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Journal of Colloid and Interface Science 345 (2010) 241–247
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Journal of Colloid and Interface Science
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Layer by layer chitosan/alginate coatings on poly(lactide-co-glycolide)nanoparticles for antifouling protection and Folic acid binding to achieveselective cell targeting
Jie Zhou a,b, Gabriela Romero b, Elena Rojas b, Lie Ma a, Sergio Moya b,*, Changyou Gao a,*
a Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, Chinab CIC BiomaGUNE, Paseo Miramón 182 Ed. Emp. C, San Sebastián, Spain
a r t i c l e i n f o a b s t r a c t
Article history:Received 4 December 2009Accepted 4 February 2010Available online 12 February 2010
Keywords:Poly(lactide-co-glycolide) nanoparticlesChitosaneAlginateLayer by layerAntifouling protectionTargetingCellular uptake
0021-9797/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.jcis.2010.02.004
* Corresponding authors. Fax: +86 571 87951108Moya).
E-mail addresses: [email protected] (S.(C. Gao).
Polyelectrolyte multilayers (PEMs) composed of two natural polysaccharides-chitosan (Chi) and alginate(Alg) were deposited by Layer by layer (LbL) assembly on top of biocompatible poly(lactide-co-glycolide)(PLGA) nanoparticles (NPs). Folic acid (FA) or FA grafted poly(ethylene glycol) (PEG–FA) were covalentlybounded to the PEMs via carbodiimide chemistry. The assembly of biocompatible PEMs was monitoredon planar surfaces by means of the quartz crystal microbalance with dissipation (QCM-D) techniqueand on top of PLGA NPs by means of f-potential measurements. BSA was used as model protein to char-acterize protein adsorption on PEMs. QCM-D showed protein deposition could not be observed on theChi/Alg multilayer, for both Chitosan and Alginate as top layers. Finally, cellular uptake experiments werecarried out by co-culture of HepG2 cells in presence of NPs. Flow Cytometry and confocal laser scanningmicroscopy (CLSM) were used to investigate the influence of the surface chemistry of the NPs on uptake.For the HepG2 cell line significantly less uptake of PLGA NPs coated with Chi/Alg than the bare NPs wasobserved but the uptake increased after attachment of FA molecules.
� 2010 Elsevier Inc. All rights reserved.
1. Introduction
Polyelectrolyte multilayer (PEMs) fabricated by means of theLayer by layer technique (LbL) provide a very versatile tool forthe noncovalent engineering of surfaces [1,2]. LbL involves theelectrostatic interaction between oppositely charged polyelectro-lytes, which are stepwise assembled on top of a charged surfaceto form a thin polymer film [3–6]. Both synthetic and natural poly-electrolyte molecules have been used for the LbL assembly. Naturalpolyelectrolytes are useful in those situations, where biocompati-bility is required; for example, in the fabrication of delivery devices[7,8]. There has been indeed a significant amount of research de-voted to the templation of capsules for drug delivery based onPEMs [9–15]. For capsule fabrication PEMs are assembled on topof sacrificial colloids, which are dissolved after the LbL coating[15,16]. After core dissolution, the PEM remains, forming an emptyclosed shell, which can be loaded with different materials [17,18].Besides the loading of the capsule interior, materials for deliveryi.e. DNA or proteins can be assembled between the layers in thePEMs. The loaded material will be released from the PEM, or the
ll rights reserved.
(C. Gao), +34 943005311 (S.
Moya), [email protected]
PEMs will be eventually peeled off liberating the entrapped mate-rial [12]. In this delivery strategy PEMs can also be assembled ontop of nano and micro particles, and not form capsules. Also, PEMscan be deposited on particles with encapsulated materials as bar-riers for release control or as a functionalization tool, to providethe nanoparticle with an specific recognition function, which canbe assembled through a noncovalent route [19,20].
