Advanced Noninvasive Strategies for the Brain Delivery of Therapeutic Proteins and Peptides.

Recent years have witnessed rapid progress in the discovery of therapeutic proteins and peptides for the treatment of central nervous system (CNS) diseases. However, their clinical applications have been considerably hindered by challenges such as low biomembrane permeability, poor stability, short circulation time, and the formidable blood-brain barrier (BBB). Recently, substantial improvements have been made in understanding the dynamics of the BBB and developing efficient approaches for delivering proteins and peptides to the CNS, especially by using various nanoparticles. Herein, we present an overview of the up-to-date understanding of the BBB under physiological and pathological conditions, emphasizing their effects on brain drug delivery. We summarize advanced strategies and elucidate the underlying mechanisms for delivering proteins and peptides to the brain. We highlight the developments and applications of nanocarriers in treating CNS diseases via BBB crossing. We also provide critical opinions on the limitations and obstacles of the current strategies and put forward prospects for future research.

Subcellular targeting strategies for protein and peptide delivery.

Cytosolic delivery of proteins and peptides provides opportunities for effective disease treatment, as they can specifically modulate intracellular processes. However, most of protein-based therapeutics only have extracellular targets and are cell-membrane impermeable due to relatively large size and hydrophilicity. The use of organelle-targeting strategy offers great potential to overcome extracellular and cell membrane barriers, and enables localization of protein and peptide therapeutics in the organelles. Although progresses have been made in the recent years, organelle-targeted protein and peptide delivery is still challenging and under exploration. We reviewed recent advances in subcellular targeted delivery of proteins/peptides with a focus on targeting mechanisms and strategies, and highlight recent examples of active and passive organelle-specific protein and peptide delivery systems. This emerging platform could open a new avenue to develop more effective protein and peptide therapeutics.

Mechanical release of homogenous proteins from supramolecular gels.

A long-standing challenge is how to formulate proteins and vaccines to retain function during storage and transport and to remove the burdens of cold-chain management. Any solution must be practical to use, with the protein being released or applied using clinically relevant triggers. Advanced biologic therapies are distributed cold, using substantial energy, limiting equitable distribution in low-resource countries and placing responsibility on the user for correct storage and handling. Cold-chain management is the best solution at present for protein transport but requires substantial infrastructure and energy. For example, in research laboratories, a single freezer at -80 °C consumes as much energy per day as a small household1. Of biological (protein or cell) therapies and all vaccines, 75% require cold-chain management; the cost of cold-chain management in clinical trials has increased by about 20% since 2015, reflecting this complexity. Bespoke formulations and excipients are now required, with trehalose2, sucrose or polymers3 widely used, which stabilize proteins by replacing surface water molecules and thereby make denaturation thermodynamically less likely; this has enabled both freeze-dried proteins and frozen proteins. For example, the human papilloma virus vaccine requires aluminium salt adjuvants to function, but these render it unstable against freeze-thaw4, leading to a very complex and expensive supply chain. Other ideas involve ensilication5 and chemical modification of proteins6. In short, protein stabilization is a challenge with no universal solution7,8. Here we designed a stiff hydrogel that stabilizes proteins against thermal denaturation even at 50 °C, and that can, unlike present technologies, deliver pure, excipient-free protein by mechanically releasing it from a syringe. Macromolecules can be loaded at up to 10 wt% without affecting the mechanism of release. This unique stabilization and excipient-free release synergy offers a practical, scalable and versatile solution to enable the low-cost, cold-chain-free and equitable delivery of therapies worldwide.

Laponite nanoclays for the sustained delivery of therapeutic proteins.

Protein therapeutics hold immense promise for treating a wide array of diseases. However, their efficacy is often compromised by rapid degradation and clearance. The synthetic smectite clay Laponite emerges as a promising candidate for their sustained delivery. Despite its unique properties allow to load and release proteins mitigating burst release and extending their effects, precise control over Laponite-protein interactions remains challenging since it depends on a complex interplay of factors whose implication is not fully understood yet. The aim of this review article is to shed light on this issue, providing a comprehensive discussion of the factors influencing protein loading and release, including the physicochemical properties of the nanoclay and proteins, pH, dispersion buffer, clay/protein concentration and Laponite degradation. Furthermore, we thoroughly revise the array of bioactive proteins that have been delivered from formulations containing the nanoclay, highlighting Laponite-polymer nanocomposite hydrogels, a promising avenue currently under extensive investigation.

