International Journal of Biological Macromolecules 282 (2024) 137029

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Impact of polycaprolactone, alginate, chitosan and zein nanofiber physical

properties on immune cells for safe biomedical applications
Anže Zidar a , Špela Zupančič a , Julijana Kristl a , Matjaž Jeras b,*
a
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia
b
Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia

A R T I C L E I N F O A B S T R A C T

Keywords: Nanofiber safety, especially immunogenicity, is important for their successful translation to clinical setting. This
Nanofibers study provides a comprehensive evaluation of how nanofiber physical properties influence immune cells cultured
Immunogenicity on them, specifically peripheral blood mononuclear cells (PBMCs). We prepared nanofibers with a wide range of
Safety
physical properties including various diameters, interfibrillar pore sizes and mat thicknesses, using four main
Biocompatibility
Electrospinning
polymers: polycaprolactone, alginate, chitosan, and zein. Our findings show that nanofiber diameters had only a
Polymers marginal influence on the activity of immune cells, whereas interfibrillar nanofiber pore sizes had a significant
Leukocytes effect, and mat thickness proved to have the greatest impact. Cells that penetrated deeper into the thick nanofiber
mats ceased to proliferate but did not experience cytotoxicity. Moreover, we discovered that PBMCs penetrating
the zein/PVP nanofiber mesh exhibited increased metabolic activity, indicating potential immunogenicity,
whereas the other tested non-immunogenic nanofibers reduced it. To best of our knowledge, this study is the first
to report on the impact of various nanofiber physical properties on in vitro immune cell behavior, thereby
expanding the knowledge in the relatively unexplored field of nanofiber immunological safety. It underscores the
need for rigorous preclinical nanofiber assessment and setting new standards for designing nanofiber-based
biomedical products.

1. Introduction trigger inflammation or even cell death, thereby compromising thera­

peutic efficacy. Current research investigates the in vitro immune re­
Physical properties of nanofibers play a crucial role in determining sponses to nanofibers, focusing on their pro- or anti-inflammatory and
their functionality, applicability and safety [1,2]. Their key properties immunoregulatory effects, which may stem both from the nanofibers
include nanofiber diameters, mechanical properties, specific surface themselves and from incorporated drugs [10–12].
area, interfibrillar pore sizes, and mat thickness [3]. The ability to tailor As Goonoo N. et al. pointed, different nanofiber compositions can
these parameters for specific applications is particularly valuable in exert diverse effects on different cell types [13]. Therefore, it is impor­
biomedical applications, where nanofibers are investigated for drug tant to consider their chemical properties, such as hydrophobicity,
delivery, wound healing and tissue engineering [4–6]. However, nano­ charge in aqueous media and polymer structure. A few examples of
fibers must be comprehensively assessed in terms of their safety and common polymers used for production of nanofibers for biomedical
biocompatibility in biological environment, due to their nanometer di­ applications are polycaprolactone (PCL), alginate (ALG), chitosan (CS),
ameters and large specific area [7,8]. polylactic acid, poly lactic-co-glycolic acid, collagen, zein, and their
According to the ISO 10993 International Standard (Biological blends [14–19]. However, to successfully electrospin certain polymers
Evaluation of Medical Devices), a material is considered biocompatible like ALG, CS and zein nanofibers, an additional electrospinnable poly­
if it does not cause unacceptable toxic, immunogenic, thrombogenic, or mer is needed, e.g. polyethylene oxides (PEO), polyvinylpyrrolidone
carcinogenic reactions [9]. This underscores the importance of studying (PVP), polyvinyl alcohol, etc. [20,21].
and understanding immune cell - nanofiber interactions, as the wide­ We selected PCL, ALG, CS, and zein for nanofiber preparation due to
spread presence of nanosized structures in tissues could potentially their varying surface chemical properties, including hydrophobicity

* Corresponding author.
E-mail addresses: [email protected] (A. Zidar), [email protected] (Š. Zupančič), [email protected] (J. Kristl), [email protected]
(M. Jeras).

https://doi.org/10.1016/j.ijbiomac.2024.137029
Received 1 August 2024; Received in revised form 14 October 2024; Accepted 27 October 2024
Available online 29 October 2024
0141-8130/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
A. Zidar et al. International Journal of Biological Macromolecules 282 (2024) 137029

