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AIMS: To evaluate feasibility of intradermal (i.d.) adalimumab administration using hollow microneedles, and to compare a single i.d. dose of adalimumab using a hollow microneedle with a single subcutaneous (s.c.) dose using a conventional needle. METHODS: In this single-centre double-blind, placebo-controlled, double-dummy clinical trial in 24 healthy adults we compared 40 mg adalimumab (0.4 mL) administered i.d. using a hollow microneedle with a s.c. dose using a conventional needle. Primary parameters were pain, acceptability and local tolerability; secondary parameters safety, pharmacokinetics and immunogenicity. We explored usability of optical coherence tomography, clinical photography, thermal imaging, and laser speckle contrast imaging to evaluate skin reaction after i.d. injections. In vitro protein analysis was performed to assess compatibility of adalimumab with the hollow microneedle device. RESULTS: While feasible and safe, injection pain of i.d. adalimumab was higher compared to s.c. adalimumab (35.4 vs. 7.9 on a 100-point visual analogue scale). Initial absorption rate and relative bioavailability were higher after i.d. adalimumab (time to maximum plasma concentration = 95 h [47-120]; Frel = 129% [6.46%]) compared to s.c. adalimumab (time to maximum plasma concentration = 120 h [96-221]). Anti-adalimumab antibodies were detected in 50% and 83% of the subjects after i.d. and s.c. adalimumab, respectively. We observed statistically significantly more erythema and skin perfusion after i.d. adalimumab, compared to s.c. adalimumab and placebo injections (P < .0001). Cytokine secretion after whole blood lipopolysaccharide challenge was comparable between administration routes. CONCLUSIONS: Intradermal injection of adalimumab using hollowing microneedles was perceived as more painful and less accepted than s.c. administration, but yields a higher relative bioavailability with similar safety and pharmacodynamic effects.
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Agujas , Piel , Adalimumab , Adulto , Humanos , Inyecciones Intradérmicas , Inyecciones Subcutáneas , Dimensión del DolorRESUMEN
In this study, we developed a microfluidics method, using a so-called H-cell microfluidics device, for the determination of protein diffusion coefficients at different concentrations, pHs, ionic strengths, and solvent viscosities. Protein transfer takes place in the H-cell channels between two laminarly flowing streams with each containing a different initial protein concentration. The protein diffusion coefficients are calculated based on the measured protein mass transfer, the channel dimensions, and the contact time between the two streams. The diffusion rates of lysozyme, cytochrome c, myoglobin, ovalbumin, bovine serum albumin, and etanercept were investigated. The accuracy of the presented methodology was demonstrated by comparing the measured diffusion coefficients with literature values measured under similar solvent conditions using other techniques. At low pH and ionic strength, the measured lysozyme diffusion coefficient increased with the protein concentration gradient, suggesting stronger and more frequent intermolecular interactions. At comparable concentration gradients, the measured lysozyme diffusion coefficient decreased drastically as a function of increasing ionic strength (from zero onwards) and increasing medium viscosity. Additionally, a particle tracing numerical simulation was performed to achieve a better understanding of the macromolecular displacement in the H-cell microchannels. It was found that particle transfer between the two channels tends to speed up at low ionic strength and high concentration gradient. This confirms the corresponding experimental observation of protein diffusion measured via the H-cell microfluidics.
