FreshPatents.com Logo
stats FreshPatents Stats
3 views for this patent on FreshPatents.com
2011: 2 views
2009: 1 views
Updated: March 31 2014
newTOP 200 Companies filing patents this week


    Free Services  

  • MONITOR KEYWORDS
  • Enter keywords & we'll notify you when a new patent matches your request (weekly update).

  • ORGANIZER
  • Save & organize patents so you can view them later.

  • RSS rss
  • Create custom RSS feeds. Track keywords without receiving email.

  • ARCHIVE
  • View the last few months of your Keyword emails.

  • COMPANY DIRECTORY
  • Patents sorted by company.

AdPromo(14K)

Follow us on Twitter
twitter icon@FreshPatents

High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them

last patentdownload pdfimage previewnext patent


Title: High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them.
Abstract: The present invention relates to novel fusogenic vesicles as highly efficient and versatile encapsulation systems for delivering a substance of choice, such as nucleic acids, proteins, peptides, antigens, pharmaceutical drugs and cosmetic agents to cells and tissues. ...


USPTO Applicaton #: #20090280163 - Class: 424450 (USPTO) - 11/12/09 - Class 424 
Drug, Bio-affecting And Body Treating Compositions > Preparations Characterized By Special Physical Form >Liposomes

view organizer monitor keywords


The Patent Description & Claims data below is from USPTO Patent Application 20090280163, High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them.

last patentpdficondownload pdfimage previewnext patent

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/428,435, filed Nov. 21, 2002, the entire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to novel fusogenic vesicles as highly efficient and versatile encapsulation systems for delivering a substance of choice, such as nucleic acids, proteins, peptides, antigens, pharmaceutical drugs and cosmetic agents to cells and tissues.

BACKGROUND OF THE INVENTION

Various publications or patents are referred to in parentheses throughout this application to describe the state of the art to which the invention pertains. Each of these publications or patents is incorporated by reference herein.

One of the paramount goals of medical therapy is the efficient delivery of therapeutic substances to the site of disease. While some therapeutic substances can be delivered in free form, others require a carrier or vector in order to reach and enter their final destination, either due to their rapid clearance from the area of introduction or their inability to cross biological barriers, or due to their systemic toxicity. Delivery of substances to cells and tissues requires vectors which are efficient, flexible, easy to prepare and safe. Currently available methods for delivering substances to eukaryotic cells involve the use of either viral or non-viral vectors. Viral vectors are replication-defective viruses with part of their coding sequences replaced by that of a therapeutic gene. Although recombinant viruses are highly efficient gene delivery and expression vectors, they are currently limited to the delivery of nucleic acids and their safety profiles have not yet been established for medical use in humans.

Most non-viral delivery systems operate at the following levels: loading of the delivery vector with a substance of interest (e.g. proteins, peptides, nucleic acids, pharmaceutical or other therapeutic drugs), endocytosis, and in the case of gene delivery, nuclear targeting and entry. The major drawback of non-viral systems, such as liposomes, is their low delivery efficiency to cells (Chu et al., id.; Legendre and Szoka, 1992, Pharmaceut. Res. Vol. 9, P. 1235), presumably due to the absence of fusion-mediating molecules on the surface of the liposomes. A hybrid type of delivery system, the virosome, combines the efficient delivery mechanism of viruses with the versatility and safety of non-viral delivery systems. Virosomes are reconstituted envelopes without the infectious nucleocapsids and the genetic material that can be derived from a variety of viruses. These virosomes are functional, in that their membrane fusion activity closely mimics the well-defined low-pH-dependent membrane fusion activity of the intact virus, which is solely mediated by the viral fusion protein. Like viruses, virosomes are rapidly internalized by receptor-mediated endocytosis. In contrast to viral systems virosomes are safe, since the infectious nucleocapsid of the virus has been removed. Thus, virosomes represent a promising carrier system for the delivery of a wide variety of different substances, either encapsulated in their aqueous interior or co-reconstituted in their membranes. Co-reconstitution of different receptors within the virosomal membrane, furthermore, allows the targeting of virosomes to different cells or tissues. So far, virosomes are mainly used as vaccines by adding antigen onto the surface of the virosomes.

