Covalent and oriented immobilization of scFv antibody fragments via an engineered glycan moiety.

Antibody-derived fragments have enormous potential application in solid-phase assays such as biomarker detection and protein purification. Controlled orientation of the immobilized antibody molecules is a critical requirement for the sensitivity and efficacy of such assays. We present an approach for covalent, correctly oriented attachment of scFv antibody fragments on solid supports. Glycosylated scFvs were expressed in Escherichia coli and the C-terminal, binding pocket-distal glycan tag was oxidized for covalent attachment to amine-functionalized beads. The glycosylated scFvs could be immobilized at salt concentrations that precluded nonspecific adsorption of unglycosylated molecules and the covalently attached antibody fragments exhibited 4-fold higher functional activity than ionically adsorbed scFvs. The glyco-tethered scFvs were stable in NaCl concentrations that removed greater than 90% of adsorbed scFvs and they exhibited improved stability of antigen binding over both adsorbed scFvs and soluble, nonimmobilized scFvs in accelerated degradation tests. The simple expression and immobilization approach reported is likely to find broad application in in vitro antibody tests.

Thermal unfolding and refolding of lysozyme in deep eutectic solvents and their aqueous dilutions.

The stability of hen's egg white lysozyme in different choline chloride-based pseudo-concentrated and neat deep eutectic solvents (DESs) has been studied by means of intrinsic fluorescence and CD spectroscopy. Thermal unfolding experiments carried out in non-diluted urea:choline chloride and glycerol:choline chloride eutectic solvents (UCCl-DES and GCCl-DES, respectively) showed the accumulation at certain temperatures of discrete, partially folded intermediates that displayed a high content of secondary structure and a disrupted tertiary structure. Reversibility of the unfolding process was incomplete in these circumstances, with the urea-based DES showing higher protein structure destabilization upon thermal treatment. On the other hand, aqueous dilution of the eutectic mixtures allowed the recovery of a reversible, two-state denaturation process. Lysozyme activity was also affected in neat and pseudo-concentrated GCCl-DES, with an increasing recovery of activity upon aqueous dilution, and full restoration after DES removal through extensive dialysis. These results suggest that protein interactions at room temperature are reversible and depend on the DES components and on the aqueous content of the original DES dilution.

Improved detection of domoic acid using covalently immobilised antibody fragments.

Antibody molecules, and antibody fragments in particular, have enormous potential in the development of biosensors for marine monitoring. Conventional immobilisation approaches used in immunoassays typically yield unstable and mostly incorrectly oriented antibodies, however, resulting in reduced detection sensitivities for already low concentration analytes. The 2H12 anti-domoic acid scFv antibody fragment was engineered with cysteine-containing linkers of two different lengths, distal to the antigen binding pocket, for covalent and correctly oriented immobilisation of the scFvs on functionalised solid supports. The Escherichia coli-produced, cysteine-engineered scFvs dimerised in solution and demonstrated similar efficiencies of covalent immobilisation on maleimide-activated plates and minimal non-covalent attachment. The covalently attached scFvs exhibited negligible leaching from the support under acidic conditions that removed almost 50% of the adsorbed wildtype fragment, and IC50s for domoic acid of 270 and 297 ng/mL compared with 1126 and 1482 ng/mL, respectively, for their non-covalently adsorbed counterparts. The expression and immobilisation approach will facilitate the development of stable, reusable biosensors with increased stability and detection sensitivity for marine neurotoxins.

Self-assembly of a barnacle cement protein into intertwined amyloid fibres and determination of their adhesive and viscoelastic properties.

The stalked barnacle Pollicipes pollicipes uses a multi-protein cement to adhere to highly varied substrates in marine environments. We investigated the morphology and adhesiveness of a component 19 kDa protein in barnacle cement gland- and seawater-like conditions, using transmission electron microscopy and state-of-the art scanning probe techniques. The protein formed amyloid fibres after 5 days in gland-like but not seawater conditions. After 7-11 days, the fibres self-assembled under gland-like conditions into large intertwined fibrils of up to 10 µm in length and 200 nm in height, with a distinctive twisting of fibrils evident after 11 days. Atomic force microscopy (AFM)-nanodynamic mechanical analysis of the protein in wet conditions determined E' (elasticity), E'' (viscosity) and tan δ values of 2.8 MPa, 1.2 MPa and 0.37, respectively, indicating that the protein is a soft and viscoelastic material, while the adhesiveness of the unassembled protein and assembled fibres, measured using peak force quantitative nanomechanical mapping, was comparable to that of the commercial adhesive Cell-Tak™. The study provides a comprehensive insight into the nanomechanical and viscoelastic properties of the barnacle cement protein and its self-assembled fibres under native-like conditions and may have application in the design of amyloid fibril-based biomaterials or bioadhesives.

