Payload Distribution & Encapsulation Efficiency Analysis

Many drugs have poor solubility or low absorption rates and are susceptible to degradation, chemical reactions, or rapid clearance when administered directly. Encapsulation within nanoparticles can enhance drug solubility and provides protection to the drug of interest, improving drug stability, absorption, bioavailability, and therapeutic efficacy. In addition, drug delivery vehicles can be designed to release the encapsulated drug in a controlled manner and selectively target specific cells, maintaining therapeutic levels and reducing toxicity.

In general, the manufacturing process and maintaining product shelf-life of therapeutics is not without challenges. Sub-optimal conditions, such as introduction of contaminants, shear stress, freeze-thaw, light exposure etc. can affect the product’s quality attributes and can not only result in formulations that contain impurities, particles that are misassembled, have lost their integrity, or show aggregation, it can also result in formulations that contain (too many) nanoparticles without payload. The number of empty delivery vehicles influences the concentration of the therapeutic agent delivered, and hence, its efficacy and safety. Therefore, characterizing the gene or drug encapsulation – or more adequately the drug loading fraction – of nanoparticles in a formulation is imperative.

Cryo-TEM offers significant advantages despite requiring specialized equipment and expertise. It enables for direct visualization of the presence and location of encapsulated payload within the delivery vehicle at a per-particle basis (complementing bulk-based measurements obtained using orthogonal techniques such as dynamic light scattering (DLS), spectroscopic methods, or fluorescence-based assays. Another huge advantage of cryo-TEM is that multiple quality attributes can be evaluated simultaneously; in addition to assessing gene or drug encapsulation, purity, aggregation, integrity, morphology, and size distribution can all be characterized using the same cryo-TEM image set. A few examples of assessing drug and gene encapsulation or loading fraction are described here.

Virus Like Particle (VLP) capsids: automated empty-partial-full ratio analysis

Adenovirus (AdV) and adeno-associated virus (AAV) are examples of well-established platforms for gene delivery. AAV in particular is popular due to the cell tropism of the various AAV serotypes and its lack of pathogenicity. Empty capsids or capsids that only contain a fraction of the DNA are considered impurities because they affect the efficacy, safety, and immunogenicity of the product. Cryo-TEM can be used to directly assess empty, partially full, and full capsids in a formulation. Cryo-TEM is superior to negative stain TEM as it allows for visualization of AAV particles in their near-native state and avoids any stain and/or drying artifacts that can potentially affect the DNA encapsulation counting results.

Determining the encapsulation efficiency of AAVs can be performed by NIS using an automated image analysis approach. Please contact us to receive an example report showing our automated analysis of full, partially full, and empty capsid ratios.

Liposomal drug encapsulation

Liposomes are vesicles composed of one or multiple lipid bilayers enclosing an aqueous core and are used to overcome some limitations of traditional therapeutics, such as low solubility and dose dependent toxicity issues. Liposomes are biocompatible, show improved bioavailability, and can be manipulated to improve specific cell-targeting. Besides characterizing size, morphology, and lamellarity, cryo-TEM is an excellent method to determine the encapsulation efficiency of liposomes for drugs that are electron dense due to precipitation, metal complexation, or simply size. One such example is liposomal doxorubicin. This anticancer drug forms fibrous crystals within the liposomes that can be visualized using cryo-TEM. Analysis of these cryo-TEM images can subsequently provide information on the ratio of empty and loaded vesicles in a formulation, possibly in combination with lamellarity, shape, or other characteristics of interest.

Lipid Nanoparticle (LNP) payload

A new class of gene delivery products has gained much traction following the recent COVID pandemic. Lipid nanoparticles, generally a mixture of an ionizable lipid, a helper lipid, cholesterol, a PEG lipid, and therapeutic nucleic acids, are being developed as vaccines, cancer treatments, and other disease treatments. One of the largest open questions in the field is related to determining the presence and distribution of nucleic acid molecules at the single-particle level.

A widely used technique to quantify nucleic acid encapsulation is the Ribogreen ® dye assay. This method, however, provides results of bulk measurements instead of per-particle data. Other indirect methods such as SEC-MALS-UV-dRI and nano Flow Cytometry can be used to perform size-dependent RNA content analysis of lipid nanoparticles. Unfortunately, these techniques do not take into account that there are particles that have fewer nucleic acid molecules encapsulated or none at all compared to others. Currently, the only technique to study per-particle payload (without the need for labeling) is cryo-TEM. Cryo-TEM images can reveal the existence and distribution of nucleic acid molecules throughout the oily (or lipid) phase as well as the aqueous phase in individual LNP particles, with – not surprisingly - larger molecules such as DNA having a much more pronounced presence than mRNA.