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.

Thermally Triggered Triazolinedione-Tyrosine Bioconjugation with Improved Chemo- and Site-Selectivity.

A bioconjugation strategy is reported that allows the derivatization of tyrosine side chains through triazolinedione-based "Y-clicking". Blocked triazolinedione reagents were developed that, in contrast to classical triazolinedione reagents, can be purified before use, can be stored for a long time, and allow functionalization with a wider range of cargoes and labels. These reagents are bench-stable at room temperature but steadily release highly reactive triazolinediones upon heating to 40 °C in buffered media at physiological pH, showing a sharp temperature response over the 0 to 40 °C range. This conceptually interesting strategy, which is complementary to existing photo- or electrochemical bioorthogonal bond-forming methods, not only avoids the classical synthesis and handling difficulties of these highly reactive click-like reagents but also markedly improves the selectivity profile of the tyrosine conjugation reaction itself. It avoids oxidative damage and "off-target" tryptophan labeling, and it even improves site-selectivity in discriminating between different tyrosine side chains on the same protein or different polypeptide chains. In this research article, we describe the stepwise development of these reagents, from their short and modular synthesis to small-molecule model bioconjugation studies and proof-of-principle bioorthogonal chemistry on peptides and proteins.

Lipid Nanoparticle Encapsulation Empowers Poly(I:C) to Activate Cytoplasmic RLRs and Thereby Increases Its Adjuvanticity.

Poly(I:C) is a synthetic analogue of dsRNA capable of activating both TLR3 and RLRs, such as MDA-5 and RIG-I, as pathogen recognition receptors. While poly(I:C) is known to provoke a robust type I IFN, type III IFN, and Th1 cytokine response, its therapeutic use as a vaccine adjuvant is limited due to its vulnerability to nucleases and poor uptake by immune cells. is encapsulated poly(I:C) into lipid nanoparticles (LNPs) containing an ionizable cationic lipid that can electrostatically interact with poly(I:C). LNP-formulated poly(I:C) triggered both lysosomal TLR3 and cytoplasmic RLRs, in vitro and in vivo, whereas poly(I:C) in an unformulated soluble form only triggered endosomal-localized TLR3. Administration of LNP-formulated poly(I:C) in mouse models led to efficient translocation to lymphoid tissue and concurrent innate immune activation following intramuscular (IM) administration, resulting in a significant increase in innate immune activation compared to unformulated soluble poly(I:C). When used as an adjuvant for recombinant full-length SARS-CoV-2 spike protein, LNP-formulated poly(I:C) elicited potent anti-spike antibody titers, surpassing those of unformulated soluble poly(I:C) by orders of magnitude and offered complete protection against a SARS-CoV-2 viral challenge in vivo, and serum from these mice are capable of significantly reducing viral infection in vitro.

Relevance of controlled cooling and freezing phases in T-cell cryopreservation.

When cells are cryopreserved, they go through a freezing process with several distinct phases (i.e., cooling until nucleation, ice nucleation, ice crystal growth and cooling to a final temperature). Conventional cell freezing approaches often employ a single cooling rate to describe and optimize the entire freezing process, which neglects its complexity and does not provide insight into the effects of the different freezing phases. The aim of this work was to elucidate the impact of each freezing phase by varying different process parameters per phase. Hereto, spin freezing was used to freeze Jurkat T cells in either a Me2SO-based or Me2SO-free formulation. The cooling rates before ice nucleation and after total ice crystallization impacted cell viability, resulting in viability ranging from 26.7% to 52.8% for the Me2SO-free formulation, and 22.5%-42.6% for the Me2SO-based formulation. Interestingly, the degree of supercooling upon nucleation did not exhibit a significant effect on cell viability in this work. However, the rate of ice crystal formation emerged as a crucial factor, with viability ranging from 2.4% to 53.2% for the Me2SO-free formulation, and 0.3%-53.2% for the Me2SO-based formulation, depending on the freezing rate. A morphological study of the cells post-cryopreservation was performed using confocal microscopy, and it was found that cytoskeleton integrity and cell volume were impacted, depending on the formulation-process parameter combination. These findings underscore the importance of scrutinizing all cooling and freezing phases, as each phase impacted post-thaw viability in a distinct way, depending of the specific formulation used.

