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Colloids and Surfaces A: Physicochem. Eng. Aspects 452 (2014) 173–180

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

journa l h om epage: www.elsev ier .com/ locate /co lsur fa

ne step poly(quercetin) particle preparation as biocolloidnd its characterization

urettin Sahinera,b,∗

Faculty of Science & Arts, Chemistry Department, Canakkale Onsekiz Mart University, Terzioglu Campus, 17100 Canakkale, TurkeyNanoscience and Technology Research and Application Center (NANORAC), Canakkale Onsekiz Mart University, Terzioglu Campus,7100 Canakkale, Turkey

i g h l i g h t s

Highly negatively chargedpoly(quercetin) particles viamicroemulsion crosslinking.Antibacterial and antioxidant p(QC)particle with fluorescence properties.Natural biopolymeric microgels froma flavonoid, quercetin.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 30 December 2013eceived in revised form 6 March 2014ccepted 28 March 2014vailable online 5 April 2014

a b s t r a c t

Quercetin (QC) was reacted with glycerol diglycidyl ether (GDE) to obtain poly(quercetin) particles(p(QC)) for the first time via a simple microemulsion polymerization/crosslinking method using l-�lecithin as surfactant and cyclohexane as organic phase. The prepared p(QC) particles were highly neg-atively charged (−48.2 mV), were thermally more stable in comparison to QC and degradable in PBS atpH 7.4., e.g., 10 wt% can degrade in about 15 h. The prepared p(QC) particles showed antibacterial char-

eywords:luorescent particlesrosslinked quercetinicrogel/nanogel

ntioxidant particles

acteristics against common bacteria such as Bacillus subtilis ATCC 6633, Escherichia coli ATCC 8739 andStaphylococcus aureus ATCC 25323. Additionally, p(QC) was found to have significant antioxidant proper-ties that is equivalent to 82.5 ± 9.6 mg/L gallic acid. More interestingly p(QC) particles retain some of theirfluorescence and can be used both as antibacterial and antioxidant materials providing great potentialfor biomedical use.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Quercetin (QC), as a flavonoid, is found in many plants including

ruits, vegetables, leaves and grains. It has been reported that QCossesses numerous therapeutic properties including the ability tocavenge free radicals against oxidative stress and even to provide

∗ Correspondence to: Faculty of Science & Arts, Chemistry Department, Canakkalensekiz Mart University, Terzioglu Campus, 17100 Canakkale, Turkey.el.: +90 286 2180018x2041; fax: +90 286 2181948.

E-mail address: sahiner71@gmail.com

ttp://dx.doi.org/10.1016/j.colsurfa.2014.03.097927-7757/© 2014 Elsevier B.V. All rights reserved.

blood pressure reduction in people who have hypertension, inaddition to many more health benefits. QC is an active anti-inflammatory with anti-cancer activity and has been extensivelyinvestigated in both in vitro and in vivo studies [1]. To increasethe effectiveness and delivery of QC various methods have beendeveloped due to the low solubility and bioavailability of QC, suchas liposomal formulations [2], micelles derived from polymericmaterials [3,4], polymeric films [5], microspheres [6], nanoparti-

cles [7] and so on. The use of QC in the medical field for differentpurposes is steadily increasing. Interestingly, QC was found tobe effective against oxidative stress and apoptosis and againstneuronal damage from cerebral ischemia [8]. The other important

1 ysicoc

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74 N. Sahiner / Colloids and Surfaces A: Ph

iomedical use for QC is as anticarcinogenic and antioxidant [8–15].lthough there is only one report of enzymatic polymerizationy QC [16], there are no reports on p(QC) and/or p(QC) particles.he enzymatic polymerization of QC was performed under normal

ig. 1. (a) The schematic representation of the poly(quercetin) particle formation from quearticles (2) crosslinked with glycerol diglycidyl ether.

