Development of Polymer Coacersome Structure with Enhanced Colloidal Stability for Therapeutic Protein Delivery

Poly(ethylene arginyl aspartate diglyceride) (PEAD) polycation is widely used to prepare coacervate particles by electrostatic complexation with an anionic heparin (HEP) in aqueous environments, for controlled release of therapeutic proteins. However, coacervate complexes aggregate randomly due to particle–particle charge interactions. Herein, a new term “coacersome” is introduced to represent a stable polyplex formed by complexation of mPEGylated PEAD and HEP. Methoxy polyethylene glycol (mPEG)‐b‐cationic PEAD diblock copolymers are synthesized and complexed with HEP to create a stable “coacersome” structure. Water‐soluble mPEG moiety assembles on the surface of coacersomes in aqueous conditions and creates a steric barrier to avoid aggregation of coacersomes. The coacersomes are able to maintain their initial spherical morphology and size for longer durations in the presence of competing ions, such as 0.3 m NaCl. Additionally, the coacersomes exhibit biocompatibility toward human dermal fibroblasts, a high loading efficiency (>96%) for encapsulation of bone morphogenetic protein 2 (BMP‐2), and a sustained release profile up to 28 days. The BMP‐2‐loaded coacersomes further exhibit increased osteogenic differentiation of human mesenchymal stem cells (hMSCs). The developed coacersome structures have the potential to be utilized as effective carriers for therapeutic protein delivery.     

Relative retention of the fibroblast growth factors FGF-1 and FGF-2 on strong cation-exchange sorbents

The isocratic retention of two heparin-binding fibroblast growth factors, FGF-1 (acidic FGF) and FGF-2 (basic FGF), was compared on a set of six preparative strong cation-exchange adsorbents. The FGFs comprise a solute pair that are structurally equivalent, yet differ in protein parameters of potential importance in cation-exchange chromatography, such as isoelectric point, net charge, and the number and distribution of basic amino acids. The cation-exchange adsorbents comprise a diverse set of materials in common use for protein purification, with physical and chemical properties that have been characterized and described previously. Isocratic k' values for the two proteins obtained on each adsorbent at several different [NaCl] are compared with one another and with corresponding data for hen egg lysozyme, which is also strongly retained on cation-exchangers. Of the six adsorbents examined, three showed strong retention of both FGFs, with equivalent k' values for FGF-1 and FGF-2. Three others, which showed weaker overall retention for the FGF pair, showed much larger retention differences between FGF-1 and FGF-2. The trends in retention order among the stationary phases are very similar to those seen previously with other unrelated proteins. However, retention differences between the two FGFs, and between the FGFs and lysozyme, do not correlate well with simple charge properties such as net charge, indicating, as in some previous studies, the importance of local regions on the protein surface in determining retention. These observations are interpreted in terms of the structural features of the proteins and the physicochemical properties of the adsorbents.     

Selective inhibition of amino-terminal methionine processing by TNP-470 and ovalicin in endothelial cells

BACKGROUND: The angiogenesis inhibitors TNP-470 and ovalicin potently suppress endothelial cell growth. Both drugs also specifically inhibit methionine aminopeptidase 2 (MetAP2) in vitro. Inhibition of MetAP2 and changes in initiator methionine removal in drug-treated endothelial cells have not been demonstrated, however. RESUTLS: Concentrations of TNP-470 sufficient to inactivate MetAP2 in intact endothelial cells were comparable to those that inhibited cell proliferation, suggesting that MetAP2 inhibition by TNP-470 underlies the ability of the drug to inhibit cell growth. Both drug-sensitive and drug-insensitive cell lines express MetAP1 and MetAP2, indicating that drug sensitivity in mammalian cells is not simply due to the absence of compensating MetAP activity. With a single exception, detectable protein N-myristoylation is unaffected in sensitive endothelial cells treated with TNP-470, so MetAP1 activity can generally compensate when MetAP2 is inactive. Analysis of total protein extracts from cells pulse-labeled with [(35)S]-methionine following TNP-470 treatment revealed changes in the migration of several newly synthesized proteins. Two of these proteins were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin A. Purification and amino-terminal sequencing of GAPDH from TNP-470-treated cells revealed partial retention of its initiator methionine, indicating that methionine removal from some, but not all, proteins is affected by MetAP2 inactivation. CONCLUSIONS: Amino-terminal processing defects occur in cells treated with TNP-470, indicating that inhibition of MetAP2 by the drug occurs in intact cells. This work renders plausible a mechanism for growth inhibition by TNP-470 as a consequence of initiator methionine retention, leading to the inactivation of as yet unidentified proteins essential for endothelial cell growth.     

