Non-toxic Protein Research Articles

Activity of botulinum neurotoxin X and its structure when shielded by a non-toxic non-hemagglutinin protein

Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex and the crystal structure of the isolated NTNH protein. Unexpectedly, the BoNT/X complex is stable and protease-resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency both in vitro and in vivo. Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents very weak ganglioside binding and exposed hydrophobic surfaces.

From a Global Perspective Towards an Integrated Approach for Ophitoxaemia

Ophitoxaemia is the medical term that describes an injury induced by a snake bite which can be fatal in the case of venomous snakes. Bleeding, allergic response, necrosis, and bradycardia are symptoms of venomous bite. Snake venom is from a complex mixture of proteins- enzymes, nonenzymatic polypeptide toxins, and nontoxic proteins. Based on the nature of the toxin and type of snake, the toxins are classified into neuro, cardio, and cytotoxins. This comparative study aims to explore and compare the practice of the traditional Ayurveda system and the modern system of medicine for snakebite management. By examining the existing literature and research on both systems, this article will provide insights into their efficacy, safety, and potential for integration.

VISH-Pred: an ensemble of fine-tuned ESM models for protein toxicity prediction.

Peptide- and protein-based therapeutics are becoming a promising treatment regimen for myriad diseases. Toxicity of proteins is the primary hurdle for protein-based therapies. Thus, there is an urgent need for accurate in silico methods for determining toxic proteins to filter the pool of potential candidates. At the same time, it is imperative to precisely identify non-toxic proteins to expand the possibilities for protein-based biologics. To address this challenge, we proposed an ensemble framework, called VISH-Pred, comprising models built by fine-tuning ESM2 transformer models on a large, experimentally validated, curated dataset of protein and peptide toxicities. The primary steps in the VISH-Pred framework are to efficiently estimate protein toxicities taking just the protein sequence as input, employing an under sampling technique to handle the humongous class-imbalance in the data and learning representations from fine-tuned ESM2 protein language models which are then fed to machine learning techniques such as Lightgbm and XGBoost. The VISH-Pred framework is able to correctly identify both peptides/proteins with potential toxicity and non-toxic proteins, achieving a Matthews correlation coefficient of 0.737, 0.716 and 0.322 and F1-score of 0.759, 0.696 and 0.713 on three non-redundant blind tests, respectively, outperforming other methods by over $10\%$ on these quality metrics. Moreover, VISH-Pred achieved the best accuracy and area under receiver operating curve scores on these independent test sets, highlighting the robustness and generalization capability of the framework. By making VISH-Pred available as an easy-to-use web server, we expect it to serve as a valuable asset for future endeavors aimed at discerning the toxicity of peptides and enabling efficient protein-based therapeutics.

Eco-friendly disposable porous absorbents from gluten proteins through diverse plastic processing techniques

The production of biodegradable gluten-based protein foams showing complete natural degradation in soil after 26 days is reported, as an alternative to commercial foams in disposable sanitary articles that rely on non-biodegradable materials. The foams were developed from an extensive evaluation of different foaming methodologies (oven expansion, compression moulding, and extrusion), resulting in low-density foams (ca. 400 kg/m3) with homogenous pore size distributions. The products showed the ability to absorb 3–4 times their weight, reaching ranges for their use as absorbents in single-use disposable sanitary articles. An additional innovative contribution is that these gluten foams were made from natural and non-toxic wheat protein, glycerol, sodium and ammonium bicarbonate, making them useful as fossil-plastic-free replacements for commercial products without the risk of having micro-plastic and chemical pollution. The impact of different processing conditions on forming the porous biopolymer network is explained, i.e., temperature, pressure, and extensive shear forces, which were also investigated for different pH/chemical conditions. The development of micro-plastic-free foams mitigating environmental pollution and waste while using industrial co-products is fundamental for developing large-scale production of single-use items. A sanitary pad prototype is demonstrated as an eco-friendly material alternative that paves the way for sustainable practices in manufacturing, and contributes to the global effort in combating plastic pollution and waste management challenges, Sustainable Development Goals: 12, 13, 14, and 15.

