Controlled Delivery Of Proteins Research Articles

Polyphenol-Assisted Biomineralization of Metal-Organic Framework Nanoparticles for Precision Delivery of Therapeutic Proteins to Cancer Cells.

The delivery of proteins into the cytosol holds great promise for cell signaling manipulation and the development of precision medicine. However, this potency is challenged by achieving targeted and controlled delivery, specifically within diseased cells. In this study, we introduce a versatile and effective method for the precision delivery of therapeutic proteins to cancer cells by designing polyphenol-assisted biomineralization of zeolite imidazole framework-8 (ZIF-8). We demonstrate that by leveraging the strong noncovalent binding affinity of epigallocatechin gallate (EGCG) with both proteins and ZIF-8, our approach significantly enhances the biomineralization of ZIF-8, which in turn improves the efficiency of protein encapsulation and intracellular delivery. Moreover, the incorporation of EGCG within ZIF-8 enables controlled degradation of the nanoparticles and the selective release of the encapsulated proteins in cancer cells. This selective release is triggered by the oxidation of EGCG in response to the high levels of reactive oxygen species (ROS) found within cancer cells that destabilize the EGCG/ZIF-8 nanoparticles. We have further demonstrated the ability of EGCG/ZIF-8 to deliver a wide range of proteins into cancer cells, including bacterial virulence protein, to rewire cell signaling and prohibit tumor cell growth in a mouse xenograft model. Our strategy and findings underscore the potential of designing the EGCG/ZIF-8 interface for specific and controlled protein delivery for targeted cancer therapy.

Orientational Pathways during Protein Translocation through Polymer-Modified Nanopores.

Protein translocation through nanopores holds significant promise for applications in biotechnology, biomolecular analysis, and medicine. However, the interpretation of signals generated by the translocation of the protein remains challenging. In this way, it is crucial to gain a comprehensive understanding on how macromolecules translocate through a nanopore and to identify what are the critical parameters that govern the process. In this study, we investigate the interplay between protein charge regulation, orientation, and nanopore surface modifications using a theoretical framework that allows us to explicitly take into account the acid-base reactions of the titrable amino acids in the proteins and in the polyelectrolytes grafted to the nanopore surface. Our goal is to thoroughly characterize the translocation process of different proteins (GFP, β-lactoglobulin, lysozyme, and RNase) through nanopores modified with weak polyacids. Our calculations show that the charge regulation mechanism exerts a profound effect on the translocation process. The pH-dependent interactions between proteins and charged polymers within the nanopore lead to diverse free energy landscapes with barriers, wells, and flat regions dictating translocation efficiency. Comparison of different proteins allows us to identify the significance of protein isoelectric point, size, and morphology in the translocation behavior. Taking advantage of these insights, we propose pH-responsive nanopores that can load proteins at one pH and release them at another, offering opportunities for controlled protein delivery, separation, and sensing applications.

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.

Electrically responsive release of proteins from conducting polymer hydrogels.

Electrically modulated delivery of proteins provides an avenue to target local tissues specifically and tune the dose to the application. This approach prolongs and enhances activity at the target site whilst reducing off-target effects associated with systemic drug delivery. The work presented here explores an electrically active composite material comprising of a biocompatible hydrogel, gelatin methacryloyl (GelMA) and a conducting polymer, poly(3,4-ethylenedioxythiophene), generating a conducting polymer hydrogel. In this paper, the key characteristics of electroactivity, mechanical properties, and morphology are characterized using electrochemistry techniques, atomic force, and scanning electron microscopy. Cytocompatibility is established through exposure of human cells to the materials. By applying different electrical-stimuli, the short-term release profiles of a model protein can be controlled over 4h, demonstrating tunable delivery patterns. This is followed by extended-release studies over 21 days which reveal a bimodal delivery mechanism influenced by both GelMA degradation and electrical stimulation events. This data demonstrates an electroactive and cytocompatible material suitable for the delivery of protein payloads over 3 weeks. This material is well suited for use as a treatment delivery platform in tissue engineering applications where targeted and spatio-temporal controlled delivery of therapeutic proteins is required. STATEMENT OF SIGNIFICANCE: Growth factor use in tissue engineering typically requires sustained and tunable delivery to generate optimal outcomes. While conducting polymer hydrogels (CPH) have been explored for the electrically responsive release of small bioactives, we report on a CPH capable of releasing a protein payload in response to electrical stimulus. The composite material combines the benefits of soft hydrogels acting as a drug reservoir and redox-active properties from the conducting polymer enabling electrical responsiveness. The CPH is able to sustain protein delivery over 3 weeks, with electrical stimulus used to modulate release. The described material is well suited as a treatment delivery platform to deliver large quantities of proteins in applications where spatio-temporal delivery patterns are paramount.

