Experimental data for ethidium bromide displacement was obtained from Lobo'1

1
Objectives / Goals
Correlations

• Collect solubility data for DNA complexed with
• 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)
  at various charge ratios.
• Correlate a relationship to calculate ethidium
  bromide displacement based on structure
  descriptors of the complex.
• Determine if a linear relationship exists between
  connectivity and shape descriptors to predict
  circular dichroism.
• Inquire about a method to compute accessible
  surface area of molecules.
• Experience the techniques and methods associated
  with research at the graduate level. Develop a
  confidence within the laboratory and utilize
  computational methodologies to accurately
  describe molecular behavior.

• Experimental data for ethidium bromide
  displacement was obtained from Lobo.1
• Peptoids and lipitoids possess physical and
  chemical characteristics that make them unique
  candidates for gene delivery.
• The data for cationic lipids and other polymers
  are used to generate correlations using the
  statistical software SPSS.
• The Ethidium Bromide Relative Florescence
  Intensity correlates to lipid DNA binding.

• Circular dichroism (CD) data for complexes
  containing cationic lipids and peptides which are
  currently the most promising non-viral gene
  delivery vehicles candidates were provided by
  Wiethoff. 3
• The data for cationic lipids and peptides are
  used to generate correlations using the
  statistical software SPSS.
• Although there are changes in the spectra upon
  complexation, the DNA actually remains in the
  ß-form.

Background and Definitions

• Non-Viral Gene Delivery is a technique that
  involves the use of plasmid DNA alone or
  sometimes complexed with lipids or polymers that
  are then inserted into cells to produce
  beneficial effects. Non-viral gene therapy often
  has low transfection efficiency because of the
  inability to overcome barriers within the body.
• Transfection Efficiency defines the capability of
  gene delivery vehicles to transfer plasmid DNA
  into the nucleus. It can be measured using
  fluorescence techniques.
• SPSS is a statistical software package used to
  comprehensively analyze data from spreadsheet
  input.
• PEG Complexation Study is a laboratory technique
  that uses polyethylene glycol to encourage
  solubility and to then test for net absorbance.
  
• Molar Ellipticity T is the comparison of
  ellipticity (T) to accurately compare secondary
  structure of proteins.
• T T / (10 x c x l) where c is the molar
  concentration
• of the sample (mole/L) and l is the
  pathlength in cm. 5

Future Work

• Improving transfection efficiency is the ultimate
  goal for gene delivery. Unfortunately, it is
  extremely difficult to correlate transfection
  efficiency directly with descriptors for various
  physical properties.
• Ethidium Bromide Relative Florescence Intensity
  corresponds to different levels of lipid binding,
  by describing the amount of complexation to the
  polymer. This can be further researched by
  expanding experimental data.
• It would be beneficial to apply more descriptors
  to accurately describe the DNA complexes. One
  such descriptor that may improve correlations
  would be the inclusion of accessible surface
  area.

DNA Complex Solubility Data

• Plasmid DNA must first be complexed with either
  cationic lipids or peptides (DOTAP was used in
  this experiment). The complex is only stable for
  72 hours.
• The equivalent amount of DNA complex is then
  mixed with various amounts of PEG and 10 mM Tris
  buffer to incubate for multiple hours at room
  temperature.
• Place DNA and PEG solution into centrifuge to
  encourage precipitate to gather at bottom.
• Using UV Spectroscopy, measure the net absorbance
  of the complex solution.
• The PEG procedure was not useful in determining
  the solubility of DNA complexes since the
  complexes are unstable for unknown extended
  periods of time. One also can not accurately
  measure absorbance because the mixture acts like
  a colloidal dispersion rather than a
  solvent/solute system.

Conclusion

• The summer research focused on solubility, charge
  ratio (CR), shape indices, and connectivity index
  values in attempt to describe properties of the
  unknown DNA complex.
• The solubility experiments for DNA complexes
  based on the PEG method were unsuccessful in the
  manner that they did not present any useful
  information regarding the solubility properties
  of the complexes, but helpful in the fact that a
  PEG lab performed with DNA has never been
  developed before, and now the results are known
  when working with the unstable complexes.
• Computational analysis of complex properties
  based on various descriptors of the assorted
  complexes with different polymers, developed more
  meaningful results in the research aspect.
• Correlations for several properties of non-viral
  gene delivery polymers have been created using
  structural descriptors.
• Correlations between the ethidium bromide
  extrusion and charge ratio, kappa, and chi values
  were determined.
• Circular dichroism data was more difficult to
  manipulate which created various barriers to
  conquer. The data available from different
  researchers was recorded in inconsistent form,
  which made analysis problematic.
• The greater the understanding of DNA complexes
  and the higher accuracy associated with
  predicting unknown molecules will optimize the
  speed and efficiency of the creation of future
  non-viral gene delivery vehicles.

References / Acknowledgments
Cationic lipid

• 1. Lobo, Brian A., Ph.D. Thesis, University
  of Kansas (2002)
• 2. Choosakoonkriang, Sirirat, Ph.D. Thesis,
  University of Kansas (2002)
• 3. Wiethoff, Christopher, Ph.D. Thesis,
  University of Kansas (2002)
• 4. Morel, Elizabeth L., University of Kansas
  (Fall 2003-Spring 2004)
• 5. www.astbury.leeds.ac.uk/Facil/cdpage.htm

Cationic Lipid is Complexed with DNA
Dr. Kyle V. Camarda and Hui Zhao provided
consistent support, advice, and knowledge
throughout the research experience. Dr. Russell
Middaugh, Chad Braun, and Haihong Fan of
Pharmaceutical Chemistry aided in the development
of the solubility experiment and interpretation
of results.