Inclusion of Quercetin in Gold Nanoparticles Decorated with

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Inclusion of Quercetin in Gold Nanoparticles Decorated with Supramolecular Hosts Amplifies its Tumor Targeting Properties Mustafa Yilmaz, Apostolos A Karanastasis, Maria V Chatziathanasiadou, Mehmet Oguz, Anastasia Kougioumtzi, Nausicaa Clemente, Tahsin F Kellici, Nikolaos Evangelos Zafeiropoulos, Apostolos Avgeropoulos, Thomas Mavromoustakos, Umberto Dianzani, Serdar Karakurt, and Andreas G. Tzakos ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00748 • Publication Date (Web): 04 Mar 2019 Downloaded from http://pubs.acs.org on March 5, 2019

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ACS Applied Bio Materials

Inclusion of Quercetin in Gold Nanoparticles Decorated with Supramolecular Hosts Amplifies its Tumor Targeting Properties Mustafa Yilmaza, Apostolos A. Karanastasisb, Maria V. Chatziathanasiadouc, Mehmet Oguza,h, Anastasia Kougioumtzid, Nausicaa Clementee, Tahsin F. Kellicic, Nikolaos E. Zafeiropoulosb, Apostolos Avgeropoulosb, Thomas Mavromoustakosf, Umberto Dianzanie, Serdar Karakurtg and Andreas G. Tzakosc* aDepartment

of Chemistry, Selcuk University Konya, 42075, Turkey

bDepartment

of Materials Science Engineering, University of Ioannina, University Campus,

45110 Ioannina, Greece cLaboratory

of Organic Chemistry and Biochemistry, Department of Chemistry, University of

Ioannina Ioannina, Greece dDepartment

of Biomedical Research, Institute of Molecular Biology and Biotechnology,

Foundation of Research and Technology-Hellas, University of Ioannina Ioannina, Greece eDipartimento

fDepartment

di Scienze della Salute, Università del Piemonte OrientaleNovara, Italy

of Chemistry, National and Kapodistrian University of Athens, Greece

ACS Paragon Plus Environment

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ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

gDepartment

of Biochemistry Selcuk University Konya, Turkey

hDepartment

of Advance material and nanotechnology, Selcuk University, Konya, Turkey

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Keywords Nanoparticles, natural products, drug delivery, antitumor agents, calixarene, quercetin, in vivo, cancer

Abstract

Despite the anticancer potential of natural products (NPs), their limited bioavailability necessitates laborious derivatization or covalent conjugation to delivery vehicles. To unleash their potential we developed a nanohybrid delivery platform with noncovalently tunable surface. Initially, the active compound was encapsulated in a macrocycle, p-sulphonatocalix[4]arene, enabling a 62,000-fold aqueous solubility amplification as also a 2.9-fold enhancement in its cytotoxicity with respect to the parent compound in SW-620 colon cancer cells. A pH stimuli responsive behavior was recorded for this formulate, where a programmable release of quercetin from the macrocycle was monitored in acidic environment. Then, a nanoparticle gold core was decorated with calixarene hosts to accommodate non-covalently NPs. The loaded nanocarrier with the NP quercetin dramatically enhanced the cytotoxicity (>50 fold) of the parent NP in colon cancer and altered its cell membrane transport mode. In vivo experiments in a mouse 4T1 tumor model showed a


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reduction of tumor volume in mice treated with quercetin-loaded nanoparticles without apparent toxic effects. Further analysis of the tumor-derived RNA highlighted that treatment with quercetinloaded nanoparticles altered the expression of 27 genes related to apoptosis.

1. Introduction Natural products have constantly served as an inexhaustible reservoir for the discovery of drugs or inspired the synthesis of potent bioactive molecules. However, despite their intrinsic therapeutic capacity, their often poor solubility, low bioavailability, metabolism and the associated challenge to form suitable derivatives have severely hampered their intense exploitation or integration in drug development pipelines 1. Quercetin (Qrc) (Scheme S1), a flavonoid, has been extensively studied and its efficacy has been evidenced in a broad therapeutic window of diseases probably due to its ability to orchestrate a polypharmacological profile 2. Recently we revealed that Qrc interacts with the BH3 binding site of Bcl-xL/Bcl-2 proteins, thus inhibiting their activity and promotes apoptosis 3 and this interaction was further probed intracellularly with in cell NMR 4. The low aqueous solubility of Qrc is responsible for its poor absorption through the gastrointestinal tract after oral administration

5-6.

