Synergic fabrication of dual drug-loaded polymeric nanoparticles system to improve the in vitro treatment of glioma metastasis specific targeting therapy

ABSTRACT

Poor drug delivery to the tumour site and harmful drug effects on the nearby healthy tissue are the two main issues with chemotherapeutic treatments for glioma. This study tests the efficacy of a chitosan dual delivery system loaded with rutin and aucubin to treat glioma. It was shown in vitro that rutin/aucubin loaded chitosan nanoparticlesignificantly inhibited U87 cells and promoted apoptosis, and the combination therapy results were superior to those of each compound used alone. The rutin-aucubin combination decreased the glioma cell’s ability to migrate. The chitosan rutin-aucubin nanocomposites showed time dependent cell uptake in U87 cells when labelled with RBITC ANG. At a low dose of 0.156 μg/mL chitosan rutin-aucubin successfully inhibited tumour growth and adhesion and ~ 43% necrosis was observed in mitochondrial membrane potential (ΔΨm) assay. In conclusion, rutin and aucubin drugs were codelivered which inhibited tumour proliferation by inducing cellular apoptosis at a low dose. This showed the system’s potential to effectively enhance glioma therapy.

KEYWORDS:

• Chitosan
• rutin
• aucubin
• glioma
• metastasis

Introduction

Gliomas are the most typical primary brain tumours. The majority of deaths from primary brain tumours are caused by gliomas, which make up nearly 30% of all primary brain tumours and 80% of all malignant ones [1]. Gliomas generally occur more frequently in people over the age of 50, with a 1 in 100,000 prevalence rate. Children and young adults can also develop glioma, but this is less common [2]. Due to the lack of knowledge about the disease and its recurrence, the current therapies for glioma treatment are limited. Despite decades of research and findings, there is still no permanent cure [3]. Glioma treatment is challenging due to the disease’s ability to metastasis (spread to nearby tissues) and the need for precise drug delivery to prevent side effects. Malignant glioma requires more research and efficient treatment to cure [4].

Rutin is a naturally occurring polyphenolic flavonoid that is found in food plants like citrus fruits and vegetables. Rutin is also referred to as rutoside or vitamin P [5]. The substance possesses an extensive quality, including antimicrobial, anti-inflammatory, anticancer, cytoprotective, and anticancer. According to [6] it is also acclimated to cure treat cardiovascular diseases and disorders of the nervous system. By modulating p53 expression, rutin administration decreases ischaemic neural apoptosis. Rutin was found to reduce dementia and neuroinflammation in Alzheimer mouse models [7, 8]. Evidence suggests that rutin-based nanoparticles increase the effectiveness of drug delivery to the site-specific delivery [9].

Aucuba japonica and Plantago asiatica are just two examples of medicinal herbs that contain aucubin, an iridoid glycoside [10]. Aucubin has anti-inflammatory, anti-cancer, and antioxidant properties. Aucubin also has hepatoprotective and neuroprotective properties. According to [11] the substance has been unveiled to protect the liver and pancreas from diabetic encephalopathy [12]. Aucubin lessens free radical damage in cases of brain injuries as well [13]. In an array of cancers, including lung cancer, liver cancer, and cervical cancer, aucubin demonstrated its anticancer properties [14, 15].

Drug nano formulations based on nanotechnology offer fresh perspectives on the pharmaceutical sector’s future. According to [16] nanoparticles have low surface potential, stable, slow drug release, and effectively accumulate drug in target cells without affecting neighbouring cells. Chitosan is a naturally occurring biopolymer that makes an alluring target in cancer therapy due to its biodegradability, biocompatibility, lack of toxicity, and ease of preparation [17,18]. Because of these characteristics, chitosan nanoparticles are an excellent candidate for treating a variety of cancer types, including glioma (Scheme 1).

Scheme 1. The schematic representation of the present investigation established that Rutin-Aucubin loaded Chitosan NPs inducing sustained drug release and apoptosis of cancer cells.

