pH-degradable, bisphosphonate-loaded nanogels attenuate liver fibrosis by repolarization of M2-type macrophages.

Immune-suppressive (M2-type) macrophages can contribute to the progression of cancer and fibrosis. In chronic liver diseases, M2-type macrophages promote the replacement of functional parenchyma by collagen-rich scar tissue. Here, we aim to prevent liver fibrosis progression by repolarizing liver M2-type macrophages toward a nonfibrotic phenotype by applying a pH-degradable, squaric ester­based nanogel carrier system. This nanotechnology platform enables a selective conjugation of the highly water-soluble bisphosphonate alendronate, a macrophage-repolarizing agent that intrinsically targets bone tissue. The covalent delivery system, however, promotes the drug's safe and efficient delivery to nonparenchymal cells of fibrotic livers after intravenous administration. The bisphosphonate payload does not eliminate but instead reprograms profibrotic M2- toward antifibrotic M1-type macrophages in vitro and potently prevents liver fibrosis progression in vivo, mainly via induction of a fibrolytic phenotype, as demonstrated by transcriptomic and proteomic analyses. Therefore, the alendronate-loaded squaric ester­based nanogels represent an attractive approach for nanotherapeutic interventions in fibrosis and other diseases driven by M2-type macrophages, including cancer.

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

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.

Monitoring Translation Activity of mRNA-Loaded Nanoparticles in Mice.

Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.

Physicochemical and Preclinical Evaluation of Spermine-Derived Surfactant Liposomes for in Vitro and in Vivo siRNA-Delivery to Liver Macrophages.

Herein we report on a liposomal system for siRNA delivery consisting of cholesterol (Chol), distearoylphosphatidylcholine (DSPC), and surfactant TF (1-hydroxy-50-amino-3,4,7,10,13,16,19,22-octaoxa-37,41,45-triaza-pentacontane), a novel spermine derivative (HO-EG8-C12-spermine) which has shown improved siRNA delivery to cells in vitro and in vivo. Predominantly single-walled liposomes with reproducible sizes and moderately broad size distributions were generated with an automated extrusion device. The liposomes remained stable when prepared in the presence of siRNA at N/P ratios of 17-34. However, when mixed with human serum in equal volumes, larger aggregates in the size range of several hundred nanometers were observed by dynamic light scattering. These larger aggregates could potentially limit prolonged in vivo applications. Aggregate formation could be reduced by the addition of a cholesterol-hyperbranched polyglycerol surfactant (hbPG) that sterically shields the liposomal surface against serum induced aggregation. In vitro experiments with murine macrophages utilizing macrophage-specific anti-CD68 siRNA loaded liposomes showed potent and sequence specific reduction of CD68 transcript levels without cytotoxicity. Experiments in mice using intravenous application of CW800 NHS ester labeled liposomes, near-infrared in vivo imaging, and fluorescent assisted cell sorting of inflammatory cells demonstrated an almost quantitative accumulation of these liposomes, with and without hbPG, in the liver and a specific knockdown of CD68 mRNA of up to 70% in liver resident macrophages. It was found that aggregate formation of TF liposomes in serum does not significantly affect in vivo siRNA delivery to these central inflammatory cells of the liver.

Reductive Decationizable Block Copolymers for Stimuli-Responsive mRNA Delivery.

Messenger ribonucleic acids (mRNAs) are considered as promising alternatives for transient gene therapy, but to overcome their poor pharmacokinetic properties, smart carriers are required for cellular uptake and stimuli-responsive release. In this work, a synthetic concept toward reductive decationizable cationic block copolymers for mRNA complexation is introduced. By combination of RAFT block copolymerization with postpolymerization modification, cationic block copolymers are generated with disulfide-linked primary amines. They allow effective polyplex formation with negatively charged mRNA and subsequent release under reductive conditions of the cytoplasm. In first in vitro experiments with fibroblasts and macrophages, tailor-made block copolymers mediate cell-specific mRNA transfection, as quantified by polyplex uptake and mRNA-encoding gene expression. Furthermore, RAFT polymerization provides access to heterotelechelic polymers with orthogonally addressable endgroup functionalities utilized to ligate targeting units onto the polyplex-forming block copolymers. The results exemplify the broad versatility of this reductive decationizable mRNA carrier system, especially toward further advanced mRNA delivery applications.

Profiling target engagement and cellular uptake of cRGD-decorated clinical-stage core-crosslinked polymeric micelles.

