Identifying Highly Active and Selective Cobalt X-Ides for Electrocatalytic Hydrogenation of Quinoline.

Earth-abundant Co X-ides are emerging as promising catalysts for the electrocatalytic hydrogenation of quinoline (ECHQ), yet challenging due to the limited fundamental understanding of ECHQ mechanism on Co X-ides. This work identifies the catalytic performance differences of Co X-ides in ECHQ and provides significant insights into the catalytic mechanism of ECHQ. Among selected Co X-ides, the Co3O4 presents the best ECHQ performance with a high conversion of 98.2% and 100% selectivity at ambient conditions. The Co3O4 sites present a higher proportion of 2-coordinated hydrogen-bonded water at the interface than other Co X-ides at a low negative potential, which enhances the kinetics of subsequent water dissociation to produce H*. An ideal 1,4/2,3-H* addition pathway on Co3O4 surface with a spontaneous desorption of 1,2,3,4-tetrahydroquinoline is demonstrated through operando tracing and theoretical calculations. In comparison, the Co9S8 sites display the lowest ECHQ performance due to the high thermodynamic barrier in the H* formation step, which suppresses subsequent hydrogenation; while the ECHQ on Co(OH)F and CoP sites undergo the 1,2,3,4- and 4,3/1,2-H* addition pathway respectively with the high desorption barriers and thus low conversion of quinoline. Moreover, the Co3O4 presents a wide substrate scope and allows excellent conversion of other quinoline derivatives and N-heterocyclic substrates.

Enhanced N-Glycan Profiling of Therapeutic Monoclonal Antibodies through the Application of Upper-Hinge Middle-Up Level LC-HRMS Analysis.

Therapeutic monoclonal antibodies (mAbs) are crucial in modern medicine due to their effectiveness in treating various diseases. However, the structural complexity of mAbs, particularly their glycosylation patterns, presents challenges for quality control and biosimilarity assessment. This study explores the use of upper-hinge middle-up (UHMU)-level ultra-high-performance liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis to improve N-glycan profiling of mAbs. Two specific enzymes, known as IgG degradation enzymes (IGDEs), were used to selectively cleave therapeutic mAbs above the hinge region to separate antibody subunits for further Fc glycan analysis by means of the UHMU/LC-HRMS workflow. The complexity of the mass spectra of IGDEs-digested mAbs was significantly reduced compared to the intact MS level, enabling reliable assignment and relative quantitation of paired Fc glycoforms. The results of the UHMU/LC-HRMS analysis of nine approved therapeutics highlight the significance of this approach for in-depth glycoform profiling.

Multicompartment Polyion Complex Micelles Based on Triblock Polypept(o)ides Mediate Efficient siRNA Delivery to Cancer-Associated Fibroblasts for Antistromal Therapy of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and the third leading cause for cancer-related death worldwide. The tumor is difficult-to-treat due to its inherent resistance to chemotherapy. Antistromal therapy is a novel therapeutic approach, targeting cancer-associated fibroblasts (CAF) in the tumor microenvironment. CAF-derived microfibrillar-associated protein 5 (MFAP-5) is identified as a novel target for antistromal therapy of HCC with high translational relevance. Biocompatible polypept(o)ide-based polyion complex micelles (PICMs) constructed with a triblock copolymer composed of a cationic poly(l-lysine) complexing anti-MFAP-5 siRNA (siMFAP-5) via electrostatic interaction, a poly(γ-benzyl-l-glutamate) block loading cationic amphiphilic drug desloratatine (DES) via π-π interaction as endosomal escape enhancer and polysarcosine poly(N-methylglycine) for introducing stealth properties, are generated for siRNA delivery. Intravenous injection of siMFAP-5/DES PICMs significantly reduces the hepatic tumor burden in a syngeneic implantation model of HCC, with a superior MFAP-5 knockdown effect over siMFAP-5 PICMs or lipid nanoparticles. Transcriptome and histological analysis reveal that MFAP-5 knockdown inhibited CAF-related tumor vascularization, suggesting the anti-angiogenic effect of RNA interference therapy. In conclusion, multicompartment PICMs combining siMFAP-5 and DES in a single polypept(o)ide micelle induce a specific knockdown of MFAP-5 and demonstrate a potent antitumor efficacy (80% reduced tumor burden vs untreated control) in a clinically relevant HCC model.

Identification of bacterial protease domains that cleave human IgM.