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Dispositivos Laboratorio en un Chip , Proteínas/química , Animales , Difusión , Concentración de Iones de Hidrógeno , Muramidasa/química , Concentración Osmolar , Solventes/química , ViscosidadRESUMEN
OBJECTIVES: Therapeutic antibodies can provoke an antidrug antibody (ADA) response, which can form soluble immune complexes with the drug in potentially high amounts. Nevertheless, ADA-associated adverse events are usually rare, although with notable exceptions including infliximab. The immune activating effects and the eventual fate of these 'anti-idiotype' complexes are poorly studied, hampering assessment of ADA-associated risk of adverse events. We investigated the in vitro formation and biological activities of ADA-drug anti-idiotype immune complexes using patient-derived monoclonal anti-infliximab antibodies. METHODS: Size distribution and conformation of ADA-drug complexes were characterised by size-exclusion chromatography and electron microscopy. Internalisation of and immune activation by complexes of defined size was visualised with flow imaging, whole blood cell assay and C4b/c ELISA. RESULTS: Size and conformation of immune complexes depended on the concentrations and ratio of drug and ADA; large complexes (>6 IgGs) formed only with high ADA titres. Macrophages efficiently internalised tetrameric and bigger complexes in vitro, but not dimers. Corroborating these results, ex vivo analysis of patient sera demonstrated only dimeric complexes in circulation.No activation of immune cells by anti-idiotype complexes was observed, and only very large complexes activated complement. Unlike Fc-linked hexamers, anti-idiotype hexamers did not activate complement, demonstrating that besides size, conformation governs immune complex potential for triggering effector functions. CONCLUSIONS: Anti-idiotype ADA-drug complexes generally have restricted immune activation capacity. Large, irregularly shaped complexes only form at high concentrations of both drug and ADA, as may be achieved during intravenous infusion of infliximab, explaining the rarity of serious ADA-associated adverse events.
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Anticuerpos/inmunología , Formación de Anticuerpos/efectos de los fármacos , Complejo Antígeno-Anticuerpo/inmunología , Antirreumáticos/inmunología , Infliximab/inmunología , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática , Humanos , Suero/inmunologíaRESUMEN
PURPOSE: Personalized peptide-based cancer vaccines will be composed of multiple patient specific synthetic long peptides (SLPs) which may have various physicochemical properties. To formulate such SLPs, a flexible vaccine delivery system is required. We studied whether cationic liposomes are suitable for this purpose. METHODS: Fifteen SIINFEKL T cell epitope-containing SLPs, widely differing in hydrophobicity and isoelectric point, were separately loaded in cationic liposomes via the dehydration-rehydration method. Particle size and polydispersity index (PDI) were measured via dynamic light scattering (DLS), and zeta potential with laser Doppler electrophoresis. Peptide loading was fluorescently determined and the immunogenicity of the formulated peptides was assessed in co-cultures of dendritic cells (DCs) and CD8+ T-cells in vitro. RESULTS: All SLPs were loaded in cationic liposomes by using three different loading method variants, depending on the SLP characteristics. The fifteen liposomal formulations had a comparable size (< 200 nm), PDI (< 0.3) and zeta potential (22-30 mV). Cationic liposomes efficiently delivered the SLPs to DCs that subsequently activated SIINFEKL-specific CD8+ T-cells, indicating improved immunological activity of the SLPs. CONCLUSION: Cationic liposomes can accommodate a wide range of different SLPs and are therefore a potential delivery platform for personalized cancer vaccines.
