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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.)

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., Competing Interests: Declaration of Competing Interest The authors report there are no competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors 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 The Authors. Published by Elsevier B.V. All rights reserved.)

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.)

Background: 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., Scope of Review: 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., Major Conclusions: 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., General Significance: 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

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.

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.

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.)

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.

Regulation of protein activity is important in their applications for biomedicine and therapeutics. Here, an approach for the regulation of protein bioactivity through molecular confinement provided by oligoethylene glycol (OEG)-based dendronized chitosan (DCS) hydrogels is reported. Structural effects on their thermoresponsiveness are investigated. The highly transparent hydrogels are formed from thermoresponsive DCSs through their thermal dehydration and exhibit an intriguing reversible sol-gel transition property when triggered at physiological temperatures. The thermo-gelling behavior and mechanical strength of these hydrogels are investigated, and possible effects from hydrophobicity of the OEG dendrons, grafting rates of the dendrons on the chitosan main chain, and solid content of polymers are examined. These DCS hydrogels are found to have lamellar morphologies and can provide characteristic hydrophobicity microenvironments formed through the crowded OEG dendrons, which show a higher level of confinement to guest proteins. This allows the DCS hydrogels remarkable activity protection capability to proteins. Furthermore, these DCS hydrogels inherit the degradability from chitosan, allowing protein release from these hydrogels through the controllable ways without impairing their activities.

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.

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., Competing Interests: Declaration of Competing Interest The authors 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 © 2022 Elsevier Inc. All rights reserved.)

Bioorthogonal catalysis (BC) generates chemical reactions not present in normal physiology for the purpose of disease treatment. Because BC catalytically produces the desired therapy only at the site of disease, it holds the promise of site-specific treatment with little or no systemic exposure or side effects. Transition metals are typically used as catalytic centers in BC; however, solubility and substrate specificity typically necessitate a coordinating enzyme and/or stabilizing superstructure for in vivo application. We report the use of self-assembling, porous exoshells (tESs) to encapsulate and deliver an iron-containing reaction center for the treatment of breast cancer. The catalytic center is paired with indole-3-acetic acid (IAA), a natural product found in edible plants, which undergoes oxidative decarboxylation, via reduction of iron(III) to iron(II), to produce free radicals and bioactive metabolites. The tES encapsulation is critical for endocytic uptake of BC reaction centers and, when followed by administration of IAA, results in apoptosis of MDA-MB-231 triple negative cancer cells and complete regression of in vivo orthotopic xenograft tumors ( p < 0.001, n = 8 per group). When Renilla luciferase (rLuc) is substituted for horseradish peroxidase (HRP), whole animal luminometry can be used to monitor in vivo activity.

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.

Engineering a system with a high mass fraction of active ingredients, especially water-soluble proteins, is still an ongoing challenge. In this work, we developed a versatile surface camouflage strategy that can engineer systems with an ultrahigh mass fraction of proteins. By formulating protein molecules into nanoparticles, the demand of molecular modification was transformed into a surface camouflage of protein nanoparticles. Thanks to electrostatic attractions and van der Waals interactions, we camouflaged the surface of protein nanoparticles through the adsorption of carrier materials. The adsorption of carrier materials successfully inhibited the phase transfer of insulin, albumin, β-lactoglobulin, and ovalbumin nanoparticles. As a result, the obtained microcomposites featured with a record of protein encapsulation efficiencies near 100% and a record of protein mass fraction of 77%. After the encapsulation in microcomposites, the insulin revealed a hypoglycemic effect for at least 14 d with one single injection, while that of insulin solution was only ∼4 h.