Immunoglobulin-degrading proteases are secreted by pathogenic bacteria to weaken the host immune response, contributing to immune evasion mechanisms during an infection. Proteases specific to IgG and IgA immunoglobulin classes have previously been identified and characterized, and only a single report exists on a porcine specific IgM-degrading enzyme. It is unclear whether human pathogens also produce enzymes that can break down human IgM. Here, we have identified four novel IgM-degrading proteases from different genera of human-infecting bacterial pathogens. All four protease domains cleave human IgM at a conserved and unique site in the constant region of IgM. These human IgM proteases may be a useful biochemical tool for the study of early immune responses and have therapeutic potential in IgM-mediated disease.

The impact of IdeS (imlifidase) on allo-specific, xeno-reactive, and protective antibodies in a sensitized rhesus macaque model.

BACKGROUND: Highly sensitized patients face many barriers to kidney transplantation, including higher rates of antibody-mediated rejection after HLA-incompatible transplant. IdeS, an endopeptidase that cleaves IgG nonspecifically, has been trialed as desensitization prior to kidney transplant, and successfully cleaves donor-specific antibody (DSA), albeit with rebound. METHODS: IdeS was generated and tested (2 mg/kg, IV) in two naïve and four allosensitized nonhuman primates (NHP). Peripheral blood samples were collected at regular intervals following IdeS administration. Total IgG, total IgM, and anti-CMV antibodies were quantified with ELISA, and donor-specific antibody (DSA) and anti-pig antibodies were evaluated using flow cytometric crossmatch. B cell populations were assessed using flow cytometry. RESULTS: IdeS successfully cleaved rhesus IgG in vitro. In allosensitized NHP, robust reduction of total, DSA, anti-pig, and anti-CMV IgG was observed within one day following IdeS administration. Rapid rebound of all IgG antibody populations was observed, with antibody levels returning to baseline around day 14 post-infusion. Total IgM level was not affected by IdeS. Interestingly, a comparable reduction in antibody populations was observed after the second dose of IdeS. However, we have not observed any significant modulation of B cell subpopulations after IdeS. CONCLUSIONS: This study evaluated efficacy of IdeS in the allosensitized NHP in IgG with various specificities, mirroring antibody kinetics in human patients. The efficacy of IdeS on preexisting anti-pig antibodies may be useful in clinical xenotransplantation. However, given the limitation of IdeS on its durability as a monotherapy, optimization of IdeS with other agents targeting the humoral response is further needed.

IdeS, a secreted proteinase of Streptococcus pyogenes, is bound to a nuclease at the bacterial surface where it inactivates opsonizing IgG antibodies.

The important bacterial pathogen Streptococcus pyogenes secretes IdeS (immunoglobulin G-degrading enzyme of S. pyogenes), a proteinase that cleaves human immunoglobulin G (IgG) antibodies in the hinge region resulting in Fc (fragment crystallizable) and F(ab')2 (fragment antigen-binding) fragments and protects the bacteria against phagocytic killing. Experiments with radiolabeled IdeS and flow cytometry demonstrated that IdeS binds to the surface of S. pyogenes, and the interaction was most prominent in conditions resembling those in the pharynx (acidic pH and low salt), the habitat for S. pyogenes. SpnA (S. pyogenes nuclease A) is a cell wall-anchored DNase. A dose-dependent interaction between purified SpnA and IdeS was demonstrated in slot binding and surface plasmon resonance spectroscopy experiments. Gel filtration showed that IdeS forms proteolytically active complexes with SpnA in solution, and super-resolution fluorescence microscopy revealed the presence of SpnA-IdeS complexes at the surface of S. pyogenes. Finally, specific IgG antibodies binding to S. pyogenes surface antigens were efficiently cleaved by surface-associated IdeS. IdeS is secreted by all S. pyogenes isolates and cleaves IgG antibodies with a unique degree of specificity and efficiency. These properties and the finding here that the proteinase is present and fully active at the bacterial surface in complex with SpnA implicate an important role for IdeS in S. pyogenes biology and pathogenesis.

Micro-interdigitated electrodes genosensor based on Au-deposited nanoparticles for early detection of cervical cancer.

