Cintia Marques

Plinio Maroni

Lionel Maurizi

Olivier Jordan

Gerrit Borchard

Understanding protein-nanoparticle interactions leading to protein corona formation: In vitro -in vivo correlation study

Keywords: nanoparticle, protein corona, protein structure, binding affinity, conformational changes

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

When nanoparticles (NPs) come into contact with biological fluids, they interact with proteins, lipids, and sugars, forming a biomolecular corona on the NP surface. These interactions result in the development of a new "biological identity" for the nanoparticles, accompanied by the acquisition of new physicochemical properties [START_REF] Lima | Understanding the Lipid and Protein Corona Formation on Different Sized Polymeric Nanoparticles[END_REF][START_REF] Anand | Human serum albumin interaction, in silico and anticancer evaluation of Pine-Gold nanoparticles[END_REF][START_REF] Gagner | Effect of gold nanoparticle morphology on protein structure and function[END_REF][START_REF] Falahati | A health concern regarding the protein corona, aggregation and disaggregation[END_REF].

Protein corona studies evolved over the last decade [START_REF] Xiao | The effects of protein corona on in vivo fate of nanocarriers[END_REF]. Initial experimental studies attempted to understand its mechanisms, which proved to be difficult. The focus shifted to trying to avoid protein corona formation by developing stealth NPs, and more recently, the efforts switched to exploiting key proteins to design the protein corona. Numerous ex vivo, in vivo, and in vitro studies have been carried out during this evolution. However, the comparison and translation between these studies are still challenging, mainly due to the lack of standardization [START_REF] Mahmoudi | The need for improved methodology in protein corona analysis[END_REF].

Moreover, it is important to discuss the relevance and challenges of in vivo vs. in vitro correlation studies as well as single protein-NPs vs. all blood proteins-NPs interaction experiments. In vivo, the characteristics of the protein corona display a high complexity since they will be affected by the dynamic interactions between nanoparticles and various biological components (biological fluids, interaction with cells, different physiological flow, immune response, etc.). Similarly, in vitro blood proteins-NPs studies offer data that are supposedly closer to the physiological behavior than single protein-NPs studies. The most common studies consist of having a library of NPs of different physicochemical properties, which would be correlated to the protein corona composition, such as NP size [START_REF] Lima | Understanding the Lipid and Protein Corona Formation on Different Sized Polymeric Nanoparticles[END_REF][START_REF] Yadav | Size-dependent interaction of silica nanoparticles with lysozyme and bovine serum albumin proteins[END_REF][START_REF] Lundqvist | Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts[END_REF][START_REF] Giri | Pore structure and particle shape modulates the protein corona of mesoporous silica particles[END_REF], morphology [START_REF] Gagner | Effect of gold nanoparticle morphology on protein structure and function[END_REF][START_REF] Giri | Pore structure and particle shape modulates the protein corona of mesoporous silica particles[END_REF][START_REF] Vincent | The Combination of Morphology and Surface Chemistry Defines the Immunological Identity of Nanocarriers in Human Blood[END_REF][START_REF] Nandakumar | Dynamic Protein Corona of Gold Nanoparticles with an Evolving Morphology[END_REF], surface chemistry [START_REF] Vincent | The Combination of Morphology and Surface Chemistry Defines the Immunological Identity of Nanocarriers in Human Blood[END_REF][START_REF] Kokkinopoulou | Visualization of the protein corona: towards a biomolecular understanding of nanoparticle-cell-interactions[END_REF], hydrophobicity [START_REF] Vincent | Surface chemistry-mediated modulation of adsorbed albumin folding state specifies nanocarrier clearance by distinct macrophage subsets[END_REF][START_REF] Bertrand | Mechanistic understanding of in vivo protein corona formation on polymeric nanoparticles and impact on pharmacokinetics[END_REF], and charge [START_REF] Lundqvist | Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts[END_REF][START_REF] Vincent | The Combination of Morphology and Surface Chemistry Defines the Immunological Identity of Nanocarriers in Human Blood[END_REF][START_REF] Vincent | Surface chemistry-mediated modulation of adsorbed albumin folding state specifies nanocarrier clearance by distinct macrophage subsets[END_REF]). However, under physiological conditions, the variables are not only NP characteristics but protein corona can also be influenced by gender [START_REF] Bertrand | Mechanistic understanding of in vivo protein corona formation on polymeric nanoparticles and impact on pharmacokinetics[END_REF] and by health conditions [START_REF] Hajipour | Sex as an important factor in nanomedicine[END_REF][START_REF] Liu | Nano-Bio Interactions in Cancer: From Therapeutics Delivery to Early Detection[END_REF], adding an extra layer of complexity to the analysis and making it more difficult to connect protein corona patterns with NP properties. Thus in vitro and single protein-NPs studies are important to have better control over the different variables that influence protein corona formation and obtain direct results that can be correlated to in vivo studies. While the direct comparison between in vivo and in vitro data has limitations, integrating findings from both types of experiments can provide a comprehensive understanding of the protein corona phenomenon.

In a previous study, superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyvinyl alcohol (PVAL) displaying either neutral ([0] NPs), positive (+ NPs), or negative (-NPs) surface charges were injected into the tail vein of rats, and the protein corona composition was tracked over time [START_REF] Marques | Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles[END_REF]. Depending on their surface chemistry/charge and resulting corona composition, NPs showed different retention times in blood circulation. Positively charged PVAL-SPIONs showed the shortest circulation time, which was attributed to high hemoglobin adsorption. Moreover, neutral NPs had the longest circulation time, which might be related to the presence of several proteins, such as alpha-2-HS-glycoprotein (fetuin-A).

This work explores the interaction between these previously identified key proteins and the PVAL-coated SPIONs, investigating binding affinity and structural changes of protein upon binding to NPs. Besides hemoglobin (Hb) and fetuin-A (Ft), albumin (BSA) and transferrin (Tf) interaction with NPs was studied, as well since they are two of the most abundant proteins in plasma. To do so, fluorescence quenching and quartz crystal microbalance (QCM) were used to determine binding affinity, while potential changes of the secondary protein structure were characterized by circular dichroism (CD). The results were compared to data previously published, obtained from in vivo experiments [START_REF] Marques | Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles[END_REF], in order to determine how simple protein-NP experiments can be correlated to the in vivo complexity of the protein corona.

Methods

Materials

Polyvinyl alcohol (PVAL-OH; Kuraray Poval 3-85) and carboxyl modified polyvinyl alcohol (PVAL-COOH; Kuraray Poval 3-86 SD) were supplied by Kuraray Co., Ltd. Polyvinyl/Vinyl amine copolymer (PVAL-NH2; Selvol Ultiloc 5003) was supplied by Sekisui Chemical Co., LTD. BSA (66 kDA, A9647), holo-transferrin human (T0665), hemoglobin human (H7379), fetuin from fetal bovine serum (F3385) and phosphate buffered saline 10x (PBS) and other chemicals were purchased from Sigma-Aldrich (Buchs, Switzerland). PVAL-SPIONs were produced according to Marques et al [START_REF] Marques | Characterization Challenges of Self-Assembled Polymer-SPIONs Nanoparticles: Benefits of Orthogonal Methods[END_REF]. Hydrodynamic diameter (Dh), and zeta potential were measured by Dynamic Light Scattering (DLS) and Electrophoretic Light Scattering (ELS). The obtained NPs had the following characteristics in PBS 0.1x: PVAL-COOH SPIONs (hydrodynamic diameter (Dh) of 130 ± 2 nm, -13.1 ± 0.2 mV), PVAL-OH SPIONs (Dh 71 ± 4 nm, -4 ± 0.3 mV) and PVAL-NH2 (Dh 81 ± 1 nm, +0.5 ± 0.3 mV) [START_REF] Marques | Characterization Challenges of Self-Assembled Polymer-SPIONs Nanoparticles: Benefits of Orthogonal Methods[END_REF].

Preparation of Quartz Crystal Microbalance (QCM) sensor chips Silica sensor ships (Q Sense E1/E4) were supplied from China by Renlux Tek Co., Limited. The sensors were initially cleaned with piranha solution for 15 min, which also increases OH availability. The wash was followed by an aminosilanization and glutaraldehyde treatment to functionalize the sensors with aldehyde groups, subsequently bound to proteins' positively charged amino acids (arginine, histidine and lysine).