Fabrication of Multiresponsive Magnetic Nanocomposite Double‐Network Hydrogels for Controlled Release Applications (Small 52/2021)

Nanocomposite Double-Network Hydrogels A multi-responsive magnetic nanocomposite double-network hydrogel is developed for controlled protein delivery in article number 2105997 by Cheng-Hsun Lu and Yi-Cheun Yeh. Proteins are rapidly released from the hydrogel network at acidic pH and high temperature. This study provides a new strategy for hydrogel design to present multi-stimuli responsiveness for biomedical applications.

Robust Angiogenesis and Arteriogenesis in the Skin of Diabetic Mice by Transient Delivery of Engineered VEGF and PDGF-BB Proteins in Fibrin Hydrogels

Non-healing ulcers are a serious complication of diabetes mellitus and a major unmet medical need. A major cause for the lack of healing is the impairment of spontaneous vascularization in the skin, despite mostly normal blood flow in deeper large vessels. Therefore, pro-angiogenic treatments are needed to increase therapeutic perfusion by recruiting new arterial connections (therapeutic arteriogenesis). Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis in physiology and disease, but exploitation of its therapeutic potential requires careful control of its dose distribution in tissue. Co-delivery of platelet derived growth factor-BB (PDGF-BB) has been shown to expand the therapeutic window of VEGF and also improve associated arteriogenesis. We used a highly controlled protein delivery system, based on a clinically applicable fibrin-based platform, to investigate the angiogenic and arteriogenic potential of engineered versions (TG-) of VEGF and PDGF-BB proteins in the skin of diabetic and obese db/db mice. Intradermal delivery of therapeutically relevant doses of TG-VEGF and TG-PDGF-BB induced robust growth of new microvascular networks with similar efficacy as in normal littermate control mice. Further, TG-PDGF-BB prevented the formation of aberrant vascular enlargements by high TG-VEGF levels. As fibrin was degraded after the first week, the induced angiogenesis mostly regressed by 4 weeks, but it promoted effective arteriogenesis in the dermal layer. Therefore, controlled co-delivery of TG-VEGF and TG-PDGF-BB recombinant proteins is effective to induce angiogenesis and arteriogenesis in diabetic mouse skin and should be further investigated to promote diabetic wound healing.

3D-printed construct from hybrid suspension as spatially and temporally controlled protein delivery system.

Protein delivery systems have been extensively applied in controlled releasing of protein or polypeptides for therapeutic treatment or tissue regeneration. While 3 D printing technology shows great promise in novel dosage form with tailoring dose size and drug release profile, 3 D printable protein delivery system has to face many difficult challenges. In this study, we developed a hybrid suspension combining Eudragit polyacrylate colloid as matrix material and Pluronic polyether hydrogel as diffusion channel for protein release. This hybrid suspension can be 3 D-printed into construct with complex shape and inner structures thanks to its pseudoplastic and thixotropic rheological properties. The protein can be incorporated in hybrid suspension either in its original or nanoparticle capsulated form. The experiment shows that the protein release from construct is a function of drying time, molecular weight (MW) of chitosan, as well as their own structural/diffusional properties. Also, the theoretical derivation suggests polyacrylate matrix tortuosity, chitosan erosion rate as well as hydrogel diffusion coefficient all contributed to the extended duration of release profile. In addition, cytotoxicity test through cell culture confirmed that the construct fabricated from hybrid suspension exhibit relative good bio-compatibility. Finally, heterogeneous constructs with zoned design were fabricated as protein delivery system, which demonstrated the capability of hybrid suspension technique for spatial and temporal release of macromolecular drugs to realize pharmaceutical effectiveness or guild cell organization.