Genosensor-based electrodes mediated with nanoparticles (NPs) have tremendously developed in medical diagnosis. Herein, we report a facile, rapid, low cost and highly sensitive biosensing strategy for early detection of HPV 18 using gold-nanoparticles (AuNPs) deposited on micro-IDEs. This study represents surface charge transduction of micro-interdigitated electrodes (micro-IDE) alumina insulated with silica, independent and mini genosensor modified with colloidal gold NPs (AuNPs), and determination of gene hybridization for early detection of cervical cancer. The surface of AuNPs deposited micro-IDE functionalized with optimized 3-aminopropyl-triethoxysilane (APTES) followed by hybridization with deoxyribonucleic acid (DNA) virus to develop DNA genosensor. The results of ssDNA hybridization with the ssDNA target of human papillomavirus (HPV) 18 have affirmed that micro-IDE functionalized with colloidal AuNPs resulted in the lowest detection at 0.529 aM. Based on coefficient regression, micro-IDE functionalized with AuNPs produces better results in the sensitivity test (R2 = 0.99793) than unfunctionalized micro-IDE.

Recombinant Endopeptidases IdeS and IdeZ and Their Potential Application.

Endopeptidases IdeS and IdeZ (streptococcal virulence factors that specifically cleave IgG heavy chains) are of particular interest because of their potential use in biotechnology, medicine, and veterinary. Genes encoding these enzymes were cloned and expressed in Escherichia coli heterologous expression system (ideS was cloned from a Streptococcus pyogenes collection strain; ideZ from Streptococcus zooepidemicus was synthesized). The 6His-tag was introduced into the amino acid sequence of each endopeptidase, and IdeS and IdeZ were purified by metal affinity chromatography to an apparent homogeneity (according to polyacrylamide gel electrophoresis). Purified enzymes were active against human and animal IgGs; their specificity toward human IgGs was confirmed by polyacrylamide gel electrophoresis. Recombinant IdeZ was used for immunological analysis of equine strangles infection (diagnostics and determination of the titer of specific antibodies in blood). Hence, IdeZ can be used in veterinary and sanitary microbiology to diagnose infections caused by Streptococcus equi and S. zooepidemicus in addition to its application in medicine and biotechnology.

Chemiresistors with In2O3 Nanostructured Sensitive Films Used for Ozone Detection at Room Temperature.

Detection of greenhouse gases is essential because harmful gases in the air diffuse rapidly over large areas in a short period of time, causing air pollution that will induce climate change with catastrophic consequences over time. Among the materials with favorable morphologies for gas detection (nanofibers, nanorods, nanosheets), large specific surfaces, high sensitivity and low production costs, we chose nanostructured porous films of In2O3 obtained by the sol-gel method, deposited on alumina transducers, with gold (Au) interdigitated electrodes (IDE) and platinum (Pt) heating circuits. Sensitive films contained 10 deposited layers, involving intermediate and final thermal treatments to stabilize the sensitive film. The fabricated sensor was characterized using AFM, SEM, EDX and XRD. The film morphology is complex, containing fibrillar formations and some quasi-spherical conglomerates. The deposited sensitive films are rough, thus favoring gas adsorption. Ozone sensing tests were performed at different temperatures. The highest response of the ozone sensor was recorded at room temperature, considered to be the working temperature for this specific sensor.

Implication of COVID-19 pandemic on the incidence of Clostridioides difficile infection in a Greek tertiary hospital.

Introduction. C. difficile infection (CDI) represents an important global threat. In the COVID-19 era, the multifactorial nature of CDI has emerged.Hypothesis - Aim. The aim was to assess the impact of COVID-19 pandemic on the incidence of CDI in a Greek hospital.Methodology. A retrospective study was performed throughout a 51 month period (January 2018 to March 2022), divided into two periods: pre-pandemic (January 2018 to February 2020) and COVID-19 pandemic (March 2020 to March 2022). The effects of the pandemic compared to the pre-pandemic period on the incidence of CDI [expressed as infections per 10 000 bed days (IBD)] were studied using interrupted time-series analysis.Results. Throughout the study, there was an increase in the monthly CDI incidence from 0.00 to 11.77 IBD (P<0.001). Interrupted time-series disclosed an increase in CDI incidence during the pre-pandemic period from 0.00 to 3.36 IBD (P<0.001). During the COVID-19 pandemic period the linear trend for monthly CDI rose from 2.65 to 13.93 IBD (P<0.001). The increase rate was higher during the COVID-19 pandemic period (r2 = +0.47) compared to the pre-pandemic period (r1 = +0.16).Conclusion. A significant increase of CDI incidence was observed, with the rate of the rise being more intense during the COVID-19 pandemic.

Expression of the Bacterial Enzyme IdeS Using a GFP Fusion in the Yeast Saccharomyces cerevisiae.

