Molecular dynamics study of host–guest interactions between anticancer drugs and Cucurbit[8]uril nanocontainer

Abstract

Cucurbit[n]urils (CB[n]s) are synthetic molecular containers with unique structural features, containing hydrophobic cavities and polar carbonyl portals. They have widespread applications in various scientific fields, such as drug delivery systems. Paclitaxel (PTX) and Camptothecin (CPT) are natural compounds extensively used as chemotherapy drugs due to their antitumor activity. However, their application is limited by severe side effects and poor aqueous solubility. Consequently, numerous studies have been conducted to overcome these limitations. Several studies have shown that the CB[n] family, especially CB[7], and their derivatives can form stable inclusion complexes with hydrophobic drugs, thereby increasing their solubility in aqueous environments. In this research, molecular docking procedure and molecular dynamics (MD) simulations were utilized to consider the interaction details of PTX and CPT with CB[8]. The results indicate that CB[8] is capable of forming inclusion complexes with both PTX and CPT not only in a 1:1 ratio but also, due to the appropriate cavity size, in a 1:2 stoichiometry, primarily driven by the release of high-energy water molecules from within the CB[8] cavity into the bulk phase. Furthermore, the results suggest the possibility of π−π interactions between the two trapped drugs within the CB[8] cavity during the MD simulation.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-27424-9.

Introduction

Despite considerable progress in medicine and treatment, cancer is one of the main cause of death globally^1–4. Depending on the type and stage of cancer malignancy, there are various treatment options, such as surgery, radiation, chemotherapy, photodynamic, stem cell therapy, and immunotherapy^1,5–8. Due to the simplicity and ease of use, chemotherapy has been one of the most common treatment methods for both primary and metastatic cancers. However, nonspecific distribution, systemic toxicity, nonselective targeting capability, evolution of multidrug resistance (MDR), and low water solubility of most anticancer drugs present significant challenges associated with conventional therapeutic agents, substantially limiting the therapeutic benefits of chemotherapy^9–11. For instance, paclitaxel (PTX) and camptothecin (CPT) are chemotherapeutic agents used to treat various cancers, but their clinical applications are significantly constrained by serious challenges^12–17. Therefore, one of the main concerns of researchers in recent decades has been finding ways to increase the effectiveness of treatments. Nanotechnology-based approaches hold potential to overcome the limitations associated with conventional chemotherapies^18–20. In this regard, various nanocarriers such as solid lipid nanoparticles (SLNs), liposomes, micelles, dendrimers, and polymer nanoparticles (PNPs) have been extensively examined for cancer-therapy drug delivery systems^21–23. In preclinical studies, nanomedicine formulations have advantages such as good biocompatibility, reducing side effects, better accumulation in tumor tissue due to the enhanced permeability and retention (EPR) effect^24–26. Accordingly, several nanopharmaceuticals have received FDA approval and are available for clinical use in cancer therapy, among which Genexol-PM (PTX encapsulated polymeric micelles), Doxil (liposomal doxorubicin), and Abraxane (albumin-bound PTX) can be mentioned^27–29. However, due to the complex nature of cancer, even nanotechnology-based treatments are not highly effective in clinical settings; therefore, efforts to discover new therapeutic approaches continue^11,30,31. Host-guest chemistry based on macrocyclic compounds like cyclodextrins, calixarenes, cucurbiturils, and pillararenes has attracted increasing attention in pharmacology and biomedicine^32,33. Since these compounds can encapsulate guest molecules within their cavity, protecting them from degradation and/or deactivation^34–36. Cucurbit[n]urils (CB[n]s), n = 5–8, 10, 13–15, are a family of synthetic pumpkin-shaped macrocycles comprising n glycoluril units connected by pairs of methylene bridges. Figure 1 shows a schematic representation of CB[8].

Fig. 1.

Schematic representation of CB[8]. The 3D molecular structure was generated using UCSF Chimera version 1.15 (https://www.cgl.ucsf.edu).

Their characteristic, including the rigid structure, hydrophilic cavity, polar carbonyl portals, and high binding affinities towards guests, make CB[n]s superior candidate among macrocyclic cavitands for a variety of applications, such as biomolecular recognition, drug delivery, sensing ensembles, and catalysis^37–41. CB[7] is the most commonly used homologue of the CB[n] family for use in drug delivery vehicles due to its high water solubility, low in vitro and in vivo toxicity profiles, and suitable cavity size^41,42. Therefore, they have been extensively considered (both experimentally and computationally) to evaluate the enhancement of the chemical and physical stability of drugs, improvement of their solubility in aqueous environments, and control of drug release^40,41,43,44. Today, computational methods are considered powerful approaches since they have great potential to investigate the host–guest interaction mechanism^43–46. In our previous works, the binding modes of PTX and CPT anticancer drugs with CB[7] and its acyclic CB[4] derivatives (aCB[4]s) were explored via computational approaches. The results revealed that aCB[4]s are capable of forming stronger inclusion complexes with PTX and CPT, particularly using hydrophobic interactions. Due to their flexible nature and aromatic sidewalls, aCB[4]s have also demonstrated the ability to bind to CPT via π−π interactions^43,44. Compared to CB[7], which has a smaller cavity and high water solubility, CB[8] exhibits a distinct advantage in forming 1:2 host–guest complexes with strong binding affinities due to its larger cavity. Despite the broader experimental application of CB[7], CB[8] has attracted significant attention from researcher, providing a unique platform for cooperative binding and dual-drug delivery systems^47–49. For example, Dang et al. demonstrated that CB[8] can induce caspase dimerization and thereby control its enzymatic activity⁵⁰. Ding et al. developed a CB[8]-based hydrogel for oral colon-targeted delivery of berberine, showing high stability in the stomach and enhanced treatment of colitis in mice⁵¹. In this study, molecular docking combined with MD simulations were employed to evaluate the binding mechanisms of PTX and CPT to CB[8] at two distinct stoichiometries: 1:1 (PTX@CB[8] and CPT@CB[8]) and 1:2 (2PTX@CB[8] and 2CPT@CB[8]). At the molecular level, we revealed that CB[8] compared to CB[7], due to the larger cavity size, not only enables the simultaneous encapsulation of the aromatic rings of two guest molecules, but also facilitates π–π interactions between the two molecules within its hydrophobic cavity.

