Manon Debayle

Thomas Marchandier

Xiangzhen Xu

Nicolas Lequeux

Thomas Pons

pH Sensitive visible or SWIR Quantum Dot Nanoprobes using Conformation-Switchable Copolymeric Ligands

Keywords: quantum dots, biosensors, pH, functional coatings, bionanotechnology, photoluminescence

Intracellular and extracellular pH are key parameters in many physiological processes and diseases. For example, the extracellular pH of the tumor micro-environment is slightly more acidic than in healthy tissue. In vivo mapping of the extracellular pH within the tumor would therefore improve our understanding of the tumor physiology. Fluorescent semiconductor quantum dots (QDs) represent interesting probes for in vivo imaging, in particular in the shortwave infrared range (SWIR). Here, pH-sensitive QD nanoprobes are developed using a conformation-switchable surface chemistry. The central fluorescent QD is coated with a copolymer ligand and conjugated to gold nanoparticle quenchers. As the pH decreases from physiological (7.5) to slightly acidic (5.5-6), the copolymer reversibly shrinks, which increases the energy transfer between the QD and the gold quenchers and modulates the QD fluorescence signal. This enables the design of ratiometric QD probes for biological pH ranging emitting in the visible or SWIR range. In addition, these probes can be easily encapsulated and remain functional within ghost erythrocyte membranes, which facilitates their in vivo application.

INTRODUCTION

Fluorescence imaging is a low cost and flexible in vivo imaging modality that has become a standard tool for preclinical imaging. In addition to its ease of use, it offers several advantages compared to other imaging techniques such as positron emission tomography or magnetic resonance imaging, including easy multiplexing and the development of functional probes reporting on their bio-chemical environment. Due to the absorption and scattering of light by superficial tissues, fluorescence signal and spatial resolution decrease however very quickly, and the imaging depth is limited to a few mm. During the past decade, the development of fluorescent imaging in the shortwave infrared range (SWIR), also known as the near infrared (NIR) II range, from 1000 to 1700 nm, have pushed the limits of penetration depth and resolution, thanks to a much lower light scattering compared to the visible or even NIR range. This enables, for example, imaging of the blood vasculature through the skull of mice with ca. 6 µm resolution at nearly 3 mm depth, an unprecedented performance. 1 However, water-soluble SWIR emitting organic fluorophores still lack brightness, with fluorescence quantum yields that usually remain below 1-2 %.

In contrast, fluorescent probes based on quantum dots (QDs) are attracting increasing attention for in vivo imaging. 2 These semiconductor nanocrystals display unique optical properties compared to conventional organic fluorophores including high extinction cross sections for linear and multiphoton excitation, 3 high quantum yields, prolonged photostability, 3,4 long fluorescence lifetimes for timegated detection [5][START_REF] Bouccara | Time-Gated Cell Imaging Using Long Lifetime near-Infrared-Emitting Quantum Dots for Autofluorescence Rejection[END_REF][START_REF] Pons | In Vivo Imaging of Single Tumor Cells in Fast-Flowing Bloodstream Using Near-Infrared Quantum Dots and Time-Gated Imaging[END_REF] ... Most, if not all, QDs share a common basic architecture based on a core-shell semiconductor hetero-structure, typically 2-6 nm in diameter, coated with organic ligands. By changing their size and composition, their emission can be tuned throughout the visible (500-700 nm), near infrared (NIR, 700-1000 nm) and short wave infrared (SWIR, 1000-1500 nm) range. [START_REF] Aharoni | Synthesis of InAs/CdSe/ZnSe Core/Shell1/Shell2 Structures with Bright and Stable near-Infrared Fluorescence[END_REF][START_REF] Dong | Facile Synthesis of Highly Photoluminescent Ag 2 Se Quantum Dots as a New Fluorescent Probe in the Second Near-Infrared Window for in Vivo Imaging[END_REF] With fluorescence quantum yields of 5 to 10 %, SWIR QDs thus appear as promising probes, in particular given the generally poor brightness of other fluorophores in this spectral region. [START_REF] Tsukasaki | Synthesis and Optical Properties of Emission-Tunable PbS/CdS Core-shell Quantum Dots for in Vivo Fluorescence Imaging in the Second near-Infrared Window[END_REF][START_REF] Benayas | PbS / CdS / ZnS Quantum Dots : A Multifunctional Platform for In Vivo Near-Infrared Low-Dose Fluorescence Imaging[END_REF][START_REF] Bruns | Next-Generation in Vivo Optical Imaging with Short-Wave Infrared Quantum Dots[END_REF] So far most applications of QDs for in vivo imaging have consisted in providing contrast to reveal targeted cells or tissues in small animal models, for example using QDs that are bioconjugated to targeting moieties (peptides, antibodies…) via their surface chemistry coating. [START_REF] Kim | Near-Infrared Fluorescent Type II Quantum Dots for Sentinel Lymph Node Mapping[END_REF][START_REF] Cai | Peptide-Labeled near-Infrared Quantum Dots for Imaging Tumor Vasculature in Living Subjects[END_REF][START_REF] Smith | Real-Time Intravital Imaging of RGD-Quantum Dot Binding to Luminal Endothelium in Mouse Tumor Neovasculature[END_REF] Fewer studies have focused on developing surface chemistry coatings to design QDbased probes that would report on their physicochemical environment, such as pH, enzymatic activity or local concentration of specific analytes. pH, for example, is a critical biochemical and physiological parameter. In particular, the pH of the microenvironment of many tumors is slightly more acidic (pH ≈5. 5-6) than in healthy tissue (pH ≈7.4). [START_REF] Tannock | Acid PH in Tumors and Its Potential for Therapeutic Exploitation[END_REF] This acidic pH plays a critical role in many processes such as metabolism, angiogenesis and tumor growth, and may affect drug delivery as well. Developing a SWIRemitting and pH-sensitive QD for in vivo fluorescence imaging would therefore enable mapping the acidity of the tumor micro-environment in depth with a high spatial resolution, as well as monitoring its response to treatments to improve our understanding of these biochemical processes.

