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Extracellular Vesicles (EVs) have been proposed as a promising tool for drug delivery because of their natural ability to cross biological barriers, protect their cargo, and target specific cells. Moreover, EVs are not recognized by the immune system as foreign, reducing the risk of an immune response and enhancing biocompatibility. Herein, we proposed an alternative therapeutic strategy to restore STAT3 signaling exploiting STAT3 loaded EVs. This approach could be useful in the treatment of Autosomal Dominant Hyper-IgE Syndrome (AD-HIES), a rare primary immunodeficiency and multisystem disorder due to the presence of mutations in STAT3 gene. These mutations alter the signal transduction of STAT3, thereby impeding Th17 CD4+ cell differentiation that leads to the failure of immune response. We set up a simple and versatile method in which EVs were loaded with fully functional STAT3 protein. Moreover, our method allows to follow the uptake of STAT3 loaded vesicles inside cells due to the presence of EGFP in the EGFP-STAT3 fusion protein construct. Taken together, the data presented in this study could provide the scientific background for the development of new therapeutic strategy aimed to restore STAT3 signaling in STAT3 misfunction associated diseases like AD-HIES. In the future, the administration of fully functional wild type STAT3 to CD4+ T cells of AD-HIES patients might compensate its loss of function and would be beneficial for these patients, lowering the risk of infections, the use of medications, and hospitalizations. [Display omitted] • EVs are a promising tool for drug delivery. • EVs have been loaded with fully functional EGFP-STAT3. • EGFP-STAT3 allows to track EVs and to follow STAT3 trafficking in cells. • EVs loaded with fully functional STAT3 might be useful to restore STAT3 signaling. [ABSTRACT FROM AUTHOR]

The layer-by-layer (LbL) assembling of oppositely charged polyelectrolytes was studied using semi-synthetic polysaccharide derivatives, namely the polycations 6-aminoethylamino-6-deoxy cellulose (ADC) and cellulose (2-(ethylamino)ethylcarbamate (CAEC), as well as the polyanion cellulose sulfate (CS). The synthetic polymers poly(allylamine) (PAH) and poly(styrene sulfonate) (PSS) were employed as well for comparison. The stepwise adsorption process was monitored by whispering gallery mode (WGM) experiments and zeta-potential measurements. Distinct differences between synthetic- and polysaccharide-based assemblies were observed in terms of the quantitative adsorption of mass and adsorption kinetics. The LbL-approach was used to prepare µm-sized capsules with the aid of porous and non-porous silica particle templates. The polysaccharide-based capsule showed a switchable permeability that was not observed for the synthetic polymer materials. At ambient pH values of 7, low-molecular dyes could penetrate the capsule wall while no permeation occurred at elevated pH values of 8. Finally, the preparation of protein-loaded LbL-capsules was studied using the combination of CAEC and CS. It was shown that high amounts of protein (streptavidin and ovomucoid) can be encapsulated and that no leaking or disintegration of the cargo macromolecules occurred during the preparation step. Based on this work, potential use in biomedical areas can be concluded, such as the encapsulation of bioactive compounds (e.g., pharmaceutical compounds, antibodies) for drug delivery or sensing purposes. [ABSTRACT FROM AUTHOR]

The layer-by-layer (LbL) assembling of oppositely charged polyelectrolytes was studied using semi-synthetic polysaccharide derivatives, namely the polycations 6-aminoethylamino-6-deoxy cellulose (ADC) and cellulose (2-(ethylamino)ethylcarbamate (CAEC), as well as the polyanion cellulose sulfate (CS). The synthetic polymers poly(allylamine) (PAH) and poly(styrene sulfonate) (PSS) were employed as well for comparison. The stepwise adsorption process was monitored by whispering gallery mode (WGM) experiments and zeta-potential measurements. Distinct differences between synthetic- and polysaccharide-based assemblies were observed in terms of the quantitative adsorption of mass and adsorption kinetics. The LbL-approach was used to prepare µm-sized capsules with the aid of porous and non-porous silica particle templates. The polysaccharide-based capsule showed a switchable permeability that was not observed for the synthetic polymer materials. At ambient pH values of 7, low-molecular dyes could penetrate the capsule wall while no permeation occurred at elevated pH values of 8. Finally, the preparation of protein-loaded LbL-capsules was studied using the combination of CAEC and CS. It was shown that high amounts of protein (streptavidin and ovomucoid) can be encapsulated and that no leaking or disintegration of the cargo macromolecules occurred during the preparation step. Based on this work, potential use in biomedical areas can be concluded, such as the encapsulation of bioactive compounds (e.g., pharmaceutical compounds, antibodies) for drug delivery or sensing purposes.

Despite clinical advances in immunotherapy, still many therapeutics cause dose‐limiting (auto)immune‐mediated toxicities. Nanoparticle‐based drug delivery systems (DDS) can improve cancer immunotherapy through site‐specific delivery and controlled release of immunotherapeutics in the tumor microenvironment (TME). However, DDS face several challenges, including unspecific release. To address this, vaterite nanoparticles (VNPs) that selectively release immunotherapeutic proteins at low pH conditions find in the TME, are established previously. In the current study, these VNPs are further modified for active targeting without affecting the loaded protein activity, exemplified with Tumor Necrosis Factor α (TNF). Specifically, VNPs are coated with gelatin, a matrix‐metalloprotease sensitive polymer which provides functional groups for further conjugation. Subsequently, streptavidin is covalently linked to the gelatin shell by amine‐epoxy chemistry, enabling coupling of any biotinylated ligand. Exemplified by biotinylated cetuximab and rituximab, targeted VNPs selectively bind to cells expressing epidermal growth factor receptor (EGFR) or CD20, respectively. Importantly, TNF remains functionally active after the modification steps, as VNP treatment increased ICAM‐1 expression on FaDu cells and activated NFκB signaling in a Jurkat.NFκB‐luciferase cell line model. In conclusion, a targetable vaterite‐based DDS is produced that allows for easy surface modification with any biotinylated ligand that may find broad applications in tumor‐selective immunotherapy. [ABSTRACT FROM AUTHOR]

