Michael Caves, India Business Development Manager, Malvern Instruments, elaborates on how nanoparticle tracking analysis (NTA) has been able to accurately measure size and concentration of nanoparticles
The use of nanoparticles in drug delivery continues to grow rapidly. Nanoparticles offer excellent pharmacokinetic properties, controlled and sustained release, and targeting of specific cells, tissues or organs. Interest in nanoparticle drug delivery is also driven by the diminishing rate of discovery of new biologically active compounds that can be exploited therapeutically to treat disease. With fewer new drugs entering the market, interest in the use of nanoparticles’ versatile and multifunctional structures for the delivery of drugs is swiftly increasing. All these features can improve the efficacy of existing drugs.
Nanoparticles used in drug delivery have been defined as colloidal systems of submicron size that can be constructed from a large variety of materials in a large variety of compositions. Commonly defined nanoparticle vectors include: liposomes, micelles, dendrimers, solid lipid nanoparticles, metallic nanoparticles, semiconductor nanoparticles and polymeric nanoparticles. In their many guises, nanoparticles have been extensively employed to deliver drugs, genes, vaccines and diagnostics into specific cells/ tissues.
When considering a nanomaterial drug delivery system, size of the nanoparticle is a key parameter as it directly influences the processes of delivery, uptake, degradation and clearance from the body. For example, nanoparticles in the range of 30 nm to a few hundred nm in diameter can passively accumulate at the site of tumours due to leaky vasculature, phagocytosis favours particles >500 nm, whilst biliary and renal clearance occurs with particles <30 nm and <8 nm respectively. In addition, the liver has a lower uptake of smaller particles (25 nm and 50 nm) compared to larger particles (200 nm and 300 nm). Accurate measurement of the size and concentration of particles being administered is therefore imperative to many systems and processes.
Analysis of Iiposomal drug delivery systems by NTA
Liposomes (Figure 1) have been the subject of significant research and development efforts for many years and are currently the most common targeted drug delivery system. Liposomes have been approved as a delivery system for amphotericin B for fungal or protozoal infections, doxorubicin for breast cancer treatment, and for vaccines for hepatitis A and influenza. The use and potential of liposomes in drug delivery continues to grow in importance. The reasons are clear:
- Therapeutics delivered via liposomes may be protected from the actions of metabolising enzymes
- Lipophilic substances may be made soluble by the use of liposomes
- Therapeutics can be targeted to specific areas by attaching specific ligands to the liposome
- Liposomes are readily absorbed by cells
- The rate of release may be controlled by the selection of liposome
- Using liposomes as a delivery vehicle allows potentially lower or less frequent doses to be used, potentially reducing toxicity and side-effects
- Liposomes can carry biological substances such as proteins and DNA
In addition to concentration, the size of the liposomes used is increasingly being recognised as an important factor in treatment efficacy. A drug delivery liposome’s size may affect its circulation and residence time in the blood, the efficacy of the targeting, its rate of cell absorption (or endocytosis) and, ultimately, the successful release of its payload. Such size considerations are hugely important to all nanoscale drug delivery systems.
Sizing liposomes with NTA
Malvern’s NanoSight instrument range accurately and rapidly sizes and measures concentration of liposomes in water and other solvents. Only small volumes and very little sample preparation is required. The instruments enable individual liposomes in suspension to be visualised and their Brownian motion tracked – enabling concentration-based particle size distributions, based on individual particles, to be built up in a matter of seconds.
Simultaneous multi-parameter analysis of nanoparticles in real-time using 21 CFR part 11 compliant software
In addition to size and concentration, NTA also provides the following parameters, simultaneously and particle-by-particle:
- Scatter intensity, which resolves adjacent populations of particles and differentiates materials of sufficiently-differing refractive indices.
- This unique ability potentially allows the user to probe whether nanoscale drug delivery structures such as liposomes vary in their contents, i.e. empty liposomes may have a lower refractive index (light scattering power) than those loaded with a higher refractive index material. This would allow them to be differentiated even though they may be of very similar sizes.
Examples of drug delivery systems understood using NTA
Poly(ß-amino ester)s (PBAEs) are potential delivery systems for genetic therapies to treat various cancers. They have an advantage over some other systems in that many combinations of polymer with DNA can be made through the combinatorial route. They also have rapid release properties due to hydrolytic degradation, but this creates challenges for dosing, production and storage. Lyophilisation is a typical storage method and NTA is to evaluate the effect of lyophilisation on aggregation (increase in size) and destruction (decrease in size) of PBAE-DNA nanoparticles.
Poly(lactic-co-glycolic acid) (PLGA) is an FDA-approved drug delivery system. It breaks down into lactic acid and glycolic acid, both of which are endpoints of metabolic pathways in the body. PLGA has been used as a drug delivery system for amoxicillin and also for gonodotropin-releasing hormone for the treatment of advanced prostate cancer. The immunosuppressant mycophenolic acid has been encapsulated in PLGA with a view to decrease dosing levels and thus reduce toxic side effects. NTA is used to determine the size and concentration of these nanoparticles, a critical parameter to ensure good delivery and allow researchers to compare results across studies.
The successful transport of molecules across the cell membrane is a key point in their delivery. In many cases, molecules alone cannot penetrate the cell membrane, therefore an efficient carrier is needed. Calcium phosphate nanoparticles (diameter: 100 nm – 250 nm, depending on the functionalisation) have been investigated as versatile carriers for small and large molecules across cell membranes using a number of techniques including NTA, Dynamic Light Scattering (DLS) and Electron Microscopy (EM).
NTA is also used to control the effect of modifications to the chemistry of lipopolyamines and spermines in various non-viral plasmid DNA and siRNA delivery systems, in addition to the characterization of dendritic nanocarriers for siRNA delivery and gene delivery polymers in cell culture. As a robust method for the accurate characterisation of nanoparticle size and concentration, NTA Is used to maintain low levels of nonspecific cytotoxicity and to increase stability during storage.
Size and concentration of drug delivery systems must be understood as these parameters directly influence the processes of delivery, uptake, degradation and clearance from the body. This explains the value of NTA, as a technique unique in being able to accurately measure size and concentration of nanoparticles, to the development and quality-testing of such systems.
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