Endothelial cell migration on surface-density gradients of fibronectin, VEGF, or both proteins

Cell migration is essential to many physiological processes, including angiogenesis, which is critical to the success of implanted biomaterials and tissue-engineered constructs. Gradients play an important role in cell migration. Previous work on cell migration has been mostly executed either in the concentration gradients of stimuli (e.g., VEGF) in bulk or hydrogels or on the surface-density gradients of ECM proteins (e.g., fibronectin) or small ligands (e.g., RGD). Little work has been done to investigate how cell migration responds to the surface-density gradients of growth factors. No work has been done to study how the surface gradients of both adhesive proteins and growth factors influence cell migration. In this work, we studied the effect of the surface-density gradients of fibronectin (FN), VEGF, or both proteins on endothelial cell migration. Gradients with different slopes were prepared to study how the gradient slope affects cell migration. The gradients were generated by first forming a counter-propagating C15COOH/C11OH self-assembled monolayer (SAM) gradient using a surface electrochemistry approach, followed by activating the -COOH moieties and covalently immobilizing proteins onto the surface. Fourier transform infrared spectra and X-ray photoelectron spectroscopy were used to characterize the SAM and protein gradients, respectively. A free cell migration assay using bovine aortic endothelial cells was performed on various gradient surfaces or on surfaces with uniform protein density. Results showed that cells on the surface-density gradients of FN, VEGF, or both proteins moved faster along the gradient direction than on the respective uniform control surface after 24-h cell culture. It is also shown that for each protein or protein combination, the directional cell displacement was not statistically different between two gradients with different slopes. Results show that the directional cell migration was increased by about 2-fold on the VEGF gradient as compared to the FN gradient and was further increased by another 2-fold on the combined gradients of both proteins as compared to the VEGF gradient alone. This is the first work to create surface-density gradients of VEGF and the first study to generate a combined surface gradient of growth factor and ECM protein to investigate their effect on cell migration on surfaces. This work broadens our understanding of the directional movement of endothelial cells. Our findings provide useful information for directing cell migration into tissue-engineered constructs and can be potentially used for those applications where cell migration is critical, such as angiogenesis.     

A polymer scaffold for protein oligomerization

We report the design and synthesis of well-defined polymers for the noncovalent oligomerization of proteins. The reported scaffolds, which were generated by atom-transfer radical polymerization (ATRP), take advantage of the well-characterized interaction between a Ni2+ complex and an oligohistidine sequence (His tag). Thus, our polymers are designed to facilitate the oligomerization of any protein possessing a His tag. We demonstrate that they can oligomerize fibroblast growth factor-8b (FGF-8b) and promote FGF-8b-mediated cell proliferation in the absence of heparin.     

A Versatile Method to Prepare Protein Nanoclusters for Drug Delivery

Nanocarriers based on natural biomaterials such as peptides and proteins have shown great advantages in the field of nanomedicine. However, the complicated preparation process and possible denaturation of proteins may limit their further applications. Herein, a novel method is developed to prepare protein nanocluster drug delivery system based on the self‐aggregated property of proteins under the isoelectric point condition. The crosslinked protein nanoclusters, prepared by adding modified natural crosslinking agent polysaccharide, exhibit excellent stability and autofluorescent property in physiological conditions. Hemoglobin, a model protein, is chosen for preparation of drug‐loaded nanoclusters. The as‐prepared nanoclusters demonstrate a pH‐responsive drug release behavior and can successfully deliver drugs into cancer cells. Moreover, this approach can be extended to various proteins, exemplifying the universal applicability of our new preparation method for protein‐based nanoparticles.     

pH-sensitive polymer-assisted refolding of urea-denatured fibroblast growth factor

A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of FGF-2(fibroblast growth factor-2) denatured with 8 mol/L urea and 10 mmol/L dithiothreitol at pH 7.2.The refolding of FGF-2 was performed by directly diluting denatured FGF-2 into a refolding buffer containing Eudragit S-100.The ability of Eudragit S-100 to enhance protein refolding level was investigated using MTr method,fluorescence emission spectroscopy and reverse phase HPLC.On the other hand,the result shows the ab...     

