Method of monitoring the release from liposomes of a product of interest using superparamagnetic nanoparticles
The invention pertains to a method of monitoring the membrane permeabilization of a liposome and the incidental release of a compound of interest. The method utilizes liposomes comprising a thermosensitive lipidic membrane encapsulating the product of interest and superparamagnetic nanoparticles having the electrostatic surface charge below −20 mV or above +20 mV when measured in an aqueous medium at physiological pH. In one embodiment, the method comprises the steps of: a) measuring relaxation time (T2*); b) heating the liposome at Tm or above Tm; c) measuring T2* after step b); d) obtaining the transverse relaxivity (r 2* ) values from the T2* values obtained from step a) and step c); and e) determining the ratio of r 2* before and after the heating step b). A ratio above 1.5 indicates the liposome membrane permeabilization and the incidental release of the product of interest.
1. A method of monitoring a liposome membrane permeabilization and the incidental release of a product of interest encapsulated in the liposome, wherein the liposome comprises a thermosensitive lipidic membrane encapsulating the product of interest and superparamagnetic nanoparticles, the superparamagnetic nanoparticles having an electrostatic surface charge below −20 mV or above +20 mV when measured in an aqueous medium between pH 6 and 8, and wherein the method comprises:
a) measuring T2* allowed by the superparamagnetic nanoparticles,
b) heating the liposome at phase transition temperature (Tm) or above Tm,
c) measuring T2* allowed by the superparamagnetic nanoparticles after step b),
d) obtaining the transverse relaxivity (r 2* ) values from the T2* values obtained from step a) and step c), and
e) determining the ratio of r 2* before the heating step at Tm or above Tm/r 2* after the heating step at Tm or above Tm, a ratio above 1.5 being indicative of the liposome membrane permeabilization and of the incidental release of the product of interest, thereby monitoring the liposome membrane permeabilization.
2. The method according to claim 1 , wherein the method further comprises at least one step of measuring T2*, or T2 and T2* allowed by the superparamagnetic nanoparticles, during the heating step b).
3. The method according to claim 1 , wherein Tm is between 39° C. and 45° C.
4. The method according to claim 1 , wherein the thermosensitive lipidic membrane comprises at least a phosphatidylcholine.
5. The method according to claim 4 , wherein the thermosensitive lipidic membrane further comprises cholesterol.
6. The method according to claim 4 , wherein the thermosensitive lipidic membrane further comprises distearylphosphatidylethanolamine-methoxypolyethylene glycol.
7. The method according to claim 5 , wherein the thermosensitive lipidic membrane further comprises distearylphosphatidylethanolamine-methoxypolyethylene glycol.
8. The method according to claim 1 , wherein the largest size of the liposome is between 50 and 500 nm.
9. The method according to claim 1 , wherein the nanoparticles are covalently or electrostatically fully coated with an agent selected from a carboxylic acid, a phosphate and an amine.
10. The method according to claim 1 , wherein the nanoparticles are prepared from an iron oxide selected from magnetite and/or maghemite.
11. The method according to claim 1 , wherein the nanoparticle largest size is between 2 and 30 nm.
12. The method according to claim 1 , wherein the product of interest is selected from a therapeutic nucleic acid, a cytostatic compound, and a cytotoxic compound.
13. The method according to claim 1 , wherein said thermosensitive lipidic membrane comprises dipalmitoylphosphatidylcholine (DPPC), hydrogenated soybean phosphatidylcholine (HSPC), cholesterol and distearylphosphatidylethanolamine (DSPE)-methoxypolyethylene glycol (PEG) in a molar ratio of 100:50:30:6 or 100:33:27:7.
14. The method according to claim 1 , wherein said thermosensitive lipidic membrane comprises dipalmitoylphosphatidylcholine (DPPC), monopalmitoylphosphatidylcholine (MPPC) and distearylphosphatidylethanolamine (DSPE)-methoxypolyethylene glycol (PEG) in a molar ratio of 100:12:5.
15. The method according to claim 1 , wherein said thermosensitive lipidic membrane comprises dipalmitoylphosphatidylcholine (DPPC), monostearylphosphatidylcholine (MSPC), and distearylphosphatidylethanolamine (DSPE)-methoxypolyethylene glycol (PEG) in a molar ratio of 100:12:5.
16. The method according to claim 1 , wherein the method consists of:
a) measuring T2* allowed by the superparamagnetic nanoparticles,
b) heating the liposome at phase transition temperature (Tm) or above Tm,
c) measuring T2* allowed by the superparamagnetic nanoparticles after step b),
d) obtaining the transverse relaxivity (r 2* ) values from the T2* values obtained from step a) and step c), and
e) determining the ratio of r 2* before the heating step at Tm or above Tm/r 2* after the heating step at Tm or above Tm, a ratio above 1.5 being indicative of the liposome membrane permeabilization and of the incidental release of the product of interest, thereby monitoring the liposome membrane permeabilization.
17. A method of monitoring a liposome membrane permeabilization and the incidental release of a product of interest encapsulated in the liposome, wherein the liposome comprises a thermosensitive lipidic membrane encapsulating the product of interest and superparamagnetic nanoparticles, the superparamagnetic nanoparticles having an electrostatic surface charge below −20 mV or above +20 mV when measured in an aqueous medium between pH 6 and 8, and wherein the method consists of:
a) measuring T2* and longitudinal relaxation time (T1) allowed by the superparamagnetic nanoparticles,
b) heating the liposome at Tm or above Tm,
c) measuring T2* and T1 allowed by the superparamagnetic nanoparticles after step b),
d) obtaining r 2* and longitudinal relaxivity (r 1 ) values from the T2* and T1 values obtained from steps a) and c), and
e) determining the ratio of r 2* /r 1 before and after the heating step at Tm or above Tm, a ratio of r 2* /r 1 before the heating step b) and of r 2* /r 1 after the heating step b) above 2 being indicative of the liposome membrane permeabilization and of the incidental release of the product of interest, thereby monitoring the liposome membrane permeabilization.
18. The method according to claim 17 , wherein the method further comprises at least one step of measuring T2* and T1 allowed by the superparamagnetic nanoparticles, and optionally T2 allowed by the superparamagnetic nanoparticles, during the heating step b).