Emerging Trends in nanotechnology-based delivery approaches: Hexakis (m-PE) macrocycles as a novel therapeutics

: Hexakis (m-phenylene ethynylene) (m-PE) macrocycles, with aromatic backbones and multiple hydrogen-bonding side chains, had a very high propensity to self-assemble via H-bond and π-π stacking interactions to form nanotubular structures with defined inner pores. Such stacking of rigid macrocycles is leading to novel applications that enable the researchers to explored mass transport in the sub-nanometer scale. Herein, we performed density functional theory (DFT) calculations to examine the drug delivery performance of the hexakis dimer as a novel carrier for doxorubicin (DOX) agent in the chloroform and water solvents. Based on the DFT results, it is found that the adsorption of DOX on the carrier surface is typically physisorption with the adsorption strength values of -115.14 and -83.37 kJ/mol in outside and inside complexes, respectively, and so that the essence of the drug remains intact. The negative values of the binding energies for all complexes indicate the stability of the drug molecule inside and outside the carrier's cavities. The energy decomposition analysis (EDA) has also been performed and shown that the dispersion interaction has an essential role in stabilizing the drug-hexakis dimer complexes. To further explore the electronic properties of dox, the partial density of states (PDOS and TDOS) are calculated. The atom in molecules (AIM) and Becke surface (BS) methods are also analyzed to provide an inside view of the nature and strength of the H-bonding interactions in complexes. The obtained results indicate that in all studied complexes, H-bond formation is the driving force in the stabilization of these structures, and also chloroform solvent is more favorable than the water solution. Overall, our findings offer insightful information on the efficient utilization of hexakis dimer as drug delivery systems to deliver anti-cancer drugs.


Introduction:
Nano-carriers based delivery platforms have developed as a promising candidate for cancer treatment. However, their quality problems (i.e., the inability to tune the pore diameters and difficult production), carrier-related toxicity, and poor drug loading capacity issues have restricted their clinical utilization [1][2][3] . Nevertheless, the research interest has shifted to focus on the design and fabrication of novel drug-delivery vehicles [4][5][6][7][8][9] . Among the different ring-shaped macrocycles, those with non-deformable cavities and rigid backbones are of particular interest. Because the stacking of such macrocycles creates structures with strictly defined inner and outer diameters, along with the internal pores 10,11 . One kind of these ring-like structures is based on oligo-(phenylene ethynylene) rigid backbones that have also been widely studied 12 . These oligomers are molecular-scale (< 2 nm) porous structures, called nanopores, with fixed sizes and interesting mass-transporting properties, which are commonly known as useful biocompatible materials. Until now, several different classes of planar and rigid macrocycles have been reported that provide attractive building blocks for the formation of nanotube assemblies containing internal pores with defined diameters 13 . Despite carbon nanotubes (CNT) and inorganic porous materials, the rigid building blocks of macrocyclic are endowed with the ability to be functionalized at defined locations inside the tube 14 . One class of these structures is Hexakis (m-PE) macrocycles that are a series of rigid macromolecular structures consisting of m-phenylene-ethynylene units. The (m-PE) macrocycles exhibit completely different self-assembling behaviors that are influenced by multiple factors. For example, Moore et al. 15 revealed that the Hexakis macrocycles are able to form a self-assembly tubular structure in low-polar solvents due to π-stacking and intermolecular H-bonding interactions. As well as, the results from our previous study 16 via molecular dynamics (MD) simulations indicated that Hexakis macrocycles undergo a self-assembly process forming a nanotubular structure that can be the biocompatible potential sensor for drug delivery applications. Doxorubicin (DOX), an anthracycline anti-cancer antibiotic, is among the most potent antitumor agents used for the treatment of a variety of malignancies, including breast, prostate, brain, cervix, and lung cancers 17 . Despite extensive clinical utilization, the DOX application is severely limited because of the risk of severe cardiotoxicity 18 . Besides, this anti-cancer drug also affects other organs like the kidney, liver, and brain 19,20 . Therefore, to improve the therapeutic effect and minimize the side effects, it is necessary to develop new therapeutic strategies for the selective delivery of DOX to tumors. Many research groups have tried to design a new generation of intelligent drug delivery systems to efficiently carry doxorubicin to destroy cancer cells [21][22][23][24] . The present study aimed to provide an efficient approach for using hexaxis dimer as a novel nanocarrier for DOX anti-cancer drug loading. For this purpose, the hexakis dimer's ability for doxorubicin delivery is examined by using density functional theory (DFT) studies to determine the structural parameters, binding energy, and electronic properties of DOX/hexakis dimer complexes in chloroform and water solvents. By studying the hexakis dimer, we intend to answer the question: Do hexakis dimer, similar to tubular stacks, serve as a host molecule and form an inclusion complex with the guest drug?

