Controlled Release Of Proteins Research Articles

Self-healing and injectable chitosan/konjac glucomannan hydrogel with pH response for controlled protein release

Dynamic hydrogels with the features of injection, self-healing, and remodeling at the target site have been developed as smart multifunctional biomaterials for drug delivery. However, most self-healing injectable hydrogels are difficult to control protein release after implantation, owing to the deficiency of pH responsiveness, which reduces the bioavailability of proteins. Herein, we propose a facile strategy to endow pH responsiveness into a dynamic hydrogel with both self-healing and injectable capabilities, by crosslinking biomacromolecular backbones via dual pH sensitive dynamic covalent bond. Particularly, oxidized konjac glucomannan (OKGM) can be crosslinked with poly (aspartic hydrazide) (PAHy) and N-carboxyethyl chitosan (CEC) to form dynamic acylhydrazone bonds and imide bonds, respectively, endowing the hydrogel with pH responsiveness and dynamic behaviors. Specifically, PAHy facilitates the formation of acylhydrazone bonds, improving the mechanical properties and pH sensitivity while reducing the degradation behavior of the hydrogels under physiological conditions. Kinetics indicate that the release of bovine serum albumin follows Fick diffusion under different pH conditions. The pH responsive hydrogel with self-healing injectable capabilities has the potential to be used as a controllable and sustain release carrier for protein drugs.

Turning fishery waste into aquafeed additives: Enhancing shrimp enzymes immobilization in alginate-based particles using electrohydrodynamic atomization

Alternative protein ingredients are gradually replacing fish meal in aquafeeds. However, farmed animals often struggle to digest the provided feed, which leads to inefficient nutrient assimilation. This results in a high feed conversion ratio and in water quality deterioration. Fish waste-derived proteases show great potential as bioactive ingredients to enhance protein digestibility. To achieve this, a suitable carrier is essential to include exogenous proteases in the fish feed formulation. Therefore, this research aimed to immobilize a shrimp enzyme extract into different alginate-based microcapsules through electrodynamic atomization and compare their structural and functional properties. Morphology, physicochemical and thermal properties, and in vitro protein release were evaluated for the following microcapsules made from various materials: alginate (A), alginate-chitosan (AC), alginate-bentonite (AB), alginate-bentonite previously subjected to a sonication treatment (ABs), and a combination of all materials (ABsC or ABC). The average sizes ranged from 360 to 790 μm (wet) and 260–516 μm (freeze-dried), confirming the expected differences, with larger sizes for those including chitosan and bentonite. ABsC proved to be the most suitable system for immobilizing exogenous proteases, as these particles exhibited high encapsulation efficiency in both wet and freeze-dried versions and displayed controlled protein release under typical conditions of the proximal intestine. This was further confirmed through an in vivo trial. Two bioassays were conducted with tilapia (Oreochromis niloticus) (n = 42, 2.6 ± 0.58 g) to evaluate the distribution of ABsC-rodamine labeled particles in digestive organs and digestive enzyme activity. ABsC remained in the intestine for over 7 h, and a significant increase in total proteolytic activity was observed 60 min after feeding. These findings suggest that ABsC microcapsules could serve as effective carriers for exogenous proteases in feeds for aquaculture.

Polydopamine-Coated Polymer Nanofibers for In Situ Protein Loading and Controlled Release.

Nanofibrous polymeric materials, combined with protein therapeutics, play a significant role in biomedical and pharmaceutical applications. However, the upload of proteins into nanofibers with a high yield and controlled release has been a challenging issue. Here, we report the in situ loading of a model protein (bovine serum albumin) into hydrophilic poly(vinyl alcohol) nanofibers via ice-templating, with a 100% protein drug loading efficiency. These protein-loaded nanofibers were further coated by polydopamine in order to improve the nanofiber stability and achieve a controlled protein release. The mass ratio between poly(vinyl alcohol) and bovine serum albumin influenced the percentage of proteins in composite nanofibers and fiber morphology. More particles and less nanofibers were formed with an increasing percentage of bovine serum albumin. By varying the coating conditions, it was possible to produce a uniform polydopamine coating with tunable thickness, which acted as an additional barrier to reduce burst release and achieve a more sustained release profile.

