Transmembrane gradient driven phase transitions within vesicles: lessons for drug delivery

Abstract

Phase transitions in closed vesicles, i.e., microenvironments defined by the size of the vesicle, its contents, and permeability of its membrane are becoming increasingly important in several scientific disciplines including catalysis, growth of small crystals, cell function studies, and drug delivery. The membrane composed from lipid bilayer is in general impermeable to ions and larger hydrophilic ions. Ion transport can be regulated by ionophores while permeation of neutral and weakly hydrophobic molecules can be controlled by concentration gradients. Some weak acids or bases, however, can be transported through the membrane due to various gradients, such as electrical, ionic (pH) or specific salt (chemical potential) gradients. Upon permeation of appropriate species and reaction with the encapsulated species precipitation may occur in the vesicle interior. Alternatively, these molecules can also associate with the leaflets of the bilayer according to the transmembrane potential. Efficient liposomal therapeutics require high drug to lipid ratios and drug molecules should have, especially when associated with long circulating liposomes, low leakage rates. In this article we present very efficient encapsulation of two drugs via their intraliposomal precipitation, characterize the state of encapsulated drug within the liposome and try to fit the experimental data with a recently developed theoretical model. Nice agreement between a model which is based on chemical potential equilibration of membrane permeable species with experimental data was observed. The high loading efficiencies, however are only necessary but not sufficient condition for effective therapies. If adequate drug retention within liposomes, especially in the case of long-circulating ones, is not achieved, the therapeutic index decreases substantially. Anticancer drug doxorubicin precipitates in the liposome interior in a form of gel with low solubility product and practically does not leak out in blood circulation in the scale of days. With an antibiotic, ciprofloxacin, the high loading efficacy and test tube stability is not reproduced in in vitro plasma leakage assays and in vivo. We believe that the reasons are higher solubility product of precipitated drug in the liposome, larger fraction of neutral molecules due closer pK values of the drug with the pH conditions in the solutions and high membrane permeability of this molecule. High resolution cryoEM shows that encapsulated anticancer agent doxorubicin is precipitated in the form of bundles of parallel fibers while antibiotic ciprofloxacin shows globular precipitate. Doxorubicin gelatin also causes the change of vesicle shape.

Virosomes of hepatitis B virus envelope L proteins containing doxorubicin: synergistic enhancement of human liver-specific antitumor growth activity by radiotherapy

“…Total lipid concentration was estimated from the cholesterol concentration, which was determined by a Cholesterol E-Test Wako kit (Wako, Osaka, Japan) utilizing cholesterol oxidase, peroxidase, 4-aminoantipyrine, and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline. 20 To introduce DOX (Sigma-Aldrich, St Louis, MO, USA) into the virosomes by a remote loading method, 21,22 360 μL of 5 mg/mL DOX-HCl solution was added to 1.4 mL of virosome solution (15 mg as total lipids, prewarmed at 60°C), and then incubated at 60°C for 20 minutes with gentle stirring. After the removal of free DOX by a Sephadex G-25 gel-filtration column equilibrated with 10 mM HEPES buffer (pH 7.4) containing 100 mM NaCl and 3.4% (w/v) sucrose, virosomes containing DOX were obtained.…”

Release of Drugs from Liposomes Varies with Particle Size

“…[6][7][8][9][10] The effectiveness of this formulation approach is dependent on the rate of drug release from the liposomes. Liposomes that rapidly release their contents in vivo will not improve delivery of drugs to target sites 2); for therapeutic value, it is important that drugs are retained in liposomes in vivo for an appropriate time.11,12) Weakly basic drugs can be actively concentrated inside liposomes using a transmembrane pH gradient [13][14][15] or an ammonium sulfate gradient.16) However, the retention of drugs in liposomes is drug-dependent and can vary dramatically. For example, the anticancer drugs doxorubicin and epirubicin are well retained inside liposomes, [17][18][19] whereas the anticancer drug vincristine and the antibiotic ciprofloxacin tend to leak out rapidly.…”

“…11,12) Weakly basic drugs can be actively concentrated inside liposomes using a transmembrane pH gradient [13][14][15] or an ammonium sulfate gradient.…”

“…For example, the anticancer drugs doxorubicin and epirubicin are well retained inside liposomes, [17][18][19] whereas the anticancer drug vincristine and the antibiotic ciprofloxacin tend to leak out rapidly. 14,19,20) In order to obtain homogenous preparations, liposomes are often extruded through polycarbonate filters of 0.4, 0.2 and 0.1 mm pore size. A high proportion of liposomes that are passed through 0.2 mm filters remain as multilamellar vesicles.…”

“…11,12) Weakly basic drugs can be actively concentrated inside liposomes using a transmembrane pH gradient [13][14][15] or an ammonium sulfate gradient. 16) However, the retention of drugs in liposomes is drug-dependent and can vary dramatically.…”