Moxifloxacin interacts with lipid bilayer, causing dramatic changes in its structure and phase transitions

doi:10.1016/j.chemphyslip.2020.104891

Chemistry and Physics of Lipids

Volume 228, May 2020, 104891

https://doi.org/10.1016/j.chemphyslip.2020.104891 Get rights and content

Highlights

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Moxifloxacin interacts with lipid bilayer, causing changes in its structure and phase transitions especially for anionic liposomes.
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For the first time, the fine structure of bilayer complexes with moxifloxacin in all details was determined.
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Combined spectral approach to study drug-to-lipid interaction and analyze fine structure of complexes is developed.
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Cardiolipin in lipid matrix composition provides stronger binding of moxifloxacin.
•
Mox interacts with two microphases of DPPC/CL liposomes esulting formation of two types of the complexes.

Abstract

Most drugs besides their intended activity, express undesired side effects, including those with the engagement of cell membrane. Previously, such undesired nonspecific effects on the membrane have been shown for a number of widely used nonsteroidal anti-inflammatory drugs. In this paper, we study the mechanism of interaction between moxifloxacin (Mox), antibacterial drug of broad specificity, with lipid bilayer of the liposomes of various compositions as a model of cell membrane using a combination of spectroscopy methods, including ATR-FTIR spectroscopy, circular dichroism, UV and fluorescence spectroscopy. The fine structure of the moxifloxacin-liposome complex, localization of the drug in bilayer and the main sites of Mox interaction with lipid membrane were determined. Lipid composition of the liposome plays a key role in the interaction with moxifloxacin, drastically affecting the loading efficiency, strength and character of drug binding, lipid phase segregation and phase transition parameters. In case of anionic liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and cardiolipin (CL²⁻) the electrostatic interaction of negatively charged nitrogen in heterocycle moiety of moxifloxacin with cardiolipin phosphate groups is a crucial factor for stable complex formation. The study of moxifloxacin-liposome complex behavior at phase transition in bilayer by DSC method revealed that in DPPC/CL²⁻ liposomes system two microphases with different content of CL^2- coexist and Mox interacts with both of these microphases resulting in the formation of two types of complexes with different structure and phase transition temperature. This binding stabilized the gel-state of the lipid bilayer with increasing the phase transition temperature Tm up to 3−5 °C. A different situation is observed for neutral DPPC liposomes: drug interaction with bilayer results in defects formation and a fluidization effect in lipid bilayer, resulted to decrease the Tm value by 2−4 °C. Moxifloxacin is not firmly binding in the membrane of DPPC and drug releases rapidly.

Graphical abstract

Introduction

Moxifloxacin (Mox) is a fourth-generation antibacterial preparation from the fluoroquinolone (FQ) group, which is highly effective antibacterial agent with wide indications for use. On the level of activity and the spectrum of antibacterial action, Mox is superior to many antibiotics (Gillespie, 2016; Fouad and Gallagher, 2011). However, like most antibiotics, Mox is not devoid of side effects, primarily phototoxicity, hemodynamic disorders, thrombosis, hepatotoxicity and neurotoxicity. In some cases, the mechanism for the development of side effects is because drugs, in addition to affecting their molecular target, cause nonspecific changes of the cell membrane. Previously, such non-specific effects have been shown for a number of widely used nonsteroidal anti-inflammatory drugs (Manrique-Moreno et al., 2009). Obtained results are indicative of a strong effect of the drugs on the hydrocarbon chains inducing a reduction of the chain–chain interactions, i.e., a fluidization effect and defects formation in lipid bilayer. Due to presence of the negatively charged groups in the molecules, the nonsteroidal anti-inflammatory drugs induce changes in the packing of the polar head group region, by modifying the surface-bond water molecules in the bilayer, which effects the lipid bilayer structure and induces a decrease in the phase transition temperature (T_m) of dimyristoylphosphocholine. Concerning fluoroquinolone drugs, such as moxifloxacin, the electrostatic binding of secondary amine group in pipyrazol heterocycle to negatively charged cell surface probably could also produce the rearrangement in the lipid bilayer, which could lead to formation of hexagonal lipids mesophase and disruption of cell integrity. These changes could finally cause higher membrane thrombogenicity and other non-specific effects of the drugs.

Bensikaddour et al.Bensikaddour et al. (2008a), b obtained the valuable data for moxifloxacin and ciprofloxacin interaction with bilayer by the computer modeling. Authors have shown that drug incorporates into circumpolar area of bilayer, but still direct evidence of the mechanism is required. Thus, better understanding of drug – cell membrane interaction may lead to improved drugs and/or their delivery systems, especially where such drug is intended for chronic or life-long application.

