Nanoparticle Characterization
Therapeutic nanoparticles used in drug formulation & development, vaccine design and development, and gene therapy delivery are complex, and vary in composition, shape, and size. As structure can change under stress, characterization of nanoparticles’ physicochemical properties is required to understand how they interact with their environment and can provide insight into the potential safety and efficacy of the final formulation. Nanoparticle size, shape, lamellarity and morphology (such as visualizing bleb formulation, or seeing if your lipid nanoparticle (LNP) is unilamellar or multilamellar) affect drug incorporation, stability, and release, which in turn affect cell toxicity, targeting, and therapeutic efficacy. In addition, the study of drug cargo encapsulation (full/empty liposomes, lipid nanoparticles, or capsids) when looking at gene or drug delivery systems is crucial, as the presence of many empty particles or impurities (or non-intact capsid for virus-like particles (VLPs) or adeno-associated viruses (AAVs)) affects immunogenic response. Cryo-TEM can also help evaluate the influence of storage conditions (pH, temperature, and time) on nanoparticle morphology.
Cryo-TEM nanoparticle characterization is an essential technique for evaluating the Critical Quality Attributes (CQAs) of your formulation as it provides important structural details on a per-particle basis that cannot be captured by other methods, quickly visualizing individual nanoparticles in the size range of ~5-500 nm.
NIS also offers GMP compliant cryo-TEM and TEM services to support you in your regulatory journey.
Expertly-Performed Cryo-TEM Provides the Whole Picture, Earlier
Cryo-TEM can unveil many aspects of a sample simultaneously, in a single image (for example particle size distribution, drug encapsulation, shape, morphology, impurities, and integrity), all with a tiny amount of sample. This makes cryo-TEM a powerful orthogonal technique to complement your characterization toolkit. Where a sample contains multiple particle types, for example in vaccine drug products, cryo-TEM outperforms. Similarly, when a sample needs to be analyzed at a concentration similar to drug preparation for IV administration, cryo-TEM is the solution, as it can handle relatively low sample concentrations.
Access the power of cryo-TEM through NIS to get a more in-depth view earlier, fast-tracking your therapeutic development.
We provide quick, flexible and cost-effective access to the answers cryo-TEM can reveal. Partner with the original and most experienced team in cryo-TEM and access cutting edge instruments to help accelerate your projects.
Expertly-Performed Cryo-TEM Provides the Whole Picture, Earlier
Cryo-TEM can unveil many aspects of a sample simultaneously, in a single image (for example particle size distribution, drug encapsulation, shape, morphology, impurities, and integrity), all with a tiny amount of sample. This makes cryo-TEM a powerful orthogonal technique to complement your characterization toolkit. Where a sample contains multiple particle types, for example in vaccine drug products, cryo-TEM outperforms. Similarly, when a sample needs to be analyzed at a concentration similar to drug preparation for IV administration, cryo-TEM is the solution, as it can handle relatively low sample concentrations.
Types of Nanoparticles We've Imaged
Frequently Asked Questions
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Is cryo-TEM imaging with NIS GMP compliant?
NIS has established an internal Quality Unit, with the assistance of third party experts, to oversee quality systems and regulatory compliance to assist us in establishing cGMP compliant nanoparticle characterization testing services. Learn more about our GMP compliant cryo-TEM nanoparticle characterization services here.
Can I see an example report of nanoparticle characterization and analysis?
Yes! Please contact us to get an example report showing services including lipid nanoparticle (LNP) cryo-TEM imaging at three magnifications along with our Qualitative Assessment, Particle Size Distribution, and Fraction Counting Analysis.
What can cryo-TEM tell me about my nanoparticles or nanomedicines?
Cryo-TEM is a one of a kind analytical tool for nanoparticle characterization. A single cryo-TEM imaging study can visualize individual particles in your formulation, and can simultaneously provide information on particle size, morphology, lamellarity, drug encapsulation, homogeneity, and purity making cryo-TEM analysis essential for characterizing your nanoparticle therapeutic or vaccine.
NIS also offers GMP compliant cryo-TEM and TEM services to support you in your regulatory journey.
Why should I use cryo-TEM instead of Dynamic Light Scattering (DLS) to measure particle size?
Dynamic Light Scattering (DLS) is a powerful, non-destructive, fast technique to measure both particle size and evaluate the presence of aggregation. It also analyzes vastly more particles than cryo-TEM. However, DLS results are heavily affected by factors such as buffer, temperature and the presence of air bubbles, dust, or other impurities in the sample. In addition, DLS measures the hydrodynamic radius of the particles and doesn't do well with heterogeneous samples as the signal of the larger particles weigh more than that of smaller particles resulting in over-estimation of the particle size. Cryo-TEM captures the particles in their native state enabling the determination of true particle size. Cryo-TEM is also well-equipped to deal with heterogeneous samples. Cryo-TEM captures specimens in their natural hydrated state and is forgiving of heterogeneous mixtures of particles, making cryo-TEM an important analysis technique to measure particle size and size distribution, as well as lamellarity, drug encapsulation, and more, with as little as 3 microliters of sample. Cryo-TEM provides more answers than any other nanoparticle characterization technique in only one study.
Read the paper in Science Direct describing the differences between lipid nanoparticle characterization done by DLS and cryo-TEM, including imaging of lipid nanoparticles (LNP’s) done by NIS.
Read a paper in the Journal of Physics: Conference Series comparing TEM and DLS methods to characterize size distribution of ceramic nanoparticles.
