Jean-Baptiste Coty

Magali Noiray

Dr Christine Vauthier
email: christine.vauthier@u-psud.fr

Assessment of Complement Activation by Nanoparticles: Development of a SPR Based Method and Comparison with Current High Throughput Methods

Keywords: Complement system -Nanoparticles -Surface plasmon resonance -Automated method ASTM, American Society for Testing and Materials CARPA, Complement Activation Related Pseudo Allergy CVF, Cobra venom factor ELISA, Enzyme-linked immunosorbent assay HAGG, Heat-activated gamma globulin IBCA, Isobutylcyanoacrylate Multi-crossed 2D immunoelectrophoresis

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I. Introduction

The complement system is a major effector of the human innate and adaptive immunity. Consisting of 35 soluble blood proteins, this system can be activated through proteolytic cleavages that are arranged in a cascade when a foreign body is detected (bacteria, virus, nanomaterial). The triggering of this cascade leads to inflammation reactions by the release of anaphylatoxins (C3a, C5a) as well as enhancement of phagocytic activity and formation of lysis complex [START_REF] Merle | Complement System Part I -Molecular Mechanisms of Activation and Regulation[END_REF][START_REF] Merle | Complement System Part II: Role in Immunity[END_REF]. In the case of nanomedicines (NMs) used upon intravenous administration, complement proteins are the first barrier of immunity encountered by the nanomaterial in human blood. The biodistribution, circulation time, or the possible induced toxicity of these nanomaterials can be influenced by their interaction with the complement system [START_REF] Dobrovolskaia | Preclinical Studies To Understand Nanoparticle Interaction with the Immune System and Its Potential Effects on Nanoparticle Biodistribution[END_REF][START_REF] Nilsson | The role of complement in biomaterial-induced inflammation[END_REF]. Indeed, the interaction of NMs with complement proteins triggers this cascade that can lead to severe adverse reactions referenced in the CARPA (Complement Activation Related Pseudo Allergy) [START_REF] Szebeni | Complement activation-related pseudoallergy: a new class of drug-induced acute immune toxicity[END_REF][START_REF] Szebeni | Hemocompatibility testing for nanomedicines and biologicals: predictive assays for complement mediated infusion reactions[END_REF][START_REF] Szeto | Materials design at the interface of nanoparticles and innate immunity[END_REF] and has even been linked to an enhancement of cancer tumor growth [START_REF] Markiewski | Modulation of the anti-tumor immune response by complement[END_REF][START_REF] Moghimi | Cancer nanomedicine and the complement system activation paradigm: Anaphylaxis and tumour growth[END_REF]. Despite these facts, complement activation has not been positioned as a priority assessment in NMs design and in their clinical batch release. While assays are recommended for complement activation of material devices by ISO (International Organization for Standardization) [START_REF]Biological evaluation of medical devices -Part 4: Selection of tests for interactions with blood[END_REF] and ASTM (American Society for Testing and Materials) [START_REF] Astmf | Standard practice for testing for whole complement activation in serum by solid materials[END_REF], only few recommendations for the complement activation evaluation of intravenous drug are existing [START_REF]Immunotoxicology Evaluation of Investigational New Drugs[END_REF][START_REF]EMA Reflection paper on the data requirements for intravenous liposomal products developed with reference to an innovator liposomal product[END_REF]. The development of a direct and automatable method on this purpose could favor its assessment hence improving the safety and knowledge about NMs links with the complement system. Today, the reference method used for in vitro complement activation assessment is ELISA (Enzyme-linked immunosorbent assay) [START_REF] Szebeni | Hemocompatibility testing for nanomedicines and biologicals: predictive assays for complement mediated infusion reactions[END_REF][START_REF] Harboe | Advances in assay of complement function and activation[END_REF][START_REF] Salvador-Morales | Complement activation. Handbook of immunological properties of engineered nanomaterials[END_REF][START_REF] Mollnes | Complement analysis in the 21st century[END_REF][START_REF] Neun | NCL Method ITA-5.2: Analysis of Complement Activation by EIA[END_REF]. This method was standardized and marketed available kits allow the detection of different specific fragments of the complement cascade (iC3b, Bb, C4d, SC5b9). However, this method suffers from its high cost. A method such as two-dimensional immunoelectrophoresis (2D-IE) allows a cheaper assessment of complement activation assessment with a global vision of C3 and its fragments [START_REF] Salvador-Morales | Complement activation. Handbook of immunological properties of engineered nanomaterials[END_REF][START_REF] Coty | Serial multiple crossed immunoelectrophoresis at a microscale: A stamp-sized 2D immunoanalysis of protein C3 activation caused by nanoparticles: Proteomics and 2-DE[END_REF]. This method was recently improved to be applicable on a higher throughput (multi-crossed 2D immunoelectrophoresis: µC-IE), but still remains time consuming. The use of a fully automatable technique such as surface plasmon resonance (SPR) to detect complement fragments in serum is another way to deal with complement assessment. SPR is widely used for affinity and kinetics studies between a ligand and a given substrate. NM-protein interaction has already been studied by SPR by grafting the NMs onto the gold chip before flowing the biological medium and looking for interactions and kinetics of association-dissociation [START_REF] Schneider | Surface plasmon resonance as a high throughput method to evaluate specific and non-specific binding of nanotherapeutics[END_REF][START_REF] Patra | Component-Specific Analysis of Plasma Protein Corona Formation on Gold Nanoparticles Using Multiplexed Surface Plasmon Resonance[END_REF][START_REF] Cedervall | Understanding the nanoparticleprotein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles[END_REF].

However, a major hurdle when working with complement system is the matrix complexity such as encountered in human serum where the non-specific protein binding is a real problem faced in SPR. Development of strategies to overcome this major drawback are emerging but require the engineering of complex chips [START_REF] Masson | Reduction of nonspecific protein binding on surface plasmon resonance biosensors[END_REF][START_REF] Seokheun | Methods of reducing non-specific adsorption in microfluidic biosensors[END_REF][START_REF] Breault-Turcot | Développement d'outils analytiques pour la détection de biomolécules directement dans des fluides sanguins[END_REF]. This typically prevents many applications from being studied by SPR. Nevertheless, some groups already worked on complement detection by SPR with different approaches where the tested biomaterial is generally coated on the chip [START_REF] Arima | Surface plasmon resonance in monitoring of complement activation on biomaterials[END_REF][START_REF] Hirata | Study of complement activation on well-defined surfaces using surface plasmon resonance[END_REF][START_REF] Ricklin | Exploring the Complement Interaction Network Using Surface Plasmon Resonance[END_REF], that limits the possibilities of high throughput analysis of several distinct biomaterials.

Added to these aspects, antibody regeneration is another critical issue when working with SPR. In previously mentioned studies, specific antibodies were flowed on the surface and their binding on complement proteins interacting with the biomaterial surface increased the signal. This procedure supposes a high consumption of antibodies, hence a high cost. The other way is to coat specific antibodies on the chip and then flow activated serum for binding of specific fragments. The difficulty of this procedure is to regenerate coated antibodies to keep their functionality for other samples [START_REF] Drake | A strategic and systematic approach for the determination of biosensor regeneration conditions[END_REF][START_REF] Andersson | Exploring buffer space for molecular interactions[END_REF][START_REF] Makaraviciute | Development of a reusable protein G based SPR immunosensor for direct human growth hormone detection in real samples[END_REF]. This was a challenge taken into account in our method development.

Finally, the way to detect complement activation by looking for a specific fragment is prone to possible artefacts such as adsorption of this specific component on the nanoparticle (NP) thus preventing its detection. A fragment of protein C3 that is quickly released is the C3a fragment (9000 Da), which acts as an anaphylatoxin, triggering inflammatory reactions. Several works reported a possible adsorption of this fragment on polymer surfaces, thus biasing their quantification when assessed in the serum supernatant [START_REF] Szebeni | and technical review[END_REF]. The phenomenon was first evidenced on polymers forming dialysis membrane such as polyacrylonitrile or cellulose acetate [START_REF] Mares | Specific adsorption of some complement activation proteins to polysulfone dialysis membranes during hemodialysis[END_REF][START_REF] Kandus | Anaphylatoxins C3a and C5a adsorption on acrylonitrile membrane of hollow-fiber and plate dialyzer--an in vivo study[END_REF]. Then, their adsorption onto different types of grafted polymers were studied by Montdargent et al. [START_REF] Montdargent | Complement activation and adsorption of protein fragments by functionalized polymer surfaces in human serum[END_REF]. They showed that adsorption on polymer surface was occurring with anaphylatoxins C3a and C5a for neutral, anionic and cationic polymer surfaces. The adsorption greatly depended on the chemical nature of the polymer substituent group. In the present work, we tested different NPs with the same chemical composition but different surface conformations and showed that this parameter was of importance too regarding C3a adsorption. These results were further put in relation with possible bias occurring with current established protocols for complement activation assessment.

The developed SPR method was confronted to standardized methods already used for complement activation assessment. Thus, the approach of this paper allowed a comparison between three methods (ELISA, SPR and µC-IE). Such a comparison has never been reported in the literature. Indeed, complement activation is generally evaluated only with commercially available assays, mostly ELISA.

The purpose of this work was to develop a SPR-based method for a simplified and cost-effective assessment of complement activation by NMs. Results obtained with this method were challenged with standardized and robust methods, namely ELISA and µC-IE. Finally, a complementary study aimed to evaluate the possible bias introduced by the presence of NPs in the medium and thus adapt the optimal conditions for reliable assays. To support this study, a series of five model NPs of the same chemical composition but bearing distinct surface density and conformation chains were used.

II. Material and methods

II.1. Materials

Chemicals: Isobutylcyanoacrylate (IBCA) was provided by Orapi (Saint-Vulbas, France). Tricine, Tris base (Sigma 7-9®), sodium chloride, EDTA sodium salt, dextran 66.7 kDa, and bromophenol blue were purchased from Sigma (Saint-Quentin-Fallavier, France). Dextran 17.7, cerium (IV) ammonium nitrate, and hydrochloric acid were supplied by Fluka (Saint-Quentin-Fallavier, France). Nitric acid was provided by Prolabo (Paris, France). Calcium lactate, glacial acetic acid, coomassie brilliant blue R-250 were supplied by Thermo Fisher Scientific (Villebon-sur-Yvette, France). Gel-Fix™ for agarose (265 x 150 mm) was obtained from Serva Electrophoresis (Heidelberg, Germany). Amine coupling kit, Series S Sensor Chip CM5, HBS-EP+ buffer 10X, NSB reducer and HCl-Glycine pH 2.5 were obtained from GE Healthcare (Velizy-Villacoublay, France). All chemicals were of reagent grade and used as purchased.