Towards a Rational Design of Antibody-Recruiting Molecules through a Computational Microscopy View of their Interactions with the Target Antibody.

Antibody recruiting molecules (ARMs) are an innovative class of chimeric molecules, consisting of an antibody-binding ligand (ABL) and a target-binding ligand (TBL). ARMs mediate ternary complex formation between a target cell of interest for elimination and endogenous antibodies that are present in human serum. Clustering of fragment crystallizable (Fc) domains on the surface of antibody-bound cells mediate destruction of the target cell by innate immune effector mechanisms. ARMs are typically designed by conjugating small molecule haptens to a (macro)molecular scaffold, without considering the structure of the respective anti-hapten antibody. Here we report on a computational molecular modeling methodology that allows for studying the close contacts between ARMs and the anti-hapten antibody, considering (1) the spacer length between ABL and TBL; (2) the number of ABL and TBL, and (3) the molecular scaffold onto which these are positioned. Our model predicts the difference in binding modes of the ternary complex and predicts which ARMs are optimal recruiters. Avidity measurements of the ARM-antibody complex and ARM-mediated antibody recruitment to cell surfaces in vitro confirmed these computational modeling predictions. This kind of multiscale molecular modelling holds potential for design of drug molecules that rely on antibody binding for their mechanism of action.

Visible Light Conjugation with Triazolinediones as a Route to Degradable Poly(ethylene glycol)-Lipids for mRNA Lipid Nanoparticle Formulation.

Polyethylene glycol (PEG) is considered as the gold standard for colloidal stabilization of nanomedicines, yet PEG is non-degradable and lacks functionality on the backbone. Herein, we introduce concomitantly PEG backbone functionality and degradability via a one-step modification with 1,2,4-triazoline-3,5-diones (TAD) under green light. The TAD-PEG conjugates are degradable in aqueous medium under physiological conditions, with the rate of hydrolysis depending on pH and temperature. Subsequently, a PEG-lipid is modified with TAD-derivatives and successfully used for messenger RNA (mRNA) lipid nanoparticle (LNP) delivery, thereby improving mRNA transfection efficiency on multiple cell cultures in vitro. In vivo, in mice, mRNA LNP formulation exhibited a similar tissue distribution as common LNPs, with a slight decrease in transfection efficiency. Our findings pave the road towards the design of degradable, backbone-functionalized PEG for applications in nanomedicine and beyond.

ER stress in antigen-presenting cells promotes NKT cell activation through endogenous neutral lipids.

CD1d-restricted invariant natural killer T (iNKT) cells constitute a common glycolipid-reactive innate-like T-cell subset with a broad impact on innate and adaptive immunity. While several microbial glycolipids are known to activate iNKT cells, the cellular mechanisms leading to endogenous CD1d-dependent glycolipid responses remain largely unclear. Here, we show that endoplasmic reticulum (ER) stress in APCs is a potent inducer of CD1d-dependent iNKT cell autoreactivity. This pathway relies on the presence of two transducers of the unfolded protein response: inositol-requiring enzyme-1a (IRE1α) and protein kinase R-like ER kinase (PERK). Surprisingly, the neutral but not the polar lipids generated within APCs undergoing ER stress are capable of activating iNKT cells. These data reveal that ER stress is an important mechanism to elicit endogenous CD1d-restricted iNKT cell responses through induction of distinct classes of neutral lipids.

Tumor-Selective Activation of Toll-Like Receptor 7/8 Agonist Nano-Immunomodulator Generates Safe Anti-Tumor Immune Responses upon Systemic Administration.

Agonists of innate pattern recognition receptors such as toll-like receptors (TLRs) prime adaptive anti-tumor immunity and hold promise for cancer immunotherapy. However, small-molecule TLR agonists cause immune-related adverse effects (irAEs) after systemic administration. Herein, we report a polymeric nano-immunomodulator (cN@SS-IMQ) that is inactive until it is selectively metabolized to an active immunostimulant within the tumor. cN@SS-IMQ was obtained via self-assembly of a cyclo(Arg-Gly-Asp-D-Phe-Lys)-modified amphiphilic copolymeric prodrug. Upon systemic administration, cN@SS-IMQ preferentially accumulated at tumor sites and responded to high intracellular glutathione levels to release native imidazoquinolines for dendritic cell maturation, thereby enhancing the infiltration of T lymphocytes. Collectively, cN@SS-IMQ tends to activate the immune system without irAEs, thus suggesting its promising potential for safe systemic targeting delivery.

