Bicontinuous Nanospheres Research Articles

Dampened Transient Actuation of Hydrogels Autonomously Controlled by pH-Responsive Bicontinuous Nanospheres.

The fabrication of a soft actuator with a dampened actuation response is presented. This was achieved via the incorporation into an actuating hydrogel of urease-loaded pH-responsive bicontinuous nanospheres (BCNs), whose membrane was able to regulate the permeability and thus conversion of fuel urea into ammonia. The dampened response of these nanoreactors to the enzymatically induced pH change was translated to a pH-responsive soft actuator. In hydrogels composed of a pH-responsive and nonresponsive layer, the transient pH gradient yielded an asymmetric swelling behavior, which induced a bending response. The transient actuation profile could be controlled by varying the external fuel concentrations. Furthermore, we showed that the spatial organization of the BCNs within the actuator had a great influence on the actuation response. Embedding the urease-loaded nanoreactors within the active, pH-responsive layer resulted in a reduced response due to local substrate conversion in comparison to embedding them within the passive layer of the bilayer hydrogel. Finally, we were able to induce transient actuation in a hydrogel comprising two identical active layers by the immobilization of the BCNs within one specific layer. Upon addition of urea, a local pH gradient was generated, which caused accelerated swelling in the BCN layer and transient bending of the device before the pH gradient was attenuated over time.

One-component lipidic bicontinuous nanospheres as a smart drug loading platform to eradicate candida biofilms in oral and vaginal infection

Bicontinuous nanospheres (BCNs) with a highly twisted network of hydrophobic phases interwoven with a network of hydrated hydrophilic parts are essential for the loading and releasing of therapeutics. However, a facile methodology to construct BCNs with a single lipid molecule is still challenging. Here we report the synthesis of X-shaped lipids that can self-assemble into stable one-component sponge-phase BCNs without the assistance of any stabilizers as a platform for drug delivery. The X-shaped lipids with two hydrophilic heads and two hydrophobic tails are formed via dynamic covalent boronate between the phenylboronic acid-modified polar head and the catechol-containing lipids with different carbon chain lengths. This methodology provides an efficient and convenient way to synthesize lipids with different architectures, and their possibility in fabrication BCNs was further screened. The representative miconazole-loaded prodrug BCNs possess high drug loading content, up to 41.6%, and a fast, zero-order drug release profile after exposure to an acidic/oxidative microenvironment, such as Candida albicans biofilm. Also, the carbon chain lengths of lipids affect the prodrug BCNs' formation and interactions with fungal membranes. The optimal prodrug BCNs with 10-carbon chains hold superb Candida biofilm eradication efficacy in vitro and in vivo without affecting the normal tissues. Therefore, our strategy provides a facile methodology to construct BCNs as a smart drug loading platform for biomedical applications.

Vaccination with mycobacterial lipid loaded nanoparticle leads to lipid antigen persistence and memory differentiation of antigen-specific T cells

Mycobacterium tuberculosis (Mtb) infection elicits both protein and lipid antigen-specific T cell responses. However, the incorporation of lipid antigens into subunit vaccine strategies and formulations has been underexplored, and the characteristics of vaccine-induced Mtb lipid-specific memory T cells have remained elusive. Mycolic acid (MA), a major lipid component of the Mtb cell wall, is presented by human CD1b molecules to unconventional T cell subsets. These MA-specific CD1b-restricted T cells have been detected in the blood and disease sites of Mtb-infected individuals, suggesting that MA is a promising lipid antigen for incorporation into multicomponent subunit vaccines. In this study, we utilized the enhanced stability of bicontinuous nanospheres (BCN) to efficiently encapsulate MA for in vivo delivery to MA-specific T cells, both alone and in combination with an immunodominant Mtb protein antigen (Ag85B). Pulmonary administration of MA-loaded BCN (MA-BCN) elicited MA-specific T cell responses in humanized CD1 transgenic mice. Simultaneous delivery of MA and Ag85B within BCN activated both MA- and Ag85B-specific T cells. Notably, pulmonary vaccination with MA-Ag85B-BCN resulted in the persistence of MA, but not Ag85B, within alveolar macrophages in the lung. Vaccination of MA-BCN through intravenous or subcutaneous route, or with attenuated Mtb likewise reproduced MA persistence. Moreover, MA-specific T cells in MA-BCN-vaccinated mice differentiated into a T follicular helper-like phenotype. Overall, the BCN platform allows for the dual encapsulation and in vivo activation of lipid and protein antigen-specific T cells and leads to persistent lipid depots that could offer long-lasting immune responses.