In addition, its extensive metabolism through enzymatic processes such as

glucuronidation, sulfation and methylation that take place mainly in the liver and the small intestine lead to the formation of metabolites which are less bioactive

5, 7.

During intravenous

injection Qrc may cause local toxicity and other side effects that limit the administered dose8. Therefore, in order to take advantage of the full Qrc therapeutic potential one has to enhance its solubility and prevent its metabolism, leading to a more efficient delivery to the tumor site. To this


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end, we developed a nanohybrid carrier, which is constructed from gold nanoparticles (GNPs) decorated with calixarenes 9-12, into which Qrc was loaded. The fist step in the construction of our nanocarrier involded the selection of an appropriate host moleclule. Calixarenes, a family of macrocycles, were proven ideal candidates in merit of their reported efficacy in the construction of molecular recognition systems, functional group versatility and relative availability thereof

9-11.

More specifically, we chose the water soluble p-

sulphonatocalixarene (6 coded as Calix, Scheme 1) due to its high stability, aqueous solubility, lower toxicity and ability to mitigate the toxicity of positively charged nanoparticles

13-15.

In the

second step we integrated Calix in the formulation of a gold nanoparticle-based nanocarrier (5 coded as GNPs@Calix, Scheme 1), harnessing the excellent biocompatibility, tunable size and affinity towards thiols of GNPs. As shown in Scheme 1, the facile incorporation of a thiolated spacer in 6 gave 4 which in turn was used to decorate the GNPs’ nanocarrier surface. To achieve the characterization of the Calix-Qrc complex as also of the GNPs@ Calix-Qrc an array of different physicochemical techniques was recruited including in silico studies, solid and solution state Nuclear Magnetic Resonance (NMR), fluorescence spectroscopy, Dynamic Transmission Electron Microscopy (TEM)- Electrophoretic Light Scattering (ELS), FTIR and Differential Scanning Calorimetry (DSC). The cytotoxic activity of the two complexes was evaluated in DLD1, SW-620 and 4T1 cells and the mechanism of cell entrance in DLD1 cells utilizing confocal microscopy. The safety and therapeutic efficacy of GNPs@ Calix-Qrc complex were examined in vivo in 4T1 BALB/c female mice. The expression levels of 86 genes, which are involved in apoptosis, were estimated through qRT-PCR in tumor-derived RNA samples isolated from the mice.


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OH

i OH

HO3S

HO OH OH

HO OH OH

SO3H

HO3S

SO3H

6

ii SH

1

O

Br

HS

OH OH

O

O

iii

Br

3

2 iv

SH

O

HO3S HO3S

O

HS

OH OH

O H O O H O

GNPs

O

HO3S

SO3H SO3H

HO OH O S

S S

S

GNPs

S

SO3H

O H O

S

O H O

S

S O

HO3S

O

OH OH

S

S HO OH O

O

OH HO

O

SO3H

5

4

Scheme 1. The schematic route for the synthesis of the calix[4]arene derivatives 1,3bis(bromopropoxy)-tert-butylcalix[4]arene 5,11,17,23-tetra(tert-butyl)-calix[4]arene

(2), (3),

25,27-bis(3-thiopropoxy)-26,28-dihydroxy25,27-bis(3-thiopropoxy)-26,28-dihydroxy-

5,11,17,23 tetra(sulphanato)-calix[4]arene (4), complexed with GNPs, GNPs@Calix-Qrc (5), the water-soluble calix[4]arene derivative, Calix (6) from p-tert-butylcalix[4]arene (1). Experimental


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conditons: (i) H2SO4 at 50 °C for 5 h, (ii) Dibromopropane/K2CO3 in acetone, reflux, 48 h (iii) Thiourea in acetonitrile, reflux, 6 h; then KOH deionized water, reflux (iv) H2SO4 at 30°C for 4h.