Computational details

Molecular docking calculations

All of the docking calculations of corresponding drugs to CB[8] nanocontainer were carried out using AutoDock Vina (http://vina.scripps.edu/). The initial structure of CB[8] were determined by X-ray crystallography data⁵² The initial coordinates of CPT was obtained from PubChem database (https://pubchem.ncbi.nlm.nih.gov), PubChem CID: 2538. The chemical structure of PTX was provided using RCSB Protein Data Bank, PDB ID TA1. Figure 2 represents the initial structures of CB[8], PTX, and CPT, which were obtained from X-ray crystallography data. All molecular visualizations were generated using UCSF Chimera version 1.15.

Fig. 2.

X-ray structure of (a) CB[8] (top and side view), (b) PTX, and (c) CPT.

AutoDockTools 1.5.7 graphic user interface was utilized to produce the PDBQT format files for all of the drugs and the CB[8] nanocontainer⁵³. For a 1:1 stoichiometry between drugs and CB[8], a three-dimensional grid box of 3 × 3 × 3 nm³ xyz points, centered at the center of mass of CB[8], was set to cover the entire molecule. In the 1:2 stoichiometry of CB[8] with PTX or CPT, an appropriate frame from the simulation trajectory of their 1:1 complex was first extracted and used as the receptor for docking calculations in the next step. Then, PTX and CPT were docked to their respective 1:1 complex frames to obtain 1:2 stoichiometric complexes of CB[8] with two PTX and two CPT molecules. Here, it should be noted that the grid box size for each receptor was determined based on its structure, including the drug (PTX or CPT) and CB[8], while ensuring that there was enough space for the second drug to explore within the cavity. The number of binding modes and exhaustiveness was 20 and 8, respectively, for all docking calculations. The grid spacing of 0.375 Å was also applied.

Molecular dynamics simulations

GROMACS software (version 2023.3)⁵⁴ was used to simulate all the complex models and each molecule individually. CHARMM36 all-atom force field⁵⁵ was selected to use for each system. The required topology parameters for all of the corresponding drugs and CB[8] nanocontainer were created with the SwissParam webserver⁵⁶ (http://www.swissparam.ch). All the complex models obtained from docking calculations were evaluated to select the best-scoring model from each complex for MD simulation. For each simulation system, a cubic box was defined in which the corresponding systems were placed at the center. All the boxes were hydrated using the TIP3P three-point water model⁵⁷. According to the size of each box, Na⁺ and Cl⁻ ions were added to perform simulations under physiological conditions. The steepest descent optimization method was employed to minimize the energy of all systems, with a maximum force of 100 kJ/mol/nm. Afterwards, 200-ps NVT and 500-ps NPT equilibrations were done to keep temperature at 300 K and pressure at 1 bar, respectively. The velocity-rescale thermostate⁵⁸ and the Parrinello–Rahman barostat^59,60 were used for NVT and NPT ensembles, respectively. The cutoff value for the short-range van der Waals interactions and coulombic terms was determined to be 1.2 nm. The particle mesh Ewald (PME) algorithm⁶¹ was also applied to evaluate the long-range electrostatic interactions. All bonds except the hydrogen bond were constrained using the linear constraint solver (LINCS) algorithm⁶². MD simulation were carried out for all equilibrated systems using the leap-frog algorithm⁶³ with a time step of 2 fs under the periodic boundary conditions and NPT ensemble.

Results and discussion

MD simulation of the CB[8] nanocontainer

To evaluate the structural characteristics and dynamic behavior of CB[8], its free form was first simulated for 200 ns. All analyses were done on the last 180 ns of the simulation trajectories. The root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values were examined to evaluate the structural stability and flexibility of the CB[8] molecule, respectively. Both RMSD and RMSF values were determined to be approximately 0.05 ± 0.01 nm, supporting the high stability of the structure and the rigid nature of these macrocyclic compounds^52,64,65. The radius of gyration (Rg), signifying a measure of compactness, was calculated to be 0.60 ± 0.00 nm. The standard deviation (SD) of 0.0 nm indicates low fluctuations in the size and shape of free CB[8] during the MD simulation. The RMSF and Rg values for CB[8] are slightly higher than those obtained for the CB[7] nanocontainer studied in the previous research^43,44. This difference is expected, considering the larger size of CB[8].

