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

Anne Huppertsberg,Christian Czysch,Bruno G De Geest,Detlef Schuppan,Jana De Vrieze,Stefan Lienenklaus,Leonard Kaps,Sunil A David,Niek N Sanders,Kaloian Koynov,Adrian Klefenz,Judith Stickdorn,Sascha Schmitt,Francis Combes,Pia Winterwerber,Chaojian Chen,Zifu Zhong,Kim Deswarte,Sabah Kasmi,Matthias Bros,Lutz Nuhn,Niklas Choteschovsky,Carolina Medina‐Montano ,Bart Lambrecht

doi:10.1021/jacs.1c03772

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 PEG-derived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are amine-reactive 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.

Highlights

Nanosized drug carriers can improve the pharmacokinetics of potent but systemically toxic small-molecular drugs; key challenges remain to establish robust fabrication processes, prevent premature drug release, and guarantee sufficient carrier integrity under physiological conditions
We first aimed to synthesize a polymerizable squaric ester amide monomer that can be polymerized under controlled conditions into well-defined homo- and block copolymers using reversible addition−fragmentation chain transfer (RAFT) agents
While squaric ester amides have already been studied as amine-reactive groups for atom transfer radical polymerization (ATRP)[40] or RAFT41 chain transfer agents, to the best of our knowledge, the use of squaric esters as functional side groups of monomers and corresponding polymers has not been introduced before

Summary

■ INTRODUCTION

Nanosized drug carriers can improve the pharmacokinetics of potent but systemically toxic small-molecular drugs; key challenges remain to establish robust fabrication processes, prevent premature drug release, and guarantee sufficient carrier integrity under physiological conditions. Tietze et al reported on squaric alkyl esters for selective amine coupling reactions with high functional group tolerance as well as excellent yields, even under mild conditions in aqueous media.[37] Due to their homo-bifunctionality squaric alkyl esters can consecutively be aminolyzed by two different amines, whereby an enhanced aromatic stability after first amidation is obtained, providing reduced reactivity for another amidation It assures a controlled sequential squaric bisamide formation.[38] Compared to other reactive esters (including pentafluorophenyl) this alternative linking chemistry allows conjugation in biocompatible solvents (e.g., water, ethanol) while releasing alcohols as nontoxic byproducts.[39]. These observations demonstrate the broad versatility of amine-reactive squaric ester-based precursor polymers and how they provide access to a modular pH-degradable nanogel platform for safe systemic administration of immune modulators

■ RESULTS AND DISCUSSION

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES