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Colloids and Surfaces B: Biointerfaces 143 (2016) 206–212

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

jo ur nal ho me p ag e: www.elsev ier .com/ locate /co lsur fb

evelopment and evaluation of decorated aceclofenac nanocrystals

eong-Joo Park, Nilesh Meghani, Jin-Seok Choi ∗, Beom-Jin Lee ∗∗

ollege of Pharmacy, Ajou University, Suwon-si, Gyounggi-do 443-749, Republic of Korea

r t i c l e i n f o

rticle history:eceived 11 November 2015eceived in revised form 18 February 2016ccepted 7 March 2016vailable online 10 March 2016

eywords:ceclofenac (ACF)anocrystals (NC)olubilityrug release

a b s t r a c t

This study was aimed at achieving enhanced solubility of aceclofenac (ACF) in nanocrystaline forms(ACF-NC) and evaluating the effects of ACF-NC on cell viability. Decorated ACF-NC were prepared bynano-precipitation with stabilizers. Three kinds of stabilizers were investigated: Tween 80, Poloxamer407, and PEG 6000. The crystal structure and morphology of ACF-NC were characterized by field emissionscanning electron microscopy (FE-SEM) and differential scanning calorimetry (DSC). The solubility of ACF-NC and ACF (pure) was evaluated in different media (pH 1.2 and pH 6.8 buffers and distilled water [DW]).A drug release study was performed in PBS for 24 h. Cell viability was evaluated for 24 h using a humancolon cancer cell-line (HCT-116) and a human breast cancer cell-line (MCF-7).

Decorated ACF-NC with a mean size of 725 nm were successfully prepared. The solubility of the deco-rated ACF-NC were 4–7 times higher than that of ACF in DW and pH 6.8 buffer. A peak shift from 153.1 ◦C

◦

ell viability to 150.5–151.0 C was observed in the DSC thermogram of decorated ACF-NC versus ACF. In terms of drugrelease, there was an initial burst in decorated ACF-NC within 1 h followed by slow release for up to 4 h.Decorated ACF-NC exhibited viability approximately 63.9% of HCT-116 cells and also showed viability in58.3% of MCF-7 cells at 15 �g/mL of drug concentration. In conclusion, decorated ACF-NC proved to be apromising approach for enhancing drug solubility and cytotoxicity.

© 2016 Elsevier B.V. All rights reserved.

. Introduction

According to the Biopharmaceutics Classification System (BCSI), aceclofenac (ACF) is a non-steroidal anti-inflammatory drugNSAID) with poor water solubility (0.058 �g/mL) and high per-

eability [1] (Fig. 1). Despite the high permeability of the BCS classI drugs, they often result in low oral bioavailability due to theirlow and limited release of drug in gastrointestinal fluid [2]. Thearket of ACF is limited to Asia, therefore, attempts to develop ACF

ormulations with high efficacy have to be made in order to expandts market.

Approximately 40% of drugs in the development pipelines are

oorly soluble in aqueous solvents and some of them in the organicolvents as well [3]. Low aqueous solubility and dissolution ratef API is one of the most prevalent problem that formulation sci-

Abbreviations: ACF, aceclofenac; NC, nanocrystals; ACF-NC, aceclofenacanocrystals; FE-SEM, field emission scanning electron microscopy; DSC, differen-ial scanning calorimetry; DW, distilled water.∗ Corresponding author at: College of Pharmacy, Ajou University, 206, World

upro, Yeongtong-gu, Suwon-si, Gyenggi-do, 443-749, Republic of Korea.∗∗ Co-corresponding author. Tel.: +82 31 219 3442; fax: +82 31 212 3653.

