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Polymer vesicles are of profound interest for designing delivery vehicles and nanoreactors toward a variety of biomedical and catalytic applications, yet robust synthesis of stable and permeable vesicles remains challenging. Here, we propose an electrostatic-templated polymerization that enables fabrication of polyelectrolyte vesicles with simultaneously regulated stability and permeability. In our design, cationic monomers were copolymerized with cross-linkers in the presence of a polyanionic-neutral diblock copolymer as a template. By properly choosing the block length ratio of the template, we fabricated a type of polyion complex vesicle consisting of a cross-linked cationic membrane, electrostatically assembled with the template copolymer which can be removed by sequential dissociation and separation under concentrated salt. We finally obtained stable polyelectrolyte vesicles of regulated size, membrane permeability, and response properties by tuning the synthesis factors including ionic strength, cross-linker type, and fraction as well as different monomers and concentrations. As a proof-of-concept, lipase was loaded in the designed cationic vesicles, which exhibited enhanced enzyme stability and activity. Our study has developed a novel and robust strategy for controllable synthesis of a new class of stable and permeable polymer (polyelectrolyte) vesicles that feature great potential applications as functional delivery carriers and nanoreactors.

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In this study, we investigated detailedly the contribution of catechol in tuning the formation and adhesive properties of coacervates. We have constructed a series of catechol-grafted Chitosan (Chitosan-C), and investigated their coacervation with gum arabic (GA) and the corresponding adhesion. We demonstrate that, increasing catechol grafting ratio from 0 %-44 % impacted the coacervation moderately, while enhanced the adhesion of the coacervate up to 438 % when the catechol faction was 37 %. Further increasing the grafting ratio to 55 % led to precipitated coacervates associated with a declined adhesion. Our findings identify the optimal grafting threshold for coacervation and adhesion, providing insights into the underlying mechanism of coacervate binding. Moreover, the catechol enhancement on adhesion of coacervates tolerates different substrates and diverse polyelectrolyte pairs. The revealed principles shall be helpful for designing adhesive coacervates and boosting their applications in various industrial and biomedical areas.

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Enzyme immobilization in nanoparticles is of interest for boosting their catalytic applications, yet rational approaches to designs achieving both high enzyme loading and activation remain a challenge. Herein, we report an electrostatically mediated in situ polymerization strategy that simultaneously realizes enzyme immobilization and activation. This was achieved by copolymerizing cationic monomers with a cross-linker in the presence of the enzyme lipase (anionic) as the template, which produces enzyme-loaded nanogels. The effects of different control factors such as pH, lipase dosage, and cross-linker fraction on nanogel formation are investigated systematically, and optimal conditions for enzyme loading and activation have been determined. A central finding is that the cationic polymer network of the nanogel creates a favorable environment that not only protects the enzyme but also boosts enzymatic activity nearly 2-fold as compared to free lipase. The nanogels improve the stability of the lipase to tolerate a broader working range of pH (5.5-8.5) and temperature (25-70 °C) and allow recycling such that after six cycles of reaction, 70% of the initial activity is conserved. The established fabrication strategy can be applied generally to different cationic monomers, and most of these nanogels exhibit adequate immobilization and activation of lipase. Our study confirms that in situ polymerization based on electrostatic interaction provides a facile and robust strategy for enzyme immobilization and activation. The wide variety of ionic monomers, therefore, features great potential for developing functional platforms toward satisfying enzyme immobilization and demanding applications.

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Protein-polyelectrolyte complex coacervation is of particular interest for mimicking intracellular phase separation and organization. Yet, the challenge arises from regulating the coacervation due to the globular structure and anisotropic distributed charges of protein. Herein, we fully investigate the different control factors and reveal their effects on protein-polyelectrolyte coacervation. We prepared mixtures of BSA (bovine serum albumin) with different cationic polymers, which include linear and branched polyelectrolytes covering different spacer and charge groups, chain lengths, and polymer structures. With BSA-PDMAEMA [poly(N,N-dimethylaminomethyl methacrylate)] as the main investigated pair, we find that the moderate pH and ionic strength are essential for the adequate electrostatic interaction and formation of coacervate droplets. For most BSA-polymer mixtures, excess polyelectrolytes are required to achieve the full complexation, as evidenced by the deviated optimal charge mixing ratios from the charge stoichiometry. Polymers with longer chains or primary amine groups and a branched structure endow a strong electrostatic interaction with BSA and cause a bigger charge ratio deviation associated with the formation of solid-like coacervate complexes. Nevertheless, both the liquid- and solid-like coacervates hardly interrupt the BSA structure and activity, indicating the safe encapsulation of proteins by the coacervation with polyelectrolytes. Our study validates the crucial control of the diverse factors in regulating protein-polyelectrolyte coacervation, and the revealed principles shall be instructive for establishing other protein-based coacervations and boosting their potential applications.