Biologicals: Goat IgG (purity > 80%) was obtained from Sigma (Saint-Quentin-Fallavier, France). Polyclonal anti-human C3 antibody raised in goat was purchased from Fitzgerald antibodies (Acton, USA). Monoclonal anti-human C3a neoepitope produced in mouse came from antibodies-online GmbH (Aachen, Germany). Serum was prepared from human plasma obtained from healthy donors from Etablissement Français du Sang (EFS, agreement # 14/EFS/041) (Rungis, France). Heat-activated gamma globulin (HAGG) and Cobra Venom Factor (CVF) were purchased from TECO Medical (Rambouillet, France) to be used as positive controls for the activation of the complement system. Proteins C3b (purity > 90%) and C3c (purity > 80%) were provided by Acris Antibodies GmbH (Herford, Germany). Human complement C3a (purity > 98%) was purchased from Merck Calbiochem  (Fontenay-sous-Bois, France). Human complement C3 protein (purity > 85%) and Microvue complement iC3b EIA kit were purchased from Teco Medical (Rambouillet, France).

II.2. Methods

II.2.1. NPs synthesis and characterization

Five types of nanoparticles were prepared from well described emulsion polymerization methods of IBCA with dextran. The method and characteristics of the nanoparticles were thoroughly described in our previous works [START_REF] Bertholon | Characterization of Dextran-Poly(isobutylcyanoacrylate) Copolymers Obtained by Redox Radical and Anionic Emulsion Polymerization[END_REF][START_REF] Couvreur | Polycyanoacrylate nanocapsules as potential lysosomotropic carriers: preparation, morphological and sorptive properties[END_REF][START_REF] Coty | Tuning complement activation and pathway through controlled molecular architecture of dextran chains in nanoparticle corona[END_REF]. In this work, three NPs were obtained from radical redox emulsion polymerization (R1, R2 and R3), two others were prepared by anionic emulsion polymerization (A1 and A2). They differ by the density and conformation of dextran chains grafted on their surface as illustrated later in this article. They display different capacities to activate the complement system [START_REF] Coty | Tuning complement activation and pathway through controlled molecular architecture of dextran chains in nanoparticle corona[END_REF]. The table 1 depicts the different characteristics of nanoparticles used in this work and the figure 1 shows transmission electron micrographs of the nanoparticles. The characteristics of the nanoparticles were consistent with those presented in previous work [START_REF] Bertholon | Characterization of Dextran-Poly(isobutylcyanoacrylate) Copolymers Obtained by Redox Radical and Anionic Emulsion Polymerization[END_REF][START_REF] Couvreur | Polycyanoacrylate nanocapsules as potential lysosomotropic carriers: preparation, morphological and sorptive properties[END_REF][START_REF] Coty | Tuning complement activation and pathway through controlled molecular architecture of dextran chains in nanoparticle corona[END_REF].

The hydrodynamic diameter of nanoparticles was measured at 20°C by dynamic light scattering (DLS) using a ZetaSizer Nano ZS90 (Malvern Instruments, Orsay, France). Prior analysis, dispersions were diluted in MilliQ ® water at 1/200 (NPs R1 and R2), 1/100 (NPs A1), 1/50 (NPs R3) and 1/20 (NPs A2). Size distributions are shown on figure S1 in the supplemental information. Surface charge was evaluated through the determination of an apparent zeta potential (zeta potential) deduced from measurements of the electrophoretic mobility of the nanoparticles by electrophoresis light scattering (ELS) using the ZetaSizer Nano ZS90 (Malvern Instruments, Orsay, France). The Smoluchowski's model was used to calculate the zeta potential without applying a correction for the surface conductivity. Nanoparticle dispersions were diluted in 1mM NaCl solution to perform this analysis. Transmission electron micrographs were obtained from observations of grids with a Jeol JEM 1400 electron microscope operating at 120 KV and equipped with a camera ORIUS SC1000A2 (Electron Microcopy Facility of I2BC, Gif sur Yvette, France). A 2µL sample of nanoparticle dispersion diluted in filtered MilliQ ® water was deposited on a formvar-carbon cupper grid for electron microscopy and let to dry in a dust free environment. No staining was applied.

Table 1: Physicochemical characteristics of model nanoparticles used for complement activation.

Hydrodynamic diameter, zeta potential, concentration, and schematic representation of the NPs.

Figure 1: Transmission electron micrographs of the 5 dispersions of nanoparticles. Samples were deposited on carbon-formvar coated grids and let to dry in a dust free environment. Observations were performed with a Jeol JEM 1400 electron microscope operating at 120 KV and equipped with a camera ORIUS SC1000A2 (Electron microcopy facility of I2BC). No staining was applied during preparation of the grids.

Sample preparation for complement activation assessments

The five types of NPs were assessed at two different concentrations (1000 and 3000 cm²/mL) for complement activation. The incubation was prepared as described previously [START_REF] Patra | Component-Specific Analysis of Plasma Protein Corona Formation on Gold Nanoparticles Using Multiplexed Surface Plasmon Resonance[END_REF] by diluting human serum (1:4) with VBS 2+ buffer (1:4) and NPs aqueous dispersion at the desired concentration (1:2) or water for controls. The final incubation volume was 100 µL. For the negative control, VBS 2+ was replaced by VBS EDTA, that inhibits complement activation. The positive control was obtained by introducing CVF (5 µL), a strong activator of the complement cascade, in the incubation medium filled with water (45 µL) to reach the final volume of 100 µL. Medium activation was obtained by introducing HAGG (5 µL) in the incubation medium as described above for CVF control.

The incubation mixture was left at 37°C for an hour. After incubation, samples were kept in ice few minutes until analysis. Each

II.2.2.2. Specificity assays and control serums

Pure C3 (185 kDa), C3b (176 kDa) and C3c (140 kDa) proteins were tested at 140 µg/mL in HBS-EP+ buffer. Pure C3a (9,1 kDa) was analyzed at 70 µg/mL in HBS-EP+ buffer. Control serum samples incubated as described above were then diluted by 12.5 in the buffer (HBS-EP+/NSB reducer 9:1) to reach a final dilution factor of 50 for human serum (4 x 12.5). All samples were analyzed sequentially on the different channels at 25°C in the following conditions: flow rate: 40µL/min; contact time: 60 sec; dissociation time: 120 sec. Channels were then regenerated with a two steps procedure: (i) Glycine/HCl pH 2.5: contact time: 30 sec at 40 µL/min, (ii) NaOH 1 mM (30 sec at 40 µL/min). The SPR signal obtained is proportional to the mass of analyte interacting with the surface. The quantification of analyte binding on the channel was expressed in RU (1 RU = 1 pg/mm²) and the report point was taken 10 seconds after the beginning of the dissociation. An example of SPR sensorgram is available in supplementary information S2.

II.2.2.3. NP-serum studies

All serums were prepared and analyzed as described in the previous paragraph. Analyzes were performed on the samples directly after incubation without additional step to remove the nanoparticles. In few experiments, nanoparticles were removed from the samples prior to analysis to study the bias possibilities caused by the removal of the NPs. In this part, the incubated serums were centrifuged at 4°C for 45 min at 16.1 rcf (relative centrifugal force) with an Eppendorf centrifuge 5415R (Eppendorf, Montesson, France) prior to the dilution. Then, the supernatant was recovered for dilution preparation and analysis. Analyzed sera corresponding to these samples were identified as "samples without nanoparticles" in the results and discussion section.

II.2.3. Multi-crossed 2D Immuno-electrophoresis (µC-IE)
Multi-crossed 2D immune-electrophoresis was performed as described previously [START_REF] Patra | Component-Specific Analysis of Plasma Protein Corona Formation on Gold Nanoparticles Using Multiplexed Surface Plasmon Resonance[END_REF][START_REF] Nguyễn | New nanoparticles obtained by co-assembly of amphiphilic cyclodextrins and nonlamellar single-chain lipids: Preparation and characterization[END_REF]. In summary, a 1% agarose gel plate was prepared on Gel-Fix™ alternating plain agarose gel bands (0.8 cm wide) and agarose gel bands containing polyclonal anti-C3 antibody (2.2 cm wide) as explained in a previous work [START_REF] Patra | Component-Specific Analysis of Plasma Protein Corona Formation on Gold Nanoparticles Using Multiplexed Surface Plasmon Resonance[END_REF]. Incubated samples were deposited on wells formed in the agarose gel bands to run the first dimension allowing the separation of proteins according to their molecular weight (600 V,16 mA). Then, the plate was turned by 90° to allow the second-dimension electrophoresis leading to the immunoprecipitation of C3 protein and its fragments in the gel bands containing the polyclonal anti-C3 antibody (500 V, 12 mA). Gel was then dried and stained with Coomassie blue prior to analysis of %C3 activation.

II.2.4. ELISA

Elisa was performed following the instructor procedure. Briefly, the sample was diluted by 100 in the specimen diluent provided by the supplier. Then, the sample was deposited and incubated at room temperature for 30 min. The plate was thoroughly washed with the wash solution. The iC3b reagent was then added and left for 30 min before being washed. Substrate was added for 30 min before adding the stop solution. The ELISA plate was read at 405 nm using LT-5000MS Elisa plate reader (Labtech France, Palaiseau).

III. Results

III.1.

Setup of a regenerable SPR chip for the direct assessment of complement activation by nanoparticles

III.1.1. Antibody immobilization

The SPR chip was grafted with a goat IgG with no affinity for complement fragments on channel 1 to give a non-specific reference channel. Antibody directed against human C3a neo-epitope was coated on channel 2 at around 10,000 RU. Anti-human C3 polyclonal antibody was coated on the third channel at the same level of immobilization. Channel 4 exposed the raw surface of the chip for another reference with no affinity. In this chip, channels 1 and 4 could highlight possible non-specific adsorption while channels 2 and 3 could evaluate specific fragments of the complement system. It was assumed that the non-specific adsorption behavior monitored on channel 1 grafted with the non-specific goat IgG was consistent to that of channels 2 and 3, grafted with antibodies specific to complement fragments.