E-mail addresses: c34281@gmail.com ( J.-S. Choi), beomjinlee@gmail.comB.-J. Lee).

ttp://dx.doi.org/10.1016/j.colsurfb.2016.03.022927-7765/© 2016 Elsevier B.V. All rights reserved.

entists are facing, and it is more common among the new drugcandidates due to the use of high throughput and combinatorialscreening processes during the drug discovery and selection of thenew molecular entity (NME) [4,5]. Poorly water-soluble drugs aredifficult to develop with commercially available techniques and arefrequently abandoned early in the drug development pipeline. Themain goal of formulation development for these drugs is to improvesolubility. To achieve solubility enhancement, several techniqueshave been developed, such as solid dispersion [1,6], salt forma-tion [7], co-solvent [8] and particle size reduction (micronizationand nanoparticle formation) [9,10]. However, often they cannotsolve the bioavailability problem for many drugs. For example,micronization does not create the sufficiently large surface toenhance dissolution rates and therefore, the bioavailability of themany poorly soluble drugs.

To overcome the challenge of poor solubility, nanomaterialssuch as nanocrystals, solid lipid nanoparticles (SLN), nanoemul-sions, and self-assembled nanoparticles have been employedto enhance drug solubilization [11]. Among the various nan-otechniques, drug nanocrystal formulation has been successfullyexplored for delivery of various anti-cancer drugs [12–15]. Nan-

onization of these drug molecules have increased the solubilityand dissolution rate owing to increased surface area [16]. Drugnanocrystals ideally exist in a stable crystalline state with high

J.-J. Park et al. / Colloids and Surfaces B: B

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Fig. 1. Chemical structure of Aceclofenac.

rug loading which can be formulated as the oral dosage dormsith improved dose-bioavailability, proportionality and increasedatient compliance, and can also be injected intravenously as thequeous nanosuspensions [17]. Drug nanocrystals can be producedainly by applying two approaches, top-down and bottom-up

18,19], which utilize different techniques. Top-down approachestilize high-energy processes whereby pure drugs are broken down

nto smaller-sized particles by the use of technologies such asearl milling and high-pressure homogenization [20–22]. Prod-cts on the pharmaceutical market such as Emend®, Tricor®,ridlide®, Naprelan®, and Theodur® were produced by the top-own approach utilizing milling technology. Bottom-up approachomprises of the precipitation of the dissolved drug by a non-olvent and controlled crystallization during freeze drying [23]. Theottom-up approach was applied for the development of productsuch as Gris-Peg® and Cesamet® [18].

In this study, nanocrystals were prepared by a nanoprecipitationethod to reduce the particle size of the drug, thereby enhancing

he surface area for solubilization and cellular viability. DecoratedCF nanocrystals (ACF-NC) were evaluated with respect to physicalroperties of morphology, solubility, and drug release. In addition,he effects of ACF and decorated ACF on cell viability were investi-ated in colon and breast cancer cell lines.

. Materials and methods

.1. Materials

ACF was obtained from Korea United Pharm., INC. Tetrazoliumye 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bro-ide (MTT) was purchased from Sigma–Aldrich (Saint Louis, MO,SA). HCT-116 cells and MCF-7 cells were obtained from the Collegef Pharmacy, Chosun University. Fetal bovine serum (FBS), antibi-tics, and Roswell Park Memorial Institute (RPMI1640) mediumere from Gibco (Billings, MT, U.S.A.). Organic solvents were pur-

hased from Samchun Pure Chemicals.

.2. Preparation of decorated ACF-NC

ACF-NC were obtained by the following nanoprecipitationethod. ACF (20 mg) was dissolved in acetone (5 mL), in which

0 mg of the polymers, i.e., Tween80, Poloxamer 407, and PEG-6000ere subsequently dissolved as stabilizers. The resulting ACF andolymer-containing solutions were added to an aqueous medium.ize of the drug crystals were controlled by probe-sonication (Son-cs & Materials INC, VCX 500, USA) at 200 W for 1 min while stirringt 300 rpm. The nanocrystals were collected by filtration with aylon filter (0.2 �m pore size) and washed two times with DW.

