Functionalized Carbon Nanotubes for Delivery of Ferulic Acid and Diosgenin Anticancer Natural Agents

It was investigated whether loading multi-wall carbon nanotubes (CNTs) with two natural anticancer agents: ferulic acid (FUA) and diosgenin (DGN), may enhance the anticancer effect of these drugs. The CNTs were functionalized with carboxylic acid (CNTCOOH) or amine (CNTNH2), loaded with the above pro-drugs, as well as both combined and coated with chitosan or chitosan–stearic acid. Following physicochemical characterization, the drug-loading properties and kinetics of the drug’s release were investigated. Their effects on normal human skin fibroblasts and MCF-7 breast carcinoma cells, HepG2 hepatocellular carcinoma cells, and A549 non-small-cell lung cancer cells were evaluated in vitro. Their actions at the molecular level were evaluated by assessing the expression of lncRNAs (HULC, HOTAIR, CCAT-2, H19, and HOTTIP), microRNAs (mir-21, mir-92, mir-145, and mir-181a), and proteins (TGF-β and E-cadherin) in HepG2 cells. The release of both pro-drugs depended on the glutathione concentration, coating, and functionalization. Release occurred in two stages: a no-burst/zero-order release followed by a sustained release best fitted to Korsmeyer–Peppas kinetics. The combined nanoformulation cancer inhibition effect on HepG2 cancer cells was more pronounced than for A549 and MCF7 cells. The combined nanoformulations had an additive impact followed by a synergistic effect, with antagonism demonstrated at high concentrations. The nanoformulation coated with chitosan and stearic acid was particularly successful in targeting HepG2 cells and inducing apoptosis. The CNT functionalized with carboxylic acid (CNTCOOH), loaded with both FUA and DGN, and coated with chitosan–stearic acid inhibited the expression of lncRNAs and modulated both microRNAs and proteins. Thus, nanoformulations composed of functionalized CNTs dual-loaded with FUA and DGN and coated with chitosan–stearic acid are a promising drug delivery system that enhances the activity of natural pro-drugs.


Study of size distribution of nanoparticles and zeta potential
. Size distribution measurement of the samples as evaluated using the DLS method.The samples description is given in Table 1 Figure S2.Zeta potential measurement of CNT materials and nanoformulations.The meaning of the symbols F1-F10 are explained in Table S1.It is seen that the non-coated with polymer samples show a bi-modal size distribution, except for the F6 -CNTNH 2 FUA sample.The coated samples show significantly larger size than the noncoated samples.
For interpretation of these results is to consider that the DLS method is not suitable for samples of fibrous structure, as CNTs.The size of such samples depends rather on their length than diameter and in addition, the fibres are entangled, as seen on FE-SEM images (Fig 1 in the main text).However, large size of the coated samples may indicate their agglomeration.The samples without polymer coating display a mean size in the range 235 -585 nm, while the coated ones in the range 792 -1191 nm.
No systematic trend is seen as far as the Zeta-potential is concerned.

Thermogravimetric characterization
Pure DGN and FUA decomposed nearly completely (mass losses of 98 wt.% and 92 wt.%, respectively).Supplementary material describes in detail decomposition of these substances.We did not find any significant differences between the non-coated nor loaded two types of The significant differences in thermal properties between non-loaded and loaded materials is related to decomposition of the anticancer agents and chitosan-stearic acid coating complex.The temperature range of the decomposition process was determined from the peaks visible in the DTG curves (Figure S3C,D).In the case of nanotubes with DGN, the temperature corresponded to the decomposition of the pure anticancer substance: 160-520℃ (min.at 290℃) for CNTOOHDGN and 200-560℃ (min.at 300℃) for CNTNH 2 DGN.In the case of CNTs with FUA, the results were not so evident.Intensive mass loss of CNTOOHFUA occurred at lower temperatures than for pure (min.~250℃), respectively.For CNTCOOHFUADGN@CSFISA, an additional peak was visible in the range 350-520°C (min.at 430°C).For all investigated nanoformulations, there were no melting peaks, which were recorded for pure anti-cancer substances (Figure S3E,F).The reason may be the  interaction of nanotubes and drugs [3], an insufficient amount of natural agent, and changes in the structure of drugs during the preparation of the nanoformulations.

