Honokiol–camptothecin loaded graphene oxide nanoparticle towards combinatorial anti‐cancer drug delivery

Abstract

Honokiol (HK) is a natural product isolated from the bark, cones, seeds and leaves of plants belonging to the genus Magnolia. It possesses anti‐cancer activity which can efficiently impede the growth and bring about apoptosis of a diversity of cancer cells. The major concerns of using HK are its poor solubility and lack of targeted drug delivery. In this study, a combinatorial drug is prepared by combining HK and camptothecin (CPT). Both CPT and HK belong to the Magnolian genus and induce apoptosis by cell cycle arrest at the S‐phase and G1 phase, respectively. The combinatorial drug thus synthesised was loaded onto a chitosan functionalised graphene oxide nanoparticles, predecorated with folic acid for site‐specific drug delivery. The CPT drug‐loaded nanocarrier was characterised by X‐ray diffractometer, scanning electron microscope, transmission electron microscope, UV–vis spectroscopy and fluorescence spectroscopy, atomic force microscopy. The antioxidant properties, haemolytic activity and anti‐inflammatory activities were analysed. The cellular toxicity was analysed by 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT assay) and Sulforhodamine B (SRB) assay against breast cancer (MCF‐7) cell lines.

1 Introduction

Honokiol (HK) is a polyphenol isolated from the barks, leaves, seeds and cones of plants of Magnolia genus. It is commonly obtained from Magnolia officinalis, a herb commonly used in Chinese medicine. Previous studies demonstrated that HK possess antioxidant properties [1], anti‐inflammatory properties [2], anti‐thrombosis and anti‐cancer activities [3, 4], induction of apoptosis, inhibition of tumour growth and anti‐angiogenic properties [5]. It is widely used for different forms of cancer, including lymphocytic leukaemia, multiple myeloma [6], lung, colorectal and angiosarcoma [7, 8, 9].

It also plays a vital role in the treatment of breast cancer. It was reported to reverse the multidrug resistance via inhibiting the expression of P‐glucoprotein and inhibiting the phosphorylation and degradation induced by tumour necrosis factor‐α (TNF‐α) of the cytosolic nuclear factor kappa‐B (NF‐kB) thus suppressing the intrinsic IkB kinase activities in MCF‐7 breast cancer cell line [10].

Major concerns of using a single drug are ease renal and blood clearance, which in turn decreases the bioavailability, to overcome this a high dosage is administered which leads to severe side effects [11]. Also, a single chemotherapeutic drug is often not potent enough due to inhomogeneous spreading of cancer cells. To overcome this and multidrug resistance, combinatorial chemotherapy is developed, which can produce a desirable effect in a low dose [12]. Another anti‐cancer drug camptothecin (CPT), isolated from the bark of Camptotheca acuminate, belonging from the genus Magnolia was combined with HK. CPT exerts its anti‐cancer activity by the development of a ternary complex, which in turn prevents the binding between topoisomerase I and DNA thus checking relegation [13, 14].

Chemotherapeutic agents are often loaded onto nanoparticles, which can easily invade the tumour tissue, thus ensuring a more specific drug delivery. In drug delivery, the most commonly used nanoparticles are dendrimers, gold nanoparticles, silver nanoparticles, fullerenes, liposomes, carbon nanotubes etc. [15, 16]. One such form of carbon, graphene (G) and the derivative products of graphene viz. graphene oxide (GO), and reduced GO (rGO) have grown the attention from the scientific community because to its astonishing properties such as massive surface area, tranquil functionalisation by π–π interactions and high conductivity [17]. The major fears of the biomedical application of graphene are its size‐dependent cytotoxicity and sharp edges. These limitations can be effectively used for the destruction of cancerous cells by masking the sharp edges of graphene, thus preventing its haemolytic activity [18, 19]. The polymers commonly used for drug delivery are polyethylene glycol, polyvinylpyrrolidone, polyacrylic acid, polyvinyl alcohol, chitosan, cellulose etc. [20]. Chitosan is preferred among the polymers mentioned above due to its low cost, easy availability, polyelectrolyte and mucoadhesive nature and solubility in several media [21].