hem. Eng. Aspects 452 (2014) 173–180

conditions (1 atm, 25 ◦C) and a common enzyme, horseradishperoxidase (HRP), was used to catalyze to QC [16]. Although manybiomedical benefits are inherently offered by QC as medicineitself, with antioxidant (radical scavenging ability), anti-bacterial

rcetin and digital camera images. (b) FT-IR spectra of quercetin (1), and p(quercetin)

ysicoc

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Fo

N. Sahiner / Colloids and Surfaces A: Ph

roperties and anti-inflammatory and anticancer activity, therere no reports in the literature on the use of p(QC) as biomaterialor multi-purpose advanced drug delivery devices. Preparation of

icron or nano sized particles from a natural source such as QC, aersatile and resourceful polyphenol, has a paramount significancen the design of advanced multitalented biomedical devices forpplications in food, pharmacy/drug, cosmetics, and environmen-al industries. Therefore, the purpose of this investigation was torepare particles from a natural flavonoid as p(QC). Microemulsionolymerization is one of the most widely employed techniques

n the preparation of microgels using different surfactants foronomers with different functionality [17–20]. By modifying

mulsion polymerization, which is a versatile technique, narrowr broad size distribution of particles can be obtained. Generally,mulsion polymerization is carried out in the presence of an addedurfactant or in the absence of the surfactant so the growing struc-ure should stabilize the particles. However, the use of commonurfactants with anionic, cationic and neutral forms in microemul-ion polymerization also provides particles with different sizes forifferent functional group-containing monomers [21–24].

This is the first report of polymeric particles based on quercetinreated in a single step by microemulsion polymerization/rosslinking techniques. The prepared particles were demonstratedo be inherently antioxidant, antibacterial and are expected to haventi carcinogenetic characteristics. Moreover, as QC has fluorescent

roperties, the particle form of QC (p(QC)) was shown to retainome of its fluorescent intensity. This particle has great potentials polymeric drug carrier to carry other medications in combinationith QC.

ig. 2. (a) Particle size distribution of p(quercetin) particles (422.4 ± 38 nm), and zeta potf p(QC) particles.

hem. Eng. Aspects 452 (2014) 173–180 175

2. Experimental

2.1. Materials

Quercetin (QC) dihydrate (Sigma, ≥95% powder), glycerol digly-cidyl ether (GDE) (Sigma-Aldrich, tech grade), l-� lecithin as surfac-tant (granular form, Acros Organic, 98%), and cyclohexane (Merck,99.9%) as solvent were used. Ethanol (ultrapure, Kimetsan), sodiumhydroxide (Sigma-Aldrich), gallic acid (Sigma, 99%), and molyb-dotungstophosphoric (3H2O·P2O5·13WO3·5MoO3·10H2O) as Folin& Ciocalteu’s phenol reagent (Sigma-Aldrich, 2N) were used asreceived. Gallic acid (Sigma-Aldrich, 98%) and Na2CO3 were alsoobtained from Sigma-Aldrich. Bacteria (Escherichia coli ATCC 8739,Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25323)were obtained from the Microbiology Department of the Schoolof Medicine at Canakkale Onsekiz Mart University.

2.2. Methods

2.2.1. Synthesis of p(quercetin) particlesA solution of QC was prepared by dissolving 100 mg QC in

1 mL 1 M NaOH, and 0.4 mL of this solution was added into 15 mL0.1 M of lecithin solution in cyclohexane under constant vortexmixing. Upon mixing this solution for 1 h at 1200 rpm at room

temperature, glycerol diglycdiyl ether, 100% of QC by mole, wasadded. After continued mixing of this particle solution for 1 h, theobtained p(QC) particles were washed with cyclohexane once,and the precipitate was washed three times with ethanol:water

ential of p(quercetin) particles (−48.24 ± 6.34 mV). (b) The optical and SEM images

1 ysicoc

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76 N. Sahiner / Colloids and Surfaces A: Ph

50:50) by centrifugation at 10,000 rpm for cleaning at 20 ◦C. Theleaned particles were dried in oven at 40 ◦C for further use.

.2.2. Particle characterizationThe size of the particles were determined by using a dynamic

ight scattering (DLS, Brookhaven Ins. and Cor. 90 plus) parti-le size analyzer. The measurements were carried out with a0◦ angle detector using 35 mW solid state laser detector oper-ting at a wavelength of 658 nm. The results were the averagealues of ten consecutive measurements with an integrationime of 2 s. Zeta potential measurements of p(QC) particlesere completed with Zeta-Pals Zeta Potential Analyzer (BIC,rookhaven Inst. Corp.) using particle suspension in 0.01 M KClolution in water. Each measurement was repeated at least fourimes.

