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Pharmaceutics
Chronic wound infections colonized by bacteria are becoming more difficult to treat with current ... more Chronic wound infections colonized by bacteria are becoming more difficult to treat with current antibiotics due to the development of antimicrobial resistance (AMR) as well as biofilm and persister cell formation. Synthetic antibacterial and antibiofilm peptide (SAAP)-148 is an excellent alternative for treatment of such infections but suffers from limitations related to its cationic peptidic nature and thus instability and possible cytotoxicity, resulting in a narrow therapeutic window. Here, we evaluated SAAP-148 encapsulation in nanogels composed of octenyl succinic anhydride (OSA)-modified hyaluronic acid (HA) to circumvent these limitations. SAAP-148 was efficiently (>98%) encapsulated with high drug loading (23%), resulting in monodispersed anionic OSA-HA nanogels with sizes ranging 204–253 nm. Nanogel lyophilization in presence of polyvinyl alcohol maintained their sizes and morphology. SAAP-148 was sustainedly released from lyophilized nanogels (37–41% in 72 h) upon reco...
Antibiotics, May 17, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Cancer Research
Vaccines based on synthetic long peptides, targeting immunogenic region(s) of tumor associated pr... more Vaccines based on synthetic long peptides, targeting immunogenic region(s) of tumor associated proteins, are becoming increasingly used in oncology. Synthetic long peptides require endogenous processing by antigen presenting cells to be presented to T cells, thus reducing the risk of antigen presentation by non-professional antigen presenting cells lacking co-stimulation receptors. The formulation and delivery strategies require further development as peptides degrade rapidly in vivo. Herein we make use of a novel targeting and adjuvant strategy to deliver peptides to antigen presenting cells. Tumor-derived peptides are coupled to a B cell epitope element (the MTTE sequence, a tetanus toxin-derived universal B cell epitope) which induce the formation of peptide-conjugate -complex formation in vivo. This strategy, further named the TET technology, makes use of pre-existing antibodies to improve the bioavailability, stability, and immunogenicity of tumor-derived peptide therapeutics. ...
Kidney injury is a complication frequently encountered in hospitalized patients. Early detection ... more Kidney injury is a complication frequently encountered in hospitalized patients. Early detection of kidney injury prior to loss of renal function is an unmet clinical need that should be targeted by a protein-based biomarker panel. In this study, we aim to quantitate urinary kidney injury biomarkers at the picomolar to nanomolar level by liquid chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode (LC-MRM-MS). Proteins were immunocaptured from urinary samples, denatured, reduced, alkylated, and digested into peptides before LC-MRM-MS analysis. Stable-isotope-labeled peptides functioned as internal standards, and biomarker concentrations were attained by an external calibration strategy. The method was evaluated for selectivity, carryover, matrix effects, linearity, and imprecision. The LC-MRM-MS method enabled the quantitation of KIM-1, NGAL, TIMP2, IGFBP7, CXCL9, nephrin, and SLC22A2 and the detection of TGF-β1, cubilin, and uromodulin. Two to three peptides were included per protein, and three transitions were monitored per peptide for analytical selectivity. The analytical carryover was <1%, and minimal urine matrix effects were observed by combining immunocapture and targeted LC-MRM-MS analysis. The average total CV of all quantifier peptides was 26%. The linear measurement range was determined per measurand and found to be 0.05−30 nmol/L. The targeted MS-based method enables the multiplex quantitation of low-abundance urinary kidney injury biomarkers for future clinical evaluation.
