Recent Advances in Dietary Sources, Health Benefits, Emerging Encapsulation Methods, Food Fortification, and New Sensor-Based Monitoring of Vitamin B12: A Critical Review.

In this overview, the latest achievements in dietary origins, absorption mechanism, bioavailability assay, health advantages, cutting-edge encapsulation techniques, fortification approaches, and innovative highly sensitive sensor-based detection methods of vitamin B12 (VB12) were addressed. The cobalt-centered vitamin B is mainly found in animal products, posing challenges for strict vegetarians and vegans. Its bioavailability is highly influenced by intrinsic factor, absorption in the ileum, and liver reabsorption. VB12 mainly contributes to blood cell synthesis, cognitive function, and cardiovascular health, and potentially reduces anemia and optic neuropathy. Microencapsulation techniques improve the stability and controlled release of VB12. Co-microencapsulation of VB12 with other vitamins and bioactive compounds enhances bioavailability and controlled release, providing versatile initiatives for improving bio-functionality. Nanotechnology, including nanovesicles, nanoemulsions, and nanoparticles can enhance the delivery, stability, and bioavailability of VB12 in diverse applications, ranging from antimicrobial agents to skincare and oral insulin delivery. Staple food fortification with encapsulated and free VB12 emerges as a prominent strategy to combat deficiency and promote nutritional value. Biosensing technologies, such as electrochemical and optical biosensors, offer rapid, portable, and sensitive VB12 assessment. Carbon dot-based fluorescent nanosensors, nanocluster-based fluorescent probes, and electrochemical sensors show promise for precise detection, especially in pharmaceutical and biomedical applications.

Semisynthesis of Aminomethyl-Insulin: An Atom-Economic Strategy to Increase the Affinity and Selectivity of a Protein for Recognition by a Synthetic Receptor.

Advancements in the molecular recognition of insulin by nonantibody-based means would facilitate the development of methodology for the continuous detection of insulin for the management of diabetes mellitus. Herein, we report a novel insulin derivative that binds to the synthetic receptor cucurbit[7]uril (Q7) at a single site and with high nanomolar affinity. The insulin derivative was prepared by a four-step protein semisynthetic method to present a 4-aminomethyl group on the side chain of the PheB1 position. The resulting aminomethyl insulin binds to Q7 with an equilibrium dissociation constant value of 99 nM in neutral phosphate buffer, as determined by isothermal titration calorimetry. This 6.8-fold enhancement in affinity versus native insulin was gained by an atom-economical modification (-CH2NH2). To the best of our knowledge, this is the highest reported binding affinity for an insulin derivative by a synthetic receptor. This strategy for engineering protein affinity tags induces minimal change to the protein structure while increasing affinity and selectivity for a synthetic receptor.

A beginner's guideline for low-cost 3D printing of macromolecules usable for teaching and demonstration.

The structure and function of biomolecules relationship is the hallmark of biochemistry, molecular biology, and life sciences in general. Physical models of macromolecules give students the possibility to manipulate these structures in three dimensions, developing a sense of spatiality and a better understanding of key aspects such as atom size and shape, bond lengths and symmetry. Several molecular model systems were developed specifically to represent particular classes or groups of molecules and hence lack the flexibility of a universal solution. Three-dimensional printing could nevertheless provide such a universal solution, as it can be used to create physical models of biomolecular structures based on the teacher's or demonstrator's needs and requirements. Here, insulin was used as a model molecule and several depiction and printing parameters were tested in order to highlight the technical limitations of the approach. In the end, a set of settings that worked is provided which could serve as a starting point for anyone wishing to print his or her own custom macromolecular model on the cheap.

Regulatory considerations in biosimilars: Latin America region.

Development of biotherapeutic products has experienced steady growth over the past three decades. Expiration of patents on many biotherapeutics such as insulin, human growth hormone, and erythropoietin has opened the door for the development of biosimilars. The high cost of biotherapeutics has limited their accessibility, particularly in developing countries. Biosimilars offer the much- needed affordability and hence improved accessibility. Global agencies such as the World Health Organization are engaged in developing a prequalification program in order to help countries that do not have strong regulatory systems. This article summarizes the prospects of biosimilars in the Latin American market. Legal framework in various countries is also discussed.

