Cellulose/Exosome Nanocarrier for Improved Prolonged Release of 5-Fluorouracil: Preparation, Characterization, Drug Release Behavior and Cytotoxicity

doi:10.21203/rs.3.rs-1102085/v1

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Cellulose/Exosome Nanocarrier for Improved Prolonged Release of 5-Fluorouracil: Preparation, Characterization, Drug Release Behavior and Cytotoxicity

https://doi.org/10.21203/rs.3.rs-1102085/v1

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Designing of nanoparticle drug delivery systems and improving the efficacy of anticancer drugs are a great deal of effort in the recent years. In this study, a novel biocompatible nanocarrier based on bacterial cellulose (BC) in presence of exosome (Exo) was prepared to controlled release of 5-fluorouracil (5-FU), ([email protected]). The physicochemical properties of [email protected] was characterized using field emission scanning electron microscopy (FESEM), Differential Scanning Calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR), and X-ray Diffraction (XRD) techniques that confirmed the successful preparation of [email protected] The release behavior of [email protected] compared to 5-FU and [email protected] demonstrated a significant sustained release during 162 h. The release mechanism of the above three systems followed Korsmeyer-peppas with non-Fickian diffusion for [email protected] and [email protected] In addition, the viability of HT-29 cells (human colon cancer cell line), towards BC, [email protected] and [email protected] indicated the promising efficacy of 5-FU into [email protected] Subsequently, the prepared bio-nanocomposite could be proposed as a potential drug delivery system with effective controlled-release function.

Polymer Science

Drug Release

Cellulose

Exosome

anticancer

Release mechanism

Cancer treatment is one of the prospective uses of nanotechnology [1]. Natural polymers have over the past decades been thoroughly examined for use in drug delivery, in regard to their biocompatibility and biodegradability, and their potential to deliver drugs and genes to targeted organs [2, 3]. Exosomes as a promising nanocarriers for drug delivery could be made of various kinds of biomolecule such as proteins, carbohydrates, lipids, and nucleic acids [4]. Exosomes can present improved blood stability, enabling them to move long distances under both physiological and pathological conditions inside the body. Moreover, having a hydrophilic core renders exosomes favorable to host water-soluble drugs [5].

An anti-metabolite, 5-Fluorouracil (5-FU) exhibits broad-spectrum anti-cancer activity against solid tumors [6]. 5-FU, a pyrimidine analog, is an exclusively ‘S-phase’ active chemotherapeutic agent and acting as a thymidylate synthase inhibitor and thus impeding DNA synthesis [7]. Over the last forty years, 5-FU has been exploited against cancers like colon cancer, playing practically the role as a thymidylate synthase inhibitor. However, drug resistance remains a major obstacle in the clinical application of 5-FU [8]. Apart from its limited clinical uses, it is vital to develop effective carriers to enhance 5-FU delivery, leading to better anticancer efficacy [9].

There has been great effort on developing an efficient cellulose carrier system to control the drug concentration and release rate. Bacterial cellulose (BC) has drawn considerable attention in medical, pharmaceutical, and other related areas due to biocompatibility and non-toxicity besides physicochemical properties such as its intrinsic physical, mechanical, and biological qualities [10]. Cellulose can be produced by various acetic acids producing bacterial strains of the genera Acetobacter, Gluconobacter, Gluconacetobacter and Komagateibacter. [11]. Recently, BC has attracted special consideration in biomedical applications because the polymer is nontoxic, biocompatible, moldable, and transparent [12]. Moreover, its high-porosity geometry and hydrophilic structure facilitate the absorption and carrying of high quantities of liquid drug for the polymer. This is vital in wound dressing and tissue engineering [13]. BC has been thoroughly examined for medical applications; still, rather little effort has been detailed in cancer treatment. Regarding its physicochemical features and efficiency in developing composite materials, the research focus is turning to extending the role of BC in cancer treatment [14, 15]. In the other hand, exosomes (Exo), the endogenous nanocarriers that can deliver biological information between cells, were recently introduced as a new kind of drug delivery system [16].

