Timing of Heparin Addition to the Biomolecular Corona Influences the

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Timing of Heparin Addition to the Biomolecular Corona Influences the Cellular Uptake of Nanocarriers Carole Champanhac, Johanna Simon, Katharina Landfester, and Volker Mailänder Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00777 • Publication Date (Web): 26 Aug 2019 Downloaded from pubs.acs.org on August 27, 2019

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Timing of Heparin Addition to the Biomolecular Corona Influences the Cellular Uptake of Nanocarriers Carole Champanhac a, Johanna Simon a,b, Katharina Landfester a. Volker Mailänder* a,b a – Max-Planck-Institute for Polymer Research, Ackermannweg 10, 55122 Mainz, Germany b –Department of Dermatology, University Medical Center of the Johannes GutenbergUniversity Mainz, Langenbeckstr. 1, 55131 Mainz, Germany

KEYWORDS: drug interaction, nanocarrier, heparin, protein corona, immune system, cellular uptake.

ABSTRACT

Few studies have considered the interaction of nanocarriers with drugs and the implications for their individual efficiency. Here, we demonstrate that heparin, a common anticoagulant, interacts with nanocarriers. Hence, nanocarriers, pre-coated with heparin and plasma in different conditions, were incubated with cancer cells, as well as primary cells from human blood. The relation between

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the timing of the heparin’s addition to the nanocarrier and the cellular uptake extent was assessed by flow cytometry. Through proteomics the effect of heparin on the biomolecular corona composition was determined. We found that HeLa cells, monocytes and macrophages reacted differently to the presence of heparin: the uptake of the pre-coated nanocarriers decreased for HeLa and primary monocytes, while it increased for macrophages. Heparin induced no obvious change in the protein corona composition; thus, we suggest that heparin itself, through its adsorption on the nanocarrier, was responsible for the change of uptake.

INTRODUCTION The development of the nanomedicine field has been driven by the need for safer and targeted delivery of a payload, usually a drug, to a specific cell. Yet, the environment that surrounds these nanomedicines is a key parameter for their successful interaction with cells. The presence of systemic drugs in the blood, such as heparin, induces changes to this environment. Heparin is a glycosaminoglycan extensively used in the clinic to prevent blood coagulation before and after surgery. Usually, fully heparinized patients are given 5,000 IU unfractionated heparin in one shot followed by a continuous infusion, leading to a blood concentration of 1.0 IU/mL.1 The polysaccharide heparin is characterized by its high negative charge, which arises from the heavy sulfonation on the sugar moiety (Fig. 1A).2 Due to its widespread use, we wondered how the presence of heparin would influence the biomolecular corona composition of the NCs and ultimately their uptake by cells. Which interferences, if any, must be considered during concomitant treatment of a patient? Polystyrene nanoparticles are used as models of nanocarrier (NC) for studying the interaction of future nanomedicines with their environment. They have the advantage of being easily


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functionalized with amino groups by co-polymerization,3,4 in addition, multiple surfactants4,5 can be used to stabilize the NCs providing the opportunity to choose from a vast array of molecules, and thus, finding the best fit for each application. The nanocarriers are usually injected intravenously, where they get in contact with a complex biological environment. There, they rapidly acquire a new biological identity making them more susceptible to be recognized by cells from the immune system.6,7 This change of identity implies difficulties to maintain targeting or stealth properties.8,9 A comprehensive understanding of the biomolecular corona composition is primordial for successful use of NCs. The biomolecular corona refers to proteins, lipids, metabolites and other small molecules present in the blood that absorb on the NCs. It forms in a matter of seconds to minutes10,11 around any foreign entities, such as implants, nanoparticles, bacteria or viruses. In particular, the composition of the proteins in the corona has been studied extensively over the past decade.12-14 The surface of a NC defined by its chemical identity,15 and the environment produced by the protein source,16-17 are the two main factors influencing the corona composition. Changes in the composition between individuals could be both a blessing and a curse. Indeed, some cancer markers are present in the blood and through their adsorption on NCs, cancer detection would be easier and less invasive.16-18 However, this personalized corona is also a burden as the type and amount of proteins or lipids absorbed influence heavily the fate of the NCs. For example, clusterin is known for its stealth effect while immunoglobulin G promotes clearance from the blood by macrophages.9,19 This change of the protein corona composition leads to concerns over the influence that systemic drugs, such as heparin, could have on the expected uptake by target cells and the immune system.