he nanocrystals were first dried in a drying oven at 60 ◦C for 1 h,ollowed by further drying in a desiccator under vacuum for 1 day.

iointerfaces 143 (2016) 206–212 207

2.3. Characterization of decorated ACF-NC

2.3.1. Particle size analysisMorphological evaluations of ACF-NC were conducted by scan-

ning electron microscopy (FE-SEM, JSM-6700F, JEOL, Japan). ACFand ACF-NC powders were dropped onto carbon tape. The car-bon tape was coated with gold for 2 min under vacuum. Sampleswere viewed at an acceleration voltage of 5.0 kV, and the sizes of atleast 50 particles were measured manually using ImageJ software(National Institute of Health).

2.3.2. Differential scanning calorimetry (DSC)Thermographs of the decorated ACF-NC and ACF samples were

obtained by using a differential scanning calorimetry (DSC) instru-ment (DSC 200 F3, NETZSCH, Germany). Samples equivalent to 1 mgof ACF were placed in aluminum pans and heated from 10 ◦C to250 ◦C at a scanning rate of 10 ◦C/min under a nitrogen purge of40 mL/min.

2.3.3. Solubility testThe solubility of decorated ACF-NC and ACF samples was deter-

mined by using a water bath shaker (BS-06, Lab Company, Korea).ACF powder (5 mg) and ACF-NC (equivalent to 5 mg ACF) were dis-persed in 5 mL of pH 1.2 buffer, pH 6.8 buffer, and D.W, followedby shaking at 100 rpm at 37 ◦C. After 1 h, the samples were sub-jected to centrifugation at 20,000g for 30 min and then filteredthrough a PVDF syringe filter (0.22 �m pore size). The ACF con-tent of the sample was determined using high-performance liquidchromatography (HPLC).

2.3.4. HPLC analysisACF for solubility analysis was subjected to HPLC (Waters 2695

Alliance system, Waters, USA) with an ultraviolet detector (Waters2484, Waters, USA) set at a wavelength of 275 nm. A C18 column(Gemini 5u 110A analytical, Intersil OSD-3) operated at 30 ◦C wasused as the analytical column The mobile phase was composedof methanol–0.02% orthophosphoric acid (70:30) at a flow rate of1 mL/min [24].

2.3.5. Drug releaseIn vitro ACF release studies were performed in pH 7.4

phosphate-buffered saline (PBS) at 48 h. The shaking speed was setto 100 rpm. Samples (1 mL) were withdrawn at 0.5, 1, 2, 4, 8, 12,24, 36 and 48 h. The samples were subjected to centrifugation at20,000g for 30 min and then filtered through a PVDF syringe filter(0.22 �m pore size). The ACF content of the sample was determinedusing high performance liquid chromatography (HPLC).

2.3.6. Cell viabilityHuman colon cancer cells (HCT-116) and human breast can-

cer cells (MCF-7) were grown in RPMI 1640 supplemented with10% (v/v) fetal bovine serum and (5%) antibiotics (100 IU/mL ofpenicillin G sodium and 100 �g/mL of streptomycin sulfate). Cellswere maintained in an incubator under a humidified 5% CO2/95%air atmosphere at 37 ◦C. They were seeded in 96-well plates ata density of 3 × 104 cells/well and grown to 70–80% confluence.ACF and decorated ACF-NC equivalent to an ACF concentration of0.15, 1.5 and 15 �g/mL were added and cells were incubated for 12and 24 h. Cytotoxicity was assayed using MTT and detected usinga microplate reader (Synergy H1Hybrid Multi-Mode MicroplateReader, BioTek, USA). The cytotoxicity percentage was calculatedby subtracting% cell viability from 100%.

2.3.7. Statistical analysisStatistical analysis was performed using the paired t-test in

SigmaPlot 10.0 (SYSTAT, Inc., Chicago, IL, USA). The data were

208 J.-J. Park et al. / Colloids and Surfaces B: Biointerfaces 143 (2016) 206–212

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ig. 2. SEM images of ACF and ACF-NC. (a) Pure ACF, (b) ACF-TW80, (c) ACF-P407 a

xpressed as mean ± standard deviation. Statistical significanceas accepted for p values < 0.1.