Protein enzymes have shown great potential in numerous technological applications. However, the design of supporting materials is needed to preserve protein functionality outside their native environment. Direct enzyme-polymer self-assembly offers a promising alternative to immobilize proteins in an aqueous solution, achieving higher control of their stability and enzymatic activity in industrial applications. Herein, we propose a modeling-based design to engineering hydrogels of cytochrome P450 and of PETase with styrene/2-vinylpyridine (2VP) random copolymers. By tuning the copolymer fraction of polar groups and of charged groups via quaternization of 2VP for coassembly with cytochrome P450 and via sulfonation of styrene for coassembly with PETase, we provide quantitative guidelines to select either a protein-polymer hydrogel structure or a single-protein encapsulation. The results highlight that, regardless of the protein surface domains, the presence of polar interactions and hydration effects promote the formation of a more elongated enzyme-polymer complex, suggesting a membrane-like coassembly. On the other hand, the effectiveness of a single-protein encapsulation is reached by decreasing the fraction of polar groups and by increasing the charge fraction up to 15%. Our computational analysis demonstrates that the enzyme-polymer assemblies are first promoted by the hydrophobic interactions which lead the protein nonpolar residues to achieve the maximum coverage and to play the role of the most robust contact points. The mechanisms of coassembly are unveiled in the light of both protein and polymer physical-chemistry, providing bioconjugate phase diagrams for the optimal material design.

Polyethyleneimine (PEI) polymers are known to compact DNA strands into spheroid, toroid, or rod structures. A formulation with mannose-grafted PEI (PEIm), however, was reported to compact DNA into ~100 nm spheroids that indented like thin-walled pressurized shells. The goal of the study is to understand why mannose bristles divert the traditional pathway of PEI-DNA compaction to produce shell-like structures, and to manipulate the process so that proteins can be packed into the core of the assembling shells for co-delivering DNA and proteins into cells. DLS, AFM, and TEM imaging provide a consistent picture that BSA proteins can be packed into the shells without altering the shell architecture, as long as the proteins were added during the time course of shell assembly. Force spectroscopy studies reveal that DNA shells that buckle also have a rich surface-coating of mannose, indicating that a micelle-like partitioning of hydrophobic and hydrophilic layers governs shell assembly. When HEK293T cells are spiked with BSA-laden DNA shells, co-transfection of DNA and BSA is observed at higher levels than control formulations. Distinct micron-sized features appear having both green fluorescence from BSA-FITC and blue fluorescence from NucBlue DNA stain, suggesting BSA release in nucleus and secretory granules. With DNA nanocontainers, proteins can take advantage of the efficiency of PEI-based DNA transfection for hitchhiking into cells while being shielded from the challenges of the intracellular route. DNA nanocontainers are rapid to assemble, not dependent on the DNA sequence, and can be adapted for different protein types; thereby having potential to serve as a high-throughput platform in scenarios where DNA and protein have to be released at the same site and time within cells (e.g., theranostics, multiplexed co-delivery, gene editing)., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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., (© 2021 Wiley-VCH GmbH.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Endolysins are a novel class of antibacterials with proven efficacy in combating various bacterial infections, in vitro and in vivo. LysMR-5, an endolysin derived from phage MR-5, demonstrated high lytic activity in our laboratory against multidrug-resistant S. aureus (MRSA) and S. epidermidis strains. However, endolysin and proteins in general are associated with instability and short in vivo half-life, consequently limiting their usage as pharmaceutical preparation to treat bacterial infections. Nanoencapsulation of endolysins could help to achieve better therapeutic outcome, by protecting the proteins from degradation, providing sustained release, thus could increase their stability, shelf life, and therapeutic efficacy. Hence, in this study, the feasibility of alginate-chitosan nanoparticles (Alg-Chi NPs) to serve as drug delivery platform for LysMR-5 was evaluated. LysMR-5-loaded nanoparticles were prepared by calcium ion-induced pre-gelation of alginate core and its complexation with chitosan. The formation of nanoparticles was confirmed on the basis of DLS, zeta potential, and electron microscopy imaging. The LysMR-5-loaded nanoparticles presented a hydrodynamic diameter of 276.5 ± 42, a PDI of 0.342 ± 0.02, a zeta potential - 25 mV, and an entrapment efficiency of 62 ± 3.1%. The potential ionic interaction between alginate, chitosan, and LysMR-5 was investigated by FT-IR and SEM-EDX analysis. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), nano-sized particles with characteristic morphology were seen. Different antibacterial assays and SDS-PAGE analysis showed no change in endolysin's structural integrity and bioactivity after entrapment. A direct antibacterial effect of blank Alg-Chi Nps, showing enhanced bactericidal activity upon LysMR-5 loading, was observed against S. aureus. At physiological pH (7.2), the release profile of LysMR-5 from Alg-Chi NPs showed a biphasic release and followed a non-Fickian release mechanism. The biocompatible nature as revealed by cytocompatibility and hemocompatibility studies endorsed their use as drug delivery system for in vivo studies. Collectively, these results demonstrate the potential of Alg-Chi NPs as nano-delivery vehicle for endolysin LysMR-5 and other therapeutic proteins for their use in various biomedical applications.