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Vacunas contra el Cáncer/administración & dosificación , Portadores de Fármacos/química , Epítopos de Linfocito T , Liposomas/química , Oligopéptidos/administración & dosificación , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/inmunología , Cationes , Composición de Medicamentos , Liberación de Fármacos , Colorantes Fluorescentes/química , Humanos , Activación de Linfocitos , Oligopéptidos/química , Oligopéptidos/inmunología , Ovalbúmina/química , Tamaño de la Partícula , Fragmentos de Péptidos/química , Biblioteca de Péptidos , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/química , Vacunas de Subunidad/inmunologíaRESUMEN
PURPOSE: To examine the immunogenicity of diphtheria toxoid (DT) loaded mesoporous silica nanoparticles (MSNs) after coated and hollow microneedle-mediated intradermal immunization in mice. METHODS: DT was loaded into MSNs and the nanoparticle surface was coated with a lipid bilayer (LB-MSN-DT). To prepare coated microneedles, alternating layers of negatively charged LB-MSN-DT and positively charged N-trimethyl chitosan (TMC) were coated onto pH-sensitive microneedle arrays via a layer-by-layer approach. Microneedle arrays coated with 5 or 3 layers of LB-MSN-DT were used to immunize mice and the elicited antibody responses were compared with those induced by hollow microneedle-injected liquid formulation of LB-MSN-DT. Liquid DT formulation with and without TMC (DT/TMC) injected by a hollow microneedle were used as controls. RESULTS: LB-MSN-DT had an average size of about 670 nm and a zeta potential of -35 mV. The encapsulation efficiency of DT in the nanoparticles was 77%. The amount of nano-encapsulated DT coated onto the microneedle array increased linearly with increasing number of the coating layers. Nano-encapsulated DT induced stronger immune responses than DT solution when delivered intradermally via hollow microneedles, but not when delivered via coated microneedles. CONCLUSION: Both the nano-encapsulation of DT and the type of microneedles affect the immunogenicity of the antigen.
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Toxoide Diftérico/administración & dosificación , Nanopartículas/química , Dióxido de Silicio/química , Animales , Toxoide Diftérico/química , Toxoide Diftérico/inmunología , Composición de Medicamentos , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Inmunización , Inmunogenicidad Vacunal , Inyecciones Intradérmicas , Ratones , Ratones Endogámicos BALB C , Tamaño de la Partícula , Porosidad , Propiedades de SuperficieRESUMEN
PURPOSE: The aim of this study was to investigate the depth-dependent intradermal immunogenicity of inactivated polio vaccine (IPV) delivered by depth-controlled microinjections via hollow microneedles (HMN) and to investigate antibody response enhancing effects of IPV immunization adjuvanted with CpG oligodeoxynucleotide 1826 (CpG) or cholera toxin (CT). METHODS: A novel applicator for HMN was designed to permit depth- and volume-controlled microinjections. The applicator was used to immunize rats intradermally with monovalent IPV serotype 1 (IPV1) at injection depths ranging from 50 to 550 µm, or at 400 µm for CpG and CT adjuvanted immunization, which were compared to intramuscular immunization. RESULTS: The applicator allowed accurate microinjections into rat skin at predetermined injection depths (50-900 µm), -volumes (1-100 µL) and -rates (up to 60 µL/min) with minimal volume loss (±1-2%). HMN-mediated intradermal immunization resulted in similar IgG and virus-neutralizing antibody titers as conventional intramuscular immunization. No differences in IgG titers were observed as function of injection depth, however IgG titers were significantly increased in the CpG and CT adjuvanted groups (7-fold). CONCLUSION: Intradermal immunogenicity of IPV1 was not affected by injection depth. CpG and CT were potent adjuvants for both intradermal and intramuscular immunization, allowing effective vaccination upon a minimally-invasive single intradermal microinjection by HMN.
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Adyuvantes Inmunológicos/farmacología , Formación de Anticuerpos/inmunología , Vacuna Antipolio de Virus Inactivados/inmunología , Adyuvantes Farmacéuticos/farmacología , Animales , Anticuerpos Antivirales/inmunología , Femenino , Inmunoglobulina G/inmunología , Inyecciones Intradérmicas/métodos , Inyecciones Intramusculares/métodos , Microinyecciones/métodos , Oligodesoxirribonucleótidos/inmunología , Ratas , Ratas Wistar , Vacunación/métodosRESUMEN
PURPOSE: Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics. METHODS: Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (<3% of total protein) of sub-visible aggregates. The effect of stimulating human dendritic cells (DC) and CD4(+) T cells with the aggregates was measured in vitro using cytokine secretion, proliferation and confocal microscopy. RESULTS: Due to its intrinsic high clinical immunogenicity, aggregation of rituximab had minimal effects on DC activation and T cell responses compared to monomeric rituximab. However, in the case of trastuzumab (low clinical immunogenicity) small quantities of aggregates led to potent CD4(+) T cell proliferation as a result of strong cytokine and co-stimulatory signals derived from DC. Consistent with this, confocal studies showed that stir-stressed rituximab was rapidly internalised and associated with late endosomes of DC. CONCLUSIONS: These data link minute amounts of aggregates with activation of the innate immune response, involving DC, resulting in T cell activation. Thus, when protein therapeutics with little or no clinical immunogenicity, such as trastuzumab, contain minute amounts of sub-visible aggregates, they are associated with significantly increased potential risk of clinical immunogenicity.
