A multistep in vitro hemocompatibility testing protocol recapitulating the foreign body reaction to nanocarriers

Abstract

The development of drug nanocarriers based on polymeric, lipid and ceramic biomaterials has been paving the way to precision medicine, where the delivery of poorly soluble active compounds and personalized doses are made possible. However, the nano-size character of these carriers has been demonstrated to have the potential to elicit pathways of the host response different from those of the same biomaterials when engineered as larger size implants and of the drugs when administered without a carrier. Therefore, a specific regulatory framework needs to be made available that can offer robust scientific insights and provide safety data by reliable tests of these novel nano-devices. In this context, the present work presents a multistep protocol for the in vitro assessment of the hemocompatibility of nanocarriers of different physicochemical properties. Poly (ethyl butyl cyanoacrylate) nanoparticles and lipid-based (LipImage™ 815) nanoparticles of comparable hydrodynamic diameter were tested through a battery of assays using human peripheral blood samples and recapitulating the main pathways of the host response upon systemic administration; i.e., protein interactions, fibrinogen-platelet binding, cytotoxicity, and inflammatory response. The data showed the sensitivity and reproducibility of the methods adopted that were also demonstrated to determine individual variability as well as to discriminate between activation of pathways of inflammation and unintended release of inflammatory signaling caused by loss of cell integrity. Therefore, this multistep testing is proposed as a reliable protocol for nanoparticle development and emerging regulatory frameworks. Graphical abstract

In Vitro Hemocompatibility and Genotoxicity Evaluation of Dual-Labeled [99mTc]Tc-FITC-Silk Fibroin Nanoparticles for Biomedical Applications

“…LDH levels increased significantly after 4 h of incubation compared to the control, indicating a loss of cell integrity, as described by other authors [ 13 , 30 ]. In our case, the increase in LDH levels was found to be ~50 U/L, while other authors reported an increase of about 100 U/L, probably due to the different nature and size of the NP studied [ 13 ].…”

“…Combining the unique properties with being biocompatible would accelerate the application for molecular imaging. Furthermore, understanding cell–nanoparticle interactions is critical not only to develop effective nanosized drug delivery systems [ 12 ], but also to predict their biocompatibility, especially in those cases where systemic administration is pursued [ 13 ]. In vivo and in vitro studies described cell toxicity after NP exposure depending on type of the material, size, shape, charge, or solubility.…”

Standardization of an in vitro assay matrix to assess cytotoxicity of organic nanocarriers: a pilot interlaboratory comparison

“…The PACA materials tested for cytotoxicity in this study were subcellularly detected by combining darkfield microscopy, confocal Raman microscopy, and ToF–SIMS analysis in NR8383 cells, confirming the uptake of these materials by macrophages [ 12 ]. A recent study on the same organic nanocarriers tested herein demonstrates the strong interaction of the particles with the immune system by a multistep in vitro hemocompatibility test [ 42 ]. The authors quantify similar trends in LDH release from donor blood as quantified in the LDH assay described above.…”

Parenteral Lipid-Based Nanoparticles for CNS Disorders: Integrating Various Facets of Preclinical Evaluation towards More Effective Clinical Translation

“…For the in vitro analysis of platelet aggregation, the procedure recommended by Neun and Dobrovolskaia [ 107 ] was the most frequently used: platelet-rich plasma obtained from freshly derived human whole blood is incubated with the tested nanoformulation, positive (collagen) and negative (PBS or RPMI cell culture medium) controls; plasma is then examined using a particle count and size analyzer to measure the number of active platelets, whereas the percentage of aggregation is determined by the number of active platelets in the sample treated with the nanoparticle, relative to the applied controls. Similar to other discussed methodologies, a wide variety of test protocols were utilized in the literature to study platelet aggregation by parenteral LNs (e.g., [ 108 , 109 , 110 ]), limiting the comparison of the obtained data. For the evaluation of the nanoparticle-induced activation of the coagulation system, platelet-poor plasma from human whole blood should be exposed to the tested nanoformulation (or positive/negative control) in vitro, and analyzed using prothrombin (PT), activated partial thromboplastin (APTT) and thrombin time assays [ 107 ].…”