Docking of PTX and CPT to CB[8]

The results of our molecular docking calculations reveal that in all twenty scoring models, PTX is positioned within the hydrophobic cavity of CB[8] using its exterior aromatic rings (named as A, B, and C), with binding energies found to range from − 18.66 to − 22.81 kJ mol⁻¹ (Fig. 3a–c). CPT in all twenty best docking models was found to form inclusion complexes with CB[8], with binding energies ranging from − 25.97 to − 32.98 kJ mol^− 1 (Fig. 3d, e).

Fig. 3.

Best-scoring docking models of (a–c) PTX and (d, e) CPT into the CB[8] nanocontainer.

However, the best-scoring models of both drugs docked to CB[8] were selected as the initial complex models for MD simulations. From now on, the complexes in which PTX enters the CB[8] cavity through its ring A, ring B, and ring C will be referred to as complex models A, A′, and A″, respectively. The two complexes in which CPT is positioned inside the cavity (Fig. 3d, e) will be briefly referred to as complex models B and B′, respectively.

MD simulation of complex models A, A′, A″, B, and B′ in water

First, the distance between the center of mass of CB[8] and that of the encapsulated ring of each drug in the complex models was measured. As shown in Fig. 4, PTX, and CPT remain inside the cavity throughout the 200 ns MD simulations.

Fig. 4.

Distance between the center of mass of CB[8] and that of the encapsulated ring of PTX and CPT in the complex models over the last 180 ns MD simulation.

Monitoring the snapshots along the trajectories of the complexes also reveals that CB[8] successfully retains PTX and CPT within its cavity during the simulations. Figure 5 displays snapshots taken every 50 ns from the MD simulation trajectories.

Fig. 5.

Side view of snapshots taken every 50 ns for the complex models (a) A, (b) A′, (c) A″, (d) B and (e) B′ in water during 200 ns MD simulation.

Considering that greater coverage of the hydrophobic regions of a drug by a carrier can increase its solubility in an aqueous environment, the solvent-accessible surface area (SASA) was determined to evaluate the CB[8] potential in improving the solubility of PTX and CPT. Table 1 shows the total SASA values of the corresponding drugs in their free and complex forms.

Table 1.

Total SASA values of the drugs, the number of water-drug contacts, and the free energy of solvation of the drugs in their free and complex forms at the 1:1 stoichiometry of CB[8] with PTX or CPT.

	Total SASA (nm2)	ΔGsolv (kJ.mol− 1)	Num. of contacts
PTX free	10.43 ± 0.41	10.26 ± 1.57	5832 ± 235
Model A	7.63 ± 0.35	5.83 ± 1.33	4959 ± 213
Model A’	6.39 ± 0.27	4.88 ± 1.07	4385 ± 213
Model A”	7.78 ± 0.32	7.50 ± 1.34	4952 ± 174
CPTfree	5.61 ± 0.22	5.27 ± 1.21	2723 ± 107
Model B	2.57 ± 0.19	4.61 ± 1.04	1745 ± 105
Model B’	2.70 ± 0.16	− 0.92 ± 1.25	1879 ± 103

The total SASA values of free PTX and CPT in water were found to be 10.43 ± 0.23 and 5.61 ± 0.22 nm², respectively. It is clear that the complex formation with CB[8] in all the complex models under study leads to a significant reduction in the total SASA values of PTX and CPT. To thermodynamically assess solvation, the free energy of solvation (ΔGsolv) for both the free and complexed forms of the drugs was calculated based on the SASA prediction. The ΔGsolv values of PTX and CPT in their free forms were positive, with average values of 10.26 ± 1.57 and 5.27 ± 1.21 kJ/mol, respectively. Their incorporation into the hydrophobic cavity of CB[8] results in a significant reduction in ΔGsolv values, as the part interacting with the hydrophobic cavity predominantly exhibits hydrophobic characteristics. Moreover, the number of water-PTX, and water-CPT contacts in both free and complex forms were calculated. The results indicate that the contacts between water molecules and the drugs decrease significantly upon complex formation (Table 1). Table S1 shows the number of CB[8]-PTX and CB[8]-CPT contacts in the related complex models. The results clearly show that the accessibility of water molecules to PTX and CPT decreases due to the formation of inclusion complexes with CB[8], increasing their solubility in aqueous environments. In the next step, the average contribution of CB[8] and the corresponding drugs in their free and complex form to the formation of hydrogen bonds was calculated. As shown in Table 2, the average number of hydrogen bonds for the free form of the CB[8] nanocontainer, PTX, and CPT is 26.22, 13.4, and 6.18, respectively.

Table 2.

The average contributions of CB[8], PTX, and CPT to the total hydrogen bonds in each of the free and 1:1 complex systems of CB[8] with PTX and CPT.