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Linfocitos T CD4-Positivos/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Agregado de Proteínas/inmunología , Rituximab/inmunología , Trastuzumab/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Citocinas/inmunología , Células Dendríticas/inmunología , Estabilidad de Medicamentos , Humanos , Inmunidad Innata/efectos de los fármacosRESUMEN
PURPOSE: The high prevalence of pure red cell aplasia in Thailand has been associated with the sharp increase in number of recombinant human erythropoietin (rhEPO) copy products, based on a classical generic regulatory pathway, which have entered the market. This study aims to assess the quality of rhEPO copy products being used in Thailand. METHODS: Twelve rhEPO copy products were purchased from pharmacies in Thailand, shipped under controlled cold chain conditions to the Netherlands and characterized using (1) high performance size-exclusion chromatography, (2) asymmetrical flow field-flow fractionation, (3) sodium dodecyl sulfate polyacrylamide gel electrophoresis in combination with (4) Western blotting and additionally tested for (5) host cell protein impurities as well as (6) endotoxin contamination. RESULTS: Some of the tested rhEPO copy products showed high aggregate levels and contained a substantial amount of protein fragments. Also, one of rhEPO copy products had a high endotoxin level, exceeding the FDA limit. CONCLUSIONS: Our observations show that some of the tested copy products on the Thai market differ significantly from the originator rhEPO product, Epogen®. This comparison study supports a link between the quality attributes of copy rhEPO products and their immunogenicity.
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Eritropoyetina/farmacología , Western Blotting , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Eritropoyetina/aislamiento & purificación , Fraccionamiento de Campo-Flujo , Humanos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , TailandiaRESUMEN
PURPOSE: The aim of the study was to develop a cheap and fast method to produce hollow microneedles and an applicator for injecting vaccines into the skin at a pre-defined depth and test the applicability of the system for dermal polio vaccination. METHODS: Hollow microneedles were produced by hydrofluoric acid etching of fused silica capillaries. An electromagnetic applicator was developed to control the insertion speed (1-3 m/s), depth (0-1,000 µm), and angle (10°-90°). Hollow microneedles with an inner diameter of 20 µm were evaluated in ex vivo human skin and subsequently used to immunize rats with inactivated poliovirus vaccine (IPV) by an intradermal microinjection of 9 µL at a depth of 300 µm and an insertion speed of 1 m/s. Rat sera were tested for IPV-specific IgG and virus-neutralizing antibodies. RESULTS: Microneedles produced from fused silica capillaries were successfully inserted into the skin to a chosen depth, without clogging or breakage of the needles. Intradermal microinjection of IPV induced immune responses comparable to those elicited by conventional intramuscular immunization. CONCLUSIONS: We successfully developed a hollow microneedle technology for dermal vaccination that enables fundamental research on factors, such as insertion depth and volume, and insertion angle, on the immune response.