2. Materials and Methods

2.1 General apparatus and reagents. Reagents used herein were from Merck or Aldrich and were of standard analytical grade. Qrc (98%) was from Sigma–Aldrich (St. Louis, MO, USA). Reactions were conducted under inert atmosphere (unless otherwise noted). 2.2. Synthetic procedures. 2.2.1. Preparation of Calix-Qrc inclusion complex. A 10 ml aqueous solution of 100 mg of Calix (Scheme 1, compound 6) was prepared and the pH was set up to 7.5. An equimolar quantity of Qrc (Scheme S1) was added to a 50 mL Erlenmayer reaction flask containing Calix. The reaction mixture was magnetically stirred at room temperature for 1 hr. The contents were filtered through nylon filter with 0.45 μm pore size pore size (Millipore®, USA). Filtrates were frozen at -40 °C for 24 h, before the freeze-drying was started (FreeZone 2.5, USA).

2.2.2. Synthesis of Calix and complexation with GNPs. 5,11,17,23-tetra-tert-butyl-2526,27,28-tetrahydroxycalix[4]arene (1),1,3-bis(bromopropoxy)-tert-butylcalix[4]arene (2), 25,27bis(3-thiopropoxy)-26,28-dihydroxy-5,11,17,23-tetra(tert-butyl)-calix[4]arene (3) and 25,27bis(3-thiopropoxy)-26,28-dihydroxy-5,11,17,23

tetra(sulphanato)-calix[4]arene(4)

were

synthesized according to previously reported method 16-20.


6

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More specifically: (2) 1,3-bis(bromopropoxy)-tert-butylcalix[4]arene: A sample of p-tert-butylcalix[4]arene (0.332 g, 0.511 mmol) and 60 mL of CH3CN was placed in three necked flask. To this 0.221 g (1.6 mmol) of anhydrous K2CO3 was added and the reaction mixture was refluxed for 1 h. Then 0.8 mL (1.59 g, 7.7 mmol) of 1,3-dibromopropane was added using a dropping funnel over 7 min, and the mixture, at reflux, was stirred for 48 h. The solvent was removed under reduced pressure to give a white solid residue. To the residue 37 mL of 5% HCl was added and the mixture was extracted with 40 mL of CH2Cl2.The organic layer was washed with deionized water (3 x 35 mL), brine (20 mL) and was then dried (MgSO4). The solvent was removed to afford 0.315 g of a white solid product. The product was recrystallized from CH2Cl2/CH3OH (1:4) to give 0.22 g (50% yield) of a precipitate which was assigned as 24. The NMR characterization is provided on the supporting information, Section I. (3) 25,27-bis(3-thiopropoxy)-26,28-dihydroxy-5,11,17,23-tetra(tert-butyl)-calix[4]arene: In a 250 mL two necked flat bottom flask containing 40 mL CH3CN, 0.5 g of p-tertbutylcalix[4]arene (0.561 mmol), 0.5 g of dialkyl bromide and 0.14 g of thiourea (0.561 mmol) were added. The contents were heated to reflux for 6 h. Solvent was removed under reduced pressure. Crude product was treated with 0.19 g of KOH (3.423 mmol) and aliquot of 21 mL of deionized water and allowed to reflux for 2 h. After neutralization with 0.1 M HCl and product was extracted with CHCl3, dried over MgSO4 to afford pure Calix-SH. Yield 40 %. The NMR characterization is provided on the supporting information, Section I. (4) 25,27-bis(3-thiopropoxy)-26,28-dihydroxy-5,11,17,23 tetra(sulphanato)-calix[4]arene: Calix-SH (0.8 g, 1.00 mmol) in 5 mL of sulfuric acid (98%) was carefully added and the suspension was stirred at 30 °C for 4 h. Small amount of suspension was added to water and the reaction was


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stopped when no precipitation was observed. The product was cooled, and the precipitate was filtered with fritted glass filter. Then, the residue on the filter was washed with cold methanol and the supernatant was collected in a clean vessel. In order to remove excess sulfuric acid, the aliqued was then mixted with 500 mL of ethyl acetate under stirring. Ethylacetate was added and decantated several times to wash precipitates. Finally, the product was dried at 40° C,Yield: 65 %. The NMR characterization is provided on the supporting information, Section I.