Vaccination with mycobacterial lipid loaded nanoparticle leads to lipid antigen persistence and memory differentiation of antigen-specific T cells.

Mycobacterium tuberculosis (Mtb) infection elicits both protein and lipid antigen-specific T cell responses. However, the incorporation of lipid antigens into subunit vaccine strategies and formulations has been underexplored, and the characteristics of vaccine-induced Mtb lipid-specific memory T cells have remained elusive. Mycolic acid (MA), a major lipid component of the Mtb cell wall, is presented by human CD1b molecules to unconventional T cell subsets. These MA-specific CD1b-restricted T cells have been detected in the blood and disease sites of Mtb-infected individuals, suggesting that MA is a promising lipid antigen for incorporation into multicomponent subunit vaccines. In this study, we utilized the enhanced stability of bicontinuous nanospheres (BCN) to efficiently encapsulate MA for in vivo delivery to MA-specific T cells, both alone and in combination with an immunodominant Mtb protein antigen (Ag85B). Pulmonary administration of MA-loaded BCN (MA-BCN) elicited MA-specific T cell responses in humanized CD1 transgenic mice. Simultaneous delivery of MA and Ag85B within BCN activated both MA- and Ag85B-specific T cells. Notably, pulmonary vaccination with MA-Ag85B-BCN resulted in the persistence of MA, but not Ag85B, within alveolar macrophages in the lung. Vaccination of MA-BCN through intravenous or subcutaneous route, or with attenuated Mtb likewise reproduced MA persistence. Moreover, MA-specific T cells in MA-BCN-vaccinated mice differentiated into a T follicular helper-like phenotype. Overall, the BCN platform allows for the dual encapsulation and in vivo activation of lipid and protein antigen-specific T cells and leads to persistent lipid depots that could offer long-lasting immune responses.

Nonlinear Transient Permeability in pH-Responsive Bicontinuous Nanospheres

We demonstrate the construction of pH-responsive bicontinuous nanospheres (BCNs) with nonlinear transient permeability and catalytic activity. The BCNs were assembled from amphiphilic block copolymers comprising pH-responsive groups and were loaded with the enzymes urease and horseradish peroxidase (HRP). A transient membrane permeability switch was introduced by employing the well-known pH-increasing effect of urease upon conversion of urea to ammonia. As expected, the coencapsulated HRP displayed a transiently regulated catalytic output profile upon addition of urea, with no significant product formation after the pH increase. This transient process displayed a nonlinear "dampening" behavior, induced by a decrease in membrane permeability as a result of significant local ammonia production. Furthermore, the catalytic output of HRP could be modulated by addition of different amounts of urea or by altering the buffer capacity of the system. Finally, this nonlinear dampening effect was not observed in spherical polymersomes, even though the membrane permeability could also be inhibited by addition of urea. The specific BCN morphology therefore allows to optimally control catalytic processes by pH changes in the nanoreactor microenvironment compared to bulk conditions due to its unique permeability profile.

Hydrogen Sulfide-Responsive Bicontinuous Nanospheres.

Block copolymers (BCPs) that can self-assemble into particles and be triggered by disease-specific molecules such as hydrogen sulfide (H2S) have the potential to impact on drug delivery, decreasing off-target toxicities while increasing drug efficacy. However, the incorporation of H2S-responsive aryl azides into BCPs for self-assembly has been limited by heat, light, and radical sensitivities. In this study, a robust activator regenerated by the electron-transfer atom-transfer radical polymerization reaction was used to synthesize aryl-azide-containing BCPs under ambient conditions. Conditions controlling self-assembly of the BCPs into 150-200 nm particles and the physicochemical properties of the particles were investigated. The use of nanoprecipitation with tetrahydrofuran to promote self-assembly of the BCPs resulted in vesicle structures, while dimethylformamide or dimethylsulfoxide resulted in polymeric bicontinuous nanospheres (BCNs). Triggering of the BCPs and particles (vesicles or BCNs) via exposure to H2S revealed that unsubstituted aryl azides were readily reduced (by HS-), resulting in particle disruption or cross-linking. The relative polar nature of the particle bilayers containing unsubstituted aryl azides and the open structure of the BCNs did however limit encapsulation of small hydrophilic and hydrophobic payloads. Incorporation of a benzylamide substituent onto the aryl azide group increased the hydrophobicity of the particles and encapsulation of hydrophilic cargo but reduced sensitivity to H2S, likely due to the reduced penetration of HS- into the bilayer.

