Harnessing Guanidinium and Imidazole Functional Groups: A Dual-Charged Polymer Strategy for Enhanced Gene Delivery.

Histidine and arginine are two amino acids that exhibit beneficial properties for gene delivery. In particular, the imidazole group of histidine facilitates endosomal release, while the guanidinium group of arginine promotes cellular entry. Consequently, a dual-charged copolymer library based on these amino acids was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The content of the N-acryloyl-l-histidine (His) monomer was systematically increased, while maintaining consistent levels of methyl N-acryloyl-l-argininate hydrochloride (ArgOMe) or N-(4-guanidinobutyl)acrylamide hydrochloride (GBAm). The resulting polymers formed stable, nanosized polyplexes when complexed with nucleic acids. Remarkably, candidates with increased His content exhibited reduced cytotoxicity profiles and enhanced transfection efficiency, particularly retaining this performance level at lower pDNA concentrations. Furthermore, endosomal release studies revealed that increased His content improved endosomal release, while ArgOMe improved cellular entry. These findings underscore the potential of customized dual-charged copolymers and the synergistic effects of His and ArgOMe/GBAm in enhancing gene delivery.

Noble Metal-Free Light-Driven Hydrogen Evolution Catalysis in Polyampholytic Hydrogel Networks.

Future technologies to harness solar energy and to convert this into chemical energy strongly rely on straightforward approaches to prepare versatile soft matter scaffolds for the immobilization of catalysts and sensitizers in a defined environment. In addition, particularly for light-driven hydrogen evolution, a transition to noble metal-free photosensitizers and catalysts is urgently required. Herein, we report a fully organic light-harvesting soft matter network based on a polyampholyte hydrogel where both photosensitizer (a perylene monoimide derivative) and a H2 evolution catalyst ([Mo3S13]2-) are electrostatically incorporated. The resulting material exhibits sustained visible-light-driven H2 evolution in aqueous ascorbic acid solution, even at rather low loadings of photosensitizer (0.4%) and catalyst (120 ppm). In addition, we provide initial insights into the long-term stability of the hybrid hydrogel. We believe that these results pave the way for a generalized route toward the incorporation of noble metal-free light-driven catalysis in soft matter networks.

Chitosan functionalized doxorubicin loaded poly(methacrylamide) based copolymeric nanoparticles for enhanced cellular internalization and in vitro anticancer evaluation.

Doxorubicin (Dox), a chemotherapeutic agent, encounters challenges such as a short half-life, dose-dependent toxicity, and low solubility. In this context, the present study involved the fabrication of N-(2-hydroxypropyl)methacrylamide (HPMA) and N-(3-aminopropyl)methacrylamide (APMA) bearing P(HPMA-s-APMA) copolymeric nanoparticles (P(HPMA-s-APMA) NPs) and their investigation for efficient delivery of Dox. Furthermore, the synthesized nanoparticles (NPs) were coated with chitosan (Cht) to generate positively charged nanoformulations. The prepared formulations were evaluated for particle size, morphology, surface charge analysis, percentage encapsulation efficiency (EE%), and drug release studies. The anticancer activity of Cht-P(HPMA-s-APMA)-Dox NPs was assessed in the HeLa cancer cell line. The prepared P(HPMA-s-APMA)-Dox NPs exhibited an average particle size of 240-250 nm. Chitosan decorated P(HPMA-s-APMA)-Dox NPs displayed a significant increase in particle size, and the zeta potential shifted from negative to positive. The EE% for Cht-P(HPMA-s-APMA)-Dox NPs was calculated to be 68.06 %. The drug release studies revealed a rapid release of drug from Cht-P(HPMA-s-APMA)-Dox NPs at pH 4.8 than pH 7.4, demonstrating the pH-responsiveness of nanoformulation. Furthermore, the cell viability assay and internalization studies revealed that Cht-P(HPMA-s-APMA)-Dox NPs had a high cytotoxic response and significant cellular uptake. Hence, the Cht-P(HPMA-s-APMA)-Dox NPs appeared to be a suitable nanocarrier for effective, and safe chemotherapy.

Diiodo-BODIPY Sensitizing of the [Mo3S13]2- Cluster for Noble-Metal-Free Visible-Light-Driven Hydrogen Evolution within a Polyampholytic Matrix.

