Squaric Ester-Based, pH-Degradable Nanogels: Modular Nanocarriers for Safe, Systemic Administration of Toll-like Receptor 7/8 Agonistic Immune Modulators

Abstract

Small-molecular Toll-like receptor 7/8 (TLR7/8) agonists hold promise as immune modulators for a variety of immune therapeutic purposes including cancer therapy or vaccination. However, due to their rapid systemic distribution causing difficult-to-control inflammatory off-target effects, their application is still problematic, in particular systemically. To address this problem, we designed and robustly fabricated pH-responsive nanogels serving as versatile immunodrug nanocarriers for safe delivery of TLR7/8-stimulating imidazoquinolines after intravenous administration. To this aim, a primary amine-reactive methacrylamide monomer bearing a pendant squaric ester amide is introduced, which is polymerized under controlled RAFT polymerization conditions. Corresponding PEGderived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are aminereactive and can sequentially be transformed by acid-sensitive cross-linkers, dyes, and imidazoquinolines. Remaining squaric ester amides are hydrophilized affording fully hydrophilic nanogels with profound stability in human plasma but stimuli-responsive degradation upon exposure to endolysosomal pH conditions. The immunomodulatory behavior of the imidazoquinolines alone or conjugated to the nanogels was demonstrated by macrophages in vitro. In vivo, however, we observed a remarkable impact of the nanogel: After intravenous injection, a spatially controlled immunostimulatory activity was evident in the spleen, whereas systemic off-target inflammatory responses triggered by the small-molecular imidazoquinoline analogue were absent. These findings underline the potential of squaric ester-based, pH-degradable nanogels as a promising platform to permit intravenous administration routes of small-molecular TLR7/8 agonists and, thus, the opportunity to explore their adjuvant potency for systemic vaccination or cancer immunotherapy purposes.

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

“…Among the various types of block copolymer-based nanogels already developed by Nuhn et al, 12 , 58 − 61 blood-stable nanogels derived from amine-reactive methacrylamides with pendant squaric ester amides were introduced recently. 55 They are fabricated from RAFT-polymerized PEG block copolymers that are self-assembled into precursor micelles and sequentially transformed by amine-bearing acid-sensitive ketal crosslinkers, drugs, dyes, and short oligo(ethylene glycol)s into fully hydrophilic nanogels ( Supporting Information , Figures S1–S6). Due to their high degree of PEGylation, these squaric ester-based nanogels (so-called “squarogels”) provide profound stability in human plasma while being equipped with a stimuli-responsive degradation property upon exposure to endolysosomal pH conditions ( Supporting Information , Figure S7).…”

“…Intravenously injected squarogels circulate in the blood stream with an even distribution all over the body and progressive accumulation mainly in the liver, spleen, or kidney. 55 …”

“…These findings correspond with previously observed properties of the squarogels, such as poor uptake by phagocytes and other cells, and long residence in the blood stream, properties that can be correlated to their high degree of PEGylation. 55 …”

“…The squaric ester amide methacrylamide (MA-SQ) monomer and the macro chain transfer agent were synthesized following an earlier report. 55 Fluorescent dyes Alexa 647, Alexa 488, and Oregon Green 488 cadaverine were obtained from Thermo Fisher Scientific (Waltham, MA, USA).…”

“…By preventing the blood cells from entering the FCS detection volume, we can now perform precise FCS experiments with molecular species as small as individual dye molecules in a single droplet of whole blood with volume below 50 μL. This allowed us to follow the fate of a pH-degradable nanogel NC derived from ketal-crosslinked squaric ester amide precursor block copolymers 55 over 3 days after injection into live mice.…”