Optimization of Mixed Micelles Based on Oppositely Charged Block Copolymers by Machine Learning for Application in Gene Delivery.

The COVID-19 mRNA vaccines represent a milestone in developing non-viral gene carriers, and their success highlights the crucial need for continued research in this field to address further challenges. Polymer-based delivery systems are particularly promising due to their versatile chemical structure and convenient adaptability, but struggle with the toxicity-efficiency dilemma. Introducing anionic, hydrophilic, or "stealth" functionalities represents a promising approach to overcome this dilemma in gene delivery. Here, two sets of diblock terpolymers are created comprising hydrophobic poly(n-butyl acrylate) (PnBA), a copolymer segment made of hydrophilic 4-acryloylmorpholine (NAM), and either the cationic 3-guanidinopropyl acrylamide (GPAm) or the 2-carboxyethyl acrylamide (CEAm), which is negatively charged at neutral conditions. These oppositely charged sets of diblocks are co-assembled in different ratios to form mixed micelles. Since this experimental design enables countless mixing possibilities, a machine learning approach is applied to identify an optimal GPAm/CEAm ratio for achieving high transfection efficiency and cell viability with little resource expenses. After two runs, an optimal ratio to overcome the toxicity-efficiency dilemma is identified. The results highlight the remarkable potential of integrating machine learning into polymer chemistry to effectively tackle the enormous number of conceivable combinations for identifying novel and powerful gene transporters.

Efficient Gene Delivery of Tailored Amphiphilic Polypeptides by Polyplex Surfing.

Within this study, an amphiphilic and potentially biodegradable polypeptide library based on poly[(4-aminobutyl)-l-glutamine-stat-hexyl-l-glutamine] [P(AB-l-Gln-stat-Hex-l-Gln)] was investigated for gene delivery. The influence of varying proportions of aliphatic and cationic side chains affecting the physicochemical properties of the polypeptides on transfection efficiency was investigated. A composition of 40 mol% Hex-l-Gln and 60 mol % AB-l-Gln (P3) was identified as best performer over polypeptides with higher proportions of protonatable monomers. Detailed studies of the transfection mechanism revealed the strongest interaction of P3 with cell membranes, promoting efficient endocytic cell uptake and high endosomal release. Spectrally, time-, and z-resolved fluorescence microscopy further revealed the crucial role of filopodia surfing in polyplex-cell interaction and particle internalization in lamellipodia regions, followed by rapid particle transport into cells. This study demonstrates the great potential of polypeptides for gene delivery. The amphiphilic character improves performance over cationic homopolypeptides, and the potential biodegradability is advantageous toward other synthetic polymeric delivery systems.

The impact of anionic polymers on gene delivery: how composition and assembly help evading the toxicity-efficiency dilemma.

Cationic polymers have been widely studied for non-viral gene delivery due to their ability to bind genetic material and to interact with cellular membranes. However, their charged nature carries the risk of increased cytotoxicity and interaction with serum proteins, limiting their potential in vivo application. Therefore, hydrophilic or anionic shielding polymers are applied to counteract these effects. Herein, a series of micelle-forming and micelle-shielding polymers were synthesized via RAFT polymerization. The copolymer poly[(n-butyl acrylate)-b-(2-(dimethyl amino)ethyl acrylamide)] (P(nBA-b-DMAEAm)) was assembled into cationic micelles and different shielding polymers were applied, i.e., poly(acrylic acid) (PAA), poly(4-acryloyl morpholine) (PNAM) or P(NAM-b-AA) block copolymer. These systems were compared to a triblock terpolymer micelle comprising PAA as the middle block. The assemblies were investigated regarding their morphology, interaction with pDNA, cytotoxicity, transfection efficiency, polyplex uptake and endosomal escape. The naked cationic micelle exhibited superior transfection efficiency, but increased cytotoxicity. The addition of shielding polymers led to reduced toxicity. In particular, the triblock terpolymer micelle convinced with high cell viability and no significant loss in efficiency. The highest shielding effect was achieved by layering micelles with P(NAM-b-AA) supporting the colloidal stability at neutral zeta potential and completely restoring cell viability while maintaining moderate transfection efficiencies. The high potential of this micelle-layer-combination for gene delivery was illustrated for the first time.