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Microinyecciones/instrumentación , Agujas , Poliomielitis/prevención & control , Vacunas contra Poliovirus/administración & dosificación , Vacunación/instrumentación , Animales , Diseño de Equipo , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inyecciones Intradérmicas/instrumentación , Poliomielitis/sangre , Poliomielitis/inmunología , Poliovirus/inmunología , Vacunas contra Poliovirus/inmunología , Ratas , Ratas WistarRESUMEN
In the field of healthcare logistics, the reliance on conventional transport methods such as cars for the delivery of monoclonal antibodies (mAbs) is susceptible to challenges posed by traffic and infrastructure, leading to increased and unpredictable transport times. Recognizing the potential role of drones in mitigating these challenges, we aimed to investigate the impact of medical drone transport on the stability of mAbs. Compromised stability could lead to aggregation and immunogenicity, thereby jeopardizing the efficacy and safety of mAbs. We studied the transportation of vials as well as ready-to-administer infusion bags with blinatumomab, tocilizumab, and daratumumab. The methodology involved the measurement of both temperature and mechanical shock during drone transport. Moreover, the analytical techniques High Performance Size-Exclusion Chromatography (HP-SEC), Dynamic Light Scattering (DLS), Light Obscuration (LO), Micro-Flow Imaging (MFI), and Nanoparticle Tracking Analysis (NTA) were employed to comprehensively assess the presence of aggregates and particle formation. The key findings revealed no significant differences between car and drone transport, indicating that the stability of mAbs in both vials and infusion bags was adequately maintained during drone transport. This suggests that medical drones are a viable and reliable means for the inter-hospital transport of mAbs, paving the way for more efficient and predictable logistics in healthcare delivery.
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Anticuerpos Monoclonales , Estabilidad de Medicamentos , Transportes , Anticuerpos Monoclonales/química , Transportes/métodos , Humanos , Embalaje de Medicamentos/métodos , Hospitales , TemperaturaRESUMEN
Monoclonal antibodies (mAbs) can be damaged during the aseptic compounding process, with aggregation being the most prevalent form of degradation. Protein aggregates represent one of several risk factors for undesired immunogenicity of mAbs, which can potentially lead to severe adverse drug reactions and less effective treatments. Since data on aggregate and particle formation by robotic compounding is missing, we aimed to compare the antibody stability between robotic- and manual compounding of mAbs with regard to formation of (sub)visible aggregates. Infliximab and trastuzumab were compounded into infusion bags with the APOTECAchemo robot or manually by nurses or pharmacy technicians. The products were analyzed by quantifying (sub)visible particles with nanoparticle tracking analysis, dynamic light scattering (DLS), light obscuration, micro-flow imaging, high pressure size exclusion chromatography (HP-SEC), and visual inspection. HP-SEC showed high percentages monomers in trastuzumab (99.4 % and 99.4 %) and infliximab (99.5 % and 99.6 %) infusion bags for both manual and robotic compounding, respectively. DLS indicated more consistent and reproducible results with robotic compounding, and confirmed monodisperse samples with a higher polydispersity index for manual compounding (0.16, interquartile range; IQR 0.14-0.18) compared to robotic compounding (0.12, IQR 0.11-0.15). This study shows that the studied compounding methods had a minor impact on the number of aggregates and particles, and that robotic compounding of mAbs provided at least similar quality as manual compounding.
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Procedimientos Quirúrgicos Robotizados , Robótica , Humanos , Anticuerpos Monoclonales/química , Infliximab/química , Robótica/métodos , Trastuzumab/química , Composición de Medicamentos/métodosRESUMEN
Lipid conjugates have advanced the field of lipid-based nanomedicine by promoting active-targeting (ligand, peptide, antibody), stability (PEGylation), controlled release (lipoid prodrug), and probe-based tracking (fluorophore). Recent findings indicate lipid conjugates dissociating from nanomedicine upon encountering a biological environment. Yet, implications for (pre)clinical outcomes remain unclear. In this study, using the zebrafish model (Danio rerio), we investigated the fate of liposome-incorporated lipid fluorophore conjugates (LFCs) after intravenous (IV) administration. LFCs having a bilayer mismatch and relatively polar fluorophore revealed counter-predictive outcomes for Caelyx/Doxil (clearance vs. circulating) and AmBisome-like liposomes (scavenger endothelial cell vs. macrophage uptake). Findings on LFC (mis)match for Caelyx/Doxil-like liposomes were supported by translational intravital imaging studies in mice. Importantly, contradicting observations suggest to originate from LFC dissociation in vivo, which was investigated by Asymmetric Flow Field-Flow Fractionation (AF4) upon liposome-serum incubation in situ. Our data suggests that LFCs matching with the liposome bilayer composition - that did not dissociate upon serum incubation - revealed improved predictive outcomes for liposome biodistribution profiles. Altogether, this study highlights the critical importance of fatty acid tail length and headgroup moiety when selecting lipid conjugates for lipid-based nanomedicine.
