifuged, and thesupernatant was replaced by MQ-water, 0.5 M NaClsolution, or Ringer’s solution ([NaCl]=0.147 M, [KCl]=0.004 M, [CaCl2]=0.0033 M). After 2 h, the particles werecharacterized by DLS and UV–vis. In recent studies, it wasshown that DLS is a highly sensitive technique for thedetection of aggregates [33–35].

Moreover, the UV–vis spectra should show a red shift ofthe peak as bigger particles or aggregates usually show acolor change from wine red to blue. Each measurement wasrepeated for a minimum of five times.

Surface characterization by SERS and Raman measure-ments SERS spectroscopy yields information about thespecies adsorbed on the gold surface. The vibrationalRaman spectra of molecules or molecular moieties in directcontact to certain metals can be strongly enhanced [36, 37].Besides reporting the occurrence of a surface-bindingevent, SERS spectra convey information about the chemicalnature of the adsorbed species and possibly on itsorientation with respect to the metal surface. In this respect,SERS is more informative for studying adsorption on metalnanoparticles than UV–visible absorption spectroscopywhich do not yield any information about the nature ofthe adsorbate.

The adsorption of one or two PE layers onto the surface ofNG was followed by SERS measurements with a Ramansystem (Renishaw plc, Wotton-under-Edge, UK). The laser(632.8 nm He–Ne laser, Melles-Griot, Albuquerque, NM,USA) was focused by a 10× objective (0.25 NA) on thesample, consisting of a 10-μL drop of NG dispersion(previously concentrated upon centrifugation) on a CaF2 slide(OEC Optoelectronic Components GmbH, Zusmarshausen,

Colloid Polym Sci (2011) 289:269–280 271

Germany) for SERS measurements or aqueous solutions ofPSS (80 mg/mL), PAH (160 mg/mL), or sodium citrate(160 mg/mL) for normal Raman measurement. The laserpower at the sample was 15 mW. The total acquisition timewas 30 s per spectrum.

In order to understand if folic acid is binding stably tothe gold nanoparticles, NG were coated only with one PAHlayer to which folic acid was bound electrostatically afterwashing, followed by SERS. It was necessary to reduce thenumber of polyelectrolyte layers between the gold surfaceand the folic acid because SERS signals can be measuredonly close to the gold surface. However, the electrostaticfolic acid binding to polycation layers other than the first issupposed to be comparable to that to the first layer. SERSmeasurements of the Fo-PNG were performed with anexcitation wavelength of 785 nm (Renishaw HP-NIF diodelaser) and a laser power at the sample 90 mW. Forcomparison, the pure folic acid crystals were measured byRaman spectroscopy.

Cellular nanoparticle up-take For the folate receptor-mediated up-take of Fo-FPNG, VP 229 and MDA MB231 (breast cancer cell lines, ECACC, and ATCC HTB 26,Sigma-Aldrich, Milan, Italy) were seeded onto 22×22-mmcover-slips to a density of 104 cells per well in MEGM,supplemented with glutamine and 2% FCS but withoutantibiotics and DMEM medium supplemented with 10%heat-inactivated FBS, penicillin (100 U/mL), streptomycin(100 μg/mL), and gentamicin (10 μg/mL). They weregrown for 24 h at 37 °C and 5% CO2. Then, the FPNG orFo-FPNG at the concentration of 2.4 and 2.6 pM, respec-

tively, were added to the cells, and after 2 h, they werewashed twice with serum-free medium, and time lapseconfocal fluorescence microscopic studies were performed.The experiments were repeated at least three times for eachcell line.

In order to exclude other mechanisms than folate-receptor mediated up-take, a competition study with freefolic acid was performed. For this, 2×105 cells/mL breastcancer cells (MDA MB 231) were seeded on a microscopicslide and incubated for 24 h at 37 °C and 5% CO2 in serumcontaining medium with different folic acid concentrations(0.567, 2.27, 4.54, 9.08, 18.16, and 27.24 nM) and 2.23 pMFo-FPNG. After rinsing the cells with serum-free medium,confocal fluorescence microscopic studies were performed.From one slide, at least 15 regions of interest were taken forthe analysis. The experiments were repeated for at leastthree times.

