Physico-Chemical Characterization and Initial Evaluation of Carboxymethyl Chitosan-Hyaluronan Hydrocolloid Systems with Insulin Intended for Intranasal Administration.

The nasal route of administration can bypass the blood-brain barrier in order to obtain a higher concentration in the brain, thus offering a feasible alternative route of administration for diseases associated with the central nervous system. The advantages of the intranasal administration and the potential favorable therapeutic effects of intranasally administered insulin led to the formulation of carboxymethyl chitosan (CMC) and sodium hyaluronate (NaHA) hydrocolloidal systems with insulin for nasal administration, targeting nose-to-brain delivery and the initial assessment of these systems. The influence of the formulation variables on the response parameters defined as surface properties, rheology, and in vitro release of insulin were analyzed using experimental design and statistical programs (Modde and Minitab software). The systems recorded good wetting and adhesion capacity, allowing the spread of the hydrocolloidal systems on the nasal mucosa. The samples had a pseudoplastic flow and the rapid release of the insulin was according to our objective. According to the physico-chemical characterization and preliminary assessment, these formulations are appropriate for administration on the nasal mucosa, but further studies are necessary to demonstrate the beneficial therapeutic actions and the safety of using intranasal insulin.

Mucosal Penetrative Polymeric Micelle Formulations for Insulin Delivery to the Respiratory Tract.

Purpose: Effective mucosal delivery of drugs continues to pose a significant challenge owing to the formidable barrier presented by the respiratory tract mucus, which efficiently traps and clears foreign particulates. The surface characteristics of micelles dictate their ability to penetrate the respiratory tract mucus. In this study, polymeric micelles loaded with insulin (INS) were modified using mucus-penetrative polymers. Methods: We prepared and compared polyethylene glycol (PEG)-coated micelles with micelles where cell-penetrating peptide (CPP) is conjugated to PEG. Systematic investigations of the physicochemical and aerosolization properties, performance, in vitro release, mucus and cell penetration, lung function, and pharmacokinetics/pharmacodynamics (PK/PD) of polymeric micelles were performed to evaluate their interaction with the respiratory tract. Results: The nano-micelles, with a particle size of <100 nm, exhibited a sustained-release profile. Interestingly, PEG-coated micelles exhibited higher diffusion and deeper penetration across the mucus layer. In addition, CPP-modified micelles showed enhanced in vitro cell penetration. Finally, in the PK/PD studies, the micellar solution demonstrated higher maximum concentration (Cmax) and AUC0-8h values than subcutaneously administered INS solution, along with a sustained blood glucose-lowering effect that lasted for more than 8 h. Conclusion: This study proposes the use of mucus-penetrating micelle formulations as prospective inhalation nano-carriers capable of efficiently transporting peptides to the respiratory tract.

Deciphering the Molecular Dance: Exploring the Dynamic Interplay Between Mouse Insulin B9-23 Peptides and their Variants.

Type 1 diabetes results from the autoimmune destruction of pancreatic insulin-producing ß-cells, primarily targeted by autoreactive T cells that recognize insulin B9-23 peptides as antigens. Using drift tube ion mobility spectrometry-mass spectrometry, transmission electron microscopy, and two-dimensional infrared spectroscopy, we characterized mouse insulin 1 B9-23 (Ins1 B9-23), insulin 2 B9-23 (Ins2 B9-23), along with two of their mutants, Ins2 B9-23 Y16A and Ins2 B9-23 C19S. Our findings indicate that Ins1 B9-23 and the Ins2 Y16A mutant exhibit rapid fibril formation, whereas Ins2 B9-23 and the Ins2 C19S mutant show slower fibrillization and a structural rearrangement from globular protofibrils to fibrillar aggregates. These differences in aggregation behaviors also manifest in interactions with (-)epigallocatechin gallate (EGCG), a canonical amyloid inhibitor. EGCG effectively disrupts the fibrils formed by Ins1 B9-23 and the Y16A mutant. However, it proves ineffective in preventing fibril formation of Ins2 B9-23 and the C19S mutant. These results establish a strong correlation between the aggregation behaviors of these peptides and their divergent effects on anti-islet autoimmunity.

