PEGylated Recombinant Aplysia punctata Ink Toxin Depletes Arginine and Lysine and Inhibits the Growth of Tumor Xenografts.

In recent years, a novel treatment method for cancer has emerged, which is based on the starvation of tumors of amino acids like arginine. The deprivation of arginine in serum is based on enzymatic degradation and can be realized by arginine deaminases like the l-amino acid oxidase found in the ink toxin of the sea hare Aplysia punctata. Previously isolated from the ink, the l-amino acid oxidase was described to oxidate the essential amino acids l-lysine and l-arginine to their corresponding deaminated alpha-keto acids. Here, we present the recombinant production and functionalization of the amino acid oxidase Aplysia punctata ink toxin (APIT). PEGylated APIT (APIT-PEG) increased the blood circulation time. APIT-PEG treatment of patient-derived xenografted mice shows a significant dose-dependent reduction of tumor growth over time mediated by amino acid starvation of the tumor. Treatment of mice with APIT-PEG, which led to deprivation of arginine, was well tolerated.

Bioresponsive Cytokine Delivery Responding to Matrix Metalloproteinases.

Cytokines are regulated in acute and chronic inflammation, including rheumatoid arthritis (RA) and myocardial infarction (MI). However, the dynamic windows within which cytokine activity/inhibition is desirable in RA and MI change timely and locally during the disease. Therefore, traditional, static delivery regimens are unlikely to meet the idiosyncrasy of these highly dynamic pathophysiological and individual processes. Responsive delivery systems and biomaterials, sensing surrogate markers of inflammation (i.e., matrix metalloproteinases - MMPs) and answering with drug release, may present drug activity at the right time, manner, and place. This article discusses MMPs as surrogate markers for disease activity in RA and MI to clock drug discharge to MMP concentration profiles from MMP-responsive drug delivery systems and biomaterials.

PEGylation of Human Vascular Endothelial Growth Factor.

Vascular endothelial growth factor A-165 (VEGF-A165) positively modulates neointimal hyperplasia, lumen stenosis, and neovascularization. One challenge for the use of VEGF-A165 for potential therapy is its short serum half-life. Therefore, we are designing VEGF-A165 bioconjugates carrying polyethylene glycol (PEG). The purity of the recombinantly expressed human VEGF-A165 exceeded 90%. The growth factor had a half-maximal effective concentration of 0.9 ng/mL (EC50) and induced tube formation of human umbilical vein endothelial cells. PEGylation was conducted by Schiff base reaction followed by reductive amination. After purification, two species were obtained, with one or two PEG attached per VEGF-A165 dimer. Both resulting bioconjugates had a purity exceeding 90%, wild-type bioactivity, and increased hydrodynamic radii as required for prolonging the half-life.

Matrix Metalloproteinase-Responsive Delivery of PEGylated Fibroblast Growth Factor 2.

Attachment of polyethylene glycol (PEG) chains is a common, well-studied, and Food and Drug Administration-approved method to address the pharmacokinetic challenges of therapeutic proteins. Occasionally, PEGylation impairs the activity of pharmacodynamics (PD). To overcome this problem, disease-relevant cleavable linkers between the polymer and the therapeutic protein can unleash full PD by de-PEGylating the protein at its target site. In this study, we engineered a matrix metalloproteinase (MMP)-responsive fibroblast growth factor 2 (FGF-2) mutant that was site-specifically extended with a PEG polymer chain. Using bioinspired strategies, the bioconjugate was designed to release the native protein at the desired structure/environment with preservation of the proliferative capacity in vitro on NIH3T3 cells. In vivo, hepatic exposure was diminished but not its renal distribution over time compared to unconjugated FGF-2. By releasing the growth factor from the PEG polymer in response to MMP cleavage, restored FGF-2 may enter hard-to-reach tissues and activate cell surface receptors or nuclear targets.

Site-specific PEGylation of recombinant tissue-type plasminogen activator.