The unspecific up-take in cells without folate-receptorwere tested in macrophages as they are also the firstresponder in immune response and can give a hint about theimmunogenicity of the coated particles. The macrophagescell line, J774.2 (ECACC, Mouse BALB/C monocytemacrophage; a gift from Prof. A. Nistri, Neurobiologysector, SISSA; 4×104 cells/mL), was grown on 22×22-mmcover-slips in Petri dishes. After 2 h, the adherent cells wererinsed three times with the medium containing FCS. Theywere grown for 18 h at 37 °C and 5% CO2. Then, cellswere rinsed with DMEM medium (without FBS andantibiotics), followed by incubation for 4 h with Fo-FPNG0.5 (2.4 pM) or with FPNG0.5 (2.9 pM). The cellswere rinsed three times, and fluorescence confocal micros-

PAH0.5

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Electrostatic binding of Folic acid to PNG0.5

PNG

Fig. 1 Scheme of multilayer PE coating according to the novel protocol and electrostatic binding of folic acid as targeting molecule for cancercell

272 Colloid Polym Sci (2011) 289:269–280

copy was performed. Each experiment was repeated fivetimes (for each experiment, ∼1,000 cells were studied).Statistical analysis was performed by SigmaStat andOrigin8 softwares.

Fluorescence microscopy Image acquisition was performedwith a Nikon C1si laser scanning confocal unit (Nikon D-eclipse C1, Japan) attached to an inverse fluorescencemicroscope (Nikon D-eclipse C1, Japan) with 100×/1.49 oilApo TIRF objective (Nikon, Japan). Excitation wasperformed with an air-cooled argon ion laser emitting at488 nm and appropriate filter sets were used to collect thefluorescence emission. Images were acquired and processedusing the NIKON software EZ-C1.

Results and discussion

SERS studies for surface interaction of polyelectrolyteswith gold nanoparticles

In order to understand the binding of the first polyelectro-lyte layer, surface-enhanced Raman spectroscopy wasperformed. SERS experiments will give information aboutthe chemical structure and orientation of moleculesadsorbed directly onto the metal surface of nanoparticles(most commonly Ag or Au).

In Fig. 2a, the normal Raman spectra of sodium citrate,PSS, and PAH aqueous solutions are shown, together withthe average SERS spectra of citrate-stabilized gold nano-particles and NG coated with PAH or PSS (Fig. 2b).Additionally, a normal Raman spectrum of PAH mixed with

PSS in a 1:1 ratio was measured to understand better thenature of binding between the two oppositely chargedpolymers (Fig. S1 in ESM). The SERS spectrum of citrate-stabilized NG is in substantial agreement with thosepreviously reported in literature [38]. The intense SERSbands corresponding to the anti-symmetric (1,640 and1,533 cm−1) and symmetric (1,380 cm−1) stretching ofcitrate carboxylates are down-shifted with respect to thenormal Raman spectrum, indicating an adsorption on themetal surface via the COO− moieties. The presence ofintense bands for both anti-symmetric and symmetricstretching suggests a variability of orientations for theadsorbed citrate, in which the carboxylate moieties areoriented with the line joining the two oxygen atoms bothparallel and perpendicular to the metal surface [38].

The SERS spectrum of PSS-coated NG (NG/PSS) showsa striking similarity with that of citrate-stabilized NG,indicating the persistence of the adsorbed citrate layer onthe gold surface upon the addition of PSS. In spite of thisresemblance, some significant differences are observed,such as the emerging of a band at 1,591 cm−1 and thedecrease in intensity of the band at 1,640 cm−1. The band at1,591 cm−1 can be attributed to the aromatic C–C stretchingof the PSS benzene ring, which is present as an intense andnarrow band at 1,598 cm−1 in the normal Raman spectrumof PSS [39]. The occurrence of this band suggests thepresence of the PSS near the metal surface. Moreover,SERS spectra of NG/PSS present less variability than thoseof citrate-stabilized NG, as indicated by the respectiveintensity standard deviations in the spectral regions of theanti-symmetric and symmetric carboxylate stretching(depicted as grey lines in Fig. 2, right panel). Thisobservation suggests that the adsorption of PSS perturbs

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Fig. 2 a Normal Raman spectraof citrate, PSS, and PAH (bottomto top) measured in water. bSERS spectra of uncoated andcoated NG. For SERS spectra,average spectra (over four sam-ples) are reported as black linesand standard deviations as greylines. The excitation wavelengthwas 632.8 nm, and the power atthe sample was 15 mW. Acqui-sition time was 30 s

Colloid Polym Sci (2011) 289:269–280 273

the pre-existing citrate layer, inducing citrate molecules toadsorb on gold with the carboxylates having a preferentialorientation (i.e., with the line joining the two oxygen atomsparallel to the surface).

