Detection of Peptide-Based Nanoparticles in Blood Plasma by ELISA

Aims The aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma. Materials & Methods A convenient enzyme linked immunosorbent assay (ELISA) was developed for the detection of peptides functionalized with biotin and fluorescein groups. As a proof of principle, polymerized pentafluorophenyl methacrylate nanoparticles linked to biotin-carboxyfluorescein labeled peptides were intravenously injected in Wistar rats. Serial blood plasma samples were analyzed by ELISA and by liquid chromatography mass spectrometry (LC/MS) technology. Results The ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS. Conclusions We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions

Detection of peptide-based nanoparticles in blood plasma by ELISA

Aims The aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma. Materials & Methods A convenient enzyme linked immunosorbent assay (ELISA) was developed for the detection of peptides functionalized with biotin and fluorescein groups. As a proof of principle, polymerized pentafluorophenyl methacrylate nanoparticles linked to biotin-carboxyfluorescein labeled peptides were intravenously injected in Wistar rats. Serial blood plasma samples were analyzed by ELISA and by liquid chromatography mass spectrometry (LC/MS) technology. Results The ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL.We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS. Conclusions We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions

Dermal PK/PD of a lipophilic topical drug in psoriatic patients by continuous intradermal membrane-free sampling

Background: Methodologies for continuous sampling of lipophilic drugs and high-molecular solutes in the dermis are currently lacking. We investigated the feasibility of sampling a lipophilic topical drug and the locally released biomarker in the dermis of non-lesional and lesional skin of psoriatic patients over 25hrs by means of membrane-free dermal Open-Flow Microperfusion probes (dOFM) and novel wearable multi-channel pumps. Methods: Nine psoriatic patients received a topical p-38 inhibitor (BCT194, 0.5% cream) on a lesional and a non-lesional application site once daily for 8 days. Multiple dOFM sampling was performed for 25 hours from each site on day 1 and day 8. Patients were mobile as dOFM probes were operated by a novel light-weight Push-Pull pump. Ultrasound was used to verify intradermal probe placement, cap-LC-MS/MS for BCT194 and ELISA for TNFα analysis. Results: dOFM was well tolerated and demonstrated significant drug concentrations in lesional as well as non-lesional skin after eight days but did not show significant differences between tissues. On day 8 TNFα release following probe insertion was significantly reduced compared to day 1. Conclusions: Novel membrane-free probes and wearable multi-channel pumps allowed prolonged intradermal PK/PD profiling of a lipophilic topical drug in psoriatic patients. This initial study shows that dOFM overcomes limitations of microdialysis sampling methodology and it demonstrates the potential for PK/PD studies of topical products and formulations in a clinical setting

LC/MS chromatograms comparing different concentrations of peptide 5A in blood plasma matrix.

<p>Peptide 5A peaks in plasma matrix spiked with 0, 13.6 and 45.2 ng/mL (SRM trace: 877.7 → (412.2 + 341.1 + 946.4).</p

Schematic representation of the nanoparticle and ELISA design.

<p>Peptide 5A contains biotin and carboxyfluorescein, lower case letters denote D-amino acids. Nanoparticles were synthesized by a free-radical polymerization method in a microemulsion system. N-isopropylacrylamide, N,N-dimethylacrylamide and acrylic acid were used as monomers with methylenebisacrylamide as cross-linker. Pentafluorophenyl methacrylate (PFM) was added and the nanoparticles were adorned with peptide 5A (B). An ELISA was designed to detect nanoparticles in biological fluids using the biotin and carboxyfluorescein groups present on peptide 5A (C). Streptavidin coated plates were used to capture the peptides by binding the biotin group and bound peptides were detected with a HRP conjugated mAb anti-fluorescein.</p

An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake

Nanobiopharmaceutic

Systemic Inflammation and Normocytic Anemia in DOCK11 Deficiency

[BACKGROUND] Increasing evidence links genetic defects affecting actin-regulatory proteins to diseases with severe autoimmunity and autoinflammation, yet the underlying molecular mechanisms are poorly understood. Dedicator of cytokinesis 11 (DOCK11) activates the small Rho guanosine triphosphatase (GTPase) cell division cycle 42 (CDC42), a central regulator of actin cytoskeleton dynamics. The role of DOCK11 in human immune-cell function and disease remains unknown.[METHODS] We conducted genetic, immunologic, and molecular assays in four patients from four unrelated families who presented with infections, early-onset severe immune dysregulation, normocytic anemia of variable severity associated with anisopoikilocytosis, and developmental delay. Functional assays were performed in patient-derived cells, as well as in mouse and zebrafish models.[RESULTS] We identified rare, X-linked germline mutations in DOCK11 in the patients, leading to a loss of protein expression in two patients and impaired CDC42 activation in all four patients. Patient-derived T cells did not form filopodia and showed abnormal migration. In addition, the patient-derived T cells, as well as the T cells from Dock11-knockout mice, showed overt activation and production of proinflammatory cytokines that were associated with an increased degree of nuclear translocation of nuclear factor of activated T cell 1 (NFATc1). Anemia and aberrant erythrocyte morphologic features were recapitulated in a newly generated dock11-knockout zebrafish model, and anemia was amenable to rescue on ectopic expression of constitutively active CDC42.[CONCLUSIONS] Germline hemizygous loss-of-function mutations affecting the actin regulator DOCK11 were shown to cause a previously unknown inborn error of hematopoiesis and immunity characterized by severe immune dysregulation and systemic inflammation, recurrent infections, and anemia. (Funded by the European Research Council and others.)Supported by the European Research Council under the European Union’s Horizon 2020 Research and Innovation Program (820074, to Dr. Boztug), the Vienna Science and Technology Fund (WWTF-LS16-060, to Drs. Boztug, Dupré, and Menche; WWTFLS14-031, to Drs. Platzer and Huppa), the Deutsche Forschungsgemeinschaft (KU 1240/13-1, to Dr. Kutsche), the Doctoral Fellowship Program of the Austrian Academy of Sciences (25590, to Ms. Block; 24486, to Dr. Ardy), grants from the Centre National de la Recherche Scientifique (International Research Project program, SysTact project, to Dr. Dupré) and Programa Institucional de Internacionalização da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES–PrInt) (to Ms. Chaves), the Wellcome Trust (207556/Z/17/Z, to Dr. Hambleton), the Netherlands Organization for Health Research and Development and the Dutch Research Council (ZonMW NWO Vici grant 91819632, to Drs. van Buul and Schoppmeyer), the Austrian Research Promotion Agency (project 7940628, Danio4Can, to Dr. Distel), a German Academic Exchange Service postdoctoral fellowship and a European Molecular Biology Organization fellowship (to Dr. Distel), and the Research Funding for Longevity Sciences from the National Center for Geriatrics and Gerontology (21-27-2, to Dr. Nishikimi). This study makes use of data shared through the PhenomeCentral repository, funded by Genome Canada and the Canadian Institute of Health Research.Peer reviewe