Synthesis, 99mTc-radiolabeling, and biodistribution of new cellulose nanocrystals from Dorema kopetdaghens

Journal Pre-proof Synthesis, 99mTc-radiolabeling, and biodistribution of new cellulose nanocrystals from Dorema kopetdaghens

Elahe Kamelnia, Adeleh Divsalar, Majid Darroudi, Parichehr Yaghmaei, Kayvan Sadri PII:

S0141-8130(19)36084-2

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.179

Reference:

BIOMAC 14207

To appear in:

International Journal of Biological Macromolecules

Received date:

2 August 2019

Revised date:

30 November 2019

Accepted date:

20 December 2019

Please cite this article as: E. Kamelnia, A. Divsalar, M. Darroudi, et al., Synthesis, 99mTc-radiolabeling, and biodistribution of new cellulose nanocrystals from Dorema kopetdaghens, International Journal of Biological Macromolecules(2018), https://doi.org/ 10.1016/j.ijbiomac.2019.12.179

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© 2018 Published by Elsevier.

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Synthesis, 99mTc-radiolabeling, and biodistribution of new cellulose nanocrystals from Dorema kopetdaghens Elahe Kamelniaa, Adeleh Divsalarb, Majid Darroudic,d,* Parichehr Yaghmaeia, Kayvan Sadric,** a

Department of Biology, Faculty of Science, Science and Research Branch, Islamic Azad University,

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Tehran, Iran b

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Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University,

Tehran, Iran

Nuclear Medicine Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

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c

d

Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of

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Medical Sciences, Mashhad, Iran

* Corresponding author at: Department of Modern Sciences and Technologies, School of Medicine, Mashhad

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University of Medical Sciences, Mashhad, Iran. Tel.: +98 513 8002286; fax: +98 513 8002287.

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E-mail addresses: [email protected], [email protected] (M. Darroudi).

*Co-Corresponding author: Nuclear Medicine Research Center, Mashhad University of Medical Sciences,

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Mashhad, Iran. Tel.: +98 513 Tel.: +98 513 8012783; fax: +98 513 8012783. E-mail: [email protected]

[email protected] (09155595867), [email protected] (09126170823), [email protected] (09153064830), [email protected] (09122010222), [email protected] (09151564406)

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Abstract Cellulose nanocrystals (CNCs) are known as nano-biomaterials that can be achieved from the different sources. The designated CNCs have been successfully fabricated from the roots of Dorema kopetdaghens (Dk) plant by sulphuric acid hydrolysis method. Structural analysis has been carried out by the means of XRD, FTIR, and TGA/DTG procedures. The XRD

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results have indicated that the crystalline structure of CNCs had been cellulose I with the

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crystallinity index of 83.20% and size of 4.95 nm. The FTIR spectra have shown that the resulting samples have been related to the cellulose species. The thermal properties of CNCs

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have exhibited a lower thermal stability in comparison to the untreated roots. It has been

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indicated by the morphological analyses of FESEM, TEM, and AFM that the nanoparticles

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had contained a spherical shape. Also, the cytotoxicity of CNCs against A549 cell line has not exhibited any cytotoxic effects. The analysis of labeling efficiency in regards to

99m

Tc-

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CNCs has been observed to be above 98%, while the biodistribution of radioactivity has

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displayed a high uptake by the kidneys and blood circulation. Therefore, it is possible to transform the low-cost by-product into a beneficial substance such as CNCs that can be

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utilized in bioimaging applications. Keywords: Cellulose nanocrystals; technetium-99m; Dorema kopetdaghens

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1. Introduction Cellulose stands as one of the most plentiful substances that is available throughout the world, which can be mostly discovered in plants (e.g., wood, hemp, cotton, linen, and etc.) and several other natural products [1, 2]. This particular material has captured the attention of many due to being biodegradable, renewable, sustainable, non-toxic, and biocompatible, as

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well as being amenable for adjustments [3, 4], which have qualified cellulose for being

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utilized in varying applications related to different research fields. [5-8]. Cellulose is an insoluble polysaccharide that is comprised of linear chains of glucopyranose units, which are

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apparently linked by β-1, 4-glycosidic bond. The crystalline regions of cellulose, defined as

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cellulose nanocrystals (CNCs), have displayed remarkable mechanical and physical-chemical

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properties, which have been intensively and potentially studied to be applied in biomedical industries

[9].

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Currently, the assistance of radioisotopes medical uses has been great throughout the

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diagnosis and treatment of diseases, since radioisotopes can be beneficial for evaluating the biodistribution of molecules in the body. The significance of imaging functionality

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throughout the diagnosis of primary stages of diseases is quite evident [10, 11]. Technetium99m (99mTc) is the most commonly used radionuclide among the diagnostic methods of nuclear medicine, which has been often used for labeling radiopharmaceuticals due to its suitable chemical and physical characteristics and inexpensive isotope cost [12]. The importance of this study lies in obtaining information about the in vivo functions of cellulose nanocrystals and thereafter, produce radiopharmaceutical drugs that could be capable of being utilized as radiotracer in diagnosis and bioimaging applications. Currently, cellulose has attracted the attention of many owing to its outstanding properties and has been

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extensively used in different industries such as medicine, pharmaceuticals, composites etc. [13]. The main components of plant fibers are majorly consisted of cellulose, lignin, and hemicellulose. The existing cellulose (40-50%) in plants, combined with hemicellulose (3020%) and lignin (35- 10%), play essential roles in the strength of cell wall [14]. Lignin is known as an amorphous heteropolymer that contains a complex structure, while Hemicellulose has been observed to be one of the branched multiple polysaccharide polymers

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[15]. As an specific material, Cellulose has been detected to accommodate a general formula

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of (C6H10O5)n. Moreover, each single unit of cellulose chain seems to hold three hydroxyl

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groups that can supply the required reactive platforms for chemical alteration purposes [16,

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17]. Cellulose molecules can arrange themselves as amorphous and crystalline regions [6].