Study of Insulin Aggregation and Fibril Structure under Different Environmental Conditions.

Protein amyloid aggregation is linked with widespread and fatal neurodegenerative disorders as well as several amyloidoses. Insulin, a small polypeptide hormone, is associated with injection-site amyloidosis and is a popular model protein for in vitro studies of amyloid aggregation processes as well as in the search for potential anti-amyloid compounds. Despite hundreds of studies conducted with this specific protein, the procedures used have employed a vast array of different means of achieving fibril formation. These conditions include the use of different solution components, pH values, ionic strengths, and other additives. In turn, this variety of conditions results in the generation of fibrils with different structures, morphologies and stabilities, which severely limits the possibility of cross-study comparisons as well as result interpretations. In this work, we examine the condition-structure relationship of insulin amyloid aggregation under a range of commonly used pH and ionic strength conditions as well as solution components. We demonstrate the correlation between the reaction solution properties and the resulting aggregation kinetic parameters, aggregate secondary structures, morphologies, stabilities and dye-binding modes.

Structural insights into i-motif DNA structures in sequences from the insulin-linked polymorphic region.

The insulin-linked polymorphic region is a variable number of tandem repeats region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of tandem repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the insulin linked polymorphic region form different DNA structures in vitro. Additionally, reporter genes in cellulo indicate that insulin expression may change depending on which DNA structures form. We report the crystal structure and dynamics of an intramolecular i-motif, which reveal sequences within the loop regions forming additional stabilising interactions that are critical to formation of stable i-motif structures. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to target i-motif DNA.

Optimization of a mass spectrometric analytical method for the quality assessment of insulin and its analogs.

The principal aim of this study was to optimize analytical methodology based on mass spectrometry for the evaluation of the quality of recombinant human insulin and its analogs. In this study ESI-MS was used to assess the quality of human insulin, short acting insulin analogs, insulin lispro, insulin aspart and insulin glulisine and long acting analogs including insulin glargine, insulin degludec, and insulin detemir, in respective pharmaceutical formulations. In this study, with the aimed to optimize analytical conditions, different factors influencing the analytical performance such as pH, ionic strength, sample dilution, organic solvent addition were addressed. The study results demonstrated that MS is a suitable technique for the analysis of biotechnological compounds like insulin and its analogs. Although the obtained results provide an important information regarding this methodology, further studies are needed to validate this analytical approach and check for its suitability to be used in the regulatory environment.

Exploring the amyloid degradation potential of nanoformulated carrageenan-bridging in vitro and in vivo perspectives.

Amyloids, with their ß-sheet-rich structure, contribute to diabetes, neurodegenerative diseases, and amyloidosis by aggregating within diverse anatomical compartments. Insulin amyloid (IA), sharing structural resemblances with amyloids linked to neurological disorders, acts as a prototype, while compounds capable of degrading these fibrils hold promise as therapeutic agents for amyloidosis intervention. In this research, liposomal nanoformulated iota carrageenan (nCG) was formulated to disrupt insulin amyloids, demonstrating about a 17-20 % higher degradation efficacy compared to conventional carrageenan through thioflavin T fluorescence, dynamic light scattering analysis, and turbidity quantification. The biocompatibility of the nCG and nCG-treated insulin amyloids was evaluated through MTT assay, live-dead cell assay on V79 cells, and hemolysis testing on human blood samples to establish their safety for use in vitro. Zebrafish embryos were utilized to assess in vivo biocompatibility, while adult zebrafish were employed to monitor the degradation capacity of IA post subcutaneous injection, with fluorescence emitted by the fish captured via IVIS. This demonstrated that the formulated nCG exhibited superior anti-amyloid efficacy compared to carrageenan alone, while both materials demonstrated biocompatibility. Furthermore, through docking simulations, an exploration was conducted into the molecular mechanisms governing the inhibition of the target protein pancreatic insulin by carrageenan.

