Hyper-branched multifunctional carbon nanotubes carrier for targeted liver cancer therapy

2.1 Materials

MWCNT was obtained from SRL Chemicals Ltd, Mumbai, India. Polyethylene glycol (PEG, Mw = 6000), Thionyl chloride (SoCl₂), Adipic acid (AA), L-lysine hydrochloride, and Potassium hydroxide (KOH) were purchased from Sigma Aldrich Co., Ltd, Mumbai, India. Tetrahydrofuran (THF), N, N-dimethylformamide (DMF), Triethylamine (TEA), Methanol (MeOH), and sulfuric acid were purchased from Merck Co., Ltd, Mumbai, India. Folic acid (FA) and Doxorubicin (DOX) were received from Sigma Aldrich, Mumbai, India. The other reagents were analytical grade, and all reagents were used without any further purification.

2.2 Synthesis of MWCNT-PEG polymer from MWCNT-COCl

The carboxylated multiwalled carbon nanotubes (MWCNT-COOH) (100 mg) were dispersed in 25 mL of DMF solvent and subjected to probe ultra-sonication (SONICS Vibra-Cell) for 30 min for 130 W with 5 sec on 2 sec off and the frequency of 20 kHz. Then, the homogeneous carboxylated MWCNT solution was transferred into 250 mL round bottom flask, further mixed with 20 mL of thionyl chloride and addition of 5 mL N, N-dimethylformamide (DMF) as a catalyst and heated to 75 °C under magnetic stirring for 24 h. The solution was then washed with anhydrous tetrahydrofuran (THF) to remove the unreacted SOCl₂ and DMF solvent. The obtained compound was dried at 60 °C under a hot air oven overnight to get MWCNT-COCl. Further, the samples were used for further characterizations and reactions. The MWCNT-COCl (50 mg) was added into 200 mg containing PEG polymer in 50 mL of (v/v = 3/1) benzene/water mixture, and the mixture was stirred under 80 °C at 40 h. After the reaction was completed, the black precipitate of MWCNT-PEG polymer was centrifuged at 3000 rpm for 15 min, further washed with distilled water. Finally, the brownish-black substance was dried in a hot air oven for 24 h (Wei et al., 2018). Additionally, the product was collected and stored.

2.3 Synthesis of MWCNT-PEG-AA

The MWCNT-PEG-AA was synthesized by the condensation reaction with MWNT-PEG and adipic acid (AA), and the reaction was followed by a previously reported procedure with slight modifications (Ahmadi Azghandi et al., 2017). Typically, 50 mg of MWNT-PEG was dispersed in 50 mL of distilled water. Then 50 mg of adipic acid was added to the system, further 5 mL of concentrated H₂SO₄ were added slowly and heated to 120 °C for 12 h with continuous stirring on the magnetic stirrer. Further, the product was washed away with distilled water and dried at 60 °C in a hot air oven for 24 h. Then, the final compounds were collected and used for further experimental sections.

2.4 Synthesis of hyperbranched poly-L-lysine (HBPLL)

Hyperbranched poly-L-lysine (HBPLL) was prepared as per the procedure previously reported by Guangyue et al. (Guangyue et al., 2016). Briefly, L-lysine hydrochloride (5.479 g, 30.0 mmol) and potassium hydroxide (1.52 g, 27.05 mmol) were thoroughly mixed with mortar until a consistent mixture was formed. The substance was moved into a 500 mL three-necked RB flask and added 25 mL of paraffin oil,; the compound was kept at 150 °C for 48 h under N₂ atmosphere during the whole polymerization process remove the water. Then, the substance was cooled at room temperature (27 ℃). Afterward, eliminating paraffin oil, several times washed with petroleum ether; further, the excess of petroleum ether was evaporated. The attained brownish-yellow solid compound was then fully dissolved in methanol solution, the obtained by products of KCl were removed by filtration using Whatman filter paper 2.5 μm size (G.No-42). Further, the THF solvent was added to obtain the yellow-brown precipitate, and the residue was recovered and dissolved in distilled water. Then, the precipitate solution was adjusted pH value to 5.0. Finally, the compound was dialyzed using a dialysis bag (MWCO-12000) against double distilled water and then lyophilized (SSIPL-LYF/065/071216) under −40 ℃.