Silk Fibroin Nanoparticles (SFNs) for nanoencapsulation of bioactive molecules

Silk Fibroin Nanoparticles (SFNs) have become a reliable and effective biomaterial for nanoencapsulation in several fields such as food, biocosmetics, and medical products due to their physicochemical characteristics. Recently, it has also been developed for application in biomaterials and regenerative medicine, also for cellular nanoencapsulation, and drug delivery systems. Silk fibroin is a natural biomaterial relatively not expensive, biocompatible, eco-friendly, and non-toxic FDA-approved protein derived from the Bombyx mori silkworm cocoon. Fibroin has recently been investigated in the drug field carrier for controlled release. Their application has also become widespread in regenerating therapy as a support for regenerating tissues, as well as in nanomedicine. Furthermore, SFNs have been studied as a carrier for single bioactive molecules such as resveratrol, quercetin, or curcumin for volatile compounds, and for vegetal oils in the form of emulsions. The nanoencapsulation of bioactive compounds in a biopolymer of silk fibroin can be efficiently protected from harmful environmental agents like light, oxygen, or water. The present review reveals that silk fibroin nanoparticles constitute a useful biomaterial to protect, stabilize, and deliver bioactive components. This is a promising combination in safety food, biocosmetic products, nanomedicine, and healthcare.

Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons.

Antigen processing is critical for therapeutic vaccines to generate epitopes for priming cytotoxic T cell responses against cancer and pathogens, but insufficient processing often limits the quantity of epitopes released. We address this challenge using machine learning to ascribe a proteasomal degradation score to epitope sequences. Epitopes with varying scores were translocated into cells using nontoxic anthrax proteins. Epitopes with a low score show pronounced immunogenicity due to antigen processing, but epitopes with a high score show limited immunogenicity. This work sheds light on the sequence-activity relationships between proteasomal degradation and epitope immunogenicity. We anticipate that future efforts to incorporate proteasomal degradation signals into vaccine designs will lead to enhanced cytotoxic T cell priming by these vaccines in clinical settings.

Updated review on neuromodulators

As botulinum toxin is increasingly used cosmetically and medically, it is important to understand the differences between each formulation of this product. While the active molecule in each is a 150 kDa protein that prevents neurotransmitter release, there are currently five products FDA-approved for clinical use, with a sixth currently in its Phase 3 trial and a seventh applied under FDA review, each with different nontoxic accessory proteins and varying amounts of human serum albumin. These properties affect diffusion rates and storage methods, which are outlined in this review. Common complications and recommendations to avoid them are discussed.

A non-toxic recombinant protein rSUMO-CPBm4 as a potential vaccine candidate against Clostridium perfringens type C beta enterotoxemia.

Beta toxin (CPB) is a lethal toxin and plays a key role in enterotoxemia of ruminants caused by Clostridium perfringens type C strain. The existing vaccines based on crude CPB need time-consuming detoxification and difficult quality control steps. In this study, we synthesized the rCPBm4 of C. perfringens type C strain and small ubiquitin-like modifier (SUMO)-tag CPBm4 (rSUMO-CPBm4) by introducing four amino acid substitutions: R212E, Y266A, L268G, and W275A. Compared with rCPBm4, rSUMO-CPBm4 was expressed with higher solubility in Escherichia coli BL21 (DE3). Neither rCPBm4 nor rSUMO-CPBm4 was lethal to mice. Although rCPBm4 and rSUMO-CPBm4 were reactogenic with polyclonal antibodies against crude CPB, rabbits vaccinated with rSUMO-CPBm4 developed significant levels of toxin-neutralizing antibody (TNA) titers that conferred protection against crude toxin challenge. These data suggest that genetically detoxified rSUMO-CPBm4 is a promising subunit vaccine candidate for C. perfringens type C beta enterotoxemia.

Enhanced Vaccine Immunogenicity Enabled by Targeted Cytosolic Delivery of Tumor Antigens into Dendritic Cells.

Molecular vaccines comprising antigen peptides and inflammatory cues make up a class of therapeutics that promote immunity against cancer and pathogenic diseases but often exhibit limited efficacy. Here, we engineered an antigen peptide delivery system to enhance vaccine efficacy by targeting dendritic cells and mediating cytosolic delivery. The delivery system consists of the nontoxic anthrax protein, protective antigen (PA), and a single-chain variable fragment (scFv) that recognizes the XCR1 receptor on dendritic cells (DCs). Combining these proteins enabled selective delivery of the N-terminus of lethal factor (LFN) into XCR1-positive cross-presenting DCs. Incorporating immunogenic epitope sequences into LFN showed selective protein translocation in vitro and enhanced the priming of antigen-specific T cells in vivo. Administering DC-targeted constructs with tumor antigens (Trp1/gp100) into mice bearing aggressive B16-F10 melanomas improved mouse outcomes when compared to free antigen, including suppressed tumor growth up to 58% at 16 days post tumor induction (P < 0.0001) and increased survival (P = 0.03). These studies demonstrate that harnessing DC-targeting anthrax proteins for cytosolic antigen delivery significantly enhances the immunogenicity and antitumor efficacy of cancer vaccines.