Protein encapsulation is a growing area of interest, particularly in the fields of food science and medicine. The sequestration of protein cargoes is achieved using a variety of methods, each with benefits and drawbacks. One of the most significant challenges associated with protein encapsulation is achieving high loading while maintaining protein viability. This difficulty is exacerbated because many encapsulant systems require the use of organic solvents. By contrast, nature has optimized strategies to compartmentalize and protect proteins inside the cell-a purely aqueous environment. Although the mechanisms whereby aspects of the cytosol is able to stabilize proteins are unknown, the crowded nature of many newly discovered, liquid phase separated "membraneless organelles" that achieve protein compartmentalization suggests that the material environment surrounding the protein may be critical in determining stability. Here, encapsulation strategies based on liquid-liquid phase separation, and complex coacervation in particular, which has many of the key features of the cytoplasm as a material, are reviewed. The literature on protein encapsulation via coacervation is also reviewed and the parameters relevant to creating protein-containing coacervate formulations are discussed. Additionally, potential opportunities associated with the creation of tailored materials to better facilitate protein encapsulation and stabilization are highlighted., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intracellular delivery of functional proteins is a promising, but challenging, strategy for many therapeutic applications. Here, we report a new methodology that overcomes drawbacks of traditional mesoporous silica (MSi) particles for protein delivery. We hypothesize that engineering enhancement in interactions between proteins and delivery vehicles can facilitate efficient encapsulation and intracellular delivery. In this strategy, surface lysines in proteins were modified with a self-immolative linker containing a terminal boronic acid for stimulus-induced reversibility in functionalization. The boronic acid moiety serves to efficiently interact with amine-functionalized MSi through dative and electrostatic interactions. We show that proteins of different sizes and isoelectric points can be quantitatively encapsulated into MSi, even at low protein concentrations. We also show that the proteins can be efficiently delivered into cells with retention of activity. Utility of this approach is further demonstrated with gene editing in cells, through the delivery of a CRISPR/Cas9 complex.

Efficient protein delivery into the target cell is highly desirable for protein therapeutics. Current approaches for protein delivery commonly suffer from low-loading protein capacity, poor specificity for target cells, and invisible protein release. Herein, we report a protein@inorganic nanodumpling (ND) system as an intracellular protein delivery platform. Similar to a traditional Chinese food, the dumpling, ND consists of a protein complex "filling" formed by metal-ion-directed self-assembly of protein cargos fused to histidine-rich green fluorescent proteins (H 39 GFPs), which are further encapsulated by an external surface "wrapper" of manganese dioxide (MnO 2 ) via in situ biomineralization. This ND structure allows for a high loading capacity (>63 wt %) for protein cargos with enhanced stability. NDs can be targeted and internalized into cancer cells specifically through folic acid receptors by surface-tailored folic acid. The protein cargo release is in a bistimuli-responsive manner, triggered by an either reductive or acidic intracellular microenvironment. Moreover, the MnO 2 nanowrapper is an efficient fluorescence quencher for inner fused GFPs and also a "switch-on" magnetic resonance imaging (MRI) agent via triggered release of Mn 2+ ions, which enables activatable fluorescence/MRI bimodal imaging of protein release. Finally, the ND is highly potent and specific to deliver functional protein ribonuclease A (RNase A) into cultured target cells and the tumor site in a xenografted mouse model, eliminating the tumor cells with high therapeutic efficacy. Our approach provides a promising alternative to advance protein-based cancer therapeutics.