Analytical Chemistry, 2020
Comprehensive determination of primary sequence and identification of post-translational modifica... more Comprehensive determination of primary sequence and identification of post-translational modifications (PTMs) are key elements in protein structural analysis. Various mass spectrometry (MS) based fragmentation techniques are powerful approaches for mapping both the amino acid sequence and PTMs; one of these techniques is matrix-assisted laser desorption/ ionization (MALDI), combined with in-source decay (ISD) fragmentation and Fourier-transform ion cyclotron resonance (FT-ICR) MS. MALDI-ISD MS protein analysis involves only minimal sample preparation and does not require spectral deconvolution. The resulting MALDI-ISD MS data is complementary to electrospray ionization-based MS/MS sequencing readouts, providing knowledge on the types of fragment ions is available. In this study, we evaluate the isotopic distributions of z′ ions in protein top-down MALDI-ISD FT-ICR mass spectra and show why these distributions can deviate from theoretical profiles as a result of co-occurring and isomeric z and y-NH 3 ions. Two synthetic peptides, containing either normal or deuterated alanine residues, were used to confirm the presence and unravel the identity of isomeric z and y-NH 3 fragment ions ("twins"). Furthermore, two reducing MALDI matrices, namely 1,5diaminonaphthalene and N-phenyl-p-phenylenediamine were applied that yield ISD mass spectra with different fragment ion distributions. This study demonstrates that the relative abundance of isomeric z and y-NH 3 ions requires consideration for accurate and confident assignments of z′ ions in MALDI-ISD FT-ICR mass spectra.
Antibiotics, May 17, 2022
Recently, using a deep learning approach, the novel antibiotic halicin was discovered. We compare... more Recently, using a deep learning approach, the novel antibiotic halicin was discovered. We compared the antibacterial activities of two novel bactericidal antimicrobial agents, i.e., the synthetic antibacterial and antibiofilm peptide (SAAP)-148 with this antibiotic halicin. Results revealed that SAAP-148 was more effective than halicin in killing planktonic bacteria of antimicrobial-resistant (AMR) Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus, especially in biologically relevant media, such as plasma and urine, and in 3D human infection models. Surprisingly, SAAP-148 and halicin were equally effective against these bacteria residing in immature and mature biofilms. As their modes of action differ, potential favorable interactions between SAAP-148 and halicin were investigated. For some specific strains of AMR E. coli and S. aureus synergism between these agents was observed, whereas for other strains, additive interactions were noted. These favorable interactions were confirmed for AMR E. coli in a 3D human bladder infection model and AMR S. aureus in a 3D human epidermal infection model. Together, combinations of these two novel antimicrobial agents hold promise as an innovative treatment for infections not effectively treatable with current antibiotics.
Pharmaceutics
Chronic wound infections colonized by bacteria are becoming more difficult to treat with current ... more Chronic wound infections colonized by bacteria are becoming more difficult to treat with current antibiotics due to the development of antimicrobial resistance (AMR) as well as biofilm and persister cell formation. Synthetic antibacterial and antibiofilm peptide (SAAP)-148 is an excellent alternative for treatment of such infections but suffers from limitations related to its cationic peptidic nature and thus instability and possible cytotoxicity, resulting in a narrow therapeutic window. Here, we evaluated SAAP-148 encapsulation in nanogels composed of octenyl succinic anhydride (OSA)-modified hyaluronic acid (HA) to circumvent these limitations. SAAP-148 was efficiently (>98%) encapsulated with high drug loading (23%), resulting in monodispersed anionic OSA-HA nanogels with sizes ranging 204–253 nm. Nanogel lyophilization in presence of polyvinyl alcohol maintained their sizes and morphology. SAAP-148 was sustainedly released from lyophilized nanogels (37–41% in 72 h) upon reco...
Antibiotics, May 17, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Cancer Research
Vaccines based on synthetic long peptides, targeting immunogenic region(s) of tumor associated pr... more Vaccines based on synthetic long peptides, targeting immunogenic region(s) of tumor associated proteins, are becoming increasingly used in oncology. Synthetic long peptides require endogenous processing by antigen presenting cells to be presented to T cells, thus reducing the risk of antigen presentation by non-professional antigen presenting cells lacking co-stimulation receptors. The formulation and delivery strategies require further development as peptides degrade rapidly in vivo. Herein we make use of a novel targeting and adjuvant strategy to deliver peptides to antigen presenting cells. Tumor-derived peptides are coupled to a B cell epitope element (the MTTE sequence, a tetanus toxin-derived universal B cell epitope) which induce the formation of peptide-conjugate -complex formation in vivo. This strategy, further named the TET technology, makes use of pre-existing antibodies to improve the bioavailability, stability, and immunogenicity of tumor-derived peptide therapeutics. ...
