Patent Application
20210030690
Self-assembly is ubiquitous in biological systems and underlies the formation of complex structures from simple components. Compared to the capabilities of natural polymers, such as polypeptides (1) and DNA (2), for dynamic assembly and reorganization via noncovalent interactions, synthetic polymer-based systems are relatively primitive (3). Such controlled aqueous self-assembly of single component supramolecular systems has remained a challenge for synthetic polymers, which typically require amphiphilicity or direct incorporation of naturally occurring peptide/nucleic acid monomers or derivatives thereof for controlled aggregation in aqueous systems. The most common synthetic self-assembling systems employ amphiphilic copolymers that are limited to separate hydrophobic and hydrophilic segments, which do not allow the complex dynamic self-assembly behavior observed in nature while also maintaining aggregate stability required for a broad range of applications. In terms of nanofabrication in aqueous solution, focus has been placed on the design of amphiphilic block (4) or random (5) copolymers wherein adjustment of the volume fraction of the hydrophobic/hydrophilic components permits fabrication of various nanostructures including spheres, cylinders, and vesicles. The multicomponent nature and dauntingly wide range of chemical compositions that have been developed for these self-assembling systems can present difficulties during chemical synthesis (6) and practical application (7), often requiring expertise in synthetic polymer chemistry and delaying translation of useful technologies.
There is a need for a simple, efficient method of producing monodisperse nanostructures using a single mixture that can be loaded with cargo at high efficiencies and can be used to carry cargo to a target.
The present disclosure provides novel nanostructures and nanogels of poly(propylene sulfone) homopolymers, specifically PPSU20 homopolymers, DMSO/water systems for making them, and applications for use of such homopolymer for delivery of cargo. In some aspects, the PPSU20 homopolymers are capable of self-assembling into nanostructures, both vesicle-like nanogels (nanoparticles) and bundle-like nanogels (nanobundles) that can be used for encapsulating both hydrophobic and water-soluble cargo, having exceptionally high loading efficiencies (greater than 90%, preferably greater than 95%) and also having high loading capacities (greater than 75%, preferably greater than 80% loading capacity m/m). The nanostructures are soluble and crystalizable within the same solvent, and using a DMSO/water system, wherein changes to the ratio of DMSO to water ratio and the stepwise addition of the water to the DMSO solution (containing the PPSU20) allows for dynamic and reproducible formation of specific nanostructures as described in the Examples below. PPSU20 is soluble in DMSO, and by the addition of water to the DMSO solution containing PPSU20, the ability to form collapsed compact nanogels (e.g., bundle-like nanogels to vesicle-like nanogels).
The present disclosure provides a PPSU20 DMSO/water system which, depending on the ratio of DMSO:water and the stepwise addition of water to the system, forms different sizes and structures of PPSU20 homopolymer nanogels and nanostructures. These PPSU20 nanostructures and nanogels can be used for delivery cargo/agents including for both hydrophobic and water-soluble agents.
In one aspect, the disclosure provides a synthetic self-assembling homopolymer for producing nanostructures comprising about 20 repeat units of poly(polyethyle sulfonate) (PPSU20).
In another aspect, the disclosure provides a system for delivery of cargo to a target, the system comprising: (a) a nanostructure of PPSU20; and (b) a cargo, wherein the nanostructure is loaded with the cargo with a loading efficiency of at least 90%, preferably at least 95%.
In another aspect, the disclosure provides a method of producing a nanostructure capable of carrying a cargo, the method comprising: mixing water to a solution of DMSO:PPSU20 (20 repeat units of poly(propylene sulfonate)) at a ratio from about 2:1 to about 10:1 (by volume), preferably about 1:2 to about 1:5 to form a monodisperse nanostructure of PPSU20 within the solution. In some aspects, the mixing the water to DMSO:PPSU20 solution is performed stepwise in 1 to about 80 steps, wherein the solution is mixed after each addition of the water.
In yet another aspect, the present disclosure provides a PPSU20 nanostructure made by the methods described herein.
In yet another aspect, the present disclosure provides a method of loading a cargo in a nanostructure capable of being delivered to a cell or target, the method comprising: (a) mixing water with a solution of DMSO and PPSU20 (20 repeat units of poly(propylene sulfonate)) at a ratio from about 2:1 to about 10:1 (by volume), preferably about 1:2 to about 1:5, wherein either the water or the solution comprises the cargo before mixing, and wherein the mixing forms a monodisperse nanostructure of PPSU20 comprising the cargo.
The foregoing and other aspects and advantages of the invention will appear from the following description. In the description, reference is made to the accompanying drawings which form a part hereof, and in which there is shown by way of illustration a preferred embodiment of the invention. Such embodiment does not necessarily represent the full scope of the invention, however, and reference is made therefore to the claims and herein for interpreting the scope of the invention.
