Improving ruthenium nanoparticle physicochemical properties and chemotherapeutic efficacy by dual-encapsulating with new amphiphilic chitosan and imidazolium ionic liquid

2.1 Preparation of IIL-coated RuNPs (RNBC1)

A full stepwise protocol used for the preparation of the starting materials and intended RNBCs is depicted in Fig. 1. IIL-coated RuNPs were prepared using a protocol modified from Parida et al work (Parida et al., 2022). Briefly, the preparation was carried out by adding 31.11 mg of RuCl₃ (0.15 mmol) to 3 mL of IIL (2b) and 15 mL of dry THF in a Schlenk flask at room temperature (RT), and then stirring the mixture for 2 h at 750 rpm. The resulting dark yellow solution was then degassed, heated to 80 °C, and stirred for a further 2 h. After cooling the reaction mixture to RT, NaBH₄ (30.26 mg, 0.8 mmol) was added while stirring vigorously. The development of RuNPs was indicated by the dark yellow solution's gradual blackening. For the full conversion of ruthenium salt to Ru(0) and the generation of NPs, stirring at room temperature was continued for 24 h. This was verified by UV–Vis spectroscopic analysis. If the broad absorption band in the precursor's spectrum disappears after reduction, it means all the Ru⁺³ ions have been converted to Ru(0) (see Fig. 1).

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Fig. 1

Step by step synthesis of ALMC (A); IIL (2b) (B); and as well thier utlization in the prepration of stablized RuNPs-based NBCs (C).

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Afterward, the reaction mixture was centrifuged for 15 min at 8000 rpm and 15 °C to collect the IIL-coated RuNPs (RNBC1). Following this initial centrifugation, RuNPs were re-suspended in a mixture containing 15 mL of acetone and 20 mL of diethyl ether, and the combined mixture was re-centrifuged. One more time, this procedure was repeated. The subsequent step was dissolving the resultant sticky, black substance in 5 mL of methanol and centrifuging it with 25 mL of diethyl ether. At 15 °C, all centrifugations were conducted for 15 min at 8000 rpm. Finally, the resulting glossy black product was vacuum-dried at 35 °C until it reached a constant weight.

2.2 Preparation of ALMC/IIL-coated RuNPs (RNBC2)

50 mg of ALMC (0.1 % W/V) and 500 mg of IIL (2b) were dissolved in 50 mL of dry THF and magnetically agitated for 60 min to prepare a homogenous suspension. The solution was then subjected to a 30-minute ultrasonic treatment. After that, 177 mg of RuCl₃ is added to the aforementioned solution and stirred at 80 °C for 2 h. Following cooling to RT, 378 mg of NaBH₄ was slowly added to the ALMC/IIL/RuCl₃ mixture while vigorously stirring. The content was subsequently subjected to a 6-hour ultrasonic treatment to form a homogeneous dispersion. Afterward, the reaction mixture was centrifuged for 15 min at 8000 rpm and 15 °C to collect the Ru-IIL/ALMC (RNBC2). After that, the RNBC2 was washed three times with ultrapure water, ethanol, and finally diethyl ether to remove the unreacted materials. After washing, the obtained product was vacuum-dried at 40 °C until it reached a consistent weight.

2.3 Cytotoxicity performance

Two medically significant cancer cells (hepatocellular (HepG2) and colorectal (CaCo-2) carcinomas) as well as normal human liver cells (THLE2) were used to test the novel Ru-nanocomposites' cytotoxic effects. According to our previous studies, we used the MTT assay to perform these analyses, which are described in detail in the ESM†(Elshaarawy et al., 2017).

2.4 Gene expression assessment

The expression levels of the P53, BAX, and Bcl2 genes in untreated and NBC2-treated HepG2 cells were compared using the real-time PCR (RT-PCR) method. This was accomplished by culturing HepG2 cells in tissue culture dishes and then treating them with NBC2 at an inhibitory dose (2.16 g/mL) for 24 h. Following the manufacturer's protocol, RNA was initially extracted using a Qiqamp mini kit (Qiagen; USA). The total cellular RNA was then subjected to RT-PCR analysis using one-step QuantiTecht SYBR green (Qiagen; USA) to quantify gene expression of P53, Bax, and Bcl2 before and after NBC2 treatment, according to a previous work (Abdel-Megeed et al., 2022). Table S1 (ESM†) provides the primer sequences and experimental settings used in this analysis. The comparative CT (2^-ΔΔCT) method was applied to determine the relative expression for each gene (Abdel-Megeed et al., 2022).