We report on a photocatalytic setup that utilizes the organic photosensitizer (PS) diiodo-BODIPY and the non-precious-metal-based hydrogen evolution reaction (HER) catalyst (NH4)2[Mo3S13] together with a polyampholytic unimolecular matrix poly(dehydroalanine)-graft-poly(ethylene glycol) (PDha-g-PEG) in aqueous media. The system shows exceptionally high performance with turnover numbers (TON > 7300) and turnover frequencies (TOF > 450 h-1) that are typical for noble-metal-containing systems. Excited-state absorption spectra reveal the formation of a long-lived triplet state of the PS in both aqueous and organic media. The system is a blueprint for developing noble-metal-free HER in water. Component optimization, e.g., by modification of the meso substituent of the PS and the composition of the HER catalyst, is further possible.

Photocatalytic Reduction of Nicotinamide Co-factor by Perylene Sensitized RhIII Complexes.

The ambitious goal of artificial photosynthesis is to develop active systems that mimic nature and use light to split water into hydrogen and oxygen. Intramolecular design concepts are particularly promising. Herein, we firstly present an intramolecular photocatalyst integrating a perylene-based light-harvesting moiety and a catalytic rhodium center (RhIII phenPer). The excited-state dynamics were investigated by means of steady-state and time-resolved absorption and emission spectroscopy. The studies reveal that photoexcitation of RhIII phenPer yields the formation of a charge-separated intermediate, namely RhII phenPer⋅+ , that results in a catalytically active species in the presence of protons.

Ultrasensitive aptasensor for arsenic detection using quantum dots and guanylated Poly(methacrylamide).

The present study demonstrate the first time usage of poly (HPMA-s-GPMA) copolymer for the fabrication of three-component based aptasensor for simple, selective, rapid and label free detection of arsenite (As3+). For this purpose, guanidinium bearing poly (HPMA-s-GPMA) copolymer and MPA-CdTe@CdS quantum dots (QDs) was employed in conjunction with As3+ specific aptamer. This protocol utilizes the quenching phenomena displayed by QDs due to the competitive binding of As3+ ions and cationic copolymer to the aptamer. In particular, the As3+ bind to the specific aptamer, leaving poly (HPMA-s-GPMA) freely available for its electrostatic intercations with QDs, which quenches the fluorescent signal. Contrarily, in the absence of As3+ ions, the aptamer can electrostatically bind to poly (HPMA-s-GPMA); making copolymer inactive to affect the fluorescence signal of the QDs. The efficiency of the proposed fluorescence nanoprobe was further tested using linear calibration curves. The obtained data in the range of 0.01-100 nM showed excellent specificity for As3+ ions with the limit of detection (LOD) of 246.77 pM. Moreover, the "on-off" fluorescent aptasensor is highly selective for As3+ ions in the presence of other interfering metal ions by utilizing As3+ specific aptamer. Furthermore, the reported study showed outstanding applicability in the real-world samples (water, food and soil) containing preservatives, metal ions, minerals, and other moieties. The proposed sensing platform not only exhibits the trace level detection of As3+ ions in cost-effective manner but also opens a pathway for the development of state-of-art device fabrication for on-site detection of arsenic.

In-depth characterization of self-healing polymers based on π-π interactions.

The self-healing behavior of two supramolecular polymers based on π-π-interactions featuring different polymer backbones is presented. For this purpose, these polymers were synthesized utilizing a polycondensation of a perylene tetracarboxylic dianhydride with polyether-based diamines and the resulting materials were investigated using various analytical techniques. Thus, the molecular structure of the polymers could be correlated with the ability for self-healing. Moreover, the mechanical behavior was studied using rheology. The activation of the supramolecular interactions results in a breaking of these noncovalent bonds, which was investigated using IR spectroscopy, leading to a sufficient increase in mobility and, finally, a healing of the mechanical damage. This scratch-healing behavior was also quantified in detail using an indenter.

Supramolecular Reorientation During Deposition Onto Metal Surfaces of Quasi-Two-Dimensional Langmuir Monolayers Composed of Bifunctional Amphiphilic, Twisted Perylenes.