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However, its nano-crystals (CNCs) have been perceived to be the crystalline regions that can be commonly prepared by utilizing the acid hydrolysis (e.g., Hydrochloric, sulfuric, and

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phosphoric acid) of any natural cellulose source at various temperatures, whereas it is

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proceeded with the mechanical or ultrasound disintegration of acid-treated cellulose within

[18-20].

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water in order to disintegrate the aggregates of liberated crystalline cellulose nanoparticles

The width and length of CNCs are mainly dependent on the cellulose source and the available conditions of acid hydrolysis. The common diameter of CNCs has been observed to be around 2–20 nm with a vast range of length distribution that varies from 100 to 600 nm [21, 22]. CNCs are abled to react with fluorophores and various radiolabels for the purpose of constructing nanoscale markers to quantify and localize nanoparticles within a cell [23]. This particular process has become possible due to the large surface area and nonporous structure of CNCs, as well as their accommodation of numerous hydroxyl groups that can be further

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modified with different functionalities [16]. The surface charge and conjugation of noncytotoxic elements, in regards to CNCs, stand as a significant factor for their application in the role of a natural carrier, which can perform bioimaging and drug delivery throughout the designated cell [24]. Recently, many researches have been conducted in an effort to develop and improve imaging techniques. Images can be captured either by measuring the amount of external radiation absorbed or by using a small amount of the radioactive compound and

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detecting the radiation that escapes the body [25].

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In this study, we have illustrated how cellulose, in the form of a nanocrystal, could be utilized

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as an agent throughout the diagnostic and bioimaging processes. We have composed CNCs

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through the usage of Dk plant roots (an endemic species of Persian mountains) and H2SO4

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hydrolysis. Dorema kopetdaghens (Dk) is a plant that grows in the western mountains of Iran, which is used as a traditional medicine in some regions of this country (Fig. 1). The

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synthesized CNCs have been distinguished by structural, morphological, and thermal

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examinations, as well as toxicological characterization. In the following, they have been labeled with Tc-99m for identifying their capability in targeting diagnosis throughout

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imaging, which can function in the role of a biomedical device; this functionality can be considered as a novel approach since it has not been exhibited in any other work. To the best of our knowledge, there are no reports on the plant roots of Dk that could be found in literature, indicating that an original and relatively green technique has been proposed for the fabrication of CNCs.

2. Experimental

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2.1. Materials The plant roots of Dk have been procured from the submontane region of Iran. All of the utilized chemicals such as sodium chlorite, sodium hydroxide, glacial acetic acid, and sulphuric acid (98%) have been obtained from Merck. In order to perform the cytotoxicity assessment, we have cultured the A549 (Human lung adenocarcinoma epithelial cell line) within Dulbecco's medium (with 10% FBS), streptomycin (100 mg/ml), penicillin (100

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μg/ml), and MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide, Sigma99m

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Aldrich, 98%, USA). For carrying out the labeling experiments,

Tc, which is obtained in

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physiological saline as Na99mTcO4, has been eluted from a commercial

99m

Mo/99mTc

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generator (Kimia Pakhsh), Methanol (98%), and the acetone (99%) that had been procured

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from Sigma-Aldrich, Germany. The radionuclide purity has been observed to be >99.99%. We have acquired Ketamine 10% and Xylazin 2% (Merck) for animal studying purposes.

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Double distilled water has been used throughout all of the involved experiments.

2.2. Preparation of CNCs

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Despite a bit of modification, we have composed the CNCs in accordance with the procedure of Moriana et al. and Mounir El Achaby et al.

[26-28]. The roots of Dk have been

thoroughly cleansed by the usage of double distilled water for the purpose of removing the residual material, which have been afterwards mechanically crushed.

2.2.1. Alkaline treatment

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Journal Pre-proof A minor pre-treatment with NaOH 5% w/w at 80 oC for 1 h has been done for removing the non-cellulosic materials, which had been afterwards centrifuged at 4,158 g-force for 5 min at 10 oC for three times.

2.2.2. Acid hydrolysis

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After drying process, the conduction of acid hydrolysis has been carried out at the

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temperature of 45 °C while being mechanically stirred by the utilization of H2SO4 (64 wt.%)

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for 2 h. In the following, we have diluted the suspension through the usage of cold double

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distilled water for terminating the reaction and thus, had it centrifuged at 10,397 g-force for

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the duration of 10 min at 10 °C. This particular operation has been performed in triplicate to remove the existing acid, whereas in each time, the supernatant has been decanted until its

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pH reached a neutral state.

2.2.3. Bleaching treatment

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A subsequent bleaching treatment has been organized for the purpose of detaching the remaining residual lignin, hemicelluloses, and extractives, which resulted in leaving cellulose nanocrystal as the final product. The solution that had been employed in this particular treatment has been composed of NaOH (2.5 g), glacial acetic acid (7.5 ml), and sodium chlorite (1.7% w/v) in 100 ml of double distilled water. We have carried out the bleaching treatment at 80 °C for a period of 3 h while it was being mechanically stirred. Afterwards, the available suspension has been centrifuged in triplicate for the duration of 10 min at 10,397 gforce and the temperature of 10 °C.

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2.2.4. Dialysis and ultra-sonication In due course, the aqueous suspension has been dialyzed against double distilled water for at least 24 h until a constant pH has been attained. As the next step, for the purpose of dispersing nanocrystals, the designated suspension has been positioned within a sonicator bath (Sonorex Digitec, Model: Andelin; 35 kHz, Germany) for the duration of 15-30 min.

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The samples have been kept in a refrigerator that accommodated a temperature of 7 °C, for

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the upcoming stability evaluations and Atomic force microscopy (AFM, JPK instrument AG,

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Stage LS, Olympus IX 71, Germany), as well as being freeze-dried (Alpha 1-4 LD plus,

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Martin Christ, Germany) for performing field emission scanning electron microscopy

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(FESEM, TESCAN MIRA3, Czech Republic), thermogravimetric analysis (TGA/DTA, Mettler Toledo, SDTA 851e model), x-ray diffraction (XRD, D8-AdvanceBruker Cu/Kα;

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λ=15406 nm), fourier transform infrared spectroscopy (FTIR, Avatar 370, Thermo Nicolet,



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USA), and toxicity analyses (Fig. 2).