Lactoferrin (Lf) is a globular glycoprotein found mainly in milk. It has a very high affinity for iron(III) ions, and its fully saturated form is called holoLf. The antimicrobial, antiviral, anticancer, and immunomodulatory properties of Lf have been studied extensively for the past two decades. However, to demonstrate therapeutic benefits, Lf has to be efficiently delivered to the intestinal tract in its structurally intact form. This work aimed to optimize the encapsulation of holoLf in a system based on the versatile Eudragit® RS polymer to protect Lf against the proteolytic environment of the stomach. Microparticles (MPs) with entrapped holoLf were obtained with satisfactory entrapment efficiency (90–95%), high loading capacity (9.7%), and suitable morphology (spherical without cracks or pores). Detailed studies of the Lf release from the MPs under conditions that included simulated gastric or intestinal fluids, prepared according to the 10th edition of the European Pharmacopeia, showed that MPs partially protected holoLf against enzymatic digestion and ionic iron release. The preincubation of MPs loaded with holoLf under conditions simulating the stomach environment resulted in the release of 40% of Lf from the MPs. The protein released was saturated with iron ions at 33%, was structurally intact, and its iron scavenging properties were preserved. [ABSTRACT FROM AUTHOR]

The development and production of innovative protein-based therapeutics is a complex and challenging avenue. External conditions such as buffers, solvents, pH, salts, polymers, surfactants, and nanoparticles may affect the stability and integrity of proteins during formulation. In this study, poly (ethylene imine) (PEI) functionalized mesoporous silica nanoparticles (MSNs) were used as a carrier for the model protein bovine serum albumin (BSA). To protect the protein inside MSNs after loading, polymeric encapsulation with poly (sodium 4-styrenesulfonate) (NaPSS) was used to seal the pores. Nano differential scanning fluorimetry (NanoDSF) was used to assess protein thermal stability during the formulation process. The MSN-PEI carrier matrix or conditions used did not destabilize the protein during loading, but the coating polymer NaPSS was incompatible with the NanoDSF technique due to autofluorescence. Thus, another pH-responsive polymer, spermine-modified acetylated dextran (SpAcDEX), was applied as a second coating after NaPSS. It possessed low autofluorescence and was successfully evaluated with the NanoDSF method. Circular dichroism (CD) spectroscopy was used to determine protein integrity in the case of interfering polymers such as NaPSS. Despite this limitation, NanoDSF was found to be a feasible and rapid tool to monitor protein stability during all steps needed to create a viable nanocarrier system for protein delivery. [ABSTRACT FROM AUTHOR]

Lactoferrin (Lf) is a globular glycoprotein found mainly in milk. It has a very high affinity for iron(III) ions, and its fully saturated form is called holoLf. The antimicrobial, antiviral, anticancer, and immunomodulatory properties of Lf have been studied extensively for the past two decades. However, to demonstrate therapeutic benefits, Lf has to be efficiently delivered to the intestinal tract in its structurally intact form. This work aimed to optimize the encapsulation of holoLf in a system based on the versatile Eudragit® RS polymer to protect Lf against the proteolytic environment of the stomach. Microparticles (MPs) with entrapped holoLf were obtained with satisfactory entrapment efficiency (90–95%), high loading capacity (9.7%), and suitable morphology (spherical without cracks or pores). Detailed studies of the Lf release from the MPs under conditions that included simulated gastric or intestinal fluids, prepared according to the 10th edition of the European Pharmacopeia, showed that MPs partially protected holoLf against enzymatic digestion and ionic iron release. The preincubation of MPs loaded with holoLf under conditions simulating the stomach environment resulted in the release of 40% of Lf from the MPs. The protein released was saturated with iron ions at 33%, was structurally intact, and its iron scavenging properties were preserved.

Recently, various innovative technologies have been developed for the enhanced delivery of biologics as attractive formulation targets including polymeric micro and nanoparticles. Combined with personalized medicine, this area can offer a great opportunity for the improvement of therapeutics efficiency and the treatment outcome. Herein, a novel manufacturing method has been introduced to produce protein-loaded chitosan particles with controlled size. This method is based on an additive manufacturing technology that allows for the designing and production of personalized particulate based therapeutic formulations with a precise control over the shape, size, and potentially the geometry. Sprayed multi adsorbed-droplet reposing technology (SMART) consists of the high-pressure extrusion of an ink with a well determined composition using a pneumatic 3D bioprinting approach and flash freezing the extrudate at the printing bed, optionally followed by freeze drying. In the present study, we attempted to manufacture trypsin-loaded chitosan particles using SMART. The ink and products were thoroughly characterized by dynamic light scattering, rheometer, Scanning Electron Microscopy (SEM), and Fourier Transform Infra-Red (FTIR) and Circular Dichroism (CD) spectroscopy. These characterizations confirmed the shape morphology as well as the protein integrity over the process. Further, the effect of various factors on the production were investigated. Our results showed that the concentration of the carrier, chitosan, and the lyoprotectant concentration as well as the extrusion pressure have a significant effect on the particle size. According to CD spectra, SMART ensured Trypsin's secondary structure remained intact regardless of the ink composition and pressure. However, our study revealed that the presence of 5% (w/v) lyoprotectant is essential to maintain the trypsin's proteolytic activity. This study demonstrates, for the first time, the viability of SMART as a single-step efficient process to produce biologics-based stable formulations with a precise control over the particulate morphology which can further be expanded across numerous therapeutic modalities including vaccines and cell/gene therapies. [ABSTRACT FROM AUTHOR]