In our previous work, we concluded that chitosan polymerised GO was more biocompatible and efficient in targeted drug delivery [22]. In this study, a combinatorial chemotherapeutic agent is prepared by combining HK and CPT and its activity against breast cancer cell lines (MCF‐7) is evaluated. The combinatorial drug was further loaded onto chitosan functionalised GO nanoparticles. Also, to achieve a more specific targeted drug delivery, the functionalised nanoparticle was predecorated with folic acid (FA), which specifically binds to the folate receptors overexpressed on the surface of cancer cells.

2 Experimental section

2.1 Materials

FA, HK, ethanol, dimethyl sμlfoxide (DMSO), foetal bovine serum (FBS), N‐(3‐dimethylaminopropyl‐N‐ethylcarbodiimide) hydrochloride (EDC), potassium ferricyanide, graphite powder, chloroacetic acid, N‐Hydroxysuccinimide (NHS), chitosan (CS), CPT, 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) reagent, Sulforhodamine B (SRB), Dulbecco's modified Eagles medium (DMEM), chloroform, folin‐Ciocalteu's reagent, acetic acid, sodium carbonate, ferric chloride, 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) were obtained from Sigma Aldrich.

2.2 Methods

2.2.1 Synthesis of the combinatorial drug (HK–CPT) loaded functionalised nanocarrier

HK and CPT were loaded onto presynthesised GO nanoparticles. GO was synthesised following modified hummers method [23]. The GO accordingly synthesised was functionalised with chitosan. Briefly, sodium hydroxide (NaOH) and chloroacetic acid (ClCH₂ COOH) were added for carboxylation of GO (GO‐COOH). This carboxylation makes it suitable for polymerisation. 1% chitosan was dissolved in 10% acetic acid followed by stirring overnight to achieve a viscous solution. A GO solution of 0.5% was then added to the viscous solution of chitosan and subjected to sonication for 1 h. The functionalised GO thus synthesised was activated by the addition of EDC and NHS (1 mg/ml) to the GO‐CS solution. The activated GO‐CS solution was then decorated with FA by adding 0.5% FA to the GO‐CS solution followed by stirring for an overnight period. The chitosan–FA, functionalised GO (GO‐CA‐FA) thus synthesised was loaded with combinatorial drug HK–CPT. Briefly, HK–CPT was dissolved in DMSO followed by dropwise addition to the predecorated GO‐CS‐FA solution and kept for overnight stirring. The unbound HK–CPT was further removed by frequent washing and centrifugation [24, 25].

2.2.2 Characterisation of GO‐CS‐FA‐CPT–HK

The nanocarrier thus synthesised was characterised by UV–vis spectroscopy and X‐ray diffractometer (XRD) (Bruker, D8 advance, Germany), using CuKα, Ni filtered radiation for the pure phase identification of the nanocarrier. The morphological analysis of the nanocarrier was carried out by scanning electron microscope (SEM) (CARL ZEISS) and transmission electron microscope (TEM) (FEI Tecnai, G2 20 Twin). The surface roughness of the drug‐loaded nanocarrier was further characterised by atomic force microscopy (AFM) (Nanosurf Easy Scan 2). The drug loading was confirmed using UV–vis spectroscopy and fluorescence spectroscopy (F‐7000 FL Spectrophotometer).

2.2.3 Anti‐inflammatory and haemolytic activity

The membrane stabilisation method was employed to determine the anti‐inflammatory activity of GO and GO‐CA‐FA‐CPT–HK. Herein whole human blood was collected and transferred to tubes containing equal volume Alsever's solution and centrifuged at 3500 rpm for 10 min. Post centrifugation the obtained pellet was washed using isosaline and resuspended again. 2 ml of hypotonic saline and 1 ml of phosphate buffer saline were then added to various concentrations of GO and GO‐CS‐FA‐CPT–HK and the reaction initiated by adding 0.5 ml of blood followed by 30 min incubation. The solution was then centrifuged for 5 min at 3000 rpm and the absorbance of the supernatant was recorded using ELISA reader (Lark Pvt. Ltd) at a wavelength of 560 nm. Herein diclofenac sodium salt was used as a standard drug [26].