Computational details
In the present work, the structures of the monomer/dimer of the hexakis and dimer/guest inclusion complexes are optimized using the DFT (M06-2X) and DFT-D3 (M06-2X-D3) functionals 25,26 by employing the 6-31G** basis set 27,28 in the chloroform and water solvents. The initial geometry of the hexakis carrier is taken from the X-ray data, reported by Zhong and et al. 13 . In our work, the initial structures of hexakis dimer are built by using Gauss View software 29 , and the size of the side chain is derived from the experimental work, which is proposed by Zhong and co-workers.
Therefore, in our model, the distances between aromatic stacking and H-bonded side-chain are 3.46 Å and 4.9 Å, respectively, where two monomers of the hexakis are placed at an angle of 20° respect together. Besides, the van der Waals diameter of this monomer is about 6.4 Å. Schematic representation of the hexakis monomer and guest molecule is illustrated in Figure S1. It is worth noting that for choosing the most stable configuration of the drug-carrier complexes, the conformational search is carried out by using the Spartan software package 30 . To explore the hexakis dimer's ability in response to organic an aqueous solvent, the polarizable continuum model (PCM) is applied 31 . All the above calculations are performed by employing the Gaussian 03 package 32 . The strength of the adsorption is determined by computing the binding energy (∆Eads) that can be obtained from the following relation: The analysis of bonding interaction between the DOX and hexakis dimer carrier has been performed using the energy decomposition analysis (EDA) via the Amsterdam Density Functional theory (ADF) package 33 The total density of states (TDOS) and partial density of states (PDOS) of the DOX-hexakis dimer are calculated by using the MultiWFN 3.8 program 37 . Topology analysis, i.e., atoms in molecules (AIM) 38 and Becke surface (BS) 39 methods are carried out using the MultiWFN 3.8 program to confirm the existence, evaluation of hydrogen bonding (HB). Also, to assess the type of interactions between the DOX and the hexakis dimer, the non-covalent interaction (NCI) calculation is carried out by the NCIPLOT code 40 .

Geometry optimization and adsorption energies
In this study, the hexakis dimer is investigated as a novel nano-carrier to predict its ability the deliver DOX to the tumor cells. The monomer and dimer structures of hexakis are optimized using the DFT method to evaluate the dimerization process of m-PE macrocycles. Moreover, all structures of the drug molecule, hexakis dimer, and DOX/hexakis dimer complexes are investigated in chloroform and water solvents to study the drug-carrier interacting system.
After the investigation of the dimerization process, it is found that the internal pores of hexakis dimer are well-preserved during the optimization. For both solvents, the calculated inner and outer diameters remained at 10.78 Å and 22.9 Å, respectively. It is worth noting that the (m-PE) macrocycles have a rigid backbone and do not allow free rotation about the axis of peripheral phenyl rings, thus making nondeformable cavities and a planar ring system. Our results is in good agreement with experimental data by Moore and coworkers 15 .  41,42 . Moreover, the calculated N⋯O distances in the two-neighboring macrocycle are approximately equal to 2.94 Å in both solvents, which these values are close to the distance between these atoms in the X-ray crystal structure (2.93 Å). Based on the obtained results, the N-H⋯O hydrogen bonds (HBs) between two plates of hexakis are perfectly formed, and also there is a strong π−π stacking interaction with a distance of 3.46 Å. The rigidity of this macrocyclic backbone gains its origin from the inter-planar attractions, mostly in form of π−π stacking interactions. The equilibrium HBs distances between carbonyl groups at the backbones with amide side chains of the adjacent is in the range of 1.  43 . According to the above results, the dimerization is more favorable in the chloroform than water solvent. Therefore, it can be concluded that the hexakis dimer is more stable in chloroform, which is in agreement with our previous MD simulation 16 . In this study, two different positions are considered for the interaction of the DOX molecule with the hexakis dimer nanoporous. In one case, the DOX molecule is placed inside the hexakis dimer pores, and in the second case, the DOX molecule is placed outside the hexakis dimer near the entrance of the pores. It is worth noting that to study the absorption at the outside of the hexakis dimer, a distance of 2.5Å between the carrier and the nearest atom of the DOX molecule is considered before optimizing the whole system. The optimized structures of complexes are displayed in Figure 1. In addition, the structural parameters for all complexes are listed in Table S2. Due to the complex formation between the DOX molecule and hexakis dimer nanoporous, the drug molecule is able to participate in various types of intermolecular hydrogen bonds such as conventional (N−H⋯O and N−H⋯N) and non-conventional (C−H⋯N and C−H⋯O) interaction (see Fig.1.).
As it is evident from Figure 1, the drug molecule in inside complexes only involves non- Outside complex conventional intermolecular HB with the carrier. In contrast, both non-conventional and conventional intermolecular HB are observed between DOX and carrier in the outside complexes.
As can be seen in Table S2, the conventional HBs are significantly shorter and consequently stronger than that non-conventional ones in the studied complexes. Therefore, it should be noted that the hydrogen bonds could be an essential driving force for stabilizing the DOX/hexakis dimer in outside complexes. To investigate the strength of interaction of DOX molecule with the hexakis dimer nanostructure, the binding energy values are computed for the studied complexes with different functionals, regular and dispersion-corrected, and the results tabulated in Table 1.
where Esolvent and Egas are the total energies of the complexes in water solution and the gas phase, respectively. The solvation energies reported in Table 1 show that the solvation is a spontaneous process. The results show that the adsorption of DOX molecule at the outside and inside of the cavities is a physisorption process. Authors previously applied theoretical methods to investigate the sensitivity of CNTs as smart drug delivery systems 44 . Compared to our previous work on CNT nanotube, the obtained results indicate that hexakis macrocycles have much better performance for the load and delivery of anti-cancer agents. Moreover, both our former and current work emphasize on the importance of solvent effects in the process of complex formation.