In this work, we employ a biodegradable pair of polyelectrolytes:alginate (Alg) and chitosane (Chi) to coat poly(lactide-co-glycolide)(PLGA) nanoparticles (NPs) [21–23] stabilized with the protein bo-vine serum albumin (BSA). These NPs are excellent candidates fordrug delivery due to their biocompatibility and degradability [24–27]. Also, they can be easily loaded for delivery [28–30]. The coatingof these NPs with a chitosane/alginate PEM has two scopes. First, wewill show that the multilayer has antifouling properties, showinglow protein adsorption and then, we will show that the unspecificcell uptake of PEM coated NPs will be reduced by the coating. Thechitosane/alginate coating can therefore be used as an alternativefor PEGylation [30] in the surface modification of NPs. The PEMs pro-vide many functional groups that can be modified or used as bindingsites for a further modification of the NPs via carbodiimide chemis-try. Also, in this way covalent chemistry would be applied to the PEMwithout affecting the stability of the NPs. We will show that the func-tional groups present in the chitosane/alginate PEM can be used toattach specific recognition functions like Folic acid (FA) or PEG
242 J. Zhou et al. / Journal of Colloid and Interface Science 345 (2010) 241–247
modified Folic acid (PEG–FA). PEG will be used as a linker here to im-prove the accessibility of FA molecules. The coating with FA andPEG–FA will be employed to increase NP uptake in selected cellcancer lines since it is known that cancer cells overexpress folatereceptors [31–34] The PEM assembly, the covalent attachment ofFA and PEG–FA as well as the interaction of the coatings with modelproteins will be studied on planar model surfaces and NPs by meansof the quartz crystal microbalance with dissipation technique (QCM-D), UV–Vis spectroscopy, and f-potential. The influence of thecoating on the cell targeting will be studied by Flowcytometry andconfocal laser scanning microscopy (CLSM) using the HepG2 cellline.
2. Experimental section
2.1. Materials
Poly(lactide-co-glycolide) (PLGA) (D,L-lactide 90: glycolide 10),average molecular weight of 100 kDa, was purchased from the Chi-na Textile Academy. 5-Dodecanoylaminolfluorescein (DAF) wasobtained from Invitrogen. Chitosan (Chi, Mw 100–300 kDa), so-dium alginate (Alg, Mw �25 kDa) were purchased from ACROS. Fo-lic acid (FA), phosphate buffered saline (PBS), ethylcarbodiimidehydrochloride (EDC), N-hydroxysuccinimide (NHS), bovine serumalbumin (BSA), Dulbecco’s Modified Eagle’s Medium (DMEM), fetalbovine serum (FBS) and 4’,6-diamidino-2-phenyindole (DAPI) werepurchased from Sigma–Aldrich. Folic acid grafted PEG was made bycarbodiimide chemistry [17]. The cell line HepG2 was purchasedfrom the American Type Culture Collection (ATCC).
2.2. PLGA NPs preparation
PLGA NPs were prepared by means of a W/O emulsion-solventevaporation method. Firstly, 1 mL of 20 mg/mL PLGA dichloro-methane solution (organic phase) was added to 4 mL of a 2% BSAsolution (water phase) and then emulsified with an ultrasonicator(SONICS� VCX 500) for 20 s. This emulsion was poured into 100 mLdistilled water, and stirred for 3 h with a magnetic stirrer until theorganic solvent was totally evaporated. The PLGA NPs were col-lected by centrifugation at 10,000g for 5 min, and washed withMilliQ water 5 times to remove free BSA initially presented inthe water phase. PLGA NPs containing rhodamine 6G (Rd6G) weresimilarly prepared by addition of 0.5 mg/mL Rd6G into the PLGAsolution before mixing with the water phase.
The NP morphology as measured with transmission electronmicroscopy (TEM) (JEOL JEM-2100F, Japan) can be observed inFig. S1a. NPs have spherical shape and can be observed as individ-ual particles with diameters of around 200 nm. Dinamic Light Scat-tering experiments (DLS) (Malvern NanoZS, UK) show that PLGANPs display a narrow distribution, being the average diametermeasured 400 nm (Fig. S1b).
2.3. LbL assembly on planar surface and NPs
Chitosan and alginate were assembled in a concentration of1 mg/mL in 0.5 M NaCl. The pH value of the polyelectrolyte solu-tion was adjusted to 5 by addition of either 1 M HCl or NaOH.For the assembly of Chi/Alg multilayers on the PLGA NPs the incu-bation time of each polyelectrolyte layer was 15 min. NPs werecentrifuged after each polyelectrolyte deposition and washed in0.5 M NaCl 3 times before deposition of the next layer.