Combination therapy is receiving attention these days compared to single therapy owing to their increased potency on cancer cells. In breast and pancreatic cancer cells in vitro, the combination of rutin and orlistat increased apoptosis [19]. On gastric cell lines, rutin and oxaliplatin similarly reported high apoptosis rates via p53 [20]. Through the downregulation of p53 and Bcl-2, 5-fluorouracil and rutin had a synergistic effect that increased apoptosis [21]. As a result, dual therapy works on cancer cells better than monotherapy [22].

Materials and methods

Chemicals

The FDA-approved medications Rutin (CAS No. 479-98-1) and Aucubin (CAS No. 479-98-1) were bought from Sigma Aldrich – Merck, U.S.A. PI, rhodamine 123, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, rhodamine B isothiocyanate, aprotinin, and Hoechst 33,342 were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). An Annexin V FITC/PI Apoptosis Detection Kit was purchased from Sigma-Aldrich, Darmstadt, Germany. Haematoxylin and eosin stains were purchased from Sigma-Merck (Darmstadt, Germany).

Cell lines

U87 glioma and L929 cell lines were purchased from the ATCC (Manassas, VA) in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Laboratories, U.S.A.). The medium contains high percentage of glucose and 10% foetal bovine serum (FBS) heat inactivated (HyClone), penicillin (100 U/mL), and streptomycin (10 μg/mL) at 37°C with 10% CO₂ in a humidified atmosphere. Before the experiment, the cells should be grown to confluence. Three individual experiment data were calculated.

Preparation of chitosan nanoparticle

Chitosan, with a molecular weight of 400 kDa and a 12.7% acetylation degree, was dissolved in 10 mL of acetic acid (6%). After that, 10 mL of sodium nitrate (0.1%) was added, and slow shaking was given at 37°C for around two h and the pH was raised to 9 using sodium hydroxide (4 M). The chitosan precipitate was filtered out, washed three times with acetone, and then dissolved in 40 mL of acetic acid (0.1 N). Using a 12 kDa cut-off dialysis bag, the solution was dialysed and then stored at 4°C in desiccators [23].

Preparation of rutin and aucubin nanocomposites

In 1 mL of distilled water (pH 5.5), the prepared chitosan nanoparticle (10 mg) was dissolved. Using 1N acetic acid, the pH was adjusted, and the solution was continuously shaken at room temperature for 3 h. 5 mg of rutin and 5 mg of aucubin were combined and briefly vortexed for 15 seconds to create the drug nanocomposite. Rutin and aucubin prepared stocks were combined with a 1 mL chitosan solution and kept undisturbed for half an hour at 37°C. Rutin and aucubin single therapy nanocomposites were made by the same protocol.

Nanoparticle drug encapsulation kinetics

Drug loading (DL) and Encapsulation efficiency (EE) of chitosan rutin-aucubin nanoparticle was determined by high-performance liquid chromatography (HPLC) method. 5 mg of lyophilised sample was dissolved in 0.1 ml of methanol. A reversed-phase C 18 column (4.6 × 250 mm, 5 μm, Thermoscientific Hypurity analysis column) and waters 2966 detector was used for analysis. Methanol-water (70:30) eluent with a flow rate of 1 mL/min was performed.

The DL and EE was calculated using the following formula:

DL = Drug/polymer+drug x 100%

EE = Experimental DL/Theoretical drug loading x 100%.

Characterization of the chitosan rutin-aucubin nanocomposite

The size and electrokinetic potential of the synthesised nanocomposites were evaluated using DTS Zetasizer Nano (Malvern, Worcestershire, UK). TEM (Hitachi, H-7650, Japan) analysis was done to find the nanoparticle size and morphology. The samples were diluted with distilled water and placed on a carbon coated copper grid for analysis.

Drug release study

The synthesised nanocomposites drug release mechanism was evaluated using the in vitro dialysis bag diffusion method. The dialysis bag was put in a beaker with 30 mL of phosphate buffer (pH 7.4) containing Tween-80 (0.5% w/w), chitosan rutin-aucubin nanoparticles. The beaker was kept under slight shaking at 37°C. At specific time intervals the samples were taken for analysis and replaced with fresh phosphate buffer. The amount of rutin and aucubin in the medium was measured using UV-Visible spectrophotometer @ 230 nm.

