Synthesis and biological evaluation of surface-modified nanocellulose hydrogel loaded with paclitaxel

Journal Pre-proof Synthesis and biological evaluation of nanocellulose hydrogel loaded with paclitaxel

surface-modified

Like Ning, Chaoqun You, Yu Zhang, Xun Li, Fei Wang PII:

S0024-3205(19)31065-3

DOI:

https://doi.org/10.1016/j.lfs.2019.117137

Reference:

LFS 117137

To appear in:

Life Sciences

Received date:

7 September 2019

Revised date:

27 November 2019

Accepted date:

1 December 2019

Please cite this article as: L. Ning, C. You, Y. Zhang, et al., Synthesis and biological evaluation of surface-modified nanocellulose hydrogel loaded with paclitaxel, Life Sciences(2019), https://doi.org/10.1016/j.lfs.2019.117137

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© 2019 Published by Elsevier.

Journal Pre-proof

Synthesis and biological evaluation of surface-modified nanocellulose hydrogel loaded with paclitaxel Like Ning, Chaoqun You *, Yu Zhang, Xun Li, Fei Wang * (College of Chemical Engineering, Nanjing Forestry University, Jiangsu Key Lab for the Chemistry and Utilization of Agro-Forest Biomass, Nanjing 210037, P. R. China. E-mail: [email protected]; Fax: +86 25 85427649; Tel: +86 25 85427649)

Abstract: Hydrogel for various applications, such as cell encapsulation and

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controlled release of drugs, has attracted the field of biomaterials in the past decades.

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Specially, research on the surface-modified nanocellulose hydrogel has grown rapidly on account of the importance of target delivery in the anti-cancer therapy. In this work,

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surface-modified nanocellulose was mixed with hexadecyl amine as long chains to

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blend to build a network to produce hydrogel, which was successfully developed for controlled and targeted delivery of paclitaxel. The pH-stimuli surface-modified

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nanocellulose hydrogel was characterized and biological evaluated in vitro. The hydrogel was stable at pH 7.4 and paclitaxel was released by the shape change of

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hydrogel in an acidic environment (pH 5.5), and the sustained release of paclitaxel was observed at pH 5.5. The vitro cytotoxicity studies indicated that the drug

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accumulation and the inhibition of A549 and HepG2 cells was effectively increased by the surface-modified nanocellulose hydrogel as compared with free paclitaxel. The inhibitory effect of A549 cells was improved by nearly 30% as compared with free paclitaxel and the apoptosis rate was up to 90.5% after 12 hours incubation. In addition, the inversion test and the results of a series of cell experiments in vitro demonstrated a good performance of the surface-modified nanocellulose hydrogel. Keywords: nanocellulose; hydrogel; paclitaxel; anti-tumor; pH response 1. Introduction Due to dose-dependent characteristics of anti-cancer formulations, frequent administration brings threatened complications in anti-cancer therapies, control release technologies have become one of the most effective measures of new 1

Journal Pre-proof pharmaceutical agents[1-4]. Moreover, for many new active anticarcinogens, the poor solubility in water make them fail to be well utilized[5]. To solve this problem, there are already some formulations on the market that have been produced and applied to patients for several years. For instance, Taxol as a widely applicated preparation to against tumors, which was mainly composed of paclitaxel (PTX) that mixed with polyethoxylated castor oil (Cremophor EL, CrEL)[6]. However, this existing formulation was delivered via intravenous injection after preventive medication and dilution, may results in serious severe neurotoxicity and allergic reactions. Scientists

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have been devoted to resolve these side effects in recent years. Therefore, the way to

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produce a more appropriate drug release system which enables both control release and improvement of solubility would greatly have a promising prospect[7, 8].

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Recent research on control release system has explored various vehicles to load

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drugs, some examples includes nanoparticles[9], liposomes[10], implants, and hydrogels[11]. For their inherent hydrated nature and excellent applications on control

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release make hydrogels became an attractive carrier in drug release system[12-16].

