Molecularly Imprinted Polymer Nanoparticles as Potential Synthetic

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Biological and Medical Applications of Materials and Interfaces

Molecularly Imprinted Polymer Nanoparticles as Potential Synthetic Antibodies for Immunoprotection against HIV Jingjing Xu, Franck Merlier, Bérangère Avalle, Vincent Vieillard, Patrice Debré, Karsten Haupt, and Bernadette Tse Sum Bui ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b22732 • Publication Date (Web): 13 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019

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ACS Applied Materials & Interfaces

Molecularly Imprinted Polymer Nanoparticles as Potential Synthetic Antibodies for Immunoprotection against HIV Jingjing Xu¥,†, Franck Merlier†, Bérangère Avalle†, Vincent Vieillard‡, Patrice Debré‡, Karsten Haupt†,*, Bernadette Tse Sum Bui†,* ¥Shanghai

University, School of Life Sciences, Center for Molecular Recognition and Biosensing, Shanghai 200444, P. R. China

†Sorbonne

Universités, Université de Technologie de Compiègne, CNRS Enzyme and Cell

Engineering Laboratory, Rue Roger Couttolenc, CS 60319, 60203 Compiègne Cedex, France ‡Sorbonne

Universités, UPMC Paris 6, INSERM U1135, CNRS ERL8255, Centre d’Immunologie

et des Maladies Infectieuses (CIMI-Paris), Boulevard de l’hôpital, 75013 Paris, France *e-mail: [email protected] (B. Tse Sum Bui) and [email protected] (K. Haupt) ABSTRACT We describe the preparation and characterization of synthetic antibodies based on molecularly imprinted polymer nanoparticles (MIP-NPs), for the recognition and binding of the highly conserved and specific peptide motif SWSNKS (3S), an epitope of the envelope glycoprotein 41 (gp41) of human immunodeficiency virus type 1 (HIV-1). This motif is implicated in the decline of CD4+ T cells and leads to the deterioration of the immune system during HIV infection. Therefore, the development of MIP-NPs that can target and block the 3S peptide to prevent subsequent cascade interactions directed toward the killing of CD4+ T cells is of prime importance. Since most antibodies recognize their protein antigen via a conformational or structured epitope (as opposed to a linear epitope commonly used for molecular imprinting), we employed protein molecular modeling to design our template epitope so that it mimics the 3D structure fold of 3S in gp41. The resulting template peptide corresponds to a cyclic structure composed of CGSWSNKSC, with the 3S motif well orientated for imprinting. MIP-NPs with a size of 65 nm were obtained by solid-phase synthesis and were water-soluble. They were prepared by a judicious combination of multiple functional monomers affording hydrogen bonding, ionic, π-π and hydrophobic interactions, conferring high affinity and selectivity toward both the cyclic peptide and the whole gp41 protein. These results suggest that our MIPs could potentially be used for blocking the function of the 3S motif on the virus. KEYWORDS: 3S peptide, epitope, gp41, HIV, molecularly imprinted polymer, solid-phase synthesis

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INTRODUCTION In 2017, 37 million people was globally living with the human immunodeficiency virus type 1 (HIV1), the infectious agent causing acquired immune deficiency syndrome (AIDS).1 The same year, 940,000 people died from AIDS-related causes worldwide, which makes HIV infection a serious global health concern worth billions of US$ investment. Infection by HIV-1 leads to the deterioration of the immune system due to the progressive depletion of CD4+ T lymphocytes. Particularly, research on the highly conserved peptide SWSNKS (3S), an epitope of the transmembrane glycoprotein 41 (gp41) present on the virus could contribute significantly to AIDS pathogenesis (Figure 1).2-4 When 3S interacts with its specific receptor gC1qR on CD4+ T cells, a cellular ligand of the natural killer (NK) cells, NKp44L, is induced at the surface of CD4+ T cells. The NKp44L in turn, interacts with its receptor NKp44 expressed on NK cells, leading to the killing of CD4+ T cells and consequently, an increase in viral load. Following the identification of the 3S peptide, further studies have shown that anti-3S antibodies inhibited the expression of NKp44L and the activation and cytotoxicity of NK cells in both in vitro studies and in in vivo immunization in macaques, thus preventing CD4+ T cell depletion.3,4 The synthetic peptide used to generate the anti-3S antibodies was a linear peptide with the following sequence NH2-PWNASWSNKSLDDIW-COOH. However, biological antibodies have shortcomings such as low physical and (bio)chemical stability outside their natural environment, thus they require storage at low temperatures, and animals are needed for their production, explaining their high cost. Herein, we propose to use a synthetic antibody, based on a molecularly imprinted polymer (MIP) to target the 3S peptide, in the hope of blocking the function of this peptide and stop NKp44L overexpression, thus preventing CD4+ T cell depletion and restoring immune protection.


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Figure 1. Implication of gp41 3S motif in the destruction of CD4+ T cells. (Inset) Proposed mechanism of the action of MIP targeting and blocking 3S, preventing interaction with gC1qR on CD4+ T cells and subsequently haltering further cascade interactions involved in the killing of the lymphocytes.

