Papain bioinspired gold nanoparticles augmented the anticancer potency of 5-FU against lung cancer

2.1. Materials

Chemicals and reagents tetrachloroauric [III] acid (HAuCl₄), 5-fluorouracil and papain were bought from Sigma-Aldrich. Unless stated otherwise, all chemicals and solvents were of analytical grade and were utilised as received.

2.2. In vitro synthesis of PpGNPs

Papain-capped GNPs were synthesised by the biological method using papain as a reducing as well as a capping agent. The synthesis of PpGNPs was performed at 6 °C for 48 H in a 3-mL reaction mixture containing 1 mM tetrachloroauric [III] acid (H[AuCl₄]) and 2 mg/mL papain in 100 mM HEPES buffer at pH 6.0. The same reaction was also performed at different temperatures (such as 16 °C, 25 °C, 37 °C and 40 °C) and in buffer solutions of different pH (such as pH 5.0, 6.0, 7.0 and 8.0), but favourable results were obtained only at conditions described above. The completion of the reaction was confirmed by UV–vis spectroscopy when the colour changed from colourless to pink. After completion, the mixture was treated with 50% ethanol to remove the unbound papain and then utilised for further purposes.

2.3. Bioconjugation of PpGNPs with 5-FU

In vitro, the above-synthesised gold nanoparticles (PpGNPs) were bioconjugated with the anti-cancer drug, 5-fluorouracil (5-FU). The coupling agent 1-ethyl-3-(3-dimethyl) carbodiimide (EDC) was used to establish a covalent bond between the free amino groups (secondary amine) of 5-FU and the carboxylate groups of papain in the PpGNPs [Citation25]. For this purpose, 250 µg of 5-FU was incubated with 250 µg of PpGNPs in a 50 mM HEPES buffer solution at a temperature of 30 °C. The solution of 5 mM EDC was added in aliquots in the given reaction mixture within 3H.

2.4. Characterisation of PpGNPs and 5F-PpGNPs

The UV–vis spectroscopic analysis of PpGNPs and 5F-PpGNPs was performed in the range of wavelengths 220–800 nm, in a quartz cuvette with a path length of 1 cm. The study was conducted on a double-beam spectrophotometer (Shimadzu, model UV-1601 PC). The transmission electron microscope (TEM) was used to analyse the size and morphology of PpGNPs and 5F-PpGNPs. The FEI Tecnai^TM G² Spirit BioTWIN from FEI Company was operated at an accelerating voltage of 80 kV. For the preparation of TEM samples, 1 µL droplets of the PpGNPs and 5F-PpGNPs were deposited and dried overnight onto a carbon-coated copper disc. The prepared samples were then kept in a vacuum chamber (∼4 × 10⁻³Torr) for 12 h. The Gatan digital micrograph was used to manually estimate the size range of the PpGNPs and 5F-PpGNPs from the obtained TEM images. The dynamic light scattering (DLS) method was used to analyse the mean particle size (MPS) of PpGNPs and 5F-PpGNPs by employing a particle size analyzer (Zetasizer Nano-ZS, Model ZEN3600, Malvern Instrument Ltd, Malvern, UK). The sample powder was diluted in deionised water to a concentration of 0.5% (w/v) and sonicated for 1 min before analysis. The sample was taken in a DTS0112-low volume disposable sizing cuvette of 1.5 mL. MPS was taken as the average of measurements performed in triplicate for a single sample. Zeta potential was also measured using a Zetasizer Nano-ZS, Model ZEN3600 (Malvern Instrument Ltd, Malvern, UK). To validate the binding of 5-FU with PpGNPs, Fourier transform infrared spectroscopy (FTIR) was used. Films of PpGNPs and 5F-PpGNPs were encrusted by putting a single drop of each on a Si (111) substrate and moderate heating was used to evaporate the water over the coated surface. The Shimadzu FTIR-8201 PC instrument, operated at a resolution of 4 cm⁻¹, in the diffuse reflectance mode, was used to record the FTIR spectra of the film. In order to achieve good signal-to-noise ratios, 256 different scans were obtained from the analysed films, in the range of 400–4000 cm⁻¹.