Scanning electron microscopy (SEM) images of p(QC) particlesere obtained using an SEM (JEOL JSM-5600) with an operating

oltage of 20 kV. The images were acquired from p(QC) particleslaced onto carbon tape-attached aluminum SEM stubs at ambientemperature after coating with gold to a few nanometer thicknessnder vacuum.

The Fourier Transform Infrared Spectra (FT-IR) of QC and p(QC)articles were recorded in the 650–4000 cm−1 range via a Perkin-lmer FT-IR spectrometer using attenuated total reflectance (ATR)ith 4 cm−1 resolution.

Thermogravimetric analysis (TGA) of QC and p(QC) were carriedut using a TGA analyzer (SII TG/DTA 6300). TGA measurements

ere performed under nitrogen atmosphere with 100 mL/min flow

ate with a heating rate of 10 ◦C/min heating up to 1000 ◦C.

(b)

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40

60

80

100

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80.0

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ig. 3. The TGA thermogram of (2) quercetin (QC), and (1) poly(quercetin) p(QC),b) the degradation of p(QC) particles in PBS (pH 7.4) at room temperature.

hem. Eng. Aspects 452 (2014) 173–180

2.2.3. Degradation of p(quercetin) particlesThe degradation of the prepared p(QC) particles was investi-

gated in phosphate buffered saline (pH = 7.4) solution. In a dialysissack (molecular weight cut off ≥12,000 Da, Aldrich) 50 mg p(QC)particles were suspended in 2 mL PBS. Then the dialysis membranewas placed into a closed beaker containing 250 mL of PBS buffer andstirred at 200 rpm at room temperature. The amount of degradedQC in the PBS buffer was determined from a calibration curve con-structed for QC at 318 nm via UV–vis spectrometer (T80 + UV/VISSpectrometer, PG Ins. Ltd.).

2.2.4. Folin–Ciocalteu (FC) methodMolybdotungstophosphoric acid (MTFA) is a hetereopoly acid

and was used as Folin–Ciocalteu reagent (FCR). In the reagent theactive center is Mo(VI), and the reaction is given as;

Mo(VI) (yellow) + e-(anti oxidant) → Mo(V) (blue)

FCR can be reduced by non phenolic compounds such asvitamin C, aromatic amines and Cu(I), therefore it is not specificfor phenolic compounds. The FC method measures the reductioncapability of a compound. In this technique, electron transferdonation from the antioxidant to the oxidant probe occurs andthe degree of color change of the probe is proportional to theantioxidant concentration [25,26]. In FC assay, the oxidant isMTFA, a heteropolyanion, and the absorbance is measured at about750 nm. Phenolic compounds only react with FCR under basicconditions e.g., pH is adjusted to 10 by Na2CO3 solution. By thedissociation of phenolic protons the generated phenolate anion,that has the ability to reduce FCR, and the blue compounds thatare formed between phenolate and FCR are independent from thestructure of the phenolic compounds. Therefore, the possibility offormation of coordination complexes between the central metalatom and the phenolic compounds is negligent. The formation ofblue complex can be measured spectrophotometrically at about765 nm. Generally, gallic acid (GA) is used as standard and theresults are given as GA equivalent mg/L.

2.2.4.1. Antioxidant tests. To test the antioxidant ability of p(QC)particles, 170 mg p(QC) particles were placed in 0.2 N 1.25 mLFolic-Ciocalteu (FC) solution and vortexed. After 4 min, 1 mLNa2CO3 solution (7.5 g in 100 mL water) was added to this mixtureand mixed. After 2 h, the absorption value at 760 nm from UV–visspectra was determined from a calibration curve constructed forgallic acid (GA), as a standard. The calibration curve was obtainedby reaction of 0.1 mL of GA at 1 mM, 400 �M, 200 �M, and 25 �Mconcentrations reacted with FC solutions in a similar way, and theirabsorbance values were measured at 760 nm to generate a calibra-tion curve. To compare the non-particle form, QC was also testedin a similar way.