Optimizing protein delivery rate from silk fibroin hydrogel using silk fibroin-mimetic peptides conjugation

Controlled release of proteins, such as growth factors, from biocompatible silk fibroin (SF) hydrogel is valuable for its use in tissue engineering, drug delivery, and other biological systems. To achieve this, we introduced silk fibroin-mimetic peptides (SFMPs) with the repeating unit (GAGAGS)n. Using green fluorescent protein (GFP) as a model protein, our results showed that SFMPs did not affect the GFP function when conjugated to it. The SFMP-GFP conjugates incorporated into SF hydrogel did not change the gelation time and allowed for controlled release of the GFP. By varying the length of SFMPs, we were able to modulate the release rate, with longer SFMPs resulting in a slower release, both in water at room temperature and PBS at 37 °C. Furthermore, the SF hydrogel with the SFMPs showed greater strength and stiffness. The increased β-sheet fraction of the SF hydrogel, as revealed by FTIR analysis, explained the gel properties and protein release behavior. Our results suggest that the SFMPs effectively control protein release from SF hydrogel, with the potential to enhance its mechanical stability. The ability to modulate release rates by varying the SFMP length will benefit personalized and controlled protein delivery in various systems.

3D Printing Cervical Implant Scaffolds Incorporated with Drug-Loaded Carboxylated Chitosan Microspheres for Long-Term Anti-HPV Protein Delivery.

As attempting personalized medicine, 3D-printed tissue engineering scaffolds are employed to combine with therapeutic proteins/peptides especially in the clinical treatment of infectious diseases, genetic diseases, and cancers. However, current drug-loading methods, such as immersion and encapsulation, usually lead to the burst release of the drugs. To address these issues, we proposed an integrated strategy toward the long-term controlled release of protein. In this study, patient-customized 3D scaffolds incorporated with drug-loaded microspheres were printed to realize the effective delivery of the anti-human papillomavirus (anti-HPV) protein after cervical conization in the treatment of cervical cancer. The 3D-printed scaffold could provide mechanical support to the defect site and ensure local release of the drug to avoid systemic administration. Meanwhile, microspheres serve as functional components to prevent the inactivation of proteins, as well as regulate their release period to meet the time requirement of different treatment courses. The research also utilized bovine serum albumin as a model protein to validate the feasibility of these scaffolds as a generic technology platform. The bioactivity of the released anti-HPV protein was validated using a pseudovirus model, which demonstrated that the microsphere encapsulation would not cause protein denaturation during the scaffold fabrication process. Besides, 3D-printed scaffolds incorporated with carboxylated chitosan microspheres were biocompatible and beneficial for cell attachment, which have been demonstrated by favorable cell viability and better coverage results for HeLa and HFF-1. This study highlights the great potential of scaffolds incorporated with microspheres to serve as tissue engineering candidate products with the function of effective protein delivery in a long-term controlled manner and personalized shapes for clinical trials.

Role of Polymer Concentration on the Release Rates of Proteins from Single- and Double-Network Hydrogels

Controlled delivery of proteins has immense potential for the treatment of various human diseases, but effective strategies for their delivery are required before this potential can be fully realized. Recent research has identified hydrogels as a promising option for the controlled delivery of therapeutic proteins, owing to their ability to respond to diverse chemical and biological stimuli, as well as their customizable properties that allow for desired delivery rates. This study utilized alginate and chitosan as model polymers to investigate the effects of hydrogel properties on protein release rates. The results demonstrated that polymer properties, concentration, and crosslinking density, as well as their responses to pH, can be tailored to regulate protein release rates. The study also revealed that hydrogels may be combined to create double-network hydrogels to provide an additional metric to control protein release rates. Furthermore, the hydrogel scaffolds were also found to preserve the long-term function and structure of encapsulated proteins before their release from the hydrogels. In conclusion, this research demonstrates the significance of integrating porosity and response to stimuli as orthogonal control parameters when designing hydrogel-based scaffolds for therapeutic protein release.

