2019 int. j. pharm f.friess et al phagocytosis of
TRANSCRIPT
Final Draft of the original manuscript:
Friess, F.; Roch, T.; Seifert, B.; Lendlein, A.; Wischke, C.: Phagocytosis of spherical and ellipsoidal micronetwork colloids from crosslinked poly(Epsilon-caprolactone). In: International Journal of Pharmaceutics. Vol. 567 (2019) 118461. First published online by Elsevier: June 24, 2019
DOI: 10.1016/j.ijpharm.2019.118461 https://dx.doi.org/10.1016/j.ijpharm.2019.118461
1
Phagocytosis of spherical and ellipsoidal micronetwork colloids from crosslinked poly(ε-1
caprolactone) 2
Fabian Friess1,2, Toralf Roch1, Barbara Seifert1, Andreas Lendlein1,2 and Christian Wischke1* 3
1 Institute of Biomaterial Science and Berlin-Brandenburg Centre for Regenerative Therapies, 4
Helmholtz-Zentrum Geesthacht, 14153 Teltow, Germany 5
2 Institute of Chemistry, University of Potsdam, 14476 Potsdam, Germany 6
* corresponding author: Christian Wischke, Institute of Biomaterial Science, Helmholtz-7
Zentrum Geesthacht, 14153 Teltow, Germany. E-mail: [email protected], Phone: 8
+49(0)3328-352-450, Fax: +49(0)3328-352-452. 9
10
Abstract 11
The effect of non-spherical particle shapes on cellular uptake has been reported as a general 12
design parameter to control cellular recognition of particulate drug carriers. Beside shape, also 13
size and cell-particle ratio should mutually effect phagocytosis. Here, the capability to control 14
cellular uptake of poly(ɛ-caprolactone) (PCL) based polymer micronetwork colloids (MNC), a 15
carrier system that can be transferred to various shapes, is explored in vitro at test conditions 16
allowing multiple cell-particle contacts. PCL-based MNC were synthesized as spheres with a 17
diameter of ~6, ~ 10, and 13 µm, loaded with a fluorescent dye by a specific technique of 18
swelling, re-dispersion and drying, and transferred into different ellipsoidal shapes by a 19
phantom stretching method. The boundaries of MNC deformability to prolate ellipsoid target 20
shapes were systematically analyzed and found to be at an aspect ratio AR of ~ 4 as obtained 21
by a phantom elongation ph of ~150%. Uptake studies with a murine macrophages cell line 22
showed shape dependency of phagocytosis for selected conditions when varying particle sizes 23
(~6 and 10 m), and shapes (ph: 0,75 or 150%), cell-particle ratios (1:1, 1:2, 1:10, 1:50), and 24
time points (1-24 h). For larger-sized MNC, there was no significant shape effect on 25
phagocytosis as these particles may associate with more than one cell, thus increasing the 26
possibility of phagocytosis by any of these cells. Accordingly, controlling shape effects on 27
phagocytosis for carriers made from degradable polymers relevant for medical applications 28
requires considering further parameters besides shape, such as kinetic aspects of the exposure 29
and uptake by cells. 30
31
2
Keywords: Particle shape, phagocytosis, macrophage, polymer micronetwork colloids, poly(ɛ-32
caprolactone) 33
34
1. Introduction 35
The phagocytosis of particles by specialized cells such as macrophages or dendritic cells 36
aims at removal of potentially pathogenic particulate matter from the organism. This process is 37
highly relevant in pharmaceutics as it may, on the one hand, impede the targeting or long-term 38
release function of micro-/nanoparticulate drug carriers [1] [2]. On the other hand, uptake can 39
facilitate the delivery of proteins and/or adjuvants into antigen presenting cells, thereby 40
allowing to initiate or modulate immune responses, such as for improved vaccination strategies 41
[3] [4]. 42
In the last decade, the role of particle shape on phagocytosis has been identified in model 43
studies with isolated cells and particles, where non-spherical ellipsoidal polymer particles 44
showed reduced engulfment [5] [6]. In model studies analyzing the interaction of single cells 45
and single particles, particle surfaces with low curvatures at the contact point between cell and 46
