From the results shown in Figure 6, we can observe that, with the increase of shear rate, the apparent viscosity of several kinds of solutions decreased obviously to show shear thinning, which is typical of non-Newtonian fluids. The entangled macromolecules began to align in a directional manner, showing a decrease in apparent viscosity. When cellulose was completely dissolved in the IL, the van der Waals forces produced physical crosslinking between the molecules [49,50]. Hydrogen bonds existed between the cellulose macromolecules and macromolecules, and between cellulose the macromolecules and ions. The Brownian motion of each component in the viscose system made the physical crosslinking and hydrogen bonds exist in a dynamic equilibrium of constant destruction and regeneration, which made the viscose form a network structure. With the increase of shear force, the dynamic equilibrium was destroyed; the physical crosslinking and hydrogen bonds were not ready to recombine; and there appeared a relative slip and rearrangement between the components, resulting in a decrease in viscosity that eventually reached a constant value [51,52,53]. The shear rate had little influence on the viscosity of RCF-CNF1-SiO21; that is to say, the state of the whole system was more stable, even during extrusion, and the tensile strength of the fiber was better.

Figure 7 shows the storage modulus G' and loss modulus G" of the testing viscose. For the viscose system without nanomaterials, G' was greater than G'' at 40 °C, indicating that the elastic modulus was greater than the viscous modulus, which showed typical solid-like properties. The main forces of the system were based on the entanglement between cellulose molecules and the network structure between cellulose/AMIMCl. At temperatures of 60 and 80 °C, G'' was greater than G', indicating that the viscous modulus was greater than the elastic modulus, showing fluid properties, which also indicated that the properties of viscose were sensitive to the temperature. As the temperature changed, the viscoelasticity also changed. With the increase of temperature, the cellulose

macromolecules were more active in AMIMCL, making the viscose appear fluid.

characterized by SEM, which was also the reason for the higher fiber strength.

**Figure 7.** Influence of temperature and angular frequency on G' and G" of RCF (**a**), RCF-CNF2 (**b**), RCF-SiO22 (**c**), RCF-CNF1-SiO21 (**d**), and RCF-CNF2-SiO22 (**e**). **Figure 7.** Influence of temperature and angular frequency on G' and G" of RCF (**a**), RCF-CNF2 (**b**), RCF-SiO2 2 (**c**), RCF-CNF1 -SiO2 1 (**d**), and RCF-CNF2 -SiO2 2 (**e**).

The viscoelasticity of viscose mainly depends on the properties of each component and their interactions. It is generally believed that the addition of nano-materials can change the viscoelasticity of viscose. For viscose with added CNF and nano-SiO2, when the temperature was constant, with the increase of frequency, the solution G' and the

moved toward the high-frequency area, where modulus transposition occurs, and the sample experienced structure reconstruction and an elasticity larger than the viscosity, which are typical characteristics of an entangled polymer solution. Under the same oscillation frequency, when the temperature increased, G' and G" decreased, which

At 80 °C, G' and G'' of RCF-CNF2 were the largest among all of the samples, which indicated that the entanglement degree between the fiber molecules in the viscose system was higher, and the physical entanglement and hydrogen bonds among the cellulose macromolecules, CNF, and IL formed a tangled network structure that affected the relative motion and stretching of the molecules. Therefore, the viscose showed an obvious elasticity. The intersection point of G' and G'' experienced a minimal oscillation frequency, indicating that as adding CNF increased the cellulose content within the viscose system, the gel point of the whole system moved forward. Thus, in the regeneration process, solidification was easier. The G' and G values of RCF-SiO22, RCF-CNF1-SiO21, and RCF-CNF2-SiO22 were all lower than those of the blank samples,

indicated that the viscoelasticity decreased [54,55,56].

**Table 6.** Modulus values and frequency at the crossover point of G' and G" of cellulose viscose.

#### **4. Conclusions**

In this study, conifer pulp was used as a raw material to make regenerated fiber, an ionic liquid AMIMCl was used as a solvent for cellulose, and organic CNF fiber and inorganic nano-SiO2 were added to regenerate composite cellulose fibers via a dry-jet wetspinning process. The mechanical properties of the composite cellulose can be improved by adding nanomaterials. Adding 1% CNF and 1% nano-SiO2 to the pulp/AMIMCl improved the mechanical properties of the composite cellulose by 47.46%. The regenerated fibers and nanocomposite fibers were typical type-II cellulose crystalline forms, and the crystallinity of the composite fibers decreased. The surface of the regenerated fiber exhibited a scaly structure with pores, and the pores could be reduced by adding 1% CNF and 1% nano-SiO2. The addition of nanomaterials changed the degradation rate of the regenerated fibers. From the perspective of the residual amount, the loss rate of nanomaterials in the preparation process was low. The viscosities of all five samples were pseudoplastic fluids with a yield value of stress and with shear thinning. The yield stress and viscosity sequences were as follows: RCF-CNF2 > RCF-CNF2 -SiO2 2 > RCF-SiO2 2 > RCF > RCF-CNF1 -SiO2 1 . Under the same oscillation frequency, when the temperature increased, the G' and G" decreased, indicating a decrease in viscoelasticity.

**Author Contributions:** Y.X. and L.Q. contributed equally to this work. L.Q., G.Y. and M.H. conceived and designed the experiments; Y.X., L.Q. and Z.L. performed the experiments; Y.X., L.Q. and Z.L. analyzed the data; G.Y. and M.H. contributed reagents, materials, and analysis tools; Y.X., L.Q. and Z.L. wrote the study; G.Y., M.H. and J.C. reviewed the manuscript and made comments. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors are grateful for the financial support received from the National Natural Science Foundation of China (Grant Nos. 31770628, 31901267 and 22108134), the Provincial Key Research and Development Program of Shandong (Grant Nos. 2019JZZY010326 and 2019JZZY010328), Foundation of State Key Laboratory of Biobased Material and Green Papermaking (No. ZZ20190214), and the Pilot Project for Integrating Science, Education and Industry (Grant No. 2020KJC-ZD14).

**Data Availability Statement:** All date used during the study appear in the submitted article.

**Acknowledgments:** The authors are grateful for the financial support received from the National Natural Science Foundation of China (Grant Nos. 31770628, 31901267 and22108134), the Provincial Key Research and Development Program of Shandong (Grant Nos. 2019JZZY010326 and 2019JZZY010328), Foundation of State Key Laboratory of Biobased Material and Green Papermaking (No. ZZ20190214), and the Pilot Project for Integrating Science, Education and Industry (Grant No. 2020KJC-ZD14).

**Conflicts of Interest:** The authors declare that they do not have any conflict of interest.

#### **References**

## *Article* **5-Fluorouracil Encapsulated Chitosan-Cellulose Fiber Bionanocomposites: Synthesis, Characterization and In Vitro Analysis towards Colorectal Cancer Cells**

**Mostafa Yusefi 1 , Hui-Yin Chan 2 , Sin-Yeang Teow 2 , Pooneh Kia 3 , Michiele Lee-Kiun Soon 2 , Nor Azwadi Bin Che Sidik 1,\* and Kamyar Shameli 1,\***

**Citation:** Yusefi, M.; Chan, H.-Y.; Teow, S.-Y.; Kia, P.; Lee-Kiun Soon, M.; Sidik, N.A.B.C.; Shameli, K. 5-Fluorouracil Encapsulated Chitosan-Cellulose Fiber Bionanocomposites: Synthesis, Characterization and In Vitro Analysis towards Colorectal Cancer Cells. *Nanomaterials* **2021**, *11*, 1691. https://doi.org/10.3390/nano11071691

Academic Editors: Carla Vilela, Carmen S. R. Freire and Eleonore Fröhlich

Received: 4 April 2021 Accepted: 19 May 2021 Published: 28 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Abstract:** Cellulose and chitosan with remarkable biocompatibility and sophisticated physiochemical characteristics can be a new dawn to the advanced drug nano-carriers in cancer treatment. This study aims to synthesize layer-by-layer bionanocomposites from chitosan and rice straw cellulose encapsulated 5-Fluorouracil (CS-CF/5FU BNCs) using the ionic gelation method and the sodium tripolyphosphate (TPP) cross-linker. Data from X-ray and Fourier-transform infrared spectroscopy showed successful preparation of CS-CF/5FU BNCs. Based on images of scanning electron microscopy, 48.73 ± 1.52 nm was estimated for an average size of the bionanocomposites as spherical chitosan nanoparticles mostly coated rod-shaped cellulose reinforcement. 5-Fluorouracil indicated an increase in thermal stability after its encapsulation in the bionanocomposites. The drug encapsulation efficiency was found to be 86 ± 2.75%. CS-CF/5FU BNCs triggered higher drug release in a media simulating the colorectal fluid with pH 7.4 (76.82 ± 1.29%) than the gastric fluid with pH 1.2 (42.37 ± 0.43%). In in vitro cytotoxicity assays, cellulose fibers, chitosan nanoparticles and the bionanocomposites indicated biocompatibility towards CCD112 normal cells. Most promisingly, CS-CF/5FU BNCs at 250 µg/mL concentration eliminated 56.42 ± 0.41% of HCT116 cancer cells and only 8.16 ± 2.11% of CCD112 normal cells. Therefore, this study demonstrates that CS-CF/5FU BNCs can be considered as an eco-friendly and innovative nanodrug candidate for potential colorectal cancer treatment.

**Keywords:** cellulose; chitosan nanoparticles; bionanocomposites; 5-fluorouracil; in vitro drug release; cytotoxicity assay; colorectal cancer

#### **1. Introduction**

Currently, conversion of natural-based residues into useful and novel materials may tackle financial and environmental issues [1]. In this regard, rice straw is an abundant lignocellulosic residue, which contains a high ratio of cellulose to use in a myriad of research fields [2,3]. As a nature gifted material, cellulose fiber is an important component of the wood cell wall and the most abundant organic polymer on earth [4]. Plant cellulose possesses advantageous and unique mechanical, optical and rheological characteristics, along with a sensitivity to the particular molecular structure of the antigen and pH-sensitivity for synthesis of novel polymeric nanodrug formulations [4]. The cellulose, however, does possess some drawbacks, including poor crease resistance and low solubility in solvent fluids. Its physiochemical characteristics suitably can be modified by esterification, etherification, de-polymerization, radical grafting and alkali treatments to impart exogenous groups over the cellulose structure without damaging its advantageous intrinsic characteristics [5].

Chitosan is the second most popular biopolymer after cellulose, with production of over 100 million tons per year [6]. It may be derived from chitin and is a cationic linear and natural amino-polysaccharide containing -(1-4)-linked d-glucosamine and *N*-acetyldglucosamine in deacetylated and acetylated form, respectively [7]. Among diverse methods to synthesize layer-by-layer chitosan-based composites and nanoparticles (NPs), ionic gelation approach is an organic solvent-free solution, straightforward and a facile method with minimal toxicity [8]. In this method, the phosphate groups of sodium tripolyphosphate (TPP) may act as a physical crosslinking agent, which has advantages over emulsifying and chemical crosslinking agents, such as less toxicity to the organs and no destruction to the structure of the loaded-drugs in chitosan nanoparticles [9]. Polymer blends are increasingly important to fabricate innovative composites for various applications [10]. The blend of degradable polymers can merge the desirable properties of the polymers [11]. In addition, the crosslinking procedure might considerably improve physiochemical properties of the polymer composites [12]. In medically-related applications, the most popular antimicrobial coating agent on cellulose is currently chitosan to synthesize composites with suitable biocompatibility and water-rich structures to encapsulate both hydrophilic and hydrophobic drugs [13]. Furthermore, the bionanocomposites or nanohybrids of chitosan and cellulose possess intermolecular interactions, owing to H-bonds and Van der Waals forces [14]. Most importantly, chitosan-cellulose bionanocomposites or nanohybrids may possess a tremendous swelling capacity and water absorption ability to release the drug at the targeted cells [15]. Therefore, the biocompatibility and physiochemical properties of both cellulose and chitosan can be modified by using cellulose as a reinforcement and chitosan as a coating agent to construct a double polysaccharide composite biomaterial.

Colorectal cancer (CRC) is the third most diagnosed cancer with death of six lakhs per year [16]. 5-Fluorouracil (5FU) as an antimetabolite and fluorinated pyrimidine has been used for CRC therapy since three decades ago [17]. Albeit, the unwanted side-effects of using 5FU-based chemotherapy might cause diarrhea, stomatitis and gastrointestinal mucosal injury. The above side-effects can be abated by encapsulation of a minimal drug dosage into a biocompatible drug carrier. Of this, 5FU has been encapsulated in many drug carrier platform, such as, chitosan [18] and cellulose [19,20]. In this matter, some polysaccharides, including cellulose, chitosan, starch, xanthan gum, hyaluronic acid, and carrageenans can display pH/thermos-responsive properties to control the drug release dosage with effective anticancer actions [21].

Nano-sized medicines may favorably enhance circulation time, targeted activities, and safe drug delivery to decrease the issues of side effects from the chemotherapy treatments [22–24]. For this aim, biopolymer-based NPs may ideally avoid the immune system to increase circulation time of the NPs in blood to enhance the anticancer actions [25]. The drug delivery systems may affect the microenvironment of tumors, which is leaky and has a higher sensitivity to macromolecules compared to the normal cells [26]. Thus, drug encapsulation in natural polymers can be considered as an emerging research area for different pharmaceutical applications [24,25,27]. Furthermore, being impressively biodegradable and possessing an ability for the enzymatic degradation by colonic microbial agents, biopolymers (such as lignin-based NPs, glycogen and gelatin and cellulose fibers) may trigger probes for novel polymeric–anticancer nanodrug conjugates with advanced antiproliferative actions [24,25,27,28].

For example, 5FU-loaded composites of nanocellulose (obtained from commercial α-cellulose), chitosan and sodium alginate eliminated HT29 CRC cells, however, it unfavorably killed the normal HEK293 cells at concentrations between 50 to 100 µg/mL [14]. In a separate experiment, curcumin loaded nanocellulose caused three times higher effect compared to curcumin alone against HT29 CRC cells, albeit, the biocompatibility of the sample was not studied [29]. In recent investigations, the release of 5FU was controlled through nanocellulose/gelatin composites [30] and also 3D printed composites of cellulose nanofibrils and CaCO3 [31]. Both studies did not examine the anticancer action of the samples.

In this present study, cellulose fibers were extracted from rice straw waste. In addition, chitosan NPs and bionanocomposites of chitosan-cellulose were fabricated by the ionic gelation method using TPP as a cross-linker. Above all, 5FU was encapsulated in the bionanocomposites of chitosan as a coating agent and cellulose as a reinforcement for CRC analysis. To the best of our knowledge, this is the first time that rice straw cellulose was coated with chitosan for anticancer drug 5FU carrier in potential CRC treatments. The physiochemical properties of the synthesized samples were evaluated by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), energy dispersion X-ray spectroscopy (EDX), thermogravimetric analysis (TGA), dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), and the swelling analysis. The drug absorbance and release of 5FU-loaded in the bionanocomposites were evaluated by using ultraviolet–visible (UV) spectroscopy. In vitro cytotoxicity assays evaluated the biocompatibility and anticancer activity of the fabricated samples against CCD112 colon normal and HCT116 CRC cell lines.

