Sonoporation

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Sonoporation

• Ultrasound creates transient permeability of cell
  membranes in the presence of microbubbles.
• Effect allows foreign molecules to enter cell.
• Ultrasound has been found to increase the
  effectiveness of almost any gene transfer
  technique.
• Effective both in vitro and in vivo
• Greatly increases transfer of DNA into cells (up
  to 7000 fold over naked DNA alone (1))

(1) Yoshiaki Taniyama, MD, PhD et. al. Local
Delivery of Plasmid DNA into Rat Carotid Artery
Using Ultrasound. Circulation March 12, 2002
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Microbubbles

• The key to transfection using naked DNA and
  ultrasound alone.
• Enhance effectiveness of gene transfer using
  naked DNA
• Air or inert gas encapsulated in a microscopic
  protein, lipid, or polymer sphere
• Commonly used, and FDA approved, to improve
  contrast in diagnostic ultrasound
• Example Optisontm (Octafluoropropane gas
  encapsulated in a human albumin sphere) is a
  commonly used brand of microbubble, found to be
  very effective when used for gene transfer

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Microbubble Effects

• The mechanism is not completely understood
• Micrographs show bubble size to oscillate under
  ultrasound until disintegration occurs.
• Recent research suggests a cascade of cavitation
  events initiated by microbubble collapse.

Cheri X. Deng, Fred Seiling, Hua Pan, and Jianmin
Cui. Ultrasound-Induced Membrane Porosity.
Ultrasound in Medicine and Biology 2004 Vol 30
No. 4519-526
James Chomas, Paul Dayton, Donovan May, Kathy
Ferrara. Threshold of fragmentaion for ultrasonic
contrast agents. Journal of Biomedical Optics.
April 2001 6(2)141-150
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Microbubbles, Continued

• 1 Mhz ultrasound results in the greatest
  expansion of microbubbles prior to destruction.
• Cell walls show small pores following application
  of ultrasound in the presence of microbubbles

James Chomas, Paul Dayton, Donovan May, Kathy
Ferrara. Threshold of fragmentation for
ultrasonic contrast agents. Journal of Biomedical
Optics. April 2001 6(2)141-150
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Lipids and Liposomes

• Effectiveness enhanced by ultrasound
  irradiation(2)
• Many types commercially available as transfection
  reagents
• Increase transfection rates over naked pDNA
  both in vitro and in vivo
• Liposomes can behave similarly to microbubbles
• Little research is available on why non-liposomal
  transfection reagents are also enhanced by
  ultrasound.

(2) Shaoling Huang, et al. Combined use of
Ultrasound and Acoustic Cationic Liposomes
Results in Improved Gene Delivery into Smooth
Muscle Cells. American Society of Gene Therapy
Annual Meeting. June 2002.
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Simplified in vitro protocol

• Culture cells in appropriate medium
• Construct or purchase plasmid containing gene of
  interest
• Replace medium with serum free medium containing
  plasmid DNA and Optisontm or a lipid based
  transfection reagent
• Apply ultrasound
• Restore optimal growth medium

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Simplified in vivo protocol

• Prepare solution of plasmid DNA containing gene
  of interest and Optisontm or a lipid based
  transfection reagent
• Inject solution into organism
• Apply ultrasound to area of tissue or organ to be
  transfected

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Ultrasound for drug delivery

• Ultrasound is also an effective method of
  targeted drug delivery.
• Drugs are encapsulated in Liposomes or
  Microbubbles
• Application of ultrasound fragments the Liposome
  or Microbubble and drugs are released at the
  target site.

Shao-Ling Huang, David D. McPherson, Robert C.
MacDonald Acoustically Active Liposomes for
Oligonucleotide/Drug Encapsulation Delivery
Agents with Capacity for Ultasound-Triggered
Release. Molecular Therapy, Supplement 1 May 2004
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Typical Ultrasound Parameters

• 1 MHz is most commonly used frequency
• Microbubbles and Liposomes show greatest relative
  expansion at 1MHz
• Intensity of 2 W/cm2 power output or less for in
  vitro use, higher power may improve results in
  vivo
• Exposure duration of 1 minute or less for most
  application
• Intensity, duration and duty cycle vary greatly
  by application

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Cell Viability

• Cell damage is far less extensive than with
  electroporation
• Limiting microbubble concentrations, reduces
  associated cell damage(3)
• Membrane recovery time in healthy cells is shown
  to be as short as 3 to 10 seconds(4)

(3) Tiell Li, PhD et al. Gene Transfer with
Echo-enhanced Contrast Agents Comparison between
Albunex, Optison and Levovist in Mice-Initial
Results. Radiology November 2003
(4) Cheri X. Deng, Fred Seiling, Hua Pan, and
Jianmin Cui. Ultrasound-Induced Membrane
Porosity. Ultrasound in Medicine and Biology
2004 Vol 30 No. 4519-526
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The Sonitron 2000

• Designed for research
• Dual probes allow multiple probe sizes on single
  unit
• Power up to 5W/cm2
• Can be customized to suit individual needs
• Proven results in labs worldwide!

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Wide Range of Probes

• Probe sizes from 0.5mm to 38mm
• Two probes per unit allows a single Sonitron to
  be used for multiple protocols

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• Industry leader in therapeutic ultrasound
• 1 Year warranty, including probes and cables
• Hardware and Application support readily
  available
• Experienced staff to respond to your individual
  needs, including custom devices