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Among these control release systems, stimuli-responsive hydrogels have been widely developed in cancer targeting. Because of the specific acidic situation in the tumor

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site while in the normal human body is a weakly alkaline physiological environment, this difference in pH values provide a type of thought to design targeting[17]. Moreover, the pH-responsive hydrogel as a typically stimuli-responsive hydrogel which has been widely used in targeting method at present[18]. Therefore, they provided a relatively complete platform to explore in the further study on anti-cancer therapies. Cellulose is the most widely distributed and most abundant polysaccharide in nature[19]. Its nanocrystals called cellulose nanocrystals (CNCs) are organized in a structure of strongly ordered crystalline particles which have been engineered by nature to be inherently strong. CNCs was obtained through chemical reagents, enzyme, or mechanical methods from natural material (wood, sugarcane, cotton, etc.)[20]. Due to its outstanding properties, such as hydrophilicity, biocompatibility, and surface tunability, CNCs have been proved to be a promising material[21-25]. 2

Journal Pre-proof Cellulose hydrogels with chemically modification have been utilized in various aspects of anti-cancer therapies. Sry D. Hujaya et al.[26] utilized chemically modified cellulose nanofibrils to make polyion complex hydrogels, while Jiao Li et al.[27] synthesized nanocellulose templated growth of ultra-small bismuth nanoparticles. Meanwhile, Hai Jun et al.[28] developed a new class of ClO-/SCN- reversibly responsive nanocellulose hydrogel for cancer spheroids culture and release. Moreover, Chaobo Huang et al.[29] developed cellulose acetate phthalate (CAP) fibers by electrospinning, this pH-responsive CAP endowed the fibers potential for “semen

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sensitive” (intravaginal) drug delivery.

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In this paper, a pH-responsive nanocellulose gel loaded with paclitaxel (PTX) was developed based on the previous research to improve the effect of anti-cancer

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(Scheme 1). A network with a large solvophobic area that can retain PTX, which was

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built with carboxylic-modified nanocellulose and long-chain hexadecyl amine (HDA).

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This method was previously reported by Manali Banerjee et al.[30], they have developed acid-responsive nanocellulose hydrogel for three different hydrophobic

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drugs successfully, thus we take this work to load PTX and have a further biological evaluation. Based on the result of biological evaluation, this work provided a potential

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approach to enhance the solubility and control release effect.

Scheme 1 Schematic illustration of formation of gel loaded with PTX and the efficient delivery to cancer cells.

2. Materials and Methods 3

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2.1 Materials CNCs were obtained from ScienceK Co., Ltd. (Shanghai, China). Hexadecyl amine (HDA) was obtained from Macklin Co., Ltd. (Shanghai, China). Paclitaxel (PTX) and Indocyanine green (ICG) was obtained from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) was obtained from Nanjing chemical reagent co., Ltd. (Nanjing, China). Dulbecco's Modified Eagle's Medium (DMEM) and 1-(4,5-dimethylthiazol-2-yl)-3.5-diphenylfarmazan

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(MTT) were purchased by Gibco Laboratories (NY, USA). Fetal bovine serum (FBS) was purchased by Zhejiang Tianhang Biotechnology Co., Ltd. (Hangzhou, China).

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The adenocarcinomic human alveolar basal epithelial cells (A549), human HCC cells

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(HepG2) were purchased by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All the dye kits were obtained from Beyotime Biotechnology Co.,

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Ltd. (China). All the other chemical reagents were purchased by Sinopharm Chemical

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Reagent Co., Ltd. (China). All of these materials were used as received. 2.2 Surface modification – Acetic anhydride oxidation

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A total of 5 mL of triethylamine and 5 mL of acetic anhydride were added to 20 mL of anhydrous DMSO supplemented with 100 mg CNCs, and the suspension was

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further stirred at room temperature (25 ℃) at least for 2 h. After the reaction was completed, a proper amount of ice water was added followed by centrifugation at 10,000 g for 15 min. Saturated sodium carbonate solution was then added to destroy the residual acetic anhydride and the pH was adjusted to neutral. The obtained precipitation was collected to freeze-dried and finally obtained the dryness oxidized nanocellulose (O-CNC). 2.3 Gelation with PTX (PG) The gelation was performed according to a previously published research[30]. In brief, in the network formation step after O-CNC (10 mg·mL-1) and HDA (30 mg·mL-1) had been properly dissolved and dispersed into DMSO, the PTX (40 mg·mL-1) were added to the mixture and sonicated for 10 min. To disrupt 4