#### **2. Materials and Methods**

#### *2.1. Materials*

Rice straw waste was obtained from Malaysian Agricultural Research and Development Institute (MARDI), Selangor, Malaysia. Potassium hydroxide (KOH, 85%), sodium chlorite (NaClO2, 80%), acetic acid glacial (CH3COOH) (98%), chitosan (low molecular weight, 190,000–310,000 degree of acetylation), Tween-80 and TPP were all purchased from Sigma Aldrich (St. Louis, MO, USA). 5FU, 99%, 5-Fluoro-2,4(1H,3H)-pyrimidinedione (ACD CODE MFC D00006018) with a molecular weight of 130.08 g/mol was purchased from ACROS ORGANICS part of Thermo Fisher Scientific, Branchburg, NJ, USA. The chemicals were used without further purification. All glassware used was washed with distilled water and dried before used.

#### *2.2. Synthesis*

2.2.1. Extraction of Cellulose Fibers from Rice Straw Waste

CF was extracted from rice straw waste through a series of chemical treatments. The rice straw powder (30 g) was dewaxed by using a soxhlet instrument as it contained a 450 mL solution of toluene/ethanol 2:1 (*v*/*v*) for 12 h at 70 ◦C. After that, the sample was washed with distilled water three times and mixed with a sodium chloride solution (1.4%), in which dropwise adding acetic acid adjusted the pH to around 4 at 70 ◦C under 100 rpm magnetic stirring for 5 h. The sample was washed three times and then treated with KOH solution (5%) for 12 h, followed by pouring 10-fold ice cubes into the sample solution to obtain cellulose. The cellulose solution was washed and centrifuged (Tabletop Centrifuge Kubota, Model: 2420) three times at 4000 rpm for 8 min. Finally, the suspension was freeze-dried (FreeZone 1.0 L Benchtop Freeze Dry System, Kansas City, MO, USA) at −45 ◦C for 48 h and termed as CF, which stored at −4 ◦C for further analysis.

2.2.2. Synthesis of Chitosan-Cellulose Fiber Bionanocomposites to Encapsulate 5-Fluorouracil

Ionic gelation method was performed to synthesize layer-by-layer of three chitosanbased samples: (i) chitosan-cellulose fibers bionanocomposites encapsulated 5FU (CS-CF/5FU BNCs), (ii) chitosan-cellulose fibers bionanocomposites (CS-CF BNCs), and (iii) chitosan nanoparticles (CS NPs). First, three 250 mL beakers respectively contained 80 mL mixture solution of 1.0% acetic acid and 0.250 g of chitosan powder (low molecular weight). Then, 2% (*v*/*v*) of Tween-80 as a stabilizer was respectively added to each solution and mixed gently for 45 min to obtain the three chitosan solutions. To prepare CS-CF/5FU BNCs, 0.125 g CF and 0.01 g 5FU were mixed with one of the prepared chitosan solutions and then homogenized vigorously (DAIHAN-brand Homogenizer, Wonju-Si, Gang-Won -Do, Korea) at 9000 rpm for 7 min. After that, 0.50 g of TPP cross-linker was dissolved in 15 mL deionized water and added dropwise to the CS-CF/5FU solution under the continuously

vigorous stirring of the homogenizer for another 45 min. The mixture solution was washed with distilled water and centrifuged three times at 2500 rpm for 7 min at 25 ◦C. Finally, the sample was freeze-dried for 16 h and stored at −4 ◦C for further analysis.

To prepare CS-CF BNCs, the above procedure was performed, however, without adding 5FU to the CS-CF solution.

To prepare CS NPs, the above method was used, however, without adding CF and 5FU to the CS solution.

#### *2.3. Characterization*

#### 2.3.1. Physicochemical Analysis

XRD (Philips, X'pert, Cu Ka, Amsterdam, North Holland, The Netherlands) at an ambient condition was used to evaluate the structure of the samples. The sample was compressed between two smooth glass films and the XRD analysis was carried out in dispersion 2 angles of 5◦– 80◦ at a step size of 0.02◦ with 2 s/step as scanning rate using a voltage of 45 kV, a Ni-filtered Cu K radiation (=1.5406 A)◦ and a filament current of 40 mA. The SEM images were taken via using an Electro-Scan SEM instrument (model JSM 7600 F SEM, Tokyo, Kanto, Japan) attached to EDX to study the elemental composition ¯ of the sample. A low-acceleration voltage (10 kV) was used to prevent the degradation of the sample. Thermal analysis was carried out though using TGA (STA F3 Jupiter, Selb, Bavaria, Germany) Q50 V20 at a heating rate of 10 ◦C/min under a nitrogen atmosphere (10 mL/min) from 10 ◦C to 800 ◦C. For DLS analysis, an Anton Paar instrument (Litesizer 500, Graz, Styria, Austria) was used to measure the hydrodynamic size of the synthesized samples in buffer solutions (100 µg/mL) at various pH values of 1.2, 7.4 and 12 at 37 ◦C. Hydrochloric acid (HCl) and sodium hydroxide (NaOH) were used to adjust the pH 1.2 and 12, respectively. FTIR spectroscopy (ThermoNicolet, Waltham, MA, USA) determined the chemical and super-molecular structural analysis of the samples under an ambient condition. First, crushing and mixing of the sample with KBr at a ratio of 1:100 *w*/*w* to prepare a transparent pellet and the spectra of the plate was evaluated under a transmittance mode in a range between 4000 cm−1 to 400 cm−1 with a 4 cm−1 resolution and an accumulation of 128 scans. The swelling ratio of the sample was measured. For this aim, the sample was immersed in the solutions with various pH values of 1.2, 7.4, and 12 at 37 ◦C under 75 rpm magnetic stirring [32]. At each interval, the sample was collected from the solution and blotted on a filter paper to eliminate excess water and it was immediately weighed to determine the weight of the wet sample. The swelling ratio of the sample was measured as Wt/W0, in which Wt and W0 are the obtained wet weights of the sample at an arbitrary and initial time, respectively. For the DLS analysis and swelling experiment, individual tests were repeated three times, which the data were indicated as mean ± standard deviation for all triplicates within an independent test.

#### 2.3.2. Encapsulation Efficiency Study of 5-Fluorouracil

UV–vis spectrophotometry (UV-1600, Shimadzu, Kyoto, Kansai, Japan) provided a calibration curve at λmax = 266 nm from known concentrations of 5FU (5–25 ppm). This followed by calculating the amount of the drug-loaded into CS-CF/5FU BNCs by using Equation (1) to obtain drug encapsulation efficiency% [33].

$$\text{Enaporation efficiency} \left( \% \right) = \frac{\left( \text{Initial drug amount in formulation (mg)} - \text{Unentrapped drug} \left( \text{mg} \right) \right)}{\text{Initial drug amount in formulation (mg)}} \times 100 \quad (1)$$

2.3.3. A Comparative Study of In Vitro Release of 5-Fluorouracil Drug from CS-CF/5FU BNCs

For the drug release performance of CS-CF/5FU BNCs, a 5 mL dialysis bag (molecular weight cut-off between 12,000 and 14,000 Da) was used according to reported studies with slight modification [20,34]. Before the experiment, the bag was soaked for 12 h in the release media of the simulated colorectal fluid (phosphate-buffered saline (PBS) at pH 7.4). Then, the dialysis bag with the two ends tied as contained the solution mixture of 5 mg of CS-CF/5FU BNCs and 2 mL of the release media. The bag was completely immersed into a 40 mL of the release media maintained under constant stirring of 100 rpm at 37 ◦C in the stoppered bottle. A 1 mL aliquot was withdrawn from the system at the selected time and characterized by UV–vis spectrophotometry at λmax = 266 nm. The same study was performed in HCl buffer solution at pH 1.2 for the simulated release of the 5-FU drug in the gastric fluid without enzymes. The following Equation (2) calculated and compared the drug release results from the fluids with the two different pH values:

Drug release (%) = Released drug at time 0 t 0 Total drug in the sample (CS − CF/5FU BNCs) × 100 (2)

#### 2.3.4. Cell Lines and Reagents

Human HCT116 colorectal cancer (CRC) (ATCC CCL-247) and CCD112 normal colon (ATCC CRL-1541) cell lines were purchased from ATCC (VA, USA) and cultured according to ATCC's recommendation [35,36]. The cell lines were maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco) under standard cell culture conditions.

#### 2.3.5. In Vitro Cytotoxicity Assay

In vitro cytotoxicity assays were performed using CellTiter 96 Aqueous One Solution kit (MTS reagent) (#G3582, Promega), according to the manufacturer's instruction with slight modification as previously described [37]. Briefly, 5000 HCT116 and CCD112 cells per well (100 µL/well) were separately seeded onto a 96 well plate and incubated overnight at 37 ◦C in a 95% humidified incubator with 5% CO2. The next day, 2-fold serially diluted samples at concentrations of 0, 7.81, 15.62, 31.25, 62.53, 125, 250 and 500 (100 µL/well) were added into the wells and the plate was incubated for 72 h at 37 ◦C in the 5% CO2 incubator. Then, 20 µL of the MTS reagent per well was added into the plate and incubated for an additional 3 h at 37 ◦C in the 5% CO2 incubator. The optical density (OD) was then measured at 490 nm using a multimode microplate reader (Tecan). The dose–response graph was plotted by calculating the percent cell viability using Equation (3) [38]:

$$\% \text{ Cell viability} = \frac{\text{OD of sample well (mean)}}{\text{OD of control well (mean)}} \times 100\tag{3}$$

The inhibitory concentration causing 50% growth inhibition (IC50) was determined through an online calculator (https://www.aatbio.com/tools/ic50-calculator (accessed on 5 May 2021)) as previously described [35].

#### 2.3.6. Statistical Analysis

Independent experiments were performed three times and data were expressed as mean ± standard deviation for all triplicates within an individual experiment. Data were analyzed with a Student's *t* test using SPSS version 26.0. *p* < 0.05 was considered significant.

#### **3. Results and Discussion**

Scheme 1 depicts the synthesis of a novel nanocomposites of rice straw CF and CS encapsulated 5FU for the in vitro CRC analysis. CF was successfully extracted from rice straw waste by a series of chemical modifications, such as, bleaching, delignification, and alkali treatments. Sodium chloride solution and potassium hydroxide degraded lignin and hemicelluloses of the rice straw waste, respectively [39]. Then, the delignificated rice straw waste was treated with 5% potassium hydroxide to isolate CF containing nano-scale diameter and almost a uniform structure. This method was used by different studies, which found similar results from the extracted cellulose of the newspaper waste [40] and rice straw [41]. In a separate investigation [42], an increase in the ratio of potassium hydroxide from 5 to 15 weight% decreased the size of the abaca fibers; whereas, the 10 and 15 weight% of the potassium hydroxide undesirably decreased the strength of fibers comprising a twisted structure. Thus, we used proper treatments of 5% potassium hydroxide solution to extract cellulose from the rice straw waste. 15 weight% of the potassium hydroxide undesirably decreased the strength of fibers comprising a twisted structure. Thus, we used proper treatments of 5% potassium hydroxide solution to extract cellulose from the rice straw waste.

Scheme 1 depicts the synthesis of a novel nanocomposites of rice straw CF and CS encapsulated 5FU for the in vitro CRC analysis. CF was successfully extracted from rice straw waste by a series of chemical modifications, such as, bleaching, delignification, and alkali treatments. Sodium chloride solution and potassium hydroxide degraded lignin and hemicelluloses of the rice straw waste, respectively [39]. Then, the delignificated rice straw waste was treated with 5% potassium hydroxide to isolate CF containing nano-scale diameter and almost a uniform structure. This method was used by different studies, which found similar results from the extracted cellulose of the newspaper waste [40] and rice straw [41]. In a separate investigation [42], an increase in the ratio of potassium hydroxide from 5 to 15 weight% decreased the size of the abaca fibers; whereas, the 10 and

*Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 6 of 22

**3. Results and Discussion**

**Scheme 1.** A schematic process of synthesis of CS-CF/5FU BNCs for in vitro cytotoxicity assays towards CRC cell line. **Scheme 1.** A schematic process of synthesis of CS-CF/5FU BNCs for in vitro cytotoxicity assays towards CRC cell line.

Incorporation of dual polysaccharides of CF and CS in a layer-by-layer complex encapsulated the anticancer drug 5FU by using the ionic gelation method, where TPP, Tween-80, and acetic acid served as a cross-linking agent, stabilizer and hydrogen ion formation, respectively. The ionic gelation method is facile and responsible for the increased degree of cross-linking between the components of the CS-based composites [43]. CF as a reinforcement can be compatible with CS as a coating agent to synthesize dual biocompatible composites [7]. The characteristics of CS based complexes might be enhanced by reinforcement of cellulose. Also, CS as a coating agent could possibly show inherently biocompatibility, whereas its physiochemical characteristics could be enhanced by CF as a matrix to modulate the drug release and improve anticancer effects Incorporation of dual polysaccharides of CF and CS in a layer-by-layer complex encapsulated the anticancer drug 5FU by using the ionic gelation method, where TPP, Tween-80, and acetic acid served as a cross-linking agent, stabilizer and hydrogen ion formation, respectively. The ionic gelation method is facile and responsible for the increased degree of cross-linking between the components of the CS-based composites [43]. CF as a reinforcement can be compatible with CS as a coating agent to synthesize dual biocompatible composites [7]. The characteristics of CS based complexes might be enhanced by reinforcement of cellulose. Also, CS as a coating agent could possibly show inherently biocompatibility, whereas its physiochemical characteristics could be enhanced by CF as a matrix to modulate the drug release and improve anticancer effects [44].

