High-performance peptide and disulfide mapping by direct injection of intact proteins using on-line coupled UV-liquid chromatography microdroplet mass spectrometry (UVLC-MMS).

Microdroplet mass spectrometry (MMS), achieving ultra-fast enzyme digestion in the ionization source, holds great promises for innovating protein analysis. Here, in-depth protein characterization is demonstrated by direct injection of intact protein mixtures via on-line coupling MMS with capillary C4 liquid chromatography (LC) containing UV windows (UVLC-MMS) through an enzyme introduction tee. We showed complete sets of peptides of individual proteins (hemoglobin, bovine serum albumin, and ribonuclease A) in a mixture could be obtained in one injection. Such full (100%) sequence coverage, however, could not be achieved by conventional nanoLC-MS method using bottom-up approach with single enzyme. Moreover, direct injection of a chaperone α-crystalline (α-Cry) complex yielded identification of post-translational modifications including novel sites and semi-quantitative characterization including 3:1 stoichiometry ratio of αA- and αB-Cry sub-units and ∼1.4 phosphorylation/subunit on S45 (novel site) and S122 (main site) of αA-Cry, ∼0.7 phosphorylation/subunit on S19 (main site) and S45 of αB-Cry, as well as 100% acetylation on both N-termini of each subunits by matching the mass and retention time of the intact and its digested peptides. Furthermore, trifluoroacetic acid was able to be used in the mobile phase with UVLC-MMS to improve the separation of differentially reduced intact species and detectability of the droplet-digested products. This allowed us to completely map four disulfide linkages of ribonuclease A based on collision-induced dissociation of disulfide clusters, some of which would otherwise not be detected, preventing scrambling or shuffling errors arising from lengthy bulk solution digestion by the bottom-up approach. Integration of UVLC and MMS greatly improves droplet digestion efficiency and MS detection, enabling highly efficient workflow for in-depth and accurate protein characterization.

The inhibitory effect of curcumin loaded poly (vinyl caprolactam) nanohydrogel on insulin fibrillation.

Amyloidosis refers to a group of diseases caused by the deposition of abnormal proteins in tissues. Herein, curcumin was loaded in a nanohydrogel made of poly (vinylcaprolactam) to improve its solubility and was employed to exert an inhibitory effect on insulin fibrillation, as a protein model. Poly (vinyl caprolactam), cross-linked with polyethylene glycol diacrylate, was synthesized by the reversible addition-fragmentation chain transfer method. The release profile of curcumin exhibited a first-order kinetic model, signifying that the release of curcumin was mainly dominated by diffusion processes. The study of curcumin release showed that 78% of the compound was released within 72 h. The results also revealed a significant decline in insulin fibrillation in the presence of curcumin-loaded poly (vinyl caprolactam). These observations confirmed that increasing the ratio of curcumin-loaded poly (vinyl caprolactam) to insulin concentration would increase the hydrogel's inhibitory effect (P-value < 0.05). Furthermore, transmission electron and fluorescence microscopies and Fourier-transform infrared spectroscopy made it possible to study the size and interaction of fibrils. Based on the results, this nanohydrogel combination could protect the structure of insulin and had a deterrent effect on fibril formation.

Instability Challenges and Stabilization Strategies of Pharmaceutical Proteins.

Maintaining the structure of protein and peptide drugs has become one of the most important goals of scientists in recent decades. Cold and thermal denaturation conditions, lyophilization and freeze drying, different pH conditions, concentrations, ionic strength, environmental agitation, the interaction between the surface of liquid and air as well as liquid and solid, and even the architectural structure of storage containers are among the factors that affect the stability of these therapeutic biomacromolecules. The use of genetic engineering, side-directed mutagenesis, fusion strategies, solvent engineering, the addition of various preservatives, surfactants, and additives are some of the solutions to overcome these problems. This article will discuss the types of stress that lead to instabilities of different proteins used in pharmaceutics including regulatory proteins, antibodies, and antibody-drug conjugates, and then all the methods for fighting these stresses will be reviewed. New and existing analytical methods that are used to detect the instabilities, mainly changes in their primary and higher order structures, are briefly summarized.

