Yaelle Schilt1, Tal Berman2, Xiaohui Wei2, Einat Nativ-Roth3, Yechezkel Barenholz4, Uri Raviv5. 1. Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; The Center for Nanoscience and Nanotechnology of the Hebrew University of Jerusalem, Jerusalem, Israel. 2. Laboratory of Membrane and Liposome Research, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem, Israel; The Center for Nanoscience and Nanotechnology of the Hebrew University of Jerusalem, Jerusalem, Israel. 3. Ilse Katz Institute for Nanoscale Science & Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel. 4. Laboratory of Membrane and Liposome Research, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem, Israel; The Center for Nanoscience and Nanotechnology of the Hebrew University of Jerusalem, Jerusalem, Israel. Electronic address: chezyb@gmail.com. 5. Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; The Center for Nanoscience and Nanotechnology of the Hebrew University of Jerusalem, Jerusalem, Israel. Electronic address: uri.raviv@mail.huji.ac.il.
Abstract
BACKGROUND: In Doxil®, PEGylated nanoliposomes are created by hydration of the lipids in ammonium sulfate, and are remotely loaded with doxorubicin by a transmembrane ammonium gradient. The ammonium sulfate is then removed from the external aqueous phase, surrounding the liposomes, and replaced by an isoosmotic sucrose solution in 10 mM histidine buffer at pH 6.5. METHODS: We prepared PEGylated liposomal doxorubicin (PLD) with a series of ammonium monovalent salts that after remote loading became the intraliposome doxorubicin counteranions. We analyzed the liposomes by solution X-ray scattering, differential scanning calorimetry, and electron micropscopy. RESULTS: PLDs prepared with sulfonic acid derivatives as counteranion exhibited chemical and physical stabilities. We determined the effect of these ammonium salt counteranions on the structure, morphology, and thermotropic behavior of the PEGylated nanoliposomes, formed before and after doxorubicin loading, and the bulk properties of the doxorubicin-counteranion complexes. By comparing the structure of the doxorubicin complexes in the bulk and inside the nanoliposomes, we revealed the effect of confinement on the structure and doxorubicin release rate for each of the derivatives of the ammonium sulfonic acid counteranions. CONCLUSIONS: We found that the extent and direction of the doxorubicin confinement effect and its release rate were strongly dependent on the type of counteranion. The counteranions, however, neither affected the structure and thermotropic behavior of the liposome membrane, nor the thickness and density of the liposome PEG layers. In an additional study, it was demonstrated that PLD made with ammonium-methane sulfonate exhibit a much lower Hand and Foot syndrome. GENERAL SIGNIFICANCE: The structure, physical state, and pharmacokinetics of doxorubicin in PEGylated nanoliposomes, prepared by transmembrane remote loading using gradients of ammonium salts, strongly depend on the counteranions.
BACKGROUND: In Doxil®, PEGylated nanoliposomes are created by hydration of the lipids in ammonium sulfate, and are remotely loaded with doxorubicin by a transmembrane ammonium gradient. The ammonium sulfate is then removed from the external aqueous phase, surrounding the liposomes, and replaced by an isoosmotic sucrose solution in 10 mM histidine buffer at pH 6.5. METHODS: We prepared PEGylated liposomal doxorubicin (PLD) with a series of ammonium monovalent salts that after remote loading became the intraliposome doxorubicin counteranions. We analyzed the liposomes by solution X-ray scattering, differential scanning calorimetry, and electron micropscopy. RESULTS: PLDs prepared with sulfonic acid derivatives as counteranion exhibited chemical and physical stabilities. We determined the effect of these ammonium salt counteranions on the structure, morphology, and thermotropic behavior of the PEGylated nanoliposomes, formed before and after doxorubicin loading, and the bulk properties of the doxorubicin-counteranion complexes. By comparing the structure of the doxorubicin complexes in the bulk and inside the nanoliposomes, we revealed the effect of confinement on the structure and doxorubicin release rate for each of the derivatives of the ammonium sulfonic acid counteranions. CONCLUSIONS: We found that the extent and direction of the doxorubicin confinement effect and its release rate were strongly dependent on the type of counteranion. The counteranions, however, neither affected the structure and thermotropic behavior of the liposome membrane, nor the thickness and density of the liposome PEG layers. In an additional study, it was demonstrated that PLD made with ammonium-methane sulfonate exhibit a much lower Hand and Foot syndrome. GENERAL SIGNIFICANCE: The structure, physical state, and pharmacokinetics of doxorubicin in PEGylated nanoliposomes, prepared by transmembrane remote loading using gradients of ammonium salts, strongly depend on the counteranions.
Authors: Giuseppina Ioele; Martina Chieffallo; Maria Antonietta Occhiuzzi; Michele De Luca; Antonio Garofalo; Gaetano Ragno; Fedora Grande Journal: Molecules Date: 2022-08-25 Impact factor: 4.927
Authors: Emma Grabarnick Portnoy; Alexander V Andriyanov; Hadas Han; Sara Eyal; Yechezkel Barenholz Journal: Pharmaceutics Date: 2021-12-17 Impact factor: 6.321