Applications of Nanoparticle Tracking Analysis in Drug and Gene Delivery

Nanoparticle tracking analysis (NTA) is a unique method that provides fast and multi-parameter information about nanoparticles. With the help of this technique, users can acquire number frequency distributions of particle sizes in polydisperse nanoparticulate systems.

NTA represents a novel technique, and as such has been implemented in many different sectors within the pharmaceutical sciences. This article describes some of the recent work published in the literature, where NTA has been recommended, employed, and evaluated for studying nanoparticle-based drug delivery systems.


The application of nanotechnology in medicine, particularly in drug and gene delivery, has become increasingly popular. This trend can be attributed to the fact that fewer novel drugs are entering into the market, and the discovery of novel biologically active compounds, which can be therapeutically utilized to treat many diseases, has come down drastically. As a result, the use of versatile and multifunctional structures of nanoparticles has grown quickly.

Nanoparticles provide improved pharmacokinetic properties, ensure sustained and controlled release, and target only certain tissues, cells, or organs. These aspects enhance the pharmacological efficacy of current drugs. Some of the commonly defined nanoparticle vectors are as follows:

  • liposomes
  • dendrimers
  • micelles
  • solid lipid nanoparticles
  • semiconductor nanoparticles
  • metallic nanoparticles
  • polymeric nanoparticles

Therefore, nanoparticles are widely used to deliver drugs, vaccines, genes, and diagnostics into particular cells and tissues. Conversely, while these particles are increasingly being utilized to bring down the side effects and toxicity levels of the drugs, it has been found that the so-called carrier systems themselves can pose hazards to patients.

In fact, the type of risks posed by utilizing nanoparticles for drug delivery are greater than the risks posed by chemicals used in traditional delivery matrices. As such, many different substances are being analyzed to formulate nanoparticles for drug delivery. These substances range from biological substances such as phospholipids, gelatin and albumin for liposomes through to substances of chemical nature such as metal containing nanoparticles and polymers.

Nanoparticles in Drug Delivery

Considering the various applications of nanoparticles, nanoparticle-based drug delivery and targeting has been the topic of a recent review, which discusses the advantages of nanotechnology and also provides warnings regarding the physical nature of the nanoparticles, and how they can impede with standardized and conventional immunotoxicity and biocompatibility testing procedures.

Yet another comprehensive review describes many assays that are needed to find out the chemical and physical characteristics of nanoparticles such as MALDI-TOF, batch-mode dynamic light scattering, TEM, AFM, zeta potential measurement, and SEM X-ray microanalysis of nanoparticles existing in cultured cell thin sections or tissues.

Since NTA is a newly developed method, it was not accounted for in this review. However, NTA is being used in the characterization of nanoparticulate suspensions, which are being formulated for drug delivery application. Before introducing nanoparticles to cellular systems for cytotoxicity testing, a better understanding of nanoparticle size distribution is very important. In this context, the NTA technique has been shown to be useful when compared to DLS and other nanoparticle characterization techniques.

Earlier Applications of NTA

Maher et al., 2009 used NTA for studying sodium caproate mediated promotion of oral drug absorption; Bult et al., 2010 utilized NTA for studying holonium; and Bhuiyan (2010) demonstrated that drug release from thermosensitive liposomes can be caused by hypothermia by utilizing the NTA technique to define the liposome preparations.

Recently, Sunshine et al. (2012) utilized the NTA technique to measure the particle size before subretinal injection. The effective transfection of the RPE in vivo indicated that nanoparticles can possibly be used for studying genetic diseases to treat various eye diseases.

Nanoparticles in Targeting

Molecular structures with an affinity for particular cell surface biomarkers are generally added during the targeting of drug delivery nanoparticles to certain sites. However, adding these capture molecules to the surface of the drug delivery nanoparticles can be quite challenging.

To ensure optimum performance, adequate loading, retention of activity, and reduced aggregation are very important. Likewise, when other biochemical species, developed to stabilize the functional structures, are added to the nanoparticles, it may cause similar toxic effects.

In contrast, when macromolecules are added to nanoparticles, NTA can detect even trace-level changes in hydrodynamic diameter and can even detect and specify aggregates that may occur during such changes. As such, NTA has been employed in many of these studies, including the influence of conjugating polymer-alendronate-taxane complexes used for targeting bone metastases (Miller et al., 2009).

NTA was used by the same group to demonstrate that conjugation for the targeting of angiogenesis-dependent calcified neoplasms utilizing varied polymers led to polydispersities of narrower and smaller sizes. This along with a cathepsin-K-cleavable system, a more specific drug release was eventually obtained. As a result, the group directed the toxicity of the free drugs to the bone cancer.

