RESUMEN
The foundation of a biosimilar manufacturer's regulatory filing is the demonstration of analytical and functional similarity between the biosimilar product and the pertinent originator product. The excipients in the formulation may interfere with characterization using typical analytical and functional techniques during this biosimilarity exercise. Consequently, the producers of biosimilar products resort to buffer exchange to isolate the biotherapeutic protein from the drug product formulation. However, the impact that this isolation has on the product stability is not completely known. This study aims to elucidate the extent to which mAb isolation via ultrafiltration-diafiltration-based buffer exchange impacts mAb stability. It has been demonstrated that repeated extraction cycles do result in significant changes in higher-order structure (red-shift of 5.0 nm in fluorescence maxima of buffer exchanged samples) of the mAb and also an increase in formation of basic variants from 19.1 to 26.7% and from 32.3 to 36.9% in extracted innovator and biosimilar Tmab samples, respectively. It was also observed that under certain conditions of tertiary structure disruptions, Tmab could be restabilized depending on formulation composition. Thus, mAb isolation through extraction with buffer exchange impacts the product stability. Based on the observations reported in this paper, we recommend that biosimilar manufacturers take into consideration these effects of excipients on protein stability when performing biosimilarity assessments.
Asunto(s)
Anticuerpos Monoclonales , Biosimilares Farmacéuticos , Anticuerpos Monoclonales/química , Biosimilares Farmacéuticos/química , Biosimilares Farmacéuticos/uso terapéutico , Excipientes/químicaRESUMEN
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.
Asunto(s)
Procedimientos Quirúrgicos Robotizados , Robótica , Humanos , Anticuerpos Monoclonales/química , Infliximab/química , Robótica/métodos , Trastuzumab/química , Composición de Medicamentos/métodosRESUMEN
A survey performed by the AAPS Drug Product Handling community revealed a general, mostly consensus, approach to the strategy for the selection of surfactant type and level for biopharmaceutical products. Discussing and building on the survey results, this article describes the common approach for surfactant selection and control strategy for protein-based therapeutics and focuses on key studies, common issues, mitigations, and rationale. Where relevant, each section is prefaced by survey responses from the 22 anonymized respondents. The article format consists of an overview of surfactant stabilization, followed by a strategy for the selection of surfactant level, and then discussions regarding risk identification, mitigation, and control strategy. Since surfactants that are commonly used in biologic formulations are known to undergo various forms of degradation, an effective control strategy for the chosen surfactant focuses on understanding and controlling the design space of the surfactant material attributes to ensure that the desired material quality is used consistently in DS/DP manufacturing. The material attributes of a surfactant added in the final DP formulation can influence DP performance (e.g., protein stability). Mitigation strategies are described that encompass risks from host cell proteins (HCP), DS/DP manufacturing processes, long-term storage, as well as during in-use conditions.