A major limitation of the protocol currently used to prepare virosomes is that it does not result in high encapsulation efficiency. At the lipid concentration at which virosomes are produced (˜1 mM lipid), and given their mean diameter of approximately 200 nm, less than 1% of the aqueous phase will be entrapped within the virosomes (Schoen et al., J. Liposome Res. 3: 767-792, 1993). Such low entrapment rates reduce virosome-mediated efficiency of drug or gene delivery to cells. The development of new/novel, more efficiently loaded vesicles that retain the advantageous fusion properties of virosomes, as well as methods of making, loading, and delivering them would thus be a highly desirable goal in the field of therapeutic drug, protein and gene delivery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: This figure shows the simplified scheme representing the overall strategy of the invention. In a first fusion step, chimeric virosomes are fused at 4° C. (pH 5.0) with liposomes containing the substance to be delivered, (in this example DNA) mediated by X-31 HA. Subsequently, the resulting neutralized fusion products are incubated with cells. After receptor-mediated endocytosis, the virosome-like proteoliposomes now containing the substance to be delivered undergo a second round of fusion, triggered by the low pH within endosomes and mediated by PR8/34 HA, releasing the entrapped substance into the cytosol.

FIG. 2: This figure shows the presence of both types of fusion proteins in the membrane of the chimeric virosomes. SDS-PAGE of X-31 and PR8 virus and virosomes. Approx. 10 μg virus and 3 μg virosomes were loaded under non-reducing conditions on a SDS-Tris/Tricine 10% (v/v) polyacrylamide gel (Schäigger et al., Anal. Biochem. 166: 368-379, 1987). The polyacrylamide gel was stained with Coomassie blue. Lane 1: X-31 virus. Lane 2: X-31 virosomes. Lane 3: chimeric virosomes. Lane 4: PR8 virosomes. Lane 5: PR8 virus.

FIG. 3: This figure shows the morphology of the chimeric virosomes by cryo-TEM. They are unilamellar vesicles with diameters ranging from 150-250 nm. Scale bar 100 nm.

FIG. 4: This figure shows the fusion activity of X-31, PR8 viruses, virosomes and X-31/PR8 chimeric virosomes with liposomes. Fluorescence was recorded continuously at excitation and emission wavelengths of 465 nm and 530 nm, respectively. Measurements were carried out with a SPEX-Fluorolog-2 fluorometer (SPEX Industries, Inc., Edison, N.J., USA) equipped with a thermostated cuvette holder and a magnetic stirring device. For calibration of the fluorescence scale the initial residual fluorescence of the liposomes was set to zero and the fluorescence at infinite probe dilution to 100%. The latter value was determined by addition of Triton X-100 (0.5% v/v). 1) X-31 virus and virosomes fused with POPC/POPG-LUVs. 2) PR8/34 virus and virosomes incubated with POPC/POPG-LUVs. 3) Chimeric virosomes incubated with POPC/POPG-LUVs. a) fusion measurements at 4° C.; b) Virus and virosomes were incubated at 4° C. and pH 5.0 for 1 h with POPC/DDAB-LUVs before fusion measurements at 37° C.

FIG. 5: This figure shows the encapsulation efficiency of DNA into POPC/DDAB-liposomes. DNA-containing liposomes were prepared by the freeze/thaw method as described in the examples and digested for 3 h at 37° C. with DNase I/Exonuclease III. After that, encapsulated DNA was phenol/chloroform extracted and subjected to gel electrophoresis on a 1% agarose gel. In lane M 1 Kb plus Ladder marker was loaded. As control 100 ng (lane 1) and 200 ng plasmid-DNA (lane 2) were directly loaded on the gel. As another control untreated (not DNase I digested) DNA-liposomes corresponding to 20 ng (lane 3), 50 ng (lane 4), 100 ng (lane 5) and 200 ng (lane 6) plasmid were phenol/chloroform extracted. The aqueous phase was loaded on the gel. DNase I digested liposomes corresponding to an initial DNA-amount of 50 ng (lane 7) and 100 ng (lane 8) were treated like the samples before. Lane 9 and 10 correspond to the same amount of liposomes, except that liposomes had been solubilized with 1% (v/v) Triton X-100 before DNase I treatment. In lane 11 and 12 DNA was not encapsulated, but added to empty liposomes. Also these liposomes were treated as described above.