Lipid environment modulates processivity and kinetics of a presenilin homolog acting on multiple substrates in vitro

Intramembrane proteases (IPs) hydrolyze peptides in the lipid membrane where they participate in a number of cellular pathways including immune response and surveillance, cholesterol biosynthesis, and they are exploited by viruses for replication. Despite their broad importance across biology, how activity is regulated in the cell to control protein maturation and release of specific bioactive peptides at the right place and right time remains largely unanswered, particularly for the intramembrane aspartyl protease (IAP) subtype. At a molecular biochemical level, different IAP homologs can cleave non-biological substrates and there is no sequence recognition motif among the nearly 150 substrates identified for just one IAP, presenilin-1, the catalytic component of γ-secretase known for its involvement in production of amyloid-β plaques associated with Alzheimer disease. Here we used gel-based assays combined with quantitative mass spectrometry and FRET-based kinetics assays to probe the cleavage profile of the presenilin homolog from the methanogen Methanoculleus marisnigri JR1 as a function of the surrounding lipid-mimicking environment, either detergent micelles or bicelles. We selected four biological IAP substrates that have not undergone extensive cleavage profiling previously, namely, the viral core protein of Hepatitis C virus, the viral core protein of Classical Swine Fever virus, the transmembrane segment of Notch-1, and the tyrosine receptor kinase ErbB4. Our study demonstrates a proclivity toward cleavage of substrates at positions of low average hydrophobicity and a consistent role for the lipid environment in modulating kinetic properties.

Managing Diabetes with Hydrogel Drug Delivery

AbstractDiabetes is one of the most pressing healthcare challenges facing society. Dysfunctional insulin signaling causes diabetes, leading to blood glucose instability and many associated complications. While the administration of exogenous insulin is then essential for achieving glucose control, issues with dosing accuracy and timing remain. Hydrogel‐based drug delivery systems have been broadly explored for controlled protein release, including for applications in long‐lasting and oral insulin delivery. More recently, efforts have focused on injectable hydrogels with glucose‐directed controlled release of insulin and glucagon, aiming for more autonomous and biomimetic approaches to blood glucose control. These materials typically use protein‐based sensing mechanisms or glucose binding by synthetic aryl boronates for glucose‐directed release. Despite advancements in this area, there remains a need for more precise timing of therapeutic availability to afford healthy blood glucose homeostasis, providing an opportunity for further research and innovation. This review summarizes the current state of hydrogel‐based delivery of insulin and glucagon, with insights into the potential benefits, future directions, and challenges that must be overcome to achieve clinical impact.

Near-infrared light triggered degradation of metal−organic frameworks for spatiotemporally-controlled protein release

Metal−organic frameworks (MOFs) have emerged as a promising platform for protein delivery due to their high loading capacity and tunable functionality. However, spatiotemporally-controlled release of loaded proteins from MOFs-based nanocarriers remains an outstanding challenge. Herein, we report a MOFs-based protein delivery system that is capable of precisely controlling protein release with deep tissue penetrable near-infrared (NIR) light. The responsive system was constructed by encapsulating a protein of interest and upconversion nanoparticles (UCNPs) within zeolitic imidazolate framework-8 (ZIF-8) MOFs and further trapping a photoacid generator (PAG) in the pores of ZIF-8. Upon NIR light irradiation, UCNPs emit ultraviolet light that activates PAG to produce protons, which enables local pH acidification and thus degradation of ZIF-8 for spatiotemporally-controlled protein release. By employing insulin as a model protein, we demonstrate that the system allows for on-demand control over its therapeutic efficacy against diabetes with NIR light. This work illustrates a general strategy to regulate MOFs-based nanocarriers for controlled drug delivery.

Two-phase matrices for the controlled release of therapeutic proteins.

Controlled and sustained delivery of therapeutic proteins is crucial for achieving desired effects in wound healing applications. Yet, this remains a challenge in growth factor delivery for bone tissue engineering. Current delivery systems can lead to negative side effects, such as ectopic bone growth and cancer, due to the over administration of growth inducing proteins. Here, we have developed a two-phase system for the controlled release of therapeutic proteins. The system consists of protein-loaded poly(methacrylic acid)-based nanoparticles conjugated to chitosan scaffolds. The effect of co-monomer hydrophilicity and crosslinking density on nanoparticle properties was evaluated. It was found that the release kinetics of model therapeutic proteins were dependent on nanoparticle hydrophilicity. The chitosan scaffold, chosen for its biocompatibility and osteogenic properties, provided additional barriers to diffusion and promoted nanoparticle retention, leading to more sustained protein delivery. Additionally, the ability of MC3T3-E1 pre-osteoblast cells to proliferate on scaffolds with and without conjugated nanoparticles was evaluated and all scaffolds were shown to promote cell growth. The results demonstrate that the two-phase scaffold system presents a superior strategy for the sustained and controlled release of therapeutic proteins for bone tissue engineering applications.

