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Applied Surface Science 352 (2015) 103–108

Contents lists available at ScienceDirect

Applied Surface Science

jou rn al h om ep age: www.elsev ier .com/ locate /apsusc

orphological changes in gold core–chitosan shell nanostructures athe interface with physiological media. In vitro and in vivo approach

.M. Popescua,∗, L. Hritcub, D.A. Pricopa, D. Creangaa

Department of Physics, University “Alexandru Ioan Cuza”, Carol I Bd., No. 11, Iasi 700506, RomaniaDepartment of Biology, University “Alexandru Ioan Cuza” of Iasi, Carol I Bd., No. 20A, Iasi 700506, Romania

r t i c l e i n f o

rticle history:eceived 27 January 2015eceived in revised form 19 May 2015ccepted 22 May 2015vailable online 31 May 2015

eywords:hitosan–AuNPsrain

a b s t r a c t

Chitosan–gold nanoparticles (AuNPs) were prepared to investigate the behavior of such nanosys-tems at the interface with biological media. Microstructural characterization by Transmission ElectronMicroscopy, Atomic Force Microscopy, and Optical Microscopy was carried out in order to provide infor-mation regarding the morphology features and size distribution.

In vivo studies showed no morphological changes within the brain tissue in rats after the administrationof AuNPs. However, nanoparticles size distribution in the in vivo localized tissue areas indicated betterdispersion than in the in vitro colloidal solution. Also the size of the AuNPs that reached the brain tissueseemed to decrease compared with their size in the colloidal solution.

urface plasmonH influenceeighbors interaction

In order to understand the factors that contribute to the increase of AuNPs dispersion degree withinthe brain tissue, this study was focused on simulating the pH conditions from the hemato-encephalicmedium. A theoretical model was also applied in order to correlate the intensity of the interactionbetween two AuNPs and their volume ratio to further explain the absence of the agglomerated AuNPsand their high degree of dispersion within the brain tissue.

. Introduction

Over the last few years AuNPs became extremely attractive forargeting drug delivery in cancer therapy, mainly regarding theirse as vector systems for pharmaceutical molecules toward variousrain diseases. Meanwhile scientists’ interest for studying the opti-al properties and surface chemistry of functionalized AuNPs hasncreased as well as for improving the functionalization mechanismf AuNPs – crucial for their interaction with living tissues.

There are certainly significant differences between in vitro andn vivo approaches as well as between the impacts of uncoated orurface modified AuNPs on neural system. Previous study on in vitroingle neuron reported citrate-AuNPs uptake followed by bioeffectsuch as increased excitability in normal physiological condition andamaging influence under pathological conditions, such as seizure1] as well as the tendency of AuNPs to agglomerate in the pres-nce of cerebral glucose. In [2] the authors reported that uncoateduNPs dispersion degree in the in vitro model of cerebrospinal

uid has not changed compared with the colloidal solution whenhey were coated with a polymer. Regarding the in vivo studies onuNPs dispersion in tissues, the restrictions imposed by the natural

∗ Corresponding author. Tel.: +44 0121 414 5634.E-mail address: carmen.mariana.popescu13@gmail.com (C.M. Popescu).

ttp://dx.doi.org/10.1016/j.apsusc.2015.05.145169-4332/© 2015 Elsevier B.V. All rights reserved.

© 2015 Elsevier B.V. All rights reserved.

selectivity of blood–brain barrier (BBB) were emphasized in [3],related to the endothelial cells sustaining the cerebral capillarieswhich are interconnected through protein bridges, thus blockingthe free circulation of molecules toward the cerebral parenchyma– and possibly AuNPs circulation if the case. The in vivo studies haveto face the issues of BBB limitations [3] that significantly compro-mise the bioavailability and therapeutic targeting of AuNPs to thebrain as also discussed in [4] – where the investigation was basedon uncoated AuNPs and in [5] – where polymer coated AuNPs wereused. Some authors have emphasized [6] that citrate-AuNPs caneither actively pass across the BBB (transcellular transport) or pas-sively (endothelial adsorption) through endocytosis mediated byreceptors or carrier mediated transport [7] – although the parti-cle concentration in brain seems to be remarkably lower comparedwith other organs [8].