Hydrogen bonds	Drug-water (A)	CB[8]-Water (B)	Drug-CB[8] (C)	Complex form - Free form (A + B + C) - (A + B)
(CB[8])Free	–	26.22	–	–
(PTX)Free	13.40	–	–	–
Model A	11.57	21.31	1.48	− 5.26
Model A’	10.75	20.69	1.18	− 6.74
Model A”	11.00	21.20	0.94	− 6.48
(CPT)Free	6.18	–	–	–
Model B	2.87	21.21	0.16	− 8.16
Model B’	7.13	20.29	0.02	− 4.96

Complex formation between CB[8] and the drugs leads to a reduction in the number of their hydrogen bonds with water. The total number of hydrogen bonds in all the complex models also decreases compared to those in the free forms, and this reduction is not compensated by hydrogen bond formation between the drugs and CB[8]. This confirms that the release of entrapped water from the CB[8] cavity has a crucial role for their complex formation in an aqueous environment, which support pervious experimental and computational studies^43,66,67. Radial distribution function (RDF), g(r), was employed to evaluate the hydration behavior around the relevant molecules. To achieve this, RDF analyses were estimated between the hydrogen atoms of water molecules and the oxygen atoms of CB[8], PTX, and CPT in all simulated systems (Fig. 6).

Fig. 6.

RDF curves between the hydrogen atoms of water molecules and the oxygen atoms of (a) CB[8], (b) PTX, and (c) CPT in both free and 1:1 inclusion complex models.

The RDF profile in the free CB[8] system shows a sharp and intense peak around 0.18–0.25 nm, which remains almost largely upon encapsulation of either PTX or CPT. This indicates the presence of strong and stable hydrogen bonding interactions between CB[8] and nearby water molecules. The second and following shoulder-like third peaks at longer distances exhibited a remarkable decrease in intensity in PTX@CB[8] and CPT@CB[8] complexes, compared to the free CB[8]. The dominant factor in this reduction is most likely attributed to steric hindrance caused by drug binding, followed by the release of internal water molecules from the CB[8] cavity (Fig. 6a). The RDF profile of PTX in both free and complex models A, A′, and A″ displays a distinct first peak with the relatively lower intensity around 0.18–0.25 nm, originating from the limited number of hydrogen bonding between the accessible oxygen atoms of PTX and surrounding water molecules. Since in all three complexes, PTX is retained inside the cavity via its one of aromatic rings, the majority of the molecule remains exposed to bulk water. Therefore, no significant reduction is observed in the first RDF peak upon complexation with CB[8] (Fig. 6b). As shown in Fig. 6C, the first RDF peak of free CPT is also sharp and relatively intense, reflecting the formation of hydrogen bonds between the oxygen atoms of CPT and surrounding water molecules. In the first complex (red curve), CPT enters the CB[8] cavity through its hydrophilic side, containing several oxygen atoms. This binding mode results in a moderate reduction in the first peak intensity, attributed to the decreased accessibility of these oxygen atoms as well as the partial displacement of water molecules from the cavity. Moreover, the RDF profile of the longer-range hydration shells shows a notable reduction upon complexation, likely due to steric shielding imposed by CB[8], which disrupts the spatial arrangement of surrounding water molecules. In contrast, in complex model B′, the hydrophilic side of the drug inserts into the CB[8] cavity. This localization exposes the oxygen atoms of the hydrophilic side of CPT, thereby enhancing their hydration and increasing the number of hydrogen bonds (Fig. 6c and Table 2). The RMSD, Rg, and RMSF values of CB[8], PTX, and CPT of in both free and complex forms were determined to assess their structural characteristics. As shown in Table S2, the average Rg values of CB[8] and CPT in their corresponding complexes are similar to those observed in their free states. The negligible changes in the average Rg values reflect that CB[8] and CPT maintained their overall size and compactness upon complex formation. It should be noted that monitoring the simulation frames reveals that the changes in the size and shape of CB[8] are slightly greater than those of CB[7] in our previous studies^43,44, which is expected due to its larger size. In contrast to complex models A and A″, the average Rg value of PTX showed a notable decrease in complex model A′ (from 0.53 ± 0.02 to 0.48 ± 0.01 nm). This suggests that PTX adopted a more compact conformation in this system through the folding of its flexible parts, likely due to interactions with the CB[8] cavity. As depicted in Fig. 7, the RMSD values of CB[8] were very low (about 0.05 ± 0.01 nm) in all models except model B′, indicating its rigid structure.

Fig. 7.

RMSD values of (a) CB[8], (b) PTX, and (c) CPT in both free and inclusion 1:1 complex models during the last 180 ns MD simulation.