Strong Inhibition of Ice Growth by Biomimetic Crowding Coacervates

The freezing of biological fluids is intensively studied but remains elusive as it is affected not only by the various components but also by the crowding nature of the biological fluids. Herein, we constructed spherical crowders, fibrous crowders, and coacervates by various components ranging from surfactants to polymers and proteins, to mimic three typical crowders in biological fluids, i.e., globular proteins, fibrous networks, and condensates of biomolecules. It is elucidated that the three crowders exhibit low, moderate, and strong ice growth inhibition activity, respectively, resulting from their different abilities in slowing down water dynamics. Intriguingly, the coacervate consisting of molecules without obvious ice growth inhibition activity strongly inhibits ice growth, which is firstly employed as a highly-potent cryoprotectant. This work provides new insights into the survival of freezing-tolerant organisms and opens an avenue for the design of ice-controlling materials.     

Protein–polysaccharide interactions and aggregates in food formulations

The protein–polysaccharide combinations that lead to electrostatic complex and coacervate formation are the object of extensive research using both layer-by-layer and mixed emulsion approaches. The protein–polysaccharide conjugates demonstrated interesting physicochemical properties as stabilizers and emulsifiers, as well as texture modifiers in food products. Furthermore, they are potential optimal nutrient delivery systems. Their complex behavior due to several factors such as pH, ionic strength, concentration, heat, and mechanical treatments is the main reason behind the continuous growth of the research field. The review is reporting some recent advances on the topic, along with an overview of the possible interactions between protein and polysaccharide, from Maillard reaction to enzymatic cross-linking passing through coacervates.     

Polymeric Micelles Loading Proteins through Concurrent Ion Complexation and pH‐Cleavable Covalent Bonding for In Vivo Delivery

Protein drugs have great potential as targeted therapies, yet their application suffers from several drawbacks, such as instability, short half‐life, and adverse immune responses. Thus, protein delivery approaches based on stimuli‐responsive nanocarriers can provide effective strategies for selectively enhancing the availability and activation of proteins in targeted tissues. Herein, polymeric micelles with the ability of encapsulating proteins are developed via concurrent ion complexation and pH‐cleavable covalent bonding between proteins and block copolymers directed to pH‐triggered release of the protein payload. Carboxydimethylmaleic anhydride (CDM) is selected as the pH‐sensitive moiety, since the CDM? amide bond is stable at physiological pH (pH 7.4), while it cleaves at pH 6.5, that is, the pathophysiological pH of tumors and inflammatory tissues. By using poly(ethylene glycol)‐poly(l ‐lysine) block copolymers having 45% CDM addition, different proteins with various sizes and isoelectric points are loaded successfully. By using myoglobin‐loaded micelles (myo/m) as a model, the stability of the micelles in physiological conditions and the dissociation and release of functional myoglobin at pH 6.5 are successfully confirmed. Moreover, myo/m shows extended half‐life in blood compared to free myoglobin and micelles assembled solely by polyion complex, indicating the potential of this system for in vivo delivery of proteins.     

A receptor-binding-based bioassay to determine the potency of a plasmid biopharmaceutical encoding VEGF-C

Over the last decade biological assays (bioassays) have gained much importance for quality control in biopharmaceutical development and manufacturing. Here we describe the development and validation of a bioassay to determine the biological activity (potency) of the plasmid biopharmaceutical pVGI.1 which encodes the VEGF-C (VEGF-2) protein. This assay was developed to test drug substance and drug product for release and stability testing for phase I and II clinical trials. The main focus was on fast assay development and easy handling of the assay, combined with valid results representing the specific therapeutic mechanism. The method includes the expression of the VEGF-C protein in mammalian cells and its binding to the cell surface receptor VEGFR-3. The binding activity of VEGF-C to its immobilized receptor is quantified in a colorimetric assay. IC₅₀ values of VEGF-C expressed after transfection with sample plasmid and an in-house standard plasmid are determined. The ratio of the IC₅₀ value of the test article to that of the reference standard reflects the potency of the sample. The potency assay meets the criteria generally requested by authorities for precision, linearity, accuracy, and range. Therefore the assay can be used in pharmaceutical quality control and is a suitable basis for development of related bioassays.     