Physically stimulus-responsive nanoparticles for therapy and diagnosis.

Nanoparticles offer numerous advantages in various fields of science, particularly in medicine. Over recent years, the use of nanoparticles in disease diagnosis and treatments has increased dramatically by the development of stimuli-responsive nano-systems, which can respond to internal or external stimuli. In the last 10 years, many preclinical studies were performed on physically triggered nano-systems to develop and optimize stable, precise, and selective therapeutic or diagnostic agents. In this regard, the systems must meet the requirements of efficacy, toxicity, pharmacokinetics, and safety before clinical investigation. Several undesired aspects need to be addressed to successfully translate these physical stimuli-responsive nano-systems, as biomaterials, into clinical practice. These have to be commonly taken into account when developing physically triggered systems; thus, also applicable for nano-systems based on nanomaterials. This review focuses on physically triggered nano-systems (PTNSs), with diagnostic or therapeutic and theranostic applications. Several types of physically triggered nano-systems based on polymeric micelles and hydrogels, mesoporous silica, and magnets are reviewed and discussed in various aspects.

A review on biofilms and the currently available antibiofilm approaches: Matrix-destabilizing hydrolases and anti-bacterial peptides as promising candidates for the food industries.

Biofilms are communities of microorganisms that can be harmful and/or beneficial, depending on location and cell content. Since in most cases (such as the formation of biofilms in laboratory/medicinal equipment, water pipes, high humidity-placed structures, and the food packaging machinery) these bacterial and fungal communities are troublesome, researchers in various fields are trying to find a promising strategy to destroy or slow down their formation. In general, anti-biofilm strategies are divided into the plant-based and non-plant categories, with the latter including nanoparticles, bacteriophages, enzymes, surfactants, active peptides and free fatty acids. In most cases, using a single strategy will not be sufficient to eliminate biofilm, and consequently, two or more strategies will inevitably be used to deal with this unwanted phenomenon. According to the analysis of potential biofilm inhibition strategies, the best option for the food industry would be the use of hydrolase enzymes and peptides extracted from natural sources. This article represents a systematic review of the previous efforts made in these directions.

Mechanisms behind the Fibrillation and Toxicity of Insulin Fibrils on Neuron Cells by Engineered Curcumin Analogs.

Among foods, the use of plant derivatives as promising drugs and/or excipients has been considered from various perspectives. In the present study, curcumin, which is one of the most important plant derivatives for biological uses, and four curcumin-based pyrido[2,3-d]pyrimidine analogs (C2-C5) were used for investigating the mechanism of insulin fibrillation and evaluating the cytotoxicity of insulin fibrils. The synthesized analogs differed in terms of hydrophobicity and electrostatic charge. The analogs with more hydrophobicity (C1 and C4) in both acidic and neutral environments were able to reduce the rate of insulin fibrillation and the degree of cross-linking in the produced fibrils. Additionally, the toxicity of these fibrils for neural cells (N2a cell line) was very low. However, they did not show any significant effects on the toxicity of non-neural cells (HEK293 cell line), indicating the effect of the biochemical surface diversity on determining the vulnerability to fibrils and even the mechanism of action of additives on cell line survival. Although negatively charged analogs were able to reduce insulin fibrillation in the acidic environment, they indicated an opposite effect in the neutral environment. The resultant fibrils in the acidic medium appeared with a well-distinguished filament, but they were very close at neutral pH levels. Moreover, such fibrils indicated very poor toxicity against the N2a cell line and had no significant effects on HEK293 cells. Considering the docking studies, by creatively using the size exclusion chromatography, it was suggested that analogs C2 and C3 were capable of binding to the C-terminal end of the insulin B chain (low affinity) and HisB10 (high affinity). Hence, it was suggested that different compounds could play different protecting and/or destroying roles in cell toxicity by blocking some ligands at the surface of neuron cells.