Quinoline yellow acts as a novel amyloid fibrillation probe by using surface-enhanced Raman spectroscopy.

Protein amyloid fibrillation is linked to a wide range of neurodegenerative diseases. Protein oligomer is an intermediate substance in the process of fibrillation, which is neurotoxic and formed by the aggregation of protein molecules under physiological stress. Early detection of protein oligomers could make timely intervention of protein fibrillation related diseases. Therefore, it is crucial to develop efficient inhibitors and probes for monitoring amyloid fibril formation. In this study, we developed a novel amyloid inhibitor quinoline yellow (QY), which was proved to be effective in inhibiting insulin protein fibrillation as demonstrated by fluorescence, morphology characterization and circular dichroism. When QY binds to insulin, it exerts inhibitory effects on the nucleation process and effectively impedes the formation of fibrillar fibrils. In addition, we present the application of surface-enhanced Raman spectroscopy (SERS) as an extremely sensitive technique for identifying amyloid oligomers. The investigation employed the probe QY, which demonstrated a linear reaction for identifying oligomers in the concentration range of 1.0-58.0 µM. Impressively, it showcased an exceptionally sensitive detection threshold of 0.2 µM. And also illustrating the binding sites and interaction mechanisms between small molecules of QY and insulin by SERS. The aforementioned methodology was also employed for the identification of insulin oligomers in human serum samples. Thereby, the proposed approach presenting a promising avenue with extensive implications in the realms of pharmaceutical exploration and disease diagnosis.

Martini 3 OliGoÌ mers: A Scalable Approach for Multimers and Fibrils in GROMACS.

Martini 3 is a widely used coarse-grained simulation method for large-scale biomolecular simulations. It can be combined with a Go̅ model to realistically describe higher-order protein structures while allowing the folding and unfolding events. However, as of today, this method has largely been used only for individual monomers. In this article, we describe how the Go̅ model can be implemented within the framework of Martini 3 for a multimer system, taking into account both intramolecular and intermolecular interactions in an oligomeric protein system. We demonstrate the method by showing how it can be applied to both structural stability maintenance and assembly/disassembly of protein oligomers, using aquaporin tetramer, insulin dimer, and amyloid-ß fibril as examples. We find that addition of intermolecular Go̅ potentials stabilizes the quaternary structure of proteins. The strength of the Go̅ potentials can be tuned so that the internal fluctuations of proteins match the behavior of atomistic simulation models, however, the results also show that the use of too strong intermolecular Go̅ potentials weakens the chemical specificity of oligomerization. The Martini-Go̅ model presented here enables the use of Go̅ potentials in oligomeric molecular systems in a computationally efficient and parallelizable manner, especially in the case of homopolymers, where the number of identical protein monomers is high. This paves the way for coarse-grained simulations of large protein complexes, such as viral protein capsids and prion fibrils, in complex biological environments.

pH-sensitive semi-interpenetrating network of microcrystalline cellulose and methacrylic acid hydrogel for the oral delivery of insulin.

