# A multi-step In Vitro Hemocompatibility Testing Protocol Recapitulating the Foreign Body Reaction to Nanocarriers

Valeria Perugini, Ruth Schmid, Ýrr Mørch, Isabelle Texier-Nogues, Martin Brodde, Matteo Santin

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## To cite this version:

Valeria Perugini, Ruth Schmid, Ýrr Mørch, Isabelle Texier-Nogues, Martin Brodde, et al.. A multi-step In Vitro Hemocompatibility Testing Protocol Recapitulating the Foreign Body Reaction to Nanocarriers. Drug Delivery and Translational Research, 2022, 12, pp.2089-2100. 10.1007/s13346-022-01141-6. cea-04465755

HAL Id: cea-04465755

https://cea.hal.science/cea-04465755v1

Submitted on 25 Sep 2024

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

Valeria Perugini<fnref n="1" /> · Ruth Schmid<fnref n="2" /> · Ýrr Mørch<fnref n="2" /> · Isabelle Texier<fnref n="3" /> · Martin Brodde<fnref n="4" /> · Matteo Santin<fnref n="1" />

Accepted: 18 February 2022 / Published online: 22 March 2022

© The Author(s) 2022

## 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** Nanobiomaterials · Drug nanocarriers · Polymeric nanoparticles · Lipid nanoparticles · Hemocompatibility · In vitro tests · Host response · Protein corona · Thrombogenicity · Cytotoxicity · Inflammatory response

## Introduction

The clinical availability of nanocarriers for the delivery of therapeutic agents to target diseased tissues more efficiently depends on the accurate assessment of their bio-compatibility [1]. It is widely accepted that nanocarriers, typically not exceeding 200 nm, induce interactions with the biochemical and cellular components that are different

from those of relatively larger particles or implants made of biomaterials bearing the same chemical moieties (e.g., hydrophobic domains and electrostatic charges) [2]. These differences can be ascribed to the nanoparticle (NP) size that increases the surface available to the adsorption and conformational changes of proteins and that are in the range favorable to their internalization by immunocompetent and tissue cells [3–5]. Indeed, rapid protein adsorption and contact with different types of cells take place soon after entering the body, regardless of the route of administration, upon contact with body fluids. In the case of systemic injection, NP will undergo an early interaction with circulating proteins and immune cells and later accumulate in tissues where a range of cells have the ability to internalize them [4, 6].

Therefore, while intravenous administration is a route that has the advantage of delivering NP systemically escaping first-pass metabolism and improving the therapeutic profile, it is also an environment where the interactions of NP with proteins and cells are enhanced [7]. In particular, through

<figure><img src="image_3.png" /></figure>

<footnote>Matteo Santin
m.santin@brighton.ac.uk</footnote>

<footnote n="1">¹ Centre for Regenerative Medicine and Devices, School of Applied Sciences, University of Brighton - Huxley Building, Lewes Road, Brighton BN2 4GJ, UK</footnote>

<footnote n="2">² Department of Biotechnology and Nanomedicine, SINTEF, Trondheim, Norway</footnote>

<footnote n="3">³ Univ. Grenoble Alpes, CEA, LETI-DTBS, 38000 Grenoble, France</footnote>

<footnote n="4">⁴ OxProtect GmbH, Munster, Germany</footnote>
this route, the immunological response to the injected nano-
carriers is increased significantly by their direct exposure
to the circulating humoral and cellular elements key to the
host response to a foreign body. Indeed, it is widely recog-
nized that protein adsorption and cell contacts may activate
pathways of the host response such as thrombogenicity and
inflammation, which may be due either to the direct recogni-
tion of the nano-biomaterials or as a result of the unintended
release of pro-inflammatory cytokines caused by cytotoxic-
ity and consequent loss of cell integrity [8].