MIPs are tailor-made receptor materials,5-7 synthesized by a templating process at the molecular level. Monomers carrying functional groups self-assemble around a template molecule (the target or a derivative), followed by copolymerization with cross-linking monomers, which result in the formation of a polymeric mould around the template. Subsequent removal of the template reveals 3D-binding sites in the polymer that are complementary to the template in size, shape and position of


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the functional groups. Consequently, MIPs exhibit binding affinities and specificities comparable to those of antibodies but in contrast to the latter, their production is reproducible, relatively fast and economic. Moreover, they are physically and chemically stable and are not degraded by hydrolytic enzymes. Recently, we and others have applied an innovative solid-phase synthesis approach to prepare water-soluble ‘monoclonal’-type MIP-nanoparticles (MIP-NPs), which exhibit antibody-like affinities (dissociation constants in the nM-pM range).8-10 Most antibodies recognize their protein antigen via a structured non-linear epitope or a conformational epitope.11,12 Our 3S peptide target with the motif SWSNKS being an internal sequential epitope, we used protein molecular modeling to design our template peptide. This peptide corresponds to a cyclic structure composed of CGSWSNKSC, that is, two cysteine residues were added to the two sides of the 3S motif, so as to form a disulfide bond and generate a cyclic structure, and a glycine residue was added between the N-terminal serine and cysteine to make this cyclic structure more stable (Figure 2). The cyclic 3S peptide was immobilized on (3-aminopropyl)triethoxysilane (APTES)-functionalized glass beads via its –COOH group so as to expose the SWSNKS motif in a freely accessible and orientated conformation, for targeting by the MIP. Thermoresponsive MIP-NPs were synthesized against the immobilized 3S peptide, and the binding performance of the MIP for both the cyclic 3S peptide and gp41 protein was studied in vitro. Results show that this synthetic antibody is promising for potentially counteracting the virus in in vivo applications.

Figure 2. Solid-phase synthesis of thermoresponsive MIP-NPs on immobilized cyclic 3S peptide. Polymerization was done at 38 °C. After washing away non-reacted monomers and low-affinity polymers, the MIP was eluted at 6 °C.


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EXPERIMENTAL SECTION Materials and Methods. All chemicals and solvents were of analytical grade and purchased from VWR International (Fontenay sous Bois, France) or Sigma-Aldrich (St Quentin Fallavier, France), unless otherwise stated. HPLC solvents were purchased from Biosolve Chimie (Dieuze, France). Cyclic 3S peptide (purity 95%, ESI-MS (m/z): [M+H]+ 969.3558 g/mol) was custom-synthesized by GL Biochem (Shanghai, China). Recombinant (E. Coli) HIV-1 gp41 was purchased from Gentaur (Belgium). Glass beads (GBs) of diameter 0.1 mm were obtained from Roth Sochiel E.U.R.L (Lauterbourg, France). Polystyrene 48-well microplates (PolySorp) were purchased from Becton Dickinson (Le Pont De Claix, France). Bicinchoninic acid (BCA) protein assay kit was purchased from Sangon Biotech Co., Ltd. (China). Buﬀers were prepared with Milli-Q water, purified using a Milli-Q system (Millipore, Molsheim, France). Fluorescence measurements were performed on a FluoroLog-3 spectrofluorimeter (Horiba Jobin Yvon, Longjumeau, France). Energy-dispersive X-ray (EDX) spectroscopy was performed on an XFlash 6 | 30 detector – Quantax EDS (Bruker, The Netherlands). For scanning electron microscopy (SEM) imaging, polymer particles were sputter coated with gold prior to the analysis, and images were taken by a Quanta FEG 250 (Philips, The Netherlands). Boc protection/deprotection of primary amines on the 3S peptide. Immobilization of the cyclic 3S peptide was done via its –COOH, so as to leave the SWSNKS motif exposed (Figure 2). The carboxyl

group

hydroxysuccinimide

was

activated

(EDC/NHS),

with

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-

followed

by

covalent

attachment

of

(3-

aminopropyl)triethoxysilane (APTES)-functionalized glass beads, via its amino group. Since the 3S peptide also contains primary amines, to prevent coupling between themselves, the amino groups of the peptide were protected by the tert-butyloxycarbonyl group (Boc), prior to coupling on the GBs. The protection and deprotection protocol was verified on the free peptide in solution before any coupling to the GBs. Protection by tert-butyloxycarbonylation of amines was done as described.13 Briefly, to a solution of 100 mg (103 µmol) cyclic 3S peptide dissolved in 20 mL of a mixture of tetrahydrofuran/water (1/1), 840 mg (10 mmol) of NaHCO3 and 100 mg (458 µmol) of di-tert-butyl dicarbonate (Boc2O) was added at 0 °C. The mixture was stirred in an ice bath for 30 min and the reaction left to proceed overnight at room temperature. The reaction mixture was extracted twice with 20 mL of diethyl ether (Et2O). The Boc-3S peptide, found in the aqueous phase (~10 mL) was adjusted to ~pH 6 by addition of 2-(N-morpholino)ethanesulfonic acid (MES) at 0 °C. This Boc-3S peptide in MES buffer constituted our stock working solution. It was kept as 1 mL aliquots at -20 °C until use. The Boc-3S peptide product was analyzed by liquid chromatography-electrospray ionization high resolution mass spectrometry (LC-ESI/HRMS). The HPLC system (Infinity 1290, Agilent ACS Paragon Plus Environment

