Bovine serum albumin: An efficient biomacromolecule nanocarrier for improving the therapeutic efficacy of chrysin

Accepted Manuscript Bovine serum albumin: An efficient biomacromolecule nanocarrier for improve therapeutic efficacy of chrysin

Hamed Nosrati, Marziyeh Salehiabar, Saeed Afroogh, Akram Rakhshbahar, Hamidreza Kheiri Manjili, Hossein Danafar, Soodabeh Davaran PII: DOI: Reference:

S0167-7322(18)30845-6 doi:10.1016/j.molliq.2018.06.066 MOLLIQ 9264

To appear in:

Journal of Molecular Liquids

Received date: Revised date: Accepted date:

15 February 2018 1 May 2018 17 June 2018

Please cite this article as: Hamed Nosrati, Marziyeh Salehiabar, Saeed Afroogh, Akram Rakhshbahar, Hamidreza Kheiri Manjili, Hossein Danafar, Soodabeh Davaran , Bovine serum albumin: An efficient biomacromolecule nanocarrier for improve therapeutic efficacy of chrysin. Molliq (2018), doi:10.1016/j.molliq.2018.06.066

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ACCEPTED MANUSCRIPT Bovine Serum Albumin: An efficient biomacromolecule nanocarrier for improve therapeutic efficacy of chrysin

Hamed Nosrati 1,2, Marziyeh Salehiabar 1, Saeed Afroogh 1, Akram Rakhshbahar 1, Hamidreza Kheiri Manjili 3, Hossein Danafar 2, 4*, Soodabeh Davaran 5*

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1- Zanjan Pharmaceutical Nanotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.

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2- Department of pharmaceutical biomaterials, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran. 3- Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.

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4- Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.

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5- Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

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*Corresponding Author's E-mail: [email protected] and [email protected]

Abstract

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This study manipulated a chrysin loaded bovine serum albumin nanoparticles (BSA NPs),

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which could solubilize the poorly water-soluble drug and increase the therapeutic efficacy of the drug. Chrysin (5, 7-dihydroxyflavone) is a natural flavonoid which have some significant biological effects on the processes of chemical defense. Chrysin loaded bovine serum

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albumin nanoparticles (Chrysin-BSA NPs) were synthesized by a simple desolvation procedure. The resultant Chrysin-BSA NPs showed a spherical shape, with a diameter of

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97.5±5.75 nm (mean ± SD) nm and a ζ-potential of - 11 mV. The in vitro drug release study of chrysin presented a sustained and controlled release pattern. Cellular toxicity of BSA NPs was also investigated on HFF2 cell lines. Additionally, a hemolysis test of as prepared NPs were performed. Hemolysis assay and cytotoxicity study results on HFF-2 cell line show that as prepared BSA NPs are biocompatible. The in vitro cytotoxicity of the nanoparticles were performed by MTT assay on MCF-7 cancer cells. These results suggest that Chrysin-BSA NPs are a new drug delivery system for cancer therapy.

Keywords: Albumin, BSA, Drug delivery, Cancer, Chrysin, Protein

ACCEPTED MANUSCRIPT 1. Introduction

At this time, over than 100 new natural products are in clinical progress, mostly for anticancer activities [1-3]. Chrysin (5, 7-dihydroxyflavone) is a natural flavonoid extracted from medicinal herbs [4]. Chrysin is a flavonoid which has been testified to have some significant biological effects on the processes of chemical defense, nitrogen fixation,

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inflammation, and oxidation [5]. However, the low solubility decreases its bioavailability and consequently disrupts the medical and pharmaceutical benefits [6, 7].

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Nanoparticulate systems are widely studied as a drug delivery approach in the biomedical and pharmaceutical research [8-14]. Among the common categories of biomolecules used for

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drug delivery [15-17], albumin protein has attracted consideration of scientists for its selective delivery capabilities, biodegradable, nontoxic, metabolized in vivo to produce safe

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degradation products, easy to purify and soluble in water, and non-immunogenicity [18-21]. Also, albumin-based drug delivery systems represent an attractive approach, since a

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noteworthy amount of drug can be combined into the particle matrix due to the different drug binding sites present in the albumin molecule [22, 23]. Human and bovine serum albumins (HSA and BSA) are the main available albumins widely used in the biopharmaceutical