Bacterial proteases are important enzymes used in several technical applications where controlled cleavage of proteins is needed. They are challenging enzymes to express recombinantly as parts of the proteome can be hydrolyzed by their activity. The eukaryotic model organism Saccharomyces cerevisiae is potentially a good expression host as it tolerates several stress conditions and is known to better express insoluble proteins compared to bacterial systems. In this chapter we describe how the protease IdeS from Streptococcus pyogenes can be expressed in S. cerevisiae. The expression of IdeS was followed by constructing a fused protein with GFP and measuring the fluorescence with flow cytometry. The protease presence was confirmed with a Western blot assay and activity was measured with an in vitro assay. To reduce potentially toxic effect on the host cell, the growth and production phases were separated by using the inducible promoter GAL1p to control recombinant gene expression. The protocol provided may be adopted for other bacterial proteases through minor modifications of the fused protein.

Design of a concept vehicle for future-oriented urban mobility using design-driven methodologies.

Due to ever-increasing technological acceleration leading to rapid changes in society and its needs, just as today's habits and needs turn out to be completely different from those of only a few years ago, likewise it is reasonable to assume that the same trend will continue in its growth path, making today's solutions rapidly obsolete as time passes and technological innovations follow. This study aims to investigate possible solutions in search of a futuristic and breakthrough response to what is present today. The idea concerns the design of a new type of means of transportation that can best interface with what today are the various criticalities given by vehicular traffic mainly urban but also suburban, going to solve by generating new opportunities from previous problems. This system will be able to go alongside and gradually replace a substantial portion of the current means of transport going to conceptually redefine some elements taken for granted today. In this regard, the application of the IDeS (Industrial Design Structure) methodology has been found to be of great use, which, thanks to the scientific and repeatable methods contained within, has made it possible to arrive at a very clear visualization of the problem, a precise definition, and a level of innovation that is fully satisfactory with respect to the contemporary scenario, while always keeping an eye on feasibility while taking into account the conceptual and therefore deliberately very driven nature of the solution being designed.

Complex Structures Made Simple - Continuous Flow Production of Core Cross-Linked Polymeric Micelles for Paclitaxel Pro-Drug-Delivery.

Translating innovative nanomaterials to medical products requires efficient manufacturing techniques that enable large-scale high-throughput synthesis with high reproducibility. Drug carriers in medicine embrace a complex subset of tasks calling for multifunctionality. Here, the synthesisof pro-drug-loaded core cross-linked polymeric micelles (CCPMs) in a continuous flow processis reported, which combines the commonly separated steps of micelle formation, core cross-linking, functionalization, and purification into a single process. Redox-responsive CCPMs are formed from thiol-reactive polypept(o)ides of polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine) and functional cross-linkers based on dihydrolipoic acid hydrazide for pH-dependent release of paclitaxel. The precisely controlled microfluidic process allows the production of spherical micelles (Dh  = 35 nm) with low polydispersity values (PDI < 0.1) while avoiding toxic organic solvents and additives with unfavorable safety profiles. Self-assembly and cross-linking via slit interdigital micromixers produces 350-700 mg of CCPMs/h per single system, while purification by online tangential flow filtration successfully removes impurities (unimer ≤ 0.5%). The formed paclitaxel-loaded CCPMs possess the desired pH-responsive release profile, display stable drug encapsulation, an improved toxicity profile compared to Abraxane (a trademark of Bristol-Myers Squibb), and therapeutic efficiency in the B16F1-xenotransplanted zebrafish model. The combination of reactive polymers, functional cross-linkers, and microfluidics enables the continuous-flow synthesis of therapeutically active CCPMs in a single process.

Π-Π interactions stabilize PeptoMicelle-based formulations of Pretomanid derivatives leading to promising therapy against tuberculosis in zebrafish and mouse models.

Tuberculosis is the deadliest bacterial disease globally, threatening the lives of millions every year. New antibiotic therapies that can shorten the duration of treatment, improve cure rates, and impede the development of drug resistance are desperately needed. Here, we used polymeric micelles to encapsulate four second-generation derivatives of the antitubercular drug pretomanid that had previously displayed much better in vivo activity against Mycobacterium tuberculosis than pretomanid itself. Because these compounds were relatively hydrophobic and had limited bioavailability, we expected that their micellar formulations would overcome these limitations, reduce toxicities, and improve therapeutic outcomes. The polymeric micelles were based on polypept(o)ides (PeptoMicelles) and were stabilized in their hydrophobic core by π-π interactions, allowing the efficient encapsulation of aromatic pretomanid derivatives. The stability of these π-π-stabilized PeptoMicelles was demonstrated in water, blood plasma, and lung surfactant by fluorescence cross-correlation spectroscopy and was further supported by prolonged circulation times of several days in the vasculature of zebrafish larvae. The most efficacious PeptoMicelle formulation tested in the zebrafish larvae infection model almost completely eradicated the bacteria at non-toxic doses. This lead formulation was further assessed against Mycobacterium tuberculosis in the susceptible C3HeB/FeJ mouse model, which develops human-like necrotic granulomas. Following intravenous administration, the drug-loaded PeptoMicelles significantly reduced bacterial burden and inflammatory responses in the lungs and spleens of infected mice.