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Technologies, France) was equipped with a diode array detector coupled to a quadrupole time of flight mass spectrometer (Q-TOF) (Agilent 6538, Agilent Technologies, France). The chromatographic column was a Hypersil GOLD C18 reversed phase (150 x 2.1 mm, 3 µm, 100 Å) (Thermo Scientific, France). The mobile phase consisted of water containing 0.1% formic acid (eluent A) and acetonitrile (eluent B). The gradient program began with 5% B, ramped to 95% B at 20 min, was held at 95% for 5 min, increased to 100% B at 30 min and was kept constant at 100% B until 35 min. The flow rate was set at 0.4 mL/min. Detection was monitored at 280 nm. An aliquot of 10 µL of Boc-3S peptide was injected. Positive ion electrospray (ESI) mass spectra was acquired by scan mode in the range m/z 100 to m/z 2000 at electrospray voltage of 3800 V and fragmentor voltage of 180 V. Nitrogen was used as the dry gas at a flow rate of 10.0 L/min and a pressure of 30.0 psi. The nebulizer temperature was set to 300 °C. Boc deprotection was performed by adding 1 M HCl to the Boc-3S peptide solution, followed by extracting twice with 10 mL of Et2O.14 The cyclic peptide was collected in the aqueous phase of which 1 mL was loaded on an Oasis HLB cartridge (1 cc Vac Cartridge, 30 mg Sorbent per Cartridge, 30 µm Particle Size, Waters, France), which was previously conditioned twice with 1.2 mL acetonitrile (ACN) and three times with 1 mL water. After loading, 3 mL water was passed through the column to reach the pH of water. The pure cyclic peptide was collected by eluting with 1 mL ACN. After drying under N2, the peptide was dissolved in 1 mL water and analyzed by mass spectrometry. The peptide was directly injected on the Infinity 1290 HPLC/Q-TOF, without chromatographic separation. Positive ESI mass spectra was acquired by scan mode in the range m/z 100 to m/z 3200 at electrospray voltage of 3800 V and fragmentor voltage of 130 V. Nitrogen was used as the dry gas at a flow rate of 10.0 L/min and a pressure of 30.0 psi. The nebulizer temperature was set to 300 °C. Solid-phase synthesis of MIP-NPs. Immobilization of peptide on APTES-functionalized GBs. First, 100 g of glass beads (GBs) were activated by boiling in 100 mL of 4 M NaOH for 10 min. After washing with water and drying in an oven at 50 °C, the activated GBs were incubated with 100 mL of 2% (v/v) APTES in toluene overnight, washed with acetone and dried at 50 °C.8,15 The APTESfunctionalized GBs were coupled to Boc-3S peptide via its free carboxyl group by EDC/NHS coupling procedure. Briefly, 10 mL of Boc-3S peptide was activated by adding 191.7 mg (1 mmol) EDC and 287.5 mg (2.5 mmol) NHS for 15 min, then this solution was diluted to 100 mL with 25 mM sodium phosphate buffer pH 7 (referred as buffer A) and incubated with APTES-functionalized GBs, with shaking overnight at room temperature. After the immobilization of Boc-3S peptide, Boc deprotection was done by immersing the GBs in 120 mL of 1 M HCl in 100 mL of Et2O for 5 h at room temperature. Synthesis of polymers. The solid-phase synthesis of MIP-NPs was carried out in a glass column ACS Paragon Plus Environment