Regarding the potential of BC and Exo to sustained drug release, in the present study, the combination of bacterial cellulose (BC) and exosome (Exo) was prepared as a nanocarrier for prolonged release of 5-Flurouracil (5-FU). The characterization was performed using field emission scanning electron microscopy (FESEM), Differential Scanning Calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR), and X-ray Diffraction (XRD) techniques. After that, the drug release and the release mechanism were examined and the results were compared to 5-FU and [email protected] To confirm of improvement effectiveness, the cytotoxicity of nanocarriers containing 5-FU against colon cancer cell line, HT-29 was evaluated.

Phosphate buffer saline tablet (PBS), and dimethyl sulfoxide (DMSO), RPMI1640, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Germany). Exosome (24.3 µM) derived from human Amniotic Fluid, were isolated by differential sedimentation (Ultra-centrifugation) method according to previous report [17]. 5-Fluorouracil (5-FU, 50 mg/ml) were received from Ebewe Co., Austria. Bacterial cellulose was purchased from Nano Zist Polymer Pars Co. Tehran, Iran that was prepared according to the previous report [18, 19], with 1 mm thickness. The human colon cancer cell line, HT-29, was obtained from the Cell Bank of the Histogenotech institute in Tehran (Iran). Deionized water (DI) was used in all of the experiments. The other reagents and solvents were analytical grad and purchased from Merck company (Germany).

The Fourier transform infrared (FT-IR) spectra were recorded using a Perkin Elmer company (spectrum400-model) FT-IR spectrometer in a KBr disk in the range of 4000-400 cm⁻¹. Differential Scanning Calorimetry (DSC), DSC-1 (TA Instruments, Mettler Toledo, Switzerland) Model was used to investigation of the crystallinity, purity, degradation of a variety of samples. The samples were weighed in a standard open aluminum pan, while an empty pan of the same type was used as a reference. The heat running for each sample was set from 23°C to 400°C at a 10°C/min increment rate, using nitrogen as a purge gas with the rate of nitrogen atmosphere being 40 ml/min. Calibration of temperature and heat flow was performed with indium and zinc. Absorption spectroscopy was adopted to study the drug release behavior using a T80 + UV-Vis Spectrophotometer, PG Instruments Ltd. Topographical and elemental information at specific magnifications with a virtually unlimited depth of samples was studied using field emission scanning electron microscopy (FESEM) where each sample was spin-coated on a microscope slide, which remained for 1 day at room temperature to dry. Then, the slides were sputter-coated with a layer of gold, and images were taken by TESCAN MIRA3, (voltage of 15.0 Kv, current intensity of 30 A). The XRD pattern of samples was recorded using a Powder X-ray diffractometer (PW 3710 mpd control) with the copper line as the source of radiation at the voltage of 40.0 (kV) and current of 30.0 (mA). The samples were scanned from 5.0100° to 79.9900° (diffraction angle 2θ) with a rate of one second per step, using a zero-background sample holder.

2.1. Preparation of samples

BC sheets (approximately 1⋅1 cm² area, 2.2±0.2 mg weight) were put in 10 ml DI water at the incubator shaker under the constant condition of 30°C for 48 h.

2.1.1. [email protected]

22 µl 5-FU (50 mg/ml) was added to 978 ml DI water and then, a BC sheet was placed in the solution (the ratio of 5-FU to BC was 2:4 w/w). After that, the sample was put in the incubator shaker at 30°C for 24 h.

2.1.2. Preparation of [email protected]

First, 22 µl 5-FU (50 mg/ml) and 0.55 mg Exo were mixed under continues stirring (100 rpm) for 1 h at room temperature and then, 978 ml DI water was added into prepared 5-FU.Exo solution. After that, according to the above sample, a BC sheet was placed into the solution (the ratio of 5-FU to Exo and BC was 2:1:4 w/w) and incubated in the incubator shaker at 30°C for 24 h.

Finally, the BC sheet was removed with forceps from all of the above prepared samples and dried at incubator 37°C for characterization. However, three samples of each system were separately transferred to the dialysis bag containing 1 ml PBS for the release experiments. The remaining solution was stored for determination of unloaded drug.