Porous polymer scaffolds are widely investigated as temporary implants in regenerative medicine to repair damaged tissues. While biocompatibility, degradability, mechanical properties comparable to the native tissues and controlled porosity are prerequisite for these scaffolds, their loading with pharmaceutical or biological active ingredients such as growth factors, in particular proteins, opens up new perspective for tissue engineering applications. This implies the development of scaffold loading strategies that minimize the risk of protein denaturation and allow to control their release profile. This work reports on a straightforward method for preparing bioactive dextran-based scaffolds from high internal phase emulsion (HIPE) templates containing poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) serving both as co-stabilizers for the emulsion and nanocarriers for drug or therapeutic protein models. Scaffold synthesis are achieved by photocuring of methacrylated dextran located in the external phase of a HIPE stabilized by the NPs in combination or not with a non-ionic surfactant. Fluorescent labelling of the NPs highlights their integration in the scaffold. The introduction of NPs, and even more so when combined with a surfactant, increases the stability and mechanical properties of the scaffolds. Cell viability tests demonstrate the non-toxic nature of these NPs-loaded scaffolds. The study of the release of a model protein from the scaffold, namely lysozyme, shows that its encapsulation in nanoparticles decreases the release rate and provides additional control over the release profile., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Frank Boury reports financial support was provided by INSERM. Antoine Debuigne reports financial support was provided by Fund for Scientific Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The efficient delivery of therapeutic proteins to tumor sites is a promising cancer treatment modality. Hydrogen-bonded organic frameworks (HOFs) are successfully used for the protective encapsulation of proteins; however, easy precipitation and lack of controlled release of existing HOFs limit their further application for protein delivery in vivo. Here, a hypoxia-responsive HOF, self-assembled from azobenzenedicarboxylate/polyethylene glycol-conjugated azobenzenedicarboxylate and tetrakis(4-amidiniumphenyl)methane through charge-assisted hydrogen-bonding, is developed for systemic protein delivery to tumor cells. The newly generated HOF platform efficiently encapsulates representative cytochrome C, demonstrating good dispersibility under physiological conditions. Moreover, it can respond to overexpressed reductases in the cytoplasm under hypoxic conditions, inducing fast intracellular protein release to exert therapeutic effects. The strategy presented herein can be applied to other therapeutic proteins and can be expanded to encompass more intrinsic tumor microenvironment stimuli. This offers a novel avenue for utilizing HOFs in protein-based cancer therapy., (© 2024 Wiley‐VCH GmbH.)

[Display omitted] • A robust approach for preparing core–shell polyelectrolyte nanogels was developed. • The structure and property of the polyelectrolyte nanogels can be easily tuned. • Nanogels can be used as a carrier for efficient protein encapsulation and delivery. Designing rational carriers for protein encapsulation and delivery is of great interest in a wide range of applications. Herein, we develop a type of PEGylated polyelectrolyte nanogel that enables efficient protein loading, protection and intracellular delivery. Our design relies on a one pot process involving cooperative electrostatic assembly and polymerization, which constructs polyion complex (PIC) micelles containing cross-linked cationic PDMAEMA network associated with anionic PAA. Further removing PAA chains leads to core–shell nanogels with a cationic network surrounded by poly (ethylene glycol) (PEG) blocks. The established strategy allows to fabricate well-defined nanogels with regulated size and swelling ability depending on pH, salt concentration and cross-link degree. More notably, the abundant positive charges and the thermo-response of PDMAEMA network realize sufficient encapsulation and protection of BSA proteins, while the PEG blocks endow appreciable biocompatibility and successful intracellular protein delivery. Our study establishes a facile and robust approach for preparing core–shell polyelectrolyte nanogels with regulated size and properties, as well as prominent capabilities for protein encapsulation and delivery. [ABSTRACT FROM AUTHOR]

The development and production of innovative protein-based therapeutics is a complex and challenging avenue. External conditions such as buffers, solvents, pH, salts, polymers, surfactants, and nanoparticles may affect the stability and integrity of proteins during formulation. In this study, poly (ethylene imine) (PEI) functionalized mesoporous silica nanoparticles (MSNs) were used as a carrier for the model protein bovine serum albumin (BSA). To protect the protein inside MSNs after loading, polymeric encapsulation with poly (sodium 4-styrenesulfonate) (NaPSS) was used to seal the pores. Nano differential scanning fluorimetry (NanoDSF) was used to assess protein thermal stability during the formulation process. The MSN-PEI carrier matrix or conditions used did not destabilize the protein during loading, but the coating polymer NaPSS was incompatible with the NanoDSF technique due to autofluorescence. Thus, another pH-responsive polymer, spermine-modified acetylated dextran (SpAcDEX), was applied as a second coating after NaPSS. It possessed low autofluorescence and was successfully evaluated with the NanoDSF method. Circular dichroism (CD) spectroscopy was used to determine protein integrity in the case of interfering polymers such as NaPSS. Despite this limitation, NanoDSF was found to be a feasible and rapid tool to monitor protein stability during all steps needed to create a viable nanocarrier system for protein delivery.

[Display omitted] Podocytes, cells of the glomerular filtration barrier, play a crucial role in kidney diseases and are gaining attention as potential targets for new therapies. Brain-Derived Neurotrophic Factor (BDNF) has shown promising results in repairing podocyte damage, but its efficacy via parenteral administration is limited by a short half-life. Low temperature sensitive liposomes (LTSL) are a promising tool for targeted BDNF delivery, preserving its activity after encapsulation. This study aimed to improve LTSL design for efficient BDNF encapsulation and targeted release to podocytes, while maintaining stability and biological activity, and exploiting the conjugation of targeting peptides. While cyclic RGD (cRGD) was used for targeting endothelial cells in vitro, a homing peptide (HITSLLS) was conjugated for more specific uptake by glomerular endothelial cells in vivo. BDNF-loaded LTSL successfully repaired cytoskeleton damage in podocytes and reduced albumin permeability in a glomerular co-culture model. cRGD conjugation enhanced endothelial cell targeting and uptake, highlighting an improved therapeutic effect when BDNF release was induced by thermoresponsive liposomal degradation. In vivo, targeted LTSL showed evidence of accumulation in the kidneys, and their BDNF delivery decreased proteinuria and ameliorated kidney histology. These findings highlight the potential of BDNF-LTSL formulations in restoring podocyte function and treating glomerular diseases. [ABSTRACT FROM AUTHOR]