Energy decomposition analysis
The energy decomposition analysis (EDA) is an effective procedure for a quantitative interpretation of chemical bonds' interactions between molecules 45 . The EDA partition the total interaction energy into the most relevant terms such as dispersion energy (ΔEdis), electrostatic energy (ΔEelect), orbital (covalent) energy (ΔEorb), and repulsive exchange (Pauli) energy (ΔEPauli).
In this work, EDA analysis is considered to describe further the intermolecular interactions between the drug and nanocarrier in DOX-hexakis dimer complexes. For performing EDA analysis, the hexakis dimer is generally considered as one fragment, and the drug molecule as the other fragment, and the results are presented in Table 2. As it is seen from the results in Table 2, the dispersion term is the dominant interaction in the stabilization of all complexes. The ∆Edis for outside complexes is higher than the inside complexes, which this result conforms with the binding energies. In addition to the ∆Edis energy, the electrostatic term (-63.68 kJ mol -1 ) also has a considerable contribution to stabilizing these complexes. Our findings are in remarkable agreement with previous studies 46,47

AIM and BS analysis for the strength of H bonding
AIM representations of studied complexes, including bond critical point (BCP) and the bond paths, are presented in Figure S2. The calculated values of total electronic density ρ(r), Laplacian electronic density ▽ 2ρ(r), and also energetic AIM parameters (kinetic energy density (G), the total energy density (H), potential energy density (V)) for considered complexes are presented in Table   S3. According to this Table, the range of (r) and  2 (r) at BCP in the interaction sites of inside complexes are from 0.001 to 0.019 a.u. and from 0.026 to 0.061 a.u., respectively, while these values are in the range of 0.0201-0.0508 a.u. and 0.012-0.098 a.u., respectively, in outside complexes in water and chloroform solvents. The N34⋯H283 interaction with  2  > 0, Hb < 0, 0.

NCI plot analysis
The use of NCI index enable the understanding of the interactions in a complex since each method can recognize regions of the weak and strong electron pairing respectively. The NCI index of Yang and co-workers is based on the reduced density gradient (RDG = |∇ρ|/2(3π 2 ) 1/3 ρ 4/3 ), and also is suitable to define the nature of the weak bonds involved in the structures. The NCI isosurfaces of the reduced electron density gradient between the DOX molecule and hexakis dimer for the most stable complex is depicted in Figure 3.

Electronic structure of DOX and hexakis dimer complexes
Changes in the electronic structure of hexakis dimer upon adsorption of DOX molecule are examined by calculating the energy gaps of frontier molecular orbital and partial density of states.
The calculated energy gap (ΔELUMO-HOMO) and the conceptual DFT-based reactivity descriptors are reported in Table 3. As it could be observed in Table 3, (i) the ΔELUMO-HOMO gap of outside complexes are higher than that of inside complexes. This means that the outside complexes are more stable than the inside complexes. (ii) The negative values of the chemical potential reveal the stability of DOX-hexakis dimer complexes. (iii) While the global hardness (η) is decreased, the electronegativity parameter is increased after the formation of complexes. (iv) The calculated electrophilicity of outside complexes are significantly higher than the value of electrophilicity of inside complexes, especially in chloroform solvent. From these results, we can conclude that these changes could be attributed to the stronger H-bond interactions between DOX and hexakis dimer. To understand the interaction of DOX with hexakis dimer, the electronic structure has been calculated through total density of states and projected density of states, as shown in Figures

Conclusions
In the present investigation, the interaction of DOX molecules with hexakis dimer as a novel nanocarrier is studied in chloroform and water solvents via density functional theory calculations, and the following conclusions have been made.
All the ΔEad values are negative, which indicated that the adsorption of the DOX drug on the hexakis dimer spontaneously proceeded. However, the ΔEads values for adsorption of DOX drug in the outside of the carrier are greater than those inside it, especially in chloroform solvent. These results can be attributed to the strong hydrogen bond interaction that forms in the outside DOXhexakis dimer complexes. Regarding the obtained results of EDA, the significant contributions to the total bonding energy are dispersion and electrostatic energies. QTAIM and BS analysis suggested that the existence of hydrogen bonds and non-covalent intermolecular interactions create a reaction of host-guest and keep the stability of complexes. In addition, the calculation results of quantum molecular descriptors revealed that the adsorption of DOX drug on hexakis dimer nanocarrier enhanced the chemical reactivity. Overall, the results obtained from this study provide the nature of the interaction between DOX molecule and hexakis dimer as a novel nanocarrier, which may be useful for making targeted decisions about cancer treatment.

Conflicts of interest
There are no conflicts to declare.