Biological aspects in controlling angiogenesis: current progress.

All living beings continue their life by receiving energy and by excreting waste products. In animals, the arteries are the pathways of these transfers to the cells. Angiogenesis, the formation of the arteries by the development of pre-existed parental blood vessels, is a phenomenon that occurs naturally during puberty due to certain physiological processes such as menstruation, wound healing, or the adaptation of athletes' bodies during exercise. Nonetheless, the same life-giving process also occurs frequently in some patients and, conversely, occurs slowly in some physiological problems, such as cancer and diabetes, so inhibiting angiogenesis has been considered to be one of the important strategies to fight these diseases. Accordingly, in tissue engineering and regenerative medicine, the highly controlled process of angiogenesis is very important in tissue repairing. Excessive angiogenesis can promote tumor progression and lack of enough angiogensis can hinder tissue repair. Thereby, both excessive and deficient angiogenesis can be problematic, this review article introduces and describes the types of factors involved in controlling angiogenesis. Considering all of the existing strategies, we will try to lay out the latest knowledge that deals with stimulating/inhibiting the angiogenesis. At the end of the article, owing to the early-reviewed mechanical aspects that overshadow angiogenesis, the strategies of angiogenesis in tissue engineering will be discussed.

Nanoparticles for Coronavirus Control.

More than 2 years have passed since the SARS-CoV-2 outbreak began, and many challenges that existed at the beginning of this pandemic have been solved. Some countries have been able to overcome this global challenge by relying on vaccines against the virus, and vaccination has begun in many countries. Many of the proposed vaccines have nanoparticles as carriers, and there are different nano-based diagnostic approaches for rapid detection of the virus. In this review article, we briefly examine the biology of SARS-CoV-2, including the structure of the virus and what makes it pathogenic, as well as describe biotechnological methods of vaccine production, and types of the available and published nano-based ideas for overcoming the virus pandemic. Among these issues, various physical and chemical properties of nanoparticles are discussed to evaluate the optimal conditions for the production of the nano-mediated vaccines. At the end, challenges facing the international community and biotechnological answers for future viral attacks are reviewed.

Bioactive Peptides: Synthesis, Sources, Applications, and Proposed Mechanisms of Action.

Bioactive peptides are a group of biological molecules that are normally buried in the structure of parent proteins and become active after the cleavage of the proteins. Another group of peptides is actively produced and found in many microorganisms and the body of organisms. Today, many groups of bioactive peptides have been marketed chemically or recombinantly. This article reviews the various production methods and sources of these important/ubiquitous and useful biomolecules. Their applications, such as antimicrobial, antihypertensive, antioxidant activities, blood-lipid-lowering effect, opioid role, antiobesity, ability to bind minerals, antidiabetic, and antiaging effects, will be explored. The types of pathways proposed for bioactive applications will be in the next part of the article, and at the end, the future perspectives of bioactive peptides will be reviewed. Reading this article is recommended for researchers interested in various fields of physiology, microbiology, biochemistry, and nanotechnology and food industry professionals.

A novel method for the chaperone aided and efficient production of human proinsulin in the prokaryotic system.