Cytocompatability and cytotoxicity of rutin and aucubin in U87 and L929 cells

For the MTT test, 400 cells per well of U87 and L929 cells were plated onto titre plates. Rutin-aucubin was present in concentrations of 0, 0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, and 10 μg/mL, respectively. Cell viability was assessed by adding 20 µl of MTT solution (5 mg/mL; Sigma, St. Louis, MO, U.S.A.) to each well at different time points (24, 48, and 72 h) and incubated for an additional 4 h. 150 μl of DMSO was added after carefully removing the medium and further shaked for 5 minutes to read the absorbance at 570 nm in a titre plate reader. Experiments were performed in triplicates for accuracy.

Cell apoptosis assay

An Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich) was used in accordance with the manufacturer’s instructions to measure cell apoptosis. Cells were co-incubated with chitosan rutin, chitosan aucubin, and chitosan rutin-aucubin for 24 h after transfection. After 48 h, the glial cells were retrieved and stained twice with FITC-Annexin V/PI (fluorescein isothiocyanate)-conjugated Annexin V. The cell apoptosis was performed using flow cytometry (CyFlow® Cube, Sysmex, Japan), and the results were examined using VenturiOne software. U87 cells were co-incubated with chitosan rutin, chitosan aucubin, and chitosan rutin-aucubin in 12-well plates overnight. The membrane potential assay was performed after 48 h by staining the cells.

Wound scratch analysis

To measure cell motion, the gliomal cells were transferred into 6 well flat bottom plates (10% CO2 at 37°C) until confluent in a humid environment. At concentrations of 0, 0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, and 10 μg/mL, the nanoparticles were administered. Artificial wounds were made on the cell monolayer. To stop cell proliferation, the cells were washing in PBS before being incubated in DMEM without serum. Using a 10× objective, the corresponding images were collected at 0 h, 24 h, and 48 h. Image J was used to find the wound location.

U87 glioma cells cellular uptake and competitive assay

15×10⁶ glioma cells per well were cultivated in a 24-well plate for a day. Following a 24-h incubation period, the glial cells were co-incubated with RBITC-labelled chitosan rutin-aucubin nanocomposite for 30, 60, and 120 minutes at 37°C. The cells PBS washed with ice-cold Phosphate buffer (pH 7.4) before being observed under a fluorescent microscope (Olympus CX43, Japan).

Prior to the gliomal cells binding assay, angiopep-2 or aprotinin (50 μg/mL) was included in the plate and kept undisturbed (30 minutes). Angiopep-2 or aprotinin (50 μg/mL) and 200 μg/mL of RBITC-labelled chitosan rutin-aucubin nanocomposite were added and kept for an additional 120 minutes. At 4°C, the procedure was completed.

Statistical analysis

All data were represented using Mean ± SEM. Statistical analysis of the comparison groups was performed by GraphPad Prism 6. The student’s t-test and the ANOVA were used, respectively, to test for significance and analyse the variance of the comparison data. Kaplan-Meier method provides the survivance rate. Data that were statistically significant were indicated by the symbols ***p < 0.001, **p < 0.01, *p < 0.05.

Results

Rutin and aucubin nanocomposites characterization

In rutin-aucubin, the drug loading (DL) and drug encapsulation efficiency (EE) was 5% and 98.4% for rutin and 5% and 93.4% for aucubin, respectively. TEM images showed the size of the nanoparticles as 100 nm and zeta potential of −1.51 mV (Figure 1).

Figure 1. Characterization of nanocomposites (a) TEM image of chitosan rutin-aucubin (b) size distribution spectrum of chitosan rutin-aucubin (c) Zeta potential of chitosan rutin-aucubin.

In vitro drug release kinetics

Rutin and aucubin were released from chitosan rutin-aucubin over a period of time, peaking at 67% and 50% of the total drug over the course of 96 h, respectively (Figure 2). The nanocomposite is stable, and its release within the body is constant to effectively accumulate drug in the tumour.