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equipped with a thermostated jacket (XK 26/40, GE Healthcare, France), connected to a circulating thermostated water-bath (Bioblock Scientific Polystat 5, Fischer Scientific, France). The solvents were pumped through the column using a peristaltic pump at a flow rate of 2.5 mL/min.8,15 The column was packed with 33 g of 3S peptide-APTES-functionalized GBs, corresponding to a bed height of 4.2 cm. The GBs were washed with 100 mL water and then equilibrated with 100 mL of buffer A. The polymerization solution was prepared by mixing N-isopropylacrylamide (NIPAm) (181.1 mg, 80 mol %), 2-(trifluoromethyl)acrylic acid (TFMAA) (14 mg, 5 mol %), Nphenylacrylamide (PAA) (14.7 mg, 5 mol %), N-tert-butylacrylamide (TBAm) (12.7 mg, 5 mol %) and N,N′-methylenebis(acrylamide) (Bis) (15.4 mg, 5 mol %) in 48 mL of buffer A, so that the total monomer concentration is 0.5% (w/w). The solution was purged with nitrogen for 30 min. Afterward, the initiation couple composed of potassium persulfate (KPS) (18 mg in 500 µL buffer A) and N,N,N′,N′-tetramethylethylenediamine (TEMED) (1.3 µL) (7.5/1 molar ratio, where the amount of KPS was 3% mol/mol with respect to polymerizable double bonds) was added. After overnight polymerization at 38 °C, the column was washed with 50 mL buffer A at 38 °C, and then cooled down to 6 °C. Polymers were eluted with two to three aliquots of 5 mL buffer A (i.e. until there were no longer particles detected in the eluent by dynamic light scattering (DLS)). Non-imprinted polymer was prepared using the same polymerization mixture and processing as for the MIP on 33 g of APTES-GBs, i.e. in the absence of peptide. Characterization of polymers. Physicochemical characterization. The eluted fractions in buffer A were analyzed by DLS on a Zetasizer NanoZS (Malvern Instruments Ltd., Worcestershire, UK). Fractions with similar sizes and polydispersity index (PDI) were mixed together to constitute a working stock solution. Zeta potential determination was carried out with a desalted sample of the stock solution. The desalting was performed on 1 mL of the stock solution by repeated concentration and dilution (5 times) with 3 mL of water, on an Amicon Ultra-15 centrifugal filter unit (MW cut-off 30 kDa, Merck Chimie SAS, France). For SEM imaging, 1-mL aliquot of the stock solution was dialyzed overnight against 1 L of Milli-Q water. Determination of polymer yield. In order to facilitate MIP precipitation, the repulsive forces within the negatively charged polymer, caused by the presence of TFMAA, had to be suppressed. 1 M HCl was added to 3 mL of MIP until the pH reaches 1-2. The polymers were centrifuged at 40,000g for 1 h at 38 °C so that they precipitate. After discarding the supernatant, the precipitate was suspended in 4 mL water and lyophilized. The dry MIPs were weighed with a precision balance; this allows to determine the concentration of the MIPs (mg/mL) and the yield of synthesis, calculated as mg of MIPs per g of GBs. LCST analysis of MIP. 1 mL of MIP solution was pipetted in a glass cuvette and 100 µL of 1 M HCl was added to adjust the pH to 1-2. The lower critical solution temperature (LCST) of the MIP was ACS Paragon Plus Environment

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determined via turbidity measurements by reading the absorbance at 600 nm.16 MIP was heated at a heating rate of 1 °C/min, from 25 to 50 °C on a Cary60 UV-Vis spectrophotometer, connected to a circulation bath and single cell Peltier Accessory (Agilent Technologies, France). The reversibility of polymer transition was determined by repeated heating/cooling cycles. Binding characteristics of MIP with cyclic 3S peptide. Preparation of rhodamine 123-cyclic 3S peptide. Cyclic 3S peptide coupled to rhodamine 123 (pep-rho) was used as a fluorescently-labeled target for monitoring binding. 5 mL of Boc-3S peptide was activated by adding 95.8 mg (0.5 mmol) EDC and 143.8 mg (1.29 mmol) NHS and incubated for 15 min at room temperature. The mixture was then added to 45 mL of buffer A containing 19.1 mg (0.05 mmol) rhodamine 123 and incubated overnight to enable conjugation via its amino group.17 Boc deprotection was performed by adding 37 % HCl to reach 1 M final concentration, followed by extraction with 50 mL Et2O. The sample was purified on PD MidiTrap G-10 columns (exclusion limit: 700 Da) by injecting portions of 1 mL. Fractions of 1.2 mL were collected, pooled together to form 12 mL-aliquots and stored at -20 °C until use. The concentration of rhodamine 123 in the pep-rho complex was determined via its absorption at 495 nm using a molar extinction coefficient of 75,000 M-1cm-1,18 and this measurement was performed on each pooled 12-mL aliquot before use so as to know the concentration of pep-rho in each aliquot. Concentrations from 15-22 µM were obtained. The fluorescence of pep-rho was verified by constructing a calibration curve using concentrations ranging from 2 – 225 nM (Figure S1). Fluorescence measurements were recorded on a spectrofluorimeter, using ex = 290 nm and em = 520 nm. Binding isotherm of immobilized polymers toward pep-rho. 0.5 mL of MIP and NIP (5 – 400 µg/mL) was pipetted into a 48-well microplate. The polymers were immobilized by physical adsorption after overnight solvent evaporation at 37 °C. MIP nanoparticles contain 5% TBAm, 5% PAA, and 80% NIPAm which are hydrophobic at 37 °C; since the interaction between MIP nanoparticles and the “PolySorp” hydrophobic surface of the wells is mainly driven by hydrophobic interactions, the MIP particles are not washed away during processing, which was conducted in aqueous media. This was verified by DLS analysis of the solutions. Each well was conditioned by washing three times with 0.5 mL of buffer A, then blocked with 0.5 mL of buffer A containing 0.1% of bovine serum albumin (BSA) and 1% of Tween 20,19 followed by washing again with buffer A. Afterward, 0.5 mL of 1.5 µM pep-rho was added into the wells and incubated with immobilized polymers overnight at 37 °C. For the measurements, the supernatant was withdrawn and diluted to fall within the calibration curve (Figure S1), then read on the spectrofluorimeter using ex = 290 nm and em = 520 nm. The amount of pep-rho bound was calculated by subtracting the unbound from the amount added to the well. To investigate the binding capacity of MIP toward pep-rho, 0.2 mL of 0.5 mg/mL (100 µg) was taken as a constant amount of MIP immobilized on the microplate. After washing, blocking and ACS Paragon Plus Environment