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applications [24-28]. Bovine serum albumin (BSA), having a molecular weight of 69,323 Da

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and an isoelectric point (pI) of 4.7 in water (at 25 °C), is broadly used for drug delivery due to its medical status, richness, low cost, ease of purification, and its wide acceptance in the biomedical industry [29-31]. Furthermore, albumin nanoparticles can be simply synthesized

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under mild conditions by coacervation, controlled desolvation or emulsion formation [29]. In the coacervation process, a desolvation agent such as acetone or ethanol was added to the

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aqueous solution of albumin under a constant stirring condition until the solution became turbid. After that, glutaraldehyde or EDC as cross-linkers must be added in order to stabilize the nanoparticles by crosslinking of amino acids residues in the protein [32-34]. Previously, our research group reported a simple improved desolvation method for the rapid preparation of albumin nanoparticles and also, several factors of the preparation process were evaluated [34]. In the present investigation, with optimal conditions in hand, the drug carrier capability of optimized albumin nanoparticles were studied by design, synthesis and characterization of a chrysin loaded bovine serum albumin nanoparticles (Chrysin-BSA NPs). In this study, a novel chrysin delivery carrier with BSA NPs were synthesized and the release profile of the drug from the nanoparticles were evaluated. The application of BSA NPs as carriers of

ACCEPTED MANUSCRIPT chrysin were evaluated by measuring its drug content, encapsulation rate and in vitro anticancer activities.

2. Materials and methods

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2.1. Materials

Bovine serum albumin (BSA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC),

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RPMI1640, 3-(4,5-dimethylthialzol-a-yl)-2,5-diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS), penicillin, streptomycin and trypsin-EDTA were purchased from

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Sigma-Aldrich Co. (St Louis, MO, USA). PBS was prepared in our laboratory. Dimethyl

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sulfoxide (DMSO) and acetone were purchased from Mojallali Company, Iran.

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2.2. Synthesis of Chrysin loaded BSA NPs (Chrysin-BSA NPs)

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BSA NPs and Chrysin-BSA NPs were prepared using desolvation method as reported before [35]. As shown in Fig. 1. 0.2 g BSA was added to 3.2 mL deionized water and kept at room temperature for 15 min under vigorous stirring. Then, 6.4 mL chrysin solution (12 mg chrysin

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in 1 mL acetone) was added to above solution drop wisely at a rate of 2 mL/min under constant stirring at room temperature in dark conditions. For stabilization and cross linking of the BSA NPs, EDC (4 mg) was added to above solution. The stirring condition was continued

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for 2 h to ensure the cross linking of amino acid residues. The resulting chrysin loaded BSA NPs (Chrysin-BSA NPs) were cleansed by three cycles of centrifugation at 18,000 rpm for 15 min and washed with deionized water three times and then dried in a vacuum oven overnight. BSA NPs were synthesized using the same method with acetone instead of chrysin solution.

2.3. Characterization of nanoparticles

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Particle size and zeta potential evaluation

The size distribution and ζ-potential of Chrysin-BSA NPs were measured by dynamic lightscattering method (DLS) using the nano/zetasizer (Malvern Instruments, Worcestershire, UK, model Nano ZS). The samples were diluted with distilled water and measured at room

were used for this analysis. Each sample was done in triplicate.

Morphology study by Atomic Force microscopy (AFM) and scanning

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2.3.2.

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temperature. An aqueous solution (0.1 mg in 20 mL deionized water) of Chrysin-BSA NPs

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electron microscopy (SEM)

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The morphology of the nanoparticles was observed by using an atomic force microscopy (AFM) (JPK, Berlin, Germany, model Nano Wizard). An aqueous solution (0.1 mg in 20 mL

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deionized water) of Chrysin-BSA NPs were used for these analysis. One droplet of sample was placed onto a freshly cleaved mica substrate (1 cm2) and air-dried. AFM measurements were done in intermittent contact mode. Also, the morphology of samples were confirmed

2.3.3.

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using scanning electron microscopy (SEM; Vega Tescan, Czech Republic).

