RÉSUMÉ
Nanoparticle (NP) formulations are inherently polydisperse making their structural characterization and justification of specifications complex. It is essential, however, to gain an understanding of the physico-chemical properties that drive performance in vivo. To elucidate these properties, drug-containing poly(lactic acid) (PLA)-poly(ethylene glycol) (PEG) block polymeric NP formulations (or PNPs) were sub-divided into discrete size fractions and analyzed using a combination of advanced techniques, namely cryogenic transmission electron microscopy, small-angle neutron and X-ray scattering, nuclear magnetic resonance, and hard-energy X-ray photoelectron spectroscopy. Together, these techniques revealed a uniquely detailed picture of PNP size, surface structure, internal molecular architecture and the preferred site(s) of incorporation of the hydrophobic drug, AZD5991, properties which cannot be accessed via conventional characterization methodologies. Within the PNP size distribution, it was shown that the smallest PNPs contained significantly less drug than their larger sized counterparts, reducing overall drug loading, while PNP molecular architecture was critical in understanding the nature of in vitro drug release. The effect of PNP size and structure on drug biodistribution was determined by administrating selected PNP size fractions to mice, with the smaller sized NP fractions increasing the total drug-plasma concentration area under the curve and reducing drug concentrations in liver and spleen, due to greater avoidance of the reticuloendothelial system. In contrast, administration of unfractionated PNPs, containing a large population of NPs with extremely low drug load, did not significantly impact the drug's pharmacokinetic behavior - a significant result for nanomedicine development where a uniform formulation is usually an important driver. We also demonstrate how, in this study, it is not practicable to validate the bioanalytical methodology for drug released in vivo due to the NP formulation properties, a process which is applicable for most small molecule-releasing nanomedicines. In conclusion, this work details a strategy for determining the effect of formulation variability on in vivo performance, thereby informing the translation of PNPs, and other NPs, from the laboratory to the clinic.
Sujet(s)
Nanoparticules , Polyéthylène glycols , Souris , Animaux , Polyéthylène glycols/composition chimique , Distribution tissulaire , Polymères/composition chimique , Polyesters/composition chimique , Nanoparticules/composition chimique , Taille de particule , Vecteurs de médicaments/composition chimiqueRÉSUMÉ
A deep and detailed understanding of drug-dendrimer conjugates key properties is needed to define the critical quality attributes that affect drug product performance. The characterization must be executed both in the formulation media and in biological matrices. This, nevertheless, is challenging on account of a very limited number of suitable, established methods for characterizing the physicochemical properties, stability, and interaction with biological environment of complex drug-dendrimer conjugates. In order to fully characterize AZD0466, a drug-dendrimer conjugate currently under clinical development by AstraZeneca, a collaboration was initiated with the European Nanomedicine Characterisation Laboratory to deploy a state-of-the-art multi-step approach to measure physicochemical properties. An incremental complexity characterization approach was applied to two batches of AZD0466 and the corresponding dendrimer not carrying any drug, SPL-8984. Thus, the aim of this work is to guide in depth characterization efforts in the analysis of drug-dendrimer conjugates. Additionally, it serves to highlight the importance of using the adequate complementary techniques to measure physical and chemical stability in both simple and biological media, to drive a complex drug-dendrimer conjugate product from discovery to clinical development.
Sujet(s)
Dendrimères , Dendrimères/composition chimique , Nanomédecine/méthodesRÉSUMÉ
The recent emergence of drug-dendrimer conjugates within pharmaceutical industry research and development introduces a range of challenges for analytical and measurement science. These molecules are very high molecular weight (100-200kDa) with a significant degree of structural complexity. The characteristics and quality attributes that require understanding and definition, and impact efficacy and safety, are diverse. They relate to the intact conjugate, the various building blocks of these complex systems and the level of the free and bound active pharmaceutical ingredient (API). From an analytical and measurement science perspective, this necessitates the measurement of the molecular weight, impurity characterisation, the quantitation of the number of conjugated versus free API molecules, the determination of the impurity profiles of the building blocks, primary structure and both particle size and morphology. Here we report the first example of a global characterisation of a drug-dendrimer conjugate - PEGylated poly-lysine dendrimer currently under development (AZD0466). The impact of the wide variety of analytical and measurement techniques on the overall understanding of this complex molecular entity is discussed, with the relative capabilities of the various approaches compared. The results of this study are an essential platform for the research and development of the future generations of related dendrimer-based medicines.