[44]. The ratio of the ingredients in the polymer composites is an important concern. The ratio between the chitosan powder to TPP cross-linker was 1:2 (*v*/*v*). Similarly, different research indicated that this ratio was suitable in which a reaction yield (75.0%), drug loading content (16.7%), and encapsulation efficiency (66.7%) were optimized to obtain chitosan NPs with desirable physiochemical properties. The study also used 1.0% acetic acid The ratio of the ingredients in the polymer composites is an important concern. The ratio between the chitosan powder to TPP cross-linker was 1:2 (*v*/*v*). Similarly, different research indicated that this ratio was suitable in which a reaction yield (75.0%), drug loading content (16.7%), and encapsulation efficiency (66.7%) were optimized to obtain chitosan NPs with desirable physiochemical properties. The study also used 1.0% acetic acid and 2% (*v*/*v*) Tween-80 in the chitosan solution. For the CS-CF composite, we use CF and chitosan with the ratio of 1:2 to obtain the spherical nanocomposite with size below 50 nm, as indicated in the SEM images. In a different study, 2.0% (*w*/*v*) chitosan, 1.2% (*w*/*v*) carboxymethyl-cellulose and 1.0% (*w*/*v*) scleroglucan were used to synthesize a nanocomposite hydrogels [45]. Based on SEM images, this sample was not as a spherical composite. In addition, the chitosan did not show homogeneous coating structure on the carboxymethyl-cellulose. This could be possibly due to using higher ratio of the cellulose and scleroglucan than chitosan. Studies by Samy et al. (2020) have shown that the ratio between chitosan and cellulose was 1:1 and epichlorohydrin acted as a cross-linker [46]. This composite showed size above 100 nm, according to the SEM images. In our study, therefore, the successful fabrication of CS-CF composites with size below 50 nm could be owing to blending the proper ratio of CS coating agent and CF reinforcement under high-speed homogenizing (9000 rpm), whereas TPP desirably acted as a crosslinking agent.

Further, the CS-CF composites may possess van der Waals interactions with the anticancer drug 5FU. For the CS-CF/5FU sample preparation, only 0.01 g 5FU was added in the CS-CF solution. It is worth to mention that using the low amount of drug in the nano-carrier system may cause high drug encapsulation efficiency and prolonged drug release to obtain effective anticancer action. Studies by Nguyen et al. (2017) showed that using the same amount of 5FU (0.01 g) in chitosan solution and ionic gelation method for the improved drug encapsulation and drug release [47]. Also, studies by Hosokawa et al. (1991) also showed that encapsulated the same amount of 5FU (0.01 g) with poly (ethylene glycol) methyl ether-chitosan nano-gels [48]. Therefore, the ratio of the ingredients in CS-CF/5FU could be suitable to obtain a nanodrug complex with relevant physiochemical properties and anticancer effects.

Scheme 2 indicates the possible intermolecular chemical interactions in the bionanocomposites. The presence of hydrogen and Van der Waals forces in CS-CF/5FU BNCs could trigger intermolecular interactions in the composites [14,19]. During the sample preparation, cationic groups of chitosan (NH+ 3 ) could react with the -OH− and phosphoric ions in the TPP solution to support the deprotonation of CS. As reported [49], the formation of CS cross-linked CF could be obtained from Schiff base reaction between primary amino and carbonyl groups of CS and CF, respectively. Furthermore, 5FU was encapsulated and conjugated into the composites potentially by hydrogen bonding and van der Waals interactions [7]. The freeze-drying process was performed for the synthesized samples. It was reported that the freeze-drying process might trigger a gentle vaporization of the bound water and also sublimation of the ice crystals upon the samples to increase the presence of –OH groups and produce white fluffy fibrous materials [50]. *Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 8 of 22

**Scheme 2.** The possible intermolecular chemical interactions between active functional groups in CS-CF/5FU BNCs. **Scheme 2.** The possible intermolecular chemical interactions between active functional groups in CS-CF/5FU BNCs.

#### *3.1. Physicochemical Characterization of CS-CF/5FU BNCs Using X-ray Powder Diffraction 3.1. Physicochemical Characterization of CS-CF/5FU BNCs Using X-ray Powder Diffraction*

XRD was used to evaluate the crystallographic structure of the fabricated samples. Figure 1a–e show the XRD results of rice straw waste, CF, CS NPs, CS-CF BNCs, and CS-CF/5FU BNCs, respectively. The samples containing CF exhibited a similar XRD pattern, representing that the chemical treatments did not destroy the cellulose structure. The diffraction peaks of CF was approximately at 2θ = 15.51°, 22.23° and 35.32°, similar to the normal cellulose-I structure [39]. The main crystalline region was at 22.23° with a strong intensity, presenting the proper crystallinity of CF. Therefore, the delignification, bleaching as well as alkali treatments on the rice straw waste effectively degraded the amor-XRD was used to evaluate the crystallographic structure of the fabricated samples. Figure 1a–e show the XRD results of rice straw waste, CF, CS NPs, CS-CF BNCs, and CS-CF/5FU BNCs, respectively. The samples containing CF exhibited a similar XRD pattern, representing that the chemical treatments did not destroy the cellulose structure. The diffraction peaks of CF was approximately at 2θ = 15.51◦ , 22.23◦ and 35.32◦ , similar to the normal cellulose-I structure [39]. The main crystalline region was at 22.23◦ with a strong intensity, presenting the proper crystallinity of CF. Therefore, the delignification, bleaching as well as alkali treatments on the rice straw waste effectively degraded the

peaks of crystallinity since the crystalline structure of CS was eliminated after its crosslinking with TPP [51]. CS-CF BNCs and CS-CF/5FU BNCs performed a similar pattern attributed to both CF and CS peaks approximately at 2θ = 22.23°, 15.78° and 9.04°, which is in a good agreement with the JCPDS card no. 04-0784 [52]. Noticeably, the peak at 15.78° is an overlapping peak between CF and CS. Compared to CF, the CS-CF/5FU BNCs sample showed lower crystallinity peaks, due to presence of the TPP cross-linker to abate the crystallinity [51]. Further results indicated that CS-CF/5FU BNCs did not have any sharp peak related to 5FU, since the drug was possibly entrapped within the carrier composites [53]. The 5FU alone could display a crystalline structure with a sharp diffraction peak at around 2θ = 28.71° [53]. Moreover, the crystalline structure of the drug alone could possibly become a non-crystalline after its encapsulation within the carrier [54]. The XRD results indicated that CS-CF/5FU BNCs possessed a structure as polysaccharides to entrap

5FU.

amorphous regions and liberated the crystal regions. From Figure 1c, CS NPs presented no peaks of crystallinity since the crystalline structure of CS was eliminated after its crosslinking with TPP [51]. CS-CF BNCs and CS-CF/5FU BNCs performed a similar pattern attributed to both CF and CS peaks approximately at 2θ = 22.23◦ , 15.78◦ and 9.04◦ , which is in a good agreement with the JCPDS card no. 04-0784 [52]. Noticeably, the peak at 15.78◦ is an overlapping peak between CF and CS. Compared to CF, the CS-CF/5FU BNCs sample showed lower crystallinity peaks, due to presence of the TPP cross-linker to abate the crystallinity [51]. Further results indicated that CS-CF/5FU BNCs did not have any sharp peak related to 5FU, since the drug was possibly entrapped within the carrier composites [53]. The 5FU alone could display a crystalline structure with a sharp diffraction peak at around 2θ = 28.71◦ [53]. Moreover, the crystalline structure of the drug alone could possibly become a non-crystalline after its encapsulation within the carrier [54]. The XRD results indicated that CS-CF/5FU BNCs possessed a structure as polysaccharides to entrap 5FU. *Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 9 of 22

**Figure 1.** XRD spectra of (**a**) rice straw waste, (**b**) CF, (**c**) CS NPs, (**d**) CS-CF BNCs, (**e**) CS-CF/5FU BNCs. **Figure 1.** XRD spectra of (**a**) rice straw waste, (**b**) CF, (**c**) CS NPs, (**d**) CS-CF BNCs, (**e**) CS-CF/5FU BNCs.

#### *3.2. Physicochemical Characterization of CS-CF/5FU BNCs Using Scanning Electron Microscopy and Energy Dispersive X-ray Analyses 3.2. Physicochemical Characterization of CS-CF/5FU BNCs Using Scanning Electron Microscopy and Energy Dispersive X-ray Analyses*

SEM provides information about surface topography of the samples. Figure 2a–d show SEM images and EDX results for CF, CS NPs and CS-CF/5FU BNCs, respectively. The extracted CF was mostly in a rod-shaped structure comprised of individual and organized nano-fibrils with an average width of 50.81 ± 3.6 nm. The size of the rice straw waste gradually decreased after degradation of the hemicellulose and lignin through dewaxing, delignification and alkali treatments to liberate CF with nearly a uniform structure. CS NPs presented spherical shapes (24.17 ± 1.1 nm) and CS-CF/5FU BNCs demonstrated that CS NPs coated the CF network. An average diameter of CS-CF/5FU BNCs was found to be 48.73 ± 1.5 nm. The size of CS NPs is related to the ratio between the TPP and the chitosan powder. According to a separate report [55], the chitosan and TPP with a ratio around 1:2 could cause the formation of CS NPs with appropriate properties and nano-scale dimension, therefore, it was used in this current study. The use of the layerby-layer synthesis potentially caused that CF was coated with CS and it was almost homogenously distributed in the multi-layered particles of the BNCs with low agglomeration. This was similarly found in different investigations [44,56]. It can be possibly noticed SEM provides information about surface topography of the samples. Figure 2a–d show SEM images and EDX results for CF, CS NPs and CS-CF/5FU BNCs, respectively. The extracted CF was mostly in a rod-shaped structure comprised of individual and organized nano-fibrils with an average width of 50.81 ± 3.6 nm. The size of the rice straw waste gradually decreased after degradation of the hemicellulose and lignin through dewaxing, delignification and alkali treatments to liberate CF with nearly a uniform structure. CS NPs presented spherical shapes (24.17 ± 1.1 nm) and CS-CF/5FU BNCs demonstrated that CS NPs coated the CF network. An average diameter of CS-CF/5FU BNCs was found to be 48.73 ± 1.5 nm. The size of CS NPs is related to the ratio between the TPP and the chitosan powder. According to a separate report [55], the chitosan and TPP with a ratio around 1:2 could cause the formation of CS NPs with appropriate properties and nanoscale dimension, therefore, it was used in this current study. The use of the layer-by-layer synthesis potentially caused that CF was coated with CS and it was almost homogenously distributed in the multi-layered particles of the BNCs with low agglomeration. This was similarly found in different investigations [44,56]. It can be possibly noticed from two

spherical CS particles; thus, CS-CF/5FU BNCs mainly indicated the spherical shape. Whereas, a few CF assembled clusters of CS particles. The CF structure as a reinforcement could potentially trigger a positive influence to host 5FU and increase the drug conjugation and absorbance within the composite; moreover, the CS coating on CF possibly en-

It can be understood from the EDX results of CF that the chemical treatments on rice straw waste dissolved the silica in aqueous ions to be subsequently replaced by carbon. Compared to the EDX results of CF, the composites of CS-CF showed increasing and decreasing carbon and oxygen content, respectively (Figure 2a–c). A separate study stated that decreasing the amount of oxygen in CS-CF composites may increase the biodegradability of the CS-based composites [2]. As shown in EDX layered image (Figure 2d), CS-CF/5FU BNCs showed elements related to CS and CF as well as a negligible ratio of the

hanced the drug entrapment and encapsulation in the composites.

images of the composites that most of the rod-shaped CF was entangled in the spherical CS particles; thus, CS-CF/5FU BNCs mainly indicated the spherical shape. Whereas, a few CF assembled clusters of CS particles. The CF structure as a reinforcement could potentially trigger a positive influence to host 5FU and increase the drug conjugation and absorbance within the composite; moreover, the CS coating on CF possibly enhanced the drug entrapment and encapsulation in the composites.

It can be understood from the EDX results of CF that the chemical treatments on rice straw waste dissolved the silica in aqueous ions to be subsequently replaced by carbon. Compared to the EDX results of CF, the composites of CS-CF showed increasing and decreasing carbon and oxygen content, respectively (Figure 2a–c). A separate study stated that decreasing the amount of oxygen in CS-CF composites may increase the biodegradability of the CS-based composites [2]. As shown in EDX layered image (Figure 2d), CS-CF/5FU BNCs showed elements related to CS and CF as well as a negligible ratio of the fluorine (F) from 5FU. The above SEM and EDX results indicated that the nanocomposites successfully were synthesized to entrap anticancer drug 5FU.

#### *3.3. Physicochemical Characterization of CS-CF/5FU BNCs Using Dynamic Light Scattering*

DLS was used to estimate the size distribution of the particles in the solution. Figure 3a–c show the DLS results with values for hydrodynamic size of the synthesized samples in distilled water. The suspensions of CF, CS NPs, and CS-CF/5FU BNCs showed the hydrodynamic size of 162.58 ± 4.31, 133.18 ± 3.46, and 197.56 ± 4.12 nm, respectively. The obtained size was larger in DLS compared to that from SEM. This can be explained that DLS indicates the size of the particle and the surrounding diffuse layer of the particle, however, the SEM size is attributed to the particle itself. For the colloidal stability in the aqueous media, the presence of particle–particle interactions affected the hydrophobic attraction energy between the particles to attract each other.

The dimensional effect of the prepared nanofluid samples under various conditions was demonstrated. In order to deliver nanodrug complex into the target cells, the pH sensitivity of the fabricated samples is a vital concern. As depicted in Table 1, effects of pHs on hydrodynamic size of the nanofluid sample were evaluated using the samples at the solution with different pH values of 1.2 (strong acid), 7.4 (neutral) and 12 (strong base). All the synthesized samples indicated the size below 220 nm in the solution at the harsh conditions of strong acidic (pH 1.2) and basic (pH 12). This may possibly show that the harsh conditions did not damage the nano-dimensional of the examined samples. The results indicated that an increase in the pH of the CF solution from 1.2 to 12 slightly increased the hydrodynamic size of CF from 135.56 ± 2.87 nm to 203.17 ± 4.81 nm. Compared to CF, both CS-CF BNCs and CS-CF/5FU BNCs showed a higher enhancement of the size with increasing the pH value. This may address the pH-sensitive nature of chitosan as also stated in a different study [57]. At the solution with pH 1.2, CS NPs, CS-CF BNCs and CS-CF/5FU BNCs showed hydrodynamic size of 71.61 ± 5.54, 109.03 ± 4.12, and 112.51 ± 4.09 nm, respectively; whereas these samples indicated the size of 246.09 ± 5.10, 275.34 ± 4.59 and 274.23 ± 5.11 nm at the solution with pH 12. This increase in the hydrodynamic size of the samples could be attributed to the particle agglomeration. Particle aggregation might trigger reduced repulsive on the particles surface, due to the rise in the pH of the CF and CS solution [57]. It could be understood from the above results that the CS coating could enhance the pH sensitivity of the CF solid support to synthesize CS-CF/5FU BNCs as a pH-sensitive nanodrug formulation.

fluorine (F) from 5FU. The above SEM and EDX results indicated that the nanocomposites

successfully were synthesized to entrap anticancer drug 5FU.

**Figure 2.** SEM image, the average diameter and EDX of (**a**) CF, (**b**) CS NPs and (**c**) CS-CF/5FU BNCs; (**d**) EDX layered image of CS-CF/5FU BNCs.

(**d**) EDX layered image of CS-CF/5FU BNCs.

traction energy between the particles to attract each other.