2.5. Drug-loading efficiency

The percentage loading of 5-FU on PpGNPs was calculated using EquationEquation (1)(1) $$\text{Percent loading of }5-\text{FU on PpGNPs}=\left[\left(A-B\right)\mathrm{*}\frac{100}{A}\right]$$(1) , where the values of A and B were substituted. The characteristic absorbance of 5-FU was recorded at a wavelength of 265 nm. (1) $$\text{Percent loading of }5-\text{FU on PpGNPs}=\left[\left(A-B\right)\mathrm{*}\frac{100}{A}\right]$$(1) where A is the absorbance value of total 5-FU (50 µg) added along with PpGNPs (prior to bioconjugation) and B is the absorbance value of 5-FU after bioconjugation in the supernatant of 5F-PpGNPs [Citation26].

The efficiency of drug loading was calculated by determination of the UV–vis absorbance spectra at a wavelength of 265 nm after calibration with the absorbance obtained for the standard curve of 5-FU. The standard graph was drawn by increasing the concentration of 5-FU from 1.56 to 25 µg. (2) $$\text{Percent bioconjugation}=\frac{\text{Amount of drug bioconjugated}}{\text{Total drug added}}\mathrm{*}100$$(2)

Further, diameters (d) of the particles in the range of 35–100 nm were calculated from the peak position [Citation27] according to EquationEquation (3)(3) $$d=\frac{\ln \left(\frac{\lambda \mathit{spr}-\lambda o}{L_1}\right)}{L_2}A=\pi r^2$$(3) : (3) $$d=\frac{\ln \left(\frac{\lambda \mathit{spr}-\lambda o}{L_1}\right)}{L_2}A=\pi r^2$$(3)

For theoretical value $\mathrm{ }\mathit{\text{of d}}>25 \mathrm{nm} (\lambda o=512; L_1=6.34;L_2=0.0216; \lambda \mathit{spr}=332),$ fit parameters were determined accurately with only a 3% error against experimental values. Therefore, the given equation can be precisely used to determine particle size in the range of 35–110 nm. $$d=\frac{\ln \left(\frac{\lambda \mathit{spr}-\lambda o}{L_1}\right)}{L_2}$$ $$d=\frac{\ln \left(\frac{532-512}{6.53}\right)}{0.0216}$$ $$d=51.77\mathrm{ }n$$

Considering the authenticity of the above equation, it was successfully used to determine the number density of the particles (N) from the absorbance of the hydrosol. (4) $$N=\frac{A_{450}{\times 10}^{14}}{d^2\left[-0.295+1.36\mathrm{e}\mathrm{xp}\left(-{\left(\frac{d-96.8}{78.2}\right)}^2\right)\right]}$$(4) $A_{450}$- is the absorbance at 450 nm wavelength and d is the particle diameter in nanometre $$A_{450}=0.829;d=51.77\mathrm{ }\mathrm{nm}$$ $$N=\frac{A_{450}{\times 10}^{14}}{d^2\left[-0.295+1.36\mathrm{e}\mathrm{xp}\left(-{\left(\frac{d-96.8}{78.2}\right)}^2\right)\right]}$$ $$N=\frac{0.829{\times 10}^{14}}{{\left(51.77\right)}^2\left[-0.295+1.36\mathrm{e}\mathrm{xp}\left(-{\left(\frac{51.77-96.8}{78.2}\right)}^2\right)\right]}$$ $$N=12\times {10}^9$$

2.6. In vitro anticancer studies of PpGNPs and 5F-PpGNPs

2.7. Statistical analysis

The result was portrayed as the mean ± S.E.M. of three independent studies performed in triplicate. One-way ANOVA utilising Dunnett’s multiple comparison test, and two-tailed, paired Student’s t-test (*p < 0.01, **p < 0.001, ***p < 0.0001 represent a significant difference compared with control) were employed for statistical analysis.