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In this work, we investigate the effect of heparin on the uptake of positively charged NCs by tumor and immune cells. The timing of the heparin addition in relation to the protein source is at the center of the study. Thus, we decided to set three pre-coating conditions with the introduction of heparin either before, during or after the protein corona formation. The pre-coated NCs were incubated with cells to assess, by flow cytometry, the influence of each coating condition on the cellular uptake.

MATERIALS AND METHODS All reagents unless otherwise specified were purchased from Sigma-Aldrich (St Louis, MO) or Thermo Fisher Scientific (Waltham, MA).

Nanoparticles synthesis: The polystyrene nanoparticles used in this work were synthesized by free-radical miniemulsion polymerization.3,4 A detailed procedure is available in supplementary information.

Cell lines and buffer: HeLa (human cervix adenocarcinoma (CCL-2)) cells were purchased from American Type Culture Collection (ATCC) and maintained in Eagle’s Minimum Essential Medium (EMEM). RAW 264.7 (murine macrophage (TIB-71)) were purchased from ATCC and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM). THP-1 (human monocyte (TIB-202)) were purchased from ATCC and maintained in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 2-mercaptoethanol to a final concentration of 0.05 mM. All media were supplemented with fetal bovine serum (10 % v/v (Gibco)) and penicillin-streptomycin (100 U/mL


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(Gibco)). All cells were cultured at 37 ˚C in a 5 % CO2 atmosphere. To prevent mutation of the cells, passaging was kept under 30.

Primary cells isolation from Buffy Coat: Primary monocytes and macrophages were isolated by the gradient density layer method and cultured in RPMI-1640 media supplemented with 2 % (v/v) human serum for respectively 18 h and 7 days. A detailed protocol of the isolation, differentiation and characterization of the cells is available in supplementary information.

Preparation of protein-heparin pre-coating of nanocarriers: Aliquots of plasma or sera were thawed in the water bath. The surface area of the nanocarriers was kept constant (A = 0.05m2) between cellular uptake and proteomics experiments. Five concentrations of Na-heparin were prepared by successive 2-fold dilution, as described in details in Table S1. Three coating conditions were tested: Pre(hep)Pre(hPc), Pre(hep+hPc) and Pre(hPc)Pre(hep). A detailed step-by-step procedure is available in supplementary information. Briefly, the coating was carried in one (Pre(hep+hPc), Pre(hep+hS), & Pre(hep+FBS)) or two steps (Pre(hep)Pre(hPc) & Pre(hPc)Pre(hep)).

For the one step coating, heparin was diluted in

plasma/sera, the NCs were added to the mixture and incubated for 1 hour at 37 ˚C under agitation. The NCs were then spun down, washed with PBS and spun down again. For the two steps coating, the NCs were incubated first in either in a heparin-PBS solution (Pre(hep)Pre(hPc)) or in pure plasma (Pre(hPc)Pre(hep)). They were then spun down, washed with PBS, and spun down again. The pellet was dispersed, in a second step, either in pure plasma (Pre(hep)Pre(hPc)) or in a heparinPBS solution (Pre(hPc)Pre(hep)). They were then spun down, washed with PBS, and spun down


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again. The NCs pellet was finally dispersed in PBS for characterization or in DMEM without serum for cellular uptake.