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washing steps as described above, 0.5 mL of pep-rho concentrations ranging from 5 – 1500 nM was added. After overnight incubation at 37 °C, the supernatant was withdrawn and read on the spectrofluorimeter using ex = 290 nm and em = 520 nm. The amount of pep-rho bound was calculated in the same way as mentioned above. Selectivity studies. 100 µg of MIP was immobilized in 48-wells microplate. After washing, blocking and washing steps as described above, 100 nM of pep-rho was added together with other peptides at concentrations ranging from 1 to 100 nM. Cyclic 3S peptide, linear 3S peptide, two different peptides referred as competitor 1 and competitor 2, and a 1:1 mixture of competitor 1 and 2, were used to compete with pep-rho. The fluorescence of pep-rho displaced by competing peptides is measured in the supernatants. Binding characteristics of MIP with gp41. Preparation of FITC-gp41urea. The complex fluorescein isothiocyanate and gp41 (FITC-gp41urea), was synthesized by following the same protocol as for FITC-trypsin conjugation as previously reported.20 Stock solutions of 100 µg/100 µL of gp41 was prepared in buffer A. Gp41 was denatured to surface-expose its internal 3S motif; therefore 120 µg (2.9 nmol; MW of gp41 is 41 kDa)21 was denatured by adding 100 µL of 8 M urea. After dialysis with a membrane of MWCO 6-8 kDa in 1 L water overnight, the protein solution was added to 2 mL of 100 mM sodium bicarbonate buffer pH 9.2, followed by 50 µL of FITC solution (10 mg/mL in dimethyl sulfoxide). The reaction mixture was incubated for 2 h at room temperature, then the labeled protein was separated by gel filtration on a PD-10 column (GE Healthcare), equilibrated with buffer A. FITC-gp41urea was aliquoted and stored at -20 °C. To determine FITC/gp41urea ratio, gp41urea concentration was measured with the enhanced BCA protein assay (calibration curve in Figure S2), whereas the bound fluorescein was determined via its absorption at 495 nm using a molar extinction coefficient of 68000 M-1 cm-1.20 The concentration of gp41urea was found to be 28.7 µg/mL, corresponding to a concentration of FITC-gp41urea of 0.7 µM, and a ratio of FITC/gp41urea of 6.8. A fluorescence calibration curve of FITC-gp41urea over the concentration range of 10 – 700 nM is shown in Figure S3. The fluorescence intensity was read on a spectrofluorimeter using ex = 288 nm and em = 515 nm.22 Binding capacity of MIP for gp41. The binding capacity of MIP for gp41urea was evaluated with MIP immobilized in a 48-well microtiter plate using FITC-gp41urea to monitor binding by fluorescence. Briefly, each well was filled with 0.2 mL of 0.5 mg/mL MIP (100 µg) and the polymers were physically coated on the plate by leaving them to dry at 37 °C overnight. Then, immobilized MIP was conditioned by washing three times with 0.5 mL of buffer A, blocked with 0.5 mL of buffer A containing 0.1% of bovine serum albumin (BSA) and 1% of Tween 20,19 followed by washing again with buffer A. Afterward, 0.5 mL of FITC-gp41urea at concentrations ranging from 10 – 700 nM was added to each well. After overnight incubation, the supernatant was withdrawn for fluorescence ACS Paragon Plus Environment

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measurements, using ex = 288 nm and em = 515 nm with slit: 2 nm. The FITC-gp41urea bound to MIP was determined by subtracting the amount of unbound from the amount incubated in wells containing no MIP. Competitive binding test. 100 µg of MIP was immobilized in 48-wells microplate. After washing, blocking and washing steps as aforementioned, 350 nM of FITC-gp41urea was added together with either gp41urea, or human serum albumin (HSA) or human transferrin, at concentrations ranging from 1 to 700 nM. Gp41urea was obtained by denaturing 80 µg of native gp41 using 60 µL of 8 M urea, followed by two dialysis in 1 L water overnight. The amount of FITC-gp41urea displaced by denatured gp41, or HSA, or human transferrin, was measured in the supernatant.

