Tsvetelina Baryakova

All Publications

• Novel Approaches to Label the Surface of S. aureus with DBCO for Click Chemistry-Mediated Deposition of Sensitive Cargo. Bioconjugate chemistry Baryakova, T. H., Hsu, C. C., Segatori, L., McHugh, K. J. 2025; 36 (6): 1157-1168

  Abstract

  The strain-promoted alkyne-azide cycloaddition (SPAAC) reaction can be used to modify the surface of bacteria for a variety of applications including drug delivery, biosensing, and imaging. This is usually accomplished by first installing a small azide group within the peptidoglycan and then delivering exogenous cargo (e.g., a protein or nanoparticle) modified with a cyclooctyne group, such as dibenzocyclooctyne (DBCO), for in situ conjugation. However, DBCO is comparatively bulky and hydrophobic, increasing the propensity of some payloads to aggregate. In this study, we sought to invert this paradigm by exploring two novel strategies for incorporating DBCO into the peptidoglycan of Staphylococcus aureus and compared them to an established approach using DBCO-vancomycin. We demonstrate that DBCO-modified small molecules belonging to all three classes─a sortase peptide substrate (LPETG), two d-alanine derivatives, and vancomycin─can selectively label the S. aureus surface to varying degrees. In contrast to DBCO-vancomycin, the DBCO-d-alanine variants do not adversely affect the growth of S. aureus or lead to off-target labeling or toxicity in HEK293T or RAW 264.7 cells. Finally, we show that, unlike IgG3-Fc labeled with DBCO groups, IgG3-Fc labeled with azide groups is stable (i.e., remains water-soluble) under normal storage conditions, retains its ability to bind the immune receptor CD64, and can be successfully attached to the surface of DBCO-modified S. aureus. We believe that the labeling strategies explored herein will expand the paradigm of specific, nontoxic SPAAC-mediated labeling of the surface of S. aureus and other Gram-positive bacteria, opening the door for new applications using azide-modified cargo.

  View details for DOI 10.1021/acs.bioconjchem.4c00575

  View details for PubMedID 40398634

• A TLR7 Agonist Conjugated to a Nanofibrous Peptide Hydrogel as a Potent Vaccine Adjuvant ADVANCED HEALTHCARE MATERIALS Euliano, E. M., Pogostin, B. H., Agrawal, A., Yu, M. H., Baryakova, T. H., Graf, T. P., Kunkel, A. A., Cahue, K. A., Hartgerink, J. D., McHugh, K. J. 2025; 14 (3): e2402958

  Abstract

  Toll-like receptors (TLRs) recognize pathogen- and damage-associated molecular patterns and, in turn, trigger the release of cytokines and other immunostimulatory molecules. As a result, TLR agonists are increasingly being investigated as vaccine adjuvants. Many of these agonists are small molecules that quickly diffuse away from the vaccination site, limiting their co-localization with antigens and, thus, their effect. Here, the small-molecule TLR7 agonist 1V209 is conjugated to a positively-charged multidomain peptide (MDP) hydrogel, K2, which was previously shown to act as an adjuvant promoting humoral immunity. Mixing the 1V209-conjugated K2 50:50 with the unfunctionalized K2 produces hydrogels that retain the shear-thinning and self-healing physical properties of the original MDP while improving the solubility of 1V209 more than 200-fold compared to the unconjugated molecule. When co-delivered with ovalbumin as a model antigen, 1V209-functionalized K2 produces a robust Th2 immune response and an antigen-specific Th1 immune response superior to alum, a widely used vaccine adjuvant. Together, these results suggest that K2 MDP hydrogels functionalized with 1V209 are a promising adjuvant for vaccines against infectious diseases, especially those benefiting from a combined Th1 and Th2 immune response.