2.8. Study of synergistic effects of PpGNPs and 5-FU

Chou and Talalay’s method was used to determine the synergism and antagonism of PpGNPs and 5-FU in the given study [Citation30] using the ‘combination index’ (CIA is considered for drugs whose responses are linear to their doses). In the given study, PpGNPs and 5-FU were combined with doses (d₁ – concentration of PpGNPs and d₂ - concentration of 5-FU) to get a 50% effect. The combined effect was calculated by: $$\mathrm{CIA}=\frac{d_1}{{\mathrm{ED}50\mathrm{ }}_1\mathrm{ }}+\frac{d_2}{{\mathrm{ED}50}_2}$$ $$=\left\{\frac{P}{\left(P+Q\right){\mathrm{ED}50}_1}+\frac{Q}{\left(P+Q\right){\mathrm{ED}50}_2}\right\}{\mathrm{ED}50}_c$$ $$\mathrm{here},\mathrm{ }{d}_1=\left(\frac{P}{P+Q}\right){\mathrm{ED}50}_c$$ $$d_2=\left(\frac{Q}{P+Q}\right){\mathrm{ED}50}_c$$ where ED50_c is the combined effect of 50% inhibition; Q and P are concentrations of PpGNPs and 5-FU required to get a 50% effect, respectively. For the combination to be synergistic in action, CIA < 1; for antagonistic action, CIA > 1; and at CIA = 1, the combination becomes additive.

3.1. Results

The present investigation deals with papain-inspired (a cysteine protease involved in antitumor/anti-angiogenesis) bioengineering of gold nanoparticles (PpGNPs). Figure 1 explains the synthesis and functionalisation of gold nanoparticles and their subsequent role in the delivery of 5-FU into the A549 lung cancer cell line. Proteases, such as bromelain and trypsin [Citation21–22], are the ideal candidates for the synthesis and stabilisation of nanomaterials.

Figure 1. Schematic representation of papain mediated synthesis of gold nanoparticles and their ioconjugation with 5-fluorouracil (5-FU) so that 5-FU could be delivered through caveolae-mediated endocytosis to the nucleus of lung cancer cells.

Display full size

3.1.1. Synthesis and characterisation of PpGNPs

Papain could not produce stable and mono-dispersed nano-emulsions at the optimum temperature of its activity. Therefore, an ideal set of parameters was standardised by performing reactions at different temperatures, pH and concentrations of papain. Astonishingly, it was found that a mono-dispersed and a stable emulsion was produced only at a temperature of 6 °C when HEPES buffer of pH 6.0 was used at 2 mg/mL concentration of papain. Nucleation of nanoparticles took place in 48 h at 6 °C, leading to the synthesis of a mono-dispersed and a stable nano-emulsion. Papain acted as a reducing as well as a capping agent. The synthesis of PpGNPs was confirmed by recording the characteristic surface plasmon resonance (SPR) spectra of GNPs at a wavelength of 530 nm (Figure 2A). Being a 23.6 kDa protease, papain contains 345 amino acids and three disulfide bridges; thus, making it an ideal template to encapsulate seeding nanoparticles. Different types of interactions including thiol bridges, electrostatic interactions, hydrogen, and dative bonds, and Van-der Waals forces [Citation36] are formed due to the presence of –OH, –OOC, –NH₂, –CH₃, –(–CH₂)_n and –SH groups. With the DLS technique, the hydrodynamic diameter of PpGNPs was found to be 49.53 nm (Figure 2B); this was further confirmed by EquationEquation (1)(1) $$\text{Percent loading of }5-\text{FU on PpGNPs}=\left[\left(A-B\right)\mathrm{*}\frac{100}{A}\right]$$(1) , where size was found to be 51.77 nm and the number of particles was calculated to be 12 × 109. Topographical studies with absolute size determination were performed under TEM by manually using the Gatan digital micrograph. The micrograph confirmed the average sizes of PpGNPs to be ∼16 nm (Figure 2C) and they had a uniform distribution with spherical shapes. The nano-emulsion of PpGNPs was found to be highly stable and anionic in nature with a zeta potential of −10.5 mV (Figure 2D). The stability of the nano-emulsion is described by zeta potential as well as the Hamaker constant [Citation37] by calculating electrostatic interaction and Van der Waal forces, respectively. Smaller values of the Hamaker constant with lower values of zeta potential can produce stable nanoemulsion [Citation37].