For the aminosilanization step, sensors were immersed in acetone, followed by immersion in an aminosilane solution -(3-Aminopropyl)triethoxysilane 2 % v/v. The chips were dipped again in the acetone jar to remove excess, weakly physisorbed aminosilane and baked for 1 h at 110 °C. Then the sensors were sonicated for 10 min in ethanol, another 5 min in milliQ® water, rinsed briefly with milliQ® water and dried with nitrogen gas.

Finally, the sensors were placed in a "humidity chamber", covered with 1 to 2 drops of 8 % glutaraldehyde solution, and incubated at room temperature for 30 min. Then sensors were washed with water and dried with nitrogen gas.

Nanoparticles' binding studies with QCM Q-sense E4 (Gothenburg, Sweden) with a flow-through cell was used to measure the change in mass on the sensor, and a peristaltic pump was used to pump solutions through the circular thin-layer cell, which features two eccentric holes as the inlet and outlet.

First, the sensors were cleaned with milliQ® water, followed by PBS 1x. Resonance frequency was monitored and when equilibrium was reached, in around 20 min, a protein solution of 0.05 mg/ml in PBS 1x (BSA, Tf, Hb or Ft) was applied until reaching equilibrium again. The protein mass on the sensor was calculated with the equation 1 [START_REF] Höök | Development of a novel QCM technique for protein adsorption studies[END_REF]:

Where 𝛥𝑚 corresponds to the protein mass on the sensor, 𝐶= 17.7 ng Hz -1 m -2 is the mass sensitivity constant, and 𝑛 is the overtone number (in this case 𝑛 = 5).

Protein coated sensors were then washed with PBS 1x before applying the NPs solution (0.1 mg Fe/ml). Once NPs reached an equilibrium, the sensor was washed with PBS 1x to check for loose protein-NPs interaction. All measurements were made in triplicate, at 25 °C.

Fluorescence spectroscopy measurements BSA, Tf and Ft 4 μM were incubated with increasing concentration of coated PVA-SPIONs (100 μM, 150 μM, 200 μM, 250 μM, 300 μM and 350 μM of Fe2O3), for 1h at 25 °C or 37 °C, with agitation (80 rpm). To ensure a good fluorescence signal, 6 μM of Hb were used, keeping the same protein/SPIONs ratio. All samples were formulated in PBS 1x (Sigma-Aldrich, Switzerland). Steady-state fluorescence measurements were performed on Synergy Mx multimode microplate reader (BioTek Instruments, Inc., USA), on a 96 well plate (150 ul of samples /well). The excitation wavelength was 295 nm (to excite tryptophan residue) [START_REF] Santos | Binding of RuCp complexes with human apotransferrin: fluorescence spectroscopy and molecular docking methods[END_REF], and the fluorescence emission was scanned from 320 to 500 nm, with a 2 nm step, at 25 °C and 37 °C.

To calculate the affinity constants, the intensity of fluorescence was considered at the maximum emission of fluorescence of each protein (BSA = 338 nm, Tf = 326 nm, BSA = 334 nm and Hb = 326 nm). To compare the quenching effect of coated SPIONs, the area under the curve (AUC) from each spectrum was plotted on a bar graph. All samples were measured in triplicates (n=3). UV-visible absorption measurements 6 μM of Hb were incubated with 150 μM, 225 μM, 300 μM, 375 μM, 450 μM and 525 μM of PVA-coated SPIONs (concentration expressed in terms of of Fe2O3), for 1h at 25 °C, with agitation (80 rpm). All samples were solubilized in PBS 1x (Sigma-Aldrich, Switzerland). Uv-visible measurements were performed on Synergy Mx multimode microplate reader (BioTek Instruments, Inc., USA), on a 96 well plate (150 ul of samples /well). The absorption spectra were acquired from 230 nm to 700 nm, with a 10 nm step, at 25 °C.

Circular Dichroism

To study the protein's secondary structure while interacting with NPs of different charges, 0.4 µM of protein solution (BSA, Tf, Hb or Ft) was incubated with 80 µM of coated SPIONs, for 1h, at 37°C, with agitation. All solutions were prepared in milliQ ® water. CD measurements were carried out at 25 °C using a spectro-polarimeter (JASCO J-810, Japan), with a 5 mm path length quartz cell. Spectra were recorded from 190 nm to 260 nm (far-UV region) with a 1nm bandwidth under a constant flow of nitrogen gas. Scanning speed was 20 nm/min and each spectrum resulted from 4 accumulations. Spectra were smoothed by applying the Savitzkx-Golay filter. The measurements were made in triplicates (n=3). CD spectra were evaluated to estimate the proportions of secondary structure using the on-line DichroWeb server [START_REF] Whitmore | DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data[END_REF].

Results and discussion

3.1. Binding affinity 3.1.1. Quartz Crystal Microbalance To study the interaction between NPs and proteins in a dynamic mode, we used a quartz microbalance to monitor the mass of NPs flowing above a protein-coated surface and able to bind to this surface (Figure 1a). Silica sensors were coated with the different proteins of interest -albumin, transferrin, hemoglobin, and fetuin. The coating process was monitored to ensure the sensor's total coverage, which is shown in Figure S1 and Table S1. It is important to note that the calculations consider that proteins are spherical, with the diameter corresponding to the longest dimension of the protein (Table S1 and Figure S2). While the shape of hemoglobin is close to spherical, that is not the case for the other proteins, which have a more ellipsoidal appearance. [START_REF] Kragh-Hansen | Structure and ligand binding properties of human serum albumin[END_REF][START_REF] Rosseneu-Motreff | Size and shape determination of apotransferrin and transferrin monomers[END_REF][START_REF] Moustaoui | A protein corona study by scattering correlation spectroscopy: a comparative study between spherical and urchin-shaped gold nanoparticles[END_REF]. Figure 1. QCM scheme (a) represents the sensor coated with proteins and its interaction with the NPs. After the protein-NPs interaction reached an equilibrium, the sensor was washed with PBS. The NPs remaining attached to the sensor were classified as having established a "strong interaction" with the protein, while the NPs removed by the buffer were classified as establishing a "reversible interaction" with the proteins. The change in frequency derived from the interaction between the protein sensors and the NPs of different charges is presented in (b), where the solid color bar corresponds to a reversible interaction, and the cross-hatched bars indicates strong interaction.

Proteins should bind with a random orientation to the aldehyde functionalized QCM sensor (side on or end on), as considered in the calculations. It cannot be excluded that some orientations might be more favorable, meaning that the surface coverage (Figure S1) might be over-or underestimated. Moreover, QCM measures "wet" mass, since the measurement includes the water trapped in the film, which can also lead to an overestimation of the adsorbed amount. According to this simple model, transferrin and fetuin-A would tend to form a multilayer (with surface coverage > 100 %), while albumin and hemoglobin form a monolayer. Independently of the number of layers, the goal was to completely cover the surface to avoid unspecific interaction with the aldehyde functionalized QCM sensor. Since calculated hemoglobin coverage was below 100 %, the NP adsorption profiles on the Hb sensor were compared with the NP adsorption on the aldehyde sensor (blank measurement) to rule out potential unspecific binding (Figure S3). Because aldehyde is negatively charged, it is expected to interact the most with the positively charged NPs (+ NPs), followed by the neutral [0] NPs. Figure S3 reveals that the affinity of + NPs and [0] NPs to the aldehyde sensor is about 10 and 6 times higher, respectively, than the NP interaction with the Hb-coated sensor. These results support full hemoglobin coverage of the sensor.

Once the full coverage of the sensor by proteins was ensured, we studied the interaction between the fixed proteins and the circulating NPs, which would pass over the sensor at a continuous flow. The change in mass on the sensor is summarized in Figure 1b, which shows that protein-NPs interactions resulted in a low change in frequency, suggesting low binding affinities due to the unspecific interaction between the NPs and the proteins.

To determine the binding affinity constant with QCM, it was necessary to increase the NP concentrations, which would increase the mass change on the sensor, allowing to build a binding isotherm curve. However, to obtain the results displayed in Figure 1b, the concentration of NPs was already high, making it technically impossible to quantify the binding affinity. Still, the data gives us a qualitative picture of the interactions between the NPs and the proteins.

Positively and negatively charged NPs had the highest and lowest interaction with the proteins, respectively (Figure 1b). Moreover, all proteins are negatively charged at pH 7.4 (excepted for the two alpha chains of the hemoglobin, Table S2). Thus, the results suggest that electrostatic interactions direct protein-NP interaction.

