Water Soluble Nanoparticles from PEG-Based CationicHyperbranched Polymer and RNA That Protect RNA fromEnzymatic Degradation
Jameel Ahmad Khan,
Rajesh Kumar Kainthan,
Jayachandran N. Kizhakkedathu,
and Souvik Maiti*
Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi 110 007, India, and Department of Pathology and Lab Medicine,Centre for Blood Research, 2350 Health Sciences Mall, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada Received December 27, 2005; Revised Manuscript Received February 14, 2006
Recent advances in understanding biological systems have proven that RNA is not merely the carrier of geneticinformation, but also a key molecule in regulation of gene expression and other crucial metabolic processes.Therefore, it is being considered as an ideal therapeutic candidate both for metabolic and genetic disorders. However,research involving RNA molecules faces a practical limitation since RNA is highly labile. We have developed anovel method to protect RNA from cleavage by complexing it with a hyperbranched cationic polymer. It wasfound that total cellular RNA isolated from yeast spontaneously interacts with the positively charged polymer toform a spherical nanoparticle morphology. This interaction protects the RNA against enzymatic degradation.This methodology can be easily adapted for long-term storage of RNA, long distance transfer of RNA, and geneticengineering using RNA as a building block.In the post-genomic era, genetic engineering has opened upnew challenges and opportunities for development of medicineand biomedical research. However, genetic engineering technol-ogy is limited to the DNA level, primarily due to practicallimitations of handling RNA, a labile molecule, which is difficultto study and manipulate, especially when it comes from an invivo source.
Considerable research, such as addition of inhibitors in DNA solutions, entrapping in liposome, or formingnanaoparticles, etc., has been done to devise methods to protectnucleic acids from cleavage.
These methods are successfulin protecting DNA to a considerable extent, but have not attainedsimilar success in protecting RNA from degradation.
Recentadvances in understanding of the role of RNA molecules haveestablished that RNA is not merely a carrier of geneticinformation but also an important molecule that plays a centralrole in regulation of gene expression and other crucial metabolicprocesses, indicating its potential as a therapeutic agent bothfor metabolic and genetic disorders.
Therefore, efforts arerequired to develop methodologies to make RNA moleculesstable enough to carry out experimental studies and manipula-tion. It will be an added advantage if such methods includeversatility and potential of nanoparticles, since in that case, itmight be possible to deliver multiple therapeutic agents to thecell at the same time. Utilizing RNA, Khaled et al. has shownthe self-assembly of RNA nanoparticles of different shapes andsizes from pRNA of bacteriophage phi-29, which were resistantto environmental stresses.
They were also able to engineerthese nanoparticles for delivery of multiple therapeutic agents.
However, better ways to give any RNA sequence a nanoparticleshape and protect it from degradation by enzymes in the bodyduring its delivery process need to be found. In this communica-tion, we report a concept of using a cationic hyperbranchedpolymer to complex with RNA, resulting in water solublenanoparticles, which then protect RNA strands from enzymaticcleavage.An amino-modified poly(ethylene glycol) (PEG) basedhyperbranched polymer (HP; Scheme 1) was synthesized byring opening anionic polymerization of glycidol and MPEGepoxide initiated from tris(hydroxymethyl) propane using potas-sium methylate (see Supporting Information). Aminated PG-PEG polymers were obtained by post modification of PG-PEGby mesytylation, amination with tris(2-aminoethyl)amine, andmethylation. The synthesized polymer was characterized by
HNMR and conductometric titration for the determination of amine content. The tertiary amine groups in PG-PEG aminewere quaternized using ethyl bromide. Due to the presence of quaternized amino groups at the surface (Scheme 1), this
* To whom correspondence should be addressed. Phone:
91-11-2766-7471. E-mail: firstname.lastname@example.org.
Institute of Genomics and Integrative Biology, CSIR.
University of British Columbia.
Schematic Representation of the CationicHyperbranched Polymers Used in This Study
All of the amines were quaternized as described in the ExperimentalSection in the Supporting Information.
10.1021/bm050999o CCC: $33.50 © 2006 American Chemical SocietyPublished on Web 04/08/2006