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Mode of PEG Coverage on Carbon Nanotubes Affects Binding of Innate Immune Protein C1q Agathe Belime, Edmond Gravel, Sophie Brenet, Sarah Ancelet, Charlotte Caneiro, Yanxia Hou, Nicole Thielens, Eric Doris, and Wai Li Ling J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b06596 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 20, 2017

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The Journal of Physical Chemistry

Mode of PEG Coverage on Carbon Nanotubes Affects Binding of Innate Immune Protein C1q

Agathe Belimea, Edmond Gravela, Sophie Brenetc, Sarah Anceletb, Charlotte Caneirob, Yanxia Houc, Nicole Thielensb, Eric Dorisa, Wai Li Lingb*

a

Service de Chimie Bioorganique et de Marquage (SCBM), CEA, Université Paris-Saclay,

91191 Gif-sur-Yvette, France. b

Univ. Grenoble Alpes, CEA, CNRS, IBS, F-38000 Grenoble, France.

c

Univ. Grenoble Alpes, CNRS, CEA, INAC, SyMMES, F-38000 Grenoble, France.

* Corresponding author: [email protected]

ACS Paragon Plus Environment


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ABSTRACT

Surface modification of nanoparticles with poly(ethylene glycol) PEG is used in biomedicines to increase the circulation time of the particles after intravenous injection. Here, we study the interaction of PEG-covered carbon nanotubes (CNTs) with the serum complement protein C1q. Besides being the target-recognizing unit of the initiating complex for the classical pathway of complement in our innate immune system, C1q is involved in a range of important physiological processes. We modified the surface of multi-walled CNTs with covalently grafted PEG and physically adsorbed PEG. Transmission electron microscopy revealed the interaction of these PEG-coated CNTs with C1q. We found abundant C1q coverage on the PEG-grafted CNTs but not on the CNTs with adsorbed PEG. We tested the ability of these CNTs to activate the complement system using in vitro complement activation assays. None of the CNTs studied activated the C1q-dependent classical complement pathway. These findings are pertinent to the safe design and novel biomedical applications of PEGylated CNTs.


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INTRODUCTION

Carbon nanotubes (CNTs) have shown promises in a number of therapeutic and diagnostic applications.1,2 Due to their specific chemical and physical properties, CNTs are especially suitable for biomedical imaging and innovative cancer therapy.3–5 Recent results have also demonstrated the potential of CNTs to cross the blood-brain barrier to deliver drugs to the brain.6 Many of these applications are designed to be injected intravenously because systemic administration remains the most efficient way for drug delivery and is generally employed for scarce therapeutics. In order to reach their targets, therapeutics that are delivered through systemic administration need to circulate in the bloodstream without being detected by the mononuclear phagocytic system (MPS). Tagging, or opsonization, by serum immune proteins in the MPS recruits phagocytes, which promptly remove the opsonized targets from circulation. Without protection, CNTs are rapidly cleared from systemic blood circulation.7

It has been

demonstrated that particles coated with poly(ethylene glycol) (PEG) can successfully evade the MPS.8 Decoration of nanoparticle surface by PEG (PEGylation) through covalent grafting or physical adsorption blocks the binding of opsonins in the blood, thus rendering the particles stealth to the MPS. Besides the MPS, proteins belonging to the complement system also patrol the serum. The complement system is part of our innate immune system, which launches an immediate response against intruders to our body.9

Complement activation prompts cytokine and

chemokine secretion, causing inflammation.

