Today, nanotechnology has occupied almost every domain on this earth. It has revolutionized the world of medicines too. Medical scientists are putting their efforts and letting no stone unturned using the nanotechnology platform so as to make the best use of drug therapies. Nanomedicines refer to drugs, medical devices and health products developed using nanotechnology with the aim of diagnosing, monitoring and treating diseases at the molecular level. Numerous nanomedicines are currently being explored and some have already found their way into the clinical trials and market shelves (Table 1).
Nanomedicines are of diverse types, mainly, liposomes, dendrimers, quantum dots, nanoemulsions, nanocrystals-based formulations, etc., (Figure 1) which are employed primarily for therapeutics and diagnosis of diseases. The numerous advantages of nanomedicines over the conventional medicines have flabbergasted the researchers. Figure 2 shows the immense potential applications of nanomedicines. Enumerated are some of important ones including clinical applications like reduction in dose, effective targeting of difficult-to-reach sites, reduction in adverse side effects and pharmaceutical application like improved solubility, bioavailability and permit the drug to resist metabolic deactivation and degradation in the body until it binds with the target cells.
Despite the fact that the nanomedicines have proved to be far more efficient than conventional medicines, the toxicity potential of nanomedicine is still uncertain. Nanomedicines should be characterized suitably with apt and rigorous methods so as to get reproducible and precise results. However, characterization of nanomedicines is a daunting task because of the associated problems, difficulties and expertise required. The commonly used in vitro, ex vivo and in vivo techniques used for characterization of nanomedicines are summarized in Table 2 and pictorial description of characterization is shown in Figure 3.
Characterization of nanomedicines Physicochemical characterization
To assess the physicochemical properties like particle shape, size and size distribution various techniques are available including visualization of nanoparticles by microscopy (atomic force microscopy, transmission electron microscopy and scanning electron microscopy); laser diffractometry, analytical ultracentrifugation, capillary electrophoresis and field flow fractionation. Zeta potential plays an important role in the stability of nanomedicines and also in their interaction with the cells and biological fluids, drug loading and circulation time in blood. Zeta potential can be determined by Laser Doppler Electrophoresis. Whatever the method used, it should be accurate and sensitive enough to give reproducible results. Indeed proper justification of using a particular method should be given at the time of analysis. The more appropriate approach in these cases is to get the results verified by conducting analysis by other methods and then driving meaningful conclusions.
Drug loading and drug content
Quantification of active materials entrapped or encapsulated in the nanomedicine is also a crucial analytical test. Dialysis bag and nano-precipitation method has been commonly used for this purpose. The problem arises in case of nanomedicines when besides the active material, quantification of certain inactives also becomes important; in case of nanomedicines having additional complex components such as proteins or nucleic acids which may be conjugated to the nanomedicines just for site direction or targeting to a particular type of cells. For these types of systems, multiple orthogonal characterizations become essential to ensure that the nanomedicines have all the desired properties for the intended therapeutic purpose.
Drug release kinetics
Measuring the amount of drug released from the nanomedicines is necessary so as to get the release kinetics of drug. No official method is available for assessing the release kinetics of nanomedicines. Therefore, drug release characterization methods differ from lab to lab leading to variation in the results. Different methods experimentally applied for quantifying drug release in vitro are side by side diffusion cells with artificial or biological membranes, equilibrium dialysis technique, reverse dialysis sac technique, ultracentrifugation, ultra filtration, centrifugal ultra- filtration technique. More scientific approach in this case will involve a strong rationale which should be given for each and every component of the applied method like selection of medium or adjusting the pH of the selected medium to a specific value.
Sterility and Pyrogenicity Assessment
Limulus amebocyte lysate (LAL)-based assay and rabbit pyrogen test are most commonly used methods for testing sterility and pyrogenicity. However the major concern with the use of these two methods is the probability of interference of nanomedicines with components of the tests giving false positive or negative results. So there is a need to develop alternate methods or interference should be taken into account if any, before coming to a conclusion when using the current methods.
Biodistribution and toxicity
Biodistribution mainly involves the ADME i.e., absorption, distribution, metabolism and elimination. The biodistribution and toxicity characterization of pharmaceuticals meant for human use involves many in vitro and in vivo studies.
