Binding behavior and in vitro cytotoxicity of inclusion complexes between aminopterin and cucurbit[7]uril

Aminopterin (AMT) is a kind of universal antineoplastic drugs, but it has severe toxic and side effects, leaving it rarely used in clinic. Herein, we found that cucurbit[7]uril (CB[7]) and AMT can form stable inclusion complexes, and the formation of CB[7]-AMT 2:1 supramolecular inclusion complex was confirmed by UV-visible absorption spectra, fluorescence spectra, 1H NMR, and molecular modeling calculations in aqueous solution. Binding stability constants (Ks) were determined by UV-visible and fluorescence spectra method, with 3.88 × 1010 L2 mol–2 and 5.24 × 1010 L2 mol–2, respectively. The binding energy was calculated to be 102.6 kcal mol–1 for the CB[7]-AMT complex. And then, through a series of cell experiments of CCK8 assay, DAPI staining and hoechst33342/PI double staining, we fully proved that the CB[7]-AMT complex can reduce the toxicity of AMT to normal cells such as hepatocyte line LO2, and improve its anticancer effect on cancer cells overexpressing spermine, typically like human colon cancer cell line HCT116. It confirmed that the CB[7]-AMT complex had the effect of reducing toxicity and increasing efficiency. These results indicated that CB[7]-AMT inclusion complex might be a promising novel formulation of AMT for its clinical development.


Introduction
Aminopterin (AMT, Fig. 1), also known as 4-aminopterin acid, is a well-known folate antagonist. AMT and its active metabolites could inhibit several folate-mediated reactions, especially the synthesis of nucleotides, thereby inhibiting DNA synthesis. In clinical practice, AMT has been widely used chemotherapeutic agent. Specific applications include either alone or in combination therapy for the treatment of acute lymphocytic leukaemia, choriocarcinoma, breast carcinoma, head and neck cancer, oat cell carcinoma, mycosis fungoides and osteogenic sarcoma [1]. However, severe side effects and toxicity limit its clinical use, such as severe bone marrow suppression with leukopenia, thrombocytopenia, gastrointestinal disorders, hepatic injury and so on [2][3][4][5]. At present, several ongoing research programs focus on the preparation of new analogues possessing better tumour-cell selectivity, lower toxicity, better transport properties, and improved lipid solubility and membrane permeability.
With the development of supramolecular macrocyclic chemistry, cucurbit[n]urils (CB[n]s, n = 5-8, 10) macrocyclic compounds have been the focus of considerable attention in the fields of chemistry, physics, biology and others due to their stronger binding constants and their higher selectivity [6][7][8][9][10][11]. CB[n]s posses hydrophobic inner cavity and two portals with polar carbonyl groups, which confers them in part the host-guest properties. It is negative charge density located at the carbonyl groups that stabilizes positively charged guests by ion-dipole and hydrogen-bonding interactions [12], complemented by the hydrophobic effect of the cavity which leads to the formation of inclusion complexes. CB[n] has stable chemical structure and good biocompatibility, which lays a foundation for its application in biomedicine. Compared with other CB[n] members, cucurbit [7]uril (CB [7], Fig. 1) has excellent properties of good water solubility, high chemical stability and low biological toxicity [13][14][15]. Day et al. reported that dinuclear platinum complex binded inside CB [7] with slow exchange kinetics did not significantly affect the cytotoxicity, but reactivity at the platinum centre was reduced [16]. Kim et al.prepared the inclusion complex of CB [7] and oxaliplatin, which could not only increase the stability of the drug but also may reduce side effects caused by protein binding of the platinum drug [17]. Complex of CB [7] and camptothecin synthesized by Zhang et al. could inhibit the systemic toxicities of the free drug, while its antitumor/anti-angiogenic activities were well preserved [18,19]. However, to the best of our knowledge, the inclusion of AMT with CB [7] has never been reported.
In this paper, the formation of CB [7]-AMT inclusion complex was confirmed by UV-Vis absorption spectrum, fluorescence spectrum, and molecular modeling calculation. Furthermore, a study of the biological activity of the inclusion complex showed that it has the effect of reducing toxicity and increasing efficiency.