. Results and discussion

.1. Characterization of decorated ACF-NC

.1.1. Particle size and morphology of decorated ACF-NCACF-NC was successfully prepared using a nanoprecipitation

ethod. The particle size of ACF-NC was reduced approximately 50imes compared to that of ACF. SEM was employed to examine theurface morphology of decorated AFC-NC for comparison with ACF.he mean particle size of ACF (pure drug) was 48.2 ± 13.6 �m. Theean particle size of ACF-NC using Tween 80, Poloxamer 407, and

EG 6000 was 716 ± 65 nm, 730 ± 71 nm and 729 ± 61 nm, respec-ively (Fig. 2; Table 1). The particle size data showed the controlledrystal formation of ACF-NC. The zeta-potential of decorated ACF-Cs were found to be −0.48 mV (Tween 80), −17.39 mV (Poloxamer07), and −18.35 mV (PEG 6000) compared to −1.66 mV for ACF. Allf the stabilizers used in the study were non-ionic polymers, and,hus, there was a slight change in the overall zeta potential of theecorated ACF-NC. This electric charge may help to stabilize theanocrystals through the steric effect of the stabilizers [25].

.1.2. Differential scanning calorimetry (DSC)The DSC thermograms of ACF, ACF-TW80 (Tween 80), ACF-P407

Poloxamer 407), and ACF-PEG6000 (PEG6000) are presented inig. 3. The DSC profiles of ACF showed a sharp endothermic peakt 153.1 ◦C corresponding to the melting point with an enthalpyf −139.7 J/g. The thermogram of ACF-TW80 showed a shift of the

elting point to 150.5 ◦C with an enthalpy of −131.5 J/g, while that

f ACF-P407 showed a shift of the melting point to 150.6◦ with annthalpy of −129.5 J/g. The ACF-PEG6000 thermogram showed ahift of the melting point to 151.0 ◦C with an enthalpy of −131.9 J/g.

ACF-PEG6000. The magnification of (a) is 200× and (b), (c), and (d) are 10,000×.

This reduction in enthalpy may be due to the reduced crystallinityof ACF in ACF-NC.

3.1.3. Solubility testThe solubility test was performed in pH 1.2 and pH 6.8

buffers and DW and decorated ACF-NC (ACF-TW80, ACF-P407,ACF-PEG6000) versus pure ACF were evaluated. The solubilityof ACF-TW80 (15.9 �g/mL), ACF-P407 (14.2 �g/mL), and ACF-PEG6000 (13.3 �g/mL) were found to be 3–4 fold higher than thatof ACF (4.6 �g/mL) in DW. The solubility of ACF-TW80 (1.4 �g/mL),ACF-P407 (1.3 �g/mL), and ACF-PEG6000 (1.2 �g/mL) decreasedmoderately relative to that of ACF (2.3 �g/mL) in pH 1.2 buffer.In addition, the solubility of ACF-TW80 (125.3 �g/mL), ACF-P407(215.0 �g/mL), and ACF-PEG6000 (123.1 �g/mL) increased approx-imately 4–7 times in comparison with that of ACF (31.1 �g/mL) inpH 6.8 buffer. The solubility of decorated ACF-NC were higher thanthat of ACF in DW and pH 6.8 buffer, indicating drastic solubilityenhancement when the nanocrystal technique was employed. Incontrast, the solubility of both ACF-NC and ACF were very low inpH 1.2 buffer, with no significant differences in solubility betweenthe nanocrystal samples and the pure drug (Fig. 4). It has beenreported that the solubility of ACF nanocpmposites with Solupluswas increased in DW [26]. The results of all the samples were sta-tistically compared to ACF (pure) at all concentrations using pairedt-test (p value < 0.05).