Figure 2. In vitro release kinetics of chitosan-rutin-aucubin.

Cytotoxicity of rutin and aucubin in U87 and L929 cells

The cytotoxicity of chitosan rutin, chitosan aucubin, and chitosan rutin-aucubin has revealed various growth responses at different time intervals 24, 48, and 72 h and concentrations. The study’s control group consisted of cells that had not been treated. At a specific concentration range from 0 to 0.312 μg/mL rutin and aucubin monotherapy and rutin-aucubin combination therapy showed significant cell viability decrease in a time and dose dependent manner. At 0.156 μg/mL chitosan rutin-aucubin and chitosan rutin/chitosan aucubin exhibited cytotoxicity of 98% and 50% respectively on U87 cells with a p-value of 0.001 (Figure 4). The cytotoxicity assessment of chitosan rutin/aucubin at different concentrations on U87 cells revealed that even both chitosan rutin/aucubin monotherapy are toxic against glioma cells. Chitosan rutin/chitosan aucubin and chitosan rutin-aucubin NPs started having a significant toxic effect on glioma cells at 0.156 μg/mL during 48 h of treatment (Figure 5). Moreover, the drug-loaded chitosan rutin/chitosan aucubin NPs combination therapy have shown more cytotoxicity against glioma cells even at a low dose of 0.156 μg/mL compared to single therapy chitosan rutin/aucubin. The nanocomposite’s maximum potential required to inhibit the glioma cells was the lowest dose specified in this assay. The cytocompatibility of L929 cells kept as controls did not exhibit any toxicity when treated with either rutin or aucubin alone or in combination (Figure 3). The findings suggest that the actions of rutin and aucubin when combined in a nanocomposite on glioma cells are complementary.

Figure 3. Cytocompatibility of chitosan, chitosan-rutin, chitosan rutin-aucubin in L929 cells at different incubation hours (0 h, 24 h and 48 h); scale bar = 100 µm.

Figure 4. Cell viability MTT study in U87 glioma cells treated with chitosan rutin, chitosan aucubin and chitosan rutin-aucubin at various concentrations for 24, 48 and 72 h. The data are indicative of at least three individual studies and are shown as mean ± sem. ANOVA and Student’s t-test are used; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. Cell viability study in U87 glioma cells AO/EB-stained (a) control (b) chitosan (c) chitosan-rutin (d) chitosan-aucubin (e) chitosan rutin-aucubin; scale bar = 100 µm.

Glioma apoptotic assay

The induction of apoptotic activity was a significant sign of the medication’s influence on those cells and is found using flow cytometry and annexin (V-FITC)/propidium iodide (PI) double staining method. Phosphatidylserine in the external membrane binding to annexin/PI is a marker for cell death analysis. Even at lower concentrations, cells that are extremely sensitive to the treated nanocomposites experience apoptosis. Both single therapy and combination therapy induced apoptosis, but the activity was directly correlated with drug dosage. Only 2.61% and 19.8%, respectively, of the 0.312 μg/mL dosage of chitosan rutin and aucubin were seen in the monotherapy, whereas 37.14% was attained in the dual therapy with chitosan rutin and aucubin (p < 0.001) (Figures 6 & 7). The findings support the hypothesis that rutin and aucubin together promote glial cell apoptosis in a synergistic manner even in a low concentration.

Figure 6. Flow cytometry images of apoptosis study in U87 cells with chitosan rutin, chitosan aucubin and chitosan rutin-aucubin at various concentrations for 48 h.

Figure 7. Apoptosis study in U87 cells with chitosan rutin, chitosan aucubin and chitosan rutin-aucubin at various concentrations for 48 h. The data are indicative of at least three individual studies and are shown as mean ± sem. ANOVA and Student’s t-test are used; *p < 0.05, **p < 0.01, ***p < 0.001.