Computer-Aided Multi-Epitope Based Vaccine Design Against Monkeypox Virus Surface Protein A30L: An Immunoinformatics Approach.

Monkeypox, a viral zoonotic disease resembling smallpox, has emerged as a significant national epidemic primarily in Africa. Nevertheless, the recent global dissemination of this pathogen has engendered apprehension regarding its capacity to metamorphose into a sweeping pandemic. To effectively combat this menace, a multi-epitope vaccine has been meticulously engineered with the specific aim of targeting the cell envelope protein of Monkeypox virus (MPXV), thereby stimulating a potent immunological response while mitigating untoward effects. This new vaccine uses T-cell and B-cell epitopes from a highly antigenic, non-allergenic, non-toxic, conserved, and non-homologous A30L protein to provide protection against the virus. In order to ascertain the vaccine design with the utmost efficacy, protein-protein docking methodologies were employed to anticipate the intricate interactions with Toll-like receptors (TLR) 2, 3, 4, 6, and 8. This meticulous approach led the researchers to discern an optimal vaccine architecture, bolstered by affirmative prognostications derived from both molecular dynamics (MD) simulations and immune simulations. The current research findings indicate that the peptides ATHAAFEYSK, FFIVVATAAV, and MNSLSIFFV exhibited antigenic properties and were determined to be non-allergenic and non-toxic. Through the utilization of codon optimization and in-silico cloning techniques, our investigation revealed that the prospective vaccine exhibited a remarkable expression level within Escherichia coli. Moreover, upon conducting immune simulations, we observed the induction of a robust immune response characterized by elevated levels of both B-cell and T-cell mediated immunity. Moreover, as the initial prediction with in-silico techniques has yielded promising results these epitope-based vaccines can be recommended to in vitro and in silico studies to validate their immunogenic properties.

Structural and functional validation of a cloned parasporin from Bacillus thuringiensis isolate KAU 41 native to Western Ghats of India.

Parasporins of Bacillus thuringiensis (Bt) exhibit specific toxicity to cancer cells. PCR based mining has identified apoptosis inducing parasporin in KAU41 Bt isolate from the Western Ghats of India. The study aimed to clone and overexpress the parasporin of native KAU41 Bt isolate for determining structural and functional characteristics of the protein. Parasporin gene was cloned in pGEM-T, sequenced, sub-cloned in pET30+ and overexpressed in Escherichia coli. The expressed protein was characterized by SDS-PAGE and in silico methods. Cytotoxicity of cleaved peptide was assessed by MTT assay. SDS-PAGE displayed a 31 kDa protein (rp-KAU41) overexpressed. Upon proteinase K digestion, the protein was cleaved into 29 kDa peptide which was found to be cytotoxic to HeLa cells. The protein has a deduced sequence of 267 amino acids with β-strands folding pattern of crystal protein. Even though rp-KAU41 shared a 99.15% identity to chain-A of non-toxic crystal protein, it only showed a less similarity to the existing parasporins like PS4 (38%) and PS5 (24%) in UPGMA analysis, emphasizing the novelty of rp-KAU41. The protein is predicted to have more similarity to the pore forming toxins of Aerolysin superfamily and an additional loop in rp-KAU41 may be contributing towards its cytotoxicity. The molecular docking with caspase 3 resulted in higher Z dock and Z rank score substantiating its role in the activation of intrinsic pathway of apoptosis. The recombinant parasporin protein, rp-KAU41 is presumed to belong to the Aerolysin superfamily. An interaction with caspase 3 substantiates its role in activating the intrinsic pathway of apoptosis in cancer cells.

Characterization of Pairs of Toxic and Nontoxic Misfolded Protein Oligomers Elucidates the Structural Determinants of Oligomer Toxicity in Protein Misfolding Diseases.