particle were identified to suppress phagocytosis, while larger curvatures enhance phagocytosis 47
[5] [7] [8]. For prolate ellipsoidal particles, a cell-particle contact with the flat and less curved 48
site is statistically more likely. Computational analysis of nanoparticle endocytosis based on 49
curvature energy of lipid – bilayer membrane and particle-membrane contact adhesion energy 50
suggest that a reorientation of adhered ellipsoids moving the long axis from parallel towards 51
perpendicular (relative to the cell membrane) is possible but energetically not preferred [9]. 52
Therefore, according to the current understanding, ellipsoids should be taken up by cells with 53
reduced efficiency compared to spherically shaped particles. This relationship of macrophage 54
uptake patterns is supported by studies with various types of particles including, e.g., spherical 55
metal-organic framework (MOF) particles compared to rod-shaped MOFs of a different 56
composition [10], L-cystine based composite particles loaded with CdTe quantum dots with 57
preference of spheres over plates over needles [11], or poly(lactide-co-glycolide) (PLGA) 58
nanoparticles and microparticles comparing spheres and prolate ellipsoids [12]. Accordingly, 59
particle shape is expanding the list of particle features like size, surface properties and 60
mechanical properties that influence cellular recognition and phagocytosis [13] [14] [15] [16]. 61
However, the cellular uptake of particles is a kinetic process typically approaching a distinct 62
maximum level, which theoretically can be either identical or different for specific particle 63
3
types/shapes and may be independent from the respective uptake velocities. For different types 64
of latex beads, microscopic analysis of single uptake events after cell contact suggested slower 65
and more broadly distributed uptake rates for ellipsoids (5 to 55 min; mean 25 min) compared 66
to spheres (2 to 10 min; mean 3 min) [17]; still those values are on a similar time scale and 67
make the time point of observation in in vitro studies highly relevant. Studies with both 68
nanoparticles and microparticles of ellipsoidal and spherical shapes revealed a practically 69
identical uptake by dendritic cells within each size class (150 nm; 2 µm) as examined after 70
overnight incubation [18]. For PLGA nanospheres vs. -ellipsoids with or without surface 71
functionalization with an invasive protein, the uptake by an epithelial cell line was not 72
influenced by particle shape when observed at different time points over 5 hours [19]. In some 73
studies, sharp tipped particles were even preferentially engulfed compared to particles of more 74
compact shapes [20] [21]. It also needs to be considered that, at a given size distribution of 75
particles, individual size fractions may behave differently since major effects of small size 76
changes on phagocytosis efficiency are well known at least for spherical particles. Often, 77
emulsion techniques applied, e.g. for polylactide-based degradable particles lead to relatively 78
broad size distributions. For such polylactide-based particles, a higher uptake of spheres 79
compared to prolate ellipsoids was observed after 4 hours, the time point of data analysis used 80
in this study [6]. Furthermore, one has to note that mechanistic studies exploring the interaction 81
of single cells and particles may not represent the conditions as present in vivo. In some 82
experimental conditions that enable multiple cell-particle contacts, it was observed that prolate 83
ellipsoidal model particles fabricated from polystyrene were initially recognized/attached more 84
efficiently than spheres [22], which is a relevant factor contributing to the probability of overall 85
engulfment particularly at a dense particle/cell packing. Therefore, beside shape, cell-particle 86
ratio, time-point of observation and size should mutually affect phagocytosis rates. As 87
