Advances in polymeric nanoparticles as novel nanomedicines have opened a new class of diagnostic and therapeutic tools for many diseases. However, although the benchtop research studies in the nanoworld are numerous, their translation to currently marketed products is still limited. This lack of transference can be attributed, among other factors, to problems with nanomedicine characterization. Characterization techniques at the nanoscale could be divided in three categories: characterization of physicochemical properties (e.g., size and surface charge), characterization of nanomaterials interactions with biological components (e.g., proteins from the blood), and analytical characterization and purification methods. Currently available literature of this last group only describes methodologies applied for a specific type of nanomaterial or even methods used for bulk materials, which are not completely applicable to nanomaterials. For this reason, the current review aims to become a scholastic guide for those scientists starting in the nanoworld, giving them a description of analytical characterization techniques aimed to analyze polymers forming nanoparticles and possible forms to purify them before being used in preclinical and clinical applications.
The field of nanotechnology and, more specifically, nanomedicine emerged about 20 years ago and since then, it experienced an exponential progress both in the fundamental study of nanosystems and in their multiple applications. Specifically, studies on polymeric nanoparticles have gained attention due to the multiple advantages that are attributed to this kind of nanosystems in terms of safety, versatility, and robustness [
Schematic representation of the process of nanomedicine characterization before translating to pharmaceutical production.
These methods have been classified not as a function of what is characterized but as a function of the technique: chromatographic, spectroscopic, calorimetric, and purification techniques. Authors will guide the reader through them with the objective to help in the selection of one or other technique depending on the parameter to study. Physicochemical techniques, mainly used to characterize size (e.g., light scattering or microscopy), are out of the scope of the present review [
Chromatographic techniques, in general, are a group of techniques devoted to the separation of various compounds [
Analytical chromatographic techniques.
Technique | Characteristics that analyses | Advantages | Disadvantages | References |
---|---|---|---|---|
Gel permeation chromatography (GPC) | Molecular weight | Rapid and simple | Interaction sample with column filling | Williams [ |
High resolution | Neverova and Van Eyk [ | |||
Cho et al. [ | ||||
High-performance liquid chromatography (HPLC) | Quantification of actives | High resolution | Interaction sample with column filling | Neverova and Van Eyk [ |
Purification | Rapid and easy performance | Sapsford et al. [ | ||
Low cost/sample | ||||
Small sample volumes | ||||
Size exclusion chromatography (SEC) | Molecular weight | High resolution | Interaction sample with column filling | Kostanski et al. [ |
Rapid and simple | Need of a labelling tag | Sapsford et al. [ | ||
Rebolj et al. [ |
The gel permeation chromatography (GPC) is a widely used technique to determine the molecular weight of materials dissolved in organic solvents as well as the physical stability of assembled nanomaterials [
This technique could be useful, for example, to study the stability of a polymer dissolved in an organic solvent. After various periods of time, the dissolved polymer should be analyzed through the GPC and its molecular weight assessed by using a calibration curve. Other examples of the use of GPC in the nanoscale could be the assessment of the polymer molecular weight, the degree of polymerization of a synthesized polymer, or even the number of monomer subunits that a polymer contains [
GPC is advantageous in terms of short-time experiments. However, an important drawback of this technique is the possible interaction between the nanomaterials and the column filling, which could interfere the size assessment [
High-performance liquid chromatography (HPLC) is the most used type of chromatography not only for colloidal nanosystem studies but also for other type of materials (e.g., proteins). In the vast majority of studies, it is used for the fine quantification and separation (purification) of actives, such as drugs [
Schematic representation of a HPLC system.