2.5 Topoisomerase inhibitory effect

The most potent anticancer agent (RNBC2) was tested for its capacity to inhibit human DNA topoisomerase II (Topo II) using an enzyme-linked immunosorbent assay (ELISA) kit, following the kit's instructions. All of the samples and reagents were set up according to the work processes (Topcu, 2001, Asghar et al., 2023). The protocol used to perform these analyses was described in detail in the ESM†.

2.6 Statistical methods

The results of this study were graphically presented, statistically analyzed, and mathematically treated using the programs OriginPro 9.1.32 and SPSS v17. A result was considered to be statistically significant if its P value was < 0.05.

3.1 Synthesis

Intially, a three-step protocol is established to synthesize the desired imidazolium ionic liquids (IILs, 2a,b) from tert-butylbenzene (TBT) (see Fig. 1). To begin, a mixture of dimethoxymethane, chlorosulfonic acid, and zinc iodide was used to chloromethylate TBT, producing 4-(tert-butyl)benzyl chloride (TBBC, 1). Then, in an inert atmosphere, 3-(4‘tert-butyl benzyl)-1,2-dimethyl imidazolium chloride (2a) was prepared by quaternizing 1,2-dimethylimidazole with the TBBC under refluxing conditions. Submitting 2a to anion metathesis with BF₄^- at under ambient conditions yielded the corresponding imidazolium tetrafluoroborate ionic liquid (2b). On the other hand, the amphiphilic low molecular weight chitosan (ALMC) was prepared by O-quaternization of LMC with glycidyldimethylhexadecyl ammonium chloride (GDMHAC) through three consecutive reactions: NH₂-protection by Schiff-base condensation, O-quaternization, and NH₂-deprotection by acidic hydrolysis. Eventually, the single-layered IIL-encapsulated RuNPs (RNBC1) and dual-layered IIL/ALMC-encapsulated RuNPs (RNBC2) were prepared by the thermal reduction of its chloride precursor (RuCl₃) using NaBH₄ as a reducing agent and mediated with IIL/THF or IIL/ALMC/THF, as a green solvent and stabilizing agent, in an inert environment.

3.2 Physicochemical characterization

3.2.2

3.2.2 Structural characterization

At first, we used the outcomes of the titrimetric and elemental analyses (EA) to determine the deacetylation degree (DD%) of LMC and the quaternization degree (QD) of ALMC as reported in our previous work (El-Sayed et al., 2021a,b). The findings of these calculations were represented in Table S2 (ESM†). Noteworthy, ALMC's N/C ratio is lower than that of LMC's, indicating that the quaternization process was successful. Moreover, the EA results for ALMC are inconsistent with a full quaternization of the amino group for LMC with the GDMHAC fragment, implying a partial quaternization.

Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analysis was used to measure the amounts of Ru present in RNBC1 and RNBC2. The Ru contents in the RNBC1 and RNBC2 were 165300 ppm (16.53 w%) and 199200 ppm (19.92 %), respectively. The higher content of RuNPs in the hybrid IIL/ALMC -based nanobiocomposite (RNBC2) compared to the only IIL-based nanobiocomposite (RNBC1) can be attributed to the combined effects of the stabilizing properties of both components, the increased surface area and binding sites for nanoparticle deposition provided by chitosan, and the synergistic interactions between the IIL and ALMC.

ESI-MS (electrospray ionization mass spectrometry) is an essential technique for studying the structural characteristics of ionic liquids. In this context, the ESI-MS of IIL (2b) was measured, and the spectrum was given in the ESM† (Fig. S2). 2b exhibits a major peak at m/z 243.2 a.m.u., corresponding to the molar mass of a monovalent cation formed owing to the departure of the BF₄ anion [M - BF₄^-]⁺. In addition, several fragmentation peaks were observed at m/z 187.1, 172.3, and 157.1 a.m.u. assignable to the consecutive departure of isobutene [M - BF₄^- - C₄H₈]⁺ and methyl radicals [M - BF₄^- - CH₃̇]⁺ & [M - BF₄^- − 2 (CH₃̇)]⁺ from the native IIL, respectively.