additional parameters, e.g. the protein adsorption that depends on polymer properties [23], can 88
contribute to biological interaction and recognition [24], it may not be possible to predict shape 89
dependency of phagocytosis for all types of particle materials without studying each specific 90
carrier system. 91
A degradable material with relevance for biomedical applications is poly(ɛ-caprolactone) 92
(PCL). Using oligo(ɛ-caprolactone) [oCL] based precursors, polymer micronetwork colloids 93
(MNC) were recently reported, which allow the deformation from spheres to ellipsoids with 94
memory of the original shape [25], thus enabling to create different shapes from otherwise 95
identical particles. Here, the maximum aspect ratios (AR) that can be created from identical 96
stock particles as well as the role of shape and size on MNC phagocytosis were investigated. 97
4
Conditions were applied allowing multiple particle-cell contacts by systematically altering cell-98
particle ratios and time points of observation. Out of the prepared MNC with narrow size 99
distributions, MNC with a diameter of ~6 and ~10 µm were selected for phagocytosis studies, 100
which provide a high internal volume for future payloads. These particles are in the mean and 101
upper range of sizes accepted by phagocytic cells, thus enabling to explore shape, size and 102
kinetic aspects of phagocytosis with expected distinction between phagocytizing and non-103
phagocytizing cells. By loading with a fluorescent model compound for particle visualization, 104
cellular uptake of spheres and ellipsoids of different prolate ellipsoidal shapes by macrophages 105
could be explored. 106
107
2. Materials and Methods 108
2.1 Synthesis of MNC 109
oCL-diol (Mn = 8 kDa, Perstop, UK Ltd., United Kingdom) was functionalized with 2-110
isocyanatoethylmethacrylate to oCL-IEMA (97% functionalization, melting temperature 111
Tm = 56 °C). Spherical oCL-IEMA particles prepared by microfluidic emulsification and 112
solvent evaporation were photocrosslinked internally to MNC in the molten state in aqueous 113
dispersion by UV irradiation (308 nm; XeCl-Excimer, Heraeus, Germany). The semi-114
crystalline MNC 6 (volume-moment mean D[4,3] main peak 6.3 µm; Uniformity U 0.688), 115
MNC 10 (9.7 µm; U 0.338) and MNC 13 (D[4,3] 12.5 µm; U 0.02) had Tm`s of 53, 48, and 116
45 °C, respectively (DSC 204 F1, Netzsch, Germany; nitrogen atmosphere, second heating 117
cycle from -100 to 150 °C, 10 Kꞏmin-1). MNC-powder was obtained by washing with water on 118
0.45 µm nylon filters (NL17, Whatman®, United Kingdom) and lyophilization (Alpha 1-2 LD 119
plus, Martin Christ, Osterode, Germany; 0.08 mbar, ice condensor -55 °C). 120
121
2.2 MNC loading with model compound 122
1 ml dichloromethane (DCM, 5 mL, ρ=1.33 gꞏmL-1) supplemented with Rhodamin B (RB, 123
0.05 wt.%, 3.3 mg) was added to dry MNC powder (35 mg) in 50 ml vials at room temperature. 124
MNC swollen in DCM were dispersed in water by sonication (Sonoplus HD 3200, MS 72 125
Sonotrode, Bandelin, Germany, 30% amplitude, 30 s) after careful overlaying the organic phase 126
with 10 mL of 5 wt.% polyvinylalcohol in water (PVA; Mowiol® 4-88, Mn = 12.6 kDa; 127
5
PD = 2.5). Three of such aliquots were added to 60 mL PVA-solution (2.5 wt.%) for solvent 128
evaporation (7 h, 500 rpm overhead stirrer), followed by washing with water and lyophilization. 129
130
2.3 Preparation of ellipsoids by MNC deformation 131
Suspensions of 10 mgꞏmL-1 MNC in 22.5 wt.% PVA solution (Mowiol® 3-85, Mn = 5.6 kDa; 132
PD = 2.5) were casted in molds and dried (24 h RT, 4 h 60 °C) to obtain flat phantoms. 133
Phantoms were uniaxially stretched (7.5 mmꞏmin-1) up to 290 % in a tensile tester (ZP 20, 134
Zwick, Germany) at 80 °C with subsequent cooling to 20 °C (heating/cooling rates 10 Kꞏmin-1). 135
Prolate ellipsoid MNC were harvested by phantom dissolution and MNC washing with water 136
(Ampuwa® Plastipur, Fresenius, Germany). 137
138
2.4 Particle size characterization 139