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.

Engineering Strategies for Lymph Node Targeted Immune Activation.

Development of vaccine technology that induces long lasting and potent adaptive immune responses is of vital importance to combat emerging pathogens and to design the next generation of cancer immunotherapies. Advanced biomaterials such as nanoparticle carriers are intensively explored to increase the efficacy and safety of vaccines and immunotherapies, based on their intrinsic potential to focus the therapeutic payload onto the relevant immune cells and to limit systemic distribution. With adaptive immune responses being primarily initiated in lymph nodes, the potency of nanoparticle vaccines in turn is tightly linked to their capacity to reach and accumulate in the lymph nodes draining the immunization site. Here, we discuss the main strategies applied to increase nanoparticle delivery to lymph nodes: (1) direct lymph node injection, (2) active cell-mediated transport through targeting of peripheral dendritic cells, and (3) exploiting passive transport through the afferent lymphatics.The intralymph nodal injection is obviously the most direct way for nanoparticles to reach lymph nodes, and multiple studies have demonstrated its capability in enhancing immunostimulant drugs' immune activation and increasing the therapeutic window. However, the requirement of using ultrasound guidance for mapping lymph nodes in patients renders intranodal administration unsuited for mass vaccination campaigns. As lymph nodes are fine structured organs with lymphocytes and chemokine gradients arrayed in a highly ordered fashion, the breakdown of such formats by the intralymph nodal injection is another concern. The exploitation of dendritic cells as live vectors for transporting nanoparticles to lymph nodes has intensively been studied both ex vivo and in vivo. While ex vivo engineering of dendritic cells in theory can achieve 100% dendritic cell-specific selectivity, a scenario impossible to be achieved in vivo, this procedure is usually laborious and complicated and entails the participation of professional staff and equipment. In addition, the poor efficiency of dendritic cell migration to the draining lymph node is another significant limitation following the injection of ex vivo cultured dendritic cells. Thus, in vivo targeting of surface receptors, particularly C-type lectin receptors, on dendritic cells by conjugating nanoparticles with antibodies or ligands is intensively studied by both academia and industry. Although such nanoparticles in vivo still face nonspecific engulfment by various phagocytes, multiple studies have shown its feasibility in targeting dendritic cells with high selectivity. Moreover, through optimizing the physicochemical properties of nanoparticles, nanoparticles can passively drain to lymph nodes carried by the interstitial flow. Compared to dendritic cell-mediated transport, passive draining is much faster and of higher efficiency. Of all such properties, size is the most important parameter as large particles (>500 nm) can only reach lymph nodes by an active cell-mediated transport. Other surface properties, such as the charge and the balance of hydrophobicity-vs-hydrophilicity, strongly influence the mobility of nanoparticles in the extracellular space. In addition, albumin, a natural fatty acid transporter, has recently been demonstrated capable of binding the amphiphiles through their lipid moiety and subsequent transporting them to lymph nodes.

Covalent Cell Surface Conjugation of Nanoparticles by a Combination of Metabolic Labeling and Click Chemistry.

Conjugation of nanoparticles (NP) to the surface of living cells is of interest in the context of exploiting the tissue homing properties of ex vivo engineered T cells for tumor-targeted delivery of drugs loaded into NP. Cell surface conjugation requires either a covalent or non-covalent reaction. Non-covalent conjugation with ligand-decorated NP (LNP) is challenging and involves a dynamic equilibrium between the bound and unbound state. Covalent NP conjugation results in a permanently bound state of NP, but the current routes for cell surface conjugation face slow reaction kinetics and random conjugation to proteins in the glycocalyx. To address the unmet need for alternative bioorthogonal strategies that allow for efficient covalent cell surface conjugation, we developed a 2-step click conjugation sequence in which cells are first metabolically labeled with azides followed by reaction with sulfo-6-methyl-tetrazine-dibenzyl cyclooctyne (Tz-DBCO) by SPAAC, and subsequent IEDDA with trans-cyclooctene (TCO) functionalized NP. In contrast to using only metabolic azide labeling and subsequent conjugation of DBCO-NP, our 2-step method yields a highly specific cell surface conjugation of LNP, with very low non-specific background binding.

Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Lipid Amphiphile Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine*.

The search for vaccines that protect from severe morbidity and mortality because of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Here we describe an amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile. It is water-soluble and exhibits massive translocation to lymph nodes upon local administration through binding to albumin, affording localized innate immune activation and reduction in systemic inflammation. The adjuvanticity of IMDQ-PEG-CHOL was validated in a licensed vaccine setting (quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS-CoV-2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model.

Potent and Prolonged Innate Immune Activation by Enzyme-Responsive Imidazoquinoline TLR7/8 Agonist Prodrug Vesicles.

Synthetic immune-stimulatory drugs such as agonists of the Toll-like receptors (TLR) 7/8 are potent activators of antigen-presenting cells (APCs), however, they also induce severe side effects due to leakage from the site of injection into systemic circulation. Here, we report on the design and synthesis of an amphiphilic polymer-prodrug conjugate of an imidazoquinoline TLR7/8 agonist that in aqueous medium forms vesicular structures of 200 nm. The conjugate contains an endosomal enzyme-responsive linker enabling degradation of the vesicles and release of the TLR7/8 agonist in native form after endocytosis, which results in high in vitro TLR agonist activity. In a mouse model, locally administered vesicles provoke significantly more potent and long-lasting immune stimulation in terms of interferon expression at the injection site and in draining lymphoid tissue compared to a nonamphiphilic control and the native TLR agonist. Moreover, the vesicles induce robust activation of dendritic cells in the draining lymph node in vivo.

Zebrafish Embryos Allow Prediction of Nanoparticle Circulation Times in Mice and Facilitate Quantification of Nanoparticle-Cell Interactions.

The zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real-time observations of vascular flow of nanoparticles and their interactions with cells throughout the body. As a consequence, this system enables the acquisition of quantitative data that are difficult to obtain in rodents. Until now, a few studies using the zebrafish model have only described semiquantitative results on key nanoparticle parameters. Here, a MACRO dedicated to automated quantitative methods is described for analyzing important parameters of nanoparticle behavior, such as circulation time and interactions with key target cells, macrophages, and endothelial cells. Direct comparison of four nanoparticle (NP) formulations in zebrafish embryos and mice reveals that data obtained in zebrafish can be used to predict NPs' behavior in the mouse model. NPs having long or short blood circulation in rodents behave similarly in the zebrafish embryo, with low circulation times being a consequence of NP uptake into macrophages or endothelial cells. It is proposed that the zebrafish embryo has the potential to become an important intermediate screening system for nanoparticle research to bridge the gap between cell culture studies and preclinical rodent models such as the mouse.

Multivalent Antibody-Recruiting Macromolecules: Linking Increased Binding Affinity with Enhanced Innate Immune Killing.

Antibody-recruiting molecules (ARMs) are a novel class of immunotherapeutics. They are capable of introducing antibodies onto disease-relevant targets such as cancer cells, bacterial cells or viruses. This can induce antibody-mediated immune responses such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent phagocytosis (ADCP), which can kill the pathogen. In contrast to the classic ARMs, multivalent ARMs could offer the advantage of increasing the efficiency of antibody recruitment and subsequent innate immune killing. Such compounds consist of multiple target-binding termini (TBT) and/or antibody-binding termini (ABT). Those multivalent interactions are able to convert low binding affinities into increased binding avidities. This minireview summarizes the current status of multivalent ARMs and gives insight into possible benefits, hurdles still to be overcome and future perspectives.

Molecular Imaging for Cancer Immunotherapy: Seeing Is Believing.

The importance of the immune system in cancer therapy has been reaffirmed by the success of the immune checkpoint blockade. The complex tumor microenvironment and its interaction with the immune system, however, remain mysteries. Molecular imaging may shed light on fundamental aspects of the immune response to elucidate the mechanism of cancer immunotherapy. In this review, we discuss various imaging approaches that offer in-depth insight into the tumor microenvironment, checkpoint blockade therapy, and T cell-mediated antitumor immune responses. Recent advances in the molecular imaging modalities, including magnetic resonance imaging (MRI), positron electron tomography (PET), and optical imaging (e.g., fluorescence and intravital imaging) for in situ tracking of the immune response, are discussed. It is envisaged that the integration of imaging with immunotherapy may broaden our understanding to predict a particular antitumor immune response.