Determination of the protein corona composition: The NCs (A = 0.05 m2) were incubated in 1.0 mL heparin solution for 1 h at 37 ˚C, under constant agitation following the steps described in the previous paragraph. Prior to the last centrifugation step, the pellet was transferred to a new Lobind tube and spun down. The NC pellet was incubated in Tris-HCl (87.5 mM) containing 2 % sodium dodecyl sulfate (SDS) for 5 min at 95 ˚C in order to dissociate the proteins from the NCs. They were spun down at 20,000 g for 1 h at 4 ˚C and the supernatant was recovered and stored at -20 ˚C. The protein concentration was determined by Pierce 660 nm assay following the manufacturer recommendation.20 The recovered proteins were denatured by heating the sample (40µg) in a mixture of running buffer, and denaturing agent at 70 ˚C for 10 min. The mixture was then loaded onto a Bolt Tris-NuPAGE gel and run in MES buffer for 1 h at 120 V. As molecular weight marker, 10 µL SeeBlue Plus2 Pre-stained standard was added in the first or last lane. The gel was stained either with SafeBlue staining solution for 4 h and de-stained overnight in Milli-pore water.

Liquid chromatography – mass spectrometry For LC-MS analysis, SDS was removed from the protein sample via Pierce Detergent Removal Spin Columns. Subsequently, proteins were precipitated using the ProteoExtract Protein Precipitation kit according to the manufacturer instruction. Furthermore, protein digestion was carried out as described in details in previous reports.21,22 Finally, the isolated peptide solutions were spiked with 50 fmol/µL Hi3 Ecoli (Waters, Milford, MA) for absolute protein


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quantification23 and diluted with 0.1 % formic acid. Measurements were performed on a nanoACQUITY UPLC system coupled to a Synapt G2-Si mass spectrometer and data was analyzed with MassLynx 4.1 software. For protein identification, Progenesis QI (2.0) was used and a reviewed human database was downloaded from Uniprot. An extended protocol describing the settings for peptides and proteins identification can be found elsewhere.24-26

Characterization of the pre-coated NCs: For the multiple angle DLS measurements, 250 µg NCs were suspended in 300 µL pure plasma or plasma with 1.0 IU/mL heparin. The measurements were conducted according to the protocol described by Rausch and al.27 For the surface charge and fix angle size measurements, a batch of 250 µg pre-coated NCs was prepared. For the size measurement, 100 µg of the pre-coated NCs was dispersed in Milli-Pore water and measurements were carried out on a Malvern Zetasizer S90 instrument. For the surface charge measurement, 150 µg of the pre-coated NCs was dispersed in potassium chloride solution (10-3 M) and measurements were carried out on a Malvern Zetasizer Nano Z instrument.

Quantification of the cellular uptake by flow cytometry: The cells were seeded as a triplicate in 24-wells plates one day before the experiment. One hour prior to the experiment, the cell culture media was replaced with DMEM without serum. One milliliter of the pre-coated NC was added to the cells to a final concentration of 75 µg/mL, and incubated for 2 h at 37 ˚C in 5 % CO2 atmosphere. Then, the supernatant was removed and the cells were washed three times with PBS. They were recovered with 0.5 % Trypsin solution and washed with PBS before being suspended in reading buffer (PBS supplemented with propidium


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iodide and glucose (1 g/L)). Twenty thousand events were recorded on Partec ML flow cytometer using two channels: FL1 (exc: 488 nm / em: 527 nm) and FL3 (exc: 488 nm / em: 685 nm). Data analysis was performed using FCS Express V4 software. On the FSC-SSC dot plot, dead cells were excluded from the cell population based on their propidium iodide staining. The median fluorescence intensity of the gated cells was calculated based on the signal from the FL1 channel. The primary data were exported on OriginPro 9.1 for the calculation of the mean and standard deviation of the triplicate, and plotting the resulting values. P-values were calculated using the one-way ANOVA function on GraphPad Prism 8.0.2. The “no heparin” condition was taken as reference value.

