A pH-sensitive delivery system based on N-succinyl chitosan-ZnO nanoparticles for improving antibacterial and anticancer activities of curcumin

Journal Pre-proof A pH-sensitive delivery system based on N-succinyl chitosanZnO nanoparticles for improving antibacterial and anticancer activities of curcumin

Seyed-Behnam Ghaffari, Mohammad-Hossein Maryam Salami, M.Reza Khorramizadeh

Sarrafzadeh,

PII:

S0141-8130(19)39790-9

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.02.141

Reference:

BIOMAC 14758

To appear in:

International Journal of Biological Macromolecules

Received date:

28 November 2019

Revised date:

12 February 2020

Accepted date:

13 February 2020

Please cite this article as: S.-B. Ghaffari, M.-H. Sarrafzadeh, M. Salami, et al., A pHsensitive delivery system based on N-succinyl chitosan-ZnO nanoparticles for improving antibacterial and anticancer activities of curcumin, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/j.ijbiomac.2020.02.141

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© 2020 Published by Elsevier.

Journal Pre-proof

A pH-sensitive delivery system based on N-succinyl chitosan-ZnO nanoparticles for improving antibacterial and anticancer activities of curcumin

Seyed-Behnam Ghaffaria, Mohammad-Hossein Sarrafzadeha*, Maryam Salamib, M.Reza

of

Khorramizadehc,d

School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran.

b

Transport Laboratory Phenomena (TPL), Department of Food Science and Technology, College

-p

ro

a

re

of Agriculture and Natural Resources, University of Tehran, Karaj, Iran. c

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Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical

Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran. d

na

Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences

ur

Institute, Tehran University of Medical Sciences, Tehran, Iran.

*

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Corresponding author:

Dr. Mohammad-Hossein Sarrafzadeh Associate Professor: School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran P.O. Box 11155-4563, Tehran, IRAN Tel: +98-21-61112185 Fax: +98-21-66957784 E-mail address: [email protected]

E-mail address of the other authors: Dr. Seyed-Behnam Ghaffari: [email protected] Dr. Maryam Salami: [email protected] Dr. M.Reza Khorramizadeh: [email protected]

1

Journal Pre-proof Abstract Inherent selective cytotoxicity, antibacterial activity and unique physicochemical properties of ZnO nanostructures and chitosan (CS) make them promising candidates for drug delivery. In this study, ZnO nanoparticles functionalized by N-succinyl chitosan as a pH-sensitive delivery system were synthesized to enhance the therapeutic potential of curcumin (CUR). CS coated-ZnO nanoparticles were synthesized by a co-precipitation

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method in the presence of CS. Chemical modification of CS-ZnO particles was

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performed by succinic anhydride for introducing –COOH functional groups which were

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then activated using 1,1′-carbonyldiimidazole for CUR conjugation. The spherical-like CUR-conjugated system (CUR-CS-ZnO) with the average particle size of 40 nm

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presented significantly enhanced water dispersibility versus free CUR. The experimental

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study of CUR release from the system showed a pH-sensitive release profile, which

na

enabled drug delivery to tumors and infection sites. MTT and Annexin-V FITC/PI assays revealed the superior anticancer activity of CUR-CS-ZnO compared to free CUR against

ur

breast cancer cells (MDA-MB-231) by inducing the apoptotic response with no cytotoxic

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effects on HEK293 normal cells. Moreover, CUR conjugation to the system notably dropped the MIC (25 to 50-fold) and MBC values (10 to 40-fold) against S. aureus and E. coli. The features qualify the formulation for anticancer and antimicrobial applications in the future.

Keywords: Zinc oxide nanostructures, Chitosan, stimuli-responsive drug delivery systems.

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Journal Pre-proof 1. Introduction

Curcumin (CUR), a hydrophobic polyphenol, is the active ingredient of turmeric, which has relieved significant pharmacological activities mainly related to its anti-oxidant and anti-inflammatory properties. The most studied therapeutic activity of the CUR over past decades is the anticancer effects. Extensive evidence verifies the utility of CUR in cancer

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treatment as a chemopreventive, a chemosensitizer and a Chemoprotector for normal

ro

cells [1-2]. CUR cans suppress all three main phases of carcinogenesis: initiation,

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promotion, and progression. The preferential anti-tumor activities of CUR are primarily mediated via its up-regulation and down-regulation of diverse molecular targets including

re

transcription factors (i.e. NF-κB, AP-1, STAT), enzymes (i.e. COX-2, iNOS, PLA2),

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growth factors (i.e. TGF-α, EGFR, HER), inflammatory cytokines (i.e. TNF-α, IL-1β and

na

IL-6), receptors and protein kinases (i.e. AK, FAK, CDPK) [3-4]. Why CUR kills cancer cells and not normal cells is not completely understood, but several reasons have been

ur

suggested by researchers. Higher CUR cellular uptake and lower the glutathione level in

reasons [5].

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tumor cells and expressed constitutively active NF-κB in them are among the suggested

Numerous investigations have also reported the broad-spectrum antimicrobial effects of CUR against bacterial, viral and fungal [6-9]. Furthermore, CUR also showed noticeable activity against bacterial when applied at a subinhibitory dose in combination with different other antibiotics [10-12]. Antibacterial activity of curcumin is also very valuable for cancer treatment. Invasive infections in patients with cancer throughout their illness stay one of the most challenging problems, which can finally be lethal [13]. Thus, due to

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Journal Pre-proof the extensive anticancer and antimicrobial activities of CUR and also non-toxic effect at active doses (12 g/day orally [14]), CUR was considered for the development of the innovative anticancer and antimicrobial agents. However, there are some serious challenges to use CUR as an antibiotic or anticancer agent. Most importantly, CUR is sparingly soluble in water, resulting in poor bioavailability and rapid metabolism [12]. With the advent of nanotechnology, various nanoscale carries have been developed which

ro

from the rapid clearance and decomposition [15-17].