Therefore, the regulatory approval of novel nanocarriers
depends on the successful demonstration of NP biocompati-
bility through a sequence of in vitro and in vivo tests that can
assess any cytotoxic or immunological effect elicited upon
their administration [8]. In vitro tests are also particularly
important to optimize nanocarriers as they enable scientists
in both academia and industry to screen a wide range of
formulations before progressing the best candidates to the
pre-clinical animal models. To this end, the availability of a
range of relatively rapid and operator-friendly in vitro tests
able to recapitulate the main biochemical and cellular path-
ways characterizing the host response to nano-biomaterials
is advocated. In those cases where a systemic administra-
tion is pursued, hemocompatibility tests are required. Rel-
evant to NP designed for systemic administration are the
many in vitro models and assays that have been published
where polymeric, lipid, metallic and ceramic nanocarriers
and nanoparticles have been incubated in whole blood and
tested for their thrombogenicity, immunological potential,
and cytotoxicity [9–11]. While offering valuable scientific
insights, these tests tend to focus on specific pathways of
hemocompatibility, and they are not always designed to pro-
vide a clear overview informing scientists and regulators
about the hemocompatibility of a nano-biomaterial in a man-
ner that recapitulates the sequence of the main events trigger-
gered by its systemic administration [10]. It is also argued
that the reliability of any in vitro model of hemocompat-
ibility does not rely only on its reproducibility but also on
its potential to provide data of individual variability as well
as adaptability to nano-biomaterials with different physico-
chemical properties.

In this work, a series of interrelated tests based on incu-
bation of either whole human blood or platelet-rich plasma
(PRP) with nano-biomaterials is proposed. The combined
tests aimed to recapitulate the sequence of events occurring
upon systemic administration; from the early protein adsorp-
tion to the overall cytotoxicity, to thrombogenicity potential,
and the release of pro-inflammatory cytokines evaluated in
terms either of active release from immunocompetent cells
or of passive release caused by the loss of cell integrity.
The resulting multistep hemocompatibility protocol was
validated by the use of nano-biomaterials with different
physicochemical properties; i.e. polymeric [poly(ethyl butyl

byanoacrylate), PEBCA] and lipid (LipImage™ 815) nano-
carriers were selected because of their previously defined
physicochemical and biocompatibility properties [12–15].

PEBCA-based NP is biodegradable drug nanocarriers
that have been exhaustively characterized in vitro as well
as by pre-clinical in vivo models and clinical trials [12–14].
PEBCA NP used in this study typically show a hydrody-
namic diameter ranging between 100 and 200 nm with a
polydispersity index ranging between 0.1 and 0.18. Their
cytotoxic effects have been assessed at a concentration
ranging from 0.3 to 285µg/mL within 24 h showing large
variations between different cell lines and showing to be
internalized by various cell lines. LipImage™ 815 are lipid
nanoparticles with a hydrodynamic diameter of approxi-
mately 50 nm, a polydispersity index lower than 0.25, and
carrying the IR780 fluorophore [15]. Their proposed use for
tumor imaging has been supported by in vitro cytotoxicity
studies on fibroblast cell lines as well as by in vivo imaging
determining their biodistribution [16, 17]. The assessment
of the cytotoxicity showed that fibroblasts well tolerate these
nanoparticles with a half-maximal inhibitory concentration
being 1 mg/mL (i.e., 1.5 × 10¹³ particles/mL), while a higher
accumulation in the liver than other organs was observed
within 24 h. These data were considered suitable bench-
marks for the validation of a multistep, operator-friendly
in vitro hemocompatibility testing protocol based on the use
of freshly isolated human peripheral blood. The protocol
sequential steps aimed to provide interrelated information
about the main checkpoints of the host response to NP with-
out the need to undergo more convolute methods of protein
adsorption and cell isolation. In addition, the use of donors’
blood made the protocol suitable to discriminate individual
host response variability.

**Materials and methods**

Tests were designed to study the hemocompatibility of the
nanocarriers to reflect their early (minutes) and protracted
(24 h) interaction with blood proteins and cells. An assay of
platelet-fibrinogen binding was used as early thrombogenic
potential, while cell lysis and pro-inflammatory cytokine
release were measured over a longer period of time.

Nanocarrier preparation

PEBCA and LipImage suspensions were prepared as previ-
ously reported [12]. Briefly, PEBCA nanoparticles were syn-
thesized under aseptic conditions at SINTEF (Trondheim,
Norway) by mixing an oil phase consisting of PEBCA con-
taining 0.2% w/v Nile Red with an aqueous phase consisting
of 0.1M HCl containing the two PEG stabilizers (Brij®L23
and Kolliphor®HS15). The oil in water mini-emulsion was

