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.

Substitution-Induced Mechanistic Switching in SNAr-Warheads for Cysteine Proteases.

The aim of this study was to investigate the transition from non-covalent reversible over covalent reversible to covalent irreversible inhibition of cysteine proteases by making delicate structural changes to the warhead scaffold. To this end, dipeptidic rhodesain inhibitors with different N-terminal electrophilic arenes as warheads relying on the SNAr mechanism were synthesized and investigated. Strong structure-activity relationships of the inhibition potency, the degree of covalency, and the reversibility of binding on the arene substitution pattern were found. The studies were complemented and substantiated by molecular docking and quantum-mechanical calculations of model systems. Furthermore, the improvement in the membrane permeability of peptide esters in comparison to their corresponding carboxylic acids was exemplified.

Efficient Self-Immolative RAFT End Group Modification for Macromolecular Immunodrug Delivery.

The reversible addition-fragmentation chain-transfer (RAFT) polymerization provides access to a broad variety of biocompatible and functional macromolecules for diverse polymer-drug conjugates. Due to thiocarbonylthio groups at the ends of each growing polymer chain, they can straightforwardly be converted into disufilde-containing self-immolative motives for reversible drug conjugation by traceless linkers. This may be relevant for RAFT-polymerized poly(N,N-dimethylacrylamide) (pDMA), which has been demonstrated to provide similar properties as poly(ethylene glycol) (PEG) in terms of improving the drug's poor pharmacokinetic profile or enhancing its bioavailability. For that purpose, we established a highly efficient one-pot reaction procedure for introducing various functionalities including both primary and secondary amines and primary alcohols and demonstrated their reversible conjugation and traceless release from pDMA's polymer chain end. Next, a first polymer-drug conjugate with a Toll-like receptor agonist exhibited significantly increased activity in vitro compared to conventional irreversibly covalently fixed variants. Finally, α-ω-bifunctional dye or drug conjugates could be generated by a cholesterol-modified RAFT chain-transfer agent. It facilitated the polymer-drug conjugate's internalization at the cellular level monitored by flow cytometry and confocal imaging. This approach provides the basis for a variety of potentially impactful polymer-drug conjugates by combining versatile small molecular drugs with a plethora of available RAFT polymers through reductive-responsive self-immolative linkers.

End Group Dye-Labeled Polycarbonate Block Copolymers for Micellar (Immuno-)Drug Delivery.

Defined conjugation of functional molecules to block copolymer end groups is a powerful strategy to enhance the scope of micellar carriers for drug delivery. In this study, an approach to access well-defined polycarbonate-based block copolymers by labeling their end groups with single fluorescent dye molecules is established. Following controlled polymerization conditions, the block copolymers' primary hydroxy end group can be converted into activated pentafluorophenyl ester carbonates and subsequently aminolyzed with fluorescent dyes that are equipped with primary amines. During a solvent-evaporation process, the resulting end group dye-labeled block copolymers self-assemble into narrowly dispersed ∼25 nm-sized micelles and simultaneously encapsulate hydrophobic (immuno-)drugs. The covalently attached fluorescent tracer can be used to monitor both uptake into cells and stability under biologically relevant conditions, including incubation with blood plasma or during blood circulation in zebrafish embryos. By encapsulation of the toll-like receptor 7/8 (TLR7/8) agonist CL075, immune stimulatory polymeric micelles are generated that get internalized by various antigen-presenting dendritic cells and promote their maturation. Generally, such end group dye-labeled polycarbonate block copolymers display ideal features to permit targeted delivery of hydrophobic drugs to key immune cells for vaccination and cancer immunotherapy.

Beyond p-Hexaphenylenes: Synthesis of Unsubstituted p-Nonaphenylene by a Precursor Protocol.