Aerosol Delivery of a Novel Recombinant Modified Superoxide Dismutase Protein Reduces Oxidant Injury and Attenuates Escherichia coli Induced Lung Injury in Rats.

Background: Acute respiratory distress syndrome (ARDS) is a life-threatening respiratory failure syndrome with diverse etiologies characterized by increased permeability of alveolar-capillary membranes, pulmonary edema, and acute onset hypoxemia. During the ARDS acute phase, neutrophil infiltration into the alveolar space results in uncontrolled release of reactive oxygen species (ROS) and proteases, overwhelming antioxidant defenses and causing alveolar epithelial and lung endothelial injury. Objectives: To investigate the therapeutic potential of a novel recombinant human Cu-Zn-superoxide dismutase (SOD) fusion protein in protecting against ROS injury and for aerosolized SOD delivery to treat Escherichia coli induced ARDS. Methods: Fusion proteins incorporating human Cu-Zn-SOD (hSOD1), with (pep1-hSOD1-his) and without (hSOD1-his) a fused hyaluronic acid-binding peptide, were expressed in E. coli. Purified proteins were evaluated in in vitro assays with human bronchial epithelial cells and through aerosolized delivery to the lung of an E. coli-induced ARDS rat model. Results: SOD proteins exhibited high SOD activity in vitro and protected bronchial epithelial cells from oxidative damage. hSOD1-his and pep1-hSOD1-his retained SOD activity postnebulization and exhibited no adverse effects in the rat. Pep1-hSOD1-his administered through instillation or nebulization to the lung of an E. coli-induced pneumonia rat improved arterial oxygenation and lactate levels compared to vehicle after 48 hours. Static lung compliance was improved when the pep1-hSOD1-his protein was delivered by instillation. White cell infiltration to the lung was significantly reduced by aerosolized delivery of protein, and reduction of cytokine-induced neutrophil chemoattractant-1, interferon-gamma, and interleukin 6 pro-inflammatory cytokine concentrations in bronchoalveolar lavage was observed. Conclusions: Aerosol delivery of a novel recombinant modified SOD protein reduces oxidant injury and attenuates E. coli induced lung injury in rats. The results provide a strong basis for further investigation of the therapeutic potential of hSOD1 in the treatment of ARDS.

Identification of cell surface-specific markers to target human nucleus pulposus cells: expression of carbonic anhydrase XII varies with age and degeneration.

OBJECTIVE: Back pain is a major cause of disability, affecting millions of people worldwide. One cause of axial back pain is degeneration of the nucleus pulposus (NP) of the intervertebral disc. This study was undertaken to investigate associations of NP cells with cell surface-specific proteins that differ from proteins in closely related cell types, i.e., intervertebral disc anulus fibrosus (AF) cells and articular cartilage (AC) chondrocytes, in order to identify potential surface molecules for directed delivery of therapeutic agents. METHODS: We conducted a complementary DNA microarray analysis of 16 human samples from 6 donors, followed by gene list reduction using a systematic approach. Genes that were more highly expressed in NP than AC cells, contained transmembrane domains, and appeared attractive for targeting were assessed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). As a viable candidate, carbonic anhydrase XII (CAXII) was analyzed at the protein level by immunohistochemistry and functional study. RESULTS: Microarray results demonstrated a clear divide between the AC and AF and between the AC and NP samples. However, the transcriptomic profile of AF and NP samples displayed a greater intersubject similarity. Of the 552 genes with up-regulated expression in NP cells, 90 contained transmembrane domains, and 28 were quantified by RT-PCR. Most intense CAXII labeling was observed in the NP of discs from young subjects and in degenerative tissue. CONCLUSION: CAXII may be considered for detection or targeting of degenerating disc cells. Furthermore, CAXII may be involved in pH regulation of NP cells. Its potential for directed delivery of regenerative factors and its functional role in NP cell homeostasis warrant further investigation.