(PCL) [22], negative charge (ALG) [15], positive charge (CS) [23], and 2. Methods
the polypeptide structure of zein, which could confer potential immu­
nogenicity [24]. Macroscale safety of these polymers has been previ­ 2.1. Materials
ously evaluated. For example, PCL has been used for more than 70 years
and is considered safe for human application [25,26]. Alginate is For nanofiber preparation the following materials and reagents were
recognized by the United States Food and Drug Administration as used: poly(ε-caprolactone) (PCL) (MW 80 kDa), poly(ethylene oxide)
“Generally regarded as safe” [27]. Chitosan has low oral and local (MW 900 kDa) (PEO900kDa), poly(ethylene oxide) (MW 2 MDa)
toxicity, but parenteral application still needs to be further investigated (PEO2MDa), low molecular weight chitosan (MW 50–190 kDa; 1 % chi­
[28]. Zein is also regarded as safe and low immunogenic protein [29]. tosan in 1 % acetic acid solution has a viscosity of 122 CPS) (CS) with 76
However, their nanoscale safety has not yet been thoroughly % deacetylation, polyvinylpyrrolidone (PVP) (MW 1.3 MDa) and zein,
investigated. all from Sigma-Aldrich (Steinheim, Germany); sodium alginate (ALG)
Fadeel et al. recommend that for obtaining robust in vitro data (MW 138 kDa; Protanal LF 10/60) defined by the manufacturer as
regarding nanomaterials safety profiles, relationships between their 65–75 % α-l-guluronate and 25–35 % β-d-mannuronate, purchased from
physicochemical properties and safety should be carefully evaluated FMC BioPolymer (Haugesund, Norway); dichloromethane (DCM)
[30]. Most scientific reports on in vitro cell responses to nanofibers are (≥99.8 % pure) and anhydrous acetic acid for analysis, both from Merck
mostly limited to specific efficacy and cytotoxicity assessments (Darmstadt, Germany), N,N-dimethylformamide (DMF) (≥99.9 % pure),
[11,31–33]. Various human cell types, including endothelial cells, purchased from Honeywell (Riedel de Haën, Germany). Deionized water
chondrocytes, smooth muscle cells, keratinocytes, fibroblasts, neural was purified using a Milli-Q system, equipped with a 0.22 μm Millipak
cells, and others, were studied by evaluating various parameters, such as 40 filter (Millipore, Ireland).
cell attachment, viability, differentiation, and growth [34,35]. Some The following materials and reagents were used for in vitro immune
studies also report in vitro correlations between nanofiber properties and cell assays: Lympholyte-H Cell Separation Media, from Cedarlane
cell responses, showing that physical properties of nanofibers can (Burlington, Ontario, Canada); BioTarget® defined serum-free cell cul­
significantly affect functions of certain cell types [13,36,37]. Our ture medium, from Biological Industries (Haemek, Israel); Antibiotic
research group has previously demonstrated that thicker PCL nanofiber antimycotic solution (100×; containing penicillin, streptomycin, and
mats reduce in vitro metabolic activity of peripheral blood mononuclear amphotericin B, from Sigma-Aldrich (Burlington, USA) which was
cells (PBMCs) [38]. However, to our knowledge, aside from this study, added at 1 % to BioTarget®; GlutaMAX™ GIBCO Life Technologies
there are no other published reports on how nanofiber physical prop­ (Grand Island, NY, USA), added to BioTarget® at 1 %;
erties affect immune cells in vitro. phytohemagglutinin-L (PHA-L), from Roche (Basel, Switzerland);
PBMCs are most commonly used in preclinical studies of immuno­ Triton® X-100 and parafilm all from Sigma-Aldrich (Darmstadt, Ger­
logical safety and efficacy, as they are composed of all relevant cells that many), fetal bovine serum from Gibco (Great Britain); sodium dodecyl
can respond to immune stimulation or inhibition. They include 70–90 % sulfate (≥85 % pure), from Merck (Darmstadt, Germany); CytoTox 96®
lymphocytes (T cells, B cells and natural killer cells), 10–20 % mono­ Non-Radioactive Cytotoxicity Assay (the LDH test) and CellTiter 96®
cytes, and 1–2 % dendritic cells, which all play crucial roles in body's Aqueous One Solution Cell Proliferation Assay (the MTS test), both from
immune defense against pathogens and foreign materials [39]. The Promega (Madison, WI, USA). ProLong™ Gold antifade reagent with
response of PBMCs to immunogenic substances can be detected in vitro at DAPI was purchased from Thermo Fisher (Eugene, OR, USA).
various levels of their activation, i.e. secretion of specific cytokines,
expression of cell surface activation or suppression markers, identifica­ 2.2. Nanofiber preparation by electrospinning
tion of HLA class I and II bound antigenic peptides, signal transduction
events, phagocytosis exerted by antigen-presenting cells and prolifera­ All nanofibers tested in our in vitro immune cell experiments un­
tion [40]. By simulating both homeostatic and inflammatory conditions derwent careful preparation steps under aseptic conditions. Both, the
on PBMCs in vitro, with the latter induced by phytohemagglutinin-L electrospinning and the laminar air flow chamber were fitted with high-
(PHA-L), researchers can investigate how a substance of interest, for efficiency particle airflow filters (0.22 μm). Additionally, to prevent
example nanofibers, affect immune cell responses. These can be assessed microbiological contamination, all working surfaces were disinfected
by measuring the extent of PBMC proliferation in terms of their meta­ with 70 % ethanol and then irradiated with UV light for at least 30 min
bolic activity. Cells respond to the physical properties of their environ­ before use. Compositions of polymer dispersions for nanofiber electro­
ment by converting mechanical cues into intracellular chemical signals, spinning were prepared by dissolving PCL, ALG/PEO2MDa, CS/PEO900kDa
which in turn, control cell behavior [41]. Such in vitro testing approach or zein/PVP in appropriate solvents, as presented in Table 1. All dis­
has been proven to closely correlate with in vivo clinical immunogenic persions were stirred overnight at room temperature on a magnetic
outcomes, as described by Joubert et al., where they analyzed immu­ stirrer, at 400 rpm.
nogenicity of antibodies using this method [40]. Namely, immunogenic Nanofibers were prepared with the electrospinning apparatus
substances increased the metabolic activity of PBMCs compared to equipped with climate control unit (Fluidnatek LE100, BioInicia SL,
controls [42]. Valencia, Spain). Polymer dispersions were loaded into a plastic syringe
The aim of this study is to investigate how the key physical properties and connected to a nozzle that was set at 15 cm from the flat collector
of nanofibers, namely their diameters, pore sizes, and mat thicknesses, covered with aluminum foil. Round glass cover slips with a 10 mm
influence the behavior of immune cells, thereby addressing a significant diameter were positioned over the aluminum foil and served as a carrier
knowledge gap. By studying PCL, ALG/PEO2MDa, CS/PEO900kDa, and for nanofiber mats during in vitro experiments. For optimal production of
zein/PVP nanofibers with well-defined properties, we aim to provide nanofibers, the process and environment parameters were adapted
valuable insights into their immunological safety and thereby support appropriately, to ensure a stable electrospinning process (Table 1).
the development of safer, more effective nanofiber-based biomedical During nanofiber deposition a cycling option was used, in which the
products. Additionally, we used fluorescent microscopy to investigate nozzle was moving at a speed of 10 mm/s in both the x- and y-axes over
immune cell activation and penetration into nanofiber mats of varying an area of 100 cm2 to obtain a uniformly thick nanofiber mat. Addi­
thickness. These findings will help to improve the design of nanofiber- tionally, ALG/PEO2MDa nanofibers were crosslinked with 2 % (m/V)
based biomedical products and raise safety standards for their clinical CaCl2 solution for 2 h and then thoroughly rinsed with purified water
applications. and airdried in a LAF chamber to ensure they do not dissolve in the cell
culture medium. Additionally, PEO900kDa only nanofibers were prepared
in the same way.