Recently, various innovative technologies have been developed for the enhanced delivery of biologics as attractive formulation targets including polymeric micro and nanoparticles. Combined with personalized medicine, this area can offer a great opportunity for the improvement of therapeutics efficiency and the treatment outcome. Herein, a novel manufacturing method has been introduced to produce protein-loaded chitosan particles with controlled size. This method is based on an additive manufacturing technology that allows for the designing and production of personalized particulate based therapeutic formulations with a precise control over the shape, size, and potentially the geometry. Sprayed multi adsorbed-droplet reposing technology (SMART) consists of the high-pressure extrusion of an ink with a well determined composition using a pneumatic 3D bioprinting approach and flash freezing the extrudate at the printing bed, optionally followed by freeze drying. In the present study, we attempted to manufacture trypsin-loaded chitosan particles using SMART. The ink and products were thoroughly characterized by dynamic light scattering, rheometer, Scanning Electron Microscopy (SEM), and Fourier Transform Infra-Red (FTIR) and Circular Dichroism (CD) spectroscopy. These characterizations confirmed the shape morphology as well as the protein integrity over the process. Further, the effect of various factors on the production were investigated. Our results showed that the concentration of the carrier, chitosan, and the lyoprotectant concentration as well as the extrusion pressure have a significant effect on the particle size. According to CD spectra, SMART ensured Trypsin’s secondary structure remained intact regardless of the ink composition and pressure. However, our study revealed that the presence of 5% (w/v) lyoprotectant is essential to maintain the trypsin’s proteolytic activity. This study demonstrates, for the first time, the viability of SMART as a single-step efficient process to produce biologics-based stable formulations with a precise control over the particulate morphology which can further be expanded across numerous therapeutic modalities including vaccines and cell/gene therapies.

We report here on protein–antibody conjugates (PACs) that are used for antibody‐directed delivery of protein therapeutics to specific cells. PACs have the potential to judiciously combine the merits of two prolific therapeutic approaches—biologics and antibody–drug conjugates. We utilize spherical polymer brushes to construct PACs using the combination of two simple and efficient functionally orthogonal click chemistries. In addition to the synthesis and characterization of these nanoparticles, we demonstrate that PACs are indeed capable of specifically targeting cells based on the presence of target antigen on the cell surface to deliver proteins. The potentially broad adaptability of PACs opens up new opportunities for targeted biologics in therapeutics and sensing. [ABSTRACT FROM AUTHOR]

Protein encapsulation into nanocarriers has been extensively studied to improve the efficacy and stability of therapeutic proteins. However, the chemical modification of proteins or new synthetic carrier materials are essential to achieve a high encapsulation efficiency and structural stability of proteins, which hinders their clinical applications. New strategies to physically incorporate proteins into nanocarriers feasible for clinical uses are required to overcome the current limitation. Here we report the spontaneous protein-induced reorganization of 'pre-formed' unilamellar lipid vesicles to efficiently incorporate proteins within multilamellar protein-lipid hybrid vesicles without chemical modification. Epidermal growth factor (EGF) binds to the surface of cationic unilamellar lipid vesicles and induces layer-by-layer self-assembly of the vesicles. The protein is spontaneously entrapped in the interstitial layers of a multilamellar structure with extremely high loading efficiency, ~99%, through polyionic interactions as predicted by molecular dynamics simulation. The loaded protein exhibits much higher structural, chemical, and biological stability compared to free protein. The method is also successfully applied to several other proteins. This work provides a promising method for the highly efficient encapsulation of therapeutic proteins into multilamellar lipid vesicles without the use of specialized instruments, high energy, coupling agents, or organic solvents. Multilamellar Protein-Lipid Vesicles (MPLVs) fabricated in a mild environment with low pressure and no organic solvent at room temperature were devised for linker-free and highly-efficient protein encapsulation. MPLVs have a high loading efficiency and long-term dispersion stability. The morphological changes, such as folding and engulfing steps, of unilamellar lipid vesicles by protein-induced lipid molecular asymmetry evolve into a multilamellar structure. Unlabelled Image • Proteins can induce the folding and engulfing of unilamellar lipid vesicles. • Proteins can be efficiently intercalated into multilamellar lipid structures. • Multilamellar protein-lipid vesicles (MPLVs) are prepared by simple mixing. • The protein loading yield and efficiency are ~33% and ~99%, respectively. • MPLVs exhibit long-term structural, chemical, and biological integrities of proteins. [ABSTRACT FROM AUTHOR]

Given the increasing interest in the use of peptide- and protein-based agents in therapeutic strategies, it is fundamental to develop delivery systems capable of preserving the biological activity of these molecules upon administration, and which can provide tuneable release profiles. Electrohydrodynamic (EHD) techniques, encompassing electrospinning and electrospraying, allow the generation of fibres and particles with high surface area-to-volume ratios, versatile architectures, and highly controllable release profiles. This review is focused on exploring the potential of different EHD methods (including blend, emulsion, and co−/multi-axial electrospinning and electrospraying) for the development of peptide and protein delivery systems. An overview of the principles of each technique is first presented, followed by a survey of the literature on the encapsulation of enzymes, growth factors, antibodies, hormones, and vaccine antigens using EHD approaches. The possibility for localised delivery using stimuli-responsive systems is also explored. Finally, the advantages and challenges with each EHD method are summarised, and the necessary steps for clinical translation and scaled-up production of electrospun and electrosprayed protein delivery systems are discussed. Unlabelled Image • Electrospinning and electrospraying can be used for protein encapsulation. • Use of biocompatible, FDA-approved materials may facilitate clinical translation. • Coaxial architectures offer advantages over blend and emulsion techniques. • Preservation of bioactivity and sustained release profiles can be achieved. • Potential for scaled-up manufacturing paves the way for product commercialisation. [ABSTRACT FROM AUTHOR]