However, in complex model B′, an increase in the RMSD value of CB[8] was observed (approximately 0.12 ± 0.02 nm), suggesting minor conformational changes during the simulation, as confirmed by visual inspection of the trajectory. The average RMSD value of free PTX was found to be 0.21 ± 0.07 nm (black graph in Fig. 7b), which is expected due to its structural flexibility. It is obvious that the RMSD values of PTX in complex models A, A′, and A″ do not follow a consistent increasing or decreasing trend. This suggests that the dynamic behavior—and even the conformational state—of PTX may be significantly influenced depending on its binding modes within the CB[8] cavity. The low RMSD values of CPT in complex models B and B′, compared to that of free CPT, also highlight that no significant conformational change occurred, which is expected given its structural rigidity. To evaluate the flexibility of CB[8], PTX, and CPT in both free and complex forms, RMSF analysis was performed. As shown in Table S3, the average RMSF values for CB[8] remained nearly unchanged across its free and complex states. However, considering RMSF values and comparison with those of CB[7] revealed that the range of atomic fluctuations in CB[8] was greater, which is not surprising as it has larger size. In the case of PTX, the average RMSF increased from 0.16 ± 0.11 nm in the free form to 0.63 ± 0.28, 0.26 ± 0.11, and 0.35 ± 0.21 nm in complexes A, A′, and A″, respectively. These results imply that PTX exhibits greater fluctuations upon complexation with CB[8], particularly in models A and A″. Such an increase in RMSF values was also observed for CPT, from 0.05 ± 0.04 nm in the free form to 0.23 ± 0.14 and 0.19 ± 0.07 nm in B and B′ forms, respectively, although the RMSD remained low in both complex systems. It seems that complex formation with CB[8] leads to increased flexibility in some regions of CPT, while its overall shape is retained. Additionally, the binding free energies between CB[8] and both PTX and CPT were obtained using the Poisson–Boltzmann surface area (MM-PBSA) method^68,69. Table 3 shows the binding free energy and its contributing components, including van der Waals, electrostatic, polar solvation, and nonpolar solvation energies for complex models A, A′, A″, B, and B′ using the MM-PBSA approach.

Table 3.

Binding free energy and its contributing components, including Van der Waals, electrostatic, Polar solvation, and nonpolar solvation energies for complex models A, A′, A″, B, and B′ using the MM-PBSA approach.

Energy unit: (kJ mol− 1)	Complex A	Complex A′	Complex A″	Complex B	Complex B′
Van der Waals interactions	− 118.23 ± 9.62	− 145.31 ± 17.53	− 153.26 ± 14.02	− 152.88 ± 12.97	− 142.67 ± 9.50
Electrostatic interactions	− 60.54 ± 15.48	− 73.09 ± 22.18	− 71.71 ± 22.47	55.86 ± 16.61	− 79.62 ± 19.46
Polar solvation energy	85.60 ± 11.71	118.03 ± 15.36	81.13 ± 22.51	− 3.97 ± 12.68	125.60 ± 14.69
Nonpolar solvation energy	− 9.87 ± 0.92	− 12.38 ± 0.75	− 11.25 ± 1.21	− 9.25 ± 0.63	− 7.49 ± 0.75
Total binding free energy	− 103.09 ± 11.76	− 112.76 ± 27.70	− 155.14 ± 16.86	− 110.25 ± 11.80	− 104.14 ± 10.17

Negative binding free energies, ranging from − 103.09 to − 155.14 kJ/mol, were observed for all computed 1:1 inclusion complex models. These findings indicate thermodynamically favorable inclusion complex formation between the molecules involved, even in the case of complex model B, in which CPT was inserted into the CB[8] cavity through its polar head. These results highlight the notable encapsulation ability of the CB[8] nanocontainer in an aqueous environment, despite its low solubility, and confirming again the great tendency of trapped water molecules to be released into the bulk.

Docking of PTX to PTX@CB[8] and CPT to CPT@CB[8]

To evaluate the ability of simultaneous binding of two PTX and two CPT molecules to the CB[8] nanocontainer, an appropriate frame was first selected from the simulation trajectories of each 1:1 drug-CB[8] complex. Subsequently, each drug was docked into its corresponding complex. In the case of PTX docking, to obtain all possible models for the 1:2 stoichiometry, PTX was docked into the selected frames from each of the three PTX@CB[8] complexes. The binding energy range of all the complex models obtained from docking calculations was found to be approximately from − 17.62 to -23.41 kJ mol⁻¹. The energetically best-scoring model of each complex was selected for a 200 ns MD simulation (Fig. 8).

Fig. 8.

Best-scoring model of PTX docked into three complex models of PTX@CB[8].

Similar to PTX, docking of CPT to CPT@CB[8] complexes was also calculated. All twenty best-scoring models of docking calculations mainly show two docking poses with binding energies from − 19.89 to -31.96 kJ mol⁻¹ (Fig. 9a, b). Furthermore, to investigate all possible binding modes, two CPT were first docked to each other. Then, the appropriate binding models were selected, and in the next step, CB[8] was docked to these models with binding energies from − 14.63 to -28.48 kJ mol⁻¹. Figure 9c shows the only acceptable binding mode obtained using this method, which could be further analyzed through molecular dynamics simulation.

Fig. 9.

Best-scoring models of (a, b) CPT docked into CPT@CB[8] complexes, (c) CB[8] docked into CPT-CPT complexes.

Considering that complexes A, A′, and A″ correspond to the incorporation of rings A, B, and C of PTX into the cavity, respectively, the six 2PTX@CB[8] complex models obtained from molecular docking calculations were sequentially named as complex models AA, AA′, AA″, A′A′, A′A″, and A″A″. Similarly, following the naming of CPT@CB[8] complexes, 2CPT@CB[8] complexes were named (B′B′)₁, BB′, and (B′B′)₂.