The complement cascade terminates in the

production of a cell-killing membrane attack complex (MAC). The MAC is meant to target bacteria but also causes collateral damage to host cells. Inappropriate activation of the complement system can be grave and even life-threatening.10 Indeed, complement has been shown to be involved in the acute hypersensitivity reactions against PEGylated liposomal doxorubicin (Doxil®) approved for cancer chemotherapy.11 The complement system is activated through three pathways, namely, the classical, lectin, and alternative pathways. Distinct PEG configurations on nanoparticles have been shown to switch activation pathways between classical and lectin.12 Both of these pathways are activated by pattern-recognition complexes that identify invaders to the body. The lectin pathway is initiated by lectins (mannose-binding lectin and ficolins) that bind sugar and carbohydrate moieties commonly found in pathogens.13 The classical pathway, on the other hand, is initiated by the C1 complex. The C1 complex consists of C1q and a hetero-tetramer C1r2C1s2. The



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subunit C1q is the target-recognizing unit, which senses targets through their surface charge motifs.14 Upon specific binding, C1q activates the proenzyme C1r, which in turn activates C1s to initiate the classical complement activation pathway. The protein C1q has been shown to bind tightly to various CNTs with or without activating the complement system.15–17 Besides, CNTs pre-coated with C1q have been shown to down-regulate inflammation and enhance clearance by phagocytes.18 Apart from complement activation, C1q is involved in a range of other important physiological functions.19,20

C1q can bind pathogens directly or indirectly and activate

complement in innate immunity.21–23 It is also crucial in adaptive immunity as it triggers complement activation by recognizing antigen-bound antibodies and is involved in modulating the adaptive immune response.24,25 Besides, C1q is important in many physiological processes, including apoptotic cell clearance, autoimmunity, pregnancy, and cancer.26–29 Notably, C1q is involved in normal and pathological processes in the brain, such as neural plasticity, ischemia, and Alzheimer’s disease.30–33 Understanding the binding of C1q onto nanoparticles is therefore highly relevant in safety as well as in the development of biomedical applications. Whereas PEG-coated CNTs have been studied with respect to their complement activities, C1q binding properties have not been investigated.34–36 Here, using transmission electron microscopy (TEM), we compare the C1q binding properties of multi-walled CNTs with different PEG coatings. Surface plasmon resonance imaging experiments have also been carried out to probe the binding affinity. The implementation of PEG chains at the surface of CNTs was carried out through two different strategies: i) covalent binding of amine-terminated PEG through peptide coupling with surface carboxylic groups, yielding partial PEG coverage of the CNT surface, and ii) supramolecular assembly of PEG-terminated amphiphiles allowing extensive coverage of the CNT surface with PEG chains. In the latter strategy, the obtained assemblies were stabilized by photopolymerization through diacetylene groups incorporated in the molecular struture of the amphiphiles. The results were compared with those obtained with pristine CNTs and CNTs with surface carboxylic groups. Lastly, we present the complement activation properties of the CNT samples using an in vitro enzyme immunoassay and a C1 activation assay.

EXPERIMENTAL METHODS

CNTs with surface carboxylic groups


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To generate carboxylic acids at open-ends and side-wall defect sites, 50 mg of Nanocyl3150 multi-walled CNTs were stirred in a H2SO4/HNO3 3:1 mixture (10 mL) during 1 h at 60 °C. The reaction mixture was then diluted with water and centrifuged for 10 min at 8000 rpm. The pellet was collected and repeatedly washed with water until neutral pH was reached and the residue was dried under vacuum to yield multi-walled CNT-COOH (~15-20 nm in diameter and ~1-10 µm in length).

PEG-grafted CNTs Multi-walled CNT-COOH was activated by reacting 50 mg of the oxidized CNTs (as prepared above) with SOCl2 (20 mL) and anhydrous dimethylformamide (1 mL) at 70 °C for 24 h. The CNTs were recovered by centrifugation and the pellet was washed with THF. The activated nanotubes (CNT-COCl) were dried under vacuum. They were then mixed with mPEG-NH2 (150 mg, MW = 516 ± 44 g mol-1) and triethylamine (1 mL) in THF (15 mL) and stirred at 60 °C under nitrogen atmosphere for 24 h. The resulting solution was centrifuged and the pellet was repeatedly washed (× 5) with methanol to remove PEG excess. A black residue (PEG-grafted CNTs) was obtained after drying under vacuum.