Biomaterials capable of delivering therapeutic proteins are relevant in biomedicine, yet their manufacturing relies on centralized manufacturing chains that pose challenges to their remote implementation at the point of care. This study explores the viability of confined cell-free protein synthesis within porous hydrogels as biomaterials that dynamically produce and deliver proteins to in vitro and in vivo biological microenvironments. These functional biomaterials have the potential to be assembled as implants at the point of care. To this aim, we first entrap cell-free extracts (CFEs) from Escherichia coli containing the transcription-translation machinery, together with plasmid DNA encoding the super folded green fluorescence protein (sGFP) as a model protein, into hydrogels using various preparation methods. Agarose hydrogels result in the most suitable biomaterials to confine the protein synthesis system, demonstrating efficient sGFP production and diffusion from the core to the surface of the hydrogel. Freeze-drying (FD) of agarose hydrogels still allows for the synthesis and diffusion of sGFP, yielding a more attractive biomaterial for its reconstitution and implementation at the point of care. FD-agarose hydrogels are biocompatible in vitro, allowing for the colonization of cell microenvironments along with cell proliferation. Implantation assays of this biomaterial in a preclinical mouse model proved the feasibility of this protein synthesis approach in an in vivo context and indicated that the physical properties of the biomaterials influence their immune responses. This work introduces a promising avenue for biomaterial fabrication, enabling the in vivo synthesis and targeted delivery of proteins and opening new paths for advanced protein therapeutic approaches based on biocompatible biomaterials.

Neurotoxins present a substantial threat to human health and security as they disrupt and damage the nervous system. Their potent and structurally diverse nature poses challenges in developing effective countermeasures. In this study, a unique nanoparticle design that combines dual-biomimicry mechanisms to enhance the detoxification efficacy of neurotoxins is introduced. Using saxitoxin (STX), one of the deadliest neurotoxins, and its natural binding protein saxiphilin (Sxph) as a model system, human neuronal membrane-coated and Sxph-loaded metal-organic framework (MOF) nanosponges (denoted "Neuron-MOF/Sxph-NS") are successfully developed. The resulting Neuron-MOF/Sxph-NS exhibit a biomimetic design that not only emulates host neurons for function-based detoxification through the neuronal membrane coating, but also mimics toxin-resistant organisms by encapsulating the Sxph protein within the nanoparticle core. The comprehensive in vitro assays, including cell osmotic swelling, calcium flux, and cytotoxicity assays, demonstrate the improved detoxification efficacy of Neuron-MOF/Sxph-NS. Furthermore, in mouse models of STX intoxication, the application of Neuron-MOF/Sxph-NS shows significant survival benefits in both therapeutic and prophylactic regimens, without any apparent acute toxicity. Overall, the development of Neuron-MOF/Sxph-NS represents an important advancement in neurotoxin detoxification, offering promising potential for treating injuries and diseases caused by neurotoxins and addressing the current limitations in neurotoxin countermeasures., (© 2023 Wiley‐VCH GmbH.)

Despite considerable advances in recent years, challenges in delivery and storage of biological drugs persist and may delay or prohibit their clinical application. Though nanoparticle-based approaches for small molecule drug encapsulation are mature, encapsulation of proteins remains problematic due to destabilization of the protein. Reverse micelles composed of decylmonoacyl glycerol (10MAG) and lauryldimethylamino-N-oxide (LDAO) in low-viscosity alkanes have been shown to preserve the structure and stability of a wide range of biological macromolecules. Here, we present a first step on developing this system as a future platform for storage and delivery of biological drugs by replacing the non-biocompatible alkane solvent with solvents currently used in small molecule delivery systems. Using a novel screening approach, we performed a comprehensive evaluation of the 10MAG/LDAO system using two preparation methods across seven biocompatible solvents with analysis of toxicity and encapsulation efficiency for each solvent. By using an inexpensive hydrophilic small molecule to test a wide range of conditions, we identify optimal solvent properties for further development. We validate the predictions from this screen with preliminary protein encapsulation tests. The insight provided lays the foundation for further development of this system toward long-term room-temperature storage of biologics or toward water-in-oil-in-water biologic delivery systems.

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Biocompatible, environmentally responsive, and scalable nanocarriers are needed for targeted and triggered delivery of therapeutic proteins. Suitable polymers, preparation methods, and crosslinking chemistries must be considered for nanogel formation. Biocompatible dendritic polyglycerol (dPG) is used in the mild, surfactant-free inverse nanoprecipitation method for nanogel preparation. The biocompatible, fast, and bioorthogonal inverse electron demand Diels-Alder (iEDDA) crosslinking chemistry is used. In this work, the synthesis of pH-degradable nanogels, based on tetrazine, norbonene, and bicyclo[6.1.0]nonyne (BCN) functionalized macromonomers, is reported. The macromonomers are non-toxic up to 2.5 mg mL−1 in three different cell lines. Nanogels are obtained in the size range of 47 to 200 nm and can be degraded within 48 h at pH 4.5 (BA-gels), and pH 3 (THP-gels), respectively. Encapsulation of asparaginase (32 kDa) yield encapsulation efficiencies of up to 93% at 5 wt.% feed. Overall, iEDDA-crosslinked pH-degradable dPG-nanogels from inverse nanoprecipitation are promising candidates for biomedical applications. [ABSTRACT FROM AUTHOR]

L-asparaginase (ASNase) has played a key role in the management of acute lymphoblastic leukaemia (ALL). As an amidohydrolase, it catalyzes the hydrolysis of L-asparagine, a crucial step in the treatment of ALL. Various ASNase variants have evolved from diverse sources since it was first used in paediatric patients in the 1960s. This review describes the available ASNase and approaches being used to develop ASNase as a biobetter candidate. The review discusses the Glycosylation and PEGylation techniques, which are frequently used to develop biobetter versions of the majority of the therapeutic proteins. Further, it explores current ASNase biobetters in therapeutic use and discusses the protein engineering and chemical modification approaches that were employed to reduce immunogenicity, extend protein half-life, and enhance protease stability of ASNase. Emerging strategies like immobilization and encapsulation are also highlighted as potential pathways for improving ASNase properties. The purpose of the development of ASNase biobetter is to achieve a novel therapeutic candidate that could improve catalytic efficiency, in vivo stability with minimum glutaminase (GLNase) activity and toxicity. Modification of ASNase by immobilization and encapsulation or by fusion technologies like Albumin fusion, Fc fusion, ELP fusion, XTEN fusion, etc. can be exploited to develop a novel biobetter candidate suitable for therapeutic approaches. This review emphasizes the importance of biobetter development for therapeutic proteins like ASNase. Improved ASNase molecules have the potential to significantly advance the treatment of ALL and have broader implications in the pharmaceutical industry. [Display omitted] • Comprehensive analysis of L-Asparaginase (ASNase) biobetters since the 1960s. • Glycosylation, PEGylation, and protein engineering optimize ASNase for better clinical outcomes. • Chemical modifications reduce immunogenicity, enhance plasma half-life and protease stability. • Immobilization and encapsulation methods hold potential for further advancing ASNase biobetters. [ABSTRACT FROM AUTHOR]

