Boosting The Anticancer Activity Of Aspergillus Favus “endophyte Of Jojoba” Taxol Via Conjugation With Gold Nanoparticles Mediated By γ‑Irradiation Ⅱ
May 30, 2023
Chemical Identity and Anticancer Activity of A. favus Taxol
The putative TLC spots of A. favus Taxol were scrapped of and dissolved in methanol, and the chemical properties of the compound were resolved from the different HPLC, HNMR, and FT-IR chromatographic and spectroscopic analyses. Proton nuclear magnetic resonance signals (1 H NMR) of A. favus extracted Taxol were distributed between 1.0 and 8.0 ppm, with three resolved proton signals at 1.0–3.0 ppm corresponding to methyl, acetate, and acetylene groups, whereas proton signals at 6.5–9.0 ppm corresponding to the aromatic moieties (Fig. 3). The pattern and distribution of protons signals of A. favus Taxol authenticate the structural identity and similarity of the sample with the standard Taxol (Fig. 3). Moreover, Taxol of A. favus has the same FTIR spectra of authentic Taxol. The peak at 3393.3 cm−1 was assigned for the hydroxyl (OH), while the peak at 2923.5 was assigned to the aliphatic CH stretch, the peaks at 1661.0 cm−1 correspond to C=O stretching frequency. The observed peaks at 1452.0 cm−1 and 1404.0 were referenced to the NH stretching frequency, while the peak at 1109.0 cm−1 refers to the carbonyl groupoxygen stretching frequency. The peaks in the range between 1020.0 and 979.77 cm−1 were referenced to the presence of aromatic C and H bends. Conclusively, the spectral pattern of FTIR regarding to the functional groups of A. favus Taxol was identical to the authentic Taxol (Fig. 3). Conclusively, from the chromatographic and spectral analyses, the putative sample has been resolved and authenticated as Taxol compounds compared to the authentic compound.
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Effect of Different Types of Medium on Taxol Production by A. favus
The influence of medium composition on the Taxol production by A. favus has been evaluated by growing on PDB, M1D, Czapek’s-Dox, and malt extract. After cultural incubation, Taxol was extracted and quantified as described in Materials and Methods. From the results in Fig. 4, the highest Taxol yield by A. favus was obtained by growing on malt extract (170 µg/l) and Czapek’s-Dox (166 µg/l), compared to PDB growth medium as control (89 µg/l). Thus, the yield of Taxol by A. favus was increased by approximately 2 folds by its growing on Czapek’s-Dox and malt extract media, compared to the control medium. So, further optimization studies to maximize the Taxol yield by A. favus by the response surface methodology statistical approach have been implemented.
Optimization of Bioprocess Variables Using Response Surface Methodology Plackett–Burman Design Screening for Critical Factors
The yield of Taxol by A. favus has been optimized by the statistical experimental design of Plackett–Burman [18, 28]. The minimum and maximum ranges of the selected parameters selected by the Plackett–Burman design were summarized in Table 2. Taxol concentration was determined by the TLC and HPLC. All the experiments were
the average Taxol concentration was considered. Statistical analysis of this design illustrates that the model F-value of 5.98 implies the model is significant and values of “Prob>F” less performed in triplicate and than 0.0500 as reported in Table 3. The actual and predicted yield of Taxol in response to the experimental parameters as revealed from the Plackett–Burman design was shown from the matrix in Table 4. The “Pred R-Squared” of 0.3061 is not as close to the “Adj R-Squared” of 0.5760 as one might normally expect. The maximum actual (255.35 µg/l) and predicted (280.4 µg/l) yield of Taxol by A. favus upon a factorial design by Plackett–Burman design was obtained at run # 8. From the obtained data (Fig. 4), the highest Taxol yield by A. favus (225 µg/l) was obtained using malt extract (20 g/l), peptone (2 g /l), sucrose (20 g /l), soytone (2 g/l), cysteine (0.5 g/l), glutamine (0.5 g/l), beef extract (1 g/l), at incubation temperature 30 °C, initial pH 6.0, and after 12 days of incubation at shaking speed (150 rpm). At this cultural run (12), the yield of Taxol by A. favus was significantly increased by about 1.4 folds, compared to the control medium (170 µg/l). The model reduction, response transformation, and Adeq Precision measure the signal-to-noise ratio, and a ratio greater than 4 is desirable [46]. Based on the parameter estimated and by applying multiple regression analysis on the experimental data, the test variables and the response variable were related by the following model equation: first-order equation in terms of coded factors — Taxol Con.= +11.51+1.91 E+2.92 J+0.81 K. The actual and predicted levels of Taxol by A. favus upon Plackett–Burman optimization bioprocess are summarized in Table 4 and Fig. 4. The maximum actual (255.35 µg/l) and predicted (280.4 µg/l) yield of Taxol by A. favus upon a factorial design by Plackett–Burman design was obtained at run # 8 at medium component. The effect of individual parameters as revealed from the Plackett–Burman design was represented in the Pareto Chart depicting the order of significance of variables involved in Taxol production Fig. 4. Thus, the most significant variables affecting Taxol productivity by A. favus were cysteine, pH, and incubation time as revealed from the Plackett–Burman design.