The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.
Nature exploits self-assembly processes to promote the formation of structurally dynamic hierarchical structures. The ability to design this dynamic behavior in synthetic materials is a central challenge of nanotechnology.
The present disclosure provides poly(propylene sulfone) (PPSU), a synthetic homopolymer capable of supramolecular self-assembly that is biomimetic of peptides and DNA. Experiments and simulations demonstrate that while PPSU is an extended solubilized chain in dimethyl sulfoxide (DMSO, (CH3)2SO), the addition of water greatly decreases its stiffness and induces bundling of PPSU chains into networks linked by short crystalline zipper segments. The PPSU zipper networks (ZipNets) trend towards collapse upon further hydration, allowing formation of dynamically controlled nanostructures with diverse morphologies and sizes. Drug loading efficiencies of the nanostructures for a wide range of polar payloads are nearly 100% with exceptionally high loading capacity (greater than 75% loading capacity, preferably greater than 90% load capacity, more preferably greater than 95% load capacity). This simple, nontoxic and facilely synthesized homopolymer presents a versatile platform for nanofabrication with potential applications in biomedicine, diagnostics, catalysis and purification.
The present disclosure provides synthetic self-assembling homopolymer for producing nanostructures comprising about 20 repeat units of poly(propylene sulfonate) (PPSU20). These nanostructures are produced by dissolving PPSU20 in DMSO (at 5 mg/ml to about 100 mg/ml, preferably 25 mg/ml to about 50 mg/ml). Water is then mixed at varying ratios (e.g., 2:1 to 10:1 by volume) with the solution of DMSO/PPSU20 allowing for the formation of differing nanostructures depending on the ratio and the method of mixing of the water. Ratio of water to DMSO solution include ratio from 2:1 to 10:1, including ranges inbetween (e.g., about 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 7.5:1, 8:1, 9:1, 10:1, etc.). Nanostructures formed include, for example, nanobundles (i.e. bundle-like nanogels) and vesicle-like nanogels (nanovesicles), for example, as depicted in
Employing Dynamic Sulfone-Sulfone Interactions for Facile Self-Assembly of Poly(Propylene Sulfone) Homopolymers.
Materials that can reversibly adapt to their environment have the potential to revolutionize technologies in diverse fields, ranging from sensors and actuators to applications in biomedicine. The design of such adaptive materials involves reversible chemistries that take advantage of weak and reversible molecular interactions. In the present disclosure, we demonstrate a new type of dynamic noncovalent bond, sulfone-sulfone interactions, that is susceptible to solvent polarity. As far as we are aware, we are the first to fully synthesize poly(propylene sulfone) (PPSU) from complete oxidation of poly(propylene sulfide), which is known for its hydrophobic-hydrophilic transition upon oxidation. However, complete oxidation of PPSU had not been previously achieved, since standard oxidation of PPS generates random copolymers of poly(propylene sulfoxide)-co-poly(propylene sulfone). We found that only the fully converted PPSU networks could undergo physical cross-linking via sulfone-sulfone bonds that can respond dynamically across length scales, allowing formation of both macro- and nanoscale hydrogels from crystalline-coil supramolecular amphiphiles. Following dissolution of PPSU in dimethylsulfoxide (DMSO), the stepwise addition of water provides a simple and highly controllable means of inducing changes in solvent polarity and thus directing dynamic supramolecular self-assembly of PPSU homopolymers. By specifying the hydration history, crystalline frameworks, macroscale hydrogels or uniform nanostructured hydrogels of spherical, vesicular, or cylindrical morphologies can be rapidly fabricated. These hydrogels and nanostructures can be used to encapsulate cargo at a high rate of efficiency and used for drug delivery and targeting.
The present disclosure also provides a system for delivering cargo to a target, e.g., a cell or organ within a subject. The system comprises (a) a nanostructure of PPSU20; and (b) a cargo, wherein the nanostructure is loaded with the cargo with a loading efficiency of at least 75%, more preferably at least 80%, still more preferably at least 90%, preferably at least 95%, more preferably at least 98%, and in some instances with a loading efficiency of about 100%. Further, the system provides that the loading capacity of the PPSU20 nanostructures is greater than 75%, preferably at least 80%. The systems are able to be loaded with both hydrophobic and hydrophilic (e.g., aqueous) cargo (e.g., a target molecule). The cargo can be added to the water or aqueous solution prior to mixing or can be added to the DMSO solution prior to mixing. It is also envisioned that one or more cargo can be added to both the DMSO solution and the water resulting in the loading of multiple cargoes into the nanostructures. In some embodiment, the system comprises nanostructure of PPSU20 is a bundle-like nanostructure. In other embodiments, the system comprises nanostructure of PPSU20 which are vesicle-like nanostructure (nanovesicles). In some embodiments, the vesicle-like nanostructure have a diameter of less than 240 nm, preferably less than 180 nm, alternatively a diameter of less than 100 nm, in alternative embodiments, a diameter of less than 50 nm, and comprise nanovesicles with diameters anywhere inbetween (e.g., 240 nm, 230 nm, 220 nm, 210 nm, 200 nm, 175 nm, 150 nm, 125 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, (i.e., between 250-50 n, between 100-20 nm, between 90-10 nm, between 80-15 nm, between 70-20 nm, and other ranges contemplated therein). The average size is on a weight basis and is measured by light scattering, microscopy, or other appropriate methods.