CNTs fully coated with PEG The preparation of the CNTs fully coated with PEG chains started with the aqueous selfassembly of PEG-based amphiphiles on the CNTs. These amphiphiles, which incorporated a diacetylenic lipophylic chain, were synthesized with PEG as previously described.37 Two types of amphiphiles (that differ only by PEG size) were studied. The first one incorporated a PEG chain (PEG550) similar to the one used for PEG-grafted CNTs, and the second one incorporated a longer PEG (PEG2000). Upon interaction with the amphiphiles, a stable suspension was produced. While the hydrophobic portion of the amphiphile was adsorbed onto the CNT surface, its hydrophilic PEG head was oriented toward the aqueous phase. To promote further stability of the PEG coating, the diyne motif incorporated in the lipophylic chain was photopolymerized by ultraviolet irradiation at 254 nm, which reinforced cohesion of the assembly.38 The PEG-coated CNTs were finally recovered by centrifugation.

Characterizations of CNTs Thermogravimetric analysis (TGA) experiments were performed with Q50 (TA instrument) under N2 flow at a heating rate of 10 °C min-1.



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X-ray photoelectron spectroscopy (XPS) analyses were performed with a Kratos Axis Ultra DLD using a high-resolution monochromatic Al-Kα line X-ray source at 1486.6 eV. Fixed analyzer pass energy of 20 eV was used for core level scans. Survey spectra were captured at pass energy of 160 eV. The photoelectron take-off angle was always normal to the surface, which provided an integrated sampling depth of approximately 15 nm. All spectra were referenced with an external gold substrate with a binding energy of 84.0 eV for Au 4f.

Purification of protein C1q and its globular region Details of the reagents and proteins were described previously.39–41 The C1q recognition subunit of C1 was purified from human plasma. The globular region (GR) of human C1q was generated essentially as described by Tacnet et al.39 Briefly, C1q was treated with collagenase (100 units collagenase from Clostridium histolyticum type III (Sigma) per mg of C1q) for 16 h at 37 ºC, and purification was achieved by high-pressure gel permeation on a TSK-G3000 SW column (Tosoh Bioscience) equilibrated in 50 mM Tris-HCl, 250 mM NaCl at pH 7.4. The proteins were dialyzed in Tris-Buffered Saline (TBS) (Euromedex). Concentrations of the purified proteins were determined as previously described42 and were around 0.8 mg mL1

.

The homogeneity of the purified proteins was assessed by sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE) analysis under reducing and non-reducing conditions and by TEM.

Transmission electron microscopy For TEM observation, CNT samples (~ 0.3 mg mL-1) and C1q (~ 0.4 mg mL-1) or GR (~ 0.6 mg mL-1) were mixed in equal volume (~ 6 µL) and incubated at room temperature or at 37 ºC with mild agitation for ~ 5 mins. To remove excess proteins, the CNTs were centrifuged at 6000 rpm for ~ 1 min and resuspended in TBS. Around 4 µL of the solution was then applied to the edge of a mica sheet covered with a film of evaporated carbon. The carbon film was subsequently floated off the mica in 2% sodium silicotungstate (SST) and retrieved onto a 400mesh copper electron microscopy grid. Imaging was performed on an FEI T12 microscope at 120 kV or an FEI F20 microscope at 200 kV. Images were recorded on an Orius 832 CCD camera (T12), an Eagle digital camera (F20), or a One View camera (F20).

Surface plasmon resonance imaging Analyses of CNTs were performed by SPR imaging (SPRi). The SPRi apparatus (Horiba Scientific) was placed in a temperature regulated incubator (Memmert, Germany) at 25 °C. It


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was coupled to a microfluidic system, including a computer controlled syringe pump (Cavro XLP 6000, Cavro scientific instruments, USA), a degassing system (Alltech, France), a PEEK flow cell with hexagonal configuration with the volume of 10 µL, and a 6-port medium pressure injection valve (Upchurch Scientific, USA). A protein chip was prepared using a prism covered with a thin gold layer, which was purchased from GenOptics (Horiba Scientific, Orsay, France). Before use, the prism was cleaned with a Femto plasma cleaner (Diener Electronic, Germany) under these conditions: 0.6 mbar, 75% Oxygen, 25% Argon, power 40 W, 3 min. It was functionalized with 1 mM 12-Mercaptododecanoic acid NHS ester for 18 h at room temperature by the formation of self-assembled monolayers (SAMs). Then, protein solutions of C1q (0.2 µM) and human serum albumin (HSA) (0.2 µM) were deposited on the prism in form of drops in quintuplicate.