2.3. CNCs characterization 2.3.1. Chemical composition The chemical composition of untreated Dk and produced CNCs has been determined in accordance with the relevant ASTM standard [ASTM D 1103-55T], hemicellulose (ASTM D 1104-56), and lignin (ASTM D 1106-56).

2.3.2. FTIR spectroscopy

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The functional groups of untreated Dk roots, hydrolysis sample, and CNCs have been analyzed through the utilization of FTIR (FTIR, Avatar 370, Thermo Nicolet, USA) within the infrared region that existed between 400–4000 cm−1, which involved a resolution of 4.0

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cm−1.

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2.3.3. XRD patterns

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We have obtained the XRD outcomes through the usage of a Rigaku Mini Flex II, Japan,

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which functioned at 30 kV and 15 mA. The scanning procedure of specimens has been

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carried out in a step-wise manner throughout the operational range of scattering angle (2θ) that existed between 2 and 90°, along with a step size of 0.02° and the utilization of CuKα

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radiation of λ=1.541Å wavelength. In the following, we have calculated the crystallinity

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index (CrI) of the untreated, hydrolysis and CNCs through the application of an empirical

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method that involved eq. 1, as it has been expressed by Segal [29].

( )

(

)

( )

in which I002 stands for the maximum intensity of crystalline region and Iam represents the lowest intensity of amorphous region in regards to the sample. The crystal size (Dhkl) of the appointed sample has been calculated by the employment of Scherrer’s equation (Eq. 2):

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( )

considering how Dhkl would be the perpendicular crystal dimensions of diffracting planes of miller indices that belong to hkl, λ represents the radiation wavelength (λ=1.5406 A), θ stands for the diffraction angle of radians, 0.9 would be the correction factor of K, and β1/2 is

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the full width of diffraction peak at the half maximum height (FWHM) in prior to the

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smoothing.

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2.3.4. Thermogravimetric analysis

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We have conducted the thermal stability of cellulose nano-crystals through the utilization of a Shimadzu TGA-DTG 50, Japan instrument (TGA, Mettler Toledo, SDTA 851e model).

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Under a heating rate of 10 °C min-1, the samples have been heated starting from room

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2.3.5. Zeta potential

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temperature until reaching 800 °C within an air atmosphere.

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The zeta potential of CNCs has been performed by a Zeta-sizer instrument (Nano ZS, Malvern Instruments, Malvern, UK). In order to steer clear of the effects of multiple scattering, we have diluted the samples by the utilization of double distilled water to function as an appropriate (concentration equalized to 0.005 wt.%), in prior to carrying out the zeta potential measurements. Each of the single measurements have contained an average of 13 runs, while being performed at room temperature.

2.3.6. FESEM and EDX

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Through an accelerating voltage of 20 kV, we have detected the surface morphology of CNCs (Freeze dried powder) by the usage of a FESEM (TESCAN MIRA 3, Czech Republic). In prior to noting down the observations, we have positioned and dried the samples on a metallic substrate and had them coated with gold within an ion sputter, while being under vacuum for the duration of 180 s. For the purpose of determining the elemental composition of freeze-dried samples, we have employed the FESEM that had been supplied

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with Energy Dispersive Spectroscopy (EDS) of X-rays at high magnification and

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2.3.7. Transmission electron microscopy (TEM)

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accommodated an accelerating voltage of 20 kV for the duration of about 60 min.

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The morphology and dimensions of CNCs were assessed using a Zeiss (EM10C -Germany)

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Transmission Electron Microscope operating at 100kV. These images were prepared as follows: The dilute aqueous solution of the CNCs was sonicated for 10 min. Then, a portion

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of solution (20 μl) was dropped onto carbon film on copper grid (Mesh: 300, EMS-USA) and dried thoroughly at room temperature. The dimension of cellulose nanocrystals were

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determined using ImageJ® software.

2.3.8. Atomic force microscopy (AFM) We have investigated the morphology of nano-crystals through the exploit of AFM scanning probe system (JPK instrument AG, Stage LS, Olympus IX 71 (Germany). For the production of the sample, we have positioned a drop of diluted nano-crystals suspension (0.001 wt.%) on the surface of a glass slide and allowed it to dry at room temperature in prior to being analyzed.

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2.3.9. Cytotoxicity assay The cellular compatibility of CNCs samples has been assessed by the utilization of A549 cells (passage 25–30), which have been cultured in Dulbecco’s Modified Medium (DMEM) and incubated at a temperature of 37 °C and 5% CO2. In regards to the cell viability assay, A549 cells have been seeded onto 96-wells plates (ELISA), which accommodated a density of 10,000 cells per well, and have been allowed to adhere overnight. Subsequently, the cells

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have been permitted to face the intensifying concentrations of CNCs after shifting the

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medium and had them incubated (37 °C in 5% CO2) for the duration of 24 h. A blank

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(DMEM without cells) and a positive control sample (DMEM with cells) have been

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evaluated as well, while every single treatment has been analyzed in quadruplicate. We have

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substituted the spent medium with 100 μl of fresh medium and thus, have appended 10 μl of MTT solution (5 mg/mL PBS, sterile) to each well while incubating them for a period of 4 h

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(37 °C, 5% CO2); this procedure resulted in the fabrication of purple formazan crystals. As

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the next step, the obtained crystals have been dissolved through the addition of 100 μl of DMSO to each well subsequent to conveying the medium. The enzymatic reduction of

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yellow tetrazolium MTT to purple formazan has been measured by the application of a Synergy™ HT Multi-mode Microplate Reader (Biotek Instruments, Winooski, VT, USA) at 570 nm. The percentage of cell viability has been determined through the utilization of Eq. (3): ( )

( )

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In which, AExp. stands as the absorbance value of the experiment, ACtrl. represents the absorbance value of blank (DMEM without cells), and A

positive

would be the absorbance

value of positive control (DMEM cells).