Differential scanning calorimetry (DSC)

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The thermal properties of chrysin, BSA, and the freeze-dried powder of Chrysin-BSA NPs were investigated utilizing a DSC apparatus (Mettler Toledo, model Star SW 9.30,

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Schwerzenbach, Switzerland). The scanning temperature for each powder sample was set from 30 °C to 350 °C (rate of 15 °C min−1).

2.3.4.

FTIR analysis

The chemical structure of samples were known by Fourier transform infrared spectroscopy (FT-IR) (Bruker, Tensor 27). Standard disks were collected by mixing 2 mg of selected samples with 200 mg of KBr medium by grinding to a fine powder and then compressing the resulting powder into transparent pellets (pressure, 12 Ton). The FTIR spectra of the KBr

ACCEPTED MANUSCRIPT disks were recorded using the aforementioned instrument from 400 to 4000 cm-1. Each spectrum was obtained using 16 scans.

2.4. Entrapment efficiency (EE) and drug loading (DL)

To assess the loading efficiency of the chrysin in the Chrysin-BSA NPs the drug loading ratio

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was appraised. For determination of the drug loading ratio, 2 mg of the final freeze-dried Chrysin-BSA NPs

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were first suspended in 2 mL acetone, followed by sonication for 10 min. The samples were shaken in a shaker incubator at 37 °C for 24 h, after which samples were removed from the

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shaker incubator and centrifugation at 18,000 rpm for 30 min to allow the nanocarriers to settle.

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The supernatant was collected and were diluted, then the chrysin content was measured using UV-visible spectroscopy (Thermo Fisher Scientific, USA, Madison, model GENESYS-TM

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10S) at 348 nm. The measurement was performed at least three times. Drug loading (DL) was

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calculated as follows:

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Where weight of drug in nanoparticles is weight of the encapsulated drug and weight of nanoparticles is the total weight of the corresponding drug-encapsulated nanoparticles. DL%

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is the drug loading ratio (%).

2.5. In vitro drug release study

The in vitro drug release of chrysin from nanoparticles was find out in phosphate buffer saline (PBS, 0.15 M, pH 7.4 and 5.8) containing 2% (v/v) tween 80. 2 mg/mL of the ChrysinBSA NPs were transported to a dialysis tube (molecular cut off = 12 000). The dialysis tube

ACCEPTED MANUSCRIPT was submerged fully in 20 mL of the release medium and kept in an incubator at 37 °C under shaking at 100 rpm. At predetermined time intervals, 2 mL of the release medium was reserved and replaced with an equal fresh release medium to keep sink conditions. The amount of chrysin was detected by UV-Vis. The results of triplicate evaluations were used to calculate cumulative drug release.

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2.6.Hemolysis assay

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Since the BSA NPs are to be used for biomedical applications, the issue of cytotoxicity has to be addressed. In order to investigate the hemocompatibility, the in vitro hemolysis assay was

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accomplished [18]. Briefly, 1 mL of the human red blood cells (HRBCs) HRBCs were obtained via removing the serum from the human blood via centrifugation at 3000 rpm for 10

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min, then washed four times with sterile PBS solution. The HRBCs were diluted with PBS solution and 0.5 mL of the mixture was mixed with 0.5 mL of sterile deionized water as a

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positive control, 0.5 mL of PBS as a negative control and 0.5 mL of nanocarrier suspensions at concentration of 10 mg/mL. The samples were shaken in a shaker incubator at 37 ᵒC for 4 h. Finally, the samples were centrifuged at 13000 rpm for 15 min, the supernatant was taken,

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and its absorbance was measured by Eppendorf Bio Photometer (λ = 540 nm). The

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percentage hemolysis was calculated using the following relationship:

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Where A is absorbance. Whereas, A negative, and A

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are the absorbance of the negative

control and the positive control samples, respectively. The percentage of hemolysis was determined based on the average of three replicates.

2.7. Cell culture

2.7.1.

Cell line and cell culture

ACCEPTED MANUSCRIPT To assess the toxicity and anti-tumor activity of Chrysin-BSA NPs, the potential formulations were examined against cancer tumor cell lines including human breast cancer cell lines (MCF-7) of that acquired from Pasteur Institute (Tehran, Iran). The study was performed using MTT assay. Also, the toxicity of BSA NPs were examined against normal cell lines including human fibroblast cell lines (HFF-2) of that acquired from Pasteur Institute (Tehran, Iran).