Supramolecular dye structures, which are often ruled by π-π interactions between planar chromophores, crucially determine the optoelectronic properties of layers and interfaces. Here, we present the interfacial assembly of perylene monoanhydride and monoimide that do not feature a planar chromophore but contain chlorine substituents in the bay positions to yield twisted chromophores and hence modified π-stacking. The assembly of the twisted perylene monoanhydride and monoimide is driven by their amphiphilicity that ensures proper Langmuir layer formation. The shielding of the hydrophilic segment upon attaching an alkyl chain to the imide moiety yielded a more rigid Langmuir layer, even though the degrees of freedom were increased due to this modification. For the characterization of the Langmuir layer's supramolecular structure, the layers were deposited onto glass, silver, and gold substrates via Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) techniques and were investigated with atomic force microscopy and surface-enhanced resonance Raman spectroscopy (SERRS). From the similarity between all SERR spectra of the LS and LB layers, we concluded that the perylenes have changed their orientation upon LB deposition to bind to the silver surface of the SERRS substrate via sulfur atoms. In the Langmuir layer, the perylenes, which are π-stacked with half of the twisted chromophores, must already be inclined and cannot achieve full parallel alignment because of the twisting-induced steric hindrance. However, upon rotation, the energetically most favorable antiparallel aligned structures can be formed and bind to the SERRS substrate. Thus, we present, to the best of our knowledge, the first fabrication of quasi-two-dimensional films from twisted amphiphilic perylene monoimides and their reassembly during LB deposition. The relation between the molecular structure, supramolecular interfacial assembly, and its adoption during adsorption revealed here is crucial for the fabrication of defined functionalizations of metal surfaces, which is key to the development of organic (opto)electronic devices.

PEGylation of Guanidinium and Indole Bearing Poly(methacrylamide)s - Biocompatible Terpolymers for pDNA Delivery.

This study describes the first example for shielding of a high performing terpolymer that consists of N-(2-hydroxypropyl)methacrylamide (HPMA), N-(3-guanidinopropyl)methacrylamide (GPMA), and N-(2-indolethyl)methacrylamide monomers (IEMA) by block copolymerization of a polyethylene glycol derivative - poly(nona(ethylene glycol)methyl ether methacrylate) (P(MEO9 MA)) via reversible addition-fragmentation chain transfer (RAFT) polymerization. The molecular weight of P(MEO9 MA) is varied from 3 to 40 kg mol-1 while the comonomer content of HPMA, GPMA, and IEMA is kept comparable. The influence of P(MEO9 MA) block with various molecular weights is investigated over cytotoxicity, plasmid DNA (pDNA) binding, and transfection efficiency of the resulting polyplexes. Overall, the increase in molecular weight of P(MEO9 MA) block demonstrates excellent biocompatibility with higher cell viability in L-929 cells and an efficient binding to pDNA at N/P ratio of 2. The significant transfection efficiency in CHO-K1 cells at N/P ratio 20 is obtained for block copolymers with molecular weight of P(MEO9 MA) up to 10 kg mol-1 . Moreover, a fluorescently labeled analogue of P(MEO9 MA), bearing perylene monoimide methacrylamide (PMIM), is introduced as a comonomer in RAFT polymerization. Polyplexes consisting of labeled block copolymer with 20 kg mol-1 of P(MEO9 MA) and pDNA are incubated in Hela cells and investigated through structured illumination microscopy (SIM).

Lysosome-targeting pH indicator based on peri-fused naphthalene monoimide with superior stability for long term live cell imaging.

Lysosomes, the acidic degradation compartments of eukaryotic cells, play an essential role in many physiological processes. Their dysfunction is associated with a number of diseases, which are often related to an altered localization or luminal pH. Thus, the in-depth characterization of lysosomes within the intact eukaryotic cell is of utmost interest. For microscopic evaluation of lysosomal distribution and acidity, a number of labels have been developed, but many showed poor organelle specificity or rapid clearing from lysosomes, rendering them unsuitable for long-term observations. Here, we describe the synthesis and spectroscopic properties of a novel small molecule marker for lysosomes based on naphthalene monoimide with reversible, pH-dependent spectral shifts in both the absorption and the emission spectrum and acidity-associated changes in fluorescence lifetime. The dye can be excited either with single- or two-photon excitation and appears to be very stably associated with lysosomes for several days. We used this chromophore to detect chemically-induced changes of lysosomal pH in HeLa cells by ratiometric and FLIM imaging.