2.3.10. 99mTc-CNCs labeling Although it involved a bit of modification, yet the prepared CNCs have been labeled with 99m

99m

Tc-labeled carboxymethyl-cellulose [30]. 500 µl of the stock CNCs (430 µg/ml) has

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for

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Tc-CNCs in the same manner that had been specified by the previously mentioned process

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been appended to 200 µl of stannous chloride dehydrate solution (2 mg/ml in saline solution

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0.10 M HCl) in the role of a reducing agent, while being stirred for the duration of 10 s. 99m

TcO4- (185 MBq / 500 µl) to the appointed

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Subsequently, we have added 5 mCi of

reaction mixture and had it incubated while being mixed for 20 min at room temperature.

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2.3.11. Quality Control

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We have assessed the labeling efficiency of 99mTc-CNCs through the utilization of ITLC-SG chromatography sheets (Agilent Technologies, Santa Clara, CA, USA) in both 85% aqueous

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methanol and Acetone solvent systems [31]. Subsequent to being developed, the ITLC sheets have been dried and cut into three pieces, which have been positioned within the standard RIA tubes in order to perform the radioactive measurement by the usage of a gamma counter (Well Gamma Count Delshid, DL 100). We have enquired into the stability of radiolabel throughout the time intervals of 0, 1, 4, and 24 h.

2.3.12. Cell binding assay

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Journal Pre-proof In the course of cell binding examinations, we have plated the cells at 106 cells/well within a 6-well plate, which has been permitted to adhere for a period of 72 h at 37 °C throughout a humidified atmosphere of 5% CO2 and 95% air. The cells have been cleansed by the means of PBS, while 500 µl of culture medium had been substituted. Five mCi of

99m

Tc-labeled

CNCs [1250 µl stock suspension of CNCs (430 µg/ml)] have been added to each well (n=3) in two concentrations of 30 and 90 µg/ml, which have been later on incubated for 3 h.

99m

Tc-CNCs. For the purpose of removing all the adhesive cells from each

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tubes as bound

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Thereafter, we have removed the cells from each well and poured them in gamma counter

99m

Tc-CNCs. A gamma counter has been used for carrying out the

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tubes as unbound

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well, we have cleansed the cells (triplicate) by the utilization of PBS and gamma counter

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radioactive measurement.

2.3.13. Animal biodistribution and imaging studies

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To evaluate the bio-distribution of

m99

Tc-CNCs in vivo, we have employed 9 white Wistar

male rats (3 groups each with 3 rats) that weighted 200–250 g. The m99Tc-CNCs [7.4 MBq in 200 µl for six rats (1 and 4 h); 1.85 MBq in 200 µl for three rats (24 h) ~ 11 µg/ml] have been administered into the tail vein of the nine designated star, which have been subsequently sacrificed in 1, 4, and 24 h after the injection. Blood has been drawn via the aortal function while the organs of interest (ttsas of stomach, muscle, intestine, lung, and liver, along with the complete pieces of heart, kidney, brain, thyroid, and tail) have been anatomized and dth metrsseh sehgdar adehs. We have considered radioactivity as a well gamma counter in

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regards to

99m

Tc-CNCs .The results have been illustrated hn the mean percentage of

administered dose per gram of tissue, which can be obtained by dividing the accumulated activity of each organ by its weight (%ID/g; Injected Dose per gram organ). In regards to the imaging studies, a sta has been anesthetized by the usage of ketamine and xylene in 6 and 24 h subsequent to the injection procedure. We have captured the static images for 5 min and counts of 250000 with a zoom factor of 2 by planar imaging, through the employment of a

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gamma camera (Siemens Dual head, Germany) that had contained a low energy/high-

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resolution collimator. The outcomes have demonstrated a tetp at 140 keV with ±20%

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window. All of the measurements have been corrected for the background and radioactive

3. Results and discussion

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3.1. Chemical composition

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decay.

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Tab. 1 displays the chemical composition of untreated Dk and produced CNCs. As it can be observed, the untreated Dk has the highest percentage of hemicellulose and lignin, along with

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the lowest percentage of cellulose. This fact indicates the occurrences of substantial breakdown of lignocellulosic structure, partial hydrolysis of the hemicellulose fraction, and depolymerization of the lignin components during the mechanical and chemical treatments. These inducements had given rise to sugars and phenolic compounds which are soluble in water as it has been reported by other authors [32]. The yield of CNCs have been observed to be 17.2%. This value is higher than the ones that are reported in the works of F. Jiang et al. with a yield of 15.7% and C. Coelho et al. with a

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yield of 20.9% [33, 34], while Lu et al,. has reported a yield of 6.4% for the CNCs that were obtained from rice straw cellulose [35]. Many factors can influence the CNCs yield, such as acid concentration, reaction time, and temperature [36].

3.2. FTIR spectroscopic analysis A chemical construction can be distinguished through the employment of FTIR, since this

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method can help in determining the functional groups that exist within each of the available

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samples [37]. The FTIR spectra of untreated, hydrolysis, and CNCs are demonstrated in Fig.

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3. We assume that the apparent absorbance peaks throughout the 3400–3300 cm-1 have been

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assigned to the stretching and bending of the OH groups of cellulose, which is indicative of

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the reflectance that is relative to the hydrophilic property [38]. Fig. 3c illustrates the increase in the intensity of the band in comparison to the untreated Dk, which had been related to the

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elimination of non-cellulosic components. The observed peaks around 2930–2800 cm-1 seem

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to correspond with the stretching vibration of C–H throughout the lignin polysaccharides, which include cellulose and hemicellulose [39, 40]. The decrease in this peak suggests that

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subsequent to performing hydrolysis and bleaching, the non-cellulosic components have been reduced and omitted.