The quantification of the actives is required in any study of the encapsulation efficiency of drugs in the nanosystems or their release kinetics, as well as the percentage of conjugation to some nanosystems [
The resolution of the HPLC depends on the filling of the column (on the stationary phase properties), which is commonly composed of silica with attached alkyl chains, being the reversed phase C18-type columns the most widely used, since it enables a differential retentionship depending on the polarity of the compounds [
The advantages of HPLC are the high resolution, the low volumes required, and an easy, rapid, and economic manipulation [
In 2004, it appeared the ultra-HPLC (UHPLC) technique, with many advantages among the traditional HPLC. It uses a column filling of particles of sub-2 micron size, while conventional HPLC uses particles between 2.5 and 5 microns. This reduction on the filling particle size enables a finer separation of similar compounds. In addition, the working pressure of UHPLC equipment is markedly higher than that supported by conventional HPLC, which enables more rapid flow rates, resulting in shorter elution times and decrease on the solvent amount used [
Size exclusion chromatography (SEC), together with ion-exchange and affinity chromatography are classified as low-pressure liquid chromatography [
Spectroscopic techniques are those that give information on the interaction of an electromagnetic radiation with a sample, thus resulting in an absorption that depends on the excitation wavelength. Therefore, a wavelength spectrum with absorption/emission peaks that depend on the material is produced [
These techniques, summarized in Table
Summary of the main advantages and disadvantages of spectroscopic techniques.
Technique | Use | Advantages | Disadvantages | References |
---|---|---|---|---|
Fourier transformed infrared (FTIR) | Chemical composition | Fast and inexpensive | Complicated sample preparation | Alben and Fiamingo [ |
Characteristic of each material | Interference of water | |||
Possible quantification | Relatively low sensitivity | |||
Requires dried samples | ||||
Destructive | ||||
UV-Vis | Quantification of concentration | Cost effective | Interference between materials | Sapsford et al. [ |
Size and shape determination | Simple and fast | |||
Useful for a variety of compounds | ||||
Fluorimetry | Quantitative fluorescence determination | High sensitivity Compound specificity | Limited to fluorescent compounds | Lakowicz [ |
Limited fluorescence lifetime |
Fourier transformed infrared spectroscopy (FTIR) is a spectroscopic technique based on the measurement of vibrational transitions between different excitation states of molecules [
It is a widely used technique, not only in the nanomedicine field but also in a variety of scientific fields. It has many advantages (Table
UV-Visible spectroscopy is a spectroscopy type that emits radiation of wavelength between 190 and 800 nm, widely used for the quantification of compounds concentration, and, in some cases, even size and shape, since each material absorbs at a determined wavelength and changes in the spectra could be related with changes in the aggregation of nanomaterials [
It is a simple, fast, and cost-effective technique that can be applied to a variety of nanomaterials (Table
Fluorimetry is a very sensitive spectroscopic technique that consists in the quantitative measurement of fluorescent signals, usually produced by aromatic molecules, for the detection and characterization of organic and inorganic compounds thanks to the application of a fluorescent laser to a sample (Figure Excitation: it is produced by the absorption of the electromagnetic radiation by the sample to study. Losses of vibrational energy: they are produced after the absorption of energy and before the emission of fluorescence, due to the internal collision between the molecules of the sample. Emission: it consists of the energy produced by a molecule of the sample when it drops to a lower vibrational level (corresponding to lower energy and longer wavelengths), thus emitting energy in the form of fluorescence. The fluorescence is obtained as a spectrum and not a single band because not all the molecules of the sample drop to the same vibrational level.
Schematic representation of a fluorescence spectrophotometer model.
This technique has been used for various applications, all of them taking advantage of the capacity of the studied materials to be excited under a fluorescent laser and emit fluorescence of another wavelength. For example, Lin et al. [
Although the varied uses of fluorescence, it has two main drawbacks (Table
Recently, advanced techniques combining the advantages of fluorescent signal have appeared. Of special importance is the Förster resonance energy transfer or FRET technology. It consists in the combination of two fluorescent probes labeling pairs of two compounds, the first one called the donor and the second one called the acceptor. Fluorescence of excitation wavelength of the donor is directed to the compound, which, under the specific fluorescence will emit fluorescence in another wavelength. The acceptor is excited specifically by the emission wavelength of the donor (energy transference) and emits fluorescence in another wavelength. Therefore, if both compounds are very close (<10 nm), when exciting the donor, only fluorescence signal of the acceptor emission wavelength will be detected. In contrast, if both molecules are not close enough, when exciting the donor, fluorescence emission of this donor wavelength will be detected. Therefore, this technique is very useful to study two compound aggregation, and it is starting to play an important role in nanosystem studies [
Calorimetric techniques are a type of techniques that apply a temperature change to the samples to study physical phenomena, such as the crystalline transition, fusion, vaporization, sublimation, absorption, adsorption, and desorption and chemical phenomena, such as chemisorptions, desolvation, decomposition, oxidative degradation, solid-state, and solid-gas reactions [
Summary of the main advantages and disadvantages of calorimetric techniques.