Studies of the passive transport of the AuNPs across the BBBshowed a size-dependent permeation profile with respect to Aucore size as well as coating polymer length [9]. Regarding the roleof coating polymer nature, chitosan was found to have high affin-ity for the cellular membranes [10] since protonated amino groupsfrom chitosan can interact with carboxylic groups of biomembrane

molecular components. The strength of chitosan interaction withcells depends on its de-acetylation degree and molecular mass; itwas reported that higher molecular mass results in lower deacety-lation degree [11,12]. Related studies reported no changes in size

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nd nonsignificant agglomeration of AuNPs coated by chitosan shelln alkaline pH conditions specific to the cerebrospinal fluid [13,14].

In this paper the biodistribution of AuNPs coated in chitosanithin rats’ brain tissue was analyzed. The intraperitoneal supply of

old nanoparticles was less studied as mentioned in [8]; our studyas designed based on similar administration protocol, meaningot only the pathway of AuNPs delivery into the body but also thedministration on a regular basis for about one week.

A theoretical model [15] was applied in order to determinehe degree of interaction between AuNPs within brain tissue andxplain their increased degree of dispersion compared with theolloidal solution.

. Materials and methods

.1. Materials and devices

Chitosan, chloroauric acid precursor (III) (H[AuCl4] × 3H2O),cetic acid (CH3-COOH), sodium hydroxide (NaOH), sodium pento-arbital (C11H18N2O3), formaldehyde solution (36.5–38% in H2O),emathoxylin–eosine (HE staining reagent), crystallized glucoseC6H12O6) and sodium chloride (NaCl) were purchased fromigma–Aldrich, Germany. All necessary solutions were preparedsing 18.2 M� deionized Mili-Q water.

Barnstead EasyPureII purification system was used for waterample deionization.

Varian PL-GPC 120 gel chromatograph was utilized for estimat-ng chitosan molar mass, gravimetric molar mass, polydispersityndex and mass distribution.

Sonoplus Bandelin device was used to sonicate nanocolloidalamples.

Malvern Zetasizer Nano ZS Zen-3500 device was used to deter-ine the size distribution of the nanoparticles within the colloidal

olution; Dynamic Light Scattering (DLS) measurements were per-ormed at room temperature. The colloidal solution stability was

onitored based on Zeta potential measurements by using theame device.

AFM NT-MDT Solver Pro-M device with XY resolution of 2 nmnd Z resolution of 0.1 nm and Transmission Electron MicroscopeTEM device) CM100 Philips, were used to investigate gold particleize, morphology and shape.

Nikon Ti-Eclipse Optical Microscope working in dark field (DF)nd differential interference contrast (DIC) techniques was alsosed for imaging nanoparticles.

NIS Elements BR (NIS-BR) specialized software was utilized toeasure and compare AuNPs size data provided by AFM images,

EM micrographs and optical microscope recordings.UV–vis Spectrophotometer Shimadzu type Pharma Speck, pro-

ided with 1 cm quartz cells was used to acquire UV–vis spectra.pHeasurements were performed using Sartorius professional meter

p – 50 device.Wister rats (3–4 months old) weighing 200 ± 10 g were used in

he experiment. The animals were kept inside a room with temper-ture and light controlled (22.0 ± 0.5 ◦C, 12-h light cycle starting at8:00 a.m.). Food and water access was ad libitum permitted.

Cryomicrotome type Leica Criostat CM 1850 was used for sec-ioning tissue samples in the process of probes preparation for

icroscope investigation.

.2. Methods

.2.1. Gold/chitosan nanosystem synthesisSolution A was prepared by adding 1 L of deionized water to 1 g

f HAuCl4 × 3H2O thus obtaining a solution with molar concentra-ion of 2.59 × 10−3 M. Further 196.6 mL of solution A was diluted

Science 352 (2015) 103–108

with deionized water up to 500 mL to obtain final solution B with10−3 M concentration.