Improved gene delivery to K-562 leukemia cells by lipoic acid modified block copolymer micelles.

Although there has been substantial progress in the research field of gene delivery, there are some challenges remaining, e.g. there are still cell types such as primary cells and suspension cells (immune cells) known to be difficult to transfect. Cationic polymers have gained increasing attention due to their ability to bind, condense and mask genetic material, being amenable to scale up and highly variable in their composition. In addition, they can be combined with further monomers exhibiting desired biological and chemical properties, such as antioxidative, pH- and redox-responsive or biocompatible features. By introduction of hydrophobic monomers, in particular as block copolymers, cationic micelles can be formed possessing an improved chance of transfection in otherwise challenging cells. In this study, the antioxidant biomolecule lipoic acid, which can also be used as crosslinker, was incorporated into the hydrophobic block of a diblock copolymer, poly{[2-(dimethylamino)ethyl methacrylate]101-b-[n-(butyl methacrylate)124-co-(lipoic acid methacrylate)22]} (P(DMAEMA101-b-[nBMA124-co-LAMA22])), synthesized by RAFT polymerization and assembled into micelles (LAMA-mic). These micelles were investigated regarding their pDNA binding, cytotoxicity mechanisms and transfection efficiency in K-562 and HEK293T cells, the former representing a difficult to transfect, suspension leukemia cell line. The LAMA-mic exhibited low cytotoxicity at applied concentrations but demonstrated superior transfection efficiency in HEK293T and especially K-562 cells. In-depth studies on the transfection mechanism revealed that transfection efficiency in K-562 cells does not depend on the specific oncogenic fusion gene BCR-ABL alone. It is independent of the cellular uptake of polymer-pDNA complexes but correlates with the endosomal escape of the LAMA-mic. A comparison of the transfection efficiency of the LAMA-mic with structurally comparable micelles without lipoic acid showed that lipoic acid is not solely responsible for the superior transfection efficiency of the LAMA-mic. More likely, a synergistic effect of the antioxidative lipoic acid and the micellar architecture was identified. Therefore, the incorporation of lipoic acid into the core of hydrophobic-cationic micelles represents a promising tailor-made transfer strategy, which can potentially be beneficial for other difficult to transfect cell types.

Differential capability of metabolic substrates to promote hepatocellular lipid accumulation.

PURPOSE: Excessive storage of triacylglycerides (TAGs) in lipid droplets within hepatocytes is a hallmark of non-alcoholic fatty liver disease (NAFLD), one of the most widespread metabolic disorders in Western societies. For the purpose of exploring molecular pathways in NAFLD development and testing potential drug candidates, well-characterised experimental models of ectopic TAG storage in hepatocytes are needed. METHODS: Using an optimised Oil Red O assay, immunoblotting and real-time qRT-PCR, we compared the capability of dietary monosaccharides and fatty acids to promote lipid accumulation in HepG2 human hepatoma cells. RESULTS: Both high glucose and high fructose resulted in intracellular lipid accumulation after 48 h, and this was further augmented (up to twofold, as compared to basal levels) by co-treatment with the lipogenesis-stimulating hormone insulin and the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-α), respectively. The fatty acids palmitic and oleic acid were even more effective than these carbohydrates, inducing significantly elevated TAG storage already after 24 h of treatment. Highest (about threefold) increases in lipid accumulation were observed upon treatment with oleic acid, alone as well as in combinations with palmitic acid or with high glucose and insulin. Increases in protein levels of a major lipid droplet coat protein, perilipin-2 (PLIN2), mirrored intracellular lipid accumulation following different treatment regimens. CONCLUSIONS: Several treatment regimens of excessive fat and sugar supply promoted lipid accumulation in HepG2 cells, albeit with differences in the extent and rapidity of steatogenesis. PLIN2 is a candidate molecular marker of sustained lipid accumulation in HepG2 cells.

How To Tune the Gene Delivery and Biocompatibility of Poly(2-(4-aminobutyl)-2-oxazoline) by Self- and Coassembly.