Why should I use cryo-TEM instead of analytical ultracentrifugation sedimentation velocity (AUC-SV) to measure full/empty capsids for samples like Adeno-associated viruses (AAVs)?
Adeno-associated viruses (AAVs) are often used as vehicles for gene therapy. During the production process, many empty or partially filled capsids may be produced, which do not provide any benefits in the therapeutic process, but can impact immunogenicity and efficacy. The full/empty/partially full ratio of capsid is one of the most important Critical Quality Attributes (CQAs)of gene delivery vehicles to characterize. Analytical ultracentrifugation sedimentation velocity (AUC-SV) can measure the ratio of full and empty capsids by separation based on mass and buoyancy of full and empty particles. Cryo-TEM on the other hand can directly visualize capsids with various payloads. In addition, cryo-TEM allows you to also determine additional critical quality attributes such as particle size distribution, particle titer, and more. Another significant consideration is that cryo-TEM requires only 3μl of sample per experiment, whereas AUC-SV can require up to 400μl of sample.
Why should I use cryo-TEM in addition to other RNA encapsulation methods?
The most commonly used assay to determine RNA encapsulation in lipid nanoparticles (LNPs) is the ribogreen assay. SEC-MALS, or size exclusion chromatography with multi-angle light scattering, is another technique that can be used characterize large nanoparticles, including lipid nanoparticles (LNPs), for determining the amount of full vs. empty particles when combined with UV and dRI detectors.
Cryo-TEM can provide data on the distribution of encapsulation and size of individual particles in a formulation. In addition, cryo-TEM can visualize bleb formation in LNPs and the lamellarity of your lipid nanoparticle, showing if your LNP is unilamellar or multilamellar. Additionally, a cryo-TEM study uses much less sample than SEC-MALS, requiring only 3ul vs 300ul.
This paper in Science shows orthogonal characterization of lipid nanoparticles done by cryo-TEM and SEC-MALS.
How is cryogenic transmission electron microscopy (cryo-TEM) different than transmission electron microscopy (TEM)?
Cryo-TEM refers to a technique using transmission electron microscopes where the sample will be frozen in vitreous ice in order to preserve its native hydrated state, allowing for deeper insight into your nanoparticle.
What kind of samples do you have experience with?
NIS has imaged 2,500+ liposomes and lipid nanoparticles (LNPs), characterized 880+ virus, vaccine, and virus-like particles (VLPs), and has imaged 200+ adeno-associated virus (AAV) samples as well as solved the structure of AAV to 1.8 Å. NIS also has experience with extracellular vesicles and exosomes, iron nanoparticles, micelles, nanotubes, polymeric nanoparticles, and protein unit based nanoparticles.
What is the size range of nanoparticles you can image with cryo-TEM?
We can image and analyze nanoparticles from 5 nm to 1 micron with cryo-TEM direct imaging and negative stain screening.
What is the likelihood for success in characterizing my nanoparticle sample?
Each nanoparticle characterization project comes with its own set of challenges. NIS recommends scheduling a meeting with our experts to help determine if your project is suitable for a cryo-TEM study, and to discuss possible solutions to roadblocks that may appear during your project. NIS will always be fully transparent in regard to samples that might need more optimization or if a project might not be suitable for cryo-TEM.
What quantity of my sample is required?
Cryo-EM has an advantage over other methods in that only a small quantity of your sample is required. We typically request duplicates of ~50μl of material for each sample, at approximately ~1mg/mL protein, 10-30 mg/mL lipid, or E12 particles/mL. Your project manager will review this with you before sample submission.
Are all reagents and buffer conditions amenable to cryo-EM?
Any standard buffer is allowed (i.e. PBS,Tris, HEPES, etc.). If specific additives are known to be necessary for sample stability, these should be present in the minimum amount possible. The dilution buffer composition can be your formulation buffer, but preferably without sucrose, glycerol, trehalose (or other cryo-protectants), or high salt. These components negatively affect the particle contrast In the images. If you cannot get around adding any of these, NIS would suggest keeping the cryo-protectant below 2% and the salt concentration below 500mM.
What are the turnaround times to receive images and a report?
For nanoparticle characterization projects, you will typically receive your images and written report 4 weeks or less after sample submission & PO receipt. For rapid negative stain screening, images will be available to view in approximately 1 week from sample submissions & PO receipt. 5, 10, and 15 day rush services are available, ask your account manager for details.
Please contact us to get an example report showing services including lipid nanoparticle (LNP) cryo-TEM imaging at three magnifications along with our Qualitative Assessment, Particle Size Distribution, and Fraction Counting Analysis.
How long does it take for NIS to begin imaging our samples?
There is usually a two week queue to begin imaging your project. It is best to ship your sample to us as soon as possible after your sample submission meeting, as the microscope schedule is fluid and we may be able to begin imaging your project earlier. If your sample is unstable, please contact your project manager for more details.
Contact us to see an example of our Starter Pack, or feasibility assessment, performed on B-amylase, which includes negative stain screening, 2D classifications, and an initial 3D structure reconstruction.
Does NIS handle documentation or paperwork?
At NIS, we respect the confidentiality of our clients' intellectual property. Our internal contracts team collaborates closely with your legal team to complete all contracts. Typically, a CDA or NDA can be finalized within a week or less, while an MSA or MTA may take 2-3 weeks. In order to facilitate an open discussion of project-specific details during our introductory meeting, we strongly recommend having a CDA/NDA in place beforehand.
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