A fast, ultrasensitive SERS immunoassay to detect SARS-CoV-2 in saliva.

The COVID-19 pandemic has emphasized the need for accurate, rapid, point-of-care diagnostics to control disease transmission. We have developed a simple, ultrasensitive single-particle surface-enhanced Raman spectroscopy (SERS) immunoassay to detect the SARS-CoV-2 spike protein in saliva. This assay relies on the use of single chain Fv (scFv) recombinant antibody expressed in E. coli to bind the SARS-CoV-2 spike protein. Recombinant scFv labeled with a SERS-active dye in solution is mixed with unlabeled scFv conjugated to gold-coated magnetic nanoparticles and a sample to be tested. In the presence of the SARS-CoV-2 spike protein, immunocomplexes form and concentrate the labeled scFv close to the gold surface of the nanoparticles, causing an increased SERS signal. The assay detects inactivated SARS-CoV-2 virus and spike protein in saliva at concentrations of 1.94 × 103 genomes mL-1 and 4.7 fg mL-1, respectively, making this direct detection antigen test only 2-3 times less sensitive than some qRT-PCR tests. All tested SARS-CoV-2 spike proteins, including those from alpha, beta, gamma, delta, and omicron variants, were detected without recognition of the closely related SARS and MERS spike proteins. This 30 min, no-wash assay requires only mixing, a magnetic separation step, and signal measurements using a hand-held, battery-powered Raman spectrometer, making this assay ideal for ultrasensitive detection of the SARS-CoV-2 virus at the point-of-care.

Rapid, Point-of-Care scFv-SERS Assay for Femtogram Level Detection of SARS-CoV-2.

Rapid, sensitive, on-site identification of SARS-CoV-2 infections is an important tool in the control and management of COVID-19. We have developed a surface-enhanced Raman scattering (SERS) immunoassay for highly sensitive detection of SARS-CoV-2. Single-chain Fv (scFv) recombinant antibody fragments that bind the SARS-CoV-2 spike protein were isolated by biopanning a human scFv library. ScFvs were conjugated to magnetic nanoparticles and SERS nanotags, followed by immunocomplex formation and detection of the SARS-CoV-2 spike protein with a limit of detection of 257 fg/mL in 30 min in viral transport medium. The assay also detected B.1.1.7 ("alpha"), B.1.351 ("beta"), and B.1.617.2 ("delta") spike proteins, while no cross-reactivity was observed with the common human coronavirus HKU1 spike protein. Inactivated whole SARS-CoV-2 virus was detected at 4.1 × 104 genomes/mL, which was 10-100-fold lower than virus loads typical of infectious individuals. The assay exhibited higher sensitivity for SARS-CoV-2 than commercial lateral flow assays, was compatible with viral transport media and saliva, enabled rapid pivoting to detect new virus variants, and facilitated highly sensitive, point-of-care diagnosis of COVID-19 in clinical and public health settings.

Video-based learning to enhance teaching of practical microbiology.

Video-based learning is an increasingly important methodology in higher education and has particular value in practical teaching. In order to enhance learning and promote student engagement in our undergraduate microbiology programme, we designed and produced a suite of teaching videos which demonstrate laboratory techniques core to the syllabus. The methods were demonstrated by Ph.D. students and the professionally-produced videos were made widely available via the free YouTube channel Microbiology teaching videos at NUI Galway (https://www.youtube.com/channel/UCsP4xz5aq7sWfR9eXSCd_QQ/), which accumulated over 40 000 views across 47 countries in its first 15 months online. A survey of students who used the videos in their teaching and learning identified a greatly increased understanding of experimental principles and ability to carry out techniques; greater engagement with practical teaching sessions; particular benefits for visual learners; and increased confidence in teaching and in communicating science amongst undergraduate teaching assistants. The videos will be central to microbiology teaching at NUI Galway over the coming decade and will benefit many third-level institutions exploring online and blended learning approaches in the coming years.

Nature-Based Biomaterials and Their Application in Biomedicine.

Natural polymers, based on proteins or polysaccharides, have attracted increasing interest in recent years due to their broad potential uses in biomedicine. The chemical stability, structural versatility, biocompatibility and high availability of these materials lend them to diverse applications in areas such as tissue engineering, drug delivery and wound healing. Biomaterials purified from animal or plant sources have also been engineered to improve their structural properties or promote interactions with surrounding cells and tissues for improved in vivo performance, leading to novel applications as implantable devices, in controlled drug release and as surface coatings. This review describes biomaterials derived from and inspired by natural proteins and polysaccharides and highlights their promise across diverse biomedical fields. We outline current therapeutic applications of these nature-based materials and consider expected future developments in identifying and utilising innovative biomaterials in new biomedical applications.