Sujet(s)
Antinéoplasiques , Dendrimères , Dendrimères/composition chimique , Lysine , Antinéoplasiques/composition chimique , Polyéthylène glycols/composition chimiqueRÉSUMÉ
Lipid nanoparticles (LNPs) are important delivery systems for RNA-based therapeutics, yet the mechanism of their interaction with endosomal membranes remains unclear. Here, the interactions of nucleic acid-loaded LNPs that contain an ionizable lipid with models of the early and late endosomal membranes are studied, for the first time, using different reflectometry techniques. Novel insight is provided with respect to the subphase pH, the stage of the endosome, and the nature of the nucleic acid cargo. It is found that the insertion of lipids from the LNPs into the model membrane is greatest at pH 6.5 and 5.5, whereas at higher pH, lipid insertion is suppressed with evidence instead for the binding of intact LNPs, demonstrating the importance of the pH in the fusion of LNPs undergoing the endosomal pathway. Furthermore, and independently of the pH, the effect of the early- versus late-stage endosomal models is minimal, suggesting that the increased fluidity and anionic nature of the late endosome has little effect on the extent of LNP interaction. Last, there is greater nucleic acid delivery from LNPs containing mRNA than Poly(A), indicating that the extent of interaction can be tuned according to the nature of the nucleic acid cargo. Such new information on the relative impact of factors influencing nucleic acid delivery by LNP interactions with endosomal membranes is important in the design and tuning of vehicles with improved nucleic acid delivery capacities.
Sujet(s)
Lipides , Nanoparticules , Endosomes/métabolisme , Liposomes , ARN messager/métabolisme , Petit ARN interférentRÉSUMÉ
Microphysiological systems (MPS), often referred to as "organ-on-chips," are microfluidic-based in vitro models that aim to recapitulate the dynamic chemical and mechanical microenvironment of living organs. MPS promise to bridge the gap between in vitro and in vivo models and ultimately improve the translation from preclinical animal studies to clinical trials. However, despite the explosion of interest in this area in recent years, and the obvious rewards for such models that could improve R&D efficiency and reduce drug attrition in the clinic, the pharmaceutical industry has been slow to fully adopt this technology. The ability to extract robust, quantitative information from MPS at scale is a key requirement if these models are to impact drug discovery and the subsequent drug development process. Microscopy imaging remains a core technology that enables the capture of information at the single-cell level and with subcellular resolution. Furthermore, such imaging techniques can be automated, increasing throughput and enabling compound screening. In this review, we discuss a range of imaging techniques that have been applied to MPS of varying focus, such as organoids and organ-chip-type models. We outline the opportunities these technologies can bring in terms of understanding mechanistic biology, but also how they could be used in higher-throughput screens, widening the scope of their impact in drug discovery. We discuss the associated challenges of imaging these complex models and the steps required to enable full exploitation. Finally, we discuss the requirements for MPS, if they are to be applied at a scale necessary to support drug discovery projects.