These solutions were left for 2h at room temperature for the protein

immobilization.

Finally, the rest of the active SAMs surface was blocked with 1 M

ethanolamine for 1 h, followed with a thorough wash. Binding of the CNTs to immobilized C1q and HSA was measured by SPRi at a fixed work angle (50.3°) and at a flow rate of 10 μL min-1 in TBS, 0.005% Tween20, pH 7.4. Two concentrations (10 µg mL-1 and 20 µg mL-1) were used for each CNT sample in this study (CNT, CNT-COOH, PEG-grafted CNT, and CNTs fully coated with PEG). Before injection, all solutions of CNTs were sonicated in a water bath for 1 h to avoid agglomeration of CNTs. After each analysis, the protein chip was regenerated with 10 mM NaOH.

Complement activation assay The ability of CNTs to trigger complement activation was measured using the Wieslab® complement system screen COMPL300 (Euro Diagnostica), an enzyme immunoassay based on the detection of a neoantigen expressed during the MAC formation. The CNT solutions were diluted in TBS at a ratio of 6:1 (v/v) to yield a final concentration of 380 µg/ml. The diluted solution was then incubated with normal human serum for 45 min at 37 °C with occasional shaking. The residual functional activities of the classical pathway, the MBL-dependent lectin pathway, and the alternative pathway were measured according to the kit instructions. IgGovalbumin aggregates (immune complexes, IC) were prepared as described previously43 and used as a positive control for the activation of the classical complement pathway (final concentration 1 mg mL-1). Zymosan (1 mg mL-1) was used as a positive control for the activation of both the lectin and alternative pathways. The control incubations for complement activation contained only the buffer TBS and define the reference (100%) complement activity of the serum. Each assay was performed in duplicate during three independent experiments.



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The means of the multiple measurements are presented and the error bars represent the standard deviations of the measurements.

C1 activation assay The in vitro C1 activation assay used in this work has been described previously.44 Briefly, the C1 complex was reconstituted from C1q and proenzyme C1r2C1s2 purified from normal human serum. The complex was incubated in 50 mM triethanolamine-HCL, 145 mM NaCl, 2 mM CaCl2 at pH 7.4 for 90 min at 37 ºC in the presence of 1 µM C1 inhibitor (C1inh) and various CNT samples. The extent of activation was measured from the amounts of the cleavage products of activated C1s generated, following Western blot analysis using an anti-C1s antibody.

RESULTS AND DISCUSSION

The surface functionalization of the CNTs was characterized by TGA and XPS. Thermogravimetric analysis was performed on the samples of CNT-COOH and the PEG550grafted CNT. The results are shown in Figure 1. The data acquired in TGA provide information on the level of functional groups grafted onto the surface of the CNTs. The PEG550-grafted CNTs (blue curve in Figure 1) undergo a significant weight loss (ca. 15%) at around 350 °C, which corresponds to the degradation temperature of the PEG chain (green curve). The oxidized CNTs (red curve) do not show this behavior, further confirming that the weight loss of PEG550-grafted CNTs is due to the grafting of a PEG moiety. The CNTs were also characterized using XPS. As seen in Table 1, the significant increase of the oxygen content after oxidation of the CNTs reflects the introduction of carboxylic acids at the surface. Upon conjugation of the PEG-NH2 unit, the oxygen content further increases (+ 2.3%) and nitrogen is present in the sample (+ 1.0 %). These XPS results are in good agreement with the weight loss observed by TGA.


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Figure 1.