Nano/micro-structured poly(ϵ-caprolactone)/gelatin nanofibers with biomimetically-grown hydroxyapatite spherules: High protein adsorption, controlled protein delivery and sustained bioactive ions release designed as a multifunctional bone regenerative membrane

Bioactive and biodegradable fibrous membranes are highly attractive for periodontal bone regeneration. Herein, we demonstrate a new approach for fabrication of a novel nano/micro-structured fibrous membrane made of biodegradable poly(ϵ-caprolactone)/gelatin (PCL/GEL) nanofibers and biomemtically-grown hydroxyapatite spherules (HAs). The proposed approach includes electrospinning fabrication of PCL/GEL nanofibers containing nanobioglass (NBG) agglomerates and their biomimetic transformation into HAs. The NBG agglomerates (~1.9 μm in diameter) enabled the growth of biomimetic HAs (~4 μm in diameter) around the PCL/GEL nanofibers and generated a unique nano/micro-structure. Interestingly, the biomimetically-grown HAs imparted the PCL/GEL-HAs nanofibrous membrane with several remarkable properties including nano/micro-topography, bone-mimetic composition (Ca/P = 1.60), large specific surface area (~31 m2/g), high protein adsorption capacity (~157 μg protein/mg membrane) and controlled protein delivery with zero-order release kinetics; along with sustained release of therapeutic ions (Ca2+ ~ 37 ppm, PO43− ~ 24 ppm, and SiO44− ~ 61 ppm). Furthermore, the PCL/GEL-HAs membrane exhibited enhanced wettability, good biodegradability and adequate mechanical properties. Collectively, the PCL/GEL-HAs demonstrated unique properties and it can be considered as a novel multifunctional bioactive/biodegradable membrane for periodontal bone regeneration.

(Keynote) Biomimetic Preservation of Protein and Cell Surface Markers with Metal-Organic Frameworks for Controlled Protein Delivery and Room Temperature Storage and Shipping

Biological specimines from histology, protein-based therapeutics, and many biomacromolecules are unstable at room temperature. This instability necessitates considerable infrastructure be in place to allow the spoilage-free transportation of a number of important medicines including vaccines, insulin, antibody-based therapeutics, etc. This cold-chain infrastructure can increase the cost of these drugs by as much as 80%. Further to these problems, the instability of protein-based materials time-delayed or slow release formulations difficult. I will present a general method to "biomimetically" encarceate biomacromolecular assemblies into crystalline thermodynamically stable crystalline frameworks that readily dissolve in vivo to release intact and natively folded proteins. I will discuss strategies to incorporate these into prosthetics and implants for long-term delivery with a focus toward methodology in improving patient compliance with drugs and patient outcomes following prosthetic implants.

Preparation and in vitro release profiling of PLGA microspheres containing BSA as a model protein

Conventional drug formulations are incapable of adequate delivery of proteins and peptides for therapeutic purposes. As these molecules have very short biological half-life, multiple dosing is required to achieve the desirable therapeutic effects. Microspheres are able to encapsulate proteins and peptide in the polymeric matrix while protecting them from enzymatic degradation. In this study Bovine Serum Albumin (BSA) matrix type microspheres were fabricated using Polylactide-co-glycolide (PLGA) by double emulsion solvent evaporation method. The effects of variables such as homogenizer speed, molecular weight of polymer and the effect of pH of the water phases, were investigated against factors such as drug loading, encapsulation efficiency, morphology, size, drug distribution and release profile of the microspheres. Results, suggested that an increase in homogenization speed leads to a decrease in microsphere size. The increase in homogenization speed also caused a significant effect on the release profile only when higher molecular weight of polymer had been used.. The pH change of the internal aqueous phase led to modification of surface morphology of spheres to a porous structure that significantly increased the total amount of released protein. Integrity of protein structure was intact as shown by SDS-PAGE. According to the results, it can be concluded that we achieved a reproducible method regarding controlled protein delivery for different sizes of particles.