Magnetic Nanostructure-Loaded Bicontinuous Nanospheres Support Multicargo Intracellular Delivery and Oxidation-Responsive Morphological Transitions.

Magnetic nanostructures (MNS) have a wide range of biological applications due to their biocompatibility, superparamagnetic properties, and customizable composition that includes iron oxide (Fe3O4), Zn2+, and Mn2+. However, several challenges to the biomedical usage of MNS must still be addressed, such as formulation stability, inability to encapsulate therapeutic payloads, and variable clearance rates in vivo. Here, we enhance the utility of MNS during controlled delivery applications via encapsulation within polymeric bicontinuous nanospheres (BCNs) composed of poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-b-PPS) copolymers. PEG-b-PPS BCNs have demonstrated versatile encapsulation and delivery capabilities for both hydrophilic and hydrophobic payloads due to their unique and highly organized cubic phase nanoarchitecture. MNS-embedded BCNs (MBCNs) were thus coloaded with physicochemically diverse molecular payloads using the technique of flash nanoprecipitation and characterized in terms of their structure and in vivo biodistribution following intravenous administration. Retention of the internal aqueous channels and cubic architecture of MBCNs were verified using cryogenic transmission electron microscopy and small-angle X-ray scattering, respectively. MBCNs demonstrated improvement in magnetic resonance imaging (MRI) contrast enhancement (r2 relaxivity) as compared to free MNS, which in combination with scanning transmission electron microscopy and energy-dispersive X-ray spectroscopy evidenced the clustering and continued access to water of MNS following encapsulation. Furthermore, MBCNs were found to be noncytotoxic and able to deliver their hydrophilic and hydrophobic small-molecule payloads both in vitro and in vivo. Finally, the oxidation sensitivity of the hydrophobic PPS block allowed MBCNs to undergo a unique, triggerable transition in morphology into MNS-bearing micellar nanocarriers. In summary, MBCNs are an attractive platform for the delivery of molecular and nanoscale payloads for diverse on-demand and sustained drug delivery applications.

Employing bicontinuous-to-micellar transitions in nanostructure morphology for on-demand photo-oxidation responsive cytosolic delivery and off-on cytotoxicity.

Bicontinuous nanospheres (BCNs) are underutilized self-assembled nanostructures capable of simultaneous delivery of both hydrophilic and hydrophobic payloads. Here, we demonstrate that BCNs assembled from poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-b-PPS), an oxidation-sensitive copolymer, are stably retained within cell lysosomes following endocytosis, resisting degradation and payload release for days until externally triggered. The oxygen scavenging properties and enhanced stability of the bicontinuous PEG-b-PPS nanoarchitecture significantly protected cells from typically cytotoxic application of pro-apoptotic photo-oxidizer pheophorbide A and chemotherapeutic camptothecin. The photo-oxidation triggered transition from a bicontinuous to micellar morphology overcame this stability, allowing on-demand cytosolic delivery of camptothecin for enhanced control over off-on cytotoxicity. These results indicate that inducible transitions in the nanostructure morphology can influence intracellular stability and toxicity of self-assembled nanotherapeutics.

On the advancement of polymeric bicontinuous nanospheres toward biomedical applications.

Self-assembled soft nanocarriers that are capable of simultaneous encapsulation of both lipophilic and water soluble payloads have significantly enhanced controlled delivery applications in biomedicine. These nanoarchitectures, such as liposomes, polymersomes and cubosomes, are primarily composed of either amphiphilic polymers or lipids, with the polymeric variants generally possessing greater stability and control over biodistribution and bioresponsive release. Polymersomes have long demonstrated such advantages over their lipid analogs, liposomes, but only recently have bicontinuous nanospheres emerged as a polymeric cubic phase alternative to lipid cubosomes. In this review, we summarize the current state of the field for bicontinuous nanosphere formulation and characterization and suggest future directions for this nascent delivery platform as it is adopted for biomedical applications.