The existing prominent peak at 1732-1730 cm-1 within the spectrum that had been relative to the untreated samples has been attributed either to the C=O stretching that belonged to the acetyl group and uronic ester groups of the hemicellulose or to the ester linkage of the carboxylic group in the ferulic and p-coumaric acids of lignin and/or hemicellulose, which had been removed subsequent to carrying out the various chemical treatments. This fact

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suggests that the existing hemicellulose and lignin have been entirely eradicated from the sample [41, 42]. It is also evident that the varying treatments have almost nearly split the involved ester bond from the non-cellulosic components. The observed characteristic peak at around 1617 cm-1 (H-O-H stretching vibration), which is commonly associated with water absorption, has apparently faced a decrease after the chemical treatments due to the loss of water samples. This change has been due to the larger surface area of CNCs in contrast to the

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untreated and hydrolyzed Dk [43]. Throughout the spectrum of untreated Dk, the detected

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peak at 1507-1508 cm−1 has been correlated with the aromatic C=C stretch of lignin aromatic

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rings. The existing insufficiency of this particular peak in regards to the hydrolyzed samples

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can be assigned to the elimination of lignin that had been caused by acid hydrolysis [44]. Due

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to the eradication of hemicellulose substances, the peak intensity that had been observed at around 1235-1233 cm-1 has faced a sharp reduction subsequent to the treatment. The apparent

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peaks that exist throughout the range of 1200–950 cm−1 have been caused by the C–O

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stretching, whereas the observed peak at 1020 cm−1 has been induced by the C–O–C vibration that had belonged to the pyranose ring skeletal of cellulose [45]. The torsional

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vibration of pyranose ring has originated the other perceptible peaks at around 530 cm-1 [32]. It had been confirmed by the achieved outcomes that most of the lignin and hemicellulose components have been dissolved in the course of hydrolysis and further bleaching procedure. Moreover, the performed treatments and enrichment in cellulose have eliminated the noncellulosic amorphous constituents, which is in compliance with all of the detected changes and XRD results.

3.3. XRD Studies

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We have conducted the XRD studies for the purpose of examining the crystallinity of CNCs that had been extracted from the roots of Dk plant and followed by chemical treatment. Cellulose is known to be originally composed of both crystalline and amorphous regions, while on the other side, hemicellulose and lignin are acknowledged as amorphous substances [46]. In accordance with the XRD patterns, the crystalline nature of cellulose with an intensive peak at 2θ = 22.6° (002) is quite perceptible, while an stretch of two weak peaks in

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the regions of 2θ = 15.4° (101) and 34.5° (004) is indicative of a typical cellulose I pattern

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[33, 47]. The apparent peak in this curve that can be observed at around 22.6° can be ascribed

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to the typical lattice construction of cellulose and considered as a sign of diffuse

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characterization pattern of the amorphous phase [48]. The CrI of the untreated, hydrolysis,

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and CNCs have been detected to be 30.11, 68.18, and 83.20%, respectively.

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As it specified by Fig. 4, the percentage of crystallinity have faced an increase from 30.11%,

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regarding the untreated (curve a), up to 83.20 % that is relative to the CNCs of curve c, which had been expected since the untreated Dk accommodates a high content of amorphous

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regions. Also, the higher crystallinity that have been observed in CNCs has been supposedly caused by the more organized eradication of no cellulosic polysaccharides, as well as the dissolution of amorphous zones [32, 49]. However, the degree of crystallinity is dependent on the type of performed chemical treatment on the material and the applied conditions [50]. Various authors have discussed the inducement of this particular increase in crystallinity subsequent to the acid treatment in their works [51, 52]. It has been suggested by the increment in CrI that the contaminants, hemicellulose, and lignin substances have been entirely eradicated from the untreated Dk. It can also be concluded that in the course of

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hydrolysis and involved treatments, there has not been any alterations in the crystalline structure of cellulose, considering how all of the diffractograms have displayed a pattern characteristic of cellulose I. The crystalline size has been estimated by the usage of Scherrer’s equation. The values of hydrolysis Dk and CNCs have been perceived to be 82.1 and 4.95 nm, respectively, which could be relative to the removal level of amorphous

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materials [53].

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3.4. TGA/DTG analyses

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The thermal stability of untreated Dk, along with the CNCs that had been extracted from it

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under different conditions, has been investigated by the utilization of TGA. It has been

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confirmed in many ways that the primary thermal decomposition of cellulosic materials can be procured at a temperature between 200 and 450 °C [54]. All of the involved samples have

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exhibited a small weight loss of approximately 5-9% throughout the region of 25–150 °C,

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which accord with the vaporization and removal of moisture (Fig. 5) [55]. Typically, it is known that hemicellulose, cellulose, and lignin decompose at different temperatures since

Jo

they contain various and dissimilar chemical structures [56].

The decomposition process of hemicellulose has initially began at around 250 °C and proceeded up to 315 °C. In the case of untreated Dk, the decomposition peak has been perceived to obtain its maximum mass loss rate at 289 °C, while displaying a 62 wt.% of solid residuals at the temperature of 700 °C [37]. It is assumed that the reason behind the low thermal stability of hemicellulose is the existence of acetyl groups. As expected, it is a common fact that the decomposition of hemicellulose happened in prior to lignin and

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cellulose [37, 41]. The mentioned peak has not been observed in the case of treated Dk, suggesting that the treated sample did not accommodate any hemicellulose, which has been in accordance with the obtained FTIR results. The beginning of cellulose decomposition has been triggered at 315 °C and had been observed to continue up to 460 °C. In regards to the untreated and treated samples, the maximum weight loss rate has been detected at the temperatures of 453 °C and 422 °C, respectively. Almost all of the existing cellulose has been

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pyrolyzed at the temperature of 455 °C as the solid residuals had been observed to be