The addition of PAH as second layer in NG/PSS/PAHsamples significantly perturbs the pre-existing layers, asshown by its SERS spectrum. The spectral features due tothe adsorbed citrate are still present, indicating thepersistence of the citrate layer, but the intensity standarddeviation increases upon PAH binding. Also, the PSS layeris still present, as suggested by the PSS band at 1,591 cm-1.Interestingly, a band appears at 1,128 cm−1 upon PAHbinding. Due to the uncertain assignment of this band, it isdifficult to put forward a hypothesis about the nature of thisinteraction, which needs further investigation. The presenceof PSS on the citrate-coated particle surface is inferred fromboth indirect (Fig. 2, NG/PSS) and direct (NG/PSS/PAH)experimental evidence. The electrostatic repulsion betweenthe citrate-coated surface and PSS is likely to be shieldedby the well-known “counter ion condensation” effect,which can even lead to a counter-intuitive phenomenon ofan attractive interaction between like charged polyelectro-lytes or nanoparticles [40–42].

In case of PAH as first layer, the citrate layer appears tobe displaced by PAH in NG/PAH samples, as indicated bythe SERS spectrum. In fact, in the spectra of NG/PAH, thecharacteristic carboxylate stretching bands due to citratedisappear and are replaced by a group of weak and broadbands, which are in agreement with previously reportedPAH SERS spectra [43, 44]. These bands are difficult toassign to PAH vibrations. The two broad bands at 1,370and around 1,600 cm−1 could be due to the formation ofamorphous carbon upon laser irradiation which are oftenobserved in SERS spectra of polymers [45]. On the otherhand, the 1,300–1,700-cm−1 region in the SERS spectrumof PAH bears some resemblance to the PAH normal Ramanspectrum, with the SERS bands at 1,600, 1,443, and1,370 cm−1 corresponding to the normal Raman bands at1,595, 1,463, and 1,353 cm-1. Indeed, the band at1,600 cm−1 could be due to the vibrations of the –NH2

groups of PAH that interact with the metal surface. In fact,amino groups are known to have a stronger affinity formetals than carboxylates [46], and therefore, PAH is likelyto displace citrate by adsorbing on the gold surface via its –NH2 group. This latter hypothesis is supported by the SERSband at 1,443 cm−1, which can be assigned to the bendingor the –CH2– groups on PAH backbone [47].

The SERS spectrum of NG/PAH/PSS is almost identicalto that of NG/PAH, indicating that the addition of PSS doesnot significantly perturb the pre-existing PAH layer. Thepresence of an additional PSS layer, however, is inferredfrom the appearance of the two weak bands at 1,128 and1,591 cm−1, which are attributed to the S = O stretching of

the undissociated form of PSS and to the aromatic C–Cstretching of the PSS benzene ring. It is interesting to notethat in all the samples in which PSS is associated with PAH(i.e., in NG/PSS/PAH and NG/PAH/PSS), the band at1,128 cm−1 is present, whereas in NG/PSS, it is absent.This observation is in good agreement with what wasobserved in the case of NG/PSS/PAH.

As to our knowledge, for the first time, a detailedorientation and binding of the first polycation (PAH) orpolyanion (PSS) layer to the curved gold surface ofnanoparticles was described. In the presented SERS study,we confirmed the counter-indicative binding of the nega-tively charged PSS to the negatively charged core surfaceand studied the underlying binding mechanism. Thechanges observed in the spectrum of the aromatic systemof PSS and the fact that some of the peaks from the PSSspectrum are visible in the citrate-gold spectrum indicatethat PSS binds but without replacing the citrate shell. BySERS, it was shown that the poly-allylamine is orientedwith the primary amine groups toward the gold surface,which was expected and is in accordance to the results ofother groups [48].