In methods according to embodiments of the present invention, the methods produce a nanostructure capable of carrying one or more cargo, the method comprising mixing water or a aqueous solution to a solution of DMSO:PPSU20 (20 repeat units of poly(propylene sulfonate)) at a ratio from about 2:1 to about 10:1 (by volume), preferably about 1:2 to about 1:5 to form a monodisperse nanostructure of PPSU20 within the solution. The water:DMSO solution comprising the nanostructures of PPSU20 can further undergo dialysis in order to remove the remaining DMSO and produce an aqueous solution (e.g., water or other physiologically suitable solution that can be administered to a subject, e.g., phosphate buffer saline).
The mixing the water to DMSO:PPSU20 solution may be performed by methods known in the art, including, for example, vortexing the sample to make a homogeneous solution. In some embodiments, the water is added stepwise in equal or unequal amounts, for example 1 to about 100 steps, wherein the solution is mixed after each addition of the water. In some embodiments, the water or aqueous solution is added stepwise in equal volumes. In some embodiments, the water is an aqueous solution comprising an aqueous cargo (e.g., an aqueous target molecule). In some embodiments, the method comprises adding a water soluble cargo to the water or aqueous solution before being mixed with the solution of DMSO:PPSU20.
Suitable solutions of DMSO:PPSU20 for use in the methods of the present invention include from about 5 mg/ml to about 100 mg/ml, preferably 25 mg/ml of PPSU20. In some embodiments, the solution of DMSO:PPSU20 may further comprise a hydrophobic cargo, for example, a hydrophobic target molecule, that one wishes to be loaded into the nanostructures of the present invention.
As used herein, the term “cargo” or “target molecule” are used interchangeably to refer to any molecule to be loaded into the nanostructures according to embodiments of the present invention. The cargo may be hydrophobic, hydrophilic, lipophilic or amphiphilic. Suitable cargo, e.g., target molecules that can be loaded into the nanostructures of the present invention include, but are not limited to, for example, hydrophilic macromolecules (e.g. water-soluble cargo) including, for example, a drug, therapeutic, protein, peptide, DNA, RNA, siRNA, plasmid, antibody, compounds, among others, lipophilic molecules, hydrophobic drug or molecules, and the like. For example, it is envisioned that if the cargo is selected from the group consisting of a DNA molecule, an RNA molecule, and a protein molecule, the water or aqueous solution will have a proper pH and salinity such that the cargo will maintain proper folding and stability while in solution. In some embodiments, the water or aqueous solution will have a physiologically relevant pH and salinity appropriate for loading biological macromolecules into the nanostructures. In one embodiment, the aqueous solution comprises approximately 150 nM salt.
Examples of other cargo that may be added to nanostructures by this process can be selected from, but are not limited to, the known classes of drugs including immunosuppressive agents such as cyclosporins (cyclosporin A), immunoactive agents, analgesics, anti-inflammatory agents, anthelmintics, anti-arrhythmic agents, antibiotics (including penicillins), anticoagulants, antidepressants, antidiabetic agents, antiepileptics, antihistamines, antihypertensive agents, antimuscarinic agents, antimycobacterial agents, antineoplastic agents, immunosuppressants, antithyroid agents, antiviral agents, anxiolytic sedatives (hypnotics and neuroleptics), astringents, beta-adrenoceptor blocking agents, blood products and substitutes, cardiac inotropic agents, contrast media, corticosteroids, cough suppressants (expectorants and mucolytics), diagnostic agents, diagnostic imaging agents, diuretics, dopaminergics (antiparkinsonian agents), haemostatics, immunological agents, lipid regulating agents, muscle relaxants, parasympathomimetics, parathyroid calcitonin and biphosphonates, prostaglandins, radio-pharmaceuticals, sex hormones (including steroids), anti-allergic agents, stimulants and anoretics, sympathomimetics, thyroid agents, vasodilators, xanthines, anti-oxidants, preservatives, vitamins, and nutrients. Cargo may also include combinations of, complexes of, mixtures of or other associations of any of the cargo molecules listed.