The size distributions of MNC were determined by static light scattering (SLS, Hydro 2000 140
µP-sample unit, Multiple Narrow Mode, Mastersizer 2000, Malvern Panalytica, Kassel, 141
Germany) and given sizes correspond to the observed main peak of D[4,3]. Particle suspensions 142
were dropwise added to ~20 ml degassed water as measurement medium until a laser 143
obscuration of 1-10% was reached. Typically, three analysis runs were performed for each 144
sample. 145
Automated light microscopy analysis and determination of AR was performed for 180-1300 146
single particles of each aqueous MNC-suspension, which were observed between a microscope 147
slide and a cover slip with a Morphology G3 instrument (Malvern, United Kingdom). 148
Ir-coated (4 nm; Sputter coater Q150TES, LOT Quantum Design, Darmstadt, Germany) 149
samples were prepared on thin glass plates and analyzed by scanning electron microscopy 150
(SEM, Gemini SupraTM 40 VP, Carl Zeiss NTS, Oberkochen, Germany) at acceleration 151
voltages of 2 kV with an Everhart-Thornley (SE2) detector in high vacuum at room 152
temperature. 153
154
155
2.5 Phagocytosis studies 156
Contaminations of particles with endotoxins were investigated by an established protocol 157
with HEK-BlueTM reporter cell lines [26]. Briefly, 5x104 HEK-BlueTM-hTLR4 or HEKBlueTM-158
Null2 cells (InvivoGen, San Diego, USA) were cultured directly in 100 µL VLE-RPMI 159
6
(Biochrom®, Berlin, Germany) in the presence of the MNC (4x104) for 24 hours at 37 °C (5% 160
atmospheric CO2). Cell culture supernatants were subjected to the QuantiBlueTM assay 161
(InvivoGen) with determination of absorbance at 620 nm (Tecan plate reader) after 3 h of 162
incubation with the reagent at 37 °C (5% atmospheric CO2). Pure medium and 163
lipopolysaccharide (LPS) from E.coli strain O111:B4 served as negative and positive controls, 164
respectively. 165
In phagocytosis studies, several specific cell–to–MNC ratios (1:1, 1:5, 1:10, 1:50) were 166
applied as ensured by particle counting using a Neubauer chamber and adjusting MNC stock 167
suspensions (108 MNCꞏmL-1; 1 wt.% PVA; Mowiol® 4-88). A final PVA concentration of 168
0.25 wt.% allowed reproducible MNC aliquotation and was shown to be non-toxic (Supp. 169
Information, Supp. Fig. 2 and 3). RAW264.7 macrophages (1ꞏ105/well) were fluorescently 170
labeled (eFluor670, eBiosciences, Germany, 10 µM) before MNC addition to clearly 171
discriminate cells and MNC when analyzed by flow cytometry. The cells were cultivated in 172
DMEM-medium (150 µL, Biochrom AG, Germany; 10% FBS, Sigma) in 96 well plates 173
(Corning, Sigma-Aldrich, MO, USA) at 37 °C and 5% CO2, to which 50 µL MNC suspensions 174
were added. Gentle centrifugation (400∙g, 5 min at room temperature) of the cell culture plate 175
facilitated instantaneous cell–MNC contact. 176
The viability (staining with 1 µgꞏml-1 4,6-diamidino-2-phenylindole (DAPI) directly before 177
measurement) and particle uptake (RB signal) were analyzed by flow cytometry (MACSQuant 178
flow cytometer, Miltenyi Biotec, Bergisch-Gladbach, Germany) at different time points. 179
Confocal laser scanning microscopy (CLSM; LSM 510, Zeiss, Germany) was applied after cell 180
fixation (paraformaldehyde), membrane permeabilization (Triton-X100) and staining of actin 181
(5 U∙ml-1 Alexa 488 phalloidin, Invitrogen, CA, USA) and nuclei (0.5 µg∙ml-1 DAPI, 182
Invitrogen). 183
184
2.6 Statistical evaluation 185
Statistical analysis was conducted by one-way ANOVA analysis with post-Tukey’s multiple 186
comparison test, comparing for each MNC type the groups with different shapes (ɛph) and cell–187
to–MNC ratio at a given time point (GraphPad Prism software v6.01). Experimental errors of 188
analytical techniques are typically < 3%. 189
190
3. Results and Discussion 191
7
Preparation of MNC and creation of ellipsoidal shapes 192
Polymer MNC based on oCL were synthesized from oCL-IEMA precursor as previously 193
