Providing The Full Picture: A Mandate for Standardizing Nanoparticle-Based Drug Delivery

Keywords::

• drug delivery
• nanoparticles
• physicochemical characterization
• standardization
• structural characterization
• surface characterization

Unique properties at the nanoscale can be utilized for improving the effectiveness of drug delivery for medical applications. Submicron particles exhibit vascular migration and extravasation into tissue and cellular uptake. Furthermore, the large surface:volume ratio potentiates surface-engineered properties such as steric coats and targeting functionality. Size, however, is not the exclusive parameter for determining the biological performance of a nanoparticle (NP) system. Shape, surface topography, chemistry and reactive group density, and physiological stability all contribute towards the pharmacokinetic profile of NPs and can be intimately linked. Size and shape, for example, influence phagocytic capture by cellular components of the mononuclear phagocyte system; however, deposition of opsonin proteins, and consequent conformational-induced recognition by phagocytes or the complement cascade, is determined by particle surface chemistry and curvature that, in turn, is controlled by geometry [1]. In addition, surface charge can also determine uptake as well as initiate intracellular danger signals and activation of proinflammatory signals, including cytokine induction, interferon response and lymphocyte activation [2–6].

All too often in the scientific literature, inadequate information is given on particle physicochemical characteristics. Of the 13,800 papers identified on PubMed with the search term ‘nanoparticles for drug delivery‘, more than 4000 had insufficient information regarding the physicochemical properties of the NPs tested. In approximately 3200 manuscripts only size distribution data were given without any information about the particle charge and shape, or the encapsulated drug or its release profile. What is needed in the field is a set of NP physicochemical characterization and biological evaluation criteria that allow not only the author, but also the reader, to fully understand the mechanism of activity, interpret experimental data and bring standardization that is required for clinical translation to the field.

Hydrodynamic particle diameter, morphology and surface charge are often the only physicochemical characteristics provided, usually determined by dynamic light scattering, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and ζ potential, respectively. Careful consideration has to be given to the techniques used. Dynamic light scattering is an ensemble intensity-weighted method that shows bias towards larger particles, restricting its use for polydispersed sample measurements. SEM and TEM can be subject to aggregation during sample preparation, while ζ values vary depending on buffer and pH conditions [7], which leads to inconsistencies in the literature. What is needed is the development of new and complementary techniques that overcome some of the drawbacks associated with ‘traditional’ methods.

The use of high-resolution cryo-TEM with environmental-SEM techniques can provide measurements in more natural states that, together with atomic force microscopy, can provide greater insights into the topography, structure, shape and stability of 3D particles. Nanoparticle tracking analysis based on video capture of the trajectory of individual particles provides high-resolution particle size discrimination in polydispersed samples and is gaining popularity [8]. The outermost surface of NPs determines the biological interactions; therefore, advanced surface characterization methods, such as x-ray photoelectron spectroscopy, to measure the elemental surface composition are needed. We have used a combination of x-ray photoelectron spectroscopy and nuclear magnetic resonance to distinguish between bulk and surface PEG and determination of PEGylated ‘nanoshields’ at the outmost 10-nm surface [7]. Precise quantitative determination of surface composition, rather than the indirect commonly employed change in surface ζ potential, is a more accurate approach to determine hydrophilic coatings and target moieties needed to optimize and further develop ‘stealth’ and targeted systems. Advanced surface characterization techniques may provide more information on surface-bound material commonly found with polymer-based polyplexes [9] that have been shown to influence biological activity [10].

Characterization of NP cargo is a requirement that should include its spatial location, for example, whether it is surface bound or incorporated within the NP. Drug loading can be determined by various analytical methods, such as mass spectrometry, HPLC and UV-visible spectroscopy, dependent on the intrinsic physicochemical and structural properties of the entrapped drug or imaging agent. Drug potency/functionality, its release profile from the NP and release mechanism, such as stimuli-triggered disassembly [11,12], need to be described to provide the reader with the design rationale for the intended application.

Following physicochemical characterization, there are numerous methods to determine the interaction of a drug-loaded NP with the biological environment or ‘nano–bio’ interface, which varies depending on the route of NP administration. Characterization of the protein corona to identify, for example, complement factors important in the immune recognition process, can be determined by surface-sensitive mass spectroscopy techniques, such as TOF secondary ion mass spectroscopy and MALDI-TOF, to complement traditional gel electrophoretic methods.

Understanding of cellular interactions, such as mechanisms of uptake (phagocytosis, endocytosis, or receptor-mediated clathrin or caveole-aided endocytosis) by which NPs enter cell subsets should be provided in addition to tracking the NP intracellular trafficking [13,14]. The use of confocal scanning microscopy is often detailed in manuscripts, but only seldom do authors describe the mechanistic process by which a particular NP system interacts with cells. Binding at equilibrium conditions and in nonequilibrium conditions must be tested, and blockers for several uptake mechanisms should be used to determine the cellular uptake in a nonbiased manner. General endocytosis blockers and pathway dependent blockers, which are known in other disciplines, should be applied in the research of NP–cell interactions. These include, for example, dynasore, a cell-permeable inhibitor of dynamin [15,16], and other pathway-dependent inhibitors. Many of the reported NP systems lack appropriate toxicity assays. Although most of the reports do show 24–48 h viability assays (which are often in static condition that, in turn, will never simulate the in vivo environment), they are usually very limited immune–NP interaction assays.

The pharmacokinetics and circulatory half-life can be determined by detection of an intrinsic NP component or a postmodified label with, for example, γ-scintigraphy. MRI [17], and luminescence and fluorescence bioimaging [18] are now gaining prominence. Robust labels covalently attached or incorporated during NP formation that are nonsusceptible to removal in the biological environment should be used. Techniques with the ability to directly monitor NP physicochemical changes resulting from its interactions in situ, rather than interpretation based on the biological fate are lacking in the field. The use of relevant animal models to study the particular biological property of the NP is a necessity. The majority of anti-tumor studies are conducted in nude mice to enable investigations on human xenografts; however, these animals possess a compromised adaptive immune system that reduces the effect of macrophage-mediated NP clearance. NP pharmacokinetic studies should be conducted in immune-competent animals. The rapidly induced animal tumors that potentiate the enhanced permeability and retention effect may not be representative of all human tumors; therefore, caution must be observed in predicting the translational capability of certain NPs evaluated in these models.

NPs made from various materials, with different geometry, size and charge, may interact with cells of the innate immune arm, such as macrophages or dendritic cells, in a similar manner to a pathogen in order to initiate a host immune response. It is, therefore, important to investigate this. There are numerous assays that can probe these interactions, including cytokine profiling, interferon response, lymphocyte activation and complement activation [2,4,19,20]. Some examples that have investigated these interactions include a targeted lipid-based NP, delivering RNAi payloads to subsets of gut leukocytes [21], and nontargeted NPs delivering RNAi payloads to hepatocytes [22].

To summarize, there is a dramatic increase in the number of reports on the use of NPs in medical applications. Authors need to make efforts to comprehensively characterize their NP system, which may require interdisciplinary research at the nanoscience/pharmaceutical interface. While, it is beyond the capability of many research groups to perform all the methods outlined in this article, the scientific community must generate some level of standardization of physicochemical characterization and biological evaluation of various NP systems. This calls for a global revision in scientific journals and for institutions such as the US FDA and the EMA to take measures that will generate standardization criteria for all reported NPs systems, not only in clinical testing, but also for publication purposes.

Financial & competing interests disclosure

D Peer has a financial interest in Quiet Therapeutics. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

D Peer has a financial interest in Quiet Therapeutics. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.