<figure><img src="image_4.png" /></figure>
prepared using a tip sonifier. The polymerization was then
carried out overnight and unreacted monomer and surplus
of surfactants were removed by extensive dialysis. Batches
of LipImage™ 815 were prepared by high-pressure homog-
enization (HPH), the lipid phase comprising soybean oil,
Suppocire™ NB, lecithin, and IR-870 oleyl. The aqueous
phase comprised Myrj™ S40 and NaCl. Mixtures of lipid
and aqueous phases were pre-emulsified and then processed
with a high-pressure homogenizer (Panda Plus 2000, GEA
Niro Soavi, Italy) operated for 16 cycles with a total pres-
sure of 1250 bars. NP was then purified through a 5-µm filter
followed by tangential flow filtration, dispersed to a concen-
tration of 100 mg/mL, and filter-sterilized through a 0.22-
µm Millipore membrane before storage and use. Both NP
types were pre-equilibrated at room temperature for 15 min
and re-suspended by careful vortexing to prepare standards.
These were prepared by the serial dilution of the mother
solution in sterile phosphate-buffered saline (PBS) pH=7.2
at different concentrations in the range of 1 to 1000µg/mL.
Samples were stored at room temperature and used within
2 h of preparation.

Human peripheral blood collection

Human peripheral blood (4 mL) was collected from 6
healthy volunteers from both genders using the Vacutain
system into heparinized tubes (BD Vacutainer™ UK). In
the case of platelet-fibrinogen binding experiments, blood
was collected by Monovette tubes containing sodium cit-
rate. Blood collection was authorized by the University of
Brighton Ethics Committee and performed under the Human
Tissue Act license no. 12583.

Protein concentration

Protein concentration levels in donors' blood samples incu-
bated with different NP concentrations (6.25, 12.5, 25, 50,
100, 250, 500, and 1000 µg/mL) were performed as an
indirect method to assess the early interactions between NP
and blood components. Samples (50 µL) of freshly isolated
human peripheral blood were added to the wells of 96-well
tissue culture plates (TCP, Corning UK) and spiked with
10µL of NP at different concentrations within 2 h from
phlebotomy. Each concentration was tested in duplicate
for each donor. Samples were incubated in a humidified
37 °C, 5% CO₂ cell incubator for 24 h under static condi-
tions. After 24 h, blood was removed, and the supernatants
were immediately tested for protein concentration. Protein
concentrations were measured by a Bradford assay (BioRad,
UK) on 5 µL of blood supernatant and data expressed as µ
g/mL mean ± standard deviation from n=6 as calculated by
a bovine serum albumin standard curve.

Thrombogenicity

A method for the measurement of platelet-fibrinogen bind-
ing was adopted to assess the thrombogenicity potential of
the tested nano-biomaterials. PRP was prepared from 5 mL
of whole blood by centrifugation at 200 × g for 10 min.
Fluorisothiocyanate (FITC)-labeled fibrinogen (OxProtec,
Germany) was added to the PRP to a final concentration of
150 µg/mL, and the platelet count was adjusted to 25,000µ
g/µL to get the stock solution. A volume (10µl) of either
NP at different concentrations or positive controls (i.e., 2 µ
g/mL collagen in PBS, OxProtec) were added to 100 µL
of the stock solution and incubated for exactly 5 min. PBS
was used as a negative control. The reaction was stopped
by adding 110 µL of 1% (v/v) formaldehyde solution to fix
the platelets for 30 min. After a washing step, the binding
of FITC-labeled fibrinogen to the platelets was measured
by flow cytometry by a BD Accuri C6 flow cytometer, and
results were analyzed using the instrument software. Data
were expressed as a percentage of the positive control.

Cytotoxicity

Overall blood cell toxicity induced by the different concen-
trations of NP was assessed both in the blood supernatants
and on cells deposited on the bottom of the well. The former
was quantitatively determined by the release of the lactate
dehydrogenase enzyme (LDH) activity, while the latter was
qualitatively assessed by a hematoxylin/eosin (HE) staining
method.

LDH activity was measured on aliquots obtained from
the same blood samples tested for protein concentration lev-
els. Aliquots of 50µl were tested by the CytoTox 96® non-
radioactive cytotoxicity assay (Promega, UK), and data were
expressed as mean percentage of the positive control ± stand-
ard deviation from n=6 according to the formula:

LDH activity (% O.D. 490 nm) = [(experimental sample - background)/(positive control - background)] × 100.

where the maximum LDH release is the enzymatic activity
measured in the supernatant of samples treated with 10 µL
of manufacturer's lytic solution for 45 min prior to addi-
tion of the kit substrate solution. Blood basal LDH activ-
ity and the LDH activity of blood samples treated with the
lytic solution (positive control) were expressed as arbitrary
units<sub>Abs490nm</sub>/mL.

