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.

Keywords: Cytotoxicity; Drug nanocarriers; Hemocompatibility; Host response; In vitro tests; Inflammatory response; Lipid nanoparticles; Nanobiomaterials; Polymeric nanoparticles; Protein corona; Thrombogenicity.

Fig. 1

Levels of protein concentrations in human donors’ peripheral blood incubated with increasing concentrations of PEBCA A and LipImage™ 815 B NP. Protein levels are expressed as mean $\mu$g/mL from n = 6 donors, each tested in duplicate. Data are shown as mean + standard deviation from n = 3 replicates for each donor. * indicates p < 0.05

Fig. 2

Levels of fibrinogen-platelet binding in human donors’ peripheral blood incubated with increasing concentrations of PEBCA A and LipImage™ 815 B NP. Data are expressed as mean ± standard deviation of the percentage of the positive control from n = 6 donors each tested in duplicate. Data are shown as mean + standard deviation from n = 2 replicates for each donor. * indicates p < 0.05

Fig. 3

Levels of cytotoxicity in human blood induced by increasing concentrations of PEBCA A and LipImage™ 815 B NP. Release LDH activity is expressed as mean ± standard deviation of the percentage of the positive control from n = 6 donors each tested in duplicate. Data are shown as mean + standard deviation from n = 2 replicates for each donor. * indicates p < 0.05

Fig. 4

Cytotoxicity of deposited human blood cells induced by increasing concentrations of PEBCA (panel A) and LipImage™ 815 (panel B) NP. Cells were stained by HE method, and photos were taken by light microscopy at × 20 magnification. Images were taken at × 10 magnification

Fig. 5

Expression of blood inflammatory cells’ membrane markers upon spiking with increasing concentrations of PEBCA A, C and LipImage™ 815 B, D NP. Flow cytometry data for CD14 A and B and CD11b C and D expression are expressed as mean ± standard deviation of the percentage of the positive control from n = 6 donors each tested in duplicate. Data are shown as mean + standard deviation from n = 2 replicates for each donor. * indicates p < 0.05

Fig. 6

Levels of MPO activity released by granulocytes in human blood spiked by increasing concentrations of PEBCA A and LipImage™ 815 B NP. Flow cytometry data are expressed as mean ± standard deviation of the percentage of the positive control from n = 6 donors each tested in duplicate. Data are shown as mean + standard deviation from n = 2 replicates for each donor. * indicates p < 0.05

Fig. 7

Semi-quantitative assessment of the levels of cytokine released inflammatory cells (granulocytes and mononuclear cells) in human blood spiked by increasing concentrations of PEBCA (Panel A) and LipImage^TM 815 (Panel B). Flow cytometry plot of control sample from Donor n. 1 reports the Th1/Th2 signalling markers analysed in the assay; $\mathrm{IFN}\gamma$, TNF, IL-10, IL-5, IL-4. Flow cytometry plots are representative of donors showing comparable or different release NP response profiles to those of those of the relative control.