The synthesis of unsubstituted oligo-para-phenylenes (OPP) exceeding para-hexaphenylene-in the literature often referred to as p-sexiphenyl-has long remained elusive due to their insolubility. We report the first preparation of unsubstituted para-nonaphenylenes (9PPs) by extending our precursor route to poly-para-phenylenes (PPP) to a discrete oligomer. Two geometric isomers of methoxylated syn- and anti-cyclohexadienylenes were synthesized, from which 9PP was obtained via thermal aromatization in thin films. 9PP was characterized via optical, infrared and solid-state 13 C NMR spectroscopy as well as atomic force microscopy and mass spectrometry, and compared to polymeric analogues. Due to the lack of substitution, para-nonaphenylene, irrespective of the precursor isomer employed, displays pronounced aggregation in the solid state. Intermolecular excitonic coupling leads to formation of H-type aggregates, red-shifting emission of the films to greenish. 9PP allows to study the structure-property relationship of para-phenylene oligomers and polymers, especially since the optical properties of PPP depend on the molecular shape of the precursor.

Self-Immolative RAFT-Polymer End Group Modification.

Reversible modifications of reversible addition-fragmentation chain transfer (RAFT)-polymerization derived end groups are usually limited to reductive degradable disulfide conjugates. However, self-immolative linkers can promote ligation and traceless release of primary and secondary amines as well as alcohols via carbonates or carbamates in ß-position to disulfides. In this study, these two strategies are combined and the concept of self-immolative RAFT-polymer end group modifications is introduced: As model compounds, benzylamine, dibenzylamine, and benzyl alcohol are first attached as carbamates or carbonates to a symmetrical disulfide, and in a straightforward one-pot reaction these groups are reversibly attached to aminolyzed trithiocarbonate end groups of RAFT-polymerized poly(N,N-dimethylacrylamide). Quantitative end group modification is confirmed by 1 H NMR spectroscopy, size exclusion chromatography, and mass spectrometry, while reversible release of attached compounds under physiological reductive conditions is successfully monitored by diffusion ordered NMR spectroscopy and thin layer chromatography. Additionally, this concept is further expanded to protein-reactive, self-immolative carbonate species that enable reversible bioconjugation of lysozyme and α-macrophage mannose receptor (MMR) nanobodies as model proteins. Altogether, self-immolative RAFT end group modifications can form the new basis for reversible introduction of various functionalities to polymer chain ends including protein bioconjugates and, thus, opening novel opportunities for stimuli-responsive polymer hybrids.

Chemical Vapor Deposition Synthesis and Terahertz Photoconductivity of Low-Band-Gap N = 9 Armchair Graphene Nanoribbons.

Recent advances in bottom-up synthesis of atomically defined graphene nanoribbons (GNRs) with various microstructures and properties have demonstrated their promise in electronic and optoelectronic devices. Here we synthesized N = 9 armchair graphene nanoribbons (9-AGNRs) with a low optical band gap of ∼1.0 eV and extended absorption into the infrared range by an efficient chemical vapor deposition process. Time-resolved terahertz spectroscopy was employed to characterize the photoconductivity in 9-AGNRs and revealed their high intrinsic charge-carrier mobility of approximately 350 cm2·V-1·s-1.

Monitoring the On-Surface Synthesis of Graphene Nanoribbons by Mass Spectrometry.

We present a mass spectrometric approach to characterize and monitor the intermediates of graphene nanoribbon (GNR) formation by chemical vapor deposition (CVD) on top of Au(111) surfaces. Information regarding the repeating units, lengths, and termini can be obtained directly from the surface sample by a modified matrix-assisted laser desorption/ionization (MALDI) method. The mass spectrometric results reveal ample oxidative side reactions under CVD conditions that can be drastically diminished by the introduction of protective H2 gas at ambient pressure. Simultaneously, the addition of hydrogen extends the lengths of the oligophenylenes and thus the final GNRs. Moreover, the prematurely formed cyclodehydrogenation products during the oligomer growth can be assigned by the mass spectrometric technique. The obtained mechanistic insights provide valuable information for optimizing and upscaling the bottom-up fabrication of GNRs. Given the important role of GNRs as semiconductors, the mass spectrometric analysis provides a readily available tool to characterize and improve their structural perfection.