Alternatives for strain-promoted azide-alkyne cycloaddition (SPAAC) chemistries are needed because of the employment of expensive and not easily scalable precursors such as bicyclo[6.1.0]non-4-yne (BCN). Inverse electron demand Diels Alder (iEDDA)-based click chemistries, using dienophiles and tetrazines, offer a more bioorthogonal and faster toolbox, especially in the biomedical field. Here, the straightforward synthesis of dendritic polyglycerin dienophiles (dPG-dienophiles) and dPG-methyl-tetrazine (dPG-metTet) as macromonomers for a fast, stable, and scalable nanogel formation by inverse nanoprecipitation is reported. Nanogel size-influencing parameters are screened such as macromonomer concentration and water-to-acetone ratio are screened. dPG-norbonene and dPG-cyclopropene show fast and stable nanogel formation in the size range of 40-200 nm and are thus used for the coprecipitation of the model protein myoglobin. High encapsulation efficiencies of more than 70% at a 5 wt% feed ratio are obtained in both cases, showing the suitability of the mild gelation chemistry for the encapsulation of small proteins., (© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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%., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

In vitro investigation of the interaction between proteins and positively curved membranes can be performed using a classic nanotube pulling method. However, characterizing protein interaction with negatively curved membranes still represents a formidable challenge. Here, we describe our recently developed approach based on laser-triggered Giant Unilamellar Vesicles (GUVs) fusion. Our protocol allows sequential addition of proteins to a negatively curved membrane, while at the same time controlling the buffer composition, lipid composition and membrane tension. Moreover, this method does not require a step of protein detachment, greatly simplifying the process of protein encapsulation over existing methods., Competing Interests: Competing interestsThe authors declare no conflict of interest., (Copyright © 2019 The Authors; exclusive licensee Bio-protocol LLC.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bacterial microcompartments have significant potential in the area of industrial biotechnology for the production of small molecules, especially involving metabolic pathways with toxic or volatile intermediates. Corynebacterium glutamicum is an established industrial workhorse for the production of amino acids and has been investigated for the production of diamines, dicarboxylic acids, polymers and biobased fuels. Herein, we describe components for the establishment of bacterial microcompartments as production chambers in C. glutamicum. Within this study, we optimized genetic clusters for the expression of the shell components of the Citrobacter freundii propanediol utilization (Pdu) bacterial compartment, thereby facilitating heterologous compartment production in C. glutamicum. Upon induction, transmission electron microscopy images of thin sections from these strains revealed microcompartment-like structures within the cytosol. Furthermore, we demonstrate that it is possible to target eYFP to the empty microcompartments through C-terminal fusions with synthetic scaffold interaction partners (PDZ, SH3 and GBD) as well as with a non-native C-terminal targeting peptide from AdhDH (Klebsiella pneumonia). Thus, we show that it is possible to target proteins to compartments where N-terminal targeting is not possible. The overproduction of PduA alone leads to the construction of filamentous structures within the cytosol and eYFP molecules are localized to these structures when they are N-terminally fused to the P18 and D18 encapsulation peptides from PduP and PduD, respectively. In the future, these nanotube-like structures might be used as scaffolds for directed cellular organization and pathway enhancement.

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 120nm (O-F68), and in the other case, a polydisperse system of NPs with diameters between 100 and 500nm 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Inspired by biological systems, many biomimetic methods suggest fabrication of functional materials with unique physicochemical properties. Such methods frequently generate organic-inorganic composites that feature highly ordered hierarchical structures with intriguing properties, distinct from their individual components. A striking example is that of DNA-inorganic hybrid micro/nanostructures, fabricated by the rolling circle technique. Here, a novel concept for the encapsulation of bioactive proteins in DNA flowers (DNF) while maintaining the activity of protein payloads is reported. A wide range of proteins, including enzymes, can be simultaneously associated with the growing DNA strands and Mg 2 PPi crystals during the rolling circle process, ultimately leading to the direct immobilization of proteins into DNF. The unique porous structure of this construct, along with the abundance of Mg ions and DNA molecules present, provides many interaction sites for proteins, enabling high loading efficiency and enhanced stability. Further, as a proof of concept, it is demonstrated that the DNF can deliver payloads of cytotoxic protein (i.e., RNase A) to the cells without a loss in its biological function and structural integrity, resulting in highly increased cell death compared to the free protein., (© 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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, 12μm and 45μm, 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Encapsulation of proteins in nanoparticles (NPs) can greatly improve the properties of proteins such as their stability against denaturation and degradation by proteases, and branches out the applications of natural proteins from their intrinsic localizations and functions in living organisms for biomedical and industrial applications. We recently developed several methods to armor proteins in NPs with sizes from nanometers up to >100nm, batch by batch or one by one, covalently or noncovalently, for a wide range of applications from biocatalysis to bioimaging and drug delivery. In this chapter, we provide detailed protocols on these methods. Key steps of specific protocols are explained with particular examples to help other laboratories to adopt and modify these methods for their own purposes. The advantages and disadvantages of each method are summarized, and guidelines for choosing the right method for a given application, as well as the current challenges and future directions of this field, are discussed., (© 2017 Elsevier Inc. All rights reserved.)