  View details for DOI 10.1002/adhm.202402958

  View details for Web of Science ID 001341200200001

  View details for PubMedID 39460390

  View details for PubMedCentralID PMC11774675

• Intra-lymph node crosslinking of antigen-bearing polymers enhances humoral immunity and dendritic cell activation BIOENGINEERING & TRANSLATIONAL MEDICINE Euliano, E. M., Agrawal, A., Yu, M. H., Graf, T. P., Henrich, E. M., Kunkel, A. A., Hsu, C., Baryakova, T., McHugh, K. J. 2024; 9 (6): e10705

  Abstract

  Lymph node (LN)-resident dendritic cells (DCs) are a promising target for vaccination given their professional antigen-presenting capabilities and proximity to a high concentration of immune cells. Direct intra-LN injection has been shown to greatly enhance the immune response to vaccine antigens compared to traditional intramuscular injection, but it is infeasible to implement clinically in a vaccination campaign context. Employing the passive lymphatic flow of antigens to target LNs has been shown to increase total antigen uptake by DCs more than inflammatory adjuvants, which recruit peripheral DCs. Herein, we describe a novel vaccination platform in which two complementary multi-arm poly(ethylene glycol) (PEG) polymers-one covalently bound to the model antigen ovalbumin (OVA)-are injected subcutaneously into two distinct sites. These materials then drain to the same LN through different lymphatic vessels and, upon meeting in the LN, rapidly crosslink. This system improves OVA delivery to, and residence time within, the draining LN compared to all control groups. The crosslinking of the two PEG components also improves humoral immunity without the need for any pathogen-mimicking adjuvants. Further, we observed a significant increase in non-B/T lymphocytes in LNs cross-presenting the OVA peptide SIINFEKL on MHC I over a dose-matched control containing alum, the most common clinical adjuvant, as well as an increase in DC activation in the LN. These data suggest that this platform can be used to deliver antigens to LN-resident immune cells to produce a stronger humoral and cellular immune response over materials-matched controls without the use of traditional adjuvants.

  View details for DOI 10.1002/btm2.10705

  View details for Web of Science ID 001268826900001

  View details for PubMedID 39545089

  View details for PubMedCentralID PMC11558197

• A Scalable Platform for Fabricating Biodegradable Microparticles with Pulsatile Drug Release. Advanced materials (Deerfield Beach, Fla.) Graf, T. P., Qiu, S. Y., Varshney, D., Laracuente, M. L., Euliano, E. M., Munnangi, P., Pogostin, B. H., Baryakova, T., Garyali, A., McHugh, K. J. 2023; 35 (22): e2300228

  Abstract

  Pulsatile drug delivery systems have the potential to improve patient adherence and therapeutic efficacy by providing a sequence of doses in a single injection. Herein, a novel platform, termed Particles Uniformly Liquified and Sealed to Encapsulate Drugs (PULSED) is developed, which enables the high-throughput fabrication of microparticles exhibiting pulsatile release. In PULSED, biodegradable polymeric microstructures with an open cavity are formed using high-resolution 3D printing and soft lithography, filled with drug, and sealed using a contactless heating step in which the polymer flows over the orifice to form a complete shell around a drug-loaded core. Poly(lactic-co-glycolic acid) particles with this structure can rapidly release encapsulated material after delays of 10 ± 1, 15 ± 1, 17 ± 2, or 36 ± 1 days in vivo, depending on polymer molecular weight and end group. The system is even compatible with biologics, releasing over 90% of bevacizumab in its bioactive form after a two-week delay in vitro. The PULSED system is highly versatile, offering compatibility with crystalline and amorphous polymers, easily injectable particle sizes, and compatibility with several newly developed drug loading methods. Together, these results suggest that PULSED is a promising platform for creating long-acting drug formulations that improve patient outcomes due to its simplicity, low cost, and scalability.

  View details for DOI 10.1002/adma.202300228

  View details for PubMedID 36862114

  View details for PubMedCentralID PMC10247432

• Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen. Journal of visualized experiments : JoVE Graf, T. P., Kadasia, K., Melhorn, S., Kessler, E., Yang, H., Baryakova, T., Brady, S., McHugh, K. J. 2023

  Abstract

  The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately burdensome to those living in low- and middle-income countries (LMICs), where the majority of deadly exposures to rabies occur. Different drug delivery strategies have been explored to condense vaccine regimens to a single injection by encapsulating antigens into polymeric particles. However, harsh stressors during the encapsulation process can cause denaturation of the encapsulated antigen. This article describes a method for encapsulating the rabies virus (RABV) antigen into polymeric microparticles that exhibit tunable pulsatile release. This method, termed Particles Uniformly Liquified and Sealed to Encapsulate Drugs (PULSED), generates microparticles using soft lithography to create inverse polydimethylsiloxane (PDMS) molds from a multi-photon, 3D-printed master mold. Poly(lactic-co-glycolic acid) (PLGA) films are then compression-molded into the PDMS molds to generate open-faced cylinders that are filled with concentrated RABV using a piezoelectric dispensing robot. These microstructures are then sealed by heating the top of the particles, allowing the material to flow and form a continuous, nonporous polymeric barrier. Post-fabrication, an enzyme-linked immunosorbent assay (ELISA) specific to the detection of intact trimeric rabies virus glycoprotein is used to confirm the high recovery of immunogenic antigen from the microparticles.