2.4. UV–Vis spectroscopy
After the binding of FA or PEG–FA on planar Chi/Alg multilayersdeposited on glass slides, the UV absorbance was measured with a
Cary 5000 UV–Vis-NIR Spectrophotometer. Glass slides coatedwith (Chi/Alg)2/Chi were used as control sample. The absorbancebetween 350 and 600 nm of the glass slides covered with FA orPEG–FA grafted (Chi/Alg)2/Chi was recorded between 350 and600 nm. The absorbance of FA was measured in solution using aquartz cuvette.
2.5. Quartz crystal microbalance
The quartz crystal microbalance with dissipation technique(QCM-D) (E4 Q-Sense, Sweden) was used to monitor mass and dis-sipation changes during multilayer assembly. Gold coated quartzcrystals (5 MHz) were used as substrates. Chitosan and alginatelayers were alternatively deposited on the crystals. For the coating,the incubation time was 15 min for each layer, followed by wash-ings with 0.5 M NaCl for 15 min. On total, up to three bilayers ofAlg/Chi were assembled on the crystals. The mass deposited onthe crystal was calculated using the Sauerbrey equation.
Dm ¼ �C � Dfn
C = 17.7 ng Hz�1 cm�2 for a 5 MHz quartz crystal, n = 1,3,5,7 and isthe overtone number.
FA or PEG–FA were immobilized on the PEMs with chitosan asthe outmost layer. The immobilization process was monitoredwith QCM-D. In a first step, the carboxylic groups of FA from1 mg/mL FA or 5 mg/mL PEG–FA were activated with 10 mMNHS and 10 mM EDC at pH 5.3 for 30 min, then the pH value ofthese solutions were adjusted to 8.6. The solutions were subse-quently injected into the different QCM-D chambers, which wereinitially stabilized for 5 min in MilliQ water. After incubated for40 h the samples were rinsed with MilliQ water until the all thefrequencies became stable.
2.6. f-potential measurements
The LbL assembly and FA or PEG–FA immobilization on the NPswere characterized by f-potential measurements (Malvern Na-noZS, UK) using 10 mM NaCl (pH 7.4) as medium.
2.7. Protein adsorption on multilayers
The BSA protein (1 mg/mL in 10 mM PBS) was used as modelprotein to study protein adsorption on the PEMs by means of tQCM-D. PEMs of the following composition were assembled onthe QCM crystals following the above mentioned protocols: (Chi/Alg)2/Chi, (Chi/Alg)3, (Chi/Alg)2/Chi-FA and (Chi/Alg)2/Chi-PEG–FA. The QCM-D chamber was initially incubated with MilliQ waterfor 5 min, and then replaced with PBS. The BSA solution was in-jected into the QCM chambers. Five minutes later, the injectionwas stopped and the PEMs were incubated in the BSA solutionfor 1 h. Finally, the BSA solution was sequentially replaced byPBS and water.
2.8. Cellular uptake
HepG2, an immortalized liver cancer cell line, was cultured inDMEM with 10% FBS and 100U penicillin, 100 lg/mL streptomycinat 37 �C and in 5% CO2. When the cell confluence was around 70%,all the cells were trypsinized and resuspended in culture medium.300,000 cells were seed into each well of a 24-well culture plate.PLGA NPs labeled with Rd6G were added into the culture medium24 h later. The final NPs concentration was adjusted to 100 lg/mL.After cultured for 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h, the cellswere washed twice with PBS, trypsinized and studied with flowcytometry (BD FACSCanto™ II). The cell uptake ratio was calcu-
J. Zhou et al. / Journal of Colloid and Interface Science 345 (2010) 241–247 243
lated from the dotplot graph of forward scattering (FCS) vs. fluores-cence intensity (PE-A). Data were analyzed with the WinMDI pro-gram. First, a threshold of fluorescence was generated using acontrol sample, i.e. the HepG2 cells without exposure to the NPs.All dots corresponding to the control sample are located at intensi-ties below this threshold. The number of cells carrying fluores-cently labeled NPs is obtained from the area corresponding tothe dots located at higher intensities than the threshold. The cellu-lar uptake ratio is equal to the (No. of the dots over the threshold/total no. of the dots) � 100%.