PPSU networks demonstrated a remarkable ability for molecular encapsulation, achieving >90% encapsulation efficiency for diverse organic solutes that include large hydrophilic molecules like DNA and protein that are typically quite difficult to encapsulate (Table 1). To the best of our knowledge, this work is the first demonstration of dynamic sulfone-sulfone bonding leading to supramolecular self-assembly.
The present disclosure also provides in some embodiments methods of loading a cargo in a nanostructure capable of being delivered to a cell, the method comprising mixing water or an aqueous solution with a solution of DMSO and PPSU20 (20 repeat units of poly(propylene sulfonate)) at a ratio from about 2:1 to about 10:1 (by volume), preferably about 1:2 to about 1:5, wherein the cargo is present in either the water or aqueous solution or the DMSO solution prior to mixing, and wherein after mixing, a monodisperse nanostructure of PPSU20 comprising the cargo is produced.
The loading efficiency of the cargo in the nanostructures by the methods of the present invention is measure as the ratio of the cargo encapsulated within the nanostructure to the total amount of cargo available for loading in the initial solution. The loading efficiency of the monodisperse nanostructure of PPSU20 is greater than 75%, preferably greater than 80%, more preferably greater than 90%, even more preferably at least 95%. Further, the loading capacity of the monodisperse nanostructures of PPSU20 is greater than 75%, more preferably greater than 80%.
In one example, the method comprises mixing a 1:2 ratio of DMSO solution of PPSU20 (25/mg/ml) to water (or aqueous solution), producing nanostructures with vesicle-like nanogels with an average diameter of about 230 nm, preferably with an average diameter of less than 180 nm, preferably with an average diameter of less than 100 nm. In another example, the method comprises mixing a ratio of about 1:5 of DMSO solution of PPSU20 (25/mg/ml) to water, producing nanoparticles with an average diameter of less than 170 nm.
Various nanogel structures in aqueous solution can be produced by the methods described herein by dynamically controlling the collapse of ZipNets from DMSO solutions using different ratio of components and different number of stepwise addition of the water to the DMSO solution. For example, as demonstrated in the Example, using the same total volume of water, adjusting only the number of water addition steps, resulting in different nanostructures with different morphologies. For example, a DMSO:PPSU20 solution (25 mg/mL PPSU in DMSO) were mixed stepwise with 1:2 ratio (by volume) with water using different number of steps with different step volumes to arrive at the ratio. Each step was followed by overtaxing to thoroughly mix the samples. Dialysis can be used to remove residual DMSO, and representative examples of the representative size and morphology of the nanogels are shown in
Depending on the number of water additions, the uniform fabrication of nanogels with micellar, vesicular and bundle nanostructures could be specified (
The nanostructures can be post processed to yield a sterile aqueous or non-aqueous solution or dispersion or could be isolated, such as via lylophilization and autoclaving, to yield a sterile powder for reconstitution into sterile injectable solutions or dispersions. The nanostructures can be combined with other acceptable compounds for parenteral injection such as but not limited to one or more of the following: water, ethanol, propyleneglycol, polyethyleneglycol, glycerol, vegetable oils, and ethyl oleate. Supplemental additives suitable for parenteral injection can also be used to tailor the composition to that suitable for a specific purpose.
In one embodiment, the nanostructure compositions produced by the methods of the present invention are formulated into a solid dosage form for oral administration such as capsules, tablets, pills, powders, and granules, or the like. In such solid dosage forms, the composition is admixed with one or more supplemental additives falling into the following classes such as, but not limited to, lubricants, buffering agents, wetting agents, adsorption, inert excipients, binders, disintegrating agents, solution retarders, accelerators, adsorbents, or fillers or extenders or other components commonly used by those skilled in the art for production of solid dosage forms.
In an embodiment, a composition comprising nanostructures of the present invention is a potent pharmaceutical containing one or more target molecules produced by methods of the present invention. In some embodiments, the composition is a solid dosage form and due to its nanoparticulate size it is evenly dispersed throughout said solid dosage form admixture and yields a high content uniformity (quantity of material in each dose) not obtained if the drug was microparticulate.
In one embodiment, the nanostructure compositions produced by the methods of the present invention are formulated into a pharmaceutically acceptable liquid dosage form for oral administration such as a syrup, solution, emulsion, suspension, or elixir. The liquid dosage forms may further comprise inert diluents, solubilizing agents, oils, emulsifiers, adjuvants suspending agents, sweeteners, wetting agents, flavoring agents, perfuming agents or other compounds commonly used by those skilled in the art. In another embodiment, the nanostructure compositions may be a dry capsule state for oral delivery. The particles are crystalline in solution and more so when dried and thus retain their payloads after desiccation for facile rehydration at a later time. In some examples, the nanostructure compositions may be a pill capsule containing the dried particles for oral administration.