Thermogravimetric analysis (TGA) of functionalized carbon nanotubes

(CNTs). The TGA signal obtained with pristine CNTs (black curve) is given as a reference. The oxidized CNTs (CNT-COOH, red curve) show no significant weight loss up to 600 °C. The CNTs grafted with poly(ethylene glycol) (CNT-PEG550, blue curve), on the other hand, lost ~15% of their weight at 350 °C. This temperature corresponds to the degeneration temperature of PEG (green curve), confirming the presence of PEG on the surface of the PEGgrafted CNTs.

Table 1. Contents (%) of carbon (C), oxygen (O), and nitrogen (N) in samples of CNT, CNTCOOH, and PEG-grafted CNT based on the X-ray photoelectron spectroscopy analysis (see Figure S1-S3 in Supporting Information). The increase in O content in CNT-COOH confirms the presence of carboxylic groups on the CNT surface. Further increase in O content and the detection of N confirms the presence of PEG on PEG-grafted CNTs. C 1s

O 1s

N 1s

CNT

98.3

1.7

-

CNT-COOH

93.4

6.6

-

PEG-grafted CNT

90.1

8.9

1.0



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Carbon nanotubes fully coated with PEG were prepared by self-assembly of PEG chains with a diyne motif. The self-assembled PEG coatings were photopolymerized via the diyne motif to enhance their stability. Figure 2 shows TEM images of the CNTs incubated with the protein C1q with mild agitation. Structurally, C1q resembles a bouquet with a collagen stem that branches out into six flexible arms, each ending in a globular head domain (GR) (see Figure 2c). Our previous results have shown that C1q binds CNTs through its GR.16,17 Similar binding is observed for CNT-COOH and PEG-grafted CNT, as shown by the fully covered CNTs in Figure 2a and Figure 2b, which are representative in these samples. Incubation at room temperature and 37 °C yielded the same results. The interaction between the CNTs and C1q molecules is illustrated in Figure 2c. Interestingly, we occasionally observed uncovered CNTs next to CNTs fully covered by C1q molecules in the resuspended samples (see Figure S3 in Supporting Information. Our previous finding that C1q molecules organize on straight CNTs suggests that C1q binding may be stabilized by the interaction among bound molecules, which might have been the reason in this occasion where CNTs compete for C1q molecules in dynamic equilibrium after the removal of excess protein.16 Nevertheless, even though unlikely, we cannot exclude the possibility that the uncovered CNT has a different surface chemistry.) We have also imaged CNT-COOH and PEG-grafted CNT incubated with isolated GR and found the CNTs fully covered by GR (not shown).


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Figure 2. Transmission electron micrographs of CNTs incubated with C1q. Molecules of C1q are found attached to the surface of (a) acid treated CNTs (CNT-COOH) and (b) PEG550grafted CNTs. Some C1q molecules, assumedly detached from the CNTs are found in the background. (c) Schematic diagrams (not-to-scale) of C1q (depicted in grey) binding onto CNTs (thick black lines). A lone C1q molecule is also shown. (d) Carbon nanotubes fully coated with PEG550 incubated with C1q shows rough sidewalls from the supramolecular assembly of PEG but no C1q molecules on the surface, which would be recognized by their protruding collagen stems. (e) Aggregates that are quasi-organized found occasionally in sample of C1q mixed with CNTs fully coated with PEG550. Unbound proteins have been removed by centrifugation and re-suspension in buffer for all samples.