Preparation of GNPs@Calix (5) and inclusion complex with Qrc An aqueous solution of HAuC14 .3H2O (88.5 mg, 0.22 mmol) in 250 mL water was brought to reflux while stirring. A trisodium citrate (38.8 mM, 25 mL) solution was added and color change was observed from pale yellow to deep red. The solution was heated for an additional period of 20 min. An aqueous solution of compound 4 (Scheme 1) (0.2 g, 0.22 mmol) containing an equivalent amount of sodium hydroxide was added to the colloidal gold suspension. The studies were carried out in dark conditions. The mixture was stirred overnight, volume was reduced to 80 mL and pH was adjusted to 7.5. Qrc (101 mg in 20 ml MeOH) was added and stirred for another 1 hr. MeOH was removed under vacuum and the content was filtered through 0.45 μm pore size nylon filter.

2.3. Determination of stoichiometry by Job’s method. The validation of the stoichiometric ratio of Calix-Qrc complex was carried out Job’s method. Fixed concentration (1×10−4 M) solutions of Calix and Qrc were mixed in varying molar ratios from 0.1 to 1. Fig. S1 represents the Job’s plot. The maximum value (330 nm) was obtained at 0.5 molar ratio, demonstrating an 1:1 stoichiometry.


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2.4. Determination of binding constant. To determine the binding constant (Kα) of Calix-Qrc complex the Benesi–Hildebrand equation21 (Equation 1) was used. For this, different concentrations of Calix at pH=7.5 were prepared and mixed with fixed concentration of Qrc.

[𝑄𝑟𝑐] 𝐴𝑏𝑠

1

1

1

= [𝐶𝑎𝑙𝑖𝑥]𝜀𝑘𝑎 + 𝜀 (1)

In the above equation [Qrc] represents Qrc’s concentration, Abs depicts the maximum absorbance of Calix-Qrc at specific wavelength and [Calix] stands for the concentration of the host (Calix). Finally, the molar absorptivity of Calix-Qrc at maximum absorbance is represented by . The value of the Ka (8.89×103 l mol−1) was acquired from the ratio of (1/ε) to (1/Kaε), i.e intercept and slope of equation that were obtained from linear regression plot between 1/ [Calix] and [Qrc]/Abs. The value of Ka indicates the effective binding of Qrc in the Calix cavity.

2.5. Phase solubility study. The solubilizing capacity of Calix for Qrc was carried out according to the method described by Higuchi and Conners

22.

For this, aqueous solutions of Calix with

concentration ranging from 0.01 mM to 0.1 mM were prepared and pH was adjusted to 7.0. In 10 mL solution of Calix for each concentration, 10 mg of Qrc were mixed and agitated for 14 hr in orbit shaker at 50 rmp. The mixtures were filtered and the concentration of Qrc in solution was analysed with UV-Vis spectroscopy. Ks 

Slope (2) S o  (1  Slope)


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A linear curve was obtained from the plot of concentration of Calix vs the quantity of dissolved Qrc (Fig. S3). A correlation coefficient square (R2=0.949) and slope < 1(0.105) in linear regression equation (y = 0.1054x + 0.0056) suggest 1:1 stoichiometric ratio for the complexation. Slope value was used in equation (2) to calculate the Ks (714 mM-1), that indicates that the formed Calix-Qrc complex is quite stable.