In one embodiment, the nanostructure compositions produced by the methods of the present invention are formulated into a pharmaceutically acceptable liquid dosage form for intravenous administration such that it is a biologically compatible aqueous solution at relevant salt concentration, composition, pH, and temperature. The liquid dosage forms may further comprise inert diluents, solubilizing agents, oils, emulsifiers, adjuvants suspending agents, wetting agents or other compounds commonly used by those skilled in the art.
The nanostructure PPSU20 compositions with or without one or more target molecules produced by the methods of the present invention can be administered to a subject, for example, humans and animals, via a number of means including, but not limited to, orally, rectally, parenterally (intravenous, intramuscular, or subcutaneous), intracisternally, intravaginally, intraperitoneally, locally (in the form of powders, ointments or drops) or as a buccal or nasal spray. In one embodiment, the nanostructure compositions are administered orally. In one embodiment, the nanostructure compositions are administered intravenously.
The PPSU20 nanostructures produced by the methods of the present invention can also be used in immunotherapy, immunotheranostic or theranostic application which combine immunotherapy and diagnostics. It is envisioned that target molecules used for immunotherapy can be incorporated along with target molecules used for imagine and diagnostics into a single nanostructure or a composition comprising nanostructures for theranostic treatments.
The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as “including,” “comprising” or “having” certain elements are also contemplated as “consisting essentially of” and “consisting of” those certain elements.
As used herein, “about” means within 5-10% of a stated concentration range or within 5-10% of a stated number.
It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as “comprising” certain elements are also contemplated as “consisting essentially of” and “consisting of” those elements. The term “consisting essentially of” and “consisting of” should be interpreted in line with the MPEP and relevant Federal Circuit's interpretation. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. “Consisting of” is a closed term that excludes any element, step or ingredient not specified in the claim. The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”
The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.
The following Examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the scope of the appended claims.
Introduction
Self-assembly is ubiquitous in biological systems and underlies the formation of complex structures from simple components. Compared to the capabilities of natural polymers, such as polypeptides (1) and DNA (2), for dynamic assembly and reorganization via noncovalent interactions, synthetic polymer-based systems are relatively primitive (3). Such controlled aqueous self-assembly of single component supramolecular systems has remained a challenge for synthetic polymers, which typically require amphiphilicity or direct incorporation of naturally occurring peptide/nucleic acid monomers or derivatives thereof for controlled aggregation in aqueous systems. The most common synthetic self-assembling systems employ amphiphilic copolymers that are limited to separate hydrophobic and hydrophilic segments, which do not allow the complex dynamic self-assembly behavior observed in nature while also maintaining aggregate stability required for a broad range of applications. In terms of nanofabrication in aqueous solution, focus has been placed on the design of amphiphilic block (4) or random (5) copolymers wherein adjustment of the volume fraction of the hydrophobic/hydrophilic components permits fabrication of various nanostructures including spheres, cylinders, and vesicles. The multicomponent nature and dauntingly wide range of chemical compositions that have been developed for these self-assembling systems can present difficulties during chemical synthesis (6) and practical application (7), often requiring expertise in synthetic polymer chemistry and delaying translation of useful technologies.
Noncovalent weak/reversible interactions such as π-π stacking (8), hydrogen bonding (9, 10) and certain metal-ligand coordination bonds (11) provide strategies for construction of structurally dynamic architectures. Here, we report on a single-component homopolymer system that assembles through dynamic noncovalent sulfone-sulfone bonding. Crystalline frameworks and nanoscale hydrogels of spherical, vesicular, and cylindrical morphology were controllably assembled from solely a poly(propylene sulfone) (PPSU) homopolymer when transitioning from dimethylsulfoxide (DMSO) solution to aqueous system. Semi-flexible PPSU chains formed electrostatically bound networks that reorganize dynamically as interactions among sulfone repeat units increased. This system mimics the dynamic self-assembly of proteins (12) and DNA (13), allowing the design and fabrication of diverse superstructures with an unprecedented capability for molecular encapsulation.