Applications of Biomolecular Nanostructures for Anti-Angiogenic Theranostics.

Angiogenesis is a physiological process of forming new blood vessels that has pathological importance in seemingly unrelated illnesses like cancer, diabetes, and various inflammatory diseases. Treatment targeting angiogenesis has shown promise for these types of diseases, but current anti-angiogenic agents have critical limitations in delivery and side-effects. This necessitates exploration of alternative approaches like biomolecule-based drugs. Proteins, lipids, and oligonucleotides have recently become popular in biomedicine, specifically as biocompatible components of therapeutic drugs. Their excellent bioavailability and potential bioactive and immunogenic properties make them prime candidates for drug discovery or drug delivery systems. Lipid-based liposomes have become standard vehicles for targeted nanoparticle (NP) delivery, while protein and nucleotide NPs show promise for environment-sensitive delivery as smart NPs. Their therapeutic applications have initially been hampered by short circulation times and difficulty of fabrication but recent developments in nanofabrication and NP engineering have found ways to circumvent these disadvantages, vastly improving the practicality of biomolecular NPs. In this review, we are going to briefly discuss how biomolecule-based NPs have improved anti-angiogenesis-based therapy.

Covalent conjugation and non-covalent complexation strategies for intracellular delivery of proteins using cell-penetrating peptides.

Therapeutic proteins provided new opportunities for patients and high sales volumes. However, they are formulated for extracellular targets. The lipophilic barrier of the plasma membrane renders the vast array of intracellular targets out of reach. Peptide-based delivery systems, namely cell-penetrating peptides (CPPs), have few safety concerns, and low immunogenicity, with control over administered doses. This study investigates CPP-based protein delivery systems by classifying them into CPP-protein "covalent conjugation" and CPP: protein "non-covalent complexation" categories. Covalent conjugates ensure the proximity of the CPP to the cargo, which can improve cellular uptake and endosomal escape. We will discuss various aspects of covalent conjugates through non-cleavable (stable) or cleavable bonds. Non-cleavable CPP-protein conjugates are produced by recombinant DNA technology to express the complete fusion protein in a host cell or by chemical ligation of CPP and protein, which ensures stability during the delivery process. CPP-protein cleavable bonds are classified into pH-sensitive and redox-sensitive bonds, enzyme-cleavable bonds, and physical stimuli cleavable linkers (light radiation, ultrasonic waves, and thermo-responsive). We have highlighted the key characteristics of non-covalent complexes through electrostatic and hydrophobic interactions to preserve the conformational integrity of the CPP and cargo. CPP-mediated protein delivery by non-covalent complexation, such as zippers, CPP adaptor methods, and avidin-biotin technology, are featured. Conclusively, non-covalent complexation methods are appropriate when a high number of CPP or protein samples are to be screened. In contrast, when the high biological activity of the protein is critical in the intracellular compartment, conjugation protocols are preferred.

Protein nanoparticles as drug delivery systems for cancer theranostics.

Protein-based nanoparticles have garnered significant attention in theranostic applications due to their superior biocompatibility, exceptional biodegradability and ease of functionality. Compared to other nanocarriers, protein-based nanoparticles offer additional advantages, including biofunctionality and precise molecular recognition abilities, which make them highly effective in navigating complex biological environments. Moreover, proteins can serve as powerful tools with self-assembling structures and reagents that enhance cell penetration. And their derivation from abundant renewable sources and ability to degrade into harmless amino acids further enhance their suitability for biomedical applications. However, protein-based nanoparticles have so far not realized their full potential. In this review, we summarize recent advances in the use of protein nanoparticles in tumor diagnosis and treatment and outline typical methods for preparing protein nanoparticles. The review of protein nanoparticles may provide useful new insights into the development of biomaterial fabrication.

[Improvement of Intestinal and Transmucosal Absorption of Peptide and Protein Drugs].