Preparation and properties of hydrophobic films based on acetylated broken-rice starch nanocrystals for slow protein delivery

Native and acetylated broken-rice starches (nanocrystals) with different degrees of substitution (DS) and their corresponding films were individually prepared, and the drug release profiles, weight loss, solubility and dispersion and surface morphology were comparatively studied. Bovine serum albumin (BSA) was used as a model drug. Acetylated native starch (ANS) DS 2.58, acetylated starch nanocrystals (ASN) DS 0.98, ASN DS 1.86, and ASN DS 2.72 were observed to be very soluble in chloroform. BSA was released rapidly from the native rice starch (NS) and ANS DS 2.58 films. ASN with high DS significantly slowed down the release of BSA from films, the percentages of BSA released from film ASN DS 2.72 only reached to 13% after 3.5 weeks release, and the release data followed Korsmeyer-Peppas equation. Further studies reveal that the particle size of ASN DS 2.72 was smallest, and the weight loss of ASN DS 2.72 film was lowest. The results demonstrate that acetylation and nanometer particle form of rice starch film can effectively retard protein drug release, and the prepared films based on ASN with high DS from broken rice may be suitable for the controlled protein delivery.

Critical parameters for the controlled synthesis of nanogels suitable for temperature-triggered protein delivery

Macromolecular bioactives, like proteins and peptides, emerged as highly efficient therapeutics. The main limitation for their clinical application is their instability and potential immunogenicity. Thus, controlled delivery systems able protect the proteins prior release are highly on demand. In the present study, we developed hydrophilic thermo-responsive nanogels with tunable volume phase transition temperatures (VPTTs) and suitable features for controlled protein delivery by the use of multifunctional, dendritic polyglycerol (dPG) as macromolecular cross-linker and temperature-sensitive polymers poly(N-isopropylacrylamide) (NIPAM) and poly(N-isopropylacrylmethacrylate) as linear counterpart. We comprehensively studied the impact of the initiator, monomers and cross-linker on the nanogel structure during the synthesis. Careful analysis of the polymerization process revealed importance of balanced reactions kinetics to form particles with diameters in the range 100–200 nm and low polydispersity. We can control the cross-linking density of the nanogels mainly by the dPG feed and its degree of acrylation. In addition, our screenings revealed that the hydrophilic character of dPG enables it to stabilize the growing particles during the polymerization and thereby reduces final particle size. Co-polymerization of NIPAM and NIPMAM allows precise tuning of the VPTT of the nanogels in the desired range of 34–47 °C. Our nanogels showed outstanding high protein encapsulation efficiency and triggered cargo release upon a temperature change. The delivery efficiency of these nanogels was investigated on excised human skin demonstrating efficient dermal penetration of encapsulated proteins dependent on a temperature triggered release mechanism.

Aptamer Functionalized DNA Hydrogel for Wise-Stage Controlled Protein Release

With the simple functionalization method and good biocompatibility, an aptamer-integrated DNA hydrogel is used as the protein delivery system with an adjustable release rate and time by using complementary sequences (CSs) as the biomolecular trigger. The aptamer-functionalized DNA hydrogel was prepared via a one-pot self-assembly process from two kinds of DNA building blocks (X-shaped and L-shaped DNA units) and a single-stranded aptamer. The gelling process was achieved under physiological conditions within one minute. In the absence of the triggering CSs, the aptamer grafted in the hydrogel exhibited a stable state for protein-specific capture. While hybridizing with the triggering CSs, the aptamer is turned into a double-stranded structure, resulting in the fast dissociation of protein with a wise-stage controlled release program. Further, the DNA hydrogel with excellent cytocompatibility has been successfully applied to human serum, forming a complex matrix. The whole process of protein capture and release were biocompatible and could not refer to any adverse factor of the protein or cells. Thus, the aptamer-functionalized DNA hydrogel will be a good candidate for controlled protein delivery.

Chaperone Copolymer-Assisted Aptamer-Patterned DNA Hydrogels for Triggering Spatiotemporal Release of Protein.