Benchmarking Bicontinuous Nanospheres against Polymersomes for in Vivo Biodistribution and Dual Intracellular Delivery of Lipophilic and Water-Soluble Payloads.

Bicontinuous nanospheres (BCNs) are polymeric analogs to lipid cubosomes, possessing cubic liquid crystalline phases with high internal surface area, aqueous channels for loading hydrophilic molecules, and high hydrophobic volume for lipophilic payloads. Primarily due to difficulties in scalable and consistent fabrication, neither controlled delivery of payloads via BCNs nor their organ or cellular biodistributions following in vivo administration have been demonstrated or characterized. We have recently validated flash nanoprecipitation as a rapid method of assembling uniform monodisperse 200-300 nm diameter BCNs from poly(ethylene glycol) -b-poly(propylene sulfide) (PEG -b-PPS) co-polymers. Here, we compare these BCNs both in vitro and in vivo to 100 nm PEG -b-PPS polymersomes (PSs), which have been well characterized as nanocarriers for controlled delivery applications. Using a small molecule fluorophore and a fluorescently tagged protein as respective lipophilic and water-soluble model cargos, we demonstrate that BCNs can achieve significantly higher encapsulation efficiencies for both payloads on a per unit mass basis. At time points of 4 and 24 h after intravenous administration to mice, we found significant differences in organ-level uptake between BCNs and PSs, with BCNs showing reduced accumulation in the liver and increased uptake in the spleen. Despite these organ-level differences, BCNs and PSs displayed strikingly similar uptake profiles by immune cell populations in vitro and in the liver, spleen, and blood, as assayed by flow cytometry. In conclusion, we have found PEG -b-PPS BCNs to be well suited for dual loading and delivery of molecular payloads, with a favorable organ biodistribution and high cell uptake by therapeutically relevant immune cell populations.

Front Cover: Polyphosphazenes – Synthetically Versatile Block Copolymers (“Multi‐Tool”) for Self‐Assembly (Eur. J. Inorg. Chem. 22/2018)

The Front Cover shows the synthesis of polyphosphazene block copolymers (“Polymer Synthesis Building”) and the nanostructured morphologies created by their self-assembly (“Self-Assembly Department”). The opportunities offered by a decisive macromolecular substitution of the chlorine atoms of the [N=PCl2]n blocks by selected nucleophiles (“Chemical Modification”) can be exploited to achieve block copolymers that can self-assemble to afford a variety of nanomorphologies such as toroidal micelles, bicontinuous nanospheres, vesicles, porous films, spherical micelles, or helical nanoarchitectures. More information can be found in the Microreview by A. P. Soto et al. For more on the story behind the cover research, see the Cover Profile.

Flash nanoprecipitation permits versatile assembly and loading of polymeric bicontinuous cubic nanospheres.

Polymeric bicontinuous nanospheres (BCNs) that are analogous to lipid cubosomes possess high internal surface area and porosity that can accommodate the loading of a wide range of hydrophobic and hydrophilic molecules for diverse applications. Self-assembly of BCNs has been reported using complex amphiphilic polymeric structures, with co-solvent dispersion being the only documented method of formation. Here, we report a simple amphiphilic diblock copolymer, poly(ethylene glycol)17-block-poly(propylene sulfide)75 (PEG17-bl-PPS75), to form BCNs using the rapid and scalable technique of flash nanoprecipitation (FNP). Dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryoTEM) verified low polydispersity and the formation of bicontinuous structures with internal aqueous channels, respectively. Small-angle X-ray scattering (SAXS) confirmed a primitive cubic (Im3m) internal organization for BCNs assembled by FNP. Both hydrophobic and hydrophilic molecules were effectively loaded into BCNs via FNP, and encapsulated payloads were found to release in controlled manner in aqueous solutions. Due to the oxidation-sensitivity of PPS, biologically relevant concentrations of reactive oxygen species could trigger payload release on demand. BCNs were found to be non-toxic and endocytosed by phagocytic cells. Furthermore, an in vitro functional assay showed BCNs co-loaded with antigen ovalbumin and adjuvant monophosphoryl lipid A (MPL) to promote peptide/MHCI surface presentation by dendritic cells, a critical step for vaccine formulations during immunization. In conclusion, FNP supports the facile and scalable assembly and loading of PEG-bl-PPS BCNs, making them an attractive nanoscale delivery vehicle for both hydrophilic and hydrophobic molecules.