Verification of the cellular uptake by confocal laser scanning microscopy: The cells were seeded in 8-wells Ibidis one day before the experiment. During the last washing step of the NC, the cell culture media was replaced with DMEM without serum. FITC-labelled heparin was used for the pre-coating of the NC. Two hundred microliters of the pre-coated NC were added to the cells to a final concentration of 75 µg/mL, and incubated for 2 h at 37 ˚C in 5 % CO2 atmosphere. Then, the supernatant was removed and the cells were washed three times with PBS. Finally, the cell membrane was stained with Cell Mask Deep Red solution (1:1000 dilution of the stock solution) for 3 min. The cells were washed once before imaging. To ensure minimal cross-talk between the fluorophore, three channels were set up: Argon laser was used to detect the heparin adsorbed on the NC (exc: 488 nm / em: 510-540 nm), DSPP diode was used to detect the nanocarrier (exc: 561 nm / em: 575-590 nm) and HeNe laser was used for the cell mask staining (exc: 633 nm /em: 660-680 nm). The images were acquired using an “in-between line” acquisition protocol using the 63x oil objective of a Leica LSM5 instrument. For the heparin channel, exposure


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was enhanced post-acquisition from 0 to 1.5 to ensure proper visualization of the heparin absorbed on the NCs.

Quantification of the amount of heparin adsorbed on the NC: One milliliter of NC was pre-coated with FITC-heparin and plasma as described above. The samples were dispersed in water and measured as a quadruplet in a Tecan, Infinite M1000 plate reader. In addition, calibration curves for the NC concentration (300 µg -18.75 µg) and FITCheparin concentration (2.0 µg – 0.125 µg) were measured in triplicate. The heparin amount was assessed by fluorescence intensity emitted at 527 nm (exc: 488 nm), and the nanoparticles were detected at 588 nm (exc: 574 nm). The fluorescence measured for the heparin was normalized according to the amount of NC; also, the fluorescence intensity of NC coated only with plasma was subtracted from the intensity of the NC coated with heparin. The normalized fluorescence intensity from the FITC-heparin obtained was plotted against the heparin concentration.

RESULTS 1) Pre-coating strategy and characterization There are three possible ways in which heparin and nanocarrier could come together as schematically described in Fig. 1B: 1) pre-coating of NCs with a mixture of human citrate plasma (hPc) and heparin (hep): Pre(hep+hPc). This would be a patient who has been heparinized before the nanocarrier-based-drug has been administered, 2) Pre-coating of the NCs with plasma followed by coating with heparin is termed accordingly Pre(hPc)Pre(hep) and would represent a patient who first has been given a nanomedicine and was heparinized afterwards. 3) Pre-coating of the NCs with heparin followed by coating with plasma is termed Pre(hep)Pre(hPc). This condition


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corresponds to an unwanted clinical scenario in which heparin and NC would come together in an infusion line before entering the human body. Nevertheless, it is important for a basic understanding of the heparin-protein interaction to include this case. In order to verify the successful adsorption of heparin on the NCs, we measured the surface charge of heparin pre-coated NCs. The amino functionalized PS NCs were originally positively charged (+36 mV). Upon heparin addition, the surface charge slowly decreased to +28 mV at 1.0 IU/mL, and finally to -26 mV at 4.0 IU/mL (Fig. 1C, Table S2), showing a dose-dependent response. In the presence of plasma, a constant surface charge around -20 mV was measured even in the presence of heparin. Thus, another technique was used to estimate the presence of heparin on the NCs surface in the presence of plasma. Using FITC-labelled heparin and Bodipy-labelled NCs, we detected the fluorescence intensity of heparin on the pre-coated NCs. The presence of the Bodipy-fluorophore in the NCs enabled the normalization of the fluorescence intensity of FITC-heparin. Hence, for all conditions, an increase of the fluorescence intensity from around 40-50 a.u. to 75-90 a.u. was noticeable as the heparin concentration increased from 0.5 IU/mL to 2.0 IU/mL (Fig. S1). The original NCs had a narrowly distributed diameter of 110 nm. Upon incubation in plasma, the NCs tended to aggregate as can be seen with multiple angle dynamic light scattering measurements (Table S3). At a 90˚ angle, the diameter of the Pre(hep+hPc) NCs increased to around 400-700 nm (Fig. 1D, Table S4). For Pre(hPc)Pre(hep) coated NCs, a monodisperse sample was obtained with a size distribution centered around 500 nm. Unfortunately, the Pre(hep)Pre(hPc) coated NCs formed, in the presence of heparin, aggregates above one micron. Thus, their potential for reliable cellular uptake across cell type was limited. This also demonstrates that heparin and some nanocarriers should not be administered over the same line of an infusion system.