NPs interacted with the proteins in a continuous flow, and the maximum interaction was achieved once the mass adsorbed onto the sensor was stable (example in Figure S3a andb), then the sensor was cleaned with PBS, which often led to the partial removal of NPs from the sensor. The remaining NPs mass on the sensor after wash was considered to have a "strong interaction" with the proteins, while the mass of NPs washed away was classified as having a "reversible interaction" (Figure 1). Following this rationale, BSA had the highest interaction with NPs in general, but while its interaction with + NPs was mainly strong, albumin's interaction with [0] NPs was mainly reversible. This tendency was followed by the other proteins since the interaction with + NPs was mainly irreversible, the interaction with [0] NPs was a mix between strong and reversible interactions, while interactions with -NPs were mainly reversible. Furthermore, even though the protein were bound to the sensor, one can hypothesize that a "strong interaction" translates into a higher chance of the protein suffering structural changes in vivo, which might lead to the exposure of more epitopes, increasing the potential interaction with the NPs surface and with other proteins.

Fluorescence quenching (quantitative analysis)

Fluorescence quenching has been widely applied to study protein binding affinity and to predict potential structural changes [START_REF] Vincent | Surface chemistry-mediated modulation of adsorbed albumin folding state specifies nanocarrier clearance by distinct macrophage subsets[END_REF][START_REF] Santos | Binding of RuCp complexes with human apotransferrin: fluorescence spectroscopy and molecular docking methods[END_REF][START_REF] Yu | Albumin-coated SPIONs: an experimental and theoretical evaluation of protein conformation, binding affinity and competition with serum proteins[END_REF][START_REF] Wang | Two different protein corona formation modes on Soluplus® nanomicelles[END_REF][START_REF] Yang | Bovine hemoglobin adsorption onto modified silica nanoparticles: Multi-spectroscopic measurements based on kinetics and protein conformation[END_REF]. Tryptophan moieties were selectively excited at 295 nm, so that a change in the polarity on the amino acid microenvironment would lead to a shift in the maximum wavelength of emission (ʎmax) [START_REF] Wang | Two different protein corona formation modes on Soluplus® nanomicelles[END_REF][START_REF] Lakowicz | Principles of fluorescence spectroscopy[END_REF][START_REF] Wang | Bovine Serum Fetuin Is Unfolded through a Molten Globule State[END_REF][START_REF] Zaidi | pH-Dependent Differential Interacting Mechanisms of Sodium Dodecyl Sulfate with Bovine Serum Fetuin: A Biophysical Insight[END_REF][START_REF] Wrobel | Influence of core and maltose surface modification of PEIs on their interaction with plasma proteins-Human serum albumin and lysozyme[END_REF].

To define the protein/NPs ratios, we took the albumin/NPs ratio used in a previous in vivo experiment as reference [START_REF] Marques | Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles[END_REF], where 0.559 mg Fe/ml of coated SPIONs (corresponding to 5000 µM of Fe2O3, defined as "SPION concentration") were injected into rats. Thus, this concentration corresponds to the highest SPIONs concentration found in plasma. Then we took as reference the albumin concentration in plasma (600 µM [START_REF] Peters | Serum Albumin[END_REF]) to define the lowest protein/SPIONs ratio in this study, which corresponds to a molar ratio of 0.11 (Table 1). Keeping the same protein concentration, we decreased the amount of coated SPIONs, mimicking the decrease in NP circulation in vivo over time [START_REF] Marques | Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles[END_REF]. To be able to compare the effect of NPs on albumin with those of the other proteins of this study (transferrin, fetuin-A, and hemoglobin), we kept the same ratios for all proteins, even though the protein/SPIONs ratios are higher than the physiological ones (Table 1).

When protein interacted with the coated SPIONs, the protein fluorescence decreased with increased NPs concentration, indicating NPs act as a quencher (Figure S4-S7).

In the present study, none of the proteins induced a shift in the maximum fluorescence with the increasing concentration of NPs, showing that protein adsorption onto NPs did not lead to significant structural changes around the tryptophane residues. This is in contrast to a previous study of bovine serum albumin (BSA) adsorption onto SPIONs, which revealed a small red shift in BSA maximum emission wavelength, suggesting some structural changes [START_REF] Yu | Albumin-coated SPIONs: an experimental and theoretical evaluation of protein conformation, binding affinity and competition with serum proteins[END_REF]. [17] Ratio protein/SPIONs (µM/µM) Albumin 66 600 [32] 5000 0.11 Transferrin 80 25.5 [33] 0.0051 Hemoglobin 64.5 4.8 [34] 0.00096 Fetuin-A 49 10.20 [35] 0.0020

Then, we proceeded to calculate the protein binding affinity (Ka) and number of binding sites (n) by applying the modified Stern-Volmer equation [START_REF] Bakar | A critical view on the analysis of fluorescence quenching data for determining ligand-protein binding affinity[END_REF], where F0 and F correspond to the fluorescence intensity of protein in the absence and presence of the quenching NPs, respectively:

𝑙𝑜𝑔 𝐹 0 -𝐹 𝐹 = log 𝐾 𝑎 + 𝑛 log([𝑁𝑃𝑠]
) Equation 1The results obtained for the different proteins are summarized in Figure 2 and Table S3. The first two columns show the binding affinity values at two different temperatures (25 °C and 37 °C). The highest Ka was found at 25 °C for both transferrin and fetuin adsorbed onto positively and negatively charged NPs. There is an overall decrease in Ka values at 37 °C, with the highest binding affinities corresponding to fetuin adsorbed onto positively charged NPs, similar to the behavior at 25 °C. However, transferrin has a different behavior since its interaction with positively charged NPs seems less favorable at a higher temperature, suggesting that temperature is a key factor in protein-NPs interaction, as has been mentioned by other authors [START_REF] Mahmoudi | Temperature: The "Ignored" Factor at the NanoBio Interface[END_REF].

As mentioned, the highest binding affinity corresponded to transferrin binding to the positively and negatively charged NPs at 25 °C, with Ka values around 30*10 3 M -1 . These values correspond to low binding affinities, typical for non-specific interactions.

In comparison, Tf binding to its receptor would lead to a much higher affinity of 10 8 M - 1 [START_REF] Schüler | Interaction Kinetics of Tetramethylrhodamine Transferrin with Human Transferrin Receptor Studied by Fluorescence Correlation Spectroscopy[END_REF]. This also explains the difficulties with the QCM experiment since the low binding affinities justify the need to apply a high NP concentration to measure protein-NPs interaction.

Following the same tendency as the binding affinity, the number of binding sites decreased with increase in temperature. The conclusions drawn from the number of binding sites need to be considered carefully. While some papers interpret n as the number of protein binding sites [START_REF] Santos | Binding of RuCp complexes with human apotransferrin: fluorescence spectroscopy and molecular docking methods[END_REF][START_REF] Yu | Albumin-coated SPIONs: an experimental and theoretical evaluation of protein conformation, binding affinity and competition with serum proteins[END_REF][START_REF] Chi | Binding of Oxytetracycline to Bovine Serum Albumin: Spectroscopic and Molecular Modeling Investigations[END_REF][START_REF] Wang | Spectroscopic studies on the interaction of azelnidipine with bovine serum albumin[END_REF], it is important to consider that n represents the Hill coefficient [START_REF] Bakar | A critical view on the analysis of fluorescence quenching data for determining ligand-protein binding affinity[END_REF]. The values shown in Figure 2 indicate that the Hill coefficient varied between 0.98 and 1.21, which corresponds to no cooperativity [START_REF] Grabarek | Appendix. On the applicability of Hill type analysis to fluorescence data[END_REF], meaning that the binding of one ligand molecule does not affect the binding of adjacent molecules. In this case, the protein still might have one or multiple non-cooperative ligand-binding sites, which cannot be deduced from the data obtained [START_REF] Cattoni | Cooperativity in Binding Processes: New Insights from Phenomenological Modeling[END_REF][START_REF] Van De Weert | Fluorescence quenching and ligand binding: A critical discussion of a popular methodology[END_REF].

Finally, while QCM results suggest that protein-NPs binding is mainly electrostatically driven, this is not so clear from the analysis of the binding constants. Focusing on the data at 25 °C (the same temperature as the QCM experiment), the + NPs have the strongest interaction with Tf and Ft. Contrarily, BSA seemed to have the highest interaction with + NPs in QCM experiments. The difference in the results might be related to the technique since QCM required the previous fixation of protein onto the sensor, while fluorescence emission was measured after the protein established an equilibrium with NPs in solution. The differences in the affinity stress the need to apply orthogonal techniques to understand protein-NPs interactions better.