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have the ability to chemically conjugate or physically load the drugs and protect them

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Besides various organic carries, metal and metal oxide nanoparticles have recently

re

attracted attention [18-22]. Such carriers have advantages such as the ability for easy physicochemical manipulation to make the preferred features for therapeutic or

lP

diagnostic goals. Among all the metal and metal oxide nanoparticles, inherent

na

antimicrobial and selective anticancer activities of zinc oxide nanoparticles (ZnO NPs) together with their other biomedical potentials (including imaging and photo dynamic

ur

therapy (PDT)) and unique properties such as appropriate biocompatibility and easy

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synthesis make them promising applicants as antimicrobials or anticancer agents [23-24]. ZnO NPs have been revealed to have a wide range of antibacterial effects against both Gram-positive and Gram-negative bacteria [25-27]. Several researchers have also studied the preferential anticancer activities of various ZnO nanostructures [28-29]. These activities against both bacterial and turmeric cells are mainly attributed to the ability of ZnO NPs to generate reactive oxygen species (ROS), zinc-mediated protein activity disequilibrium and their capability to release zinc ions [23, 30]. A suggested mechanism behind the preferential cytotoxicity of ZnO NPs is the greater ROS generation in rapidly

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Journal Pre-proof proliferating cells including cancer cells. The generation of more ROS by ZnO NPs, resulting in huge oxidative stress that can finally kill the cells [24]. However, for such biological applications, the surface of ZnO NPs must be modified to improve their poor water dispersibility and introduce the nanoparticles the ability to attach drugs or probes [31]. Among various compounds, chitosan (CS), a hydrophilic polysaccharide, possess great biodegradability, appropriate biocompatibility and low

of

toxicity [32-35]. Furthermore, CS has chemical functional groups (amino and hydroxyl

ro

groups), making it a worthy choice to use as a stabilizer and a linker to attach different

-p

drugs to a metal oxide core [36-39]. CS also has a wide-ranging antimicrobial spectrum

re

to gram-negative and gram-positive bacteria and fungi [40-41], which make it a candidate to be used in antibacterial systems. The combination of ZnO and CS and co-utilization of

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the antibacterial activity of both compounds were investigated by several researchers [42-

na

45]. Therefore, the modification of ZnO through CS will provide an opportunity to enhance the antimicrobial activity of ZnO via synergistic effects.

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In this study, to improve targeting, bioavailability, and anticancer and antibacterial

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activity of curcumin (CUR), an innovative functionalized pH-sensitive delivery system composed of ZnO and CS was designed. Stimuli-responsive drug delivery systems can target drugs or release their host molecules in the needed site in response to endogenous (such as pH) or exogenous stimuli [46]. Being pH-sensitive enabled drug delivery to the acidic environment of tumors and infection sites. Because of specific stimuli response, stimuli-responsive drug delivery systems can regulate drug release, so as to progress the therapeutic outcomes, lower the damage of healthy tissues, reduce adverse side effects of conventional anti-tumor or antibacterial agents, and enhanced patient comfort [47]. We

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Journal Pre-proof modified CS by succinic anhydride to form N-succinyl CS that can react with various types of drugs including CUR because of having –COOH groups in its structure. Nsuccinyl CS has beneficial features including improved aqueous solubility and long-term retention in the body as a drug delivery vehicle [48]. The physicochemical characteristics and the CUR release behavior of CUR-conjugated N-succinyl CS-ZnO were studied and the formulation was examined for the anticancer and antibacterial properties using breast

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cancer cells (MDA-MB-231) and two gram-positive and gram-negative bacteria

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(Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli)), respectively.

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2. Material and methods

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2.1. Chemicals, cell lines and cell culture

Zinc chloride, sodium hydroxide, 1, 1’-carbonyldiimidazole (CDI), methanol, and

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curcumin (CUR) were purchased from Merck Chemical Co. Dried dimethyl sulfoxide

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(DMSO) and acetic acid were purchased from Ameretat Shimi Chemical Co., Iran. Chitosan (low molecular weight) and succinic anhydride were supplied by Sigma Aldrich Chemical Co. MDA-MB-231 breast cancer and Human Embryonic Kidney (HEK-293) cell lines (Pasteur Institute, Tehran, Iran) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillinstreptomycin and mentioned in a humidified 5% CO2 chamber at 37°C.

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Journal Pre-proof 2.2. Synthesis of CS-ZnO and N-succinyl CS-ZnO

Chitosan (CS) coated ZnO was synthesized by a co-precipitation method using zinc chloride as the zinc source in the presence of CS. 1 g of zinc chloride and 0.1 g of CS were dissolved in 50 mL of 1% acetic acid. Next, 2 M NaOH aqueous solution was added drop by drop until the solution attained pH 11. After 24 h stirring at room temperature,

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the produced particles were washed several times with distilled water and then re-

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suspended in 50 mL of methanol containing 5% acetic acid. This formulation was

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considered as CS-ZnO. The procedure without the presence of CS was also carried out.

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The product was considered as ZnO.

To synthesis N-succinyl CS-ZnO, 0.1 g of succinic anhydride dissolved in methanol was

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added to the solution and stirred for 24 h at room temperature. The obtained particles

na

were centrifuged, washed by methanol and re-dispersed in dry DMSO. This formulation

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was considered as N-succinyl CS-ZnO.

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2.3. Conjugation of curcumin to N-succinyl chitosan-ZnO

0.5 gr 1, 1’-Carbonyldiimidazole (CDI) was added slowly to the previously prepared suspension to activate the carboxyl groups on the surface of N-succinyl CS-ZnO particles. After refluxing for 2 h, the powders were separated and re-dispersed in DMSO. Subsequently, 0.2 gr CUR was added to the solution. The suspension was continuously stirred for 24 h at room temperature. The as-synthesized product, named as CUR-CSZnO, separated by centrifugation, washed frequently with DMSO to eliminate unattached

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Journal Pre-proof CUR and ultimately dried at 65 °C overnight.