III.1.2. Specificity assays and control serums

At first, the specificity of the different channels towards different protein C3 fragments was assessed to ensure their relevance for the intended use. Results are depicted in figure 2A. The channel 1 showed no affinity for any of the tested proteins. Channel 2 was specific to C3a released fragment, confirming the ability of the antibody to specifically detect the neo-epitope of C3a. Channel 3 could detect both C3 protein, C3b and C3c fragments, but not C3a. Then, these channels were tested for their ability to detect these specific fragments in sera with different levels of C3 activation obtained from control samples made with known activators (HAGG, CVF) and inhibitors (EDTA) of complement activation. Flow conditions, buffer and sample dilution described in the previous part were optimized in order to give a relevant signal on the response for all channels. Results are presented in figure 2B. CVF is a positive control that produces complement C3 full fragmentation releasing C3a and C3b fragments. The results indicated that non-specific binding was quite important with serum, showing an important background level on channel 1. Nevertheless, channel 2 showed an ability to distinguish activated C3 samples from negative controls. Non-specific binding found on negative control was around 100 RU while the fully activated serum gave a response above 300 RU. In contrast with our previous hypothesis, we have found that the level of non-specific signal depended on each channel according to the grafted antibody. The variation of non-specific binding between the different channels was nevertheless similar. On channel 3, we observed a decrease of the signal for C3 activated samples meaning a decrease of the mass of protein binding to the immobilized antibody. The decrease is substantial, but the amplitude is less important than for channel 2 between negative and positive full activated controls.

III.1.3. Chip longevity

The regeneration was optimized to permit a sustained use of coated antibodies. The longevity of channel 2 was assessed with the fully activated sample (CVF) containing a maximum amount of C3a. The channel 3 longevity was monitored with the negative control (VBS EDTA) containing maximum of C3 native protein (Figure 2C). A quite constant value for C3a detection in a fully activated control serum was obtained on Channel 2 along the analysis of almost 150 samples. A slight variation was observed between the different runs, but the relative standard deviation (RSD) for the positive control on channel 2 over the 150 analyses was < 10%. The behavior of channel 3 was similar to channel 2 showing good performance after regeneration. The signal tended to decrease slightly after nearly 100 analyses. A similar RSD value below 10% was also obtained on channel 3 for the longevity study.

III.2. SPR, µC-IE and ELISA comparison of the assessment of complement activation produced by NPs

The activation profile of five poly-IBCA NPs already well described were compared using three methods for complement activation assessment: SPR developed in this paper, µC-IE [START_REF] Patra | Component-Specific Analysis of Plasma Protein Corona Formation on Gold Nanoparticles Using Multiplexed Surface Plasmon Resonance[END_REF] and ELISA. The results of activation at two given surface concentrations of NPs (1000 and 3000 cm 2 /mL) are presented in figure 3.

The activation of the complement cascade was respectively assessed by the detection of C3a fragment by SPR (channel 2), iC3b fragment by ELISA and the balance between native C3 and released fragment (C3b, C3c) by µC-IE. All these determinations were linked to the cleavage of the C3 protein of the complement system.

Figure 3: Complement activation by five types of NPs assessed by µC-IE, SPR (channel 2) and ELISA. Serum was exposed to 1000 cm2/mL (A) and 3000 cm2/mL (B) of nanoparticles. indicates the activation of C3 protein ( ), leading to the release of protein fragments C3b ( ) and C3a (

).

µC-IE was used as our reference method, giving reproducible results of complement activation based on the monitoring of the cleavage of the C3 protein as demonstrated in previous works [START_REF] Patra | Component-Specific Analysis of Plasma Protein Corona Formation on Gold Nanoparticles Using Multiplexed Surface Plasmon Resonance[END_REF][START_REF] Nguyễn | New nanoparticles obtained by co-assembly of amphiphilic cyclodextrins and nonlamellar single-chain lipids: Preparation and characterization[END_REF]. At 1000 cm 2 /mL, the level of activation expressed in µC-IE was quite low (below 30%) for all tested NPs. The same level was obtained for C3a fragment detected by SPR when comparing with values of negative and positive controls shown in figure 2. At 3000 cm 2 /mL where A1, A2, R2 and R3 NPs are known to strongly activate the complement system [START_REF] Nguyễn | New nanoparticles obtained by co-assembly of amphiphilic cyclodextrins and nonlamellar single-chain lipids: Preparation and characterization[END_REF], SPR measurements of C3a fragments released gave consistent results with C3 cleavage observed by µC-IE. The activation reached the same level as fully activated CVF control for NPs A1, A2, R2, R3 while R1 was non-activating. The ELISA detection of iC3b fragment gave high values of absorbance in all cases. Tendencies that might emerge from this method were counterbalanced by standard deviations that prevent from a possible distinction in terms of iC3b released.

III.3.

Investigation of possible bias involved in complement activation evaluation methods

A study about sample preparation's influence was done with the SPR technique to evaluate possible bias involved in complement activation evaluation. To perform this investigation, nanoparticles were removed from the incubation medium by centrifugation prior to SPR analysis and the results were compared with those obtained analyzing the incubation medium still containing the NPs. The SPR allows the sample to be flowed onto the channels for protein specific recognition. Released proteins are thus captured. In this part, we compared the effect of removal of NPs prior to SPR analysis. Results were depicted in figure 4A.

Figure 4: Comparison of C3a level detection by SPR before and after removal of NPs by centrifugation. A: on samples incubated with NPs for one hour. B: On samples fully activated by CVF and then incubated with NPs for one hour.

An important decrease of C3a signal was observed after removal of most NPs excepted for A1, which seemed to not disturb the determination of the C3a level by SPR (Figure 4A). The observed effect was particularly pronounced for A2, R2 and R3 NPs where C3a signal dramatically felt after removal of NPs. NP R1, while producing only a little amount of C3a fragment also seemed to impact on its detection when they were removed prior to analysis.

Another assay was carried out with serum that was first fully activated with CVF, leading to a full conversion into C3a, and then incubated for one hour with nanoparticles (Figure 4B). The purpose of this experiment was to investigate whether results from analysis of this fully activated serum could be disturbed by incubation and removal of nanoparticles due to possible adsorption of the C3a protein.

The signal on Channel 2 for C3a response was monitored before and after removal of the NPs (Figure 4B). Observations made from the first study depicted in figure 4A were confirmed with this experiment. A strong decrease of C3a level was observed upon removal of NPs for A2, R1, R2, and R3, whereas A1 removal from the incubation medium did not significantly influence the level of detected C3a. A strong adsorption of the C3a fragment onto NPs was thus evidenced for A2, R1, R2 and R3. It is worth noting that A2 does not reduce the signal at the same level as R1, R2 and R3 NPs. Conversely, the response on channel 1, reference for non-specific binding, and channel 3, bearing anti-C3 polyclonal antibody, were almost unchanged between these two types of samples (see Supplementary Information S3). This meant that a strong adsorption of random serum proteins onto NPs was not at the root of the signal decrease observed on channel 2, but rather a very specific adsorption of C3a onto NPs.

IV. Discussion

A SPR chip bearing C3 specific antibodies for the assessment of complement activation by nanoparticles was optimized in this work. The detection of complement activation focused on C3 protein that is at the heart of the complement cascade. Monitoring the activation of this protein allows the assessment of global complement activation. In the developed chip, an antibody against C3a neoepitope and a polyclonal anti-C3 antibody were used. The first allowed a specific detection of a released fragment resulting from C3 activation. The second permitted assessment of native C3 variation. We could observe a decrease of the signal on channel 3 grafted with polyclonal anti-C3 antibody upon complement activation (Figure 2B), meaning a decrease of the mass of protein captured on the surface. This was consistent with the fragmentation of the native C3 (185 kDa) into smaller fragments recognized by this channel (C3b: 176 kDa and C3c: 139 kDa). Then, the C3a fragment was used as a finest revelator of C3 cleavage and thus complement cascade activation.

The aim was to develop an automatable method for a high throughput analysis of samples. The requirements for such an application were firstly to check the antibody specificity with pure proteins before testing their response to different level of activated serums. Specificity of antibodies grafted on channels 2 and 3 could be established using purified C3 proteins and various corresponding fragments produced during activation. Another requirement was the wish to limit costs associated with the analysis. Usually, SPR analysis require the grafting of an antibody on the chip for each sample measurement. The regeneration steps destroy all proteins and then the primary antibody grafted on the chip is removed as well. The regeneration of the chip for removing all specific or non-specific bound proteins was optimized to ensure the antibody functionality and allow a longevity of the chip without the need to recoat the chip prior to each analysis. We showed that the grafted antibody was able to resist for at least 100 analytical cycles including sample analysis and chip regeneration. This can be considered as a good level of longevity. Moreover, no sandwich detection was used in this case. The amount of antibody used was thus extremely low, limiting the costs associated to their use. From that, we estimated the cost of consumables for one analysis by the three methods studied here, in regards with their respective characteristics of throughput, handling complexity and time consumption (cf. Table 2). The estimated price per sample was calculated excluding the instruments prices, it comprised reagents, antibodies, buffers and supports (chip for SPR, gel support for µC-IE, ELISA plate). The price of a SPR analysis was found to be significantly lower than ELISA's, for an automated method allowing the detection of several fragments. The throughput of SPR is lower than other methods but presents the advantage to be easy-to-use and fully automated. The recently renovated µC-IE [START_REF] Coty | Serial multiple crossed immunoelectrophoresis at a microscale: A stamp-sized 2D immunoanalysis of protein C3 activation caused by nanoparticles: Proteomics and 2-DE[END_REF] offering a high throughput use (35 samples per plate) allows a direct visualization of the native C3 and its fragments, providing a global vision of C3 status, in contrast with the ELISA method. Among other advantages, the µC-IE method is much less expensive than the ELISA test. The inherent drawback of this method is its higher time consumption. However, it still can be applied for a deeper understanding of complement activation behavior and concentration studies [START_REF] Coty | Serial multiple crossed immunoelectrophoresis at a microscale: A stamp-sized 2D immunoanalysis of protein C3 activation caused by nanoparticles: Proteomics and 2-DE[END_REF][START_REF] Coty | Tuning complement activation and pathway through controlled molecular architecture of dextran chains in nanoparticle corona[END_REF][START_REF] Nguyễn | New nanoparticles obtained by co-assembly of amphiphilic cyclodextrins and nonlamellar single-chain lipids: Preparation and characterization[END_REF]. The SPR method was challenged to monitor complement activation triggered by a series of 5 nanoparticles for which we had previously established their complement activation status using the µC-IE [START_REF] Coty | Tuning complement activation and pathway through controlled molecular architecture of dextran chains in nanoparticle corona[END_REF]. All nanoparticles were composed of a core of poly(isobutylcyanoacrylate) covered by a neutral polysaccharide, dextran, which conferred almost neutral charge on nanoparticle surface as acknowledged by the low value of the zeta potential. The analysis of complement activation of our five well known NPs showed consistent results between µC-IE and SPR measurements. In contrast, the ELISA methods showed a very high background for all serum samples incubated with nanoparticles independently of the concentration and type of nanosystem. This unexplained result hampered the evaluation of complement activation produced by the different type of nanoparticles considered in this work. The ELISA test measured the protein iC3b which is a different fragment from those detected by the two other methods. Evaluating this fragment that results from the cleavage of the C3 protein into an inactivated C3b fragment could provide a very different insight of complement activation. However, the results obtained with the ELISA method could not be compared those obtained with SPR and µC-IE.

