Engineering of a phosphorylatable tag for specific protein binding on zirconium phosphonate based microarrays

A phosphorylatable tag was designed and fused at the C-terminal end of proteins, which allowed efficient and oriented immobilization of capture proteins on glass substrates coated with a zirconium phosphonate monolayer. The concept is demonstrated using Nanofitin directed against lysozyme. This peptide tag (DSDSSSEDE) contains four serines in an acidic environment, which favored its in vitro phosphorylation by casein kinase II. The resulting phosphate cluster at the C-terminal end of the protein provided a specific, irreversible, and multipoint attachment to the zirconium surface. In a microarray format, the high surface coverage led to high fluorescence signal after incubation with Alexa Fluor 647 labeled lysozyme. The detection sensitivity of the microarray for the labeled target was below 50 pM, owing to the exceptionally low background staining, which resulted in high fluorescence similar to signal to noise ratios. The performance of this new anchoring strategy using a zirconium phosphonate modified surface compares favorably with that of other types of microarray substrates, such as nitrocellulose-based or epoxide slides, which bind proteins in a nonoriented way

Le dosage par spectroscopie proche infrarouge, Application au test de l'uniformité de teneur des comprimés II

The first part of this article [1] described the methodology to follow to develop a technique for the quantitative analysis of active substance in a solid tablet type formulation using near infrared spectroscopy. It seems appropriate to complete this description by detailing the different steps involved through illustrated examples and by proposing useful guidelines for new users. The quantitative analysis example chosen is that of content uniformity (CU) of tablet type solid forms (cf. Ph. Eur. 2.9.6/2.9.40) [2], a test required in the pharmaceutical industry and for which this alternative quantitative analysis could offer substantial gains. It should be noted that the approach described applies to other types of quantitative analysis (mean assay, water content in raw materials, polymorph assay within a tablet, etc.)

Analyse quantitative par spectroscopie proche infrarouge: Application au test d'unifomité de teneur des comprimés

La spectroscopie Proche infrarouge présente un large potentiel d'applications dans l'industrie pharmaceutique par sa capacité à transmettre des informations reliées à la fois à la structure chimique des échantillons et à leur état physique. Elle peut être mise à profit pour des analyses qualitatives, telles l'identification des matières premières, et également pour des analyses quantitatives telles le dosage d'un principe actif dans un comprimé. Avec pour avantage une rapidité inégalée pour délivrer un résultat comparée aux techniques classiques en vigueur. Attrayante par ses performances, cette spectroscopie nécessite cependant une prise en main qui doit respecter une démarche minutieuse et souvent déroutante par son aspect heuristique (allers-retours) et à la mise en oeuvre d'outils d'analyse statistique multivariée. Si de nombreuses publications témoignent de la puissance de cet outil, il est apparu opportun de rassembler en un même texte la démarche de développement à suivre et les repères à connaître pour permettre son utilisation en analyse quantitative dans un contexte de libération pharmaceutique. Le travail de cette commission a été de rassembler les éléments issus de la pratique industrielle en se concentrant sur l'un des essais les plus intéressants: celui de l'uniformité de teneur des comprimés. Ce test est en effet l'une des applications où la rapidité de mise en oeuvre est le plus appréciable, tout en étant celui qui peut poser le plus de questions quant à la validité des résultats et la prise de décision pharmaceutique

Le dosage par spectroscopie proche infrarouge, Application au test de l'uniformité de teneur des comprimés, I. Méthodologie

This first document describes guidelines to develop a quantification method for active pharmaceutical ingredient content in solid dose formulation, using near-infrared spectroscopy. This new approach offers several advantages over classical techniques, in particular for tablets content uniformity determination. This document is intended to help users addressing the numerous questions pertaining to number and types of samples, usable spectral range for quantification, importance of the reference method, development of a model and potential pitfalls, use of chemometrics tools and method validation. It also offers a statistical support that used as an aid for decision for routine controls, when a sample falls near acceptance limits. The document gathers basic principles and will be complemented by a second article with practical examples to illustrate the methodology

Assembly of Therapeutic pRNA-siRNA Nanoparticles Using Bipartite Approach

The 117-nucleotide (nt) RNA, called the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor, has been shown to be an efficient vector for the construction of RNA nanoparticles for the delivery of small interfering RNA (siRNA) into specific cancer or viral-infected cells. Currently, chemical synthesis of 117-nt RNA is not feasible commercially. In addition, labeling at specific locations on pRNA requires the understanding of its modular organization. Here, we report multiple approaches for the construction of a functional 117-base pRNA using two synthetic RNA fragments with variable modifications. The resulting bipartite pRNA was fully competent in associating with other interacting pRNAs to form dimers, as demonstrated by the packaging of DNA via the nanomotor and the assembly of phi29 viruses in vitro. The pRNA subunit assembled from bipartite fragments harboring siRNA or receptor-binding ligands were equally competent in assembling into dimers. The subunits carrying different functionalities were able to bind cancer cells specifically, enter the cell, and silence specific genes of interest. The pRNA nanoparticles were subsequently processed by Dicer to release the siRNA embedded within the nanoparticles. The results will pave the way toward the treatment of diseases using synthetic pRNA/siRNA chimeric nanoparticles