The intrinsic apoptotic pathway’s involvement in the process was well-indicated by the mitochondrial membrane potential (ΔΨm) assay. With chitosan rutin-aucubin, the predicted percentage of necrotic cells was 43.9%, whereas with chitosan rutin and aucubin alone, the percentages were 4.47% and 16.06%, respectively (chitosan rutin-aucubin vs chitosan rutin, p < 0.001) (Figure 8). However, no discernible apoptotic activity in the control (untreated, vehicle) was seen. The results also showed that an intrinsic apoptotic pathway was in charge for the antiproliferative properties of chitosan rutin-aucubin.

Figure 8. Changes in mitochondrial membrane potential (ΔΨm) of chitosan rutin-aucubin.

Wound healing assay

Finding the potential for metastasis in gliomas is made easier by the cell’s motility. From a concentration of 0.156 μg/mL and up, the chitosan rutin-aucubin nanocomposites did not show any proliferation and the width of the wound did not show any change. The single therapy rutin and aucubin cells also showed the same results like chitosan rutin-aucubin (Figure 9). Additionally, the concentration of the nanodrug directly affects the anti-proliferation activity. This indicated that the synergistic combination therapy of rutin and aucubin in a carrier inhibits metastasis.

Figure 9. In vitro wound scratch assay for chitosan rutin-aucubin at different incubation time (0 h, 6 h, 12 h, 24 h & 48 h); scale bar = 100 µm.

Intake characteristic of in vitro chitosan rutin-aucubin nanocomposite by U87 cells

Fluorescent based procedure was accomplished to examine the cellular intake mechanism of the U87 cells using the RBITC-labelled chitosan rutin-aucubin nanocomposite. The chitosan rutin-aucubin nanocomposites had a higher level of cellular intake than the individual rutin-aucubin therapies. The fluorescence images showed that the nanodrugs were consumed in a time-based manner (Figure 10).

Figure 10. Cell uptake fluorescent microscopy study of RBITC-labelled NP (a, c, e) and ANG-NP (b, d, f) at different time intervals. A & b 30 min, c & d 60 min, e & f 120 min. Red: RBITC; scale bar = 100 µm.

The uptake of RBITC-labelled chitosan rutin and aucubin was decreased in the competition assay by the addition of LRP ligands (angiopep-2, aprotinin). In addition, compared to 37°C, the intake was inhibited at 4°C. Therefore, LRP ligands competitively inhibit the intake of nanocomposites by cells in a time-dependent manner (Figure 11).

Figure 11. Cell uptake fluorescent microscopy study of RBITC-labelled NP after 120 min. A &b ANG-NP and ANG-NP at 4°C, c & d 200 mg/ml free angiopep-2 ANG-NP and aprotinin. Red: RBITC; scale bar = 100 µm.

Discussion

The malignant brain tumour called glioma arises from glial cells and spreads to nearby brain regions. About 30% of tumours that develop in the brain regions are gliomas. Inadequate access to suitable drugs and their effectiveness contributes to the prognosis and recurrence of gliomas. Due to BBB, the drugs find difficult to navigate the layer and treating gliomas or any disease becomes challenging [24]. The drug’s therapeutic potential is decreased by the brain barrier, which prevents the drug from crossing the membrane [25, 26]. Glioma cannot be completely cured with conventional chemotherapy, and recurrences are more frequent [27]. Using chitosan as a nanocarrier, rutin and aucubin were both redeemed to the glioma cells in this study.

By cause of traditional non-specific chemotherapy, the evolution of novel molecular targets and therapies are in need to treat malignant gliomas [28]. For glioma therapy new technologies are under development, including hybrid, lipids, and polymers-based nanoparticle carriers [29, 30]. A significant advance in targeted glioma therapy will be made by peptides and gene therapy. These drug-targeted therapies aim to reduce drug side effects, improve drug delivery, and kill tumour cells at specific sites. To facilitate site-specific drug delivery, minimise side effects, and ascertain the medications’ synergistic effects, chitosan was amalgamated with a number of distinct drugs [31, 32]. Since the nanoparticles are biodegradable, water soluble, and small in size, they enable molecules to pass through endothelial junctions. Chitosan helps slow drug release to the tumour site by holding the drug for a very long time. So, the ideal drug delivery system is the chitosan nanocarrier.