2.4. Characterization of the products

An X-ray diffractometer (XRD; Panalytical, X’ Pert Pro) using copper Kα1 (λ=1.54056 Å) radiation was used for the phase characterization of the samples. The particle size and

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morphology of the particles were evaluated by a transmission electron microscope (TEM;

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Zeiss-EM10C-100 kV) and a field-emission scanning electron microscope equipped with

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an EDS energy dispersive spectroscopy (SEM; TESKAN MIRA3).

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The hydrodynamic diameter (HD) of the particles was analyzed by dynamic light scattering (DLS; ZetaPlus, Brookhaven Instruments). DLS test preparation contained the

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dispersion of the particles in distilled water (0.5 mg/mL) and subsequent sonication for

na

18 min (bath sonication, 100 w). To study the effect of the cell culture and serum on the HD of the particles, they were dispersed first in DMED+10% FBS, stirred for 2 h and

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subsequently re-dispersed in water.

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Fourier Transform Infrared (FTIR) spectra were taken by a Perkin Elmer FT-IR Spectrophotometer by KBr pellets in the region between 400 and 4000 cm-1. Ultraviolet– visible

(UV–Vis)

spectra

were

scanned

via

an

OPTIZEN

2120UV

plus

spectrophotometer in the range of 200–600 nm. Thermo gravimetric analysis and differential scanning calorimetry (TGA-DSC, TA Q600) were taken under Ar atmosphere at a heating rate of 10 ◦C/min.

2.5. Assessment of curcumin content in CUR-CS-ZnO

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Journal Pre-proof

The standard calibration curve of CUR in DMSO was prepared by measuring the absorption intensity using UV-Vis spectroscopy at 430 nm. The concentration of CUR in CUR-CS-ZnO particles was calculated by determining the content of free CUR (nonconjugated CUR) in the supernatant of the CUR conjugation procedure. The amount of conjugated CUR and the efficiency of conjugation were estimated via the formula:

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Amount of CUR conjugated = Total amount of CUR used – the amount of CUR in the

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supernatant

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Efficiency of conjugation (%) = [(weight of the conjugated CUR in the formulation) /

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2.6. Sedimentation study

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(weight of CUR used)] 100

The sedimentation rate of CUR-CS-ZnO particles was examined through measuring the

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optical absorbance (at 400 nm) as a function of time by UV–Vis spectrophotometry [31].

2.7. In vitro curcumin release study and kinetics analysis

In vitro CUR release from the CUR-CS-ZnO particles was studied at 37 °C in buffer solutions with two pH (PBS+ 0.1% W/V Tween-80, pH 7.4 and pH 5.2) under shaking (150 rpm) [49]. 2 mg of the test sample was dispersed in 3 mL of the buffer solution. At predetermined intervals, the particles were centrifuged (12000 rpm for 20 min) and the particles were re-dispersed by the fresh buffer. CUR concentration in the supernatant was

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Journal Pre-proof measured by UV–Vis spectroscopy based on the absorption intensity at 430 nm. The release kinetics of CUR from the CUR-CS-ZnO under both neutral and acidic buffer solutions were further studied by fitting the release data into five kinetic models: zero-order, first-order, Higuchi, Korsmeyer–Peppas and Hixson–Crowell [49].

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2.8. In vitro cytotoxicity assay

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Cytotoxicity activity of the free CUR, N-succinyl CS-ZnO and CUR-CS-ZnO particles

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was evaluated by determining the cell viability of the cell lines using the MTT reagent based colorimetric assay [31]. In brief, cells (MDA-MB-231 or HEK-293) were cultured

re

into 96 well plates at a density of 104 cells/well in 2 mL DMEM medium and incubated

lP

overnight. Then, the cells were treated with the desired concentrations of samples for 48

na

h. The dispersion of N-succinyl CS-ZnO and CUR-CS-ZnO samples in the cell culture was done by a bath sonicator for 15 min. Moreover, DMSO was used as the co-solvent of

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CUR. The maximum DMSO percentage in DMEM was 0.5% (v/v). 0.5% DMSO

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exhibited an absorbance value similar to that of the untreated cells showing that 0.5% DMSO has no cytotoxic activity on the cells. After 48 h post-treatment, the particlescontaining medium was replaced with 20 μL of MTT reagent (3-(4,5-dimethylthiazolyl)2,5-diphenyltetrazolium bromide) (Sigma Aldrich) dissolved in PBS (4 mg/mL). Then, the cells were incubated for 3 h at 37°C. The supernatant was then replaced with 60 μL of DMSO to dissolve the formed formazan crystals. The color intensity was measured at 570 nm with background subtracted at 660 nm by a microplate reader (Anthos, Austria). The absorption is directly related to the number of viable cells in the culture media. The

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Journal Pre-proof results were stated as percentage viability compared to the untreated cells as the negative control. The data presented are from three replicates.

2.9. Cell apoptosis assay

Cell apoptotic was studied using Annexin-V FITC/PI staining assay by flow cytometry

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[50]. Briefly, MDA-MB-231 cells were cultured in 6-well culture plates (1×105

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cells/well) for 24 h. Then, the cells were incubated with free CUR and CUR-CS-ZnO at

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two different equivalent CUR concentrations (3 and 5 μg/mL) for 48 h. The treated cells were then washed with PBS, harvested and centrifuged. The cells were resuspended in

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100 µl of binding buffer and 5 µl of PI/Annexin V-FITC solution was added and mixed at

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room temperature in the dark. Following incubation for 15 min, the cells were diluted

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with 100 µl of binding buffer. The apoptosis rate was calculated by a BD Accuri C6 flow

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cytometer and the results were investigated via the flow cytometer software.

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2.10. Determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC)

The MIC and MBC of free CUR, CS, ZnO Np (Sigma-Aldrich, particle size <100 nm) and CUR-CS-ZnO were determined using a broth susceptibility test method against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) [49]. In brief, the test tubes contained 1 ml Muller-Hinton (MH) broth with ~1×106 bacterial cells with a specific concentration of each antibacterial sample, were incubated aerobically at 37°C

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Journal Pre-proof for 24 h. Different concentrations of the samples (0.5 to 1000 μg/ml) were made through the two-fold serial dilution. The concentration that did not permit any visible growth of the bacterial cells was considered as the MIC. To measure the MBC, 10 μL of bacterial suspension from each test tube showing no apparent growth was sub-cultured and incubated on an MH agar plate for another 24 h at 37°C under aerobic conditions. The

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concentration of the sample with no growth was taken as the MBC.