Materials
AMT was provided by Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). CB [7] was synthesized and characterized according to the literature [20,21] Stock standard solution of AMT (100 µg mL −1 ) was prepared in ultrapure water. Stock solutions of CB [7] was prepared by adding ultrapure water to a final concentration of 1.0 × 10 − 3 mol L −1 . CCK-8, DAPI staining solution, and Hoechst33342/PI kit were purchased from Beijing Soleibao. The cell strains of human normal liver cell LO2, human colon cancer cell HCT116 and human hepatoma cell HepG2, were all obtained from Shanghai Cell Institution. All other reagents were of analytical reagent grade.

Instruments
The absorption spectra analyses were performed by a U-3010 UV-visible spectrophotometer (Hitachi, Japan). Fluorescence measurements were monitored with a Perkin Elmer LS-45 spectrofluorometer (America). Fluorescence spectra was operated at a scanning rate of 1200 nm min -1 with width of excitation slit is 5 nm, and the width of emission slit is 10 nm. The electronic structures of CB [7]-AMT inclusion complex were completely optimized and calculated at B3LYP/6-31G(d) level.

Preparation of inclusion complex
The complex of CB [7]-AMT was prepared by a suspension method. In brief, CB [7] and AMT were added to water in a molar ratio of 2:1, and the resulting solutions were allowed to complex under ultra-sound for 30 min and then placed in the ambient temperature for 2 h to cool down.

Encapsulation efficiency(EE)
In order to determine the drug loading and encapsulation efficiency of AMT, we first drew the standard curve of AMT. Stock standard solution of AMT (3.00 mg mL −1 ) was prepared in water, and dilute it into different concentrations. The fluorescence intensities of the solutions were measured at 460 nm using an excitation wavelength of 343 nm. And then, concentration standard curve was established with fluorescence intensities: F = 661.31 C + 63.616 (0.15 ~ 6.00 µg mL −1 , R 2 = 0.9995).
The inclusion complexes prepared under 2.3 (9.68 mg) were weighed and dissolved in 10 mL of water. Then diluted by 100 times, the working solution of 9.68 µg mL −1 (3.5 µmol L −1 ) was made. 3 mL of the working solution was placed into an ultrafiltration centrifuge tube with a molecular cut-off of 1000 Da, centrifuged for 30 min at 10,000 r/min, and then the filtrate determined by fluorescence spectrum. The assay was repeated three times with different samples. The concentration of free drug was obtained by substituting the fluorescence intensity value into the above standard curve. The mass of AMT encapsulated in CB [7] was equal to this value, that is, the total mass of AMT minus the mass of free AMT obtained by centrifugation. According to formula (1), the encapsulation efficiency of the inclusion compound is (62.73 ± 1.96)%. Fig. 1 The chemical structure of AMT and CB [7] In vitro cytotoxicity studies

Cell lines and cell culture
The HCT116 colorectal cancer, HepG2 liver cancer, and LO2 normal hepatocytes were chosen for the initial experiments. HCT116 and HepG2 were placed in DMED highglucose complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. LO2 cells were placed in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Before and during the experiments all cell cultures were incubated under a humidified atmosphere with 5% CO 2 at 37 °C.

Cell inhibition rate
For assessing the effect of drugs on cellular viability, cell proliferation assays were carried out using Cell Counting Kit-8 (CCK-8) assay. HCT116, HepG2, and LO2 cells were seeded on 96-well plates at a density of 5 × 10 3 in 100 µL of growth medium/well. The experimental groups were added to the drug-containing medium containing different concentrations of AMT and CB [7]-AMT, the control groups were added to the complete medium, and 10 µL of CCK-8 reagent was added after culturing for 24 and 48 h. After culturing for 1 h at 37 °C, the culture media were measured the absorbance at 450 nm using a microplate reader. Each experiment was repeated 6 times.