Synthesis of polyelectrolyte multilayer-coated goldnanoparticles stable in the presence of small ions

Though PNG are good candidates for targeted drug deliveryor therapeutic approaches as in local radio- and hyper-thermal therapy for cancer treatment, the major obstacle intheir use is the low stability against agglomeration in thepresence of ions.

In order to overcome this obstacle, which we hypothe-size to be due to bridging flocculation induced by amismatch of particle size and PE length or incomplete PEattachment to the particle surface, the PEs were pre-incubated in a 0.5 M NaCl solution. This should collapseweak polyions such as PAH from extended chains (purewater induces fully charged monomers in the chain andhence repulsion) to random coils. Moreover, we observedin preliminary experiments that if ions were added in theencapsulation process for the first five layers, immediateagglomeration took place deduced from a color changefrom red to blue indicative for aggregates. But if the firstfive layers were deposited from aqueous polyelectrolytesolutions, then the following layers can be deposited from0.5 M NaCl containing polyion solutions with a reducedaggregation (small amount of aggregates are detected byUV–vis (shoulder at 650 nm, Fig. 5d, e), SEM (84±20%single particles, Fig. 3c) and a slight color change to bluishred (data not shown)).

The diagrams in Fig. 3 depict surface charge (Fig. 3a)and hydrodynamic diameter (Fig. 3b) versus the number oflayers for the PNG0.5 measured by DLS and ζ-potential. In

274 Colloid Polym Sci (2011) 289:269–280

comparison to particles coated with the same polyelectro-lytes but from pure water (Fig. S2 in ESM), the PNG0.5 arelarger. This is in good agreement to the hypothesis that thePE is binding as a collapsed random coil to the NG surfaceand that more polyelectrolyte is needed to overcompensatethe charge of the underlying layer because of a lowercharge of the single polyelectrolyte chain due to shielding.That the large increase in diameter is not due to particleaggregation can be seen from the SEM image (Fig. 3c),which showed that most particles (84%, counted particles128 (aggregates count as 1)) exist as single coated particles.Here, we depict exemplarily nanogold coated with thesequence [(PAH/PSS)2/PAH/(PSS0.5/PAH0.5) or 7A PNG0.5.In the following, these particles are called 7A PNG0.5 inwhich 7 gives the number of layers, the index 0.5 indicatesthat the last two layers were deposited from polyelectrolytesolutions containing 0.5 M NaCl, and the A that theoutermost layer is the positively charged PAH while S willindicate a negative PSS outermost layer. By SEM, thethickness of the collapsed polymeric shell (bright rimaround the NG) completely deprived by water can beestimated to be 8 nm (data not shown).

We found that with the deposition of PSS0.5 as sixth layer,the surface charge significantly decreases from −60 mV forthe fourth layer to −40 mV, while for PAH0.5, the value of+50 mV is the same for the fourth and the sixth layers(Fig. 3a). A possible explanation can be the nature of theunderlying, fifth layer is composed of PAH in case of PSS0.5as the sixth layer. PAH is a weak polyion and hence moreprone to counter-ion shielding. Once in contact with thePSS0.5 solution, chloride ions penetrate the layer and shieldsome of positive charges of PAH, which will lead to areduced polyanion binding in the next layer.

For PAH0.5 as sixth layer, the situation is different. PSS(fifth layer) is a strong PE and is not very prone to counter-ion binding [49]. Thus, the surface negative charge remains

high. But here, the shielding ions in the collapsed polycationreduce the number of free charges in PAH0.5, and hence, asignificantly higher amount of PAH0.5 must bind toovercompensate the negative charge of the underlying layer.This is supported by the size determination in DLS thatshowed a significant increase in particle diameter for thesixth layer (PAH0.5; arrow in Fig. 3b).

In accordance to the results found by Schneider andDecher [14], the presence of ions (NaCl or Ringer’ssolution) causes immediate agglomeration of NG andPNG if all layers are deposited from pure water (Fig. 4).