  View details for DOI 10.3791/65147

  View details for PubMedID 37246855

• Overcoming barriers to patient adherence: the case for developing innovative drug delivery systems NATURE REVIEWS DRUG DISCOVERY Baryakova, T. H., Pogostin, B. H., Langer, R., McHugh, K. J. 2023; 22 (5): 387-409

  Abstract

  Poor medication adherence is a pervasive issue with considerable health and socioeconomic consequences. Although the underlying reasons are generally understood, traditional intervention strategies rooted in patient-centric education and empowerment have proved to be prohibitively complex and/or ineffective. Formulating a pharmaceutical in a drug delivery system (DDS) is a promising alternative that can directly mitigate many common impediments to adherence, including frequent dosing, adverse effects and a delayed onset of action. Existing DDSs have already positively influenced patient acceptability and improved rates of adherence across various disease and intervention types. The next generation of systems have the potential to instate an even more radical paradigm shift by, for example, permitting oral delivery of biomacromolecules, allowing for autonomous dose regulation and enabling several doses to be mimicked with a single administration. Their success, however, is contingent on their ability to address the problems that have made DDSs unsuccessful in the past.

  View details for DOI 10.1038/s41573-023-00670-0

  View details for Web of Science ID 000956753600001

  View details for PubMedID 36973491

  View details for PubMedCentralID PMC10041531

• Computationally Aided Discovery of LysEFm5 Variants with Improved Catalytic Activity and Stability. Applied and environmental microbiology Baryakova, T. H., Ritter, S. C., Tresnak, D. T., Hackel, B. J. 2020; 86 (4)

  Abstract

  Bacteriophage-derived lysin proteins are potentially effective antimicrobials that would benefit from engineered improvements to their bioavailability and specific activity. Here, the catalytic domain of LysEFm5, a lysin with activity against vancomycin-resistant Enterococcus faecium (VRE), was subjected to site-saturation mutagenesis at positions whose selection was guided by sequence and structural information from homologous proteins. A second-order Potts model with parameters inferred from large sets of homologous sequence information was used to predict the average change in the statistical fitness for mutant libraries with diversity at pairs of sites within the secondary catalytic shell. Guided by the statistical fitness, nine double mutant saturation libraries were created and plated on agar containing autoclaved VRE to quickly identify and segregate catalytically active (halo-forming) and inactive (non-halo-forming) variants. High-throughput DNA sequencing of 873 unique variants showed that the statistical fitness was predictive of the retention or loss of catalytic activity (area under the curve [AUC], 0.840 to 0.894), with the inclusion of more diverse sequences in the starting multiple-sequence alignment improving the classification accuracy when pairwise amino acid couplings (epistasis) were considered. Of eight random halo-forming variants selected for more sensitive testing, one showed a 1.8 (±0.4)-fold improvement in specific activity and an 11.5 ± 0.8°C increase in melting temperature compared to those of the wild type. Our results demonstrate that a computationally informed approach employing homologous protein information coupled with a mid-throughput screening assay allows for the expedited discovery of lysin variants with improved properties.IMPORTANCE Broad-spectrum antibiotics can indiscriminately kill most bacteria, including commensal species that are a part of the normal human flora. This can potentially lead to the proliferation of drug-resistant bacteria upon elimination of competing species and to unwanted autoimmune effects in patients. Bacteriophage-derived lysin proteins are an alternative to conventional antibiotics that have coevolved alongside specific bacterial hosts. Lysins are capable of targeting conserved substrates in the bacterial cell wall essential for its viability. To engineer these proteins to exhibit improved therapeutically relevant properties, homology-guided statistical approaches can be used to identify compelling sites for mutation and to quantify the functional constraints acting on these sites to direct mutagenic library creation. The platform described herein couples this informed approach with a visual plate assay that can be used to simultaneously screen hundreds of mutants for catalytic activity, allowing for the streamlined identification of improved lysin variants.

  View details for DOI 10.1128/AEM.02051-19

  View details for PubMedID 31811034

  View details for PubMedCentralID PMC6997734