We use fluorescence correlation spectroscopy (FCS) to measure the hydrodynamic radius, fluorescence brightness, and local concentration of fluorescently labeled colloids and macromolecules (polymers, copolymers, proteins, and DNA). These measurements allow us to investigate conformational changes, mutual interactions, and aggregation. As discussed in our recent Reviews [1, 2], such FCS studies are particularly useful for characterizing drug nanocarriers (NCs) at all stages of the delivery process, enabling monitoring of carrier formation, drug loading efficiency, stability, and kinetics of drug release [3–8]. To reach their target sites, NCs need to circulate in the bloodstream for prolonged periods without aggregation, degradation, or cargo loss. However, identifying and monitoring small NCs and their cargo in the dense, highly complex, and opaque blood environment is extremely challenging. To address this problem, we have developed two FCS approaches that allow direct characterization of drug NCs in blood:
Monitoring drug NCs in a blood droplet [9]
In the first approach we place a blood droplet of about 30 µL on a plasma separation membrane positioned directly above the FCS observation volume. The membrane retains the large blood cells, but lets the liquid part of the blood and the NCs pass through, thus enabing their FCS characterization (Figure 1). We applied this approach to monitor changes in the size, concentration, and loading efficiency of pH-degradable fluorescent cargo-loaded squarogel NCs in the blood of live mice (Figure 2) for periods of up to 72 h after NCs injection.
Figure 1. Schematic representation of the FCS experiments in a blood droplet. (Left) Optical setup and sample chamber. (Middle) Schematic ilustrating how the membrane prevents the blood cells from reaching the detection volume. (Right) SEM images showing top view and bottom view of the membrane.
Figure 2. Monitoring the fate of drug NCs in blood stream of a mouse. (Left) Normalized autocorrelation curves recorded in blood samples taken from a mouse 0 (green), 6 (blue), 24 (orange) and 72 h (magenta) after injection of NCs. (Right) Hydrodynamic radius of the NCs versus time after injection. Data from 3 mice experiments are shown. In cooperation with L. Nuhn (MPIP, AK Weil) and L. Kaps (Mainz University Medical Center, TIM).
Monitoring drug NCs in flowing blood [10]
In the second approach we monitor the NCs directly in whole blood, i.e. without removing the blood cells. To this end we use a fully near infrared FCS setup and perform experiments in slowly flowing blood to ensure time intervals, in which the FCS probing volume is free of blood cells, and thus accessible for the studied fluorescent species (Figure 3). Using this approach, we reported the first FCS based measurements of the size, loading efficiency and stability of nanocarriers in whole blood [Figure 4].
Figure 3. Overview of the NIR-FCS experiments and data analysis in flowing blood. (Left) Blood containing fluorescently labeled polymer brushes (as a model for drug nanocarriers) is pumped through a flow channel. The FCS observation volume is consequently either free (schematics 1) or occupied (schematics 2) by a blood cell. Correspondingly, the fluorescence intensity time trace revealed high (1) and low (2) intensity time segments. (Right up) The experimental autocorrelation curve (squares) is fitted (line) with analytical model combining standard and inverse FCS, thus taking into account contributions of fluorescent species and blood cells, respectively. (Right down) The information extracted from the fit in panel (b) is used to subtract the cells’ contribution and obtain an autocorrelation curve (squares) resembling that of a standard FCS experiment.
Figure 4. Loading stability of core-crosslinked micelle nanocarriers in blood. Normalized autocorrelation curves (symbols) and the corresponding fits (lines) are shown for core-crosslinked micelles that were either covalently (blue color) or non-covalently (green color) loaded with IRDye®800CW. In water (left) the dye is mainly loaded in the core-crosslinked micelles and only a small fraction of free dye was detected for both systems. In blood (right) the covalently loaded dye is still in the micelles even after 30 hours incubation, but the non-covalently (hydrophobically) loaded dye is fully released after 30 minutes. In cooperation with M. Barz (Uni Mainz and MPIP, AK Weil).
1.
van den Hoven, L.; Goddaer, S.; Mirzahossein, E.; Vermonden, T.; Koynov, K.; Remaut, K.; van Ravensteijn, B. G.P.
Unravelling drug delivery using in vitro Fluorescence Correlation Spectroscopy (FCS).
Journal of Controlled Release, 2025, 388, 114311
2.
Schmitt S, Nuhn L, Barz M, Butt H-J, Koynov K.
Shining Light on Polymeric Drug Nanocarriers with Fluorescence Correlation Spectroscopy.