Engineering of a fungal beta-galactosidase to remove product inhibition by galactose.

Beta-galactosidase is an enzyme administered as a digestive supplement to treat lactose intolerance, a genetic condition prevalent in most world regions. The gene encoding an acid-stable beta-galactosidase potentially suited for use as a digestive supplement was cloned from Aspergillus niger van Tiegh, sequenced and expressed in Pichia pastoris. The purified recombinant protein exhibited kinetic properties similar to those of the native enzyme and thus was also competitively inhibited by its product, galactose, at application-relevant concentrations. In order to alleviate this product inhibition, a model of the enzyme structure was generated based on a Penicillium sp. beta-galactosidase crystal structure with bound beta-galactose. This led to targeted mutagenesis of an Asp(258)-Ser-Tyr-Pro-Leu-Gly-Phe amino acid motif in the A. niger van Tiegh enzyme and isolation from the resultant library of a mutant beta-galactosidase enzyme with reduced sensitivity to inhibition by galactose (K (i) of 6.46 mM galactose, compared with 0.76 mM for the wildtype recombinant enzyme). The mutated enzyme also exhibited an increased K (m) (3.76 mM compared to 2.21 mM) and reduced V (max) (110.8 micromol min(-1) mg(-1) compared to 172.6 micromol min(-1) mg(-1)) relative to the wild-type enzyme, however, and its stability under simulated fasting gastric conditions was significantly reduced. The study nevertheless demonstrates the potential to rationally engineer the A. niger van Tiegh enzyme to relieve product inhibition and create mutants with improved, application-relevant kinetic properties for treatment of lactose intolerance.

Insights into the mode of action of a putative zinc transporter CzrB in Thermus thermophilus.

The crystal structures of the cytoplasmic domain of the putative zinc transporter CzrB in the apo and zinc-bound forms reported herein are consistent with the protein functioning in vivo as a homodimer. NMR, X-ray scattering, and size-exclusion chromatography provide support for dimer formation. Full-length variants of CzrB in the apo and zinc-loaded states were generated by homology modeling with the Zn2+/H+ antiporter YiiP. The model suggests a way in which zinc binding to the cytoplasmic fragment creates a docking site to which a metallochaperone can bind for delivery and transport of its zinc cargo. Because the cytoplasmic domain may exist in the cell as an independent, soluble protein, a proposal is advanced that it functions as a metallochaperone and that it regulates the zinc-transporting activity of the full-length protein. The latter requires that zinc binding becomes uncoupled from the creation of a metallochaperone-docking site on CzrB.

An Engineered Pathway for Production of Terminally Sialylated N-glycoproteins in the Periplasm of Escherichia coli.

Terminally sialylated N-glycoproteins are of great interest in therapeutic applications. Due to the inability of prokaryotes to carry out this post-translational modification, they are currently predominantly produced in eukaryotic host cells. In this study, we report a synthetic pathway to produce a terminally sialylated N-glycoprotein in the periplasm of Escherichia coli, mimicking the sialylated moiety (Neu5Ac-α-2,6-Gal-ß-1,4-GlcNAc-) of human glycans. A sialylated pentasaccharide, Neu5Ac-α-2,6-Gal-ß-1,4-GlcNAc-ß-1,3-Gal-ß-1,3-GlcNAc-, was synthesized through the activity of co-expressed glycosyltransferases LsgCDEF from Haemophilus influenzae, Campylobacter jejuni NeuBCA enzymes, and Photobacterium leiognathi α-2,6-sialyltransferase in an engineered E. coli strain which produces CMP-Neu5Ac. C. jejuni oligosaccharyltransferase PglB was used to transfer the terminally sialylated glycan onto a glyco-recognition sequence in the tenth type III cell adhesion module of human fibronectin. Sialylation of the target protein was confirmed by lectin blotting and mass spectrometry. This proof-of-concept study demonstrates the successful production of terminally sialylated, homogeneous N-glycoproteins with α-2,6-linkages in the periplasm of E. coli and will facilitate the construction of E. coli strains capable of producing terminally sialylated N-glycoproteins in high yield.