Cells deposited at the bottom of the wells used for pro-
tein concentration and LDH activity measurements were
washed twice in 50 µL of PBS, fixed by 50 µL of 3.7% v/v
paraformaldehyde for 10 min at room temperature, washed
three times with PBS, and stained by hematoxylin solution
(Sigma, UK) adding 50 µL of dye into each well for 5 min at
room temperature. Samples were rapidly rinsed three times
with distilled water and in 0.5% v/v acetic acid for 40 s,
washed again in distilled water, and counterstained in 50 µL eosin solution (Sigma, UK) for 2 min at room temperature. The samples were finally rinsed three times in water. Light microscopy images were taken from samples of the two types of nanoparticles at ×20 magnification and qualitatively analyzed for their area coverage.

## Inflammatory cell levels and response

The levels and activation of inflammatory cells involved in the early inflammatory response, granulocytes, and monocytes/macrophages, were assessed in the whole blood samples challenged by different concentrations of NP (1, 2, 5, 10, 100, and 1000µg/mL) by flow cytometry. In particular, the analysis of typical cell membrane markers as well as that of the release of pro-inflammatory agents, including myeloperoxidase enzyme activity for granulocytes and cytokines for monocytes/macrophages, were performed.

**Granulocyte levels and myeloperoxidase (MPO) activity** PBS (10 µL) was used as a negative control, while the TSP-1 peptide RFYVVMWK (10 µL, 200µM in PBS) was used as a positive control. NP was added to flow cytometer vials, to which 100 µL of whole blood from individual donors was added and incubated, at room temperature, for 30 min. Samples were fixed by the addition of 110 µL of 0.4% (v/v) formaldehyde solution for 30 min. Samples were washed by the addition of 1 mL of PBS followed by centrifugation for 10 min at 500×g. The supernatants were discarded, leaving approximately 100 µL volume, including the cell pellet. Anti-myeloperoxidase PE-labeled antibodies (5 µL, Becton Dickinson) were added to each sample and incubated for 1 h at room temperature and washed twice. Lysing solution (1 mL) was added for 10 min at room temperature. PBS (2 mL) was added, and samples were centrifuged at 500×g for 10 min. The supernatants were aspirated and the cell pellets suspended in 500 µL of PBS prior to analysis by flow cytometry (Accuri C6, BD, UK).

## Monocyte/macrophage levels and cytokine release

For each 100 µL of whole blood required, 5 µL of FITC-labeled mouse anti-human CD14 (Becton Dickinson) and 5 µL of PE-labeled anti-human CD11b antibody (BioLegend) were added. Aliquots of 10 µL of either NP or TSP-1 peptide (positive control) or PBS (negative control) were added to 100 µL of antibody-labeled whole blood and incubated for 30 min, room temperature. Samples were fixed by the addition of 110 µL of 0.4% (v/v) formaldehyde solution (Sigma-Aldrich) to the cultures for 30 min. Samples were washed by the addition of 1 mL of PBS followed by centrifugation for 10 min at 500×g. The supernatants were discarded, leaving approximately 100 µL volume, including the cell pellet.

Lysing solution (1 mL, Beckman Coulter) was added for 10 min at room temperature. Two washes in PBS (2 mL) were performed. The supernatants were aspirated and the cell pellets suspended in 500 µL of PBS prior to analysis by flow cytometry (Accuri C6, BD, UK).

Human Th1/Th2 cytokine standards (Cytometric Bead Array, CBA, BD, UK) were reconstituted in assay diluent and incubated for 15 min at room temperature (0–2500 pg/mL). After incubation, 6 human Th1/Th2 cytokine capture beads were treated with 50 µL PE-conjugated detection antibodies (BD, UK) before being mixed with 10 µL plasma/ standards diluted in 40 µL of serum enhancement (BD, UK). Both samples and standards were then incubated for 3 h. After 3 washing steps with 1 mL of washing buffer, samples and standards were assessed by BD Accuri C6 flow cytometer, and results were analyzed using a specific BD analysis software. Data of blood samples from individual donors after spiking with different concentrations of NP were assessed for relative levels of cytokines compared to the control.