Tissue-type plasminogen activator (tPA) is the gold standard for emergency treatment of ischemic stroke, which is the third leading cause of death worldwide. Major challenges of tPA therapy are its rapid elimination by plasminogen activator inhibitor-1 (PAI-1) and hepatic clearance, leading to the use of high doses and consequent serious side effects, including internal bleeding, swelling and low blood pressure. In this regard, we developed three polyethylene glycol (PEG)ylated tPA bioconjugates based on the recombinant human tPA drug Alteplase using site-specific conjugation strategies. The first bioconjugate with PEGylation at the N-terminus of tPA performed by reductive alkylation showed a reduced proteolytic activity of 68 % compared to wild type tPA. PEGylation at the single-free cysteine of tPA with linear and branched PEG revealed similar proteolytic activities as the wild-type protein. Moreover, both bioconjugates with PEG-cysteine-modification showed 2-fold slower inhibition kinetics by PAI-1. All bioconjugates increased in hydrodynamic size as a critical requirement for half-life extension.

Bioconjugation of a Fibroblast Activation Protein Targeted Interleukin-4.

Interleukin-4 (IL-4) is an immune-modulating therapeutic with growing potential for the treatment of inflammatory diseases. Current challenges of IL-4 therapy include a low serum half-life and pleiotropic activity, suggesting effective targeting of IL-4. To develop an interleukin-4 bioconjugate with rapid targeting to inflammatory disease sites, we report the chemical synthesis, bioconjugation, and in vitro characterization of a murine interleukin-4 (mIL-4) conjugate decorated with a fibroblast activation protein inhibitor (FAPI). The FAPI targeting moiety features 2,2',2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) to allow future biodistribution and imaging studies of the FAPI-mIL-4 bioconjugate. We demonstrated site-specific coupling of mIL-4 and FAPI-DOTA deploying chemo-enzyme and enzyme chemistries with a high purity exceeding 95%. The FAPI-DOTA modified mIL-4 was bioactive with polarization of murine macrophages into the M2 state while maintaining specific binding to FAP on fibroblast cells. Together, these results point to future in vivo use of the FAPI-mIL-4 bioconjugate to assess biodistribution and biological effects in animal models of inflammatory joint disease.

Amber Light Control of Peptide Secondary Structure by a Perfluoroaromatic Azobenzene Photoswitch.

The incorporation of photoswitches into the molecular structure of peptides and proteins enables their dynamic photocontrol in complex biological systems. Here, a perfluorinated azobenzene derivative triggered by amber light was site-specifically conjugated to cysteines in a helical peptide by perfluoroarylation chemistry. In response to the photoisomerization (trans→cis) of the conjugated azobenzene with amber light, the secondary structure of the peptide was modulated from a disorganized into an amphiphilic helical structure.

PEtOxylated Interferon-α2a Bioconjugates Addressing H1N1 Influenza A Virus Infection.

Influenza A viruses (IAV), including the pandemic 2009 (pdm09) H1N1 or avian influenza H5N1 virus, may advance into more pathogenic, potentially antiviral drug-resistant strains (including loss of susceptibility against oseltamivir). Such IAV strains fuel the risk of future global outbreaks, to which this study responds by re-engineering Interferon-α2a (IFN-α2a) bioconjugates into influenza therapeutics. Type-I interferons such as IFN-α2a play an essential role in influenza infection and may prevent serious disease courses. We site-specifically conjugated a genetically engineered IFN-α2a mutant to poly(2-ethyl-2-oxazoline)s (PEtOx) of different molecular weights by strain-promoted azide-alkyne cyclo-addition. The promising pharmacokinetic profile of the 25 kDa PEtOx bioconjugate in mice echoed an efficacy in IAV-infected ferrets. One intraperitoneal administration of this bioconjugate, but not the marketed IFN-α2a bioconjugate, changed the disease course similar to oseltamivir, given orally twice every study day. PEtOxylated IFN-α2a bioconjugates may expand our therapeutic arsenal against future influenza pandemics, particularly in light of rising first-line antiviral drug resistance to IAV.