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A. Zidar et al. International Journal of Biological Macromolecules 282 (2024) 137029

Table 1
Summary of electrospinning solution compositions and electrospinning parameters. The data for each parameter used are presented as a range from minimal to
maximal value.
Polymer Polymer Polymer concentration Solvent Spinning Relative Voltage Flow rate Time of spinning
composition ratio (% m/V) temperature (◦ C) humidity (%) (kV) (μL/h) (min)

PCL / 10–14 DCM/DMF 25 ± 2 25 ± 2 18–28 2000 5–80

(1:1, V/V)
ALG/PEO2MDa 80:20 3.75–4.75 Water 42 ± 2 12 ± 2 15–24 500 15–240
CS/PEO900kDa 60:40 3–4 50 % (V/V) acetic 37 ± 2 12 ± 2 20–27 500 20–320
acid in water
zein/PVP 92.5:7.5 21.5–25.5 100 % acetic acid 25 ± 2 25 ± 2 15–24.5 1000 5–80

2.3. Characterization of nanofiber morphology and mat thickness Accordingly, two controls were used, i.e. the negative, consisting of
non-stimulated PBMCs and the positive one where PBMCs were stimu­
To visualize the morphology of the electrospun nanofiber mats lated by PHA-L, both being cultured in the absence of nanofiber mats on
scanning electron microscope (SEM) 235 Supra 35VP-24-13 SEM in­ round glass cover slips fixed in the nanofiber in vitro cell model inserts
strument (Carl Zeiss, Jena, Germany) was used. Collected samples were that were placed in a 48 well microtiter plate, as described previousely
cut and attached to metal pins with double-sided conductive tape. High- [38].
resolution SEM images were captured without sputter coating, with the In all experiments, PBMCs were suspended in BioTarget®, a defined
SEM microscope operating at an accelerating voltage of 1 kV and the use serum-free medium to obtain the density of 5 × 105 cells/mL and then
of a secondary detector. To determine the average nanofiber diameters seeded in a 200 μL volume on all nanofiber mat samples or glass cover
and the appertaining standard deviations (SDs), at least 50 randomly slips (controls), immobilized in the nanofiber in vitro model. Addition­
selected nanofibers were measured in each mat and analyzed using ally, PHA-L solution in a final concentration of 10 μg/mL was added
ImageJ software (National Institutes of Health, Bethesda, USA). immediately after seeding PBMCs to appropriate wells.
Nanofiber pore sizes were also quantified using the ImageJ software, To determine relative metabolic activity (RMA) of PBMCs, cultures
wherein the SEM images were processed with a 30 % threshold setting to were incubated for 72 h under standard conditions (37 ◦ C, 95 % relative
measure the pore surface area. The option to exclude the pores at the humidity and 5 % CO2) always in triplicates. After incubation, MTS
edges was chosen, in order to ensure accuracy. To improve the precision reagent was added and further incubated for 4 h, after which 25 μL of 10
of the average pore size measurement, a weighted average calculation, % sodium dodecyl sulphate solution was added to each culture to lyse
considering the relative importance of each pore's area, was utilized (Eq. the cells and release any trapped formazan. Then 100 μL samples of
(1)). supernatants were transferred to 96 well flat bottom microtiter plate and
∑ absorbances were measured at 490 nm, using the BioTek Synergy H4
(Si × wi ) Si
Sw = ∑ wi = ∑ (1) microplate reader (BioTek, Winooski, Vermont, USA). All absorbances
wi Si.
were corrected for the absorbance of the cell culture medium alone
Equation 1. Calculation of the weighted average pore size (Sw ), (blank) and normalized to the negative control to obtain RMA values as
where Si represents the size of the ith pore, and wi is the weight of the ith shown in Eq. (2).
pore. The weight is determined by the ratio of the ith pore's size to the Asample
sum of all pore's sizes, reflecting its proportionate contribution to the Relative metabolic activity (RMA) = (2)
Aneg.control
total pore area.
To measure the nanofiber mat thickness, a previously described Equation 2. Calculation of relative metabolic activity (RMA) by
method was employed described by Zupančič et al. [43]. Briefly, the normalizing the absorbance of a sample (Asample ) against the absorbance
mats were quickly frozen in liquid nitrogen, cut with a razor blade and of a corresponding negative control (Aneg.control ), both corrected for the
attached to a vertical mount with double-sided tape to ensure stability absorbance of the blank culture medium.
during the measurement. Images were taken with a stereomicroscope
(Olympus SZX12, Japan) where we determined mat thickness in three 2.6. Acute cytotoxicity
parallels each consisting of at least 5 measurements using imageJ
software. Acute cytotoxicity of nanofiber mats against PBMCs was assessed
using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (LDH
2.4. Isolation of peripheral blood mononuclear cells assay), following the manufacturer's instructions [45]. Briefly, PBMCs in
our recently developed test model were incubated on samples with or
Peripheral blood mononuclear cells (PBMCs) were isolated from without (controls) nanofibers for 6 h, after which LDH assay was per­
buffy coats of healthy donors and used according to the approval of the formed and absorbances were measured at 490 nm using the BioTek
Medical Ethics Committee of the Republic of Slovenia (No. 0120-279/ Synergy H4 microplate reader. For Triton controls wells without nano­
2017-3). Briefly, PBMCs were isolated by density gradient centrifuga­ fibers, 20 μL of 9 % Triton X-100 was added after 5.5 h incubation to lyse
tion, using Lympholyte-H solution, according to manufacturer's in­ all cells providing the maximal LDH release value, corresponding to 100
structions. The cells were then washed, counted, aliquoted and stored in % cell death. The results were presented as cytotoxicity relative to Triton
liquid nitrogen until use. control, corrected for spontaneous LDH release from negative controls,
calculated with Eq. (3). All absorbance readings were corrected for the
2.5. Relative metabolic activity (RMA) of PBMCs absorbance of the blank (cell culture medium alone).
Asample − Aneg.control
The commonly used in vitro method for testing immunogenicity of Cytotoxicity = × 100% (3)
Atriton control − Aneg.control
various materials is the leukocyte proliferation assay, as described by
Potter et al., 2020 [44]. In of our experiments we set two groups of cell Equation 3. Percentage of cytotoxicity, calculated by subtracting the
culturing conditions, i.e. the homeostatic, where PBMCs were cultured absorbance of the negative control (Aneg. control) from the absorbance of
on nanofiber mats without any stimulation and the inflammatory one the sample (Asample) divided by the difference between the absorbance of
where PBMCs grown on nanofiber mats were stimulated with PHA-L. Triton control (Atriton control) and the absorbance of the negative control