It is well known that the oral bioavailability of hydrophilic and macromolecular drugs is generally very poor due to their poor membrane permeability characteristics. Among these poorly absorbed drugs, peptide and protein drugs are typical poorly absorbed drugs which have low stability and poor permeability in the gastrointestinal tract. Consequently, the clinical administration of peptide and protein drugs is presently limited to administration by injection. However, such frequent administration subjects the patients to considerable pain, and there is also the possibility of the manifestation of serious side effects. Therefore, various approaches have been examined to overcome the poor absorption characteristics of these drugs. These approaches include (1) to use additives including absorption enhancers and protease inhibitors, (2) to modify the chemical structure of peptide and protein drugs, and (3) to apply dosage forms to these drugs, (4) to develop a novel administration method for these drugs that can serve as an alternative to oral and injection administration. We demonstrated that intestinal and transmucosal absorption of peptide and protein drugs could be improved by using these approaches. These approaches may give us useful basic information to improve the intestinal and transmucosal absorption of peptide and protein drugs.

Biodynamer Nano-Complexes and -Emulsions for Peptide and Protein Drug Delivery.

Background: Therapeutic proteins and peptides offer great advantages compared to traditional synthetic molecular drugs. However, stable protein loading and precise control of protein release pose significant challenges due to the extensive range of physicochemical properties inherent to proteins. The development of a comprehensive protein delivery strategy becomes imperative accounting for the diverse nature of therapeutic proteins. Methods: Biodynamers are amphiphilic proteoid dynamic polymers consisting of amino acid derivatives connected through pH-responsive dynamic covalent chemistry. Taking advantage of the amphiphilic nature of the biodynamers, PNCs and DEs were possible to be prepared and investigated to compare the delivery efficiency in drug loading, stability, and cell uptake. Results: As a result, the optimized PNCs showed 3-fold encapsulation (<90%) and 5-fold loading capacity (30%) compared to DE-NPs. PNCs enhanced the delivery efficiency into the cells but aggregated easily on the cell membrane due to the limited stability. Although DE-NPs were limited in loading capacity compared to PNCs, they exhibit superior adaptability in stability and capacity for delivering a wider range of proteins compared to PNCs. Conclusion: Our study highlights the potential of formulating both PNCs and DE-NPs using the same biodynamers, providing a comparative view on protein delivery efficacy using formulation methods.

New perspective on protein-based microcapsules as delivery vehicles for sensitive substances: A review.

Sensitive substances have attracted wide attention due to their rich functional activities, such as antibiosis activities, antioxidant activities and prevent disease, etc. However, the low stability of sensitive substances limits their bioavailability and functional activities. Protein-based microcapsules can encapsulate sensitive substances to improve their adverse properties due to their good stability, strong emulsifying ability and wide source. Therefore, it is necessary to fully elaborate and summarize protein-based microcapsules to maximize their potential benefits in nutritional interventions. The focus of this review is to highlight the classification of protein-based microcapsules. In addition, the principles, advantages and disadvantages of preparation methods for protein-based microcapsules are summarized. Some novel preparation methods for protein-based microcapsules are also emphasized. Moreover, the mechanism of protein-based microcapsules that release sensitive substances in vitro is elucidated and summarized. Furthermore, the applications of protein-based microcapsules are outlined. Protein-based microcapsules can effectively encapsulate sensitive substances, which improve their bioavailability, and provide protective effects during storage and gastrointestinal digestion. In addition, microcapsules can improve the sensory quality of food and enhance its stability. The performance of protein-based microcapsules for delivering sensitive substances is influenced by factors such as protein type, the ratio between protein ratio and the other wall material, the preparation process, etc. Future research should focus on the new composite protein-based microcapsule delivery system, which can be applied to in vivo research and have synergistic effects and precise nutritional functions. In summary, protein-based microcapsules have broader research prospects in the functional foods and nutrition field.

Anti-Drug Antibody Incidence Comparison of Therapeutic Proteins Administered Via Subcutaneous vs. Intravenous Route.