Results
PPSU can be synthesized from complete oxidation of poly(propylene sulfide) (PPS) (14, 15), which is known for its hydrophobic-hydrophilic transition upon oxidation (16-18). However, complete oxidation of PPS had not been previously achieved, since standard oxidation of PPS generates random copolymers of poly(propylene sulfoxide)-co-poly(propylene sulfone) (19). We successfully synthesized PPSU20 (20 sulfones,
a MeOH = methanol, AA = acetic acid, CAN = acetonitrile, NMP = 1-methyl-2-pyrrolidinone, DMSO = dimethylsulfoxide, DMF = dimethylformamide, Py = pyridine, THF = tetrahydrofuran, Diox = dioxane.
b Relative polarity (S2).
c Using PPSU20 crystals for the solubility tests. X = insoluble, ✓ = soluble.
d Mixing a DMSO solution of PPSU20 (25 mg/mL) with 9 times the volume of other solvents, then checking the turbidity of the mixtures. C = cloudy. M = miscible.
e The mixture was clear at the beginning, but became cloudy in 1 hour.
f Solubility of PPSU20 in 1 mL of NMP is ~23 mg.
g Solubility of PPSU20 in 1 mL of DMSO is >250 mg.
We anticipated that the weak intra-chain associations of PPSU in DMSO would be accompanied by high chain stiffness. In order to establish that the structural features of PPSU can give rise to chain expansion and solubilization, we carried out all-atom explicit solvent molecular dynamics (AAMD) simulations for PPSU20 in DMSO (simulation parameters are given in Table 3 and snapshots of initial and final conformations are shown in
aThree parallel simulations for both DMSO and water solvents.
bThe simulation box length in the production simulations of 50 ns.
cTime in the equilibration simulation + time in production simulation.
a All calculations are based on the sulfur atoms on PPSU20. The GROMACS program gmx polystat was employed.
b End-to-end distance, in the unit of nm. Ree = 7.9 nm for a fully extended PPSU20 chain.
c Persistence length, in the unit of repeat units.
We further investigated the solubility of PPSU20 by mixing its DMSO solution with an excess amount of other solvents (Table 2). Interestingly, aggregates were visible in highly polar solvents including water and aprotic acetonitrile. In contrast, solvents with lower polarity than DMSO did not induce aggregation. These observations indicate that the highly polar PPSU polymer is not soluble in water as widely predicted (14). Characterized by a high dipole moment (22) and partially charged groups, the sulfones show a formal similarity to zwitterions, and it seemed possible that PPSU, like zwitterionic polymers (23), may have the ability to form physically cross-linked networks. Although formation of such sulfone-sulfone bonded networks would be inhibited by electrostatic repulsion among oxygen atoms, we anticipated PPSU to overcome this limitation when exposed to highly polar mediums such as water. AAMD simulations of PPSU20 in water provided insight into the mechanism and kinetics of aggregate formation. We observed in the simulations that the initially extended PPSU20 chains collapsed within 1 ns of the simulations, followed by chain folding and association into three-dimensional (3D) superstructures (
Because the AAMD simulations predicted that PPSU20 can form crystalline superstructures in water (
In an effort to control the formation of specific nanostructures, we developed a simple strategy (
a corresponding to FIG. 15.
b Determined by CryoTEM.
c Measured by SAXS.
d Measured by DLS.
Regardless of the concentrations investigated, one-step hydration of PPSU20 solutions in DMSO using an excess amount of water generated ultra-small (<15 nm) nanogels (
The work described above using kinetically controlled self-assembly of PPSU20 hydrogels results in crystalline frameworks or nanostructured hydrogels of spherical, vesicular and cylindrical morphologies in aqueous solution. These hierarchical superstructures are achieved via multiple levels of sulfone organization. The first level involves the inter-chain interactions that lead to initial network formation, which is followed by a second level of sulfone-sulfone bonding that locks the polymer chains into a stable conformation. In the latter, the hydration history from multiple small additions of water leads to bundling of PPSU20 chains into highly organized low curvature ribbons that increase persistence length, similar to bundling of DNA origami filaments (28). Alternatively, quick hydration generates disordered spherical hydrogels which contain coiled PPSU20 chains that experience greatly decreased electrostatic repulsion and strong interactions. Further hydration triggers the collapse and rearrangement of these sulfone-sulfone bonded into compact structures but imparts little change in overall chain stiffness. The formation of nanogel vesicles may be explained by taking into account the diffusivity of water into nanoscale aggregates that are stabilized by dynamic sulfone-sulfone bonding. The proximity of the outer layers of large spherical hydrogels to the water interface will promote stable cross-linking and prevent further structural rearrangement, resulting in an outer shell encapsulating an inner lumen of lower density sulfone-bonding. Taken together, the diverse sulfone-sulfone bonded networks formed by PPSU20 network self-assembly provided a strong basis to infer distinct chain conformations specified by hydration histories. We therefore explored through coarse-grained molecular dynamics (CGMD) simulations (
The sulfone-sulfone bonded networks of PPSU20 are mimetic of electrostatic networks in dimethylsulfone, a high-temperature polar solvent that can dissolve a wide range of organic solutes and is miscible with many other solvents. We investigated the use of PPSU20 nanostructured hydrogels to capture organic molecules from DMSO or aqueous solution using the stepwise hydration strategy. The results are shown in Table 1, wherein exceptionally high encapsulation efficiencies (EE) of >95% were observed for a wide range of molecules, including hydrophobic Nile red and water-soluble fluorescein isothiocyanate (FITC), doxorubicin hydrochloride (DOX/HCl), dextran, green fluorescent protein (GFP), DNA, and RNA. We also investigated the loading of FITC-labeled albumin (FITC-BSA), a model protein, at varying concentrations (
a100 μL of aqueous drug (except Nile red) solutions were mixed stepwise with 50 μL of PPSU20 solution (25 mg/ml in DMSO), followed by a one-time quick hydration using 400 μL of water. Nile red was loaded using its DMSO solution.