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2.11. Statistical analysis

All data were processed by SPSS Statistics 22.0 and illustrated as mean ± SD for

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triplicates. Statistical analyses were performed using unpaired two-tailed Student’s t-test

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to find the significant differences. The levels of P < 0.05 (*) and P < 0.01 (**) were

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defined to be statistically significant and highly significant, respectively.

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3. Results and discussion

In the current study, with the goal of producing a system for aqueous delivery of CUR, a simple chemical technique was designed where a Zinc oxide core was precipitated in the presence of CS. After the preparation of the CS-ZnO formulation, CS chains were modified by succinic anhydride to form N-succinyl CS. ZnO crystals were precipitated in a zinc-containing chloride medium using adding NaOH solution as the neutralizing agent. Adjusting the pH value in the range of 10–12 is essential to the direct formation of ZnO crystals instead of the creation of a precursor (Zn5(OH)8Cl2. (H2O)). The comprehensive

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Journal Pre-proof formation mechanism has been argued elsewhere [51]. Due to the ability of NH2 and OH groups of CS to form coordination bonds with metal ions [52], the stable composite between ZnO particles and CS was created in the reaction environment. Among various hypothesis for the complex formation between CS and metal ions, two models have been experimentally confirmed: Coordination of metal ions to one NH2 or OH groups of CS

groups of one or more CS (bridge structure) [53].

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(pendant structure), and coordination of metal ions to two or more NH2 and/or OH

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The X-ray diffraction (XRD) analysis was done to confirm the formation of the ZnO

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phase and the study of CS structure after the attachment to ZnO crystals and modification

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with succinic anhydride. Fig. 1a demonstrates the XRD patterns of the test samples obtained from the CUR-CS-ZnO synthesis procedure. CS pattern shows two distinct

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peaks at around 10° and 20° arising for its orthorhombic crystalline structure. The certain

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structural regularity appeared probably due to the strong intermolecular hydrogen bonds

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between NH2 or OH groups of CS [54]. In the case of CS-ZnO, both peaks related to crystalline regions of CS disappeared and all the diffraction peaks were in good agreement with the JCPDS file of hexagonal wurtzite zinc oxide (JCPDS #36-1451). The coordination of zinc ions to hydroxyl and amino groups might result in the destruction of the intermolecular hydrogen bonds. Interestingly, a sharp peak at around 7° and a number of short ones appeared in the N-succinyl CS-ZnO diffraction profile, indicating the formation of the structural regularity.

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Journal Pre-proof In the XRD pattern of free CUR, several peaks were seen in the range of 10–30◦ inferring its crystalline nature. However, after CUR conjugation, no such crystalline peaks can be observed. In the DSC (Fig. 1b) analysis, Free CUR exhibited an endothermic peak at a temperature around 180 ◦ C, because of the crystalline nature of CUR, whereas the delivery system (N-Succinyl CS-ZnO) and CUR-CS-ZnO displayed no peak in this region. Thus, the XRD and DSC results showed the amorphous or disordered-crystalline

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nature of the conjugated CUR.

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FTIR spectroscopy as an analytical technique was performed to study the chemical

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structure of the synthesized products in the steps of synthesis procedure stages of CUR-

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CS-ZnO. FTIR spectra of ZnO (the formation of ZnO was evidenced by XRD, data not

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shown), CS, CS-ZnO, N-succinyl CS-ZnO, and CUR-CS-ZnO along with free CUR were illustrated Fig. 2.

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For ZnO, the peak at 423 cm-1 is assigned to the Zn-O band. This characteristic is observed in all ZnO-based formulations (at 423 to 467 cm-1) indicating the presence of

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ZnO in the structures. The peak at 1398 cm-1 is related to the C-OH bonding. Moreover, a

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broad peak can be clearly seen at 3400 cm-1 due to the presence of -OH groups. CS spectrum indicated characteristic bands at 3453 cm-1 due to NH2 and ν(O-H), at 2865 cm-1 related to ν(CH3, CH2, CH, NH), at 1656 cm-1 attributed to ν(C=O), at 1599 cm-1 correspond to the scissoring vibration of -NH2, at 1382 cm-1 due to ν(COO) and at 1092 cm-1 associated to ν(C-O) [54-55]. The FTIR spectrum of CS-ZnO provides evidence for the incorporation of CS in the sample by the observation of the characteristic peaks of CS with peak position shifting at 2922, 1650, 1572, 1409 and 1083 cm-1. After the modification with succinyl anhydride, the intensity of the peak at 1409 cm-1

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Journal Pre-proof corresponding to symmetric stretching of the COO groups increased greatly and shifted to 1400 cm-1. Moreover, the peak at 1572 cm-1 disappeared and a sharp peak assigned to amide II appeared at 1556 cm-1. These two bands provide evidence of N-succinyl CSZnO formation [55]. The modification was carried out by substituting the hydrogen of amino groups of CS by succinyl groups. In the designed system, CUR molecules were covalently bonded to the surface of N-

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succinyl CS-ZnO particles via a chemical reaction between the carboxylic groups of the

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succinic bonded molecules to CS and the hydroxylic groups of CUR molecules (ester

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formation). The direct conversion of a carboxylic acid group to an ester is a challenging process and it would be significantly simpler if the carboxylic groups are first activated

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by an activating agent (in this study 1,1’-carbonyldiimidazole (CDI)). By the activation

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process, a reactive intermediate, N-acylimidazole, is created, which can then react with a

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hydroxyl group to form the ester linkage [56]. CUR can also bond to the surface of ZnO NPs via metal ion-ligand coordination (the formation of a chelate ring with ZnO) [57].