The aberrant misfolding and aggregation of peptides and proteins into amyloid aggregates occurs in over 50 largely incurable protein misfolding diseases. These pathologies include Alzheimer's and Parkinson's diseases, which are global medical emergencies owing to their prevalence in increasingly aging populations worldwide. Although the presence of mature amyloid aggregates is a hallmark of such neurodegenerative diseases, misfolded protein oligomers are increasingly recognized as of central importance in the pathogenesis of many of these maladies. These oligomers are small, diffusible species that can form as intermediates in the amyloid fibril formation process or be released by mature fibrils after they are formed. They have been closely associated with the induction of neuronal dysfunction and cell death. It has proven rather challenging to study these oligomeric species because of their short lifetimes, low concentrations, extensive structural heterogeneity, and challenges associated with producing stable, homogeneous, and reproducible populations. Despite these difficulties, investigators have developed protocols to produce kinetically, chemically, or structurally stabilized homogeneous populations of protein misfolded oligomers from several amyloidogenic peptides and proteins at experimentally ameneable concentrations. Furthermore, procedures have been established to produce morphologically similar but structurally distinct oligomers from the same protein sequence that are either toxic or nontoxic to cells. These tools offer unique opportunities to identify and investigate the structural determinants of oligomer toxicity by a close comparative inspection of their structures and the mechanisms of action through which they cause cell dysfunction.This Account reviews multidisciplinary results, including from our own groups, obtained by combining chemistry, physics, biochemistry, cell biology, and animal models for pairs of toxic and nontoxic oligomers. We describe oligomers comprised of the amyloid-β peptide, which underlie Alzheimer's disease, and α-synuclein, which are associated with Parkinson's disease and other related neurodegenerative pathologies, collectively known as synucleinopathies. Furthermore, we also discuss oligomers formed by the 91-residue N-terminal domain of [NiFe]-hydrogenase maturation factor from E. coli, which we use as a model non-disease-related protein, and by an amyloid stretch of Sup35 prion protein from yeast. These oligomeric pairs have become highly useful experimental tools for studying the molecular determinants of toxicity characteristic of protein misfolding diseases. Key properties have been identified that differentiate toxic from nontoxic oligomers in their ability to induce cellular dysfunction. These characteristics include solvent-exposed hydrophobic regions, interactions with membranes, insertion into lipid bilayers, and disruption of plasma membrane integrity. By using these properties, it has been possible to rationalize in model systems the responses to pairs of toxic and nontoxic oligomers. Collectively, these studies provide guidance for the development of efficacious therapeutic strategies to target rationally the cytotoxicity of misfolded protein oligomers in neurodegenerative conditions.

Protoplast Technology and Somatic Hybridisation in the Family Apiaceae.

Species of the family Apiaceae occupy a major market share but are hitherto dependent on open pollinated cultivars. This results in a lack of production uniformity and reduced quality that has fostered hybrid seed production. The difficulty in flower emasculation led breeders to use biotechnology approaches including somatic hybridization. We discuss the use of protoplast technology for the development of somatic hybrids, cybrids and in-vitro breeding of commercial traits such as CMS (cytoplasmic male sterility), GMS (genetic male sterility) and EGMS (environment-sensitive genic male sterility). The molecular mechanism(s) underlying CMS and its candidate genes are also discussed. Cybridization strategies based on enucleation (Gamma rays, X-rays and UV rays) and metabolically arresting protoplasts with chemicals such as iodoacetamide or iodoacetate are reviewed. Differential fluorescence staining of fused protoplast as routinely used can be replaced by new tagging approaches using non-toxic proteins. Here, we focused on the initial plant materials and tissue sources for protoplast isolation, the various digestion enzyme mixtures tested, and on the understanding of cell wall re-generation, all of which intervene in somatic hybrids regeneration. Although there are no alternatives to somatic hybridization, various approaches also discussed are emerging, viz., robotic platforms, artificial intelligence, in recent breeding programs for trait identification and selection.

A non-toxic recombinant bivalent chimeric protein rETXm3CSAm4/TMD as a potential vaccine candidate against enterotoxemia and braxy.