Asunto(s)
Excipientes , Tensoactivos , Estabilidad Proteica , LipoproteínasRESUMEN
The purpose of this study was to optimize the manufacturing of dissolving microneedles (dMNs) and to increase the antigen loading in dMNs to investigate the effect on their physicochemical properties. To achieve this, a novel single-array wells polydimethylsiloxane mold was designed, minimizing antigen wastage during fabrication and achieving homogeneous antigen distribution among the dMN arrays. Using this mold, hyaluronan (HA)-based dMNs were fabricated and tested for maximal ovalbumin (OVA) content. dMNs could be fabricated with an OVA:HA ratio as high as 1:1 (w/w), without compromising their properties such as shape and penetration into the ex vivo human skin, even after storage at high humidity and temperature. High antigen loading did not induce protein aggregation during dMN fabrication as demonstrated by complementary analytical methods. However, the dissolution rate in ex vivo human skin decreased with increasing antigen loading. About 2.7⯵g OVA could be delivered in mice by using a single array with an OVA:HA ratio of 1:3 (w/w). Intradermal vaccination with dMNs induced an immune response similar as subcutaneous injection and faster than after hollow microneedle injection. In conclusion, results suggest that (i) the polydimethylsiloxane mold design has an impact on the manufacturing of dMNs, (ii) the increase in antigen loading in dMNs affects the microneedle dissolution and (iii) dMNs are a valid alternative for vaccine administration over conventional injection.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Composición de Medicamentos/métodos , Sistemas de Liberación de Medicamentos/instrumentación , Vacunación/instrumentación , Vacunas/administración & dosificación , Adyuvantes Inmunológicos/farmacocinética , Animales , Antígenos/administración & dosificación , Antígenos/inmunología , Dimetilpolisiloxanos/química , Sistemas de Liberación de Medicamentos/métodos , Liberación de Fármacos , Femenino , Humanos , Ácido Hialurónico/administración & dosificación , Ácido Hialurónico/inmunología , Ácido Hialurónico/farmacocinética , Inmunogenicidad Vacunal/inmunología , Inyecciones Intradérmicas/instrumentación , Ratones , Ratones Endogámicos BALB C , Microinyecciones/instrumentación , Modelos Animales , Agujas , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Ovalbúmina/farmacocinética , Vacunación/métodos , Vacunas/inmunologíaRESUMEN
Microneedle technologies have been developed for dermal drug and vaccine delivery, including hollow-, solid-, coated-, and dissolving microneedles. Microneedles have been made in many different geometries and of many different materials, all of which may influence their skin-penetrating ability. To ensure reproducible and effective drug and vaccine delivery via microneedles, the optimal insertion parameters should be known. Therefore, a digitally-controlled microneedle applicator was developed to insert microneedles into the skin via impact insertion (velocity) or via pressing force insertion. Six microneedle arrays with different geometries and/or materials were applied onto ex vivo human skin with varying velocities or pressing forces. Penetration efficiency and delivered antigen dose into the skin after application of microneedles were determined. In general, microneedles pierced the skin more efficiently when applied by impact application as compared to application via pressing force. However, the angle of application of the applicator on the skin can affect the velocity of the impact, influencing the penetration efficiency of microneedles. Regarding the antigen delivery into the skin, the delivered dose was increasing by increasing the velocity or pressure, and thus, increasing the penetration efficiency. These data demonstrate that an applicator is an important tool to determine optimal application conditions with ex vivo human skin.
RESUMEN
Skin is an attractive but also very challenging immunisation site for particulate subunit vaccines. The aim of this study was to develop hyaluronan (HA)-based dissolving microneedles (MNs) loaded with PLGA nanoparticles (NPs) co-encapsulating ovalbumin (OVA) and poly(I:C) for intradermal immunisation. The NP:HA ratio used for the preparation of dissolving MNs appeared to be critical for the quality of MNs and their dissolution in ex vivo human skin. Asymmetrical flow field-flow fractionation and dynamic light scattering were used to analyse the NPs released from the MNs in vitro. Successful release of the NPs depended on the drying conditions during MN preparation. The delivered antigen dose from dissolving MNs in mice was determined to be 1⯵g OVA, in NPs or as free antigen, by using near-infrared fluorescence imaging. Finally, the immunogenicity of the NPs after administration of dissolving MNs (NP:HA weight ratio 1:4) was compared with that of hollow MN-delivered NPs in mice. Immunization with free antigen in dissolving MNs resulted in equally strong immune responses compared to delivery by hollow MNs. However, humoral and cellular immune responses evoked by NP-loaded dissolving MNs were inferior to those elicited by NPs delivered through a hollow MN. In conclusion, we identified several critical formulation parameters for the further development of NP-loaded dissolving MNs.