Apoptotic morphology
HCT116 cells were seeded in 6-well plates at a density of 1 × 10 6 cells per well. After culturing for 24 h, the medium was aspirated and discarded, and 3.5 µmol L -1 of AMT and CB [7]-AMT medicated medium 2 mL were added respectively. There were 3 replicate wells in each group. After the medium was aspirated, the cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Then, DAPI was added at room temperature for 5-10 min. The cells were observed and pictured of the changes of HCT116 nucleus after AMT and AMT-CB [7] effects.

Detection of cell necrosis
In order to observe the apoptosis of HCT116 cells after the effect of drugs, Hoechst33342/PI staining was used, and the cell slides were placed in 6-well plates, and the cells were digested and counted at 5000 cells/well. 24 h later, and 2 mL of AMT and CB [7]-AMT medicated medium were added with the same dose (3.5 µmol L -1 ), three replicate wells in each group. Then, the fixative solution was discarded and HCT116 cells were washed with PBS three times for 3 min each time. Next, 4% paraformaldehyde fixative was added to fix the cells for 10 min, washed three times with PBS, and then stained with Hoechst33342. Thereafter, cells were observed under a fluorescence microscope. Representative photographs were captured. The red fluorescence-positive cells were counted and the percentage of necrotic cells was calculated. Therefore, we speculate that the pteridine and glutamate groups of AMT molecules may be embedded in the cavity of CB [7], resulting in a significant chemical shift. These observations suggest that CB [7]-AMT inclusion complex is formed.

Detection of stoichiometry between CB[7] and AMT
The stoichiometric ratio of CB [7] and AMT was determined in pH 7.4 phosphate buffer by UV-vis spectroscopy. As can be seen from Fig. 3A, with the increase of the concentration of CB [7] added into AMT solution, the absorption intensity of the whole system increased, and an isoabsorptive point was formed at 368 nm, indicating that there was interaction between CB [7] and AMT. Another evidence for the formation of 2:1 CB[7]-AMT inclusion complex was the data curve of absorbance versus the molar ratio of CB [7] and AMT (N CB [7] /N AMT ) shown in Fig. 3B. And the Job curve [22] (embedded graph) also validated the stoichiometry. Under the determined experimental conditions, assuming that the composition ratio of CB [7]-AMT inclusion complex was 2:1, the expression is as follows: The binding constant (K) was calculated as following formula: The K-value of CB [7]-AMT inclusion complex could be determined by the typical Benesi-Hildebrand equation [23]:

CB[7]
Where C AMT 0 , C CB [7] 0 are the initial concentrations; A 0 and A are the absorbance of AMT within and without CB [7], respectively; and ε 0 and ε are the molar absorptivity of AMT within and without CB [7], respectively. A good linear relationship was obtained by plot- with the correlation coefficients (R 2 ) = 0.9994 Fig. 3C, which proves the existence of 2:1 inclusion complex. And the K-value of the 2:1 inclusion complex was 3.88 × 10 10 L 2 mol -2 .

Fluorescence spectrometric titration
Quantitative investigation of the inclusion complexation binding behavior of host CB [7] with AMT was also  [7] molecule, the complex stability constant (K) was determined, as shown in Fig. 4A. The results show that a good linear relationship was obtained by plotting 1/(F 0 −F) against 1/C 2 CB [7] with the correlation coefficients (R 2 ) = 0.9993 Fig. 4B, but has no linear relationship with 1/C CB [7] . which proves the existence of 2:1 inclusion complex. And the K-value of the 2:1 inclusion complex was 5.24 × 10 10 L 2 mol -2 . This is consistent with the result of UV spectrum.