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A. Zidar et al. International Journal of Biological Macromolecules 282 (2024) 137029

(Aneg. control). All absorbances were corrected for the absorbance of the diameters, interfibrillar pore sizes and mat thickness. To evaluate the
blank (cell culture medium alone). impact of these physical properties on the immunological safety of
nanofibers, we measured relative metabolic activity (RMA) of PBMCs
2.7. Relative number of viable PBMCs cultured on nanofibers. They were investigated without stimulation, i.e.,
homeostatic environment and with stimulation using PHA-L, a poly­
Relative number of viable cells was also determined using the LDH clonal activator of T lymphocytes, to simulate immune cell response in
assay. In this case, after 72 h incubation, the cells in all cultures were an inflamed environment. Additionally, we investigated the influence of
lysed with Triton X-100, after which the LDH assay was performed ac­ nanofiber mat thickness through cytotoxicity and relative number of
cording to manufacturer's instructions and absorbances were measured viable cell assays. To validate the obtained results, we visualized PBMC
at 490 nm using the BioTek Synergy H4 microplate reader [45]. Results proliferation and nanofiber mat penetration using fluorescence micro­
were presented as the relative number of viable cells, normalized to scopy. Primary PBMCs from a healthy human donor, encompassing all
negative control, using the Eq. (4). All absorbance readings were cor­ major immune cell subtypes, were used to ensure a comprehensive im­
rected for the absorbance of the blank (cell culture medium alone). mune response.