## Statistical analysis

Statistical analysis was performed by ANOVA *t*-test (Tukey Kramer test) from *n*=6 blood donors. Significant differences of samples treated with different concentrations of NP from those of each donor's control were tested at *p* < 0.05.

## Results and discussion

The measurement of the protein levels in the blood samples exposed to different concentrations of PEBCA and LipImage™ 815 was adopted as indirect testing of the early interactions of the NP with blood components. Protein levels in freshly isolated whole heparinized human peripheral blood showed a statistically significant reduction of protein levels in all donors when relatively high concentrations (≥ 100µg/mL) of both PEBCA and LipImage™ 815 were tested (Fig. 1A, B, individual donors' data, Suppl. Fig. 1, pooled donors' data, *p* < 0.05). These decreased levels indicated possible adsorption of blood proteins onto the NP surface or the possible precipitation of proteins induced by high concentrations of NP. The incubation time was chosen to evaluate both early protein adsorption and possible precipitation events. Indeed, while protein interactions with biomaterial surfaces are known to take place in milliseconds after the exposure of biomaterial surfaces to body fluids [18], over a longer period of time, the so-called Vroman's effect takes place, whereby proteins with a relatively high molecular weight displace those with low molecular weight, leading to the formation of a stable protein layer [18]. In addition, serum protein depletion by NP incubation has been previously
<figure><img src="image_5.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**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</figcaption></figure>

µg/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

demonstrated, particularly in the case of NP with a negatively charged surface [19].

The consequent adsorption of this protein film at the bio-
material surface is also considered the main cause of the
host response to implants, including thrombogenicity and
inflammatory response [3]. Among the proteins relevant to
the host response and able to adsorb onto biomaterial sur-
faces, fibrinogen plays a key role in the clot activation upon
NP systemic administration [20].

The data showed that fibrinogen-platelet binding was
dependent on the type of NP tested but not exceeding 40%
of the positive control (Fig. 2A, B). In the case of PEBCA,
a binding level of ca 40% was observed in all donors in the
range of concentrations 1 to 100µg/mL (Fig. 2A individual
donors' data, Suppl. Fig. 2A, pooled donors' data, p<0.05).
This binding significantly dropped to ca 20% at 1000 µg/mL,
following a pattern similar to that observed for the protein
concentration levels. It can therefore be speculated that, in
the case of these polymeric NP, the binding of platelets is
limited and possibly reduced by the reduced concentrations
of adsorbed fibrinogen. A different behavior was observed

when different concentrations of LipImage™ 815 NP were
studied (Fig. 2B, individual donors’ data, Suppl. Figure 2B,
pooled donors’ data, *p* < 0.05). This lipid-based nanocarrier
showed a slight increase of fibrinogen-platelet binding at
increasing NP concentrations but never exceeding the 30%
level. In addition, higher individual variability was observed,
with two out of 6 donors showing significantly lower levels
of binding up to concentrations of 100 µg/mL. These obser-
vations are supported by previous studies that have demon-
strated that the binding of fibrinogen sequences to liposomes
induces the binding and activation of platelets [25, 26].

Fibrinogen binding to NP with different physicochemical
properties has been widely documented and related to both
the NP surface electrostatic charges and size [21, 22]. In
the present protocol, the assay adopted assessed the binding
of fibrinogen to platelets by spiking freshly isolated PRP
with labeled fibrinogen. The assay provides indications of
(i) the NP-induced activation of platelets leading to a higher
expression of their receptors for fibrinogen and consequently
of thrombogenicity through fibrinogen-platelet complex
formation, (ii) the binding of the formed fibrinogen to the
<figure><img src="image_7.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**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</figcaption></figure>