Secondary structure Circular dichroism

Besides the binding affinity, it is also important to check the structural integrity of the proteins when interacting with the NPs, which can be performed applying circular dichroism (CD). This technique allowed us to assess changes in the protein structure upon interaction with the NPs in solution.

When designing the experimental conditions, the first criteria was that NP CD spectral changes would be negligible, achieved for NP concentration below 80 µM. Then, the protein concentration needed to be high enough to obtain a high-intensity spectrum, but low enough to ensure that all proteins in the solution interacted with NPs. The latter was essential to ensure that possible protein structure alterations were not masked by the spectra of free protein in solution (which would assume its native structure). Therefore, the lowest protein/NPs ratio applied for fluorescence was divided by half, meaning that protein/SPIONs solutions at a molar ratio of 0.005 were prepared.

Figure 3a shows the CD protein spectra obtained for proteins interacting with the differently charged NPs. Transferrin maintained its native structure when interacting with the different NPs, while the fetuin structure was the most affected by + NPs. On the other hand, the interaction with the three different NPs affected both Hb and BSA conformations.

Furthermore, to quantify the different types of content of secondary structures, far-UV CD spectra were analyzed by the algorithm SELCON3 [START_REF] Whitmore | DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data[END_REF], as shown in Figure 3b (1st column). Then, the structure variation (Δ fraction) was calculated to better understand the NPs' influence on the protein structure, as shown in Figure 3b (2nd column). BSA, Hb, and Ft structures suffered the greatest changes when interacting with the positively charged NPs. This is in agreement with the data obtained by the QCM measurements, indicating that higher affinity might be related to higher changes in the secondary structure. However, this was not the case for transferrin, which had one of the highest affinities to the NPs according to QCM and fluorescence quenching, although it did not translate into a loss of its native structure. Consequently, we may hypothesize that the Tf/NPs interaction occurs in a region that does not affect the protein structure.

A decrease in albumin's α-helix content to values around 0.35 was previously correlated to protein denaturation [START_REF] Vincent | Surface chemistry-mediated modulation of adsorbed albumin folding state specifies nanocarrier clearance by distinct macrophage subsets[END_REF], while an increase is linked to higher protein stability [START_REF] Lai | A Protein Corona Adsorbed to a Bacterial Magnetosome Affects Its Cellular Uptake[END_REF][START_REF] Yang | Conformationaltransited protein corona regulated cell-membrane penetration and induced cytotoxicity of ultrasmall Au nanoparticles[END_REF]. Our results showed a small decrease in α-helix structure upon interaction with the different NPs, with the minimum α-helix content being registered to the BSA/+NPs interaction of around 0.6, suggesting that the studied NPs do not denature albumin in solution. interaction with the different NPs, while the graphs on the right represent the change in the secondary structure (Δ fraction) during interaction with NPs.

Fetuin-A is characterized by small α-helix content [START_REF] Zaidi | pH-Dependent Differential Interacting Mechanisms of Sodium Dodecyl Sulfate with Bovine Serum Fetuin: A Biophysical Insight[END_REF][START_REF] Gejyo | Characterization of the B-chain of human plasma alpha 2HSglycoprotein. The complete amino acid sequence and primary structure of its heteroglycan[END_REF]. If solubilized in a urea solution, Ft suffered an increase in random conformation, while dissolution in chloroethanol (organic solvent) increased its α-helix structure content [START_REF] Gejyo | Characterization of the B-chain of human plasma alpha 2HSglycoprotein. The complete amino acid sequence and primary structure of its heteroglycan[END_REF]. The increase in α-helix shown for Ft/[0] NPs and Ft/-NPs interactions has been associated with an exposition of the protein's hydrophobic regions, which would also lead to a shift of the maximum of fluorescence after tryptophan excitation [START_REF] Zaidi | pH-Dependent Differential Interacting Mechanisms of Sodium Dodecyl Sulfate with Bovine Serum Fetuin: A Biophysical Insight[END_REF][START_REF] Gejyo | Characterization of the B-chain of human plasma alpha 2HSglycoprotein. The complete amino acid sequence and primary structure of its heteroglycan[END_REF]. However, the results discussed previously showed that the increase in α-helix did not change the maximum wavelength of emission (Figure S7). On the other hand, the increase in βsheet confirmed for + NPs interaction has been associated with the formation of amyloid fibrils [START_REF] Nelson | Structural Models of Amyloid-Like Fibrils[END_REF], which has been identified as a generic danger signal to the immune system [START_REF] Gustot | Activation of innate immunity by lysozyme fibrils is critically dependent on cross-β sheet structure[END_REF]. However, fibril formation would have to be proven by complementary measurements, e.g., by transmission electron microscopy (TEM) [START_REF] Zaidi | pH-Dependent Differential Interacting Mechanisms of Sodium Dodecyl Sulfate with Bovine Serum Fetuin: A Biophysical Insight[END_REF].

Finally, hemoglobin interaction with the different NPs lead to a slight decrease in the α-helix structure, associated with its partial denaturation [START_REF] Ikai | Partially Helical Conformation of Hemoglobin in Aqueous 2-Chloroethanol[END_REF][START_REF] Mondal | Contrasting effects of pH on the modulation of the structural integrity of hemoglobin induced by sodium deoxycholate[END_REF][START_REF] Fotouhi | Application of merged spectroscopic data combined with chemometric analysis for resolution of hemoglobin intermediates during chemical unfolding[END_REF]. Still, it is possible to observe peaks at wavelengths around 210 nm and 220 nm for the Hb/NPs interaction spectrum (Figure 3a), which indicates that the unfolding of Hb did not achieve the hemichrome state, leading to Hb dimerization [START_REF] Fotouhi | Application of merged spectroscopic data combined with chemometric analysis for resolution of hemoglobin intermediates during chemical unfolding[END_REF].

Overall discussion

This study aimed to investigate the interactions between different proteins (albumin, transferrin, hemoglobin, and fetuin) and NPs of different surface charges and compare the data obtained with the protein corona data obtained from an in vivo study performed with similar NPs [START_REF] Marques | Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles[END_REF]. QCM and fluorescence quenching explored the binding affinity, while potential changes in the secondary structure were investigated by CD.

BSA and transferrin were previously identified as part of the top 10 most abundant proteins adsorbed in vivo [START_REF] Marques | Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles[END_REF] independently of their charge. This can be related to the affinity of these proteins to the NP surface and their high abundance in the blood [START_REF] Xiao | The effects of protein corona on in vivo fate of nanocarriers[END_REF]. Fetuin-A, identified as one of the most abundant constituents of the hard corona [START_REF] Lai | A Protein Corona Adsorbed to a Bacterial Magnetosome Affects Its Cellular Uptake[END_REF][START_REF] Mohammad-Beigi | Mapping and identification of soft corona proteins at nanoparticles and their impact on cellular association[END_REF][START_REF] Pinals | Quantitative Protein Corona Composition and Dynamics on Carbon Nanotubes in Biological Environments[END_REF], was correlated with the increase in the circulation time of [0] NPs and -NPs. Contrarily, hemoglobin, which has a low concentration in the blood (Table 1), was identified as a key protein that shortens + NPs circulation time since it was one of the most abundant components of the protein corona of these short-lived NPs [START_REF] Marques | Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles[END_REF].

Previous studies have shown that negatively charged NPs interact with Hb leading to reversible changes in its secondary structure, preserving its functionality [START_REF] Devineau | Structure and Function of Adsorbed Hemoglobin on Silica Nanoparticles: Relationship between the Adsorption Process and the Oxygen Binding Properties[END_REF]. However, Hb interaction with positively charged NPs leads to heme degradation and promotes NP aggregation [START_REF] Yang | Bovine hemoglobin adsorption onto modified silica nanoparticles: Multi-spectroscopic measurements based on kinetics and protein conformation[END_REF][START_REF] Mobasherat Jajroud | Human hemoglobin adsorption onto colloidal cerium oxide nanoparticles: a new model based on zeta potential and spectroscopy measurements[END_REF]. Thus, it was hypothesized that hemoglobin would have a high affinity to the + NPs, inducing protein structure changes and ultimately exposing new epitopes, stimulating + NPs elimination by the immune system.

Focusing on the data obtained at 25 °C, Hb showed highest binding affinity to positive NPs on both QCM and fluorescence quenching measurements, compared to [0] NPs and -NPs. Accordingly, this interaction led to the highest loss in α helix structure (Figure 3). While the Hb/[0] NPs and Hb/-NPs interactions lead to an increase of both β-sheets and turns, Hb/+ NPs interaction lead to an increase of unordered structures. Thus, we hypothesized that this might indicate that + NPs lead to higher changes in Hb secondary structure. In order to confirm this assumption, we performed UV spectroscopy to investigate Hb degradation, since the consequent hemichrome release should lead to a shift of the maximum absorption.

