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Optimizing the Binding/Release Efficiency of siRNA/Hydrophilic-block-Cationic
Copolymer Complexes
Andrew C. Holley,‡ Adam W. York,‡ DeeDee Smith,‡ Charles L. McCormick‡§
‡The
Department of Polymer Science and §Department of Chemistry and Biochemistry
The University of Southern Mississippi, Hattiesburg MS 39406
Folic Acid Targeted Complexes
Silencing Ribonucleic Acid - siRNA
• Double stranded ribonucleic acid consisting of 19-25
base pairs possessing a 2-nucleotide 3’-overhang
• RISC, an enzyme, facilitates both the unwinding of
siRNA and complexation of its mRNA counterpart
• Once complexed with its mRNA counterpart, RISC
cleaves
the
mRNA
strand
which
deregulates/eliminates the specific protein leading to
the desired therapeutic effect (i.e. gene knockdown)
Synthesis of Targeted Cationic Polymer by RAFT
Copolymer Characterization
Fluorescence Microscopy Images of siRNA Delivery
KB (FA overexpressed)
A549 (FA low)
Pol1
Pol1EC
Pol1ECFA
1.0
Abs
Project Goals
Lipofectamine
0.5
0.0
Advantages:
• siRNA as a therapeutic has the ability for specific
knock down any gene of interest.
Disadvantages:
• Short-half life in serum (i.e. premature degradation).
• Cellular recognition is nonexistent
Fire, A.; Montgomery, M.K.; Kostas, S.A.; Driver, S.E.; Mello, C.C. Nature 1998, 391, 806-811
Dorsett, Y.; Tuschl, T. Nature 2004, 3, 318-329
Polymer
York, A.W.; Zhang, Y.; Holley, A.C.; Guo, Y.; Huang, F.; McCormick, C.L. Biomacromolecules 2009, 10, 936-943
• Molecular weights were in the range of 50 kDa with PDIs < 1.2
• 1H NMR, Uv-Vis, and MALDI-ToF confirm the incorporation of ~ 11
FA moieties into the copolymer side-chain.
Gene Knockdown Efficiency of Complexes in KB Cells
Synthetic approach:
Utilize ARAFT polymerization in the construction of
biocompatible-cationic block copolymers for the complexation and subsequent
delivery of siRNA
The proposed RAFT main equilibrium.
500
UV-Vis spectra of Pol1 before end capping
(black), after end capping (red), and after FA
and SATA conjugation (blue).
• ASEC-MALLS of the synthesized
polymers are in agreement with theorized
molecular weights, and posses narrow
molecular weight distributions.
• Folic acid and SATA content were
determined via UV-Vis spectroscopy and
1H NMR spectroscopy. Four to five folic
acid residues are incorporated along the
polymer. In addition, approximately 18
SATA residues are present in the polymer.
siRNA-polymer Complex
• Lipofectomine (control) shows nonspecific delivery
to both cell lines
• siRNA-polymer complexes demonstrate specific
delivery to KB cells (high Folic Acid expression)
compared to A549 cells (low Folic Acid expression)
• Control (unprotected siRNA) shows no gene knockdown
• KB cells treated with FA-siRNA-polymer complexes show 60% gene knockdown
efficiency
• Cells pretreated with excess folic acid (KB/free FA) and then FA-siRNA-polymer
complexes had negligible knockdown due to competitive binding of free folic acid
• A549 cells, that have low folic acid expression, did not exhibit gene knockdown
400
Wavelength (nm)
ASEC-MALLS chromatograms of narrowly
dispersed, synthesized polymers (Pol1-Pol4).
Design a vehicle for the effective and specific delivery of siRNA to cancer cells
Approach
300
1H
NMR spectra of SATA (blue, top), di-NHS
Folic acid (middle, green), and Pol2 after
SATA and FA conjugation (bottom, red).
Folic acid conjugated siRNA/copolymer complexes show target-specific delivery.
Copolymer molecular weights agree well with theory. The presence of FA
and SATA were confirmed utilizing 1H NMR and UV-Vis spectroscopies with
4-5 and 18 residues per polymer, respectively
Advantages of RAFT:
• Performed under mild reactions conditions
• Tolerant to a wide range of functional groups including free amines
• Yields polymers with controlled molecular weights and narrow distributions
Tailored Binding of siRNA
RAFT Monomers
• N-(2-hydroxypropyl)methacrylamide (HPMA) provides hydrophilicity
and biocompatibility to the polymer.
• N-(3-aminopropyl)methacrylamide (APMA) allows for conjugation of
biologically relevant compounds (e.g. fluorescent compounds,
cellular receptor targeting, etc).
• N,N’-(3-dimethylaminopropyl)methacrylamide (DMAPMA) is cationic
at physiological pH and provides a site for siRNA complexation.
HPMA
APMA
DMAPMA
Moad et al. Aust .J. Chem. 2005, 58, 379
Ringsdorf, H.J. J. Polym. Sci. Symp. 1975, 51, 135-153
siRNA-Cationic Polymer Complex
• The cationic charge density is varied through
the hydrophilic incorporation of HPMA in the
cationic block.
• The binding and release of siRNA to/from these
polymeric carriers is vital for gene knockdown
efficacy.
• The varied charge density should weaken
siRNA binding allowing for a more facile release
mechanism, while varying the incorporation of
targeting moieties allows for an increased or
decreased cellular response. Combining these
approaches will lead to increased gene
knockdown efficacy.
Conclusions
In the proposed research, Reversible addition-fragmentation chain transfer polymerization (RAFT) is
used to polymerize hydrophilic, biocompatible N-(2-hydroxpropyl)methacrylamide (HPMA) that is chain
extended with a cationic monomer N,N’-(dimethylaminopropyl)methacrylamide (DMAPMA). The
charge density is mitigated by varying the hydrophilic nature of the cationic block through the
incorporation of varying molar percentages of HPMA in the feed. These (HPMA-statistical-APMA)block-(HPMA-statistical-DMAPMA) copolymers are complexed with tRNA and their binding efficiency
monitored utilizing gel electrophoresis. The release efficiency will be monitored utilizing gene
knockdown experiments involving siRNA/copolymer complexes. In the near future thermodynamic
experiments (DSC, ITC, and CD Spectroscopy) will be utilized to further elucidate the mechanism
involved in binding and release of siRNA to/from (co)polymers which will lead to the development of
more efficient siRNA delivery vehicles.
Acknowledgements
Statistical incorporation of additional HPMA into the cationic block should
disrupt siRNA binding and provide more efficient release
NSF EPSCoR EPS-0903787
MRSEC DR-0213883

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