2.3. Drug loading and release experiment

2.3.1. Standard Equation

The absorption of the various concentrations of 5-FU (0-0.34 mg/ml) was recorded at characteristic wavelength of 5-FU (325 nm). Then, the recorded absorption versus concentration was plotted and the best linear equation was determined. The concentration of unknown samples was estimated [20] by the obtained standard equation as A=mC, where A was absorption of each sample, m was slope and C was concentration.

2.3.3. determination of Drug Loading and Entrapping Percentage

The drug loading (%DL) and encapsulation efficiency (%EE) were determined by the following equations [21]:

%DL = (M₀/M) × 100 (1)

%EE = (M₀/M₁) × 100 (2)

Where M₀, M₁, and M were weight of loaded 5-fluorouracil, the total weight of 5-fluorouracil, and weight of carrier, respectively.

The release of AZT from LPN was studied using dialysis bag method. The hybrid nanoparticles con- taining 5 mg drug were dispersed in 5 mL PBS solution (pH 7.4) in preheated dialysis membrane (Spectra/Poresize; 12000 Da cutoffs, CA, USA). This dialysis bag was immersed in a beaker containing PBS (50 mL) and stirred at 37°C. The sample (3 mL) was withdrawn from the beaker at predetermined intervals to estimate released AZT. An equal volume of fresh buffer was introduced to the beaker containing the dialysis bag. The AZT release was estimated specrophotometrically at 265 nm.

2.3.4. Drug Release experiment

The study of 5-FU release from designed carriers was performed by the dialysis bag method. So that, the three systems of 5-FU, [email protected] and [email protected] (1 ml) was separately embedded into a dialysis bag (12 kDa cutoffs) and immersed in a release medium containing 6 ml of PBS solution. Then, each system put in shaker at 37 °C and 500 µl sample was withdrawn from it at the certain time intervals and an equal volume of fresh buffer was introduced to the beaker. This experiment was performed two times. After that, the amount of drug release was analyzed with absorption spectroscopy at 325 nm and the cumulative drug release percentage was determined by the following equation [21]:

%cumulative release= M_t/M₀⋅100 (3)

The release rate (r) was also determined using Eq. 5 [22, 23]:

r = M_t/t (4)

where, M_t was weight of released drug in time (t) and M₀ was initial weight.

2.3.5. Release Mechanism

The release mechanism was evaluated using the following three mathematical models [24]:

- Zero-order model

M_t = M₀ + k₀ t (5)

where M_t is the amount of released drug in time t, M₀ is the starting amount of the drug in the solution (in most cases, M₀ = 0) and k₀ is the zero-order release constant. The zero-order kinetic model and the drug concentration are independent.

- First-order model

logM_t/M₀ = -k₁ t/2.303 (6)

where M_t, M₀, and t are as in the Zero-order model, and k₁ is the first-order release constant. The first-order kinetic model is independent of drug concentration.

- Korsmeyer-Peppas model

logM_t/M = logk_p + nlogt (7)

where M_t/M is the fraction of drug release at time t, n is the release index and the value to predict the mechanism of release (n < 0.43 was Fickian diffusion and n between 0.43-1 was non-Fickian diffusion in the drug release process) and k_p is the Korsmeyer-Peppas release rate constant.

furthermore, the release kinetic parameters were determined as follow [22, 23]:

t/C_t = α + βt (8)

C_t is the amount of drug released at time t, β = 1/C_max is the inverse of the maximum amount of drug released, α = 1/(C_max)² k_rel, k_rel is the kinetic constant of release and equals the inverse of the initial release rate (r₀) [22].