Indole, Phenyl, and Phenol Groups: The Role of the Comonomer on Gene Delivery in Guanidinium Containing Methacrylamide Terpolymers.

This report highlights the importance of hydrophobic groups mimicking the side chains of aromatic amino acids, which are tryptophan, phenylalanine, and tyrosine, in guanidinium bearing poly(methacrylamide)s for the design of non-viral gene delivery agents. Guanidinium containing methacrylamide terpolymers are prepared by aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization with different hydrophobic monomers, N-(2-indolethyl)methacrylamide (IEMA), N-phenethylmethacrylamide (PhEMA), or N-(4-hydroxyphenethyl)methacrylamide (PhOHEMA) by aiming similar contents. The well-defined polymers are obtained with a molar mass of ≈15 000 g mol-1 and ≈1.1 dispersity. All terpolymers demonstrate almost comparable in vitro cell viability and hemocompatibility profiles independent of the type of side chain. Although they all form positively charged, enzymatically stable polyplexes with plasmid DNA smaller than 200 nm, the incorporation of the IEMA monomer improve these parameters by demonstrating a higher DNA binding affinity and forming nanoassemblies of about 100 nm. These physicochemical characteristics are correlated with increased transfection rates in CHO-K1 cells dependent on the type of the monomer and the nitrogen to phosphate (N/P) ratio of the polyplexes, as determined by luciferase reporter gene assays.

1,7,9,10-Tetrasubstituted PMIs Accessible through Decarboxylative Bromination: Synthesis, Characterization, Photophysical Studies, and Hydrogen Evolution Catalysis.

In this work, we present a new synthetic strategy for fourfold-substituted perylene monoimides via tetrabrominated perylene monoanhydrides. X-ray diffraction analysis unveiled the intramolecular stacking orientation between the substituents and semicircular packing behavior. We observed the remarkable influence of the substituent on the longevity and nature of the excited state upon visible light excitation. In the presence of poly(dehydroalanine)-graft-poly(ethylene glycol) graft copolymers as solubilizing template, the chromophores are capable of sensitizing [Mo3 S13 ]2- clusters in aqueous solution for stable visible light driven hydrogen evolution over three days.

Incorporation of Indole Significantly Improves the Transfection Efficiency of Guanidinium-Containing Poly(Methacrylamide)s.

A highly efficient transfection agent is reported that is based on terpolymer consisting of N-(2-hydroxypropyl)methacrylamide (HPMA), N-(3-guanidinopropyl) methacrylamide (GPMA), and N-(2-indolethyl)methacrylamide monomers (IEMA) by analogy to the amphipathic cell-penetrating peptides containing tryptophan and arginine residues. The incorporation of the indole-bearing monomer leads to successful plasmid DNA condensation even at a nitrogen-to-phosphate (N/P) ratio of 1. The hydrodynamic diameter of polyplexes is determined to be below 200 nm for all N/P ratios. The transfection studies demonstrate a 200-fold increase of the transgene expression in comparison to P(HPMA-co-GPMA) with the same guanidinium content. This study reveals the strong potential of the indole group as a side-chain pendant group that can increase the cellular uptake of polymers and the transfection efficiency of the respective polyplexes.

Tackling the Limitations of Copolymeric Small Interfering RNA Delivery Agents by a Combined Experimental-Computational Approach.