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Lípidos , Liposomas , Nanomedicina , Pez Cebra , Animales , Nanomedicina/métodos , Lípidos/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/farmacocinética , Ratones , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Doxorrubicina/análogos & derivadosRESUMEN
Antigenic peptide-loaded cationic liposomes have shown promise as cancer vaccines. Quantification of both peptides and lipids is critical for quality control of such vaccines for clinical translation. In this work we describe a reversed phase ultra-performance liquid chromatography (RP-UPLC) method that separates lipids (DOTAP, DOPC and their degradation products) and two physicochemically different peptides within 12 min. Samples were prepared by dilution in a 1:1 (v/v) mixture of methanol and water. Peptide quantification was done via UV detection and lipids were quantified by an evaporative light scattering detector (ELSD), both coupled to the RP-UPLC system, with high precision (RSD < 3.5%). We showed that the presence of lipids and peptides did not mutually influence their quantification. Limit of detection (LOD) and limit of quantification (LOQ), as determined in the ICH guidelines, were 6 and 20 ng for DOTAP, 12 ng and 40 ng for DOPC, 3.0 ng and 8.0 ng for peptide A and 2.4 ng and 7.2 ng for the more hydrophobic peptide B. Finally, lipid degradation of DOTAP and DOPC was monitored in peptide loaded DOTAP:DOPC liposomes upon storage at 4 °C and 40 °C.
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Cromatografía de Fase Inversa , Liposomas , Cationes , Cromatografía Líquida de Alta Presión/métodos , Luz , Lipólisis , Liposomas/química , Péptidos , Dispersión de RadiaciónRESUMEN
Opalescence measurements are broadly applied to assess the quality and stability of biopharmaceutical products at all stages of development and manufacturing. They appear to be simple and straight forward but detect complex light scattering phenomena. Despite a routine calibration step, opalescence values obtained with the same biopharmaceutical sample but on different instruments and/or with different methods may vary significantly. Since the reasons for this high variability are generally not well understood, comparison of opalescence results from different biopharmaceutical laboratories is difficult. Here, we characterized a comprehensive set of biopharmaceutically relevant samples with five opalescence methods to illustrate fundamental differences in method performance and explore the reasons for poor comparability. In addition, we developed a high-throughput method for measuring opalescence in a conventional light scattering plate reader that yields opalescence values in the same range as compendial methods. The presented results underline the impact of sample properties, instrument type, and calibration standards on the determined opalescence value. Based on our findings we provide recommendations for the appropriate application of each method during biopharmaceutical drug product development. Overall, our study contributes to an improved understanding of opalescence measurements in the biopharmaceutical field.
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Productos Biológicos , IridiscenciaRESUMEN
The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.