Journal Pre-proof intermolecular interactions, the mixtures were heated to 70 ℃. Then the mixtures were slow cooled at 4 ℃ to initiate gelation. Finally, the samples were dried overnight after water filtration. The gelation with ICG (IG) was prepared as the same steps as the gels with PTX in them. 2.4 Pharmaceutical characterization and biological evaluation 2.4.1 Fourier transform infrared spectroscopy (FTIR)

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Based on our previous method[31], Fourier transform infrared spectrometer

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(Nicolet 380, Thermo, Waltham, MA, USA) was performed to observe the degree of the oxidized nanocellulose (O-CNC) qualitatively. The samples were ground into

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powders and dried prior to analyses. Then, the samples were blended with potassium

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bromide powder and compressed to form a disk. The spectrum for each sample was recorded from 4000 to 400 cm-1 at a resolution of 2 cm-1 and an accumulation of 64

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times.

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2.4.2 Differential scanning calorimetry (DSC) The melting points and phase transformations of the samples were performed on

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the Differential scanning calorimetry (DSC 200 F3, Netzsch, GER). Samples were heated using a temperature width ranging from 40 to 350 ℃ at 10 ℃·min-1. 2.4.3 Transmission electron microscopy (TEM) The morphology of samples was observed on a Transmission Electron Microscope (JEM-1400, JEOL, JPN). The samples were suspended with water and sonicated in an ultrasonic crusher for 20 min. After the diluted samples suspensions were stewing overnight at 4 ℃, a drop of the suspensions was put on the surface of a carbon-coated copper grid. TEM images were taken at an accelerating voltage of 180 kV at 25 ℃. 2.4.4 In vitro drug release Drug dissolution studies were performed using dialysis method based on a 5

Journal Pre-proof previous studies[32]. PG were suspended (10 mL) in dialysis bag and then put in PBS (with pH 7.4, 6.8 and 5.5, respectively) at room temperature. Then, PG and free PTX for comparison was further suspended in dialysis and put in PBS (pH 5.5), respectively. Samples were taken at specific time intervals between 2 minutes and 72 hours and analyzed at 278 nm on a UV-Vis Spectrophotometer (UV-2450, SHIMAODZU, JPN) ranging from 200 to 400 nm. The dissolution measurements were obtained by comparing with the standard curve.

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2.4.5 In vitro cytotoxicity study The characterization of vitro cytotoxicity was referenced to previous literature

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using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)

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assay[33]. In brief, HepG2 cells and A549 cells were seeded at a density of 5.0×103 cells per well in 96-well plates in DMEM, respectively. After 24 h of incubation, the

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medium was replaced with fresh DMEM (100 µL) containing samples with six serial

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dilutions (3.125, 6.25, 12.5, 25 and 50 µg·mL-1). After 48 h of another incubation, 20 µL of the MTT solution (5 mg·mL-1) was added to each well of the culture medium.

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Then the cells were further incubated for 4 h to allow the reaction of MTT. Next, the medium was replaced with 100 µL of DMSO and thorough mixed with a microplate

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shaker. Absorbance at 490 nm was measured on a microplate reader (BioTek Cytation3, USA). Cell viability of hydrogels without PTX (NG), hydrogels with PTX (PG), free PTX and Control group were calculated with relevant equation. 2.4.6 Cellular uptake

According to the previous study[34], the cellular uptake test was performed by Confocal Laser Scanning Microscopic (CLSM LSM710, CarlZeiss, GRE). In brief, HepG2 cells and A549 cells were seeded 1.0×105 cells per dish respectively in the confocal petri dish cultured with Dulbecco’s modified Eagle medium (DMEM). After being incubated for 24 h, the medium was replaced with fresh DMEM containing an appropriate amount of gels suspension (IG) and incubated for different time durations (1.5, 3, 12 h). Then, the cells were stained by 100 µL Hoechst 33258 dye solution for 6

Journal Pre-proof 20 min. Finally, the intracellular distribution of ICG was then observed on CLSM. 2.4.7 Cell apoptosis A549 cells were cultured with DMEM in 6-well plates and incubated for 24 h. After removing the culture medium, cells were cultured with fresh DMEM containing PTX loaded gels (PG) and incubated for different time durations (3, 12 h). Untreated group was used as Control. The results were analyzed by flow cytometry (FCM) after stained with Annexin V and PI[35].