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RPMI-1640 (GIBCO, USA) containing 10% Fetal Bovine Serum (FBS) (Gibco, Germany), 2 mM L-glutamine, penicillin (50 IU/mL) and streptomycin (50 lg/mL) were selected as cell cultured medium, and incubated at 37 °C in a humidified incubator with 5% CO2

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atmosphere. Harvest of cells were don with 0.5 g/L trypsin (Gibco Laboratories) and 0.2 g/L

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EDTA (Gibco Laboratories) after 75% confluence for 3 min (min) in 37 °C. Also, the

Cytotoxicity assay on HFF-2 cells and MCF-7 cells

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2.7.2.

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concentration of the cells in the culture flasks was attuned to allow for exponential growth.

96 well plates were used, to complete the MTT assay, and cells were seeded onto plates and allowed to adhere and grow overnight in 200 µL medium with 3 × 104 cells per well. The

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cells were then incubated with fresh medium including various concentrations (30–480 µM)

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of void chrysin and equivalent doses of Chrysin-BSA NPs and BSA NPs in the solutions form for 96 h and a group for control. Then the MTT solution at proper concentrations (20 µL of 4 mg/mL MTT solution in each 100 µL media) was added to each well and the plates

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were then incubated at 37 °C for 3 h. Next, the remaining MTT solution was take out and 100 µL of DMSO was added to each well to dissolve the formazan crystals. To establish

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acceptable solubility, the plates were shaken for 10 min on a plate shaker. Absorbance readings of each well were done at 570 nm (single wavelength) using a multi scan plate reader made in the UK. All tests were done in triplicate and repeated twice. Results were expressed as mean ± S.D.

3. Results and discussion

ACCEPTED MANUSCRIPT Bovine serum albumin (BSA) has been extensively used as a delivery carrier for drug and diagnostic agents. In this study, Chrysin-BSA NPs were successfully synthesized by the desolvation method, which can be done simply with a narrow particle size distribution and polydispersity index (PI) less than 0.18. In previously study of our research group many important factors, such as the concentration of BSA, amount of acetone, acetone addition speed and so on, were optimized to reach a perfect drug delivery system [34]. Now, with

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optimal conditions in hand, the drug carrier capability of optimized BSA NPs were studied.

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3.1. Characterization of size and morphology

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The formation of BSA NPs and Chrysin-BSA NPs were convinced by AFM. In fact, ChrysinBSA NPs exhibited a globular shape and homogeneous spherical morphology, and the

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average size of these nanoparticles was ~ 97.5±5.75 nm (mean ± SD) (Fig. 1a,b). AFM images of BSA NPs (as control) was compared with Chrysin-BSA NPs (drug-loaded NPs) to make a proper document for success loading of chrysin in BSA NPs. BSA NPs exhibited a

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globular shape and homogeneous spherical morphology, and the average size of these nanoparticles was ~ 36.5±3.63 nm (mean ± SD) (Fig. 1c). Fig. 2 shows illustrative SEM

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image of Chrysin-BSA NPs. The morphology observed for Chrysin-BSA NPs reveals a low degree of agglomeration. Also, the size of Chrysin-BSA NPs were measured by dynamic

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light scattering system (DLS). As shown in Fig. 3, the hydrodynamic size of Chrysin-BSA NPs were found to be about 127 nm with their corresponding PDI being 0.18 and ζ-potential of -11 mV.

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The surface charge can mainly affect the stability of the nanoparticles distribution. Surface charge is important in decisive whether the nanoparticles will cluster in blood flow or will

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stick to or interact with oppositely charged cell membrane. The plasma and blood cells constantly had a negative charge; NPs with slight negative surface charge can minimize nonspecific contact with these components through electrostatic interactions [36]. The loading capacity of Chrysin-BSA NPs were calculated to be 2.09 ± 0.41.

Fig. 2.

Fig. 3.

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3.1.FTIR analysis

So as to confirm the components of Chrysin-BSA NPs, the FTIR spectrum analysis was used to give us some visions about the compositions of pure BSA, and Chrysin-BSA NPs. Fig. 4 compares the FTIR spectra of pure BSA, and Chrysin-BSA NPs. The characteristic

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adsorption peaks of BSA at 1644 and 1525 cm−1 were attributed to flexural vibration adsorption of Amide I ( ̶ NH2) and Amide II ( ̶ NH ̶ ), respectively [37, 38].