The faced central composite design was adopted for further optimization studies using the significant variable that from the Plackett–Burman design. The results of 20 runs of three variables with positive effects demonstrated that Taxol productions diversified markedly with the condition were tested in the range of 96.5–302.7 µg/l. The maximum yield of Taxol by A. favus (302.7 µg/l) was observed using cysteine (0.50 g/l) at pH 6.0 and incubated for 15 days as recorded in Table 5. Minimum Taxol production was recorded at run number 15, where cysteine (1.0 g/L) at pH 7.0 and incubated for 7 days. The statistical analysis of the linear model design demonstrates that the F-value is 87.08 implying the significance of the model, and the values of “Prob>F” less than 0.050 indicate that model terms are significant (Table 6). Therefore, cysteine, pH, and incubation time are significant model terms. The analysis revealed that the “Pred. R-Squared” of 0.8972 is in reasonable agreement with the “Adj. R-Squared” of 0.9315. “Adeq. Precision” ratio of 30.800 measures the signal-to-noise ratio, as the ratio greater than four is desirable, the Adeq. The results obtained from the CCD experiment were analyzed by ANOVA that yielded the following regression equation at the level of Taxol production: first-order model equation terms of coded factors — Taxol Conc.= +11.47+1.60A+2.43B+0.67C. The three-dimensional response surface curves showing the effect of interactions of the most signifcant parameters, cysteine, pH, and incubation time, were shown in Fig. 4.
Table 2 Minimum and maximum ranges of the parameters selected in P-BD for optimization of Taxol production
Table 4 Plackett–Burman design to evaluate factors affecting Taxol production by A. favus
variables with positive effects demonstrated that Taxol productions diversified markedly with the condition tested in the range of 96.5–302.7 µg/l. The maximum yield of Taxol by A. favus (302.7 µg/l) was observed using cysteine (0.50 g/l) at pH 6.0 and incubated for 15 days as recorded in Table 5. Minimum Taxol production was recorded at run number 15, where cysteine (1.0 g/L) at pH 7.0 and incubated for 7 days. The statistical analysis of the linear model design demonstrates that the F-value is 87.08 implying the significance of the model, and the values of “Prob>F” less than 0.050 indicate that model terms are significant (Table 6). Therefore, cysteine, pH, and incubation time are significant model terms. The analysis revealed that the “Pred. R-Squared” of 0.8972 is in reasonable agreement with the “Adj. R-Squared” of 0.9315. “Adeq. Precision” ratio of 30.800 measures the signal-to-noise ratio, as a ratio greater than four is desirable, the Adeq. The results obtained from the CCD experiment were analyzed by ANOVA that yielded the following regression equation at the level of Taxol production: first-order model equation terms of coded factors — Taxol Conc.= +11.47+1.60A+2.43B+0.67C. The three-dimensional response surface curves showing the effect of interactions of the most significant parameters, cysteine, pH, and incubation time, were shown in Fig. 4.
modified malt extract medium obtained from the RSM by Plackett–Burman design. After cultural incubation under standard conditions, Taxol was extracted and quantified by TLC and HPLC. From the obtained results (Fig. S1), there is no significant effect on Taxol productivity upon γ-irradiation at the different doses.
Synthesis of AuNPs and Conjugation with A. favus Taxol Mediated by γ‑ Radiation
Polyvinylpyrrolidone (PVP)-capped gold nanoparticles (AuNPs) were synthesized by mixing PVP with gold (III) chloride, mediated by gamma irradiation at doses of 0.25–10.0 kGy, and the developed PVP-AuNPs were evaluated from the UV–Vis, FT-IR, and XRD analyses. The sketch of the formation of the Taxol-PVP-AuNPs consortium was shown in Fig. 5. Size of the developed PVP-AuNPs mediated by 1.0 kGy gamma rays was monitored by DLS, and it was about 50 nm (Fig. 5). From the HRTEM image, the expected shape and size of the synthesized AuNPs was demonstrated with spherical particles within the nanoscale range from 20 to 30 nm with the average main diameter of 25 nm (Fig. 5). Similar results were observed with PVP-AuNPs mediated by gamma-irradiation [49, 50]. The spectral properties of the developed AuNPs in response to different doses of gamma rays were assessed from the UV–Vis analysis, with maximum recorded absorption spectrum at λ540 nm at 1.5–3.0 kGy. In addition, the development of AuNPs was monitored from the visual inspection, with a plausible yellowish to brown color, revealing the formation of AuNPs.