The FTIR spectrum of IIL (2b) (Fig. S3, ESM†) shows absorption bands at 3157, 2960, 1600, 1196, 874, 702, and 581 cm⁻¹, which are related to the vibrational modes of stretching associated with benzyl-imidazolium cation (alkyl C—H, C⚌N, and benzyl groups) (Ibrahim et al., 2008, Sidek et al., 2017, El-Sayed et al., 2021a,b). Additionally, the characteristic (BF₄) vibration band can be seen at 1065 cm⁻¹ (Elshaarawy and Janiak, 2014). On the other hand, typical vibration bands for LMC (O—H/N—H, 3398 cm⁻¹; bending N—H, 1649 cm⁻¹; amide C⚌O, 1589 cm⁻¹; and glycoside (C—O—C) group 837 cm⁻¹) and GDMHAC fragment (methyl groups, 2958, 2881 cm⁻¹; R₄N⁺, 1511 and 669 cm⁻¹) may be seen in the spectru of ALMC (Fig. S4, ESM†). However, with significant alterations due to the grafting reaction, is indicative of its successful (El-Sayed et al., 2021a,b). As for the ruthenium architectures (RNBC1 and RNBC2), it is clear that RuNPs have been immobilized in the IIL (2b) matrix for RNBC1 and the IIL/ALMC matrix for RNBC2 due to the profound impacts of nanoruthenium on the distinctive peaks of FTIR spectra for both matrices. Specifically, the OH, NH₂, and C⚌O peaks at 3389, 1600, and 1593 cm⁻¹ were shifted (see Fig. 2A) due to their interactions with the RuNPs.

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Fig. 2

(A) FTIR spectra of IILs (2b), IIL-coated RuNPs (RNBC1), and IIL/ALMC-coated RuNPs (RNBC2) showing the major destinctive absorbtion bands: (1), C⚌N; (2), R₄N⁺; (3), BF₄; (4), glycoside group; (5), imidazolium cation; (6), benzyl group. (B) UV–Vis spectra of IILs (2b), ALMC, RuCl₃, RNBC1, and RNBC2.

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The absence of the distinctive peak associated with the ruthenium precursor (RuCl₃) in the spectra of the nanocomposites (as shown in Fig. 2B) suggests that the reduction of ruthenium ions (Ru³⁺) has been effectively achieved through the use of NaBH4, facilitated either by IIL or an IIL-ALMC combination. As an ionic liquid, IIL has a high solubility for both polar and non-polar compounds. This property allows for better dispersion and interaction between the Ru(III) ion and the reducing agent, enhancing the efficiency of the reduction reaction. Moreover, the low viscosity of IIL facilitates mass transfer and diffusion of reactants, ensuring efficient contact between the Ru(III) ion and the reducing agent (Schmolke et al., 2019). On the other hand, the IIL-ALMC combination may facilitates the reduction of Ru(III) ions to Ru(0) in the presence of NaBH₄ in several ways. Firstly, the interactions between IIL and ALMC may lead to the formation of a stable microenvironment. This microenvironment provides a confined space where the reduction reaction can take place efficiently. Secondly, the ALMC can coordinate with the Ru(III) ions, forming complexes that enhance the reactivity of the Ru(III) ions towards the reducing agent, NaBH₄. This coordination promotes the transfer of electrons from NaBH₄ to the Ru(III) ions, facilitating the reduction process. Additionally, the presence of ALMC helps in stabilizing the reduced RuNPs formed during the reduction reaction, preventing their agglomeration and ensuring their uniform dispersion (Zein et al., 2024). According to these results, Ru³⁺ has been converted entirely to Ru(0).