The UV absorption spectra (Figure S8) showed the characteristic bands of hemoproteins, namely, the Soret band centered at 415 nm for oxy-hemoglobin [START_REF] Carvalho | Thermal denaturation and aggregation of hemoglobin of Glossoscolex paulistus in acid and neutral media[END_REF]. The blue shift observed is associated with Hb degradation [START_REF] Carvalho | Thermal denaturation and aggregation of hemoglobin of Glossoscolex paulistus in acid and neutral media[END_REF][START_REF] Samuel | The Interplay between Molten Globules and Heme Disassociation Defines Human Hemoglobin Disassembly[END_REF][START_REF] Hanson | A Blue Spectral Shift of the Hemoglobin Soret Band Correlates with the Age (Time Since Deposition) of Dried Bloodstains[END_REF], which might indicate Hb denaturation towards hemichrome formation [START_REF] Samuel | The Interplay between Molten Globules and Heme Disassociation Defines Human Hemoglobin Disassembly[END_REF][START_REF] Liu | The interaction between hemoglobin and two surfactants with different charges[END_REF]. Unfortunately, since SPIONs have a considerable UV absorbance at around 370 nm (Figure S8), it is not possible to confirm the absence of free hemin in the solution [START_REF] Fotouhi | Application of merged spectroscopic data combined with chemometric analysis for resolution of hemoglobin intermediates during chemical unfolding[END_REF], as suggested by CD data. Still, the peaks between 550 nm and 650 nm did not change with the increase of coated NPs concentration, which supports the lack of free hemin formation [START_REF] Fotouhi | Application of merged spectroscopic data combined with chemometric analysis for resolution of hemoglobin intermediates during chemical unfolding[END_REF][START_REF] Samuel | The Interplay between Molten Globules and Heme Disassociation Defines Human Hemoglobin Disassembly[END_REF][START_REF] Liu | The interaction between hemoglobin and two surfactants with different charges[END_REF]]. Namely, it was not considered how the change in Hb structure would affect its interaction with other proteins, which reminds us that this is a simplistic modularization of the in vivo complexity. Still, the in vitro Hb-NPs interaction characterization supports our initial hypothesis that + NPs lead to higher changes in Hb structure, potentially stimulating NPs elimination.

Furthermore, this study revealed a general tendency for bigger changes in the protein structure after interacting with + NPs, which could be correlated with stronger protein-NPs interaction. Similarly, the interaction of fetuin-A with + NPs lead to a significant decrease in α helix structure (> 20 %), suggesting a significant change in the secondary structure. Even though fetuin-A had one of the highest Ka values with the + NPs (Figure 2), this protein was not identified as part of the most abundant proteins on the + NPs protein corona in vivo [START_REF] Marques | Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles[END_REF]. This might have two explanations: i) according to the Vroman effect [START_REF] Vroman | Identification of rapid changes at plasma-solid interfaces[END_REF], proteins with low affinity to the surface are replaced by proteins with higher affinity to the NP surface over time. However, since + NPs have a half-life of about 2 min [START_REF] Marques | Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles[END_REF], there is not enough time to observe the enrichment in fetuin-A in the protein corona of these NPs; ii) as mentioned before, the experiment ignores the contribution of protein-protein interaction, which might affect the protein corona enrichment.

Fetuin-A has been classified as both an opsonin and dysopsonin, depending on the NPs charge [START_REF] Thiele | Competitive adsorption of serum proteins at microparticles affects phagocytosis by dendritic cells[END_REF]. Since it was classified as a protein prolonging the blood circulation time of neutral and negatively charged NPs, it was assumed that fetuin-A would have to keep its native structure to act as a dysopsonin. In terms of structure, neither [0] NPs nor -NPs lead to significant changes in the protein structure, which supports that fetuin-A native structure might be essential for its dysopsonin activity. Unfortunately, the Ka data is not so conclusive. From the fluorescence measurements it is possible to conclude that the Ka determined for -NPs is higher than for [0] NPs, even though QCM data showed the opposite tendency. This discrepancy is probably related to the technical differences between the two techniques. While fetuin is fixed on a sensor for QCM measurements, the protein is free in solution during fluorescence quenching measurements, which might affect the protein-NP binding. This paper starts by mentioning the relevance and challenges of in vivo vs. in vitro studies as well as single protein-NPs vs. all blood proteins-NPs interaction experiments. A paper discussing the in vivo protein corona differences between PVALcoated SPIONs with different charges [START_REF] Marques | Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles[END_REF] was compared to the in vitro results obtained from selected proteins throughout this study. As mentioned above, the experiments confirmed the hypothesis established from the in vivo studies for the interaction between Hb and Ft with the different NPs. Namely that: a) Hb has a higher binding affinity to + NPs, which leads to a higher increase in unordered structures, indicating that + NPs induce greater changes in Hb secondary structure. Thus, changes in Hb secondary structure potentially lead to faster elimination of + NPs by the immune system. b) Neither [0] NPs nor -NPs lead to significant changes in the fetuin-A structure, which supports that its native structure might be essential for its dysopsonin activity.

However, while simplifying the experimental design allowed us to better understand the 1:1 protein-NP interaction, there are some limitations. To improve the accuracy of translating in vitro results to in vivo conclusions, we highlighted the importance of orthogonal techniques and complementary information on protein-NPs interaction, namely binding affinity, and effect on the secondary structure, which, taken together, demonstrated to be essential. In fact, studies have shown that protein binding affinity to NPs is not directly correlated with the protein concentration in the protein corona [START_REF] Moustaoui | A protein corona study by scattering correlation spectroscopy: a comparative study between spherical and urchin-shaped gold nanoparticles[END_REF][START_REF] Pinals | Quantitative Protein Corona Composition and Dynamics on Carbon Nanotubes in Biological Environments[END_REF]. This can be explained by: a) The difficulty in considering all the physiological factors influencing protein binding affinity in the studies (media, pressure, blood flow, other proteins, etc.). b) Protein-protein contribution to the protein corona composition and protein binding affinity i.

Protein adsorbed into the surface might lead to the enrichment of certain proteins with which they have a higher affinity. ii.

Protein adsorption and alteration to its structure might expose new epitopes with higher affinity to certain proteins.

Thus, affinity studies alone are not enough to predict the protein corona formation, but it is important to include other tests, such as the effect on the protein structure and influence on cell uptake. To get the full picture, it will be important to complement the results obtained in this paper with uptake studies after NPs were coated with the different proteins studied.

Conclusions

This comprehensive study thoroughly investigated the interactions between key proteins (albumin, transferrin, hemoglobin, and fetuin-A) and nanoparticles of different surface charges (neutral, positive, and negative) using QCM, fluorescence quenching, and circular dichroism. Hb and Ft were previously found to be important components of the protein corona for NPs of different charges, affecting their circulation time in vivo.

The results could be correlated with the in vivo protein corona studied previously. Namely, the Hb structure changed the most with interaction with positive NPs, while the Ft structure did not suffer significant changes when in contact with [0] NPs and -NPs. Overall, data indicate that protein-NPs interactions are primarily driven by electrostatic interactions and that it is important to check for changes in the native structure since high Ka values do not directly translate into higher structural changes. While the combination of binding affinity and protein structure study was essential to understand protein-NP interactions, complementary cell uptake studies would be important to unveil, for example, the effect on macrophage uptake. Still, in vitro and single protein-NPs studies proved successful in obtaining direct results that be translated into in vivo studies. The established in vivo-in vitro correlation opens new and exciting possibilities for future studies on the protein corona.

  -𝐹 * 𝐶 (Equation1)

Figure 2 .

 2 Figure 2. Binding affinity (Ka) and number of binding site (n) calculated for BSA (bovine serum albumin), Tf (transferrin), Hb (hemoglobin), Ft (fetuin-A), [0] NPs (neutral NPs), + NPs (positive NPs), -NPs (negative NPs) (mean values, n=3). The values were obtained by treating the fluorescence quenching data with the modified Stern-Volmer equation.

Figure 3 .

 3 Figure 3. (a) Far-UV CD spectra of proteins alone (BSA, Tf, Hb, and Ft) and in solution in the presence of the different NPs ([0] NPs, + NPs, and -NPs), obtained at 25 °C (n=3). (b) the left graphs represent the secondary structure composition of each protein alone and during

Table 1 .