described [25], with the exception that a microfluidic particle templating was applied to produce 194
MNC with a more narrow size distribution and that linear precursors were crosslinked by 195
exposure to UV irradiation (308 nm) in the molten state. During the crosslinking step, IEMA 196
units are converted (FTIR data, see Supp. Fig. 1) and oligomethacrylate chains were formed 197
acting as covalent netpoints [27], which define the permanent shape of the MNC. These 198
netpoints are connected by coiled oCL chain segments. The obtained spherical MNC showed 199
sizes of ~6 µm (MNC 6), ~10 µm (MNC 10) and ~13 µm (MNC 13) as determined by static 200
light scattering (SLS) in aqueous dispersion. 201
Based on their composition of covalent netpoints and elastic chain segments, MNC can 202
undergo an elastic deformation upon application of external forces at least at temperatures 203
above the melting temperature Tm of PCL crystalline domains (e.g. Tm,MNC 13 = 45 °C). For 204
macroscopic materials, such deformation experiments can be performed by directly clamping 205
the material in a tensile tester. In contrast, the systematic and reproducible deformation of 206
numerous MNC required the use of phantom matrices. The MNC-loaded phantoms (films) were 207
prepared by dispersing MNC in aqueous poly(vinyl alcohol) (PVA) solution, casting the 208
dispersion into molds, and drying by evaporation of water (Fig. 1). These PVA phantoms can 209
subsequently be subjected to uniaxial stretching, e.g. at 80 °C (T > Tm, MNC) to the desired 210
degrees of phantom elongation εph [5] [25], enabling elastic deformation of numerous MNC in 211
parallel within the phantoms. By subsequent cooling below Tm, MNC, the MNC adapted a semi-212
crystalline morphology with PCL crystallites fixing the ellipsoidal shape. Subsequently, MNC 213
can be collected by phantom dissolution in water, while their ellipsoidal shape remains stable. 214
In order to allow identification of samples originating from a distinct phantom elongation, 215
εph is indicated in the sample code, e.g., MNC 6(150) for εph = 150%. In order to exclude any 216
unknown handling effects and thus allow a direct comparison of MNCs in phagocytosis studies, 217
particles indicated e.g. as MNC6 (0) were also subjected to phantom embedding and stretching, 218
while only a marginal deformation of εph = 0.1% was applied that practically did not change the 219
spherical particle shape. 220
As the particles are composed of a covalent network structure limiting infinite deformation, 221
the possible range of aspect ratios AR, i.e. the ratio of longest and smallest particle axis, should 222
be determined for MNC in a first instance. Based on their monodispersity (U 0.02), MNC 13 223
were employed in this set of experiments. In a simplified model (Fig. 1B), the stretching of 224
MNC by a factor f in one spatial direction results in a reduction in the other two spatial 225
8
directions by the factor f -0.5. When assuming full displacement of ɛph to the individual 226
incorporated MNC at T > Tm,MNC, then f = (ɛphꞏ(100%)-1+1). Under these conditions and within 227
the elastic deformation regime, the experimentally observed AR should correspond to the 228
theoretical ARth predicted by the model (AR = ARth). In contrast, AR < ARth may be observed 229
when the network resists deformation, e.g. by being maximally stretched. When MNC13 were 230
subjected to ɛph ranging from 0% to 290% and subsequently studied by automated light 231
microscopy/shape analysis, a mean AR could be determined for the isolated particle 232
populations. As expected, prolate ellipsoidal MNC shapes of increasing AR were observed (Fig. 233
1C). By plotting the experimentally determined AR over the phantom elongation (Fig. 1D), an 234
excellent agreement with ARth was illustrated in the low deformation range. As expected, at 235
large ɛph, AR adapted a maximum value when the elastic chain segments were fully stretched. 236
Based on the here observed boundary of MNC deformability at ɛph of ~150% resulting in ARth 237