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relatively small (26 and 39 wt.% for untreated and treated, respectively). The decomposition

-p

process of lignin has had a gradual speed and took place throughout the temperature range of

re

200 °C to 700 °C. Since lignin contains aromatic rings with various branches, the

lP

functionality of its chemical bonds cover a large region and cause its degradation to occur throughout a large temperature range (100–900 °C) [56]. The thermal degradation behavior of

na

Dk has shown that after being treated, a reduction had been induced in the thermal stability of

ur

cellulose nano-crystals that had been composed through the usage of sulfuric acid hydrolysis [57]. In regards to Fig. 5, the detected reduction in the thermal stability of cellulose could be

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either the resultant of dehydration reaction of cellulose crystals or the increased exposure of surface area to heat, due to the high surface area of nanoparticles [58, 59]. Besides, an expansion in the portion of short cellulose chains could be counted as the reason behind the induced reduction in the thermal stability of CNCs. This expansion has been caused by the high specific surface area and free end chains of the nano-crystals that had been procured in the course of the long duration of hydrolysis. The decomposition of these particular end chaines have been initiated at low temperatures [60].

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3.5. Zeta Potential The results have announced stronger negative charges for CNCs as the average zeta potential has been observed to be -19.7, -61.2, and -13.47 mv in the cases of untreated, CNCs, and 99m

Tc-CNCs, respectively. This outcome has been quite predictable since a hydrolysis that

incorporates H2SO4 generally conveys the sulfate groups on its surface and gives rise to stronger negativity in contrast to HCl and other mineral acids [61, 62]. Furthermore, it has

of

been indicated by the obtained results that the suspension of CNCs had contained a satisfying

ro

stability considering how the achieved value has been more than -15mV, which is recognized

re

-p

as the minimum value for determining the onset of agglomeration [9, 63].

lP

3.6. FESEM and EDX analyses

The FESEM images in Fig. 6 a-f demonstrate and discuss the hydrolysis and CNCs, along

na

with their particles size distributions and energy dispersive x-ray spectroscopy (EDX). The

ur

spherical shape of hydrolysis sample and the produced CNCs that have apparently been monodispersed (Fig. 6 a, b), which were detected after carrying out hydrolysis and the

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appointed treatment, could have been the resultants of self-assembled short cellulose rods that had been caused by the interfacial hydrogen bonds and/or the incorporated preparation procedure. There are reports that have been previously submitted by various researchers on the topic of spherical cellulose production [64, 65]. The statistical analysis of samples dimensions based upon the FESEM images have been performed by the utilization of ImageJ software. Subsequent to carrying out hydrolysis and the treatment, the diameters of hydrolysis sample

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and CNCs have been perceived to be in the range of 99.88 ± 40 nm and 35.58 ± 12 nm, respectively. Fig. 6 e, f illustrate the elemental constituent of the extracted CNCs that had been achieved through the usage of energy dispersive x-ray spectroscopy (EDX). The obtained spectrum has confirmed that the hydrolysis and CNCs derived Dk possess C and O content, which had been observed to be 49.3, 38.8, and 4.4% and 68.6, 26.6, and 0.4 %, respectively; there has not been any further impurities involved [48, 65]. The sulphur

of

impurity in CNCs is more likely originates from negatively charged sulphate groups

-p

ro

introduce on CNCs surface after acid hydrolysis as described earlier [66].

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3.7. TEM analysis

lP

The TEM images was made to precisely confirm the spherical shaped structures of CNCs. As it is maintained in TEM images (Fig. 7), the particles of CNCs have been observed to contain

na

a spherical shape in nano-dimension that had distributed uniformly and aggregated to some

ur

extent that can be by the evaporation of water, very small size of CNCs and their huge specific surface area that led to stack each other by van der Waals forces [67]. Some

69].

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investigations was done on the topic of spherical CNCs composed by different resources [68,

Either the mechanical pretreatment procedures or the outcomes of ultrasonic and dialysis treatments in the course of the acid hydrolysis could be the reason behind the obtained spherical morphology [70]. The average diameter of spherical shaped nanocrystals was 24.1 nm which was in agreement with data obtained by FE-SEM images [68].

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3.8. AFM analysis The AFM images of CNCs (Fig. 8) were used to determine the distribution and dispersion of CNCs in solution. These images were clearly revealed a relatively smooth and uniform surface of nanoparticles [69].

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3.9. Cytotoxicity assay

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A 549 cell has been selected to study the cytotoxicity of CNCs through the utilization of

-p

MTT assay. Subsequent to 24 h of treatment, the appointed A 549 cells (10,000 cells/well)

re

have been incubated with the varying doses (200, 100, 50, 25, 12.5, 6.25, 3.12, and 1.56

lP

μg/ml) of nanoparticles. The viability percentage of cell line throughout the exerted concentrations of composed CNCs has been demonstrated in Fig. 9. It has been specified by

na

the in vitro cytotoxicity investigations that the CNCs have not caused any notable cellular

ur

toxicity effects on the appointed A549 cells. The achieved outcomes have suggested that the CNCs contain the capability of being employed in varying biomedical implementations [71].

Jo

The changes of cell viability from approx. 80% to 65% might be either the result of deeper penetration of nanoparticles into the cell or the induced impact on the cell membrane integrity by high surface area and size of particles.

3.10. 99mTc-CNCs labeling assay 99m

Tc has been selected for labeling the CNCs in conforming to a formerly recounted process

that had been relative to the 99mTc-labeled nanofibrillar-cellulose [30]. The stability of 99mTc-

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CNCs has been ascertained in vitro that involved a serum and also by the usage of double distilled water. The labeling efficiency of more than 98% has been obtained for the case of 99m

Tc-CNCs that included the utilization of a novel and directly labeling approach for in vivo

targeted diagnosis in Tab. 2. After labelling carboxymethyl cellulose with technetium-99m, Schade et.al have displayed a high labeling efficiency of about 97% as a newly developed non-digestible marker of solid phase of gastric contents [72].