Hollow vesicles made from a single or double layer of block-copolymer molecules, called polymersomes, represent an important technological platform for new developments in nano-medicine and nano-biotechnology. A central aspect in creating functional polymersomes is their combination with proteins, especially through encapsulation in the inner cavity of the vesicles. When producing polymersomes by techniques such as film rehydration, significant proportions of the proteins used are trapped in the vesicle lumen, resulting in high encapsulation efficiencies. However, because of the difficulty of scaling up, such methods are limited to laboratory experiments and are not suitable for industrial scale production. Recently, we developed a scalable polymersome production process in stirred-tank reactors, but the statistical encapsulation of proteins resulted in fairly low encapsulation efficiencies of around 0.5%. To increase encapsulation in this process, proteins were genetically fused with hydrophobic membrane anchoring peptides. This resulted in encapsulation efficiencies of up to 25.68%. Since proteins are deposited on the outside and inside of the polymer membrane in this process, two methods for the targeted removal of protein domains by proteolysis with tobacco etch virus protease and intein splicing were evaluated. This study demonstrates the proof-of-principle for production of protein-functionalized polymersomes in a scalable process.

Maltodextrin, which is obtained by partial hydrolysis of starch, is water soluble and could serve as hydrophilic carrier for the encapsulation of protein-based active pharmaceutical ingredients. We investigated three different commercial maltodextrins (Dextrose Equivalents (DE) 4.0–7.0, DE 13.0–17.0 and DE 16.5–19.5) with focus on their ability to form nanoparticles by inverse precipitation. Successful particle formation was observed for DE 13.0–17.0 and DE 16.5–19.5 but not for DE 4.0–7.0. The process was investigated using acetone as anti-solvent and poloxamer 407 as stabilizer. A tunable size between 170 nm and 450 nm was achieved by varying the type of maltodextrin and the stabilizer concentration. Particles were spherical in shape and were stable over a time period of 14 days. Maltodextrin nanoparticles (MD NPs) were tested on A549 cells and did not show any cytotoxic effects. This underlines the potential of maltodextrin as material for drug delivery systems. Bovine serum albumin (BSA) as a model protein was successfully encapsulated into MD NPs with encapsulation efficiencies of approx. 70% and loading rates of up to 20%. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Alginate hydrogel particles are promising delivery systems for protein encapsulation and controlled release because of their excellent biocompatibility, biodegradability, and mild gelation process. In this study, a facile microfluidic approach is developed for making uniform core-shell hydrogel microparticles. To address the challenge of protein retention within the alginate gel matrix, poly(ethyleneimine) (PEI)- and chitosan-coated alginate microparticles were fabricated demonstrating improved protein retention as well as controlled release. Furthermore, a model protein ovalbumin was loaded along with delta inulin microparticulate adjuvant into the water-core of the alginate microparticles. Compared to those microparticles with only antigen loaded, the antigen + adjuvant loaded microparticles showed a delayed and sustained release of antigen. This microfluidic approach provides a convenient method for making well-controlled alginate microgel particles with uniform size and controlled properties, and demonstrates the ability to tune the release profiles of proteins by engineering microparticle structure and properties. [ABSTRACT FROM AUTHOR]

Encapsulation of therapeutic proteins into particles from appropriate material can improve both stability and delivery of the drugs, and the obtained particles can serve as a platform for development of their new oral formulations. The main goal of this work was development of sol-gel/emulsion method for preparation of silica microcapsules capable of controlled release of encapsulated protein without loss of its native structure. For this purpose, the reported in literature direct sol-gel/W/O/W emulsion method of protein encapsulation was used with some modifications, because the original method did not allow to prepare silica microcapsules capable for protein release. The particles were synthesized using sodium silicate and tetraethoxysilane as silica precursors and different compositions of oil phase. In vitro kinetics of bovine serum albumin (BSA) release in buffer (pH 7.4) was studied by Fourier transform infrared (FTIR) and fluorescence spectrometry, respectively. Structural state of encapsulated BSA and after release was evaluated. It was found that the synthesis conditions influenced substantially the porous structure of the unloaded silica particles, release properties of the BSA-loaded silica particles and structural state of the encapsulated and released protein. The modified synthesis conditions made it possible to obtain the silica particles capable of controlled release of the protein during a week without loss of the protein native structure. [ABSTRACT FROM AUTHOR]

Herein, an amino‐acid‐boosted biomimetic strategy is reported that enabled the rapid encapsulation, or co‐encapsulation, of a broad range of proteins into microporous metal–organic frameworks (MOFs), with an ultrahigh loading efficiency. It relies on the accelerated formation of prenucleation clusters around proteins via a metallothionein‐like self‐assembly. The encapsulated proteins maintained their native conformations, and the structural confinement within porous MOFs endowed enzymes with excellent bioactivity, even in harsh conditions (e.g. in the presence of proteolytic or chemical agents or at high temperature). Furthermore, owing to the merits of nondestructive and protein surface charge‐independent encapsulation, the feasibility of this biomimetic strategy for biostorage, enzyme cascades, and biosensing was also verified. It is believed that this convenient and versatile encapsulation strategy has great promise in the important fields of biomedicine, catalysis, and biosensing. It's a trap: A convenient and versatile amino‐acid‐boosted biomimetic strategy is presented for the nondestructive encapsulation of biomacromolecules within metal–organic frameworks. The structural confinement within porous MOFs gave the biomacromolecules excellent stability, and endowed the composite system with novel functionality for biostorage, biocatalysis, and sensing. [ABSTRACT FROM AUTHOR]

