RG502 poly(
Pluronic F127 (20 g, 3.2 mmol of OH) was dissolved in toluene (100 mL) in a three neck round bottom flask connected to a Soxhlet (filled with dry 4 Å molecular sieves) and fitted with a reflux condenser, under dry argon atmosphere and dried by azeotropic distillation (4 h). The dried Pluronic F127 was cooled to 40 °C and NaH (15.36 mg, 0.64 mmol, corresponding to 1:0.2 Pluronic F127-OH/NaH molar ratio) added. The reaction was stirred at 40 °C for 40 min. The partially deprotonated Pluronic F127 was syphoned into another degassed reactor containing DVS (4.73 g, 40 mmol, corresponding to 80 mmol of double bonds, i.e. to 1:0.2:25 Pluronic F127-OH/NaH/double bonds molar ratio) previously dissolved in dry toluene (300 mL). The reaction was left stirring at 40 °C for 24 h. The mixture was filtered to remove any salt formed, neutralized by adding a drop of acetic acid, reduced in volume by rotary evaporation and precipitated twice in ice-cold diethyl ether. After precipitation, the polymer was washed three times with diethyl ether, dried under vacuum and stored at − 20 °C. Yield: 92% (based on the weight of recovered polymer/theoretical amount of polymer). Conversion: 63% (molar percentage of reacted OH groups), as confirmed by 1H NMR.
1H NMR was recorded on ca. 1% wt. polymer solutions in CDCl3, D2O or D2O/buffer mixture using a Bruker Avance 400 MHz spectrometer. The residual solvent signal was used as internal reference (ppm). NMR samples with a high content of non-deuterated aqueous solvent were analyzed using a pulse sequence with f1 presaturation for the selective suppression of the HOD signal.
Pluronic F127-VS-RGD preparation and characterization. (
Peptide solutions were prepared in phosphate buffer (100 mM, pH = 9), previously degassed with argon, as follow: 21.5 mg and 11.6 mg of, respectively, linear and cyclic peptide (corresponding to ≈ 20 µmol of peptide) were dissolved in 1 mL of buffer. The pH was then adjusted to ≈ 9 using a 0.1 M NaOH solution and then both peptide solution volumes adjusted to 2.5 mL. Equal volumes of peptide solution and Pluronic F127-VS solution (10% w/v in phosphate buffer, pH = 9) were then mixed (30 min under stirring) in order to have a VS:peptide molar ratio of 1:2 (10 µmol of VS : 20 µmol of peptide. 100 mg of Pluronic = 8 µmol of Pluronic F127 and 16 µmol of OH end groups. The 63% of OH group, corresponding to 10 µmol of VS, were replaced with VS group). Finally, the compound was dialyzed for a minimum of 12 h against MilliQ water using a Spectra-Por Float-A-Lyzer G2 (MWCO: 3.5 KDa, Spectrum Labs), freeze dried and stored at − 20 °C until further use.
PLGA-COOH was covalently conjugated to Lissamine Rhodamine B ethylenediamine to produce a fluorescently labeled PLGA (PLGA-Rho). All solutions were prepared in THF unless stated otherwise. Briefly, 50 mg of the polymer, PLGA-COOH (4.17 × 10−3 mmol of carboxylate) was dissolved in 10 mL by shaking overnight. After complete dissolution of the polymer, 2.5 mL of a solution containing 3.25 mg of rhodamine B (5.4 × 10−3 mmol, 1.3 eq.) was added to the polymer solution and stirred for 15 min, after which 2.5 mL of a solution containing 7 mg of DMT-MM (2.5 × 10−2 mmol, 6 eq.) was added. The reaction was stirred at 400 rpm at 25 °C for 72 h, and then quenched and precipitated using cold methanol. The polymer pellet was collected by centrifugation (10 min at 4,500 g), resuspended in dichloromethane, and precipitated again using cold methanol. The precipitate was then thoroughly washed with methanol and finally vacuum-dried (mass recovery: 80%). The degree of derivatization (0.8% mol) was determined by measuring the fluorescence intensity of the PLGA-Rho, using the rhodamine dye to relate the measured emission to the molar concentrations of the fluorophore (please note that this was later transformed in a molar ratio between functionalized and non-functionalized units in the polymer).