Molecular modeling calculation
The binding ernergy of CB[7]-AMT was calculated by the B3LYP method combined with 6-31G(d,p) basis set for all the atoms, and emperical dispersion correction of D3BJ model proposed by Grimme et al. [24][25][26]. The solvation effect of solvent water was included in the geometry optimizations and vibration analysis [27]. Under the standard state (298.15 K and 1 atm), the binding energy of CB[7]-AMT was calculated to be 102.6 (enthalpy change, ΔH°) and 56.7 (Gibbs free energy change, ΔG°) kcal/mol, respectively. This is basically consistent with the results of the 1 H NMR experiments. It can be seen from molecular simulation that, in the energy-minimized structure as shown in Fig. 5, the pteridine ring and glutamate group of AMT molecule are located in two different CB [7] hydrophobic cavities respectively. Among them, the pteridine ring is enclosed in the CB [7] cavity due to the formation of ion dipole and hydrogen bond interaction between the N-H on the pteridine ring and the carbonylation inlet of CB [7]. The glutamic acid group is fixed in CB [7] cavity because its carboxyl group formed hydrogen bond with the carbonyl group of CB [7].

In vitro cytotoxicity for cancer and normal cells
The cytotoxicity of CB [7]-AMT complexes is evaluated against two different types of human cancer cell lines HepG2 and HCT116, and the human liver cell line LO2 by the CCK-8 assay using the AMT alone as the comparator, the result is shown in Fig. 6. Cell growth curves by CCK-8 assay showed that CB [7] had no cytotoxicity. CB [7]-AMT complexes had stronger cytostatic effect on colorectal cancer HCT116 cells than AMT alone, and its IC 50 at 48 h were 1.50 µmol L −1 and 3.34 µmol L −1 (P < 0.05), respectively. Molecular CB [7]-inclusion of AMT, its efficacy is 2.2 times that of single-agent AMT, which can effectively improve the inhibition of colon cancer cell proliferation. The IC 50 of CB [7]-AMT and AMT on liver cancer HepG2 cells are respectively 1.86 µmol L −1 .and 1.82 µmol L −1 (P > 0.05), the inhibition rate did not change significantly before and after inclusion. However, the IC 50 of CB [7]-AMT and AMT on normal liver LO2 cells for 48 h were 2.74 µmol L −1 and 1.41 µmol L −1 (P < 0.05), respectively. The inhibitory effect of AMT on normal cell proliferation after inclusion is only 1/2 that of single drug AMT.
In brief, compared with AMT single drug, CB[7]-AMT can significantly reduce the toxic and side effects on normal liver cells, and CB [7]-AMT against HCT116 cells in the inhibition of tumor cell proliferation showed good results.

DAPI staining of HCT116 cells
DAPI dye is a fluorescent dye that stains the nuclear DNA of a cell and is therefore used to determine the effect of plant extracts on inducing morphological changes in the nuclei of cancer cells undergoing apoptosis. The morphological changes of HCT116 nuclei stained with DAPI are shown in Fig. 7. The nuclei of HCT116 cells treated with CB[7]-AMT and AMT groups for 48 h were darker, the density of cells became lower, and there were irregular protrusions on the edge of the nucleus, the nuclear debris increased, and the phenomenon of nuclear apoptosis was obvious. After

Hoechst33342/PI double staining of HCT116 cells
The Hoechst33342/PI double-labeling rule is based on the principles of apoptotic cell nuclear chromatin condensation and cell membrane permeability changes. The results were judged as follows: normal cells showed Hoechst 33,342 + / PI -, early apoptotic cells showed Hoechst 33,342 ++ /PI -, late apoptotic cells or necrotic cells showed Hoechst 33,342 ++ / PI + . The results of HCT116 cells' apoptosis determined by hoechst3342/PI method are shown in Fig. 8. It can be seen that after the same concentration of AMT and CB [7]-AMT acted on HCT116 cells, some cells showed a concentrated and bright blue nucleus, showing the characteristics of high blue/low red (apoptotic cells), and some cells were obviously red. Compared with the single-agent AMT group, the cell density of the CB[7]-AMT group was significantly reduced, and the proportion of dead cells was significantly increased, which showed that the CB[7]-AMT complex had a stronger anticancer effect consistent with the results of CCK-8 assay.