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Figure 1. Schematic representation of the pre-coating procedure and characterization of the pre-coated nanocarriers. A) Schematic representation of the main repeating units of heparin, for unfractionated heparin, on average, n=18. B) The pre-coating procedure is detailed for the three conditions investigated. C) Surface charge and D) size distribution of the pre-coated nanocarriers (NCs) were measured in a water-based heparin solution (Pre(hep) in water) and in the plasmabased heparin solution described above (Pre(hep)Pre(hPc), Pre(hep+hPc), and Pre(hPc)Pre(hep)).

2) Cellular uptake for hPc pre-coated NCs The uptake of fluorescently labelled NCs was quantified by measuring the median fluorescence intensity of each cell by flow cytometry. After a two-hour incubation of the pre-coated NCs at 37˚C, we observed distinct results depending on the cell line, with heparin inducing a reduced


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uptake, an enhanced uptake, or no change in the uptake. For HeLa cells, we observed a five-fold decrease of the median fluorescence intensity (MFI) from 170 a.u. in the absence of heparin to 25 a.u. in its presence (Fig. 2A). The effect was observed for all pre-coating conditions and even at the lowest heparin concentration tested of 0.25 IU/mL. This trend was confirmed by confocal microscopy. In the absence of heparin, some NCs were observed within the cells, yet in the presence of 1.0 IU/mL heparin, no NCs were observed inside the cells (Fig. 2B, Fig. S2). In addition, the effect was confirmed by microscopy for all three pre-coating procedure (Fig. S3, S4, and S5). For RAW 264.7 macrophages, the opposite effect was observed with an increase of uptake in the presence of heparin for the concomitant pre-coating, Pre(hep+hPc). In this case, in the absence of heparin a MFI around 1300 a.u. is measured, increasing to 1600 a.u. at 1.0 IU/mL of heparin (Fig. 2C). Interestingly, the effect is not significant when the NCs encountered heparin either before (Pre(hep)Pre(hPc) or after (Pre(hPc)Pre(hep)) the plasma addition. Furthermore, NCs internalization was confirmed, by confocal imaging, with the presence of multiple NCs inside the cells (Fig. 2D, Fig. S6). The presence of heparin in the corona could be also confirmed (Fig. S7, S8, S9).


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Figure 2. Determination of the cellular uptake of pre-coated NH2-PS NCs with heparin and plasma in (A+B) HeLa cells and (C+D) RAW 264.7 macrophages. The cells were incubated for 2 hours with the pre-coated NCs in serum-free media. The extent of the cellular uptake was quantified by flow cytometry (A+C). The error bars represent the standard deviation of a biological triplicate. p-values 0.01 were not significant and thus not reported.

3) Cellular uptake based on serum pre-coating


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Besides human plasma, human (hS) and fetal bovine (FBS) sera were used to evaluate the universality of the effect. Upon pre-coating of the NCs with human serum (Pre(hep+hS), the same inhibitory effect of heparin was observed for HeLa and primary monocytes (Fig. S13). The uptake was significantly (p < 0.001) reduced in the presence of heparin in the corona. However, when these NCs were incubated with RAW 264.7, THP-1, THP-1 differentiated, and primary macrophages no significant change of uptake was observed as the heparin concentration increased. This confirms that there is a strong inhibitory effect for HeLa and primary monocytes. Pre-coating with fetal bovine serum (Pre(hep+FBS)) was also tested, albeit with lower amounts of heparin. A dose-dependent response to heparin was observed for HeLa cells, with a reduction of the uptake starting from 0.16 IU/mL and reaching its maximal inhibitory effect at 0.62 IU/mL of heparin. No change of uptake was observed with THP-1 cells. For RAW 264.7 and THP-1 differentiated, a steadily increasing uptake was observed, which led to a doubling of the MFI at 2.5 IU/mL for Pre(hep+FBS)M(FBS) (Fig. S14). Thus, we can conclude, that the effect of heparin was strengthened in the presence of fetal bovine serum compared to human serum.