Functionalization with a VEGFR2-binding antibody fragment leads to enhanced endothelialization of a cardiovascular stent in vitro and in vivo.

Rapid endothelialization of cardiovascular stents is critical to prevent major clinical complications such as restenosis. Reconstruction of the native endothelium on the stent surface can be achieved by the capture of endothelial progenitor cells (EPCs) or neighboring endothelial cells (ECs) in vivo. In this study, stainless steel cardiovascular stents were functionalized with recombinant scFv antibody fragments specific for vascular endothelial growth factor receptor-2 (VEGFR2) that is expressed on EPCs and ECs. Anti-VEGFR2 scFvs were expressed in glycosylated form in Escherichia coli and covalently attached to amine-functionalized, titania-coated steel disks and stents. ScFv-coated surfaces exhibited no detectable cytotoxicity to human ECs or erythrocytes in vitro and bound 15 times more VEGFR2-positive human umbilical vein ECs than controls after as little as 3 min. Porcine coronary arteries were successfully stented with scFv-coated stents with no adverse clinical events after 30 days. Endovascular imaging and histology revealed coverage of the anti-VEGFR2 scFv-coated stent with a cell layer after 5 days and the presence of a neointima layer with a minimum thickness of 80 µm after 30 days. Biofunctionalization of cardiovascular stents with endothelial cell-capturing antibody fragments in this manner offers promise in accelerating stent endothelialization in vivo. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:213-224, 2020.

Trigger factor from the psychrophilic bacterium Psychrobacter frigidicola is a monomeric chaperone.

In eubacteria, trigger factor (TF) is the first chaperone to interact with newly synthesized polypeptides and assist their folding as they emerge from the ribosome. We report the first characterization of a TF from a psychrophilic organism. TF from Psychrobacter frigidicola (TF(Pf)) was cloned, produced in Escherichia coli, and purified. Strikingly, cross-linking and fluorescence anisotropy analyses revealed it to exist in solution as a monomer, unlike the well-characterized, dimeric E. coli TF (TF(Ec)). Moreover, TF(Pf) did not exhibit the downturn in reactivation of unfolded GAPDH (glyceraldehyde-3-phosphate dehydrogenase) that is observed with its E. coli counterpart, even at high TF/GAPDH molar ratios and revealed dramatically reduced retardation of membrane translocation by a model recombinant protein compared to the E. coli chaperone. TF(Pf) was also significantly more effective than TF(Ec) at increasing the yield of soluble and functional recombinant protein in a cell-free protein synthesis system, indicating that it is not dependent on downstream systems for its chaperoning activity. We propose that TF(Pf) differs from TF(Ec) in its quaternary structure and chaperone activity, and we discuss the potential significance of these differences in its native environment.

Use of folding modulators to improve heterologous protein production in Escherichia coli.

Despite the fundamental importance of E. coli in the manufacture of a wide range of biotechnological and biomedical products, extensive process and/or target optimisation is routinely required in order to achieve functional yields in excess of low mg/l levels. Molecular chaperones and folding catalysts appear to present a panacea for problems of heterologous protein folding in the organism, due largely to their broad substrate range compared with, e.g., protein-specific mutagenesis approaches. Painstaking investigation of chaperone overproduction has, however, met with mixed - and largely unpredictable - results to date. The past 5 years have nevertheless seen an explosion in interest in exploiting the native folding modulators of E. coli, and particularly cocktails thereof, driven largely by the availability of plasmid systems that facilitate simultaneous, non-rational screening of multiple chaperones during recombinant protein expression. As interest in using E. coli to produce recombinant membrane proteins and even glycoproteins grows, approaches to reduce aggregation, delay host cell lysis and optimise expression of difficult-to-express recombinant proteins will become even more critical over the coming years. In this review, we critically evaluate the performance of molecular chaperones and folding catalysts native to E. coli in improving functional production of heterologous proteins in the bacterium and we discuss how they might best be exploited to provide increased amounts of correctly-folded, active protein for biochemical and biophysical studies.

The expression and characterization of recombinant cp19k barnacle cement protein from Pollicipes pollicipes.