**Figure 3.** Hydrodynamic size distributions of (**a**) CF, (**b**) CS NPs and (**c**) CS-CF/5FU BNCs. **Figure 3.** Hydrodynamic size distributions of (**a**) CF, (**b**) CS NPs and (**c**) CS-CF/5FU BNCs.

The dimensional effect of the prepared nanofluid samples under various conditions was demonstrated. In order to deliver nanodrug complex into the target cells, the pH sensitivity of the fabricated samples is a vital concern. As depicted in Table 1, effects of pHs **Table 1.** Hydrodynamic size of the synthesized samples in the solutions with various pH values at 37 ◦C. Data is expressed as the mean ± standard deviation for triplicates within an individual experiment.

**Figure 2.** SEM image, the average diameter and EDX of (**a**) CF, (**b**) CS NPs and (**c**) CS-CF/5FU BNCs;

DLS was used to estimate the size distribution of the particles in the solution. Figure 3a–c show the DLS results with values for hydrodynamic size of the synthesized samples in distilled water. The suspensions of CF, CS NPs, and CS-CF/5FU BNCs showed the hydrodynamic size of 162.58 ± 4.31, 133.18 ± 3.46, and 197.56 ± 4.12 nm, respectively. The obtained size was larger in DLS compared to that from SEM. This can be explained that DLS indicates the size of the particle and the surrounding diffuse layer of the particle, however, the SEM size is attributed to the particle itself. For the colloidal stability in the aqueous media, the presence of particle–particle interactions affected the hydrophobic at-

*3.3. Physicochemical Characterization of CS-CF/5FU BNCs Using Dynamic Light Scattering*

to CF, both CS-CF BNCs and CS-CF/5FU BNCs showed a higher enhancement of the size

#### with increasing the pH value. This may address the pH-sensitive nature of chitosan as *3.4. Physicochemical Characterization of CS-CF/5FU BNCs Using Thermal Analysis*

The thermal stability of the samples was determined by TGA. The results of TGA and differential thermogravimetric analysis (DTGA) of the fabricated samples are shown in Figure 4a–e. CF, CS NPs, CS-CF BNCs, CS-CF/5FU BNCs, and 5FU showed the main thermal degradation at 308.58, 236.54, 269.56, 298.10 and 284.60 ◦C with the final residue of 12.07, 14.53, 36.81, 32.50 and 0.91 weight % at 800 ◦C, respectively. During the thermal degradation, carbonyl and carboxyl groups could cause the reduction of the chain size and rapture the bonds of the polysaccharides of CS and CF [58]. It stated that the cellulosic materials may have a single-step degradation in nitrogen atmosphere, however, it is a two-step thermal degradation in air atmosphere [59]. In this manner, CF displayed a single-step degradation. CS-CF and CS-CF/5FU BNCs indicated a higher final residue % and thermal stability than CF and 5FU alone, showing the needs for the polymer blend of chitosan-cellulose to encapsulate anticancer drug in an advanced nano-carrier system.

(**e**) 5FU.

*Spectroscopy*

**Figure 4.** TGA and DTGA traces of (**a**) CF, (**b**) CS NPs, (**c**) CS-CF BNCs, (**d**) CS-CF/5FU BNCs and **Figure 4.** TGA and DTGA traces of (**a**) CF, (**b**) CS NPs, (**c**) CS-CF BNCs, (**d**) CS-CF/5FU BNCs and (**e**) 5FU.

*3.5. Physicochemical Characterization of CS-CF/5FU BNCs Using Fourier-Transform Infrared*  So, for the percentage of the ingredient used for the preparation of CS-CF/5FU BNCs, it has been determined that approximately 64.91, 32.64, and 2.60 % of the constituent compounds are related to CS, CF and 5FU, respectively.

#### FTIR can identify changes in the chemical structure of the sample by generating an infrared absorption spectrum. The FTIR results of rice straw waste, CF, CS NPs, CS-CF *3.5. Physicochemical Characterization of CS-CF/5FU BNCs Using Fourier-Transform Infrared Spectroscopy*

BNCs, CS-CF/5FU BNCs and 5FU are indicated in Figure 5a–f, respectively. The bleaching procedure on rice straw waste potentially triggered the development of C-H aromatic hydrogen groups. From Figure 5b, the peaks at 1524 and 1750 cm−1 are for C–O–C bonds and bleaching of hemicellulose, respectively [41]. Both peaks at 760 and 491 cm−1 are attributed to elimination of silica (Si–O–Si stretching) [41]. Further results presented that the peaks at 3352, 2891 and 1100 cm−1 might determine the stretching vibrations of -OH groups, C-H stretching, and the cellulose network structure, respectively [3]. In the anomeric region (950–700 cm−1), CF indicated a minor peak at 887 cm−1, due to presence of the glycosidic – C1 –O –C4 deformation of the β-glycosidic bond [60]. This possibly indicated the efficient chemical treatments to isolate CF. The absorption peaks are different between CS NPs and CS-CF BNCs, showing contribution of OH or NH and changes in the sugar ring, Van der Waals forces, dipole moments and hydrogen bonds [61]. In CS-CF/5FU BNCs, the CO stretching vibration at 1654 cm−1 can be owed to a good interaction between the drug and its nano-carrier. Furthermore, an overlapping between OH and NH bonds of 5FU possibly demonstrated a band at 2750–3500 cm−1, comparable with another study [19]. It stated that the amine groups of CS with carbonyl groups of CF may form functional groups of imines with carbon-nitrogen double bond [62]. FTIR can identify changes in the chemical structure of the sample by generating an infrared absorption spectrum. The FTIR results of rice straw waste, CF, CS NPs, CS-CF BNCs, CS-CF/5FU BNCs and 5FU are indicated in Figure 5a–f, respectively. The bleaching procedure on rice straw waste potentially triggered the development of C-H aromatic hydrogen groups. From Figure 5b, the peaks at 1524 and 1750 cm−1 are for C–O–C bonds and bleaching of hemicellulose, respectively [41]. Both peaks at 760 and 491 cm−1 are attributed to elimination of silica (Si–O–Si stretching) [41]. Further results presented that the peaks at 3352, 2891 and 1100 cm−1 might determine the stretching vibrations of -OH groups, C-H stretching, and the cellulose network structure, respectively [3]. In the anomeric region (950–700 cm−1 ), CF indicated a minor peak at 887 cm−1 , due to presence of the glycosidic –C1 –O –C4 deformation of the β-glycosidic bond [60]. This possibly indicated the efficient chemical treatments to isolate CF. The absorption peaks are different between CS NPs and CS-CF BNCs, showing contribution of OH or NH and changes in the sugar ring, Van der Waals forces, dipole moments and hydrogen bonds [61]. In CS-CF/5FU BNCs, the CO stretching vibration at 1654 cm−1 can be owed to a good interaction between the drug and its nano-carrier. Furthermore, an overlapping between OH and NH bonds of 5FU possibly demonstrated a band at 2750–3500 cm−1 , comparable with another study [19]. It stated that the amine groups of CS with carbonyl groups of CF may form functional groups of imines with carbon-nitrogen double bond [62].

**Figure 5.** FTIR of (**a**) rice straw waste, (**b**) CF, (**c**) CS NPs, (**d**) CS-CF BNCs, (**e**) CS-CF/5FU BNCs and (**f**) 5FU. **Figure 5.** FTIR of (**a**) rice straw waste, (**b**) CF, (**c**) CS NPs, (**d**) CS-CF BNCs, (**e**) CS-CF/5FU BNCs and (**f**) 5FU.

As indicated in Figure 5d, CS-CF/5FU present a peak at 3385 cm−1, because of –NH2 and –OH stretching groups. The interaction between NHଷ ା groups of CS and phosphate groups of TPP led to presence of CONH2 and NH2 groups with peaks at 1639 and 1548 cm−1, respectively [63]. Whereas, in the spectra of CS-CF/5FU, these peaks shifted to 1654 and 1541 cm−1, respectively. The bands at 1411 and 1016 cm−1, respectively, presented – CH2 and P=O stretching vibrations from phosphate groups [63]. Furthermore, the primary amino group of CS reacted with the carbonyl groups of CF, whereas acetic acid dissolved CS to form hydrogen ion and affect the crosslinking structure from the Schiff base bond [64]. As stated that the primary amine group in CS could trigger the in situ gelation in the CS-based composites [65]. As indicated in Figure 5d, CS-CF/5FU present a peak at 3385 cm−1 , because of –NH2 and –OH stretching groups. The interaction between NH+ 3 groups of CS and phosphate groups of TPP led to presence of CONH2 and NH2 groups with peaks at 1639 and 1548 cm−1 , respectively [63]. Whereas, in the spectra of CS-CF/5FU, these peaks shifted to 1654 and 1541 cm−1 , respectively. The bands at 1411 and 1016 cm−1 , respectively, presented –CH2 and P=O stretching vibrations from phosphate groups [63]. Furthermore, the primary amino group of CS reacted with the carbonyl groups of CF, whereas acetic acid dissolved CS to form hydrogen ion and affect the crosslinking structure from the Schiff base bond [64]. As stated that the primary amine group in CS could trigger the in situ gelation in the CS-based composites [65].

In addition, the development of the Schiff's base probably reduced the aldehyde groups on the CF surface for bonding with CS. From the spectrum of 5FU, the C–F stretching vibration is probably at 1244 cm−1. This peak was shifted to 1249 cm−1 in the spectra of the CS-CF/5FU sample [19]. As reported [66], two protonated amide groups in the 5FU molecule may lead to a positive charge of 5FU and hydrogen bonds with carboxyl bonds, albeit, there were nearly ionized together to form a hydrogen bond. In turn, the 5FU molecules were possibly conjugated to the CS structure [66]. The FTIR spectrum of CS-CF/5FU BNCs indicated only a few peaks attributed to the drug, due to an efficient entrapping of the drug within the polysaccharide composites. In addition, the development of the Schiff's base probably reduced the aldehyde groups on the CF surface for bonding with CS. From the spectrum of 5FU, the C–F stretching vibration is probably at 1244 cm−1 . This peak was shifted to 1249 cm−1 in the spectra of the CS-CF/5FU sample [19]. As reported [66], two protonated amide groups in the 5FU molecule may lead to a positive charge of 5FU and hydrogen bonds with carboxyl bonds, albeit, there were nearly ionized together to form a hydrogen bond. In turn, the 5FU molecules were possibly conjugated to the CS structure [66]. The FTIR spectrum of CS-CF/5FU BNCs indicated only a few peaks attributed to the drug, due to an efficient entrapping of the drug within the polysaccharide composites.

#### *3.6. Swelling Analysis of CS-CF/5FU BNCs*

*3.6. Swelling Analysis of CS-CF/5FU BNCs*  The swelling analysis may determine the amount of the absorbed liquid by the polymer materials. The swelling properties of composites can act importantly in the drug loading and drug release performance. Figure 6a-d show the swelling kinetic of the fabricated samples in media at different pHs at 37 °C. The samples in the solutions at different pHs showed the main swelling ratio in the first 4 h. The increased swelling index ratio could be related to the natural property of the sample containing porous interconnected fibers [67]. The sample approximately showed its equilibrium state after 12 h, comparable with different studies [32,67]. With an increasing time and pH value, the swelling increased that CF, CS NPs, CS-CF BNCs and CS-CF/5FU BNCs indicated the maximum swelling ratio of 2.91 ± 0.11, 3.49 ± 0.09, 3.77 ± 0.07 and 3.85 ± 0.10 in media at pH 12, respectively, after 36 h. Similarly, in different studies, the pH of solution controlled the swelling performances of polymer complexes, including polyvinyl alcohol/chitosan/TiO2 nanofibers [68], chitosan cross-linked poly (acrylic acid) hydrogels [69] and hydrogel composites of The swelling analysis may determine the amount of the absorbed liquid by the polymer materials. The swelling properties of composites can act importantly in the drug loading and drug release performance. Figure 6a-d show the swelling kinetic of the fabricated samples in media at different pHs at 37 ◦C. The samples in the solutions at different pHs showed the main swelling ratio in the first 4 h. The increased swelling index ratio could be related to the natural property of the sample containing porous interconnected fibers [67]. The sample approximately showed its equilibrium state after 12 h, comparable with different studies [32,67]. With an increasing time and pH value, the swelling increased that CF, CS NPs, CS-CF BNCs and CS-CF/5FU BNCs indicated the maximum swelling ratio of 2.91 ± 0.11, 3.49 ± 0.09, 3.77 ± 0.07 and 3.85 ± 0.10 in media at pH 12, respectively, after 36 h. Similarly, in different studies, the pH of solution controlled the swelling performances of polymer complexes, including polyvinyl alcohol/chitosan/TiO2 nanofibers [68], chitosan cross-linked poly (acrylic acid) hydrogels [69] and hydrogel composites of carboxymethylstarch-g-poly (acrylic acid)/palygorskite/starch/sodium algi-

carboxymethylstarch-g-poly (acrylic acid)/palygorskite/starch/sodium alginate [70]. As

nate [70]. As presented in Figure 6a–d, CS NPs and the CS-CF composites displayed higher swelling properties compared to CF. Therefore, the CS structure domain the swelling properties of the CS-CF composites. The increasing swelling properties of the tested samples were in line with the hydrodynamic size in the solution with various pH values. The above swelling results could indicate the necessity of using CS to coat the CF matrix for increasing pH sensitivity of the CS-CF composites. presented in Figure 6a–d, CS NPs and the CS-CF composites displayed higher swelling properties compared to CF. Therefore, the CS structure domain the swelling properties of the CS-CF composites. The increasing swelling properties of the tested samples were in line with the hydrodynamic size in the solution with various pH values. The above swelling results could indicate the necessity of using CS to coat the CF matrix for increasing pH sensitivity of the CS-CF composites.

**Figure 6.** Swelling kinetic of (**a**) CF, (**b**) CS NPs, (**c**) CS-CF BNCs, and (**d**) CS-CF/5FU BNCs initiated from the wet state in three different media at pH 1.2, 7.4, and 12. Data is expressed as the mean ± standard deviation for triplicates within an individual experiment. **Figure 6.** Swelling kinetic of (**a**) CF, (**b**) CS NPs, (**c**) CS-CF BNCs, and (**d**) CS-CF/5FU BNCs initiated from the wet state in three different media at pH 1.2, 7.4, and 12. Data is expressed as the mean ± standard deviation for triplicates within an individual experiment.