RESULTS AND DISCUSSION Design of cyclic 3S peptide. Previous studies have shown that antibodies raised against the linear peptide NH2-PWNASWSNKSLDDIW-COOH containing the highly conserved 3S motif on gp41 HIV-1 envelope protein, prevented the decline of CD4+ lymphocytes implicated in HIV-1 infection.2-4 In this work, we investigated whether a synthetic antibody based on nanosized watersoluble molecularly imprinted polymers could potentially act as a more stable and cost-effective anti3S antibody mimic to target and block this 3S motif in vitro. Generally, epitope imprinting is performed using a well-defined specific linear sequence, often a terminal peptide on the surface of a protein because of its easy definition and acquirement.23-25 However, most antibodies recognize a conformational epitope or an internal sequence epitope of a protein rather than its linear terminal peptide epitope,11,12 and it was pointed out that more attention should be paid to the internal peptide of the protein target, due to its more conserved feature and relation with immune response.26 In our case, the 3S peptide is an internal sequence of gp41 and is only exposed during the interaction of the virus with the host cell, where the surface protein gp120 (Figure 1) undergoes substantial conformational rearrangements.27,28 Therefore, to mimic closely the conformation of this 3S internal peptide (Figure 3A, highlighted), we have used protein molecular modeling29,30 to design our template peptide, which adopts a cyclic configuration. Two cysteine residues and a glycine were added to stabilize the cyclic structure (Figure 3B). The final cyclic peptide presents the following sequence: CGSWSNKSC (C-C cyclic). Its immobilization was done through the carboxyl group on aminofunctionalized GBs, so as to orient the SWSNKS motif in a fixed non-hindered position (Figure 2). Accordingly, the resulting MIP will have improved binding site homogeneity since its binding site will have the same orientation, reminiscent of ‘monoclonal’-type antibodies.


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Figure 3. (A) Structure of gp160 (consisting of gp120 et gp41, see also Figure 1) based on the 3D structure of the protein from Protein Data Bank Europe accession code 5FUU, viewed on Cn3D program version 4.3.1 3D structure viewer (National Center for Biotechnology Information), with the 3S motif (ochre) highlighted; (B) Model structure of 3S peptide (Pepfold 2.0).29,30

Analysis of Boc protected and deprotected cyclic 3S peptide. The carboxyl group of the 3S peptide was covalently attached to (3-aminopropyl)triethoxysilane (APTES)-functionalized glass beads, via its amino group, using EDC/NHS coupling method (Figure 2). Since the 3S peptide also contains primary amines, to prevent coupling between themselves, the amino groups of the peptide were protected by the tert-butyloxycarbonyl group (boc), prior to coupling. The presence of boc-3S peptide and its deprotected form was confirmed by LC-ESI/HRMS (Figure S4, Figure S5). The mass spectrum of the deprotected peptide was similar to that of its corresponding untreated cyclic 3S peptide, indicating that the boc/deboc procedure did not linearize the peptide (Figure S5). Solid-phase synthesis of MIPs. The solid-phase synthesis of the polymers is represented in Figure 2 and the first steps were done as previously reported.8,15,31 Briefly, activated GBs were prepared by boiling in NaOH so as to introduce OH groups, then silanized with APTES, followed by coupling with the boc cyclic 3S peptide, then deprotection in situ. The presence of the peptide on the GBs was confirmed by energy-dispersive X-ray spectroscopy (EDS) analysis (Figure S6). The element sulfur, ACS Paragon Plus Environment