of ~4, this condition was determined as maximum ɛph applied for samples used in phagocytosis 238
studies. 239
240
Fig 1: Preparation of MNC in spherical shape and transformation into prolate ellipsoidal shapes. 241 (A) Principle of preparation. (B) Deformation model. (C) Microscopic images of MNC 13 isolated 242 from phantoms prepared at different ɛph. (D) Effect of phantom elongation on AR for MNC 13 243 compared to theoretical ARth as predicted according to panel (B). 244
245
Loading of MNC 246
For their exploration as potential drug carriers, a suitable strategy for compound 247
incorporation is required. Since the presence of drug may not be preferred during MNC 248
synthesis to avoid drug alteration at crosslinking conditions, which comprise radical 249
polymerization of IEMA moieties under UV-irradiation, a subsequent loading by swelling was 250
9
anticipated (Fig. 2A). For such MNC, degrees of swelling in the range of 300 vol.% were 251
observed in DCM. Importantly, due to the covalent MNC structure, the integrity of the colloidal 252
particles is preserved. The swelling approach conceptually allows introducing various types of 253
substances, as here exemplarily shown for Rhodamin B (RB), which should allow intracellular 254
visualization of the particles in subsequent cell experiments. While rapid uptake of RB was 255
observed during MNC swelling in DCM, a critical technical step was the subsequent removal 256
of the swelling agent, since aggregation of swollen particles may occur. 257
10
258
Fig 2: Dye loading and characterization of obtained prolate ellipsoid MNC. (A) Principle of RB 259 loading of MNC by swelling in dried MNC in organic dye solution, followed by MNC redispersion and 260 drying. (B) Procedure of dispensing swollen MNC by sonication in a two phase system: (left) organic 261 phase of swollen MNC covered by aqueous top-layer, (middle) sonication for separation of swollen 262 MNC into aqueous dispersion, (right) powder of dye-loaded MNC after washing and lyophilization. (C) 263 Size-distribution analysis of MNC before and after loading. (E) Exemplary SEM and light microscopy 264 images of loaded spherical and deformed MNC 6 (εph = 150%). 265
266
11
By adding a PVA solution to the swollen MNC and applying sonication, an aqueous 267
dispersion of swollen MNC was obtained (Fig. 2B). The internal polymer network structure 268
allowed for MNC integrity during this process. Subsequent extraction/evaporation of DCM 269
resulted in RB-loaded, semi-crystalline MNC. SLS showed identical size distribution plots 270
compared to non-loaded MNC (Fig. 2 C). Light microscopy of aqueous dispersions confirmed 271
the absence of aggregates or ultrasound-induced fragments/debris formed in this step. The 272
minor fractions of smaller or larger particles can be assigned to the microfluidic preparation 273
process, where syringe pumps show occasional pulses in flow as known from literature [28]. 274
Overall, the suitability of the applied swelling process to create successfully loaded MNC 275
samples without major aggregation was confirmed. 276
The RB loaded particles were incorporated in phantoms, transferred to their target shapes 277
(εph = 0.1, 75, or 150%) and studied by electron microscopy, again confirming the desired 278
shapes and illustrating the presence of intact particles with smooth surfaces (Fig. 2 D). As 279
determined with a Neubauer counting chamber, ~250 million MNC were programmed in a 280
single programming step in case of MNC 6. Importantly, MNC remained fluorescent, 281
suggesting suitable persistence of RB in the MNC. 282
283
Phagocytosis of MNC depending on size, shape, cell-particle ratio and time point 284
Prior to conducting phagocytosis experiments, the presence of MNC bound and soluble 285
endotoxins should be excluded. For this, the HEK-BlueTM reporter cell lines was used 286
according to an established protocol [26]. The HEK-BlueTM-hTLR4 are genetically engineered 287