The ¹H NMR spectrum of IIL (Fig. S5, ESM†) reveals two distinct proton peak configurations. Two doublets and a multiplet in the 7.74–7.62 ppm range, which can be attributed to the the protons in imidazolium and phenyl resonances, were seen in the first down-field set. In contrast, multiple singlet peaks can be observed in the first high-field set (5.41–1.41 ppm), ascribed to the resonances of the benzylic, tert-butyl, and methyl protons. Meanwhile, the ¹³C NMR spectrum of IIL (Fig. S6, ESM†) shows two sets of carbon signals in the low-field (160.28–121.08 ppm) and high-field (50.31–9.87 ppm) regions, corresponding to the resonances of the carbon skeleton for the benzylimidazolium cation and alkyl side chains, respectively. In addition, a two singlets in the ¹¹B NMR (−148.28 ppm) and ¹⁹F NMR (−1.29 ppm) spectra of IIL (Fig. S7–8, ESM†) further verify the presence of counter tetrafluoroborate anion in this ionic liquid. On the other hand, the ¹H NMR spectrum of ALMC exhibits remarkable changes as compared to that of LMC (Fig. S9, ESM†). Specifically, the ALMC spectra almost completely lacked the NH₂ signal distinctive of LMC (5.18 ppm), indicating a great quaternization degree. In addition, a new singlet appeared at 3.22 ppm, and multiplet peaks appeared between 3.50 and 0.88 ppm, possibly representing methyl and hexadecyl proton resonances in the GDMHAC segment.

3.3 Morphological characterization

The TEM nanograph of IIL-coated RuNPs (RNBC1) illustrated in Fig. 3A shows the formation of a thin film of IIL around nanoparticles, which acts as a monolayer protective shell formed of IIL encasuplating the RuNPs. This shell effectively mitigates nanoparticle aggregation, enabling the RuNPs to maintain a homogeneous dispersion within the mixture. Moreover, the RuNPs exhibited a primarily spherical morphology, characterized by a mean particle diameter (MPD) of 2.16 nm, as depicted in Fig. 3C. This MPD was calculated by measuring the diameters of 75 individual nanoparticles from its TEM picture using ImageJ software, and finally calculating the average diameter. Additionally, the RuNPs displayed a low degree of polydispersity (PDI = 0.23), suggesting a uniform size distribution of the nanoparticles. Regarding the double-layered coated ruthenium nanoparticles (RNBC2), the presence of a stiff protective shell effectively inhibits the agglomeration of the ruthenium nanoparticles (as shown in Fig. 3B), resulting in their uniform dispersion within a homogeneous mixture. Moreover, the RuNPs exhibited a primarily spherical morphology, characterized by a mean particle diameter (MPD) of 5.19 nm as depicted in Fig. 3D. Additionally, the RuNPs displayed a low PDI value of 0.21, suggesting a uniform size distribution of the RuNPs. Overall, the TEM image provides visual evidence of the effectiveness of the protective shell in maintaining the dispersion of ruthenium nanoparticles, offering promising opportunities for their utilization in various scientific and technological domains.

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Fig. 3

TEM images of RuNPs-based nanocomposites: (A) RNBC1 and (B) RNBC2. PSD histograms of: RuNPs (C) RNBC1 and (D) RNBC2.

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3.4 Cytotoxicity studies

The cytotoxic activity of RNBCs was tested in vitro using the MTT assay on two different medically relevant cancer cell lines (HepG2 and CaCo-2). One of the most powerful anticancer drugs, staurosporine (STP), was utilized as a standard in this investigation (Chae et al., 2000). The CaCo-2 and HepG2 cell proliferation as a function of successive concentrations of these nanocomposites is shown in Fig. 4A,B. The results demonstrated a statistically significant (P < 0.05) reduction in the viability of CaCo-2 and HepG2 cells following treatment with RNBCs. Additionally, the RNBC dose and cell line type affected anticancer effectiveness differently. Meanwhile, HepG2 cells respond to treatments substantially better than CaCo-2 cells do. Furthermore, the RNBC2 nanocomposite outperformed the RNBC1. Overall, the higher cytotoxicity of RNBCs could be attributed to their ultra-small size. The findings of this study align with previous research indicating that the size of nanoparticles (NPs) plays a crucial role in determining their cytotoxicity. The smaller the NPs, the more significant the negative impact on the viability of cancer cells (Lewinski et al., 2008). The small size and special hydrophilic coating of RuNPs allow them to penetrate the cell membrane more efficiently than larger ones, which can result in more significant damage to the cellular structure. Futhermore, the higher surface area to volume ratio of smaller RuNPs, which may increase the amount of reactive oxygen species (ROS) produced upon contact with cells. ROS are known to cause cellular damage and lead to oxidative stress, which can ultimately result in cell death (Lewinski et al., 2008).