Subcutaneous (SC) administration of therapeutic proteins is perceived to pose higher risk of immunogenicity when compared with intravenous (IV) route of administration (RoA). However, systematic evaluations of clinical data to support this claim are lacking. This meta-analysis was conducted to compare the immunogenicity of the same therapeutic protein by IV and SC RoA. Anti-drug antibody (ADA) data and controlling variables for 7 therapeutic proteins administered by both IV and SC routes across 48 treatment groups were analyzed. RoA was the primary independent variable of interest while therapeutic protein, patient population, adjusted dose, and number of ADA samples were controlling variables. Analysis of variance was used to compare the ADA incidence between IV and SC RoA, while accounting for controlling variables and potential interactions. Subsequently, 10 additional therapeutic proteins with ADA data published for both IV and SC administration were added to the above 7 therapeutic proteins and were evaluated for ADA incidence. RoA had no statistically significant effect on ADA incidence for the initial dataset of 7 therapeutic proteins (p = 0.55). The only variable with a significant effect on ADA incidence was the therapeutic protein. None of the other controlling variables, including their interactions with RoA, was significant. When all data from the 17 therapeutic proteins were pooled, there was no statistically significant effect of RoA on ADA incidence (p = 0.81). In conclusion, there is no significant difference in ADA incidence between the IV and SC RoA, based on analysis of clinical ADA data from 17 therapeutic proteins.

Acrylated adhesive proteinic microneedle patch for local drug delivery and stable device implantation.

Microneedle patches have been developed as favorable platforms for delivery systems, such as the locoregional application of therapeutic drugs, and implantation systems, such as electronic devices on visceral tissue surfaces. However, the challenge lies in finding materials that can achieve both biocompatibility and stable fixation on the target tissue. To address this issue, utilizing a biocompatible adhesive biomaterial allows the flat part of the patch to adhere as well, enabling double-sided adhesion for greater versatility. In this work, we propose an adhesive microneedle patch based on mussel adhesive protein (MAP) with enhanced mechanical strength via ultraviolet-induced polyacrylate crosslinking and Coomassie brilliant blue molecules. The strong wet tissue adhesive and biocompatible nature of engineered acrylated-MAP resulted in the development of a versatile wet adhesive microneedle patch system for in vivo usage. In a mouse tumor model, this microneedle patch effectively delivered anticancer drugs while simultaneously sealing the skin wound. Additionally, in an application of rat subcutaneous implantation, an electronic circuit was stably anchored using a double-sided wet adhesive microneedle patch, and its signal location underneath the skin did not change over time. Thus, the proposed acrylated-MAP-based wet adhesive microneedle patch system holds great promise for biomedical applications, paving the way for advancements in drug delivery therapeutics, tissue engineering, and implantable electronic medical devices.

Enhancement of Oral Bioavailability of Protein and Peptide by Polysaccharide-based Nanoparticles.

Oral drug delivery is a prevalent and cost-effective method due to its advantages, such as increased drug absorption surface area and improved patient compliance. However, delivering proteins and peptides orally remains a challenge due to their vulnerability to degradation by digestive enzymes, stomach acids, and limited intestinal membrane permeability, resulting in poor bioavailability. The use of nanotechnology has emerged as a promising solution to enhance the bioavailability of these vital therapeutic agents. Polymeric NPs, made from natural or synthetic polymers, are commonly used. Natural polysaccharides, such as alginate, chitosan, dextran, starch, pectin, etc., have gained preference due to their biodegradability, biocompatibility, and versatility in encapsulating various drug types. Their hydrophobic-hydrophilic properties can be tailored to suit different drug molecules.

Rosacea treatment with mussel adhesive protein delivered via microneedling: In vivo and clinical studies.