3.2. In-vitro drug release

In-vitro ACF release studies were performed in pH 7.4 PBS for48 h. The cumulative release of ACF significantly increased withdecorated ACF-NC for the first 30 min; however, the release rate

was decreased after 30 min. After 4 h, the cumulative release ofACF significantly increased and was similar to that from deco-rated ACF-NC. After 24 h, the cumulative release of ACF was almost100% compared to ACF-TW80 (99.5%), ACF-P407 (80.6%), and ACF-

J.-J. Park et al. / Colloids and Surfaces B: Biointerfaces 143 (2016) 206–212 209

ted AC

PAT

Fig. 3. DSC images. Images compare the thermal changes of ACF and decora

EG6000 (80.4%) (Fig. 5). The increased solubility of decoratedCF-NC at earlier time points was possibly because of the polymers.his may be due to the enhanced wettability and reduced surface

F-NC with Tween 80, Poloxamer 407, or PEG 6000. (PM = physical mixture).

tension of surfactant-incorporated powder. Furthermore, smallercrystals have a larger surface area, leading to faster drug release.However, decrease in the release rate of decorated ACF-NC after

210 J.-J. Park et al. / Colloids and Surfaces B: Biointerfaces 143 (2016) 206–212

Fig. 4. Solubility test. Solubility of aceclofenac (ACF), aceclofenac-Tween80 (ACF-TW80), aceclofenac-Poloxamer407 (ACF-P407) and aceclofenac-PEG6000 (ACF-PEG6000)in different media. (a) Distilled water, (b) pH 1.2 buffer, and (c) pH 6.8 buffer.

Fig. 5. Drug release profiles. Drug release of aceclofenac (ACF), aceclofenac-Tween80 (ACF-TW80), aceclofenac-Poloxamer407 (ACF-P407) and aceclofenac-PEG6000 (ACF-PEG6000). Studiers were performed in PBS buffer (pH 7.4) and a shaking incubator for 48 h at 37 ◦C.

Table 1Physical properties of ACF (pure drug) and decorated ACF-NC (mean ± S.D.).

Properties ACF ACF-TW ACF-P407 ACF-PEG6000

716 ±−0.4

todraaaa

Particle size 48.2 ± 13.6 �m

Surface charge (mV) −1.66 mV

he initial 30 min can be explained by the possible re-precipitationf nanocrystal powders. The cumulative drug release of ACF andecorated ACF-NC over the period from 30 min to 24 h of drugelease study was found to be 68.0% (ACF-TW80), 38.1% (ACF-P407)nd 53.9% (ACF-PEG6000) and 95.5% (ACF). These results indicate

burst in drug release for ACF-NC in the first 30 min, followed by slower release afterwards. The cumulative drug release of ACFnd decorated ACF-NC were found to be 100.0% (ACF-TW80), 96.1%

65 nm 730 ± 71 nm 729 ± 61 nm8 mV −17.79 mV −18.35 mV

(ACF-P407) and 96.0% (ACF-PEG6000) and 100.0% (ACF) for 48 h,thus the data for 48 h drug release was not significantly differentthan 24 h drug release.

Previous studies have shown that, release from the paclitaxelnanocrystal was faster than pure drug [27,28]. Similarly, Fenofi-

brate and piroxicam nanocrytal also has shown faster or burst drugrelease than pure drug [29,30].

J.-J. Park et al. / Colloids and Surfaces B: Biointerfaces 143 (2016) 206–212 211

F ility.