bGFP = Recombinant A. victoria GFP protein. Dextran (Mw = 4,000), Albumin, RNA, and DNA are conjugated with FITC.
cEncapsulation efficiency is defined as the ratio of the amount of molecules in the assemblies to the total amount applied in formulation. The encapsulation efficiencies were calculated by fluorescence measurements.
dGFP has no detectable fluorescence after encapsulation.
PPSU20 nanostructures were highly stable in water and no dense aggregates or precipitating was observed by aging a suspension of vesicular nanogels (
The high stability of PPSU nanogels in water provides insight into the mechanism of drug encapsulation. Starting from a swollen network that shows high affinity for a wide range of organic solutes, subsequent hydration induces quick collapse of the sulfone-sulfone bonded network (
These results suggest that FITC-BSA is accessible on the surface of the nanogels at densities dependent upon the initial loading concentration, and one potential explanation for this is described in the schematic of
We concluded that the observed high molecular encapsulation achieved by PPSU is due to tight molecular trapping, which allows us to fabricate novel protein-based nanocarriers without requiring covalent chemistries. By adjusting formulation conditions, nanogels up to 120% protein by mass could be achieved (Table 1). Our results thus demonstrate the possibility of extensive customization for the formation of protein/PPSU nanogels. The decreased fluorescence of GFP upon loading suggests that the current methodology may partially denature the protein structure, likely due to exposure to DMSO during loading (Table 1). Further investigations will be required to determine the limits of this platform for protein loading and retention of bioactivity as well as to assess the specific influences of different protein physicochemical properties on the final products.
Our results demonstrate that sulfone-sulfone bonded networks have exceptional molecular capture capability, providing us with a new type of drug delivery platforms that would greatly enhance the ability to encapsulate drug molecules. Although there have been intensive studies on vesicle-based delivery in the past few decades, substantial challenges remain for polymeric nanocarriers due in part to their low loading of water-soluble biologics and hydrophilic small molecule therapeutics. Drug loading in these polymer vesicles (i.e. polymersomes) are lower than 20%; some are even substantially less than 5% (29). For their potential use in biomedical applications, we further demonstrated no cytotoxicity up to 0.25 mg/mL (
In summary, we demonstrate that PPSU promotes the formation of an electrostatic network via sulfone-sulfone bonds in DMSO-water mixed systems. The sulfone-sulfone bonded network undergoes kinetically dependent self-assembly upon hydration, which imparts control over the morphology and size of macro- and nanoscale hydrogels. Without the need to change polymer structure or molecular weight, this single-component homopolymer system permits fabrication of crystalline frameworks and nanoscale hydrogels of spherical, vesicular, and cylindrical morphologies, controlled easily by the addition of water. The PPSU network shows a high affinity for a wide range of organic molecules, similar to the high-temperature solvent dimethyl sulfone. Taking advantage of this capability, we are able to encapsulate diverse hydrophilic molecules, including often difficult to load large biologics like protein and DNA, with nearly 100% efficiency. These results demonstrate that noncovalent sulfone-sulfone inter- and intra-chain bonding between PPSU homopolymers presents a structurally dynamic synthetic system that is biomimetic of natural biopolymers. PPSU is therefore a versatile platform for supramolecular chemistry with potential applications in biomedicine, diagnostics, catalysis and purification.
Methods
Polymer Synthesis.
PPS20 was prepared by anionic ring-opening polymerization of propylene sulfide (20 equiv) using ethanethiol (1 equiv) initiator and benzyl bromide (5 equiv) end-capper in the presence of sodium methylate as a base in dry dimethyl formamide (30). Mixing PPS20 with 30% of hydrogen peroxide (1 g PPS20 per 100 mL of H2O2 solution) and shaking the mixtures led to a homogeneous solution overnight. Lyophilization of the obtained solution resulted in shiny solids of PPSU20 without requirement for further purification.