Figure 2. Characterisation of PpGNPs and 5F-PpGNPs under (A) UV–vis spectra, (B) dynamic light scattering (inset: 5F-PpGNPs), (C) transmission electron microscopy (inset: 5F-PpGNPs), (D) zeta potential (inset: 5F-PpGNPs).

Display full size

3.1.2. Bioconjugation of anti-cancer flutamide drug with PpGNPs

Further, the bioengineered PpGNPs were functionalised with the anti-cancer drug 5-FU by using EDC (a coupling agent) to establish physical bonding between the carboxylate groups of papain (present in surplus over the surface of PpGNPs) and the secondary amino groups of 5-FU, respectively. The functionality was confirmed by recording the SPR absorption spectra of 5F-PpGNPs (at 535 nm) in comparison to PpGNPs (at 530 nm) and 5-FU (at 265 nm) (Figure 2A). The changes in the broadening of the peaks, decrease in intensity, and shifting of the spectra towards a higher wavelength (red-shift, from 530 nm to 535 nm) were observed in UV–vis spectroscopy (Figure 2A). The attachment of any ligand on the surface of nanoparticles causes a change in the absorption intensity, with a minor change in the full width of the half maximum [Citation38]. Further, the hydrodynamic diameter of particles, observed under DLS increased to 57.53 nm after bioconjugation (Figure 2B, inset). A blurred image of 5F-PpGNPs under the TEM (Figure 2C, inset) with an increase in average sizes further confirmed the bioconjugation. The average size was found to be ∼18 nm (Figure 2C, inset) which is bigger than that of PpGNPs. After functionalisation, 5F-PpGNPs were found to be spherical, mono-dispersed, and stable. The zeta potential of the anionic nano-emulsion after functionalisation was different from the earlier one (−10.5 mV for 5F-PpGNPs); its exact value was −9.41 mV (Figure 2D, inset). Further, the bioconjugation of PpGNPs with 5-FU was confirmed by FTIR spectroscopy. The spectrum of PpGNPs (Figure 3A) was compared with that of 5F-PpGNPs (Figure 3B) and it was found that the broadband contour in both the spectra was in the range of 3600–3000 cm⁻¹, which was corresponding to the –NH stretch of the peptide bonds of papain. Further, the peaks at 1737 cm⁻¹ represent C = O anhydride stretching vibration.

Figure 3. FTIR spectra of (A) GNPs encapsulated papain, (B) papain after bioconjugation of PpGNPs with 5FU and (C) UV–vis spectra of pure 5-FU.

Display full size

3.1.5. In vitro anticancer study of PpGNPs, 5F-PpGNPs, and 5-FU

The in vitro anticancer activities of PpGNPs, 5F-PpGNPs and 5-FU were studied against lung cancer cell line (A549). The functionalised 5F-PpGNPs were found to be more effective against the A549 cells. The IC₅₀ values of PpGNPs (Figure 4A), 5F-PpGNPs (Figure 4B), 5-FU (Figure 4B) and papain (Figure 4C) were found to be 255 µM, 11.7 µM, 25.6 µM and 548 µM, respectively. The anticancer effects were calculated in a dose-dependent manner. The enhanced activity of 5F-PpGNPs in comparison with 5-FU and PpGNPs is because of the synergistic effect of both papain and 5-FU. However, PpGNPs and 5F-PpGNPs were found to be non-toxic towards the 3T3-L1 cells up to a concentration of 50 µg/mL (data not shown).

Figure 4. The cytotoxicity (dose-dependent) study of (A) PpGNPs (B) pure 5-FU and 5F-PpGNPs and (C) pure papain on A549 cells. All the data were expressed in mean ± SD of three experiments.