to express toll-like receptor 4 (TLR4), which binds endotoxins and more specifically LPS. After 288
TLR4 engagements, the HEK-BlueTM-hTLR4 secrete an alkaline phosphatase (AP), which is 289
proportional to the amount of LPS contaminations. The AP can be quantified by using 290
QUANTI-BlueTM medium containing a substrate for the AP. The HEKBlueTM-Null2 cells serve 291
as control since they bear the same genetic modifications allowing the secretion of AP, but lack 292
the capacity to express TLR4. As positive control, the HEK-BlueTM-hTLR4 were treated with 293
LPS. When both cells types were cultivated for 24 hours in the presence of the different MNC, 294
neither of them secreted AP indicating that the MNC were not contaminated with endotoxins 295
(Fig 3). 296
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297
Fig 3: Investigation of endotoxin levels of exemplary MNC with reporter cell lines using the 298 QuantiBlueTM assay. Control: HEKBlueTM-Null2 cells lacking TLR4 expression; Test cells: HEK 299 BlueTM hTLR4 cells expressing TLR4 responsive to lipopolysaccharides. 300
301
The shape-dependency of particle uptake of MNC 6 and MNC 10 (εph = 0.1, 75, or 150%) 302
was studied with RAW264.7 macrophages by systematically altering the cell–to–MNC ratios 303
as well as the time point of observation. This should allow identifying potential concentration 304
effects and understanding if distinct shapes are preferentially phagocytized in case of multiple 305
cell-particle contacts. 306
Microscopic analysis illustrated that a complete coverage of cell-culture plates by MNC was 307
given at a ratio of 1:10 for MNC 10 (1:50 for MNC 6) (Supp. Fig. 4). As quantitatively analyzed 308
by flow cytometry (Fig. 4A), the viability of macrophages was not substantially influenced by 309
the presence and concentration of MNC. Therefore, cytotoxicity as interfering factor in 310
phagocytosis experiments could be excluded. 311
When comparing the extent of phagocytosis for different particle concentrations, an 312
increasing uptake of MNC at higher particle concentrations (decreasing cell-to-MNC ratios) 313
was observed after 24 h of incubation (Fig. 4B). This was true for both particle sizes and all 314
particle shapes, suggesting that defined cell–to–MNC ratios are of significance for 315
comparability of literature reports on shape dependent phagocytosis. Generally, the extent of 316
phagocytosis after 24 h was lower for MNC 10 compared to MNC 6, which may be assigned to 317
steric reasons considering the cell size of about 10 µm. CLSM analysis after 24 h suggested 318
that numerous MNC 6 were engulfed by individual cells, while other macrophages did not 319
incorporate a single MNC (Fig. 5). Interestingly, more than one phagocytized MNC 10 per cell 320
was observed only very seldom as may be rationalized by the large volume of MNC 10 321
compared to cells. 322
13
323
Fig 4: Phagocytosis study with RAW264.7 macrophages enabling multiple cell-particle contacts and 324 employing different MNC-sizes (6 or10 µm sphere diameter), degrees of elongation (εph = 0, 75, 150%) 325 and cell–to–MNC ratios (1:1, 1:5, 1:10, 1:50). (A) Viability, (B) MNC uptake after 24 h, and (C) kinetics 326 of uptake, all determined by flow cytometry. Data from three independent experiments are shown (mean; 327 standard error of the mean; * p < 0.1; ** p < 0.01). 328
329
As phagocytosis is a time-dependent process, also the uptake kinetics were studied for 330
selected cell–to–MNC ratios. A significantly higher phagocytosis was detected for spherical 331
MNC 6(0) compared to ellipsoids, particularly after 1 h as assumed (Fig. 4C). At later time 332
14
points, the differences in engulfment for MNC 6 spheres compared to ellipsoids were limited 333
to selected conditions only (1:5 cell–to–MNC ratio). Beyond this, for MNC 10, the effect of 334
shape on uptake was less clear, as data unexpectedly suggested a higher extent of phagocytosis 335