An automated microfluidic Asia 320 flow reactor (Syrris, Royston UK) was used for all nanoparticle preparations. Nanoparticles were prepared by mixing a 0.015%wt. aqueous surfactant solution (Pluronic F127 mixtures containing 0, 1, 10, 50 or 100% of RGD derivatives) with a 0.31%wt. PLGA (RG502) acetone solution using an Asia 1,000 μL 3-input reaction chip (Syrris part number: 2100146). For fluorescently labelled particles, the acetone solution contained a 10% wt. PLGA-Rho:PLGA mixtures. In all experiments acetone/water flow rate ratio of 0.2 and a total flow of 2 mL/min. Nanoparticles were left at 30 °C for 12 h under stirring in order to evaporate the acetone, filtered through a 0.22 µm PES membrane, divided in 1 mL aliquots and centrifuged in sterile 2 mL round-bottom plastic tubes. Particles were then resuspended in 1 mL of distilled water; five of these aliquots were combined and freeze dried to determine the dry weight and from there the mass recovery of the process. Typical mass recovery values: 90–95%. For cell culture purposes, the nanoparticles were further centrifuged and re-dispersed in culture media, typically at a concentration of 0.5 mg/mL.
The Z-average (apparent hydrodynamic) size, polydispersity (PDI), count rates and ζ-potential of nanoparticle dispersions were obtained by DLS/electrophoretic mobility measurements at 25˚C using a Zetasizer Nano ZS (Malvern Instrument, UK). Samples were measured as prepared immediately after evaporating the organic phase.
A Synergy 2 multi-mode Biotek microplate reader (NorthStar Scientific Ltd, Leeds, UK) equipped with Gen5 software was used to characterize the fluorescence intensity of the nanoparticles prepared using different PLGA-Rho blends (excitation: 540/25 nm; emission: 620/40 nm; optical position: top 50%, light source: xenon flash).
The QuantiPro bicinchoninic acid (BCA) Assay Kit was used to assess the amount of RGD exposed at the surface of the RGD-functionalized PLGA nanoparticles. Briefly, nanoparticles (prepared from 0, 1, 10, 50 or 100% Pluronic F127-RGD mixtures) were centrifuged at 13,000 rpm for 15 min, concentrated 5 to 10 times (i.e. concentration range 5–10 mg/mL), and washed with distilled water (three steps of centrifugation/washings to ensure removal of unbound RGD-surfactant) before being incubated with the BCA solution overnight at 37 °C, following manufacturer recommendations for highest sensitivity. Prior to any measurements, each nanoparticle suspension was thoroughly centrifuged (at 13,000 rpm for 1 h at 4 °C) to eliminate any background contribution to the UV–Vis reading from light scattering of the particles. Finally, the amount of RGD peptide at the surface of the nanoparticles was estimated by using a calibration of soluble Pluronic F127-RGDx (linear or cyclic RGD peptides as appropriate) in water in the concentration range 0.12–125 µg/mL using a Synergy2 Biotek plate reader (excitation: 540/25 nm; emission: 620/40 nm) equipped with Gen5 software (sensitivity of the instrument adjusted to wells with the highest nanoparticle concentration of the calibration curve; optical position: top 50%; light source: xenon flash). Note that in this quantification we assumed an identical BCA assay response for both the Pluronic F127-RGD (“calibration curve standard”) and the RGD-functionalized nanoparticles (“sample”).
A2780 and U87MG cells were grown in MEM or RPMI 1640 respectively under standard conditions (5% (v/v) CO2, 37 °C), regularly tested for mycoplasma, and used at passage numbers below 20. The RPMI 1640 and MEM cell culture growth media were supplemented with 10% (v/v) FBS, 2 mM L-glutamine, and 1% (v/v) penicilin-streptomycin.