 1 Summary of protein/NPs ratios for the fluorescence quenching study, and comparison with physiological ratios expected from the previous in vivo study[START_REF] Marques | Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles[END_REF]

	Fluorescence	quenching	BSA/Tf/Ft (µM) 4.00	SPIONs (µM) 350 300 250 200	Hb (µM) 6.00	SPIONs (µM) 525 450 375 300	Ratio protein/SPIONs (µM/µM) 0.011 0.013 0.016 0.020
				150		225	0.027
				100		150	0.040
	Physiological	concentration	Protein	MW (kDa)	Average concentration in plasma (µM)	SPIONs injected in vivo (µM)

Acknowledgements

For the purpose of open access, the authors have applied a CC BY-ND public copyright license to any Author Accepted Manuscript version arising from this submission.

Conflict of interest

The authors declare no competing financial interest.

Author contributions

Conceptualization


MD Joris Pensier

MD Lucas Deffontis

MD Amélie Rollé

MD Yassir Aarab

MD Mathieu Capdevila

MD Clément Monet

MD,* † Julie Carr

MD Emmanuel Futier

PhD Nicolas Molinari

MD Samir Jaber

PhD,* †∥ Audrey De Jong
email: a-de_jong@chu-montpellier.fr.

Review With Meta-analysis and Trial Sequential Analysis

come   L'archive ouverte pluridisciplinaire

METHODS:

In this meta-analysis and TSA, we searched for randomized controlled trials (RCTs) comparing intraoperative HES intravascular volume replacement to crystalloid intravascular volume replacement in adult patients undergoing major abdominal surgery. Primary outcome was 30-day AKI, defined as a binary outcome according to Kidney Disease Improving Global Outcomes (KDIGO) criteria, combining stages 1, 2, and 3 into an AKI category versus no AKI category (stage 0). Secondary outcomes included rates of intraoperative blood transfusion and postoperative intra-abdominal complications. We used random effects models to calculate summary estimates. We used relative risk (RR) as summary measure for dichotomous outcomes, with corresponding 95% confidence intervals (CIs) for the primary outcome (P value <.05 was considered statistically significant) and 99% CI after Bonferroni correction for the secondary outcomes (P value <.01 was considered statistically significant).

RESULTS:

Seven RCTs including 2398 patients were included. HES intravascular volume replacement was not associated with an increased risk of 30-day AKI (RR = 1.22, 95% CI, 0.94-1.59; P = .13), when compared to crystalloid intravascular volume replacement. According to TSA, this analysis was underpowered. HES intravascular volume replacement was associated with higher rates of blood transfusion (RR = 1.57 99% CI, 1.10-2.25; P = .001), and similar rates of postoperative intra-abdominal complications (RR = 0.76 99% CI, 0.57-1.02; P = .02).

CONCLUSIONS:

In this meta-analysis to focus on HES intravascular volume replacement in major abdominal surgery, HES intravascular volume replacement was not associated with a higher risk of 30day AKI when compared to crystalloid intravascular volume replacement. However, CI and TSA do not exclude harmful effects of HES intravascular volume replacement on the renal function Crystalloid solutions are the most commonly used perioperative fluids.1 Crystalloid solutions accumulate in tissues and are inclined to promote edema and prolonged recovery.2,3 Contrary to crystalloids, colloids produce faster and more persistent hemodynamic effects on account of their higher oncotic pressure.4 Nevertheless, colloids are disparaged for their suspected adverse effects on coagulation by reducing clot formation and strength, implying hematological complications. 5 In addition, colloids might also impair kidney function.6 This is the reason that in 2013, the Food and Drug Administration issued warnings about a risk of death and acute kidney injury (AKI) with hydroxyethyl starch (HES) solutions in critically ill patients, while the European Medicine Agency prohibited them in intensive care units.7 However, these data were obtained exclusively from critically ill patients.8,9 Applying them directly to a surgical population needs a specific evaluation, especially in the modern area of goal-directed fluid strategies.10

In major abdominal surgery, fewer postoperative complications have been reported with a colloid-based goal-directed fluid strategy compared to a goal-directed fluid strategy using only crystalloids.11 However, more AKI,12 altered coagulation and subsequent bleeding and blood transfusion13,14 have also been reported when using a colloid-based goal-directed fluid strategy during the perioperative period.

We hypothesized that, similarly than in critically ill patients, the use of HES during major abdominal surgery was associated with increased 30-day AKI. We also hypothesized that the use of HES would be associated with increased renal replacement therapy (RRT), increased 30-day mortality, increased intraoperative red blood cell (RBC) transfusion, increased intraoperative need for norepinephrine, and decreased 30-day postoperative intra-abdominal complications. Therefore, we designed this systematic review with meta-analysis of randomized controlled trials (RCTs) and trial sequential analysis (TSA) to assess the effect of a HES intravascular volume replacement strategy versus a crystalloid intravascular volume replacement strategy on AKI, RRT, 30-day mortality, intraoperative RBC transfusion, intraoperative need for norepinephrine, and 30-day postoperative intra-abdominal complications in patients undergoing major abdominal surgery.

METHODS

We conducted a systematic review and meta-analysis of RCTs comparing HES versus crystalloids for major abdominal surgery, in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement. [START_REF] Liberati | The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration[END_REF] The meta-analysis was registered on the International Prospective Register of Systematic Reviews (PROSPERO) register of systematic review (CRD42020163450).

Search Strategy

We performed a computerized search of MEDLINE (2000-2020), the Cochrane Centre Register of Controlled Trials (CENTRAL) (2000-2020), and Web of Science (2000-2020) for RCTs comparing a HES intravascular volume replacement strategy to a crystalloid intravascular volume replacement strategy.

We included non-English publications. We searched abstracts of selected conferences from 2010 to 2020, including those of the American Society of Anaesthesiologists, the Canadian Anaesthesiologists' Society, the International Anaesthesia Research Society, the American Thoracic Society, the European Society of Anaesthesiology and the "Société Française d'Anesthésie Réanimation."

For the bibliographic review, keywords ("Colloid," "Hydroxyethyl starches," "HES," "Abdominal surgery," "Crystalloid," "Intravascular volume replacement," and "Fluidloading") and medical subject headings ("Perioperative care" and "adult") were used in our search strategy. References in the retrieved articles were also examined for relevant publications.

We identified and deleted any duplicate papers. All potential eligible papers were retrieved in full.

Selection Criteria and Outcome Measures

We screened for relevant RCTs that enrolled adult patients undergoing major abdominal surgery, defined as open or laparoscopic-assisted abdominal surgery expected to last at least 2 hours and associated with a high risk of postoperative complications.12,16

Then we made a quantitative synthesis performing a meta-analysis and systematic review. The main outcome was all stages AKI within 30 days (30-day AKI) defined as a binary outcome according to Kidney Disease Improving Global Outcomes (KDIGO) criteria, 17 combining stages 1, 2, and 3 into an AKI category versus no AKI category (stage 0). Whenever data on 30-day AKI were not available, we selected 28-day AKI or 14-day AKI as end point, in this order. The secondary outcomes were 30-day need for RRT, to assess postoperative renal failure, 30-day mortality, intraoperative RBC transfusion and intraoperative need for norepinephrine to assess potential impaired coagulation and subsequent bleeding and transfusion, and 30-day postoperative intra-abdominal complications (defined as anastomotic leakage, intraabdominal sepsis, or reoperation). The composite outcome was deemed appropriate, as the 3 components of the composite outcome "30-day postoperative intra-abdominal complications" (anastomotic leakage, abdominal sepsis, and reoperation) may have similar severity, frequency and might equally be affected by the intervention. 18,19 Severity and frequency of anastomotic leakage, intra-abdominal sepsis, or reoperation may be considered equivalent, as a reoperation is often driven by an intraabdominal sepsis, consecutive to an anastomotic leakage,20 and as an anastomotic leakage often triggers an intra-abdominal sepsis followed by a reoperation.20,21 By avoiding massive fluid administration, the colloids may similarly decrease the 3 components of the composite outcome: primary anastomotic leakage, second, the rate of intra-abdominal sepsis (because of less anastomotic leakage), and third, reoperation (because of less anastomotic leakage and intra-abdominal sepsis).20,21

Data Collection and Analysis

First, 2 authors (L.D. and J.P.) independently screened the retrieved studies by title and then by abstract for exclusion. They assessed the full text of the possible relevant studies for inclusion and exclusion criteria. Disagreement was resolved by discussion and arbitrated, if necessary, by a third author (A.D.J.). Data were then added to an excel database, specifically designed for this review and analyzed in RevMan 5.3 software and TSA viewer version 0.9.5.10 Beta.22

Statistical Analysis

Data were extracted as they were reported in the original paper or based on the answers of the authors to our queries. Queries were sent via e-mail to corresponding authors of each study included, and data on both primary and secondary outcomes were asked when not reported in the original paper. Included studies were appraised for their risk of bias by 3 independent authors (J.P., L.D., A.D.J.) using the Cochrane Collaboration's tool for assessing risk of bias in RCTs. We used random effects models to calculate summary estimates. [START_REF] Pensier | Effect of lung recruitment maneuver on oxygenation, physiological parameters and mortality in acute respiratory distress syndrome patients: a systematic review and meta-analysis[END_REF] We used relative risk (RR) as the summary measure for dichotomous outcomes, and mean difference (MD) as the summary measure for continuous outcomes, with corresponding confidence intervals (CIs). Statistical heterogeneity was quantified by the Q-Cochrane heterogeneity test (Q statistic with the degrees of freedom [df]) and the I2 statistic. [START_REF] Liberati | The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration[END_REF] In the case of heterogeneity, the cause of heterogeneity was then explored in sensitivity analyses.