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2.5 Statistical analysis All analyses were performed in triplicate and mean ± SD values are reported.

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The results indicated that the analyses are similar and no significant difference was

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observed in each set of experiments. Furthermore, one-way ANOVA and the Tukey's multiple comparisons test (p < 0.05) was applied to reveal the statistically significant

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differences between the experiments and to compare the results.

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3.1 Surface modification

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3. Results and discussions

CNCs were oxidized to produce a surface containing carboxyl groups (O-CNC).

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The degree of oxidation can be confirmed qualitatively using FTIR. As shown in Fig. 1A, both CNC and O-CNC showed the typically appearance of 3403 cm-1, 2899 cm-1, 1623 cm-1, 1375 cm-1, 1224 cm-1, and 1051 cm-1, corresponding to O-H telescopic absorption peak, C-H stretching modes, in-plane vibrations of sp2-hybridized carbon atoms (C=C), hydroxyl stretching vibrations in alcohols (C-OH), C-O-C asymmetric bridge stretching, and C-O-C stretching modes, respectively. Furthermore, a strong C=O telescopic peak after oxidation at 1736 cm-1 corresponding to the carboxyl group on the O-CNC was observed, indicating the good degree of the oxidation with CNCs. The morphology of CNCs and O-CNC were observed by TEM under normal conditions. As shown in Fig. 1B, the image of O-CNC demonstrates more slender and uniform fiber after oxidation. This corresponds to the oxidation which was shortened the long chain of the fiber. Therefore, the result of FTIR and TEM demonstrate the 7

Journal Pre-proof oxidation of nanocellulose has been react relatively completely.

Fig. 1. (A) Oxidation of nanocellulose characterized via FTIR with CNCs (brown), O-CNC (yellow). (B) TEM

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images of oxidized nanocellulose.

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3.2 Gelation and characterization

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The stability of nanocellulose hydrogel was characterized by the inversion test. In the result of the test, at low concentrations the gel was not strong enough to keep its

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origin form, while sample with 10 mg·mL-1 O-CNC and 10 mg·mL-1 of ODA could

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not shape into gel (Fig. 2A). Furthermore, a 3: 1 ratio of ODA to O-CNC was kept a well gelation, and the gelation could also keep well when the gel further loaded with

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paclitaxel. The drug loading ratio and loading efficacy was investigated on UV-Vis, as shown in Table S1, the drug loading capacity of the prepared nanoparticles was about

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13.7%, and entrapment efficacy was about 58.9%. The hydrogel loaded with PTX (PG) was dried and characterized on DSC, the significant difference was showed on the result as compared with the ordinary PTX. The free PTX showed a melting peak at 216 ℃, as the same as the literature of PTX (Fig. 2B, green curve), while the PTX recovered from the dried gel did not show a melting peak, indicating that the PTX in the gel was not in a crystalline form (Fig. 2B, black curve). There is an exothermic peak at 87 ℃, which arises due to the melting of the HDA. The degradation exotherm of the PTX at 243 ℃ did not present, indicating that the morphology of PTX likely had changed. X-ray diffraction (XRD) measurements also proved this (Supporting Information, Fig. S1). The abnormal performance of PTX in PG was probably due to the limited nanocellulose space. The morphology of samples was demonstrated by SEM and TEM under normal 8

Journal Pre-proof conditions. The SEM image (Fig. 2C, SEM) showed the spatial structure of the gel. The TEM image (Fig. 2C, TEM) showed the morphology of reticular form, and the high-resolution image further exhibited the morphological characteristics of gel with a reticular structure. This reticular stereoscopic structure provided a favorable space to

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incorporate drugs and would improve the effect of water hydration.