Several pH sensitive QD probes have been previously demonstrated, mostly to image endosomal or intracellular pH. For example, QD-dye constructs have been designed where a central visible-emitting QD acts as an antenna for one-or two-photon excitation and is engaged in energy transfer with a pHsensitive fluorescence dye. [START_REF] Preston | A Ratiometric CdSe / ZnS Nanocrystal PH Sensor[END_REF][START_REF] Dennis | Quantum Dot-Fluorescent Protein FRET Probes for Sensing Intracellular PH[END_REF] An alternative strategy relies on ratiometric changes in the fluorescence of large "dot-in-bulk" CdSe/CdS core/shell QDs, where emission from the core provides a reference signal and emission from the large CdS shell is pH-sensitive due to changes in surface passivation. [START_REF] Bruni | Two-Color Emitting Colloidal Nanocrystals as Single-Particle Ratiometric Probes of Intracellular PH[END_REF] However each of these strategies presents limitations. pH-sensitive organic fluorophores suffer from low photostability. In addition, these probes emit in the visible range, which is of limited use for in vivo imaging, and neither of these strategies are easily translatable to the more favorable NIR and SWIR range. Finally, the useful pH range of these probes cannot be easily tuned for optimal response in specific applications. For example, Wang et al. recently reported pH sensitive SWIR organic probes, which operate within the pH 2-4 range, far from physiological pH. [START_REF] Wang | Anti-Quenching NIR-II Molecular Fluorophores for in Vivo High-Contrast Imaging and PH Sensing[END_REF] Herein, we present a novel strategy to design QDbased pH sensors using a copolymeric coating that reversibly changes conformation around a specific pH (Figure 1a). These copolymeric surface ligands are composed of lipoamide and sulfamethazine functions. Lipoamides enable strong binding to the central QD emitter and to small peripheral gold nanoparticle (AuNP) quenchers, while sulfamethazine provides pH-sensitivity by switching reversibly between a charged hydrophilic state and a neutral hydrophobic state around a critical pH value. This transition translates into reversible polymer conformation changes, which modulate the separation distance, and thus the nonradiative energy transfer efficiency, between the QD donor and the AuNP quenchers as a function of pH. This, in turn, modulates the fluorescence signal to provide an optical readout of the environmental pH (Figure 1). Careful design of the pH-sensitive functions and polymer lengths enables us to tune the pH sensitivity range around pH 6-7 under physiological conditions. We characterize the physicochemical and optical response of our QD sensors and demonstrate two strategies to perform ratiometric pH measurements, based on dual color imaging or fluorescence lifetime measurements. We demonstrate that our QD sensors can be easily integrated within erythrocyte ghosts to translate these probes to in vivo imaging. Finally, we show that this strategy is adapted to a broad spectral range of QDs, including visible and SWIR emitting QDs, thus paving the way to high resolution fluorescence pH imaging in deep tissues.

EXPERIMENTAL SECTION

2.1 Materials: N-(3-Aminopropyl)methacrylamide hydrochloride (APMA) was purchased from Tebu-bio and 2.2'-Azobis(2.4-dimethyl)valeronitrile from Molekula. All other chemicals were purchased from Sigma-Aldrich and used without further purification.

Synthesis of visible-emitting QDs:

Core/shell CdSe/CdS/ZnS nanoparticles were synthesized according to previously published protocols. CdSe cores were synthesized from cadmium myristate and Se powder in octadecene at 240 °C, with reinjections of cadmium oleate and Se dissolved in octadecene at 300 °C to make larger, redder quantum dots if needed. [START_REF] Yang | Synthesis of CdSe and CdTe Nanocrystals without Precursor Injection[END_REF] After purification by precipitation in ethanol and resuspension in hexane, a shell composed of typically three monolayers of CdS, two monolayers of CdZnS and two monolayers of ZnS was grown using cadmium oleate, zinc oleate and sulfur precursors in octadecene, according to the SILAR procedure. [START_REF] Xie | Synthesis and Characterization of Highly Luminescent CdSe-Core CdS/Zn 0[END_REF] The obtained core/shell QDs were precipitated in ethanol and stored in hexane until use. Their concentration was determined using the absorbance of the CdSe cores at 350 nm. [START_REF] Leatherdale | On the Absorption Cross Section of CdSe Nanocrystal Quantum Dots[END_REF]

Synthesis of SWIR-emitting QDs:

The synthesis of PbS and PbS/CdS QDs was adapted from previous reports. [START_REF] Cademartiri | Solventless , and Diffusion-Controlled Route to Highly Monodisperse PbS Nanocrystals[END_REF][START_REF] Neo | Synthesis of PbS/CdS Core-Shell QDs and Their Nonlinear Optical Properties[END_REF] Briefly, a sulfur-oleylamine (S-OAm) precursor solution was prepared by heating sulfur (0.16 g) and oleylamine (15 mL) at 125°C under argon for 30 min. Separately, PbCl2 (0.834 g) was dissolved in oleylamine (7.5 mL) and degassed under vacuum for 30 min at 125 °C. It was then heated for 30 min at 150 °C under argon atmosphere. The temperature was lowered to 60 °C and the S-OAm solution (2.25 mL) was injected. The temperature was maintained at 60 °C for typically 4-10 min before cooling down to room temperature, depending on the desired final emission wavelength. The PbS QDs were precipitated in ethanol and resuspended in 10 mL hexane. 3 mL of this PbS core solution was precipitated in ethanol and resuspended in octadecene (3 mL). Separately, a mixture of cadmium oleate (3.9 mL, 0.5 M in oleic acid) and octadecene (2.6 mL) was degassed under vacuum at 110°C then cooled down to 100 °C under argon. The PbS QD solution in octadecene was then injected and maintained at 100 °C for 10 minutes. The solution was then cooled down to room temperature, precipitated in ethanol and resuspended in 10 mL hexane. The core/shell QD concentration was estimated from absorbance and TEM size measurements on the PbS core solution. [START_REF] Moreels | Size-Dependent Optical Properties of Colloidal PbS Quantum Dots[END_REF] 2.4 Synthesis of gold seeds: Gold seeds nanoparticles, hereafter referred to as AuNP, were synthesized following a previously reported protocol. [START_REF] Duff | A New Hydrosol of Gold Clusters[END_REF] To a solution of 91 mL of pure milliQ water, NaOH (3 mL, 0.2 M) and then tetrakis(hydroxymethyl)phosphonium chloride (THPC, 24 µL, 80 %wt.) were added. After 5 min of stirring, hydrogen tetrachloroauric acid (HAuCl4, 6 mL, 20 mM) was injected in the solution dropwise. The solution turns brown during 15 min of stirring and is stored at 4°C until used. Their concentration was determined using the extinction cross section of the particles estimated from Mie scattering calculations and their absorbance at 400 nm. [START_REF] Haiss | Determination of Size and Concentration of Gold Nanoparticles from UV-Vis Spectra[END_REF]