MD simulation of complex models AA, AA′, AA″, A′A′, A′A″, A″A″, (B′B′)₁, BB′, and (B′B′)₂ in water

Monitoring the trajectories of all 2PTX@CB[8] simulations reveals that in complex models AA, AA′, AA″, and A″A″, CB[8] retains both PTX molecules within its hydrophobic cavity during the 200 ns MD simulations. However, in complex models A′A′ and A′A″, one PTX molecule leaves the cavity and separates from CB[8] within the first few seconds of the simulation. The observation of the simulation trajectories for 2CPT@CB[8] complexes reveals that in the complex model BB′, one CPT molecule separates from the cavity. In contrast, in the complex models (B′B′)₁ and (B′B′)₂, both CPT remain within the cavity during the simulation.

Hereafter, further analyses will be conducted on the complex models AA, AA′, AA″, A″A″, (B′B′)₁, and (B′B′)₂. Representative snapshots of the corresponding complex models were extracted at 50 ns time intervals (Fig. 10).

Fig. 10.

Side view of snapshots taken every 50 ns for the complex models (a) AA, (b) AA′, (c) AA″, (d) A″A″, (e) (B′B′)₁, and (f) (B′B′)₂ in water during 200 ns MD simulation.

The distance between the center of mass of CB[8] and the center of the incorporated ring of two drugs was measured over the final 180 ns of the simulation to gain more molecular insights. Considering the distance between two drugs and CB[8] in each complex model, it can be concluded that C8[8] is capable of simultaneously localizing two PTX or two CPT molecules in its hydrophobic cavity throughout the MD simulation (Fig. 11).

Fig. 11.

Distance between the center of mass of CB[8] and that of the encapsulated rings of two PTX and two CPT in the corresponding complex models over the last 180 ns MD simulation.

Compared to 2CPT@CB[8] complexes, the distance between CB[8] and the two PTX molecules exhibits greater fluctuations during the simulation. The larger size of PTX appears to be effective in this behavior. CB[8]’s ability to retain both PTX molecules under such conditions emphasizes its unique and exceptional capability. In the following, the average SASA and ΔGsolv values were evaluated for both PTX and CPT molecules within the CB[8] nanocontainer (Table 4). Our results clearly demonstrate a significant reduction in water accessibility and an increase in the solubility of the corresponding drugs, consistent with the 1:1 stoichiometry of PTX and CPT with CB[8]. As shown in Table 4, the number of contacts between the drugs and water molecules significantly decreases in the complex models compared to the free drugs. Taken together, these findings align with experimental studies on the ability of CB[8] to form inclusion complexes with two molecules simultaneously, particularly through interactions involving their hydrophobic regions.

Table 4.

Total SASA values of the drugs, the number of water-drug contacts, and the free energy of solvation for the drugs in their free and complex forms at the 1:2 stoichiometry of CB[8] with PTX or CPT.

	Total SASA (nm2)	ΔGsolv (kJ.mol− 1)	Num. of Contacts
Model AA	7.67 ± 0.33 7.61 ± 0.39	5.02 ± 1.41 5.05 ± 1.53	4859 ± 217 4788 ± 195
Model AA′	7.70 ± 0.33 7.34 ± 0.39	5.38 ± 1.40 5.54 ± 1.23	4824 ± 192 4798 ± 245
Model AA″	7.33 ± 0.40 7.59 ± 0.31	4.14 ± 1.18 6.18 ± 1.28	4835 ± 228 4808 ± 190
Model A″A″	7.60 ± 0.30 7.62 ± 0.31	6.21 ± 1.27 6.16 ± 1.28	4770 ± 190 4779 ± 180
Model (B′B′)1	2.69 ± 0.16 2.69 ± 0.16	− 1.55 ± 0.62 − 1.52 ± 0.63	1784 ± 97 1780 ± 98
Model (B′B′)2	1.98 ± 0.13 1.97 ± 0.12	− 1.48 ± 0.49 − 1.46 ± 0.49	1477 ± 77 1477 ± 73

As shown in Table 5, the number of hydrogen bonds formed between CB[8] and the drugs in the 2PTX@CB[8] and 2CPT@CB[8] complexes is relatively low and does not compensate for the reduction in hydrogen bonding between CB[8] and the drugs with water compared to their free forms.

Table 5.

The average contributions of CB[8], PTX, and CPT to the total hydrogen bonds in the 1:2 complex systems of CB[8] with PTX or CPT.

Hydrogen Bonds	Drug-Water (A)	CB[8]-Water (B)	Drug-CB[8] (C)	Drug-Drug (D)	Complex form (2 A + B + 2 C + D) −  Free form (2 A + B)
Model AA	11.01 11.09	14.29	1.30 2.03	0.00	− 13.3
Model AA’	10.73 11.88	14.76	1.50 1.26	0.00	− 12.89
Model AA”	11.38 12.24	15.67	1.16 0.92	0.00	− 11.65
Model A"A”	12.13 12.18	13.53	0.96 0.95	0.00	− 13.27
Model (B′B′)1	6.75 6.62	18.72	0.64 0.67	0.00	− 5.18
Model (B′B′)2	4.72 4.67	19.93	0.00 0.00	1.84	− 7.42

Therefore, similar to the PTX@CB[8] and CPT@CB[8] complexes, a significant decrease in the total number of hydrogen bonds is observed. This finding further emphasizes the role of water molecules trapped within the hydrophobic cavity of CB[8] in the inclusion complex formation. Figure 12 demonstrates the RDF analysis between oxygen atoms of CB[8], PTX, and CPT in their respective 1:2 complexes with hydrogen atoms of water molecules, compared to their free states.