confocal laser scanning microscopy (CLSM, Carl-Zeiss LSM 10META) was applied to image the HepG2 cells after incubation withNPs. 100,000 cells were seeded on a coverslip, which was placedinto a 3.5 cm culture plate. 24 h later, the PLGA NPs containingRd6G were added into the culture medium with a final concentra-tion of 50 lg/mL. After incubation for another 12 h the culturemedium was removed and the cells were rinsed 3 times with ster-ilized PBS. The cells were subsequently fixed with 3.7% formalde-hyde solution in PBS for 30 min. Finally, the cells were incubatedwith DAF (500 ng/mL) and DAPI (100 ng/mL) in 10 mM PBS for20 min to stain the cell membrane and nucleus, respectively. Afterwashed 5 times with PBS, the cells on these coverslips were ob-served under CLSM employing a 63� oil objective.
3. Results and discussion
The assembly of chitosan and alginate was first monitored onplanar substrates by means of QCM-D.
Fig. 1a shows the assembly of Chi/Alg PEM followed with QCM-D. 3 bilayers of chitosan and alginate were assembled following theLbL procedure. Previous to the Chi/Alg coating poly ethylene imine(PEI) was assembled since it results on a good coating of a gold sur-face and then on top of PEI BSA was deposited. BSA was assembledto reproduce the surface of the BSA stabilized PLGA NPs. When thefirst layer of chitosan was assembled, frequency almost did notchange but after assembling the next alginate layer a prominentfrequency change could be observed (Fig. 1a). The 2nd layer ofAlg showed a decrease in frequency of 40 Hz and the third layerof 50 Hz while for the Chitosan only a shift of 20 Hz was observed.The assembly of alginate always induced a bigger frequency shiftthan chitosane. Also, dissipation was always larger after alginateadsorption than after chitosan, hinting that more water moleculesentrapped in the alginate layer than in the chitosan one. QCM-Dexperiments combined with ellipsometry showed that the alginate
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Fig. 1. Frequency and dissipation changes monitored by QCM-D for (a) the Layer by la
layer is highly hydrated while the dry and wet mass of chitosan arealmost coincident.
After the last chitosan layer was deposited, the frequency de-creased �22 Hz, which would correspond to 0.39 lg/cm2 chitosanaccording to Sauerbrey equation, assuming that the mass increasecomes from the chitosan alone and there is no water entrapped.This would represent a maximal possible density amino groupsof 2.4 nmol/cm2. These amino groups are reactive for further mod-ification through carboimine chemistry.
The binding of FA and PEG–FA to (Chi/Alg)2/Chi in presence ofEDC/NHS was also studied by means of QCM-D. The mixture ofEDC/NHS is used to catalyze the amidation of amine groups fromthe multilayers and the carboxylic groups from FA or PEG–FA(Fig. 1b). After the reaction and the rinsing with MilliQ water a finalfrequency changes of �6 Hz and �10 Hz were observed for thebinding of FA and PEG–FA, respectively. Since only small changesin dissipation were observed after grafting FA and PEG–FA it is pos-sible to apply the Sauerbrey equation to calculate the density of thegrafted molecules. A density of 0.24 nmol/cm2 and 0.08 nmol/cm2
were observed for FA and PEG–FA respectively. Assuming a densityof 2.4 nmol/cm2 for the amine groups of the chitosan a graftingyield of 10% for FA and of 3.3% for PEG–FA can be calculated.
The differences in the grafting yield must be due to the changesin the chemical structure of FA after being conjugated with PEG.For PEG–FA the available number of carbolic acid decreases eventhough the feeding molar ratio is almost the same. Moreover, thePEG chain may also bring a certain spatial hindrance, which re-duces the reactivity of the carboxylic acid, being unfavorable forthe immobilization of the PEG–FA.
