Scalable microfluidic method for tunable liposomal production by a design of experiment approach.

Liposomes constitute a widespread drug delivery platform, gaining more and more attention from the pharmaceutical industry and process development scientists. Their large-scale production as medicinal products for human use is all but trivial, especially when parenteral administration is required. In this study an off-the-shelf microfluidic system and a methodological approach are presented for the optimization, validation and scale-up of highly monodisperse liposomes manufacturing. Starting from a Doxil®-like formulation (HSPC, MPEG-DSPE and cholesterol), a rational approach (Design of Experiments, DoE) was applied for the screening of the process parameters affecting the quality attributes of the product (mainly size and polydispersity). Additional DoEs were conducted to determine the effect of critical process parameters "CPPs" (cholesterol concentration, total flow rate "TFR" and flow rate ratio "FRR"), thus assessing the formulation and process robustness. A scale-up was then successfully accomplished. The procedure was applied to a Marqibo®-like formulation as well (sphingomyelin and cholesterol) to show the generality of the proposed formulation, process development and scale-up approach. The application of the system and method herein presented enables the large-scale manufacturing of liposomes, in compliance with the internationally recognized regulatory standards for pharmaceutical development (Quality by Design).

Anaerobic demethylation of guaiacyl-derived monolignols enabled by a designed artificial cobalamin methyltransferase fusion enzyme.

Lignin-derived aryl methyl ethers (e.g. coniferyl alcohol, ferulic acid) are expected to be a future carbon source for chemistry. The well-known P450 dependent biocatalytic O-demethylation of these aryl methyl ethers is prone to side product formation especially for the oxidation sensitive catechol products which get easily oxidized in the presence of O2. Alternatively, biocatalytic demethylation using cobalamin dependent enzymes may be used under anaerobic conditions, whereby two proteins, namely a methyltransferase and a carrier protein are required. To make this approach applicable for preparative transformations, fusion proteins were designed connecting the cobalamin-dependent methyltransferase (MT) with the corrinoid-binding protein (CP) from Desulfitobacterium hafniense by variable glycine linkers. From the proteins created, the fusion enzyme MT-L5-CP with the shortest linker performed best of all fusion enzymes investigated showing comparable and, in some aspects, even better performance than the separated proteins. The fusion enzymes provided several advantages like that the cobalamin cofactor loading step required originally for the CP could be skipped enabling a significantly simpler protocol. Consequently, the biocatalytic demethylation was performed using Schlenk conditions allowing the O-demethylation e.g. of the monolignol coniferyl alcohol on a 25 mL scale leading to 75% conversion. The fusion enzyme represents a promising starting point to be evolved for alternative demethylation reactions to diversify natural products and to valorize lignin.

Thiols Act as Methyl Traps in the Biocatalytic Demethylation of Guaiacol Derivatives.

Demethylating methyl phenyl ethers is challenging, especially when the products are catechol derivatives prone to follow-up reactions. For biocatalytic demethylation, monooxygenases have previously been described requiring molecular oxygen which may cause oxidative side reactions. Here we show that such compounds can be demethylated anaerobically by using cobalamin-dependent methyltransferases exploiting thiols like ethyl 3-mercaptopropionate as a methyl trap. Using just two equivalents of this reagent, a broad spectrum of substituted guaiacol derivatives were demethylated, with conversions mostly above 90 %. This strategy was used to prepare the highly valuable antioxidant hydroxytyrosol on a one-gram scale in 97 % isolated yield.

Thiols Act as Methyl Traps in the Biocatalytic Demethylation of Guaiacol Derivatives.

Demethylating methyl phenyl ethers is challenging, especially when the products are catechol derivatives prone to follow-up reactions. For biocatalytic demethylation, monooxygenases have previously been described requiring molecular oxygen which may cause oxidative side reactions. Here we show that such compounds can be demethylated anaerobically by using cobalamin-dependent methyltransferases exploiting thiols like ethyl 3-mercaptopropionate as a methyl trap. Using just two equivalents of this reagent, a broad spectrum of substituted guaiacol derivatives were demethylated, with conversions mostly above 90 %. This strategy was used to prepare the highly valuable antioxidant hydroxytyrosol on a one-gram scale in 97 % isolated yield.