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Proteins used as building blocks to template nanostructures with manifold morphologies have been widely reported. Understanding their self-assembly and reassembly mechanism is important for designing functional biomaterials. Herein, we show that enzyme-hydrolyzed α-lactalbumin (α-lac) can self-assemble into either nanotubes in the presence of Ca2+ ions or nanospheres in the absence of Ca2+ in solution. Remarkably, such assembled α-lac nanotubes can be elongated by adding preassembled α-lac nanospheres and Ca2+ solution, which suggests that the self-assembled α-lac nanospheres undergo disassembly and reassembly processes into existing nanotube nuclei. By performing atomic force microscopy (AFM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM), it indicates that there is an equilibrium among nanotubes, nanospheres, hydrolyzed α-lac, and Ca2+ in solution. The structural transition between nanotubes and nanospheres is driven from a less stable structure into a more stable structure determined by the conditions. During the transition from nanospheres into nanotubes, the hydrolyzed α-lac in nanospheres transfers into helical ribbon form at both nanotube extremities. Then helical ribbons close into mature nanotubes, extending the length of the initial nuclei. Besides, by dilution or adding ethylene glycol bis(2-aminoethyl ether) tetraacetic acid (EGTA), the decreased Ca2+ concentration in solution drives the Ca2+ dissociating from nanotubes into solution, leading to the transitions from nanotubes into nanospheres. The reversible transformation between nanotubes and nanospheres is achieved by adjusting the pH value from 7.5 to 5.0 and back to 7.5. This is because the stability of nanotubes decreases from pH 7.5 to 5 but increases from 5 to 7.5. Significantly, this approach can be used for the fabrication of various responsive nanomaterials from the same starting material.

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In food manufacturing and particular biomedical products selected proteins are often required. Obtaining the desired proteins in a pure form from natural resources is therefore important, but often very challenging. Herein, we design a sequential coacervation process that allows to efficiently isolate and purify proteins with different isoelectric points (pIs) from a mixed solution, namely Bovine Serum Albumin (BSA, pI = 4.9) and Peroxidase from Horseradish (HRP, pI = 7.2). The key to separation is introducing a suitable polyelectrolyte that causes selective complex coacervation at appropriate pH and ionic strength. Specifically, polyethyleneimine (PEI), when added into the mixture at pH 6.0, produces a coacervation which exclusively contains BSA, leading to a supernatant solution containing 100 % HRP with a purity of 91 %. After separating the dilute and dense phases, BSA is recovered by adding poly(acrylic acid) (PAA) to the concentrated phase, which displaces BSA from the complex because it interacts more strongly with PEI. The supernatant phase after this step contains approximately 75 % of the initial amount of BSA with a purity of 99 %. Our results confirm that coacervation under well-defined conditions can be selective, enabling separation of proteins with adequate purity. Therefore, the established approach demonstrates a facile and sustainable strategy with potential for protein separation at industrial scale.

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Incorporating copper (Cu) ions into polymeric particles can be a straightforward strategy for mimicking copper enzymes, but it is challenging to simultaneously control the structure of the nanozyme and of the active sites. In this report, we present a novel bis-ligand (L2) containing bipyridine groups connected by a tetra-ethylene oxide (4EO) spacer. In phosphate buffer the Cu-L2 mixture forms coordination complexes that (at proper composition) can bind polyacrylic acid (PAA) to produce catalytically active polymeric nanoparticles with well-defined structure and size, which we refer to as 'nanozymes'. Manipulating the L2/Cu mixing ratio and using phosphate as a co-binding motif, cooperative copper centres are realized that exhibit promoted oxidation activity. The structure and activity of the so-designed nanozymes remain stable upon increasing temperature and over multiple cycles of application. Increasing ionic strength causes enhanced activity, a response also seen for natural tyrosinase. By means of our rational design we obtain nanozymes with optimized structure and active sites that in several respects outperform natural enzymes. This approach therefore demonstrates a novel strategy for developing functional nanozymes, which may well stimulate the application of this class of catalysts.

