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Review
. 2022 Oct 10:13:984252.
doi: 10.3389/fimmu.2022.984252. eCollection 2022.

Lessons learned from immunological characterization of nanomaterials at the Nanotechnology Characterization Laboratory

Marina A Dobrovolskaia  1
Affiliations
Review

Lessons learned from immunological characterization of nanomaterials at the Nanotechnology Characterization Laboratory

Marina A Dobrovolskaia. Front Immunol. .

Abstract

Nanotechnology carriers have become common in pharmaceutical products because of their benefits to drug delivery, including reduced toxicities and improved efficacy of active pharmaceutical ingredients due to targeted delivery, prolonged circulation time, and controlled payload release. While available examples of reduced drug toxicity through formulation using a nanocarrier are encouraging, current data also demonstrate that nanoparticles may change a drug's biodistribution and alter its toxicity profile. Moreover, individual components of nanoparticles and excipients commonly used in formulations are often not immunologically inert and contribute to the overall immune responses to nanotechnology-formulated products. Said immune responses may be beneficial or adverse depending on the indication, dose, dose regimen, and route of administration. Therefore, comprehensive toxicology studies are of paramount importance even when previously known drugs, components, and excipients are used in nanoformulations. Recent data also suggest that, despite decades of research directed at hiding nanocarriers from the immune recognition, the immune system's inherent property of clearing particulate materials can be leveraged to improve the therapeutic efficacy of drugs formulated using nanoparticles. Herein, I review current knowledge about nanoparticles' interaction with the immune system and how these interactions contribute to nanotechnology-formulated drug products' safety and efficacy through the lens of over a decade of nanoparticle characterization at the Nanotechnology Characterization Laboratory.

Keywords: characterization; immunotoxicity; methods; nanoparticles; nucleic acids; trends.

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Conflict of interest statement