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However, our evaluations by FTIR and UV-Vis indicated that the amount of CUR-loaded

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by physical attachments or the mentioned chelate complex formation was not remarkable (data not shown).

Successful CUR conjugation was evidenced by FTIR. The FTIR spectrum of free CUR exhibited peaks at 1630, 1507, 1427, 1277 and 963 cm-1 corresponding to C=C symmetric aromatic ring stretching, C=O of the benzene ring, C–H bending vibration, aromatic C–O stretching vibrations and C–O–C stretching vibrations, respectively. The spectrum of CUR-CS-ZnO showed peaks related to N-succinyl CS-ZnO as well as CUR

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Journal Pre-proof characteristic peaks; especially the main peak at 1507 cm-1, attributed to C=O of the benzene ring. The concentration of CUR in the formulation was estimated at about 130 μg per mg of the complex (about %13 w/w of the formulation). The efficiency of conjugation was found to be 69.6%. The EDS analysis was also performed on the synthesized products in the steps of the

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synthesis procedure stages of CUR-CS-ZnO (shown in table 1). Although hydrogen is not

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present in EDS results, the results are helpful for a better interpretation during the

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synthesis procedure steps. The EDS analysis results revealed a decrease in the proportion

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of zinc (from 49.3% in ZnO to 26.1% in the CUR-CS-ZnO) and an increase in the proportion of carbon (from 3.9% in ZnO to 28.2% in the CUR-CS-ZnO) after each step

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that confirmed the bonding of the organic molecules. The presence of nitrogen also

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indicated the presence of CS in the formulations. TGA analysis of free CUR, CS and the products in the synthesis steps of CUR-CS-ZnO

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particles was investigated up to 800°C as shown in Fig. 3. It can be seen from the results

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that ZnO particles show small weight loss probably due to the removal of absorbed water. The thermal degradation of CS was a two-step degradation with a rapid weight loss (about 40%) happens between 260∘C and 340∘C due to the degradation of CS [58]. At the end of 800∘C, the total weight loss was ~65%. The composite of ZnO and CS (CSZnO) also exhibited two steps of weight loss, with about ~20% weight loss up to 800∘C. According to the CS and CS-ZnO TGA results, ~21% of CS gets loaded on the ZnO particles. The total weight loss increased after the modification with succinyl anhydride to ~32%.

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Journal Pre-proof TGA result for free CUR, with a total weight loss of ~83%, presented a relatively rapid weight loss in the range of 240-420°C that may be related to the degradation of CUR. The CUR-conjugated sample (CUR-CS-ZnO) did not show the weight loss with a high rate at the mentioned temperature range (occurred at a higher temperature range between 400∘C and 600∘C) indicating that CUR conjugation enhanced the thermal stability of free CUR probably due to the covalent bonding. At the end of 800∘C, the total weight loss

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was ~41%. Based on the TGA results, the estimated CUR content in the complex was

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about 11% w: w which is reasonably close to the measured 13% w: w by the chemical

-p

method.

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The light absorption behavior of free CUR and CUR-CS-ZnO was studied in methanol.

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CUR has an absorption maximum in the wavelength range of 340–535 nm, based on the

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used solvent and CUR structural modifications [59]. According to Fig. 4, the spectrum of free CUR exhibited a strong maximum at 430 nm. The spectra of CUR-CS-ZnO

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formulation demonstrated two absorption maxima at 358 nm related to the characteristic

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absorption edge of the ZnO crystal, and 480 nm, corresponding to the adsorption behavior of CUR. The appearance of CUR characteristic absorption maximum in the formulation obviously showed the successful CUR conjugation to the N-succinyl CSZnO particles. The observed red-shift of the absorption maxima of CUR could be related to the covalent bonding of CUR molecules to the particles. It is remarkable that the efficacy of the most drug or antibiotic delivery systems and the inherent activity of nanoparticles are directly correlated to particle size. Nanoscale structures are materials with at least one dimension in the nanometer scale range (1–100 nm). However, for nanomedical potential applications, the favored particle size is less 17

Journal Pre-proof than 200 nm [60]. The smaller particles, the greater specific surface areas and the higher possibility of being in touch with and passing across the cell membrane [61]. The smaller particles (<~200 nm but >~10 nm) are also desirable for transporting an anticancer compound to the site of action due to the enhanced circulation time and the benefits of the passive targeting strategy [62]. Typical SEM images and TEM micrographs of CURCS-ZnO particles are presented in Fig, 5. Based on both microscopic images, the

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particles have a spherical-like shape. The average diameter size of CUR-CS-ZnO

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particles was about 40 nm, according to the TEM images of > 100 particles and 50 nm,

-p

based on SEM images. Therefore, the formulation can be considered as nanoparticles.

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SEM and TEM give the size of particles in dried form and do not appropriately predict the size of particles in body fluids. Agglomeration of particles in liquids increases the

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efficient volume of the particles. Moreover, the attachment of molecules in body fluids

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including proteins and lipids can additionally increase the size of the particles and consequently affect their size-dependent properties [63]. Therefore, the assessment of the

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particle sizes in solutions, especially in serum-containing culture media, as a model for

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body fluids, is critical.

DLS is a valuable method to assess particle size and size distribution in solutions. In the present study, DLS together with the sedimentation study was carried out to evaluate the size and stability of CS-ZnO, N-succinyl CS-ZnO and CUR-CS-ZnO particles (Fig. 6). The Average hydrodynamic size of CS-ZnO particles was 140 nm in de-ionized water. After the modification with succinyl anhydride, the hydrodynamic size decreased to 77 nm with a narrower dispersity index. The decrease in hydrodynamic size also accompanied by zeta potential alteration. The zeta potential of CS-ZnO particles was

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Journal Pre-proof −7.5± 0.8 mV while it converted to -26.1 ± 1.35 mV after the modification, which can be related to the negative charge of the carboxyl groups in water [49]. The higher surface charge of N-succinyl CS-ZnO particles allows lower agglomeration phenomenon and thus better stability of particles in water, compared to CS-ZnO particles. According to the sedimentation study results (Fig. 6c), coating with CS and afterward the modification with succinyl anhydride enhanced the water dispersibility of ZnO particles using inducing

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steric and electrostatic stabilization. Regarding ZnO, about 54 and 64% of the particles

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precipitated after 24 and 48 h in water, respectively. Synthesis of ZnO NPs in the

-p

presence of CS results in a reduction in the sedimentation rate. The mentioned values reduced to 42 and 49%, respectively. After modifying with succinyl anhydride, only 17%

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of the particles precipitated within 24 h. This value was 23% after 48 h.

