Oligonucleotide therapeutics have seen rapid progress in recent years due to their ability to modulate gene expression and therefore control protein production, often critical to a specific disease state. However, like many medicines born from nature, stability and delivery have been particularly challenging attributes to address. Biosynthetically, such modulators are produced at the site of action and often intentionally created with short half-lives.
Ideally, therapeutic delivery must be both specific and efficient. For oligonucleotides, these are particularly demanding attributes, given their inherent chemical characteristics, which tend to lack targeting capability and have poor membrane permeability. A number of diverse strategies have been developed in attempts to overcome these delivery challenges.
To date, two broad strategies have emerged to support oligonucleotide therapeutic delivery:
The first strategy has, in part, been discussed in earlier blogs of this series, describing modifications such as:
While many of these changes are primarily aimed at improving stability, other modifications, usually involving conjugation, form the basis for targeting and delivery. These are further detailed in a separate blog but briefly summarized below:
Design effective structural characterization studies for oligonucleotide therapeutics.
Example data from the analysis of unmodified Inotersen and Inotersen.
This blog further explores vector-based approaches that aim to both target and protect oligonucleotide drugs during administration.
First-generation vectors such as liposomes, colloidal vesicles often composed of phospholipid and cholesterol bilayers, use the endosomal pathway for intracellular delivery.
Specific “facilitator” molecules such as chloroquine and DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine) have been incorporated into the liposomes to allow endosomal escape by inducing endosomal membrane destabilization. This facilitates oligonucleotide leakage, which then appears to be actively transported in high concentration to the nucleus.
PEG has also been utilized in the liposome construction. Adjusting the length and structure of these ethoxylates, along with the lipid charge and component ratios, has enabled targeting away from the liver to alternative organs such as the spleen and lungs.
A range of organic polymers, often serving dual roles in protection and targeting, have been used to create nanospheres for oligonucleotide delivery. These include:
In addition, nanoparticle carriers made from a range of inorganic materials have also been explored for oligonucleotide delivery, including those manufactured from silica, silver, gold and calcium phosphate.
These structures have recently been investigated for delivering several therapeutic types, including oligonucleotides. The structural properties of DNA allow the formation of organized 2D and 3D architectures, which can be combined with targeting capabilities using aptamers directly integrated into the nanostructure. While still relatively new and with limited data on their in vivo biodistribution and stability, these systems show great potential across a wide range of applications.
Since the delivery system plays a role as critical as the API itself, its analytical characterization should be given equal importance. The broad range and diversity of molecular types used in delivery platforms requires an equally diverse set of analytical methodologies and techniques, particularly when the final formulation includes multiple chemical types. In addition to these complexities, there is often a requirement for delineating the size, shape and distribution of the nanoparticles, as well as the abundance of internalized API. As such, both qualitative and quantitative methodologies are typically necessary to fully understand the system's performance and stability.
The wide applicability and versatility of techniques such as GC-MS and LC-MS offer valuable analytical solutions for complex delivery systems composed of heterogeneous molecular type populations. In addition, a range of complementary technologies, such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) for particle sizing and distribution, and electron microscopy for evaluating encapsulation, may also be employed.
Such delivery platforms often require bespoke analytics with expertise from a range of disciplines. Such challenges have long been successfully addressed by BioPharmaSpec’s dynamic and innovative services, which continue to lead the way in solving such complex analytical problems.
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