Synthesis of pSMZ-LA:

The synthesis of monomers is described in the Supporting Information. The statistic copolymers were prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. In those syntheses, we used: 4-cyano-4[(dodecyl-sulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDP) as the chain transfert agent (CTA) and 2,2'-azobis(2,4-dimethylvaleronitrile) (V65) as the initiator. As a typical example, sulfamethazine methacrylamide monomer (3 mmol, 300 eq), dihydrolipoïc acid methacrylamide (0.3 mmol, 30 eq), CDP (0.01 mmol, 1 eq) and V65 (0.005 mmol, 0.5 eq) were combined in a 50 mL round-bottomed flask and dissolved in DMF (7 mL). The mixture was purged by argon bubbling for 1 h and thereafter heated in an oil bath at 70°C during 10 h. The polymer was precipitated in methanol, then purified by two cycles of dissolution in DMF and precipitation in methanol and finally dried under vacuum. The polymerization efficiency was evaluated by 1 H NMR, corresponding to a degree of polymerization of approximatively DPn = 80 and a molar mass Mn of approx. 29 000 g/mol for this specific example. As a consequence, this polymer will be referred to as pSMZ65/LA15. Other polymers with different DPn and SMZ/LA ratios were prepared in a similar fashion.

Ligand Exchange:

The nanoparticles were transferred in aqueous solution after (i) substitution of the initial organic oleate/oleylamine ligands by 3mercaptopropionic acid (MPA) and then (ii) subsequent MPA ligand exchange with pSMZ/LA statistic copolymers. [START_REF] Tasso | Sulfobetaine-Vinylimidazole Block Copolymers: A Robust Quantum Dot Surface Chemistry Expanding Bioimaging's Horizons[END_REF] 4 nmol QDs in hexane were precipitated by ethanol addition followed by centrifugation. QDs were resuspended in pure MPA (400 µL) and left overnight at 60°C. The MPAmodified QDs were thereafter precipitated in chloroform.

They were redispersed in dimethylformamide (1 mL) and reprecipitated with potassium tert-butoxide (typ. 50 mg). QDs were centrifuged and the obtained precipitate was rinsed with ethanol and centrifuged again. The 4 nmol of MPA-modified QDs were then resuspended in sodium bicarbonate buffer solution (400 µL, 0.1 M, pH 9). In parallel, 10 mg of the statistic copolymer ligand pSMZ/LA were reduced by sodium borohydride (10-20 mg) in water (2 mL) during 1 h. The ligand and nanoparticle solutions were then mixed together and left to react for one night at 60°C. Then, the polymer exchanged nanoparticles were filtered (100 kDa cutoff Vivaspin membrane) and rinsed with NaCl solution (20 mM, 500 µL, 4 times) to remove excess copolymer and MPA. The resulting nanoparticles were size-purified via ultracentrifugation (268,000 g, 25 min) in a 10-40 wt % sucrose gradient (20 mM NaCl). The nanoparticle band formed after ultracentrifugation was extracted (BioComp Instruments, Inc., Fredericton, NB, Canada). The excess sucrose was removed by a series of filtration through a Vivaspin 100 kDa membrane. The purified nanoparticles were finally resuspended in buffer solution (1 mL, HEPES/Na 100 mM, NaCl 40 mM, pH 7.5) and stored at 4°C until use. The resulting QDs capped with pSMZ/LA are hereafter referred to as QD-pSMZ/LA.

In addition, QDs insensitive to pH were prepared to provide a reference signal. These QDs underwent a ligand exchange following the same procedure as above with a poly(vinylimidazole-b-sulfobetaine) block copolymer instead of pSMZ/LA. Synthesis of this block copolymer is described in Ref. 29 and in the Supporting Information. The resulting QDs are referred to as SB-QDs.

2.7

Anchoring gold nanoparticles onto QD-pSMZ/LA: Quantum Dots coated with pSMZ/LA as described above (1 molar equivalent, typ. 0.5-1 nmol) were dissolved in NaCl solution (1 mL, 150mM) at pH=7.5 and gold nanoparticles (typ. 5 molar equivalents) were added. The solution was left for 10 min, so that the disulfide bridge of LA from the polymer was reduced on AuNP surface allowing the AuNP to be attached onto QD-pSMZ/LA. Then, a short molecular ligand, lipoic acid-sulfobetaine (LASB, see Supporting Information for the description of the synthesis, 1000 equivalents) was added in order to saturate the gold nanoparticles surface. The resulting QD-gold complexes are referred to as QD-SMZ/Au for brevity.

Incorporation into erythrocytes:

Erythrocytes were drawn from healthy volunteers and centrifuged in 1 mL saline buffer (HBS: 10 mM HEPES, 150 mM NaCl, pH 7.4) supplemented with 2 mg/mL Na2(EDTA) (600 g, 5 min, 3 times). A 20 μL pellet containing erythrocytes was mixed with 60 µL of SMZ/Au-and SB-QDs (typical concentration 80 µM) in hypotonic HEPES saline buffer (HEPES/Na 2.2 mM, NaCl 33 mM, pH 7.5) for 20 minutes. 1 mL of 100% HBS was then added, and erythrocytes were purified by three rounds of centrifugation (14,000 g, 5 min) and resuspension in HBS. In parallel, a solution at 2 mg/mL of amphotericin B5 was prepared and 2 µL and were added to the labeled erythrocytes.