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The hydrodynamic size of CUR-CS-ZnO particles dispersed in de-ionized water was

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approximately 130 nm. Obtained larger size, compared to the mean particle size estimated from TEM results, indicated the agglomeration of the particles. As it was

ur

shown, N-succinyl CS coating of ZnO particles can reduce the particle agglomeration

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phenomenon. However, CUR conjugation can again increase the agglomeration of the particles because of the hydrophobic nature of CUR and the reduction of zeta potential of the particles (to -16± 1.1 mV) [31]. Based on the sedimentation study results, approximately 29 and 35% of the particles precipitated after 24 and 48 h in water, respectively. The attachment of serum molecules such as proteins to the nanoparticles (formation of protein corona) can affect the hydrodynamic sizes of particles and consequently their agglomeration trend. A decrease was observed in the hydrodynamic size (to 75 nm) when the particles were firstly suspended in DMEM supplemented with

19

Journal Pre-proof 10% FBS. The bonded layer decreased the agglomeration behavior of the particles owing to the steric and electrostatic stabilization. The obtained hydrodynamic size is within the reported efficient size range for the goal of passive targeting to tumor cells (<200 nm but >10 nm) [62]. The particles in DMEM + 10% FBS displayed a significantly slower deposition rate than those in water. Almost only 13% of the particles deposited after 24 h. This value was 17%

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after 48 h. Thus, the pre-dispersion of the particles in serum-containing solutions might be

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a stabilization strategy for biological studies. Considering the fact that free CUR

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precipitates immediately in aqueous solutions, CUR conjugation to the designed delivery

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system significantly enhanced water dispersibility of CUR. To evaluate the time-specific and pH-dependent release profile of CUR from the

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formulation, the particles were incubated in buffers with different pH (PBS with pH=7.4

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and pH=5.2, mimicking the physiological and extracellular acidic environment of tumor conditions or infection sites, respectively) at 37C. Then the percentage of CUR was

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monitored as a function of time (shown in Fig. 7). There was a rapid release (burst

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release) under both pH conditions during the first hour, followed by a slow release. The initial rapid release occurs by desorption of CUR molecules which are weakly bound to the surface of the particles. Moreover, the CUR release from CUR-CS-ZnO particles was significantly pH-dependent and CUR released at a remarkably increased pace under mildly acidic conditions. In neutral pH, 20% of CUR molecules were released within 1 h and after a 16 h period, about 60% of CUR was not released. This showed the efficiency of the CUR-conjugation strategy to diminish the amount of CUR loss to the blood circulation.

20

Journal Pre-proof In contrast to that, in the acidic buffer, 45% of CUR molecules were liberated after the first hour and only about 5% of CUR remained after 16 h. This release behavior at acidic pHs would be contributed to the degradation of CS [64], the hydrolysis of esters (reverse of esterification which is catalyzed by acidic situations) to the parent carboxylic acid and the alcohol groups [65] and the breaking of the chelate complex between CUR and ZnO [57]. This result is in good agreement with other studies mentioned the pH-dependent

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swelling and controlled drug release properties of CS [66-67].

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The release profiles were fitted to the five kinetic models for a better understanding of the

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release mechanisms of CUR. The highest correlation coefficient (R2) value was

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considered as the best fitted model. In neutral pH, mathematical modeling was performed for the fraction of CUR released within 8 h. After 8 h, no significant CUR released. The

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best fit was observed in the case of first order and Korsmeyer-Peppas models (R2=0.98)

na

indicating that CUR release rate depends on its concentration. Calculated ‘n’ value in Korsmeyer-Peppas equation was 0.3 which is lower than 0.45 showing that the release

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mechanism can be described as a quasi Fickian diffusion mechanism [49]. In acidic pH,

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the best fit during the first hour (burst release) was seen in the case of zero order model (R2=0.99). Zero order model describes the procedure of constant drug release from a delivery system (independent of CUR concentration). The correlation coefficient of Korsmeyer-Peppas equation was also satisfactory (R2=0.98). The estimated ‘n’ value was 0.88 indicating case-2 relaxation or non-Fickian diffusion mechanism which involves a mixture of erosion and diffusion mechanisms. The best fit during the next stage after burst release was again the first order and Korsmeyer-Peppas models (R2=0.98) with the ‘n’ value of 0.25, indicating a quasi Fickian diffusion mechanism.

21

Journal Pre-proof The pH-sensitive drug release is considered as a smart strategy for drug delivery and targeting in cancer therapy and bacterial infection. In solid tumors, the cells metabolize glucose abnormally, resulting in producing lactic acids. Acid transfer to the extracellular fluid leads to the alteration of pH that is valuable for pH-sensitive nanocarriers [68]. Same as the cancer cells, some bacteria, such as S. aureus, are known to produce acidity at infection sites. Therefore, recently the potential of pH-sensitive drug-

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delivery systems for the treatment of infections that are associated with localized acidity

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has been studied. [69-71].