Fig. 2. (A) State of hydrogel with different conditions in Gel with PTX (PG), Gel with 10 mg·mL-1 O-CNC and 30

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mg·mL-1 of ODA (NG), Gel with 10 mg·mL-1 O-CNC and 20 mg·mL-1 of ODA (NG1), Gel with 10 mg·mL-1 O-CNC and 10 mg·mL-1 of ODA (NG2), respectively. (B) DSC curve of dried gel comparied with O-CNC (red),

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HDA (blue), free PTX (green), PG (black). (C) SEM and TEM Images of dried PG in different resolution ratios.

3.3 In vitro drug release

Owing to the acidic conditions of tumors internally, the drug release efficiency of prepared hydrogel was investigated under different pH conditions and the results were showed in Fig. 3[36]. Due to the pH targeting function of the gel system, the dissolution rates are expected to be different in various pH conditions. As shown in the Fig. 3A, the concentration of PTX at pH 5.5 was obviously increased during the first 10 hours and showed a smooth increase in the following times. However, the PTX was not showed an obvious release at pH 7.4, indicating gel was stable enough at a normal human physiological environment. In addition, the ability of release was between the two above at pH 6.8 suggesting an acidic-stimuli of the gel. This result 9

Journal Pre-proof indicated the structure of hydrogel was destroyed by the acidic condition and resulted in the release of PTX, which suggested that the stability of hydrogel decreased along with the acidity. Moreover, dissolution test was further carried out at pH 5.5 with PG and free PTX for comparison. The experimental result showed that at pH 5.5 PG had a better release performance than free PTX (Fig. 3B). This result correlates with the encapsulation of PTX by nanocellulose networks. The hydrogel with nanocellulose networks was disassembled at pH 5.5 first and PTX released subsequently, thus PG has the sustained release performance. Therefore, this result confirmed the function of

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pH targeting of the gel.

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Fig. 3. (A) Acid sensitivity and durg release of hydrogel in pH 5.5 (blue), pH 6.8 (green), pH 7.4 (red). (B)Drug

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release performance of PG and free PTX at pH 5.5.

3.4 In vitro cytotoxicity study

The cytotoxicity of samples was characterized by using standard MTT method[37]. The efficacy of hydrogel to inhibit tumor cells was presented by the cytotoxicity for tumor cells and the results were showed in Fig. 4. In HepG2 cells, when the concentration up to 50 µg·mL-1, the cell viability of PG was significantly lower than NG for which cell viability of PG was reduced to 36.7% contrasting with 56.6% of the cell viability in free PTX (Fig. 4A). These results indicated that the present gel loaded with PTX showed good inhibition on HepG2 cells, and the anticancer effect was improved as compared with free PTX. Meanwhile, HepG2 cells treated with NG suggested the cell viability was more than 80%, indicating the low cytotoxicity of the gel without drugs. Moreover, PG treated A549 cells also indicated a low cell viability 10

Journal Pre-proof (30.9%) while free PTX treated cells indicated cell viability in 40.2% (Fig. 4B). Therefore, the cell viability with HepG2 and A549 cells were both decreased along with the concentration of hydrogel in the experimental groups, and no significant cytotoxicity was observed in the control group. In addition, NG showed the more stable low-toxic property and higher cell inhibition rate incubated in A549 cells than in HepG2 cells. Furthermore, when the concentration increased to 50 µg·ml-1, PG treated HepG2 cells and A549 cells showed low cell viability than that treated with free PTX, with the ratio of 34.2% and 30.5%, respectively. Thus, the surface-modified

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nanocellulose hydrogel was preliminarily confirmed to be an ideal vehicle in target

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release system.

Fig. 4. Cell viabilities of different cancer cells after incubated in PG (green), NG (red), free PTX (blue), and

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control group (black). (A) HepG2 cells. (B) A549 cells. Pairwise comparisons between treatments and control groups were made using the Student’s t-test. Statistical significance was defined as *P < 0.05, and n.sP > 0.05.