4) Protein corona composition Proteomics analysis was conducted to determine whether the addition of heparin changes the composition of the corona. On polyacrylamide gels, we can observe that the same pattern was obtained for different amounts of heparin within the same pre-coating condition with an intense band at 62 kDa indicating a high amount of albumin present in the protein corona (Fig. 4A, and Fig. S15). The addition of heparin before plasma led to a more complex protein corona with a higher abundance of heavy protein, larger than albumin (> 62 kDa). The protein pattern obtained


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for Pre(hep+hPc) and Pre(hPc)Pre(hep) was similar with most visible bands located below the albumin band. A more precise analysis of the protein corona by mass spectrometry, led to the same findings. The top 25 most abundant proteins were identical in the absence or presence of 1.0 IU/mL of heparin for all three conditions. Serum albumin represented about 40 % of the total amount of protein found in the corona; for Pre(hPc)pre(hep) the relative abundance of albumin appears to be higher (around 50 %). The abundance of some proteins differed depending on the timing of the heparin addition (Fig. 4B). The introduction of heparin after the plasma led to a two-fold enrichment in vitronectin counter-balanced by a two-fold depletion in fibrinogen for Pre(hPc)Pre(hep) (Fig. S16, and Table S5). As expected, antithrombin III (AT III) was highly enriched for Pre(hep+hPc) with a four-fold change, and for Pre(hep)Pre(hPc) with a six-fold change, meanwhile only a 1.5-fold change was measured for Pre(hPc)Pre(hep). On the other hand, hyaluronan-binding-protein 2 stood out by its important depletion in the different coronas. A matching trend to AT III, was observed for Pre(hep+hPc) and Pre(hep)Pre(hPc) with depletion of up to 80%, compared to 5% for Pre(hPc)Pre(hep). Small changes (under 1.2-fold) could be explained by different washing steps for the Pre(hPc)Pre(hep) condition compared to Pre(hep)Pre(hPc) rather than the presence of heparin. This is the case for immunoglobulins with a depletion observed for the Pre(hPc)Pre(hep) compared to the Pre(hep+hPc) condition. For more marked alterations such as antithrombin, vitronectin and HABP-2, heparin was most likely responsible for the changes.


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Figure 4. Characterization of the protein corona formed in three conditions in the presence and absence of heparin. (A) The proteins were recovered from the NCs and run in denaturing conditions in 10% SDS-Tris polyacrylamide gel. The gel was stained with SimplyBlue Safe Staining. M = marker, hPc = human citrate plasma. (B) The protein mixture was analyzed by LCMS. The 25 most abundant proteins and their relative percent in the total corona were summarized in the heatmap.

DISCUSSION The presence of heparin was determined by two methods. First, we took advantage of the negative charge of heparin to measure the decrease of the ζ-potential of the otherwise positively charged NCs. However, regardless of the original surface charge, plasma pre-coated NCs had generally a surface charge around -20 mV. Thus, we used FITC-labelled heparin and measured the