Microfluidic Formation of Membrane‐Free Aqueous Coacervate Droplets in Water

We report on the formation of coacervate droplets from poly(diallyldimethylammonium chloride) with either adenosine triphosphate or carboxymethyl‐dextran using a microfluidic flow‐focusing system. The formed droplets exhibit improved stability and narrower size distributions for both coacervate compositions when compared to the conventional vortex dispersion techniques. We also demonstrate the use of two parallel flow‐focusing channels for the simultaneous formation and co‐location of two distinct populations of coacervate droplets containing different DNA oligonucleotides, and that the populations can coexist in close proximity up to 48 h without detectable exchange of genetic information. Our results show that the observed improvements in droplet stability and size distribution may be scaled with ease. In addition, the ability to encapsulate different materials into coacervate droplets using a microfluidic channel structure allows for their use as cell‐mimicking compartments.     

Fuel-driven macromolecular coacervation in complex coacervate core micelles

Fuel-driven macromolecular coacervation is an entry into the transient formation of highly charged, responsive material phases. In this work, we used a chemical reaction network (CRN) to drive the coacervation of macromolecular species readily produced using radical polymerisation methods. The CRN enables transient quaternization of tertiary amine substrates, driven by the conversion of electron deficient allyl acetates and thiol or amine nucleophiles. By incorporating tertiary amine functionality into block copolymers, we demonstrate chemical triggered complex coacervate core micelle (C3M) assembly and disassembly. In contrast to most dynamic coacervate systems, this CRN operates at constant physiological pH without the need for complex biomolecules. By varying the allyl acetate fuel, deactivating nucleophile and reagent ratios, we achieved both sequential signal-induced C3M (dis)assembly, as well as transient non-equilibrium (dis)assembly. We expect that timed and signal-responsive control over coacervate phase formation at physiological pH will find application in nucleic acid delivery, nano reactors and protocell research.

We apply an allyl acetate fuelled chemical reaction network (CRN) to control the coacervation of macromolecular species at constant physiological pH without the need for complex biomolecules.     

A light-activated polymer with excellent serum tolerance for intracellular protein delivery

The design of efficient materials for intracellular protein delivery has attracted great interest in recent years; however, most current materials for this purpose are limited by poor serum stability due to the early release of cargoes triggered by abundant serum proteins. Here, we propose a light-activated crosslinking (LAC) strategy to prepare efficient polymers with excellent serum tolerance for intracellular protein delivery. A cationic dendrimer engineered with photoactivatable O-nitrobenzene moieties co-assembles with cargo proteins via ionic interactions, followed by light activation to yield aldehyde groups on the dendrimer and the formation of imine bonds with cargo proteins. The light-activated complexes show high stability in buffer and serum solutions, but dis-assemble under low pH conditions. As a result, the polymer successfully delivers cargo proteins green fluorescent protein and β-galactosidase into cells with maintained bioactivity even in the presence of 50% serum. The LAC strategy proposed in this study provides a new insight to improve the serum stability of polymers for intracellular protein delivery.

A light-activated crosslinking strategy was proposed to prepare a polymer with high robustness and serum stability for cytosol protein delivery. It can deliver proteins into cytosols with maintained bioactivities even in 50% FBS-containing medium.     

Polymeric Nanoparticles Amenable to Simultaneous Installation of Exterior Targeting and Interior Therapeutic Proteins

Effective delivery of therapeutic proteins is a formidable challenge. Herein, using a unique polymer family with a wide‐ranging set of cationic and hydrophobic features, we developed a novel nanoparticle (NP) platform capable of installing protein ligands on the particle surface and simultaneously carrying therapeutic proteins inside by a self‐assembly procedure. The loaded therapeutic proteins (e.g., insulin) within the NPs exhibited sustained and tunable release, while the surface‐coated protein ligands (e.g., transferrin) were demonstrated to alter the NP cellular behaviors. In vivo results revealed that the transferrin‐coated NPs can effectively be transported across the intestinal epithelium for oral insulin delivery, leading to a notable hypoglycemic response.     