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Polyelectrolyte nanogel consisting of charged network is a prospective platform for developing nanoreactor due to their integrated features of both polyelectrolyte and hydrogel. In this work, cationic poly (methacrylatoethyl trimethyl ammonium chloride) (PMETAC) nanogels with regulated size (30-82 nm) and crosslinking degree (10-50%), has been synthesized by Electrostatic Assembly Directed Polymerization (EADP) method and applied to load gold nanoparticles (AuNPs). Based on the typical reduction reaction of 4-nitrophenol (4-NP), the catalytic performance of the constructed nanoreactor was examined by studying their kinetic process, where the loaded AuNPs exhibited dependent activity on crosslinking degree of nanogels, while independent catalytic activity on nanogel size. Our results validate that, polyelectrolyte nanogels are capable of loading metal NPs and regulating their catalytic performance, therefore demonstrates potential for developing functional nanoreactors.

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Targeted transfection of siRNA to preosteoclasts features the potential of anti-osteoporosis, yet challenge arises from the development of satisfied delivery vehicles. Here, we design a rational core-shell nanoparticle (NP) composed of cationic and responsive core for controlled load and release of small interfering RNA (siRNA) and compatible polyethylene glycol shell modified with alendronate for enhanced circulation and bone-targeted delivery of siRNA. The designed NPs perform well on transfection of an active siRNA (siDcstamp) that interferes Dcstamp mRNA expression, leading to impeded preosteoclast fusion and bone resorption, as well as promoted osteogenesis. In vivo results corroborate the abundant siDcstamp accumulation on bone surfaces and the enhanced trabecular bone mass volume and microstructure in treating osteoporotic OVX mice by rebalancing bone resorption, formation, and vascularization. Our study validates the hypothesis that satisfied transfection of siRNA enables preserved preosteoclasts that regulate bone resorption and formation simultaneously as potential anabolic treatment for osteoporosis.

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Achieving organized assembly structures with high complexity and adjustable functionalities is a central quest in supramolecular chemistry. In this report, we study what happens when a discotic benzene-1,3,5-tricarboxamide (BTA) ligand containing three dipicolinic acid (DPA) groups is allowed to coordinate with lanthanide (Ln) ions. A multi-BTA coordination cluster forms, which behaves as a type of "supramolecular monomer", stacking into fibers via hydrogen bonds enabled by multiple BTA cores. The fibrous morphology and size, as well as the packing unit and the process by which it grows, were investigated by light scattering measurements, luminescence spectra, TEM images and molecular simulation data. More notably, by selecting the kind of lanthanide or mixture of lanthanides that is incorporated, tunable luminescence and magnetic relaxation properties without compromising the fibrous structure can be realized. This case of hierarchical self-assembly is made possible by the special structure of our BTA-like building block, which makes non-covalent bond types that are different along the radial (coordination bonds) and axial (H-bonds) directions, respectively, each with just the right strength. Moreover, the use of lanthanide coordination leads to materials with metal-derived optical and magnetic properties. Therefore, the established approach demonstrates a novel strategy for designing and fabrication of multi-functional supramolecular materials.

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Zwitterionic polyelectrolyte nanogels are prospective nanocarriers due to their soft loading pocket and regulated charges. We here report a facile strategy, namely, electrostatic-templated polymerization (ETP) for synthesizing zwitterionic nanogels with controlled size and properties. Specifically, with anionic-neutral diblock polymers as the template, zwitterionic monomers such as carboxybetaine methacrylate (CBMA) or carboxybetaine acrylamide (CBAA) are polymerized together with a cross-linker at pH 2 where the monomers exhibit only positive charge due to the protonation of the carboxyl group. The obtained polyelectrolyte complex micelles dissociate upon introducing a concentrated salt. The subsequent separation yields the released template and zwitterionic nanogels with regulated size and swelling ability, achieved by tuning the salt concentration and cross-linker fraction during polymerization. The obtained PCBMA nanogels exhibit charges depending on the pH, which enables not only the selective loading of different dye molecules, but also encapsulation and intracellular delivery of cytochrome c protein. Our study develops a facile and robust way for fabricating zwitterionic nanogels and validates their potential applications as promising nanocarriers for load and delivery of functional charged cargos.

