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PDZ2-conjugated-PLGA nanoparticles are tiny heroes in the battle against SARS-CoV-2 – Scientific Reports

Synthesis of PLGA-PEG co-polymer

The synthesis of PLGA-PEG copolymer followed the method outlined by Zumaya et al., with minor modifications47.

PLGA-NHS activation

PLGA-COOH (1 g, MW 10kDa) (Nanosoft polymers, Winston-Salem, NC, USA) was activated and converted to PLGA-NHS with excess of N,N′-Dicyclo-hexylcarbodiimide (DCC) and N-Hydroxysuccinimide (NHS). Quickly, PLGA-COOH was dissolved in DCM (10 ml, obtaining a solution of 0.01 mM) (Merck, Darmstadt, DE) followed by the addiction of 2.5 equivalent of DCC (52 mg, 0.025 mM) and NHS (29 mg, 0.025 mM) (both from Fluorochem Ltd, Glossop, UK). The reaction was left under magnetic stirring at room temperature overnight. Insoluble dicyclohexyl urea was filtered and the activated PLGA was dried under vacuum at room temperature to be later conjugated to PEG (see Fig. 2).

PLGA-NHS conjugation through NH2-PEG-NH2

The resultant PLGA-NHS was dissolved in 10 ml of DCM before the addiction of 2 equivalent of NH2-PEG-NH2 (MW 3 kDa) (Sigma-Aldrich, Saint Louis, MO, USA) obtaining a solution of 0.02 mM. Again, the reaction was left under magnetic stirring overnight and the resultant polymer was dried under vacuum. To separate NH2-PEG-NH2 from PLGA-PEG copolymer, the obtained dried product was purified through solid–liquid extraction with methanol (Fluka Chemicals, Buchs, CH) in order to get ready for the next reaction (see Fig. 2). Subsequently the compound was characterized by Fourier-transform infrared spectroscopy (FT-IR) (see Fig. S1).

Bis-sulfone activation

100 mg of Bis-sulfone (Fluorochem Ltd, Glossop, UK) were dissolved in 10 ml of DCM (0.02 mM). Both DCC and NHS were added in a stoichiometric excess of 2 times (0.04 mM) compared to Bis-sulfone. The reaction was left under gentle stirring for about 2 h and the resulting product was filtered, dried under vacuum, and lastly purified via solid–liquid extraction using diethyl ether (Merck, Darmstadt, DE). This process effectively removed unreacted Bis-sulfone, separating it from the desired product (see Fig. 2).

Preparation of PLGA-PEG-Bis-sulfone

After the activation of bis-sulfone, PLGA-PEG-NH2 were dissolved in 10 ml of DCM (0.007 mM). Once solubilized, 1.1 equivalents of activated bis-sulfone were added to the solution (0.0077 mM). The reaction was allowed to proceed under magnetic stirring over-night. The resulting polymer was purified via solid–liquid extraction with methanol and dried under vacuum to be later finally used to prepare NPs (see Fig. 2). The purity of compound was characterized by FT-IR and Nuclear Magnetic Resonance (NMR) (see Figs. S1, S2).

Preparation of PLGA-PEG-Bis-sulfone nanoparticles

To synthetize PLGA-PEG-Bis-sulfone nanoparticles (PPB-NPs), we performed nanoprecipitation method. PLGA-PEG-Bis-sulfone was dissolved in Tetrahydrofuran (THF) (Applied Biosystems by Thermo Fisher Scientific, Waltham, MA, USA) to reach a concentration of 100 mg/ml. Finally, 300 μl (equivalent to 30 mg of PLGA-PEG-bis-sulfone polymer) of the result solution was added dropwise to 7 ml of stirring water. The final product was freeze-dried and then analyzed through DLS and NTA.

Preparation of PEG-mono-sulfone

The synthesis of PEG-mono-sulfone was induced adding to freeze-dried-NPs an appropriate buffer (1500 mM of NaCl, 20 mM EDTA (Sigma-Aldrich, Saint Louis, MO, USA) and 500 mM of sodium phosphate (Carlo Erba, Val de Reuil, FR), pH 8)41. The reaction was incubated at 37 °C for ~ 6h.