Asunto(s)
Nanopartículas/química , Vacunación/métodos , Vacunas/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Administración Cutánea , Animales , Liberación de Fármacos , Femenino , Humanos , Ácido Hialurónico/administración & dosificación , Ácido Hialurónico/inmunología , Ácido Hialurónico/farmacocinética , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Inyecciones Intradérmicas/métodos , Ácido Láctico/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microinyecciones/métodos , Agujas , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Ovalbúmina/farmacocinética , Poli I-C/administración & dosificación , Poli I-C/inmunología , Poli I-C/farmacocinética , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Piel/metabolismo , Vacunas/inmunología , Vacunas/farmacocinéticaRESUMEN
The safety and efficacy of protein pharmaceuticals depend not only on biological activity but also on purity levels. Impurities may be process related because of limitations in manufacturing or product related because of protein degradation occurring throughout the life history of a product. Although the pharmaceutical biotechnology industry has made great progress in improving bulk and drug product manufacturing as well as company-controlled storage and transportation conditions to minimize the level of degradation, there is less control over the many factors that may subsequently affect product quality after the protein pharmaceuticals are released and shipped by the manufacturer. Routine handling or unintentional mishandling of therapeutic protein products may cause protein degradation that remains unnoticed but can potentially compromise the clinical safety and efficacy of the product. In this commentary, we address some potential risks associated with (mis)handling of protein pharmaceuticals after release by the manufacturer. We summarize the environmental stress factors that have been shown to cause protein degradation and that may be encountered during typical handling procedures of protein pharmaceuticals in a hospital setting or during self-administration by patients. Moreover, we provide recommendations for improvements in product handling to help ensure the quality of protein pharmaceuticals during use.
Asunto(s)
Preparaciones Farmacéuticas/química , Proteínas/química , Contaminación de Medicamentos , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Humanos , Preparaciones Farmacéuticas/administración & dosificación , Fotólisis , Agregado de Proteínas , Estabilidad Proteica , Proteínas/administración & dosificación , Proteolisis , AutoadministraciónRESUMEN
OBJECTIVES: Anti-citrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA). Here, we studied binding of ACPA-IgG immune complexes (IC) to individual Fc gamma receptors (FcγR) to identify potential effector mechanisms by which ACPA could contribute to RA pathogenesis. METHODS: ACPA-IgG1 and control IgG1(IgG1 depleted of ACPA-IgG1) were isolated from plasma and synovial fluid (SF) of RA patients by affinity chromatography using CCP2 peptides. Subsequently, IC were generated using fluorescently labelled F(ab')2 fragments against the F(ab')2 region of IgG, or by using citrullinated fibrinogen. IC were incubated with FcγR-transfected CHO cell lines or neutrophils from healthy donors. FcγR binding of IC was analysed by flow cytometry in the presence or absence of specific blocking antibodies. RESULTS: ACPA-IgG1 IC predominantly bound to FcγRI and FcγRIIIA on FcγR-transfected CHO cell lines, while much lower binding was observed to FcγRIIA and FcγRIIB. ACPA-IgG1 IC showed reduced binding to FcγRIIIA compared to control IgG1 IC, in line with enhanced ACPA-IgG1 Fc core-fucosylation. Neutrophils activated in vitro to induce de novo expression of FcγRI showed binding of ACPA-IgG IC, and blocking studies revealed that almost 30% of ACPA-IgG IC binding to activated neutrophils was mediated by FcγRI. CONCLUSIONS: Our studies show that ACPA-IgG1 IC bind predominately to activating FcγRI and FcγRIIIA, and highlight FcγRI expressed by activated neutrophils as relevant receptor for these IC. As neutrophils isolated from SF exhibit an activated state and express FcγRI in the synovial compartment, this IC-binding could contribute to driving disease pathogenesis in RA.