For a control quality analysis of complement activation, SPR could be the candidate of choice due to its simplicity and its possible automation. Nevertheless, while working with specific fragments, it should be verified that no bias or artefacts were interfering with the detection of the analyte. Indeed, in the present study we evaluated the influence of the preparation of a sample prior to its analysis by SPR. Indeed, in the literature, assessment of complement activation produced by nanoparticles is described on both serum samples still containing the nanoparticles [START_REF] Neun | NCL Method ITA-5.2: Analysis of Complement Activation by EIA[END_REF] and on serum samples obtained after removal of the nanoparticles [START_REF] Wibroe | Complement Sensing of Nanoparticles and Nanomedicines[END_REF]. It is generally not explained why analysis is performed on either serum although it has been described that several C3 fragments has the tendency to adsorb on polymer surfaces [START_REF] Montdargent | Complement activation and adsorption of protein fragments by functionalized polymer surfaces in human serum[END_REF]. Our study including the removal of NPs showed that the procedure when dealing with assessment of NP-protein interaction and protein detection is very important. C3a was found to adsorb strongly on R1, R2 and R3 NPs, all bearing a brush conformation of dextran. On the contrary A1 did not adsorb C3a while A2 was adsorbing in a less extent than nanoparticles of the R series. The adsorption of C3a onto certain polymer surfaces has already been mentioned [START_REF] Mares | Specific adsorption of some complement activation proteins to polysulfone dialysis membranes during hemodialysis[END_REF][START_REF] Kandus | Anaphylatoxins C3a and C5a adsorption on acrylonitrile membrane of hollow-fiber and plate dialyzer--an in vivo study[END_REF][START_REF] Montdargent | Complement activation and adsorption of protein fragments by functionalized polymer surfaces in human serum[END_REF] and was confirmed in our study. It is noteworthy that it seems hard to predict this specific adsorption from generally known characteristics of the nanoparticles (i.e. composition, size and zeta potential). Indeed, in our study, all nanoparticles were coated with dextran and only a slight modification on dextran chain architecture between A1 and A2 leads to different behaviors of C3a adsorption. This highlights the fact that careful and critical study of results must be done when dealing with detection of a specific marker for detection of complement but also in other cases when dealing with nanoparticles. As suggested by the results, the removal of NPs prior to analysis may generate bias leading to false negative results that may have dramatic consequence when evaluating the safety of a nanomaterial. When NPs were still in the medium, C3a could be recognized by antibodies grafted on the SPR chip, supposing that this fragment might be at the external surface of NPs in the soft corona and could be readily intercepted by antibodies grafted on the chip surface. The test in presence of CVF confirmed the fact that a specific adsorption of C3a occurred on NPs. Surprisingly, A1 did not adsorb C3a at all, while these NPs are known to adsorb protein of much larger size (data not published). Here, the C3a fragment mostly adsorbed on nanoparticles showing a lower surface curvature. In the context of characterization of interactions between nanomedicine and proteins, this is an insight into the specific adsorption of a small protein of the complement system. Among the known parameters affecting the adsorption, NPs' surface curvature and architecture are known to be of importance [START_REF] Walkey | Nanoparticle Size and Surface Chemistry Determine Serum Protein Adsorption and Macrophage Uptake[END_REF][START_REF] Gref | Stealth" corona-core nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption[END_REF][START_REF] Pozzi | Effect of polyethyleneglycol (PEG) chain length on the bio-nano-interactions between PEGylated lipid nanoparticles and biological fluids: from nanostructure to uptake in cancer cells[END_REF]. Here, these two parameters have a strong influence on C3a deposition onto nanoparticle surface. Results obtained from this part of the work brought new evidences that confirm the high complexity of phenomena governing protein adsorption onto NPs and the need for a better understanding of these interactions influencing the in vivo fate of nanoparticles.

V. Conclusion

A SPR based method for the evaluation of complement activation by nanoparticles through C3 protein fragmentation has been developed in this work. The optimized conditions allowed for a prolonged use of the chip after a single antibody immobilization. Results provided by this method were consistent with those of the multi-crossed 2D immune-electrophoresis method, previously validated for complement activation evaluation. In contrast, no comparison with ELISA could be established due to a high non-specific background found with this method. The evidence of an artefact based on possible fragment adsorption onto NPs was shown by SPR considering analysis of samples containing or not the nanoparticles. This warn about caution required when performing complement activation analysis evaluating nanoparticles. Finally, the SPR method is a rapid and automatable which gave consistent results in terms of complement activation upon direct sample analysis without need of nanoparticle removal. This method could be the method of choice for a daily assessment of complement activation by nanomedicines and nanomaterials in general.

S3: Effect of NPs removal on channels 1, 2 and 3

Figure 2 :

 2 Figure 2: Evaluation of the SPR chip performance. A: Evaluation of antibodies specificities on SPR chip channels. B: channel response to control sera with different level of complement activation. C: Study of the longevity of specific antibodies to C3 fragments grafted on channels 2 and 3. Channel 1: Goat IgG; Channel 2: Anti-C3a monoclonal antibody; Channel 3: Anti-C3 polyclonal antibody.

Figure S3 :

 S3 Figure S3: Responses obtained on channels 1, 2 and 3 for the study on removal with positive controls.

  sample was analyzed in triplicate by SPR and µC-IE and in duplicate by ELISA.

	II.2.2. Surface Plasmon Resonance (SPR)
	II.2.2.1.	Antibody immobilization
	SPR measurements were performed on a Biacore T100 instrument with a Series S sensor chip CM5

equipped with 4 channels (GE healthcare Life Sciences, Vélizy, France). Antibodies were immobilized onto the chip via a standard amine coupling consisting in a successive injection of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) (75 mg/mL, 420 sec, 5 µL/min), Nhydroxysuccinimide (NHS) (11.5 mg/mL, 420 sec, 5 µL/min) and antibody (20 µg/mL) in 10 mM acetate buffer pH 4.5 (420 sec, 10 µL/min) to reach an immobilization level of 10,000 RU (Resonance Unit). The remaining blank were filled with ethanolamine-HCl pH 8.5 (1 M) to prevent for residual non-specific binding (NSB). NSB was assessed on a channel bearing a non-relevant antibody and another channel with a blank surface.

Table 2 :

 2 Comparison of SPR, µC-IE and ELISA methodological aspects towards a routine assessment of complement activation by nanomedicines. Advantages and drawbacks.

		SPR	µC-IE	ELISA
	Cost/sample	1.75 €	0.14 € (20)	6 €
	Throughput	7 samples/hour	35 samples/6 hours 96 samples/3 hours
	Handling	Easy	Technical for gel preparation (45 min)	Not complex. Pipetting can be automated
			Gel preparation -No
	Time consumption	Fully automated	manipulation during electrophoresis (5h)	Full 3 hours
			-Gel staining
	Information	Multiplex fragment	Balance	Single fragment
	obtained	quantitation (C3a, C3)	C3/fragments	quantitation (iC3b)

Acknowledgements

The authors acknowledge: The financial support from BPI-France, Project NICE. IPSIT Platform, Intermol (Chatenay-Malabry, France) for SPR analysis. This work has benefited from the platform and expertise of the Electron Microscopy Facility if I2BC, CNRS, Gif sur Yvette, France.

Supplementary information

S2: Example of a sensorgram obtained by SPR


Christine Vauthier
email: christine.vauthier@u-psud.fr

Denis Labarre

Christine Vauthier Physicochimie

Modular biomimetic drug delivery systems

Keywords:

come

I. INTRODUCTION

Over the last thirty years, considerable efforts have been made to develop new types of drug delivery systems for improving the efficacy and specificity of treatments of serious diseases. A new era has started by the introduction of nanotechnology which makes it possible to produce small drug delivery devices theoretically capable of being diffused in the body and carrying the loaded drug down to target organs. So far, several platforms have emerged making considerable improvements of efficacy and precision of treatments possible and solving difficulties for the delivery of poorly soluble drugs, easy biodegradable molecules and therapeutic compounds with high systemic toxicity [START_REF] Allen | Pharmacokinetics and pharmacodynamics of lipidic nano-particles in cancer[END_REF][START_REF] Couvreur | Nanotechnology: intelligent design to treat complex disease[END_REF][START_REF] Minko | New generation of liposomal drugs for cancer[END_REF][START_REF] Nishiyama | Current state, achievements, and future prospects of polymeric micelles as nanocarriers for drug and gene delivery[END_REF][START_REF] Toub | Innovative nanotechnologies for the delivery of oligonucleotides and siRNA[END_REF][START_REF] Karanth | -pH Sensitive liposomes -Principle and application in cancer therapy[END_REF][START_REF] Devalapally | Role of nanotechnology in pharmaceutical product development[END_REF][START_REF] Torchilin | Micellar nanocarriers: pharmaceutical perspectives[END_REF][START_REF] Vauthier | Nanomedicine: a new approach for the treatment of serious diseases[END_REF].

Basically, the different drug delivery nanotechnologies developed until now can be divided into two categories, whether they are existing biological entities diverted as drug carriers, e.g. inactivated viruses, ghost of red blood cells and bacteria and low density lipoproteins, or artificial constructs [START_REF] Berkel | Drug targeting by endogenous transport vehicles[END_REF][START_REF] Rossi | Erythrocyte-based drug delivery[END_REF][START_REF] Paukner | Bacterial ghosts as a novel advanced targeting system for drug and DNA delivery[END_REF][START_REF] Campos | Current advances and future challenges in adenoviral vector biology and targeting[END_REF][START_REF] Hamidi | Applications of carrier erythrocytes in delivery of biopharmaceuticals[END_REF][START_REF] Riedmann | Bacterial ghosts as adjuvant particles[END_REF]. The latter category has attracted much more work because they are considered to be safer for in vivo uses. Progresses in polymer chemistry and in colloid science have greatly contributed to their development as well as understanding amphiphilic copolymers behavior and macromolecule selfassembly phenomenon [START_REF] Nishiyama | Current state, achievements, and future prospects of polymeric micelles as nanocarriers for drug and gene delivery[END_REF][START_REF] Torchilin | Micellar nanocarriers: pharmaceutical perspectives[END_REF][START_REF] Singh | Recent advances in vaccine adjuvants[END_REF][START_REF] O'hagan | Novel approaches to vaccine delivery[END_REF][START_REF] Wu | Biofabrication: using biological materials and biocatalysts to construct nanostructured assemblies[END_REF][START_REF] Tu | Bottom-up design of biomimetic assemblies[END_REF][START_REF] Sethuraman | Stimuli-sensitive polymeric micelles as anticancer drug carriers[END_REF][START_REF] Qiu | Polymer architecture and drug delivery[END_REF][START_REF] Torchilin | Multifunctional nanocarriers[END_REF].