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Conjugation of drug molecules to the delivery system particles might change their

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functionality because of the engaging of the main therapeutic groups of CUR [72]. Therefore, it is essential to evaluate the anticancer effects of the drug-conjugated

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formulation. In order to evaluate the anticancer activity of the formulation, we used the

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MTT assay and flow cytometry to determine cell cytotoxicity and apoptosis against the MDA-MB-231 breast cancer cells. MTT assay for 48 h demonstrated a dose-dependent

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effect of free CUR, N-Succinyl CS-ZnO and CUR-CS-ZnO on cancer cells. A relatively

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sharp decrease in the viability of the cells is obvious in MTT results in Fig. 8. The halfmaximal inhibitory concentration value (IC50: the concentration of drug which relived 50% cell viability) was calculated as 5.0, 40.1 and 64.2 µg/mL for free CUR, CUR-CSZnO and N-Succinyl CS-ZnO treatment, respectively. The anticancer activity of CUR-CS-ZnO could be compared with that of free CUR based on the concentration of CUR. The IC50 value of CUR-CS-ZnO, measured from the CUR concentration base curve, was about 5.2 µg/mL, which is approximately equal to that of free CUR. In other words, the therapeutic potential of CUR has been protected during the

22

Journal Pre-proof conjugation procedure presented in this study. The preferential cytotoxic activity of all samples was further investigated using HEK293 normal cells. MTT results confirmed that, with concentrations up to 100 µg/mL, no significant cytotoxic activity was observed. Therefore, the anticancer agent (CUR), the designed delivery system (N-Succinyl CSZnO) and the CUR-conjugated formulation (CUR-CS-ZnO) can be considered as a selective anticancer agent.

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The possible mechanism of death (apoptosis and necrosis) of the MDA-MB-231 cells

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after exposure to free Cur and CUR-CS-ZnO was further investigated through Annexin-

-p

V/PI assay. Necrosis and apoptosis are commonly regarded as the two main types of cell

re

death with different features in incidence, mechanisms, and morphology. Apoptosis as a programmed process is considered to be a regulated and controlled procedure. Moreover,

lP

most of the resistances that are seen in cancer cell lines are owing to apoptotic-related cell

na

death. Therefore, its occurrence during cancer treatment has received great attention [73]. Moreover, the inherent anti-apoptotic mechanisms of diverse types of cancer cells

ur

regularly need aggressive treatments, limiting the effectiveness of the therapy.

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Preferential induction of apoptosis would be also beneficial for therapeutic purposes which are sensitive to inflammation that can occur owing to uncontrolled leakage of cell content [74]. Induction of apoptosis in cancer cell lines by ZnO NPs and CUR treatment was frequently reported in the literature [75-76]. Increased expression of pro-apoptotic and the inhibition of anti-apoptotic proteins were also observed in in-vitro studies [77]. Fig. 9 represents the flow cytogram of MDA-MB-231 cells after treatment at the equivalent CUR concentrations of 3 and 5 µg/mL for 48 h incubation. Moreover, the percentage of the four regions (viable, necrotic, early apoptosis and late apoptosis) was

23

Journal Pre-proof illustrated in Fig. 10. After 48 h incubation, 99.7% of untreated cells were viable (annexin V- PI-). Treatment of the cells by both free Cur and especially CUR-CS-ZnO particles resulted in a significant decreased viable cell percentage. Treatment by CURCS-ZnO restricted the viable percent to merely <5%. Furthermore, the viable percent reduced in a dose-dependent manner with increasing the CUR concentration from 3 to 5 g/mL (~75 to 53%, 55 to 9% and 5 to 2% for free CUR and CUR-CS-ZnO,

of

respectively).

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The flow cytometry results stablished the induction of apoptosis by free CUR and CUR-

-p

CS-ZnO in MDA-MB-231 cells. Interestingly, remarkable induction of apoptosis happens

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even at the moderately toxic equivalent CUR concentration (3 g/mL) after treatment by CUR-CS-ZnO particles. The percentages of total apoptosis, the sum of early (annexin V+

lP

PI-) and late apoptosis (annexin V+ PI+), were ~24 and 90% after the exposure of the

na

cells by free CUR and CUR-CS-ZnO, respectively (CUR concentration of 3 g/mL). Apoptosis does not go through lysis bodies, due to the engulfment of apoptotic cells by

ur

scavengers such as macrophages. In the in vitro studies, where clearance by phagocytes

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does not operate, the created apoptotic bodies will eventually swell and the apoptotic process proceeds to an autolytic necrotic outcome. Therefore, the term late apoptosis or secondary necrosis is applied to explain the cells which have reached the mentioned state by the progression of apoptosis [78]. An increase in the total apoptosis proportion was obtained with increasing of the CUR concentration to 5 g/mL (~44 and 96% for free CUR and CUR-CS-ZnO, respectively). Additionally, no notable late necrosis (annexin VPI+) induction was observed for the treated cells. As the results show, the designed formulation induced apoptosis more than free CUR at the same CUR concentrations.

24

Journal Pre-proof The antibacterial activity of free CUR, ZnO NPs, and CUR-CS-ZnO NPs was evaluated against S. aureus and E. coli by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) values. S. aureus and E. coli (grampositive and gram-negative bacteria, respectively) are among pathogens with serious clinical effects on cancer patients [79]. Cancer treatment, in addition to the illness itself, can suppress the immune system, which makes the cancer patients susceptible to invasive

of

bacterial infections which can ultimately be fatal [80]. Free CUR, CS, ZnO NPs, and

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CUR-CS-ZnO NPs showed the antibacterial effect against both bacteria (Table 2).

-p

Although CUR is only about 13% w: w of CUR-CS-ZnO NPs, the formulation was found to have meaningfully higher antibacterial activity than free CUR, CS and ZnO NPs.

lP

with free CUR and ZnO NPs.

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Significant differences were found in MIC and MBC values of the formulation compared

na

In comparison with free CUR, the MIC value reduced 25-fold (250 to 10 µg/mL) and 50fold (250 to 5 µg/mL) against S. aureus and E. coli, respectively. The MIC value reduced

ur

25-fold against both bacteria compared to ZnO NPs. Furthermore, the combined activity

Jo

of CUR, ZnO, and CS resulted in a 10-fold (500 to 50 µg/mL against S. aureus) and a 40fold (1000 to 25 µg/mL against E. coli) reduction in MBC value, compared with free CUR. The formulation also exhibited a 20-fold reduction in the MBC value against the two bacteria, compared with ZnO NPs. Therefore, CUR combined with ZnO and CS exerts the superior antibacterial activity probably due to the synergistic effects.