Despite their promising potential in gene transfection, the toxicity and limited efficiency of cationic polymers as nonviral vectors are major obstacles for their broader application. The large amount of cationic charges, for example, in poly(ethylene imine) (PEI) is known to be advantageous in terms of their transfection efficiency but goes hand-in-hand with a high toxicity. Consequently, an efficient shielding of the charges is required to minimize toxic effects. In this study, we use a simple mixed-micelle approach to optimize the required charge density for efficient DNA complex formation and to minimize toxicity by using a biocompatible polymer. In detail, we coassembled mixed poly(2-oxazoline) nanostructures ( d ≈ 100 nm) consisting of a hydrophobic-cationic block copolymer (P(NonOx52- b-AmOx184)) and a hydrophobic-hydrophilic stealth block copolymer (P(EtOx155- b-NonOx76) in ratios of 0, 20, 40, 60, 80, and 100 wt % P(NonOx52- b-AmOx184). All micelles with cationic polymers exhibited a very good DNA binding efficiency and dissociation ability, while the bio- and hemocompatibility improved with increasing EtOx content. Analytics via confocal laser scanning microscopy and flow cytometry showed an enhanced cellular uptake, transfection ability, and biocompatibility of all prepared micelleplexes compared to AmOx homopolymers. Micelleplexes with 80 or 100 wt % revealed a similar transfection efficiency as PEI, while the cell viability was significantly higher (80 to 90% compared to 60% for PEI).

Efficient Transfection via an Unexpected Mechanism by Near Neutral Polypiperazines with Tailored Response to Endosomal pH.

Cationic pH-responsive polymers promise to overcome critical challenges in cellular delivery. Ideally, the polymers become selectively charged along the endosomal pathway disturbing only the local membrane and avoiding unintended interactions or cytotoxic side effects at physiological conditions. Polypiperazines represent a novel, hydrophilic class of pH-responsive polymers whose response can be tuned within the relevant pH range (5-7.4). The authors discovered that the polypiperazines are effectively binding plasmid DNA (pDNA) and demonstrate high efficiency in transfection. By design of experiments (DoE), a wide parameter space (pDNA and polymer concentration) is screened to identify the range of effective concentrations for transfection. An isopropyl modified polypiperazine is highly efficient over a wide range of concentrations outperforming linear polyethylenimine (l-PEI, 25 kDa) in regions of low N*/P ratios. A quantitative polymerase chain reaction (qPCR) surprisingly revealed that the pDNA within the piperazine-based polyplexes can be amplified in contrast to polyplexes based on l-PEI. The pDNA must therefore be more accessible and bound differently than for other known transfection polymers. Considering the various opportunities to further optimize their structure, polypiperazines represent a promising platform for designing effective soluble polymeric vectors, which are charge-neutral at physiological conditions.

Tracking the Endosomal Escape: A Closer Look at Calcein and Related Reporters.

Crossing the cellular membrane and delivering active pharmaceuticals or biologicals into the cytosol of cells is an essential step in the development of nanomedicines. One of the most important intracellular processes regarding the cellular uptake of biologicals is the endolysosomal pathway. Sophisticated nanocarriers are developed to overcome a major hurdle, the endosomal entrapment, and delivering their cargo to the required site of action. In parallel, in vitro assays are established analyzing the performance of these nanocarriers. Among them, the release of the membrane-impermeable dye calcein has become a popular and straightforward method. It is accessible for most researchers worldwide, allows for rapid conclusions about the release potential, and enables the study of release mechanisms. This review is intended to provide an overview and guidance for scientists applying the calcein release assay. It comprises a survey of several applications in the study of endosomal escape, considerations of potential pitfalls, challenges, and limitations of the assay, and a brief summary of complementary methods. Based on this review, it is hoped to encourage further research groups to take advantage of the calcein release assay for their own purposes and help to create a database for more efficient cross-correlations between nanocarriers.

Type 1 diabetes mellitus and SARS-CoV-2 in pediatric and adult patients - Data from the DPV network.