Cycloparaphenylenes and Their Catenanes: Complex Macrocycles Unveiled by Ion Mobility Mass Spectrometry.

The insoluble product mixture obtained from cycloparaphenylene (CPP) synthesis from Suzuki coupling and reductive aromatization was analyzed. Traditional mass spectrometry suggests a homologous series of macrocycles with 12 to 84 phenylene units. Ion-mobility mass spectrometry, however, unravels an unexpected complexity of isomers with identical chemical formula, but different topologies. Whereas macrocycles containing up to 30 phenylene units show only one structure, the homologue with 36 phenylene units forms at least four different isomers with significant molecular size differences. They can be assigned to catenanes composed of CPPs with 2×18 and 12+24 phenylene units together with the ordinary [36]CPP macrocycle. Most likely, a trefoil knot of the CPP with 36 moieties is also present. For the first time, catenanes can be elucidated in a simple reaction mixture by analyzing their ions in the gas phase, an analysis which lies beyond the scope of traditional analytical methods.

Synthesis of Graphene Nanoribbons by Ambient-Pressure Chemical Vapor Deposition and Device Integration.

Graphene nanoribbons (GNRs), quasi-one-dimensional graphene strips, have shown great potential for nanoscale electronics, optoelectronics, and photonics. Atomically precise GNRs can be "bottom-up" synthesized by surface-assisted assembly of molecular building blocks under ultra-high-vacuum conditions. However, large-scale and efficient synthesis of such GNRs at low cost remains a significant challenge. Here we report an efficient "bottom-up" chemical vapor deposition (CVD) process for inexpensive and high-throughput growth of structurally defined GNRs with varying structures under ambient-pressure conditions. The high quality of our CVD-grown GNRs is validated by a combination of different spectroscopic and microscopic characterizations. Facile, large-area transfer of GNRs onto insulating substrates and subsequent device fabrication demonstrate their promising potential as semiconducting materials, exhibiting high current on/off ratios up to 6000 in field-effect transistor devices. This value is 3 orders of magnitude higher than values reported so far for other thin-film transistors of structurally defined GNRs. Notably, on-surface mass spectrometry analyses of polymer precursors provide unprecedented evidence for the chemical structures of the resulting GNRs, especially the heteroatom doping and heterojunctions. These results pave the way toward the scalable and controllable growth of GNRs for future applications.

Collision-Induced Dissociation Ion Mobility Mass Spectrometry for the Elucidation of Unknown Structures in Strained Polycyclic Aromatic Hydrocarbon Macrocycles.

Structure determination of unexpected products obtained during synthesis of large carbon nanotube sidewall segments with more than 200 carbon atoms represents a challenging task for traditional analytical methods. Herein, we investigate a homologous series of four products having the same number of carbon atoms but slightly different hydrogen numbers ranging from 168 to 162. We demonstrate that the combination of mass spectrometry, ion mobility separation, and collision-induced dissociation (CID) can be used to finally elucidate the complete structures with high certainty. The postulated 1,2-phenyl shift as origin for the side reaction could be proven by changes in the minimum fragment sizes. A combination of CID and ion mobility spectrometry was applied for the first time to prove the cyclic nature of all molecules by the significant size increase upon ring opening. Thereby, also, more compact molecules were discovered in the gas phase with thus far unknown structures. Finally, the potential presence of numerous isomers could be ruled out by drift time measurements and molecular modeling together with theoretical collision cross-section (CCS) calculations. Surprisingly, only one defined structure could be assigned to each macrocycle in the homologous series, most likely as a result of natural selection rules driven by ring strain and steric hindrance. With a decreasing hydrogen content, the macrocycles undergo a stepwise transition from a cylindrical to conical shape. Overall, ion mobility mass spectrometry together with molecular modeling shows great potential to analyze unknown structures, especially in cases where structure determination by X-ray single-crystal analysis is not applicable.

Vapor-phase transport deposition, characterization, and applications of large nanographenes.