3.5 Intracellular uptake

We further evaluated the delivery of therapeutic agents into the cytoplasm or nucleus of carcinoma cells. In this study, the gel with ICG (IG) was observed at different incubation times by CLSM using intrinsic red fluorescence of ICG to observe the cellular uptake capacity with HepG2 and A549 cells, respectively. As shown in Fig. 5, two types of fluorescence intensity were detected and recorded in A549 cells, which were blue color of Hoechst 33258 (used for staining cell nucleus) and red color of ICG (used to replace the PTX)

[38]

. As expected, no significant

fluorescence intensity could be observed in the cells in the initial 1.5 h. However, the 11

Journal Pre-proof representative red fluorescence was found in the cytoplasm around the nucleus after been incubated with nanoparticles for 3 h. Furthermore, the red fluorescence of IG could be observed in the nucleus of both HepG2 and A549 cells after been incubated for 12 h, which indicated the prepared nanoparticles had potential to overcome drug resistance as the improved drug distribution intracellularly on tumor cells. The results indicated that the fluorescence signals of ICG mainly scattered around the cytoplasm while the fluorescence signals of Hoechst 33258 was surrounded in the middle, and the cellular uptake of gel was increased along with incubation time, indicating that the

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gel was entrapped inside the lysosome vesicles[36]. Moreover, A549 cells had a more

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rapidly intracellular uptake than HepG2 cells when incubated for 3h.

Fig. 5. CLSM images of HepG2 and A549 cells treated with IG in different times. Images from left panel to right present cell nuclei stained by Hoechst 33258 (blue), ICG fluorescence in cells (red), and merge of the two images, respectively. Scale bar: 50 μm.

3.6 Cell apoptosis

Based on the results of the above analysis, the induced apoptosis effect of the gel was evaluated with A549 cells and the results were showed in Fig. 6. The gel loaded with PTX induced 52.8% apoptosis after been incubated for 3 h and the apoptosis rate of A549 cells reached 90.5% after been incubated for 12 h, a dramatical increase of apoptosis in A549 cells was showed compared with control groups (Fig. 6A). In Fig. 6B, remarkable significant differences were shown in apoptosis rate between the treated groups and the control group, and the apoptosis rate increased along with the 12

Journal Pre-proof incubation time. The obtained results indicated that the obvious impact of PG for cell apoptosis was showed in A549 cells. In addition, the group that treated gel without PTX (NG) demonstrated a low apoptosis rate (12.7%), and in which most of the apoptosis was early apoptosis (12.4%), only 0.285% was late apoptosis, indicating the remarkable biocompatibility and low-toxicity of NG. The apoptosis analysis data was consistent with the MTT results, suggesting that the design of gel loaded with PTX was a promising and high-effective strategy to accelerate the apoptosis of A549

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cells[38].

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Fig. 6. (A)The apoptosis performance of A549 cells treated with NG, PG. And PG was incubated in 3h, 12h. (B) SD value was provided in the flow cytometric curves. Pairwise comparisons between treatments and control

0.001.

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groups were made using the Student’s t-test. Statistical significance was defined as *P < 0.05, **P < 0.01, ***P <

4. Conclusions

In summary, surface-modified nanocellulose gel was successfully formulated and processed for detailed as an excellent therapeutic vehicle for drug develivery. The physicochemical characterization results indicated that the gel was highly pH-sensitive and was stable under normal physiological conditions. The surface-modified nanocellulose gel presented sufficient capacity of paclitaxel, and the PTX-loaded hydrogel showed a sustained release behavior at a mild acidic environment. Moreover, the surface-modified nanocellulose gel obviously improved the distribution of PTX in cancer cells intracellularly, and the antitumor efficacy was 13

Journal Pre-proof significantly enhanced after incubation. Based on the results of the in vitro characterization studies, this surface-modified nanocellulose gel demonstrated an improved potential as a novel drug delivery system with better stability and more efficient pH-sensitive anti-cancer drug delivery. The present study highlights the research of CNC-mediated drug delivery system. Conflict of Interest Policy The authors have declared that no competing interests exist in this study.

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Acknowledgements

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We are highly grateful to the financial support from the National Natural Science Foundation of China (Grant Nos. 21905138), the National Natural Science

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Foundation of Jiangsu Province (Grant Nos. BK20190756), the project funded by

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China Postdoctoral Science Foundation (No. 2019M651841), the National Key Research and Development Program of China (2016YFD0600801), the Top-notch

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Academic Programs Project of Jiangsu Higher Education Institutions (TAPP, Grant Nos. PPZY2015C221) and Priority Academic Program Development of Jiangsu

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Higher Education Institutions (PAPD).

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