The formulation in which therapeutic proteins are administered plays a key role in retaining their biological activity. Enzyme wrapping, using synthetic polymers, is a strategy employed to provide enzymes with lower immunogenicity, longer circulation times, and better targeting capabilities. Protein-polymer complexation methods, involving covalent, noncovalent, and electrostatic interactions, that can provide means to develop formulations for retaining enzyme stability are discussed in this chapter. Amphiphilic self-cross-linkable polymer was used to encapsulate capsase-3 enzyme in the nanogel, while inverse emulsion polymerization method was used to entrap α-glucosidase enzyme in the nanogel. These nanogels were characterized by dynamic light scattering, transmission electron microscopy, and gel electrophoresis. Upon release of caspase-3 enzyme from polymeric nanogel, it retained nearly 86% of its original activity. Similarly, α-glucosidase that was encased in the acid cleavable polymeric nanogel exhibited substantial activity after release under acidic conditions (pH 5, 48h). Nano-armoring of the enzymes were nearly complete and provided high yields of the encased enzyme., (© 2017 Elsevier Inc. All rights reserved.)

DNA hydrogel is a promising biomaterial for biological and medical applications due to its native biocompatibility and biodegradability. Herein, we provide a novel, versatile, and cost-effective approach for self-assembly of DNA hydrogel using the enzymatically polymerized DNA building blocks. The X-shaped DNA motif was elongated by terminal deoxynucleotidyl transferase (TdT) to form the building blocks, and hybridization between dual building blocks via their complementary TdT-polymerized DNA tails led to gel formation. TdT polymerization dramatically reduced the required amount of original DNA motifs, and the hybridization-mediated cross-linking of building blocks endows the gel with high mechanical strength. The DNA hydrogel can be applied for encapsulation and controllable release of protein cargos (for instance, green fluorescent protein) due to its enzymatic responsive properties. Moreover, this versatile strategy was extended to construct a functional DNAzyme hydrogel by integrating the peroxidase-mimicking DNAzyme into DNA motifs. Furthermore, a hybrid cascade enzymatic reaction system was constructed by coencapsulating glucose oxidase and β-galactosidase into DNAzyme hydrogel. This efficient cascade reaction provides not only a potential method for glucose/lactose detection by naked eye but also a promising modular platform for constructing a multiple enzyme or enzyme/DNAzyme hybrid system.

Poly(D,L-lactic-co -glycolic) acid (PLGA)-based sub-micron particles are uniquely posed to overcome limitations of conventional drug delivery systems. However, tailoring cargo/payload release profiles from PLGA micro/nanoparticles typically requires optimization of the multi-parameter formulation, where small changes may cause drastic shifts in the resulting release profiles. In this study, we aimed to establish whether refining the average diameter of sub-micron particle populations after formulation alters protein release profiles. PLGA particles were first produced via double emulsion-solvent evaporation method to encapsulate bovine serum albumin. Particles were then subjected to centrifugal fractioning protocols varying in both spin time and force to determine encapsulation efficiency and release profile of differently sized populations that originated from a single batch. We found the average particle diameter was related to marked alterations in encapsulation efficiencies (range: 36.4-49.4%), burst release (range: 15.8-49.1%), and time for total cargo release (range: 38-78 days). Our data corroborate previous reports relating PLGA particle size with such release characteristics, however, this is the first study, to our knowledge, to directly compare particle population size while holding all formulation parameters constant. In summary, centrifugal fractioning to selectively control the population distribution of sub-micron PLGA particles represents a feasible tool to tailor release characteristics. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A 104A: 688-696, 2016., (© 2015 Wiley Periodicals, Inc.)