Poly(2-ethyl-2-oxazoline) Conjugates with Salicylic Acid via Degradable Modular Ester Linkages.

Conjugation of drugs to polymers is a widely used approach to gain control over the release of therapeutics. In this contribution, salicylic acid, a multipurpose model drug, is conjugated to the biocompatible poly(2-ethyl-2-oxazoline) (PEtOx). The drug is attached to the side chains of a polymer carrier through a hydrolytically cleavable ester linker, via a sequential postpolymerization modification. The chemical modulation of this ester, i.e., by primary or secondary alcohols, is demonstrated to greatly influence the ester hydrolysis rate. This crucial parameter allows us to tune the in vitro kinetics of the sustained drug release for periods exceeding a month in phosphate-buffered saline (PBS). The synthetic accessibility of the cleavable linker, together with the modularity of the drug release rate offered by this approach, highlights the utility of this class of polymers in the field of long-lasting drug delivery systems for persistent and chronic disease treatment.

Control over Imidazoquinoline Immune Stimulation by pH-Degradable Poly(norbornene) Nanogels.

The reactivation of the innate immune system by toll-like receptor (TLR) agonists holds promise for anticancer immunotherapy. Severe side effects caused by unspecific and systemic activation of the immune system upon intravenous injection prevent the use of small-molecule TLR agonists for such purposes. However, a covalent attachment of small-molecule imidazoquinoline (IMDQ) TLR7/8 agonists to pH-degradable polymeric nanogels could be shown to drastically reduce the systemic inflammation but retain the activity to tumoral tissues and their draining lymph nodes. Here, we introduce the synthesis of poly(norbornene)-based, acid-degradable nanogels for the covalent ligation of IMDQs. While the intact nanogels trigger sufficient TLR7/8 receptor stimulation, their degraded version of soluble, IMDQ-conjugated poly(norbornene) chains hardly activates TLR7/8. This renders their clinical safety profile, as degradation products are obtained, which would not only circumvent nanoparticle accumulation in the body but also provide nonactive, polymer-bound IMDQ species. Their immunologically silent behavior guarantees both spatial and temporal control over immune activity and, thus, holds promise for improved clinical applications.

Synthetic Rhamnose Glycopolymer Cell-Surface Receptor for Endogenous Antibody Recruitment.

Synthetic materials capable of engineering the immune system are of great relevance in the fight against cancer to replace or complement the current monoclonal antibody and cell therapy-based immunotherapeutics. Here, we report on antibody recruiting glycopolymers (ARGPs). ARGPs consist of polymeric copies of a rhamnose motif, which can bind endogenous antirhamnose antibodies present in human serum. As a proof-of-concept, we have designed ARGPs with a lipophilic end group that efficiently inserts into cell-surface membranes. We validate the specificity of rhamnose to attract antibodies from human serum to the target cell surface and demonstrate that ARGPs outperform an analogous small-molecule compound containing only one single rhamnose motif. The ARGP concept opens new avenues for the design of potent immunotherapeutics that mark target cells for destruction by the immune system through antibody-mediated effector functions.

Design of pH-Degradable Polymer-Lipid Amphiphiles Using a Ketal-Functionalized RAFT Chain Transfer Agent.

Conjugation of small molecule drug to lipid-polymer amphiphiles is a powerful strategy to alter the pharmacokinetic profile of these molecules by promoting binding to albumin or other serum molecules. Incorporation of a responsive linker between the lipid anchor and the polymer chain can be of interest to avoid indefinite binding of the conjugates to hydrophobic pockets of serum proteins or phospholipid membranes when reaching a target cell or tissue. Here, the synthesis of pH-sensitive lipid-polymer conjugates by reversible addition-fragmentation chain transfer (RAFT) polymerization using a RAFT chain transfer agent that is equipped with a pH-sensitive ketal bond between a cholesterol moiety and the trithiocarbonate RAFT chain transfer group is reported. It is demonstrated that in native form these conjugates exhibit a high affinity to albumin and cell membranes but loose this ability in response to a mild acidic trigger in aqueous medium.