Designing effective drug nanocarriers that are easy to synthesize, robust, and nontoxic is a significant challenge in nanomedicine. Polyamine-multivalent molecule nanocomplexes are promising drug carriers due to their simple and all-aqueous manufacturing process. However, these systems can present issues of colloidal instability over time and cellular toxicity due to the cationic polymer. In this study, we finely modulate the formation parameters of poly(allylamine-tripolyphosphate) complexes to jointly optimize the robustness and safety. Polyallylamine was ionically assembled with tripolyphosphate anions to form liquid-like nanocomplexes with a size of around 200 nm and a zeta potential of -30 mV. We found that nanocomplexes exhibit tremendous long-term stability (9 months of storage) in colloidal dispersion and that they are suitable as protein-loading agents. Moreover, the formation of nanocomplexes induced by tripolyphosphate anions produces a switch-off in the toxicity of the system by altering the overall charge from positive to negative. In addition, we demonstrate that nanocomplexes can be internalized by bone-marrow-derived macrophage cells. Altogether, these nanocomplexes have attractive and promising properties as delivery nanoplatforms for potential therapies based on the immune system activation.

Abstract Bovine Serum Albumin (BSA) and Horseradish Peroxidase (HRP) have been encapsulated within microparticulated matrices composed of Eudragit RS100 by the water-in-oil-in-water double emulsion solvent evaporation method. Good encapsulation efficiencies were achieved for BSA and HRP, 88.4 and 95.8%, respectively. The stability of the loaded proteins was confirmed by using circular dichroism and fluorescence. The gastroresistance of the protein-loaded microparticles was evaluated under simulated gastric conditions demonstrating the preservation of the structural integrity of the proteins loaded inside the particles. The enzymatic activity of HRP after being released from the enteric microparticles was evaluated by using the peroxidase substrate, revealing that the released enzyme preserved its 100% function. The high drug loadings achieved, reduced cytotoxicity and efficient gastric protection point out towards the potential use of those carriers as oral delivery vectors of therapeutic proteins offering a more controlled targeted release in specific sites of the intestine and an enhanced gastrointestinal absorption. [ABSTRACT FROM AUTHOR]

Complex coacervates based on natural biopolymers have been explored as a protein delivery system to provide controlled release of loaded protein and circumvent the proteolytic degradation in chronic wounds. The coacervates composed of gelatin A (GA) and sodium alginate (SA) were optimized with respect to turbidity, size, zeta potential, and polydispersity index. Bovine serum albumin (BSA), a model protein, was effectively encapsulated in the coacervates, resulting in protection from trypsin digestion and controlled release. In contrast, EGF was not encapsulated in the same coacervates. Striking difference in the encapsulation efficiencies of BSA and EGF, despite their similar net charges, was attributed to their different levels of binding to GA based on the surface plasmon resonance (SPR) biosensor analysis. In conclusion, GA and SA coacervates can protect the encapsulated protein from proteolytic degradation, demonstrating its potential as a delivery system in the chronic wounds. SPR biosensor is proposed as an analytical tool to study the interactions between polymers and proteins in association with encapsulation efficiency in complex coacervation. The results of EGF studies suggested that GA was not a suitable polymer for EGF encapsulation and therefore, further investigation would be needed to find suitable polymer systems for improved encapsulation efficiency. [ABSTRACT FROM AUTHOR]

Abstract: Introduction of large pore in the primitive microporous metal–organic frameworks (MOFs) with tailorable particle size can endow them with desired properties for potential applications in the intracellular delivery of membrane‐impermeable proteins. However, no research is found to focus on this topic until now. Herein, a monocarboxylic acid (MA) and organic base comodulation strategy is developed to synthesize the hierarchically porous UiO‐66 nanoparticles. MA of dodecanoic acid is utilized to control the pore size while trimethylamine (TEA) plays a key role in modulating the nucleation of crystallization to regulate the particle size. In comparison with microporous UiO‐66, a model protein of cytochrome c (Cyt c) could be efficiently loaded into the mesoporous MOFs (mesoMOFs). The size‐dependent cellular uptake is also evaluated, and it is verified that mesoMOFs with particle size of 90 nm could be endocytosed into living cells with highest efficiency. These outstanding merits enable the current mesoMOFs not only to exhibit efficient encapsulation of Cyt c but also facilitate the protein delivery into the cytosol and subsequent endosomal escape. Given the exceptional chemical stability, hierarchically porous structure as well as tunable particle size, the elaborated mesoUiO‐66 nanoparticles might offer a promising platform for a variety of biomedical applications. [ABSTRACT FROM AUTHOR]

Chemokines are known to stimulate directed migration of cancer cells. Therefore, the strategy involving gradual chemokine release from polymeric vehicles for trapping cancer cells is of interest. In this work, the chemokine stromal cell-derived factor-1α (SDF-1α) was encapsulated into nanoparticles composed of poly-(lactic-co-glycolic acid) (PLGA) and a polyethylene glycol (PEG)-PLGA co-polymer to achieve sustained release. SDF-1α, and lysozyme as a model protein, were firstly precipitated to promote their stability upon encapsulation. A novel phase separation method utilising a non-toxic solvent in the form of isosorbide dimethyl ether was developed for the individual encapsulation of SDF-1α and lysozyme precipitates. Uniform nanoparticles of 200–250 nm in size with spherical morphologies were successfully synthesised under mild formulation conditions and conveniently freeze-dried in the presence of hydroxypropyl-β-cyclodextrin as a stabiliser. The effect of PLGA carboxylic acid terminal capping on protein encapsulation efficiency and release rate was also explored. Following optimisation, sustained release of SDF-1α was achieved over a period of 72 h. Importantly, the novel encapsulation process was found to induce negligible protein denaturation. The obtained SDF-1α nanocarriers may be subsequently incorporated within a hydrogel or other scaffolds to establish a chemokine concentration gradient for the trapping of glioblastoma cells. [ABSTRACT FROM AUTHOR]