Insulin is commonly administered to diabetic patients subcutaneously and has shown poor patient compliance. Due to this, research has been carried out extensively to find molecules that could deliver insulin orally. In this context, a new type of pH-responsive hydrogel, composed of microcrystalline cellulose and methacrylic acid-based hydrogels, has been developed and studied for the oral delivery of insulin. These hydrogels were prepared by free radical polymerization using potassium persulphate as initiator and N, N'-methylenebisacrylamide as a cross-linker. These pH-sensitive hydrogels showed swelling in distilled water as high as 5800 %. The hydrogels were investigated for swelling in saline and glucose solutions, and pH sensitivity was confirmed by swelling in solutions of different pH. The morphological shape was established by SEM, and the structure was analyzed by FTIR. Thermal degradation was investigated by TGA. In vitro release studies have confirmed pH sensitivity, showing lower insulin release at pH 1.2 than at pH 6.8. The encapsulation efficiency was determined to be 56.00 ± 0.04 %. It was further validated by in-vivo investigations for which insulin was loaded into hydrogels and administered orally to healthy and diabetic Wistar rats at 40 IU/kg, showing maximum hypoglycemic effect at 6 h, which was sustained for 24 h. In the stomach's acidic environment, the gels remained unaffected due to the formation of intermolecular polymer complexes. Insulin remained in the gel and was protected from proteolytic degradation. Thus, pH-responsive methacrylic acid-based hydrogels are promising for biomedical applications, especially oral drug delivery.

Surface-catalyzed liquid-liquid phase separation and amyloid-like assembly in microscale compartments.

Liquid-liquid phase separation is a key phenomenon in the formation of membrane-less structures within the cell, appearing as liquid biomolecular condensates. Protein condensates are the most studied for their biological relevance, and their tendency to evolve, resulting in the formation of aggregates with a high level of order called amyloid. In this study, it is demonstrated that Human Insulin forms micrometric, round amyloid-like structures at room temperature within sub-microliter scale aqueous compartments. These distinctive particles feature a solid core enveloped by a fluid-like corona and form at the interface between the aqueous compartment and the glass coverslip upon which they are cast. Quantitative fluorescence microscopy is used to study in real-time the formation of amyloid-like superstructures. Their formation results driven by liquid-liquid phase separation process that arises from spatially heterogeneous distribution of nuclei at the glass-water interface. The proposed experimental setup allows modifying the surface-to-volume ratio of the aqueous compartments, which affects the aggregation rate and particle size, while also inducing fine alterations in the molecular structures of the final assemblies. These findings enhance the understanding of the factors governing amyloid structure formation, shedding light on the catalytic role of surfaces in this process.

Carbon dots fluorescence can be used to distinguish isobaric peptide and to monitor protein oligomerization dynamics.

Carbon dots (CD) are widely investigated particles with interesting fluorescent properties which are reported to be used for various purposes, as they are biocompatible, resistant to photobleaching and with tuneable properties depending on the specific CD surface chemistry. In this work, we report on the possibility to use opportunely designed CD to distinguish among isobaric peptides almost undistinguishable by mass spectrometry, as well as to monitor protein aggregation phenomena. Particularly, cell-penetrating peptides containing the carnosine moiety at different positions in the peptide chain produce sequence specific fluorescent signals. Analogously, different insulin oligomerization states can also be distinguished by the newly proposed experimental approach. The latter is here described in details and can be potentially applied to any kind of peptide or protein.

Assessment of subcutaneously administered insulins using in vitro release cartridge: Medium composition and albumin binding.

Biotherapeutics is the fastest growing class of drugs administered by subcutaneous injection. In vitro release testing mimicking physiological conditions at the injection site may guide formulation development and improve biopredictive capabilities. Here, anin vitrorelease cartridge (IVR cartridge) comprising a porous agarose matrix emulating subcutaneous tissue was explored. The objective was to assess effects of medium composition and incorporation of human serum albumin into the matrix. Drug disappearance was assessed for solution, suspension and in situ precipitating insulin products (Actrapid, Levemir, Tresiba, Mixtard 30, Insulatard, Lantus) using the flow-based cartridge. UV-Vis imaging and light microscopy visualized dissolution, precipitation and albumin binding phenomena at the injection site. Divalent cations present in the release medium resulted in slower insulin disappearance for suspension-based and in situ precipitating insulins. Albumin-binding acylated insulin analogs exhibited rapid disappearance from the cartridge; however, sustained retention was achieved by coupling albumin to the matrix. An in vitro-in vivorelation was established for the non-albumin-binding insulins.The IVR cartridge is flexible with potential in formulation development as shown by the ability to accommodate solutions, suspensions, and in situ forming formulations while tailoring of the system to probe in vivo relevant medium effects and tissue constituent interactions.