2.9 Characterization: Transmission electron microscopy was performed on a Jeol 2010F microscope. Fluorescence spectra were recorded using a F900 spectrometer (Edinburgh Instruments). Fluorescence quantum yields of SWIR emitting QDs were measured by an integrating sphere and confirmed by comparison with a reference indocyanine green dye ( = 13% in DMSO). Absorption spectra were recorded using a Shimadzu UV-1800 spectrometer. Fluorescence microscopy images were acquired with a IX71 Olympus widefield epifluorescence microscope using a 60 × 1.2 NA water immersion ojective and a Roper Cascade 512B CCD camera. Dynamic light scattering measurements were performed on an ALV/CGS-3 goniometer system equipped with a HeNe laser (633 nm) and an ALV/LSE-5003 correlator.

RESULTS AND DISCUSSION

3.1

Copolymer ligand synthesis and characterization

The polymers were synthesized by RAFT copolymerization using 4-cyano-4[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDP) as the chain transfert agent (CTA) and 2,2'azobis(2,4-dimethylvaleronitrile) (V65) as the initiator. Polymerizations were carried out at 70°C in DMF with an initial CTA:initiator ratio of 2:1. The copolymers are composed by two methacrylamide monomers containing sulfamethazine (SMZ) and lipoamide (LA) functions as represented in Figure 1. SMZ is the pH-dependent monomer [START_REF] Abel | Tunable PHand CO 2 -Responsive Sulfonamide-Containing Polymers by RAFT Polymerization[END_REF] whereas LA ensures binding of the polymer onto both the central QD emitters and the peripheral AuNP quenchers. 1 H NMR analysis of the raw polymerization solution and of the purified polymer was used to evaluate the polymerization efficiency (typically ≈ 40%) and the SMZ:LA ratio in the purified polymer. As a typical example, we used a SMZ:LA ratio of 5:1 , and a CTA:total monomer ratio of 1:180. For this polymer, we estimate a degree of polymerization DPn = 80, (molar mass Mn ≈ 29000 g/mol) and a final SMZ:LA ratio of 5:1. As a consequence this polymer will be hereafter referred to as pSMZ65/LA15. The pKa of sulfonamides can be tuned via chemical modifications, from pH = 5 to pH = 9. [START_REF] Abel | Tunable PHand CO 2 -Responsive Sulfonamide-Containing Polymers by RAFT Polymerization[END_REF] In this work, we selected sulfamethazine (SMZ) for its pKa value in the biologically relevant pH range, around 7-8. The hydrophilic-hydrophobic transition of the free polymer is observable through turbidity measurements as a function of pH, as shown in Figure 2a. When the pH is higher than the polymer cloud point, enough sulfonamide groups are deprotonated and negatively charged to ensure water solubility of the polymer. However, when pH is lower than the cloud point, sulfonamide functions become protonated and the predominantly hydrophobic polymer becomes insoluble in water and aggregates. The solution becomes turbid and its transmittance decreases. Figure 2a shows that the cloud point strongly depends on salt concentration in the aqueous solution. Increasing the salt concentration tends to decrease polymer solubility due screening of the electrostatic charges. As a result, the cloud point shifts to higher pH, since a larger fraction of SMZ needs to be charged to ensure polymer solubility in water. Interestingly, increasing the polymer size also shifts the cloud point to higher pH, from 6 (DPn = 12) to 8 (DPn = 100), as shown in Figure 2b. Acid-base titrations show in contrast that the effective pKa* of the different SMZ polymers were around 7.9 (compared with 7.5 for the SMZ monomer), independently of size or salt concentration. This suggests that larger polymers aggregate more readily than shorter ones, even though they carry the same fraction of charged and uncharged species at a given pH. Incorporation of the small fraction of LA functions did not seem to modify the pH dependent conformation changes of these polymers.

In vitro characterization of the pH-sensitive QD probes

We next investigated the behavior of pSMZ/LA polymers bound to the surface of QDs. Visible emitting CdSe/CdS/ZnS core/multishell QDs were first solubilized in water using mercaptopropionic acid (MPA) as a short labile ligand. A second ligand exchange was then performed to replace MPA with pSMZ/LA polymers in which lipoamides have been previously reduced to dihydrolipoamides (DHLA). QDs were then purified by ultracentrifugation to eliminate MPA and excess polymer ligands. The multiplication of anchoring LA functions on the same polymer strand provides long term stability of the polymer on the QD surface, as previously demonstrated with other poly-LA based polymeric ligands. [START_REF] Yildiz | Biocompatible CdSe-ZnS Core-Shell Quantum Dots Coated with Hydrophilic Polythiols[END_REF][START_REF] Palui | Poly(Ethylene Glycol)-Based Multidentate Oligomers for Biocompatible Semiconductor and Gold Nanocrystals[END_REF][START_REF] Giovanelli | Highly Enhanced Affinity of Multidentate versus Bidentate Zwitterionic Ligands for Long-Term Quantum Dot Bioimaging[END_REF] Dynamic light scattering (DLS) measurements in basic pH yield hydrodynamic radii of 8.5-9.5 nm, which is consistent with the expected size of these inorganic QDs (ca. 7 nm in diameter, Figure S1) surrounded by a layer of polymeric ligands.

Protonation of the SMZ groups at acidic pH leads to a decrease of the hydrodynamic radius of about 2 nm compared to more basic pH (Figure 3a,b). This corresponds to shrinking of the polymer layer surrounding the nanoparticles when it becomes partially hydrophobic. It is important to note that the QDs remain however colloidally stable in water or saline solution, as demonstrated by their monomodal intensity size distribution in DLS. At even more acidic pH, below 5, the polymers become completely hydrophobic, leading to the aggregation of the QDs and to a significant increase of the apparent hydrodynamic radius. Increasing salt concentration and polymer length lead to a shift of the transition pH towards more basic values, consistent with observations on the free polymer. In subsequent experiments, we select pSMZ65/LA15 with a pH transition around 7 when dispersed in a 150 mM NaCl solution.