Clostridium perfringens epsilon toxin (ETX) and Clostridium septicum alpha toxin (CSA) are lethal and necrotizing toxins, which play key roles in enterotoxemia and braxy of ruminants, respectively. In the present study, we synthesized a bivalent chimeric protein rETXm3CSAm4/TMD comprising ETXm3 (Y30A/H106P/Y196A) and CSAm4/TMD (C86L/N296A/H301A/W342A and a deletion of residues 212 to 222). Compared with recombinant ETX and recombinant CSA, rETXm3CSAm4/TMD showed no cytotoxicity in Madin-Darby Canine Kidney cells and was not fatal to mice. Moreover, rETXm3CSAm4/TMD could protect immunized mice against 10×mouse LD100 of crude ETX or 3×mouse LD100 of crude CSA without obvious histopathologic difference. Most importantly, both rabbits and sheep immunized with rETXm3CSAm4/TMD produced high titers of neutralizing antibody which protected the animals against the challenge with crude ETX or crude CSA. These data suggest that genetically detoxified rETXm3CSAm4/TMD is a potential subunit vaccine candidate against enterotoxemia and braxy.

Inducible CRISPRi-Based Operon Silencing and Selective in Trans Gene Complementation in Borrelia burgdorferi.

To accelerate genetic studies on the Lyme disease pathogen Borrelia burgdorferi, we developed an enhanced CRISPR interference (CRISPRi) approach for isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible repression of specific B. burgdorferi genes. The entire system is encoded on a compact 11-kb shuttle vector plasmid that allows for inducible expression of both the sgRNA module and a nontoxic codon-optimized dCas9 protein. We validated this CRISPRi system by targeting the genes encoding OspA and OspB, abundant surface lipoproteins coexpressed by a single operon, and FlaB, the major subunit forming the periplasmic flagella. As in other systems, single guide RNAs (sgRNAs) complementary to the nontemplate strand were consistently effective in gene repression, with 4- to 994-fold reductions in targeted transcript levels and concomitant reductions of protein levels. Furthermore, we showed that ospAB knockdowns could be selectively complemented in trans for OspA expression via the insertion of CRISPRi-resistant, synonymously or nonsynonymously mutated protospacer adjacent motif (PAM*) ospA alleles into a unique site within the CRISPRi plasmid. Together, this establishes CRISPRi PAM* as a robust new genetic tool to simplify the study of B. burgdorferi genes, bypassing the need for gene disruptions by allelic exchange and avoiding rare codon toxicity from the heterologous expression of dCas9. IMPORTANCE Borrelia burgdorferi, the spirochetal bacterium causing Lyme disease, is a tick-borne pathogen of global importance. Here, we expand the genetic toolbox for studying B. burgdorferi physiology and pathogenesis by establishing a single plasmid-based, fully inducible, and nontoxic CRISPR interference (CRISPRi) system for transcriptional silencing of B. burgdorferi genes and operons. We also show that alleles of CRISPRi-targeted genes with mutated protospacer-adjacent motif (PAM*) sites are CRISPRi resistant and can be used for simultaneous in trans gene complementation. The CRISPRi PAM* system will streamline the study of essential Borrelia proteins and accelerate investigations into their structure-function relationships.

Crystal structures of OrfX1, OrfX2 and the OrfX1-OrfX3 complex from the orfX gene cluster of botulinum neurotoxin E1.

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A-G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1-OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs.

Preclinical evaluation of an investigational 21-valent pneumococcal conjugate vaccine, V116, in adult-rhesus monkey, rabbit, and mouse models.

Despite the widespread effectiveness of pneumococcal conjugate vaccines on the overall incidence of invasive pneumococcal disease, the global epidemiological landscape continues to be transformed by residual disease from non-vaccine serotypes, thus highlighting the need for vaccines with expanded disease coverage. To address these needs, we have developed V116,an investigational 21-valent non-adjuvanted pneumococcal conjugate vaccine (PCV),containingpneumococcal polysaccharides (PnPs) 3, 6A, 7F, 8, 9N, 10A, 11A,12F, 15A, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F, 35B, anda de-O-acetylated 15B(deOAc15B) individually conjugated to the nontoxic diphtheria toxoid CRM197 carrier protein. Preclinical studies evaluated the immunogenicity of V116 inadult monkeys, rabbits, and mice. Following one dose, V116 was found to be immunogenic in preclinical animal species and induced functional antibodies for all serotypes included in the vaccine, in addition to cross-reactive functional antibodies to serotypes 6C and 15B. In these preclinical animal studies, the increased valency of V116 did not result in serotype-specific antibody suppression when compared to lower valent vaccines V114 or PCV13. In addition, when compared with naïve controls, splenocytes from V116 to immunized animals demonstrated significant induction of CRM197-specific T cells in both IFN-γ and IL-4 ELISPOT assays, as well as Th1 and Th2 cytokine induction through in vitro stimulation assays, thus suggesting the ability of V116 to engage T cell dependent immune response pathways to aid in development of memory B cells. V116 also demonstrated significant protection in mice from intratracheal challenge with serotype 24F, a novel serotype not contained in any currently licensed vaccine.