Asunto(s)
Anticuerpos Antiproteína Citrulinada/metabolismo , Complejo Antígeno-Anticuerpo/metabolismo , Artritis Reumatoide/etiología , Receptores de IgG/metabolismo , Anciano , Artritis Reumatoide/inmunología , Femenino , Glicosilación , Humanos , Masculino , Persona de Mediana Edad , Neutrófilos/fisiología , Receptores de IgG/fisiología , Sinovitis/etiologíaRESUMEN
In this study, we investigated the potential of intradermal delivery of nanoparticulate vaccines to modulate the immune response of protein antigen using hollow microneedles. Four types of nanoparticles covering a broad range of physiochemical parameters, namely poly (lactic-co-glycolic) (PLGA) nanoparticles, liposomes, mesoporous silica nanoparticles (MSNs) and gelatin nanoparticles (GNPs) were compared. The developed nanoparticles were loaded with a model antigen (ovalbumin (OVA)) with and without an adjuvant (poly(I:C)), followed by the characterization of size, zeta potential, morphology, and loading and release of antigen and adjuvant. An in-house developed hollow-microneedle applicator was used to inject nanoparticle suspensions precisely into murine skin at a depth of about 120µm. OVA/poly(I:C)-loaded nanoparticles and OVA/poly(I:C) solution elicited similarly strong total IgG and IgG1 responses. However, the co-encapsulation of OVA and poly(I:C) in nanoparticles significantly increased the IgG2a response compared to OVA/poly(I:C) solution. PLGA nanoparticles and liposomes induced stronger IgG2a responses than MSNs and GNPs, correlating with sustained release of the antigen and adjuvant and a smaller nanoparticle size. When examining cellular responses, the highest CD8+ and CD4+ T cell responses were induced by OVA/poly(I:C)-loaded liposomes. In conclusion, the applicator controlled hollow microneedle delivery is an excellent method for intradermal injection of nanoparticle vaccines, allowing selection of optimal nanoparticle formulations for humoral and cellular immune responses.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antígenos/administración & dosificación , Nanopartículas/administración & dosificación , Agujas , Ovalbúmina/administración & dosificación , Poli I-C/administración & dosificación , Vacunación/instrumentación , Animales , Antígenos/química , Liberación de Fármacos , Femenino , Inyecciones Intradérmicas , Ácido Láctico/administración & dosificación , Ácido Láctico/química , Liposomas , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microinyecciones , Nanopartículas/química , Ovalbúmina/química , Poli I-C/química , Ácido Poliglicólico/administración & dosificación , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Vacunación/métodosRESUMEN
The release of drugs from poly(lactic-co-glycolic acid) (PLGA) microparticles depends to a large extent on the porosity of the particles. Therefore, porosity determination of PLGA microparticles is extremely important during pharmaceutical product development. Currently, mercury intrusion porosimetry (MIP) is widely used despite its disadvantages, such as the need for a large amount of sample (several hundreds of milligrams) and residual toxic waste. Here, we present a method based on the estimation of the volume of a known mass (a few milligrams) of particles using micro-flow imaging (MFI) to determine microparticle batch porosity. Factors that are critical for the accuracy of this method (i.e., density of the suspending fluid, particle concentration, and postsample rinsing) were identified and measures were taken to minimize potential errors. The validity of the optimized method was confirmed by using nonporous polymethylmethacrylate microparticles. Finally, the method was employed for the analysis of 7 different PLGA microparticle batches with various porosities (4.0%-51.9%) and drug loadings (0%-38%). Obtained porosity values were in excellent agreement with the MIP-derived porosities. Altogether, the developed MFI-based method is a valuable tool for deriving the total volume of a known mass of PLGA particles and therewith their porosity.
Asunto(s)
Preparaciones de Acción Retardada/química , Ácido Láctico/química , Ácido Poliglicólico/química , Inyecciones , Microscopía/métodos , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , PorosidadRESUMEN
A master student, who surveyed the procedures in a hospital pharmacy with regard to the handling of biologicals, identified several issues that might have jeopardized product quality. This case may be a tip of the iceberg and illustrates the urgent need for a better education of end-users about how to handle biologicals.