The discovery of insulin revisited: lessons for the modern era.

2021 to 2022 marks the one hundredth anniversary of ground-breaking research in Toronto that changed the course of what was, then, a universally fatal disease: type 1 diabetes. Some would argue that insulin's discovery by Banting, Best, Macleod, and Collip was the greatest scientific advance of the 20th century, being one of the first instances in which modern medical science was able to provide lifesaving therapy. As with all scientific discoveries, the work in Toronto built upon important advances of many researchers over the preceding decades. Furthermore, the Toronto work ushered in a century of discovery of the purification, isolation, structural characterization, and genetic sequencing of insulin, all of which influenced ongoing improvements in therapeutic insulin formulations. Here we discuss the body of knowledge prior to 1921 localizing insulin to the pancreas and establishing insulin's role in glucoregulation, and provide our views as to why researchers in Toronto ultimately achieved the purification of pancreatic extracts as a therapy. We discuss the pharmaceutical industry's role in the early days of insulin production and distribution and provide insights into why the discoverers chose not to profit financially from the discovery. This fascinating story of bench-to-beside discovery provides useful considerations for scientists now and in the future.

Estrogenization of insulin by catecholestrogen produced high affinity autoantibodies and altered the normal function of insulin in type 1 diabetes.

AIMS: Insulin (Ins) covalently modified by catecholestrogens (CEs) was commonly found in diabetic patients who have developed insulin resistance. Estrogenization of insulin altered its molecular function and effect carbohydrates metabolisms in these patients. Insulin resistance is a common phenomenon in diabetes but the exact mechanism remains unknown. In this study, binding specificity and affinity of autoantibodies against estrogenized insulin (4-hydroxyestradiol-insulin; 4-OHE2-Ins) were assayed in the serum of type 1 diabetes (T1D) patients in order to explain the phenomena behind insulin resistance. MATERIALS AND METHODS: Specificity and affinity of autoantibodies from the sera of 66 T1D patients and 41 controls were analyzed by direct binding, competition ELISA and quantitative precipitin titration. Insulin was also estimated in the serum of T1D patients by ELISA. KEY FINDING: Estrogenized insulin (4-OHE2-Ins) exhibited high affinity and specificity to T1D autoantibodies in comparison to Ins (p < .05) or 4-OHE2 (p < .001). Estrogenization of insulin alters its interaction with the insulin receptor (IR). The affinity constant of 4-OHE2-Ins with the T1D autoantibodies was found to be 1.41 × 10-7 M. SIGNIFICANCE: Estrogenization of insulin by catecholestrogen makes these molecules highly antigenic and produced high-affinity autoantibodies in T1D patients. As a result, patients develop insulin resistance and presented this molecule as a potential biomarker for T1D.

FT-IR versus EC-QCL spectroscopy for biopharmaceutical quality assessment with focus on insulin-total protein assay and secondary structure analysis using attenuated total reflection.

For the quality control of biopharmaceutical products, which contain proteins as the most important active ingredients, shelf life may be limited due to inappropriate storage conditions or mechanical stress. For insulins as representatives of life-saving pharmaceuticals, analytical methods are needed, which are providing additional information than obtained by assays for total protein quantification. Despite sophisticated formulations, the chemical stability may be challenged by temperatures deviating from recommended conditions or shear rate exposure under storage, leading to misfolding, nucleation, and subsequent fibril formation, accompanied by a decrease in bioactivity. A reliable method for insulin quantification and determination of secondary structure changes has been developed by attenuated total reflection (ATR) Fourier-transform infrared spectroscopy of insulin formulations by a silver halide fiber-coupled diamond probe with subsequent dry-film preparation. A special emphasis has been placed on the protein amide I band evaluation, for which spectral band analysis provides unique information on secondary structure fractions for intact and misfolded insulins. Quantitative measurements are possible down to concentrations of less than 0.5 mg/ml, whereas the dry-film preparation delivers high signal-to-noise ratios due to the prior water evaporation, thus allowing a reliable determination of secondary structure information. Graphical abstract.

Mechanistic Assessment of Functionalized Mesoporous Silica-Mediated Insulin Fibrillation.