Technique | Use | Advantages | Disadvantages | References |
---|---|---|---|---|
Differential scanning calorimetry (DSC) | Glass transition |
Low amounts of sample |
Requires sample preparation |
Hunt [ |
Thermogravimetry (TGA) | Weight loss | Low amounts of sample |
Requires sample preparation |
Berger et al. [ |
Differential scanning calorimetry (DSC) is a technique that continuously measures the apparent specific heat of a system as a function of the temperature [
It is a useful technique to determine the structure and stability of nanomaterials, as well as their conformation, since material transitions will change depending on the nanomaterial composition [
Thermogravimetric assays (TGA) are another type of calorimetric techniques which measure the weight loss of the samples [
It is a useful technique to determine the amount of nanoconjugation, since the change on the nanomaterial composition produces changes in the temperature weight loss [
Since both calorimetric techniques have a similar performance, their advantages and disadvantages have been summarized together. Calorimetric techniques are advantageous in terms of small amount of sample required, high precision and sensitivity. However, an appropriate reference as well as the preparation of the sample is required [
The composition of colloidal nanomaterials is a key point, since it affects not only its transport, delivery, and biodistribution
Although various methods exist for the colloidal nanomaterial purification, such as the magnetic separation for magnetic nanoparticles, for example [
Summary of the main advantages and disadvantages of the described purification techniques.
Technique | Use | Advantages | Disadvantages | References |
---|---|---|---|---|
Filtration | Purification | Useful for thermolabile compounds | Time consuming | Scopes [ |
Sterilization | ||||
Concentration | Rapid and simple | Bigger sizes determined by the cut-off size | Sapsford et al. [ | |
Dispersant changement | Commercially available devices | Single-use devices | Roy et al. [ | |
Reduce size polydispersity | Cost effective | Dobrovolskaia and McNeil [ | ||
Centrifugation | Purification | High efficiency | Special equipment required for large volumes | Scopes [ |
Concentration | Rapid, facile, and economic | Difficult to resuspend soft matter | Sapsford et al. [ | |
Dispersant changement | Low amounts of sample | |||
Appropriate for different kinds of nanomaterials | ||||
Dialysis | Purification | Rapid, facile, and economic | Limited to the membranes MWCO | Vauthier et al. [ |
Concentration | Commercially available devices | High receptor solution volumes | Sapsford et al. [ | |
Dispersant changement | No sample pretreatment | |||
Electrophoresis | Purification | Simple and economic | Postelectrophoresis purification steps | Sapsford et al. [ |
Jason [ | ||||
High resolution and sensitivity | ||||
Need of charged compounds | Scopes [ |
It is worth remarking the contamination by endotoxins. Although methodology to purify from endotoxins is out of the scope of the present review, authors would like to remark the importance of producing nanomedicines clean from this type of contamination, which could be produced from many lipopolysaccharides, of the omnipresent Gram-negative bacteria. It is important to clean all material used before starting the production (e.g., by cleaning with sodium hydroxide or heating treatments) of nanomedicines and to confirm that resulting nanomedicines are clean from endotoxins [
Filtration is a purification method, specifically used to sterilize nanomaterial colloidal dispersions. This method is advantageous as a sterilization technique for thermolabile compounds [
In addition, it can also be useful for other purposes, such as for the concentration of colloid nanomedicines or the reduction of their polydispersity. For example, Roy et al. [
Centrifugation is another technique useful for the purification of nanomaterials. It consists of the application of a centrifugal force to enhance the precipitation of nanomaterials due to the increased gravitational field [
Centrifugation is more efficient than filtration. It is a rapid, facile, and economic technique, able to be used for different kinds of nanomedicines. In addition, low amounts of sample are required. However, the centrifugation of large volumes requires special equipment and in some cases, difficulties on resuspending sediment nanomaterials appear, specifically when working with soft matter, which are not always possible to recover their dispersion liquid state [
Apart from the use of centrifugation to purify nanomedicines, it has been also used to concentrate nanomedicines, to change their dispersant, and to separate conjugated nanomaterials from those nonconjugated [