AuNPs (ca. 2-3 nm in diameter, Figure S2) were assembled around pSMZ/LA coated QDs using the remaining LA functions that were not immobilized on the QD surface by simply mixing both nanoparticles in basic solution, above the polymer pH transition. Then, a short hydrophilic ligand, lipoic acidsulfobetaine (LA-SB), was added to saturate the remaining AuNP surface (Figure S11). In its oxidized form, LA-SB is able to bind to the AuNP surface, but not to the QD surface. This prevents displacing the pSMZ/LA polymers from the QDs. Ultrafiltration was then performed to eliminate excess LA-SB and small unconjugated AuNPs. Backtitration shows that, for AuNP:QD ratio below 20, the conjugation of AuNP onto pSMZ/LA QDs reached a >95 % efficiency (Figure S11). This high assembly efficiency could be attributed to the high affinity of free LA functions for AuNPs surfaces. Figure 4a shows electron microscopy pictures of the QD-pSMZ/LA-AuNP assemblies, with central QDs surrounded by several peripheral AuNPs. Adding increasing numbers of AuNPs around the QDs leads to a progressive quenching of their photoluminescence (PL) intensity, even under basic conditions when the polymer is fully extended, as shown in Figure 4b. In this configuration, 50% quenching efficiency occurs at a AuNP:QD ratio of 5:1. This high quenching efficiency despite the large QD-AuNP center-to-center distance is consistent with previous reports of QD-AuNP nonradiative energy transfer. [START_REF] Yun | Nanometal Surface Energy Transfer in Optical Rulers, Breaking the FRET Barrier[END_REF][START_REF] Pons | On the Quenching of Semiconductor Quantum Dot Photoluminescence by Proximal Gold Nanoparticles[END_REF][START_REF] Breshike | Leaving Fo ̈ Rster Resonance Energy Transfer Behind: Nanometal Surface Energy Transfer Predicts the Size-Enhanced Energy Coupling between a Metal Nanoparticle and an Emitting Dipole[END_REF][START_REF] Hildebrandt | Energy Transfer with Semiconductor Quantum Dot Bioconjugates : A Versatile Platform for Biosensing , Energy Harvesting , and Other Developing Applications[END_REF] This interpretation is confirmed by timeresolved PL measurements, which show a progressive acceleration of the QD PL kinetics due to the additional nonradiative decay channels (Figure 4c). Quenching efficiencies derived from steady-state and time-resolved PL measurements are consistent with a progressive homogeneous quenching of the QD population, as opposed to total quenching of a QD population fraction.

When these QD-pSMZ/LA-AuNP nanoassemblies were subjected to an acidic pH below the polymer transition pH, shrinking of the polymer reduced the QD-AuNP distance, which increased the QD PL quenching. Figure 4d shows the relative changes in QD PL between basic (pH=7) and acidic (pH=5.5) conditions as a function of AuNP:QD ratio. This relative pH-induced quenching first increases with increasing AuNP:QD ratio, then reaches a maximum value at ratios around 5-10, in the region where the initial QD PL quenching is around 50%. This is also consistent with a nonradiative energy transfer mechanism since energy transfer theories predict that the 50% quenching point is where the slope of the energy transfer efficiency curve vs. separation distance is the highest. [START_REF] Yun | Nanometal Surface Energy Transfer in Optical Rulers, Breaking the FRET Barrier[END_REF][START_REF] Pons | On the Quenching of Semiconductor Quantum Dot Photoluminescence by Proximal Gold Nanoparticles[END_REF][START_REF] Breshike | Leaving Fo ̈ Rster Resonance Energy Transfer Behind: Nanometal Surface Energy Transfer Predicts the Size-Enhanced Energy Coupling between a Metal Nanoparticle and an Emitting Dipole[END_REF][START_REF] Hildebrandt | Energy Transfer with Semiconductor Quantum Dot Bioconjugates : A Versatile Platform for Biosensing , Energy Harvesting , and Other Developing Applications[END_REF] Then the relative pHinduced changes decrease, since the QD PL becomes more and more efficiently quenched even under basic conditions.

We note that in the absence of AuNP, pH-induced variations in QD PL are very limited (< 2%). This demonstrates that PL varations in QD-pSMZ/LA-AuNP assemblies are indeed due to pH-induced changes in QD-to-AuNP distances and not to other less controllable parameters such as the degree of QD surface passivation or directly to shrinking of the surrounding polymer layer. In the subsequent experiments, we selected the optimal AuNP:QD of 5.

The pH dependence of the selected nanoprobes, QD-pSMZ/LA-5AuNPs, was studied as a function of pH in 150 mM NaCl conditions to mimic biologically relevant salinity. As shown in Figure 5a, the QD PL progressively decreases between pH 7.5 and 5.5, with a more pronounced drop below pH 6.5. The total relative pH-induced variation at pH 5.5 reached about 50%. Time-resolved QD PL accelerates accordingly, as shown in Figure 5b. This progressive quenching is consistent with shrinking of the polymer layer as a progressively more important fraction of SMZ monomers become hydrophobic, while the global nanoprobes assembly remains hydrophilic and colloidally stable in solution.

We then investigate the reversibility of our pHsensitive nanoprobe. QD-pSMZ/LA-5AuNPs were subjected to several pH-cycles between basic and acidic conditions. Figure 6 and S13 show that pHinduced changes in both PL steady-state intensities and kinetics were reversible for these 3 cycles of pH variations. In contrast, the PL intensity of QD-pSMZ/LA without any conjugated AuNPs remained within +/-3 % of its initial value when subjected to the same pH cycles. This behavior stems from the fact that pH causes only reversible conformation changes of the polymer layer. This represents a critical advantage over strategies based on pH-cleavable linkers, [START_REF] Wang | Evaluating Tumor Metastatic Potential by Imaging Intratumoral Acidosis via PH-Activatable near-Infrared Fluorescent Probe[END_REF] since those would irreversibly become cleaved over time, even at moderately acidic pH.