BACKGROUND: Rosacea is a prevalent chronic dermatological condition marked by facial inflammation and erythema, significantly compromising the quality of life for affected individuals. Current treatment methods for rosacea are not considered ideal because of the complex etiology of the disease. Mussel adhesive protein (MAP) is a glycoprotein derived from the foot gland of mussels. The protein exhibits anti-inflammatory properties, relieves skin itching, and promotes wound healing. AIMS: We aimed to explore the feasibility of using MAP administered via microneedle delivery for treating rosacea and the potential molecular mechanism involved. MATERIALS AND METHODS: The therapeutic effect and mechanism of MAP microneedle delivery in an LL-37-induced rosacea-like mouse model were observed using morphological and histological methods. Twenty-seven patients with erythematotelangiectatic rosacea (ETR) underwent treatment once every 1 month, with three treatments constituting one treatment course. The therapeutic effect was evaluated by comparing the clinical images taken at baseline, after the first treatment course, and after the second treatment course. The red value, CEA, and GFSS score were also calculated. RESULTS: In response to the microneedle delivery of MAP, innate immunity, inflammatory infiltration, and abnormal neurovascular regulation improved significantly in rosacea-like mice. In the clinical experiments, the microneedle delivery of MAP significantly improved the symptoms of erythema, flushing, and telangiectasia in patients with ETR, and no obvious adverse reactions were observed. CONCLUSIONS: MAP delivered by microneedling is effective and safe for treating ETR.

Challenges and Therapeutic Approaches for the Protein Delivery System: A Review.

The protein delivery system is one of the innovative or novel drug delivery systems in the present era. Proteins play an indispensable role in our body and are mainly found in every part, like tissue and cells of our body. It also controls various functions, such as maintaining our tissue, transportation, muscle recovery, enzyme production and acting as an energy source for our body. Protein therapeutics have big future perspectives, and their use in the treatment of a wide range of serious diseases has transformed the delivery system in the pharmaceutical and biotechnology industries. The chief advantage of protein delivery is that it can be delivered directly to the systemic circulation. So far, parenteral routes, such as intravenous, intramuscular, and subcutaneous, are the most often used method of administering protein drugs. Alternative routes like buccal, oral, pulmonary, transdermal, nasal, and ocular routes have also shown a remarkable success rate. However, as with all other types of delivery, here, several challenges are posed due to the presence of various barriers, such as the enzymatic barrier, intestinal epithelial barrier, capillary endothelial barrier, and blood-brain barrier. There are several approaches that have been explored to overcome these barriers, such as chemical modification, enzymatic inhibitors, penetration enhancers, and mucoadhesive polymers. This review article discusses the protein, its functions, routes of administration, challenges, and strategies to achieve ultimate formulation goals. Recent advancements like the protein Pegylation method and Depofoam technology are another highlight of the article.

Recent Advances in the Preparation of Protein/peptide Microspheres by Solvent Evaporation Method.

Protein/peptide drugs are extensively used to treat various chronic and serious diseases. The short half-life in vivo of protein and peptide as therapeutics drug limit the realization of complete effects. Encapsulating drugs in microspheres can slow the speed of drug release and prolong the efficacy of drugs. The solvent evaporation method is widely used to prepare protein/ peptide microspheres because of its facile operation and minimal equipment requirements. This method has several challenges in the lower encapsulation efficiency, fluctuant release profiles and the stabilization of protein/peptides, which researchers believe may be solved by adjusting the preparation parameter or formulation of microspheres. The article discusses the formulation parameters that govern the preparation of protein/peptide-loaded microspheres by the solvent evaporation method, which provides an overview of the current promising strategies for solvent evaporation for protein/peptide microspheres. The article takes parameter evaluation as the framework, facilitating subsequent researchers to quickly find possible solutions when encountering problems.

The strawberry-derived permeation enhancer pelargonidin enables oral protein delivery.

Although patients generally prefer oral drug delivery to injections, low permeability of the gastrointestinal tract makes this method impossible for most biomacromolecules. One potential solution is codelivery of macromolecules, including therapeutic proteins or nucleic acids, with intestinal permeation enhancers; however, enhancer use has been limited clinically by modest efficacy and toxicity concerns surrounding long-term administration. Here, we hypothesized that plant-based foods, which are well tolerated by the gastrointestinal tract, may contain compounds that enable oral macromolecular absorption without causing adverse effects. Upon testing more than 100 fruits, vegetables, and herbs, we identified strawberry and its red pigment, pelargonidin, as potent, well-tolerated enhancers of intestinal permeability. In mice, an oral capsule formulation comprising pelargonidin and a 1 U/kg dose of insulin reduced blood glucose levels for over 4 h, with bioactivity exceeding 100% relative to subcutaneous injection. Effects were reversible within 2 h and associated with actin and tight junction rearrangement. Furthermore, daily dosing of mice with pelargonidin for 1 mo resulted in no detectable side effects, including weight loss, tissue damage, or inflammatory responses. These data suggest that pelargonidin is an exceptionally effective enhancer of oral protein uptake that may be safe for routine pharmaceutical use.