Polymeric micelles are increasingly explored for tumor-targeted drug delivery. CriPec® technology enables the generation of core-crosslinked polymeric micelles (CCPMs) based on thermosensitive (mPEG-b-pHPMAmLacn) block copolymers, with high drug loading capacity, tailorable size, and controlled drug release kinetics. In this study, we decorated clinical-stage CCPM with the αvß3 integrin-targeted cyclic arginine-glycine-aspartic acid (cRGD) peptide, which is one of the most well-known active targeting ligands evaluated preclinically and clinically. Using a panel of cell lines with different expression levels of the αvß3 integrin receptor and exploring both static and dynamic incubation conditions, we studied the benefit of decorating CCPM with different densities of cRGD. We show that incubation time and temperature, as well as the expression levels of αvß3 integrin by target cells, positively influence cRGD-CCPM uptake, as demonstated by immunofluorescence staining and fluorescence microscopy. We demonstrate that even very low decoration densities (i.e., 1 mol % cRGD) result in increased engagement and uptake by target cells as compared to peptide-free control CCPM, and that high cRGD decoration densities do not result in a proportional increase in internalization. In this context, it should be kept in mind that a more extensive presence of targeting ligands on the surface of nanomedicines may affect their pharmacokinetic and biodistribution profile. Thus, we suggest a relatively low cRGD decoration density as most suitable for in vivo application.

DNA-polymer micelles as nanoparticles with recognition ability.

The Watson-Crick binding of DNA single strands is a powerful tool for the assembly of nanostructures. Our objective is to develop polymer nanoparticles equipped with DNA strands for surface-patterning applications, taking advantage of the DNA technology, in particular, recognition and reversibility. A hybrid DNA copolymer is synthesized through the conjugation of a ssDNA (22-mer) with a poly(ethylene oxide)-poly(caprolactone) diblock copolymer (PEO-b-PCl). It is shown that, in water, the PEO-b-PCl-ssDNA(22) polymer forms micelles with a PCl hydrophobic core and a hydrophilic corona made of PEO and DNA. The micelles are thoroughly characterized using electron microscopy (TEM and cryoTEM) and small-angle neutron scattering. The binding of these DNA micelles to a surface through DNA recognition is monitored using a quartz crystal microbalance and imaged by atomic force microscopy. The micelles can be released from the surface by a competitive displacement event.

SiRNA-mediated in vivo gene knockdown by acid-degradable cationic nanohydrogel particles.

Cationic nanohydrogel particles have become an attractive tool for systemic siRNA delivery, but improvement of their in vivo tolerance is desirable, especially to prevent potential long term side effects by tissue and cellular accumulation. Here, we designed novel ketal cross-linked cationic nanohydrogel particles that were assessed for reduced tissue accumulation and robust siRNA delivery in vitro and in vivo. An oligo-amine cross-linker equipped with a ketal moiety in its core was synthesized and applied to nanohydrogel cross-linking of self-assembled reactive ester block copolymers in DMSO. The resulting acid-sensitive cationic nanoparticles spontaneously disassembled over time in acidic milieu, as investigated by dynamic light scattering. Fluorescent correlation spectroscopy showed effective complexation with siRNA as well as its release upon particle degradation at endosomal pH. These properties resulted in an enhanced in vitro gene knockdown for the acid-degradable cationic nanoparticles compared to their non-degradable spermine analogues. In a murine liver fibrosis model enhanced carrier and payload accumulation in the fibrotic tissue facilitated sequence-specific gene knockdown and prevented fibrosis progression. Long-term monitoring of the carrier in the body showed an enhanced clearance for the acid-degradable carrier, even after multiple dosing. Therefore, these acid-degradable cationic nanohydrogel particles can be considered as promising siRNA carriers for in vivo purposes towards therapeutic applications.

Dextran-based therapeutic nanoparticles for hepatic drug delivery.

AIM: Evaluation of dextran-based nanoparticles (DNP) as a drug delivery system to target myeloid cells of the liver. MATERIALS & METHODS: DNP were synthesized and optionally PEGylated. Their toxicity and cellular uptake were studied in vitro. Empty and siRNA-carrying DNP were tested in vivo with regard to biodistribution and cellular uptake. RESULTS: In vitro, DNP were taken up by cells of the myeloid lineage without compromising their viability. In vivo, empty and siRNA-carrying DNP distributed to the liver where a single treatment addressed approximately 70% of macrophages and dendritic cells. Serum parameters indicated no in vivo toxicity. CONCLUSION: DNP are multifunctional liver-specific drug carriers which lack toxic side effects and may be utilized in clinical applications targeting liver macrophages.

In Vivo Gene-Silencing in Fibrotic Liver by siRNA-Loaded Cationic Nanohydrogel Particles.

Cationic nanohydrogel particles loaded with anti-Col1α1 siRNA suppress collagen synthesis and deposition in fibrotic mice: Systemically administered 40 nm sized nanogel particles accumulate in collagen-expressing cells in the liver. Their siRNA payload induces a sequence specific in vivo gene knockdown affording an efficient antifibrotic effect in mice with liver fibrosis.