2.6. Solid state NMR spectroscopy. 13C and 1H NMR spectra were recorded on a 600 MHz Varian system. The NMR spectra were recorded using a 3.2 mm Varian Double Resonance HX MAS probe. Tetramethylsilane was used as internal standard. Larmor frequencies were 150.744 MHz for 13C nuclei and 599.556 MHz for 1H nuclei. For the 1H echo MAS and the 1H-13C CPMAS spectra the sample rotation frequencies were 20 and 16 kHz with 50 and 20 s relaxation delay, respectively. RAMP cross-polarization had a contact time of 5 ms. The obtained spectrum for Qrc is in agreement with the one recorded by Wamer and Zielinski 23 using a 300 MHz spectrometer. The spectrum obtained with our 600 MHz spectrometer and with faster sample rotation is characterized by better resolution and absence of many spinning bands interfering with the isotropic peaks. In both spectra, however, the multiplicity of the isotropic peaks indicates that Qrc either exhibits polymorphism or crystallizes with a crystallographic asymmetric unit that contains several Qrc molecules. The spectrum of Cis is almost identical to the one reported in the literature 24.

2.7. FT-IR Analysis. The inclusion of Qrc on the surface of GNPs@Calix was verified by FTIR spectroscopy on a Jasco 6200 instrument.


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2.8. HR-TEM. HR-TEM imaging was conducted with a JEOL JEM-2100 instrument operating at 200 KV. Samples were prepared by casting a minute drop of a 0.1% w/v GNP dispersion onto carbon coated grids followed by gentle solvent evaporation under ambient conditions.

2.9. Dynamic and Electrophoretic Light Scattering (DLS). DLS measurements were performed using a Malvern Instruments Zetasizer Nano ZS.

2.10. Differential Scanning Calorimetry (DSC). DSC thermograms were realized on a TA Q20 instrument under inert Ar-N2 conditions.

2.11. pH-dependent release of Qrc from Calix. For the quantitative determination of % Release of Qrc, 5 mg of Calix-Qrc complex were dissolved in 10 ml of H2O at prescribed pH values ranging from 7.5 to 4. After 15 min of equilibration, an aliquot was used for UV-Vis measurements and rest of the solution was passed through 0.45 μm PES filter. Following the same method for the determination of % Loading, the filter was washed with MeOH until colorless. The amount of Qrc in the filtrate was determined spectroscopically by applying the Beer-Lambert law. Upon variation from pH=6 to 5.5, turbidity was developed denoting pronounced release of Qrc from the complex. Control UV-Vis spectra of Qrc solutions and Calix-Qrc physical mixtures at end point pH values are provided on Fig. S6.


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2.12 In vitro studies. 2.12.1. Cell culture. HeLa (human adenocarcinoma cervical cell line) and HepG2 (hepatocellular carcinoma cell line) cells were cultured in ATCC-formulated Eagle's Minimum Essential Medium (Catalog No. 30-2003), MDA-231 (breast adenocarcinoma cell line) and SW620 (metastatic colorectal adenocarcinoma cell line) in ATCC-formulated Leibovitz's L-15 Medium, (Catalog No. 30-2008), PC-3 (metastatic prostate adenocarcinoma cell line) in ATCCformulated F-12K Medium (Catalog No. 30-2004) and HT-29 (colorectal adenocarcinoma cell line) in ATCC-formulated McCoy's 5a Medium Modified (Catalog No. 30-2007). Human colorectal adenocarcinoma cell line DLD-1 was cultured in McCoy’s 5A (Modified) Medium with 10% heat-inactivated calf serum. Cells were grown on 60 mm dishes until approximately 70% confluence at 37 °C under 5% CO2 and were passaged every 3-4 days. The human colon cancer cell line SW-620 (CCL-227) was provided from ATCC (American Type Culture Collection, ATCC, Rockville, MD, USA) and cultured in Leibovitz L-15 (30-2008, ATCC) medium. The murine breast cancer 4T1 cell line was cultured in RPMI 1640 medium. All media were supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1% penicillin/streptomycin. Cells were grown as monolayer cell cultures in an atmosphere containing 5% CO2, and 95% relative humidity at 37 °C.