Classical model to study drug – to – membrane interaction is liposome as an artificial membrane consisting phospholipids as the major component of the biomembrane, responsible for several features of the bilayer like stability and semi-permeable properties (Pinheiro et al., 2014, 2019). The interaction of FQ with phospholipids can change the physicochemical properties of the membranes, which may be essential to understand the mechanism of non-specific side effects of the drugs.

The aim of this study is to disclose details of mechanism of interaction between Mox with lipid bilayer and distinguish the fine structure of liposomal formulation of moxifloxacin (LMox) depending on the lipid matrix composition (namely single-component neutral liposomes and binary anionic liposomes, containing cardiolipin were tested).

To achieve this goal, we have applied spectral methods: florescence and UV as well ATR-FTIR spectroscopy, circular dichroism (CD) spectroscopy providing information from different sides: binding sites, phase transition and bilayer structure (ATR-FTIR), the microenvironment of functional groups (CD, UV and fluorescence spectroscopy).

A complementary method for determining the structure of a bilayer is DSC. How does the lipid bilayer respond to interaction with moxifloxacin? Does the complex formation with Mox affect structural reorganization of the bilayer? How does these effects depend on the membrane charge? These questions were examined with differential scanning calorimetry (DSC) where the influence of Mox on the phase transitions in the phospholipid membrane of different composition were analyzed. This method is highly informative to determine the microphases formation in mixed lipid bilayer (Yaroslavov et al., 2009).

The results of the work will shed light to the fine structure of the moxifloxacin-liposome complex, localization of the drug in bilayer, enable to determine the main sites of Mox interaction with lipid membrane and to reveal the behavior of moxifloxacin-liposome complex at phase transition in bilayer depending on the lipid matrix composition.

Section snippets

Materials

Moxifloxacin, (NH₄)₂SO₄, octane, DMSO, PBS tablets to prepare 0.02 M buffer solution – all from Sigma-Aldrich (USA), DPPC and CL²⁻disodium salt in chloroform (16:0 Cardiolipin 1′,3′-bis[1,2-dipalmitoyl-sn-glycero-3-phospho]-glycerol)– Avanti Polar Lipids (USA).

Liposome preparation

Liposomes were prepared using the thin film/hydration method and extrusion. DPPC and CL²⁻(25 mg\mL in chloroform) were added in desired mass ratio. The solvent was removed under low pressure. Then, the lipid film was hydrated in 0.02 M

LMox preparation

How would the membrane charge influence the drug loading and the structure of final vesicle? To address this question, we have studied the liposomal system composed of zwitterionic lipid, DPPC, with 0–30% (w/w) addition of anionic phospholipid, cardiolipin (CL²⁻) (Table 1, Fig. 1). The addition of anionic component determines the membrane’s ability for electrostatic interaction with aminogroup of Mox (Fig. 1), positively charged at pH 7.4. This interaction could also contribute to higher

Conclusions

In this paper, we show that the interaction of moxifloxacin with the lipid bilayer significantly affects the state of the polar region of the liposome. Suggested combined approach based on ATR-FTIR, CD, DSC and fluorescence spectroscopy provides evidence that Mox, via its positively charged pipyrazol cycle, can interact with phosphate and carbonyl groups of DPPC\CL²⁻ bilayer. The interaction is temperature-dependent – at higher temperature the mobility of the hydrocarbon chains in lipid bilayer

Declaration of Competing Interest

None.

Acknowledgments

This work is supported by Russian Foundation for Basic Research (RFBR) grant ID 18-33-00134 and The Developmental program of Lomonosov Moscow State University. The authors thank Dr. A.A. Vinogradov for fruitful scientific discussions.

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Moxifloxacin interacts with lipid bilayer, causing dramatic changes in its structure and phase transitions

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Liposome preparation

LMox preparation

Conclusions

Declaration of Competing Interest

Acknowledgments

// BBA - Biomembr.

Biochim. Biophys. Acta - Biomembr. Elsevier B.V.

Biophys. J.

Chem. Phys. Lipids

Colloids Surf. B Biointerfaces

// Spectrochim. Acta Part A Mol. Biomol. Spectrosc. Elsevier B.V.

Biochim. Biophys. Acta - Rev. Biomembr.

Spectrochim. Acta Part A Mol. Biomol. Spectrosc. Elsevier B.V.

J. Pharm. Biomed. Anal.

Chem. Phys. Lipids

Chem. Phys. Lipids

Chem. Phys. Lipids

J. Mol. Struct.

Eur. J. Pharm. Biopharm.

Biochim. Biophys. Acta Biomembr.