Adhesive proteins of barnacle cement have potential as environmentally friendly adhesives owing to their ability to adhere to various substrates in aqueous environments. By understanding the taxonomic breath of barnacles with different lifestyles, we may uncover commonalities in adhesives produced by these specialized organisms. The 19 kDa cement protein (cp19k) of the stalked barnacle Pollicipes pollicipes was expressed in Escherichia coli BL21 to investigate its adhesive properties. Initial expression of hexahistidine-tagged protein (rPpolcp19k-his) yielded low levels of insoluble protein. Co-overproduction of E. coli molecular chaperones GroEL-GroES and trigger factor (TF) increased soluble protein yields, although TF co-purified with the target protein (TF-rPpolcp19k-his). Surface coat analysis revealed high levels of adsorption of the TF-rPpolcp19k-his complex and of purified E. coli TF on both hydrophobic and hydrophilic surfaces, while low levels of adsorption were observed for rPpolcp19k-his. Tag-free rPpolcp19k protein also exhibited low adsorption compared to fibrinogen and Cell-Tak controls on hydrophobic, neutral hydrophilic and charged self-assembled monolayers under surface plasmon resonance assay conditions designed to mimic the barnacle cement gland or seawater. Because rPpolcp19k protein displays low adhesive capability, this protein is suggested to confer the ability to self-assemble into a plaque within the barnacle cement complex. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

Crystallization and preliminary X-ray diffraction analysis of a soluble domain of the putative zinc transporter CzrB from Thermus thermophilus.

CzrB is a putative zinc transporter from Thermus thermophilus. The protein is proposed to consist of a hexahelical transmembrane domain with a cytosolic extramembranal C-terminus. The latter 92-residue fragment may be expressed free and may function independently of the full-length integral membrane protein. A 6xHis-tagged form of the water-soluble fragment has been overexpressed in Escherichia coli and diffraction-quality crystals of the tagged and tag-free variants have been grown. Preliminary X-ray analyses of tag-free fragment crystals with (2.2 A resolution) and without zinc ions (1.7 A resolution) reveal that the former has at least two zinc ions bound per monomer.

Adsorption and activity of a domoic acid binding antibody fragment on mesoporous silicates.

The adsorption of an anti-domoic acid single-chain Fv (scFv) antibody fragment onto a range of mesoporous silicate supports was investigated. The scFv fragment adsorbed to all materials investigated, and pI had an apparently large effect on coating, with the greatest-and fastest-adsorption found on the most negatively charged silicates. Maximal coating levels attainable did not reflect the pore diameters of the materials. The immobilized antibody was functional on all materials and bound its antigen, a naturally occurring neurotoxin produced by shellfish, in a rapidly saturating manner that suggested the antibody adsorbed in a multilayer on the mesoporous particles. The antigen:antibody ratio decreased from 1:1.3 to <1:10 with increasing concentration of immobilized antibody, and the immobilized scFv exhibited no detectable reduction in domoic acid binding over a 42-day incubation period.

Poly(ethylene glycol)-Based Hyperbranched Polymer from RAFT and Its Application as a Silver-Sulfadiazine-Loaded Antibacterial Hydrogel in Wound Care.

A multifunctional branched copolymer was synthesized by Reversible Addition-Fragmentation Chain Transfer polymerization (RAFT) of poly(ethylene glycol) diacrylate (PEGDA Mn = 575) and poly(ethylene glycol) methyl methacrylate (PEGMEMA Mn = 500) at a feed molar ratio of 50:50. Proton nuclear magnetic resonance spectroscopy (1H NMR) confirmed a hyperbranched molecular structure and a high degree of vinyl functionality. An in situ cross-linkable hydrogel system was generated via a "click" thiol-ene-type Michael addition reaction of vinyl functional groups from this PEGDA/PEGMEMA copolymer system in combination with thiol-modified hyaluronic acid. Furthermore, encapsulation of antimicrobial silver sulfadiazine (SSD) into the copolymer system was conducted to create an advanced antimicrobial wound care dressing. This hydrogel demonstrated a sustained antibacterial activity against the bacterial strains Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli in comparison to the direct topical application of SSD. In addition, in vitro toxicology evaluations demonstrated that this hydrogel-with low concentrations of encapsulated SSD-supported the survival of embedded human adipose derived stem cells (hADSCs) and inhibited growth of the aforementioned pathogens. Here we demonstrate that this hydrogel encapsulated with a low concentration (1.0% w/v) of SSD can be utilized as a carrier system for stem cells with the ability to inhibit growth of pathogens and without adverse effects on hADSCs.