Institution of Metal-Organic Frameworks as a Highly Sensitive and Selective Layer In-Field Integrated Soil-Moisture Capacitive Sensor.

The ongoing global industrialization along with the notable world population growth is projected to challenge the global environment as well as pose greater pressure on water and food needs. Foreseeably, an improved irrigation management system is essential and the quest for refined chemical sensors for soil-moisture monitoring is of tremendous importance. Nevertheless, the persisting challenge is to design and construct stable materials with the requisite sensitivity, selectivity, and high performance. Here, we report the introduction of porous metal-organic frameworks (MOFs), as the receptor layer, in capacitive sensors to efficiently sense moisture in two types of soil. Namely, our study unveiled that Cr-soc-MOF-1 offers the best sensitivity (≈24,000 pF) among the other tested MOFs for any given range of soil-moisture content, outperforming several well-known oxide materials. The corresponding increase in the sensitivities for tested MOFs at 500 Hz are ≈450, ≈200, and ≈30% for Cr-soc-MOF-1, Al-ABTC-soc-MOF, and Zr-fum-fcu-MOF, respectively. Markedly, Cr-soc-MOF-1, with its well-known water capacity, manifests an excellent sensitivity of ≈450% in clayey soil, and the analogous response time was 500 s. The noted unique sensing properties of Cr-soc-MOF-1 unveils the great potential of MOFs for soil-moisture sensing application.

Imlifidase therapy: exploring its clinical uses.

INTRODUCTION: Imlifidase, the IgG-degrading enzyme derived from Streptococcus pyogenes, can cleave all four human IgG subclasses with precise specificity. All IgG molecules can be inactivated for ~1-to-2 weeks, until new IgG synthesis is detected. AREAS COVERED: Imlifidase was first studied for the desensitization of highly HLA-sensitized patients to enable kidney transplantation. It is currently being evaluated for kidney transplant recipients who have antibody-mediated rejection (AMR), those with acute kidney injury in the setting of anti-glomerular basement membrane disease, and those with Guillain-Barré syndrome. In 2020, imlifidase received conditional approval from the European Medicines Agency for use to desensitize deceased-donor kidney transplant recipients with a positive crossmatch. Literature search through PubMed revealed that so far, 39 crossmatched-positive patients, i.e. in the presence of donor-specific alloantibodies (DSA) on the transplantation day, have received imlifidase prior to kidney transplantation in four single-arm, open-label, phase II studies. Results at 3-year follow-up are good, i.e. allograft survival is 84%, despite 38% of patients presenting with acute AMR. Mean estimated glomerular filtration rate at 3 years was 55 mL/min/1.73 m2. EXPERT OPINION: The major hurdle now is how to prevent/avoid DSA rebound within days 5-15 post-transplantation. Thus, imlifidase represents a major breakthrough for highly HLA-sensitized kidney transplant candidates, particularly those that have calculated panel-reactive alloantibodies of ≥90%.

Impact of imlifidase treatment on immunoglobulins in an HLA-hypersensitized lupus nephritis patient with anti-SSA/SSB antibodies after kidney transplantation: A case report.