$$\mathrm{Percentageof}\mathrm{stabilisation}=100-[(\mathrm{O}{\mathrm{D}}_{\mathrm{sample}}/\mathrm{O}{\mathrm{D}}_{\mathrm{contorl}})\times 100]$$

To study the biocompatibility of GO and GO‐CS‐FA‐CPT–HK, haemolysis activity was carried out. This was determined by a human red blood cell (HRBC) method. Briefly, human whole blood (5 ml) was collected in ethylene diamine tetraacetic acid (EDTA) containing tubes and mixed with magnesium and calcium‐free Dulbecco's phosphate buffer. The tubes were centrifuged for 5 min at 500g and repeatedly washed to obtain serum‐free RBC. From the respective stock solutions (500 μg/ml), different concentration of GO‐CS‐FA‐CPT–HK and GO were prepared. Human blood was then added to the different drug concentration (1: 4) and subjected to incubation. Centrifugation was done followed by incubation and the absorbance of the supernatant was recorded at 544 nm in an ELISA reader (Lark Pvt. Ltd) [18].

$$\mathrm{Percentageof}\mathrm{haemolysis}=(\mathrm{O}{\mathrm{D}}_{\mathrm{sample}}=\mathrm{O}{\mathrm{D}}_{\mathrm{negative}\mathrm{control}}/\mathrm{O}{\mathrm{D}}_{\mathrm{positive}\mathrm{control}}-\mathrm{O}{\mathrm{D}}_{\mathrm{negative}\mathrm{control}})\times 100$$

2.2.4 Antioxidant studies

DPPH radical‐scavenging assay: 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) being a stable radical is used in this study to assess the free radical scavenging activity of GO and GO‐CA‐FA‐CPT–HK. Ascorbic acid was used as a standard. Briefly, various concentrations of GO and GO‐CA‐F‐ACPT–HK were prepared to which 0.1 mM DPPH (1 ml) was added, mixed properly and incubated for 30 min. The radical scavenging activity was recorded spectrophotometrically at a wavelength of 517 nm [27]

$$\text{\%}\mathrm{disappearance}=[(A_{\mathrm{control}}-A_{\mathrm{sample}})/A_{\mathrm{control}}]\times 100\text{\%}$$

2.3 In‐vitro investigation

2.3.1 MTT assay

Cytotoxicity of drug‐loaded nanocarrier and the nanocarrier was tested against breast cancer cells (MCF‐7). In this study, MCF‐7 cell lines were cultivated in 96‐well culture plates at a temperature of 37°C, CO₂ 5% and air 95%. The cells were treated with GO‐CS‐FA‐CPT–HK, GO‐CS‐FA, CPT and HK in 96‐well culture plates and subjected to 24 h incubation. Post incubation of MTT reagent (10 μl) was poured to the wells followed by incubation for 4 h. Further, the crystals were dissolved in a solubilising agent (100 μl) and cytotoxicity was recorded spectrophotometrically using a microplate reader (Lark Pvt. Ltd) at 570 nm. The morphological images of MCF‐7 cell lines post‐treatment with the drug were obtained using an inverted microscope (Nikon TS‐100) [28].

2.3.2 SRB assay

Cellular toxicity was also assayed by a colourimetric Sulforhodamine B assay (SRB). Herein cell concentration was measured based on the protein content of the cell. Briefly, MCF‐7 was cultivated in 96‐well plates under prime circumstances, such as 95% humidity, 5% CO₂ and temperature 37°C. Post‐cellular growth varying concentrations of drug‐loaded nanocarrier was added and incubated for another 48 h. The reaction was concluded by the addition of 30% w/v trichloroacetic acid (ice cold) and subjected to incubation of 60 min at 4°C. The plates were washed 4–5 times and tap dry and Sulforhodamine B solution (0.4%) was then added to each well for the development of pink colour. The residual dye thus left after removal of unbound dye by repeated washing was eluted with trizma base 10 mM and the absorbance was measured using an ELISA reader (Lark Pvt. Ltd) at a wavelength of 540 nm [29].