The chitosan stock solution was prepared by dissolving chitosanpowder 0.1% (w/v) in acetic acid 1% (v/v). Then 4 mL of solutionB (HAuCl4 ×3H2O10−3 M) were mixed with 36 mL chitosan solu-tion 0.1%, the resulting mixture being treated with ultrasounds byapplying 20 kHz ultrasonic field with 50% amplitude for 10 min.

2.2.2. Gold/chitosan nanosystem characterizationPristine colloidal solution (pH = 3.2) was directly analyzed by

means of microscopy techniques – TEM, AFM, Optical Microscopy.

2.2.3. Gold/chitosan nanoparticle administrationAnimals were divided into two groups of 5 rats each: (1) control

group (treated with chitosan in acetic solution 0.5%, pH = 6); and (2)animals treated with AuNPs. The AuNPs solution B was intraperi-toneal injected (19.7 �g/kg) daily for 8 consecutive days. The bodyweight of animals and their behavior were carefully monitored andrecorded daily before AuNPs administration and during the exper-iment. On the 9th day after the last injection of AuNPs, the ratswere sacrificed by administration of an overdose of sodium pento-barbital (100 mg/kg body weight). Rats were treated in accordancewith the guidelines of animal bioethics from the Act on AnimalExperimentation and Animal Health and Welfare from Romaniaand all procedures were in compliance with the European CouncilDirective of 24 November 1986 (86/609/EEC).

2.2.4. Brain slice preparationAfter brain extraction the tissue samples were fixed in 10%

formaldehyde solution and then coronary sectioned in 7 �m slicesusing microtome. Each slice of tissue was placed onto a glassslide and after being dyed with hemathoxylin–eosine, opticalmicroscopy investigation was carried in Dark-Field technique (DF)to reveal the presence and distribution of AuNPs within the braintissue. DIC (Differential Interference Contrast) technique was alsoused to image crystal details.

3. Results and discussion

3.1. Chitosan characterization

Chitosan measured molar mass was Mn = 97.607 g/mol, thegravimetric molar mass was Mw = 263.836 g/mol while polydisper-sity index, PI, was found equal to 2.70.

3.2. Characterization of AuNPs/chitosan solution

TEM micrographs (Fig. 1, left) enabled us to see the Au cores inthe studied colloidal dispersion which have different geometrieswith size distribution characterized by mean value of 36.26 nm.This is concordant with the results reported in [16] where fol-lowing a similar preparation method the mean incidence of goldparticles imaged by TEM was around 30 nm. The AFM scanning(Fig. 1, right) revealed chitosan–AuNPs systems having dimensionaldistribution with a maximum around 124 nm since AFM also pro-vides imaging of polymer (chitosan) coverage. The size resultedfrom AFM images is comparable with the mean value from the sizedistribution obtained from DLS measurements. The Zeta potentialvalues indicated that the AuNPs solution falls in the class of col-loidal solutions with moderate stability having Zeta potential equalto 34.1 mV.

In Fig. 2 optical microscope investigation results can be seen (DFtechnique). Gold nanoparticles imaging was favored by the metalsurface plasmon resonance phenomenon, which occurs when inci-dent light frequency matches the frequency of metal surface

C.M. Popescu et al. / Applied Surface Science 352 (2015) 103–108 105

Fig. 1. Comparing the size distribution of the AuNPs resulted from TEM data (A) with the size distribution obtained from the AFM image (B); the mean size of the AuNPs islarger in AFM imaging due to the coating polymer (image analysis with NIS-BR).

pristine colloidal solution (pH = 3.2); (B) the distribution of AuNPs/chitosan systems.

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Fig. 2. (A) Microscope DF image showing the distribution of AuNPs within the

lectrons oscillating in the nuclei field; thus, stimulated light emis-ion enhance microscope resolution enabling the observer to imageetallic objects with two orders of magnitude smaller. Indirecteasurements of nanoparticles diameter evidenced the size dis-

ribution maximum between the values obtained from the AFMnd TEM images.