Sujet(s)
Découverte de médicament/instrumentation , Laboratoires sur puces , Techniques d'analyse microfluidique/instrumentation , Microscopie/instrumentation , Analyse sur cellule unique/instrumentation , Animaux , Laboratoire automatique , Cellules cultivées , Tests de criblage à haut débit/instrumentation , HumainsRÉSUMÉ
How the cell rapidly and completely reorganizes its architecture when it divides is a problem that has fascinated researchers for almost 150 yr. We now know that the core regulatory machinery is highly conserved in eukaryotes, but how these multiple protein kinases, protein phosphatases, and ubiquitin ligases are coordinated in space and time to remodel the cell in a matter of minutes remains a major question. Cyclin B1-Cdk is the primary kinase that drives mitotic remodeling; here we show that it is targeted to the nuclear pore complex (NPC) by binding an acidic face of the kinetochore checkpoint protein, MAD1, where it coordinates NPC disassembly with kinetochore assembly. Localized cyclin B1-Cdk1 is needed for the proper release of MAD1 from the embrace of TPR at the nuclear pore so that it can be recruited to kinetochores before nuclear envelope breakdown to maintain genomic stability.
Sujet(s)
Protéine-kinase CDC2/métabolisme , Protéines du cycle cellulaire/métabolisme , Cycline B1/métabolisme , Kinétochores/métabolisme , Pore nucléaire/métabolisme , Transduction du signal/génétique , Systèmes CRISPR-Cas , Points de contrôle du cycle cellulaire/effets des médicaments et des substances chimiques , Points de contrôle du cycle cellulaire/génétique , Protéines du cycle cellulaire/antagonistes et inhibiteurs , Protéines du cycle cellulaire/génétique , Cycline B1/génétique , Exons , Édition de gène , Instabilité du génome/génétique , Cellules HeLa , Humains , Protéines Mad2/génétique , Protéines Mad2/métabolisme , Spectrométrie de masse , Mutation , Enveloppe nucléaire/métabolisme , Complexe protéique du pore nucléaire/métabolisme , Protéines nucléaires/métabolisme , Liaison aux protéines , Protein-Serine-Threonine Kinases/antagonistes et inhibiteurs , Protein-tyrosine kinases/antagonistes et inhibiteurs , Protéines proto-oncogènes/métabolisme , Transduction du signal/physiologie , Appareil du fuseau/métabolismeRÉSUMÉ
The interaction of the ionic liquid [C4 C1 Im][BF4 ] with anatase TiO2 , a model photoanode material, has been studied using a combination of synchrotron radiation photoelectron spectroscopy and near-edge X-ray absorption fine structure spectroscopy. The system is of interest as a model for fundamental electrolyte-electrode and dye-sensitized solar cells. The initial interaction involves degradation of the [BF4 ]- anion, resulting in incorporation of F into Oâ vacancies in the anatase surface. At low coverages, [C4 C1 Im][BF4 ] is found to order at the anatase(101) surface via electrostatic attraction, with the imidazolium ring oriented 32±4° from the anatase TiO2 surface. As the coverage of ionic liquid increases, the influence of the oxide surface on the topmost layers is reduced and the ordering is lost.
RÉSUMÉ
The adsorption of p-aminobenzoic acid (pABA) on the anatase TiO2(101) surface has been investigated using synchrotron radiation photoelectron spectroscopy, near edge X-ray absorption fine structure (NEXAFS) spectroscopy, and density functional theory (DFT). Photoelectron spectroscopy indicates that the molecule is adsorbed in a bidentate mode through the carboxyl group following deprotonation. NEXAFS spectroscopy and DFT calculations of the adsorption structures indicate the ordering of a monolayer of the amino acid on the surface with the plane of the ring in an almost upright orientation. The adsorption of pABA on nanoparticulate TiO2 leads to a red shift of the optical absorption relative to bare TiO2 nanoparticles. DFT and valence band photoelectron spectroscopy suggest that the shift is attributed to the presence of the highest occupied molecular orbitals in the TiO2 band gap region and the presence of new molecularly derived states near the foot of the TiO2 conduction band.