Asample 3.1. Impact of nanofiber diameter on RMA of PBMCs

Relative number of viable cells = (4)
Aneg.control

Equation 4. Determination of the relative viable cell number, Nanofibers with different diameters were prepared by adjusting the
calculated by normalizing the absorbance of a sample (Asample ) against total concentration of a specific polymer. Table 2 summarizes the elec­
the absorbance of a negative control (Aneg.control ), both corrected for the trospinning dispersion composition ranges and the measured physical
absorbance of the blank (cell culture medium alone). property value ranges of prepared nanofibers. As expected, higher
polymer concentrations resulted in larger nanofiber diameters and in
larger inter-fiber pores (Fig. 1). Complete information on individual
2.8. Fluorescence microscopy nanofibers is presented in the supplementary material. As shown in
supplementary Table S1, different polymer dispersion compositions
Proliferation and penetration of PBMCs were visualized in two yielded different nanofiber diameter ranges, where ALG/PEO2MDa (75
separate experiments using fluorescence microscopy. In the first one, nm) and CS/PEO900kDa (240 nm) had a much narrower range, than PCL
PHA-L activated PBMCs were cultured differently thick nanofiber mats (416 nm) and zein/PVP (562 nm) nanofiber mats. These differences
for 72 h to visualize the extent of PBMC proliferation. After incubation, arise from different properties of polymer dispersions and their abilities
the supernatants were carefully removed, and the inserts of in vitro to be electrospun at different concentrations.
model were disassembled. Nanofiber samples were then arranged on The effects of varying nanofiber diameters on RMAs of PBMC are
glass slides without washing and stained using 25 μL of ProLong™ Gold presented in Fig. 2. Nanofibers were considered neutral if they did not
antifade reagent with DAPI. Images were captured by the Zeiss Confocal change immune cell RMA compared to controls. An increase in RMA
microscope LSM 800 KMAT, equipped with the GaAsP-PMT detector value suggested they were potentially immunogenic, while a decrease,
(Carl Zeiss, Thornwood, NY, USA), enabling precise visualization of cell indicated they were non-immunogenic or immunomodulatory. To
distribution and density. determine the link between nanofiber diameters and RMA values in
In the second experiment, non-stimulated PBMC penetration into terms of positive or negative correlations, we used Pearson correlation
differently thick nanofiber mats was investigated after 4 h incubation. test. The significance of correlations was presented in graphs as asterisks
Following incubation, the in vitro models were disassembled, and “*”.
nanofiber mat samples thoroughly washed with phosphate buffer saline The results presented in Fig. 2 show that nanofiber diameters of PCL,
to remove all the cells that did not penetrate into mats. The samples ALG/PEO2MDa, and CS/PEO900kDa nanofibers did not significantly affect
were placed on glass slides and stained with 25 μL of ProLong™ Gold the RMAs of PBMCs, while increasing diameters of zein/PVP increased
antifade reagent with DAPI. Images were captured by Invitrogen™ RMA. Despite similar average diameter ranges of PCL and zein/PVP
EVOS™ FL Digital Inverted Fluorescence Microscope (Invitrogen, MA, nanofibers, only the latter showed a statistically significant positive
USA). correlation. As previously mentioned, zein/PVP nanofiber diameters
significantly positively correlated with RMA of non-stimulated PBMCs
2.9. Statistics (Fig. 2). The correlation is evident from the increased RMAs in cultures
on nanofibers with larger diameters, suggesting they enhance their po­
GraphPad Prism 8.4.3. (GraphPad Software, Boston, USA) software tential immunogenicity. This finding is particularly interesting, as zein is
was used for statistical analysis and graphical presentations of results. regarded as a safe and low immunogenic plant protein [29]. However, it
To statistically evaluate significant differences between groups of re­ is important to emphasize that ALG/PEO2MDa and CS/PEO900kDa nano­
sults, one-way ANOVA in conjunction with Dunnett's multiple compar­ fibers had a much narrower average diameter range than those made of
isons test was performed. The positive and negative correlations PCL and zein/PVP, which could potentially explain the lack of their
between two parameters were evaluated using the Pearson correlation significant impact on RMA.
test. Multiple linear regression was applied to evaluate the relationship However, CS/PEO900kDa nanofibers of all diameters reduced RMA
between RMA values of non-stimulated and PHA-L stimulated PBMCs values in both stimulated and non-stimulated PBMCs compared to
(analyzed separately) and the following variables: nanofiber diameter, controls (Fig. 2). This reduction is likely because of increased viscosity of
pore size, and mat thickness. The assumptions of linearity, indepen­ cell medium due to dissolved PEO900kDa [46], which likely limits
dence, homoscedasticity, and normality of residuals were verified with nutrient exchange and the access of MTS reagent to PBMCs, resulting in
diagnostic tests. Statistical significance was set at p ≤ 0.05 for all lower measured metabolic activity. We observed the reduced metabolic
analyses. activity in additional experiments using PEO nanofibers alone (see
Fig. S1 in the Supplement). Such an effect was not observed with ALG/
3. Results and discussion PEO2MDa as most of the PEO2MDa was removed during the crosslinking
process.
In this study, we prepared nanofibers from various polymer com­ However, due to the variability in pore sizes across all the tested
positions, namely, PCL, ALG/PEO2MDa CS/PEO900kDa and zein/PVP nanofiber mat samples, these subtle effects should be interpreted with
using electrospinning. They were prepared with a well-defined range of caution. To further clarify the influence of nanofiber characteristics, we

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A. Zidar et al. International Journal of Biological Macromolecules 282 (2024) 137029

Table 2
Summary of electrospinning solution compositions and the ranges of nanofiber physical properties averages.
Polymer Polymer concentration; increment (%, m/V) Nanofiber diameter (nm) Nanofiber pore size (μm2) Nanofiber mat thickness (μm)

PCL 10–14; 1 381–797 1.7–5.7 15–19

ALG/PEO2MDa 3.75–4.75; 0.25 87–162 0.2–0.4 16–19
CS/PEO900kDa 3–4.25; 0.25 56–296 0.3–0.6 9–12
zein/PVP 21.5–25.5; 1 212–774 2.0–7.5 20–24

Fig. 1. SEM images show a comparison of different nanofiber diameters. Top row - nanofibers with the smallest diameters and bottom row - nanofibers with the
largest diameters (Table S1), made from: PCL, ALG/PEO2MDa, CS/PEO900kDa and zein/PVP polymers.