The structural characterization and color stabilization of the pigment protein-phycoerythrin glycosylated with oligochitosan

Phycoerythrin (PE) is a kind of natural and nontoxic pigment protein with a distinctive phycobilin, and this pigment is easy to be destroyed in the process of food application, which makes its stability weak and application limited. In this work, PE was extracted by freeze-thaw method from Porphyra haitanensis, and the transglutaminase (10 U g−1 protein) was used to catalyze the glycosylation reaction between PE and oligochitosan with molar ratios of 1:1 to 4:1 (oligochitosan/PE) to obtain the oligochitosan-modified phycoerythrin complexes (OMPC). The glycosylation of oligochitosan to PE was proven by the polyacrylamide gel electrophoresis and was evidenced by the glucosamine and free amino analysis. When the molar ratio of oligochitosan to PE was 3:1, the glucosamine content of OMPC was the highest, which was 31.403 ± 0.004 mg g−1 protein. The OMPC samples were studied by infrared spectrum, fluorescence spectrum, and scanning electron microscopy. The results showed that the secondary structure and tertiary structure of glycosylated products changed, and a compact polymer appeared. In addition, compared with untreated PE, the surface hydrophobicity and emulsifying activity of OMPC decreased, while the solubility, emulsifying stability, thermal stability, photostability, and pH stability were significantly improved. This study reveals an innovative method to stabilize PE by a glycosylation approach, which will advance PE usage as a natural colorant in food applications.

Risk-Assessment-Based Optimization Favours the Development of Albumin Nanoparticles with Proper Characteristics Prior to Drug Loading.

Albumin nanocarrier research and development is a challenging area in the field of personalized medicine and in providing advanced therapeutic solutions. Albumin as a biocompatible, nonimmunogenic, and non-toxic protein carrier that can be exploited to conjugate drugs with poor bioavailability to improve on this feature. With many different perspectives and desired target profiles, a systematic structural approach must be used in nanoparticle development. The extended Research and Development (R&D) Quality by Design thinking and methodology proved to be useful in case of specific nanoparticle development processes before. However, the coacervation method is the most frequently applied preparation method for HSA nanoparticles; there is a lack of existing research work which has directly determined the influence of process parameters, control strategy, or design space. With a quality-management-driven strategy, a knowledge space was developed for these versatile nanoparticles and an initial risk assessment was conducted on the quality-affecting factors regarding the coacervation method, followed by an optimization process via Plackett–Burman and Box–Behnken experimental design. As a result of screening the effect of process variables on the fabrication of HSA nanoparticles, an optimized colloidal drug delivery system was engineered with desired nanoparticulate properties.

Green chemistry method for hair strengthening and setting using visible light-mediated protein crosslinking

A clean and nontoxic one-step protein crosslinking reaction method induced by visible light was developed using eco-friendly chemicals, such as riboflavin phosphate (RFP), citric acid (CA), and polyols . In this study, oxygen radicals generated by blue light (BL)-irradiated RFP replace the conventional free thiol generatio step of the alkali condition. CA and polyol were used as crosslinkers and extenders , respectively, to enhance keratin crosslinking in hair. The new one-step crosslinking method was compared with the existing permanent hair waving treatment using thioglycolic acid (TGA). Compared to TGA treatment, the newly proposed crosslinking method induced minor damage to hair and keratin. In addition, the mechanical property changes were measured after crosslinking treatment on the hair. The tensile stress values and Young's moduli of the treated hair samples were approximately 1.3 and 2.2 times higher than untreated hair. The hair-setting effect was also confirmed by the changes in the radii of the hair fibers and the length of the hair tresses after crosslinking treatment. Therefore, we believe that the clean one-step crosslinking technology can be used to develop eco-friendly products for hair strengthening and setting. • Clean one-step crosslinking of keratin is effective in strengthening and setting hair. • Keratin crosslinking is induced by exposure to blue light in the presence of RFP. • Citric acid and polyols form extender acts as a linker for keratin crosslinking.