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Particle structures comprising sterols and saponins

a technology of saponin and sterols, which is applied in the field of complexes of sterols and saponin glycosides, can solve the problems of not being able to remove by dialysis, unable to investigate the possibility that the detergent forms part of the iscom, and limited amount of intact iscom particles, so as to facilitate the uptake of polynucleotides and facilitate complex formation

Inactive Publication Date: 2011-01-27
ISKONOVA AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0056]Lipophilic moieties such as phospholipids are preferably present during the process of forming the complexes according to the invention. The presence of e.g. phospholipids facilitates complex formation over a broad concentration range. Accordingly, phosphatidyl choline may be added when mixing saponins and sterols during complex formation.
[0057]It is preferred to use a phospholipid with a positively charged headgroup such as phosphatidyl ethanolamine. One reason for this is the inclusion into the complex of an overall positive charge capable of facilitating or resulting in an interaction with negatively charged polynucleotides including ribonucleic acids. This interaction will facilitate the uptake of the polynucleotides across a cellular membrane and into a biological cell for subsequent integration and / or translation and / or polypeptide expression. Phosphatidyl choline is an example of another phospholipid capable of being used in connection with the present invention.
[0081]One principal aspect of the invention is thus the provision of a novel system that enhances the uptake of polynucleotides including nucleic acids by combining—in one preferred embodiment—i) the ability of iscom-structures to associate with, and penetrate into or through membranes containing cholesterol, with ii) the property of contacting or associating with polynucleotides including nucleic acids. In particular for the application of vaccination with naked DNA the complexes according to the invention is likely to reduce the required amount of DNA.

Problems solved by technology

Quil A is as such surface-active and hemolytic and may have adverse effects when applied in vivo.
However, it has not been investigated if the detergent forms part of the ISCOMs rendering removal by dialysis impossible.
Adding 20% of ethanol to the reaction mixture resulted in a very limited amount of intact ISCOM particles and clearly mis-shapen structures (FIG. 9B).
ISCOMs were used for immunization, but showed inefficient in inducing antibodies (personal communication) indicating the embedding approach to be inapplicable for peptide vaccines.

Method used

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  • Particle structures comprising sterols and saponins
  • Particle structures comprising sterols and saponins
  • Particle structures comprising sterols and saponins

Examples

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Effect test

example 1

Incorporation of Low-Molecular Weight Substances into ISCOMs

[0489]Immune stimulating complexes (ISCOMs) are cage-like structures of uniform size with a diameter of approximate 40 nm. In general, the term ISCOM is used when antigen is inserted into the structures, whereas ISCOM-matrix denotes structures without antigen. Several reports describe the formation of ISCOMs with amphipathic proteins like virus membrane proteins. But also, non-amphipatic proteins like BSA and OVA as well as hydrophilic proteins linked to fatty acids have been used. Two crucial components for the formation of ISCOMs are cholesterol and phospholipid that are solubilized by detergent. Many protein antigens can be brought into solution by detergents compatible with the process of ISCOM-formation whereas other compounds like lipopeptides require different solvents. The methods reported here allow the embedment of such lipopeptides into the structures of ISCOMs by employing a pre-dissolution step in organic solve...

example 2

Preparation of DNA-Binding Complexes According to the Invention

[0539]This example demonstrates how to prepare micro-particles with the capability of forming an association with DNA by means of an electrostatic interaction The complexes were prepared by substituting a fraction of cholesterol for DC-cholesterol during the formation of the complexes.

[0540]Stock solution of sterol and phospholipid. Three lipid stock solutions was prepared by dissolving Cholesterol, 3β-[N-(N′,N′-Dimet-hylaminoethane)-Carbamoyl] Cholesterol (DC-Cholesterol, Avanti Polar Lipids) and phophatidylcholine (Epikuron 200S, Lucas Meyer Gmbh, Germany) in 20% w / v Mega 10 (N-Decanoyl-N-methyl-glucamide, Sigma-Aldrich). The concentration of the three stocks were (w / v):

DC-CholesterolCholesterolPhosphaditylcholineStock A0.5%0.5%1.0%Stock B0.25%0.75%1.0%Stock C0.13%0.87%1.0%

[0541]Lipids were dissolved by agitation and heating to 40° C. Stock solutions were stored at −20° C. until used.

[0542]Complex formation. A reaction...

example 3

Degradation of Linear Plasmid DNA Bound to DNA-Binding ISCOMs

[0546]The aim of the present example is to examine whether DNA associated with DNA-binding ISCOMs are protected from degradation by DNase I. Lineraized plasmid DNA was enzymatically labeled with 32P. Labeled DNA was allowed to bind to ISCOMs and treated with or without DNAse I. ISCOMs were collected by filtration and the amount of label retained was used as a measurement of the amount of DNA protected from degradation.

[0547]Labeling of DNA. Plasmid pUC18 digested with BamH1 (Amersham-Pharmacia, pUC18 BamH1 / BAP) was labeled using DNA polymerase I Kienow Fragment by adding 10 μCi [α-32P]dGTP (Amersham-Pharmacia, AA0066) to 1 μg of DNA in 25 μl EcoPol Buffer (New England BioLabs, 10 mM Tris-HCl, 5 mM MgCl2, 5 mM DTT, pH 7.5). By further addition of 1 unit Kienow Fragment (New England BioLabs, M0212S) the reaction was initiated. After incubation for 15 minutes at room temperature the reaction was stopped by heating for 5 minut...

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Abstract

The present invention pertains to complexes comprising sterols and saponins. The complexes are capable of binding a genetic determinant including a polynucleotide. The complexes may further comprise a lipophilic moiety, optionally a lipophilic moiety comprising a contacting group and / or a targeting ligand, and / or a saccharide moiety. The complexes may further comprise an immunogenic determinant and / or an antigenic determinant and / or a medicament and / or a diagnostic compound. The complexes may in even further embodiments be encapsulated by an encapsulation agent including a biodegradable microsphere. The present invention also pertains to pharmaceutical compositions and methods of treatment of an individual by therapy and / or surgery, methods of cosmetic treatment, and diagnostic methods practised on the human or animal body.

Description

[0001]This application is a continuation of U.S. application Ser. No. 10 / 114,957, filed on Apr. 4, 2002, now allowed, which claims the benefit of U.S. provisional application Ser. No. 60 / 308,609 filed on Jul. 31, 2001; and of Danish application number PA 200100560, which was filed on Apr. 4, 2001, all incorporated herein by reference in their entireties. All patent and nonpatent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.FIELD OF THE INVENTION[0002]The present invention relates to complexes of sterols and saponin glycosides. The complexes are capable of interacting with bioactive agents including genetic determinants, such as e.g. polynucleotides. The complexes pertaining to the present invention can be used to facilitate the transport of polynucleotides, including DNA and derivatives thereof, across cellular membranes.[0003]Acting as carriers of various bioactive agents and genetic determinants, th...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K49/00A61K47/28A61K31/7088A61K38/02A61K47/42A61K9/14A61K9/00A61P37/04A61K39/39A61K48/00C12N15/87
CPCA61K39/39C12N15/87A61K48/00A61P37/04
Inventor DALSGAARD, KRISTIANKIRKBY, NIKOLAI SOREN
Owner ISKONOVA AB
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