3 Results and discussion

3.1 Synthesis and characterisation of the combinatorial drug (CPT–HK) loaded nanocarrier

GO thus synthesised following modified Hummer's method was functionalised with chitosan to make it biocompatible. Before loading CPT and HK to the chitosan functionalised nanocarrier, FA was added to achieve a targeted drug delivery to the cancerous cells. The nanocarrier loaded with the drug was characterised by XRD. Herein GO gave it representative peaks at 11.8θ (JCPDS card No: is 75‐1621). Post functionalisation with chitosan GO‐CS gave several blunt peaks at 17.71 (d  = 4.587 Å), 29.8.56 (d  = 2.189 Å) and 41.18 (d  = 2.206 Å). Further FA conjugation to the functionalised GO lead to the generation of multiple blunted and strident peaks at 2θ 15.79 (d  = 5.479 Å), 25.83 (d  = 3.226 Å), 31.35 (d  = 2.876 Å) and 37.83 (d  = 2.343). CPT loading to the functionalised nanocarrier was evident from the peaks at 15.89° (d  = 5.552 Å), 31.38° (d  = 2.89 Å), 36.83° (d  = 2.43) and 47.86° (d  = 2.125 Å). The combining of HK to CPT resulted in noticeable peaks at 16.84 (d  = 8.46 Å), 19.97 (d  = 8.17 Å), 29.92 (d  = 5.86 Å) and 34.322 (d  = 3.29 Å) (Fig. 1).

Fig. 1.

XRD patterns of GO/GO‐CS/GO‐CS‐FA/GO‐CS‐FA‐CPT/GO‐CS‐FA‐CPT–HK

UV–vis spectroscopy was done to characterise the drug‐loaded nanocarrier. A visible hump was produced in the case of GO at a wavelength of 230 nm and functionalisation of GO by chitosan was confirmed from peaks at 224 and 300 nm. Similar absorption spectrum for GO has been reported by Eda et al. [30]. Further, the conjugation of FA with functionalised GO (GO‐CS‐FA) was established by peaks at 248 and 371 nm (Fig. 2). Further, the loading of the anti‐cancer drug CPT to the nanocarrier was evident by peaks at 365, 279 and 256 nm and a rise in peaks was observed at 222, 286 and 352 nm post‐loading of HK to GO‐CS‐FA‐CPT. The surface morphology of GO after functionalisation with chitosan was observed by SEM. Post functionalisation the crumbled and wrinkled sheets of the layered GO were smoothen, thus indicating proper masking of the sharp wedges of GO (Fig. 3). The SEM data was further supported by TEM. After loading of CPT–HK, the particle size was found to be increased, as shown in Fig. 4. The morphology of GO‐CS‐FA‐CPT–HK was analysed by AFM. The average surface roughness Ra of GO‐CS‐FA‐CPT–HK was calculated to be 654.39 nm when scanned with areas of 25 × 25 μm (Fig. 5).

Fig. 2.

UV–vis absorption spectra of GO/GO‐CS/GO‐CS‐FA/GO‐CS‐FA‐CPT

Fig. 3.

SEM images showing

(a) GO, (b) GO‐CS‐FA‐CPT–HK

Fig. 4.

TEM images of

(a) GO, (b) GO‐CS‐FA‐CPT–HK

Fig. 5.