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

surface of the NP that may also trigger thrombogenicity through complexation with the platelets. Platelet activation measured as an expression of CD63 and CD62b antibodies was negligible for both NP types until concentrations of 1000 micrograms/mL were tested, levels of ca 30% of the positive controls were observed (data not shown). Therefore, it is suggested that the adsorption of fibrinogen rather than direct activation of the platelets by NP may determine mild thrombogenicity potential. It may be speculated that the different profiles of fibrinogen-platelet binding on the two types of NP could be linked to their different physicochemical properties. The data suggest that the fibrinogen adsorbed within the PEBCA NP protein corona favors platelet binding at low NP concentrations and that only the alleged depletion of proteins observed at relatively high concentrations (> 100 µg/ mL) slightly reduces this binding. It is also observed that in such a case, the presence of PEG surfactants in the PEBCA NP formulation did not prevent the adsorption of fibrinogen and consequent platelet binding. These findings are corroborated by previous studies showing that PEG reduces but does not completely prevent the formation of a protein corona and fibrinogen binding at the surface of NP and that it is rather changing its composition and it is the cause of serum protein depletion as that observed in the experiments of protein concentration [23–26]. In addition, the formation of a

protein corona at the surface of NP has been linked to their longer stability in vivo [26]. Conversely, the intrinsically more unstable nature of the lipid-based LipImage™ 815 appears to induce a reduced and more variable formation of the fibrinogen-platelet complex [26–28].

In the proposed NP testing protocol, the combination of the simple and relatively rapid Bradford's protein concentration assay and the flow cytometry study of fibrinogen-platelet binding highlights the different hemocompatibility of NP under investigation, particularly in relation to the early interaction of these NP with blood components following systemic administration.

The availability of relatively large volumes (5 mL) of blood samples also enabled a further assessment of the host response to the tested NP in relation to their cytotoxicity and inflammatory response. To this end, aliquots of the same samples used for the protein concentration measurements were used to assess the NP cytotoxicity. Indeed, these samples provided the opportunity to test the loss of cell integrity by released LDH activity (Fig. 3A, B) in the blood volume and that of cells, mainly erythrocytes, deposited at the bottom of the well (Fig. 4A, B). The basal LDH activity of the tested blood donors, measured as absorbance at 490 nm, was 0.534 ± 0.044, while the average LDH activity released by induced cell lysis (positive control) was 1.651 ± 0.270. An

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /><figcaption>**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 per-</figcaption></figure>

centage 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

increase in the LDH activity was observed only at concentrations of 100 µg/mL and above for both NP types (Fig. 3A, B) with clear individual donor variations. A direct comparison

between the two types of NP was obtained by an analysis of the donors’ pooled data showing similar cytotoxic levels for the two nanocarriers (Suppl. Fig. 3). The loss of cell

<figure><img src="image_11.png" /><figcaption>**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</figcaption></figure>
integrity was qualitatively confirmed by HE staining of the well surface that showed reduced staining of deposited cells, mainly erythrocytes, at the same concentrations for both types of NP (Fig. 4 panels A and B) in line with previously published data of erythrocyte toxicity by NP [29].

Whole human blood samples enabled the assessment of the inflammatory response to NP, another key parameter in the study of biomaterial hemocompatibility. This last batch of experiments of the multi-phase protocol was linked to the data of protein concentrations and cytotoxicity to indicate whether the incubation with the two types of NP was affecting the mononuclear cell population and its role in inducing an inflammatory response either by cell activation or by the unintended release of pro-inflammatory cytokines through loss of cell integrity. As the inflammatory response to bio-materials is triggered by the early activation of granulocytes followed by that of monocytes/macrophages [30, 31], the flow cytometry analysis of this study focused on the markers of these two types of cells as well as on the release of pro-inflammatory macromolecules. In this part of the protocol, the flow cytometry tests focused on the presence of cells in the human blood expressing CD14 and CD11b, cell surface markers common to both granulocytes and monocytes/macrophages [32, 33]. The levels of CD14 observed at increasing concentration of PEBCA NP showed a gradual reduction of the levels of these cells, suggesting that relatively high concentrations of NP, consistently with the data of cytotoxicity, led to the loss of cell integrity (Fig. 5A, Suppl. Fig. 4A, C). Similar results were observed for LipImage™ 815 (Fig. 5B, Suppl. Fig. 4B, D).

<figure><img src="image_12.png" /><figcaption>**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</figcaption></figure>

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

However, the study of the release of soluble pro-inflammatory pathways seemed to suggest that an inflammatory insult could derive from the cell death detected at the relatively high NP concentrations. Indeed, granulocyte MPO activity [34] increased with the increase of NP concentrations, with a higher release being observed in the case of PEBCA NP, where the MPO activity ranged from approximately 20 to 50% of the positive control (Fig. 6A, Suppl. Fig. 5A). In the case of LipImage™ 815, MPO activity was relatively low reaching a maximum of approximately 20% at the highest concentrations tested and being not significantly different from the negative control up to 50 µg/mL (Fig. 6B, Suppl. Fig. 5B).