Effectiveness Comparison of Polysaccharides, Proteins, and Lipids as Composite Edible Coatings on the Quality of Food Products / Comparación de la Efectividad de Polisacáridos, Proteínas y Lípidos como Recubrimientos Compuestos Comestibles en la Calidad de los Productos Alimenticios

Background: This research was motivated by the complaints of tomato farmers about their crops that quickly rotted before being sold, as well as the many research results (raw materials and methods) that edible coating films could not be applied optimally. Objectives: The research was a practical recommendation by comparing the effectiveness of raw materials (polysaccharides, proteins, and lipids) with the dipping and spray methods. Materials and methods used in the comparison process were the application of Structural Equation Modeling (SEM) with the Partial Least Square (PLS) approach. Results: Dipping has a strong effect (f2 ≥ 0.35; p<0.05), while spray had a moderate effect (f2: 0.15-0.35; p<0.05). Thus, the role of dipping as a mediator was more dominant than spray. Compared to proteins and lipids, polysaccharides had the best effectiveness (ß:0.460-0.584; f2: 0.15-0.35; p<0.05). Conclusion: the three ingredients improved the quality of tomatoes, and the dipping method was easier to apply by farmers than the spray method, which had many obstacles in its application

Antecedentes: esta investigación está motivada por las quejas de los productores de tomate sobre sus cultivos que se pudren rápidamente antes de ser vendidos, así como por los muchos resultados de la investigación (materias primas y métodos) de que las películas de recubrimiento comestibles no se pudieron aplicar de manera óptima. Objetivos: La investigación consiste en recomendaciones prácticas mediante la comparación de la eficacia de las materias primas (polisacáridos, proteínas y lípidos) con los métodos de inmersión y aspersión. Métodos: El método utilizado en el proceso de comparación es la aplicación del modelo de ecuaciones estructurales (SEM) con el enfoque de mínimos cuadrados parciales (PLS). Resultados: La inmersión tiene un efecto fuerte (f2 ≥ 0,35; p<0,05), mientras que la pulverización tiene un efecto moderado (f2: 0,15-0,35; p<0,05). Por lo tanto, el papel de la inmersión como mediador es más dominante que el del rociado. Los polisacáridos tienen la mejor eficacia (ß:0,460-0,584; f2: 0,15-0,35; p<0,05) en comparación con las proteínas y los lípidos. Conclusión: es que los tres ingredientes pueden mejorar la calidad de los tomates, y el método de inmersión es más fácil de aplicar por los agricultores que el método de aspersión, que tiene muchos obstáculos en su aplicación

Intralesional Infiltrations of Arteriosclerotic Tissue Cells-Free Filtrate Reproduce Vascular Pathology in Healthy Recipient Rats.

Lower-extremity arterial disease is a major health problem with increasing prevalence, often leading to non-traumatic amputation, disability and mortality. The molecular mechanisms underpinning abnormal vascular wall remodeling are not fully understood. We hypothesized on the existence of a vascular tissue memory that may be transmitted through soluble signaling messengers, transferred from humans to healthy recipient animals, and consequently drive the recapitulation of arterial wall thickening and other vascular pathologies. We examined the effects of the intralesional infiltration for 6 days of arteriosclerotic popliteal artery-derived homogenates (100 µg of protein) into rats' full-thickness wounds granulation tissue. Animals infiltrated with normal saline solution or healthy brachial arterial tissue homogenate obtained from traumatic amputation served as controls. The significant thickening of arteriolar walls was the constant outcome in two independent experiments for animals receiving arteriosclerotic tissue homogenates. This material induced other vascular morphological changes including an endothelial cell phenotypic reprogramming that mirrored the donor's vascular histopathology. The immunohistochemical expression pattern of relevant vascular markers appeared to match between the human tissue and the corresponding recipient rats. These changes occurred within days of administration, and with no cross-species limitation. The identification of these "vascular disease drivers" may pave novel research avenues for atherosclerosis pathobiology.