With the rapid spread of diabetes in human society, the demand for insulin and its precursor (proinsulin) continues to rise. Therefore, the introduction of new methods for their production is essential. In the present study, human proinsulin, while ligated to αB-crystallin chaperone, was effectively expressed in the prokaryotic host system and then purified by the ion-exchange chromatography at high purity (>97%). In the next step, human proinsulin with relatively high efficiency was released chemically from the hybrid protein (αB-pIns) and then purified using an appropriate gel filtration column. The SDS-PAGE and HPLC analyses confirmed the high purity, while mass spectroscopy assessment verified the exact molecular mass of the human proinsulin. Using a well-established protocol, the protein was folded in a one-step folding process with a yield of about 70%. The assessment of the secondary structures of the human proinsulin by Raman and FTIR spectroscopy suggested that this protein is rich in α-helix. Also, the conformation of disulfide bonds in the folded proinsulin was confirmed by Raman spectroscopy. The recombinant human proinsulin also demonstrated hypoglycemic activity and mitogenic action (induction of cell proliferation). The method proposed in this work for the production of human proinsulin is easy to run and does not depend on expensive and complex equipment. Thus, it can be used in the industrial production of human proinsulin.

Drug-based therapeutic strategies for COVID-19-infected patients and their challenges.

Emerging epidemic-prone diseases have introduced numerous health and economic challenges in recent years. Given current knowledge of COVID-19, herd immunity through vaccines alone is unlikely. In addition, vaccination of the global population is an ongoing challenge. Besides, the questions regarding the prevalence and the timing of immunization are still under investigation. Therefore, medical treatment remains essential in the management of COVID-19. Herein, recent advances from beginning observations of COVID-19 outbreak to an understanding of the essential factors contributing to the spread and transmission of COVID-19 and its treatment are reviewed. Furthermore, an in-depth discussion on the epidemiological aspects, clinical symptoms and most efficient medical treatment strategies to mitigate the mortality and spread rates of COVID-19 is presented.

Insulin therapy; a valuable legacy and its future perspective.

Proteins and peptides are widely used in various areas including pharmaceutical, health, food, textile and biofuel industries. At present, pharmaceutical proteins and peptides have attracted the attention of many researchers. These types of drugs are superior to chemical drugs in many ways so that every year the number of drugs with a protein or peptide moiety is increasing. Due to high performance and low side effects, the demand for these drugs has increased year by year. The beginning of the protein and peptide drug industry dates back to 1982 with the introduction of the protein hormone insulin into the field of treatment. From this year onwards, a new number of protein and peptide drugs have entered the field of treatment every year. In this article, we focus on human therapeutic insulin. First, the history of the hormone will be introduced, then-current methods for insulin therapy will be discussed and finally, the treatments by this hormone in the future will be pointed. Reading this article would be very helpful for nano researchers, biochemists, organic chemists, material scientists and other people who are interested in soft and hard matters interfaces.

Applications of Graphene and Graphene Oxide in Smart Drug/Gene Delivery: Is the World Still Flat?

Graphene, a wonder material, has made far-reaching developments in many different fields such as materials science, electronics, condensed physics, quantum physics, energy systems, etc. Since its discovery in 2004, extensive studies have been done for understanding its physical and chemical properties. Owing to its unique characteristics, it has rapidly became a potential candidate for nano-bio researchers to explore its usage in biomedical applications. In the last decade, remarkable efforts have been devoted to investigating the biomedical utilization of graphene and graphene-based materials, especially in smart drug and gene delivery as well as cancer therapy. Inspired by a great number of successful graphene-based materials integrations into the biomedical area, here we summarize the most recent developments made about graphene applications in biomedicine. In this paper, we review the up-to-date advances of graphene-based materials in drug delivery applications, specifically targeted drug/ gene delivery, delivery of antitumor drugs, controlled and stimuli-responsive drug release, photodynamic therapy applications and optical imaging and theranostics, as well as investigating the future trends and succeeding challenges in this topic to provide an outlook for future researches.

Insulin fibrillation: toward strategies for attenuating the process.