Rutin is a bioflavonoid used in the treatment of gliomas and is found in a variety of foods, including apple, tea, and others [6]. According to reports, rutin has few side effects and has been used in nano formulations to treat diverse diseases [5, 33]. Similar to this, aucubin is an iridoid glycoside that can be seen in many medicinal plants. According to reports [15,34], aucubin has been shown themselves to be effective against a diversity of cancer types. As a result, chitosan-based drug delivery efficiently encapsulates rutin and aucubin with a long drug-holding property, constant drug release, site-specific treatment, and a decrease in glioma treatment side effects.

By promoting apoptosis, the chitosan delivery system in this study’s formulation with rutin and aucubin prevented the proliferation of in vitro glioma cells. This work has confirmed the effectiveness of the glial cells’ uptake of the synthesised chitosan nanocomposites. The importance of the cell division and its modulatory effects in cancer has been emphasised by [35, 36]. Rutin and aucubin’s cytotoxicity showed that the nanocomposites are powerful enough to halt the growth of glioma cells when used in combination therapy. The results show that both drugs complement each other in enhancing the effectiveness of the synthesised nanocomposite. The apoptosis activity shows that the combined rutin and aucubin showed that even a low concentration of 0.312 μg/mL was efficient enough to increase the apoptotic activity by 37.14%. According to reports, aucubin causes apoptosis and cell cycle arrest in the A549 non-small cell lung cancer cell line [14]. Similar to this, rutin promotes miRNA-877-3p, which causes apoptosis in breast and pancreatic cancer [19, 37]. Rutin inhibits the notch signalling pathway and causes Caski cells to die in cervical cancer [38, 39]. Rutin has also been observed to have an impact on neuroblastoma and glioma CHME cells by inducing apoptosis through the upregulation of p53 [40].

The rutin and aucubin synergistic effect drastically altered the ΔΨm and the necrotic percentage appeared to be increased when rutin and aucubin were used together as a therapy. Rutin compounds have been used to treat neurotoxicity and cervical cancer in the past [41–43]. These compounds have been shown to alter membrane potential. In the meantime, aucubin was associated with the alteration of membrane potential and the promotion of apoptosis and programmed cell death in neuroblastoma and injuries [44,45].

Wound healing assay proved that the combined therapy of rutin and aucubin inhibited glioma cells migration at 0.156 μg/mL. The ability of rutin-loaded liquid crystalline nanoparticles to prevent cell migration has been demonstrated in vitro using cancer cells [46]. Similarly, it inhibits superoxide production and prevents cancer cells from adhering [47]. According to reports, aucubin has immunomodulatory properties and inhibits cell adhesion as part of its anticancer properties [48].

The BBB obstruction is a crucial consideration when developing drugs for brain diseases. The best way to deliver chemotherapeutic drugs to the brain with the fewest side effects is to identify the receptor targets on the BBB and glioma cells [49]. One such receptor, LRP, is upregulated in glioma and expressed in the BBB [50]. LRP mediates transport through the BBB’s endothelial cells by binding with numerous ligands, such as Angiopep-2 and aprotinin [51]. In vitro uptake of U87 glioma cells by the Angiopep-conjugated chitosan rutin-aucubin nanoparticles was enhanced. The uptake of nanocomposites by cells may be competitively inhibited by LRP ligands in a time-dependent manner.

Therefore rutin-aucubin’s synergistic effect encourages apoptosis and prevents cell movement and enables it to effectively inhibit glioma growth even at low dosages and with reduced toxicity.

Conclusion

For the sake of delivering rutin and aucubin to glioma cells, chitosan rutin-aucubin nanoparticles were created. This improved the drug’s absorption by the glioma cells and reduced the cancer cell’s ability to proliferate. In gliomas, the synergistic interaction between rutin and aucubin was confirmed. As a result, rutin and aucubin may be an excellent drug candidate for the clinical management of gliomas.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data used to support the findings of this study are available from the corresponding author upon request.