Asunto(s)
Productos Biológicos/administración & dosificación , Productos Biológicos/química , Estabilidad de Medicamentos , Hospitales , Humanos , Farmacia , Agregado de ProteínasRESUMEN
Nanoparticle tracking analysis (NTA) has attracted great interest for application in the field of submicron particle characterization for biopharmaceuticals. It has the virtue of direct sample visualization and particle-by-particle tracking, but the complexity of method development has limited its routine applicability. We systematically evaluated data collection and processing parameters as well as sample handling methods using shake-stressed protein samples. The camera shutter and gain were identified as the key factors influencing NTA results. We also demonstrated that sample filtration was necessary for NTA analysis if there were high numbers of micron particles, whereas the choice of filter membrane was critical for data quality. Sample dilution into corresponding formulation buffer did not affect particle size distributions in our study. Finally, NTA analysis exhibited excellent repeatability in intraday comparison of multiple measurements on the same sample and interday comparison on different batches of samples. Shaking-induced protein aggregation could also be sensitively monitored by NTA. In conclusion, NTA analysis can be used as a robust stability-indicating method for the characterization of proteinaceous submicron particles and thereby complement other analytical methods, provided that consistent sample handling and parametric settings are established for the specific case study.
Asunto(s)
Química Farmacéutica/métodos , Inmunoglobulina G/química , Nanopartículas/química , Tamaño de la Partícula , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/química , Evaluación Preclínica de Medicamentos/métodos , Inmunoglobulina G/análisis , Nanopartículas/análisis , Proteínas/análisis , Proteínas/químicaRESUMEN
This study addressed the effect of contact sliding during stirring of a monoclonal antibody solution on protein aggregation, in particular, in the nanometer and micrometer size range. An overhead stirring set-up was designed in which the presence and magnitude of the contact between the stir bar and the container could be manipulated. A solution of 0.1 mg/mL of a monoclonal antibody (IgG) in phosphate buffered saline was stirred at 300 rpm at room temperature. At different time points, samples were taken and analyzed by nanoparticle tracking analysis, flow imaging microscopy, and size-exclusion chromatography. In contrast to non-contact-stirred and unstirred samples, the contact-stirred sample contained several-fold more particles and showed a significant loss of monomer. No increase in oligomer content was detected. The number of particles formed was proportional to the contact area and the magnitude of the normal pressure between the stir bar and the glass container. Extrinsic 9-(2,2-dicyanovinyl) julolidine fluorescence indicated a conformational change for contact-stirred protein samples. Presence of polysorbate 20 inhibited the formation of micron-sized aggregates. We suggest a model in which abrasion of the potentially destabilized, adsorbed protein leads to aggregation and renewal of the surface for adsorption of a fresh protein layer.
Asunto(s)
Anticuerpos Monoclonales/química , Química Farmacéutica/métodos , Nanopartículas/química , Soluciones Farmacéuticas/química , Agregado de Proteínas , Estrés Mecánico , Adsorción , Cromatografía en Gel/métodos , Tamaño de la PartículaRESUMEN
Human serum albumin (HSA)-free formulation of Escherichia coli-derived human interferon beta (IFNß-1b) with a high percentage of monomeric protein and low immunogenicity is developed and characterized in the current study. UV spectroscopy, fluorescence spectroscopy, dynamic light scattering, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, Micro-Flow Imaging, resonant mass measurement, size exclusion, and reversed-phase high performance liquid chromatographies were applied to assess the effect of excipients on the stability of IFNß-1b to establish a HSA-free formulation. The antiviral activity of IFNß-1b was evaluated using human lung carcinoma cell line. Immune tolerant mice to hIFNß were used to assess the immunogenicity of the HSA-free formulated IFNß-1b in comparison to Betaferon drug product and Avonex drug substance as standards through IgG titering of plasma. HSA-free formulated IFNß-1b, including 200 mM L-arginine, 200 mM trehalose, and 0.1% n-dodecyl ß-D-maltoside in 10 mM sodium acetate buffer, pH 7.4, showed the highest biological activity. The stability of IFNß-1b in the HSA-free formulation was monitored for 3 weeks at 4°C and 37°C with relative humidity of 10% and 75%, respectively. Protein aggregation and immunogenicity in transgenic mice were decreased in the HSA-free formulated IFNß-1b compared to Betaferon. The stability, biological activity, and immunogenicity of the HSA-free formulation and Betaferon were evaluated. Incubation of formulations at 4°C and 37°C for 3 weeks showed that the HSA-free formulated IFNß-1b was more stable and less immunogenic in transgenic FVB/N mice. Low immunogenicity and the absence of HSA, which reduces the potential risk of viral infection (eg, HIV and HCV), are promising for clinical studies.
