The entrapment of anticancer drug ellipticine in the dipalmitoylphosphocholine (DPPC) liposome and its release by addition of three different bile salts, namely sodium deoxycholate, cholate and taurocholate, have been studied by steady state and time resolved fluorescence spectroscopy. We found that the release of the drug from a liposome depends on the degree of penetration of bile salts. Among the three bile salts, deoxycholate was most effective in releasing the drug from the hydrocarbon core of the liposome because of its high insertion ability owing to its maximum hydrophobicity. The time resolved studies revealed that with addition of bile salt to the liposome solution, ellipticine molecules were removed from the hydrocarbon core and were entrapped in an interfacial region of liposomes by electrostatic interaction. This led to an increase in the shorter lifetime component. On the other hand, the longer lifetime component decreased because bile salts wet the hydrocarbon core of the liposome by carrying hydrogen bonded water. Entrapment of ellipticine in the interfacial region was also supported by an increase in the rotational relaxation time with addition of bile salt.
Interaction of hen egg white lysozyme with different liposomes made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) was studied by circular dichroism (CD), steady state and time resolved fluorescence spectroscopy. We used anticancer drug ellipticine and studied its entrapment and release from liposomes upon interaction with lysozyme. The molecular docking study revealed that ellipticine preferably binds to the hydrophobic pocket of lysozyme (Kbinding = 1.09 × 10(6) M(-1)). The binding was also supported by spectroscopic evidence. Addition of lysozyme to the ellipticine impregnated liposomes caused quenching of the fluorescence intensity of ellipticine as lysozyme induces hydration and phospholipid rearrangement in the bilayers leading to the leakage of drug molecules. The extent of quenching depends on the prehydration level of liposomes. Maximum quenching took place in the DPPC liposome as it is the least hydrated while minimum quenching was observed in the DOPC liposome having the highest hydration level among all the lipids. The time resolved studies revealed that both the fast and slow lifetime components of ellipticine decrease significantly with addition of lysozyme. This fact is attributed to lysozyme induced hydration and rupture of bilayers. It is revealed that upon addition of lysozyme to liposomes, the amplitude of the fast component increases and that of the slow component decreases which imply that the drug molecules are released from liposomes and subsequent migration takes place to the aqueous phase. Molecular docking studies and fluorescence measurements indicate that ellipticine after removal from the liposome binds to the hydrophobic binding site of lysozyme.
The interaction of human serum albumin (HSA) with liposomes made of saturated and unsaturated phosphocholines having distinctly different phase transition temperatures has been studied using circular dichroism (CD), steady state and time resolved fluorescence spectroscopic techniques. We used 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) as the saturated lipid and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-oleoyl-1-palmitoyl-snglycero-3-phosphocholine (POPC) as the unsaturated lipid to prepare liposomes. The CD measurement reveals that the liposomes induce some kind of stabilization in HSA. The steady state and time resolved fluorescence spectra of PRODAN (6-propionyl 1,2-dimethylaminonaphthalene) were monitored to unravel the interaction between liposome and HSA. We observed that HSA partially penetrates the liposomes due to hydrophobic interaction and destabilizes the packing order of the lipid bilayer leading to leakage of the probe molecules from the liposome. It was found that HSA preferably penetrates into the liposomes, which are less prehydrated at room temperature. Thus penetration is higher in DPPC and DMPC liposomes as these liposomes are less prehydrated due to higher phase temperature (43 C and 23 C respectively). On the other hand HSA has less penetration in DOPC and POPC liposomes because these liposomes are more hydrated owing to lower phase transition temperature (À20 C and À2 C respectively). The time resolved fluorescence measurements revealed that penetration of HSA into liposomes brings about release of PRODAN molecules. Incorporation of HSA in all the liposomes results in a significant increase in the rotational relaxation time of PRODAN. This fact confirms that HSA penetrates into the liposome and forms a bigger complex.
The entrapment of neutral and cationic species of an anticancer drug, namely ellipticine and their dynamic features in different bile salt aggregates have been investigated for the first time using steady state and time-resolved fluorescence spectroscopy. Because ellipticine exists in various prototropic forms under physiological conditions, we performed comparative photophysical and dynamical studies on these prototropic species in different bile salts varying in their head groups and hydrophobic skeletons. We found that the initial interaction between ellipticine and bile salts is governed by the electrostatic forces where cationic ellipticine is anchored to the head groups of bile salts. Bile salts having conjugated head groups are better candidates to bind with the cationic species than those having the non-conjugated ones. The fact implies that binding of cationic species to different bile salts depends on the pK(a) of the corresponding bile acids. The hydrophobic interaction dominates at higher concentrations of bile salts due to formation of aggregates and results in entrapment of neutral ellipticine molecules according to their hydrophobicity indices. Thus bile salts act as multisite drug carriers. The rotational relaxation parameters of cationic ellipticine were found to be dependent on head groups and the number of hydroxyl groups on the hydrophilic surface of bile salts. Cationic ellipticine exhibits a faster rotational relaxation in the tri-hydroxy bile salt aggregates than in di-hydroxy bile salts. We interpreted this observation from the fact that tri-hydroxy bile salts hold a higher number of water molecules in their hydrophilic surface offering a less viscous environment for ellipticine compared to di-hydroxy bile salts. Surprisingly, the neutral ellipticine molecules display almost the same rotational relaxation in all the bile salts. The observation indicates that after intercalation inside the hydrophobic pocket, neutral ellipticine molecules experience similar confinement in all the bile salts.
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