#### *3.7. Drug Loading and Encapsulation Efficiency Percentage of CS-CF/5FU BNCs 3.7. Drug Loading and Encapsulation Efficiency Percentage of CS-CF/5FU BNCs*

Encapsulation efficiency was used to estimate the successful drug loading ratio in a nano-carrier system. Figure 7a(i–iii) indicate the 5FU absorbance, calibration curve at λmax = 266 nm from known concentrations of 5FU (5–25 ppm), and UV-vis spectra of the unentrapped 5FU, respectively. From the UV absorbance and Equation (1) the encapsulation efficiency value was estimated to be 86 ± 2.75%. The alkali treated CF possibly possessed open bonds and good swelling properties to host 5FU [71]. The CS-CF composites could show the OH groups for effectively binding and releasing 5FU by ionic interactions, as reported [72]. Furthermore, 5FU is a heterocyclic aromatic organic compound with a low molecular weight to be diffused well within open pores and substrate of the CF [73]; whereas the CS coated both CF and 5FU to improve encapsulation efficiency. From the value of encapsulation efficiency, the synthesized composites could be a suitable nanocarrier for the 5FU encapsulation in cancer treatment. Encapsulation efficiency was used to estimate the successful drug loading ratio in a nano-carrier system. Figure 7a(i–iii) indicate the 5FU absorbance, calibration curve at λmax = 266 nm from known concentrations of 5FU (5–25 ppm), and UV-vis spectra of the unentrapped 5FU, respectively. From the UV absorbance and Equation (1) the encapsulation efficiency value was estimated to be 86 ± 2.75%. The alkali treated CF possibly possessed open bonds and good swelling properties to host 5FU [71]. The CS-CF composites could show the OH groups for effectively binding and releasing 5FU by ionic interactions, as reported [72]. Furthermore, 5FU is a heterocyclic aromatic organic compound with a low molecular weight to be diffused well within open pores and substrate of the CF [73]; whereas the CS coated both CF and 5FU to improve encapsulation efficiency. From the value of encapsulation efficiency, the synthesized composites could be a suitable nano-carrier for the 5FU encapsulation in cancer treatment.

*Nanomaterials* **2021**, *11*, x FOR PEER REVIEW 16 of 22

**Figure 7.** (**a**) (**i**) The 5FU absorbance and (**ii**) calibration curve at λmax = 266 nm from known concentrations of 5FU (5–25 ppm); (**iii**) UV-vis spectra of the unentrapped 5FU; (**b**) UV-vis spectra and (**c**) the release kinetics of 5FU from CS-CF/5FU BNCs; (**d**) a schematic of possible drug release. Data is expressed as the mean ± standard deviation for triplicates within an individual experiment. **Figure 7.** (**a**) (**i**) The 5FU absorbance and (**ii**) calibration curve at λmax = 266 nm from known concentrations of 5FU (5–25 ppm); (**iii**) UV-vis spectra of the unentrapped 5FU; (**b**) UV-vis spectra and (**c**) the release kinetics of 5FU from CS-CF/5FU BNCs; (**d**) a schematic of possible drug release. Data is expressed as the mean ± standard deviation for triplicates within an individual experiment.

#### *3.8. In Vitro Drug Release of CS-CF/5FU BNCs 3.8. In Vitro Drug Release of CS-CF/5FU BNCs*

In vitro drug release analysis was used to determine the release kinetics of the drugloaded samples. Figure 7b,c shows UV-vis spectra and the release kinetics of 5FU from CS-CF/5FU BNCs, respectively. As seen in Figure 7c, the time taken was 8 h for the drug release of 50.60 ± 1.88% and 28.04 ± 1.14% at the release fluids with pH 7.4 and pH 1.2, respectively. After 36 h, the total 5FU release was lower in the simulated acidic fluid at pH 1.2 (42.37 ± 0.43%) compared to that in the simulated colorectal fluid at pH 7.4 (76.82 ± 1.29%). This can be explained that CS-CF/5FU BNCs became smaller in the acidic media [67]. In the human body, the above may possibly result in a safe transferring of CS-CF/5FU BNCs through the acidic fluid of the stomach and then it may begin to break up in the In vitro drug release analysis was used to determine the release kinetics of the drugloaded samples. Figure 7b,c shows UV-vis spectra and the release kinetics of 5FU from CS-CF/5FU BNCs, respectively. As seen in Figure 7c, the time taken was 8 h for the drug release of 50.60 ± 1.88% and 28.04 ± 1.14% at the release fluids with pH 7.4 and pH 1.2, respectively. After 36 h, the total 5FU release was lower in the simulated acidic fluid at pH 1.2 (42.37 ± 0.43%) compared to that in the simulated colorectal fluid at pH 7.4 (76.82 ± 1.29%). This can be explained that CS-CF/5FU BNCs became smaller in the acidic media [67]. In the human body, the above may possibly result in a safe transferring of CS-CF/5FU BNCs through the acidic fluid of the stomach and then it may begin to break

up in the colorectal fluid [67]. The low molecular weight of 5FU may cause a tendency for consecutive release, albeit, the cellulosic material as an amphipathic drug carrier and even excipient may modulate the release dosage [73,74]. The drug release showed an analogous trend with the swelling ratio of the samples, as schematically depicted in Figure 7d. An investigation stated that the controlled release ratio could be related to the interaction between 5FU and –COOH functionalities presented in the cellulosic material as it decreased when the swelling capacity increased [19]. The swelling may lead to polymer relaxation and gelling of 5FU to obtain a controlled drug release profile from CS-CF/5FU BNCs [74]. CS-CF/5FU BNCs presented a sustained and pH-sensitive release behavior, therefore, it may be considered as an attractive drug formulation candidate for modern CRC treatments.

#### *3.9. In Vitro Cytotoxicity Assays*

The biocompatibility and potential anticancer effects of the samples were studied by in vitro cytotoxicity assays and antibacterial assay (Supplementary Materials Figure S1). The cytotoxicity of CF, CS NPs, CS-CF BNCs, CS-CF/5FU BNCs against HCT116 CRC and CCD112 normal colon cell lines are shown in Figure 8a–d. CF exhibited almost no prominent killing towards both normal and cancer cell lines (Figure 8a). Different reports stated that the wood-based nanocelluloses possess no adverse toxicity and based on ISO standard 10993-5, it does not have toxicity above 30% [75]. From Figure 8b, 15.62 µg/mL of CS NPs was enough to eliminate 26.46 ± 6.52% of the CRC cells without any toxicity on the CCD112 normal cell. In addition, the highest concentration (500 µg/mL) of CF, CS NPs, and CS-CF BNCs showed an appropriate biocompatibility without any damage against the normal cells. Among the samples without the loaded-5FU, CS-CF BNCs displayed better anticancer effect (41.62 ± 3.15%), showing a suitable contribution between CS and CF in the composites. As previously stated [15], the therapeutic potency of the chitosan-based samples could be explained that the positive charge of chitosan neutralized the negative charge on the tumor cell surface [14]. A different study also reported that the strong bonding between the primary amino group of chitosan and the carbonyl of cellulose could block bacteria from crossing the biocomposites [49]. It was indicated that the anticancer effects and permeation enhancement of CS-based materials could be related to the primary amine group in CS [65].

It can be noticed from Figure 8d that certain concentrations of CS-CF/5FU BNCs gave only negligible damage on the normal cells, while exhibiting effective anti-proliferation actions towards the cancer cells.  This anticancer effect might be attributed to the properties of the synthesized double polysaccharides to encapsulate a sufficient amount of 5FU for potentially improving mobility, binding ability, and colloidal stability to attach on the cell surface, as reported earlier [75]. Besides, based on a different study [17], polysaccharide complex might conjugate onto cancer cells to improve selectivity and decrease the drug leakage from the selected cells for saving the drug from unwanted degradation and elimination [16]. The polymeric network of CS-CF entrapped 5FU to potentially obtain a prolonged release dosage of the drug, similar to a separate report [16]. In the drug-loaded chitosan materials, the primary amine groups can possibly cause the controlled drug release [65]. As shown in Table 2, no activity was seen in the synthesized samples on the normal cells which suggests its high biocompatibility. Among all the synthesized samples, the IC50 happened only for the drug loaded sample that CS-CF/5FU BNCs showed the IC50 of 228.27 µg/mL on the cancer cells. It could be understood from the results of in vitro cytotoxicity assays that the polysaccharide samples of CF and CS have a suitable biocompatibility and CS-CF/5FU BNC suggests promising anticancer actions.

ments.

*3.9. In Vitro Cytotoxicity Assays*

mary amine group in CS [65].

effects and permeation enhancement of CS-based materials could be related to the pri-

colorectal fluid [67]. The low molecular weight of 5FU may cause a tendency for consecutive release, albeit, the cellulosic material as an amphipathic drug carrier and even excipient may modulate the release dosage [73,74]. The drug release showed an analogous trend with the swelling ratio of the samples, as schematically depicted in Figure 7d. An investigation stated that the controlled release ratio could be related to the interaction between 5FU and –COOH functionalities presented in the cellulosic material as it decreased when the swelling capacity increased [19]. The swelling may lead to polymer relaxation and gelling of 5FU to obtain a controlled drug release profile from CS-CF/5FU BNCs [74]. CS-CF/5FU BNCs presented a sustained and pH-sensitive release behavior, therefore, it may be considered as an attractive drug formulation candidate for modern CRC treat-

The biocompatibility and potential anticancer effects of the samples were studied by in vitro cytotoxicity assays and antibacterial assay (Supplementary Materials Figure S1). The cytotoxicity of CF, CS NPs, CS-CF BNCs, CS-CF/5FU BNCs against HCT116 CRC and CCD112 normal colon cell lines are shown in Figure 8a–d. CF exhibited almost no prominent killing towards both normal and cancer cell lines (Figure 8a). Different reports stated that the wood-based nanocelluloses possess no adverse toxicity and based on ISO standard 10993-5, it does not have toxicity above 30% [75]. From Figure 8b, 15.62 µg/mL of CS NPs was enough to eliminate 26.46 ± 6.52% of the CRC cells without any toxicity on the CCD112 normal cell. In addition, the highest concentration (500 µg/mL) of CF, CS NPs, and CS-CF BNCs showed an appropriate biocompatibility without any damage against the normal cells. Among the samples without the loaded-5FU, CS-CF BNCs displayed better anticancer effect (41.62 ± 3.15%), showing a suitable contribution between CS and CF in the composites. As previously stated [15], the therapeutic potency of the chitosanbased samples could be explained that the positive charge of chitosan neutralized the negative charge on the tumor cell surface [14]. A different study also reported that the strong bonding between the primary amino group of chitosan and the carbonyl of cellulose could

**Figure 8.** In vitro anticancer activity of (**a**) CF, (**b**) CS NPs, (**c**) CS-CF BNCs and (**d**) CS-CF/5FU BNCs against CCD112 colon normal and HCT116 CRC cell lines. Three independent experiments were **Figure 8.** In vitro anticancer activity of (**a**) CF, (**b**) CS NPs, (**c**) CS-CF BNCs and (**d**) CS-CF/5FU BNCs against CCD112 colon normal and HCT116 CRC cell lines. Three independent experiments were performed and the data are expressed as the mean ± standard deviation for all triplicates within an individual experiment.

#### **4. Conclusions**

In this study, lignin and hemicellulose of rice straw waste were degraded by a series of chemical modifications include dewaxing, bleaching, and alkali treatments to liberate CF containing organized nanofibrils. By using the ionic gelation method, CS-CF composites encapsulated 5FU to synthesize nanodrug candidate of CS-CF/5FU BNCs for in vitro CRC analysis. The composites indicated the XRD and FTIR peaks related to the CS cross-linked CF. The SEM images of CS-CF/5FU BNCs showed that the spherical CS NPs covered the rod-shaped CF reinforcement. Based on swelling and hydrodynamic size analysis, the composites indicated a pH responsive behavior. Analogously, CS-CF/5FU BNCs exhibited a lower release ratio in media simulating the gastric fluid at pH 1.2 compared to the colorectal fluid at pH 7.4. In cytotoxicity assays, all the synthesized polysaccharide samples did not indicate the IC50 against the CCD112 normal colon cells, showing their proper biocompatibility and safety for anticancer applications. Among the fabricated samples, CS-CF/5FU BNCs only displayed the IC50 value (228.27 µg/mL) against the HCT116 cancer cells. Further results presented that the 250 µg/mL concentration of CS-CF/5FU BNCs showed better anticancer actions than other concentrations. The intriguing implication of this research is that using a very low dosage of 5-FU encapsulated in CS/CF composites could reduce the manufacturing cost and make it an effective green nanodrug candidate for anticancer actions against CRC.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/nano11071691/s1, Figure S1: Antibacterial activity of CF, CS NPs, CS-CF BNCs and CS-CF/5-FU BNCs against (a) gram-positive *Staphylococcus aureus* and (b) gram-negative *Escherichia coli.*

**Author Contributions:** The manuscript was written through contributions of all authors. Conceptualization, methodology, writing—original draft, fabrication of the samples, the physiochemical analysis, drug loading and drug release study, contribution for in vitro anticancer assays, M.Y.; review & editing, analysis of anticancer assays, H.-Y.C.; supervision, validation and writing—review & editing, analysis of anticancer assays, S.-Y.T.; contribution in SEM analysis, drug loading, and drug release study, P.K.; investigation, in vitro anticancer assays, M.L.-K.S.; supervision, validation, N.A.B.C.S.; supervision, project administration, formal analysis, sample preparation, validation and writing—review & editing, funding acquisition, K.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Takasago Thermal Engineering Co. Ltd. grants including R.K130000.7343.4B422, 4B472 and 4B631. Also, we have express gratitude to the research management center (RMC) of Universiti Teknologi Malaysia (UTM) and Malaysia-Japan International Institute of Technology (MJIIT).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** Special thanks to School of Medical and Life Sciences, Sunway University Malaysia for the cell culture and laboratory facilities.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

## *Article* **Cellulose Nanocrystals/Chitosan-Based Nanosystems: Synthesis, Characterization, and Cellular Uptake on Breast Cancer Cells**

**Ricardo J. B. Pinto 1 , Nicole S. Lameirinhas 1 , Gabriela Guedes 1 , Gustavo H. Rodrigues da Silva 1 , Párástu Oskoei 2 , Stefan Spirk 3 , Helena Oliveira 2 , Iola F. Duarte 1 , Carla Vilela 1,\* and Carmen S. R. Freire 1,\***

**Citation:** Pinto, R.J.B.; Lameirinhas, N.S.; Guedes, G.; Rodrigues da Silva, G.H.; Oskoei, P.; Spirk, S.; Oliveira, H.; Duarte, I.F.; Vilela, C.; Freire, C.S.R. Cellulose Nanocrystals/Chitosan-Based Nanosystems: Synthesis, Characterization, and Cellular Uptake on Breast Cancer Cells. *Nanomaterials* **2021**, *11*, 2057. https://doi.org/10.3390/nano 11082057

Academic Editor: Hirotaka Koga

Received: 18 June 2021 Accepted: 9 August 2021 Published: 12 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Abstract:** Cellulose nanocrystals (CNCs) are elongated biobased nanostructures with unique characteristics that can be explored as nanosystems in cancer treatment. Herein, the synthesis, characterization, and cellular uptake on folate receptor (FR)-positive breast cancer cells of nanosystems based on CNCs and a chitosan (CS) derivative are investigated. The physical adsorption of the CS derivative, containing a targeting ligand (folic acid, FA) and an imaging agent (fluorescein isothiocyanate, FITC), on the surface of the CNCs was studied as an eco-friendly methodology to functionalize CNCs. The fluorescent CNCs/FA-CS-FITC nanosystems with a rod-like morphology showed good stability in simulated physiological and non-physiological conditions and non-cytotoxicity towards MDA-MB-231 breast cancer cells. These functionalized CNCs presented a concentration-dependent cellular internalization with a 5-fold increase in the fluorescence intensity for the nanosystem with the higher FA content. Furthermore, the exometabolic profile of the MDA-MB-231 cells exposed to the CNCs/FA-CS-FITC nanosystems disclosed a moderate impact on the cells' metabolic activity, limited to decreased choline uptake and increased acetate release, which implies an anti-proliferative effect. The overall results demonstrate that the CNCs/FA-CS-FITC nanosystems, prepared by an eco-friendly approach, have a high affinity towards FR-positive cancer cells and thus might be applied as nanocarriers with imaging properties for active targeted therapy.