2.12.2. Evaluation of Qrc cytotoxic effect on cancer cell lines. Qrc was investigated on several cancer cell lines to assess its cytotoxic effect. Each cell line was treated with several concentrations of Qrc varying between 0.1 µM to 400 µM. The effect of Qrc on the viability of cancer cell lines


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was detected spectrophotometrically by using Alamar Blue (Invitrogen, California, USA) as cell viability marker. Qrc caused a dose-dependent decrease in cell viability in HeLa, HEPG2, MDA231, PC-3 and SW-620 and HT-29 cells, DLD1 and 4T1 cells with IC50 values equal to 27.1 µM, 79.2 µM, >200 µM, 153.9 µM, 13.5 µM, 172.4 µM, 17.2 µM and 23 µM, respectively.

2.12.3. Confocal Laser Scanning Microscopy. DLD-1 cells were plated on coverslips in 24 well plates (6x104 cells/well) for 24 hours. The next day, the culture medium was exchanged and the cells were then incubated at 37 °C or 4 °C for 45 minutes, with 10 μM Qrc, Calix-Qrc, GNPs@Calix-Qrc Complex in medium without serum. After the incubation with compounds, the coverslips were washed three times with Phosphate-Buffered Saline (PBS), fixed with 3.7% Paraformaldehyde for 15 min and quenched with 50 mM ammonium chloride for 15 min. For Propidium iodide (PI) staining the cells were treated with RNase (1 mg·mL-1) for 20 minutes and stained with PI (1.6 μg·mL-1 in PBS) for 4 min. Subsequently, the coverslips were washed with PBS and mounted upside-down on glass slides, with a drop of Mowiol containing DABCO (100 mg/mL). Distribution of fluorescent compounds was analyzed using a Leica TCS SP5 confocal microscope (Leica Microsystems GmbH, Mannheim, Germany) and objective HCX PL APO CS 63.0x1.4 oil UV. Qrc, Calix-Qrc, GNPs@Calix-Qrc Complex were excited at 488 nm and their emission was detected at 500-579 nm.

2.12.4. Cell viability assay. The human colon cancer cell lines SW-620 and DLD1 and the murine breast cancer cells 4T1 were cultured in 96-well plates in triplicates to optimize the cell number and the incubation time. Before treatment with compounds, 1x104 cells were incubated for 24 h at 37 °C in 5% CO2. Various concentrations of Qrc, Calix and Calix-Qrc complex, ranging from 0.1


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µM to 200 µM were used to determine the effect of Qrc, Calix and Calix-Qrc complex on proliferation of the cells and IC50 values. The Alamar blue assay was selected to observe the differences in cell viability. The change in color from blue to pink was estimated spectrophotometrically.

2.13. In vivo studies. Eight-week old female BALB/c mice were bred under sterile conditions in the Universitàdel Piemonte Orientale animal facility, and the treatment has been performed in accordance with the University Ethical Committee and European guidelines. Experimental manipulations were performed under the inhalation anesthesia induced by 4% and maintained by 2.3% mixture of isofluorane in oxygen administered through facial masks. 4T1 cells were harvested from subconfluent cultures by trypsinization. The cells (105 cells in 100 µl) were orthotopically implanted with the injection into the fat pad of the second right mammary gland. The tumor growth was allowed during the following 10 days. Then, the mice were randomized into four groups (n=6 each) receiving in total five tail intravenous injection (one every four days): control group, Qrc alone (3.3 mg·kg-1), GNPs@Calix alone (6.6 mg·kg-1), or GNPs@Calix-Qrc complex (10 mg·kg-1). The last (fifth) injection was performed with a double dose of each compound to maximize the effects. The mice body weight and tumor size were measured every injection day. Twenty-two days after the first injection mice were anesthetized and sacrificed by cervical dislocation. The mammary tumors were carefully excised, and the maximum length (a) and minimum length (b) and the thickness (c) of the tumors were measured. The volume was calculated using the formula: a*b*c. The health state of the animals was monitored throughout the experimental procedure. Data analysis was performed using one-way ANOVA and the Dunnett test using GraphPad Instat Software (GraphPad Software, San Diego, CA, USA). Data are


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expressed as mean and standard error of the mean (SEM) and statistical significance was set at p

Inclusion of Quercetin in Gold Nanoparticles Decorated with

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