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present on the peptide, was clearly observed as the S peak (Figure S6C). Neither the spectra of the NaOH-activated GBs nor the APTES-functionalized GBs contain a S peak (Figures S6A-B). A molar percentage ratio 5:5:5 of functional monomers comprising 2-(trifluoromethyl)acrylic acid (TFMAA),32 N-phenylacrylamide (PAA)33 and N-tert-butylacrylamide (TBAm),9,10,19,33,34 to enable electrostatic, π – π and hydrophobic interactions respectively with the 3S-peptide, was used. Due to the flexible conformation of the 3S peptide conformation in vivo, a low cross-linking degree of 5% (molar ratio) N,N’-methylenebis(acrylamide) (Bis) was used,8,15,20,21,35 so as to obtain a flexible MIP. Additionally, the hydrogen-bond forming N-isopropylacrylamide (NIPAm) functional monomer was added at a high molar ratio (80%), to endow the polymers with thermoresponsiveness and facilitate their elution from the solid support.8,15,20,21,35 Indeed, below its LCST (~32 °C), polyNIPAm is in a soluble state and above 32 °C, it is in a collapsed state. Thus, the MIP was synthesized at 38 °C overnight. After polymerization, the GBs were washed with buffer A at 38 °C to remove unreacted reagents and low-affinity polymers. The high affinity MIPs were eluted with buffer A at 6 °C, temperature at which they swell and detach from the peptide. A control non-imprinted polymer (NIP) was synthesized using the same protocol as for the MIP except that no template was used. Characterization of polymers. Physicochemical analyses. The size distribution of the polymers in buffer A (Figure S7A) was determined by DLS analysis. The diameters of MIP and NIP were 65 ± 4 nm (PDI: 0.384) and 72 ± 15 nm (PDI: 0.28), respectively. The morphology of MIP, as analyzed by SEM imaging is fairly homogeneous, with some polydispersity (Figure S7A, inset). The zeta potentials of both polymers were similar, with a value of -11.6 mV, due to the presence of TFMAA (Figure S7B). The yield of synthesis was found to be respectively 0.17 mg/g and 0.16 mg/g of GBs for MIP and NIP. LCST analysis of MIP. The LCST of the polymers was determined via turbidity measurement (cloud point determination) by measuring the absorbance at 600 nm versus temperature.16 With temperature increase, NIPAm-based polymers change from a hydrated hydrophilic conformation (< LCST) to a collapsed hydrophobic state (> LCST), accompanied by an increase of turbidity. The LCST value was determined by “tangent plot”36 of the data and found to be 37 °C (Figure S8). Recognition of the cyclic 3S peptide by MIP. Equilibrium binding assay. The recognition behavior of MIP and NIP for cyclic 3S peptide (1.5 µM) was evaluated by equilibrium binding studies using immobilized polymers (5 – 400 µg/mL) on micotiter plates. Figure 4A shows that MIP has a higher binding than NIP, indicating the creation of imprinted binding sites. Binding capacity. The binding capacity of MIP was studied using a fixed amount of MIP, 100 µg, and pep-rho concentrations ranging from 5 – 1500 nM. A graph of bound versus free pep-rho (Figure 4B) was plotted; non-linear fitting of the data to a single-site Langmuir binding isotherm yielded a dissociation constant (Kd) of 79.6 nM and a maximum binding capacity of 3598 pmol of peptide per ACS Paragon Plus Environment

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mg of MIP. Considering the molecular weight of the MIP to be 475 kDa,20 this corresponds to 2 nmol of MIP binding ~3.6 nmol of peptide, indicating that there are between 1 - 2 binding sites per MIP nanoparticle, which is in agreement with values found for similar materials produced by the same method.9

Figure 4. (A) Equilibrium binding isotherms of MIP (black) and NIP (grey) for 1.5 µM pep-rho; (B) binding capacity of 100 µg MIP immobilized on microplate with pep-rho concentrations varying from 5 – 1500 nM. Experiments were done in 25 mM sodium phosphate buffer pH 7. Data are means of 3 independent experiments with 3 different batches of polymers. The error bars represent standard deviations.

Binding selectivity. Various peptides were used to compete with the binding of pep-rho with immobilized MIP. The composition of the peptides is shown in Table 1. Figure 5 shows that low concentrations of cyclic 3S peptide displaces pep-rho from immobilized MIP and thus accounts for the lowest IC50 (the concentration of competing ligand required to displace 50% of pep-rho from the MIP). The IC50 values as determined from a nonlinear regression fit, are summarized in Table 1. The MIP shows the highest affinity for the cyclic 3S peptide (IC50 of 18.7 nM), indicating a slightly higher affinity than that obtained using pep-rho, reported above (Kd = 79.6 nM), which we attribute to the presence of the bulky rhodamine group. The linear 3S peptide with an IC50 of 69 nM has a 3-fold lower affinity than the cyclic 3S peptide, the original template. The observed cross-reactivity of 27% is not unexpected since the linear peptide is quite flexible so that it can nevertheless fit into the binding site. Note that the linear peptide is the one used to prepare anti-3S antibodies which prevent NKp44L expression and CD4+ lymphocyte depletion in vivo.3 Low cross-reactivities were observed with the competitor 1 and competitor 2 peptides, taken separately or as a 1:1 mixture. The sequence of


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competitor 1, though in a cyclic conformation, has no similarity with that of the cyclic 3S peptide, which explains its low cross-reactivity. The cross-reactivity of competitor 2 was found to be 13.7%, probably due to some similarity in amino acids. A mixture of competitor 1 and 2 yielded a cross reactivity of 6.4%, indicating that in a complex environment, the cross-reactivity of competitor 2 might favorably be lowered.

Figure 5. Inhibition of pep-rho (100 nM) binding to 100 µg MIP by competitor 1 (circles), competitor 2 (squares), a mixture of competitor 1 and 2 (triangles), linear 3S peptide (inverted triangles) and cyclic 3S peptide (diamonds), at concentrations varying from 1 - 100 nM. B/B0 is the ratio of the amounts of pep-rho bound in the presence and absence of displacing competitors.