2.5 FA and MWCNT-PEG-AA tagged hyperbranched HBPLL synthesis

The MWCNT-PEG-AA and FA functionalized HBPLL were prepared through previous reported one and slight changes (Wen et al., 2013). In brief, HBPLL (50 mg) dissolved in DMSO (5 mL) with an equal ratio of MWCNT-PEG-AA (10 mg) and FA (10 mg) (1:1) was separately dissolved in DMSO (5 mL) solution carboxylic acid groups were activated by EDC.HCl (10.0 mg) and NHS (10.0 mg) was magnetically stirred for 2 h. Afterward, the activated carboxylic groups were mixed dropwise into the HBPLL solution. The reaction was continued with stirring for 3 days to obtain HBPLL conjugated MWCNT-PEG-AA-FA nanocarrier. The obtained material was dialyzed for 48 h against distilled water. The final products were lyophilized (SSIPL-LYF/065/071216) under −40 °C.

2.6 Synthesis of DOX-loaded multifunctional HBPLL-functionalized MWCNT-PEG-AA-HBPLL-FA.

The DOX-loaded nanocarrier was prepared through the solvent evaporation method (Pradeepkumar et al., 2017). The anticancer drug of doxorubicin (3 mg/mL) solution was dissolved in a 5 mL ethanol solution and mixed with HBPLL-functionalized MWCNT-PEG-AA-HBPLL-FA solution (30 mg/mL) and stirred for 24 h at room temperature (27 °C). The final DOX/MWCNT-PEG-AA-HBPLL-FA was collected and washed by ultracentrifugation (5,000 rpm, 30 min) to remove the unbound DOX molecule. The drug encapsulation and loading content of DOX were determined UV–Vis spectrophotometrically at λ_max value of 494 nm of DOX.

2.7 Determination of drug encapsulation and drug loading capacity

Drug encapsulation and loading capacity were studied using the DOX/MWCNT-PEG-AA-HBPLL-FA carrier, and then free DOX was then measured after the supernatant solution was centrifuged. The loading capacity of DOX was determined by UV–Vis spectrophotometer 494 nm. Thus, the percentage (%) of drug encapsulation efficiency and loading capacity was calculated using the below formulas: $$\mathrm{E}\mathrm{E}\left(\%\right)=\frac{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}-\mathrm{F}\mathrm{r}\mathrm{e}\mathrm{e}\mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}}\mathrm{X}100$$ $$\mathrm{L}\mathrm{C}\left(\%\right)=\frac{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}-\mathrm{F}\mathrm{r}\mathrm{e}\mathrm{e}\mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}}{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{d}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{d}\mathrm{n}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{c}\mathrm{a}\mathrm{r}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{r}}\mathrm{X}100$$

2.8 In-vitro drug release study

The in-vitro drug release properties of the DOX/MWCNT-PEG-AA-HBPLL-FA carrier were evaluated by the dialysis method. Briefly, 10 mg of DOX/MWCNT-PEG-AA-HBPLL-FA carrier was placed into a dialysis bag with a molecular weight cut-off (MWD) of 10,000 kDa. Furthermore, the dialysis bag was submerged in 50.0 mL of PBS at different pHs like 2.8, 5.5, 6.8 & 7.4 at 27 °C with gentle stirring on a magnetically stirred for 150 rpm. At an every 3 h time intervals, 2 mL of the sample suspension was withdrawn from the PBS release medium and replaced with an equal volume of fresh PBS suspension. Finally, the DOX release medium was determined by the UV–vis spectrometer (Shimadzu-1600, Japan) at a λ_max value of 494 nm.

2.9 Biological studies

2.9.6

2.9.6 Western blotting

The human HepG2 liver carcinoma cells were treated with MWCNT-PEG-AA-HBPLL-FA and DOX/MWCNT-PEG-AA-HBPLL-FA carrier used to induce apoptosis for 24 h. After treatment, cells were lysed with RIPA buffer, and the protein concentration of cell lysates was measured using the DC Protein Assay Reagent (Bio-Rad, Hercules, CA, USA). Proteins were separated using gel electrophoresis (2 h at 100 V) and transferred into nitrocellulose membrane (wet transfer; 2 h at 80 V). Primary antibodies (Bcl-2: mouse monoclonal antibody, Cat# 13–8800; BAX: mouse monoclonal antibody, Cat# 336400; GAPDH: mouse monoclonal antibody, Cat# MA5-15738) and secondary antibody HRP conjugated (Goat anti-mouse HRP, Cat# A90-234P) from Thermo Fisher Scientific, (Waltham, MA, USA) were used. The target proteins were visualized using an ECL detection kit (Millipore, Burlington, MA, USA). Chemi luminescence signals were captured using Chemi-Doc Imaging Systems. Band density was quantitated using Image J Software (National Institutes of Health, Bethesda, MD, USA) (see Fig. 1).