Despite the first successful applications of nonviral delivery vectors for small interfering RNA in the treatment of illnesses, such as the respiratory syncytial virus infection, the preparation of a clinically suitable, safe, and efficient delivery system still remains a challenge. In this study, we tackle the drawbacks of the existing systems by a combined experimental-computational in-depth investigation of the influence of the polymer architecture over the binding and transfection efficiency. For that purpose, a library of diblock copolymers with a molar mass of 30 kDa and a narrow dispersity (D < 1.12) was synthesized. We studied in detail the impact of an altered block size and/or composition of cationic diblock copolymers on the viability of each respective structure as a delivery agent for polynucleotides. The experimental investigation was further complemented by a computational study employing molecular simulations as well as an analytical description of systemic properties. This is the first report in which molecular dynamics simulations of RNA/cationic polymer complexes have been performed. Specifically, we developed and employed a coarse-grained model of the system at the molecular level to study the interactions between polymer chains and small interfering RNA. We were further able to confirm a threshold lengthbinding block/lengthnonbinding block ratio, which is required for efficient complexation of siRNA, and it was possible to find a correlation between the length of the cationic block and the size of the resulting polyplex. Hence, the combined insights from the experiments and the theoretical investigation resulted in a wealth of information about the properties of cationic diblock copolymers employed as RNA delivery agents, in particular regarding the molecular and mechanistic details of the interaction between the two components of a polyplex.

Overcoming the barrier of CD8+T cells: Two types of nano-sized carriers for siRNA transport.

Bioengineering immune cells via gene therapy offers treatment opportunities for currently fatal viral infections. Also cell therapeutics offer most recently a breakthrough technology to combat cancer. These primary human cells, however, are sensitive to toxic influences, which make the utilization of optimized physical transfection techniques necessary. The otherwise commonly applied delivery agents such as LipofectamineⓇ or strongly cationic polymer structures are not only unsuitable for in vivo experiments, but are also highly toxic to immune cells. This study aimed to improve the design of polymeric carrier systems for small interfering RNA, which would allow efficient internalization into CD8+T-cells without affecting their viability and thereby removing the current limitations in the field. Here, two new carrier systems for small interfering RNA were tested. One is a cationic diblock copolymer, in which less than 10% of the monomers were modified with triphenylphosphonium cations. This moiety is lipophilic, promotes uptake and it is mostly known for its mitotropic properties. Furthermore, cationic nanohydrogel particles were synthesized in exceedingly small sizes (Rh < 14 nm). After full physicochemical characterization of the two carriers, extensive cytotoxicity studies were performed and the concentration dependent uptake into CD8+T-cells was tested in correlation to incubation time and protein content of the surrounding medium. Both carriers facilitated efficient complexation of siRNA as well as significant internalization into primary human cells in less than three hours of incubation. In addition, neither of the delivery systems reduced cell viability making them good candidates to transport siRNA into CD8+T-cells efficiently. STATEMENT OF SIGNIFICANCE: This study provides insights into the design of polymeric delivery agents as the method of choice for overcoming the limitations of cell manipulation. Until now, CD8+T-cells, which have become a treatment tool for currently fatal diseases, have not yet been made accessible for gene silencing by polymeric siRNA carrier systems. Choosing appropriate modification approaches for two chemically different polymer structures, we were, in both cases, able to achieve significant uptake in these cells even at low concentrations and without inducing cytotoxicity. These results remove current limitations and pave the way for bioengineering via gene therapy.

The influence of gradient and statistical arrangements of guanidinium or primary amine groups in poly(methacrylate) copolymers on their DNA binding affinity.

Herein, we report the first gradient guanidinium containing cationic copolymers and investigate their binding ability to plasmid DNA (pDNA). To understand the effect of different charge distributions and cationic charge sources (primary amines vs. guanidinium group) on (pDNA) binding affinity, we synthesized a library of well-defined statistical cationic copolymers comprising N-(2-hydroxy-propyl)methacrylamide (HPMA) and N-(3-aminopropyl)methacrylamide (APMA) or N-(3-guanidinopropyl)methacrylamide (GPMA) and compared them with gradient polymers containing the same monomers of similar composition. All copolymers were synthesized through aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization at various monomer ratios by aiming at similar molar masses with low dispersity indices. For the molar mass characterization, in addition to size exclusion chromatography with two different systems, hydrodynamic characterization utilizing analytical ultracentrifugation, viscometry, and accompanied density measurements was conducted. pDNA was used as a model drug to demonstrate the impact of copolymer architecture on binding efficiency. For both HPMA-APMA and HPMA-GPMA copolymers, the gradient distribution demonstrated superior binding and denser packing of pDNA than their statistical counterparts at 20% and lower cationic charge contents. With respect to charge origin, the guanidinium group represented a higher binding efficiency than primary amines with the same nitrogen to phosphate ratio (N/P ratio). Our study demonstrates the profound effect of gradient monomer arrangement on the ability of polyplex formation and reveals the potential for further investigation in gene delivery applications. Gradient guanidinium containing copolymers have great promise for gene delivery applications due to their high affinity toward pDNA even at very low degrees (<20%) of charged monomer content.