2.4. Cytotoxicity measurement

The MTT assay is a colorimetric assay for measuring cell metabolic activity. It is based on the ability of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes to reduce the tetrazolium dye MTT to its insoluble formazan, which has a purple color. This assay, therefore, measures cell viability in terms of reductive activity as the enzymatic conversion of the tetrazolium compound to water-insoluble formazan crystals by dehydrogenases occurring in the mitochondria of living cells although reducing agents and enzymes located in other organelles, such as the endoplasmic reticulum are also involved [25]. Therefore, the cell viability of 5-FU, [email protected] and [email protected] onto HT-29 cancer cell line can be evaluated by MTT assay. For this reason, the harvested HT-29 cells that grown in the complete medium (RPMI supplemented with streptomycin and penicillin (5 lg/mL), heat-inactivated fetal calf serum (10%) and 2 mM glutamine) at 37 °C in an incubator containing 5% CO₂ and 95% air atmosphere, were seeded into 24-well plates with the concentration of 5×10⁴ cells/well. Afterwards, to each one of the cultured media well containing the cancer cells were separately added 5-FU, [email protected] and [email protected] and then, incubated for 48 h. After 48 h of incubation, the cells were treated with 50 µl of MTT (5 mg/ml PBS) and incubated for 4 h at 37°C. When the incubation finished, 150 µl of DMSO was mixed to dissolve the colored crystals of produced formazan.

Finally, the absorbance was recorded at 630 nm using ELISA reader. The results reported as cell viability percentage at least three independent experiments and calculated as follow [26]:

%cell viability=(A_treated / A_control)×100 (9)

where A _treated and A _control were absorbance of the treated cells and untreated cells, respectively.

3.1. Characterization

The FTIR spectra of BC, 5-FU, 5-FU.Exo, [email protected] and [email protected] were recorded (Fig. 1a) and mentioned the peak position was changed after the conjugation. So that, the main features of 5-FU spectra were stretching and bending vibrations of –NH group around 3658 cm⁻¹ and 1649 cm⁻¹, respectively [27]. The strong bands at 1402 cm⁻¹ and 1684 cm⁻¹ were attributed to C-F and C=O amide. The main vibrational bands of BC were appeared at 1076 cm⁻¹ (strong band; C-O group) and around 3338 cm⁻¹ (broad band; -OH group). The shift of vibration bands of 5-FU and BC when the loading between them was performed, indicated that the [email protected] was formed. Besides the changes of C-F vibrational band (1428 cm⁻¹), -NH bands of 5-FU and C-O band of BC were almost disappeared that confirmed the conjugation between –NH and –CO groups. Furthermore, the FU.Exo spectrum showed the removal of –NH group of 5-FU that maybe due to the binding between 5-FU with amide groups of Exo [28]. In addition, the band shift was mentioned to 1718 cm⁻¹, 1651 cm⁻¹ and 1419 cm⁻¹ for C=O_amide, NH_bending and C-F, respectively. The comparison of [email protected] spectrum with the other spectra was also shown the main vibrational band changes that attributed to the binding formation of 5-FU.Exo and BC. Also, the broad bands of O-H stretching vibrations and C-H of methyl and methylene groups appeared in the spectral region 3600-3000 cm⁻¹ and 1000-650 cm⁻¹, respectively, for all of the spectra.

The DSC analysis of BC, [email protected] and [email protected] was performed and recorded as shown in Fig. 1b, the degradation peak of BC is visible at around 343°C and the peak at the low temperature is due to the removal of the surface water from the sample. The characteristic sharp peak of the parent [email protected] sample was shown at around 320-350°C. Thermal transformation occurred at 320-370°C, where changing the thermal behavior could be due to a change in the physical form of 5-FU in BC. Further, the degradation peak of BC showed up in [email protected] Also, a crucial result in the [email protected] was that peaks were no longer visible since degradation did not accompany the release of the drug, so the loaded drug released on the carrier. This key factor along with the positive effect of exosomes on the designed carrier demonstrated favorable thermal stability. Finally, the thermal analysis study indicates that the [email protected] designed here consisted mostly of 5-FU.

The analysis of XRD (Fig. 4c), showed that the characteristic peaks of pure BC were located at approximately 42° owing to the linear structure of semi crystalline homopolysaccharide. The XRD pattern of [email protected] was similar to that of pure BC, since the exosome as an amorphous material did not exhibit characteristic peaks. However, according to data shown in [email protected], it appears that the addition of [email protected] was shifting the diffraction angle from 22.79° to 23.18°, which was due to the increased hydrogen bonds formed between hydrogens of amide group and oxygen of the carbonyl group, forming the body of the protein. In addition, the hydrogen-bonding network within the α-helix served to stabilize the conformation and interaction between the drug and exosome with BC. Also, the crystallinity of part of BC resulted in having sharper peaks in the range of 20°-24°. Pure 5-FU has a crystalline structure, with a sharp diffraction peak at around 2θ = 28.71° [29], whereas the results here noticeably indicated that [email protected] did not have any sharp peak related to 5-FU, because the drug was possibly entrapped within the composites of the carrier. Further, the crystalline structure of the pure drug may become non-crystalline after it is encapsulated within the carrier.