Close

Fig. 1

Overall schematic illustration of DOX/MWCNT-PEG-AA-HBPLL-FA carrier synthesis.

Export to PPT

3.1 FT-IR and XRD analysis

In order to distinguish the structural changes of MWCNT-COOH, MWCNT-PEG, MWCNT-PEG-AA, MWCNT-PEG-AA-HBPLL-FA, and DOX/MWCNT-PEG-AA-HBPLL-FA carriers are investigated using FT-IR spectroscopy (Fig. 2A). FT-IR spectrum of carboxylate MWCNT is represented in Fig. 2A(a). A broad, strong peak at 3309 cm⁻¹ indicates the –OH group of the carboxylic MWCNT, and the stretching vibrations at 1740 cm⁻¹, 1641 cm^−1, and 1023 cm⁻¹ represent the functional groups of C=O, C=C, and C-O, respectively. The peak at 2925 cm⁻¹ corresponds to the presence of a C-H stretching bond (Mahinpour et al., 2018). After functionalization with PEG on the carboxylic acid group of MWCNT surface, the higher shift of carbonyl peaks at 1740 cm⁻¹ to 1743 cm⁻¹ due to the formation of ester linkage as presented in Fig. 2A(b) (Vasheghani Farahani et al., 2016). Fig. 2A(c) displays the FT-IR spectrum of adipic acid-functionalized MWCNT-PEG. A new strong attained absorption peak at the range of 1705 cm⁻¹ is observed from another ester bond that indicates adipic acid was successfully conjugated with MWCNT-PEG (Ahmadi Azghandi et al., 2017). The HBPLL polymer combined with MWCNT-PEG-AA and FA to appear in two new amide bonds at 1637 cm⁻¹ and 1420 cm⁻¹, which ascribed to the amide linkages of -C=O-NH- stretching vibration and N-H bending vibration (Nasrollahi et al., 2016) consistently (Fig. 2A(d)). It demonstrates the successful covalent conjugation of FA and MWCNT-PEG-AA into HBPLL polymer. From FT-IR spectra of Fig. 2A(e) signify the DOX/MWCNT-PEG-AA-HBPLL-FA carrier in the spectrum of the carbonyl peaks at 1641 cm⁻¹ and 1023 cm⁻¹ were shifted into higher and lower positions at 1699 cm⁻¹ and 1011 cm⁻¹, which can be introduced by the formation of hydrogen bonds and π-π stacking interaction between DOX and MWCNT carrier. A new peak appeared at 867 cm⁻¹ and 3042 cm⁻¹ due to the presence of out-of-plane N-H vibration and N-H stretching vibration of DOX molecule (Dong et al., 2013). We also well correlated with early Adeli et al., 2013 proposed poly amidoamine poly (ethylene glycol)-poly amidoamine (PAMAM-PEG-PAMAM) nanotubes shows that liposomes like structures which effectively encapsulate and deliver the drug molecules on affected areas (Adeli et al., 2013). It reveals that the prepared carrier proves the successful loading of DOX on the MWCNT surface through physical interactions.

Close

Fig. 2

(A) FTIR spectra of (a) MWCNT-COOH, (b) MWCNT-PEG, (c) MWCNT-PEG-AA, (d) MWCNT-PEG-AA-HBPLL-FA carrier and (e) DOX/MWCNT-PEG-AA-HBPLL-FA carrier and (B) XRD pattern of (a) MWCNT-COOH, (b) MWCNT-PEG, (c) MWCNT-PEG-AA, (d) MWCNT-PEG-AA-HBPLL-FA carrier and (e) DOX/MWCNT-PEG-AA-HBPLL-FA carrier.