Generally, drug carriers should hold various properties such as being capable of being loaded with a drug, possessing a very precise biodistribution and drug presentation to the target cells, delivering the drug with a defined delivery pattern and finally biodegrading into nontoxic products capable of being eliminated via natural means. The success of drug delivery can also be greatly affected by the way the device interacts with the biological surroundings encountered after in vivo administration. Finally, these interactions determine the performance of the system and its in vivo fate including the specificity of the drug targeting. To design such complex systems, several approaches have suggested integrating biomimetic methodologies [START_REF] Tu | Bottom-up design of biomimetic assemblies[END_REF][START_REF] Ranney | Biomimetic transport and rational drug delivery[END_REF][START_REF] Hilt | Nanotechnology and biomimetic methods in therapeutics: molecular scale control with some help from nature[END_REF][START_REF] Peppas | Intelligent therapeutics: biomimetic systems and nanotechnology in drug delivery[END_REF][START_REF] Mandal | Interfacing biology with nanoparticles[END_REF].

This paper attempts to review the different strategies integrating biomimetic methodologies to design drug carrier nanotechnology. The first part presents the design of antigen-presenting devices as new formulation tools for vaccines. It shows the integration of a biomimetic approach to build versatile nanotechnologies to present antigens in the correct way to immune cells, by mimicking viruses. The second part focuses on the design of drug delivery devices made of polymers whose surfaces are camouflaged by using a biomimetic approach. This part of the review shows methods that can be used to coat polymer nanoparticles with polysaccharides and how this can be applied to modulate their surface properties and interactions with biological media. Finally, the last part summarizes the recent developments in nanosystems integrating polymeric stimuli responding materials. This will give a more prospective view of the integration of another biomimetic-based methodology suggested to program and control the release of a drug associated with drug carriers.

II. VACCINES

A first field concerned by the application of biomimetic methodologies is the formulation of adjuvants used in vaccines for the prophylaxis of infectious diseases. Indeed, a key problem in vaccinology is the way antigens are presented to antigen-presenting cells such as dendritic cells and macrophages. The main role of the adjuvant added to formulations is to achieve presentation of the antigen to the immune system. The need to have efficient adjuvants is nowadays reinforced because most of the antigens entering the formulation of vaccines are synthetic proteins or peptides for safety reasons [START_REF] Singh | Recent advances in vaccine adjuvants[END_REF][START_REF] O'hagan | Novel approaches to vaccine delivery[END_REF][START_REF] Berzofsky | Progress on new vaccine strategies against chronic viral infections[END_REF][START_REF] Vajdy | Mucosal adjuvants and delivery systems for protein-,DNA-and RNA-based vaccines[END_REF]. These antigens are prepared from recombinant proteins which replace the whole pathogens, or the inactivated subunits found in the traditional vaccines. Recombinant peptide and protein antigens are generally less immunogenic than the corresponding crude inactivated microorganism used in the old vaccine formulations [START_REF] Aucouturier | Adjuvants designed for veterinary and human vaccines[END_REF][START_REF] Jiang | R -Synthetic vaccines: the role of adjuvants in immune targeting[END_REF]. In addition, all molecules of peptidic nature are difficult to administer in vivo because they are unstable in biological fluids and barely diffuse through biological barriers [START_REF] Pawar | -protein and peptide parenteral controlled delivery. -Expert opin[END_REF].

To overcome these drawbacks, it has been suggested to reconsider the way adjuvants are designed. To make the development of new antigen-presenting methods possible, new strategies started to utilize biomimetic approaches. Basically, proposals were made to build artificial viral particles presenting the antigen in the same way that it occurs on the surface of natural viruses. This was made possible due to progresses in immunology, biology, virology, and microbiology over the last thirty years which provided better understanding of the immune system. Moreover, our improved understanding of self-assembling mechanisms of lipids and proteins has made it possible to produce vesicles for reconstituting empty viral capsides [START_REF] Singh | Recent advances in vaccine adjuvants[END_REF][START_REF] O'hagan | Novel approaches to vaccine delivery[END_REF][START_REF] Aucouturier | Adjuvants designed for veterinary and human vaccines[END_REF][START_REF] Noad | Virus-like particles as immunogens[END_REF][START_REF] Huckriede | The virosome concept for influenza vaccines[END_REF]. For instance, nanotechnologies used for liposomes were rapidly integrated into this move because they provided a method for the reconstitution of adjuvants resembling the lipidic envelope of enveloped viruses. Virosomes, which are a recent technology platform, consist in liposome carriers for antigens (Figure 1A). They are already marketed as injectable vaccines against viruses responsible for major health threats in humans including hepatitis, e.g. Epaxal®, and influenza, e.g. Inflexal® (Berna Biotech, Zurich, Switzerland) [START_REF] Huckriede | The virosome concept for influenza vaccines[END_REF][START_REF] Daemen | Virosomes as an antigen delivery system[END_REF][START_REF] Esposito | Clinical and economic impact of influenza vaccination on healthy children aged 2-5 years[END_REF][START_REF] Glück | New technology platforms in the development of vaccines for the future[END_REF][START_REF] Moser | Virosomal adjuvanted antigen delivery systems[END_REF][START_REF] Westerfeld | Peptides delivered by immunostimulating reconstituted influenza virosomes[END_REF].

Figure 1 -Composition and structures of biomimetic inspired antigen presenting devices (adapted from [2]).

Virosomes are formidable antigen-presenting devices made of unilamellar lipid vesicles with a mean diameter close to the size of a real virus (150 nm). Two functional glycoproteins produced by biotechnology are intercalated in the phospholipid bilayer membrane: the influenza hemagglutinin and neuraminidase. They play a key role in the immunoadjuvant properties of Virosomes providing fusion capabilities that enable them to fuse with cell membranes of macrophages and dendritic cells and deliver antigens straight in the cytoplasm of those antigen-presenting cells [START_REF] Moser | Virosomal adjuvanted antigen delivery systems[END_REF]. The second role of the influenza glycoprotein is to enhance the immune response of the carried antigen because it also promotes opsonization of the Virosomes after administration; hence their uptake by the immune cells [START_REF] Moser | Virosomal adjuvanted antigen delivery systems[END_REF][START_REF] Westerfeld | Peptides delivered by immunostimulating reconstituted influenza virosomes[END_REF]. The method used to prepare Virosomes is based on phospholipid vesicle reconstitution by elimination of surfactant from a blend of natural lipids, synthetic lipids, the two glycoproteins and the purified antigen [START_REF] Westerfeld | Peptides delivered by immunostimulating reconstituted influenza virosomes[END_REF]. The antigen provides for specificity of the vaccine (Figure 1A). This method of preparation makes the Virosomes a versatile technology. For instance, antigens can be incorporated in the Virosomes, adsorbed on the surface, or inserted in the lipid bilayer membrane. This allows the influenza vaccine to be yearly formulated using hemagglutinin and neuraminidase as antigens taken from the viral strains recommended by the World Health Organization [START_REF] Moser | Virosomal adjuvanted antigen delivery systems[END_REF]. Although Virosomes were first proposed in vaccines administered by injections, innovations including this adjuvant also concern the development of vaccine formulation for mucosal routes of administration [START_REF] Gluck | Phase 1 evaluation of intranasal virosomal influenza vaccine with and without Escherichia coli heat-labile toxin in adult volunteers[END_REF][START_REF] Gluck | Intranasal immunization against influenza[END_REF]. Indeed, mucosal immunization represents a promising approach to protecting an individual against infections by neutralizing pathogens at its portal of entry [START_REF] Holmgren | Mucosal immunisation and adjuvants: a brief overview of recent advances and challenges[END_REF]. However, Virosomes appeared unsuitable for mucosal administration, inducing unexplained side effects [START_REF] Huckriede | The virosome concept for influenza vaccines[END_REF][START_REF] Gluck | Phase 1 evaluation of intranasal virosomal influenza vaccine with and without Escherichia coli heat-labile toxin in adult volunteers[END_REF][START_REF] Mutsch | Use of the inactivated intranasal influenza vaccine and the risk of Bell's palsy in Switzerland[END_REF]. Since safety is part of a key issue for prophylactic immunization of healthy individuals, nasal formulations of Virosomes against influenza were quickly withdrawn from the market after several months of clinical use, but the corresponding parenteral vaccines are currently applied each year in large vaccination programs without producing side effects.

The second type of adjuvant built on the basis of a biomimetic methodology is the Virus-Like Particle (VLP) [START_REF] Noad | Virus-like particles as immunogens[END_REF][START_REF] Mutsch | Use of the inactivated intranasal influenza vaccine and the risk of Bell's palsy in Switzerland[END_REF][START_REF] Garcea | Virus-like particles as vaccines and vessels for the delivery of small molecules[END_REF][START_REF] Grgacic | Virus-like particles: passport to immune recognition[END_REF][START_REF] Sin | Human papillomavirus vaccines for the treatment of cervical cancer[END_REF]. It looks very similar to empty viruses but differs fundamentally from Virosomes in the sense that it results from the reconstitution of a protein viral capside obtained by spontaneous self-assembly of recombinant viral coat proteins (Figure 1B). VLP can be prepared from recombinant proteins arising from various viral strains [START_REF] Noad | Virus-like particles as immunogens[END_REF] and using immunogenic epitopes of several pathogens such as the human papilloma virus, Ebola virus, HIV or Hepatitis B [START_REF] Garcea | Virus-like particles as vaccines and vessels for the delivery of small molecules[END_REF]. Recently, a human papilloma virus VLP for parenteral administration was approved as a prophylactic vaccine against human papilloma virus infections. It is believed that the immunogenicity of this adjuvant is due to its interactions with dendritic cells that enable the development of both humoral and cellular immunologic response in patients [START_REF] Garcea | Virus-like particles as vaccines and vessels for the delivery of small molecules[END_REF].