FIG. 6: This figure shows the morphology of loaded liposomes by cryo-TEM of. They are unilamellar and measure between 100-150 nm in diameter. Scale bar 100 nm.

FIG. 7: This figure shows the products of the fusion between the chimeric virosomes and the loaded liposomes by cryo-TEM before (upper panels) and after extrusion (lower panels). Before extrusion, the vesicles are of widely varying sizes with some vesicles exceeding 900 nm. This result indicates multiple rounds of fusion. Scale bars upper panels: 500 nm; scale bars lower panels: left: 100 nm; right: 200 nm.

FIG. 8: This figure demonstrates the fusogenicity of the extruded vesicles resulting from the fusion of chimeric virosomes with liposomes. Remarkably, the vesicles retain their fusion activity. The proteoliposomes were extruded through nucleopore membranes. Fluorescence was recorded continuously at excitation and emission wavelengths of 465 nm and 530 nm, respectively. Measurements were carried out with a SPEX-Fluorolog-2 fluorometer equipped with a thermostated cuvette holder and a magnetic stirring device. 100% corresponded to infinite probe dilution, determined after addition of Triton X-100 (0.5% v/v). Fusion measurements were carried out at 37° C. and pH 5.0 with POPC/POPG (4:1)-LUVs containing 0.6% N-NBD-PE and N-Rh-PE.

FIG. 9: This figure shows that the proteoliposomes are rapidly internalized by cells. Rhodamine-labeled proteoliposomes are detected in MDCK and HeLa cells. Fused virosomes were bound at 4° C. for 1 h to HeLa and MDCK cells. After that, unbound material was washed away. Cells were immediately fixed (A), or incubated for another 15 min at 37° C., fixed with 3.7% (v/v) formaldehyde and analyzed by fluorescence microscopy (B).

FIG. 10: This figure shows the rapid uptake of proteoliposomes loaded with Texas Red dextran by HeLa cells. HeLa cells were incubated with Texas Red dextran-containing proteoliposomes for 1 h at 4° C. After that, unbound material was washed away and fixed with 3.7% (v/v) formaldehyde (A). Otherwise, cells were further incubated for 15 min (B), 2 h (C) or 5 h (D) at 37° C. After fixation, cells were analyzed by fluorescence microscopy. Objective magnification: for (A) 63×; for (B), (C), (D) 100×.



Download full PDF for full patent description/claims.

Advertise on FreshPatents.com - Rates & Info


You can also Monitor Keywords and Search for tracking patents relating to this High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them patent application.
###
monitor keywords



Keyword Monitor How KEYWORD MONITOR works... a FREE service from FreshPatents
1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored.
3. Each week you receive an email with patent applications related to your keywords.  
Start now! - Receive info on patent apps like High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them or other areas of interest.
###


Previous Patent Application:
High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them
Next Patent Application:
Therapy for human cancers using cisplatin and other drugs or genes encapsulated into liposomes
Industry Class:
Drug, bio-affecting and body treating compositions
Thank you for viewing the High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them patent info.
- - - Apple patents, Boeing patents, Google patents, IBM patents, Jabil patents, Coca Cola patents, Motorola patents

Results in 0.73598 seconds


Other interesting Freshpatents.com categories:
Amazon , Microsoft , IBM , Boeing Facebook -g2-0.3294
     SHARE
  
           

FreshNews promo


stats Patent Info
Application #
US 20090280163 A1
Publish Date
11/12/2009
Document #
12029454
File Date
02/11/2008
USPTO Class
424450
Other USPTO Classes
514 44/R, 514 44/A
International Class
/
Drawings
9


Cosmetic Agents
Fusogenic Vesicle
Vesicle
Vesicles


Follow us on Twitter
twitter icon@FreshPatents