Export to PPT

The crystalline phase differentiation of MWCNT based carrier and DOX loaded MWCNT carrier systems was examined by XRD spectroscopy. The XRD pattern of Fig. 2B(a), denotes the MWCNT-COOH; the typical characteristic diffraction peak at 27° appeared by 002 plane exhibit the sharp peak corresponds due to the crystalline nature of the MWCNT-COOH. This peak also correlated with Egbosiuba et al., (2020), previously reported multiwalled carbon nanotubes are also crystalline in nature (Egbosiuba et al., 2020). After functionalization with the hydrophilic polymer of PEG, the MWCNT Sharp peak intensity was diminished, which indicate in Fig. 2B(b). Further, AA functionalized MWCNT-PEG also introduces the same broad-like peak at 26° as given in Fig. 2B(c). The appeared peaks at 12°, 20°, and 24° also confirmed the functionalization of FA and MWCNT-PEG-AA on HBPLL polymer (Fig. 2Bd), which also correlated with previously Chen et al., reported folic acid functionalized aminated multiwalled carbon nanotubes using targeted drug delivery system (Kayat et al., 2015). Moreover, DOX/MWCNT-PEG-AA-HBPLL-FA carrier represents the peaks such as 27°, 31°, 32°, and 44° was observed and confirms the poly amorphous natured drug-loaded carrier, which is more helpful due to the easy distribution of drug molecules on tumors sites.

3.2 Morphological analysis

The surface morphology of MWCNT-COOH, MWCNT-PEG, MWCNT-PEG-AA-HBPLL-FA, and DOX/MWCNT-PEG-AA-HBPLL-FA was characterized by using SEM and HR-TEM analysis as given in Fig. 3. The MWCNT-COOH clearly shows a tubular-like structure (Fig. 3a). After PEG reaction in MWCNT shows a cluster form of PEG attached MWCNT edges, as illustrated in Fig. 3(b). In addition, the modification of HBPLL polymer in MWCNT-PEG-AA and FA morphology was detected and given in Fig. 3(c), which displays that curled-like tubular structure also indicates the successful conjugation of successful conjugation FA and MWCNT-PEG-AA and the average scale bar range of MWCNT-PEG-AA-HBPLL-FA is 1 μm from SEM analysis. The DOX molecule was loaded on the MWCNT-PEG-HBPLL-AA-FA surface to obtain a sporadic coil-like structure. The yellow marked arrow indicates the evidence of DOX loading on the carrier through physical interactions between the DOX and MWCNT-PEG-AA-HBPLL-FA carrier (Fig. 3d).

Close

Fig. 3

SEM and HR-TEM images: MWCNT-COOH, MWCNT-PEG, MWCNT-PEG-AA-HBPLL-FA, and DOX/MWCNT-PEG-HBPLL-AA-FA carrier.

Export to PPT

HR-TEM study used analysis of the intrinsic surface morphology of as-prepared CNT-based carrier and drug-loaded carrier systems; it was well correlated with SEM analysis. HR-TEM images of (Fig. 3e) prove a tubular-like structure of MWCNT-COOH. Fig. 3f indicates the presence of hydrophilic PEG on grafted with MWCNT surface edges. Moreover, the presence of PEG on the MWCNT surface appeared in entangled MWCNT morphology, which determines the improved dispersion properties during the carbon-based drug carrier system. Further, the MWCNT-PEG-AA and FA were connected with the polymeric material of HBPLL to form an enlarged polymeric network-like structure in Fig. 3(g). Also, the drug molecule bounded on the MWCNT-PEG-AA-HBPLL-FA carrier shows an interconnected network-like structure represented in Fig. 2(h). Finally, the tubular-like morphology of the carbon-based carrier system created the ideal platform for the high loading and releasing nature of cancer drug delivery systems.