BACKGROUND: Data on patients with type 1 diabetes mellitus (T1DM) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are sparse. This study aimed to investigate the association between SARS-CoV-2 infection and T1DM. METHODS: Data from the Prospective Diabetes Follow-up (DPV) Registry were analyzed for diabetes patients tested for SARS-CoV-2 by polymerase chain reaction (PCR) in Germany, Austria, Switzerland, and Luxembourg during January 2020-June 2021, using Wilcoxon rank-sum and chi-square tests for continuous and dichotomous variables, adjusted for multiple testing. RESULTS: Data analysis of 1855 pediatric T1DM patients revealed no differences between asymptomatic/symptomatic infected and SARS-CoV-2 negative/positive patients regarding age, new-onset diabetes, diabetes duration, and body mass index. Glycated hemoglobin A1c (HbA1c) and diabetic ketoacidosis (DKA) rate were not elevated in SARS-CoV-2-positive vs. -negative patients. The COVID-19 manifestation index was 37.5% in individuals with known T1DM, but 57.1% in individuals with new-onset diabetes. 68.8% of positively tested patients were managed as outpatients/telemedically. Data analysis of 240 adult T1MD patients revealed no differences between positively and negatively tested patients except lower HbA1c. Of these patients, 83.3% had symptomatic infections; 35.7% of positively tested patients were hospitalized. CONCLUSIONS: Our results indicate low morbidity in SARS-CoV-2-infected pediatric T1DM patients. Most patients with known T1DM and SARS-CoV-2 infections could be managed as outpatients. However, SARS-CoV-2 infection was usually symptomatic if it coincided with new-onset diabetes. In adult patients, symptomatic SARS-CoV-2 infection and hospitalization were associated with age.

Correlation between Protonation of Tailor-Made Polypiperazines and Endosomal Escape for Cytosolic Protein Delivery.

Responsive polymers, which become protonated at decreasing pH, are considered a milestone in the development of synthetic cell entry vectors. Exact correlations between their properties and their ability to escape the endosome, however, often remain elusive due to hydrophobic interactions or limitations in the design of water-soluble materials with suitable basicity. Here, we present a series of well-defined, hydrophilic polypiperazines, where systematic variation of the amino moiety facilitates an unprecedented fine-tuning of the basicity or pKa value within the physiologically relevant range (pH 6-7.4). Coincubation of HEK 293T cells with various probes, including small fluorophores or functioning proteins, revealed a rapid increase of endosomal release for polymers with pKa values above 6.5 or 7 in serum-free or serum-containing media, respectively. Similarly, cytotoxic effects became severe at increased pKa values (>7). Although the window for effective transport appears narrow, the discovered correlations offer a principal guideline for the design of effective polymers for endosomal escape.

Solely aqueous formulation of hydrophobic cationic polymers for efficient gene delivery.

Cationic polymers are promising gene delivery vectors due to their ability to bind and protect genetic material. The introduction of hydrophobic moieties into cationic polymers can further improve the vector efficiency, but common formulations of hydrophobic polymers involve harsh conditions such as organic solvents, impairing intactness and loading efficiency of the genetic material. In this study, a mild, aqueous formulation method for the encapsulation of high amounts of genetic material is presented. A well-defined pH-responsive hydrophobic copolymer, i.e. poly((n-butylmethacrylate)-co-(methylmethacrylate)-co-(2-(dimethylamino) ethylmethacrylate)), (PBMD) was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. Exploiting the pH-dependent solubility behavior of the polymer, stable pDNA loaded nanoparticles were prepared and characterized using analytical ultracentrifugation (AUC), cryo-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS). This novel formulation approach showed high transfection efficiencies in HEK293T cells, while requiring 5- to 10-fold less pDNA compared to linear polyethylenimine (LPEI), in particular at short incubation times and in serum-containing media. Furthermore, the formulation was successfully adopted for siRNA and mRNA encapsulation and the commercially approved polymer Eudragit® E(PO/100). Overall, the aqueous formulation approach, accompanied by a tailor-made hydrophobic polymer and detailed physicochemical and application studies, led to improved gene delivery vectors with high potential for further applications.

Cell-Penetrating, Peptide-Based RAFT Agent for Constructing Penetration Enhancers.