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Laboratorios , Programas Informáticos , Tamaño de la PartículaRESUMEN
PURPOSE: To determine the immunogenicity of diphtheria toxoid (DT) formulated in two types of vesicles following transcutaneous immunization (TCI) of mice onto microneedle array-treated skin. METHODS: DT-containing cationic liposomes or anionic surfactant-based vesicles were prepared by extrusion and sonication. The physicochemical properties were characterized in terms of size, ζ-potential, vesicle elasticity and antigen association. TCI was performed by applying formulations onto intact or microneedle array-pretreated mice skin, using cholera toxin as an adjuvant. Subcutaneous and intradermal immunizations were as control. Immune responses were evaluated by IgG and neutralizing antibody titers, and the immune-stimulatory properties were assessed using cultured dendritic cells. RESULTS: Stable DT-containing cationic liposomes (â¼150 nm) and anionic vesicles (â¼100 nm) were obtained. Incorporation of Span 80 increased liposome elasticity. About 90% and 77% DT was associated with liposomes and vesicles, respectively. TCI of all formulations resulted in substantial antibody titers only if microneedle pretreatment was applied. Co-administration of cholera toxin further augmented the immune responses of TCI. However, vesicle formulations didn't enhance the immunogenicity on either intact or microneedle-treated skin and showed low stimulatory activity on dendritic cells. CONCLUSIONS: Microneedle pretreatment and cholera toxin, but not antigen association to vesicles, enhances the immunogenicity of topically applied DT.
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Toxoide Diftérico/administración & dosificación , Inmunización/métodos , Microinyecciones/métodos , Agujas , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Células Cultivadas , Química Farmacéutica , Células Dendríticas/inmunología , Toxoide Diftérico/inmunología , Portadores de Fármacos/química , Elasticidad , Femenino , Humanos , Inmunización/instrumentación , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inyecciones Intradérmicas , Inyecciones Subcutáneas , Liposomas , Ratones , Ratones Endogámicos BALB C , Microinyecciones/instrumentaciónRESUMEN
Sample preparation for extraction of nanoscale plastic debris (NPD, size < 1 µm) from environmental samples is a critical step to prepare NPD for further identification and quantification. Developing a NPD extraction method from soil matrices is particularly challenging due to the complexity of solid matrices. In the present study, we built upon the lessons learned from method development for extraction of microplastics and nanomaterials from environmental samples to develop a sample preparation method for extraction of NPD from soil matrices. The evaluation criteria for the extraction method are size distribution, particle number recovery, and particle mass recovery. Since there is no validated method available to trace and quantify the mass of NPD in complex matrices, we applied polystyrene particles doped with europium (Eu-PS NPs). Standard LUFA soil and field soil were spiked and mixed for 24 h with 1 mg of Eu-PS NPs and the particles were extracted from the matrices of the soils. The extraction method did not significantly influence the size distribution of the particles and the extraction agents did not degrade the Eu-PS NPs. Mass balance calculation suggested recoveries of 82 and 77% of the added Eu-PS NPs in LUFA soil and field soil, respectively. The number recoveries of the particles were 81 and 85% for LUFA soil and field soil, respectively. This method can be further optimized and used as the first building block to develop a generic sample preparation method for the extraction of NPD from soil samples. By combining this developed and verified extraction method with identification and quantification techniques, a fit-for-purpose workflow can be developed to quantify and subsequently understand the fate of NPD in soil.
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Biomaterials used as matrix for dissolving micro needles (dMNs) may affect the manufacturing process as well as the potency of the active pharmaceutical ingredient, e.g. the immunogenicity of incorporated vaccine antigens. The aim of this study was to investigate the effect of the molecular weight of hyaluronan, a polymer widely used in the fabrication of dMNs, ranging in molecular weight from 4.8 kDa to 1.8 MDa, on the dissolution of microneedles in the skin in time as well as the antibody response in mice and T-cell activation in vitro. Hyaluronan molecular weight (HA-MWs) did not affect antibody responses (when lower than 150 kDa) nor CD4+ T-cell responses against model antigen ovalbumin. However, the HA-MWs had an effect on the fabrication of dMNs. The 1.8 MDa HA was not suitable for the fabrication of dMNs. Similarly, the 4.8 kDa HA generated dMN arrays less robust compared to the other HA-MWs requiring optimization of the drying conditions. Finally, higher HA-MWs led to longer application time of dMN arrays for a complete dissolution of microneedles into the skin. Specifically, we identified 20 kDa HA as the optimal HA-MW for the fabrication of dMNs as with this MW the dMNs are robust and dissolve fast in the skin without affecting immunogenicity.