Dark days for diabetic patients were transformed into an era of hope when the therapeutic usage of insulin was discovered. However, those initial glory days changed to being somewhat gloomy, when it was discovered that insulin easily undergoes undesirable, fast, and non-reversible aggregation and fibrillation. After more than half a century of intensive attempts to limit the rate of the insulin aggregation and fibrillation, there is no clear-cut strategy for eliminating these processes once and for all. A plethora of studies focused on using various organic compounds to combat the process, whereas other researchers believe that the process can be inhibited (or altered) by well-designed nanoparticles. In an attempt to inhibit insulin aggregation, some other approaches, such as protein/peptide inhibitors, have been considered for therapeutic purposes. Beyond biological processes and interactions between biological molecules, there are also strong physicochemical laws. Therefore, the goal of this article is to provide an overview of chemical, physical, and biological studies dedicated to the analysis of approaches that attenuate and inhibit insulin aggregation and fibrillation. After a detailed characterization of the insulin fibrillation process, this review focuses on various aspects related to the inhibition and modulation of insulin fibrillation using nanoparticles, proteins/peptides, and cyclic and non-cyclic compounds. Hopefully, these findings will pave the way for scientists in various fields to increase the stability of pharmaceutical proteins and peptides.

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.

Characterization of insulin cross-seeding: the underlying mechanism reveals seeding and denaturant-induced insulin fibrillation proceeds through structurally similar intermediates.

Insulin rapidly fibrillates in the presence of amyloid seeds from different sources. To address its cross-reactivity we chose the seeds of seven model proteins and peptides along with the seeds of insulin itself. Model candidates were selected/designed according to their size, amino acid sequence, and hydrophobicity. We found while some seeds provided catalytic ends for inducing the formation of non-native insulin conformers and increase fibrillation, others attenuated insulin fibrillation kinetics. We also observed competition between the intermediate insulin conformers which formed with urea and amyloid seeds in entering the fibrillogenic pathway. Simultaneous incubation of insulin with urea and amyloid seeds resulted in the formation of nearly similar insulin intermediate conformers which synergistically enhance insulin fibrillation kinetics. Given these results, it is highly likely that, structurally, there is a specific intermediate in different pathways of insulin fibrillation that governs fibrillation kinetics and morphology of the final mature fibril. Overall, this study provides a novel mechanistic insight into insulin fibrillation and gives new information on how seeds of different proteins are capable of altering insulin fibrillation kinetics and morphology. This report, for the first time, tries to answer an important question that why fibrillation of insulin is either accelerated or attenuated in the presence of amyloid fibril seeds from different sources.

Inhibitory effect of coumarin and its analogs on insulin fibrillation /cytotoxicity is depend on oligomerization states of the protein.

Looking through a historical lens, attention to the stabilization of pharmaceutical proteins/peptides has been dramatically increased. Human insulin is the most challenging and the most widely used pharmaceutical protein in the world. In this study, the protein and coumarin as a plant-derived phenolic compound and two coumarin analogs with different moieties were investigated to evaluate the protein fibrillation and cytotoxicity. The obtained data showed that with a change in environmental pH, the behavior of the compounds on the process of insulin fibrillation will be changed completely. Coumarin (C1) and its hydrophobic analog, 7-methyl coumarin (C2), in an acidic environment, inhibit insulin fibrillation, change the oligomerization state of insulin and produce fibrils with notable lateral interactions with low cytotoxicity. However, negatively-charged 3-trifluoromethyl coumarin (C3) without significant changes in insulin structure and by altering the oligomerization state of the protein, slightly accelerates hormone fibrillation. Also, the compounds showed a disulfide protecting role during protein aggregation. Regarding the toxicity of the fibrils, it was observed that in addition to the secondary structures of proteinous fibrils, the ability to destroy the cell membrane is also related to the length of the fibrils and their degree of lateral interactions. By and large, this work can be useful in finding a better formulation for bio-pharmaceutical macro-molecules.

Modulating Insulin Fibrillation Using Engineered B-Chains with Mutated C-Termini.