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FTIR analysis of chrysin revealed peaks at: 1609 cm−1 and 1494 cm−1 due to C=C and C=O

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of benzene ring, respectively, 1462 cm−1 due to olefinic C–H bending vibration, 1358 cm−1 due to aromatic C–O stretching vibrations, and 1026 cm−1 due to C–O–C stretching

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vibrations.

As shown in Fig. 4, all the characteristic absorption peaks of BSA and chrysin could be found in the FTIR spectra of the Chrysin-BSA NPs. FTIR spectrum of the Chrysin-BSA NPs

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confirmed peaks related to chrysin, at: 1420 cm−1 due to C=O of benzene ring, 1156 cm−1 due to aromatic C–O stretching vibrations, and 1023 cm−1 due to C–O–C stretching vibrations, in

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addition to BSA related peaks, at: a strong peak 1644 and 1525 cm−1 which are assigned to the Amide I ( ̶ NH2) and Amide II ( ̶ NH ̶ ). Apparently, the FTIR spectrums provided that

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both BSA and chrysin existed in the protein nanoparticles.

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Fig. 4.

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3.2.Differential scanning calorimetry (DSC)

DSC thermograms of BSA, Chrysin and Chrysin-BSA NPs were gained as shown in Fig. 5. The thermal behavior, i.e. endothermic peak of each sample, was evaluated as a response parameter. Chrysin was characterized by the existence of endothermic peak at 288.73°C and BSA at 173.53°C. While the Chrysin-BSA NPs system showed only single peak at 202.53°C where characteristic endothermic peak of Chrysin was absent and ensure molecular binding. It is commonly preferred that the drug in the formulation is amorphous, resulting in better dissolution, absorption and bioavailability. As shown in Fig. 5, the chrysin powders showed a sharp melting peak at 288.73°C, indicating the crystalline state of chrysin. However, the peak

ACCEPTED MANUSCRIPT disappeared in the thermogram of Chrysin-BSA NPs, revealing that chrysin was not in the crystalline state after encapsulation into the nanoparticles. This result indicated the stability of the obtained drug delivery system.

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Fig. 5.

3.3.In vitro drug release study

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Fig. 6 shows the in vitro release behaviour of the Chrysin-BSA NPs over 168 h in phosphate

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buffered solution (PBS, 0.15 M, pH 7.4 and 5.8) containing 2% (v/v) tween 80. In the pH= 7.4, the Chrysin-BSA NPs pursued a generally sustained and controlled release, with an

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initial release of 20 % in 10 h, an ensuing release of 35 % in 96 h, and a plateau level was arrived after 120 h. In contrast, in the pH= 7.4, the Chrysin-BSA NPs pursued a generally sustained and controlled release, with an initial release of 20 % in 10 h, an ensuing release of

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57 % in 96 h, and a plateau level was arrived after 120 h. This is due to initially chrysin adsorbed on the BSA surface released, and then chrysin encapsulated in BSA NPs released

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gently. This controlled release phenomenon is derived from the erosion and degradation of BSA. As shown in Fig. 6, drug release was pH dependent and increased in acidic medium.

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Meanwhile the microenvironment at tumor places in vivo and inside endosome/lysosome of cells is acid (pH = 4.5–6.0) whereas the bloodstream is neutral, the favorable chrysin release

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at acid medium can decrease the unwanted danger to normal cells during the intravenously

Fig. 6.

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injected nanoparticles are circulated in vivo.

3.4.Hemocompatibility

To determine the biocompatibility of the BSA NPs, and Chrysin-BSA NPs the nanoparticles should have blood compatibility with the minimal value of hemolytic effect on the HRBCs. Hemolysis assay was useful to estimate the blood adaptability of nanoparticles. As expected, negligible effect of nanoparticles is clear in the experiment concentration. The hemolytic activity of the specimens was further quantitatively specified by evaluating the supernatant

ACCEPTED MANUSCRIPT absorbance at 540 nm (hemoglobin) with Eppendorf Bio Photometer. At a high concentration, less than 3.2 % hemolytic activity was observed each of the nanoparticles (Fig. 7). Consequently, the negligible hemolytic activity of BSA NPs as a nanocarrier confirmed the great biocompatibility, which are desirable for biological utilization especially in drug delivery.