Dialysis consist on changing the nanomaterial dispersant by means of submerging a semipermeable dialysis bag (or a dialysis dispositive) filled with the nanomaterial dispersion, in a receptor solution. Therefore, it is not only useful for the nanomaterial purification but also for nanomedicine concentration and to change the dispersant to achieve the desired properties (e.g., dialysis with PBS to achieve the physiological pH and osmolality). The liquid diffuses through the membrane from the more concentrated solution (sample or receptor solution) to the less concentrated one, to achieve an osmotic equilibrium. Therefore, osmotic conditions have to be completely controlled to avoid volume changes; except in the case of nanosystem concentration, where a decrease of the sample volume is required and it can be achieved using a hypotonic receptor solution [
This technique is advantageous in terms of minimal sample manipulation, without the need of any pretreatment, but it is limited to the existent dialysis membranes. If the sample to dialyse is expected to interact with the membrane or if the molecular weight cut-off is not appropriate, this technique cannot be used. In addition, high volumes of the receptor solution are usually required [
Electrophoretic techniques consist of the application of an electric field to a polymeric gel (composed mainly of agarose or polyacrylamide) submerged in a liquid buffer; through which charged molecules run depending on their charge and/or on their molecular weight. Although electrophoresis is used with many objective, one of the uses of this technique is the separation and purification of determined nanomaterials, which has been widely reported [
The main advantages of this technique are its economic and simple performance together with the high resolution and sensitivity. However, only charged compounds can be purified through electrophoresis, and once the compound is separated in the gel, further purification steps are required to extract it from the gel [
Although it is not specifically a technique for the purification of nanosystems, it was considered appropriate to remark an electrophoretic type widely used to characterize the formation of nanocomplexes by the electrostatic interactions between their components. This technique is called electrophoretic mobility shift assay (EMSA). EMSA consists in a native (nondenaturing) polyacrylamide electrophoresis where samples migrate depending on their charge, under an electric field [
Example of an EMSA of polymeric nanoparticles conjugated with antisense oligonucleotides at different nanoparticle/oligonucleotide (N/P) charge ratios. When the complexation is achieved (zero surface charge), the band is diffused (at 0.75/1 in this case).
Its rapid performance, simplicity, robustness, and high sensitivity make this methodology the choice for the study of electrostatic complexes formation of a wide range of compounds. Although EMSA is usually applied as a qualitative technique, under the appropriate conditions, it can be also useful to quantify the stoichiometry of complexation. It is also remarkable that multiple EMSA variants exist for various purposes, such as the time-course EMSA to measure the dissociation kinetics or the circular permutation to measure the DNA bending [
A complete characterization of nanomaterials intended for biomedical purposes (diagnostic, treatment, or theragnostic) is a must before the translation to preclinical and clinical studies. Classical chemistry analytical techniques can be applied for the characterization of some aspects of nanomaterials, since nanomaterial properties usually change from those of their components. Chromatographic techniques can be useful for the determination of the molecular weight of nanosystems, as well as for the purification of the produced nanoobject from raw materials and impurities. Spectroscopic techniques, in parallel, can be very useful for the confirmation of the formation of the nanosystems, assessing the materials of which it is composed as well as its aggregation/attachment state. Calorimetric techniques can be useful for the study of the nanomaterial behavior when submitting them to temperature changes. Finally, it is always required a purification step to ensure the obtaining of a safe nanosystem, free of impurities and raw materials. Therefore, as a general conclusion of this review, it is strongly recommended to take a look on the variety of existent techniques to look for all the aspects that must be known before the translation of a novel nanomaterial to a human diagnostic or therapeutic formulation.
The authors declare that they have no conflicts of interest.
This work was funded by Spanish Ministry of Economy and Competitivity, MINECO (grant CTQ2014-52687) and Generalitat de Catalunya (grant 2014-SGR-1655). CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Cristina Fornaguera is grateful to MINECO for their PTQ2015 grant.
Schematic representation of analytical techniques to characterize a nanoparticle dispersion, as a summary of the whole review.