Effect of cucurbit[7]uril on DPPC-containing liposomes: Interactions with the lipid bilayer

Abstract

Liposomes, which are bilayer lipidic nanocarriers, have been utilized in many pharmaceutical applications to enhance the solubility and therapeutic index of drugs. Liposomes have also been used as carriers for smaller drug carriers, such as cucurbiturils, to achieve a more controlled release of the drug into the targeted site in the body. In this study, we investigated the effects of cucurbit[7]uril, a macrocyclic organic compound, on the integrity of liposome lipid membranes. The average liposome size, measured by dynamic light scattering, increased with increasing concentrations of cucurbit[7]uril. In addition, fluorescence spectroscopy was used to calculate an association constant (K_a) between cucurbit[7]uril and cholesterol of 3 $\times {10}^6/M$. This high K_a value demonstrated the ability of cucurbit[7]uril to reduce liposome stability by extracting cholesterol molecules from the lipid bilayer. Thermogravimetric analysis demonstrated the localization of cucurbit[7]uril molecules on the surface of the liposomes. As the concentration of cucurbit[7]uril increased, the thermal stability increased, i.e. the mass loss of the liposomal suspension decreased. The biocompatibility of cucurbit[7]uril was also investigated using a hemolysis test on human red blood cells. In conclusion, the current study is the first to explain the relationship between lipid membranes and cucurbit[7]uril. The results of this study can be used to develop a new drug delivery system comprising liposomes and cucurbit[7]uril.

Keywords: Cholesterol; cucurbit[7]uril; hemolysis; liposome; stability.

Figure 1.

Representative DLS (size measurement) and TEM plots of (a) blank liposomes and (b) liposomes with 0.1698 mM CB[7] 24 hours after preparation. DLS measurements were performed at 25 °C, and the results are reported as triplicate averages ± SD.

Figure 2.

Fluorescence intensities ( $I_t$ ) and time-history of calcein release from liposomes with different CB[7] concentrations over 21.33 hours at 25 °C and different CB[7] concentrations; the results are the averages $\pm \mathrm{SE}$ (standard error of measurement; SE is 0.3 arbitrary units (a.u.)) of four independent experiments with statistical significance of P < 0.05. The relative standard deviation (RSD%) is ≤ 0.52% for all time intervals.

Figure 2.

Fluorescence intensities ( $I_t$ ) and time-history of calcein release from liposomes with different CB[7] concentrations over 21.33 hours at 25 °C and different CB[7] concentrations; the results are the averages $\pm \mathrm{SE}$ (standard error of measurement; SE is 0.3 arbitrary units (a.u.)) of four independent experiments with statistical significance of P < 0.05. The relative standard deviation (RSD%) is ≤ 0.52% for all time intervals.

Figure 3.

Chemical structure of calcein.

Figure 4.

Formation of CB[7]–Chol inclusion complexes: (a) fluorescence intensity over 13 minutes as a function of Chol concentration; (b) Benesi–Hildebrand method based on fluorescence spectroscopy by titration of the CB[7] host solution with a Chol guest solution at different ratios. The temperature was 25 °C. The measurements represent averages of five independent trials with SE and RSD% values of ≤ 0.083 a.u. and ≤ 0.033%, respectively.

Figure 5.

Stacked ¹H-NMR spectra of liposomes titrated with CB[7]. The proton peak of D₂O is distinguished by an asterisk. D₂O, 25 °C, 500 MHz.

Figure 6.

TGA thermograms representing the mass losses (%) of CB[7] and liposomes with different concentrations of CB[7] versus temperature. Heating rate: 10 °C/minute.

Figure 7.

Hemolysis rate of RBCs under the effect of different CB[7] concentrations. The temperature was 37 °C, and the data are averages of three independent experiments with RSD% ≤ 1%.