.3. In vitro study

In order to explain the changes in size of the AuNPs after crossinghe BBB, the specific pH conditions were simulated (Fig. 3). TheuNPs colloid was treated with NaOH (0.1 M) to increase the pH of

he solution. Every 10 min 0.05 mL of NaOH 0.1 M was added to theolution under continuous mechanical stirring.

According to the UV–vis investigation of the alkalized colloidalolution (Fig. 4 right), the peak corresponding to the transversalscillation mode of AuNPs plasmons presents a blue shift from73 nm to 539 nm and progressive intensity diminution; this indi-ates spherical or hexagonal systems (nanoparticle associations)ith decreasing size (segregation) to pH increasing. Simulta-eously, longitudinal mode peak – corresponding to rod shapedarticles, was recorded at 683 nm and seems to shift toward redhile relative intensity amplification with pH increasing has been

lso observed.The DF and DIC images revealed a polymer crystallization devel-

pment to the pH increasing. In Fig. 4 left the relatively largeolymer crystals can be clearly observed as having quasi-regularhape and micrometric dimensions. Thus, besides the possible dis-ociation of gold/chitosan agglomerated and symmetrical shaped

ystems, for increased pH conditions, the coagulation of coating chi-osan could also occur; consequently micrometric crystals appearnd uncovered AuNPs are released. Such uncoated or partiallyxposed gold particles tend to agglomerate in asymmetrical shaped

Fig. 3. Alkalizing curve of AuNPs colloidal solution.

nanosystems that could enlarge in concordance with transversalmode peak shifting to red.

In order to establish if AuNPs lose their coating due to the contactwith cerebral glucose, 2 mL of AuNPs colloid (solution B) was mixedwith 7 mL of Z-glucose in concentration of 4 mM, 10 mM, 20 mM,30 mM, 40 mM in the presence of 1 mL of 0.1 M NaCl.

The UV–vis investigation of the AuNPs colloidal solution mixedwith glucose in different concentrations (Fig. 5) did not indicate anysegregation process; thus, it can be concluded that the presence

106 C.M. Popescu et al. / Applied Surface Science 352 (2015) 103–108

Fig. 4. (A, left) Chitosan crystals; detailed images in the upper left corners obtained with DIC technique; (B, right) UV–VIS recording of colloidal nanoparticle solutions. Toincrease of pH solution, chitosan coagulation occurs so that crystals as well as uncovered AuNPs are released; thus gold particles tend to agglomerate because the loss ofpolymer coating favored their interaction. So, the main peak corresponding to large spherical systems moves to the left when these systems became smaller, while secondarypeak corresponding to smaller asymmetrical associations of uncoated particles moves to the red since such new associations became larger.

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ig. 5. (left) UV–vis recording of AuNPs colloidal solution for different glucose conendency of the peak intensity with increase of glucose concentration without aggl

f glucose did not induce the degradation of the polymeric chainsn the chitosan shell surrounding the AuNPs. The DF microscopymages showed a constant distribution of the AuNPs in the presencef different concentrations of glucose.

.4. In vivo study

DF images of the cerebral tissue sample (Fig. 6) showed biodis-ribution of AuNPs with 13.5 nm mean diameter.

The results from Fig. 6 showing that AuNPs have passed throughBB reaching brain tissues are concordant with other scientificeport [5] where single dose of 15 nm polyethylene glycol (PEG)oated AuNPs – equivalent to the total amount of chitosan–AuNPsdministrated in our experiment through repeated supplies, haveesulted in nanoparticle biodistribution in all mice organs – brainncluded. Also the concordance of our results with those reported

n [4] can be mentioned related to the fact that gold particles withimilar size, injected intravenously in unique dose in mice wereetected in most organs including brain. The experiment carriedut in the present study demonstrated that chitosan–AuNPs with

ations; (right) gold nanoparticle agglomerations. It can be noticed the decreasingtions formation.

relatively small size diameter (13.5 nm) supplied daily for aboutone week in doses of 19.7 �g/kg daily were absorbed into the bodyfluids and succeeded to circulate toward the brain that is concord-ant with [8] where the gold particles had 12.5 nm while the lowestdaily dose was of 40 �g/kg except in that case uncoated particleswere used and the mass spectroscopy was the investigation methodof particle biodistribution.