of non-spherical compared to spherical particles after 24 h as well as at intermediate time points. 336
337
Fig 5: Representative CLSM images of cells washed and stained after 24 h of incubation at cell–to–338 MNC ratio: MNC 6 1:50, MNC 10 1:10 (blue: nucleus, green: cell actin skeleton, red: RB-loaded MNC). 339
340
Conceptually, phagocytosis of ellipsoids may need a more extensive rearrangement of the 341
cells’ cytoskeleton than for spheres. The time required for this reorganization might justify the 342
presence of shape effects on MNC 6 phagocytosis at early time points, while being only limited 343
at later time points. This is in line with models describing first order kinetics for the 344
internalization process [29]. Accordingly, conclusions on shape-dependent particle engulfment 345
reported at distinct (early) time points may not in all cases be representative for a terminal state, 346
at least for the PCL-based MNC studied here. 347
It should be noted that in a setting with an excess of particles, i.e. multiple particles available 348
for uptake per cell, elongated and larger MNC (e.g. MNC 10) may bridge between several cells, 349
thus increasing the possibility of phagocytosis by any of these cells due to the higher number 350
of cell contacts. This suggests that, in the boundaries of parameters and particles tested in this 351
study, ellipsoidal particles may not necessarily show a reduced uptake in application relevant 352
conditions of multiple cell-particle contacts. Subsequent studies with several types of particle 353
materials may need to continue exploring this subject, whereby an important step will be to 354
investigate the particles in tissue models. 355
4. Conclusions 356
A methodology was applied to investigate phagocytosis of oCL-derived MNC as an 357
application-relevant polymer depending on shape, size and exposure time, while allowing 358
multiple cell-particle contacts. Boundaries of MNC ellipsoidal shapes were determined and a 359
15
swelling/drying technique for compound loading was successfully employed. For the PCL-360
based particles investigated, this study illustrated a limited effect of particle shape on 361
phagocytosis by a macrophage cell line in vitro at least at later time points and for larger 362
particles. Based on the different uptake pattern upon slight size shifts from MNC 6 to MNC 10, 363
the size distribution of particulate carrier systems, which typically is broad for particles 364
prepared by conventional emulsion techniques, may need to be considered in the fabrication of 365
particles for shape-selective uptake. 366
Overall, controlling shape dependent phagocytosis requires evaluating further parameters 367
besides particle shape, including, e.g. kinetic aspects of cell exposure, tissue models, and 368
particle size distribution. By applying the methodology reported here, a variety of further 369
materials may be studied in the future to gain a comprehensive view on opportunities and 370
boundaries of shape-dependent uptake. 371
372
Author contributions 373
FF: planning of experiments/setups, preparation of samples, data acquisition, analysis, 374
interpretation, article drafting and approval 375
TR: design of study, data acquisition, analysis, interpretation, article drafting and approval 376
BS: data acquisition, analysis, article drafting and approval 377
AL: design of study, supervision, data interpretation, article revision and approval, funding 378
acquisition 379
CW: design of study, supervision, data analysis, interpretation, article revision and approval, 380
funding acquisition 381
Declaration of Competing Interest 382
AL is an inventor on patents/paten applications in the field of shape-memory polymers and 383
drug delivery systems based on these. 384
Acknowledgements 385
Technical support by Anja Müller-Heyn and program-oriented funding by the Helmholtz-386
Association are acknowledged. 387
388
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