Fig. 2. The impacts of nanofiber diameters on relative metabolic activity (RMA) of non-stimulated (green circles) and PHA-L stimulated (red squares) PBMCs
incubated on PCL, ALG/PEO2MDa, CS/PEO900kDa, and zein/PVP nanofiber mats. Negative control – green dotted line; positive control – red dotted line. All presented
RMA values are relative to appropriate negative controls. Data represents average values ± SD (n > 3). Pearson correlation significance: *(p < 0.05). (For inter­
pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

performed multiple linear regression, which revealed that nanofiber 3.2. Impact of nanofiber mat interfibrillar pore sizes on RMA of PBMCs
diameter, did not have a significant effect on RMA values of PBMCs in
most cases. On the other hand, zein/PVP nanofibers showed significant As mentioned in the previous section, nanofibers produced from
influence of all three tested parameters in PHA-L stimulated cultures polymer dispersions with higher total polymer concentrations also form
while the non-stimulated cultures didn't show any significance as larger mat pores. Which makes it difficult to isolate the impact of
detailed in Table S4. nanofiber diameter or interfibrillar pore size on PBMCs. To overcome
Previous research on non-immune cells, such as the study by Pel­ this, we produced nanofiber mats with different pore sizes by increasing
ipenko et al., demonstrated that nanofiber diameters affect different cell the collector voltage in 5 kV increments, from 0 to − 20 kV and appro­
types differently, depending on their chemical composition. They found priately adjusting the nozzle voltage (Fig. 3). An absolute higher col­
that different optimal nanofiber diameters are needed for maximal lector voltage required lower nozzle voltage to ensure stable
proliferation and migration of keratinocytes and fibroblasts [36]. electrospinning conditions (see Table S2 in the Supplement) which
resulted in a narrower electrospinning cone and consequently smaller
interfibrillar pores. With this approach, we produced nanofiber mats
with different pore sizes while maintaining similar nanofiber diameters

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A. Zidar et al. International Journal of Biological Macromolecules 282 (2024) 137029

Fig. 3. SEM images showing different pore sizes of nanofiber mats made from PCL, ALG/PEO2MDa, CS/PEO900kDa and zein/PVP polymers. Top row - nanofiber mats
with the smallest pores; bottom row - nanofibers with the largest pores.

and mat thicknesses within the same nanofiber composition (Table 3). μm2 and 1–4 μm2 large, respectively. Additionally, osmotic pressure and
Methodology of nanofiber average interfibrillar pore size determi­ swelling could also have drawn cells deeper into the mesh through these
nation is rarely reported in scientific literature and when it is, it varies larger pores [16,47,48]. Similarly, as seen in Fig. 2, here again CS/
significantly between research groups [3]. We first opted to use ImageJ PEO900kDa caused a significant decrease in RMA values compared to
for automatic pore size determination in conjunction with the arithmetic controls (Fig. 4). Conversely, larger pore sizes of zein/PVP nanofibers
mean calculation. However, these calculated means were significantly increased immune cell RMA values, thereby showing a clear significant
biased downwards, as small pores were overrepresented considering the positive correlation between the two parameters, which is in line with
surface they occupy. To address this issue and increase the relevance of our previous results (Fig. 2). This outcome suggests that the potential
measurements on SEM images, we opted for a weighted pore size immunogenicity increased with larger nanofiber mat pores, which
average calculation (Methods section 2.3). Using this calculation, the probably is due to deeper cell penetration providing larger surface area
contribution of each pore to the average value was weighted according available for nanofiber-cell interactions causing greater cell activation
to its relative size in relation to the total pore area. The result of and increased RMA.
weighted average calculations was 2.5 to 3.75 times greater than those The relationship between pore size and RMA was further confirmed
obtained by the arithmetic mean calculation, and they were comparable by multiple linear regression analysis (Table S4), where nanofiber mat
to results from manual sizing methods described in the literature. By pore size consistently showed a significant effect on RMA, including all
applying this weighted average calculation, we obtained a more relevant those cases where a significant correlation was observed with a single
and accurate values of average nanofiber mat pore sizes and improved parameter (Fig. 4). Our results obtained on PCL, ALG/PEO2MDa and CS/
the objectivity compared to alternative manual size assessment methods PEO900kDa nanofiber mats are comparable with literature data, as for
described by Langwald et al. [3]. example, a similar effect was observed with human mesenchymal stem
The impact of interfibrillar pore sizes of different nanofiber mats on cells, where larger pores in the nanofiber mesh made of PCL reduced
immune cell RMAs is shown in Fig. 4. Increasing pore sizes of nanofiber their ability to proliferate, as described by Wei et al. [49].
mats made from PCL, ALG/PEO2MDa and CS/PEO900kDa all caused
similar decrease in RMAs, especially in PHA-L stimulated PBMCs. On the
other hand, the effect of pore size on non-stimulated PBMCs was much 3.3. Impact of nanofiber mat thickness on RMA, cytotoxicity and relative
less noticeable but still present. These negative correlations are most numbers of viable PBMCs
probably a consequence of deeper penetration of cells into the nanofiber
mesh, where they get entrapped. This, in turn, could lead to potential Desired thicknesses of tested nanofiber mats were achieved by
cytotoxicity, reduced proliferative capacity and/or impaired metabolic adjusting the electrospinning time, which was calibrated for each indi­
activity of individual cells, all of which we set to investigate in further vidual polymer, based on its concentration and flow rate. ALG/PEO2MDa
experiments. It is important to note that both ALG/PEO2MDa and CS/ and CS/PEO900kDa nanofiber mats had narrower mat thickness ranges
PEO900kDa nanofiber mats had small average pore sizes that do not allow due to low total polymer concentrations and flow rates required for
extensive cell penetration. However, the largest pores in these two types stable electrospinning, which significantly increased preparation time of
of nanofiber mats, which accounted for about 2 % of all pores, were 1–2 thickest mats. As expected, longer electrospinning times led to thicker
nanofiber mats with small variations in nanofiber diameter and pore