The distance between AuNPs within cerebral tissue has beenmeasured by analyzing the DF images of the tissue samples in NISElements program; the mean distance between two neighboringAuNPs plasmons was found to be of 3.46 �m. The minimum mea-sured distance between two neighboring plasmons was of 0.62 �m.In order to identify the selectivity criteria, which allowed suchdistribution of the AuNPs within the cerebral tissue, a theoreticalmodel was applied.

The theoretical model establishes that the resulting plasmon

intensity of two neighboring AuNPs depends on their volume ratio[16].

The following statement will be useful for further understand-ing the analysis that has been done: two neighboring nanoparticles

C.M. Popescu et al. / Applied Surface Science 352 (2015) 103–108 107

Fig. 6. (A) In the DF image of the rat cerebral tissue evident biodispersion of the AuNPs can be observed; (B) size distribution suggesting smaller size of the AuNPs withintissue than within the colloidal solution.

NPs (dimmers) observed in brain tissue with DF microscopy.

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Fig. 7. Intensity profile of light diffracted by coupled Au

orm a dimmer and each of the AuNPs is optically defined by its sur-ace plasmon resonance. Thus, the strength of interaction betweenuNPs dimmers within the cerebral tissue can be determined by

heir plasmon intensity that is easily observed in DF images.In Fig. 7 the three categories of AuNPs dimmers, as resulting

rom the investigation of the analyzed images, present in cerebralissue, are shown.

It can be seen that the two AuNPs forming a dimmer, havingomparable sizes, are situated at a distance that is longer than

�m – these being considered as non-interacting AuNPs. The non-nteracting AuNPs percentage in the analyzed sample of cerebralissue was of 72.7% and they mostly form symmetrical dimmers13.5 nm average size). The second category of dimmers consists ofeakly interacting AuNPs, which are situated at distances between

�m and 1.5 �m and have different sizes. A percentage of 16.6%rom the total number of AuNPs in the studied sample have beenound to be weakly coupled. Strongly interacting AuNPs result inignificant intensity enhancement of the associated plasmons ofwo AuNPs with a high volume ratio and situated at distanceshorter than 1.5 �m. In the studied sample these dimmers repre-ent 10.6% from the total number of AuNPs. For this last mentionedype of dimmers it is already difficult to distinguish between thewo component AuNPs, thus, the limit between them is mostly

efined by the color of the generated plasmon.

In Fig. 8 the results of the statistical analysis carried out on theotal number of AuNPs dimmers counted in the DF image recordedor the brain tissue sample are presented. The size of the AuNPs

Fig. 8. Types of interactions between AuNPs within the brain tissue.

and the distances between them were estimated using the mea-surement tools provided by the NIS Elements software associatedto the optical system used. The percentages of the three types ofdimmeric particle interaction were textually presented above.

4. Conclusions

In alkaline pH conditions, as across the BBB, part of the coat-ing polymer crystallizes. This was revealed by the DF images ofthe AuNPs colloidal solution treated with NaOH as shown by the

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n vitro study. However, the AuNPs inside the brain did not agglom-rate suggesting that they still keep a thin layer of chitosan on theirurface. The uniform coating with chitosan ensures good disper-ion of AuNPs. The chitosan shell also allows high affinity to theell membrane. The DF microscopy investigation showed that intraeritoneal injected AuNPs crossed the BBB and reached the brainissue. Most of the AuNPs are well dispersed being separated byistances over 2 �m. Due to the size dependence of the BBB per-eability most of the AuNPs that crossed the barrier were small

13.5 nm); these particles were found to be non-interacting (72.7%rom the total number of AuNPs that were found within the cerebralissue).

The results of this study emphasized that chitosan acts as aood coating polymer for the AuNPs, ensuring biodispersion withinat brain and hindering their agglomeration inside the tissue bydapting to the increased pH conditions and by rearranging itshains around the AuNPs. Also as a natural defense mechanism, theelectivity feature of the BBB imposes the condition of minimumnteraction between AuNPs within the neural tissue.

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