AFM images of GO‐CS‐FA‐CPT–HK

3.2 Haemolysis and anti‐inflammatory activity

The biocompatibility of GO, CPT loaded nanocarrier (GO‐CS‐FA‐CPT) and CPT–HK loaded nanocarrier (GO‐CS‐FA‐CPT–HK) was evaluated by haemolysis assay by comparing with diclofenac sodium salt, which served as a standard. The haemolytic property of GO was effectively masked after functionalisation by chitosan. It barred the interactions between the negatively charged oxygen of GO and the phosphatidylcholine lipids ( + charged) present over the exterior surface of red blood cells consequently comes up with least haemolytic activity. Also, no significant difference was found between the percentage of haemolysis of GO‐CS‐FA‐CPT and GO‐CS‐FA‐CPT–HK, which confirmed that the drugs play no role in haemolysis. In the case of GO, a concentration‐dependent haemolytic activity was observed. At a concentration of 100 μg/ml of GO, the percentage of haemolysis was found to be 59.4, at GO concentration of 100 μg/ml, whereas for GO‐CS‐FA‐CPT, GO‐CS‐FA‐CPT–HK and the standard used, the haemolytic activity declines with an escalation in concentration which might be attributed to the masking of electrostatic interactions.

The percentage of haemolysis at 100 μg/ml of GO‐CS‐FA‐CPT, GO‐CS‐FA‐CPT–HK and diclofenac sodium salt was 0.78, 0.67 and 0.43%, respectively, (Figs. 6 a, b and 7). The haemolytic property of GO and its derivatives have been reported by other researchers in determining the blood biocompatibility of graphene [18].

Fig. 6.

Biocompatibility of drug‐loaded nanocarrier

(a) Percentage of haemolysis (b) Percentage of stabilisation of GO, GO‐CS‐FA‐CPT and GO, GO‐CS‐FA‐CPT–HK

Fig. 7.

SEM images of RBC showing haemolysis

The anti‐inflammatory assay was evaluated by membrane stabilisation method, wherein RBC served a reference to lysosomal membranes. The percentage of stabilisation in the case of GO declines with an escalation in concentration, whereas for GO‐CS‐FA‐CPT and GO‐CS‐FA‐CPT–HK with an escalation in concentration the percentage of stabilisation was found to increase. The percentage of stabilisation was >95 for both GO‐CS‐FA‐CPT and GO‐CS‐FA‐CPT–HK at a concentration of 100 μg/ml. Diclofenac sodium salt served as a standard in this assay (Fig. 6 b). The topographical structure of graphene and its derivatives play an important role in its biocompatibility leading to a distinct effect on the lipopolysaccharide‐induced inflammations [31].

3.3 Antioxidant studies

3.3.1 DPPH radical‐scavenging assay

The free radical scavenging of combinatorial drug‐loaded nanocarrier (GO‐CS‐FA‐CPT–HK) was compared with GO and HK alone using ascorbic acid standard. Herein the property of GO, GO‐CS‐FA‐CPT–HK, HK and ascorbic acid all showed a concentration‐dependent activity. GO (100 μg/ml) showed 52.5% of free radical scavenging activity, whereas HK, GO‐CS‐FA‐CPT–HK and ascorbic acid showed 70.1, 82.34 and 97.7.% of radical scavenging activity, respectively. This clearly indicated that the loading of HK to the nanocarrier imparted the radical scavenging activity (Fig. 8).

Fig. 8.