2.2.4.2. Antimicrobial tests. The antimicrobial behavior of QC andp(QC) particles were investigated against three common strains ofbacteria, E. coli ATCC 8739, S. aureus ATCC 25323, and B. subtilisATCC 6633 strains. In brief, 0.1 mL stock culture and five differ-ent amounts (0.1, 0.05, 0.025, 0.01, 0.005 g) of sterile QC or p(QC)were inoculated in 9.9 mL nutrient broth. Incubation was carriedout at 35 ◦C for 18–24 h. For enumeration, the cultures in each

media were serially 5 × 105 fold diluted using sterile FTS solution,and 100 �L of each diluted sample was plated on nutrient agarand incubated for 18–24 h at 35 ◦C, and finally, the colonies werecounted.

N. Sahiner / Colloids and Surfaces A: Physicochem. Eng. Aspects 452 (2014) 173–180 177

) teste

3

3

wurdeirf[QiaGiafsr

TM

Fig. 4. Digital camera images of QC and p(QC

. Results and discussion

.1. Particle characterization

Due to the beneficial health effects of bioflavonoids such as QC,hich can be found in many common plants, they have directse as anticancer, cardioprotective agents and antioxidant mate-ial [27–29]. Different polymeric nanoparticles have been used forelivery of QC as an active agent [30,31]. For advanced drug deliv-ry systems, quantum dots with QC were used by co-encapsulationnto biodegradable nanoparticles for monitored delivery, and it waseported that QC delivery by a carrier is more effective than freeorms of QC to suppress human hepatoma HepG2 cell proliferation32]. Here, a novel concept is provided where the particle form ofC, as p(QC), can be simply prepared via a microemulsion crosslink-

ng polymerization technique using lecithin as surfactant and GDEs crosslinking agent. As illustrated in Fig. 1(a), the epoxy groups inDE can readily react with the phenol OH groups in QC, generat-

ng polymeric particles. The digital camera images of QC and p(QC)

re also given Fig. 1(a), and as can be seen the color of QC changedrom bright yellow to dark brown as an indication of some kind oftructural change. Furthermore, the particle formation via epoxyeaction of the QC crosslinking with GDE is confirmed by FT-IR

able 1BC and MIC values of quercetin (QC), and p(quercetin) p(QC) particles.

Material B. subtilis ATCC 6633 E

MIC (g/ml) MBC (g/ml) M

Quercetin (QC) 0.0005 – –p(quercetin) particles p(QC) 0.005 0.025 0

d against common bacteria for 24 h at 37 ◦C.

spectra as shown in Fig. 1(b). The reactants, QC and GDE, have theircharacteristic bands, whereas p(QC) is shown as aromatic aryl-Ostretching at about 1300–1230 cm−1 and band broadening at about3000–3700 cm−1 due to formation of free OH coming from GDE.As illustrated in Fig. 2(a), the prepared p(QC) particles have quitebroad size distribution and DLS measurements give about 422 nmparticle diameters after syringe filtration (0.8 �m). The zeta poten-tial measurements of p(QC) particles provide further corroborationof free OH functional groups, rendering negative zeta potential tothe particles of about −48.24 ± 6.34 mV, as shown in Fig. 2(a). Itis very well known that magnitude and sign of the zeta potentialof the materials is directly related to the surface functional groupsand as the free OH groups can be readily ionized in aqueous envi-ronments, negative charges developed. The sizes of particles canbe further visualized with optical microscopy, and SEM images asillustrated in Fig. 2(b), respectively.