Directed Evolution Enables Simultaneous Controlled Release of Multiple Therapeutic Proteins from Biopolymer-Based Hydrogels.

With the advent of increasingly complex combination strategies of biologics, independent control over their delivery is the key to their efficacy; however, current approaches are hindered by the limited independent tunability of their release rates. To overcome these limitations, directed evolution is used to engineer highly specific, low affinity affibody binding partners to multiple therapeutic proteins to independently control protein release rates. As a proof-of-concept, specific affibody binding partners for two proteins with broad therapeutic utility: insulin-like growth factor-1 (IGF-1) and pigment epithelium-derived factor (PEDF) are identified. Protein-affibody binding interactions specific to these target proteins with equilibrium dissociation constants (KD ) between 10-7 and 10-8 m are discovered. The affibodies are covalently bound to the backbone of crosslinked hydrogels using click chemistry, enabling sustained, independent, and simultaneous release of bioactive IGF-1 and PEDF over 7 days. The system is tested with C57BL/6J mice in vivo, and the affibody-controlled release of IGF-1 results in sustained activity when compared to bolus IGF-1 delivery. This work demonstrates a new, broadly applicable approach to tune the release of therapeutic proteins simultaneously and independently and thus the way for precise control over the delivery of multicomponent therapies is paved.

Long acting injectables for therapeutic proteins

Biotherapeutic development presents a myriad of challenges in relation to delivery, in particular for protein therapeutics. Protein delivery is complicated due to hydrophilicity, size, rate of degradation in vivo, low permeation through biological barriers, pH and temperature sensitivity, as well as the need to conserve its quaternary structure to retain function. To preserve therapeutic levels in vivo, proteins require frequent administration due to their short half-lives. Formulation strategies combining proteins with lipid carriers for parenteral administration show potential for improving bioavailability, while preserving protein activity and bypassing the mucosal barriers of the body. Encapsulating protein in long acting injectable delivery systems can improve therapeutic indices by prolonging and controlling protein release and reducing the need for repeat interventions. Two lyotropic crystal forming lipids, monoolein and phytantriol, have been formulated to produce lipidic cubic phases and assessed for their use as long acting protein eluting injectables. Three soluble proteins, cytochrome c, glyceraldehyde-3-phosphate dehydrogenase and aldehyde dehydrogenase and one membrane protein, cytochrome c oxidase, were incorporated into bulk cubic phase formulations of each lipid system to comparatively assess protein release kinetics. The activity of the soluble proteins was measured upon release from a phytantriol bulk cubic phase and phytantriol cubosomes, produced using a liquid precursor method.

Optimizing zinc-HisTag coordination remote loading of proteins in PLGA microspheres

Metal-HisTag coordination remote loading (MHCRL) of proteins in PLGA microspheres was previously developed to provide a useful tool for discovery and preclinical development of controlled release protein formulations. Here we describe optimization of MHCRL, including (1) reducing thermal stress, (2) decreasing the complexity and duration of the procedure, (3) increasing loading capacity, (4) increasing the penetration depth of protein, and (5) improving the release profile. Directly encapsulating ZnCO3as a Zn2+source for HisTag coordination, rather than remotely loading Zn2+, increased the Zn content ∼6-fold. Microspheres with directly encapsulated ZnCO3more deeply encapsulated green fluorescent protein and more efficiently encapsulated human serum albumin at protein loading solutions concentrations ≥100 μg/mL than remotely loaded Zn2+microspheres. Tributyl acetylcitrate plasticized microspheres in terms of decreasingTg, but led to a decrease in protein encapsulation efficiency. As such, the plasticizer was not deemed useful. The loading/healing cycles were reduced in time and temperature from 48 h/42 h at 43 °C to 2 h/6h at 37 °C while maintaining strong encapsulation efficiency, resulting in significantly improved protein stability. Immunoreactive protein was slowly released for months following a modest burst release. The improved microspheres and shorter, low-temperature encapsulation could be a valuable asset to drug discovery scientists interested in controlled release of delicate and/or costly biologic candidates.