Display full size

3.1.6. Measurement of cytomorphological changes in the A549 cells

The morphological changes in the A549 cells were observed at the respective IC₅₀ values of papain, PpGNPs, 5F-PpGNPs and 5-FU under phase-contrast microscopy (Figure 5). The control (untreated) cells appeared to be uniformly spread and normal in surface with no distinctive or momentous changes in morphology even after 48 h of incubation (Figure 5A). However, noteworthy changes were seen in the cells treated with papain, PpGNPs, 5F-PpGNPs and 5-FU (Figure 5B–E). After 48 h of treatment, these treated cells shrunk in size became irregular and necrotic and got detached from the surface of the wells. Some cells were found with their plasma membranes unblemished, demonstrating that apoptosis had begun. It was observed that PpGNPs induced shrinking and apoptosis in the cells, yet this phenomenon occurred at considerably higher concentrations of PpGNPs (than those required for induction of apoptosis by 5F-PGNPs).

Figure 5. Image showing cytotoxic effect of (A) untreated control, (B) papain treated, (C) PpGNPs treated, (D) 5F-PpGNPs treated, and (E) 5-FU treated A549 cells at their respective IC50 concentrations at 20× magnifications.

Display full size

3.1.7. Analysis of PpGNPs, 5F-PpGNPs and 5-FU-mediated disruption of the mitochondrial membrane in A549 cells

The process of apoptosis is elicited with the activation of caspases, a group of cysteine-aspartate proteases. Moreover, the intracellular apoptosis is stimulated by the release of the mitochondrial cytochrome C (a characteristic feature of the mitochondrial apoptotic pathway) and the loss of mitochondrial membrane potential (ΔΨm). The mitochondrial membrane disruption (Δψm) was analysed at the corresponding IC₅₀ values of the A549 cells treated with PpGNPs, 5F-PpGNPs and 5-FU in comparison with that of the untreated cells by the Mito Tracker Red CMXRos (Figure 6A). It was observed that the maximum intensity was observed for untreated cells followed by the cells treated with PpGNPs, 5-FU and 5F-PpGNPs in this order. This dye can distinguish between the changes in the Δψm in the cells; these changes were recorded under an inverted fluorescence microscope (Nikon ECLIPSE Ti-S, Japan) by the Image J software. In comparison with the untreated cells (Figure 6B), the A549 cells treated with PpGNPs (Figure 6C), 5F-PpGNPs (Figure 6D) and 5-FU (Figure 6E) showed a diminished fluorescence intensity. The minimum intensity was observed in the 5F-PpGNPs-treated cells; a gradual increase in intensity was observed for the 5-FU-treated and PpGNPs-treated A549 cells. However, untreated cells produced maximum intensity.

Figure 6. Mitochondrial depolarisation by disrupting mitochondrial membrane potential (ΔΨm) in A549 cells and images observed by staining with Mitotracker Red CMXROS. (A) Untreated control cells, (B) PpGNPs treated cells, (C) 5-FU treated cells, (D) 5F-PpGNPs treated cells. (E) Graph showing change in intensity of DCFDA stained control, PpGNPs, 5-FU and 5F-PpGNPs treated cells.

Display full size

3.1.8. Estimation of production of reactive oxygen species

In general, ROS-mediated toxicity is one of the modes by which nanomaterials act as anticancer agents. The intracellular ROS generation in A549 cells treated with PpGNPs, 5F-PpGNPs, and 5-FU was estimated using the 5 (6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) (Sigma-Aldrich) as an oxidation-sensitive fluorogenic marker of ROS in the viable cells (Figure 7). When A549 cells were treated with 5F-PpGNPs, 5-FU and PpGNPs at their respective IC₅₀ values, the generation of intracellular ROS increased gradually; maximum ROS generation was found with 5F-PpGNPs followed by 5-FU, PpGNPs and untreated cells (Figure 7). The fluorescence was quantified by the ImageJ software. Moreover, micrographs of the untreated (Figure 7A) PpGNPs- (Figure 7B), 5F-PpGNPs- (Figure 7C) and 5-FU-treated (Figure 7D) A549 cells were captured by the fluorescence microscope (Nikon ECLIPSE Ti-S, Japan). We could observe that the untreated cells showed minimum fluorescence, followed by PpGNPs-, 5-FU- and 5F-PpGNPs-treated cells (Figure 7E). The 5F-PpGNPs-treated cells were found to emanate a splendid fluorescence with a vandalised morphological structure because of the disturbing effect in the compactness of the plasma membrane brought about by the generation of the ROS. However, the untreated cells did not exhibit any fluorescence and maintained their natural morphology. The impact of cytotoxicity may be applied through the generation of oxidative pressure and apoptosis with a plausible association of overproduction of reactive oxygen species (ROS).