<figure><img src="image_13.png" /></figure>
<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>**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</figcaption></figure>

For all these markers of the inflammatory response, the reproducibility of the data across donors is highlighted when donors’ data were pooled, showing a significantly (*p* < 0.05) lower activation of the inflammatory cells when compared to the positive controls (Suppl. Fig. 4A–D and Suppl. Fig. 5A, B).

Finally, flow cytometry was applied to test the Th1/Th2 pathway that is shared by most of the blood immunocompetent cells (i.e., monocytes/macrophages and lymphocytes) [35], thus enabling us to evaluate the immune response to NP more comprehensively. The flow cytometry analysis was performed by commercially available antibody-functionalized microbead arrays to study the release of other relevant pro-inflammatory factors at different NP concentrations (Fig. 7, panels A and B). The flow cytometry panels for both PEBCA NP and LipImage™ 815 NP showed release of the tested cytokines at levels not significantly different from the negative control (blood samples not challenged by NP) when the tested NP concentrations were below the determined cytotoxic threshold. Only a slight and donor’s dependent higher release of some of the tested cytokines was observed at the cytotoxic concentrations. As observed in the case of other assays of the multistep

protocol, the testing of blood samples from individual donors highlighted that the levels of release of the cytokines of the Th1/Th2 pathway showed, once again, the importance of assessing NP hemocompatibility in the context of individual variability.

Taken together, the data obtained by this multistep protocol demonstrates its ability to discriminate between different components of the host response, different doses, and different NP physicochemical properties, as well as individual variability. The comparable size of the NP and the same range of concentration tested in this study also corroborate the comparative value offered by the protocol. The tested NP were not showing a significant activation of the host response in most of its measured parameters. The thrombogenic potential seemed to be affected slightly by both the type of NP tested and their ability to induce blood protein depletion. At the same time, while pronounced lysis of erythrocytes was observed, the integrity of the remaining blood cell population seemed to be compromised only when very high NP concentrations were tested, resulting in low levels of release of pro-inflammatory cytokines.
<figure><img src="image_16.png" /><figcaption>**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™ 815 (Panel B). Flow cytometry plot of control sample</figcaption></figure>

from Donor n. 1 reports the Th1/Th2 signalling markers analysed in the assay; IFNγ, 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.

<figure><img src="image_17.png" /></figure>
## Conclusions

Future developments and regulatory approval of nanoparticulate carriers will depend on the definition of reliable testing protocols able to identify in a user-friendly, cost-effective, and relatively fast manner the host response triggered upon administration. At the same time, it is argued that these tests should also provide reassurance about the variability of the host response of individual patients. The present work shows that a multistep protocol obtained from single collections of peripheral blood from different donors may provide a first level of safety check of NP with different physicochemical properties during their phase of product development and provide batches of in vitro data suitable for regulatory approval. In addition, this sequence of tests could be adopted to predict any adverse effect occurring on single patients and help to establish personalized safety assessments and dose regimes of nanomedicines.

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1007/s13346-022-01141-6.

**Acknowledgements** The work has been supported by the EC Horizon 2020 project "Regulatory Science Framework for Nano(bio)material-based Medical Products and Devices" REFINE, contract no. 751104.

**Author contribution** Valeria Perugini contributed to the design of the protein and cytotoxicity studies, performed all the hemocompatibility experiments, and collected and reviewed data. Isabelle Texier contributed to the synthesis of the LipImage™ 815 NP. Yrr Mørch contributed to the design and synthesis of PEBCA NP and the revision of the manuscript. Ruth Schmid and Martin Brodde contributed to the experimental design and revision and interpretation of data and manuscript. Matteo Santin contributed to the experimental design, data analysis and interpretation, and drafting of the manuscript.

**Data availability** Raw data are stored in the University of Brighton repository and made available upon request.

## Declarations

**Ethics approval** This study was approved by the University of Brighton Ethics committee.

**Consent to participate** Consents to participate were obtained in the form of signed documents from all blood donors.

**Consent for publication** Blood donors have consented to the use of the data for publication. All authors have reviewed the data and the manuscript and consented to their publication.

**Competing interests** The authors declare no competing interests.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes

were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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