Table 1. IC50 of competitive peptides IC (nM) Cross-reactivity, % 50

Competing peptides Cyclic 3S peptide: CGSWSNKSC (C-C cyclic)

18.7

100

Linear 3S peptide: CPWNASWSNKSLDDIW

69.0

27.1

Competitor 1: CQKYNTQGSDVC (C-C cyclic)

1525

1.2

Competitor 2: CWDALNDWSPSKIAS

136.3

13.7

Mixture of competitor 1 and 2

292.6

6.4

Recognition of MIP toward gp41. The competitive binding assay was also performed with the entire gp41 protein. Negligible displacement of pep-rho was observed with native gp41, indicating that gp41 does not interact with the MIP. This was not very surprising as the 3S motif is internal and is only exposed during infection by the virus.3,27,28 To make the 3S peptide in gp41 accessible, the protein ACS Paragon Plus Environment

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was denatured by adding an excess of urea. FITC-gp41urea was synthesized so as to monitor the binding of the protein by fluorescence measurements, with immobilized MIP and NIP on well-plates. Figure 6A shows that the binding of MIP is specific as it binds FITC-gp41urea to a higher extent than NIP. Increasing concentrations of free gp41urea were then made to compete with fixed amounts of FITC-gp41urea bound to the MIP, to determine its affinity (Figure 6B). The IC50 was determined from a nonlinear regression fit, yielding a value of 85.4 nM; thus the unfolded protein can bind to the MIP with a still high affinity. The interaction of the whole protein for the MIP is however lower than that of the cyclic 3S peptide (IC50: 18.7 nM), which is consistent as the 3S peptide within the protein may be sterically hindered by other amino acids sequences, making it more difficult to be targeted by the MIP as compared to the free 3S peptide. In order to investigate whether blood proteins interfere with the binding of gp41 to the MIP, we examined the effect of albumin and transferrin as representative proteins found in human blood. HSA is the most abundant protein (50-60%) in human blood and transferrin, like gp41, is a glycoprotein. Increasing concentrations of free gp41urea, HSA, or transferrin were made to compete with a fixed amount of FITC-gp41urea bound to the MIP (Figure 6B). The IC50 was determined from a nonlinear regression fit, yielding a value of 5276 nM for HSA (cross-reactivity: 1.6%) and 75685 nM for transferrin (cross-reactivity: 0.1%). These results are very promising and show that the MIP can selectively recognize the 3S peptide of gp41 in vitro. Therefore, the MIP has great potential to interact and block the peptide on the virus during its interaction with CD4+ T cells.

Figure 6. (A) Binding of FITC-gp41urea (10 to 700 nM in 25 mM sodium phosphate buffer pH 7) to 100 µg MIP (black) or NIP (grey), immobilized on microplate; (B) Inhibition of FITC-gp41urea (350 nM) binding to 100 µg MIP by free denatured gp41 (circles), HSA (triangles), transferrin (inverted triangles), at concentrations ranging from 1 to 700 nM. B/B0 is the ratio of the amounts of FITCgp41urea bound in the presence and absence of displacing competitors.


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CONCLUSION We applied a solid-phase synthesis approach to prepare ‘monoclonal’-type synthetic antibodies in the form of nanosized (65 nm) water-soluble MIP-NPs, to target the 3S motif of the gp41 envelope protein of HIV-1. This highly conserved 3S motif present in HIV-1 strains has been reported to significantly contribute to the decline of CD4+ T cells during infection. Anti-3S antibodies could prevent the depletion of the lymphocytes but as with all antibodies, they have a few shortcomings such as low stability outside their natural environments and their high production costs. Herein, MIPNPs used as antibody mimics, bind the 3S motif alone or within the protein, with a high affinity and selectivity. Our results show the great potential of this MIP to block the function of 3S peptide in vitro. However, its effectiveness has to be confirmed in the presence of HIV-1 during infection of the host cell, process which will expose the 3S motif. Our next step will include the cytotoxicity evaluation of the MIP on peripheral blood mononuclear cells and the obtention of a fluorescent MIP for future injection in HIV-infected mice for bioimaging, pharmacokinetic and antivirus studies. Our findings indicate that MIPs may potentially be used as therapeutic drugs and can eventually be engineered to become an immunoprotective HIV vaccine, constituting an important breakthrough in nanomedicine.

SUPPORTING INFORMATION The Supporting Information contains standard calibration curves for protein, pep-rho and FITCgp41urea quantification, physicochemical characterization of MIP-NPs (size, SEM, zeta potential, LCST, EDS) and chromatograms of Boc protected and deprotected cyclic 3S peptide as analyzed by LC-ESI/HRMS. Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENTS Jingjing Xu thanks the China Scholarship Council (CSC) and the China Postdoctoral Science Foundation (project No. 2018M632083) for financial support. J. Xu, F. Merlier, B. Avalle, V. Vieillard, P. Debré, K. Haupt and B. Tse Sum Bui acknowledge financial support from the Region of Picardy and the European Union (cofunding of equipment under CPER 2007-2020 and project POLYSENSE). We thank Frederic Nadaud for the SEM and EDS images.

REFERENCES (1) http://www.unaids.org/en/resources/fact-sheet, accessed August 06, 2018. (2) Vieillard, V.; Strominger, J. L.; Debré, P. NK Cytotoxicity Against CD4+ T Cells During HIV-1 ACS Paragon Plus Environment

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Molecularly Imprinted Polymer Nanoparticles as Potential Synthetic

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