Sujet(s)
Acide 4-amino-benzoïque/composition chimique , Titane/composition chimique , Adsorption , Théorie quantique , Propriétés de surfaceRÉSUMÉ
Synchrotron radiation photoelectron spectroscopy and near-edge X-ray absorption fine structure (NEXAFS) techniques have been used to study the adsorption of dopamine on a rutile TiO2 (110) single crystal. Photoemission results suggest that dopamine bonds through the oxygen molecules in a bidentate fashion. From the data, it is ambiguous whether the oxygens bond to the same 5-fold coordinated surface titanium atom or bridges across two, although based on the bonding of pyrocatechol on rutile TiO2 (110), it is likely that the dopamine bridges two titanium atoms. Using the searchlight effect, the carbon K-edge near-edge X-ray absorption fine structure NEXAFS spectra recorded for dopamine on rutile TiO2 (110) show the phenyl ring to be oriented at 78° ± 5° from the surface and twisted 11 ± 10° relative to the (001) direction.
Sujet(s)
Dopamine/composition chimique , Nanoparticules/composition chimique , Titane/composition chimique , Adsorption , Carbone/composition chimique , Cristallographie aux rayons X , Conformation moléculaire , Oxygène/composition chimique , Taille de particule , Spectroscopie photoélectronique , Propriétés de surface , ThermodynamiqueRÉSUMÉ
Myostatin, a secreted growth factor, is a member of the TGF-beta superfamily and an inhibitor of myogenesis. Previously, we have shown that myostatin gene expression is regulated at the level of transcription and that myostatin is a downstream target gene of MyoD. Here we show that myostatin gene expression is auto-regulated by a negative feedback mechanism. Northern blot analysis indicated that there are relatively higher levels of myostatin mRNA in the biceps femoris muscle of cattle that express a non- functional myostatin allele (Belgian Blue) as compared to normal cattle. In contrast, addition of exogenous myostatin decreases endogenous myostatin mRNA. Consistent with this result, wild type myostatin protein is able to repress myostatin promoter activity via Activin type IIb receptor (ActRIIB) and ALK5 (P < 0.001). However, non-functional myostatin (Piedmontese) failed to repress the myostatin promoter suggesting that myostatin auto-regulates its promoter by negative feedback inhibition. Auto-regulation by myostatin appears to be signaled through Smad7, since the expression of the inhibitory Smad7 is induced by myostatin and the over-expression of Smad7 in turn inhibits the myostatin promoter activity (P < 0.001). In contrast down regulation of Smad7 by siRNA results in increased myostatin mRNA indicating that Smad7 is a negative regulator of myostatin gene expression. Consistent with these results, a decrease in Smad7 mRNA and concomitant increase in myostatin expression is seen in myotubes that express non functional myostatin. In addition, interference with myostatin signaling prevents the induction of Smad7 promoter activity by myostatin. Based on these results, we propose that myostatin auto-regulates its gene expression through a Smad7 dependent mechanism in myogenic cells.
Sujet(s)
Rétrocontrôle physiologique , Régulation de l'expression des gènes , Protéine Smad7/métabolisme , Facteur de croissance transformant bêta/métabolisme , Animaux , Bovins , Cellules cultivées , Myoblastes/cytologie , Myoblastes/métabolisme , Myostatine , Régions promotrices (génétique) , Protéines recombinantes/génétique , Protéines recombinantes/métabolisme , Protéine Smad7/génétique , Facteur de croissance transformant bêta/génétiqueRÉSUMÉ
In mammalian cells, the Golgi apparatus is disassembled at the onset of mitosis and reassembled at the end of mitosis. This disassembly-reassembly is generally believed to be essential for the equal partitioning of Golgi into two daughter cells. For Golgi disassembly, membrane fusion, which is mediated by NSF and p97, needs to be blocked. For the NSF pathway, the tethering of p115-GM130 is disrupted by the mitotic phosphorylation of GM130, resulting in the inhibition of NSF-mediated fusion. In contrast, the p97/p47 pathway does not require p115-GM130 tethering, and its mitotic inhibitory mechanism has been unclear. Now, we have found that p47, which mainly localizes to the nucleus during interphase, is phosphorylated on Serine-140 by Cdc2 at mitosis. The phosphorylated p47 does not bind to Golgi membranes. An in vitro assay shows that this phosphorylation is required for Golgi disassembly. Microinjection of p47(S140A), which is unable to be phosphorylated, allows the cell to keep Golgi stacks during mitosis and has no effect on the equal partitioning of Golgi into two daughter cells, suggesting that Golgi fragmentation-dispersion may not be obligatory for equal partitioning even in mammalian cells.