In another experimental study, both NTA and DLS techniques were utilized to demonstrate the particle size between 1 and 50nm in aqueous solution (Dellinger et al., 2013). This was done by using a number of advanced methods such as NMR, matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF), and high performance liquid chromatography to define stabilized FcεRI-mediated mast cells, fullerene derivatives, and peripheral blood basophils.

NTA was also applied to size the calcium phosphate nanoparticles used for transporting supramolecular drugs through the cell membrane (Rotan et al., 2013). NTA was also used to size bioresorbable polymersomes for delivery of cisplatin (Petersen et al., 2013).

Gene, RNA and DNA Delivery

Novel biologically active compounds can be therapeutically exploited to treat many types of diseases. However, with fewer number of discoveries made in this regard, there is a need to bridge this critical gap. Here, the use of nanotechnology, predominantly in gene and drug delivery, represents a key milestone.

In this context, using nanoparticles to transport and deliver cargoes of various genetic materials presents an interesting approach. The use of using polymeric nanoparticles to deliver non-viral genes is one attractive method for treating genetic diseases and developing sophisticated technologies for regenerative medicine.

Viruses come with major safety issues, but polymeric nanoparticles can be formulated to be non-toxic, non-mutagenic, and non-immunogenic. Such particles are chemically versatile, environmentally responsive, biodegradable, easier to produce, and can carry larger nucleic acid cargo.

siRNA delivery to cell systems has been recently researched to improve human mesenchymal stem cell differentiation through RNA interference (RNAi). This approach may provide a suitable means for regulating cellular conditions for tissue engineering. However, this would require the development of a safe and effective delivery system.

The delivery of SiRNA has also been researched by using cell-penetrating peptides (CPPs), which have been widely studied as drug delivery systems for nanoparticles, nucleic acids, and proteins. Ezzat et al., 2012 also used NTA to show the stability of nanoparticles on drying and re-suspension. In a similar way, Troiber et al., 2012 compared four types of particle sizing techniques for the characterization of siRNA polyplex, as a standard method for measuring the particle size was not available.

As such, four types of analytical techniques, such as AFM, DLS, fluorescence correlation spectroscopy (FCS), and “nanoparticle trafficking analysis” (NTA), were assessed for their viability for studying the characteristics of both heterogeneous and homogeneous siRNA polyplexes. While larger particles measuring 120nm can be sized by all techniques, particles measuring 40nm had very low refractive index to be detected by NTA.

Recently, NTA was used to demonstrate that dendrimer structures, which were being used as siRNA delivery vehicles, experienced changes in terms of polydispersity and size at higher dendrimer concentrations. This suggested that electrostatic complexation leads to an equilibrium between complex aggregates of various sizes (Jensen, 2011). This analysis made it possible to detect the optimum dendrimer structure for subsequent delivery of nucleic acids.

The NTA technique has even been applied to develop non-viral gene delivery systems, which are based on poly(β-amino ester)s (PBAEs) (Tzeng et al., 2011), a lipophilic plasmid DNA condensate (Do et al., 2011), and in the screening of these structures in vitro (van Gael et al., 2011).

Recently, Shmueli et al., 2013 described a novel procedure to define PBEA nanoparticles through the NTA technique. These hydrolytically degradable PBAEs have been found to be effective at gene delivery to hard-to-transfect cell types, namely human brain cancer cells, human retinal endothelial cells, macrovascular endothelial cells, and mouse mammary epithelial cells. This NTA-based procedure can be used for assessing polymeric nanoparticles and any target cell type in a multiwell or 96-well plate format for transfection assay for quick screening of the transfection efficacy.

According to Witwer et al., 2013, droplet digital PCR and real-time quantitative PCR or RT-qPCR for plant miRNAs in mammalian blood does not provide significant proof for the uptake of dietary miRNAs. This is contrary to the earlier evidence that exogenous dietary miRNAs penetrate the tissues and bloodstream of ingesting animals. This evidence indicates that at least a single plant miRNA, miR168, takes part in cross-kingdom regulation of a mammalian transcript.

However, when RT-qPCR was used to determine plant and endogenous miRNAs from pigtailed macaques, which were injected with a miRNA-rich, plant-based substance, the outcomes did not support the uptake of dietary plant miRNAs. Here, NTA was applied to demonstrate a change in population and particle size after food intake.

Further, NTA technique was also used for studying chitosan-based nanoparticles for delivery of gene-and siRNA (Malmo, 2012) and for “The in vitro assessment of alkylglyceryl-functionalized chitosan nanoparticles as permeating vectors for the blood–brain barrier.”


NTA is a unique tool used for the characterization of nanoparticles. A better understanding of the distribution of nanoparticles before their introduction to cellular systems for cytotoxicity testing purposes is very important. NTA has proved handy in this regard when compared to other types of nanoparticle characterization methods, and therefore is used in a wide range of applications within the pharmaceutical sciences.


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