**Keywords:** cellulose nanocrystals; chitosan; folic acid; fluorescein isothiocyanate; nanosystems; physical adsorption; cellular uptake; cellular exometabolomics; folate receptor-positive cancer cells

#### **1. Introduction**

Polysaccharides are natural polymers composed of monosaccharides linked by glycosidic bonds that hold great promise as eco-friendly building-blocks to develop advanced functional materials [1]. Within the vast portfolio of polysaccharides, cellulose (i.e., linear homopolysaccharide composed of β-D-glucopyranose units linked by β-(1,4) glycosidic bonds) is amongst the most studied natural polymer in multiple fields of research [2–5]. This polysaccharide, and particularly its nanoscale forms, viz. cellulose nanocrystals (CNCs), cellulose nanofibrils (CNFs) and bacterial nanocellulose (BNC) [6], are showing incredible potential as precursors of nanomaterials for a deluge of applications, including in the fight against cancer [7–9]. In fact, the number of developed systems for cancer diagnosis and treatment [7,9,10] is increasing at a fast pace since this large group of diseases is the second leading cause of death globally, with ca. 10 million deaths in 2020 [11,12].

CNCs have a rod-like morphology [13] and are of particular relevance in this context. Recent studies have demonstrated that the shape of the nanocarriers is an important parameter for their efficiency, and that elongated or filamentous nanostructures present several benefits over spherical ones in terms of surface area-to-volume ratio, rate of clearance from the body and elimination mechanism, as well as an enhanced uptake rate by tumor cells [14–17]. Moreover, CNCs are rich in hydroxyl groups that enable easy coupling (covalent and non-covalent) of targeting, imaging, and therapeutic agents [9,18] to actively target cancerous cells [19].

As an example, Roman and co-workers have looked extensively into the use of CNCs as nanocarriers by the functionalization of these elongated nanostructures solely with fluorescein isothiocyanate (FITC) as an imaging agent [20–22] or with both FITC and folic acid (FA) as imaging and targeting agents [23–25], respectively.  Following the same idea, Raja et al. [26] chemically modified CNCs via covalent tethering of PEGylated biotin (targeting ligand) and perylenediimide (imaging agent) and demonstrated their aptness for cell labelling and imaging of fundamental cells of the immune system (J774A.1 macrophages and primary DCs), the connective tissue (NIH-3T3 fibroblasts), and a severe pathological state (HeLa adenocarcinoma cells).

Despite the promising results of the previously enumerated studies, the functionalization of the CNCs nanosystems was mostly performed via multiple synthesis steps and with fairly toxic reagents [20–26], and thus the need for alternative methodologies [18,27] is of utmost importance. Therefore, the use of CNCs in conjunction with a chitosan (CS, i.e., linear heteropolysaccharide obtained from chitin via *N*-deacetylation [28]) derivative with targeting and imaging ligands seems an interesting approach (inspired by their opposite surface charge) that has never been addressed before. In this scenario, the present study describes the synthesis, characterization, and cellular uptake on breast cancer cells (MDA-MB-231 cell line) of a dual polysaccharide nanosystem based on CNCs and a CS derivative with opposite surface charges. The physical adsorption of the chitosan derivative, containing targeting (FA) and imaging (FITC) ligands, on the surface of the CNCs was studied as an environmentally friendly approach to engineer CNCs nanosystems with enhanced cellular internalization by MDA-MB-231 cells. The CNCs nanosystems were characterized in terms of structure, optical properties, fluorescence, stability in simulated physiological and non-physiological conditions, morphology, in vitro cytotoxicity, cellular internalization, and extracellular metabolomic profile to evaluate their suitability for the targeting and imaging of breast cancer cells.

#### **2. Materials and Methods**

#### *2.1. Chemicals, Materials, and Cells*

Acetic acid (97.0%, Sigma-Aldrich (St. Louis, MO, USA)), chitosan (CS, purified powder, MW 15,000, >85% degree of deacetylation, viscosity >90 cPs at 1% solution, Polysciences (Warrington, PA, USA)), Azpack™ cotton wool (BP grade, Fisher Scientific (Hampton, NH, USA)), dimethyl sulfoxide (DMSO, >99%, LabScan (Bangkok, Thailand)), 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT, 98%, Sigma-Aldrich (St. Louis, MO, USA)), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, ≥98.0%, Sigma-Aldrich (St. Louis, MO, USA)), fluorescein isothiocyanate (FITC, isomer I, 90%, Sigma-Aldrich (St. Louis, MO, USA)), folic acid (FA, 97.0%, Sigma-Aldrich (St. Louis, MO, USA)), methanol (99.9%, Fisher Chemical (Hampton, NH, USA)), phosphate buffered saline (PBS, pH 7.2, Sigma-Aldrich (St. Louis, MO, USA)), sodium hydroxide (99.0%, Fisher Chemical (Hampton, NH, USA)), and phosphotungstic acid hydrate (HPW, p.a., Carl Roth (Karlsruhe, Germany)) were used as received. Other chemicals and solvents were of laboratory grade.

RPMI 1640 medium, fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, fungizone (250 U mL−1 ) and trypsin–ethylenediaminetetraacetic acid (EDTA) (0.25% trypsin and 1 mM EDTA) were purchased from Gibco® (Life Technologies, Carlsbad, CA, USA). Type 1 ultrapure water (resistivity of 18.2 MΩ cm (25 ◦C)) was filtered by a Simplicity® Water Purification System (Merck Millipore, Darmstadt, Germany). The Spectra/Por® regenerated cellulose dialysis membranes (MWCO 6–8 kDa) were purchased from Daigger Scientific (Hamilton, NJ, USA). The MDA-MB-231 breast cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

#### *2.2. Preparation of Cellulose Nanocrystals (CNCs)*

The CNCs were prepared via acid hydrolysis with phosphotungstic acid according to a modified literature protocol [29]. In a typical procedure, 322 g of HPW (111 mmol) and water (110 mL) were placed in a 500 mL three-neck round bottom flask equipped with a reflux condenser. Then, the mixture was heated to 90 ◦C and thoroughly washed cellulose cotton fibers (9.0 g, repeated washing with distilled water to remove impurities) were added in small portions. To achieve hydrolysis of the cotton fibers, the mixture was stirred for 48 h under reflux. After cooling to room temperature under vigorous stirring, the mixture was transferred to a 3 L extraction funnel. Diethyl ether (500 mL) and water (250 mL) were added, resulting in the formation of three phases (heaviest phase: HPW/ethanol/water, medium phase: aqueous, upper phase: ethanol). The medium aqueous phase was separated, and the CNCs were purified by repeated centrifugation and washing with NaOH (0.1 M) and distilled water to remove traces of tungstate. The average yield after freeze-drying (5 experiments) was 86.3 ± 3.1%.

The obtained CNCs have an elemental composition of 40.3 ± 0.4% of carbon (C), 5.4 ± 0.1% of hydrogen (H) and 54.3 ± 0.5% of oxygen (O) determined by elemental analysis (LECO TruSpec 630-200-200 CHNS, LECO Corporation, Michigan, USA), dimensions of 45 ± 12 nm (diameter) and 364 ± 74 nm (length) (micrographs acquired on a HR-SEM-SE SU-70 microscope, Hitachi High-Technologies Corporation, Tokyo, Japan), and zeta (ζ)-potential of −11.5 ± 0.7 mV at pH 7 (Zetasizer Nano ZS, Malvern Panalytical, Cambridge, UK).

#### *2.3. Synthesis of the FA-CS-FITC Derivative*

Firstly, CS (ζ-potential of +68.4 ± 2.1 mV at pH 3) was functionalized with folic acid (i.e., CS-FA derivative) following the methodology described by Lee et al. [30] with some modifications. Briefly, EDC (20 mg) was added to an FA solution (20 mg in 10 mL DMSO) and allowed to react for 90 min at room temperature to activate the carboxylic groups of FA. This solution was then added dropwise to a CS solution (100 mg in 20 mL of 1 M acetic acid aqueous solution) under magnetic stirring, in an ice bath, and the pH was adjusted to 6 using an aqueous solution of NaOH (1 M). The reaction mixture was kept under stirring over 48 h at room temperature in the dark. The CS-FA derivative was dialyzed in distilled water for three days to remove the unreacted FA, followed by freeze-drying, which generated a yellowish solid with an elemental composition of 47.9 ± 0.4% of C, 6.2 ± 0.3% of H, 33.0 ± 0.5% of O, and 12.9 ± 0.3% of nitrogen (N), and a ζ-potential value of +57.0 ± 4.4 mV at pH 3.

The FA-CS-FITC derivative was synthesized via an adapted method described by Huang et al. [31]. A solution of FITC (12 mg in 18 mL of methanol) was added dropwise to a solution of CS-FA (60 mg in 18 mL of 1 M acetic acid aqueous solution) under magnetic stirring. The reaction mixture was allowed to react for 24 h at room temperature in the dark. Then, the pH of the solution was adjusted to 10 with an aqueous solution of NaOH (1 M). The solution containing the FA-CS-FITC derivative was dialyzed in distilled water for four days, followed by freeze-drying, which produced a light orange solid with an elemental composition of 50.0 ± 0.5% of C, 5.3 ± 0.3% of H, 37.5 ± 0.4% of O, 6.6 ± 0.1% of N and 0.6 ± 0.1% of sulphur (S), and a ζ-potential value of +51.9 ± 3.6 mV at pH 3.

#### *2.4. Preparation of the CNC Nanosystems Functionalized with the FA-CS-FITC Derivative*

The CNCs were functionalized with the FA-CS-FITC derivative via electrostatic assembly. Briefly, a solution of FA-CS-FITC derivative (1, 2 or 4 mg in 40 mL of 1 M acetic acid aqueous solution) was added dropwise to a suspension of CNCs (50 mg in 10 mL of ultrapure water), as summarized in Table 1. The mixture stood for 1 h under magnetic stirring in the dark. Afterwards, the suspension was centrifuged (13,400 rpm, 10 min, Megafuge 16R centrifuge (Thermo Scientific, Waltham, MA, USA)) and washed one time with 1 M acetic acid aqueous solution and three times with ultrapure water, followed by freeze-drying, which yielded solid nanosystems with an orange coloration.

**Table 1.** Composition of the prepared CNCs/FA-CS-FITC nanosystems.

a The nominal composition is the initial mass of CNCs (WCNCs) and CS derivative (WFA-CS-FITC). b The measured composition was indirectly determined by measuring the absorbance of the washing solutions of each nanosystem at 441 nm (calibration curve: *y* = 2.2591*x* − 0.0141, *R* 2 = 0.9995), concentration range of the FA-CS-FITC derivative: 0.005–0.5 mg mL−1 ).

#### *2.5. Characterization Methods*

The Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectra of all samples were collected in the solid-state in a Perkin-Elmer FT-IR System Spectrum BX spectrophotometer (Perkin-Elmer, Waltham, MA, USA), coupled with a single horizontal Golden Gate ATR cell (Specac®, London, UK), using 32 scans at a resolution of 4 cm−1 in the wavenumber range of 600–4000 cm−1 .

The optical spectra were recorded on a Thermo Scientific Evolution 220 UV-visible spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) using 100 scans min−1 with a bandwidth of 2 nm and an integration time of 0.3 s in the wavelength range of 250–600 nm. The CS and the FA-CS-FITC samples were analyzed in acidic aqueous solutions (1 M of acetic acid), and the FA and FITC samples were examined in DMSO and ethanol (1 mg mL−1 ), respectively. For the solid samples, namely the CNCs and CNCs/FA-CS-FITC\_3 nanosystem, a Jasco V-560 UV-visible spectrophotometer (JASCO Corporation, Tokyo, Japan) was utilized also in the wavelength range of 250–600 nm.

The fluorescence emission spectra were obtained on a Horiba Jobin-Yvon FluoroMax-4 spectrofluorometer (Horiba Jobin-Yvon, Kyoto, Japan) with a 2.0 nm width of both excitation and emission slits, and an integration time of 0.1 s in the wavelength range of 500–650 nm. The appropriate excitation wavelengths (λex) were obtained as the wavelength of maximum absorption as follows: λex = 450 nm for the FITC sample in ethanol (1 mg mL−1 ) and λex = 440 nm for the FA-CS-FITC in an acidic aqueous solution (ca. 1 mg mL−1 , 1 M of acetic acid). The fluorescence spectrum of the solid CNCs/FA-CS-FITC\_3 nanosystem was recorded using a Jasco spectrofluorometer FP-8300 (JASCO Corporation, Tokyo, Japan) equipped with a xenon lamp, using a scan speed of 200 nm min−1 , bandwidth of excitation and emission of 5 nm and excitation wavelength of 495 nm, also in the wavelength range of 500–650 nm.

Zeta potential measurements were performed on a Malvern ZetaSizer Nano-ZS (Malvern Panalytical, Cambridge, UK) at room temperature and different media depending on the analyzed sample, as described above. All measurements were performed in triplicate.

Scanning transmission electron microscopy (STEM) images were acquired in a fieldemission gun (FEG) SEM Hitachi SU-70 microscope (Hitachi High-Technologies Corporation, Tokyo, Japan) operated at 15 kV. Samples were prepared by placing a drop of the suspensions of CNCs and CNCs/FA-CS-FITC\_3 nanosystem directly onto carbon-coated copper grids and allowing the solvent to evaporate. The size (diameter and length) of the

samples was determined by measuring over 100 rods (elongated nanostructures) for each sample, using the Fiji image processing software.