Sujet(s)
Cycle cellulaire/physiologie , Cellules eucaryotes/métabolisme , Appareil de Golgi/métabolisme , Mitose/physiologie , Protéines nucléaires/métabolisme , Adenosine triphosphatases , Animaux , Protéine-kinase CDC2/métabolisme , Protéines du cycle cellulaire/génétique , Protéines du cycle cellulaire/métabolisme , Lignée cellulaire , Noyau de la cellule/métabolisme , Noyau de la cellule/ultrastructure , DEAD-box RNA helicases , Cellules eucaryotes/ultrastructure , Appareil de Golgi/ultrastructure , Interphase/physiologie , Membranes intracellulaires/métabolisme , Fusion membranaire/physiologie , Microscopie électronique , Phosphorylation , Liaison aux protéines/physiologie , Transport des protéines/physiologie , Rats , Sérine/métabolisme , Protéine contenant la valosineRÉSUMÉ
Cyclin B1-Cdk1 is the key initiator of mitosis, but when and where activation occurs has not been precisely determined in mammalian cells. Activation may occur in the nucleus or cytoplasm, as just before nuclear envelope breakdown, Polo-like kinase1 (Plk1) is proposed to phosphorylate cyclin B1 in its nuclear export sequence (NES), to trigger rapid nuclear import. We raised phospho-specific antibodies against cyclin B1 that primarily recognise the active form of the complex. We show that cyclin B1 is initially phosphorylated on centrosomes in prophase and that Plk1 phosphorylates cyclin B1, but not in the NES. Furthermore, phosphorylation by Plk1 does not cause cyclin B1 to move into the nucleus. We conclude that cyclin B1-Cdk1 is first activated in the cytoplasm and that centrosomes may function as sites of integration for the proteins that trigger mitosis.
Sujet(s)
Protéine-kinase CDC2/métabolisme , Centrosome/métabolisme , Cycline B/métabolisme , Prophase/physiologie , Animaux , Anticorps/métabolisme , Protéines du cycle cellulaire , Noyau de la cellule/métabolisme , Cycline B1 , Cytoplasme/métabolisme , Activation enzymatique , Cellules HeLa , Humains , Structures macromoléculaires , Microscopie de fluorescence , Phosphorylation , Protein kinases/métabolisme , Protein-Serine-Threonine Kinases , Protéines proto-oncogènes , Sérine/métabolisme ,RÉSUMÉ
Cyclins A and E and their partner cyclin-dependent kinases (Cdks) are key regulators of DNA synthesis and of mitosis. Immunofluorescence studies have shown that both cyclins are nuclear and that a proportion of cyclin A is localized to sites of DNA replication. However, recently, both cyclin A and cyclin E have been implicated as regulators of centrosome replication, and it is unclear when and where these cyclin-Cdks can interact with cytoplasmic substrates. We have used live cell imaging to study the behavior of cyclin/Cdk complexes. We found that cyclin A and cyclin E are able to regulate both nuclear and cytoplasmic events because they both shuttle between the nucleus and the cytoplasm. However, we found that there are marked differences in their shuttling behavior, which raises the possibility that cyclin/Cdk function could be regulated at the level of nuclear import and export. In the course of these experiments, we have also found that, contrary to published results, mutations in the hydrophobic patch of cyclin A do affect Cdk binding and nuclear import. This has implications for the role of the hydrophobic patch as a substrate selection motif.