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Clinical trials involving nanotechnology-based formulations. This figure was prepared based on data downloaded from ClinicalTrials.gov (8) on August 5, 2020; the accessibility of the site was verified on March 2, 2022. The data were grouped based on disease type and the percentage of total (1,200) was calculated. The cancer category included malignancies affecting various organs and systems. Non-cancerous diseases were grouped based on the type of affected system. “Other” includes communicable diseases, congenital abnormalities, body weight, musculoskeletal, death, fibrosis, infectious and stomatognathic diseases, tissue adhesion, blister, and breast and otorhinolaryngologic diseases.
Figure 2
Figure 2
Infusion reactions to nanomedicines. IRs to nanomedicines involve multiple players (immune cells, coagulation, and complement systems) and complex, often overlapping mechanisms. Not all patients are sensitive to these responses. Interaction between one or several components of the immune system and the nanocarrier triggers these responses in sensitive individuals. Timely detection and appropriate management of IRs are critical to avoid severe health consequences for patients undergoing therapy with nanomedicines. IRs are not unique to nanomedicines and have been documented for other types of drug products (44). Rational design of nanocarriers and understanding of mechanisms underlying nanoparticle-mediated immunotoxicity are currently considered a solution to overcome the issue of IRs to nanomedicines.
Figure 3
Figure 3
The main characteristics of the complement system. The main characteristics of plasma and intracellular complement are summarized. The intracellular complement system is discussed in the figure in the context of T lymphocytes due to a better understanding of its function in the currently available literature. The intracellular complement system has also been detected in other cell types; its role in other cells is less understood and, therefore, not mentioned in the figure. Statements highlighted with an asterisk (*) were hypothesized based on the role of lymphocytes in which the intracellular complement system was described; experimental verification is still required for these statements and represents one of the future directions of research in this field.
Figure 4
Figure 4
NCL assay cascade experience with cytokine analysis. Between 2005 and 2021, NCL has characterized over 450 nanotechnology formulations using assay cascade protocols (https://ncl.cancer.gov/resources/assay-cascade-protocols) that include six assays for the assessment of cytokines (ITA-10, ITA-22, ITA-23, ITA-24, ITA-25, and ITA-27). (A) Breakdown of formulations by cytokine profile (i.e., formulations that induced broad-spectrum cytokines versus those that exclusively induced chemokines). Percentage reflects the total number of formulations subjected to cytokine analysis. The data for 2005–2015 are pooled; during this time, IL-8 was the only chemokine on the NCL cytokine panel; other cytokines in the NCL 2005–2015 panel include TNF, IL-1β, IL-6, and IFNλ. The panel was expanded and, since 2016, includes chemokines MIP-1α, MIP-1β, MCP-1, MCP-2, and RANTES, in addition to the IL-8. Other cytokines in the extended panel are TNF, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-17, IL-22, IL-23, IL-27, IFNγ, IFNα, IFNβ, and IFNλ. “Broad-spectrum” refers to all or any combination of these cytokines where the combination includes cytokines of different functional types (e.g., pro-inflammatory and chemokines; pro-inflammatory and interferons; interferons and chemokines, or all of the above). Chemokines only refer to formulations that induce all or any chemokines in the absence of other functional cytokine types. (B) Breakdown of formulations that exclusively induce chemokines by nanoparticle composition. Most of the chemokine-inducing formulations are lipid-based, polymer-based, or contain both lipids and polymers either in the nanoparticle core or as the excipient or both. NCL, Nanotechnology Characterization Laboratory; ITA, immunotoxicity assay.
Figure 5
Figure 5
NCL assay cascade experiences with nanoparticle effects on blood coagulation. NCL has characterized more than 450 nanotechnology formulations using assay cascade protocols (https://ncl.cancer.gov/resources/assay-cascade-protocols) that include two assays for the assessment of the coagulation system (ITA-2 and ITA-12). Shown on the graph is a proportion of formulations that induced prolongation of plasma coagulation time in the NCL assay ITA-12 (A) and breakdown by nanoparticle composition of formulations resulting in APTT prolongation, a feature shared with traditional blood-thinning agent heparin (B). Most of these concepts are polymer-based, contain lipids, or both lipids and polymers either as the core nanoparticle or excipient or both. NCL, Nanotechnology Characterization Laboratory; ITA, immunotoxicity assay.
Figure 6
Figure 6
Immunosuppressive properties of nanotechnology formulations characterized at NCL. NCL has characterized more than 450 nanotechnology formulations using assay cascade protocols (https://ncl.cancer.gov/resources/assay-cascade-protocols) that include two assays for the assessment of immunosuppression (ITA-6 and ITA-18). Shown on the graph is a proportion of formulations that were immunosuppressive in these in vitro assays due to either API or carrier. The immunosuppressive properties attributed to APIs included those due to small molecules: cytotoxic oncology drugs (COD), therapeutic nucleic acids (TNA), small-molecule protein kinase inhibitor (SMPKI), or nanoparticle platform (NP). API, active pharmaceutical ingredient; NCL, Nanotechnology Characterization Laboratory; ITA, immunotoxicity assay.
Figure 7
Figure 7
Considerations for selection of whole blood versus PBMC cultures for cytokine analysis. (A) NCL decision tree for model selection. The decision is influenced by the nanoparticle composition, study questions, and instrument availability. *PBMC could be used to assess pro-inflammatory cytokines and answer questions related to the risk of the cytokine storm. Whole blood, however, is a better system if type II interferon induction is of interest. (B) Differences in immune-cell populations between the whole-blood and PBMC cultures may influence the detection of various cytokines. Bullet points at the bottom list other cells or matrices present in the culture but not shown in the forward and side-scatter cytometry plots.
Figure 8
Figure 8
Complementary approaches in establishing humanized animal models for basic and translational research purposes. Key benefits (highlighted as yellow circle with a plus sign inside) versus shortcomings (highlighted as a green circle with a minus sign inside) of both strategies are listed, as well as anticipated future directions toward improved utility of these models for preclinical drug-assessment studies. This figure is reproduced with permission from (254).
Figure 9
Figure 9
A framework of mechanistic studies. Types of mechanistic studies, model nanoparticles that could be used as controls, methodologies, and intrumentation, and, whenever available, relevant biomarkers are summarized. API, active pharmaceutical ingredient; ATP, adenosine triphosphate; CRT, calreticulin; ER, endoplasmic reticulum; ELISA, enzyme-linked immunosirbent assay; FDA, fluorescein diacetate; GNP, gold nanoparticles; GSH, glutathion; Histones, Phospho-Histone H2AX (Ser139); HSP, heat shock protein; HMG, high mobility group; ID, identifier; LC, light chain; MSU, monosodium urate; NP, nanoparticles; PI, propidium iodine; ROS, reactive oxygen species; SNP, silver nanoparticles; TM, trade mark; WB, western blot.
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