Another example of antigen-presenting adjuvant built on a biomimetic approach was developed in the late nineties as formulation of mucosal vaccines to be administered by the nasal route. This adjuvant called Supra Molecular Bio-Vector (SMBV) was constituted by a polysaccharide core surrounded by a phospholipidic bilayer in which antigens can be inserted. The diameter of the whole particle, which presented similarities to empty viruses, was in the 50-100 nm range (Figure 1C). These systems were developed to phase I clinical trials for the nasal vaccination against the influenza viruses. It was demonstrated that the SMBV constituted a platform for the delivery of antigens into cells, while it did not act as an immunomodulator or simple adjuvant. The nasal vaccination with SMBV generated both local mucosal immunity and a systemic cytotoxic T lymphocyte response [START_REF] Hoegen | Synthetic biomimetic supra molecular Biovector TM (SMBV TM ) particles for nasal vaccine delivery[END_REF][START_REF] Debin | Intranasal immunization with recombinant antigens associated with new cationic particles induces strong mucosal as well as systemic antibody and CTL responses[END_REF]. Using an antigen from the group C meningococci, this immunization strategy induced an Immunoglobulin-A mediated bactericidal activity at the mucosal level which can be very effective in fighting the infection at its portal of entry [START_REF] Baudner | Enhancement of protective afficacy following intranasal immunization with vaccine plus a nontoxic LTK63 mutant delivered nanoparticles[END_REF]. All data demonstrated the high potential of SMBV for use as nasal delivery for various antigens and further developments are still expected.

Although methods from nanotechnology were introduced as early as the 1970s using liposomes and nanoparticles, the application of these systems was mainly restricted to a simple encapsulation method insuring protection of antigens against degradation by peptidases prior to their uptake by macrophages [START_REF] Kreuter | -In vitro studies of poly(methylmethacrylate) adjuvants[END_REF][START_REF] Gregoriadis | The immunological adjuvant and vaccine carrier properties of liposomes[END_REF][START_REF] Kreuter | Nanoparticles as adjuvants for vaccines[END_REF]. The introduction of biomimetic approaches in these nanotechnologies made a breakthrough possible, which led to a new generation of antigen-presenting devices to be built on the model of empty viruses and used to formulate prophylactic vaccines which were approved by the authorities. In addition to the opening of an innovative antigen-presenting method, the biomimetic adjuvant suggested so far that it has carrier capability, meaning it can be used to carry and deliver additional compounds to the immune cells [START_REF] Westerfeld | Peptides delivered by immunostimulating reconstituted influenza virosomes[END_REF][START_REF] Storni | Nonmethylated CG motifs packaged into virus-like particles induce protective cytotoxic T cell responses in the absence of systemic side effects[END_REF]. For instance, immuno-stimulating agents such as synthetic bacterial CpG oligonucleotides were incorporated into VLP together with the epitope p33 of the lymphatic choriomeningitis virus glycoprotein incorporated as antigen. The whole particle was shown to protect mice from viral challenges enhancing the immuno-stimulatory capacity of the antigen and reducing side effects of the CpG oligonucleotides [START_REF] Storni | Nonmethylated CG motifs packaged into virus-like particles induce protective cytotoxic T cell responses in the absence of systemic side effects[END_REF].

III. INTEGRATION OF BIOMIMETIC APPROACHES TO CONTROL THE FATE OF DRUG CARRIERS AFTER INTRAVENOUS ADMINISTRATION

The introduction of nanotechnology in the formulation of drugs administered by the intravenous route can considerably improve the success of treatments of serious diseases [START_REF] Couvreur | Nanotechnology: intelligent design to treat complex disease[END_REF]. Such progress can be made possible due to an increase in the precision of the biodistribution of the drug which is then delivered more specifically to targeted organs and cells. The main advantage of using synthetic drug carriers is that they can theoretically be tailor made. Thus, the way they are designed will greatly influence their in vivo fate hence the biodistribution of their cargo. Mechanisms which control the fate of drug carriers administered by the intravenous route involve surface phenomena. They mainly consist in interactions between the drug delivery device outermost layer and components of the biological surrounding medium. For instance, the first event which occurs after intravenous injection of liposomes and nanoparticles is the adsorption of serum proteins [START_REF] Brash | Exploiting the current paradigm of blood-material interactions for the rational design of blood-compatible materials[END_REF][START_REF] Montdargent | Toward new biomaterials[END_REF][START_REF] Wang | Effects of the chemical structure and the surface properties of polymeric biomaterials on their biocompatibility[END_REF][START_REF] Yampolskaya | Proteins at fluid interfaces: Adsorption layers and thin liquid films[END_REF]. This phenomenon is capital for determining whether the drug delivery device will be recognized as a foreign particle by macrophages of the host defense system or whether it will bypass this recognition barrier and remain in the blood [START_REF] Muller | Drug delivery to the brain--realization by novel drug carriers[END_REF][START_REF] Owens | biodistribution and pharmacokinetics of polymeric nanoparticles[END_REF][START_REF] Vonabourg | Parameters influencing the stealthiness of colloidal drug delivery systems[END_REF][START_REF] Labarre | Nanoparticles for site specific drug delivery: from concept to clinic[END_REF][START_REF] Lynch | The nanoparticle-protein complex as a biological entity; a complex fluids and surface science challenge for the 21st century[END_REF]. In a series of recent works, targeting specificity was suggested to be promoted by enhancing the selective adsorption of some proteins on the drug carrier surface. For instance, a higher tropism for the brain was observed in case of PEG-coated liposomes and nanoparticles due to an increase in the spontaneous adsorption of apolipoprotein-E [START_REF] Kreuter | Influence of the surface properties on nanoparticlemediated transport of drugs to the brain[END_REF][START_REF] Jones | Blood-brain barrier transport of therapeutics via receptor-mediation[END_REF][START_REF] Kim | Translocation of PEGylated nanoparticles into rat brain endothelial cells: role of apolipoproteins on receptor mediated endocytosis[END_REF]. Thus, it has been suggested that a promising strategy to promote drug carrier specificity could be based on the development of differential protein adsorption capability by modifying the drug carrier surface properties [START_REF] Muller | Drug delivery to the brain--realization by novel drug carriers[END_REF]. To achieve this aim, it is necessary to develop drug delivery systems with a wide range of controlled surface properties. A biomimetic approach was suggested to be included in this challenge. It consists in copying the surface structures of living cells such as leukocytes, bacteria and viruses which are able to achieve tissue targeting. In these structures, carbohydrates are highly exposed on the surface and constitute an original bio-addressing and signalization arsenal making communication possible between cells and components of the biological surroundings. The development of a biomimetic approach implying the coating of drug carriers with carbohydrates offers almost infinite possibilities as varied as the carbohydrates in nature [START_REF] Passirani | Interactions of nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate) with the complement system[END_REF][START_REF] Passirani | Longcirculating nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate)[END_REF][START_REF] Jaulin | Reduction of the uptake by a macrophagic cell line of nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate)[END_REF][START_REF] Rouzes | Surface modification of poly(lactic acid) nanospheres using hydrophobically modified dextrans as stabilizers in an o/w emulsion/evaporation technique[END_REF][START_REF] Chauvierre | Novel polysaccharide-decorated poly(isobutyl cyanoacrylate) nanoparticles[END_REF][START_REF] Gabius | The sugar code in drug delivery[END_REF][START_REF] Holemann | Carbohydrate diversity: synthesis of glycoconjugates and complex carbohydrates[END_REF][START_REF] Ratner | Tools for glycomics: mapping interactions of carbohydrates in biological systems[END_REF][START_REF] Labarre | Interactions of blood proteins with poly(isobutylcyanoacrylate) nanoparticles decorated with a polysaccharidic brush[END_REF][START_REF] Bertholon | Complement activation by core-shell poly(isobutylcyanoacrylate)-polysaccharide nanoparticles: influences of surface morphology, length, and type of polysaccharide[END_REF][START_REF] Lemarchand | Influence of polysaccharide coating on the interactions of nanoparticles with biological systems[END_REF]. However, it requires the development of methods that make easy integration of carbohydrates possible as surface coating material of liposomes and nanoparticles. Indeed, the surface properties of the drug carriers must be easily modulated to obtain the expected protein adsorption pattern, hence the expected targeting specificity of the drug delivery device.

III.1. Preparation of biomimetic drug delivery systems

Carbohydrate-coated liposomes can be obtained by the incorporation of either natural or synthetic glycolipids in the lipidic composition of liposomes. For nanoparticles, several options were proposed. On the one hand, copolymers can be synthesized by grafting poly(lactideco-glycolide) or poly(ecaprolactone) chains on polysaccharides. After purification, the copolymers are used to formulate nanoparticles by either nanoprecipitation or emulsification-solvent evaporation/ displacement methods [START_REF] Lemarchand | Influence of polysaccharide coating on the interactions of nanoparticles with biological systems[END_REF][START_REF] Huve | Comprendre et éviter la capture des nanoparticules de poly(acide lactique) par le système des phagocytes mononucléaires[END_REF][START_REF] Villemson | Dextran-poly-eta-caprolactone micro-and nanoparticles: preparation, characterization and tamoxifen solubilisation[END_REF]. On the other, polysaccharide-coated nanoparticles can be synthesized by emulsion polymerization of alkylcyanoacrylate monomers which can be initiated on the polysaccharide chains added to the polymerization medium [START_REF] Chauvierre | Novel polysaccharide-decorated poly(isobutyl cyanoacrylate) nanoparticles[END_REF][START_REF] Bertholon | Complement activation by core-shell poly(isobutylcyanoacrylate)-polysaccharide nanoparticles: influences of surface morphology, length, and type of polysaccharide[END_REF][START_REF] Couvreur | Tissue distribution of antitumor drugs associated with polyalkylcyanoacrylate nanoparticules[END_REF][START_REF] Douglas | Particle size and size distribution of poly(butyl 2-cyanoacrylate) nanoparticles. II. Influence of stabilizers[END_REF]. In this case, the carbohydrate-coated nanoparticles are formed at the same time as the copolymers. These polymerization methods developed with alkylcyanoacrylates are very interesting. They are versatile and can be applied using various polysaccharides. They can be used to produce a wide range of nanoparticles directly from commercially available compounds in a one pot process [START_REF] Chauvierre | Novel polysaccharide-decorated poly(isobutyl cyanoacrylate) nanoparticles[END_REF][START_REF] Bertholon | Complement activation by core-shell poly(isobutylcyanoacrylate)-polysaccharide nanoparticles: influences of surface morphology, length, and type of polysaccharide[END_REF]. Playing with the mechanism of the polymerization initiation, i.e. anionic vs. redox radical, nanoparticles with differing spatial arrangement of the polysaccharide chains can be generated in a controlled manner. Table I summarizes the different types of nanoparticles coated with polysaccharide chains that can be synthesized. As evidenced in this table, the main difference between the nanoparticles produced by the methods described above results in the spatial arrangement of the polysaccharide chains at the nanoparticle surface depending on the structure of the copolymers. It may be noted that only the nanoparticles with a hairy conformation of the polysaccharide chains are arranged the same way as oligo-and polysaccharides present on the surface of eukaryotic cells and of viruses [START_REF] Bertozzi | Chemical glycobiology[END_REF]. In contrast, the nanoparticles coated with polysaccharides arranged as loops and trains display an outermost surface structure which looks more or less like the crosslinked structure of the peptidoglycan found at the surface of Gram + bacteria cells [START_REF] Van Heijenoort | Formation of the glycan chains in the synthesis of bacterial peptidoglycan[END_REF].