Bacterial recombinant cysteine protease Ides (imlifidase, Idefirix®, Hansa Biopharma) is used to prevent humoral transplant rejection in highly HLA-sensitized recipients, and to control IgG-mediated autoimmune diseases. We report the case of a 51 years old woman suffering from lupus nephritis with end stage kidney disease, grafted for the second time and pre-treated with imlifidase. The patient was HLA-hypersensitized (calculated Panel Reactive Antibodies [Abs], cPRA>99 %) and has three preformed Donor Specific Antibodies (DSA). Circulating immunoglobulins were monitored at initiation (0, 6, 36, 72 and 96 h), and at Ab recovery one and two months following imlifidase injection. From baseline, the higher depletion was reported after 36h for total IgG (-75 %) and IgG subclasses (-87 % for IgG1, IgG2 and IgG3, -78 % for IgG4), while no significant impact on IgA and IgM was observed. Anti-SSA 60 kDa and anti-SSB auto-Abs quickly decreased after imlifidase injection (-96 % for both after 36 h) as well as post-vaccinal specific IgG (-95 % for tetanus toxoid, -97 % for pneumococcus and -91 % for Haemophilus influenzae Abs after 36 h). At the Ab recovery phase, total IgG and anti-SSA60/SSB Abs reached their initial level at two months. Regarding alloreactive Abs, anti-HLA Abs including the three DSA showed a dramatic decrease after injection with 100 % depletion from baseline after 36 h as assessed by multiplex single bead antigen assay, leading to negative crossmatches using both lymphocytotoxicity (LCT) and flow cell techniques. DSA rebound at recovery was absent and remained under the positivity threshold (MFI = 1000) after 6 months. The findings from this case report are that imlifidase exerts an early depleting effect on all circulating IgG, while IgG recovery may depend in part from imlifidase's capacity to target memory B cells.

Mass spectrometry characterization of antibodies at the intact and subunit levels: from targeted to large-scale analysis.

Antibodies are one of the most formidable molecular weapons available to our immune system. Their high specificity against a target (antigen) and capability of triggering different immune responses (e.g., complement system activation and antibody-dependent cell-mediated cytotoxicity) make them ideal drugs to fight many different human diseases. Currently, both monoclonal antibodies and more complex molecules based on the antibody scaffold are used as biologics. Naturally, such highly heterogeneous molecules require dedicated analytical methodologies for their accurate characterization. Mass spectrometry (MS) can define the presence and relative abundance of multiple features of antibodies, including critical quality attributes. The combination of small and large variations within a single molecule can only be determined by analyzing intact antibodies or their large (25 to 100 kDa) subunits. Hence, top-down (TD) and middle-down (MD) MS approaches have gained popularity over the last decade. In this Young Scientist Feature we discuss the evolution of TD and MD MS analysis of antibodies, including the new frontiers that go beyond biopharma applications. We will show how this field is now moving from the "quality control" analysis of a known, single antibody to the high-throughput investigation of complex antibody repertoires isolated from clinical samples, where the ultimate goal is represented by the complete gas-phase sequencing of antibody molecules without the need of any a priori knowledge.

Chop-Chop: The Future of Bacterial Enzymes in Transfusion Medicine.

The discovery of bacterial enzymes with specificity for IgG antibodies has led to breakthroughs in several autoantibody-mediated diseases. Two such enzymes, IdeS and EndoS, degrade IgG by different mechanisms, and have separately shown promise in numerous animal models of autoimmune diseases. Recently, imlifidase (the international nonproprietary name for IdeS) has advanced to clinical trials, where it has performed remarkably well in desensitizing patients to enable kidney transplantation, and in anti-glomerular basement membrane disease. Conversely, it performed poorly in thrombotic thrombocytopenic purpura. This review summarizes the development of antibody-degrading enzymes, with a discussion of key clinical studies involving imlifidase. The future of the field is also discussed, including the use of these enzymes in other diseases, and the potential for re-dosing.

Systematic Study of Enzymatic Degradation and Plasmid DNA Complexation of Mucus Penetrating Star-Shaped Lysine/Sarcosine Polypept(o)ides with Different Block Arrangements.

8-Arm star polypep(o)ides comprising cationic polylysine and hydrophilic polysarcosine blocks with a degree of polymerization (DP) of 30 per block are synthesized. Two different block sequences with polylysine as the inner and polysarcosine as the outer block and vice versa are obtained in addition to a statistical copolymer. Analysis of the enzymatic hydrolysis by the proteolytic enzyme trypsin demonstrates a strong dependence on structural arrangements. While polypept(o)ide disintegration is detectible after 24 h by Size Exclusion Chromatography (SEC), significant hydrolysis of the lysine blocks is only monitored after 48 h by fluorescamine labeling of the produced lysine and clearly accelerated in structures with more accessible polylysine blocks. All structures are capable of complexing plasmid DNA and form gene nanomedicines at sizes around or below 200 nm as determined by Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), and Transition Electron Microscopy (TEM). The polyplex formation is slightly enhanced for both block structures over the random copolypept(o)ide. Moreover, it is demonstrated that the polyplexes can transport through mucus. The results highlight the importance of structural control in compartmentalized polymeric gene vector candidates with hydrophilic domains for potential mucosal delivery.