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Fig. 4

Comparison of the cytotoxic effects of a clinical anticancer drug (STP) and two new Ru-nanocomposites against (A) HepG2 and (B) CaCo-2 cells as a function of dose.

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According to the data presented in Table 1, the IC₅₀ values for RNBC2 were determined to be 2.16 ± 0.17 μg/ml against HepG2 cells and 4.59 ± 0.25 μg/ml against CaCo-2 cells. These findings indicate that RNBC2 exhibits the highest efficacy as an anticancer medication against both cancer cells. Comparatively, the IC₅₀ values for RNBC1 were 9.12 ± 0.71 μg/ml against HepG2 and 11.05 ± 0.48 μg/ml against CaCo-2 cells, indicating that it was less cytotoxic toward these cancer cell lines. Most crucially, when tested for cytotoxicity against normal cells, the new RNBC2 had a high IC₅₀ value of 25.81 ± 1.45 μg/ml, indicating that they had little impact on healthy cells (THLE2). Thus, compared to normal THLE2 cells, cancer cells were more susceptible to RNBC2′s toxicity, as measured by a greater selectivity index (SI) values (SI_{HepG2/ CaCo-2} = 11.94/ 5.61. Furthermore, the RNBC2 could be a more secure and effective anticancer candidate than the commercial anticancer compound (STS) (SI_{HepG2/ CaCo-2} = 1.65/ 3.95). Therefore, Further studies on the effects of the ruthenium nanocomposite on the expression levels of enzymes P53, Bax, and Bcl2, as well as topoisomerase II activity, are crucial for gaining a comprehensive understanding of the mechanism underlying its anticancer effect. By investigating these specific aspects, we can unravel the intricate interactions between the nanocomposite and key cellular pathways involved in cancer development and progression. Such investigations will not only enhance our knowledge of the therapeutic potential of this nanocomposite but also pave the way for the development of more targeted and effective treatments for cancer.

Interestingly, the anticancer activity of the new Ru-nanobiocomposite (RNBC2) against HepG2 and CaCo-2 is significantly higher than the previously reported RuNPs and ruthenium-complexes (see Table S3, ESM†). This could be ascribed to the RuNPs possess inherent anticancer properties coupled with the ALMC/IIL vehicles provide a stable and targeted delivery system. This combination allows for the efficient uptake of RNBC2 by cancer cells, leading to enhanced therapeutic effects. The results demonstrated a remarkable improvement in anticancer activity compared to previously studied RuNPs and ruthenium-complexes. This advancement holds great promise for the development of more effective and targeted cancer therapies, potentially offering new treatment options for patients. Further research and clinical trials are needed to fully explore the potential of RNBC2 and its implications in cancer treatment.

3.5 Topoisomerase II inhibitory activity

Topoisomerase is an enzyme that helps to regulate DNA topology in cells, and its activity can affect gene expression, cellular metabolism and other important cellular functions. Therefore, it is crucial to research how topoisomerase activity is affected by RNBC2, how this biochemical reaction occurs, and how this effect may be applied for potential medicinal and therapeutic purposes. To that objective, the inhibition of topoisomerase II (Topo II) by RNBC2 was studied. When compared with control HepG2 (Topo II = 489.2 ± 23.8 ng/mL), Topo II activity is significantly decreased (p < 0.01) when HepG2 cells treated with RNBC2, with a resulting value of 181.4 ± 6.11 ng/mL (see Fig. 5A). Interestingly, RNBC2 was a more potent inhibitor of the Topo II enzyme than the chemotherapeutic inhibitor Staurosporine (STS) which can lower the enzyme to 208.9 ± 4.39 ng/mL (57.2 % reduction compared to control HepG2), while RNBC2 was able to decrease the Topo II enzyme to 181.4 ± 6.11 ng/mL, a reduction of 62.9 % compared to HepG2 control (see Fig. 5A). This great inhibitory effect was thought to be due to the direct interaction between the RuNPs and the topoisomerase enzyme, inhibiting cellular activities related to DNA replication and transcription. Furthermore, RuNPs have also been found to induce oxidative stress in cells, leading to damage to proteins, lipids, and DNA (Cai et al., 2017). These results demonstrate that RNBC2 have a significant impact on topoisomerase activity, suggesting that RNBC2 may be used as a potential chemotherapeutic tool to target topoisomerase activity. In addition, RNBC2 could potentially be used in future treatments to modulate gene expression and other cellular functions.