DPPH radical‐scavenging assay of GO/ GO‐CS‐FA‐CPT–HK/HK

3.4 In‐vitro analysis

3.4.1 MTT assay

MTT assay was performed to evaluate the percentage of cell inhibition of GO‐CS‐FA‐CPT/GO‐CS‐FA‐CPT–HK/HK/CPT in breast cancer cell lines (MCF‐7). At all the studied concentrations (10, 20, 40, 80 and 100 μg/ml), GO‐CS‐FA‐CPT–HK showed the maximum percentage of inhibition of MCF‐7 cell lines. When treated at a concentration of 100 μg/ml of CPT the percentage of inhibition was found to be 50.5, GO‐CS‐FA‐CPT inhibited the growth by 89.14% and GO‐CS‐FA‐CPT–HK exhibited a percentage of inhibition of 94.31. In the case of drug‐loaded chitosan functionalised nanocarrier (GO‐CS‐FA‐CPT), the increase in cytotoxicity of the drug might be accredited to proper loading of CPT to GO‐CS‐FA. Further, the conjugation of FA ensured a more precise delivery of the drug to the cancerous cells as they are overexpressed with folate receptors (Fig. 9). The inhibitory concentration (IC 50) and pIC (pIC 50 = −log 10 IC50) were calculated and provided in Table 1. In the case of combinatorial drug loading GO‐CS‐FA‐CPT–HK, a slight increase in the % inhibition was observed. In all the events mentioned above, a concentration‐dependent activity was observed. The increase in the cellular toxicity of the combinatorial drug might be attributed by the antioxidant properties of HK also the difference in mechanism of action employed by the two drugs helps in attaining a more targeted delivery and cytotoxicity. The results are further supported by the morphological images obtained by Nikon TS‐100 at a magnification of 10 × (Fig. 10). Research studies on the cytotoxicity have revealed that the compact sheets of graphene are more detrimental in comparison to its derivatives (GO and rGO).

Fig. 9.

Percentage of inhibition of MCF‐7 cell lines after 24 h treatment with CPT/GO‐CS FA‐CPT/ GO‐CS‐FA‐CPT–HK/HK

Table 1.

IC50 and pIC 50

	IC 50	pIC 50
HK	9.228	5.03
CPT	4.48	5.34
GO‐CS‐FA‐CPT	1.77	5.77
GO‐CS‐FA‐CPT–HK	1.604	5.76

Fig. 10.

Morphological changes of MCF‐7 cells

(a) Control, (b) After treatment with CPT, (c) After treatment with GO‐CS‐FA‐CPT, (d) After treatment with GO‐CS‐FA‐CPT–HK

Also, the cytotoxicity is highly influenced by its particulate size, charge and shape. Further, the use of biocompatible polymers like chitosan practically eliminates its haemolytic and anti‐inflammatory properties, thus making it biocompatible [18].

3.4.2 SRB assay

The SRB assay is based on the uptake of aminoxanthine by the active cells. It is considered as an important measure of cellular toxicity in comparison to MTT assay, as MTT assay is based on the mitochondrial activity, which varies for several reasons like glucose level etc. The nanocarrier (GO/GO‐CS/GO‐CS‐FA‐CPT) was found to have no cytotoxicity (cell growth ∼95%) against the MCF‐7 cells, whereas the CPT and combinatorial drug (CPT–HK) loaded nanocarrier showed a decrease in cell growth with increasing concentration (Fig. 11). The cell growth was found to be 60, 28.97, 10.78 and 7.31% for HK, CPT, GO‐CS‐FA‐CPT and GO‐CS‐FA‐CPT–HK at 100 μg/ml concentration, respectively. Thus, the SRB data was found in accordance with MTT assay data.

Fig. 11.

Percentage of cell growth of MCF‐7 cell lines with different concentrations of GO/GO‐CS/GO‐CS‐FA/GO‐CS‐FA‐CPT/CPT/GO‐CS‐FA‐CPT–HK/HK

4 Conclusion

Our study intended for generating a coordinated effect by conjoining two different natural products CPT and HK to attain a more precise treatment against breast cancer. Herein, the natural polymer chitosan was used to functionalise GO synthesised by modified Hummer's method. Chitosan masked the sharp wedges of GO and imparted solubility and biocompatibility so that it can be used as a nanocarrier. Further, the conjugation of the nanocarrier with FA is believed to impart a site‐specific drug delivery to the cancerous cells. Also, the combinatorial drug CPT–HK thus produced was loaded onto chitosan functionalised nanocarrier and was assayed for its biocompatibility by haemolysis and anti‐inflammatory assay. Chitosan successfully reduced the haemolytic and anti‐inflammatory properties of GO. It was further assayed for antioxidant studies. GO showed negligible antioxidant properties, whereas after co‐loading HK to CPT loaded nanocarrier, a significant rise was observed in the DPPH activity. Also, the in‐vitro studies showed a significant difference in the percentage of inhibition when assayed by MTT and SRB assays.