The

99m

Tc-CNCs have

of

contained a z-potential of -13 mV, while it has been detected to be -61 mV for the CNCs.

ro

This data indicated that Tc-99m has labeled to CNCs and changed their surface negative

-p

charges.

re



lP

3.11. Cell binding

Throughout the Cell binding assay, 99mTc-CNCs have been added at the concentrations of 30

na

and 90 μg/ml to each of the involved cell culture plate wells and thus, the cells have been

ur

permitted to incubate for the duration of 3 to 4 h. For the purpose of eliminating the remaining of CNCs that had not been taken up, we have washed the cells in triplicate with

Jo

PBS and covered them with fresh PBS; thereafter, we have measured the radioactivity of each tube by the usage of gamma counter. As it has been displayed by the results, the cell binding at low concentration (30 μg/ml) has been 34%, while in regards to the case of high concentration (90 μg/ml), it has been observed to be 14%. The main presumption that can be reported from the given data is that the cell binding of CNCs throughout the A549 cell lines has been more practical at a low concentration, since the high concentrations of nanoparticles can take up the cells [73, 74].

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3.12. Imaging and Biodistribution For targeted diagnosis, it is necessary to examine the distribution of in vivo administered particles throughout the organs. Fig. 10 displays the scintigraphy images that have been captured 6 h subsequent to the intravenous administration of 99mTc-CNCs into the designated rat. As it can be observed in the available scintigrams, although most of the administered dose has been consumed by the kidneys (30.24 ± 8.78, 33.78 ± 5.67, and 39.92 ± 4.96 %ID/g

of

at 1, 4, and 24 h after injection and then sacrificed the rats, respectively), yet the uptake of

ro

radioactivity has been clearly visualized throughout blood and some organs.

-p



re

Ch. Ming Fu et al., have reported the direct labeling of radioisotope 99m Tc with magnetite

lP

(Fe3O4) nanoparticles for diagnostic applications and the labeling efficiency has been above 90%, which could apparently sustain over a lifetime. The biodistribution of radioactivity has

na

shown a high uptake by the liver subsequent to intravenously injecting the radiolabeled 99m

Tc-labeled-Fe3O4 nanoparticles through

ur

ferrite [75]. R. Rahmani et al have demonstrated

the usage of Quince seeds extract. The labeling efficiency has been 90% and The

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biodistribution has displayed that the highest accumulation was in the kidneys and then throughout liver and spleen [76]. The value of the existing radioactivity in various organs has been measured and the radioisotope count of each organ has been displayed in Tab. 3, which is relative to the time intervals of 1, 4, and 24 h, respectively. The uptake of particles is associated with the physiochemical characterization of particle size, particle charge, surface hydrophobicity, and the chemical nature of functional groups [75]. Liver, lung, and spleen have been the substitution organs for where the

25

99m

Tc-CNCs have been metabolized. The

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inducement of a low uptake in the stomach and thyroid can be considered as a sign for the existence of a small amount of free 99mTc in blood.

In the work of S. Imlimthan et al., after labeling cellulose nanocrystals with In-111, it has been reported that the high uptake was first observed in liver and then, lungs and spleen. This

of

radiolabeled molecular imaging probes can stand as valuable information in regards to non-

ro

invasive imaging of intrinsic for targeted delivery [77]. P. Laurén et al., have demonstrated a

-p

reliable and efficient method for labeling 99mTc-nanofibrill cellulose, which had enabled them

re

to trace the in vivo localization of hydrogel. It is explained in their report that the labeling

lP

efficiency has been 98% with high uptake in kidneys [30]. In the work of L. Colombo et al., subsequent to synthesizing cellulose nanocrystals from Whatman filters and labeling them

na

with fluorescent dye, it is indicated that a concentration of 35 µg/ml CNCs has been well

ur

tolerated in the body of rat and the highest accumulation has belonged to liver and then

Jo

kidneys [78].

4. Conclusion

CNCs have been successfully fabricated from the roots of Dorema kopetdaghens (Dk) plant via a sulphuric acid hydrolysis as a new method for the synthesis of cellulose nanocrystals, which also stands as a reliable and efficient technique to perform the labeling procedure of radioisotope Tc-99m through the utilization of CNCs in regards to diagnostic and bioimaging implementations. The FTIR spectra of CNCs have confirmed the removal of non-cellulosic components after the chemical treatments. The XRD results have shown that after carrying

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out successive chemical treatments, CNCs had exhibited cellulose I crystalline structure with high crystallinity (83.20%) and the size of 4.95 nm. The morphological study has confirmed that the utilized chemical treatment is quite practical for obtaining a spherical shape of CNCs in the size of 35.58 ±12 nm by FESEM and 24.1 nm by TEM. The produced CNCs have been thermally less stable than the untreated Dk. These structural analyses have suggested the effectiveness of acid hydrolysis in isolating the cellulose nanocrystals from the roots of Dk.

of

The XRD crystalline structural data, along with the FTIR chemical structural compositions,

ro

have confirmed the purity of isolated CNCs. Moreover, the CNCs have not shown any

-p

significant cellular toxicity effects on the A549 cells. The labeling efficiency has been

re

observed to be above 98%. The biodistribution of radioactivity has displayed a high uptake

lP

by the kidneys. This successful approach to isolate CNCs from the Dk roots serves as an excellent prototype to extract cellulose nanocrystals from many other similar biomasses.

na

However, the potential of CNCs in being easily prepared and directly labeled with Tc-99m as

Jo

for the very first time.

ur

a marker for bioimaging and diagnostic applications has been exhibited throughout our work

Conflit of interest

The authors declare that they have no conflict of interest.

Acknowledgements The authors thankfully acknowledge the technical support for this article provided by Mashhad University of Medical Sciences and Islamic Azad University of Tehran. This research did not receive any specific grant from funding agencies in the public.

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Data availability The raw/processed data required to reproduce these findings cannot be shared at this time, as the data forms part of an ongoing study.