U87MG and A2780 cells were seeded in Costar polystyrene 12-well plates with flat bottom at a density of 1.5 × 104 and 3 × 104 cells/cm2, respectively. Cells were left to adhere overnight and grow until a confluency of ~ 60 to 70% was reached. Cells were then exposed to 0.5 mg/mL rhodamine-labelled PLGA nanoparticles (centrifuged and redispersed in the appropriate cell culture medium) differing in (1) the format of RGD peptide exposed at their surface, i.e. either RGDc or RGDl, or (2) the amount of RGD peptide exposed at their surface, i.e. 1, 10, 50 or 100% Pluronic F127-RGD/–OH mixtures, for specific time points (4 , 8 and 16 h) in a humidified 5% (v/v) CO2 air atmosphere at 37 °C. A control of nontargeted particles, i.e. particles with exposed –OH end groups on their surface was also studied. Following a protocol previously described by our group
U87MG cells were plated in Costar polystyrene 12-well plates with flat bottom at a density of 1.5 × 104 cells/cm2. Cells were left to attach and grow until a confluency of ~ 60 to 70% was reached. In order to block specific uptake pathways, cells were pre-treated for 30 min at 37 °C with growth medium containing either (1) EIPA 5 µM, (2) 5 µg/mL αvβ3 antibody, (3) 5 µg/mL α5β1 antibody, (4) excess RGDc peptide 2.5 µM, (5) excess RGDl peptide 2.5 µM, or (6) PBS. Following pre-treatment and a thorough washing with PBS, the cells were exposed to rhodamine labelled PLGA particles (nontargeted or functionalized either with 100% RGDc or RGDl mixtures) in cell media containing the aforementioned inhibitors/competitors for 1 h at 37 °C. A control at 4 °C was also run to evaluate the effect of blocking the energy dependent processes on nanoparticle uptake. Following a previously described protocol
U87MG cells were seeded with a density of 25,000 cells/cm2 in an Ibidi 8 well µ-Slide and let to attach overnight at 37 °C, 5% CO2. A volume of 200 µL/well of cell culture media containing 0.5 mg/mL of rhodamine-labelled particles (prepared from Pluronic F127-RGD 0% (i.e. nontargeted) or 100% RGDc or RGDl mixtures) were used to treat the cells for 1, 4 or 24 h. In order to analyze the endocytic pathway involved, media supplemented with 1 μg/mL Hoechst and 200 nM LysoTracker Green were added in each well for 15 min at 37 °C, 5% CO2. To analyze the macropinocytic pathway, cells were incubated with 0.25 mg/mL green labelled dextran 70 in media for 1 h at 37 °C, 5% CO2. After treatment, living cells were washed with PBS and immediately used for imaging using a confocal microscopy protocol of ours
U87MG cells were seeded at a concentration of 1,000 cells (in 0.1 mL) in a 96-well ultra-low adherent (ULA) plate. The spheroids were grown at 37 °C, 5% CO2 for 10 days, replacing 50 μL of media every 3 days, until the spheroid mass reached a diameter of 400 μm. At day 10, 100 µL/well of cell culture media containing 1 mg/mL of rhodamine labeled particles (prepared from Pluronic F127-RGD 0% (i.e. nontargeted) or 100% RGDc or RGDl mixtures) was added to each well containing a spheroid to obtain a final nanoparticle concentration of 0.5 mg/mL in a total volume of 200 µL (at least 10 spheroids per experimental condition). After 24 h, the spheroids were washed with PBS and incubated with 1 μg/mL Hoechst 33,342 for 5 min at 37 °C, 5% CO2. Spheroids were then washed with PBS and incubated with trypsin/EDTA for 10 min at 37 °C, 5% CO2, further disaggregated with the help of a pipette and the fluorescence arising from Hoechst and rhodamine determined using flow cytometry. Untreated cells were used as negative control for autofluorescence and Hoechst fluorescence gradient was used to individuate two distinct regions: Hoechst positive cells (bright, Fig.
The reason why larger spheroids (average diameter ≈400 µm) were used for flow cytometry vs confocal microscopy experiments is threefold: (1) a much higher cell number is needed to run flow experiments, (2) larger spheroids are easier to manipulate (i.e. to see and handle) in experiments involving multiple passages, and more importantly (3) to obtain a well-defined Hoechst gradient. A quick incubation with Hoechst (for 10 min) is enough to create a three-region gradient in 400 µm spheroids; smaller spheroids would encumber this operation as Hoechst would quickly penetrate from the surface down to the core.
Spheroids with diameter of about 250 μm were incubated with a final nanoparticle concentration of 0.5 mg/mL in cell media (0.2 mL/well). After a 24 h incubation, the spheroids were washed with PBS and incubated with 1 μg/mL Hoechst 33,342 and 5 μg/mL CellMask for 5 min at 37 °C, 5% CO2. The spheroids were then washed and used for imaging using a confocal microscope. Confocal acquisitions were performed on live cells using a Leica TCS SP8 AOBS inverted confocal equipped with an immersion oil objective (20×/1.40/HCX PL Apo, Leica). For the acquisitions, the confocal settings
For the avoidance of doubt, we decided not to use spheroids larger than 250 µm in diameter for microscopy studies (as opposed to the 400 µm spheroids used for flow cytometry) since the transmitted light decreases with spheroid size. The use of 250 µm spheroids turned out to be the best compromise between ease of handling and light transmission at the core of the sphere.