Ratiometric sensing in erythrocyte carriers

Quantitative pH imaging in biological samples requires a ratiometric signal. Indeed, a weak PL signal could mean either a low QD concentration in a basic or neutral environment or a higher QD concentration in an acidic environment. Variations of PL decay kinetics shown in Figure 5b could enable pH estimations based on lifetime measurements. However these variations occur mainly at pH below 6, whereas QD PL intensities vary more progressively. We therefore chose to develop a dual color system composed of pH-sensitive QD-pSMZ/LA-AuNPs emitting at 610 nm (hereafter referred to as QD610-SMZ/Au) mixed with a reference pH-insensitive QD population coated with a poly(vinylimidazole-bsulfobetaine) polymer (Figures S8,S9) and emitting at 650 nm (QD650-SB). [START_REF] Tasso | Sulfobetaine-Vinylimidazole Block Copolymers: A Robust Quantum Dot Surface Chemistry Expanding Bioimaging's Horizons[END_REF] Figure 7a shows a strong quenching of QD610-SMZ/Au PL intensity between pH 7 and 5.5 which is consistent with previous measurements. In contrast, the PL intensity of QD650-SB remained constant, providing a useful reference signal. Cycling between neutral and acidic conditions shows that the variations of the 610/650 PL ratio are fully reversible (Figure 7b). Similarly, time-resolved PL measurements shown in Figure 7c demonstrate that the PL decay kinetics of the QD650-SB do not depend on pH within this physiological range, while QD610-SMZ/Au exhibit a marked acceleration under acidic conditions. These changes in PL decay kinetics are also fully reversible when cycling between neutral and acidic conditions. Similar experiments repeated at higher salt concentrations demonstrated a similar behavior, with more pronounced variations of the QD610-SMZ/Au PL properties due to charge screening as discussed above (Figure S14).

Taken together, the above results show that a dual color system composed of QD610-SMZ/Au and QD650-SB could be used to perform ratiometric pH imaging in biological environments. For in vivo imaging, two difficulties are however expected. Once injected in the blood stream, the anionic nature of the SMZ polymer is susceptible to induce rapid adsorption of serum proteins. This would lead to subsequent opsonization and efficient uptake by macrophages from the reticuloendothelial system. In addition, two QD populations bearing different surface chemistries may exhibit considerably different biodistributions after intraveneous injection, making ratiometric imaging impossible in practice. We propose to circumvent these limitations by incorporating both pH-sensitive and reference QDs into ghost erythrocytes. As the most abundant blood circulating cells, erythrocytes are attractive natural carriers to transport functional imaging agents or drugs in the bloodstream, while ensuring excellent biocompatibility and prolonged blood circulation times. [START_REF] Han | Red Blood Cells as Smart Delivery Systems[END_REF] Over the recent years, many different systems based on erythrocyte membranes have been developed, from whole erythrocytes to smaller extruded vesicles and nanoparticle coatings. [START_REF] Han | Red Blood Cells as Smart Delivery Systems[END_REF] Erythrocytes ghosts were loaded with a mixture of QD610-SMZ/Au and QD650-SB using a hypotonic shock, as summarized in Figure 8a. [START_REF] Antonelli | New Strategies to Prolong the In Vivo Life Span of Iron-Based Contrast Agents for MRI[END_REF] After washing, erythrocytes were modified by incorporating Amphotericin B into their membranes. Amphotericin B enables small ions to cross the erythrocyte membrane barrier, ensuring that the internal pH follows the external pH. [START_REF] Deuticke | The Influence of Amphotericin B on the Permeability of Mammalian Erythrocytes to Nonelectrolytes, Anions and Cations[END_REF] The obtained erythrocyte ghosts display heterogeneity in their global QD uptake, due to the different levels of membrane permeability occurring during the hypotonic shock loading procedure. However, the 610/650 PL intensity ratio R show a monomodal distribution centered around R≈1.5 at physiological pH 7.5. In contrast, when the QD-loaded erythrocytes are exposed to a slightly acidic pH of 6 typical of tumor microenvironments, the ratio distribution shifts towards lower values (R≈1), as shown in Figure 8b,c. This demonstrates that our dual color ratiometric pH sensor remain functional inside ghost erythrocyte carriers, which opens interesting possibilities for in vivo imaging, using either full ghost erythrocytes or smaller extruded vesicles. [START_REF] Han | Red Blood Cells as Smart Delivery Systems[END_REF]

SWIR emitting pH-sensitive QDs

In our nanoprobes, the pH sensitivity originates from the pH dependent conformational changes of the polymeric surface ligands, and should not depend on the specific nature of fluorescent nanoparticle. In addition, quenching by gold nanoparticles is effective over a broad range of wavelengths. [START_REF] Riskowski | Triangulating Nucleic Acid Conformations Using Multicolor Surface Energy Transfer[END_REF] This makes our strategy potentially applicable to fluorescent nanoparticles emitting in other spectral regions. Here we demonstrate pH sensing using PbS/CdS QDs emitting in the SWIR region. This spectral range is particularly interesting for future in vivo imaging applications, since it preserves micrometer spatial resolution even several mm deep into the tissue. [START_REF] Hong | Through-Skull Fluorescence Imaging of the Brain in a New near-Infrared Window[END_REF] We synthesized PbS/CdS core/shell quantum dots emitting at 1100 nm via partial cation exchange, [START_REF] Cademartiri | Solventless , and Diffusion-Controlled Route to Highly Monodisperse PbS Nanocrystals[END_REF][START_REF] Neo | Synthesis of PbS/CdS Core-Shell QDs and Their Nonlinear Optical Properties[END_REF][START_REF] Pietryga | Utilizing the Lability of Lead Selenide to Produce Heterostructured Nanocrystals with Bright, Stable Infrared Emission[END_REF] coated these QDs with a poly(SMZ12/LA3) ligand, and conjugated these to gold nanoparticles at a 1:4 QD:AuNPs ratio, following the same rationale as for visible emitting QDs. As shown in Figure 9a, the photoluminescence signal of these SWIR emitting QD1100-SMZ/Au nanoprobes dropped below pH 6.5 to about 50 % of its neutral pH intensity. In comparison, the photoluminescence of QD1100-SMZ without any attached gold nanoparticles remained constant between pH 8.5 and 4.5 (additional photoluminescence spectra are presented in Figure S16a). This demonstrates that the pH sensitivity of QD1100-SMZ/Au originates from controlled distance changes between the AuNPs and the central QD, due to conformational switching of the polymer coating. Similarly to visible emitting QD-SMZ/Au, these probes showed reversible pH-induced PL changes, over 3 cycles of pH variations between pH=7.5 and pH=6, as shown in Figure 9b and Figure S16b. We then prepared a mix of two PbS/CdS QD populations: the QD1100-SMZ/Au that is sensitive to pH and QD1250-SB, emitting at around 1250nm and coated with a pH insensitive sulfobetaine polymer coating to provide a spectrally distinct reference signal. Both QD populations have a quantum yield of about 5-6% in saline buffer after functionalization. In vitro, the PL intensity of the QD1100-SMZ/Au population drops by about 50% when decreasing the pH from 7.5 to 5.5, compared to the PL intensity of QD1250-SB which remains nearly constant, as shown in Figure 10a. This provides a ratiometric PL signature of the environment pH, as shown in Figure 10b. We then incorporated these two QD populations simultaneously into erythrocyte ghosts and washed the QD-loaded ghosts from free QDs in the solution. The ghosts were then permeabilized to small cations using amphotericin B. The emission spectra of the SWIR QD-loaded erythrocyte ghosts were then recorded in physiological pH (7.5) or slightly acidic pH (5.5). As shown in Figure 10c, the QD1100 / QD1250 nm PL intensity ratio decrease by of about 40% between the two pH environments, similar to their in vitro behavior. This demonstrates that ratiometric pH measurements are readily translatable to SWIRemitting QDs for pH imaging in deep tissues.