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In the quest for stimuli-responsive materials with specific, controllable functions, coacervate hydrogels have become a promising candidate, featuring sensitive responsiveness to environmental signals enabling control over sol-gel transitions. However, conventional coacervation-based materials are regulated by relatively non-specific signals, such as temperature, pH or salt concentration, which limits their possible applications. In this work, we constructed a coacervate hydrogel with a Michael addition-based chemical reaction network (CRN) as a platform, where the state of coacervate materials can be easily tuned by specific chemical signals. We designed a pyridine-based ABA triblock copolymer, whose quaternization can be regulated by an allyl acetate electrophile and an amine nucleophile, leading to gel construction and collapse in the presence of polyanions. Our coacervate gels showed not only highly tunable stiffness and gelation times, but excellent self-healing ability and injectability with different sized needles, and accelerated degradation resulting from chemical signal-induced coacervation disruption. This work is expected to be a first step in the realization of a new class of signal-responsive injectable materials.

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Uniting photothermal therapy (PTT) with magnetic resonance imaging (MRI) holds great potential in nanotheranostics. However, the extensively utilized hydrophobicity-driven assembling strategy not only restricts the intramolecular motion-induced PTT, but also blocks the interactions between MR agents and water. Herein, we report an aggregation-induced emission luminogen (AIEgen)-mediated polyelectrolyte nanoassemblies (APN) strategy, which bestows a unique "soft" inner microenvironment with good water permeability. Femtosecond transient spectra verify that APN well activates intramolecular motion from the twisted intramolecular charge transfer process. This de novo APN strategy uniting synergistically three factors (rotational motion, local motion, and hydration number) brings out high MR relaxivity. For the first time, APN strategy has successfully modulated both intramolecular motion and magnetic relaxivity, achieving fluorescence lifetime imaging of tumor spheroids and spatio-temporal MRI-guided high-efficient PTT.

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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.

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Polyelectrolyte nanogels containing cross-linked ionic polymer networks feature both soft environment and intrinsic charges which are of great potential for enzyme encapsulation. In this work, well-defined poly(acrylic acid) (PAA) nanogels have been synthesized based on a facile strategy, namely, electrostatic assembly directed polymerization (EADP). Specifically, AA monomers are polymerized together with a cross-linker in the presence of a cationic-neutral diblock copolymer as the template. Effects of control factors including pH, salt concentration, and cross-linking degree have been investigated systematically, based on which the optimal preparation of PAA nanogels has been established. The obtained nanogel features not only compatible pocket for safely loading enzymes without disturbing their structures, but also abundant negative charges which enable selective and efficient encapsulation of cationic enzymes. The loading capacities of PAA nanogels for cytochrome (cyt c) and lysozyme are 100 and 125 µg/mg (enzyme/nanogel), respectively. More notably, the PAA network seems to modulate a favorable microenvironment for cyt c and induces 2-fold enhanced activity for the encapsulated enzymes, as indicated by the steady-state kinetic assay. Our study reveals the control factors of EADP for optimal synthesis of anionic nanogels and validates their distinctive advances with respect to efficient loading and activation of cationic enzymes.

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Polyion complex (PIC) vesicles prepared by polyelectrolyte assembly have attracted extensive attention as distinctive carriers and nanoreactors, particularly for biological cargoes. However, the constrained regulation of their structure and functionality at this stage hinder the application of PIC vesicles. Herein, we design a new asymmetric assembly system, namely cationic-neutral-cationic triblock copolymer co-assembly with a supramolecular ionic coordination polymer. The former creates poly(ethylene oxide) (PEO) loops upon complexation, which are favorable for vesicle fabrication, while the coordination polyelectrolyte composed of metal ions and a dipicolinic acid (DPA)-based bis-ligand features well-defined functionalities depending on the incorporated metal ions. Thus, the rational combination allows controlled fabrication of PIC vesicles with a modulated structure and functionalities. Moreover, the encapsulation and release of hydrophilic dextran based on different PIC vesicles has been realized. Our design integrates the advantages of both triblock and coordination polymers, and therefore demonstrates a novel strategy for harmonious regulation of the structure and functionality of PIC vesicles. The revealed findings and achieved properties shall be inspirational for developing functional PIC vesicles and boosting their applications towards demand encapsulation and delivery.

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Associations of amphiphiles assume their various morphologies according to the so-called packing parameter under thermodynamic control. However, one may raise the question of whether polymers can always relax fast enough to obey thermodynamic control, and how this may be checked. Here, a case of polyion complex (PIC) assemblies where the morphology appears to be subject to kinetic control is discussed. Poly (ethylene oxide)-b-(styrene sulfonate) block copolymers are combined with cationic PAMAM dendrimers of various generations (2-7). The PEO-PSS diblocks, and the corresponding PSS-PEO-PSS triblocks should have nearly identical packing parameters, but surprisingly creat different assemblies, namely core-shell micelles and vesicles, respectively. Moreover, the micelles are very stable against added salt, whereas the vesicles are not only much more sensitive to added salt, but also appear to exchange matter on relevant time scales. The small and largely quenched early-stage precursor complexes are responsible for the morphological and dynamic differences, implying that kinetic control may also be a way to obtain particles with well-defined and useful properties. The exciting new finding that triblocks produce more "active" vesicles will hopefully trigger the exploration of more pathways, and so learn how to tune PICsomes toward specific applications.