UV–Vis spectra (Fig. 2) further confirmed the immobilization ofthe FA on the multilayers. The spectra in Fig. 2 shows absorbance inthe region of 350–400 nm after immobilization of FA and PEG–FA,which is missing in the chi/alg multilayer (control). This region ofthe spectra can be assigned to the FA (Fig. 2, inset) confirming theattachment of the molecule to the PEMs. The absorbance was high-er after FA immobilization than after PEG–FA inmobilization.
For example at 360 an absorbance of 0.012 was measured for FAwhile for PEG–FA the absorvance value was only of 0.004. Thesedifferences in absorbance are roughly consistent with the QCM-Dresults and indicate a higher efficiency for the FA inmobilizationon the PEMs.
PEMs assembly and FA or PEG–FA attachment to the PEMs onPLGA NPs were also followed by f-potential measurements(Fig. 3). The PLGA NPs with BSA as stabilizer show a negativecharge with a f-potential of �27 mV due to the low pKa value of
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Fig. 2. UV–Vis spectra of chitosane/alginate PEMs after FA and PEG–FA grafting.Inset corresponds to the absorbance spectrum of the FA solution.
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Fig. 3. Changes in f-potential during the assembly of alginate and chitosan on PLGANPs.
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Fig. 4. Frequency and dissipation changes during BSA adsorption on Chi/Alg PEMsmonitored by QCM-D.
244 J. Zhou et al. / Journal of Colloid and Interface Science 345 (2010) 241–247
BSA (pKa: 4.7–4.9). After chitosan assembling, a f-potential of�15 mV was measured. Although the potential of the NPs re-mained negative, its absolute value decreased, hinting chitosandeposition but not a complete coverage of the NPs surface. Thisis consistent with QCM-D results (Fig. 1a). The adsorbed chitosanecan not form a layer dense enough to hide the influence of thecharges from the surface beneath. For the next assembled layersthe f-potentials oscillated between ��45 mV and �0 for after algi-nate and chitosane assembly respectively. After immobilization ofFA and PEG–FA, the potential dropped to ��15 mV, revealing thepartial consumption of the amino groups. After immobilization ofthe FA and PEG–FA, the NPs suspension became pale yellowish.This color remained even after three rinses with MilliQ water, indi-cating the presence of FA groups on the NPs.
DLS experiments show that Chi/Alg coated NPs as well as theparticles modified with PEG and PEG–FA have narrow size distribu-tions (Fig. S2). The coating of PLGA NPs with polyelectrolyte multi-layers does not induce changes in the morphology of thenanoparticles neither induces substantial aggregation as observedin TEM [19].
3.1. BSA adsorption on different polyelectrolytes multilayers
Protein adsorption is an important aspect to be studied takinginto consideration the possible application of NPs for drug delivery.NPs will inevitably interact with serum proteins upon contact withblood or body fluids. The circulation time of the NPs and their up-take by the cells will be significantly affected by the nature andamount of proteins deposited on their surface. BSA was used as amodel protein to study the adsorption of protein on PEMs. The
interaction of BSA with Chi/Alg multilayers was followed byQCM-D on planar surfaces. In Fig. 4 we observe the adsorptionbehavior of BSA on multilayers of (Chi/Alg)2/Chi, (Chi/Alg)2/Chi-FA, (Chi/Alg)2/Chi-PEG–FA and (Chi/Alg)3. For all the curves thefrequency decreases when passing from water to the phosphatemedia but then the subsequent addition of BSA does not induce achange in frequency. Finally, after rinsing with water the valuesof frequency and dissipation returned to their original values inwater, except for the dissipation of (Chi/Alg)2/Chi. These results re-veal that almost no BSA was adsorbed on all of the PEMs. In thecase of the (Chi/Alg)2/Chi, the phosphate molecules will probablyremain attached to the PEM even after water rinsing. When algi-nate was the outmost layer, i.e. (Chi/Alg)3, the high hydrophilicityof alginate and the repulsion between alginate and BSA should beresponsible for the lack of BSA adsorption [32,33]. The absence ofBSA adsorption on PEMs with chitosan as the outmost layer israther surprising, since it is known that BSA can interact withchitosan by electrostatic