CONCLUSION

In summary, we have proposed a novel strategy to design environment-sensitive QD nanoprobes, based on reversible conformational changes of surface polymeric ligands. We coated QDs emitting in the visible or SWIR range with pH sensitive copolymer ligands and conjugated them with peripheral gold nanoparticle quenchers. pH-induced switching of the polymer ligand conformation modifies the distance between the QD and gold nanoparticles, resulting in a reversible modulation of the QD photoluminescence. These PL changes occur between physiological pH (7.5) and a more acidic pH (5.5-6), comparable to that encountered in endosomes, tumor microenvironments or inflamed tissues. We demonstrate that these probes enable ratiometric pH measurements, using timeresolved spectroscopy or another population of pHinsensitive reference QDs. Advantages of this strategy includes reversibility over several pH changes cycles, applicability over a large spectral range of QD emission and tunability of the switching pH value via the composition and/or length of the polymer ligands. Furthermore, we demonstrate that our QD nanoprobes can be integrated and are still functional into ghost erythrocytes, opening the way to translating these probes to in vivo imaging.

Figure S3. Synthesis of the LA monomer

Synthesis of lipoamide (LA) methacrylamide. To a suspension of APMA-HCl (2 g, 11.2 mmol) in dichloromethane (20 mL) was added triethylamine (2.5 mL, 17.9 mmol). Methanol (2 mL) was introduced to obtain complete solubilization. A solution of LA (2.76 g, 13.4 mmol) in dichloromethane (5 mL) was then added, followed by NHS (1.58 g, 13.8 mmol) in one portion.

The reaction mixture was cooled down to 0°C with an ice bath and a solution of DCC (3.00 g, 14.4 mmol) in dichloromethane (10 mL) was injected dropwise. The medium was warmed up to room temperature and further stirred overnight. A pale yellow solution containing a white precipitate was obtained. The solution was washed with a 0.1 M aqueous HCl solution (2x50 mL), deionized water (1x50 mL) and a 0.2 M aqueous NaOH solution (2x50 mL). The organic phase was separated, dried over MgSO4, filtrated and concentrated under reduced pressure. The crude residue was purified by chromatography on silica (eluent: hexane/ethyl acetate 1/4, then hexane/acetone 1/1) to give DHLA (2.88 g, 8.71 mmol, 78%) as a pale yellow solid.

Poly(SB-b-VIM) Synthesis:

The zwitterionic polymer was used as macro raft agent to add a terminal poly(vinylimidazole) block (degree of polymerization of about 10). Typically, SB block (Mn = 6900 g/mol, DPn = 23, 0.39 mmol), 4VIM (0.39 mmol) and 2,2′-Azobis(2-methylpropionitrile) (AIBN, 0.39 mmol) were dissolved in 37 ml of acetic acid containing 10 vol.% of NaCl 100 mM aqueous solution. After degassing with argon, the reaction medium was heated at 70°C during 6 hours. The crude block copolymer was purified by three cycles of precipitation in ethanol and redispersion in water and finally drying under vacuum. To remove phenyl end groups, the obtained pink-colored block copolymer (500 mg) was dissolved in water (5 mL) and reacted with NaBH4 (∼50 mg). After 6 h (when the solution turned colorless), the polymer was purified by two cycles of precipitation in acetone and redispersion in water, then precipitated in methanol. The obtained white polymer with a thiol end group was finally dried before use (Figure S9,S10). Quantitative 1 H NMR of crude samples before and after polymerization indicated that about 90 mol % of the vinylimidazole monomers were incorporated into the final product. Selection of the optimal Au/QD ratio. Experiments have been realized in order to select the best AuNP/QD ratio to maximize the relative difference in fluorescence intensity between acidic (5.5) and neutral (7.0) pH. Several tubes were prepared with the physiological concentration of NaCl (i.e. 150mM). In each tube 0.05 nmol of QD-pSMZ/LA-AuNP were added with different AuNP/QD ratios of 0, 1, 2, 5, 10, 20 and 50. For each tube, a fluorescence spectrum was realized at pH 7.0 and pH 5.5. The pH was adjusted by NaOH (0.1M) or HCl (0.1M) using a pH-meter. The highest relative fluorescence differences between pH 7.0 and 5.5 occur when for AuNP/QD ratios between 5 and 10.

Figure 1 .