Protein delivery based on uncoated and chitosan-coated mesoporous silicon microparticles

Mesoporous silicon is a biocompatible, biodegradable material that is receiving increased attention for pharmaceutical applications due to its extensive specific surface. This feature enables to load a variety of drugs in mesoporous silicon devices by simple adsorption-based procedures. In this work, we have addressed the fabrication and characterization of two new mesoporous silicon devices prepared by electrochemistry and intended for protein delivery, namely: (i) mesoporous silicon microparticles and (ii) chitosan-coated mesoporous silicon microparticles. Both carriers were investigated for their capacity to load a therapeutic protein (insulin) and a model antigen (bovine serum albumin) by adsorption. Our results show that mesoporous silicon microparticles prepared by electrochemical methods present moderate affinity for insulin and high affinity for albumin. However, mesoporous silicon presents an extensive capacity to load both proteins, leading to systems were protein could represent the major mass fraction of the formulation. The possibility to form a chitosan coating on the microparticles surface was confirmed both qualitatively by atomic force microscopy and quantitatively by a colorimetric method. Mesoporous silicon microparticles with mean pore size of 35 nm released the loaded insulin quickly, but not instantaneously. This profile could be slowed to a certain extent by the chitosan coating modification. With their high protein loading, their capacity to provide a controlled release of insulin over a period of 60-90 min, and the potential mucoadhesive effect of the chitosan coating, these composite devices comprise several features that render them interesting candidates as transmucosal protein delivery systems.     

Preparation of Fe3O4@SiO2@layered double hydroxide core-shell microspheres for magnetic separation of proteins

Three-component microspheres containing an SiO(2)-coated Fe(3)O(4) magnetite core and a layered double hydroxide (LDH) nanoplatelet shell have been synthesized via an in situ growth method. The resulting Fe(3)O(4)@SiO(2)@NiAl-LDH microspheres display three-dimensional core-shell architecture with flowerlike morphology, large surface area (83 m(2)/g), and uniform mesochannels (4.3 nm). The Ni(2+) cations in the NiAl-LDH shell provide docking sites for histidine and the materials exhibit excellent performance in the separation of a histidine (His)-tagged green fluorescent protein, with a binding capacity as high as 239 μg/mg. The microspheres show highly selective adsorption of the His-tagged protein from Escherichia coli lysate, demonstrating their practical applicability. Moreover, the microspheres possess superparamagnetism and high saturation magnetization (36.8 emu/g), which allows them to be easily separated from solution by means of an external magnetic field and subsequently reused. The high stability and selectivity of the Fe(3)O(4)@SiO(2)@NiAl-LDH microspheres for the His-tagged protein were retained over several separation cycles. Therefore, this work provides a promising approach for the design and synthesis of multifunctional LDH microspheres, which can be used for the practical purification of recombinant proteins, as well as having other potential applications in a variety of biomedical fields including drug delivery and biosensors.     

Diffusivity of whey protein and gum arabic in their coacervates

Structural properties of whey protein (WP)/gum arabic (GA) coacervates were investigated by measuring the diffusivity of WP and GA in their coacervate phase as a function of pH by means of three different complementary techniques. The combination of these measurements revealed new insights into the structure of coacervates. Nuclear magnetic resonance (NMR) measured the self-diffusion coefficient of the GA in the coacervate phase prepared at various pH values. Fluorescence recovery after photobleaching (FRAP) was measured using a confocal scanning laser microscope. The WP and GA were covalently labeled with two different dyes. The time of fluorescence recovery, related to the inverse of the diffusion coefficient, was evaluated from the measurements, and the diffusivity of the WP and GA on a long time scale could be individually estimated at each pH value. Diffusing wave spectroscopy (DWS) combined with transmission measurement was carried out in the coacervate phase, and the diffusion coefficient, corresponding to the averaged diffusion of all particles that scattered in the system, was calculated as a function of pH. Independently of the technique used, the results showed that the diffusion of the WP and GA within the coacervate phase was reduced as compared to a diluted biopolymer mixture. NMR, DWS, and FRAP measurements gave similar results, indicating that the biopolymers moved the slowest in the coacervate matrix at pH 4.0-4.2. It is assumed that the diffusion of the WP and GA is reduced because of a higher electrostatic interaction between the biopolymers. Furthermore, FRAP results showed that in the coacervate phase WP molecules diffused 10 times faster than GA molecules. This result is very relevant because it shows that WP and GA move independently in the liquid coacervate phase. Finally, DWS measurements revealed that the coacervate phase rearranged with time, as evidenced by a decrease of the diffusion coefficient and a loss of the turbidity of the sample. A more homogeneous transparent coacervate phase was obtained after a few days/weeks. Faster rearrangement was obtained at pH 3.0 and 3.5 than at higher pH values.     

