Tuning the Elasticity of Nanogels Improves Their Circulation Time by Evading Immune Cells

Abstract

Peptide receptor radionuclide therapy is used to treat solid tumors by locally delivering radiation. However, due to nephro- and hepato-toxicity, it is limited by its dosage. To amplify radiation damage to tumor cells, radiolabeled nanogels can be used. We show that by tuning the mechanical properties of nanogels significant enhancement in circulation half-life of the gel could be achieved. We demonstrate why and how small changes in the mechanical properties of the nanogels influence its cellular fate. Nanogels with a storage modulus of 37 kPa were minimally phagocytosed by monocytes and macrophages compared to nanogels with 93 kPa modulus. Using PET/CT a significant difference in the blood circulation time of the nanogels was shown. Computer simulations affirmed the results and predicted the mechanism of cellular uptake of the nanogels. Altogether, this work emphasizes the important role of elasticity even for particles that are inherently soft such as nano- or microgels.

Keywords: Biodistribution; Elasticity; Nanogels; Phagocytosis; Radiolabeling.

Figure 1

Synthesis of NGs with different elasticities. A) Schematic representation of nanogels synthesis via inverse miniemulsion. B) Tuning bulk hydrogel modulus over a range of pre‐polymer concentrations by varying the number of functional thiol‐groups. C) Schematic representation of the difference in crosslink density using two different pre‐polymer molecular weights but with the same polymer volume fraction. D) Raman spectra of the nanogels which confirms NG synthesis. The thiol band at 2560 cm⁻¹ disappears upon NG formation and a disulfide band at 503 cm⁻¹ appears after NG synthesis.

Figure 2

Characterization of three different nanogels. A) Hydrodynamic radius and Zeta potential of different nanogels showing similar physicochemical properties, with R _h in the range 140–160 nm and Zeta potential ranging from −13 to −27 mV. B), C), D) Spherical morphology of the different nanogels as demonstrated by Cryo‐SEM. Scale bar: 2 μm. E), F), G) Histogram of different nanogels size analyzed from cryo‐FESEM using ImageJ.

Figure 3

Height profiles of the nanogels in dry state to determine their deformability. A), B) AFM of hard and soft reducible nanogels. C) Schematic representing the behavior of elastic NGs on the mica surface. D) Height profile of a single nanogel particle. E) Lateral size of 20 random nanogels particles. F) Average deformation of nanogels from 20 particles each. **** (p<0.0001) denotes statistical significance using student's t‐test (two‐tailed, Welch's correction).

Figure 4

AFM peak force measurement in the swollen state. A), B) Topography image obtained with peak force microscopy at the water‐gold interface and C), E) Young's modulus histograms (mapped to the topography images) of hard reducible NG (C) and soft reducible NG (E). The Young's modulus was calculated by analyzing 3000 force–distance (F)–(D) curves collected during the AFM imaging. D) Rrepresentative force–distance curves taken at the apex of the soft reducible NG relative to the solid support.

Figure 5

Cellular uptake of Alexa Fluor 488 labeled nanogels. A), B), D), and E) flow cytometry histogram of cellular uptake of different nanogels at 4 h and 24 h, respectively, by THP‐1 cells and stimulated THP‐1 cells showing higher uptake by the hard nanogels (as demonstrated by an increase in forward scatter). C), F) MFI of the nanogels uptake by THP‐1 and stimulated THP‐1, respectively (n=3). **** (p<0.0001), *** (p<0.001), ** (p<0.01), * (p<0.05) denotes statistical significance using student's t‐test (two‐tailed, Welch's correction) between hard reducible and soft reducible NG, soft reducible and soft non‐reducible NG.

Figure 6

Mechanism of nanogel uptake by stimulated THP‐1 cells. A) Flow cytometry histogram of different elastic NGs treated with or without CytD. B) Percentage of NGs cell uptake inhibited upon treating with CytD. C), D) Schematic representing the effect of actin filaments on different elastic NG uptake. C) The presence of actin filaments beneath the cell membrane adds a certain force to the membrane, which deforms the softer NGs, thereby reducing uptake. D) Blocking the actin polymerization by treating the cells with cytochalasin D slows down and reduces the kinetics of cell uptake of the NGs.

Figure 7

A), B) Simulation snapshots of single hard (A) and soft (B) nanogels at lipid membranes at different simulation times (water molecules are not shown); C), D) number of contacts between NG segments and lipid heads (C) and wrapping ratio (the ratio of NG‐membrane contact area to the total area of the NG) as D) functions of simulation time.

Figure 8

In vivo profiles of different radiolabeled nanogels. A) PET/CT images of radiolabeled NGs injected in vivo at 1 h and B) 4 h post injection (p.i.) showing an increased circulation of soft nanogels. SUV: Standardised uptake value, HU: Hounsfield units. Arrows in white indicate the heart. C) Gamma counter analysis after the last PET/CT of the harvested organs represented as % ID/g tissue. D), E), and F) Average activity (KBq/cc) of the heart, kidneys, and bladder, respectively, was obtained by drawing regions of interest (ROIs) on the whole body images. Values are mean±SD (n=3). * (p<0.05), *** (p<0.001) as determined by ANOVA.