Encapsulation of model proteins (catalase, insulin, aprotinin) into multilayer dextran sulphate/protamin capsules by templating on CaCO3 microparticles is investigated employing: (i) PRE-loading into CaCO3 particles by adsorption or co-synthesis and (ii) POST-loading into performed capsules. Protein encapsulation is governed by both its size and electrostatic interactions with the carbonate microparticles and multilayer shell. PRE-loading enables improved encapsulation compared to POST-loading (catalase content in capsules 630 and 70 mg · g(-1)). Bioactivity of encapsulated protein is not affected by interaction with multilayers but may be reduced at slightly alkaline pH due to CaCO3 hydrolysis. This study might help to successfully encapsulate fragile bio-macromolecules into multilayer capsules., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Currently, vaccination is the most efficient and cost-effective medical treatment for infectious diseases; however, each year 10 million infants remain underimmunized due to current vaccination schedules that require multiple doses to be administered across months or years. These dosing regimens are especially challenging in the developing world where limited healthcare access poses a major logistical barrier to immunization. Over the past four decades, researchers have attempted to overcome this issue by developing single-administration vaccines based on controlled-release antigen delivery systems. These systems can be administered once, but release antigen over an extended period of time to elicit both primary and secondary immune responses resulting in antigen-specific immunological memory. Unfortunately, unlike controlled release systems for drugs, single-administration vaccines have yet to be commercialized due to poor antigen stability and difficulty in obtaining unconventional release kinetics. This review discusses the current state of single-administration vaccination, challenges delaying the development of these vaccines, and potential strategies for overcoming these challenges., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The aim of this work was to encapsulate transforming growth factor β1 (TGF-β1) into PLGA microparticles for regenerative medicine applications. TGF-β1 was firstly precipitated to ensure its stability during subsequent encapsulation within microparticles. A novel emulsification/extraction process in CO2 medium under mild conditions of pressure and temperature was used to encapsulate the protein. Interestingly, non-volatile injectable solvents, isosorbide dimethyl ether (DMI) and glycofurol (GF), were employed to precipitate the protein and to dissolve the polymer. Good encapsulation efficiency was obtained with preserved bioactivity of the protein. The microparticles were characterized in terms of size and zeta potential. In addition, the morphology and surface properties were determined using scanning electron microscopy (SEM) and atomic force microscopy (AFM) respectively. In vitro release study of the protein from microparticles was presented to assess the capacity of these systems to control the protein release. Moreover, cytotoxicity study was performed and showed an excellent cytocompatibility of the obtained microparticles. Thus, we described an effective and original process for TGF-β1 encapsulation into PLGA microparticles. The obtained polymeric carriers could be used in many biomedical applications and were more specifically developed for cartilage regeneration., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Nanoparticles (NPs) are currently used as drug delivery systems for numerous therapeutic macromolecules, e.g. proteins or DNA. Based on the preparation by double emulsion solvent evaporation a sponge-like structure was postulated entrapping hydrophilic drugs inside an internal aqueous phase. However, a direct proof of this hypothesized structure is still missing today. NPs were prepared from different polymers using a double-emulsion method and characterized for their physicochemical properties. Combining ion milling with field emission scanning electron microscopy allowed to cross section single NP and to visualize their internal morphology. The imaging procedure permitted cross-sectioning of NPs and visualization of the internal structure as well as localizing drugs associated with NPs. It was observed that none of the model actives was encapsulated inside the polymeric matrix when particle diameters were below around 470 nm but predominantly adsorbed to the particle surface. Even at larger diameters only a minority of particles of a diameter below 1 μm contained an internal phase. The properties of such drug loaded NPs, i.e. drug release or the observations in cellular uptake or even drug targeting needs to be interpreted carefully since in most cases NP surface properties are potentially dominated by the 'encapsulated' drug characteristics., (Copyright © 2014 Elsevier B.V. All rights reserved.)