attraction. Indeed, Yang et al found thatthe human serum albumin adsorption was higher than 30 lg/cm2 on chitosan grafted polysulfone [34]. Mello et al observed thatalmost 100% BSA adsorbed after 1 mg/mL BSA solution was filteredthrough a membrane made by chitosan [35]. On the other hand,Goosen found that chitosan deposited on alginate microcapsulesdid not affect the concentration of BSA in solution if there wasno BSA ligand-blue dextran encapsulated [36]. From electropho-retic mobility measurements we know that the coating with chito-san resulted in a value of f-potential around 0 at pH 7.4. Therefore,it is likely that there is a limited electrostatic interaction betweenBSA and the PEMs- In a multilayer of the LbL type the properties ofthe surface are not independent of the layers underneath and thereis always a certain degree of interdigitation between layers. Forchitosan as top layer with alginate below the properties of the sur-face may be substantially different from those of a pure chitosanmonolayer. In other words, the alginate layer assembled beforethe top layer of chitosan may be protruding partially into the sur-face helping to prevent the deposition of BSA. It is also possiblethat the phosphate groups in PBS screen the charge of the aminesof chitosane. It is known that phosphates have a specific interac-tion with primary amines. For PEMs this screening of the aminegroups of the last layer may induce a recharge of the surface withthe charge of the underlying polyelectrolyte layer. For the chi/algwith chi as last layer the phosphate binding may be causing a more
J. Zhou et al. / Journal of Colloid and Interface Science 345 (2010) 241–247 245
significant influence of the alginate on the antifouling properties ofthe PEM The binding of FA or PEG–FA to the chitosane does notchange the adsorption of BSA. The grafting of FA, approximatelya 10 percent of the binding sites of the chitosan brings additionalcharges to the multilayer but they do not imply the accumulationof BSA. The Chi/Alg coatings could therefore be used as an alterna-tive for PEGylation to prevent protein adsorption on surfaces orparticles. The influence on the antifouling properties of a polyca-tion by the presence of a layer of alginate below are subject of fur-ther investigation.
3.2. Cellular uptake
In Fig. 5 we observe the cell uptake ratio (Fig. 5a) ,the fluores-cence intensity as the mean fluorescence intensity of all the cells(Fig. 5b) or only from the cells internalized with the NPs (Fig. 5c).
Two types of uptake profiles can be found in Fig. 5a. The firstprofile is evidenced by bare NPs, whose uptake ratio increased rap-idly in the first 1–2 h and reached the highest value, �90%. For allthe engineered NPs the cell uptake ratio increased continuouslyduring the first 8 h, and remained constant afterwards. Neverthe-less, the uptake ratio changed significantly with the nature of thesurface functionalization. The uptake ratios of (Chi/Alg)2/Chi-FA,(Chi/Alg)2/Chi-PEG–FA were 10–20% higher than those of the(Chi/Alg)2/Chi and (Chi/Alg)3 coated NPs. The average fluorescenceintensity per cell (Fig. 5b) shows some differences with the uptakeratio, although all the values increased initially along the culturetime as well. A highest value of intensity was also reached after2 h incubation for the bare NPs, while the highest values for the(Chi/Alg)2/Chi-FA and (Chi/Alg)2/Chi-PEG–FA coated NPs were
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Fig. 5. (a) Cell uptake ratio, (b) average fluorescence intensity normalized to all cells inincubation time. PLGA NPs concentration was fixed at 100 lg/ml.
reached 8 and 4 h, respectively. For the NPs covered with (Chi/Alg)3 multilayers, like for the uptake ratio, the fluorescence inten-sity was also the lowest, conveying the low interaction of the NPshaving alginate as the outmost layer with the cells [37]. In contrast,the NPs covered with (Chi/Alg)2/Chi-PEG–FA showed the highestfluorescence intensity with an value of around 450 a.u., which is150 a.u. higher than that of the (Chi/Alg)2/Chi-FA coated NPs, and3.5 times and 10 times of the bare NPs or (Chi/Alg)2/Chi and(Chi/Alg)3 coated NPs, respectively. After 24 h incubation, the fluo-rescence intensity decreased significantly for (Chi/Alg)2/Chi-FA and(Chi/Alg)2/Chi-PEG–FA coated NPs. The reason is not clear atpresent.