FTIR results
Bands corresponding to hydroxyl groups at approximately 3400 cm -1 and a number of signals at lower wavenumbers [1,4].In the case of DGN, the band assigned to the -OH stretching vibrations occurred at 3447 cm -1 and the peaks attributed to the CH 2 stretching and scissoring vibrations at 2950 cm -1 and 1455 cm -1 , respectively.Bands corresponding to -C-O stretching vibrations were visible at 1172 cm -1 and 1051 cm -1 , whereas those corresponding to the CH 2 twisting vibrations were visible at 896 cm -1 [5].FUA showed a signal corresponding to hydroxyl groups (-OH) at 3432 cm -1 , the alkane characteristic band at 1687 cm -1 , C=C stretching vibration at 1661 cm -1 , -C-O stretching vibrations at 1163 cm -1 , and C=C-H bond at 942 cm -1 [4].
The results obtained for both types of nanotubes were very similar, with no intense well-defined signals detected.
4. In vitro release data.To calculate CI, we do the following steps: 1.The calculation was performed with cell inhibition percent%.
As our data presented in the paper in cell viability %, we firstly calculated the cell inhibition according to this equation: Cell inhibition %= 100 -cell viability.
2. For CI calculation, we used HAS combination index calculation eq: CI= EAB-max(EA,EB) # EAB= effect producing cell inhibition% of combined two drugs (FUA combined DGN) into nanoformulation before and after polymeric coating.# EA and EB = effect producing cell inhibition% of single drug (FUA or DGN) into nanoformulation.

Molecular evaluations data
Intensive weight losses related to decomposition are visible on the TG curves in the temperature range of 250-560℃C for DGN and 170-430℃ for FEA (FigureS3A,B).The corresponding broad peaks in the DTG curves have extremes at approximately 350°C and 250°C, respectively (FigureS3C,D).The DSC curves of both anticancer agents were similar in nature (FigureS3E,F).Only endothermic peaks are visible.The first peak was intense and narrow.For DGN, it occurred between 180 and 220℃, with a minimum of approximately 205℃.For FUA, the peak occurred in the range of 160-180℃ with a minimum at approximately 175℃.This thermal effect was not accompanied by a change in the mass of the substance (no signals on the DTG curve).These peaks are related to the melting process of DGN and FUA [1, 2].At higher temperatures, wide endothermic signals were present in DSC curves of DGN and FUA, correlating with the decomposition process.In the case of DGN, a series of overlapping peaks occur in the temperature range of 290-560℃.The most intense peak had a minimum at approximately 360℃.For FUA, the endothermic effect was visible at 200-280℃ with a minimum at approximately 260℃.

FUA.
The DTG peak was presented at RT-220℃ with a minimum at 110℃.The CNT-NH 2 FUA results were very similar to those obtained for pure CNT-NH 2 .The DTG curves for nanotubes enriched with two active agents exhibited the decomposition effects of both DGN and FUA.For CNTOOHFUADGN, the first occurred at RT-160℃ (min.~120℃) and second at 180-480℃ (min.~290℃).For CN-NH2FUADGN, the first occurred at 210-350℃ (min.~300℃) and second at 350-500℃ (min.at 420℃).The TG/DTG curves of both nanoformulations with the polymer coating had a similar course (FigureS3A-D).The broad peaks in the DTG curves, corresponding to the FUA and DGN decomposition, occurred in the range of RT-160℃ (min.~95℃) and 160-400℃

Table S1 : Mean size and zeta potential of the samples.
nanotubes of CNTCOOH and CNTNH 2 .The weight losses of CNTOOH and CNTNH 2 reached

Table S2 .
In vitro release data.

Table S4 .
IC50 values for single and combined nanoformulations, as well as free DGN and FUA.

Table S5 .
Calculated combination index for single nanoformulations composed FUA or DGN and combined nanoformulation composed both agents before and after polymeric shell coating.