Recently, chemical synthesis of a range of large nanographene molecules with various shapes and sizes opened a new path to utilize them in various applications and devices. However, due to their extended aromatic cores and high molecular weight, film formation of large nanographene molecules, bearing more than 90 sp(2) carbon atoms in aromatic cores, is very challenging, which has prevented their applications such as in thin-film transistors. Here, we developed an effective approach to prepare films of such large nanographene molecules using a vapor-phase transport (VPT) technique based on molecule sublimation. The VPT of these molecules was made possible by combining the molecules and the target substrate in a small confinement of vacuum-sealed glass tube, so that a small amount of sublimation can be utilized to create films. Surprisingly, such heavy and large molecules can be deposited on any substrate by this method to create films of assembled large nanographene molecules while maintaining their aromatic cores intact, which was confirmed using mass spectrometry measurements. Moreover, field-effect transistors based on these films are depleted and show significantly improved current on/off ratio compared to previous large nanographene-based transistors fabricated using liquid-phase-based process. Our work shows that VPT deposition can be a viable technique to prepare films based on large nanographene molecules and potentially other high molecular weight compounds, which may find exciting applications in electronics and optoelectronics.

The Precise Synthesis of Phenylene-Extended Cyclic Hexa-peri-hexabenzocoronenes from Polyarylated [n]Cycloparaphenylenes by the Scholl Reaction.

The longitudinal extension of cycloparaphenylenes (CPP) towards ultrashort carbon nanotubes (CNTs) is essential for the solution based bottom-up synthesis of CNTs. Herein, the longitudinal extension of the CPP skeleton by the introduction of hexaphenylbenzene units towards polyarylated [n]CPPs is described. Further, the applicability of the Scholl reaction to selectively form graphenic sidewalls is demonstrated. The ring size and substitution patterns of the polyarylated [n]CPPs were varied to overcome strain-induced side reactions during the oxidative cyclodehydrogenation and cyclic para-hexa-peri-hexabenzocoronene trimers ([3]CHBCs) were selectively obtained. This concept is envisioned as an access to ultrashort carbon nanotubes subject to the condition that further benzene rings with the right connectivity will be inserted.

Photoinduced C-C reactions on insulators toward photolithography of graphene nanoarchitectures.

On-surface chemistry for atomically precise sp(2) macromolecules requires top-down lithographic methods on insulating surfaces in order to pattern the long-range complex architectures needed by the semiconductor industry. Here, we fabricate sp(2)-carbon nanometer-thin films on insulators and under ultrahigh vacuum (UHV) conditions from photocoupled brominated precursors. We reveal that covalent coupling is initiated by C-Br bond cleavage through photon energies exceeding 4.4 eV, as monitored by laser desorption ionization (LDI) mass spectrometry (MS) and X-ray photoelectron spectroscopy (XPS). Density functional theory (DFT) gives insight into the mechanisms of C-Br scission and C-C coupling processes. Further, unreacted material can be sublimed and the coupled sp(2)-carbon precursors can be graphitized by e-beam treatment at 500 °C, demonstrating promising applications in photolithography of graphene nanoarchitectures. Our results present UV-induced reactions on insulators for the formation of all sp(2)-carbon architectures, thereby converging top-down lithography and bottom-up on-surface chemistry into technology.

Reversible Polymer-Protein Functionalization by Stepwise Introduction of Amine-Reactive, Reductive-Responsive Self-Immolative End Groups onto RAFT-Derived Polymers.