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Fig. 5

Topo II activity and changes in p53, Bax, and Bcl-2 gene expression, as measured by polymerase chain reaction (PCR), following 24 h of treatment with RNBC2 (IC₅₀ dosage).

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3.6 Effect of the RNBC2 on P53, Bax and Bcl2 expressions in HepG2 cells

The regulation of apoptosis is a complex process intimately linked to the balance between pro- and anti-apoptotic signaling pathways. P53, Bax, and Bcl2 are major players in the regulation of this process. P53 is a tumor suppressor gene that can induce and initiate apoptosis by upregulating Bax and downregulating Bcl2. Bax is a proapoptotic protein that can induce cytochrome c release from the mitochondria and activate the caspase cascade. Bcl2 is an anti-apoptotic protein that can inhibit cytochrome c release and prevent activation of the caspase cascade. Together, P53, Bax, and Bcl2 are integral components of the apoptotic cascade and are essential for the proper regulation of cell death and survival (Brambilla et al., 1996, Basu, 2022). Therefore, investigations have been done into how RNBC2 affects the expressions of P53, Bax, and Bcl2 enzymes (See Fig. 5). After HepG2 cells were exposed to the IC₅₀ dosage of RNBC2 for 24 h, the levels of P53, Bax, and Bcl2 were assessed by ELISA. Fig. 5A,B demonstrates that, in comparison to untreated HepG2 cells, Bax and P53 levels were substantially increased in RNBC2-treated HepG2 cells (five- and six-times, respectively). Contrary, the decrease in Bcl2 (fourfold) was much smaller in RNBC2-treated HepG2 cells compared to uninfected cells. As can be seen from these data, apoptosis in HepG2 cells is induced by RNBC2 in a p53-dependent fashion. Upon being exposed to RNBC2, HepG2 cells trigger the tumor-suppressor gene p53, which in turn may alter the inner mitochondrial membrane's normal function and lead to a change in membrane potential. These results showed that activation of the pro-apoptotic gene (Bax) suppresses the release of the anti-apoptotic protein (Bcl2). Thus, apoptotic pathways are activated, and cell death occurs, due to the balance between pro- and anti-apoptotic proteins. DNA damage, oxidative stress, and mutations are just some of the things that can set cells on the path to apoptosis.

Notably, the results of zeta potential and morphological properties of Ru nanocomposites play a crucial role in supporting the findings of their superior anticancer activity as well as their capacities to up-regulate P53 and Bax, while down-regulating Bcl2. A high zeta potential indicates a strong electrostatic repulsion between particles, preventing their aggregation. This is important because the nanocomposites need to maintain their stability and remain dispersed in order to effectively interact with cancer cells. Furthermore, the morphological properties of nanoparticles, such as their size, shape, and surface characteristics, also contribute to their anticancer activity. RuNPs with a smaller size generally have a higher surface area, which translates to increased interaction with cancer cells. Additionally, nanoparticles with nanosphere shapes can exhibit enhanced cellular uptake and improved targeting of cancer cells. The ability of Ru nanocomposites to up-regulate P53 and Bax, while down-regulating Bcl2, is another crucial aspect supporting their superior anticancer activity. P53 and Bax are tumor suppressor proteins that play important roles in regulating cell cycle arrest and apoptosis, while Bcl2 is an anti-apoptotic protein that inhibits cell death. The up-regulation of P53 and Bax and the down-regulation of Bcl2 can promote apoptosis in cancer cells, leading to their death. The mechanism through which these nanoparticles achieve the up-regulation of P53 and Bax and the down-regulation of Bcl2 is likely multifaceted. The cationic chitosan/imidazolium coating of the RuNPs may facilitate their internalization into cancer cells and enable the release of the encapsulated ruthenium. Once inside the cells, ruthenium can potentially interact with cellular components, such as DNA or proteins, and trigger signaling pathways that lead to the desired changes in gene expression.