Welcome to Scribd, the world's digital library. Read, publish, and share books and documents. See more
Download
Standard view
Full view
of .
Look up keyword
Like this
1Activity
0 of .
Results for:
No results containing your search query
P. 1
Bio Macro Molecules

Bio Macro Molecules

Ratings: (0)|Views: 104|Likes:
Published by subhoiitkgp

More info:

Published by: subhoiitkgp on Sep 11, 2009
Copyright:Attribution Non-commercial

Availability:

Read on Scribd mobile: iPhone, iPad and Android.
download as PDF, TXT or read online from Scribd
See more
See less

09/10/2009

pdf

text

original

 
Water Soluble Nanoparticles from PEG-Based CationicHyperbranched Polymer and RNA That Protect RNA fromEnzymatic Degradation
Jameel Ahmad Khan,
Rajesh Kumar Kainthan,
Munia Ganguli,
Jayachandran N. Kizhakkedathu,
Yogendra Singh,
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.
1
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.
2
-
4
These methods are successfulin protecting DNA to a considerable extent, but have not attainedsimilar success in protecting RNA from degradation.
5,6
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.
7
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.
8,9
They were also able to engineerthese nanoparticles for delivery of multiple therapeutic agents.
10,11
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
1
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:
+
91-11-2766-6156. Fax:
+
91-11-2766-7471. E-mail: souvik@igib.res.in.
Institute of Genomics and Integrative Biology, CSIR.
University of British Columbia.
Scheme 1.
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.
1386
Biomacromolecules 
2006,
7,
1386 
-
1388
10.1021/bm050999o CCC: $33.50 © 2006 American Chemical SocietyPublished on Web 04/08/2006
 
polymer is positively charged and is able to interact withnegatively charged RNA molecules.Electrophoretic mobility shift assay (EMSA) was performedto visualize the interaction of RNA and HP. In brief, complexesof HP and RNA were obtained by mixing aqueous solutions of HP and RNA at different positive units of HP vs negative unitsof RNA or charge ratios (
 Z 
+
 / 
-
). Denaturing agarose gelelectrophoresis was subsequently performed. A representativegel image is presented in Figure 1. Although free RNA orincompletely neutralized RNA migrated in the electric fieldtoward the anode, full retardation occurred at and above the
 Z 
+
 / 
-
)
3. This clearly demonstrates that the cationic segmentsof the HP interact electrostatically with the negative charges of phosphate groups of RNA, thereby resulting in the formationof stable complexes. This leads to retardation in the migrationrate of RNA molecules due to the larger size of the complexand charge neutralization.Atomic force microscopy was then used to determinemorphological changes in the shape of RNA after interactionwith HP. Figure 2 shows graphs elaborating the size andmorphology of complexes formed at different
+
 / 
-
values. Anirregular elongated shape and a broad size distribution wereobserved in the images, where
+
 / 
-
is lower than 3. EMSAresults (Figure 1) showed that at these charge ratios the HP couldonly partially bind. However, as the extent of complexation isincreased by raising the charge ratio (
 Z 
+
 / 
-
>
3), where the entireRNA gets bound to the HP, the morphology of the HP/RNAcomplex changes into spherical nanoparticles. The particles arehighly monodispersed, and sizes are around 60
(
10 nm. Theseparticles have the minimum possible size for a RNA/polycationconjugate based on the partial specific volumes of the constituentcomponents. Subsequent turbidity measurements at 550 nm andsize measurements showed that turbidity of the solution andsize of the particles remained unchanged. These observationsrevealed that the systems are quite stable and remained solubleover a period of time.The stability of this spherical nanoparticle-shaped RNA wasfurther studied from the viewpoint of nuclease resistance.Addition of RNase to native RNA solution immediatelyincreases the absorbance at 260 nm due to the fragmentationof RNA, but no substantial increase in the absorbance wasobserved for the nanoparticles, where
+
 / 
-
was 3 (Figure 3).At lower
+
 / 
-
, the rate of increment of absorbance was lessthan that of the native RNA revealing the RNA was partiallyprotected by the polymer. High nuclease resistance ability wasobserved for the spherical nanoparticle shaped RNA of 
+
 / 
-
)
3. This indicates the stable and inert nature of RNA when it isfully complexed with the HP, implying that the complexformation hinders the availability of RNA for nuclease action.In summary, small stable water-soluble RNA nanoparticleswhich are resistant to nuclease action can be made from thenovel cationic HP. Our results clearly show that RNA could beeasily adsorbed onto the positively charged surface of thecationic HP and protect RNA strands from nuclease action.Ingenious methods can be designed to release RNA from HP/ RNA complex for further use. For example, a solvent systemwhich preferentially solublizes HP but not RNA can bedeveloped. An ion exchange system or buffers with differentpH may also serve the purpose. By careful and smart design of hyperbranched cationic polymers, it is quite possible to syn-
Figure1.
Electrophoretic mobility shift assay for HP/RNA complexes;lane 1: native RNA, lane 2: HP/RNA complex at
+
 / 
-
)
1, lane 3:HP/RNA complex at
+
 / 
-
)
3, lane 4: HP/RNA complex at
+
 / 
-
)
5,and lane 5: HP/RNA complex at
+
 / 
-
)
7. Complexes were preparedsimply by mixing aqueous solutions of HP and RNA at different
+
 / 
-
(see Supporting Information).
Figure 2.
Atomic force microscopy images (3000 nm
×
3000 nm)of (a) native RNA, (b) HP/RNA complex at
+
 / 
-
)
1, and (c) HP/ RNA complex at
+
 / 
-
)
3.
Communications
Biomacromolecules, Vol. 7, No. 5, 2006 
1387

You're Reading a Free Preview

Download
scribd
/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->