Fig. 12.

RDF curves between the hydrogen atoms of water molecules and the oxygen atoms of (a) CB[8], (b) PTX, and (c) CPT in 1:2 inclusion complex models.

The RDF profiles between the oxygen atoms of CB[8] and the hydrogen atoms of water in the 2PTX@CB[8] complexes exhibit a moderate decrease in the first peak relative to free CB[8] (Fig. 12a). This trend aligns with the number of hydrogen bonds formed between CB[8] and water molecules in all 1:2 complexes (Table 5), influencing by the spatial hindrance posed by the drugs near the CB[8] portals. Compared to the 1:1 complexes, the second and third RDF peaks display a more distinct decrease in the 1:2 systems, supporting the release of a greater amount of internal water molecules due to the increased steric occupation upon simultaneous binding of two drug molecules. In 1:2 PTX complexes, partial inclusion of the drug results in oxygen atoms regions remaining exposed to water, keeping oxygen–water interactions and the first RDF peak intensity, consistent with what was observed the 1:1 PTX@CB[8] complexes (Fig. 12b). Compared to free CPT, the increase in the first RDF peak in the 2CPT@CB[8] complexes—even in the (B′B′)₂ model where both drug molecules are positioned closely at the same portal of CB[8]—clearly indicates the enhanced exposure of oxygen atoms to surrounding water molecules (Fig. 12c). However, the expected reduction in the second and third RDF peaks is more pronounced in the (B′B′)₂ model than in (B′B′)₁, since steric crowding at a single portal of CB[8] disrupts the spatial organization of water molecules at longer distances. Considering the average Rg values of CB[8] and CPT in the 1:2 complexes, no significant changes were observed compared to their free forms, which is reasonable given their size and structural features. However, due to its more flexible and extended structure, PTX shows minor changes in its average Rg values, depending on its binding modes, (Table S4). The RMSD values of the corresponding molecules in the 1:2 complexes were also calculated (Fig. 13).

Fig. 13.

RMSD values of (a) CB[8], (b, c) two PTX, and (d) two CPT in 1:2 complex models during the last 180 ns MD simulation.

The RMSD values of CB[8] in all the 1:2 stoichiometric complexes remained at a low value similar to those observed in the 1:1 systems, confirming again its rigid nature (Fig. 13a). CPT molecules across both of their 1:2 complexes also exhibited low RMSD values, as expected due to their small and rigid nature (Fig. 13d). However, PTX in its 1:2 complex models including AA, AA′, AA″, and A″A″ represented a larger range of RMSD values from 0.12 ± 0.02 to 0.36 ± 0.02 nm. Owing to its structural flexibility, PTX could undergo a variable degree of structural rearrangement (Fig. 13a, b). This feature leads to a high dependency of its behavior on different binding modes and spatial constraints within the CB[8] cavity, as observed in its 1:1 complex models. In this here, aiming to evaluate the effect of simultaneous incorporation of 2PTX and 2CPT within the CB[8] cavity on their flexibility, the RMSF values of each molecules were estimated (Table S5). Our results indicate that the CB[8] atoms in 2PTX@CB[8] complexes exhibit an RMSF pattern similar to that of free CB[8], with slightly greater or lesser fluctuation amplitude. In the case of 2CPT@CB[8] complexes, all atoms of CB[8] display lower RMSF values with similar pattern respect to the free CB[8] nanocontainer. This reflects the influence of the shape, size, and number of drugs involved in the cavity on the flexibility of the CB[8] structure. It is worth noting that we are dealing with a molecule with a rigid structural nature. However, the results reveal that the structural properties of CB[8] were not significantly affected by the simultaneous incorporation of 2PTX or 2CPT within the CB[8] cavity. The CPT molecules also demonstrated low RMSF values in both corresponding complex models (around 0.04 nm). In the case of PTX, the average values of RMSF in the 1:2 complex models exhibited a significant reduction, compared with those in the 1:1 systems, with average values ranging from 0.10 to 0.15 nm. It appears that the co-encapsulation of two PTX molecules limits their atomic fluctuations. Here, to further validate the release of water molecules upon 1:1 and 1:2 complex formation, RDF analyses were estimated between the incorporated rings of drugs with oxygen atoms of water molecules (Fig. S1) in both free and complex systems. The results observation imply that the first peak for all PTX@CB[8] and CPT@CB[8] complex models significantly decreases compared to that in free forms of drugs. Moreover, the formation of inclusion complexes with 2PTX and 2CPT lead to a more reduction of first peak, suggesting more dehydration due to simultaneous inclusion complex formation with 2PTX or 2CPT. At this stage, the possibility of π-π stacking interactions between the incorporated aromatic rings of two PTX and two CPT within the CB[8] cavity was evaluated. Considering that both drugs simultaneously remain within the CB[8] cavity during the simulation via their aromatic rings in each corresponding complex, and taking into account the distance between the attached rings of each drug and CB[8] (see Fig. 11), the possibility of π-π interactions between the rings of the two drugs in the cavity is conceivable. In this regard, the distances between the incorporated rings of the two PTX or CPT molecules in each complex, along with the angles between the normal vectors of their aromatic ring planes, were calculated. Typically, for optimal π–π interactions between two aromatic rings, the angle between the normal vectors of the ring planes should be close to 0° or 180°, and the distance is ranging from 0.35 to 0.45 nm. Figure 14 illustrates the probability distribution plots of the calculated angles and distances, as well as the 2D angle–distance density plots for all 1:2 complex models.