Stress-induced unfolding and fibrillation of insulin represent serious medical and biotechnological problems. Despite many attempts to elucidate the molecular mechanisms of insulin fibrillation, there is no general agreement on how this process takes place. Several previous studies suggested the importance of the C-terminal region of B-chain in this pathway. Therefore, we generated the T30R and K29R/T30R mutants of insulin B-chain. Recombinantly produced wild-type A-chain and mutant B-chains were combined efficiently in the presence of chaperone αB-crystallin. The mutant B-chains along with the control wild-type insulin were used in a wide range of parallel experiments to compare their fibrillation kinetics, morphology of fibrils, and forces driving the fibril formation. The mutant insulins and their B-chains displayed significant resistance against stress-induced fibrillation, particularly at the nucleation stage, suggesting that the B-chain might be influencing the insulin fibrillation. The fact that the different mature insulins formed larger fibrillar bundles compared to those formed by their B-chains alone suggested the role of A-chain in the lateral association of the insulin fibrils. Overall, in addition to the N-terminal region of the B-chain, which was shown to serve as an important regulator of insulin fibrillation, the C-terminal region of this peptide is also crucial for the control of fibrillation, likely serving as an attachment site engaged in the formation of the nucleus and protofibril. Finally, two mutated insulin variants examined in this study might be of interest to the pharmaceutical sector as, to our knowledge, novel intermediate-acting insulin analogs because of their suitable biological activity and improved stability against stress-induced fibrillation.

Human αB-crystallin as fusion protein and molecular chaperone increases the expression and folding efficiency of recombinant insulin.

Low expression and instability are significant challenges in the recombinant production of therapeutic peptides. The current study introduces a novel expression and purification system for human insulin production using the molecular chaperone αB-crystallin (αB-Cry) as a fusion partner protein. Insulin is composed of A- and B-chain containing three disulfide bonds (one intarchain and two interchains). We have constructed two plasmids harboring the A- or B-chain of insulin joined with human αB-Cry. This system is suitable for cloning of the genes and for directing the synthesis of large amounts of the fusion proteins αB-Cry/A-chain (αB-AC) and αB-Cry/B-chain (αB-BC). The construction of vectors, their efficient expression in Escherichia coli and simple purification of the fusion proteins and two insulin chains are described. A large amount of the recombinant fusion proteins with high purity was obtained by applying a single step anion exchange chromatography or metal chelate affinity. The insulin A- and B-chain were released from the fusion proteins using cyanogen bromide cleavage. The insulin peptides were obtained with an appreciable yield and high purity using one-step gel filtration chromatography. To increase efficiency of chain combination to produce insulin, αB-Cry was used under oxidative conditions. The purification of natively folded insulin was performed by phenyl sepharose hydrophobic interaction chromatography. Finally, using an insulin tolerance test in mice and various biophysical methods, the structure and function of purified human recombinant insulin was compared with authentic insulin, to verify folding of insulin to its native state. Overall, the novel expression system using αB-Cry is highly demanding for producing human insulin and functional protein. The procedure for αB-Cry-mediated insulin folding could be also applicable for the large-scale production of this highly important therapeutic peptide hormone.

Chemical modifications of insulin: Finding a compromise between stability and pharmaceutical performance.

Insulin, a key peptide hormone that conjointly with its receptor regulates blood glucose levels, is used as the major means to treat diabetes. This therapeutic hormone may undergo different chemical modifications during industrial processes, pharmaceutical formulation, and through its endogenous storage in the pancreatic ß-cells. Insulin is highly sensitive to the environmental stresses and easily undergoes structural changes, being also able to unfold and aggregate. Even small changes altering the structural integrity of insulin may have important impact on the biological efficacy in relation to the physiological and pharmacological activities of this hormone. The chemical modifications of insulin may occur either randomly or based on the well-planned strategies to enhance its pharmaceutical properties. A plethora of studies have attempted to answer the fundamental questions of how chemical modifications and which environmental conditions may have either destructive effects or improve structural stability and pharmaceutical performance of insulin. The aim of this review is to highlight the impact of different modifications on structure, stability, biological activity, and pharmaceutical properties of insulin.