In vitro test may include cell based cell viability assays, hemolytic toxicity studies, thrombogenicity (i.e., platelet-aggregation tests), flow cytometry, Fluorescence activated cell sorting (FACS) or colorimetric assays. Figure 4 demonstrates the result of intracellular uptake study of fluorescence marker 6-Carboxyfluorecein loaded nanovesicular formulation in A549 cell line (Human lung adenocarcinoma epithelial cell line). These are the tests which are normally applied to conventional pharmaceuticals. In case of nanomedicines some additional tests like phagocytosis which can demonstrate the probable accumulation of nanomedicines in particular type of cells, tissues, or organs or their elimination as such need to be carried out. FACS assay is currently most widely used method to determine the intracellular uptake of drugs from the nanomedicines. Trouble can be confronted in these different characterization techniques; for instance, in platelet-aggregation test there may false results because of the nanomedicine-platelet aggregate or aggregation of nanomedicines particles itself instead of platelets.
In vivo study should include evaluation of hematology, clinical chemistry, acute and sub-acute toxicity, gross pathology, immune organ weights and histology, followed by examination of immune cell function in those cases in which the preliminary study indicated potential immunotoxicity. Tracing behavior of the nanomedicines i.e., in vivo characterization is yet another challenge as many times nanomedicines are composed of multiple components. So dissociation before reaching the target site or even failure to dissociate at target site, or incomplete/partial release of the drug from the nanomedicine will result in inadequate efficacy. Therefore if in vivo imaging is applied to track the path of nanomedicines so all the components of the nanomedicine should be tracked if there is any dissociation. Multiple imaging methods (radiolabeled studies) should be used employing multiple isotopes.
Stability
The overall stability of nanomedicines should be tested with nanoparticles in solid form, in suspension, and in biological medium, and under accelerated conditions such as higher temperature to ensure the robust performance of nanoparticles. However, the above tests may not be able to functionally differentiate between an “active” formulation and one that is “inactive” or “less active.”
In sum, owing to the complex nature of nanomedicines, it is reasonable to expect that the level of analytical characterization, testing and release required to adequately understand and define the physicochemical or biological nature of these products is more sophisticated and burdensome than for standard pharmaceutical products. Identifying the appropriate analytical tests to fully characterize nanomedicines, whether physical, chemical or biological, may be one of the more challenging aspects of nanomedicine development both from technical and regulatory perspective.
Problems and issues in characterization
The main problems which make the characterization of nanomedicines a daunting task belongs to following scientific challenges which are obstructing the outstretch use of nanomedicines at commercial level:
Applicability, appropriateness and pertinence of presently available methods to assess description, classification and characterization of the nanomedicines.
Difficulty in analysis of particle size and size distribution of free particles and agglomerates, expertise required carrying out such type of analysis and also the stability issues related to agglomeration/ aggregation/dissolution/ release from encapsulation during analysis.
The probable alteration in the performance of nanomedicines upon coming in contact with biological systems than performance assessed by conducting various in vitro tests depicting limitations of current testing methods.
Diverse chemical nature of nanomedicines like liposomes, dendrimers, quantum dots, nanocrystals also make the characterization process cumbersome.
Difficulty in assessing the properties of combinational nanomedicines.
Lack of safety protocol for personnel involved in manufacturing and testing of nanomedicines.
Today, there are several agencies which have taken initiative in this particular area of characterizing the nanomedicines. These organizations assist in developing unique tools and techniques to characterize nanoscale materials. e.g., Nanotechnology Characterization Laboratory (NCL) established by the National Cancer Institute (NCI) and Nanomedicines characterization core facility (NCore) of the University of North Carolina. The NCL characterizes nanoparticles' physical attributes, in vitro biological properties, and in vivo compatibility using animal models. The time required to characterize nanomaterials from receipt through the in vivo phase is approximately one year. NCL facilitates the clinical development and regulatory review of nanomaterials for cancer clinical trials. NCore granted researchers an opportunity for in-depth characterization of nanomaterials and carrier-mediated agents.
Epilogue
Nanomedicine offers enormous potential for improving health care and health outcomes but also may involve uncertain risks that will need to be adequately managed for this new technology to achieve its promise. The need to establish suitable standards, validated tests and safety protocol are the areas which still requires particular attention and lot of work remaining to be done. Since regulatory agencies around the world are simultaneously struggling with regulatory issues of nanomedicines , there may be benefits from attempting to harmonize national regulations. There should be facilitation between the scientists, academicians and industrial innovators so as to make this dreaming nanotechnology to come into the real world and realize its potential.
(Authors are faculty, Dept of Pharma Sciences, Guru Nanak Dev University, Amritsar (Punjab) )