Visualization, Size and Count Measurement of Drug Delivery Nanoparticles Using NTA (Nanoparticle Tracking Analysis)

The use of nanoparticles in drug delivery is rapidly increasing. Nanoparticles offer excellent pharmacokinetic properties, sustained and controlled release, and targeted delivery to specific cells, tissues, or organs. The growing interest in nanoparticle drug delivery can also be attributed to the slowing pace of discovering new biologically active compounds as therapies for disease treatment. With the decrease in new drugs introduced to the market each year, there is a swift rise in interest in utilizing the versatile, multifunctional structure of nanoparticles for drug delivery, enhancing the efficacy of existing drugs.

Nanoparticles used in drug delivery are defined as colloids smaller than one micron and can be composed of various materials with diverse compositions. Commonly defined nanoparticle vectors include liposomes, micelles, dendrimers, solid lipid nanoparticles, metal nanoparticles, semiconductor nanoparticles, and polymer nanoparticles. Nanoparticles have been extensively adopted to deliver drugs, genes, vaccines, and diagnostics to specific cells or tissues in various ways.

Considering the drug delivery systems of nanomaterials, the size of nanoparticles is a crucial parameter as it directly impacts delivery, absorption, degradation, and excretion from the body. For instance, nanoparticles with diameters ranging from 30nm to several hundred nm can passively accumulate at tumor sites due to leaky vasculature structures, while phagocytic activity favors particles >500nm, and hepatic accumulation favors particles >200nm & <300nm. Therefore, accurate measurement of the size of the particles administered in various systems and processes is important.

Analysis of Drug Delivery Systems Using NTA (Nanoparticle Tracking Analysis)

Liposomes

Liposomes (Figure 1) have been actively researched and developed over many years and are currently the most common targeted drug delivery system. Liposomes have been approved as delivery systems for amphotericin B in fungal or protozoal infections, doxorubicin in breast cancer treatment, and vaccines for Hepatitis A and influenza. The importance and potential of liposome use in delivery systems are continually increasing. The reasons are clear.

Figure 1

• Therapies delivered via liposomes can protect drugs from enzymatic activity during metabolic processes.
• Liposomes can be used to solubilize lipophilic substances.
• Connecting specific ligands to liposomes can target specific areas for treatment.
• Liposomes can be easily absorbed by cells.
• Liposomes can be selected to control release rates.
• Using liposomes as carriers can reduce dosage or frequency, reducing toxicity and side effects.
• Liposomes can carry biological materials like proteins and DNA.

The size of the liposomes used is increasingly recognized as a critical factor for therapeutic efficacy. The size of drug delivery liposomes affects circulation and retention time in the bloodstream, targeting efficacy, rate of cellular uptake (or cellular foreign uptake), and successfully releasing the administered amount. These considerations regarding size are crucial for all nanoscale drug delivery systems.

Measuring Liposome Size Using NTA

The NanoSight equipment quickly and accurately measures the size and concentration of liposomes in water and other solvents.

Only a small amount of sample and minimal sample preparation are required. This equipment allows visualization of individual liposomes in suspension, tracking their Brownian motion to determine particle size distribution in seconds.

Real-time Simultaneous Multiparameter Analysis of Nanoparticles

In addition to size and concentration, NTA can provide particle information based on the following parameters:

• Scattering intensity can identify adjacent particle populations and differentiate materials with significantly different refractive indices.
• This unique function allows users to investigate whether nanoparticle drug delivery structures such as liposomes differ from their contents. For instance, empty liposomes may have a lower refractive index (lower light scattering ability) than liposomes containing high-refractive-index materials. This allows differentiation even with very similar sizes.
• Fluorescence detection can enable proper classification of particles in complex backgrounds. The fluorescence capability of NanoSight is detailed in additional application notes.
• NTA has been used in photodynamic cancer therapy research to investigate the effect of serum on the stability of various liposome studies (Reshetov et al., Photochem Photobiol. 2012 Sep-Oct, 88(5):1256-64. doi: 10.1111/j.1751-1097.2012.01176.x).

NTA Application in Other Drug Delivery Systems

PBAE (Poly β-amino ester) could be a delivery system for gene therapy to treat various cancers. PBAE offers advantages over other systems due to its ability for diverse polymer binding with DNA through combination pathways. Although it has fast release properties due to hydrolysis, it causes problems with dose variability, production, and storage. Freeze-drying is a common storage method, and NTA was used to evaluate how freeze-drying affects PBAE-DNA nanoparticle aggregation (increased size) and disruption (reduced size) (Tzeng et al. 2011 and Sunshine et al. 2012).

PLGA (Poly-lactic-co-glycolic acid) is an FDA-approved drug delivery system. PLGA breaks down into lactic acid and glycolic acid, both of which are endpoints in the body’s metabolic pathways. PLGA has been used as a drug delivery system for gonadotropin-releasing hormone for advanced prostate cancer treatment, as well as amoxicillin. To ensure the correct delivery, and as a critical parameter for researchers to compare results across studies, NTA was used to verify the size of nanoparticles (Shirali et al. 2011).

Successful movement of molecules between cell membranes is the essence of delivery. Efficient carriers are required as molecules alone cannot typically penetrate cell membranes. Sokolova et al. (2012) investigated calcium phosphate (diameter: 100nm – 250nm, depending on functionalization) as versatile carriers for large and small molecules between cell membranes using NTA, DLS (Dynamic Light Scattering), and EM (Electron Microscopy).

Ohlsson et al. researchers (2012) used NTA to confirm the stability and integrity of liposomes in reporting solute movement within less than 100 milliseconds between lipid bilayer membranes of individual proteoliposomes.

Ghonaim and colleagues have extensively reported on modifications to the chemistry of lipopolyamines and spermines and their effects on various non-viral plasmid DNA and siRNA delivery systems as part of their study of nanoparticles as a gene delivery vehicle (Ghonaim et al., 2007a, Ghonaim et al., 2007b, Ghonaim et al., 2007c, Ghonaim, 2008, Ghonaim et al., 2009, Soltan et al., 2009, Ghonaim et al., 2010). Similarly, Ofek and several researchers (2010) employed NTA to characterize dendritic nanocarriers for siRNA delivery, and Bhise utilized NTA for particle size and size distribution measurement in polymer nanocarrier research in cell culture. Recently, Bhise expanded this research to develop an assay for quantifying the number of plasmids encapsulated in polymer nanoparticles, measuring plasmid density count per 100nm nanoparticle using NTA (Bhise et al., 2011).

Wei and colleagues (2012) have identified the need for a robust method for accurately characterizing nanoparticle size, shape, and composition to develop and advance nano drugs, as well as the necessity for particle engineering to maintain non-specific cytotoxicity at low levels and improve stability during storage.

Other examples of nanoparticle size measurement and delivery using NTA have been reported (Hsu et al., 2010, Park et al., 2010, Tagalakis et al., 2010).