Polymer selection impacts the pharmaceutical profile of site-specifically conjugated Interferon-α2a.

Conjugation of poly(ethylene glycol) (PEG) to biologics is a successful strategy to favorably impact the pharmacokinetics and efficacy of the resulting bioconjugate. We compare bioconjugates synthesized by strain-promoted azide-alkyne cycloaddition (SPAAC) using PEG and linear polyglycerol (LPG) of about 20 kDa or 40 kDa, respectively, with an azido functionalized human Interferon-α2a (IFN-α2a) mutant. Site-specific PEGylation and LPGylation resulted in IFN-α2a bioconjugates with improved in vitro potency compared to commercial Pegasys. LPGylated bioconjugates had faster disposition kinetics despite comparable hydrodynamic radii to their PEGylated analogues. Overall exposure of the PEGylated IFN-α2a with a 40 kDa polymer exceeded Pegasys, which, in return, was similar to the 40 kDa LPGylated conjugates. The study points to an expanded polymer design space through which the selected polymer class may result in a different distribution of the studied bioconjugates.

Fibrin Sealants: Challenges and Solutions.

Intraoperative bleeding and postoperative bleeding are major surgical complications. Tissue sealants, hemostats, and adhesives provide the armamentarium for establishing hemostatic balance, including the tissue sealant fibrin. Fibrin sealants combine advantages including instantaneous effect, biocompatibility, and biodegradability. However, several challenges remain. This review summarizes current fibrin product generations and highlights new trends and potential strategies for future improvement.

Merging bioresponsive release of insulin-like growth factor I with 3D printable thermogelling hydrogels.

3D printing of biomaterials enables spatial control of drug incorporation during automated manufacturing. This study links bioresponsive release of the anabolic biologic, insulin-like growth factor-I (IGF-I) in response to matrix metalloproteinases (MMP) to 3D printing using the block copolymer of poly(2-methyl-2-oxazoline) and thermoresponsive poly(2-n-propyl-2-oxazine) (POx-b-POzi). For that, a chemo-enzymatic synthesis was deployed, ligating IGF-I enzymatically to a protease sensitive linker (PSL), which was conjugated to a POx-b-POzi copolymer. The product was blended with the plain thermogelling POx-b-POzi hydrogel. MMP exposure of the resulting hydrogel triggered bioactive IGF-I release. The bioresponsive IGF-I containing POx-b-POzi hydrogel system was further detailed for shape control and localized incorporation of IGF-I via extrusion 3D printing for future applications in biomedicine and biofabrication.

Bioconjugation strategies and clinical implications of Interferon-bioconjugates.

Interferons (IFN) are immunomodulating, antiviral and antiproliferative cytokines for treatment of multiple indications, including cancer, hepatitis, and autoimmune disease. The first IFNs were discovered in 1957, first approved in 1986, and are nowadays listed in the WHO model list of essential medicines. Three classes of IFNs are known; IFN-α2a and IFN-ß belonging to type-I IFNs, IFN-γ a type-II IFN approved for some hereditary diseases and IFN-λs, which form the newest class of type-III IFNs. IFN-λs were discovered in the last decade with fascinating yet under discovered pharmaceutical potential. This article reviews available IFN drugs, their field and route of application, while also outlining available and future strategies for bioconjugation to further optimize pharmaceutical and clinical performances of all three available IFN classes.

Chemo-Enzymatic PEGylation/POxylation of Murine Interleukin-4.

Interleukin-4 (IL-4) is a potentially interesting anti-inflammatory therapeutic, which is rapidly excreted. Therefore, serum half-life extension by polymer conjugation is desirable, which may be done by PEGylation. Here, we use PEtOx as an alternative to PEG for bioconjugate engineering. We genetically extended murine IL-4 (mIL-4) with the d-domain of insulin-like growth factor I (IGF-I), a previously identified substrate of transglutaminase (TG) Factor XIIIa (FXIIIa). Thereby, engineered mIL-4 (mIL-4-TG) became an educt for TG catalyzed C-terminal, site-directed conjugation. This was deployed to enzymatically couple an azide group containing peptide sequence to mIL-4, allowing C-terminal bioconjugation of polyethylene glycol or poly(2-ethyl-2-oxazoline). Both bioconjugates had wild-type potency and alternatively polarized macrophages.

