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Lipid particles having asymmetric lipid coating and method of preparing same

a technology of lipid coating and lipid particles, which is applied in the direction of pharmaceutical delivery mechanism, biochemistry apparatus and processes, fermentation, etc., can solve the problems of limited success in intracellular delivery of liposome-entrapped agents, inherent difficulty in delivering a molecule, in particular a large and/or charged molecule, and the difficulty of delivering charged molecules intracellularly

Inactive Publication Date: 2006-07-27
ZHANG YUANPENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The invention provides a method for preparing lipid particles that have a minimal external surface charge after formation, but can develop a charge over time. The particles are composed of a lipid composition containing a charged lipid and a therapeutic agent, and are incubated under conditions effective to remove the charged lipid from the external lipid leaflet. The particles can be liposomes or have an entrapped therapeutic agent. The lipid composition has a phase transition temperature between about 34-38°C. The technical effect of this invention is the ability to prepare lipid particles with a minimal external surface charge after formation, but that can develop a charge over time."

Problems solved by technology

Success in achieving intracellular delivery of a liposome-entrapped agent has been limited for a variety of reasons.
Another reason is the inherent difficulty in delivering a molecule, in particular a large and / or a charged molecule, into the cellular cytoplasm and / or the nucleus.
Delivery of charged molecules intracellularly remains a technical challenge.
In particular, delivery of nucleic acids, both DNA and RNA, has been challenging, due to the charge and size of the molecules.
However, the presence of the positive charge on the external surface of lipid particles prepared with cationic lipids is detrimental to the goal of achieving a long blood circulation lifetime for widespread biodistribution.
The charge on the particles causes immediate binding with the tissue surfaces at or near the site of administration, substantially limiting the availability of particles for circulation and distribution to the target site.

Method used

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  • Lipid particles having asymmetric lipid coating and method of preparing same
  • Lipid particles having asymmetric lipid coating and method of preparing same
  • Lipid particles having asymmetric lipid coating and method of preparing same

Examples

Experimental program
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Effect test

example 1

Preparation of Asymmetric Lipid Particles

[0094] Cationic liposomes (small unilamellar vesicles) were prepared from a lipid composition of DMTAP / DOPE / CHOL / PEG-DS (50 / 24 / 24 / 2 mol / mol). Individual lipid stocks were made in chloroform / methanol (90:10 v / v) at 10 mg / mL for DMTAP (Avanti Polar Lipids, 890860, 20 mg / mL for DOPE (Avanti Polar Lipids, 850725), 20 mg / mL for cholesterol, and 10 mg / mL for mPEG-DS (methoxy-polyethyleneglycol-distearoyl, mPEG molecular weight 2000 Daltons, Shearwater Polymers Inc). Aliquots of solvent solutions containing appropriate amount of lipids for a final lipid suspension of 2 mL at 20 mM lipid concentration were taken using positive displacement pipettes and mixed in 10 mL round bottom flasks. The solvent was slowly removed by rotary evaporation at about 45° C. to form a thin film around the flask. The residual solvent was removed by vacuum overnight. The lipid was then hydrated by adding 2 mL of deionized water at 50° C. for 0.5-1 hour with stirring. The...

example 2

In Vitro Transfection

[0103] Asymmetric lipid-DNA particles and various controls prepared as described in Example 1 were compared in vitro. BHK cells were seeded in 6-well plates at 1.13×104 cells / well and incubated at 37° C., 5% CO2, for 48 hours with complete MEM media. Before the transfection, the cells were rinsed twice with 1.0 mL serum-free MEM media. An aliquot of the transfection sample was mixed with serum-free MEM media to achieve a desired concentration of plasmid DNA (typically 60-200 μg / mL pCC-Luc). For transfection, 1 mL was than overlayed onto the rinsed cells followed by incubation at 37° C. for 5 hours. After incubation, the sample-containing media was aspirated and replaced with 1.0 mL of complete MEM media and the incubation was continued under the same condition for an additional 16.5 hours.

[0104] The luciferease activity was assayed using Promega Luciferase Assay System (cat# E1500). The cells were rinsed twice with phosphate buffered saline (PBS) and then 250 ...

example 3

Preparation of Asymmetric Lipid Particles

[0106] Lipid particles were formed as described in Example 1, except that the temperature of the incubation solution comprised of neutral SUVs (see step 4 C in Example 1) was maintained between 0-4° C. with an ice bath.

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PUM

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Abstract

A method of preparing lipid particles having an asymmetric lipid coating is described. The lipid composition of the outer lipid coating of the particles varies from the inner to outer surfaces. The asymmetric lipid particles are formed by preparing a lipid composition containing a charged lipid and a therapeutic agent, where the particles each have an outer lipid coating with an external lipid leaflet and an internal lipid structure. The particles are then incubated under conditions effective to remove the charged lipid from the external lipid leaflet, thus rendering the lipid coating asymmetric. The particles have the ability to their regain surface charge via translocation of the lipids.

Description

[0001] This application is a divisional of U.S. application Ser. No. 10 / 814,703 filed Mar. 30, 2004, which claim the benefit of U.S. Provisional Application No. 60 / 459,305, filed Nov. 14, 2003 and U.S. Provisional Application No. 60 / 459,305 filed Mar. 30, 2003. All of these documents are incorporated by reference in their entirety.FIELD OF THE INVENTION [0002] The present invention relates to a lipid particle composition having an asymmetric lipid coating, for use in delivery of therapeutic agents to a person, and more specifically, to a cell. BACKGROUND OF THE INVENTION [0003] Lipid vesicles, or liposomes, have demonstrated utility for delivering therapeutic agents and diagnostic agents to target tissues and organs. Lipid vesicles have an aqueous interior enclosed by one or more lipid bilayers, where the therapeutic agent is entrapped in the aqueous interior spaces or within the lipid bilayer. Thus, both water-soluble and water-insoluble drugs can be transported by lipid vesicles w...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K9/127C12N15/88A61K47/48
CPCA61K9/1271A61K9/1272A61K9/1277A61K47/48815A61K47/6911A61K9/127A61K47/50
Inventor ZHANG, YUANPENG
Owner ZHANG YUANPENG
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