Fig. 14.

Probability distributions of (a) the angles between the normal vectors of the aromatic ring planes, (b) the distances between the centers of mass of the incorporated rings of the two drugs, and (c) the corresponding 2D angle–distance density plots.

The probability distribution curves show that, in most models, the highest probabilities fall within the range of 0.35–0.40 nm and 150–180° (Fig. 14 (a, b)), which are characteristic of π–π stacking interactions observed during the MD simulations. The 2D angle-distance density plots of the complex models support these findings (Fig. 14 (c)). Monitoring the plots highlights that the π − π interaction between two CPT molecules is stronger than that between two PTX molecules in the cavity. This is expected due to the small size and quasi-planar structure of CPT. Indeed, the suitable cavity size of CB[8] facilitates the alignment of the aromatic rings of the two encapsulated molecules, allowing for π − π interactions inside the cavity. The fact that the cavity size is such that it can simultaneously retain two molecules via their hydrophobic parts and establish stable π − π interactions is considered a unique advantage of CB[8], revealing its potential in the design of nanocontainer-based drug delivery systems. The binding free energies calculated using the MM/PBSA method reveal considerably negative values for the 1:2 complex models, ranging from − 193.87 to − 312.96 kJ/mol, compared to the 1:1 complex models (Table 6).

Table 6.

Binding free energy and its contributing components, including Van der Waals, electrostatic, Polar solvation, and nonpolar solvation energies for complex models AA, AA′, AA″, A″A″, (B′B′)₁, and (B′B′)₂ using the MM-PBSA approach.

Energy unit: (kJ mol− 1)	Complex AA	Complex AA′	Complex AA″	Complex A″ A″	Complex (B′B′)1	Complex (B′B′)2
Van der Waals energy	− 277.06 ± 23.68	− 257.15 ± 15.61	− 299.66 ± 19.46	− 242.04 ± 26.94	− 221.08 ± 10.88	− 235.23 ± 10.50
Electrostatic interactions	− 120.37 ± 34.43	− 134.35 ± 48.66	− 145.39 ± 31.55	− 125.10 ± 27.40	− 232.04 ± 28.28	− 182.88 ± 14.35
Polar solvation energy	173.85 ± 29.71	199.87 ± 29.46	152.21 ± 31.76	177.07 ± 21.84	272.42 ± 9.62	230.83 ± 15.44
Nonpolar solvation energy	− 19.29 ± 2.30	− 16.11 ± 1.88	− 20.17 ± 1.17	− 16.48 ± 1.72	− 13.35 ± 0.88	− 6.95 ± 0.59
Total binding free energy	− 242.92 ± 31.09	− 207.74 ± 32.76	− 312.96 ± 24.31	− 198.15 ± 35.27	− 193.87 ± 16.82	− 194.22 ± 13.43

These findings strongly suggest greater stability in the dual-guest inclusion complex formation within the CB[8] cavity. The increased binding free energy and enhanced stability of the 1:2 complexes may arise from drug–drug interactions, particularly π − π stacking, as well as the displacement of a more number of water molecules from the cavity. Overall, this study demonstrates the potential of CB[8] to simultaneously encapsulate two poorly soluble drug molecules.

Conclusion

In conclusion, the ability of the CB[8] nanocontainer to hold paclitaxel (PTX) and camptothecin (CPT), two prominent anticancer therapeutic agents, within its hydrophobic cavity was investigated via in silico approaches. Using molecular dynamics (MD) simulations, it was shown that CB[8] is capable of forming inclusion complexes with PTX and CPT at both 1:1 and 1:2 stoichiometries inside its hydrophobic cavity. The binding modes between CB[8] and the two drugs were determined using molecular docking calculations. The MD results demonstrated that the solubility of PTX and CPT in both 1:1 and 1:2 stoichiometries increases in an aqueous environment compared to the free drugs during the MD simulation. The dynamic behavior and structural characteristics of CB[8] in both free and complex states were also evaluated. The results reveal that the flexibility of the CB[8] structure is somewhat affected upon complex formation. Indeed, drugs of various sizes, shapes, and structures have different effects on the flexibility of CB[8]. Finally, the possibility of π − π bond formation between the aromatic rings of the two trapped drugs in the CB[8] cavity, positioned at an appropriate distance from each other, were considered. Overall, this study reports a molecular insight into the interaction mechanism of the CB[8] nanocontainer with PTX and CPT anticancer drugs in an aqueous environment at 1:1 and 1:2 stoichiometries using in silico approaches.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

N. A.: Data curation and formal analysis. M. A.: Investigation and data curation. F. M.: Investigation and conceptualization. M. A. E.: Project administration and supervision. All authors participated in writing, editing, and reviewing the manuscript.

Data availability

All the data generated or analyzed during this study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.