Many promising therapeutic protein or peptide drug candidates are rapidly excreted from an organism due to their small size or their inherent immunogenicity. One way to counteract these effects is PEGylation, in which the biopolymer is shielded by synthetic polymers exploiting their stealth properties. However, these modifications are often accompanied by a reduction in the biological function of the protein. By using responsive moieties that bridge the polymer to the protein, a reversible character is provided to this type of conjugation. In this regard, the reductive-responsive nature of disulfides can be exploited via self-immolative structures for reversible linkage to aminic lysine residues and the N-terminus on the protein surface. They enable a traceless release of the intact protein without any further modification and thus preserve the protein's bioactivity. In this study, we demonstrate how this chemistry can be made broadly accessible to RAFT-derived water-soluble polymers like poly(N,N-dimethylacrylamide) (pDMA) as a relevant PEG alternative. A terminal reactive imidazole carbamate with an adjacent self-immolative motif was generated in a gradual manner onto the trithiocarbonate chain transfer moiety of the polymer by first substituting it with a disulfide-bridged alcohol and subsequently converting it into an amine reactive imidazole carbamate. Successful synthesis and complete characterization were demonstrated by NMR, size exclusion chromatography, and mass spectrometry. Finally, two model proteins, lysozyme and a therapeutically relevant nanobody, were functionalized with the generated polymer, which was found to be fully reversible under reductive conditions in the presence of free thiols. This strategy has the potential to extend the generation of reversible reductive-responsive polymer-protein hybrids to the broad field of available functional RAFT-derived polymers.

Extending the limits of precision polymer synthesis: giant polyphenylene dendrimers in the megadalton mass range approaching structural perfection.

The catalyst-free Diels-Alder synthesis of polyphenylene dendrimers with a chromophore core has now been demonstrated to achieve the seventh to ninth generations upon divergent growth. Since standard analytical tools such as size-exclusion chromatography do not provide realistic molecular weights, MALDI-TOF mass spectrometry was applied to characterize the complete series of nine generations. Perfection and monodispersity were thus elucidated at such high masses. Transmission electron microscopy imaging was used to determine the size of these molecularly defined nanosized "particles" with diameters of up to 33 nm.

Photoinduced Amyloid Fibril Degradation for Controlled Cell Patterning.

Amyloid-like fibrils are a special class of self-assembling peptides that emerge as a promising nanomaterial with rich bioactivity for applications such as cell adhesion and growth. Unlike the extracellular matrix, the intrinsically stable amyloid-like fibrils do not respond nor adapt to stimuli of their natural environment. Here, a self-assembling motif (CKFKFQF), in which a photosensitive o-nitrobenzyl linker (PCL) is inserted, is designed. This peptide (CKFK-PCL-FQF) assembles into amyloid-like fibrils comparable to the unsubstituted CKFKFQF and reveals a strong response to UV-light. After UV irradiation, the secondary structure of the fibrils, fibril morphology, and bioactivity are lost. Thus, coating surfaces with the pre-formed fibrils and exposing them to UV-light through a photomask generate well-defined areas with patterns of intact and destroyed fibrillar morphology. The unexposed, fibril-coated surface areas retain their ability to support cell adhesion in culture, in contrast to the light-exposed regions, where the cell-supportive fibril morphology is destroyed. Consequently, the photoresponsive peptide nanofibrils provide a facile and efficient way of cell patterning, exemplarily demonstrated for A549, Chinese Hamster Ovary, and Raw Dual type cells. This study introduces photoresponsive amyloid-like fibrils as adaptive functional materials to precisely arrange cells on surfaces.

Nontoxic N-Heterocyclic Olefin Catalyst Systems for Well-Defined Polymerization of Biocompatible Aliphatic Polycarbonates.

Herein, N-heterocyclic olefins (NHOs) are utilized as catalysts for the ring-opening polymerization (ROP) of functional aliphatic carbonates. This emerging class of catalysts provides high reactivity and rapid conversion. Aiming for the polymerization of monomers with high side chain functionality, six-membered carbonates derived from 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) served as model compounds. Tuning the reactivity of NHO from predominant side chain transesterification at room temperature toward ring-opening at lowered temperatures (-40 °C) enables controlled ROP. These refined conditions give narrowly distributed polymers of the hydrophobic carbonate 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one (MTC-OBn) (D < 1.30) at (pseudo)first-order kinetic polymerization progression. End group definition of these polymers demonstrated by mass spectrometry underlines the absence of side reactions. For the active ester monomer 5-methyl-5-pentafluorophenyloxycarbonyl-1,3-dioxane-2-one (MTC-PFP) with elevated side chain reactivity, a cocatalysis system consisting of NHO and the Lewis acid magnesium iodide is required to retune the reactivity from side chains toward controlled ROP. Excellent definition of the products (D < 1.30) and mass spectrometry data demonstrate the feasibility of this cocatalyst approach, since MTC-PFP has thus far only been polymerized successfully using acidic catalysts with moderate control. The broad feasibility of our findings was further demonstrated by the synthesis of block copolymers for bioapplications and their successful nanoparticular assembly. High tolerability of NHO in vitro with concentrations ranging up to 400 µM (equivalent to 0.056 mg/mL) further emphasize the suitability as a catalyst for the synthesis of bioapplicable materials. The polycarbonate block copolymer mPEG44-b-poly(MTC-OBn) enables physical entrapment of hydrophobic dyes in sub-20 nm micelles, whereas the active ester block copolymer mPEG44-b-poly(MTC-PFP) is postfunctionalizable by covalent dye attachment. Both block copolymers thereby serve as platforms for physical or covalent modification of nanocarriers for drug delivery.