III.2. Strategy of formulation

So far, several strategies were followed to formulate biomimetic drug delivery systems. In the first, the use of sialic acids as coating material for nanoparticles and liposomes has been explored. The rationale behind this approach was to obtain long circulating drug delivery carriers capable of escaping the immediate capture by the host defense systems and of enabling distribution of the drug towards various organs by increasing residence time in the bloodstream. In nature, sialic acids play an important role as a biological mask hiding epitopes of antigens exposed on cells. For instance, circulating sialic acid coated red blood cells are not recognized by macrophages. After the sialic acid cover has been removed from the cell surface, they are immediately taken up by macrophages due to very efficient cooperation between the complement and phagocytic systems [START_REF] Jancik | Sialic acid--a determinant of the life-time of rabbit erythrocytes. -Hoppe Seylers Z[END_REF][START_REF] Durocher | Role of sialic acid in erythrocyte survival[END_REF][START_REF] Jancik | Sequestration of neuraminidase-treated erythrocytes[END_REF][START_REF] Schauer | Sialic acids and their role as biological masks[END_REF][START_REF] Schauer | Sialic acids regulate cellular and molecular recognition[END_REF][START_REF] Kato | Evaluation of N-succinyl-chitosan as a systemic long-circulating polymer[END_REF].

Table I. Outmost structure of drug carriers designed according to a biomimetic approach and relation with the capacity of the drug carrier to avoid the activation of the complement system (+: activator,non activator, ND: not determined) and the half-life in the blood after intravenous administration. Abbreviations: PLA; poly(lactic acid), PCL: poly(epsilon caprolactone), PACA: poly(alkylcyanoacrylate), PMMA: poly(methylmethacrylate).

By another way, sialic acids are used as surface camouflaging material by extremely virulent bacteria to fool the host defense mechanism. This method helps them bypass the barrier of complement activation and macrophage uptake. It results in severe septicemias in infected patients [START_REF] Ourth | Bacterial sialic acid modulates activation of the alternative complement pathway of channel catfish[END_REF][START_REF] Sakarya | Bacterial adhesins and the role of sialic acid in bacterial adhesion[END_REF][START_REF] Vimr | Diversity of microbial sialic acid metabolism[END_REF]. According to such a biomimetic approach, sialic acids were introduced on the surface of liposomes by incorporating ganglioside M1 (GM1) in the structure of phospholipidic vesicles. The long-circulating capacity of these liposomes was shown to be species dependent. Indeed, long circulating properties in the blood were reported after intravenous injection of GM1-coated liposomes to mice but they were more rapidly removed from the blood stream when injected to rats [START_REF] Gregoriadis | -Polysialic acids: potential in drug delivery[END_REF]. Until today, this species dependency has remained unexplained. Concerning nanoparticles, the first sialic acids coating was obtained by adsorbing orosomucoid, a sialic acid-rich glycoprotein, on the surface of poly(alkylcyanoacrylate) nanoparticles. Opsonisation of the nanoparticles was reduced as well as complement activation, but effective duration of the effects was short because the adsorbed orosomucoid was rapidly displaced by other serum proteins resulting from the Vroman effect [START_REF] Yamauchi | Effects of sialic acid derivative on long circulation time and tumor concentration of liposomes[END_REF][START_REF] Olivier | Stability of orosomucoid-coated polyisobutylcyanoacrylate nanoparticles in the presence of serum[END_REF]. Other approaches suggested using poly(sialic acids) as coating material. Lipids coupled with poly(sialic acids) designed to be incorporated in liposomes have been patented but no data about the performance of these new compounds to produce long circulating liposomes were reported in the literature [START_REF] Gregoriadis | -Polysialic acids: potential in drug delivery[END_REF]. Poly(sialic acids) coated nanoparticles obtained by the incorporation of a copolymer of poly(sialic acids) and poly(lactic acids) showed a significant longer circulation time of the nanoparticles in the blood after intravenous administration to mice when compared to the corresponding uncoated nanoparticles [START_REF] Huve | Comprendre et éviter la capture des nanoparticules de poly(acide lactique) par le système des phagocytes mononucléaires[END_REF]. The longest circulation time was shown by the nanoparticles on which poly(sialic acid) chains adopted a hair conformation. Many advantages are presented by poly(sialic acids): they are biodegradable and their catabolites, i.e. neuraminic acids, are nontoxic; they are T-independent antigens, i.e. they do not induce immunological memory; and the results obtained with poly(sialic acids) coated nanoparticles were promising. However, works were not continued further. It can be assumed that a major problem in the development of poly(sialic acids) coated drug delivery systems can be the difficulty of producing such a camouflaging material. As mentioned above, poly(sialic acids) are extracted from highly pathogenic bacteria strains. Thus, their production requires the handling of highly pathogenic bacteria which should be cultivated only in safe laboratories, generating a high cost for large-scale production.

Among the other polysaccharides of interest, heparin was proposed as the first line candidate. Heparin is a bioactive polysaccharide produced on a large scale as an anticoagulant drug. Interestingly, heparin is also an inhibitor of the activation of the complement system [9496]. Heparin coated nanoparticles did not induce activation of the complement system [START_REF] Passirani | Interactions of nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate) with the complement system[END_REF][START_REF] Chauvierre | Novel polysaccharide-decorated poly(isobutyl cyanoacrylate) nanoparticles[END_REF][START_REF] Labarre | Interactions of blood proteins with poly(isobutylcyanoacrylate) nanoparticles decorated with a polysaccharidic brush[END_REF] and remained in the bloodstream for long period of time with estimated half-life over 5 h after intravenous administration to mice [START_REF] Passirani | Longcirculating nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate)[END_REF]. Other polysaccharides extracted from mushrooms were found to inhibit the activation process of complement. They could be included as alternative polysaccharides to produce nanoparticles with a reduced capacity to activate complement [START_REF] Jeong | Characteristics of anti-complementary biopolymer extracted from Coriolus versicolor[END_REF].

In a more prospective view, several simple polysaccharides were used to modify surface properties of nanoparticles hence the interactions with blood proteins [65-69, 73, 74]. The aim was to develop suitable methods for the production of versatile polysaccharide coated nanoparticles and to investigate how such surface modifications could influence the in vivo fate of the nanoparticles. The polysaccharides used in these still ongoing studies included dextran which is an alpha1-6-poly(glucose), dextran sulfate, a negatively charged derivative of dextran, and chitosan which contains Nacetylglucosamine and deacetylated glucosamine units conferring a net positive charged to the polysaccharide. The results obtained so far have clearly demonstrated that the conformation of the polysaccharide chains present on the nanoparticle surface is one of the key parameters which control complement activation induced by the nanoparticles and opsonisation. The nature of the polysaccharide and the molecular weight were of secondary importance. The charge of the polysaccharides did not show any influence on the complement activation induced by the nanoparticles [START_REF] Gabius | The sugar code in drug delivery[END_REF]. For instance, the complement system was systematically activated in human serum in the presence of nanoparticles displaying loops and trains of polysaccharide chains at the surface, as this is the case for Sephadex which is a crosslinked dextran and for many bacteria [69, 73-75, 98, 99]. Such nanoparticles are recognized by the host defense systems and rapidly cleared from the bloodstream by macrophages of the mononuclear phagocyte systems. In contrast, nanoparticles displaying polysaccharides in the hairy conformation on their surface can avoid activation of the complement system [START_REF] Passirani | Interactions of nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate) with the complement system[END_REF][START_REF] Chauvierre | Novel polysaccharide-decorated poly(isobutyl cyanoacrylate) nanoparticles[END_REF][START_REF] Labarre | Interactions of blood proteins with poly(isobutylcyanoacrylate) nanoparticles decorated with a polysaccharidic brush[END_REF][START_REF] Bertholon | Complement activation by core-shell poly(isobutylcyanoacrylate)-polysaccharide nanoparticles: influences of surface morphology, length, and type of polysaccharide[END_REF] (Table I). Consequently, a hairy dextran-coated nanoparticle was found to escape the intense capture by the macrophages and remained in the blood stream for several hours [START_REF] Passirani | Longcirculating nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate)[END_REF]. Adsorption of blood proteins on polysaccharide coated nanoparticles was also evaluated in plasma and it was shown that the amount and nature of the adsorbed proteins were modulated by the type, conformation and length of the polysaccharidic coating [START_REF] Labarre | Interactions of blood proteins with poly(isobutylcyanoacrylate) nanoparticles decorated with a polysaccharidic brush[END_REF]. Thus, a unique protein adsorption pattern was observed for each type of nanoparticle. This is a very important result in the perspective of developing targeting strategies based a differential protein-adsorption pattern on

In the strategies considered above, interactions of nanoparticle surface with blood proteins were controlled by the polysaccharide coating. Although these included important phenomena such as discrimination between stealth and non-stealth systems, it still did not reach the high degree of specificity required to achieve recognition of the drug delivery system by the targeted cells. In nature, carbohydrates directed towards cell receptors are involved in mechanisms of cell regulation and communications either between cells or with the surrounding medium. Thus, several simple carbohydrates including monosaccharides, disaccharides and Lewis carbohydrates directed toward specific receptors were grafted at the surface of liposomes and nanoparticles [100-105]. Satisfactory results were obtained using relevant cell culture models regarding the level of targeting specificity. Convincing results were also obtained in vivo in targeting liposomes and nanoparticles towards hepatocytes after grafting galactose residues at the surface of the drug delivery system [for references see 106]. These results obtained with simple carbohydrates are very encouraging and promise very exciting developments in the future. Indeed, glycobiology is presently a very active field of investigation. It generates considerable results on the resolution of complex structures of carbohydrates and identifying the corresponding biological activities and functionalities. This will eventually contribute to increasing our knowledge in cell communication and therefore help identify new targets and carbohydrate targeting based materials [70-72, 107, 108]. For further prospective, it would seem preferable to attach the ligand receptor, i.e. carbohydrate, as the targeting moiety on the surface of the drug carrier because the targeting strategy will take advantage of differences in the expression level of the targeted receptor according to the cell physiology. Additionally, it would probably be easier to find a relatively universal method to graft various types of carbohydrate on the surface of drug delivery systems in comparison with coupling methods of protein receptors which need to be finely tuned to preserve the functionality of their active site [START_REF] Holemann | Carbohydrate diversity: synthesis of glycoconjugates and complex carbohydrates[END_REF]. However, one should keep in mind that biological recognition systems are exquisitely sensitive. For instance, they are capable of differentiating small changes in conformation of carbohydrates in antigen epitopes of pathogens. Thus, a difficulty of the method probably requires that the orientation of the carbohydrate moiety grafted at the surface of the biomimetic drug carrier would be perfectly controlled.