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fluorescence intensity of the pre-coated NCs. The fluorescence intensity increased in a dosedependent manner after the addition of heparin. In addition, through CLSM, we could confirm the co-localization of heparin on the NCs even after internalization by the cells. Rausch et al. demonstrated that NCs tend to aggregate in plasma,27 yet the formation of micron size aggregates in the case of Pre(hep)Pre(hPc) led to their exclusion from the primary cell study. Macrophages can take NCs up to several microns but the mechanism of uptake would be unspecific.19, 28 Otherwise, the size of all the NCs increased during the coating procedure, leading to hydrodynamic radii between 200 nm to 350 nm. This uniform increase of the size across the heparin concentration range could not explain the difference in cellular uptake observed. Indeed, 100 % of Pre(hPc)Pre(hep) NCs and over 80 % of Pre(hep+hPc) NCs accounted for these larger particles (Table S4). Upon cellular uptake, various effects were observed depending on the cell tested and the precoating conditions. HeLa (cervix adenocarcinoma) and primary monocytes were highly sensitive to heparin. The addition of as little as 0.25 IU/mL of heparin, well below the clinical level (1.0 IU/mL), was enough to prevent the uptake of the pre-coated NCs. For these cells, the timing of the heparin addition did not influence the uptake. THP-1 a monocyte-like cell line had a low uptake ability, which remained unchanged in the presence of heparin for both conditions tested. Two human macrophage cells were generated, one from THP-1 cells and the other from primary monocytes. Both macrophages cell types had high uptake ability; however, the impact of heparin was limited for both pre-coating conditions. It is important to note that the differentiation process is stressful for the cells and could explain the lack of clear response towards heparin. When heparin was mixed with FBS, macrophages derived from THP-1 cells showed a higher sensibility to heparin leading to an enhanced uptake of the pre-coated NCs, which could be explain by a more


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favorable environment since they were differentiated in FBS supplemented media. Finally, RAW 264.7 cells were also tested and heparin present during the corona formation led to an enhanced uptake; while its addition before (Pre(hep)Pre(hPc)) and after (Pre(hPc)Pre(hep)) the corona formation did not change the uptake extend. This effect was also observed with human serum and FBS, but with a twist: the presence of heparin initially induced a decrease of the uptake followed by an increase as the heparin concentration increased. In short, the mixed results obtained when testing the NCs with different protein sources (citrate plasma, human, and bovine serum) demonstrate once more29 the importance of careful consideration and proper documentation when carrying cellular uptake experiments. The peculiar uptake pattern observed is expected to be due to the presence of heparin itself in the corona. This hypothesis was supported by proteomics. Polyacrylamide gel electrophoresis revealed an identical protein pattern through the different coating conditions and heparin concentrations. The addition of plasma on heparin pre-coated NCs induced an apparent higher adsorption of heavy proteins. Yet, the composition of the corona remained unchanged with 99% overlap between the three conditions and 86% overlap between the presence and absence of heparin. Overall, very little changes were observed for the Pre(hPc)Pre(hep) corona, as expected since heparin was added after a stable hard corona was formed. More pronounced changes were observed for Pre(hep+hPc) and Pre(hep)Pre(hPc) coronas. In both cases, antithrombin III, a known highly specific ligand to heparin,30 alongside with HABP-2, which is relatively abundant but less specific were highly enriched. One could argue that a competition between both aforementioned proteins take place for binding to heparin. Apolipoproteins were also impacted by the presence of heparin, yet their enrichment or depletion was variable depending on the pre-coating condition, expect for clusterin which was universally depleted.


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As we have shown by proteomics, surface charge, and size distribution, Pre(hep+hPc) and Pre(hPc)Pre(hep) NCs are similar. It also appeared that the same amount of heparin absorbed per NCs for the same concentration in all three conditions. Thus, all these characteristics being identical, we are suggesting that the orientation or accessibility of the heparin chains must differ between the three conditions. Indeed, with heparin being a bulky molecule (18 kDa), electrostatically attracted to the positively charged NCs, the adsorption should be dependent on its surrounding. The kinetic of heparin adsorption should be different for Pre(hep+hPc) when heparin is surrounded by plasma compared to Pre(hPc)Pre(hep) when heparin has to interact with proteins to reach the NC’s surface in a buffered environment. The existence of a cellular receptor dedicated to clearance of heparin from the blood stream, such as hyaluronan receptor for endocytosis (HARE),31,32 is of particular interest. Macrophages, presenting such a receptor, would be sensitive to the orientation of heparin on the NCs, explaining the increased uptake of Pre(hep+hPc) NCs compared to the constant uptake of Pre(hep)Pre(hPc) and Pre(hPc)Pre(hep) NCs. The lack of such receptor on HeLa cells would point out towards a direct inhibition by the heparin molecule of the uptake, which is true for all pre-coating conditions. In addition to proteomics, lipidomics and carbohydrate analysis would be needed to have a full understanding of the biomolecular corona. Future tandem investigations of expression level of heparin related cell receptors for HeLa and RAW 264.7 cells, and heparin orientation on the NCs could explain the uptake pattern. This could also lead to a better exploitation of the natural stealth property of heparin.