#### *2.6. Stability Tests*

The stability of the CNCs/FA-CS-FITC nanosystems was evaluated in simulated nonphysiological and physiological conditions, namely at pH 2.1 (aqueous solution of 0.01 M HCl) and at pH 7.2 (PBS), for 24 h and 48 h [32]. Typically, 1 mg of CNCs/FA-CS-FITC was added to vials containing 2 mL of the two media. Then, the suspensions were placed on an orbital shaker at 37 ◦C in the dark for 24 h and 48 h. After these periods, the suspensions were centrifugated at 13,400 rpm during 5 min (Megafuge 16R centrifuge, Thermo Scientific, Waltham, MA, USA). The absorbance spectra of the corresponding supernatants were recorded on a Thermo Scientific Evolution 220 UV-visible spectrophotometer (Thermo Scientific, Waltham, MA, USA) to quantify the amount of the derivative that was released (calibration curve: *y* = 2.2591*x* − 0.0141, *R* 2 = 0.9995), concentration range of the FA-CS-FITC derivative: 0.005–0.5 mg mL−1 ).

#### *2.7. Cell Culture*

MDA-MB-231 cells were cultured in RPMI culture medium with L-glutamine, without folic acid, supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin (10,000 U mL−1 ), and 1% fungizone (250 U mL−1 ), at 37 ◦C in a humidified atmosphere with 5% CO2. Cells were daily observed for confluence and morphology using an inverted phase-contrast Eclipse TS100 microscope (Nikon, Tokyo, Japan). Sub-confluent cells were trypsinized with trypsin-EDTA (0.25% trypsin, 1 mM EDTA) when monolayers reached 70% of confluence.

#### *2.8. In Vitro Cytotoxicity Assay*

Cells were seeded in a 96-well plate at 20,000 cells/well and, after cell adhesion, the cell culture medium (in the 96 well plates) was replaced with fresh medium containing CNCs and CNCs/FA-CS-FITC nanosystems at 0, 12.5, 25, 50, 100, 200 µg mL−1 and then further incubated for 24 h at 37 ◦C, 5% CO2 humidified atmosphere.

At the end of the exposure time, 50 µL of MTT solution (1 mg mL−1 in PBS pH 7.2) were added to the medium and cells were incubated for 4 h. After that, the culture medium with MTT was removed and replaced by 150 µL of DMSO and the plate was placed in a shaker for 2 h in the dark to completely dissolve the formazan crystals. The absorbance of the samples was measured with a BioTek Synergy HT plate reader (Synergy HT Multi-Mode, BioTeK, Winooski, VT, USA) at 570 nm with blank corrections. The cell viability was calculated with respect to the control cells:

$$\text{Cell viability} \left( \% \right) = \left[ \left( \text{Abs}\_{\text{sample}} - \text{Abs}\_{\text{DMSO}} \right) / \left( \text{Abs}\_{\text{control}} - \text{Abs}\_{\text{DMSO}} \right) \right] \times 100 \tag{1}$$

where *Abssample* is the absorbance of the sample, *AbsDMSO* is the absorbance of the DMSO solvent and *Abscontrol* is the absorbance of the control.

#### *2.9. Cellular Uptake Assay*

Cells were seeded in 24-well plates at the concentration of 138,320 cells/well and then incubated for 24 h at 37 ◦C and 5% CO2, for cell adherence. The culture medium was then replaced with 500 µL of growth medium with pristine CNCs and CNCs/FA-CS-FITC nanosystems at 200 µg mL−1 , and the cells were incubated with the previously described culture conditions. Acellular medium and control cells (no treatment) were also incubated. After the 24 h incubation period, the medium was collected and 400 µL aliquots were stored at −80 ◦C. Following washing with 250 µL of PBS, the cells were trypsinized and 250 µL of growth medium was added to neutralize trypsin. Two parameters, namely side-scattered light, and side-fluorescence light (excitation at 488 nm and measurement with a 530/30 band pass filter), were measured in an Attune® Acoustic Focusing Cytometer

(ThermoFisher Scientific, Waltham, MA, USA). At least 50,000 cells were examined for each test. The data were analyzed by FlowJo software (FlowJo LLC, Ashland, OR, USA).

#### *2.10. Cellular Exometabolomics*

For the NMR analysis, the medium samples collected during the cellular uptake assay (described in Section 2.9) were processed to remove interfering proteins. Briefly, 700 µL of cold methanol were added to 350 µL of medium, followed by 30 min resting at −20 ◦C, centrifugation (13,000× *g*, 20 min) and vacuum drying of the supernatant. Dried samples were then reconstituted in 600 µL of deuterated PBS (100 mM, pH 7.4) and transferred into 5 mm NMR tubes.

The NMR spectra were acquired on a Bruker Avance III HD 500 NMR spectrometer (Bruker Corporation, Billerica, MA, USA) operating at 500.13 MHz for 1H observation using a 5 mm TXI probe. Standard 1D 1H spectra with water pre-saturation (pulse program 'noesypr1d', Bruker library) were recorded with 32 k points, 7002.801 Hz spectral width, a 2 s relaxation delay and 512 scans. Spectral processing (TopSpin 4.0.3, Bruker BioSpin) comprised cosine multiplication (ssb 2), zero-filling to 64 k data points, manual phasing, baseline correction, and calibration to the TSP-*d4* signal (δ 0 ppm). Selected signals representative of the main metabolites detected were then integrated (Amix-Viewer 3.9.15, Bruker Biospin) and, for each metabolite, the fold change relative to the acellular medium was calculated for control and exposed groups to assess the magnitude of consumptions/secretions.

#### *2.11. Statistical Analysis*

Cellular viability, uptake and exometabolomics data were analyzed using the Graph-Pad Prism Software (GraphPad Software Inc., San Diego, CA, USA) with the data presented as the mean values ± standard error. Where differences existed, the source of the differences at the *p* < 0.05 significance level was identified by all pairwise multiple comparison procedures via the Tukey's test.

#### **3. Results and Discussion**

Nanosystems based on cellulose nanocrystals (CNCs) and a multifunctional chitosan (CS) derivative were prepared via the simple (and eco-friendly) physical adsorption of the CS derivative, containing targeting (folic acid, FA) and imaging (fluorescein isothiocyanate, FITC) ligands (i.e., FA-CS-FITC), on the surface of the CNCs (Figure 1). Herein, the FA vitamin was carefully chosen as a targeting agent given the over-expression of folate receptors (FRs) in several tumor cells (and under-expression in non-tumor cells) [9,33], while the FITC fluorophore was picked for being a widely used imaging probe for the flow cytometry assays [33]. Furthermore, the CNCs were selected for their elongated nanostructure and anionic surface charge [29], whereas the CS derivative was chosen for its imaging and targeting moieties, as well as the cationic surface charge due to the presence of the characteristic protonated amine groups of CS at an acidic pH [34]. The assembly of the CNCs/FA-CS-FITC nanosystems via non-covalent interactions was preferred to covalent bonding due to the milder reaction conditions, when compared with previous studies where the functionalization of the CNC nanosystems was performed with fairly toxic reagents [20,22,23,25].

The opposite surface charge of these two polysaccharides enabled the facile physical adsorption [35] of the multifunctional CS derivative (i.e., FA-CS-FITC) on the surface of the elongated CNCs (Figure 1). The CNCs/FA-CS-FITC nanosystems were characterized in terms of structure, optical properties, fluorescence, stability in simulated physiological and non-physiological conditions, morphology, in vitro cytotoxicity, cellular internalization, and exometabolomics profile to evaluate their suitability for the targeting and imaging of breast cancer cells.

**Figure 1.** Scheme representing the preparation of the CNCs/FA-CS-FITC nanosystems.

#### *3.1. Preparation and Characterization of the CNCs/FA-CS-FITC Nanosystems*

The synthetic pathway to obtain the CNCs nanosystems with targeting and imaging functions (Figure 1) comprised three main steps. First, the CNCs were extracted from cellulose cotton fibers via acid hydrolysis with phosphotungstic acid [29]. Then, the FA-CS-FITC derivative was synthesized via a two-step pathway, where the CS was initially functionalized with FA by a carbodiimide-mediated amidation reaction, followed by the reaction of this intermediate with the isothiocyanate groups (N=C=S) of the FITC to obtain the FA-CS-FITC derivative [30,31]. Lastly, the simple and eco-friendly physical adsorption of the FA-CS-FITC derivative (ζ-potential of +51.9 ± 3.6 mV at pH 3) on the surface of the CNCs (ζ-potential of −11.5 ± 0.7 mV at pH 7) generated the CNCs/FA-CS-FITC nanosystems (Figure 1). Herein, three CNCs nanosystems with different contents of the FA-CS-FITC derivative were prepared, namely CNCs/FA-CS-FITC\_1 with a content of 13 µg of FA-CS-FITC per mg of CNCs, CNCs/FA-CS-FITC\_2 with 29 µg mg−1 and CNCs/FA-CS-FITC\_3 with 59 µg mg−1 (Table 1). The adsorption efficiencies of the FA-CS-FITC derivative on the surface of the CNCs (determined by UV-vis spectroscopy) were ca. 64% for CNCs/FA-CS-FITC\_1, and around 72% for both CNCs/FA-CS-FITC\_2 and CNCs/FA-CS-FITC\_3 (Table 1).

The first indication of the successful assembly process between the CNCs and the CS derivative was given by the color change of the elongated CNCs from white to an orange color, as illustrated in Figure 1. This was further corroborated by FTIR-ATR, UV-vis, and fluorescence spectroscopy, as exemplified in Figure 2 for the nanosystem with the higher content of the FA-CS-FITC derivative, i.e., CNCs/FA-CS-FITC\_3 (Table 1).

The FTIR-ATR spectrum of the CNCs/FA-CS-FITC\_3 nanosystem (Figure 2B) presents predominantly the characteristic vibrations of the CNCs at 3342 cm−1 (O–H stretching), 2902 cm−1 (C–H stretching), 1316 cm−1 (O–H in-plane bending), and 1032 cm−1 (C–O stretching) (Figure 2B) [36], but also those of the CS derivative, namely from: (i) chitosan at 3342 cm−1 (O–H and N–H stretching), 1636 cm−1 (C=O stretching and N–H bending), 1592 cm−1 (–NH2 bending), 1380 cm−1 (–CH2 bending), 1078 and 1032 cm−1 (C–O stretching) [37]; (ii) folic acid at 1688 cm−1 (C=O stretching), 1602 and 1481 cm−1 (C=C aromatic) cm−1 (phenyl ring) [38], and (iii) FITC at 1545, 1458 and 1378 cm−1 (xanthene ring skeletal C–C stretching) [39–41], as depicted in Figure 2A. However, the majority of these absorption bands overlap because of the common functional groups of the individual components (i.e., CNCs, CS, FA and FITC). Worth noting here is the absence of the absorption bands at 1686 cm−1 (carboxyl moiety of FA [38]) and 2018 cm−1 (isothiocyanate moiety (N=C=S) of the FITC [39]), together with the appearance of the absorption band (ca. 1650 cm−1 ) assigned to the amide thioamide bonds [42], that confirm the covalent link between CS and FA [43], and CS and FITC [44], respectively, in agreement with data reported elsewhere [45].

**Figure 2.** (**A**) FTIR-ATR spectra (vibrational modes: *ν* = stretching, *δ* = bending) of the pure CS, FA and FITC, (**B**) FTIR-ATR spectra of the pristine CNCs, FA-CS-FITC derivative and CNCs/FA-CS-FITC\_3 nanosystem, (**C**) UV-vis spectra of CNCs, CS, FA, FITC, FA-CS-FITC derivative and CNCs/FA-CS-FITC\_3 nanosystem, and (**D**) fluorescence emission spectra **Figure 2.** (**A**) FTIR-ATR spectra (vibrational modes: *ν* = stretching, *δ* = bending) of the pure CS, FA and FITC, (**B**) FTIR-ATR spectra of the pristine CNCs, FA-CS-FITC derivative and CNCs/FA-CS-FITC\_3 nanosystem, (**C**) UV-vis spectra of CNCs, CS, FA, FITC, FA-CS-FITC derivative and CNCs/FA-CS-FITC\_3 nanosystem, and (**D**) fluorescence emission spectra of the FITC, FA-CS-FITC derivative and CNCs/FA-CS-FITC\_3 nanosystem.

of the FITC, FA-CS-FITC derivative and CNCs/FA-CS-FITC\_3 nanosystem. The morphology and size of the CNCs nanosystems functionalized with the FA-CS-FITC derivative were assessed by STEM (Figure 3). As anticipated, the pristine CNCs showed the typical rod-like morphology with dimensions of 45 ± 12 nm (diameter) and 364 ± 74 nm (length), analogous to the data described in the literature [29]. After the assembly process, the CNCs maintained the rod-like morphology, but their dimensions The UV-vis spectrum of the CNCs/FA-CS-FITC\_3 nanosystem (Figure 2C) exhibits only the characteristic bands of the CS derivative, given that the CNCs do not present any absorption in the UV and visible regions, as registered elsewhere [20,46]. On the other hand, the bands of the FA-CS-FITC derivative (Figure 2C) are typical of the FA (at ca. 285 and 356 nm ascribed to the π–π\* and n–π\* transitions, respectively [38]) and FITC (at about 289, 462 and 490 nm [39,40]) ligands since the pure CS polysaccharide does not display any absorption in the UV and visible regions [47]. Predictably, some of the absorption bands were red-shifted compared to those reported for the pure FA and FITC, which is an

slightly increased to 57 ± 15 nm (diameter) and 442 ± 124 nm (length). This minor increase

on the surface of the CNCs, and it was also observed for other CNCs-based nanosystems

utmost importance due to the cell-size-dependent uptake of nanoparticles by the cells [14,17]. Thus, one can speculate that the elongated morphology of the CNCs/FA-CS-FITC nanosystems, together with their nanometric size, will not hinder the cellular internalization by the FR-positive breast cancer cells (i.e., MDA-MB-231 cell line), as will be discussed

Both the shape and size of the CNCs/FA-CS-FITC nanosystems are parameters of

[25,53].

in the following paragraphs.

indication of the covalent bond between CS, FA and FITC in the FA-CS-FITC derivative, as described for other systems such as FA-polyaniline [38] and FITC-organoclay [39].

The fluorescence emission spectrum of the CNCs/FA-CS-FITC\_3 nanosystem (Figure 2D) shows an absorption maximum at ca. 524 nm, which mimics the emission fluorescence peak of the FA-CS-FITC derivative. This peak is characteristic of the FITC fluorophore that, as reported by Ghosh et al. [40], upon excitation at a wavelength of 490 nm presents a strong emission peaking at around 520 nm. As anticipated, the pristine CNCs showed no emission in the wavelength range of the visible region. These results indicate that the CNCs nanosystems have fluorescent properties since the FITC fluorophore maintained its fluorescence. This behavior was expected given that the FITC is widely used to attach a fluorescent label mostly to polysaccharides (e.g., FITC-labelled CS [33,48], FITC-labelled CNCs [20,49], FITC-labelled dextran [40]) and proteins (e.g., FITC-labelled albumin [50]).