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ig. 6. Cell viability. Comparison of effects of ACF and decorated ACF-NC on cell viabith drug at 12 h, (b) and (d) were cell incubated with drug at 24 h. Three different

.3. Cell viability

Cell viability of decorated ACF-NC with three different kinds oftabilizers was analyzed against pure ACF. ACF is a well-knownSAID and does not have any reported anti-cancer effects. Theell viability studies of decorated ACF-NC were evaluated usinghe cancer cell-lines HCT-116 and MCF-7. ACF standard solutionas prepared by using dimethyl sulfoxide (DMSO) as the solvent.riefly, ACF was dissolved in DMSO from 0.15 mg/mL to 15 mg/mLf drug concentration and then diluted 100 times by DW from.15 �g/mL to 15 �g/mL. The blank of standard was 1.0% DMSO

n DW. The drug content of all the decorated ACF-NCs was simi-ar. Samples were dispersed in DW from 15 �g/mL to 0.15 �g/mLequivalent ACF concentration).

For MCF-7 cells, ACF-PEG6000 exhibited higher cell cytotoxicityhan all of the samples for 12 h and 24 h (Fig. 6a,b). Interestingly,ell cytotoxicity was increased depending on drug concentra-ions. Overall, decorated ACF-NC exhibited higher cell cytotoxicityhan ACF (pure). At 12 h incubation time, cell viability forecorated ACF-NCs were 58.3 ± 1.2% (ACF-PEG6000), 74.7 ± 2.9%ACF-TW80) and 78.0 ± 2.8% (ACF-P407) at 15 �g/mL, 75.0 ± 2.7%ACF-PEG6000), 81.0 ± 2.3% (ACF-TW80) and 83.3 ± 1.4% (ACF407) at 1.5 �g/mL, and 86.3 ± 2.0% (ACF-PEG6000), 88.6 ± 2.1%ACF-TW80) and 87.1 ± 0.9% (ACF-P407) at 0.15 �g/mL. Thus, the

ell viability was dose dependent in this cell line. As expected, ACFpure) showed low cell cytotoxicity, i.e., 86.4 ± 1.7%, 98.5 ± 2.3%nd 99.3 ± 4.1%, at 15 �g/mL, 1.5 �g/mL and 0.15 �g/mL respec-

Cell lines used were MCF-7 (a, b) and HCT-116 (c, d). (a) and (c) were cell incubatedg concentrations were 0.15 �g/mL, 1.5 �g/mL and 15 �g/mL.

tively. This low cytotoxicity for the drug which does not targetthe cancer cells can be attributed to the solvent effect. The resultsof all the samples were statistically analyzed against ACF (pure)at all concentrations using paired t-test (p value < 0.1), exceptfor ACF-P407 (p value was 0.12). At 24 h incubation time, cellviability was found to be 69.2 ± 4.6% (ACF-PEG6000), 80.8 ± 4.0%(ACF-TW80) and 80.8 ± 2.3% (ACF-P407) at 15 �g/mL, 77.9 ± 3.1%(ACF-PEG6000), 84.4 ± 2.4% (ACF-TW80) and 85.6 ± 2.7% (ACFP407) at 1.5 �g/mL, 88.7 ± 2.3% (ACF-PEG6000), 98.8 ± 3.3% (ACF-TW80) and 95.6 ± 4.9% (ACF-P407) at 0.15 �g/mL. similar to theeffect shown in the result of 12 h study, ACF (pure) exhibitedlow cell cytotoxicity of 90.4 ± 2.2%, 98.5 ± 1.7% and 99.1 ± 0.6%, at15 �g/mL, 1.5 �g/mL and 0.15 �g/mL respectively. The results ofall the samples were statistically compared to ACF (pure) at allconcentrations using paired t-test (p value < 0.1), except for ACF-TW (p value was 0.15) at 15 �g/mL, ACF-TW (p value was 0.88) at0.15 �g/mL, and ACF-P407 (p value was 0.58) at 0.15 �g/mL.

For HCT-116 cells, ACF-PEG6000 exhibited higher cell cytotox-icity than all of the samples for 12 h and 24 h. Similar to the trendobserved in MCF-7 cell line, cell cyototoxicity was increased andthus cell viability was reduced depending on drug concentrations(Fig. 6c,d).