Peptide-polymer conjugates represent a promising class of compounds that can be used to overcome some of the limitations associated with peptides intended for therapeutic and diagnostic applications. The efficient generation of well-defined peptide/protein-polymer conjugates can promote the development of the design and synthesis of functional drugs and gene delivery platforms. In this research, a sequence defined cell penetrating peptide (i.e., Transportan 10 (TP 10))-based chain transfer agent (TP-CTA) was designed and synthesized in an automated peptide synthesizer. Thereafter, amphiphilic block copolymers poly[oligo(ethylene glycol) methyl ether acrylate]-b-poly(n-butyl acrylate) (TP-POEGA-b-PBA) were synthesized using the TP-CTA via reversible addition-fragmentation chain transfer (RAFT) polymerization. Circular dichroism (CD) spectroscopy confirmed the preservation of α-helix structure of TP 10, which is crucial for its bioactivity. Transmission electron microscopy (TEM) revealed the formation of self-assembled rod-like and vesicle nanostructures in an aqueous environment. Finally, the obtained peptide-conjugated block copolymers were demonstrated to be effective compounds for cell penetration. This method opens up a way for accessing peptide-polymer conjugates with cell-penetrating abilities.

Tuning of endosomal escape and gene expression by functional groups, molecular weight and transfection medium: a structure-activity relationship study.

The use of genetic material by non-viral transfer systems is still in its initial stages, but there are high expectations for the development of targeted therapies. However, nucleic acids cannot enter cells without help, they must be well protected to prevent degradation and overcome a variety of biological barriers, the endosomal barrier being one of the greatest cellular challenges. Herein, the structure-property-relationship was investigated in detail, using well-defined polymers. Polyacrylamides were synthesized via RAFT polymerization resulting in a polymer library of (i) different cationic groups as aminoethyl acrylamide (AEAm), dimethylaminoethyl acrylamide (DMAEAm), dimethylaminopropyl acrylamide (DMAPAm) and guanidinopropyl acrylamide (GPAm); (ii) different degree of polymerization; and investigated (iii) in different cell culture settings. The influence of molar mass and cationic moiety on complex formation with pDNA, cytotoxicity and transfection efficiency of the polymers were investigated. The systematic approach identified a pH-independent guanidinium-containing homopolymer (PGPAm89) as the polymer with the highest transfection efficiency and superior endosomal release under optimal conditions. Since PGPAm89 is not further protonated inside endosomes, common escape theories appear unsuitable. Therefore, the interaction with bis(monoacryloylglycerol)phosphate, a lipid specific for endosomal vesicles, was investigated. Our research suggests that the interactions between amines and lipids may be more relevant than anticipated.

Escherichia coli prevents phagocytosis-induced death of macrophages via classical NF-kappaB signaling, a link to T-cell activation.

NF-kappaB is a crucial mediator of macrophage inflammatory responses, but its role in the context of pathogen-induced adaptive immune responses has yet to be elucidated. Here, we demonstrate that classical NF-kappaB activation delays phagocytosis-induced cell death (PICD) in Raw 264.7 and bone marrow-derived macrophages (BMDMs) upon ingestion of bacteria from the Escherichia coli laboratory strain Top10. By expression of a nondegradable form of IkappaBalpha (superrepressor) and pyrrolidine dithiocarbamate treatment, prolonged activation of NF-kappaB upon bacterial coculture is suppressed, whereas initial induction is only partially inhibited. This activation pattern results in partial inhibition of cellular activation and reduced expression of costimulatory CD86. Notably, suppression of classical NF-kappaB activation does not influence bacterial uptake rates but is followed by increased production of oxygen radicals and enhanced intracellular killing in Raw macrophages. This is associated with reduced expression of NF-kappaB-dependent antiapoptotic c-IAP-2 and a loss of the mitochondrial transmembrane potential. Accordingly, NF-kappaB inhibition in Raw cells and BMDMs causes increased apoptotic rates within 12 h of bacterial ingestion. Interestingly, accelerated eradication of E. coli in NF-kappaB-inhibited macrophages is associated with reduced antigen-specific T-cell activation in macrophage-lymphocyte cocultures. These data suggest that E. coli inhibits PICD of macrophages via classical, antiapoptotic NF-kappaB activation and thus facilitates signaling to T cells. Subsequently, a proper adaptive immune response is likely to be generated. Conclusively, therapeutic inhibition of classical NF-kappaB activation in macrophages may hamper the initiation of adaptive immunity.