Competitive protein adsorption in isocratic anion exchange chromatography.

Protein chromatography is the dominant method of purification of biopharmaceuticals. Although all practical chromatography involves competitive absorption and separation of M. species, competitive protein absorption has remained inadequately understood. We previously introduced the measurement of equilibrium protein adsorption isotherms with all intensive variables held constant, including competitor concentration. In this work, we introduce isocratic chromatographic retention measurements of dynamic protein adsorption in the presence of a constant concentration of a competitor protein. These measurements are achieved by establishing a dynamic equilibrium with a constant concentration of competitor (insulin) in the mobile phase flowing through an ion exchange adsorbent column and following the behavior of a test protein (α-lactalbumin) injected into this environment. We observed decreased retention times for α-lactalbumin in presence of the competitor. The presence of competitor also reduces the heterogeneity of the sites available for adsorption of the test protein. This investigation provides an approach to fundamental understanding of competitive dynamics of multicomponent protein chromatography.

Reversible Conjugation of Polypeptides and Proteins Utilizing a [3.3.1] Scaffold under Mild Conditions.

An investigation of reversible protein conjugation and deconjugation is presented. Despite numerous available protein conjugation methods, there has been limited documentation of achieving protein conjugation in a controlled and reversible manner. This report introduces a protocol that enables protein modification in a multicomponent fashion under aqueous buffer and mild conditions. A readily available mercaptobenzaldehyde derivative can modify the primary amine of peptides and proteins with a distinctive [3.3.1] scaffold. This modification can be reversed under mild conditions in a controlled fashion, restoring the original protein motif. The effectiveness of this approach has been demonstrated in the modification and quantifiable regeneration of insulin protein.

Evolution of biosynthetic human insulin and its analogues for diabetes management.

Hormones play a crucial role in maintaining the normal human physiology. By acting as chemical messengers that facilitate the communication between different organs, tissues and cells of the body hormones assist in responding appropriately to external and internal stimuli that trigger growth, development and metabolic activities of the body. Any abnormalities in the hormonal composition and balance can lead to devastating health consequences. Hormones have been important therapeutic agents since the early 20th century, when it was realized that their exogenous supply could serve as a functional substitution for those hormones which are not produced enough or are completely lacking, endogenously. Insulin, the pivotal anabolic hormone in the body, was used for the treatment of diabetes mellitus, a metabolic disorder due to the absence or intolerance towards insulin, since 1921 and is the trailblazer in hormone therapeutics. At present the largest market share for therapeutic hormones is held by insulin. Many other hormones were introduced into clinical practice following the success with insulin. However, for the six decades following the introduction the first therapeutic hormone, there was no reliable method for producing human hormones. The most common source for hormones were animals, although semisynthetic and synthetic hormones were also developed. However, none of these were optimal because of their allergenicity, immunogenicity, lack of consistency in purity and most importantly, scalability. The advent of recombinant DNA technology was a game changer for hormone therapeutics. This revolutionary molecular biology tool made it possible to synthesize human hormones in microbial cell factories. The approach allowed for the synthesis of highly pure hormones which were structurally and biochemically identical to the human hormones. Further, the fermentation techniques utilized to produce recombinant hormones were highly scalable. Moreover, by employing tools such as site directed mutagenesis along with recombinant DNA technology, it became possible to amend the molecular structure of the hormones to achieve better efficacy and mimic the exact physiology of the endogenous hormone. The first recombinant hormone to be deployed in clinical practice was insulin. It was called biosynthetic human insulin to reflect the biological route of production. Subsequently, the biochemistry of recombinant insulin was modified using the possibilities of recombinant DNA technology and genetic engineering to produce analogues that better mimic physiological insulin. These analogues were tailored to exhibit pharmacokinetic and pharmacodynamic properties of the prandial and basal human insulins to achieve better glycemic control. The present chapter explores the principles of genetic engineering applied to therapeutic hormones by reviewing the evolution of therapeutic insulin and its analogues. It also focuses on how recombinant analogues account for the better management of diabetes mellitus.