In this paper, a DNA hydrogel with low DNA concentration, short sticky end, and good mechanical strength was simply prepared via one-pot self-assembly from two kinds of DNA building block (X- and L-shaped DNA units) chaperoned by a cationic comb-type copolymer (CCC). The gelling process was completed under physiological conditions within 1 min, and the reversible sol-gel phase transition was achieved at room temperature through the continuous addition of CCC and an anionic polymer poly(sodium vinylsulfonate). Moreover, aptamer was successfully patterned into the hydrogel system via click chemistry. Upon the addition of complementary sequences (CSs) of aptamer, the aptamer was hybridized with CSs, leading to the fast dissociation of protein from aptamer with an adjustable release rate in specific regions at prospective times. The hydrogel with excellent cytocompatibility was successfully applied to human serum, a complex matrix. The aptamer-patterned DNA hydrogel is a potential candidate for controlled protein delivery.

Encapsulation of Biological Agents in Hydrogels for Therapeutic Applications.

Hydrogels are materials specially suited for encapsulation of biological elements. Their large water content provides an environment compatible with most biological molecules. Their crosslinked nature also provides an ideal material for the protection of encapsulated biological elements against degradation and/or immune recognition. This makes them attractive not only for controlled drug delivery of proteins, but they can also be used to encapsulate cells that can have therapeutic applications. Thus, hydrogels can be used to create systems that will deliver required therapies in a controlled manner by either encapsulation of proteins or even cells that produce molecules that will be released from these systems. Here, an overview of hydrogel encapsulation strategies of biological elements ranging from molecules to cells is discussed, with special emphasis on therapeutic applications.

Injectable hydrogels based on the hyaluronic acid and poly (γ-glutamic acid) for controlled protein delivery

Injectable hydrogels have great potential in minimally invasive delivery. In this work, novel injectable hydrogels were prepared via self-crosslinking of aldehyde hyaluronic acid (HA-CHO) and hydrazide-modified poly (γ-glutamic acid) (γ-PGA-ADH) for proteins delivery. The HA/γ-PGA hydrogels could be formed in situ as fast as 9s with high swelling ratios. Rheological properties illustrated a wide processing range and good mechanical properties, which were reflected by broad linear viscoelastic region and higher threshold shear stress (σc) and storage modulus (G′). Meanwhile, the gelation time, swelling ratio, rheological properties, as well as the protein release behavior could be modulated conveniently. Bovine serum albumin (BSA) was designed as a model drug to study the release behavior. We found that the release mechanisms were either diffusion or Case-II relaxation depending on the different hydrogel components. The HA/γ-PGA hydrogels also showed good biocompatibility. Therefore, the HA/γ-PGA hydrogels have great potential as promising injectable biomaterials for controlled protein delivery.

Follicle-stimulating hormone encapsulation in the cholesterol-modified chitosan nanoparticles via molecular dynamics simulations and binding free energy calculations

Follicle-stimulating hormone (FSH) is widely applied in the modern ovarian stimulation techniques. However, it must be administered daily because of its short half-life. Recently, the cholesterol (CS) modified chitosan (CTS) nanogels have attracted significant interest as promising controlled release protein delivery because of their ability to minimize the aggregation and irreversible denaturation of proteins. Herein, we report a molecular dynamics (MD) simulation investigation on the molecular mechanisms of FSH encapsulation in the CS-CTS nanogels. The MD simulations have been performed using the GROMACS software for up to 200ns simulation time. Furthermore, the binding free energy has been calculated by the molecular mechanics [MM] with Poisson-Boltzmann [PB] and surface area solvation (MM/PBSA) method by using the g_mmpbsa tool. Our findings suggest that the main driving force of the formation of the CS-CTS nanogels is the hydrophobic interactions between the CS–CS moieties in water. The results have also indicated that the CS-CTS nanogel formation can occur through the hydrogen bonding in addition to the hydrophobic interactions. The obtained data demonstrate that the FSH encapsulation into the CS-CTS nanogels is a gradual process driven by the hydrophobic interactions between the hydrophobic patch of FSH and the hydrophobic nanodomains of the nanogel. Our results also reveal that except in the hydrophobic patch region, the flexibility of FSH was reduced in the presence of the nanogel. This study provides the elucidation of the nanogel–FSH interactions at the molecular level and presents new perspective for the ideal design and applications of the CS-CTS nanogel in protein delivery.