Table 3
Overview of nozzle and collector voltages used for electrospinning of nanofibers and the ranges of nanofiber physical properties averages.
Polymer Nozzle voltage (kV) Collector voltage; increment (kV) Nanofiber diameter (nm) Nanofiber pore size (μm2) Nanofiber mat thickness (μm)

PCL 7.5–17.5 − 20–0; 5 692–926 1.93–6.65 28–33

ALG/PEO2MDa 4–15 − 20–0; 5 133–171 0.19–0.43 16–18
CS/PEO900kDa 6–15 − 20–0; 5 165–229 0.06–0.74 20–28
zein/PVP 4.5–18.5 − 20–0; 5 401–537 1.16–5.54 81–87

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A. Zidar et al. International Journal of Biological Macromolecules 282 (2024) 137029

Fig. 4. The effects of nanofiber mat pore sizes on relative metabolic activity (RMA) of non-stimulated (green circles) and PHA-L stimulated (red squares) PBMCs,
cultured on nanofibers made of: PCL, ALG/PEO2MDa, CS/PEO900kDa, and zein/PVP. Negative control – green dotted line; positive control – red dotted line. All
presented values are relative to appropriate negative controls. Data represent average values ± SD (n > 3). Pearson correlation significance: *(p < 0.05) and **(p <
0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

sizes (Table 4). However, doubling of electrospinning time did not cells. This seemingly contradictory observation could be due to greater
consistently lead to doubling of mat thickness (see Table S2 in the activation of some immune cells, which increases their RMA despite
Supplement). mechanical constraints that physically hinder their proliferation [50].
The impact of nanofiber mat thickness on immune cell RMAs is To evaluate the impact of nanofiber physical properties on PBMC
presented in top row of Fig. 5. The results show a statistically significant RMA values, cytotoxicity, and the relative number of viable cells in these
negative correlation between PCL, ALG/PEO2MDa, and CS/PEO900kDa experiments, we performed multiple linear regression analysis. We
nanofiber mat thickness and RMA values. In contrast, zein/PVP nano­ found that mat thickness was the primary factor influencing RMA,
fiber mat thickness exhibited a statistically significant positive correla­ though nanofiber diameter and pore size also showed significant effects
tion with RMA. Given these surprising outcomes, we aimed to further in specific cases (Table S4). Regarding cytotoxicity, various physical
investigate them. parameters were found to have significant impacts across different
We hypothesized that thicker nanofiber mats would enable deeper nanofibers, but the overall changes were minimal (Table S5). Notably,
PBMC penetration, leading to extensive nanofiber-cell interactions and for the relative number of viable cells, pore size and mat thickness
their physical entrapment. This could potentially result in cytotoxicity, emerged as the most influential factors (Table S6).
impaired cell proliferation, and/or altered metabolic activity of indi­ In summary, our study shows that optimal nanofiber diameters, pore
vidual cells. To test this, we performed LDH release acute cytotoxicity sizes, and mat thickness depend on the intended application and the
tests and additionally assessed relative numbers of viable cells present properties of the chosen polymer. In tissue engineering, immune
after their incubation on nanofiber mats. response and nanofiber compatibility with tissue-specific cells are key
Acute cytotoxicity assay showed no significant cytotoxic effects of all factors [53]. Based on the immunological findings of this study, we
the tested nanofiber mats on PBMCs (Fig. 5, middle row). This means recommend conducting immune assays with multiple donors to account
that the previously observed reduced RMA values were not caused by for interindividual variability and identify the optimal physical prop­
cytotoxicity. We then also determined relative number of viable cells erties for specific applications. The nanofibers should be non-cytotoxic,
cultured on nanofiber mats and found that in the non-stimulated cul­ avoid harmful immune activation (ensuring that non-stimulated PBMC
tures these values were comparable to negative controls (PBMCs without cultures do not significantly exceed the negative control), and simulta­
nanofiber mats). However, in PHA-L stimulated cell cultures with neously promote immune surveillance (maintaining RMA levels above
thicker mats the cell numbers were converging towards those found in non-stimulated cultures in PHA-L stimulated assays).
negative controls. This indicates that cell proliferation was reduced or
even stopped on/in thicker nanofiber mats (Fig. 5, lower row). 3.4. Visualization of PBMC proliferation and penetration on nanofiber
In summary, these results suggest that thicker non-immunogenic mats by fluorescent imaging
nanofiber mats hinder the proliferation of PBMCs and reduce RMA of
individual cells most likely due to their easier and deeper penetration Until now, no specific studies have discussed immune cell penetra­
into nanofibers. This reduced proliferation was evidenced by lower tion into nanofiber mats and the consequently reduced proliferation
numbers of viable cells in PHA-L stimulated cultures after 72 h incu­ ability of spatially constrained PBMCs. However, it is known that other
bation on thicker mats. Meanwhile, in non-stimulated cultures incu­ non-immune cells cease to proliferate when they lack sufficient space for
bated on thicker mats compared to thinner ones or corresponding their growth [51,52]. In this study, we used fluorescence microscopy in
controls, the same number of viable cells exhibited lower metabolic two separate experiments to visualize and better understand how
activity. This proves that the individual measured metabolic activity of nanofiber mat thickness impacts proliferation (relative number of viable
immune cells was reduced. cells) and penetration of PBMCs. First, we observed the impact of
Potentially immunogenic zein/PVP nanofiber also reduced prolifer­ nanofiber mat thickness on proliferation of PHA-L stimulated cultures
ation of PBMCs, while they simultaneously increased RMA of individual after 72 h of incubation (Fig. 6). The fluorescent images illustrate that