Asunto(s)
Anticuerpos Antivirales/sangre , Antivirales/farmacología , Virus de la Encefalomiocarditis/efectos de los fármacos , Interferon beta-1b/farmacología , Células A549 , Animales , Antivirales/inmunología , Antivirales/aislamiento & purificación , Clonación Molecular , Virus de la Encefalomiocarditis/crecimiento & desarrollo , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Tolerancia Inmunológica , Interferon beta-1b/biosíntesis , Interferon beta-1b/aislamiento & purificación , Ratones , Ratones Transgénicos , Estabilidad Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacologíaRESUMEN
The purpose of this study was to investigate the formation and growth kinetics of complexes of proteins and oppositely charged polyelectrolytes. Equal volumes of IgG and dextran sulfate (DS) solutions, 0.01 mg/ml each in 10mM phosphate, pH 6.2, were mixed. At different time points, samples were taken and analyzed by nanoparticle tracking analysis (NTA), Micro-Flow Imaging (MFI) and size-exclusion chromatography (SEC). SEC showed a huge drop in monomer content (approximately 85%) already 2 min after mixing, while a very high nanoparticle (size up to 500 nm) concentration (ca. 9 × 10(8)/ml) was detected by NTA. The nanoparticle concentration gradually decreased over time, while the average particle size increased. After a lag time of about 1.5h, a steady increase in microparticles was measured by MFI. The microparticle concentration kept increasing up to about 1.5 × 10(6)/ml until it started to slightly decrease after 10h. The average size of the microparticles remained in the low-µm range (1-2 µm) with a slight increase and broadening of the size distribution in time. The experimental data could be fitted with Smoluchowski's perikinetic coagulation model, which was validated by studying particle growth kinetics in IgG:DS mixtures of different concentrations. In conclusion, the combination of NTA and MFI provided novel insight into the kinetics and mechanism of protein-polyelectrolyte complex formation.
Asunto(s)
Sulfato de Dextran/química , Inmunoglobulina G/química , Microscopía/métodos , Nanopartículas , Nanotecnología/métodos , Tecnología Farmacéutica/métodos , Tampones (Química) , Cromatografía en Gel , Sulfato de Dextran/metabolismo , Concentración de Iones de Hidrógeno , Inmunoglobulina G/metabolismo , Cinética , Modelos Químicos , Tamaño de la Partícula , Unión Proteica , Reproducibilidad de los ResultadosRESUMEN
We assessed the potential of a suspended microchannel resonator (SMR) to measure the adsorption of proteins to nanoparticles. Standard polystyrene beads suspended in buffer were weighed by a SMR system. Particle suspensions were mixed with solutions of bovine serum albumin (BSA) or monoclonal human antibody (IgG), incubated at room temperature for 3 h and weighed again with SMR. The difference in buoyant mass of the bare and protein-coated polystyrene beads was calculated into real mass of adsorbed proteins. The average surface area occupied per protein molecule was calculated, assuming a monolayer of adsorbed protein. In parallel, dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and zeta potential measurements were performed. SMR revealed a statistically significant increase in the mass of beads because of adsorption of proteins (for BSA and IgG), whereas DLS and NTA did not show a difference between the size of bare and protein-coated beads. The change in the zeta potential of the beads was also measurable. The surface area occupied per protein molecule was in line with their known size. Presented results show that SMR can be used to measure the mass of adsorbed protein to nanoparticles with a high precision in the presence of free protein.
