By Doug Macron
Researchers from Alnylam Pharmaceuticals and collaborators at the Massachusetts Institute of Technology this week published a report describing a class of novel non-lipid nanoparticles with potential as siRNA delivery vehicles.
The publication, which appeared in the Proceedings of the National Academy of Sciences, comes just one week after the same team reported how it used combinations of new lipid materials for RNAi therapeutic delivery (GSN 7/21/2011). Notably, Alnylam in recent months has been promoting its efforts to develop new delivery approaches amid a legal battle with Tekmira Pharmaceuticals over the rights to a key lipid nanoparticle technology.
"Continued progress in delivery of RNAi therapeutics requires broad-based efforts around novel lipids, conjugates, and polymers,” Kevin Fitzgerald, senior director of research at Alnylam, said in a statement. “In the current study, core-shell nanoparticles were discovered using combinatorial approaches to identify novel materials for siRNA delivery. These findings further expand our systemic delivery platform to achieve the broadest applications of RNAi therapeutics."
In the PNAS paper, the investigators note that while “chemically diverse nanoparticles” have promise as delivery vehicles, “it is difficult to predict the optimal chemical and physical properties for delivery of a specific drug or biomolecule.” In addition, distinct synthesis, characterization, and formulation steps must be carried out before performance can be evaluated.
In light of recent advances in the controlled synthesis of polymers with functional group tolerance and in robotics, the researchers decided to evaluate an experimental process of siRNA-delivery nanoparticle preparation involving 96-well plates that are shuffled between various high-throughput instruments for synthesis, characterization, and screening, according to the paper.
“This process allows for examination of key physical and chemical properties in relation to performance,” the researchers wrote.
“We prepared a library of nanoparticles composed of cationic cross-linked nanogel cores and variable shells with precise control over particle size, chemical composition, and architecture,” they noted. In total, the library comprised 1,536 structurally distinct nanoparticles.
A process of cross-linking block copolymers prepared by reversible addition-fragmentation chain transfer polymerization was used to achieve the desired nanoparticle structure. The team chose ring-opening of epoxides with amines for cross-linking “due to the availability of a large number of amines that can incorporate cationic charge into the core, and the efficiency of this catalyst-free reaction.”
The team then measured the nanoparticles' molecular weight, diameter, RNA complexation, cellular internalization, and utility for siRNA and pDNA delivery.
“Analysis revealed structure-function relationships and beneficial design guidelines, including a higher reactive block weight fraction, stoichiometric equivalence between epoxides and amines, and thin hydrophilic shells,” the investigators wrote in PNAS. “Cross-linkers optimally possessed tertiary dimethylamine or piperazine groups and potential buffering capacity.”
Nanoparticles that performed the best in in vitro testing were selected and covalently joined with cholesterol for in vivo evaluation using a mouse factor VII gene-silencing model, according to the paper.
One nanoparticle in particular triggered a 40 percent inhibition of the clotting factor. Noncovalent encapsulation of unmodified cholesterol, however, had no measured effect, and delivery of control siRNAs using these nanoparticles resulted in no measurable silencing, “suggesting specific delivery to the liver and silencing of factor VII,” the researchers wrote.
No signs of toxicity were observed, they added.
“We believe that the development of this library of core-shell nanoparticles represents an important step forward for the [high-throughput] synthesis of polymers for intracellular delivery,” they added. “Further studies are warranted to extend this technology for the broadest applications of RNAi therapy and drug delivery.”
Looking Past Tekmira
Although Alnylam has been a close partner of Tekmira's for years and has incorporated that company's lipid-delivery technology into its phase I liver cancer drug ALN-VSP02, the companies have been embroiled in litigation since early this year over the rights to related technologies (GSN 6/9/2011).
Meanwhile, Alnylam has been showcasing the promise of alternate delivery strategies. Earlier this summer, CEO John Maraganore told investors during ThinkEquity's annual healthcare conference that conjugate-based approaches, rather than ones founded on lipids, are “the future for delivery of small interfering RNAs” (GSN 6/2/2011).
He also touted the benefits of proprietary second-generation lipid nanoparticles developed without the help of Tekmira.
And earlier this month, Alnylam and MIT reported on the use of combinations of novel lipid materials to systemically deliver RNAi therapeutics (GSN 7/21/2011).
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