Exocytosis and the release of the Rd6G dye as a result of partialdegradation of the PLGA NPs can also cause the decrease in fluores-cence intensity. Fig. 5c shows that more NPs coated with (Chi/Alg)2/Chi-PEG–FA are associated to the HepG2 cells than the onescoated with (Chi/Alg)2/Chi-FA. The use of PEG as spacer seems tobe favorable for FA recognition. When the NPs were coated with(Chi/Alg)2/Chi, the mean fluorescence intensity was also higher.An interesting feature here is that the mean fluorescence intensityfor the cells incubated with the bare NPs and (Chi/Alg)3 coated NPswas almost the same, although from the Fig. 5a the uptake ratiowas so different.
Finally, CLSM was employed to observe the distribution of theNPs in cells. The images in Fig. 6 were taken from the middle planeof the cells in the z direction. The bare NPs (Fig. 6a) and (Chi/Alg)3
coated (Fig. 6e) NPs have similar appearance. Only a few NPs couldbe recognized on the cell membranes. This is consistent with theresults from flow cytometer, few NPs clusters were found on thecell membranes when they were covered by (Chi/Alg)2/Chi
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a population, and (c) fluorescence intensity normalized to cells NPs as a function of
Fig. 6. CLSM images of hepatocytes after co-cultured with: (a) bare NPs, (b) (Chi/Alg)2/Chi, (c) (Chi/Alg)2/Chi-FA, (d) (Chi/Alg)2/Chi-PEG–FA and (e) (Chi/Alg)3 covered NPs for12 h.
246 J. Zhou et al. / Journal of Colloid and Interface Science 345 (2010) 241–247
(Fig. 6b). This is probably caused by the very weak surface chargeof the NPs (Fig. 3), which is further screened by phosphate salts inthe culture medium. As expected, more NPs coated with (Chi/Alg)2/Chi-FA (Fig. 6c) or (Chi/Alg)2/Chi-PEG–FA (Fig. 6d) were attachedonto the HepG2 cell membranes. Moreover, the (Chi/Alg)2/Chi-FAcoated NPs showed aggregation on the membrane, but the (Chi/Alg)2/Chi-PEG–FA covered NPs uniformly distributed with neglect-able aggregation.
The cellular uptake of the PLGA NPs can be controlled by theproper engineering of their surface. While the alginate covered sur-face can significantly reduce the particle attachment onto theHepG2 cells and cellular uptake, the binding of FA alone or PEG–FA to the multilayers can, on the other hand, increase both of theuptake ratio and the number of NPs per cell. The strategy of step-wise modification of the PLGA NPs is successful in achieving NPswith selective recognition functions.
4. Summary
Chitosan and alginate were Layer by layer assembled on top ofthe BSA stabilized PLGA NPs. The assembled PEMs provided a sta-ble coating for further functionalization with specific recognitionfunctions by means of covalent binding of FA and PEG–FA. Thechitosan/alginate multilayers with either alginate or chitosan theoutmost layer displayed very low interaction with albumin, whichalmost did not deposit on the multilayer and leaded to very lowassociation with cells. These data envisage the use of Chi/Alg mul-tilayers as antifouling coatings to avoid interaction with proteinsand to decrease cell uptake. A higher cell uptake ratio and particlenumber per cell were achieved after FA or PEG–FA was covalentlybounded to the multilayer. Although high uptake ratio was also ob-served for the bare NPs (with albumin on their surface), on averagethe number of NPs per cell was much lower.
To conclude, the combination of the Chi/Alg LBL coating withcarbodiimide chemistry provides a simple but effective way of tai-loring NPs surfaces in a sequential way to reduce unspecific inter-actions first and then to attach other molecules to achieve specifictargeting properties.
Acknowledgments
This work has been financially supported by the Grant MAT2007-60458 from the Spanish Ministry of Science and Innovation,
the Natural Science Foundation of China (50873087), the MajorState Basic Research Program of China (2005CB623902) and theNational High-tech Research and Development Program(2006AA03Z442, 2006AA02A140). S.E. Moya is a Ramon y Cajal Fel-low and he thanks this program of the Spanish Ministry of Scienceand Innovation for support.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jcis.2010.02.004.
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