The chemical conjugation of Taxol and PVP-AuNPs has checked from the FT-IR spectral analysis, as revealed by the slight shift of the intensity and transmission ratio of the functional groups of Taxol-PVP-AuNPs conjugates compared to Taxol as control. The intensity of the peak at 3393.3 cm−1 assigned for the hydroxyl (OH) in Taxol-PVPAuNPs consortium was increased by about 3 folds comparing native Taxol, revealing the chemical stretch on the hydroxyl groups (Fig. 5). As well as the intensity of the peaks 1661.0 cm−1 referring to C=O stretching frequency was strongly increased by about 10 folds upon conjugation with PVP-AuNPs, ensuring the stretching on the C=O bonds. The intensity of the peak 2923.5 corresponding to aliphatic CH stretching was quite stable. A slight shifting on the intensity of observed peaks at 1452.0 cm−1 and 1404.0 cm−1 has been observed due to the NH stretching frequency. The intensity of the peak 1109.0 cm−1 of carbonyl group-oxygen stretching frequency was slightly reduced upon conjugation with PVP-AuNPs.
The crystal/physical structure of the Taxol upon conjugation with PVP-AuNPs was resolved from the XRD analysis (Fig. 5). From the XRD, the Taxol-PVP-AuNPs consortium had the same structural configuration, crystal orientation, and size of the nativeTaxol, ensuring the lack of negative effect on the crystal structure of native Taxol. From the results, the amorphous and crystal structure of the Taxol-PVP-AuNPs compared to the native Taxol has been confirmed. From the XRD analysis, conjugation of Taxol and PVP-AuNPs has been resolved as revealed from the diffraction properties concerning 2ɵ=38.18°, 44.01°, 64.57°, 77.67°, and 81.74° which described the Bragg’s observations at (111), (200), (220), (311), and (222), respectively. All the peaks were related to the ideal card of the Joint Committee on Powder Diffraction Standards (JCPDS) of AuNPs (JCPDS card No. 04–0784) [51]. So, the strength of crystals of the synthesized AuNPs was shown, providing the face-centered cubic (fcc) crystalline structure. In addition, there is a simply amorphous peak at 19.25° for Taxol that is included in the organization and permanence of AuNPs. The XRD pattern confirmed the successful conjugation of Taxol and PVP-AuNPs. The mean crystallite size of the incorporated AuNPs was determined from Scherrer’s equation [52], and it was 20.2 nm for the Taxol-PVP-AuNPs consortium mediated by gamma rays as mentioned in the equation:
where D is the average crystallite size, β is the full-width at half maximum, λ is the X-ray wavelength, θ is Bragg’s angle, and k is a constant.
Anticancer Activity of Taxol‑PVP‑AuNPs Conjugates
The antiproliferative activity of Taxol-PVP-AuNPs conjugates was assessed against HEPG-2 and MCF-7 cell lines, normalizing to native Taxol and AuNPs, separately. From the results (Fig. 6), the bioactivity of Taxol was dramatically increased upon conjugation with AuNPs, compared to native Taxol as control. As revealed from the IC50 values, Taxol-PVP-AuNPs consortium displayed signifcant activity against HEPG-2 (2.2 µg/ml) and MCF-7 (3.3 µg/ml) compared to native Taxol and AuNPs separately. So, upon conjugation with AuNPs, the activity of Taxol was increased by two folds towards the tested cell lines. Regarding to the bioactivity of AuNPs, the antiproliferative activity of the Taxol-PVP-AuNPs consortium was increased by about 4 folds compared to AuNPs as control. Interestingly, the activity of the Taxol-AuNPs consortium was plausibly consistent with the authentic Taxol.
Antimicrobial Activity Taxol and Synthesized AuNPs Against Different Microorganisms
The antimicrobial activity of the extracted A. favus Taxol and Taxol-AuNPs consortium was evaluated against various multidrug-resistant bacteria and fungi (Table 7). Upon conjugation with AuNPs, the antimicrobial activity of Taxol has been strongly increased compared to native Taxol and AuNPs separately, as revealed from the diameter of inhibition zones of the tested microorganisms (Fig. 6). As shown in Fig. 6, the Taxol-AuNPS consortium displayed the highest activity against E. agglomerates (11 µg/ml), C. albicans (12 µg/ ml), and E. coli (14 µg/ml), comparing to native A. favus Taxol and authentic one. The higher activity of Taxol upon conjugation with AuNPs authenticates the bioavailability, solubility, and efficacy of Taxol to bind with the target tubulin protein in vivo.
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