Figure 7. Images showing DCFDA staining under phase contrast microscope after 48 h of treatment on A549 cells at 20× magnification. (A) Control of DCFDA, (B) PpGNPs treated cells, (C) 5F-PpGNPs treated cells, (D) 5-FU treated cells. (E) Graph showing change in intensity of DCFDA stained control, PpGNPs, 5-FU and 5FPpGNPs treated cells.

Display full size

3.1.9. Analysis of changes in the nuclear morphology

The interaction of nanomaterials with the genetic material is inevitable and size-dependant. Moreover, 5-FU is an antimetabolite drug that incorporates fluoronucleotides instead of nucleotides. This interaction ultimately inhibits thymidylate synthase (TS), an enzyme involved in nucleic acid synthesis. Therefore, 5F-PpGNPs, 5-FU, and PpGNPs were found to interact with the nuclear material and cause substantial variations in the morphology and the nuclear structure of the A549 cells. The mode of cellular internalisation and, ultimately interaction with chromatin, were assessed by utilising a fluorescent dye (4′,6-diamidino-2-phenylindole) DAPI (Figure 8). The A549 cells were treated with PpGNPs, 5F-PpGNPs and 5-FU at their corresponding IC₅₀ values for 24 h at 37 °C and stained by the DAPI dye; the untreated cells were also stained to prepare them as controls (Figure 8). The observed blue fluorescence intensities were measured under an inverted fluorescence magnifying instrument (Nikon ECLIPSE Ti-S, Japan). The apoptotic effects in comparison with the untreated cells (Figure 8A) could be easily noticed in the PpGNPs (Figure 8B), 5F-PpGNPs- (Figure 8C), and 5-FU-treated (Figure 8D) cells by observing the expanded cell membrane penetrability that produced condensed chromatin and a dark blue fluorescent consolidated nucleus. It was interesting to observe that PpGNPs also initiated apoptosis: this was further intensified by the bioconjugation of 5-FU with PpGNPs. The most critical and particular indication of the cytotoxic impact of stress is the condensation of the nucleus. The graphical representation of the observed intensities is shown in Figure 8E.

Figure 8. Images showing DAPI staining under phase contrast microscope after 48 h of treatment on A549 cells at 20× magnification. (A) Control of DAPI, (B) PpGNPs treated cells, (C) 5F-PpGNPs treated cells, (D) 5-FU treated cells. (E) Graph showing change in intensity of DAPI stained control, PpGNPs, 5-FU and 5FPpGNPs treated cells.

Display full size

3.2. Discussion

In the given study, papain – a cysteine protease – was used to reduce the gold salt into gold nanoparticles. In this process, papain also acted as an encapsulating agent. The role of the protein in the nucleation of NPs is critical as the nucleation process depends upon the redox potential of the protein. The reducing properties of a protein increase with an increase in temperature and the overall charge of the protein. The regulation of overall charge and the ability to transfer electrons are the key factors during the nucleation of NPs. The reaction was carried out at 6 °C in the present study because, at its optimum temperature (37 °C), papain acted as a very strong reducing agent and resulted in the formation of NPs with a larger size and lesser stability. At 6 °C, the overall charge of papain decreased and this restricted its electron-transferring ability. Hence, the reaction progressed in a regulated and slow manner and also provided enough time for papain to reduce and encapsulate the NPs. During the complete synthesis of the NPs, the initially formed NPs behaved as a catalyst towards the completion of the reaction.