Gel Based Formulations in Oral Controlled Release Drug Delivery

Gel-based formulations may be classified as hydrogels, emulgels, and organogels or oleogels. This categorization depends on the polarity of the liquid component present in it. Hydrogels offer excellent potential to be used in oral drug delivery due to inherent biocompatibility, diversity of both natural and synthetic components. In particular, stimuli-responsive hydrogels can meet the physiological changes along the gastro intestinal tract to achieve site-specific, controlled release of protein, peptide and many other molecules for systemic treatment. There are many different techniques for the preparation of different sort of customized hydrogel. In recent years the popularity of oleogels is also in increasing order due to the easy method of preparation and inherent long-term stability of these products to provide the ideal drug delivery matrix. Oleogels are comprised of non-polar dispersion medium like fixed oil, mineral oil, organic solvents which are gelled with organogelator. Most of the studies on oleogels has been conducted on their use in food applications. Hydrogel and oleogel both have good acceptance in the field of oral drug delivery. This review work highlights brief description about both Hydrogels and Oleogels focusing their characteristics, advantages, disadvantages, application, some major techniques of the preparations and modifications of the same in oral drug delivery.

Hypoxia-responsive hyaluronic acid nanogels with improved endo/lysosomal escape ability for tumor-targeted cytochrome c delivery

An ideal protein therapeutic effect depends on the protein drug-carrying nanocarrier to achieve high loading efficiency, excellent systemic stability, specific tumor targeting, rapid endo/lysosomal escape, and controlled protein release behavior. Herein, novel and interesting hypoxia-responsive nanogels with improved endo/lysosomal escape ability are reported in this work for tumor-targeted protein delivery. These nanogels can encapsulate a cationic protein, cytochrome C (CC), inside their cores with good stability in various media and thus protect the protein from degradation during blood circulation. In this nanogel system, monomethoxy poly(ethylene glycol) (mPEG) is conjugated to hyaluronic acid (HA) and serves as an outer shell for improving stability of the nanostructure, and the anionic HA is employed for encapsulation of cationic CC through multiphysical interactions with a high loading efficiency. The nanogel system is crosslinked using a hypoxia-responsive crosslinker for subsequent triggered release of CC. Due to presence of the hypoxia-responsive crosslinker, more than 80% of the loaded CC can be released within 24 h under hypoxic conditions. The in vitro experimental results demonstrate that these nanogels can effectively deliver CC into cancer cells via CD44-mediated tumor targeting, subsequently enable rapid endo/lysosomal escape, and trigger CC release under hypoxic conditions. In addition, cytotoxicity test confirms that the CC-loaded nanogels exhibit much higher cytotoxicity to cancer cells at hypoxic condition than that at normoxic condition, which indicates that this nano-platform is a promising candidate for tumor-targeting protein delivery and efficient cancer therapy.

Stability of proteins encapsulated in Michael-type addition polyethylene glycol hydrogels.

Degradable polyethylene glycol (PEG) hydrogels are excellent vehicles for sustained drug release due to their biocompatibility, tunable physical properties, and customizable degradation. However, protein therapeutics are unstable under physiological conditions and releasing degraded or inactive therapeutics can induce immunogenic effects. While controlling protein release from PEG hydrogels has been extensively investigated, few studies have detailed protein stability long-term or under stress conditions. Here, lysozyme and alcohol dehydrogenase (ADH) stability were explored upon encapsulation in PEG hydrogels formed through Michael-type addition. The stability and structure of the two model proteins were monitored by measuring the free energy of unfolding and fluoresce quenching when confined in a hydrogel and compared to PEG solution and buffer. Hydrogels destabilized lysozyme structure at low denaturant concentrations but prevented complete unfolding at high concentrations. ADH was stabilized as the confining mesh size approached the protein radius of gyration. Both proteins retained enzymatic activity within the hydrogels under stress conditions, including denaturant, high temperature, and agitation. Conjugation between lysozyme and PEG-acrylate was identified at long reaction times but no conjugation was observed in the time required for complete gelation. Studies of protein stability in PEG hydrogels, as the one detailed here, can lead to designer technologies for the improved formulation, storage, and delivery of protein therapeutics.