Flash NanoPrecipitation (FNP) is a rapid method for encapsulating hydrophobic materials in polymer nanoparticles with high loading capacity. Encapsulating biologics such as proteins remains a challenge due to their low hydrophobicity (logP < 6) and current methods require multiple processing steps. In this work, we report rapid, single-step protein encapsulation via FNP using bovine serum albumin (BSA) as a model protein. Nanoparticle formation involves complexation and precipitation of protein with tannic acid and stabilization with a cationic polyelectrolyte. Nanoparticle self-assembly is driven by hydrogen bonding and electrostatic interactions. Using this approach, high encapsulation efficiency (up to ~80%) of protein can be achieved. The resulting nanoparticles are stable at physiological pH and ionic strength. Overall, FNP is a rapid, efficient platform for encapsulating proteins for various applications.

Because of the biocompatible and biodegradable properties of poly (lactic-co-glycolic acid) (PLGA), nanoparticles (NPs) based on this polymer have been widely studied for drug/biomolecule delivery and long-term sustained-release. In this work, two different formulation methods for lysozyme-loaded PLGA NPs have been developed and optimized based on the double-emulsion (water/oil/water, W/O/W) solvent evaporation technique. They differ mainly in the phase in which the surfactant (Pluronic ® F68) is added: water (W-F68) and oil (O-F68). The colloidal properties of these systems (morphology by SEM and STEM, hydrodynamic size by DLS and NTA, electrophoretic mobility, temporal stability in different media, protein encapsulation, release, and bioactivity) have been analyzed. The interaction surfactant-protein depending on the formulation procedure has been characterized by surface tension and dilatational rheology. Finally, cellular uptake by human mesenchymal stromal cells and cytotoxicity for both systems have been analyzed. Spherical hard NPs are made by the two methods However, in one case, they are monodisperse with diameters of around 120 nm (O-F68), and in the other case, a polydisperse system of NPs with diameters between 100 and 500 nm is found (W-F68). Protein encapsulation efficiency, release and bioactivity are maintained better by the W-F68 formulation method. This multimodal system is found to be a promising “dual delivery” system for encapsulating hydrophilic proteins with strong biological activity at the cell-surface and cytoplasmic levels. [ABSTRACT FROM AUTHOR]

Thermosensitive liposomes are clinically-relevant nanocarriers which have been used to deliver chemotherapeutic agents to tumors in combination with local hyperthermia. However, the encapsulation and release of macromolecular therapeutic agents (proteins, nucleic acids, bioactive polymers) is often hindered by their instability during the liposome formation as well as by the low encapsulation efficiency. The objective of this study was to investigate the influence of the thermosensitive liposomal formulation on the encapsulation and release of low and high molecular weight hydrophilic drugs, in order to identify the key parameters to control during nanocarrier design, depending on the specific drug delivery application. Thermosensitive liposomes with different formulations were prepared through the combinations of different lipids, including dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), cholesterol (Chol), 1-palmitoyl-2-hydroxy-sn- glycero -3-phosphocholine (P-Lyso-PC), and the PEGylated lipid distearoyl-sn- glycero -3-phosphoethanolamine- N -methoxy(PEG)-2000 (DSPE-PEG2000). The thin film hydration method was used for liposome preparation and loading of different water soluble molecules. The encapsulation efficiency and release profiles were investigated for a low molecular weight compound such as carboxyfluorescein (CF), proteins (albumin), and hydrophilic polymers which do not interact with the lipid bilayer, such as a linear dextran and a poly(ethylene glycol)-based star polymer. An optimised liposomal formulation [DPPC/P-lyso-PC/DSPE-PEG2000 90/10/4 (mol/mol) (LTSL)] was chosen for further application in encapsulating therapeutic proteins, such as lysozyme and the brain-derived neurotrophic factor (BDNF), which are recognized as drug carriers and potential therapeutic agents for kidney diseases and neurological disorders. [ABSTRACT FROM AUTHOR]

Principles of double emulsion generation were incorporated in a glass microfluidic chip fabricated with two different surface properties in order to produce protein loaded polymer microspheres. The microspheres were produced by integrating two microfluidic flow focusing systems and a multi-step droplet splitting and mixing system into one chip. The chip consists of a hydrophobic and a hydrophilic section with two different heights, 12 μm and 45 μm, respectively. As a result, the protein is homogenously distributed throughout the polymer microsphere matrix, not just in its center (which has been studied before). In our work, the inner phase was bovine serum albumin (BSA) in phosphate buffered saline, the disperse phase was poly (lactic acid) in chloroform and the continuous phase was an aqueous solution of poly(vinyl alcohol). After solvent removal, BSA loaded microspheres with an encapsulation efficiency of up to 96% were obtained. Our results show the feasibility of producing microspheres loaded with a hydrophilic drug in a microfluidic system that integrates different microfluidic units into one chip. [ABSTRACT FROM AUTHOR]

Depending on their architectural and chemical design, microgels can selectively take up and release small molecules by changing the environmental properties, or capture and protect their cargo from the surrounding conditions. These outstanding properties make them promising candidates for use in biomedical applications as delivery or carrier systems. In this study, hollow anionic p(N-isopropylacrylamid-e-co-itaconic acid) microgels are synthesized and analyzed regarding their size, charge, and charge distribution. Furthermore, interactions between these microgels and the model protein cytochrome c are investigated as a function of pH. In this system, pH serves as a switch for the electrostatic interactions to alternate between no interaction, attraction, and repulsion. UV-vis spectroscopy is used to quantitatively study the encapsulation of cytochrome c and possible leakage. Additionally, fluorescence-lifetime images unravel the spatial distribution of the protein within the hollow microgels as a function of pH. These analyses show that cytochrome c mainly remains entrapped in the microgel, with pH controlling the localization of the protein - either in the microgel's cavity or in its network. This significantly differentiates these hollow microgels from microgels with similar chemical composition but without a solvent filled cavity., (© 2023 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)