Insulin, which is a small protein hormone consisting of 51 amino acids, rapidly fibrillates under stressogenic conditions. This biotechnological/medical problematic reaction quickly accelerates in the presence of some particles, while there are several other particles that slow down the kinetic process. To address the unexplored demand of the particles that modulate protein fibrillation, we have synthesized two amino-based particles and a chitosan-coated mesoporous silica particle (MS-NH2, MS-3NH2, and MS-chitosan) to investigate insulin fibrillation. While these particles were fairly similar in size, they are differ in their net positive charge and surface hydrophobicity. To monitor the exact role of the hydrophobic interaction between the protein and MS-chitosan during the fibrillation, we have also co- and preincubated insulin with cholesterol and the particles under stressogenic conditions. The results indicate that MS-NH2 and MS-3NH2, due to their high positive charges and lack of surface hydrophobicity, repel the positively charged unfolded insulins at pH 2.0. Moreover, MS-chitosan with 25% surface hydrophobicity stacks partially unfolded insulins to its surface and induces some α-helix to ß-sheet structural transitions to the protein. Consequently, both amino- and chitosan-based particles slow down the kinetics of the fibrillation. We also showed that cholesterol can structurally participate in insulin fibril architecture as a hydrophobic bridge, and extraction of this molecule from the preformed fibrils may disrupt the fibril structure.

Combined Experimental and Molecular Simulation Study of Insulin-Chitosan Complexation Driven by Electrostatic Interactions.

Protein-polysaccharide complexes constructed via self-assembly methods are often used to develop novel biomaterials for a wide range of applications in biomedicine, food, and biotechnology. The objective of this work was to investigate theoretically and to demonstrate via constant-pH Monte Carlo simulations that the complexation phenomenon between insulin (INS) and the cationic polyelectrolyte chitosan (CS) is mainly driven by an electrostatic mechanism. Experimental results obtained from FTIR spectra and ζ-potential determinations allowed us to complement the conclusions. The characteristic absorption bands for the complexes could be assigned to a combination of signals from CS amide I and INS amide II. The second peak corresponds to the interaction between the polymer and the protein at the level of amide II. INS-CS complexation processes not expected when INS is in its monomeric form, but for both tetrameric and hexameric forms, incipient complexation due to charge regulation mechanism took place at pH 5. The complexation range was observed to be 5.5 < pH < 6.5. In general, when the number of INS units increases in the simulation process, the solution pH at which the complexation can occur shifts toward acidic conditions. CS's chain interacts more efficiently, i.e. in a wider pH range, with INS aggregates formed by the highest monomer number. The charge regulation mechanism can be considered as a previous phase toward complexation (incipient complexation) caused by weak interactions of a Coulombic nature.

Formulary Considerations for Insulins Approved Through the 505(b)(2) "Follow-on" Pathway.

OBJECTIVE: To summarize formulary-relevant issues for follow-on insulins approved through the Food and Drug Administration (FDA) 505(b)(2) approval pathway (Basaglar and Admelog). DATA SOURCES: A search of the MEDLINE database was performed for articles pertaining to clinical and formulary considerations for follow-on insulin products through July 2018. STUDY SELECTION AND DATA EXTRACTION: All clinical trials used in the 505(b)(2) approval process for follow-on insulin glargine and insulin lispro products were included and summarized. DATA SYNTHESIS: Follow-on insulin glargine and insulin lispro products have been recently approved as the first lower-cost alternatives to innovator insulin products. The follow-on insulins were approved via the 505(b)(2) pathway, making them neither generics nor biosimilars. Current data do not suggest any clinically relevant differences between the follow-on insulins and their respective innovator products. Clinicians should be aware that follow-on insulins will be reclassified as biologic products in the year 2020. Relevance to Patient Care and Clinical Practice: This article provides information about currently available follow-on insulin products that were approved through the 505(b)(2) pathway, including product characteristics and efficacy and safety data. These products will likely be considered for both clinical use and formulary placement because of their potentially lower cost compared with innovator products. CONCLUSIONS: Follow-on insulin products approved through the 505(b)(2) pathway are supported by robust efficacy and safety data. As new follow-on insulins are approved and the regulatory change that will occur with these products in 2020 approaches, formulary decisions and clinical policies (eg, substitution) will continue to be revisited.