To explore more thoroughly the primary end point, we used TSA to assess the risk of random errors due to sparse data and multiple testing of accumulating data, and to calculate the required information size. [START_REF] Pensier | Effect of lung recruitment maneuver on oxygenation, physiological parameters and mortality in acute respiratory distress syndrome patients: a systematic review and meta-analysis[END_REF] The calculated required information size24 takes into account the control event proportion, the anticipated heterogeneity variance (D2) of the meta-analysis, and the assumption of a plausible relative risk reduction (RRR) or relative risk increase (RRI). [START_REF] Wetterslev | Estimating required information size by quantifying diversity in random-effects model meta-analyses[END_REF] We used an alpha risk of 5%,25 a beta risk of 10%,25 and a D2 calculated according to the trials in the meta-analysis,23,24,26 or a D2 of 20% if the measured heterogeneity was 0.26 As anticipated intervention effects for the primary outcome in the TSA, we used a realistic a priori RRI of 20% of the primary outcome (basal incidence rate assessed in the crystalloid group according to the data of the meta-analysis),23,26 as retrieved in recent large trials. [START_REF] Kabon | Crystalloid-Colloid Study Team. Effect of intraoperative goal-directed balanced crystalloid versus colloid administration on major postoperative morbidity: a randomized trial[END_REF][START_REF] Futier | Effect of hydroxyethyl starch vs saline for volume replacement therapy on death or postoperative complications among highrisk patients undergoing major abdominal surgery: the FLASH randomized clinical trial[END_REF] We used an O'Brien-Fleming alpha and beta spending function and a variance-based O'Brien-Fleming heterogeneity correction.

When a statistical significance was found on a dichotomous outcome, we calculated the number needed to treat/to harm.27 All tests were 2-sided. For the primary outcome, P value <.05 was considered statistically significant, and 95% CI were calculated.

For the 5 secondary outcomes, to adjust for multiple testing, we used the Bonferroni correction. P values <.01 were considered statistically significant, and 99% CI were calculated.25

RESULTS

Study Selection

We identified 4530 articles using the search strategy. We excluded 852 citations because of duplication and 3384 citations on the initial abstract screen because inclusion criteria were not met. After examination of the full text of the 293 selected papers, we included 7 RCTs for the systematic review and metaanalysis. 3,11,12,14,28-30 Figure 1 shows the study selection flowchart.

Study Description

The 7 studies3,11,12,14,28-30 involved a total of 2398 participants from 7 countries (Austria n = 1, Belgium n = 1, Denmark n = 1, France n = 1, Iran n = 1, Serbia n = 1, United Kingdom n = 1). Pooling all studies, 1197 participants were analyzed in the HES group and 1201 in the crystalloid group. The Table presents the characteristics of the studies included in the systematic review and meta-analysis.

Risk of Bias and Quality Assessment

All RCTs were identified with low to moderate risk of bias according to the Cochrane collaboration's tool. Supplemental Digital Content, Figures S1 andS2, http://links.lww.com/AA/D717, present the risk of bias assessment of included studies. Five studies3,11,12,14,29 were assessed having a low risk of bias and 2 studies29,30 having a moderate risk of bias. The primary outcome of 30-day AKI was reported in 6 studies. 3,11,12,28-30 Among the secondary end points, 30-day need for RRT was reported in 6 studies. [START_REF] Kabon | Crystalloid-Colloid Study Team. Effect of intraoperative goal-directed balanced crystalloid versus colloid administration on major postoperative morbidity: a randomized trial[END_REF] 2A: forest plot of 30-day AKI). The pooled RR across studies was 1.22 (95% CI, 0.94-1.59; P = .13), indicating no significant difference concerning the rate of 30day AKI with the use of HES, when compared to crystalloid. There was low evidence of statistical heterogeneity for this outcome (df = 5, P = .38) with a corresponding I2 statistic of 6%. In TSA (Supplemental Digital Content, Figure S3, http://links.lww.com/AA/D717), assuming a RRI of 20%, from an incidence of 30-day AKI of 8.9% in the crystalloid group to 10.7% in the HES group, neither the conventional boundaries nor the trial sequential monitoring boundaries for benefit, harm, or futility were crossed by Z curve. The required information size for 30-day AKI was estimated to be 12,351. The accrued information size was 2365. The TSA analysis results suggest that there is not nearly enough evidence in the existing meta-analyses on this research question to make a definitive conclusion. More research is needed.

Secondary Outcomes

Need for Renal Replacement Therapy. Six studies3,11,12,14,28,30 including 2314 patients reported data on 30-day need for RRT (Figure 3A: forest plot of 30-day need for RRT). The pooled RR across studies was 0.86 (99% CI, 0.28-2.61; P = .72), indicating no significant difference concerning the rate of 30day need for RRT with the use of HES, when compared to crystalloid. There was low evidence of statistical heterogeneity for this outcome (df = 3, P = .38), with a corresponding I2 statistic of 3%. 30-Day Mortality. Four studies3,11,12,30 including 2072 patients reported rate of mortality (Figure 3B: forest plot of 30-day mortality). The pooled RR across studies was 1.28 (99% CI, 0.40-4.12; P = .58), indicating no significant difference in 30-day mortality with HES when compared to crystalloid. There was low evidence of statistical heterogeneity for this outcome (df = 2; P = .26) and a corresponding I2 statistic of 26%.

Blood Loss.

Five studies11,12,14,28,30 including 1250 patients reported results for the incidence of RBC transfusion (Figure 4A: forest plot of intraoperative RBC transfusion). The pooled RR across studies was 1.57 (99% CI, 1.10-2.25; P = .001), indicating a higher rate of intraoperative RBC transfusion associated with the use of HES when compared to crystalloid. There was low evidence of statistical heterogeneity for this outcome (df = 4, P = .61), with a corresponding I2 statistic of 0%. The number needed to harm was 15 (99% CI, 6.8-85).

Five studies3,11,12,28,29 including 2285 patients reported results for the intraoperative need for norepinephrine (Supplemental Digital Content, Figure S4, http://links.lww.com/AA/D717: forest plot of intraoperative need for norepinephrine). The pooled RR across studies was 0.53 (99% CI, 0.13-2.23; P = .26), indicating no significant difference for the intraoperative need for norepinephrine associated with the use of HES when compared to crystalloid. There was a substantial heterogeneity for this outcome (df = 1, P = .002), with a corresponding I2 statistic of 90%.

Postoperative Intra-abdominal Complications.

Seven studies3,11,12,14,28-30 including 2398 patients presented results for 30-day postoperative intra-abdominal complications (Figure 4B: forest plot of 30-day postoperative intra-abdominal complications). The pooled RR across studies was 0.76 (99% CI, 0.57-1.02; P = .02) indicating no significant difference after Bonferroni correction of 30-day postoperative intraabdominal complications with HES when compared to crystalloids. There was a moderate heterogeneity for this outcome (df = 6, P = .12) with a corresponding I2 statistic of 40%. Within postoperative complications, the observed treatment effect appeared to be consistent across anastomotic leakage, reoperation, and surgical site infection but this was not tested (Supplemental Digital Content, Figures S5-S7, http://links.lww. com/AA/D717).