Nanobody-Ferritin Conjugate for Targeted Photodynamic Therapy

Ferritin is an iron-storage protein nanocage that is assembled from 24 subunits. The hollow cavity of ferritin enables its encapsulation of various therapeutic agents; therefore, ferritin has been intensively investigated for drug delivery. The use of antibody-ferritin conjugates provides an effective approach for targeted drug delivery. However, the complicated preparation and limited protein stability hamper wide applications of this system. Herein, we designed a novel nanobody-ferritin platform (Nb-Ftn) for targeted drug delivery. The site-specific conjugation between nanobody and ferritin is achieved by transglutaminase-catalyzed protein ligation. This ligation strategy allows the Nb conjugation after drug loading in ferritin, which avoids deactivation of the nanobody under the harsh pH environment required for drug encapsulation. To verify the tumor targeting of this Nb-Ftn platform, a photodynamic reagent, manganese phthalocyanine (MnPc), was loaded into the ferritin cavity, and an anti-EGFR nanobody was conjugated to the surface of the ferritin. The ferritin nanocage can encapsulate about 82 MnPc molecules. This MnPc@Nb-Ftn conjugate can be efficiently internalized by EGFR positive A431 cancer cells, but not by EGFR negative MCF-7 cells. Upon 730 nm laser irradiation, MnPc@Nb-Ftn selectively killed EGFR positive A431 cells by generating reactive oxygen species (ROS), whereas no obvious damage was observed on MCF-7 cells. Given that ferritin can be used for encapsulation of various therapeutic agents, this work provides a strategy for facile construction of nanobody-ferritin for targeted drug delivery.     

Protein charge parameters that influence stability and cellular internalization of polyelectrolyte complex micelles

Proteins are an important class of biologics, but there are several recurring challenges to address when designing protein-based therapeutics. These challenges include: the propensity of proteins to aggregate during formulation, relatively low loading in traditional hydrophobic delivery vehicles, and inefficient cellular uptake. This last criterion is particularly challenging for anionic proteins as they cannot cross the anionic plasma membrane. Here we investigated the complex coacervation of anionic proteins with a block copolymer of opposite charge to form polyelectrolyte complex (PEC) micelles for use as a protein delivery vehicle. Using genetically modified variants of the model protein green fluorescent protein (GFP), we evaluated the role of protein charge and charge localization in the formation and stability of PEC micelles. A neutral-cationic block copolymer, poly(oligoethylene glycol methacrylate-block-quaternized 4-vinylpyridine), POEGMA₇₉-b-qP4VP₁₇₅, was prepared via RAFT polymerization for complexation and microphase separation with the panel of engineered anionic GFPs. We found that isotropically supercharged proteins formed micelles at higher ionic strength relative to protein variants with charge localized to a polypeptide tag. We then studied GFP delivery by PEC micelles and found that they effectively delivered the protein cargo to mammalian cells. However, cellular delivery varied as a function of protein charge and charge distribution and we found an inverse relationship between the PEC micelle critical salt concentration and delivery efficiency. This model system has highlighted the potential of polyelectrolyte complexes to deliver anionic proteins intracellularly. Using this model system, we have identified requirements for the formation of PEC micelles that are stable at physiological ionic strength and that smaller protein–polyelectrolyte complexes effectively deliver proteins to Jurkat cells.

The stability and delivery efficiency of protein-based polyelectrolyte-complex micelles was evaluated for a panel of proteins with varying net charge and charge distribution.