Linear Polyglycerol for N-terminal-selective Modification of Interleukin-4.

Polymer conjugation to biologics is of key interest to the pharmaceutical industry for the development of potent and long acting biotherapeutics, with poly(ethylene glycol) (PEG) being the gold standard. Within the last years, unwanted PEG-related side effects (immunological reactions, antibody formation) arose, therefore creating several attempts to establish alternative polymers with similar potential to PEG. In this article, we synthesized N-terminal bioconjugates of the potential therapeutic human interleukin-4 (hIL-4 WT) with linear polyglycerol (LPG) of 10 and 40 kDa and compared it with its PEG analogs of same nominal weights. Polyglycerol is a highly hydrophilic polymer with good biocompatibility and therefore represents an alternative polymer to PEG. Both polymer types resulted in similar conjugation yields, comparable hydrodynamic sizes and an unaltered secondary structure of the protein after modification. LPG- and PEG-bioconjugates remained stable in human plasma, whereas binding to human serum albumin (HSA) decreased after polymer modification. Furthermore, only minor differences in bioactivity were observed between LPG- and PEG-bioconjugates of same nominal weights. The presented findings are promising for future pharmacokinetic evaluation of hIL-4-polymer bioconjugates.

Freeform direct laser writing of versatile topological 3D scaffolds enabled by intrinsic support hydrogel.

In this study, a novel approach to create arbitrarily shaped 3D hydrogel objects is presented, wherein freeform two-photon polymerization (2PP) is enabled by the combination of a photosensitive hydrogel and an intrinsic support matrix. This way, topologies without physical contact such as a highly porous 3D network of concatenated rings were realized, which are impossible to manufacture with most current 3D printing technologies. Micro-Raman and nanoindentation measurements show the possibility to control water uptake and hence tailor the Young's modulus of the structures via the light dosage, proving the versatility of the concept regarding many scaffold characteristics that makes it well suited for cell specific cell culture as demonstrated by cultivation of human induced pluripotent stem cell derived cardiomyocytes.

Molecular Insights into Site-Specific Interferon-α2a Bioconjugates Originated from PEG, LPG, and PEtOx.

Conjugation of biologics with polymers modulates their pharmacokinetics, with polyethylene glycol (PEG) as the gold standard. We compared alternative polymers and two types of cyclooctyne linkers (BCN/DBCO) for bioconjugation of interferon-α2a (IFN-α2a) using 10 kDa polymers including linear mPEG, poly(2-ethyl-2-oxazoline) (PEtOx), and linear polyglycerol (LPG). IFN-α2a was azide functionalized via amber codon expansion and bioorthogonally conjugated to all cyclooctyne linked polymers. Polymer conjugation did not impact IFN-α2a's secondary structure and only marginally reduced IFN-α2a's bioactivity. In comparison to PEtOx, the LPG polymer attached via the less rigid cyclooctyne linker BCN was found to stabilize IFN-α2a against thermal stress. These findings were further detailed by molecular modeling studies which showed a modulation of protein flexibility upon PEtOx conjugation and a reduced amount of protein native contacts as compared to PEG and LPG originated bioconjugates. Polymer interactions with IFN-α2a were further assessed via a limited proteolysis (LIP) assay, which resulted in comparable proteolytic cleavage patterns suggesting weak interactions with the protein's surface. In conclusion, both PEtOx and LPG bioconjugates resulted in a similar biological outcome and may become promising PEG alternatives for bioconjugation.

From Thermogelling Hydrogels toward Functional Bioinks: Controlled Modification and Cytocompatible Crosslinking.