For 12 h incubation time, cell viability for the decorated ACF-NCs was found to be 63.9 ± 2.6% (ACF-PEG6000), 74.4 ± 2.1%

(ACF-TW80) and 74.4 ± 1.5% (ACF P407) at 15 �g/mL, 79.8 ± 3.5%(ACF-PEG6000), 83.3 ± 4.3% (ACF-TW80) and 84.1 ± 1.0% (ACFP407) at 1.5 �g/mL, and 85.8 ± 1.1% (ACF-PEG6000), 87.1 ± 2.9%

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ACF-TW80) and 98.3 ± 5.6% (ACF-P407) at 0.15 �g/mL. ACF (pure)howed low cell cytotoxicity as 92.6 ± 1.3%, 94.7 ± 4.2% and8.4 ± 2.4%, at 15 �g/mL, 1.5 �g/mL and 0.15 �g/mL respectively.he results were statistically compared to ACF (pure) at alloncentrations using paired t-test (p value < 0.1), except for ACF-W (p value was 0.13) at 1.5 �g/mL. Cell viability results after4 h was found to be, 75.7 ± 3.4% (ACF-PEG6000), 73.6 ± 3.5%ACF-TW80) and 78.7 ± 3.3% (ACF-P407) at 15 �g/mL, 79.8 ± 1.1%ACF-PEG6000), 81.3 ± 4.9% (ACF-TW80) and 83.7 ± 2.7% (ACF407) at 1.5 �g/mL, 90.3 ± 5.6% (ACF-PEG6000), 91.1 ± 7.8% (ACF-W80) and 93.0 ± 3.5% (ACF-P407) at 0.15 �g/mL. Results for ACFpure) were exhibited as 92.5 ± 3.8%, 94.3 ± 2.6% and 98.6 ± 3.4%, at5 �g/mL, 1.5 �g/mL and 0.15 �g/mL respectively. The results of allhe samples were statistically compared to ACF (pure) at all con-entrations using paired t-test (p value < 0.1), except for ACF-TW (palue was 0.33) at 15 �g/mL, and ACF-P407 (p value was 0.18) at.15 �g/mL.

As visible from the data, both MCF-7 cells and HCT-116 cellsxhibited similar pattern in cell cytotoxicity as ACF-PEG6000howed the highest cell cytotoxicity, and ACF-P407 showed theowest cell cytotoxicity in all incubation times and drug concen-rations. At Low concentration of 0.15 �g/mL, both ACF (pure) andCF-NCs showed approximately similar low cell cytotoxicity. How-ver, at increasing drug concentration, cytotoxicity for ACF-NCere higher than ACF (pure) as the difference in the cytotoxic-

ty was significant. Dubey et al. [31,32] have reported that folateunctionalized BSA NPs have shown increased difference in cellularytotoxicity with increasing. Martinez et al., [33] also has providedhe similar result for Tamoxifen-loaded thiolated alginate-albuminanoparticles. Interestingly, In MCF-7 cells (breast cancer cell line),CF-NCs have exhibited higher cell cytotoxicity than in HCT-116ells (colon cancer cell line). Furthermore, upon 12 h incubation,ytotoxicity was higher than that of at 24 h. These results indicatedhat drug release has an impact on cell growth. In fact, the resultndicates that fast drug release leads to a stronger cytotoxicity thanlow drug release at initial times.

. Conclusion

ACF-NC were successfully prepared by nanoprecipitation withween 80, Poloxamer 407, or PEG 6000. The decorated ACF-NC had

mean particle size of approximately 0.725 nm. The solubility ofecorated ACF-NC were significantly enhanced compared to ACF inW and pH 6.8 buffer. Cytotoxicity was enhanced in comparisonith pure drug. Decorated ACF-NCs showed high cell cytotoxicity

t 15 �g/mL of drug concentration. Further investigations into ACF-C parameters, such as size reduction and surface modification wille employed to improve drug efficacy and ensure fewer side effectsith enhanced solubility and bioavailability.

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