Facile assembly and loading of theranostic polymersomes via multi-impingement flash nanoprecipitation

Flash nanoprecipitation (FNP) has proven to be a powerful tool for the rapid and scalable assembly of solid-core nanoparticles from block copolymers. The process can be performed using a simple confined impingement jets mixer and provides an efficient and reproducible method of loading micelles with hydrophobic drugs. To date, FNP has not been applied for the fabrication of complex or vesicular nanoarchitectures capable of encapsulating hydrophilic molecules or bioactive protein therapeutics. Here, we present FNP as a single customizable method for the assembly of bicontinuous nanospheres, filomicelles and vesicular, multilamellar and tubular polymersomes from poly(ethylene glycol)-bl-poly(propylene sulfide) block copolymers. Multiple impingements of polymersomes assembled via FNP were shown to decrease vesicle diameter and polydispersity, allowing gram-scale fabrication of monodisperse polymersomes within minutes. Furthermore, we demonstrate that FNP supports the simultaneous loading of both hydrophobic and hydrophilic molecules respectively into the polymersome membrane and aqueous lumen, and encapsulated enzymes were found to be released and remain active following vesicle lysis. As an example application, theranostic polymersomes were generated via FNP that were dual loaded with the immunosuppressant rapamycin and a fluorescent dye to link targeted immune cells with the elicited immunomodulation of T cells. By expanding the capabilities of FNP, we present a rapid, scalable and reproducible method of nanofabrication for a wide range of nanoarchitectures that are typically challenging to assemble and load with therapeutics for controlled delivery and theranostic strategies.

Formation and Reversible Morphological Transition of Bicontinuous Nanospheres and Toroidal Micelles by the Self‐Assembly of a Crystalline‐b‐Coil Diblock Copolymer

AbstractWe herein report the formation of two complex nanostructures, toroidal micelles and bicontinuous nanospheres, by the self‐assembly of the single structurally simple crystalline‐b‐coil diblock copolymer poly[bis(trifluoroethoxy)phosphazene]‐b‐poly(styrene), PTFEP‐b‐PS, in one solvent (THF) and without additives. The nature of these nanostructures in solution was confirmed by DLS and cryo‐TEM experiments. The two morphologies are related by means of a new type of reversible morphological evolution, bicontinuous‐to‐toroidal, triggered by changes in the polymer concentration. WAXS experiments showed that the degree of crystallinity of the PTFEP chains located at the core of the toroids was higher than that in the bicontinuous nanospheres, thus indicating that the final morphology of the aggregates is mostly determined by the ordering of the PTFEP core‐forming blocks.

Formation and Reversible Morphological Transition of Bicontinuous Nanospheres and Toroidal Micelles by the Self-Assembly of a Crystalline-b-Coil Diblock Copolymer.

We herein report the formation of two complex nanostructures, toroidal micelles and bicontinuous nanospheres, by the self-assembly of the single structurally simple crystalline-b-coil diblock copolymer poly[bis(trifluoroethoxy)phosphazene]-b-poly(styrene), PTFEP-b-PS, in one solvent (THF) and without additives. The nature of these nanostructures in solution was confirmed by DLS and cryo-TEM experiments. The two morphologies are related by means of a new type of reversible morphological evolution, bicontinuous-to-toroidal, triggered by changes in the polymer concentration. WAXS experiments showed that the degree of crystallinity of the PTFEP chains located at the core of the toroids was higher than that in the bicontinuous nanospheres, thus indicating that the final morphology of the aggregates is mostly determined by the ordering of the PTFEP core-forming blocks.

Semi-crystalline block copolymer bicontinuous nanospheres for thermoresponsive controlled release

We demonstrate the controlled release of pyrene, as a model hydrophobic molecule, from self-assembled bicontinuous nanospheres formed from an amphiphilic block copolymer.

Bicontinuous Nanospheres from Simple Amorphous Amphiphilic Diblock Copolymers

Bicontinuous nanospheres have been observed (although rarely) from a variety of block copolymers with architectural and compositional complexity, and often in the presence of additives. Unlocking k...