The hydrodynamic diameter (Fig. 4) shows a strongincrease in the diameter for NG and PNG indicating particleagglomeration, especially in Ringer’s solution. This isconfirmed by the PDI value which increases up to 0.7

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arrow). For both diagrams, circles are related to coatings starting withPSS and the squares to that with PAH as first layer. c Overview SEMimage of 7A PNG0.5. The image shows that 84% of the PNG0.5 aresingle particles

NG 7A PNG7S PNG 7S PNG0.5 7A PNG0.5

Fig. 4 Agglomeration of NG, PNG, and PNG0.5 in the absence or thepresence of small ions. The particle diameter was measured in MQwater (striped), 0.5 M NaCl (light grey), or Ringer’s solution (darkgrey). DLS measurement of the hydrodynamic diameter (columns) andthe PDI (circles) versus particle preparation and stored for 2 h indifferent solutions

Colloid Polym Sci (2011) 289:269–280 275

indicative for polydispersity. In general, it can be stated thatthe particles coated from pure water are more polydisperse(PDI around 0.4) compared to the PNG0.5 (PDI 0.2–0.3)even for storage in water (Fig. 4, striped column). Moreover,the UV–vis absorption spectrum for NG and PNG shows eithershoulder at 630 nm indicative for aggregates or a decrease inpeak intensity (Figs. 5 and S3). Comparing the peak maximumin the UV–vis spectra for NG (Fig. S3a in ESM) with thePNG (Fig. S3b, c in ESM)) or PNG0.5 (Fig. 5a, b), a small redshift can be seen for which the coating is responsible.

In spite of a small shoulder for small aggregates from theparticle coating, it can be clearly seen by UV–vis thatexposure of the PNG0.5 (Fig. 5a, b) to ion containingsolutions does not change the absorption spectrum, neitherin intensity nor in terms of red-shift, especially for thePSS0.5 finishing coating (Fig. 5a).

The improved stability of single PNG0.5 particles cannotbe explained by electrostatic repulsion, since the surfacecharge decreases as measured by zeta-potential. Bridgingflocculation due to the excess length of the polyelectrolyteswas the main reason Schneider and Decher identified forthe nanoparticle agglomeration caused by small ions [14]. Itwas reported that the polycation, PAH, collapse into arandom coiled structure in the presence of NaCl concen-trations >0.3 M [20]. So, the increased stability againstagglomeration of PNG0.5 can be explained by a bettermatch of the particle size and the gyration radius of thecollapsed polycation preventing bridging flocculation.

For the observed aggregation in the case of the first fivelayers containing ions, a possible explanation could be thefollowing. The model by Decher [18] for the film growth inthe layer-by-layer technique proposed that the first “pre-cursor” layers on flat surfaces differ from latter onesbecause the polyelectrolyte adsorption is influenced by thevicinity to the template. Moreover, these layers are

supposed to contain small counter ions, and they arecharged. We assume that the same is true for self-assembled layers on curved templates like gold nano-particles. From the experiments, we deduce that on NG, thefirst five layers present the precursor layers and that theyare more prone to small ion interference. For latter layers,the proposed “neutral layers from zone II” from Decher’smodel stabilize the capsule, and the influence of small ionson agglomeration decreases [20].

Electrostatic functionalization of FPNG0.5 with folic acidfor cancer cell targeting

The electrostatic binding of folic acid was tested as anexample for a promising targeting for cancer cells. Due tothe fact that the receptor recognizes the pteroic acid moiety[50] in the folic acid molecule, the molecule waselectrostatically bound via α- and γ-COOH (glutamic acidmoiety) to a positive outermost polyelectrolyte layer. Inorder to be able to follow particle up-take in real-time,fluorescence microscopy fluorescently labeled PNG werefunctionalized with folic acid. In DLS and ζ-potentialmeasurements, it was found that the hydrodynamic diam-eter of FPNG0.5 with coating sequence [(FITC-PAH/PSS)2/FITC-PAH/(PSS0.5/FITC-PAH0.5)] increases from 174±5 to213±14 nm and surface charge decreases from 32.4±0.6 to19±3.5 mV because of folic acid binding (Fig. 6a).