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On the other hand, no C1q attachment was found on the surface of the CNTs fully coated with PEG (representative image in Figure 2d). Nonetheless, we occasionally found aggregates among the CNTs (example shown in Figure 2e). Such aggregates were also found for GR incubated with these CNTs. On the contrary, they were not observed in the case of C1q or GR with the pristine or covalently functionalized CNTs, nor the case of CNTs coated with PEG alone. Notably, PEG is a common reagent in protein crystallization.45 Even though the PEGterminated molecules were photo-polymerized on the surface of the CNTs here, some may still have the degree of freedom to interact with protein molecules in a way that brings about their assemblage. We have also probed the interaction between the CNTs and C1q using SPRi. Human serum albumin was used as a reference for non-specific binding. The proteins (C1q and HSA) were immobilized onto a prism functionalized with SAMs. Areas of SAMs not covered by proteins were passivated with ethanolamine. The different samples of CNTs at concentrations 10 µg mL-1 and 20 µg mL-1 were dispersed by sonication (all CNT samples except pristine CNTs were well dispersed) and passed through the prism surface at a slow flow rate of 10 µL min-1. Only very weak signal was detected for all the CNT samples (CNT, CNT-COOH, PEG550-grafted CNT, and CNT fully coated with PEG), with comparable signals for C1q, HSA, and the passivated SAMs (see Figure S5 in Supporting Information). In other words, no strong specific binding was observed for any of the CNT samples with C1q. We speculate that the apparent inconsistency between the SPRi results and the TEM results is due to the inherent configuration of the SPRi experiment. As immobilizing the CNTs, especially the PEG coated ones, would require altering the CNT surface, we were limited to immobilizing the proteins in our experiments. The CNTs will only have tangential contact around its circumference with the flat protein covered chip. Sporadic binding of C1q along its length is likely not enough to retain the CNTs even at a slow flow rate. We next examined the serum complement activation capacities of the different CNTs. Full activation of the complement cascade was detected by labeled antibodies against a neo-antigen generated from the formation of its terminal product MAC. For completeness, we have tested the complement activities of the CNTs through all the three pathways of activation (classical, MBL-dependent lectin, and alternative). We incubated the different CNT samples with normal human serum to allow the CNTs to react with complement. The mixture was then diluted with a solution containing specific blockers that inhibit all but one activation pathway and subsequently incubated with known activators specific to the pathway under investigation. We


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then measured the quantity of antibodies bound to MAC, which corresponded to the level of residual complement activation through the investigated pathway.

Figure 3. Serum complement activation assays of CNTs. Normal human serum (NHS) was incubated for 45 min at 37°C with the CNT solutions (0.4 mg mL-1) and the residual functional activity of the classical pathway (black), the MBL-dependent lectin pathway (white), and the alternative pathway (grey) of complement was measured. IgG-ovalbumin immune complexes (IC) and Zymosan (1 mg mL-1) were used as positive controls for activation of the classical and lectin/alternative pathways, respectively. Measurement of control NHS complement activity was performed in the presence of buffer instead of CNTs. Results are expressed in percentage (%) of NHS activity (mean +/- standard deviation of three independent experiments performed in duplicate). Comparisons between control NHS activity and residual activity following incubation with IC, Zymosan or the different CNTs were made using the raw absorbance values in a paired student t test; two-tailed p values < 0.05 are considered significant. *p < 0.05, **p < 0.005.

Results are shown in Figure 3. Within experimental uncertainties, none of the CNTs activated the classical or the lectin pathways at the concentration tested. Pristine CNT and PEG550-grafted CNT did not activate the alternative pathway but slight activity was detected for CNT-COOH. Interestingly, CNTs fully covered with PEG550 had appreciable activity via the alternative pathway. Activation of the alternative complement pathway by PEGylated CNTs has not been reported in the previous studies concerning PEGylated CNTs but these



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studies have only involved single-walled CNTs with PEG of molecular weights over 1000 and thus are different from the system here in discussion.34,35 It has been reported that surface PEG chain of molecular weight of 2000 or greater have better MPS-avoidance characteristics.8 Shorter chains are thought to be less flexible, especially in a high-coverage configuration.46 To investigate the idea whether CNTs coated with PEG2000 may be inert to the alternative complement pathway, we prepared CNTs fully coated with PEG2000 in the same manner as with PEG550. As shown in Figure 3, these CNTs did not trigger complement activation. We examined these CNTs fully coated with PEG2000 for C1q binding by TEM and SPRi and no binding was observed as is in the case of PEG550.