To further substantiate that QC is crosslinked and thermal sta-bility is increased via epoxy crosslinking, TGA analysis of QC andp(QC) were carried out under nitrogen atmosphere. The degrada-

tion curve shown in Fig. 3(a) unquestionably proves that p(QC)particles have better thermal stability than QC molecules. The QCstarts to degrade about 100 ◦C, and the degradation continues upto 1000 ◦C, whereas p(QC) has thermal stability up to 320 ◦C with

. coli ATCC 8739 S. aureus ATCC 8739

IC (g/ml) MBC (g/ml) MIC (g/ml) MBC (g/ml)

– 0.005 –.0005 0.01 0.0005 0.0025

1 ysicochem. Eng. Aspects 452 (2014) 173–180

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78 N. Sahiner / Colloids and Surfaces A: Ph

lmost no degradation observed, and between 320–360 ◦C a sharpegradation. After 360 ◦C, the degradation of p(QC) continues up to000 ◦C, and the difference between the weight losses of p(QC) andC monomers up to heating 1000 ◦C is almost 47% suggesting that(QC) has better thermal stability than QC molecules.

As p(QC) particles are formed from antioxidant and antibac-erial QC molecules, its degradation products are very importantnd expected to be QC molecules. As the degradation of p(QC) wasone in PBS (pH 7.4), a calibration curve constructed for pure QColecules as control was created at 318 nm, and then the degraded

mount of QC was calculated from this calibration curve and sub-racted from the particles initial mass. Therefore, the prepared(QC) particle degradation was investigated in PBS at pH 7.4 atoom temperature. The degradation curve is given in Fig. 3(b). Asan be seen from the figure, with an initial fast degradation burst,lmost 10 wt% p(QC) particles were degraded in 15 h. This coulde very useful for controlled drug release applications in that byegrading biomaterials such as p(QC), an active drug may haveynergic effects and provide great advantages for design of dualelivery devices.

.2. Antioxidant and antibacterial test

It is known that QC has antioxidant properties [33]. Therefore,he antioxidant properties of the prepared p(QC) particles werelso determined using the FC method. Generally, the antioxidantroperty of a material is given in terms of one of the standardntioxidant materials. Among these standards gallic acid is gen-rally the most commonly used as standard antioxidant material33]. Here, we found that 170 mg QC molecules have the antioxi-ant equivalency of 69.27 ± 0.5 mg/L gallic acid, whereas p(QC) hashe antioxidant equivalency to 82.5 ± 9.6 mg/L gallic acid. It is rea-onable to assume that the formation of p(QC) from QC moleculesan cause little increase in the antioxidant property of the particlesven though some of the hydroxyl functional groups were used inhe linkage of the QC molecules for p(QC) generation. The change inntioxidant properties also confirms that the number of OH func-ionalities on the QC and p(QC) particles are different and some mayave reacted with epoxy groups of the crosslinker, GDE. The little

ncrease in the antioxidant property of the p(QC) can be attributedo the cumulative effect of QC molecules in particle form and higholume occupation of the polymeric structure in comparison tomaller QC molecules.

The antibacterial properties of the p(QC) particles were deter-ined against E. coli ATCC 8739, S. aureus ATCC 25323, and B. subtilisTCC 6633 strains. The p(QC) particles and QC molecules were con-

acted with these bacteria for 24 h at 37 ◦C and their MIC (Minimumnhibitory Concentration) and MBC (Minimum Bactericidal Con-entrations) were determined. It was found that the MIC value ofC is tenfold lesser than p(QC) and both have the same MBC values

or B. subtilis ATCC 6633. Interestingly, QC did not show any MICnd MBC values whereas the MIC and MBC values for p(QC) were.0005 and 0.01 g/mL, respectively for E. coli ATCC 8739. More inter-stingly, the MIC value for p(QC) is lower than QC for S. aureus ATCC5323 bacteria and no MBC value was obtained for QC at inves-igated concentration. So, it can be said that p(QC) has a potentntibacterial effect with 0.01 g/mL concentration against all stud-ed bacterial strains. The digital images of the investigated bacteriatrains for QC and p(QC) are given in Fig. 4, and Table 1 summarizesheir MIC and MBC values.