Protein expression and purification

Expression and purification of the PDZ2 domain of the ZO1 protein involved subcloning its encoding construct into a pET28b + plasmid vector, which was subsequently transformed into Escherichia coli BL21 (DE3) cells. Bacterial cells were cultured in LB medium with 30 μg/ml kanamycin at 37 °C until reaching an OD600 of 0.7 − 0.8. Protein expression was then induced with 0.5 mM IPTG. Following induction, cells were allowed to grow overnight at 25 °C and were subsequently harvested through centrifugation. The resulting pellet was resuspended in a buffer containing 50 mM TrisHCl, 300 mM NaCl, 10 mM imidazole (pH 8.0), along with one antiprotease tablet (Complete EDTA-free, Roche). The suspension was sonicated and centrifuged, and the soluble fraction from the bacterial lysate was applied to a Ni-charged HisTrap Chelating HP (GE Healthcare) column. The equilibration buffer used was 50 mM TrisHCl, 300 mM NaCl, 10 mM imidazole (pH 8.0). Elution was carried out using an ÄKTA-prime system with a gradient of imidazole ranging from 0 to 1 M. Fractions containing the protein were identified through SDS-PAGE. Buffer exchange to 10 mM Hepes, 150 mM NaCl (pH 7.4) was performed using a HiTrap Desalting column (GE Healthcare). The protein’s purity was assessed via SDS-PAGE. Site-directed mutagenesis was accomplished using the QuikChange mutagenesis kit (Stratagene) following the manufacturer’s instructions.

Functionalization of PLGA-PEG-Bis-sulfone NPs with wt PDZ2-ZO1

After the incubation, a solution of PDZ2-ZO1 was added in order to reach a final concentration of 200 μM of equivalent of this domain. The final product was incubated at Tamb overnight at pH 6 and finally characterized via HPLC. The whole procedure and final construct obtained, named PDZ2-f-NPs, was finally patented (provisional national patent application, No 102023000023964,, October 2023)).

Synthesis of 6-Coumarin-loaded NPs

6-Coumarin-loaded NPs were made dissolving 5 mg of 6-Coumarin (Sigma-Aldrich, Saint Louis, MO, USA) in 300 μl of a solution of 30 mg of PLGA-PEG-Bis-sulfone in THF. This solution was added dropwise to 7 ml of stirring water. The reaction was left to proceed for 3 h and then they were collected and centrifugated to be subsequently used for further analysis.

High performance liquid chromatography (HPLC)

To understand the purity of PDZ2-f-NPs, High Performance Liquid Chromatography (HPLC) was performed. All the measurements were made using a Thermo Finnigan Surveyor HPLC using an Agilent ZORBAX RRHD SB300-C8, 12.5 × 2.1 mm, 5 μm as column.

The mobile phases used consisted of 0.1% Trifluoroacetic acid (TFA) (Sigma-Aldrich, Saint Louis, MO, USA) in H2O for mobile phase A and 0.1% TFA in 80% Acetonitrile (ACN) (Merck, Darmstadt, DE) for mobile phase B. A gradient elution was conducted at a flow rate of 350 μl/min, with 100 μl of the sample injected using an autosampler set at 10 °C. The column temperature was optimized to 70 °C. Detection of the eluent’s absorbance was performed at both 214 nm and 280 nm.

Surface activation, ligand immobilization and binding (PDZ2-f-NPs vs SARS-CoV-2 E-protein)

The interaction between the C-terminal portion of the Envelope protein (ligand) and purified PDZ2-f-NPs (analyte) was assessed using the Surface Plasmon Resonance (SPR) technique, employing a Biacore X100 instrument (Biacore, Uppsala, Sweden). The N-terminal biotinylated peptides (ligand, VKNLNSSRVPDLLV12) were sourced from GenScript (Piscataway, NJ, USA), and immobilized on a Sensor Chip SA pre-coated with streptavidin from Biacore AB. The ligand immobilization procedure adhered to the manufacturer’s instructions, targeting 1000 response units for ligand immobilization. The running buffer employed was Hepes-buffered saline-EP 1× (HBS-EP 1X), containing 10 mM Hepes (pH 7.4), 0.15 M NaCl, 3 mM EDTA, and 0.05% v/v Surfactant P20 from Biacore AB.

Analytes were dissolved in the running buffer, and binding experiments were conducted at 25 °C with a flow rate of 30 μl/min. The association phase between the ligand and analyte was monitored for 180 s, followed by a dissociation phase lasting 300 s. The highest concentration used for PDZ2-f-NPs was 30 μM (in equivalents of proteins). Concentrations in the SPR assay were achieved through successive dilutions: 30 μM, 15 μM, 7.5 μM, 3.75 μM, 1.875 μM, 1 μM, 0.5 μM, 0.25 μM, and 0.0625 μM. To regenerate the chip’s surface, complete dissociation of the active complex was achieved by introducing 2M NaCl for 30 s before initiating each new cycle. When the experimental data met the quality criteria, data analysis was conducted using the Biacore X100 Evaluation Software. An affinity steady-state model was employed to fit the data since kinetic parameters fell outside the instrument’s measurement range, but an equilibrium signal of interaction was distinctly observed. As a result, the specific KD (dissociation constant) was determined, along with a confidence interval associated with a standard error value to mitigate potential biases.

