Understanding the correlation between in vitro and in vivo immunotoxicity tests for nanomedicines

Abstract

Preclinical characterization of novel nanotechnology-based formulations is often challenged by physicochemical characteristics, sterility/sterilization issues, safety and efficacy. Such challenges are not unique to nanomedicine, as they are common in the development of small and macromolecular drugs. However, due to the lack of a general consensus on critical characterization parameters, a shortage of harmonized protocols to support testing, and the vast variety of engineered nanomaterials, the translation of nanomedicines into clinic is particularly complex. Understanding the immune compatibility of nanoformulations has been identified as one of the important factors in (pre)clinical development and requires reliable in vitro and in vivo immunotoxicity tests. The generally low sensitivity of standard in vivo toxicity tests to immunotoxicities, inter-species variability in the structure and function of the immune system, high costs and relatively low throughput of in vivo tests, and ethical concerns about animal use underscore the need for trustworthy in vitro assays. Here, we consider the correlation (or lack thereof) between in vitro and in vivo immunotoxicity tests as a mean to identify useful in vitro assays. We review literature examples and case studies from the experience of the NCI Nanotechnology Characterization Lab, and highlight assays where predictability has been demonstrated for a variety of nanomaterials and assays with high potential for predictability in vivo.

Keywords: Anaphylaxis; Coagulopathy; Complement activation; Cytokines; Disseminated intravascular coagulation; Hemolysis; Nanoparticles; Phagocytosis; Procoagulant activity; Thrombosis.

Figure 1. Challenges in identifying immunotoxicity during nonclinical studies

The likelihood of identifying nanoparticle immunotoxicity increases as a drug product progresses from early in vitro models, to preclinical in vivo and into to clinical phases. However the high cost of in vivo tests coupled with increasing ethical concerns regarding animal use often impede the application of these in vivo tests despite their advantages in predictability. In contrast, the high-throughput nature and lower time and resource requirements of preclinical in vitro tests makes them an attractive alternative.

Figure 2. Correlation between in vitro and in vivo immunological tests for nanoparticle characterization

This summary highlights the experience of the NCI Nanotechnology Characterization Lab (NCL) through applying an in vitro assay cascade (http://ncl.cancer.gov/working_assay-cascade.asp) for preclinical characterization of various types of engineered nanomaterials to more than 280 nanomaterials and comparing this data to the findings of our standard in vivo toxicity tests in animals and to clinical data (when available). One limitation of the in vitro CFU-GM test is that data interpretation depends on nanoparticle biodistribution, which cannot be tested in vitro, interpretation of this test is supported by the PK profile for the given nanoformulation. DIC-disseminated intravascular coagulation; BM-bone marrow; CFU-GM- colony forming unit granulocyte macrophage; PCA – procoagulant activity; PBMC- peripheral blood mononuclear test; MAT- macrophage activation test; RPT – rabbit pyrogen test; RBC – red blood cells – HB – hemoglobin, Hct – hematocrit, downward arrow refers to a decrease, upward arrow refers to an increase.

Figure 3. In vitro-in vivo correlation of hemolysis assay results

A nanoformulation containing a cationic membrane perforating peptide was tested in vitro (top panel) and in vivo (bottom panel). The precursor formulation without the peptide was also tested as a control (data not shown). The peptide-containing formulation resulted in dose-responsive hemolysis in vitro (top panel) and caused animal death when injected intravenously. Histopathology evaluation revealed erythrophagocytosis (bottom panel) consistent with hemolysis. Images are adapted from reference [51] with permission.

Figure 4. In vitro-in vivo correlation of the complement assay

Three nanoformulations (NP1, NP2, and NP3) of metal oxide nanoparticles with identical cores and different surface chemistries were tested in vivo in rats and rabbits and in vitro using human, rat and rabbit plasma. PBS was used as a negative control (NC) and cobra venom factor was used as a positive control (PC) in vitro. Formulations causing anaphylaxis in rabbits in vivo also activated complement in rabbit plasma in vitro. Red arrows identify formulations reactive with rabbit complement in vitro.

Figure 5. In vitro-in vivo correlation of the cytokine assay

Two nanoformulations (NP1 and NP2) of metal oxide nanoparticles with identical cores and different surface chemistries were tested in vivo in rats and rabbits and in vitro using normal human peripheral blood mononuclear cells. PBS was used as a negative control (NC) and 10ng/mL K12 E.coli endotoxin was used as a positive control (PC) in vitro. Formulation 2 caused cytokine induction and related toxicity (congestion) in animals and induced inflammatory cytokines in vitro in human PBMC.

Figure 6. In vitro-in vivo correlation of phagocytosis and protein binding

A. Nanoparticles with surfaces unprotected by hydrophilic polymers are readily taken up by macrophages in vitro and in vivo. An in vitro phagocytosis assay and electron microscopy were used to detect uptake of citrate stabilized colloidal gold nanoparticles (top panel, left) and PEGylated colloidal gold nanoparticles (bottom panel, left). Nanoparticles taken up by macrophages in vitro were also retained by MPS organs in vivo (top panel, right). In vivo MPS retention image was adopted from reference [86] with permissions B. Gold shell nanoparticles showing abundant binding of plasma proteins (bottom, left) were retained by macrophages in vitro and resulted in inflammation-mediated toxicity in vivo (bottom, right). Their counterparts which did not bind proteins were not taken up by cells and were not toxic. Adopted from reference [87] with permission.

Figure 7. In vitro-in vivo correlation of the leukocyte proliferation assay

A PEGylated micelle nanoparticle was tested at non-cytotoxic concentrations in a leukocyte proliferation test in vitro and resulted in inhibition of both mitogen- and antigen-specific proliferation.. Human PBMC were used in vitro and the in vivo study was performed in rats. PC = positive control, NP = nanoparticle, WBC = white blood cells, NE = neutrophils, LY = leucocytes, MO=monocytes.