Sulfated Alginate as a Mimic of Sulfated Glycosaminoglycans: Binding of Growth Factors and Effect on Stem Cell Behavior.

Sulfated glycosaminoglycans (GAGs) are principal elements of the extracellular matrix, where they are involved in a plethora of signaling pathways mainly via interactions with diverse proteins such as growth factors and cytokines. However, the mechanisms that drive these interactions are not yet clear, mostly because of the difficulty to access large quantities of homogeneously sulfated natural GAGs. In this work, GAG mimics are synthesized from readily available alginate with different degrees of sulfation (DS, from 0.8 to 2.6) by simple process. The effect of the DS is determined on the binding of basic fibroblast growth factor (FGF-2). The enzyme-linked immunosorbent assay demonstrates that the binding of FGF-2 is significantly greater for alginates with high DS as compared to unmodified and low sulfated analogs. These results are further applied to engineer FGF-2-loaded substrates for stem cell culturing using the layer-by-layer approach. All films support the attachment and growth of adipose derived stem cells (ADSCs). Noteworthy, highly sulfated alginates maintain the stemness of the ADSCs that exhibit remarkably long filopodia. These results can be exploited in the engineering of novel substrates that induce targeted cell behavior via controlled protein delivery and for tissue engineering constructs applicable in various regenerative approaches.

Development of a Gastric Absorptive, Immediate Responsive, Oral Protein-Loaded Versatile Polymeric Delivery System.

A multifunctional platform to deliver three diverse proteins of insulin, interferon beta (INF-β) and erythropoietin (EPO), using a novel copolymeric microparticulate system of TMC-PEGDMA-MAA, was synthesised as an intelligent pH-responsive 2-fold gastric and intestinal absorptive system. Physiochemical and physicomechanical studies proved the degree of crystallinity that supported the controlled protein delivery of the microparticulate system. The copolymer was tableted before undertaking in vitro and in vivo analysis. After 2.5h in simulated gastric fluid (SGF), insulin showed a fractional release of 3.2% in comparison to simulated intestinal fluid (SIF), in which a maximum of 83% of insulin was released. Similarly, INF-β and EPO released 3 and 9.7% in SGF and a maximum of 74 and 81.3% in SIF, respectively. In vivo studies demonstrated a significant decrease in blood glucose by 54.19% within 4h post-dosing, and the comparator formulation provided 74.6% decrease in blood glucose within the same time period. INF-β peak bioavailable dose in serum was calculated to be 1.3% in comparison to an SC formulation having a peak concentration of 0.9%, demonstrating steady-state release for 24h. EPO-loaded copolymeric microparticles had a 1.6% peak bioavailable concentration, in comparison to the 6.34% peak concentration after 8h from the SC comparator formulation.

Protease-degradable microgels for protein delivery for vascularization.

Degradable hydrogels to deliver bioactive proteins represent an emerging platform for promoting tissue repair and vascularization in various applications. However, implanting these biomaterials requires invasive surgery, which is associated with complications such as inflammation, scarring, and infection. To address these shortcomings, we applied microfluidics-based polymerization to engineer injectable poly(ethylene glycol) microgels of defined size and crosslinked with a protease degradable peptide to allow for triggered release of proteins. The release rate of proteins covalently tethered within the microgel network was tuned by modifying the ratio of degradable to non-degradable crosslinkers, and the released proteins retained full bioactivity. Microgels injected into the dorsum of mice were maintained in the subcutaneous space and degraded within 2 weeks in response to local proteases. Furthermore, controlled release of VEGF from degradable microgels promoted increased vascularization compared to empty microgels or bolus injection of VEGF. Collectively, this study motivates the use of microgels as a viable method for controlled protein delivery in regenerative medicine applications.