 1 Figure 1. (a) Schematics of the QD-pSMZ/LA-AuNPs nanoprobes in basic (pH 7) or acidic (pH 6) conditions. (b) Synthesis of the SMZ/LA polymers

Figure 2 .

 2 Figure 2. (a) Influence of NaCl concentration on pHdependent solubility transition of the pSMZ65/LA15 polymer. (b) Influence of the polymer length on its pHdependent solubility transition at [NaCl] = 150 mM.

Figure 3 .

 3 Figure 3. (a) Variation of hydrodynamic radius of QDs coated with pSMZ65/LA15 as a function of the pH for [NaCl] = 0 mM and [NaCl] = 150 mM obtained by DLS measurements (initial hydrodynamic radius at pH 8, 150 mM NaCl: 9.5±0.5 nm; pH 8 without NaCl: 8.5±0.4 nm). (b) Variation of hydrodynamic radius of QDs coated with pSMZ65/LA15 or pSMZ12/LA3 as a function of the pH at [NaCl] = 150 mM (initial hydrodynamic radius of pSMZ12/LA3 coated QDs at pH 8: 6.8±0.4 nm).

Figure 4 :

 4 Figure 4: (a) TEM images of the nanoprobes (AuNP:QD = 5). (b) Steady-state and (c) time-resolved photoluminescence of the nanoprobes for different AuNP:QD ratios in basic pH. (d) Relative changes in QD PL at 610 nm between pH 7 and 5.5 normalized by the QD PL at pH 7, as a function of the number of AuNP attached per QD.

Figure 5 .Figure 6 :

 56 Figure 5. (a) PL spectrum and (b) time-resolved PL of QD-pSMZ-LA-5AuNPs as a function of pH.

Figure 7 :

 7 Figure 7: (a) PL spectrum of QD610-SMZ-Au and QD650-SB at pH 7 and 5.5 ([NaCl]=150 mM). (b) Ratio between the PL intensity at 610 nm and 650 nm over 2 cycles of pH variations between 5.5 and 7. (c) Timeresolved PL at 610 and 650 nm at pH 7 and 5.5. (d) Ratio between the average PL decay time at 610 and 650 nm over 2 cycles of pH variations between 5.5 and 7.

Figure 8 .

 8 Figure 8. (a) Principles of ghost erythrocyte QD loading and permeabilization with amphotericin. (b) Image of two QDloaded ghost erythrocytes at 610 nm (top line), 650 nm (medium line) and 610/650 ratiometric image at pH 7.5 (left column) and 6 (right column). Scale bar = 10 µm (c) Distribution histograms of 610/650 intensity ratios among the ghost erythrocyte population at pH 7.5 (black) and 6 (gray).

Figure 9 .

 9 Figure 9. (a) Variations of PL intensity of SWIR (≈1100 nm) emitting QD-pSMZ-LA as a function of pH with and without AuNP. (b) Variations of QD PL intensity over 3 pH cycles between 6 and 7.5, with and without AuNP.

Figure 10 .

 10 Figure 10. (a) Normalized photoluminescence spectrum of QD1100-SMZ/Au and QD1250-SB as a function of pH. (b) Normalized intensity ratio between 1050 nm and 1300 nm as a function of pH. (c) Photoluminescence spectra of the mixed QD1100-SMZ/Au and QD1250-SB populations incorporated within erythrocytes, at pH 7.5 and 5.5.

Figure S1 .

 S1 Figure S1. (a) Absorption and photoluminescence emission spectra of QD610 in hexane; (b) TEM image of QD610; (c) Size distribution of QD610. (d) Absorption and photo-luminescence emission spectra of QD650 in hexane; (e) TEM image of QD650; (f) Size distribution of QD650.

Figure S2 .

 S2 Figure S2. (a) Absorption spectra of AuNP in water; (b) TEM image of AuNP; (c) Size distribution of AuNP.

Figure S4 .

 S4 Figure S4. Synthesis of the SMZ monomer

Figure S5. 1 H

 1 Figure S5. 1 H NMR spectrum (DMSO-d6, 400 MHz) of SMZ (up) and SZ (down)

Figure S6. 1 H

 1 Figure S6. 1 H NMR spectrum (DMSO-d6, 400 MHz) of pSMZ65/LA15

Figure S7. 1 H

 1 Figure S7. 1 H NMR spectrum (D2O, 400 MHz) of SB monomer (up) and SB first zwitterionic block (down).

Figure S8 .

 S8 Figure S8. Synthesis steps of the SB-b-VIM block copolymer.

Figure S9. 1 H

 1 Figure S9. 1 H NMR spectrum (D2O, 400 MHz) of SB copolymer.

Figure

  Figure S12. (A) Absorbance spectrum of the PbS core, PbS/CdS core-shell and PbS/CdS covered by pSMZ/LA. (B) Absorbance spectrum of SWIRQDs, AuNPs, QD-pSMZ/LA-AuNP assemblies and final filtrate solutions.

Fluorescence spectra and fluorescence

  Figure S13. (a) Fluorescence spectrum and (b) fluorescence decay of the QD-pSMZ/LA-AuNP corresponding to Figure 4 during 3 cycles of pH variation between 7.0 and 5.5.

Figure S15 .

 S15 Figure S15. (a) Absorption and photoluminescence emission spectra of SWIRQD in water; (b) TEM image of SWIRQD; (c) Size distribution of SWIRQD.

ACKNOWLEDGMENT This work was supported partially by LocalEndoProbes grant CE16-0012 from Agence Nationale de la Recherche. The authors thank the SIMM laboratory for help with DLS measurements.

ASSOCIATED CONTENT Supporting Information. Additional synthesis protocols and characterization. The following files are available free of charge: Supporting Information (pdf)

AUTHOR INFORMATION

Corresponding Author *Thomas Pons: thomas.pons@espci.fr; Nicolas Lequeux : nicolas.lequeux@espci.fr

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

SUPPORTING INFORMATION pH Sensitive Visible or SWIR Quantum Dot Nanoprobes using Conformation-Switchable Copolymeric Ligands

Manon Debayle, a Thomas Marchandier, a Xiangzhen Xu, a Nicolas Lequeux, a,* Thomas Pons