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Legends Fig. 1. The picture of the endemic plant of Dk and its roots. Fig. 2. A flowchart with the steps from the root crushing to the final CNCs. Fig. 3. FTIR spectra of (a) untreated Dk, (b) hydrolyzed Dk, and (c) CNCs. Fig. 4. XRD patterns of the Dk; (a) untreated; (b) hydrolyzed, and (c) CNCs. Fig. 5. TGA/DTG curves of the untreated Dk (red) and CNCs (black).

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Fig. 6. FESEM images of the hydrolyzed Dk (a), CNCs (b), and their particles size

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distributions (c and d) and EDX results (e and f). Fig. 7. TEM images of CNCs.

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Fig. 8. AFM images of prepared CNCs.

Fig. 9. MTT result of A 549 after exposure to different concentrations of CNCs. Each data

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point represents the average ± SD of three viability measurements (cell density of 1×104

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cells/ml).

Fig. 10. Anterior and posterior images of rat at 6 and 24 h after injection of 99mTc- CNCs (7.4

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MBq in 200 µl, 1.85 MBq in 200 µl, respectively). Planar images were acquired using a 256 × 256 matrix for a total of 250,000 counts with a zoom factor of 2. The kidenyes was clearly

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seen in the anterior and posterior images of rat for 6h and 24 h. Table 1. Chemical composition of untreated Dk and CNCs.

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Table 2. 99mTc-CNCs stability studies in distilled water and human serum up to 24 h (ITLCSG as stationary phase and Acetone, Metanol as mobile phase).

99m

Tc-CNCs remains at the

origin and free Pertechnetate migrate to the solvent front. The labeling stability was calculated by dividing 99mTc-CNCs activity to 99mTc-CNCs + free Pertechnetate. Table 3. Biodistribution of 99mTc-CNCs formulation at different time points in rat. Values are presented as mean of injected dose per gram each tissue (%ID/g) ± SD (n=3)

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Fig. 1. M. Darroudi et al., 2019

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Fig. 2. M. Darroudi et al., 2019

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Fig. 3. M. Darroudi et al., 2019

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Fig. 4. M. Darroudi et al., 2019

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Fig. 5. M. Darroudi et al., 2019

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Fig. 6. M. Darroudi et al., 2019

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Fig. 7. M. Darroudi et al., 2019

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Fig. 8. M. Darroudi et al., 2019

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Fig. 9. M. Darroudi et al., 2019

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Fig. 10. M. Darroudi et al., 2019

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Tab. 1. M. Darroudi et al., 2019 Untreated Dk

CNCs

Cellulose

41.2 ± 1.02

78.08 ± 0.2

Hemicellulose

20.06 ± 0.4

4.50 ± 0.1

Lignin

12.1 ± 0.5

2.09 ± 1.2

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Material (%)

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Tab. 2. M. Darroudi et al., 2019

Incubation times (h) in human

water

serum

Labeling stability of 0

1

4

Acetone

99

99

100

Methanol

95

99

100

0

1

4

24

100

98

99

100

100

100

97

99

100

100

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a: Technetium-99m- Cellulose nanocrystals

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Tc-CNCs a (%)

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99m

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Incubation times (h) in deionized

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Tab. 3. M. Darroudi et al., Int. J. Biol. Macromol., 2019

99m

Tc-CNCs (%ID/g) a 4h

24 h

Muscle

0.29 ± 0.03

0.8 ±1.04

0.14 ±0.04

Stomach

1.29 ± 0.54

0.54 ±0.03

Intestine

2.14 ± 0.56

0.69 ±0.21

0.31 ±0.01

Heart

0.81 ± 0.09

0.73 ±0.33

Lungs

1.1 ± 0.17

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0.33 ±0.05

0.73 ±0.06

0.49 ±0.08

Blood

1.36 ± 0.42

0.72 ±0.25

0.48 ±0.14

Spleen

0.63 ± 0.17

1.59 ±1.61

0.79 ±0.22

Kidneys

30.89 ± 8.78

33.78 ±5.67

39.92 ±4.69

Liver

1.20 ± 0.36

0.83 ±0.25

1.19 ±0.29

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Tissue

0.52 ±0.19

0.2 ± 0.08

0.09 ±0.04

0.04 ±0.01

Tail

4.04 ± 1.06

1.21 ±0.59

3.32 ±0.58

Thyroid

0.54 ± 0.35

1.44 ±1.73

0.25 ±0.05

Ear

1.22 ± 0.26

0.72 ±0.18

0.95 ±0.04

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Brain

a: percent injected dose per gram of tissue ±SD (n=3) (%ID/g)

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Authors statement: Elahe Kamelnia, Adeleh Divsalar, Majid Darroudi, Parichehr Yaghmaei, Kayvan Sadri

E.K: First Athor, Designed, and performed experiments; analysed data A.D: Supervised the research M.D: Corresponding author; Supervised the research

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P.Y: Advisor

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K.S: Co-Corresponding author; Advisor

* Corresponding author at: Department of Modern Sciences and Technologies, School of Medicine, Mashhad

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University of Medical Sciences, Mashhad, Iran. Tel.: +98 513 8002286; fax: +98 513 8002287.

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E-mail addresses: [email protected], [email protected] (M. Darroudi).

*Co-Corresponding author: Nuclear Medicine Research Center, Mashhad University of Medical Sciences,

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Mashhad, Iran. Tel.: +98 513 Tel.: +98 513 8012783; fax: +98 513 8012783. E-mail: [email protected]

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Graphical abstract

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Highlights The production of CNCs from Dorema kopetdaghens plant by acid hydrolysis.

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CNCs have not caused any significant cellular toxicity effects on the A549 cells.



The direct labeling of CNCs by Tc-99m for diagnostic and bioimaging applications.



labeling efficiency of 99mTc-CNCs was more than 98%.



The biodistribution of radioactivity has displayed a high uptake by the kidneys.

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