The mechanism of CB[7]-AMT complex in enhancing efficacy and reducing toxicity
The inclusion of AMT by CB [7] can significantly enhance the anticancer effect of AMT. The possible mechanism is related to a certain target substance in the tumor tissue, and the target substance competes with the anticancer drug AMT for the supramolecular CB [7] host and then releases AMT to restore its anticancer activity. On the other hand, the mechanism of reducing the toxic and side effects on normal cells may be related to the masking of the activity center of AMT by the CB [7] supramolecular host [28].
To further study its mechanism of reducing toxicity and increasing efficiency, following extensive literature research, it was founded that, spermine (SPM) is a polyamine substance necessary for cell proliferation and growth [29], which tend to accumulate in rapidly growing tissues. Abnormal polyamine biosynthesis is associated with the development of a variety of pathological states, such as Alzheimer's disease and cancer [29][30][31]. Therefore, the method of using polyamines as targets to treat tumors is gradually developed [32]. For example, the SPM content of human colorectal cancer HCT116 cells [33,34] and liver cancer HepG2 cells is higher than that of normal cells with very small SPM content, the concentration order was HCT116 > HepG2 > normal cells. Secondly, SPM has a sufficient number of positively charged groups, which can be included with CB[7] [35]. Based on available data, we extrapolated that the enhancement of anticancer activity might be due to the fact that SPM competes with AMT for CB [7] and releases AMT to restore the anticancer activity of AMT, and at the same time consumes SPM necessary for cell proliferation and growth (As shown in Fig. 9).
In response, we performed in vitro observations of the SPM competitive release of AMT. As shown in Fig. 10A, since SPM and CB [7] have no fluorescence absorption in this range, we can directly observe the fluorescence changes of AMT before and after the competitive displacement. With the gradual addition of SPM, the absorbance of AMT gradually returned to a near-free state. We selected the absorbance of the fluorescence maximum absorption peak at 460 nm, as shown in Fig. 10B: 2 times the amount of SPM can competitively replace nearly 40% of AMT, and 4 times the amount of SPM can replace more than 50% of AMT. The higher the concentration, the more AMT was displaced.
To further verify spermine may displace AMT at the cell level, we took LO2 of normal hepatocytes as the research   Fig. 10 Fluorescence observation data of competitive replacement of AMT with SPM in vitro, with λex=380 nm object. As shown in Fig. 11, SPM alone can effectively improve the proliferation and growth of cells; however, when adding equal proportion of SPM to the inclusion complex of CB [7]-AMT, the cell survival rate was significantly reduced. The cytotoxicity of AMT can be partially restored, but yet a small amount of SPM expressed in normal cells is not enough to release AMT. CB [7]-AMT may exist in the form of inclusion complex in typical cell environment, this combination is not only free from the interference of amino acids, growth factors and other substances in the environment, but also can significantly reduce the toxicity of AMT to normal cells, and the effect of supramolecular chemotherapy on drug control is further verified.
Although the further in vivo antitumor activity and detailed mechanism of action for these inclusion complexes also needs to be further explored, these preliminary results could provide valuable preference to a new formulation of AMT for its clinical development.

Conclusion
In this paper, the formation of CB[7]-AMT 2:1 supramolecular inclusion complex was confirmed in an aqueous solution, based on UV-visible, fluorescence spectrum, 1 H NMR, and theoretical calculations. Secondly, we have fully demonstrated through a series of cell experiments that the inclusion complex of AMT and CB [7] can reduce the toxicity of AMT to such normal cells as LO2, and at the same time improve its anticancer effect on cancer cells overexpressing SPM such as HCT116 cells. These results verified the feasibility of the supramolecular chemotherapy delivery system and indicated that CB [7]-AMT inclusion complex might be a promising novel formulation of AMT for its uses in the pharmaceutical industry.