CONCLUSION


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We have demonstrated that the timing of the introduction of heparin into the biomolecular corona plays an important role in the cellular uptake of NCs. Indeed, when NCs are coated with a heparin-plasma mixture, HeLa and primary monocytes internalized dramatically less NCs, meanwhile RAW 264.7 and primary macrophages internalized more NCs. When heparin is added after protein corona formation, the inhibitory effect of heparin is maintained while the behavior of RAW 264.7 and primary macrophages has remained unaffected. The effect observed for each cell type is enhanced with higher heparin amount. Proteomics analysis of the corona led to the identification of identical proteins, yet some such as antithrombin III, hyaluronan-binding protein 2 or fibrinogen were strongly affected by the presence of heparin and the timing of its addition. According to these in vitro studies, a more reliable and consistent uptake of NCs would be achieved by delaying heparin injections until a stable endogenous corona is obtained.

ASSOCIATED CONTENT Supporting Information. Supporting graphs and tables (PDF) can be found online. The proteins identified are summarized in an excel file which is also available online. AUTHOR INFORMATION Corresponding Author Correspondence should be addressed to Prof. Dr. V. Mailänder ([email protected]). Author Contributions


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CC and JS performed the experiments, CC and VM designed the experiments. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by the Deutsche Forschungsgemeinschaft (DFG) for the Collaborative Research Centre 1066 “Nano-Dimensional Polymer Therapeutics for Tumor Therapy”. ACKNOWLEDGMENT The authors are grateful for the synthesis of the polystyrene nanocarriers by Katja Klein. The authors thank Christine Rosenauer for the multiple angle dynamic light scattering measurement. ABBREVIATIONS NC, nanocarrier; hep, heparin; hPc, human citrate plasma; hS, human serum; FBS, fetal bovine serum; HABP-2, hyaluronic acid binding protein-2; AT III, antithrombin III; MFI, median fluorescence intensity; a.u., arbitrary units; CLSM, confocal laser scanning microscope. REFERENCES 1 – Smythe, M.A.; Priziola, J.; Dobesh, P.P.; Wirth, D., Cuker, A.; Wittkowsky, A.K. Guidance for the practical management of the heparin anticoagulants in the treatment of venous thromboembolism. J. Thromb. Thrombolysis, 2016, 41, 165-186. 2 - Capila, I. and Lindhardt, R.J. Heparin-protein interactions. Angew. Chem. Int. Ed., 2002, 41, 390-412. 3 – Holzapfel, V.; Musyanovych, A.; Landfester, K.; Ricarda Lorenz, M.; Mailänder, V. Preparation of Fluorescent Carboxyl and Amino Functionalized Polystyrene Particles by


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29 – Schöttler, S.; Klein, K.; Landfester, K.; Mailänder, V. Protein source and choice of anticoagulant decisively affect nanoparticle protein corona and cellular uptake. Nanoscale, 2016, 8, 5526-5536. 30 – Petitou, M.; Casu, B.; Lindhal, U. 1976-1983, a critical period in the history of heparin: the discovery of the antithrombin binding site. Biochimie, 2003, 85, 83-89. 31 – Zhou, B.; Weigel, J.A.; Fauss, L.; Weigel, P.H. Identification of the hyaluronan receptor for endocytosis (HARE). J. Biol. Chem., 2000, 275, 37733-37741. 32 – Harris, E.N.; Weigel, J.A.; Weigel, P.H. The human hyaluronan receptor for endocytosis (HARE/Stabilin-2) is a systemic clearance receptor for heparin. J. Biol. Chem., 2008, 283, 1734117350.

For Table of Contents Only.


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Timing of Heparin Addition to the Biomolecular Corona Influences the

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