The SEM images of BC, 5-FU.Exo, [email protected] and [email protected] were shown in Fig. 4d. the surface of BC shows the regular and uniform fibers with an average diameter of 42.0 nm±19.2 nm and 5-FU.Exo illustrated the cylindrical structure with a width of 260±92.2 nm. However, the agglomeration between nanoparticles of 5-FU.Exo was clearly demonstrated. After the conjugation of 5-FU.Exo to BC (Fig. 4d-IV), the presence of 5-FU.Exo was confirmed as the dispersed bar into BC network structure (57.5±9.6 nm) as shown with yellow arrows with 82.0±13.0 nm. In addition, Exos were caused the more dispersity in [email protected] in comparison to [email protected] (width size of 5-FU and BC was 80.0±14.1 nm and 37.5±15 nm, respectively, comparison of III and IV of Fig. 4d).

3.2. Drug Loading and Release Studies

3.2.1. Standard plot

For calculation of unknown concentrations of 5-FU, first, the standard plot was drawn as absorbance versus the various concentrations of 5-FU in the range of 0-1.375 mg/ml (Fig. 2). Second, the standard equation was obtained by the best straight-line equation that was A=0.2218 [5-FU], R²=0.99.

3.2.2. Determination of %DL and %EE

The %DL and %EE were calculated using Eqs. 2 and 3. The results showed that %DL values were 33.56±1.18% and 34.34±0.58% for [email protected] and [email protected], respectively. Noticeably, %EE values of [email protected] and [email protected] were 83.91±2.94% and 85.85±1.45%, respectively. Considering the drug loading percentage of most nanocarriers is generally less than 10% [30], the above designed carriers were improved the drug loading that was due to conjugating the drug to BC as a polymer with various binding sites. The drug loading percentage of calcium alginate/sodium cellulose sulfate microcapsule for BSA was 38.14% [31], [email protected] was 39.1% [32] and also, [email protected] nanoparticles was 27.1% [30] that comparable with the achievement results of this study. Additionally, encapsulation efficiency values were upper than folic acid-loaded in a chitosan nanoparticles and cellulose nanocrystals (62%) [33] and zidovudine-loaded in a carboxy methyl cellulose and compritol-PEG carrier (18%) [34] and in the range of paclitaxel loaded poly(N-vinylpyrrolidone)-b-poly(epsilon-caprolactone) nanoparticles [30]. Therefore, the two nanocarrirs containing 5-FU had acceptable loading behavior. However, the presence of EXO had no significant effects on the two parameters.

3.2.3. Release behavior

Cumulative release profiles of 5-FU, [email protected], and [email protected] were presented in Fig. 3. The drug release was explosively complete at 5-FU and [email protected] system during 29 h and 53 h, respectively. In respect of the curves, the presence of BC was caused a rather slow release compared to 5-FU alone. Furthermore, [email protected] showed a significant sustained release during 168 h compared to the above mentioned systems, owing to the release of 5-FU from Exo and BC. As shown in Fig. 3b, the release rate of the novel [email protected] indicated also a slow, constant, and controlled within a specific period of time (20-168 h) that confirmed the sustained release of the novel designed system of 5-FU load into Exo and BC while release rate of only 5-FU was greater than the other systems of [email protected], and [email protected] In addition, the release rate behavior of 5-FU released from BC was similar to [email protected] with the higher rate of release. Therefore, the presence of Exo with BC led to a more extended period of 5-FU release and the slower release rate. This despite the fact that tamoxifen citrate released from ethyl cellulose during 36 h [35] and 10% of Doxorubicin was release from Cellulose nanocrystals within 100 [36].