3.3 Particle size distribution and zeta potential measurements

The average particle size of MWCNT-PEG-AA-HBPLL-FA was 365 nm, whereas the diameter range of DOX/MWCNT-PEG-AA-HBPLL-FA was 392 nm denoted in Fig. 4. The MWCNT-PEG-AA-HBPLL-FA carrier, after that the loading of DOX molecule on MWCNT surface the particle size was enlarged. The PDI for the MWCNT-PEG-AA-HBPLL-FA before and after loading with DOX was 0.168 and 0.183 correspondingly. It finds out the non-symmetric nature of MWCNT. Besides, the obtained PI values were <0.4, demonstrating the dependability of the reported size values with some homogeneity (Raza et al., 2016). The zeta potential value of DOX with and without a carrier is −18.13 mV and −0.27 mV (Fig. 4b,d). The DOX/MWCNT-PEG-AA-HBPLL-FA carrier surface charge was decreased, which designates the loading of DOX on the MWCNT surface. It also indicates that the prepared MWCNT based carrier is stable in a neutral medium. The stability of the carrier plays a vital role in newly synthesized drug cargo systems.

Close

Fig. 4

Particle size and zeta potential analysis of MWCNT-PEG-AA-HBPLL-FA (a&b) DOX/MWCNT-PEG-AA-HBPLL-FA carrier (c&d).

Export to PPT

Moreover, the zeta potential results strongly indicate the DOX/MWCNT-PEG-AA-HBPLL-FA carrier stability and use for potential drug carrier applications (Guo et al. 2018). Further, the highly negative charge of the nanocarrier was greatly interactive with the positive cell membrane. These results are also related to the previously Vinothini et al. developed a magnetic nanocarrier having a stable and suitable drug cargo system on cancer sites due to the promising biological interaction of carrier and cancer cell walls (Vinothini et al., 2020).

3.4 Thermal analysis (TGA)

Thermal stability and decomposition nature of (a) MWCNT-COOH, (b) MWCNT-PEG, (c) MWCNT-PEG-AA-HBPLL-FA, and (d) DOX/MWCNT-PEG-AA-HBPLL-FA carrier were examined through thermal gravimetric analysis, and the results are established in Fig. 5. As can be seen, From in Fig. 5 (a), is TGA spectrum of MWCNT-COOH represent the decomposition of –COOH group at 81% weight loss indicates to elimination of H₂O & CO₂ on MWCNTs-COOH surface (Bruno et al., 2017). After functionalized with PEG exhibits two distinct stages of weight losses of 14%, and 74% was observed at 200–600 °C, which describes the loss of oxygen-containing functional (H₂O, CO₂, & CO) groups grafted on MWCNT surface (Meihua Tan et al., 2020). Our TGA analysis also well correlated with previously Maria et al., 2020 proposed MWCNT-COOH and PEG functionalized MWCNT-PEG exhibits high thermal stability (Maria et al., 2020). The thermal decomposition of the MWCNT-PEG-AA-HBPLL-FA carrier displays three stages of decomposition (Fig. 5c). Significantly, the weight loss of MWCNT-PEG-AA-HBPLL-FA indicates that amino groups functionalized material was more stable, and they could involve in some thermal reaction at a temperature more than 400 °C. The first step is due to the weight loss of the moisture and H₂O groups at 90–100 °C, while the second step introduces the decomposition of polymer combined ester and CO₂ molecules up to 110–300 °C, in the third step indicated the deterioration of amide linkages at degradation temperature range at 300–700 °C, as compared to DOX-loaded carrier, the DOX molecules interacted with MWCNT surface through physical interactions, when the temperature was also increased up (Meihua Tan et al., 2020) to 700 °C, the DOX molecules were totally dissociated on the MWCNT wall surface and the weight ratio at 88%, which indicated the thermal stability of MWCNT-PEG-AA-HBPLL-FA carrier. Finally, our results concluded that the functional groups are effectively grafted with the MWCNT surface.

Close

Fig. 5

TGA thermal stability of (a) MWCNT-COOH, (b) MWCNT-PEG, (c) MWCNT-PEG-AA-HBPLL-FA, and (d) DOX/MWCNT-PEG-AA-HBPLL-FA carrier.

Export to PPT

3.5 Encapsulation efficiency and in-vitro drug release studies

The drug encapsulation efficiency and loading capacity are the two major parameters for designing and developing effective drug delivery carrier systems (Meysam et al., 2021). The DOX/MWCNT-PEG-AA-HBPLL-FA carrier in drug-releasing rates exists in Fig. 6. The DOX was encapsulated with MWCNT-PEG-AA-HBPLL-FA carrier surface through physical interactions like electrostatic and π-π stacking interactions. The UV–Vis spectrum of the DOX/MWCNT-PEG-AA-HBPLL-FA carrier shows the DOX region absorbance peak at 494 nm. The DOX encapsulation efficiency was obtained at 62.5%, and the loading capacity of DOX is 25.4%, separately. These results indicate a high amount of the drugs was successfully encapsulated on the MWCNT-PEG-AA-HBPLL-FA carrier. Finally, high encapsulating and loading efficiency bounded carriers will help and improve the in-vitro drug delivery application.