Cu(II) Specifically Disassembles Insulin Amyloid Nanostructures via Direct Interaction with Cross-ß Fibrils.

In this work, we demonstrate direct evidence of the antiamyloid potential of Cu(II) ions against amyloid formation of insulin. The Cu(II) ions were found to efficiently disassemble the preformed amyloid nanostructures into soluble species and suppress monomer fibrillation under aggregation-prone conditions. The direct interaction of Cu(II) ions with the cross-ß structure of amyloid fibrils causes substantial disruption of both the interchain and intrachain interactions, predominantly the H-bonds and hydrophobic contacts. Further, the Cu(II) ions show a strong affinity for the aggregation-prone conformers of the protein and inhibit their spontaneous self-assembly. These results reveal the possible molecular mechanism for the antiamyloidogenic potential of Cu(II) which could be important for the development of metal-ion specific therapeutic strategies against amyloid linked complications.

Challenges in Peptide Solubilization - Amyloids Case Study.

Peptide science has been a rapidly growing research field because of the enormous potential application of these biocompatible and bioactive molecules. However, many factors limit the widespread use of peptides in medicine, and low solubility is among the most common problems that hamper drug development in the early stages of research. Solubility is a crucial, albeit poorly understood, feature that determines peptide behavior. Several different solubility predictors have been proposed, and many strategies and protocols have been reported to dissolve peptides, but none of them is a one-size-fits-all method for solubilization of even the same peptide. In this review, we look for the reasons behind the difficulties in dissolving peptides, analyze the factors influencing peptide aggregation, conduct a critical analysis of solubilization strategies and protocols available in the literature, and give some tips on how to deal with the so-called difficult sequences. We focus on amyloids, which are particularly difficult to dissolve and handle such as amyloid beta (Aß), insulin, and phenol-soluble modulins (PSMs).

Expression and biochemical characterization of the putative insulinase enzyme PF11_0189 found in the Plasmodium falciparum genome.

PF11_0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11_0189 is about 27 kDa. Substrate specificity study shows PF11_0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11_0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11_0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11_0189 show inhibition with an IC50 value of 4.8 µM and 7.5 µM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11_0189 with an IC50 value of 0.75 µM suggesting PF11_0189 as a potential target for peptide inhibitors. The study implicates that PF11_0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria.

Monitoring insulin fibrillation kinetics using chromatographic analysis.

Insulin is a small protein widely used to treat patients with diabetes and is a commonly used model for protein fibrillation studies. Under specific conditions, such as low pH and high temperature, insulin monomers aggregate to form fibrils. This aggregation is problematic for manufacturing and storage of insulin. The thioflavin T (ThT) assay is commonly used to study amyloid fibrillation but suffers from several limitations, such as the effect of protein concentration, the size of the amyloid fibrillar bundles, competitive binding, and fibril aggregation, all of which hinder precise quantitative analysis. Here, we present a method for studying the kinetics of insulin fibrillation utilizing ultra-performance liquid chromatography (UPLC). This method enables the quantitative detection of soluble insulin components, including chemically modified components. The formation of a deamidated species could be monitored at the early stage of fibrillation, and this species was likely included in the fibrils. In addition, in the presence of inhibitors known to compete with ThT for binding to fibrils, UPLC analysis showed the disappearance of soluble components even though the ThT assay did not indicate the presence of fibrils. These results suggest that the UPLC-based analysis presented here can complement the ThT assay for investigating the kinetics of protein fibrillation.