The presence of folic acid was further confirmed bySERS. However, only a proof of concept for folic acidbinding was achievable because of the short range of theSERS effect. Therefore, a SERS spectrum of NG coatedwith one PAH layer, and folic acid was measured (Fig. 6b).The spectrum in Fig. 6b (top) shows intense bands, whichare very similar in both, relative intensity and frequency, tothose of the normal Raman spectrum of folic acid (Fig. 6b,

533

533

528529

ba

λ (nm) λ (nm)

Abs

orb

ance

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ance

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0.8

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0.2

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0.8

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0.4

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0.0400 450 500 550 600 650 700 400 450 500 550 600 650 700

528

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Fig. 5 Aggregation of PNG0.5 coated according to the new protocol.The first five layers are deposited from pure water while the last twoare from PE0.5 in different ionic solutions. The particles wereincubated for 2 h in water (straight line), in 0.5 M NaCl (dashed

line) or in Ringer’s solution (dotted line) before UV–vis absorptionspectroscopy. a PSS0.5 or b PAH0.5 as last layer. All particles arecoated with seven polyelectrolyte layers

276 Colloid Polym Sci (2011) 289:269–280

bottom). Moreover, it is significantly different from theSERS of pure PAH-coated nanoparticles (Fig. 2b). Thesimilarity with the Raman spectrum of folic acid clearlyindicates that the molecule is adsorbed close to the goldnanoparticle surface. Experiments with folic acid attachedto the third, fifth, and seventh layer did not give any signalin SERS. From this observation, we deduced that folic acidis not penetrating deeply in the polyelectrolyte matrix butremains attached to the polyelectrolyte shell surface.Despite some differences, most likely due to the differentnature of the surface, the SERS spectrum of folic acid onPAH-coated gold nanoparticles shares many spectral fea-tures with the SERS spectrum of folic acid on silvernanoparticles, as recently reported by Stokes et al. [51].

Furthermore, the presence of folic acid on the coatedgold nanoparticle was confirmed by absorption spectrum ofFo-PNG0.5, which shows prominent peaks at 280 and350 nm (Fig. 6c, blue line), which are characteristic forfolic acid (red line), along with an absorption at 533 nmfrom the gold core (Fig. 6c, black line). The small red shiftin the shoulder of folic acid (arrows) can be a hint that thefolic acid is associated with the PNG and that the signaldoes not come from unbound folic acid which was notcompletely removed from the solution.

Interaction of Fo-FPNG0.5 with macrophage and cancer cells

The uptake of Fo-FPNG0.5 was followed in two differentbreast cancer cell lines, i.e., VP229 and MDA MB 231,which are known to over-express the folate receptor. In thediagram in Fig. 7a, comparing the amount of cells withfluorescently marked PNG0.5 with and without folic acidfunctionalization, the folic acid binding to the polyelectrolyteshell leads in both tested breast cancer cell lines to asignificant (p≤0.001) increase in particle endocytosis,

indicating that it is folate receptor mediated. This was alsosupported by the competition assay in which increasingconcentrations of free folic acid hinder the up-take of the Fo-functionalized FPNG0.5 (Fig. 7b) as shown exemplarily forMDA MB 231 cells.

In Fig. 7c, endocytosis of FPNG0.5 into VP229 breastcancer cells was visualized by confocal fluorescencemicroscopy. Due to the fact that the cells were notsynchronized, the cells are in different stages of theendosomal pathway (scheme and tags in Fig. 7d). In thecell tagged with 1, the endosomes are still distributedhomogenously in the cytosol. The endosomes in the celltagged with 2 are accumulating FPNG0.5 close to thesurface of the nucleus, while in the cell tagged with 3, theparticles are close to the nucleus, perhaps even released tothe nucleus. It is well known that folate receptor-mediatedendocytosis guides the vesicular content directly to thenucleus [21].

The observed high up-take by cancer cells of folic acidfunctionalized gold nanoparticles is in good accordance toother studies described in the literature for folic acid-poly-ethylenglycol conjugates [29, 30].

Macrophage recognition (i.e., innate immunologicalresponse) of nanoparticles is the main reason for fastclearance of charged drug delivery systems from blood.Therefore, we evaluated by confocal fluorescence mi-croscopy the Fo-FPNG0.5 content in non-activated macro-phages after 4 h of incubation. We counted thatapproximately 30% of the macrophages incorporate theFo-FPNG0.5 (Fig. 8).

A possible explanation for this low up-take forfunctionalized and non-functionalized FPNG0.5 is anunspecific endocytosis or pinocytosis rather than receptor-mediated endocytosis because the macrophages do notexpress folate receptors.

a

FPNG0.5 Fo-FPNG0.5

c Fo-PNG0.5

PNG0.5

Folic acid

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.)