The success of the assembly process was additionally confirmed by testing the stability of the CNCs/FA-CS-FITC\_3 nanosystem under simulated non-physiological and physiological pH conditions, namely pH 2.1 for acidic medium [32] and pH 7.2 for human blood plasma [51], respectively. Since these CNC nanosystems rely on the physical adsorption between the negatively charged CNCs and the cationic CS derivative, it is important to assess the potential release of FA-CS-FITC from the nanosystems to avoid ambiguous interpretations of the results or even unnecessary cytotoxic effects, which might hinder their applicability [52]. According to the obtained data, the amount of the CS derivative released from the CNCs/FA-CS-FITC\_3 nanosystem is low at a non-physiologic pH with values of 6.6 ± 0.8% and 8.4 ± 0.5% for 24 h and 48 h, respectively. At the physiological pH 7.2, the release was slightly higher, with values of 11.0 ± 0.1% and 14.5 ± 0.1% for 24 h and 48 h, respectively. Overall, the CNCs/FA-CS-FITC nanosystems present good stability and dispersibility in both pH conditions, which can be credited to the effective electrostatic interactions established between the anionic CNCs and the cationic FA-CS-FITC derivative.

The morphology and size of the CNCs nanosystems functionalized with the FA-CS-FITC derivative were assessed by STEM (Figure 3). As anticipated, the pristine CNCs showed the typical rod-like morphology with dimensions of 45 ± 12 nm (diameter) and 364 ± 74 nm (length), analogous to the data described in the literature [29]. After the assembly process, the CNCs maintained the rod-like morphology, but their dimensions slightly increased to 57 ± 15 nm (diameter) and 442 ± 124 nm (length). This minor increase in the size range is clearly a direct result of the physical adsorption of the CS derivative on the surface of the CNCs, and it was also observed for other CNCs-based nanosystems [25,53].

Both the shape and size of the CNCs/FA-CS-FITC nanosystems are parameters of utmost importance due to the cell-size-dependent uptake of nanoparticles by the cells [14,17]. Thus, one can speculate that the elongated morphology of the CNCs/FA-CS-FITC nanosystems, together with their nanometric size, will not hinder the cellular internalization by the FR-positive breast cancer cells (i.e., MDA-MB-231 cell line), as will be discussed in the following paragraphs.

#### *3.2. Cellular Viability*

The in vitro cytotoxicity of the pristine CNCs and the CNCs/FA-CS-FITC nanosystems was evaluated in human breast adenocarcinoma cells (i.e., MDA-MB-231 cells) for 24 h at concentrations ranging from 12.5 to 200 µg mL−1 using the indirect MTT assay. The MDA-MB-231 cell line was selected for being often used as a model of triple-negative breast cancer cells [54] with over-expression of folate receptors [55], and for which the available therapeutic options are more scarce than other breast cancer subtypes [56].

**Figure 3.** STEM micrographs (**A**,**C**) with the corresponding size histograms (diameter and length, (**B**,**D**)) of the (**A**,**B**) pristine CNCs and (**C**,**D**) CNCs/FA-CS-FITC\_3 nanosystem.

According to the data presented in Figure 4A, the cell viability of the MDA-MB-231 cells after 24 h of exposure to the pristine CNCs at five different concentrations (12.5, 25, 50, 100 and 200 µg mL−1 ) remained at the level of 100%, meaning that the cell viability is not dose-dependent in the tested concentration range. This outcome for the pristine CNCs is consistent with the results found in the literature for this cell line [22], but also for other FR-positive human cancer cell lines, e.g., MDA-MB-468, KB, and PC-3 cells [22].

Regarding the CNCs/FA-CS-FITC nanosystems, the profile is the same and the cell viability is higher than 90% for all the tested concentrations. This confirms without a doubt that the three CNCs/FA-CS-FITC nanosystems are non-cytotoxic to the MDA-MB-231 cells at concentrations below 200 µg mL−1 , with an in vitro cell viability quite above the 70% threshold [57]. These results are similar to those reported for other CNCs-based nanosystems with other tumor cell lines, such as the human breast adenocarcinoma MCF-7 cell line [58], and the A375 and M14 cells (malignant melanoma cell lines) [59].

The non-cytotoxicity of the three CNCs/FA-CS-FITC nanosystems for the tested concentration range evinces their potential to act as nanocarriers of imaging and therapeutic agents for targeted therapy, without inhibiting the viability of the FR-positive breast cancer cells. In this sense, the cellular internalization assays will be performed with the highest tested concentration, namely at 200 µg mL−1 of each of the CNCs/FA-CS-FITC nanosystems, as discussed in the following paragraphs.

**Figure 4.** (**A**) Cell viability of the MDA-MB-231 cells after 24 h of exposure to the pristine CNCs and CNCs/FA-CS-FITC nanosystems, and flow cytometry data: (**B**) side-scattered light and (**C**) sidefluorescence light of the pristine CNCs and CNCs/FA-CS-FITC nanosystems for 24 h (the symbols \* and \*\*\*\* epitomize the means with a significant difference from the control at *p* < 0.05 and *p* < 0.0001 levels, respectively).

#### *3.3. Cellular Internalization*

The in vitro cellular internalization of the CNCs/FA-CS-FITC nanosystems by the MDA-MB-231 cells was studied by flow cytometry (side-scattered light (Figure 4B) and side-fluorescence light (Figure 4C)) for an exposure time of 24 h. The data compiled in Figure 4B shows that the incubation of the MDA-MB-231 cells in the presence of the pristine CNCs did not result in cell internalization, which is in line with the findings reported for this cell line [22], but also for other FR-positive human cancer cell lines, namely KB and MDA-MB-468 cells [22,24,25], and PC-3 cells [22].

In the case of the CNCs/FA-CS-FITC nanosystems, the ones with the lower content of the CS derivative, i.e., CNCs/FA-CS-FITC\_1 (13 µg of FA-CS-FITC per mg of CNCs) and CNCs/FA-CS-FITC\_2 (29 µg of FA-CS-FITC per mg of CNCs), showed a minor enhancement when compared to the control, and thus the cell internalization was minimal. In fact, only the CNCs/FA-CS-FITC\_3 nanosystem (59 µg of FA-CS-FITC per mg of CNCs) was significantly internalized by the FR-positive breast cancer cell line (*p* < 0.05).

Since these measurements are proportional to cell granularity or cell complexity and thus can be sometimes less sensitive [60], the fluorescence intensity of the cell population was also evaluated (Figure 4C) by taking advantage of the existent FITC fluorophore probe in the CNCs/FA-CS-FITC nanosystems. Once again, the cells incubated in the presence of the pristine CNCs exhibited a fluorescence intensity matching the control, which agrees with the results shown in Figure 4B. On the contrary, the CNCs/FA-CS-FITC nanosystems displayed a significant increase in the fluorescence intensity (Figure 4C). Basically, it is possible to observe the augment of the fluorescence intensity with the increasing content of the CS derivative (i.e., FA-CS-FITC, Table 1), reaching a maximum 5-fold increase compared to the control. The growing cell internalization by the FR-positive breast cancer cells is credited to the increasing content of FA.

Notably, this outcome also validates that the size of the CNCs/FA-CS-FITC nanosystems (Figure 3) did not hamper in any way the effective internalization of these FA-target nanosystems by the FR-positive breast cancer cells, as is in fact shown with other CNCsbased nanosystems with FA ligands [23,25,26].

#### *3.4. Cellular Exometabolomics*

The extracellular metabolomic profile of the MDA-MB-231 cells was studied by 1H NMR spectroscopy to assess possible changes in the cells metabolic activity induced by the 24 h exposure to the pristine CNCs and the CNCs/FA-CS-FITC nanosystems (200 µg mL−1 ). Figure 5A shows the characteristic 1H NMR spectra of the MDA-MB-231 cell-conditioned medium and of the acellular culture medium incubated under identical conditions. Integration of the NMR signals representative of the main metabolites allowed the determination of the metabolite consumption and secretion by control and exposed cells, as summarized in Figure 5B.

The MDA-MB-231 control cells mainly consumed glucose and choline, along with some amino acids (glutamine, branched-chain amino acids, aromatic amino acids, and histidine), while excreting lactate, glutamate, alanine, and acetate (Figure 5B,C). These findings are similar to those reported by Guerra et al. [61] and highlight the fact that the MDA-MB-231 cell line, like most cancer cells, exhibits the classical Warburg effect with a high glucose uptake and lactate production, together with high glutaminolytic activity [62].

Upon exposure of the MDA-MB-231 cells to the CNCs, there were no changes in their metabolic activity, which is not unexpected, given that the pristine CNCs were poorly internalized (Figure 4B,C). However, when cells were incubated with the CNCs/FA-CS-FITC nanosystems, slight but significant dose-dependent differences were found in choline uptake and acetate secretion (Figure 5B). The CNCs functionalized with intermediate and high FA concentrations (i.e., CNCs/FA-CS-FITC\_2 and CNCs/FA-CS-FITC\_3, respectively) caused a decrease in choline consumption and an increase in acetate release to the extracellular medium. Choline is an essential vitamin-like nutrient required for the de novo synthesis of membrane phospholipids, like phosphatidylcholine and sphingomyelin [63]. Various types of cancer cells, including breast cancer, display enhanced choline uptake and altered metabolism, to support fast proliferation and migratory capacity [64]. Hence, it is possible that the herein observed decrease in choline consumption by the cells incubated with the CNCs/FA-CS-FITC nanosystems reflects a lower synthesis of membrane lipids and a slower proliferation. This is consistent with the observed increase in the amount of acetate released by cells upon incubation with the nanosystems containing the highest concentrations of the FA-CS-FITC derivative (i.e., CNCs/FA-CS-FITC\_2 and CNCs/FA-CS-FITC\_3, Table 1). Indeed, besides glucose and glutamine, acetate provided as an extracellular nutrient or produced endogenously from pyruvate [65], may chiefly contribute to de novo lipid synthesis via conversion into acetyl-CoA by cytosolic and/or mitochondrial acetyl-CoA synthetases [66]. Hence, its release into the culture medium could reflect its lower intracellular utilization, possibly in relation to the downregulation of lipogenesis.

To summarize, the assembly of the CNCs/FA-CS-FITC nanosystems via physical adsorption was inspired by the possibility of using the opposite surface charge of the CNCs and the FA-CS-FITC derivative and originated non-cytotoxic nanosystems up to concentrations of 200 µg mL−1 . Furthermore, and since the cellular uptake is a limiting factor for the efficacy of countless anticancer drugs, the presence of FA in the CNCs/FA-CS-FITC nanosystems promoted a higher cellular internalization towards FR-positive MDA-MB-231 breast cancer cells. Besides, mild alterations in the cells' exometabolome upon 24 h exposure to the CNCs/FA-CS-FITC nanosystems suggest an anti-proliferative effect, which may be beneficial in the context of cancer treatment.

**Figure 5.** (**A**) 1H NMR spectra of the acellular medium (pink) and supernatant from MDA-MB-231 breast cancer cells grown for 24 h (black), (**B**) variations in metabolites consumed (negative bars) and excreted (positive bars) by the MDA-MB-231 cells, under control conditions and upon treatment with 200 µg mL−1 of CNCs, CNCs/FA-CS-FITC\_1, CNCs/FA-CS-FITC\_2 and CNCs/FA-CS-FITC\_3 for 24 h (the symbol \* represents the means with a significant difference from the control at *p* < 0.05), and (**C**) chemical structures of the consumed (black) and excreted (green) metabolites.

Henceforth, the CNCs/FA-CS- FITC nanosystems can be exploited as nanocarriers of imaging and chemotherapeutics agents for active targeted therapy. In fact, the incorporation of, for instance, gold nanoparticles (with potential for photothermal cancer therapy [67]) into the CNCs/FA-CS-FITC nanosystems could be an option to engineer a nanocarrier with combined diagnostic and therapeutic capabilities, viz. a theranostic nanosystem [68].

#### **4. Conclusions**

In the present work, nanosystems composed of CNCs and a CS derivative were successfully developed and characterized. The physical adsorption of the CS derivative, containing a targeting ligand (FA) and an imaging agent (FITC), on the surface of the CNCs was an eco-friendly methodology to obtain CNCs-based nanosystems. The ensuing nanosystems displayed good stability in two distinct simulated non-physiological and physiological conditions (pH 2.1 and 7.2, respectively), as well as non-cytotoxicity towards MDA-MB-231 cells up to a concentration of 200 µg mL−1 . The CNCs nanosystems showed a superior cellular internalization with a 5-fold increase in the fluorescence intensity for the nanosystem with the greater content of FA, viz. concentration-dependent internalization by the FR-positive breast cancer cells. Additionally, the exometabolomics of MDA-MB-231 cells exposed to the CNCs/FA-CS-FITC nanosystems revealed a mild impact on the metabolic activity of the cells, namely a decreased choline uptake and increased acetate release, which suggests an anti-proliferative effect. The overall data evidenced that the elongated CNCs/FA-CS-FITC nanosystems produced by an eco-friendly methodology have high affinity towards folate receptor-positive cancer cells with enhanced cellular internalization, and hence might be employed as nanocarriers with imaging properties for active targeted therapy.

**Author Contributions:** Conceptualization, C.S.R.F. and C.V.; investigation, R.J.B.P., N.S.L., G.G., G.H.R.d.S., P.O. and C.V.; resources, S.S., H.O., I.F.D. and C.S.R.F.; writing—original draft preparation, C.V. and R.J.B.P.; writing—review and editing, R.J.B.P., N.S.L., G.G., G.H.R.d.S., P.O., S.S., H.O., I.F.D., C.V. and C.S.R.F.; supervision, C.S.R.F.; funding acquisition, C.S.R.F., I.F.D., H.O. and S.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was developed within the scope of the projects CICECO—Aveiro Institute of Materials (UIDB/50011/2020 & UIDP/50011/2020) and CESAM—Centre for Environmental and Marine Studies (UIDB/50017/2020 & UIDP/50017/2020) financed by national funds through the Portuguese Foundation for Science and Technology (FCT)/MCTES. The research contract of R.J.B.P. was funded by national funds (OE), through FCT in the scope of the framework contract foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law 57/2016, of 29 August, changed by Law 57/2017, of 19 July. FCT is also acknowledged for the doctoral grant to N.S.L. (SFRH/BD/140229/2018) and the research contracts under Scientific Employment Stimulus to H.O. (CEECIND/04050/2017), C.V. (CEECIND/00263/2018) and C.S.R.F. (CEECIND/00464/2017). FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and the University of Aveiro are acknowledged for the doctoral grants to G.H.R.d.S. (2018/24814-0) and P.O. (BD/REIT/8623/2020), respectively. The NMR spectrometer is part of the National NMR Network (PTNMR), partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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