.3. Optical properties of QC and p(QC) particles

As QC contains aromatic phenolic groups, and it has fluores-ent properties [34], the prepared p(QC) particles are colorful andre expected to have the same optical properties. The UV–vis

Fig. 5. (a) UV–vis absorbance spectra of quercetin (QC) and poly(quercetin) p(QC),and (b) fluorescence emission spectra of QC and p(QC).

absorbance measurements of QC and p(QC) were performed andare given in Fig. 5(a). As can be seen QC has two maxima at about222 and 282 nm, whereas the p(QC) has again two absorption max-ima, one is still at about 217 nm, and the other has shifted to about321 nm. Also, as seen from the UV–vis spectra both QC and p(QC)have shoulders at 350, and 381 nm, respectively. Therefore, threeEgap values for QC and p(QC) were expected. The concentration ofQC in UV–vis 1.48 E−5 mg/mL, and p(QC) was 2.38 E−4. For UV–vismeasurements, the QC and the p(QC) stock solution were preparedas 0.0005 and 0.005 mg/mL in water and diluted with 0.02 M NaOH.It is important to note that the p(QC) concentration is about 16 foldmore than QC. The fluorescence emission spectra of QC and p(QC)are presented in Fig. 5(b). The QC solution sample was excited at370 nm, and two peaks were observed at around 562 nm (2.2 eV)and 465 nm (2.66 eV) for QC, and only one relatively weaker peakwas observed for p(QC) around 498 nm (2.48 eV). The reduction inthe intensity of the fluorescence peak for p(QC) could be due tothe crosslinking of some of the phenolic groups that can lead to areduction in the fluorescent property of QC.

The absorption coefficients ( ̨ (cm−1)) were calculated from theabsorbance (A) values of UV–vis measurements by equation:

A = ˛.d (1)

where d is the sample thickness. Here d is assumed to be the widthof the cuvette, which is 1 cm. Assuming that the energy band gapstructure of QC is a direct band gap structure, the optical energygap (Eg) of the samples were calculated from equation [35];

(˛hv)2 = B(hv − Eg) (2)

where B is a constant. The graph of (˛h�)2 as a function of h� ispresented in Fig. 6, and Eg values were determined by fitting a linearfunction to (˛h�)2 and extrapolating it to (˛h�)2 which is equal tozero value. The calculated Eg values are given in Table 2. The small

N. Sahiner / Colloids and Surfaces A: Physicoc

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30

40

50

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Energy (eV)

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Querc eti n

Fig. 6. The absorbance spectra of QC and p(QC) as (hv˛)2 (eV cm−1)2 versus energy(h�) graph for Egap determination.

Table 2The calculated Egap values for QC and p(QC).

Materials Egap,1 Egap,2 Egap,3

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Quercetin (QC) 3.0 ± 0.1 eV 3.5 ± 0.1 eV 4.8 ± 0.1 eVP(quercetin) particles p(QC) 3.2 ± 0.1 eV 3.6 ± 0.1 eV 4.6 ± 0.1 eV

ariation in band gap can also be due to the polymeric structuresf p(QC) and are relatively bigger in comparison to quantum dotsuch as CdS which was about 2.3 eV [36].

. Conclusion

In this investigation, an antibacterial, antioxidant and anti-arcinogenic molecule, QC, with much biomedical potential wasurned into particle form as p(QC) via a microemulsion polymer-zation/crosslinking polymerization technique and characterized. It

as found that the prepared p(QC) particles have negative surfaceharges and are thermally more stable than QC particles. It was alsoemonstrated that p(QC) particles do have antibacterial propertiesqual to QC molecules, and in some cases even better antimicrobialroperties, against three common bacteria strains at the investi-ated concentrations. Furthermore, p(QC) particles have very littleeduction in their antioxidant nature as compared with standardallic acid e.g., each 100 mg of QC and p(QC) have 69.27 ± 0.5 mg/L,nd 82.5 ± 9.6 mg/L gallic acid equivalency, respectively, in termsf antioxidant characteristics. With this investigation it can be sug-ested that the p(QC) particle has great potential in biomedical,ood, pharmacy, cosmetic and biosensor fields, as radical capturing

aterial, anti aging, antioxidant, anti carcinogenic and fluorescentrobe. The further use of p(QC) particles in various fields is currentlynder investigation.

cknowledgments

The author thanks Selin Sagbas, Duygu Alpasalan and Dr. Kivancel for support for some of the experiments, and optical character-zation.

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