Dynamic light scattering (DLS)

The effective average size and the polydispersity index of the development NPs were evaluated using the Zetasizer Nano S (Malvern Instruments, Malvern, UK). NPs were diluted with distilled water and the measurements were carried out using the Dynamic Light Scattering (DLS) mode at 25 °C. The obtained results are the average of at least two analyses on the same sample.

Nanoparticle tracking analysis (NTA)

NTA (nanoparticle tracking analysis) measurements were conducted using a NanoSight LM10-HS system manufactured by NanoSight (Amesbury, United Kingdom). NPs were diluted at a 1:100 ratio in PBS-1X. Subsequently, the sample was introduced into the sample chamber using sterile syringes (BD Discardit II, New Jersey, USA) until the liquid reached the nozzle’s tip. Five recordings, each lasting 60 s, were performed at room temperature. The NTA software was employed to generate high-resolution particle size distribution profiles and determine particle concentration. Dilution factors were utilized to calculate the particle concentration accurately.

Cell culture

African green monkey kidney (VERO) epithelial cells (ATCC CCL-81) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% inactivated fetal calf serum (FCS) (Euro-Clone, Milan, Italy), 1% glutamine (EuroClone, Milan, Italy), 1% streptomycin–penicillin antibiotics (EuroClone, Milan, Italy) and incubated in a humidified atmosphere (5% CO2 at 37 °C)48.

Measurement of SARS-CoV-2 infection inhibition by PDZ2-f-NPs

African green monkey kidney (VERO) epithelial cells (ATCC CCL-81) were cultured as reported elsewhere and before48. Cells were washed with sterile warm Phosphate buffer (PBS), trypsinized and counted. The monolayer was obtained seeding cells in 48 well plates (Nest) at a final concentration of 7 × 104 cell/ml. When a confluent monolayer > 90% was reached, cells were treated with serial dilutions of PDZ2-f-NPs (PDZ2 concentration in the range from 200 to 1.5 µM) in culture medium. Twenty-four hours later, cells were washed with sterile warm PBS and then infected with 0.1 ml of solution containing SARS-CoV-2 (1 × 105 PFU/ml). Two hours later, infection solution was removed, and new fresh DMEM medium (supplemented with 2% FCS, 1 mM glutamine, 1% streptomycin-penicillin antibiotics) was added. Cells were incubated as previously described and infection status was monitored daily49. All the experiments that involved SARS-CoV-2 manipulation were carried out in Biosafety level 3 laboratory (BSL3) in the Institute of Microbiology of IRCCS—Fondazione Policlinico Gemelli.

Crystal violet staining was performed to evaluate cell viability and cellular disruption following the SARS-CoV-2 infection and its replication. Briefly, cells were fixed by using 4% paraformaldehyde for 30 min and then stained by using Crystal violet for 30 min. After incubation five washes were carried out and images were acquired by using Cytation instrument48,49.

Images were analyzed using the freely available ImageJ version 1.47v (NIH, USA). Every set of tiff images corresponding to crystal violet staining were analyzed through the “Process > Batch > Macro tool”. Each image was converted to 8-bit image. Minimum and maximum thresholds were manually set for each batch of images, to correctly convert areas to white and black, respectively. Prior to perform the “Measure” tool of ImageJ, all the images were processed with the “Smooth” and “Convert to Mask”. The fraction of the area covered by cells is then automatically stored in the results file48.

Confocal microscopy

VERO cells were plated on µSlide 8 wells to reach a concentration of 6.000 cells/well and treated for 24 h with 6-coumarin-loaded-PDZ2-f-NPs. Subsequently the cells were washed three times with PBS-1× to be later fixed with 5% formalin for 20 min at room temperature. The cells were then permeabilized with methanol for 4 min at – 20 °C and then they were washed 2 times with PBS-1×. Afterward, cells were blocked with a solution of PBS with 5% Bovine serum albumin (BSA) and 0.1% Tween 20 for 1 h. For the staining, at first, we added a solution of 1:200 of primary antibody (β-Actin (D6A8) Rabbit mAb) and incubated it for 2 h. The cells were washed with blocked solution and the secondary antibody was added (CyTM3 AffiniPure Goat Anti-Rabbit IgG (H + L), 1:200, 1h). Finally, VERO cells were washed with blocking solution to be later analyzed with both confocal and fluorescence microscopy. Confocal microscopy measurements were carried out using an inverted microscope (Nikon A1 MP+, Nikon, Tokyo, Japan) equipped with a 20 × objective.