3.2.4. Kinetic behavior

Zero-order, First-order, and Korsmeyer-Peppas mathematical models were employed for the determination of mechanism mode. The correlation coefficients of three kinetic models are shown in Table 1. Based on the results, the release profile of 5-FU, [email protected] and [email protected] was fitted well with Korsmeyer-Peppas release kinetic model and the n value of 5-FU was greater than 0.85, hence, the release behavior followed by Fickian diffusion and [email protected] and [email protected] were between 0.43-0.85, thus, the drug release followed by non-Fickian diffusion [31]. Therefore, BC could affect the drug release behavior.

Furthermore, the results of other kinetic parameters (Table 1) revealed that, C_max value was approximately equal to the initial weight of drug, therefore, it was also confirmed the completion of the release. r₀ values as well as k_rel values of the three system demonstrated the distinct behavior in [email protected] that confirms the presence of Exo and the improvement of drug release behavior due to an appropriate designed carrier containing BC and Exo.

Table 1

The kinetic parameters of various mathematical models
Samples	Zero-order	First-order	Korsmeyer-Peppas			Kinetic-Parameters
Samples	R²	R²	k_P	n	R²	C_max (mg)	k_rel (h/mg)	r₀ (mg/h)	R²
5-FU	0.72	0.31	0.39	1.05	0.99	1.33	0.15	6.63	0.93
[email protected]	0.63	0.29	0.41	0.59	0.92	0.86	0.22	4.44	0.81
[email protected]	0.79	0.55	0.28	0.60	0.97	1.26	0.01	72.38	0.95

3.3. In vitro cytotoxicity study

Continuing studies of the three systems for biomedical application, the viability of HT-29 cancer cell lines towards BC, [email protected], and [email protected] was separately performed based on MTT assay. As shown in Fig. 4, the BC sheet had no toxicity and the cell viability percentage of [email protected] (50.16±0.51) was lower than [email protected] (69.32±5.94), whereas, according to release plots (Fig. 3a), after 48 h, 54% and 98% of 5-FU were released from BC and [email protected] BC. Hence, the encapsulation of 5-FU into Exo and BC may have prevented the destruction of the drug during release. Therefore, the lower concentration was needed to cell death that was a remarkable achievement to cancer treatment.

In this study, two drug delivery systems of [email protected] and [email protected] were successfully prepared and characterized by FTIR, DSC, XRD, and FESEM. The results confirmed the loading of 5-FU into the carriers. Also, the drug loading and encapsulation efficacy percentage were determined and the release behavior of [email protected] and [email protected] was studied and mentioned the significant sustained release of 5-FU in presence of Exo and BC, whereas, the release of drug from BC carrier was lower than only 5-FU. Furthermore, the release mechanism followed by Korsmeyer-peppas model with non-Fickian diffusion for [email protected] and [email protected] systems. Finally, the cytotoxicity of BC, [email protected] and [email protected] on the human colon cancer cell line, HT-29, was shown the high cytotoxicity in low concentration of [email protected] Overall, the designed nanocarriers based on cellulose can be introduced as a promising carrier for sustained released of anticancer drugs, especially in presence of exosome as a vehicle for chemotherapy agents that improve drug delivery efficacy and reduce drug side effects.

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Cellulose/Exosome Nanocarrier for Improved Prolonged Release of 5-Fluorouracil: Preparation, Characterization, Drug Release Behavior and Cytotoxicity

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Preparation of samples

2.1.1. [email protected]

2.1.2. Preparation of [email protected]

2.3. Drug loading and release experiment

2.3.1. Standard Equation

2.3.3. determination of Drug Loading and Entrapping Percentage

2.3.4. Drug Release experiment

2.3.5. Release Mechanism

2.4. Cytotoxicity measurement

3. Results And Discussion

3.1. Characterization

3.2. Drug Loading and Release Studies

3.2.1. Standard plot

3.2.2. Determination of %DL and %EE

3.2.3. Release behavior

3.2.4. Kinetic behavior

3.3. In vitro cytotoxicity study

4. Conclusion

References

Supplementary Files

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