IV. INTEGRATED STIMULI-RESPONSIVE PROPERTIES IN DRUG DELIVERY SYSTEMS

Delivery of therapeutic molecules such as DNA, RNA and proteins remain an unresolved significant challenge, as entering the cytoplasm of cells without being degraded is highly complicated. Indeed, the first barrier is crossing the outside membrane of a cell. Then such molecules are generally engulfed in early endosomes, trafficking to late endosomes and finally to lysosomes where they are degraded. Virus and pathogens have developed strategies to enhance protein and nucleic acids transport to the cell cytoplasm escaping the lysosomal compartment. They have evolved potent molecular machineries able to sense the lowered pH gradient of the endosomes and become activated to destabilize the endosomal membrane [109]. Mechanisms developed by viruses were highly used to inspire the strategy followed to design drug carriers making possible delivery of DNA and RNA in the cytoplasm of cells. Acid sensitive liposomes were developed first and have been one of the most extensively studied active triggering release strategies so far [110,111]. One of the remarkable properties of acid sensitive liposomes is that they become fusogenic at acidic pH enabling fusion of the liposomal membrane with the endosomal membrane. Consequently, the liposome content is released in the cell cytoplasm avoiding the intense enzymatic metabolisation of the lysosomal compartment [START_REF] Karanth | -pH Sensitive liposomes -Principle and application in cancer therapy[END_REF]110]. Although the first generation of pH sensitive liposomes were leaky in vivo and lost their sensing properties, these problems were solved by formulating liposomes with lipids of high transition temperature and incorporating lipid-polymer conjugates [111,112]. Interestingly, liposomes formulated with poly(ethylene glycol)-lipid conjugates were additionally less cleared by macrophages and their circulation time in the bloodstream was increased after systemic administration. It was reported that lipid-polymer conjugate liposomes can lose their pH sensitivity. However, in contrast to the previous formulations, it can be restored by incorporating a cleavable bond in the lipid-polymer conjugate so that the polymer moiety of the conjugate can be removed once the liposome has reached the targeted place. This has been obtained using a di-stearoyl glycerol phosphoryl ethanolamine coupled with PEG via a disulfide bond [113].

Several degrees of sophistication can be added to the relatively simple formulations of acid-sensitive liposomes. Peptides having fusogenic properties triggered by an acidic environment were associated with liposomes to deliver the therapeutic cargo in the cell cytoplasm either from the endosomal compartment or directly from the extracellular medium [114].

Development of stimuli-responsive drug carriers is a fast-expanding area. This exciting field of research is boosted by polymer chemistry enabling synthesis of polymeric materials responding to various types of stimuli [115][116][117][118][119]. Stimuli responding polymers have been widely used to formulate controlled drug delivery systems since the early 1990s [120]. Many of them have already been synthesized but the main difficulty is to find components with characteristics suiting the tight constraints imposed by in vivo delivery of drugs. In fact, the polymers must be safe for administration to humans and their responsive characteristics must suit those of the physiological environment in which the drug will be released [118,121]. Most pathological processes related to the methods are accompanied by local temperature increase and/or acidosis and hypoxia because of modification of the cell metabolism [122,123]. However, the ranges of these physicochemical modifications of the environment conditions are narrow and very precise. For instance, in tumors the local temperature increases by 2 to 5°C and/or the pH decreases by 0.5 to 2.5 pH units (acidosis) when compared to normal tissues. Many of the stimuli responding polymers developed so far respond to modifications in pH or temperature but only a few are suitable for in vivo applications. Polymers containing poly(amino acids) have been actively developed for this purpose and have shown great promise to be used as stimuli-responsive material suitable for in vivo applications [START_REF] Torchilin | Micellar nanocarriers: pharmaceutical perspectives[END_REF][124][125][126]. They are now on the front line of the development for various applications in this field. It can also be expected that the new area of the genetically engineered polymers will bring new insight into the development of stimuli-responsive drug carriers in the near future [118,[127][128][129]. Other stimuli-responsive polymers are sensitive to glutathione or calcium concentration modifications, specific enzymes, ultrasounds or light [118,130,131]. In general, all these stimuli-responsive polymers undergo sharp conformational modifications in response to changes in their environmental conditions. Stimuli-responsive polymers coupled to lipids or cholesterol are used to sensitize liposomes to various types of stimuli [131-133] (Figure 2). Change in conformation of polymeric chains anchored at the liposome surface destabilized the phospholipidic bilayer in response to the environmental modification. The liposome therefore becomes leaky and releases the encapsulated drug.

The other types of stimuli-responsive drug carriers are formulated with polymers only. Generally, they are obtained by using block copolymers formed with blocks of different nature. Each block is endowed with a defined function in the drug delivery system. Block copolymers synthesized in the purpose of formulating a stimuli-responsive drug carrier are composed of at least one block of stimuli responding polymer whose sensitivity will be given to the drug carrier device. Stimuli-responsive drug carriers developed with polymers included mainly polymeric micelles. These systems are formed with amphiphilic block copolymers which self-assemble in aqueous media to form spherical nanoparticles with a core-shell architecture [see for example: 4, 8, 20, 115, 130, 134-136] (Figure 2). Hydrophobic molecules can be entrapped in the hydrophobic core of the polymeric micelles. Copolymers containing ionizable groups can be used to entrap charged therapeutic proteins and peptides or DNA and RNA. A block of PEG is generally included in the copolymer structure to confer stealth properties to the stimuliresponsive polymeric micelles [START_REF] Nishiyama | Current state, achievements, and future prospects of polymeric micelles as nanocarriers for drug and gene delivery[END_REF]125]. Polymersomes are another type of drug delivery system made by self-assembly of block copolymers. They are a kind of nanocapsule in which the envelope is formed by a single layer of triblock copolymers containing pH or temperature-sensitive sequences [137-140] (Figure 2). In response to modification of the environmental conditions, polymersomes are capable of inverting their structure, resulting in a release of their content. Finally, stimuli responding dendrimers can be described as unimolecular polymeric micelles. They are formed from star-shaped polymers. Their size ranging from 10 to 40 nm in diameter is much lower than those of the previous objects. They can be sensitized with pH or temperature-sensitive block copolymers.

Although not all the stimuli-responsive drug carriers made of polymers presented in this review are strictly inspired by a biomimetic approach, they show that polymers can be used as sensor platforms to integrate stimuli-responsive functions in drug carriers. Today, polymers can be tailor made with controlled architecture, leading to almost infinite possibility to synthesize smart polymers [START_REF] Qiu | Polymer architecture and drug delivery[END_REF][141][142][143][144][145][146]. They can be used to explore new biomimetic strategies in which stimuli responding systems are at the center of biological events, such as those found in the intercellular communication mechanisms involving neurons, mast cells and endocrinal cells [147-150]. They are generally relatively complex and energetic dependent but have in common the fact that an intracellular signal triggers a massive release of biochemical mediators in the extracellular medium from secretory granules or vesicles which serve as storage containers. These containers enable transient fusion with the cell membrane under reversible structural changes in response to an external stimulus, including a pH decrease or a small variation in the concentration in glutathione or ions [147][148][149][150]. Although the mechanisms of these biological systems are still incompletely understood, they constitute a basis for future developments of stimuli responding drug carriers based on biomimetic strategies [110-112, 132, 151, 152]. For instance, the recent publication of Broz et al. [153] which described a pH switchable channel-equipped, functional polymer nanocontainer shows these very exciting developments to be promising.

V. CONCLUSION

The emergence of new treatments which may involve poorly soluble drugs, easily biodegradable molecules and some drugs endowed with systemic toxicity need highly specific delivery, i.e. targeting. Nanocarriers, including the various types of liposomes, nanoparticles, dendrimers, polymer micelles and polymersomes, are small enough drug delivery devices to serve this purpose. In addition to the usual requirements concerned with drug delivery in humans, they have to be designed in such a way that they can meet with the constraints of the in vivo environments. Together with the improved understanding of the immunological mechanisms and very diverse possibilities in chemistry of polymers and properties of colloids, such nanocarriers can be increasingly tailor made. True targeting requires sophisticated strategies, as delivery of intact drugs such as macromolecular compounds into the cytoplasm of the targeted cells is a complex task. Some bacteria and viruses have acquired such capabilities over centuries of evolution. Mimicking these pathogenic invaders for therapeutic purposes has been progressively developed through the so-called biomimetic strategies. The results obtained in the domain of vaccines are very convincing with already several biomimetic antigen presenting devices on the market. To improve the precision of drug delivery to target cells, development of smart drug carriers capable of delivering their cargo in response to an external stimulus is still in the early stages. However, it can be confidently expected that this nanotechnology will quickly expend taking advantage of the synthesis of stimuli-responding polymers based on biomimetic strategies with perfectly controlled structures and finely tunable properties. The other active area of research concerns the guidance of drug carriers to find their way from the site of administration down to the target site. It has been known for years that sugars and polysaccharides are involved in recognition phenomena, and the introduction of such compounds on the surface of nanocarriers is a promising biomimetic strategy. The results obtained for instance by coating nanoparticles with dextran or chitosan have shown that conformation of the bound polysaccharide on the surface is of prime importance, as nonspecific recognition can be either decreased or increased. As the mechanisms of specific recognition which are probably involved in specific targeting are even more complex, the "magic bullet" of Paul Ehrlich is not yet on the shelf. However, it is no longer merely a pipe dream.

Figure 2 -

 2 Figure 2 -Structures of pH and temperature-sensitive drug carriers.