Ultra-sensitive detection of malathion using quantum dots-polymer based fluorescence aptasensor.

A novel detection platform with high malathion specificity has been developed, which operates based on the signal response in the fluorescence of CdTe@CdS quantum dots (QDs). The designed nanoprobe comprises of QDs, poly(N-(3-guanidinopropyl)methacrylamide) homopolymer (PGPMA) and malathion specific aptamer. The interaction of aptamer with malathion results in switching off of the fluorescence signal of the probe due to the availability of the cationic polymer, which causes quenching of the QDs. However, in the absence of malathion, the polymer interacts with the aptamer, via electrostatic interactions thereby rendering the fluorescence of QDs unaffected. The assay exhibited excellent sensitivity towards malathion with a detection limit of 4pM. A logarithmic correlation was observed in a wide range of malathion concentrations from 0.01nm to 1µM, facilitating the potential of proposed assay in the quantitative determination of the analyte of interest. The selectivity of the designed probe was confirmed in the presence of various pesticides, commonly employed in agricultural fields.

Characterization of the interaction forces in a drug carrier complex of doxorubicin with a drug-binding peptide.

Polypeptide-based materials are used as building blocks for drug delivery systems aimed at toxicity decrease in chemotherapeutics. A molecular-level approach is adopted for investigating the non-covalent interactions between doxorubicin and a recently synthesized drug-binging peptide as a key part of a system for delivery to neoplastic cells. Molecular dynamics simulations in aqueous solution at room and body temperature are applied to investigate the structure and the binding modes within the drug-peptide complex. The tryptophans are outlined as the main chemotherapeutic adsorption sites, and the importance of their placement in the peptide sequence is highlighted. The drug-peptide binging energy is evaluated by density functional theory calculations. Principal component analysis reveals comparable importance of several types of interaction for the binding strength. π-Stacking is dominant, but other factors are also significant: intercalation, peptide backbone stacking, electrostatics, dispersion, and solvation. Intra- and intermolecular H-bonding also stabilizes the complexes. The influence of solvent molecules on the binding energy is mild. The obtained data characterize the drug-to-peptide attachment as a mainly attractive collective process with interactions spanning a broad range of values. These results explain with atomistic detail the experimentally registered doxorubicin-binging ability of the peptide and outline the complex as a prospective carrying unit that can be employed in design of drug delivery systems.

[FeFe]-Hydrogenase H-cluster mimics mediated by naphthalene monoimide derivatives of peri-substituted dichalcogenides.

Synthetic models of the active site of [FeFe]-hydrogenase containing naphthalene monoimide (NMI) of peri-substituted dichalcogenides as bridging linkers have been prepared and characterized using different spectroscopic methods. The influence of the imide functionality and the chalcogen atoms on the redox properties and the catalytic behaviour of complexes 7-10 was studied using cyclic voltammetry. The results revealed that the imide functionality has improved the chemical stability of the reduced species and the replacement of the S atoms by Se caused a cathodic shift in the oxidation peaks. Moreover, the optical properties of compounds 1, 2, 4, and 5 and the respective diiron complexes 7-10 were investigated by UV-Vis absorption and fluorescence spectroscopy assisted by quantum chemical simulations. The structures of complexes 6-9 were confirmed by X-ray diffraction analysis.

Modulation of Mitochondriotropic Properties of Cyanine Dyes by in Organello Copper-Free Click Reaction.

Cyanine (Cy) dyes show a general propensity to localize in polarized mitochondria. This mitochondriotropism was used to perform a copper-free click reaction in the mitochondria of living cells. The in organello reaction of dyes Cy3 and Cy5 led to a product that was easily traceable by Förster resonance energy transfer (FRET). As determined by confocal laser scanning microscopy, the Cy3-Cy5 conjugate showed enhanced retention in mitochondria, relative to that of the starting compounds. This enhancement of a favorable property can be achieved by synthesis in organello, but not outside mitochondria.