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HYPOTHESIS: In the quest for large but well-controlled supramolecular structures, the discotic benzene-1,3,5-tricarboxamide (BTA) has received quite some attention, because it can form hydrogen-bonded stacks that can be regarded as supramolecular polymers of which the single BTA molecule is the monomer. In this report, we consider a more complex BTA-based supramolecular polymer, namely one that is built up from supramolecular 'monomers'. EXPERIMENTS: We design a tris-ligand L3 consisting of a BTA core carrying three dipicolinic acid (DPA) groups. L3 itself is too small to form polymers, but in the presence of appropriate metal ions, each L3 can form three coordination bonds and so form (L3)n clusters that are large enough to stack successfully: at an appropriate metal dose, long and stable filaments with a cross-sectional diameter of 12 nm appear. We monitor the growth process by UV-vis spectroscopy and light scattering, and use small angle X-ray scattering (SAXS), TEM as well as molecular simulation to confirm the filamentous structure of the fibers and determine their dimensions. FINDINGS: The formation and structure of the fiber are very similar for various transition metal ions, which enables introducing different functionalities, e.g., magnetic relaxivity, by proper choice of the metal ions. Hence, we obtain a doubly supramolecular polymer, connected axially by hydrogen bonds, and radially by coordination bonds. Not only does this realize a higher level of complexity, but it also allows to easily introduce and vary metal-derived functionalities.

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Hierarchical dendrimer-based polyion complex (PIC) vesicles with multiple compartments have attracted considerable attention as functional delivery vehicles and nano-carriers. Formation of these vesicles relies on the electrostatic assembly of asymmetric polyelectrolytes, namely branched dendrimers with linear polyion-neutral diblock copolymers. However, successful incorporation of dendrimers in vesicle lamellae is challenging due to the compact structure of dendrimers, and therefore, vesicles reported so far are prepared mainly with low generation dendrimers which lack the cavity required for carrier functions. Here, we present a new assembly combination of amine-terminated dendrimer polyamidoamine (PAMAM) with polyion-neutral diblock copolymer poly (styrene sulphonate-b-ethylene oxide) (PSS-b-PEO). The strong charge interaction between the building blocks leads to stable and well-defined PIC vesicles that can tolerate not only different PSS block lengths but, more importantly, also different dendrimer generations from 2 to 7. As a consequence, high generation dendrimers with a cavity can be packed in the vesicle wall, and one obtains hierarchical PIC vesicles with multiple compartments, namely the dendrimer cavity for loading small hydrophobic cargo, and the vesicle lumen for encapsulating hydrophilic macromolecules. Our study demonstrates that combining proper building blocks enables to manipulate the charge interactions, which is essential for controlling the dendrimer packing and the formation of PIC vesicles. These findings should be helpful for understanding the assembly of asymmetric (linear / branched) polyelectrolyte complexes, as well as for designing new hierarchical PIC vesicles for controlled delivery of multiple active substances.

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Nanoscale polyelectrolyte complex materials have been extensively investigated for their promising application in protocell, drug carriers, imaging, and catalysis. However, the conventional preparation approach involving positive and negative polyelectrolytes leads to large size, wide size distribution, instability, and aggregation due to the nonhomogeneous mixing process. Herein, we employ reactive flash nanoprecipitation (RFNP) to control the mixing and preparation of the nanoscale polyelectrolyte complex. With RFNP, homogeneous mixing complexation between oppositely charged chitosan derivatives could be achieved, resulting in stable nanoscale complexes (NCs) with controllable size and narrow size distribution. The smallest size of NCs is found at specific pH due to the maximum attraction of positive and negative molecules of chitosan. The size can be modulated by altering the volumetric flow rates of inlet streams, concentration, and charge molar ratio of two oppositely charged chitosan derivatives. The charge molar ratio is also tuned to create NCs with positive and negative shells. There is no significant variation in the size of NCs produced at different intervals of time. This method allows continuous and tunable NC production and could have the potential for fast, practical translation.