Close

Fig. 6

(a) Encapsulation efficiency and (b, c, d, & e) In-vitro drug release profiles of various physiological conditions like pH-2.8, pH-5.5, pH-6.8 & pH-7.4 and (e) Cumulative drug-releasing profile of DOX/MWCNT-PEG-AA-HBPLL-FA carrier.

Export to PPT

The drug release rates of DOX from the carrier were explored at three different physiological pHs (2.8, 5.5, 6.8 & 7.4) at room temperature, and the results are assumed in Fig. 6. The UV–Visible spectroscopy analysis recorded a sustained DOX drug release nature of the DOX/MWCNT-PEG-AA-HBPLL-FA carrier. At 24 h investigation, a maximum of 92.0 % of DOX release rate was observed in acidic 2.8 pH. The physically interacted DOX molecule was easily cleavable in acidic conditions. Meanwhile, DOX molecules are readily soluble in the acidic medium because the –NH₂ group of DOX was quickly protonated (Zhang et al., 2017) on the acidic medium from MWCNT based materials are efficient drug delivery carrier system for biomedical applications. Fig. 6 (e) shows the cumulative drug release pattern of MWCNT-PEG-AA-HBPLL-FA carrier, the release rate of DOX at pH-5.5 is 84 %, pH-6.8 is 32 %, and pH −7.4 is 28% drug release with the 24 h intervals. Finally, the acidic condition was the most suitable environment for the release of drugs in tumor cells.

3.6 In-vitro cytotoxicity studies

The in-vitro cell viability of MWCNT-PEG-AA-HBPLL-FA and DOX/MWCNT-PEG-AA-HBPLL FA carriers were determined using human liver cancer (HepG2) cell line and human embryonic kidney (HEK239) cell lines subsequently. Both cell lines were treated with varying concentrations of (0–150 μg/mL) as prepared compounds, and the cytotoxic results are given in Fig. 7. The cytotoxic effect was increased with increasing concentrations of MWCNT-PEG-AA-HBPLL-FA and DOX/MWCNT-PEG-AA-HBPLL-FA carriers. After 24 h incubation periods, we have observed clear cell death in both cell lines. Fig. 7(a&b) shows that the MWCNT-PEG-AA-HBPLL-FA and DOX/MWCNT-PEG-AA-HBPLL-FA nanocarriers treated HEK239 cell line exhibits less cytotoxic effects, which proved that our prepared nanocarrier has good biocompatible nature in the normal cell line. Further, in Fig. 7(c&d), MWCNT-PEG-AA-HBPLL-FA treated HepG2 cell line shows minimum cell death. After that, the DOX/MWCNT-PEG-AA-HBPLL-FA treated liver cancer HepG2 cell line manifested a higher amount of cytotoxic effect because the anticancer drug of DOX has effectively inhibited topoisomerase I permitting DNA damages in a cancer cell line, over a 24 h incubation period. Finally, the IC₅₀ values for both normal and cancer cell line treated MWCNT-PEG-AA-HBPLL-FA and DOX/MWCNT-PEG-AA-HBPLL-FA noncarrier were observed at 170.4, 86.94 µg/mL and 210.1, 425.1 µg/mL, respectively.

Close

Fig. 7

In-vitro cell viability of HEK293 (a&b) and HepG2 (c&d) cell line incubated with varying concentrations of MWCNT-PEG-AA-HBPLL-FA and DOX/MWCNT-PEG-AA-HBPLL-FA carrier.

Export to PPT

3.7 JC-1 mitochondrial depolarization

We measured mitochondrial depolarization of the cells as an indicator of apoptosis being induced by MWCNT-PEG-AA-HBPLL-FA (DU) and DOX/MWCNT-PEG-AA-HBPLL-FA (DL) carriers in both HepG2 and HEK239 cells using the IC₅₀ concentrations. In Fig. 8, the DOX loaded MWCNT-PEG-AA-HBPLL-FA shows that membrane depolarization increased. The membrane depolarization increases were reduced cell proliferation due to increased oxidative stress and mitochondrial pathway induced apoptosis.