25

Journal Pre-proof 4. Conclusion During recent years, hybrid or composite biomaterials containing polymers, metal or metal oxide nanoparticles, and drugs have largely been developed. Their outstanding features do not only rely on the individual components but also on their synergistic effects. Considering the unique biomedical potentials of ZnO and chitosan (CS), a pHresponsive delivery system composed of ZnO nanoparticles functionalized by N-succinyl

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chitosan was successfully prepared for the delivery of curcumin (CUR). CUR

ro

conjugation to the system was performed to improve the bioavailability, targeting and

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therapeutic applicability of CUR as the main obstacle for its biomedical applications. The physicochemical properties of the conjugate product were then characterized, and its

re

anticancer and antibacterial activities were assessed using the MDA-MB-231 breast

lP

cancer cells, S. aureus and E. coli. The CUR-conjugated system showed superior toxicity

na

effect against breast cancer cells, with no effect on normal cells (HEK 293) by inducing the apoptotic response. Furthermore, the formulation remarkably lowered the MIC (25 to

ur

50-fold) and the MBC values (10 to 40-fold) against S. aureus and E. coli compared to

Jo

free CUR. The experimental study of CUR release from the system showed a pHsensitive release profile, which enabled CUR delivery to the acidic environment of tumors and infection sites. All the findings showed that the engineered delivery system can be considered as a promising multi-functional delivery candidate to maximize the delivery of drugs and enhance the anticancer and antibacterial activity of agents. The system also has the potential to utilize other ZnO biomedical capacities such as imaging and PDT therapy that are presently in progress at our lab.

26

Journal Pre-proof Acknowledgment

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Table 1 EDS analysis results of the obtained products in the steps of synthesis procedure stages of CUR-CS-ZnO. Zn (W%)

O (W%)

C (W%)

N (W%)

ZnO

49.3

46.8

3.9

0

CS-ZnO

46.4

45.0

N-succinyl CS-ZnO

32.7

CUR-CS-ZnO

26.1

2.5

52.9

11.9

2.3

28.2

4.5

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6.1

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Sample

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42.0

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Journal Pre-proof Table 2 MIC and MBC of free CUR, CS, ZnO NPs and CUR-CS-ZnO against S. aureus and E. coli.

Staphylococcus aureus

Escherichia coli

MIC (µg/mL)

MBC (µg/mL)

MIC (µg/mL)

MBC (µg/mL)

Free CUR

250

500

250

1000

CS

500

1000

250

1000

ZnO NPs

250

1000

125

500

CUR-CS-ZnO

10

50

5

25

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Sample

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Journal Pre-proof Figures captions:

Fig. 1 (A) XRD patterns of chitosan (CS), CS-ZnO, N-succinyl CS-ZnO, CUR-CS-ZnO and free curcumin powders. (B) DSC results of N-succinyl CS-ZnO, CUR-CS-ZnO and free curcumin under atmosphere of Ar.

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Fig. 2 FTIR spectra of the obtained products in the steps of synthesis procedure stages of

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CUR-CS-ZnO along with free curcumin, chitosan (CS) and ZnO powders.

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Fig. 3 TGA curves of ZnO, CS-ZnO, N-Succinyl CS-ZnO, free CUR and CUR-CS-ZnO

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under the atmosphere of Ar (up to 800∘C).

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Fig. 4 UV-Visible spectra of free CUR and CUR-CS-ZnO.

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Fig. 5 (A and B) FE-SEM image of CUR-CS-ZnO particles. (C and D) TEM images of

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CUR-CS-ZnO particles.

Fig. 6 (A) Dynamic light scattering (DLS) measurements of CS-ZnO and N-Succinyl CSZnO particles dispersed in water after sonication. (B) DLS result of CUR-CS-ZnO particles dispersed in water and the DLS result when they were firstly dispersed in DMEM + 10% FBS and then separated and re-dispersed in water. (C) Sedimentation curves of the obtained products in the steps of synthesis procedure stages of CUR-CSZnO in water along with CUR-CS-ZnO particles DMEM + 10% FBS, vertical axis

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Journal Pre-proof declared with regard to optical absorbance (using UV–Vis spectrophotometry at 400 nm) at time t (At) relative to the value at t = 0 (A0).

Fig. 7 In vitro release profile of CUR from CUR-CS-ZnO particles in PBS (pH=7.4 and 5.2).

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Fig. 8 (a) Viability of MDA-MB-231 breast cancer cells (MTT assay) after 48 h exposure

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to free CUR. (b) Viability of normal HEK293 cells after 48 h treatment with free CUR.

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(c) Viability of MDA-MB-231 and normal HEK293 cells after 48 h treatment by NSuccinyl CS-ZnO. (d) Viability of MDA-MB-231 and normal HEK293 cells after 48 h

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treatment by CUR-CS-ZnO particles. Data represented as the means ± SD of three

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identical experiments derived from three replicate. (*) p < 0.05 and (**) p < 0.01

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expression in the formulations treated cells vs. free CUR treated cells.

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Fig. 9 Flow-cytometric analysis of MDA-MB-231 cells representing apoptosis assay based

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on Annexin V-FITC and PI staining assay after the treatment with free CUR and CUR-CSZnO particles at equivalent CUR concentration of 3 and 5 g/mL.

Fig. 10 Proportion of the four regions of the results of the Annexin V-FITC and PI staining assay. Data derived from three replicate.

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CRediT author statement

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Seyed-Behnam Ghaffari: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing- Original draft preparation. Mohammad-Hossein Sarrafzadeh.: Resources, Conceptualization, Project administration, Supervision, Funding acquisition, Writing - Review & Editing. Maryam Salami: Formal analysis, Investigation, Writing Review & Editing. M-Reza Khorramizadeh: Supervision, Conceptualization, Methodology.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10