Hydrogels are key components in bioink formulations to ensure printability and stability in biofabrication. In this study, a well-known Diels-Alder two-step post-polymerization modification approach is introduced into thermogelling diblock copolymers, comprising poly(2-methyl-2-oxazoline) and thermoresponsive poly(2-n-propyl-2-oxazine). The diblock copolymers are partially hydrolyzed and subsequently modified by acid/amine coupling with furan and maleimide moieties. While the thermogelling and shear-thinning properties allow excellent printability, trigger-less cell-friendly Diels-Alder click-chemistry yields long-term shape-fidelity. The introduced platform enables easy incorporation of cell-binding moieties (RGD-peptide) for cellular interaction. The hydrogel is functionalized with RGD-peptides using thiol-maleimide chemistry and cell proliferation as well as morphology of fibroblasts seeded on top of the hydrogels confirm the cell adhesion facilitated by the peptides. Finally, bioink formulations are tested for biocompatibility by incorporating fibroblasts homogenously inside the polymer solution pre-printing. After the printing and crosslinking process good cytocompatibility is confirmed. The established bioink system combines a two-step approach by physical precursor gelation followed by an additional chemical stabilization, offering a broad versatility for further biomechanical adaptation or bioresponsive peptide modification.

Metabolic Glycoengineering in hMSC-TERT as a Model for Skeletal Precursors by Using Modified Azide/Alkyne Monosaccharides.

Metabolic glycoengineering enables a directed modification of cell surfaces by introducing target molecules to surface proteins displaying new features. Biochemical pathways involving glycans differ in dependence on the cell type; therefore, this technique should be tailored for the best results. We characterized metabolic glycoengineering in telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) as a model for primary hMSC, to investigate its applicability in TERT-modified cell lines. The metabolic incorporation of N-azidoacetylmannosamine (Ac4ManNAz) and N-alkyneacetylmannosamine (Ac4ManNAl) into the glycocalyx as a first step in the glycoengineering process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency (osteogenic and adipogenic differentiation potential) was maintained under these adapted culture conditions. In the second step, glycoengineered cells were modified with fluorescent dyes using Cu-mediated click chemistry. In these analyses, the two mannose derivatives showed superior incorporation efficiencies compared to glucose and galactose isomers. In time-dependent experiments, the incorporation of Ac4ManNAz was detectable for up to six days while Ac4ManNAl-derived metabolites were absent after two days. Taken together, these findings demonstrate the successful metabolic glycoengineering of immortalized hMSC resulting in transient cell surface modifications, and thus present a useful model to address different scientific questions regarding glycosylation processes in skeletal precursors.

A Complete and Versatile Protocol: Decoration of Cell-Derived Matrices with Mass-Encoded Peptides for Multiplexed Protease Activity Detection.

This article provides guidance toward a platform technology for monitoring enzyme activity within the extracellular matrix (ECM) assessed by quantifying reporters secreted into the cell culture supernatant and analyzed by tandem mass spectrometry. The reporters are enzymatically and covalently bound to the ECM by transglutaminases (TG) using the peptide sequence of human insulin-like growth factor I's (IGF-I) D-domain which is known to be bound to the ECM by transglutaminase. The IGF-I D-domain sequence is followed by a peptide sequence cleaved by the intended target protease. This protease-sensitive peptide sequence (PSS) is cleaved off the ECM and can be used to monitor target-enzyme activity by employing a downstream mass tag designed according to isobaric mass encoding strategies, i.e., the combination of isotopically labeled, heavy amino acids. Thereby, cleavage events are linked to the appearance of encoded mass tags, readily allowing multiplexing. This article presents the design and synthesis of these mass reporters. It further aims at detailing the search for peptide sequences responding to target proteases to facilitate future work on enzyme activity measurement for enzymatic activities of hitherto unknown enzymes. In conclusion, the goal of this article is to arm scientists interested in measurements of local enzymatic activities within the ECM with robust protocols and background knowledge.