Kidney injury is a complication frequently encountered in hospitalized patients. Early detection ... more Kidney injury is a complication frequently encountered in hospitalized patients. Early detection of kidney injury prior to loss of renal function is an unmet clinical need that should be targeted by a protein-based biomarker panel. In this study, we aim to quantitate urinary kidney injury biomarkers at the picomolar to nanomolar level by liquid chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode (LC-MRM-MS). Proteins were immunocaptured from urinary samples, denatured, reduced, alkylated, and digested into peptides before LC-MRM-MS analysis. Stable-isotope-labeled peptides functioned as internal standards, and biomarker concentrations were attained by an external calibration strategy. The method was evaluated for selectivity, carryover, matrix effects, linearity, and imprecision. The LC-MRM-MS method enabled the quantitation of KIM-1, NGAL, TIMP2, IGFBP7, CXCL9, nephrin, and SLC22A2 and the detection of TGF-β1, cubilin, and uromodulin. Two to three peptides were included per protein, and three transitions were monitored per peptide for analytical selectivity. The analytical carryover was <1%, and minimal urine matrix effects were observed by combining immunocapture and targeted LC-MRM-MS analysis. The average total CV of all quantifier peptides was 26%. The linear measurement range was determined per measurand and found to be 0.05−30 nmol/L. The targeted MS-based method enables the multiplex quantitation of low-abundance urinary kidney injury biomarkers for future clinical evaluation.
Analytical Chemistry, 2020
Comprehensive determination of primary sequence and identification of post-translational modifica... more Comprehensive determination of primary sequence and identification of post-translational modifications (PTMs) are key elements in protein structural analysis. Various mass spectrometry (MS) based fragmentation techniques are powerful approaches for mapping both the amino acid sequence and PTMs; one of these techniques is matrix-assisted laser desorption/ ionization (MALDI), combined with in-source decay (ISD) fragmentation and Fourier-transform ion cyclotron resonance (FT-ICR) MS. MALDI-ISD MS protein analysis involves only minimal sample preparation and does not require spectral deconvolution. The resulting MALDI-ISD MS data is complementary to electrospray ionization-based MS/MS sequencing readouts, providing knowledge on the types of fragment ions is available. In this study, we evaluate the isotopic distributions of z′ ions in protein top-down MALDI-ISD FT-ICR mass spectra and show why these distributions can deviate from theoretical profiles as a result of co-occurring and isomeric z and y-NH 3 ions. Two synthetic peptides, containing either normal or deuterated alanine residues, were used to confirm the presence and unravel the identity of isomeric z and y-NH 3 fragment ions ("twins"). Furthermore, two reducing MALDI matrices, namely 1,5diaminonaphthalene and N-phenyl-p-phenylenediamine were applied that yield ISD mass spectra with different fragment ion distributions. This study demonstrates that the relative abundance of isomeric z and y-NH 3 ions requires consideration for accurate and confident assignments of z′ ions in MALDI-ISD FT-ICR mass spectra.
Antibiotics, May 17, 2022
Recently, using a deep learning approach, the novel antibiotic halicin was discovered. We compare... more Recently, using a deep learning approach, the novel antibiotic halicin was discovered. We compared the antibacterial activities of two novel bactericidal antimicrobial agents, i.e., the synthetic antibacterial and antibiofilm peptide (SAAP)-148 with this antibiotic halicin. Results revealed that SAAP-148 was more effective than halicin in killing planktonic bacteria of antimicrobial-resistant (AMR) Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus, especially in biologically relevant media, such as plasma and urine, and in 3D human infection models. Surprisingly, SAAP-148 and halicin were equally effective against these bacteria residing in immature and mature biofilms. As their modes of action differ, potential favorable interactions between SAAP-148 and halicin were investigated. For some specific strains of AMR E. coli and S. aureus synergism between these agents was observed, whereas for other strains, additive interactions were noted. These favorable interactions were confirmed for AMR E. coli in a 3D human bladder infection model and AMR S. aureus in a 3D human epidermal infection model. Together, combinations of these two novel antimicrobial agents hold promise as an innovative treatment for infections not effectively treatable with current antibiotics.