Asunto(s)
Inmunoglobulina G/química , Análisis por Micromatrices/métodos , Nanopartículas/química , Tamaño de la Partícula , Albúmina Sérica Bovina/química , Adsorción , Animales , Bovinos , Humanos , Inmunoglobulina G/metabolismo , Peso Molecular , Nanopartículas/metabolismo , Poliestirenos/química , Poliestirenos/metabolismo , Albúmina Sérica Bovina/metabolismoRESUMEN
The search for alternative therapies to improve bone regeneration continues to be a major challenge for the medical community. Here we report on the enhanced mineralization, osteogenesis, and in vivo bone regeneration properties of a bioactive elastin-like recombinamer (ELR) membrane. Three bioactive ELRs exhibiting epitopes designed to promote mesenchymal stem cell adhesion (RGDS), mineralization (DDDEEKFLRRIGRFG), and both cell adhesion and mineralization were synthesized using standard recombinant protein techniques. The ELR materials were then used to fabricate membranes comprising either a smooth surface (Smooth) or channel microtopographies (Channels). Mineralization and osteoblastic differentiation of primary rat mesenchymal stem cells (rMSCs) were analyzed in both static and dynamic (uniaxial strain of 8% at 1 Hz frequency) conditions. Smooth mineralization membranes in static condition exhibited the highest quantity of calcium phosphate (Ca/P of 1.78) deposition with and without the presence of cells, the highest Young's modulus, and the highest production of alkaline phosphatase on day 10 in the presence of cells growing in non-osteogenic differentiation medium. These membranes were tested in a 5 mm-diameter critical-size rat calvarial defect model and analyzed for bone formation on day 36 after implantation. Animals treated with the mineralization membranes exhibited the highest bone volume within the defect as measured by micro-computed tomography and histology with no significant increase in inflammation. This study demonstrates the possibility of using bioactive ELR membranes for bone regeneration applications.
Asunto(s)
Materiales Biocompatibles/metabolismo , Regeneración Ósea , Sustitutos de Huesos/metabolismo , Elastina/metabolismo , Células Madre Mesenquimatosas/citología , Andamios del Tejido/química , Secuencia de Aminoácidos , Animales , Sustitutos de Huesos/química , Fosfatos de Calcio/análisis , Fosfatos de Calcio/metabolismo , Adhesión Celular , Diferenciación Celular , Células Cultivadas , Elastina/química , Masculino , Membranas Artificiales , Células Madre Mesenquimatosas/metabolismo , Datos de Secuencia Molecular , Osteogénesis , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Proteínas y Péptidos SalivalesRESUMEN
Non-covalent interactions are often regarded as insufficient to construct macroscopic materials of substantial integrity and cohesion. However, the low binding energy of such reversible interactions can be compensated by increasing their number to work in concert to create strong materials. Here we present the successful development of an injectable, cohesive nanocomposite hydrogel based on reversible bonds between calcium phosphate nanoparticles and bisphosphonate-functionalized hyaluronic acid. These nanocomposites display a capacity for self-healing as well as adhesiveness to mineral surfaces such as enamel and hydroxyapatite. Most importantly, these non-covalently cross-linked composites are surprisingly robust yet biodegradable upon extensive in vitro and in vivo testing and show bone interactive capacity evidenced by bone ingrowth into material remnants. The herein presented method provides a new methodology for constructing nanoscale composites for biomedical applications, which owe their integrity to reversible bonds.