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Hydro

dynamic diam

eter (nm)

Fig. 6 a Hydrodynamic diameter and zeta-potential of FPNG0.5

coated with the sequence [(FITC-PAH/PSS)2/FITC-PAH/(PSS0.5/FITC-PAH0.5)] before and after folic acid binding. b SERS spectrumof NG coated with PAH followed by folic acid binding (top); normalRaman spectrum of folic acid (powder) (bottom). Excitation at

785 nm, power at the sample 90 mW. c UV–vis spectra of stablePNG0.5 before (black)and after (blue) folic acid binding comparedwith the spectrum of pure folic acid (red). The arrows indicate a smallred shift possibly related to binding of folic acid to the PNG0.5

Colloid Polym Sci (2011) 289:269–280 277

a b

0

20

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% o

f m

acro

ph

ages

wit

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ocy

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Fo-FPNG0.5 FPNG0.5

Fig. 8 a Diagram of the percentage of non-activated J774.2 macrophages with endocytosed FPNG0.5. b Merged transmission and confocalfluorescence micrograph of macrophages with internalized FPNG0.5 (arrows) after 4-h incubation

50µµm

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aFPNG0.5Fo-FPNG0.5

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Fo-FPNG0.5

Folate receptormediatedendocytosis ofFo-FPNG0.5

Endocytosis

Folate receptor

Lysosome

Fusion withendosome

3

2

1

Fig. 7 a Receptor-mediated endocytosis of FPNG0.5 with and withoutfolic acid functionalization by two breast cancer cell lines, VP229 andMDA MB 231, displayed in percentage of cells with particles. bDiagram of free folic acid concentration in the presence of a constantamount of Fo-FPNG0.5 versus the percentage of MDA MB 231 cellswith particles. The competition study confirms the folate receptor-mediated up-take. Inset: Fluorescence micrograph of MDA MB 231

cells loaded with Fo-FPNG0.5 in the presence of free folic acid at agiven concentration (arrow) after 24-h incubation. c Confocalfluorescence micrograph of VP229 cells with internalized Fo-FPNG0.5 after 2-h incubation. Red numbers indicate fluorescentparticle aggregates in different stages of endocytosis. The numberscorrespond to different steps of internalization depicted in d Schemeof the proposed folate receptor endosomal pathway

278 Colloid Polym Sci (2011) 289:269–280

Conclusion

Polyelectrolyte multilayer-coated gold nanoparticles are apromising tool for diagnostics as well as non-toxic deliveryvehicle for drugs. It was shown by Schneider and Decher[14] that encapsulation in presence of ions like sodium orchloride, hence medicinal solutions, leads to gold nano-particles agglomeration. Summarizing our results, the novelencapsulation improves the stability of polyelectrolytemultilayer-coated nanoparticles in the presence of smallions and hence makes the resulting particles an interestingsystem for nanodrug delivery. The same particles allow foran easy, fast, and stable non-covalent but electrostaticfunctionalization for targeted delivery as shown by thebinding of the model targeting molecule, folic acid.

These targeted stable PNG0.5 can serve as signalenhancer and antenna for thermo- and radio-therapy incancer therapy due to the gold core or loaded electrostat-ically with drugs as delivery system. High stability in thepresence of ions and facilitated binding of targetingmoieties in addition to the possibility to incorporate ineach deposited layer a different drug or increasing amountsof the same drug and the visibility of the gold in X-ray,electron microscopy, and multi-photon fluorescence mi-croscopy promise a high impact of coated gold nano-particles in diagnostics and as drug delivery system.

As sometimes the size of the particles plus coatingmatters to have the possibility to create a coating startingwith either a positive or a negative first layer on gold is anadd-on. In the present work, we clarified with SERS theunderlying mechanism for the unexpected binding of thepolyanion and described the orientation of the polyelec-trolytes towards the gold surface.

Acknowledgments This work was financially supported by theItalian grant CIPE. SM was financially supported by a SISSA PhDfellowship. AB acknowledges support from Fondazione CRTrieste.VS acknowledges partial support from the Scientific Direction ofIRCSS Burlo Garofolo. The authors thank Dr. F. Petrera for thetechnical help with the cell culture, Dr. D. Latawiec for the manuscriptrevision, and A. Bruns and M. Kaszuba from Malvern for theirsupport with the DLS. Special thanks to Prof. G. Scoles for hisvaluable suggestions and discussions.

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