Cancer is one of the most deadly diseases worldwide and claims maximum deaths annually. Though radiotherapy, surgery and chemotherapy contribute a lot in fighting against cancer, their development is not sufficient to curb this most devastating disease of the world [Citation39]. Conventional chemotherapy has become voluntary torture to the patient, due to its non-selective action on normal cells. Target-specific drug delivery systems with trivial non-specific interactions must be evolved that may lead to a minimum or inconsequential side effects to the patients. Non-specific interactions cause huge collateral damages to patients [Citation40]. The evolution and progress of nanotechnology have extended a new archetype of potential in the development of nanomedicines that comprise of nanometre sizes and avail special treatment and behaviour from the immune system of the patient. These nanomedicines can be engineered to produce non-significant immunogenic responses, sustained release of the drug, insignificant non-specific interactions and site-specificity [Citation41]. Several drug delivery systems have been developed with huge success such as PEGylated PLGA, NP-encapsulated paclitaxel, etoposide [Citation42], and doxorubicin-conjugated bisphosphonate nanoparticles [Citation43] against osteosarcoma; methoxy-poly (ethylene glycol) aldehyde conjugated with doxorubicin and curcumin against HepG-2 cancer cells [Citation44]. In the same line of action, papain-encapsulated nanoparticles have been developed as effective drug delivery systems because they were found to be biocompatible, displayed anticancer property and had enhanced pharmacokinetics and pharmacodynamics of the bioconjugated 5-FU drug with vigorous intracellular delivery. In general, the gold nanoparticles, by the excellence of their metallic properties and size, hinder the propagation of the cancer cells by eliciting the NF-κB and Nrf-2 signalling pathways [Citation45], and they remain safe up to a concentration of 10¹² particles/mL [Citation46]. 5-FU is an antimetabolite drug that inhibits the thymidylate synthase by interacting with its metabolite, 5-fluoro-2′-deoxyuridine-5′-monophosphate, which ultimately hinders normal RNA processing. However, for the treatment of advanced stages of cancer, its bioavailability decreases to 15% and it also causes rigorous side-effects on the gastrointestinal tract, haematological, neural, cardiac and dermatological processes [Citation47]. Also, it is catabolized (more than 80%) in the liver by the dihydropyrimidine dehydrogenase (DPD) enzyme into dihydrofluorouracil (DHFU) [Citation48]. The bioconjugation protects 5-FU from degradation and prevents non-specific interactions, thus leading to a minimum or no side effects. Nanoparticles, by virtue of their sizes, avoid interactions with the reticuloendothelial system and tend to improve the concentration of drugs in the cancer cells by passive and active targeting mechanism as well as by diminishing the drug efflux from the cancer cells. In passive targeting, the nanomedicines leak from the blood vessels supplying blood to the cancer cells and accumulate in the cells by the enhanced permeability and retention (EPR) effect [Citation49]. On the other hand, in active targeting, the internalisation of nanomedicines takes place via receptor-mediated endocytosis. The anionic caveolae-mediated endocytosis and the cationic clathrin-mediated endocytosis processes rely on the net charges, shapes, and the presence of conjugated ligands over the surface of these nanoparticles, resulting in increased cellular uptake and therefore increased drug accumulation in the cancer cells. This mechanism works with the interaction between the tumour ligands conjugated on the surface of the nanoparticles and cell-surface receptors or antigens on the cancer cell surfaces [Citation28].

The selection of capping agent/s was based on its/their abilities to bioconjugate with the drug for its safe and prompt delivery to the site. Various proteins such as transferrin (a serum glycoprotein), HSA, bromelain, trypsin [Citation21–22] have been used successfully as capping and functionalising agents. Similarly, papain was selected by virtue of its reducing and anticancer properties. Papain is a sulfhydryl protease from the latex of the plant Carica papaya and displays a powerful digestive action that is stronger than that of the pancreatic enzymes. Therefore, it has been used for a long time in herbal medicine in different countries [Citation50,Citation51]; yet, very limited information on its molecular targets and anticancer effects is available. Papain can inhibit the propagation and invasion of TFK-1 and CC cells by inhibiting the phosphorylated forms of NFκB, STAT-3, AKT and ERK. It also up regulates the phosphorylated AMPK in the SZ-1 cells. After adopting multiple targeting processes using different pathways to inhibit cancer cell proliferation, this combination was found to be highly active and site-directed.