Oxygen-Free Regioselective Biocatalytic Demethylation of Methyl-phenyl Ethers via Methyltransfer Employing Veratrol-O-demethylase.

The cleavage of aryl methyl ethers is a common reaction in chemistry requiring rather harsh conditions; consequently, it is prone to undesired reactions and lacks regioselectivity. Nevertheless, O-demethylation of aryl methyl ethers is a tool to valorize natural and pharmaceutical compounds by deprotecting reactive hydroxyl moieties. Various oxidative enzymes are known to catalyze this reaction at the expense of molecular oxygen, which may lead in the case of phenols/catechols to undesired side reactions (e.g., oxidation, polymerization). Here an oxygen-independent demethylation via methyl transfer is presented employing a cobalamin-dependent veratrol-O-demethylase (vdmB). The biocatalytic demethylation transforms a variety of aryl methyl ethers with two functional methoxy moieties either in 1,2-position or in 1,3-position. Biocatalytic reactions enabled, for instance, the regioselective monodemethylation of substituted 3,4-dimethoxy phenol as well as the monodemethylation of 1,3,5-trimethoxybenzene. The methyltransferase vdmB was also successfully applied for the regioselective demethylation of natural compounds such as papaverine and rac-yatein. The approach presented here represents an alternative to chemical and enzymatic demethylation concepts and allows performing regioselective demethylation in the absence of oxygen under mild conditions, representing a valuable extension of the synthetic repertoire to modify pharmaceuticals and diversify natural products.

Regioselectivity of Cobalamin-Dependent Methyltransferase Can Be Tuned by Reaction Conditions and Substrate.

Regioselective reactions represent a significant challenge for organic chemistry. Here the regioselective methylation of a single hydroxy group of 4-substituted catechols was investigated employing the cobalamin-dependent methyltransferase from Desulfitobacterium hafniense. Catechols substituted in position four were methylated either in meta- or para-position to the substituent depending whether the substituent was polar or apolar. While the biocatalytic cobalamin dependent methylation was meta-selective with 4-substituted catechols bearing hydrophilic groups, it was para-selective for hydrophobic substituents. Furthermore, the presence of water miscible co-solvents had a clear improving influence, whereby THF turned out to enable the formation of a single regioisomer in selected cases. Finally, it was found that also the pH led to an enhancement of regioselectivity for the cases investigated.

[Professional policies: a qualitative study of perceptions and opinions of professionals nurses/midwives and students attending the Master of Science in Nursing]. / Politiche professionali: studio qualitativo sulle percezioni e le opinioni dei professionisti infermieri/ostetriche e degli studenti della Laurea magistrale in Scienze infermieristiche e ostetriche.

INTRODUCTION: Politics, the science and the art of administering public affairs, is a very important field. Dealing with professional policy means to dedicate ourselves to the good of the profession. Professional policies are an integral part of taking care; the professional family needs clear reference points: training, tutoring, promotion and institutional representation. AIM: Exploring perceptions, ideals, shared imagination and desires about the professional policies of nurses/midwives and of master's students in order to be able to have a representation of the reality and to start a critical reflection on the subject. METHOD: A qualitative study was conducted on a proactive sample of 22 nurses / midwives as qualified witnesses of the national situation. The data was collected through a questionnaire built ad hoc. RESULTS: From the analysis of the answers given to the questionnaire, we identified 10 main themes. In the training sector two of the themes that were selected are the importance of the new knowledges and inadequate differentiation of the positions; Regarding the working sector two of the themes that came out are the lack of meritocracy and of new opportunities; meanwhile, in the research field one of the main theme identified is the gap between the theory and the practice. CONCLUSION: The present study finds out that participation in policy-making processes is occasional and of little intensity: just few nurses/midwives are actively involved in the promotion and development of projects in the health sector and of healthcare policies.