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Antígenos/inmunología , Ácido Hialurónico/química , Microinyecciones , Agujas , Vacunas/administración & dosificación , Animales , Femenino , Humanos , Ácido Hialurónico/inmunología , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Ovalbúmina/inmunología , Solubilidad , Vacunas/inmunologíaRESUMEN
Regulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is often associated with auto-immune disorders. We have previously shown that antigen-loaded anionic liposomes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) and cholesterol can induce strong antigen-specific Treg responses. We hypothesized that altering the rigidity of these liposomes while maintaining their size and surface charge would affect their capability of inducing Treg responses. The rigidity of liposomes is affected in part by the length and saturation of carbon chains of the phospholipids in the bilayer, and in part by the presence of cholesterol. We used atomic force microscopy (AFM) to measure the rigidity of anionic OVA323-containing liposomes composed of different types of PC and PG, with or without cholesterol, in a molar ratio of 4:1(:2) distearoyl (DS)PC:DSPG (Young's modulus (YM) 3611 ± 1271 kPa), DSPC:DSPG:CHOL (1498 ± 531 kPa), DSPC:dipalmitoyl (DP)PG:CHOL (1208 ± 538), DPPC:DPPG:CHOL (1195 ± 348 kPa), DSPC:dioleoyl (DO)PG:CHOL (825 ± 307 kPa), DOPC:DOPG:CHOL (911 ± 447 kPa), and DOPC:DOPG (494 ± 365 kPa). Next, we assessed if rigidity affects the association of liposomes to bone marrow-derived dendritic cells (BMDCs) in vitro. Aside from DOPC:DOPG liposomes, we observed a positive correlation between liposomal rigidity and cellular association. Finally, we show that rigidity positively correlates with Treg responses in vitro in murine DCs and in vivo in mice. Our findings underline the suitability of AFM to measure liposome rigidity and the importance of this parameter when designing liposomes as a vaccine delivery system.
Asunto(s)
Liposomas , Linfocitos T Reguladores , Animales , Antígenos , Ratones , Microscopía de Fuerza Atómica , FosfolípidosRESUMEN
PURPOSE: To investigate degradation kinetics of oxytocin as a function of temperature and pH, and identify the degradation products. MATERIALS AND METHODS: Accelerated degradation of oxytocin formulated at pH 2.0, 4.5, 7.0 and 9.0 was performed at 40, 55, 70 and 80 degrees C. Degradation rate constants were determined from RP-HPLC data. Formulations were characterized by HP-SEC, UV absorption and fluorescence spectroscopy. Degradation products were identified by ESI-MS/MS. RESULTS: The loss of intact oxytocin in RP-HPLC was pH- and temperature-dependent and followed (pseudo) first order kinetics. Degradation was fastest at pH 9.0, followed by pH 7.0, pH 2.0 and pH 4.5. The Arrhenius equation proved suitable to describe the kinetics, with the highest activation energy (116.3 kJ/mol) being found for pH 4.5 formulations. At pH 2.0 deamidation of Gln(4), Asn(5), and Gly(9)-NH2, as well as combinations thereof were found. At pH 4.5, 7.0 and 9.0, the formation of tri- and tetrasulfide-containing oxytocin as well as different types of disulfide and dityrosine-linked dimers were found to occur. Beta-elimination and larger aggregates were also observed. At pH 9.0, mono-deamidation of Gln(4), Asn(5), and Gly(9)-NH2 additionally occurred. CONCLUSIONS: Multiple degradation products of oxytocin have been identified unequivocally, including various deamidated species, intramolecular oligosulfides and covalent aggregates. The strongly pH dependent degradation can be described by the Arrhenius equation.