Fluorescence Correlation Spectroscopy Monitors the Fate of Degradable Nanocarriers in the Blood Stream

The use of nanoparticles as carriers to deliver pharmacologically active compounds to specific parts of the body via the bloodstream is a promising therapeutic approach for the effective treatment of various diseases. To reach their target sites, nanocarriers (NCs) need to circulate in the bloodstream for prolonged periods without aggregation, degradation, or cargo loss. However, it is very difficult to identify and monitor small-sized NCs and their cargo in the dense and highly complex blood environment. Here, we present a new fluorescence correlation spectroscopy-based method that allows the precise characterization of fluorescently labeled NCs in samples of less than 50 μL of whole blood. The NC size, concentration, and loading efficiency can be measured to evaluate circulation times, stability, or premature drug release. We apply the new method to follow the fate of pH-degradable fluorescent cargo-loaded nanogels in the blood of live mice for periods of up to 72 h.


Additional Characterization Methods
DLS measurements of the nanogels were performed at 25 °C using a Malvern ZetaSizer Nano S purchased from Malvern Instruments Ltd. (Malvern, Great Britain) with a He/Ne Laser (λ = 633 nm) at a fixed scattering angle of 173°.
1 H NMR spectra of the precursor polymes for the nanogel synthesis were recorded at room temperature on a Bruker Avance III 300 MHz spectrometer. The chemical shifts (δ) are given in parts per million (ppm) relative to TMS. NMR spectra were processed with the software MestReNova 11.0.4 by Mestrelab Research. Samples were prepared in deuterated solvents and their corresponding signals referenced to residual non-deuterated solvent signals.
Size-exclusion chromatography of the precursor polymes for the nanogel synthesis was performed using the following set-up: a PU2080+ pump, an auto sampler AS1555, an UV detector UV2075+ and a RI-detector RI2080+ from JASCO. Hexafluoroisopropanol (HFIP) containing 3.0 g L -1 of potassium trifluoroacetate was used as eluent at a flowrate of 0.8 mL min -1 and a column temperature of 40 °C.
The column material was composed of modified silica obtained from MZ-Analysentechnik: PFG columns, particle size: 7 μm, porosity: 100 Å + 1000 Å. A calibration with poly(methyl methacrylate) (PMMA) standards was purchased from PSS (Mainz, Germany) and used for determining relative molecular weight. Toluene was used as internal standard. Samples were prepared at 1 mg mL -1 and filtered through PVDF syringe filters (0.2 μm pore size, Acrodisc) prior to injection. The data was processed with the software PSS WINGPC UniChrom.
UV/Vis spectra of the sequential bisamide formation during squarogel fabrication was recorded on a V-630 spectrophotometer equipped with a Peltier thermostatted ETC-717 single cell holder purchased from JASCO (Pfungstadt, Germany). Measurement conditions of 20 °C were guaranteed by a V50 water thermostat from Krüss Optronic (Hamburg, Germany).     Figure S5. Scheme of sequential preparation of Oregon Green-labeled squarogels.

Nanogel Synthesis
The preparation of squaric ester-based nanogels (referred to as squarogels) using mPEG 113 -b-p(MA-SQ) 43 derived block copolymers has been described recently. [1] The herein investigated Oregon-Greenlabeled, pH-responsive squarogels have been fabricated in analogy: Initially, the block copolymer mPEG 113 -p(SQ-MA) 43 (30.0 mg, 1.89 µmol polymer, 75.56 µmol reactive squaric ester amide units) was dispersed in ethanol at a final concentration of 10 mg/mL. After 1 h of ultrasonicating, the formation of self-assembled polymeric micelles was confirmed by DLS measurements. Next, the micellar solution was transferred into a Schlenk tube equipped with a stirring bar and 150 µL of Oregon Green cadaverine stock solution (2.5 mg mL -1 in DMSO, 187.6 µg, 0.38 µmol, 0.005 eq) were added to label the squarogels fluorescently. Subsequently, the pH-responsive ketal-crosslinker 2,2-bis(aminoethoxy)propane (1.8 µL, 11.3 µmol, 0.15 eq) was added and the resulting reaction mixture was stirred for 2 days at RT. To fabricate non-drug loaded fully PEGylated nanogels the remaining squaric ester amide units were reacted with mPEG 0.75kDa -NH 2 . Therefore, an excess of 8 mPEG 0.75kDa -NH 2 stock solution (2 mL, 85 mg mL -1 in DMSO,226.69 µmol,170.01 mg, 3 eq.) was added and the reaction mixture was allowed to stir at RT for another 7 days. Complete conversion of the reactive ester units was confirmed by UV-Vis absorbance (compare Figure S6).
In order to purify the obtained squarogels and remove small molecular by-products, the reaction mixture was dialyzed (molecular weight cut-off: 1 kDa) against milliQ-water containing 0.1 % ammonia (1 L) for 7 days with frequent water exchanges. Subsequent lyophilization afforded the Oregon Greenlabeled squarogels as fluffy orange powder (56 mg).
To ensure complete removal of residual unbound dye that could interfere with FCS measurements, the fluorescently labeled squarogels were further purified by spin-filtration. For this purpose, Oregon Green-labeled squarogels were redispersed in PBS (2 mg mL -1 ) and purified using centrifugal filter units (regenerated cellulose, molecular weight cut-off: 10,000 g mol -1 ). After each sequential centrifugation step PBS was added until complete disappearance of Oregon Green derived absorbance was observed by UV Vis measurements.