Nonlinear behavior during semi-quantitative analysis of thin organic layers by laser desorption mass spectrometry.

Characterization of the surface coverage and thickness of an organic thin film is particularly important in organic electronics and optoelectronics. For surface coverage down to the submonolayer level there is still a need for characterization methods which are easily applicable. In the present work we report on the evaluation of laser desorption mass spectrometry (LD-MS) for its use in thickness determination of organic thin films. Whereas LD-MS is well established as a soft ionization method for small molecules, its capability for use in quantitative analysis is nearly unexplored. We carried out experiments with two different molecules, 7,7,8,8-tetracyanoquinodimethane and hexabenzocoronene, in a series of experiments with increasing surface coverage. The obtained data were analyzed by plotting the LD signal intensities versus the relative layer thickness and they reveal a nonlinear behavior, which can be classified into regions of different desorption/ionization efficiencies. Visualization by atomic force microscopy reveals that the first efficiency change corresponds to the transition between incomplete and complete coverage of the metal surface by analyte molecules. A second transition is observed at high layer thickness where the signal intensity stays constant, independent of further thickness increments, and this is attributed to the limited penetration depth of the laser beam. The intermediate region between the two transitions shows a linear behavior and can thus be used for semi-quantitative thickness measurements. The efficiency change observed at the point of complete surface coverage is particularly useful for thin layer preparation of organic field effect transistors, where complete surface coverage is a minimum requirement.

The effects of molecular weight distribution and sample preparation on matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of petroleum macromolecules.

To date there have been no systematic, quantitative investigations of the effect of sample preparation on the matrix-assisted laser desorption/ionization time-of-flight (MALDI) mass spectrometry response for polydisperse systems. To this end, the interrelationships between sample preparation, analyte molecular weight distribution (MWD) and solubility, and signal response were investigated for mixtures of alkylated polycyclic aromatic hydrocarbon (PAH) oligomers, the constituents of petroleum pitch that serve as precursors for advanced carbon materials. These PAH oligomers served as a useful analyte system for study, as their solvent solubilities decrease significantly with each increasing oligomeric unit. Molecular weight standards consisting of relatively pure dimer and trimer cuts of the starting M-50 petroleum pitch were produced using a dense-gas/supercritical extraction (DGE/SCE) technique and were then used to produce oligomeric mixtures of well-defined composition for study. Both traditional, solvent-based and newer, solvent-free sample preparation methods were evaluated, and their effects on both homogeneity and signal response were determined. While solvent-free sample preparation methods produced homogeneous samples and reproducible results regardless of the MWD of the analyte, solvent-based samples that contained more than one oligomeric cut produced non-homogeneous samples and poor reproducibilities. The differing solubilities of dimer, trimer, and tetramer oligomers in a given solvent (e.g., CS(2) or toluene) were found to be the cause of the inhomogeneities observed in solvent-based sample preparation. A quantitative analysis study performed with dimer/trimer mixtures over a wide range of compositions via solvent-free sample preparation indicates that linear, reproducible calibration curves can be generated and used to calculate the molecular composition of unknown dimer/trimer mixtures with confidence.