Figure 4. C1 activation properties of CNTs. The ability of the various CNT species at 0.5 mg mL-1 to activate the C1 complex in the presence of C1 inhibitor (C1inh) was tested in an in vitro assay. A positive control (in the absence of C1inh and CNTs) and a negative control (in the presence of C1inh and absence of CNTs) are shown. None of the CNT samples activated the C1 complex.

Single-walled CNTs have been found to activate complement but fail to complete the complement cascade.35 We thus further tested whether the classical pathway was initiated upon the C1q binding observed. We also included the CNTs fully coated with PEG to test whether the aggregates as the ones shown in Figure 2e would activate C1. The CNT samples were incubated with the C1 complex in the presence of C1inh. Binding of C1q to targets that activate the classical pathway of complement would impart a conformational change on the C1 complex


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triggering the self-activation of C1r. The active enzyme C1r will subsequently convert the zymogen C1s to its active form, which carries on the complement cascade overcoming the control by C1inh. Results presented in Figure 4 show that none of the CNT samples activated the C1 complex, as observed in our previous study using non-functionalized CNTs.16

CONCLUSIONS

We observed that different modes of PEG functionalization on CNTs gave rise to different binding properties of the complement recognition protein C1q. The protein bound to the PEGgrafted CNTs but not to the CNTs fully coated with PEG. As C1q also bound readily to pristine CNT and CNT-COOH samples, the binding to PEG-grafted CNTs might reflect the incomplete coverage of the CNT surface. Whereas no protein binding was observed on the surface of CNTs fully coated with PEG, quasi-organized aggregates were found occasionally in the CNTs mixed with C1q or GR. Despite the different C1q binding properties, none of the CNT samples we tested activated the classical pathway of complement, in which C1q is involved. This lack of activity in the C1q-binding CNTs indicates that C1q does not bind to these samples in the specific configuration that triggers the activation of the proenzymes C1r and C1s, which are associated with C1q in the C1 complex. Comparing two different lengths of adsorbed PEG on fully covered CNTs gave the same results regarding C1q binding but yielded different results in complement activation. Similar to the pristine CNT, CNT-COOH, and PEG550-grafted CNT, PEG2000-coated CNT is inert to the complement. However, slight activity via the lectin pathway and clear activity via the alternative pathway were detected for CNTs decorated with PEG550-terminated amphiphiles. Our results indicate that CNTs fully coated with PEG do not bind C1q and thus will likely not interfere with the many physiological processes that C1q is involved in. On the other hand, PEG-grafted CNTs may bind C1q while evading complement and the MPS. Such combination in nanoparticle properties may be serviceable in novel biomedical applications. Nonetheless, the protein crystallization aspect of PEG deserves further investigation, as caution has to be exercised to avoid protein aggregation in vivo by PEG-modified nanoparticles. Lastly, our results concerning complement activities reiterate the sensitivity of the complement response towards foreign materials. It is thus imperative to test complement activities for original nanoparticle constructs intended for biomedical applications.



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Supporting Information Available: -- XPS spectra for pristine CNTs (Figure S1), oxidized CNTs (Figure S2), and PEG-grafted CNTs (Figure S3). -- TEM image of oxidized CNTs with and without C1q coverage (Figure S4). -- SPRi data for CNT, CNT-COOH, PEG-grafted-CNT, and CNT fully coated with PEG (Figure S5).

ACKNOWLDEGEMENT

We thank the Transversal Toxicology Program of the CEA for the financial support of this work (NanoImmunoTox). This work used the platforms of the Grenoble Instruct-ERIC Centre (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INSB-0502) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The IBS electron microscope facility is supported by the Rhône-Alpes Region, the Fonds Feder, and the Fondation pour la Recherche Médicale (FRM). The “Service de Chimie Bioorganique et de Marquage” belongs to the Laboratory of Excellence in Research on Medication and Innovative Therapeutics (ANR-10-LABX-0033-LERMIT). We thank the Direction Générale de l'Armement (DGA) for the financial support of a PhD thesis.

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low PEG density

high PEG density


Mode of PEG Coverage on Carbon Nanotubes ... - ACS Publications

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