Tailored Multivalent Targeting of Siglecs with Photosensitizing Liposome Nanocarriers

Abstract The modification of surfaces with multiple ligands allows the formation of platforms for the study of multivalency in diverse processes. Herein we use this approach for the implementation of a photosensitizer (PS)‐nanocarrier system that binds efficiently to siglec‐10, a member of the CD33 family of siglecs (sialic acid (SA)‐binding immunoglobulin‐like lectins). In particular, a zinc phthalocyanine derivative bearing three SA moieties (PcSA) has been incorporated in the membrane of small unilamellar vesicles (SUVs), retaining its photophysical properties upon insertion into the SUV's membrane. The interaction of these biohybrid systems with human siglec‐10‐displaying supported lipid bilayers (SLBs) has shown the occurrence of weakly multivalent, superselective interactions between vesicle and SLB. The SLB therefore acts as an excellent cell membrane mimic, while the binding with PS‐loaded SUVs shows the potential for targeting siglec‐expressing cells with photosensitizing nanocarriers.


Supporting Information
Experimental Procedures

Preparation of Small Unilamellar Vesicles (SUVs)
Batches of 1 mL of 0.5 mg/mL of DOPC SUVs in PBS pH= 7.4, with/without different molar percentages of PcSA (0.1, 0.2, 0.5, 1, 2 and 5%), were prepared using a stock solution of 25 mg/mL of DOPC in chloroform (with 0-2 mol% DOPE-biotin in case of SUVs for SLB formation), and a stock solution of 1.44 mg/mL of PcSA in PBS. After adding the required amount of DOPC in chloroform into a glass vial, a gentle nitrogen flux was used to evaporate the solvent, forming a lipidic film on the vial walls. The residual solvent was removed placing the vials into a desiccator for 1 h. 1 mL of Milli-Q water (in case of SUVs for SLB formation) or PBS (for the binding of SUVs onto SLBs; different ratios of PBS and PcSA in PBS were used to obtain the desired %) was added. After performing 10 freeze-pump-thaw cycles, the vesicles were extruded through a 100 nm polycarbonate membrane (11 times; Whatman) and were stored in the fridge for two weeks.

Insertion Approach
DOPC-SUVs (0.5 mg/mL) in PBS were mixed with different concentrations of PcSA (0.6, 1.2, 3, 6, 12 and 30 µM) adjusting the volume to 1 mL of PBS pH= 7.4, being the same concentrations used for the formation of DOPC-SUVs containing different mol% of PcSA and were incubated for 4 h at room temperature under stirring. Vesicles were stored in the fridge and used within two weeks.

Particle Size Analysis
Particle size was recorded using DLS of 1 mL of DOPC-SUVs and DOPC-Pc SUVs (0.5 mg/mL) containing 0.1, 0.2, 0.5, 1, 2 and 5% of PcSA in PBS using a Nanotrac Wave particle size analyzer. The data are expressed as the average of three measurements, for a time of 120 s at 22°C. In the insertion experiment, the particle size of the resulting vesicles was measu red using the same parameters mentioned before.

Spectroscopic Experiments
UV-Vis spectra were recorded using a Perkin Elmer Lambda 850 spectrophotometer. 1 mL of every sample of vesicles were used to record the data, in a Hellma 108-QS 1000 μL Semi-Micro Absorption Cell, 10 mm Light Path. Fluorescence spectra of the previous mentioned vesicles were recorded using a Perkin Elmer FL 6500 fluorescence spectrometer, using Hellma 105.250-QS 100 μL Ultra-Micro Fluorescence Cell with 100 μL of every sample and irradiating at 665 nm.

Z-potential Measurements
Z-potential values were obtained using a Malvern Instruments Zetasizer Nano ZS and a folded capillary Zeta Cell from Malvern Panalytical. Samples were obtained diluting 800 µl of vesicles (0.5 mg/mL) 20 times with ultra-purified water, and Zpotential values were expressed as an average of 3 measurements at 25°C of 800 µL of every sample.

QCM-D Measurements
For the fabrication of SLBs, vesicles (SUVs) were diluted to a concentration of 0.1 mg/ml in PBS directly before use. SLBs were obtained by flowing this solution on a cleaned and activated SiO2 surface, after obtaining a stable baseline. The quality of the SLBs was monitored in situ by QCM-D (where high quality SLBs are defined by Δf = -24 ± 1 Hz and ΔD < 0.5×10 -6 ). QCM-D measurements were performed with a Qsense Analyser from Biolin Scientific, and SiO2-coated sensors (QSX303, Biolin Scientific) were used throughout this work. Measurements were done at 22 ºC and operated with four parallel flow chambers, using two Ismatec peristaltic pumps with a flow rate of 20 l/min. For every measurement, the fifth overtone was used for the normalized frequency (Δf5) and dissipation (ΔD5). In a typical experiment, SLBs were formed on previously cleaned and activated SiO2 sensors. Afterwards, solutions of SAv, of siglec-10 or siglec-10/PEG (1:1) mixtures, and then DOPC-SUVs or DOPC-Pc SUVs were added until a stable plateau was reached. Each step was followed by rinsing with PBS. The SLB and SAv steps were found reproducible within a standard deviation of 5% over multiple series. The siglec steps were found reproducible within a standard deviation of below 20%. For practical reasons, this % deviation could not be determined for the vesicle steps but based on previous experience, 41,43,45 we can assume the upper limit of the siglec (20% standard deviation) to hold also in this case. Figure S1. a) Schematic cartoon representing the mechanism of internalization and subcellular behavior of the PcSA photosensitizer, according to previous PDT studies by de la Escosura, Torres and coworkers, 1 which involve the following steps.