Improvement of wound healing by the development of ECM-inspired biomaterial coatings and controlled protein release.

Implant design has evolved from biochemically inert substrates, minimizing cell and protein interaction, towards sophisticated bioactive substrates, modulating the host response and supporting the regeneration of the injured tissue. Important aspects to consider are the control of cell adhesion, the discrimination of bacteria and non-local cells from the desired tissue cell type, and the stimulation of implant integration and wound healing. Here, the extracellular matrix acts as a role model providing us with inspiration for sophisticated designs. Within this scope, small bioactive peptides have proven to be miscellaneously deployable for the mediation of surface, cell and matrix interactions. Combinations of adhesion ligands, proteoglycans, and modulatory proteins should guide multiple aspects of the regeneration process and cooperativity between the different extracellular matrix components, which bears the chance to maximize the therapeutic efficiency and simultaneously lower the doses. Hence, efforts to include multiple of these factors in biomaterial design are well worth. In the following, multifunctional implant coatings based on bioactive peptides are reviewed and concepts to implement strong surface anchoring for stable cell adhesion and a dynamic delivery of modulator proteins are discussed.

In Situ Forming iEDDA Hydrogels with Tunable Gelation Time Release High-Molecular Weight Proteins in a Controlled Manner over an Extended Time.

Off-target interactions between reactive hydrogel moieties and drug cargo as well as slow reaction kinetics and the absence of controlled protein release over an extended period of time are major drawbacks of chemically cross-linked hydrogels for biomedical applications. In this study, the inverse electron demand Diels-Alder (iEDDA) reaction between norbornene- and tetrazine-functionalized eight-armed poly(ethylene glycol) (PEG) macromonomers was used to overcome these obstacles. Oscillatory shear experiments revealed that the gel point of a 15% (w/v) eight-armed PEG hydrogel with a molecular weight of 10 kDa was less than 15 s, suggesting the potential for fast in situ gelation. However, the high-speed reaction kinetics result in a risk of premature gel formation that complicates the injection process. Therefore, we investigated the effect of polymer concentration, temperature, and chemical structure on the gelation time. The cross-linking reaction was further characterized regarding bioorthogonality. Only 11% of the model protein lysozyme was found to be PEGylated by the iEDDA reaction, whereas 51% interacted with the classical Diels-Alder reaction. After determination of the mesh size, fluorescein isothiocyanate-dextran was used to examine the release behavior of the hydrogels. When glucose oxidase was embedded into 15% (w/v) hydrogels, a controlled release over more than 250 days was achieved. Overall, the PEG-based hydrogels cross-linked via the fast iEDDA reaction represent a promising material for the long-term administration of biologics.

Experimental Studies and Modeling of the Degradation Process of Poly(Lactic-co-Glycolic Acid) Microspheres for Sustained Protein Release.

In this study, poly(lactic-co-glycolic acid) microspheres (PLGA MS)for controlled protein release by double emulsion-solvent evaporation were produced and characterized for their morphological and technological features. MS autocatalytic degradation was described by a mathematical model based on a Michaelis and Menten-like chemical balance. Here, for the first time MS degradation was correlated to the advancement of MS degradation front with respect to the degraded radius, derived from mass loss experiments. The model can satisfactorily describe the kinetics of advancement of the degradation front experimentally derived for all MS formulations, especially when produced at higher PLGA concentrations.

Magnetic Liposomes for Remote Controlled High-Molecular Drugs Release under a Low-Frequency Non-Heating Magnetic Field

Magnetic anionic liposomes (MALip) conjugated with magnetite magnetic nanoparticles (MNPs) are developed for the controlled release of a protease inhibitor (BBI) under exposure to a low-frequency nonheating magnetic field (LF AMF). It is shown that an increase of up to 35% of the protein release rate occurred when the MALip are exposed to the LF AMF (frequency 110 Hz, intensity 75–150 kA/m) for 5–15 min. The research provides prospects for the development of remotely controlled protein release from liposomes.