Close

Fig. 8

Mitochondrial depolarization effect of MWCNT-PEG-AA-HBPLL-FA (DU) and DOX/MWCNT-PEG-AA-HBPLL-FA (DL) carrier treated with IC₅₀ concentrations.

Export to PPT

3.8 Caspase activity assay

The intracellular caspase activation is one of the main tools or the apoptosis cell death pathway, which leads to the cleavage and inactivation of many cellular proteins. In the present study, the caspase activations of −8, −9, & −3 were investigated at 24 h incubation of MWCNT-PEG-AA-HBPLL-FA and DOX/MWCNT-PEG-AA-HBPLL-FA carrier. The results demonstrate that MWCNT-PEG-AA-HBPLL-FA carrier treated HepG2 cell exhibits significant upregulation caspase activity of −8 &-9, and downregulation caspase activity of −3. Moreover, the DOX/MWCNT-PEG-AA-HBPLL-FA carrier treated HepG2 cell introduce decreasing the whole caspase −8, −9, and −3 activity compared with control as shown in Fig. 9. In these results, our fabricated DOX/MWCNT-PEG-AA-HBPLL-FA carrier significantly induced cells via intrinsic apoptotic signaling pathway.

Close

Fig. 9

Caspase −8, −9 & −3 activities of MWCNT-PEG-AA-HBPLL-FA (DU) and DOX/MWCNT-PEG-AA-HBPLL-FA (DL) carriers treated with IC₅₀ concentrations.

Export to PPT

3.9 Hoechst staining

Cellular nuclear damage was analyzed using fluorescence microscopy. The bright blue fluorescent Hoechst stain is a cell internalized nucleic acid dye (Syed Abdul Rahman et al., 2013), which is widely used to analyze the nucleus damage and fragmentation of apoptotic cells. Fig. 10 shows the nuclear damage in HepG2 cells after 24 h treatment of DOX with/without nanocarrier-loaded compounds. The control cell appeared to be intact oval shapes and bright blue fluorescent dye. In contrast, the DOX/MWCNT-PEG-AA-HBPLL-FA carrier treated cells showed less fluorescent intensity, unclear cell shapes, and a damaged nucleus. These results suggested that the DOX/MWCNT-PEG-AA-HBPLL-FA carrier is highly efficient to inducing cell death in HepG2 cells.

Close

Fig. 10

The fluorescent images of HepG2 cells incubated with various concentrations of MWCNT-PEG-AA-HBPLL-FA and DOX/MWCNT-PEG-AA-HBPLL-FA nanocarrier stained with Hoechst stain.

Export to PPT

3.10 Apoptosis analysis

The pro-apoptotic and anti-apoptotic proteins are generally involved in cell proliferation and cell death. In this study, the two apoptosis-related proteins, Bcl-2 and BAX, were analyzed using western blotting. Fig. 11(a&b) shows the western blot images of Bcl-2 and BAX in DOX/MWCNT-PEG-AA-HBPLL-FA treated and control groups of HepG2 cells. DOX is widely used as a potent anticancer agent as it is a potent inducer of apoptosis. The anti-apoptotic protein Bcl-2 mainly inhibits the mitochondrial pathway (Vivek et al. 2014). BAX is a pro-apoptotic protein that promotes apoptosis in cells, although it was present in inactivating states in many cancers. The treatment with DOX/MWCNT-PEG-AA-HBPLL-FA nanocarrier upregulated the protein and gene expression of BAX and down-regulated the expression of Bcl-2. These results strongly support the observations that DOX/MWCNT-PEG-AA-HBPLL-FA is a potent induces of cell death in cancer cells by increased apoptosis.

Close

Fig. 11

(a) Representative blot of Bcl-2 and BAX levels in HepG2 cells, (b) Ratio of Bcl-2 and BAX to GAPDH. Error bars represent SEM from three replicates (*P < 0.05) (c) mRNA expression of Bcl-2 and BAX in HepG2 cells. Group 1- Control; Group II- DU; Group III- DL. Error bars represent SEM from three replicates (*p < 0.05).

Export to PPT