DISCUSSION

This systematic review, meta-analysis, and TSA of 7 RCTs3,11,12,14,28-30 suggested that a HES intravascular volume replacement strategy in patients undergoing major abdominal surgery does not increase the risk of 30-day AKI when compared to a crystalloid intravascular volume replacement strategy. However, the 95% CI of the RR (0.94-1.59) for 30-day AKI highlights the potential for HES to increase the risk of AKI by 60%. This would be clinically important and so the results cannot be accurately interpreted as reliably consistent with "no effect." Using HES might be associated with higher rates of intraoperative RBC transfusion (Figure 4A), even if no conclusion can be drawn from the analysis of secondary outcomes and no statistical difference in the need for norepinephrine was found (Supplemental Digital Content, Figure S4, http://links.lww.com/ AA/D717).

Our findings are largely consistent with the many RCTs8,18 and meta-analyses31-38 performed in the critically ill population, with results suggesting higher rates of RBC transfusion associated with the use of HES. Contrary to our results in the field of major abdominal surgery, meta-analyses in the field of critical care have also found an increased risk of need for RRT and mortality. [START_REF] Haase | Hydroxyethyl starch 130/0.38-0.45 versus crystalloid or albumin in patients with sepsis: systematic review with meta-analysis and trial sequential analysis[END_REF][START_REF] Zarychanski | Association of hydroxyethyl starch administration with mortality and acute kidney injury in critically ill patients requiring volume resuscitation: a systematic review and meta-analysis[END_REF] One must keep in mind that the population of patients undergoing major abdominal surgery differs widely from the population of critically ill patients. In the 7 studies included for analysis, the patients were mostly noncritically ill patients undergoing scheduled procedures. Indeed, only the study by Futier et al12 included patients undergoing unscheduled surgery (13 of 775 patients) and patients with American Society of Anesthesiologists (ASA) physical status of IV (11 of 775 patients). Only 2 studies11,12 reported more than 5% of AKI. In our meta-analysis, <1% of overall patients needed RRT, and <2% of overall patients died. Thus, neither any of the recent big RCTs performed in abdominal surgery3,11,12 nor our meta-analysis were powered to detect differences in AKI, RRT, or mortality. According to the TSA performed for the primary outcome (Figure 2B), 12,351 randomized patients would be needed to bring out a 20% RRI, from 8.9% to 10.7% of 30-day AKI with a highlevel of certainty. Another difference between patients undergoing major abdominal surgery and critically ill patients lies in the prominence of surgical concerns.39 Renal prognosis may be altered by postoperative surgical complications and the need for reoperation, extended antibiotic treatment, or intensive care unit stay. [START_REF] Montravers | Short-course antibiotic therapy for critically ill patients treated for postoperative intra-abdominal infection: the DURAPOP randomised clinical trial[END_REF] One of the strengths of the present systematic review and meta-analysis is the large sample size and the low statistical heterogeneity. Second, it is to our knowledge the first meta-analysis to focus on 1 type of colloid (third-generation HES, 6% HES 130/0,4) in 1 category of surgery (major abdominal surgery). This limits the discrepancies between studies and strengthens the external validity of our results, and explains why studies before 2000 were not included. Our systematic review with metaanalysis has limitations. First, if protocols for fluid management are similar, discrepancies do appear in the stroke volume and mean arterial pressure objectives, and in the protocol for use of norepinephrine. Both elements play an important role in perioperative fluid management.41 Second, due to the low number of studies in the review, the presence of publication bias by funnel plots could not be assessed,42 and the results are highly dependent on the quality of included trials, with some outcomes and variables being not comparable. Some interesting outcomes as postoperative transfusion could not be analyzed.

The clinical heterogeneity would have been too wide between studies. No postoperative transfusion strategy was defined in trials. Indeed, patients underwent different surgeries with different postoperative managements, and potentially different transfusion thresholds. Third, the TSA highlighted a lack of power to detect a statistical difference between groups for the main outcome. Fourth, normal saline may be harmful on renal function and outcomes in critically ill patients when compared to the use of balanced crystalloid solutions.43 It is worth noting that 2 studies used normal saline (Table ), the study by Futier et al12 used boluses of saline serum in the control group while using lactated Ringer's solution for maintenance fluid, and the study by Ghodraty et al29 used serum saline for maintenance fluid while using lactated Ringer's solution for boluses in the control group. However, each study but one29 used a protocol with stroke volume monitoring to guide fluid infusion, and each study respected a maximal amount of infused HES of 33 mL/kg/d.44 Finally, the results on the main outcome and need for RRT and 30-day mortality show wide 95% CI (Figures 2 and3). Those CIs include values indicating potential substantial harm in the HES group for those outcomes.

To conclude, this systematic review with metaanalysis suggests that HES intravascular volume replacement was not associated with a higher risk of 30-day AKI when compared to crystalloid intravascular volume replacement, without reaching a sample size sufficient to conclude to the lack of difference with certainty. CI and TSA do not exclude harmful effects of HES intravascular volume replacement on the renal function. Using HES for intravascular volume replacement in patients undergoing major abdominal surgery might be associated with higher intraoperative RBC transfusion. This study brings out that applying critical care data appears to be exerting caution in the surgical population, especially in major abdominal surgery. Further data from large multicenter studies or big data banks including thousands of patients with major abdominal surgery are needed to draw firm conclusions regarding the association of HES and AKI, RBC transfusion or intra-abdominal complications.

DISCLOSURES

Name:

Joris Pensier, MD.

Contribution:

This author helped with conception and design of the study, acquisition of the data, analysis of the data, drafting the submitted article, and final approval of the version to be published.

Primary Outcome: 30-Day AKI

  ,11,12,14,28,30 Thirty-day mortality was reported in 4 studies.3,11,12,30 Intraoperative RBC transfusion was reported in 5 studies. 11,12,14,28,30 Intraoperative need for norepinephrine was reported in 5 studies.3,11,12,28,29 Thirty-day postoperative intraabdominal complications was reported in 73,11,12,14,28-30 studies. The Table presents the characteristics of intravascular volume replacement for each study included. Only 6% HES 130/0.4 was used.

	Six studies3,11,12,28-30 including 2365 patients presented results for the 30-day AKI
	(Figure

Name:

Audrey De Jong, MD, PhD.

Contribution:

This author helped with conception and design of the study, analysis and interpretation of data, drafting the submitted article, and final approval of the version to be published.

Conflicts of Interest:

None.

Name:

Lucas Deffontis, MD.

Contribution:

This author helped with conception and design of the study, acquisition of the data,

Funding:

The study is an investigator-initiated trial. The study sponsor is Montpellier University Hospital, Montpellier, France. There was no industry support or involvement in the study. The funder had no role in the design or conduct of the study, data collection, analysis or interpretation, the writing of the report or in the decision to submit for publication. The corresponding author had full access to all of the data and the final responsibility to submit for publication.

Conflicts of Interest:

See Disclosures at the end of the article. analysis of the data, drafting the submitted article, and final approval of the version to be published.

Conflicts of Interest:

None.

Name:

Amélie Rollé, MD.

Contribution:

This author helped with analysis of the data, drafting the submitted article, and final approval of the version to be published.

Conflicts of Interest:

None.

Name:

Yassir Aarab, MD.

Contribution:

This author helped with analysis of the data, drafting the submitted article, and final approval of the version to be published.

Conflicts of Interest:

None.

Name:

Mathieu Capdevila, MD.

Contribution:

This author helped with analysis of the data, drafting the submitted article, and final approval of the version to be published.

Conflicts of Interest:

None.

Name:

Clément Monet, MD.

Contribution:

This author helped with analysis of the data, drafting the submitted article, and final approval of the version to be published.

Conflicts of Interest:

None.

Name:

Julie Carr, MD.

Contribution:

This author helped with analysis of the data, drafting the submitted article, and final approval of the version to be published.

Conflicts of Interest:

None.

Name:

Emmanuel Futier, MD, PhD.

Contribution:

This author helped to the conception and design of the study, to the analysis and interpretation of data, to drafting the submitted article, and to provide final approval of the version to be published.

Conflicts of Interest:

None.

Name:

Nicolas Molinari, PhD.

Contribution:

This author helped with analysis of the data, drafting the submitted article, and final approval of the version to be published.

Conflicts of Interest:

None.

Name:

Samir Jaber, MD, PhD.

Contribution:

This author helped with conception and design of the study, analysis and interpretation of data, drafting the submitted article, and final approval of the version to be published.

Conflicts of Interest:

S. Jaber reports receiving consulting fees from Drager, Medtronic, Fresenius, Baxter, and Fisher & Paykel.

Conflicts of Interest:

None.

This manuscript was handled by:

Alexander Zarbock, MD.