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Fig. 7.

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3.5.In vitro cytotoxicity

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The cytotoxicity investigation of BSA NPs were performed on HFF2 cells. The cells were incubated with nanoparticles for 96 h with concentrations of from 30, 60, 120, 240, and 480

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μM (Fig. 8a). The blank BSA NPs had no ostensible cytotoxicity on the growth of the two studied cell lines. The MTT test outcomes, proposed that these BSA NPs are highly biocompatible and do not possess a toxic effect. The cytotoxicity profile revealed that the

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blank BSA NPs could be applied as a biocompatible carrier for biomedical applications. In vitro cytotoxicity of free chrysin, BSA NPs and Chrysin-BSA NPs were assessed by MTT

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assays on MCF-7 cells. As shown in Fig. 8b, for MCF-7 cells, the inhibition rate of the Chrysin-BSA NPs increased with an increasing drug concentration from 30 μM to 480 μM.

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Compared with untreated cells, cells exposed to BSA NPs remained unchanged due to their outstanding biocompatibility of BSA NPs. In comparison, when incubated with Chrysin-BSA NPs, the amount of living cells was obviously decreased, which is attributed to the cytotoxic

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effect of chrysin released from the nanoparticles. The data exhibit that cell toxicity is directly

Fig. 8.

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commensurate to chrysin concentration in 96 h period time.

4. Conclusions

In our study, we have developed a novel approach for chrysin delivery with controllable performances. The spherical Chrysin-BSA NPs with a narrow diameter size less than 100 nm were successfully synthesized. In vitro release study confirmed its sustained release features.

ACCEPTED MANUSCRIPT The cytotoxicity of the nanoparticles in two lines of cultured cells, MCF-7 and HFF-2 cells, were studied. Blank BSA NPs were biocompatible with no obvious cytotoxicity on HFF-2 cells. Following 96 h in cell culture medium, Chrysin-BSA NPs showed a similar reduction in cellular viability in MCF-7 cells compared to the void of chrysin. Therefore, the prepared Chrysin-BSA NPs are a promising drug delivery system for chrysin and other hydrophobic drugs.

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ACKNOWLEDGEMENT

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This work was supported financially by the Faculty of Pharmacy, Zanjan University of

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Medical Sciences, Zanjan, Iran. Conflicts of interest

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The authors declare that they have no conflict of interest.

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ACCEPTED MANUSCRIPT Fig. 1. AFM height image and corresponding cross-sectional (a) and AFM phase contrast and corresponding cross-sectional (b) profiles of the Chrysin-BSA NPs. AFM phase contrast and corresponding cross-sectional profile of the BSA NPs (c). Fig. 2. SEM image of the Chrysin-BSA NPs. Fig. 3. The size distributions and ζ-potential of the Chrysin-BSA NPs.

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Fig. 4. FTIR spectrum of BSA NPs (a), Chrysin-BSA NPs (b), and pure Chrysin (c) from 400 to 4000 cm–1.

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Fig. 5. DSC thermograms of BSA (a), pure Chrysin (b), and Chrysin-BSA NPs (c) in the temperature range of 30–350 ◦C.

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Fig. 6. In vitro drug release profiles of Chrysin-BSA NPs in PBS containing 2% (v/v) Tween 80 (pH= 7.4 and 5.8).

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Fig. 7. Photographs of hemolysis of RBCs incubated with BSA NPs, and Chrysin-BSA NPs. Fig. 8. In vitro cytotoxicity of BSA NPs to HFF-2 cells (a) and BSA NPs, Chrysin-BSA NPs,

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and pure Chrysin to MCF-7 cells (b). Each bar represents the mean of five measurements ± SD.

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Fig. 1.

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Fig. 2.

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Fig. 3.

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Fig. 7.

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Fig. 8.

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 The bovine serum albumin nanoparticles were prepared through desolvation process  The resultant Chrysin-BSA NPs showed a spherical shape, with a diameter of 97.5±5.75 nm.  Hemolysis assay and cytotoxicity study results show that BSA NPs are biocompatible