Lack of larger-scale production and reproducible production for the generation of chitosan based polyplexe.
By Laurence Bérubé, April 10th 2019
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Even though science has improved greatly over the last decades, there is still unresolved problems. Among them, genetic disorders. Currently the treatments consist of minimizing the symptoms rather than treating the underlying cause, the genetic mutation in that case.
As a way to overcome this, gene therapy is investigated. It consist of the delivery of nucleotides, which after being taken up by the cell, would restore the activity of the disrupted gene. However, to be effective, those nucleotides requires a carrier in order to reach the cells without being degraded or damaged. One way to do it, is by the use of nanoparticles which have the advantages to be tunable in terms of their physical and chemical properties. However, much effort must be invest in the way of finding such nanocarriers with all the desirable properties for efficient gene delivery. Indeed, before any nanoparticle becomes available to the public there is a variety of steps that must be taken. From the publication of the discovery, the approval from the authorities, there is another challenge that requires engineering tuning. In the jargon this step is referred as the ‘scale-up’, which consist of the transition from a small-scale to a larger scale production. Usually, in the research lab, the results published comes from small, manual productions but clinical trials requires that bigger volumes are yielded to reach effective dosages. Let aside batch size problems associated with nanoparticles production, there is also batch-to-batch and inter-user reproducibility problems. That means that there is variation of the nanoparticles properties across different batches. It is an important problem for the comparability between tests, since at the nanoscale, the properties of nanoparticles influence their efficiency toward the treatment. Indeed, as an example, the literature draw a link between cellular intake and bioavailability and charge surface and size of nanoparticles.
A study published in 2017 provides new insight on how to achieve nanoparticles with consistent physical properties in bigger batches. The research team had as a goal to develop an automated system (AIMS) for the large scale production of chitosan polyplexes, a type of nanoparticle. The aim was thus principally to find an appropriate replacement of manual mixing and its inherent limitations but also study how the variation of mixing parameters such as concentration and mixing speed affect their properties. By knowing the effect of these properties of the polyplexes, they wanted to identify parameters that should be used for large-scale production of desirable sized homogeneous nanoparticles.
To truly understand how this system works and could help advance the use of nanochemistry in the biomedical field, Dr. Lavertu from Polytechnique Montréal offered his help. He was part of the research team but also have sizable knowledge on chitosan, with his previous researches directed toward this molecule and its properties as a biopolymer for gene delivery and tissue engineering.
Chitosan is currently studied for a variety of applications in the nanotechnology such as of course nanoparticles, hydrogels and also for wounds dressing. This biopolymer, is reputed for being biocompatible, and can associate with nucleic acids based on their charges, yielding a polyplexes. This makes it an interesting molecule in gene delivery purposes since the nucleotides are directly associated with the carrier.
One major drawback of the use of chitosan-based polyplexes is that in the laboratory researchers are limited to the manual method, with only a hundred of microliters yield at a time. This is a problem because the reaction is carried out in multiple tubes, then lyophilized individually and then reconstituted. With such small quantities, to have enough polyplexes to conduct animal testing for example, the researchers have to use the combination of multiple individual reactions, with each slightly different properties due to ineluctable variations introduced during their preparation. But Dr. Lavertu explains that In vivo testing requires to be able to reproduce nanoparticles with the same properties than another lot that was produced earlier to be able to compare results.
One first advantage this automated system can offer (compared to the manual production) is principally control. Indeed, the final properties of polyplexes are the result of the combination of production parameters. Thus control over these parameters and knowledge of their influence is one step towards production of polyplexes with constant properties. Investigated mixing parameters was notably concentration of chitosan and nucleic acid and speed of mixing.
In the study, they varied these parameters using AIMS on two different polyplexes both based on chitosan (plasmid-DNA and small interfering RNA with the latter being much smaller) to compare the resulting properties of the polyplexes. However, they were not equal in both polyplexes, with plasmid DNA being apparently less sensitive with varying mixing parameters. Which arise questions of limits of the system in term of achievable polyplexes properties.
However, Dr. Lavertu explained that there will always be limitation based on the molecule we are using, mainly based on their individual sizes. The only control we have is through the mixing parameters. In the case of plasmid-DNA polyplexes, their size was more sensitive to the initial concentration of the reagents but was more stable in the case of small interfering RNA. Yet, the later was more sensitive to the speed of mixing than in the case of plasmid-DNA polyplexes, which was more or less constant. These differences were associated with the difference in size in the nucleotides and their associated diffusion properties, small interfering RNA being smaller it had a higher diffusivity which resulted in a higher sensitivity in the speed of mixing but a lower sensitivity to initial concentration. This demonstrate how the molecule we are working with physically limits range of parameters achievable thus is not associated with limitations of the system.
Another advantage of the system, as explained by Dr. Lavertu, is that in manual mixing there is a limitation in the concentration of the reactants. This limitation arise from the fact that there is normally a correlation between a higher concentration and a higher polydispersity, which is not desirable since it means that there is a bigger difference in size among the polyplexes population. An achievement made possible with AIMS was that they were able to produce particles at a higher concentration without significantly increase the polydispersity with the system, which would allow to increase deliverable dose.
Overall the system was able to produce polyplexes with as good or better reproducibility (at varying parameters) than the manual mixing for a large-scale production. It was also able to use concentrated solutions without sacrificing low polydispersion thus show promises for further application not limited to chitosan. At least this study also allowed to report how the polyplexes properties change according to mixing parameters. Yet, one might ask how the development of such system can concretely help nanoparticles research moving forward beside chitosan-based polyplexes.
This study used AIMS for chitosan based polyplexes, in this case the polyplexes are forming from the electrostatic interactions between the negatively charged nucleotides (necessary for gene therapy), and the positively charged polymer, in this case chitosan. Thus the formation of the polyplexes is rather simple since it relies on the charge of the molecules. What Dr. Lavertu explained is that because of the nature of the interaction, “Yes, it would be possible to use this system on another type of polyplexes, as long as it has the same combination of [electrostatic] interactions and rapid association between the two molecules.” but has not yet being tested but has to potential to applies to other types of nanoparticles, reaching a broader purpose.
Yet, the method of nanocarrier-based gene delivery still requires optimization for clinical applications. Thus the next step toward their application would be to overcome the problem of physiological barrier, with the lysosomal sequestration the hardest barrier to overcome. This type of barrier is thought to be resolved through the design of the nanoparticle. Dr. Lavertu explains that the challenge consist of getting to the organ in question, then get internalized in the cells. The lysosomal sequestration must be minimize to have a sufficiently high portion of nanoparticles that will end up in the cytoplasm or the nucleus, were the gene therapy take effects. Thus in the future, researchers must find a strategy to minimize this effect. Some people in the community says that it is the greatest challenge of gene therapy. Solving the problem of batch size and reproducibility will help further research by providing polyplexes with constant properties and in sufficient quantity for clinical purposes in the way of developing genetic disorders treatments. The study not only showed how an automated system can help but also provided insight on how to choose mixing parameters to yield polyplexes with desired properties.
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References :
Naeini, Ashkan Tavakoli, Ousamah Younoss Soliman, Mohamad Gabriel Alameh, Marc Lavertu, and Michael D. Buschmann. 2017. “Automated in-Line Mixing System for Large Scale Production of Chitosan-Based Polyplexes.” Journal of Colloid and Interface Science. https://doi.org/10.1016/j.jcis.2017.04.013.