Table 4
Overview of electrospinning times of nanofibers and the ranges of nanofiber physical properties averages.
Polymer Electrospinning time (min) Nanofiber diameter (nm) Nanofiber pore size (μm2) Nanofiber mat thickness (μm)

PCL 5–80 550–693 3.9–5.5 19–124

ALG/PEO2MDa 15–240 103–150 0.3–0.5 5–42
CS/PEO900kDa 20–320 88–115 0.2–0.3 11–45
zein/PVP 5–80 343–428 2.0–2.9 24–148

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A. Zidar et al. International Journal of Biological Macromolecules 282 (2024) 137029

Fig. 5. The impact of nanofiber mats thicknesses on relative metabolic activity of non-stimulated (green circles) and PHA-L stimulated (red squares) PBMCs (upper
row); cytotoxicity (middle row), and relative number of viable cells in cultures (lower row). Negative control – green dotted line; positive control – red dotted line. All
values presented are relative to appropriate negative controls. Data represent average values ± SD (n > 3). Pearson correlation significance: *(p < 0.05) and **(p <
0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

proliferation and clustering are most pronounced on/in the thinnest visual evidence obtained through fluorescence microscopy imaging
mats (5–24 μm) (Fig. 6: top row), reduced on/in medium-thick mats reinforced the quantitative results from our previous experiments.
(20–67 μm) (Fig. 6: middle row), and barely observable on/in the
thickest mats (42–148 μm) (Fig. 6: bottom row). Additionally, some cells 4. Conclusion
cultured on/in the thicker mats appear to be out of focus (lower colour
intensity), indicating they are located deeper within the nanofiber mat. Varying the physical properties of nanofibers, altered the behavior of
These results confirm our quantitative observations of the impact of mat immune cells cultured on them. Specifically, we found that nanofiber
thickness on proliferation of PBMCs. As expected, thinner mats enable diameter had a minimal impact on PBMC metabolic activity. In contrast,
greater cell proliferation and clustering, due to less physical constraints interfibrillar pore size, determined using a more accurate and relevant
and better nutrient diffusion. weighted average approach, had a greater effect. Nanofiber mat thick­
Second, we examined the impact of various nanofiber mat thick­ ness was the most influential physical characteristic, primarily because
nesses on penetration of non-stimulated PBMCs after 4 h of incubation it influenced cell penetration and entrapment within the mat. These led
(Fig. 7). For this purpose, we thoroughly rinsed nanofiber mat samples to a reduced relative metabolic activity of PBMCs cultured on non-
after incubation to remove any cells remaining on their surface and then immunogenic nanofibers, namely, PCL, ALG/PEO2MDa, and CS/
visualized fluorescently labelled cells still present within the nanofiber PEO900kDa. Conversely, potentially immunogenic zein/PVP nanofibers
mat. The images indicate that, as expected, the thinnest nanofiber mats increased the relative metabolic activity of entrapped cells. Importantly,
retained the fewest cells, while the medium-thick and the thickest ones all entrapped immune cells lost their ability to proliferate without
contained increasingly more PBMCs (Fig. 7, top, middle and bottom exhibiting any signs of cytotoxicity.
row, respectively). This visual evidence serves as a compelling confir­ This study reveals that the immunological safety of nanofibers is
mation of observations and quantitative results obtained in our earlier significantly influenced by their physical properties. Therefore, these
experiments, suggesting that the cells penetrate deeper into thicker properties must be carefully considered during the development process
nanofiber mats. This confirms that PBMC penetration is one of the main for biomedical applications. Additionally, the nanofiber production
factors affecting their RMA and number of viable cells after incubation. process must be robust, reliable, and repeatable to enable consistent
We concluded that nanofiber mat thickness significantly affects the production of nanofibers with uniform physical properties, a known
behavior of immune cells cultured on them, with greater thickness challenge of electrospinning.
leading to increased PBMC penetration and reduced proliferation. The

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A. Zidar et al. International Journal of Biological Macromolecules 282 (2024) 137029

Fig. 6. Proliferation of PHA-L activated PBMCs cultured for 72 h on differently thick nanofiber mats (thickness noted in upper left corners) made of PCL, ALG/
PEO2MDa, CS/PEO900kDa, and zein/PVP. Cells were stained with DAPI. Scale bars represent 40 μm on each individual image.

Fig. 7. Fluorescent microscopy images showcasing the distribution of PBMCs retained in nanofiber mats of varying thicknesses (thickness noted in upper left corner)
and compositions (PCL, ALG/PEO2MDa, CS/PEO900kDa, and zein/PVP), post intensive rinsing. The cells were stained with DAPI. Scale bars represent 200 μm in
individual image.

Funding Development Fund (grant number: C333-19-952061 EATRIS-TRI.si).

This work was supported by the Slovenian Research Agency CRediT authorship contribution statement
[Research Core Funding No. P1-0189 and No. P3-0310, and Research
Projects J3-3062 and J7-4418], as well as the European Regional Anže Zidar: Writing – original draft, Visualization, Methodology,

9
A. Zidar et al. International Journal of Biological Macromolecules 282 (2024) 137029

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