AAMD Simulations.
Classical all-atom MD simulations were performed using the CHARMM 36m force field (31). The recommended CHARMM TIP3P water model (32) was applied with the structures constrained using the SETTLE algorithm (33). The simulations were performed using the package GROMACS (version 2016.3) (34). A detailed description of the simulation procedure is provided in the Supplementary Materials. The short-range electrostatic interactions were calculated up to 1.2 nm, and the long-range electrostatic interactions were calculated by means of the Particle Mesh Ewald algorithm (35). A time step of 2 fs (2.5 fs) was employed by constraining all the covalent bonds using the LINCS algorithm (36) in the DMSO system (water system). Annealing simulations were performed to speed up the convergence of the equilibrations (37).
Dipolar Energy Calculation.
By following a previous work (38), we calculated the dipolar interactions between the neighbor units. Each sulfone is defined as one charge-neutral unit, as well as one DMSO molecule and one H2O molecule. The calculation of dipolar interaction energy between the charge-neutral units is provided in the Supplementary Materials. The average dipole moments were calculated to be 2.347 D for the CHARMM TIP3P water model, the same as the reported value of 2.347 D (39). The dipole moment of DMSO was calculated to be 5.22 D, in consistent with the reported value of 5.11 D in the original literature where the DMSO CHARMM force field were originally presented, and around 20% larger than the experimental value (40). The dipole moment was calculated to be 6.534 D for the sulfone.
Fabrication of Nanostructured Hydrogels of PPSU20 with Varied Sizes and Morphologies.
200 μL of PPSU20 solutions (25 mg/mL in DMSO) were added stepwise with 400 μL of water and then one-time with another 400 μL of water. Each step was followed by vortexing to thoroughly mix the samples. After dialysis, the nanogels were applied for CryoTEM imaging, SAXS, DLS, WAXD, and MTT assay.
CGMD Simulations.
Each polymer is modeled by a linear bead-spring chain consisting of N=20 coarse-grained (CG) monomers. Each CG monomer carries three-point charges: a positive charge in the backbone corresponding to the S atoms and two neighboring backbone C atoms, and two negative charges corresponding to the O atoms (white spheres). Overall the monomer is charge neutral and has a net dipole as indicated by the all-atom model calculations. The relative positions of the three-point charges are maintained by harmonic springs between the S—O bonds and constraining the O—S—O angle. The non-bonded interactions between the CG monomers includes the excluded volume interaction and electrostatics. The excluded volume interaction is modeled by the Weeks-Chandler-Andersen potential, which is the Lennard-Jones potential truncated and shifted to zero at the minimum. The parameter is chosen as the length unit of the system, which corresponds to the van der Waals diameter of the S atom. The electrostatic interaction is truncated at the cutoff distance of rc=8□, which is considered adequate for our coarse-grained simulations where the solvent is treated implicitly as a uniform back ground with the dielectric constant of 47 for DMSO and 80 for water. The parameters of the CG model are calibrated against the all-atom in terms of the chain persistent length in these two solvents. To ensure that such a truncated Coulombic scheme does not affect our results, we have also performed test simulations with long-range Coulombic electrostatics with the particle-particle particle-mesh method. The simulations with long-range Coulombic electrostatics yield similar assembled morphologies. All the CGMD simulations were performed with the LAMMPS (version 19 Sep. 2019) software package (41).
Loading Experiments.
For the loading of Nile red, nanogels were prepared using pure water and Nile red-containing (0.1 mg/mL) DMSO solutions of PPSU20. For the loading of water-soluble drugs, aqueous solutions of FITC (0.05 mg/mL), doxorubicin hydrochloride (1 mg/mL), dextran (1 mg/mL), albumin (various concentrations), GFP (0.2 mg/mL), RNA (0.2 mg/mL), DNA (0.2 mg/mL), and DMSO solutions of PPSU20 (25 mg/mL) were used. Typically, 100 μL of the corresponding aqueous solution were mixed stepwise (10 μL/step or 100 μL/step) with 50 μL of PPSU20 solution under vortex, then another 400 μL of water was added. After centrifugation at 16000×g for 10 min, fluorescence of the supernatant was measured and the encapsulation efficiencies were calculated.
Trypsin Digestion of FITC-BSA-Loaded PPSU20 Vesicular Nanogels.
DMSO solutions of PPSU20 (200 μL, 25 mg/mL) were hydrated using 800 μL of aqueous FITC-BSA solutions (0, 0.1, 1.0, 10.0 mg/mL). DMSO was removed by dialysis after self-assembly. Negatively stained FITC-BSA-loaded PPSU20 vesicular nanogels were characterized by TEM. After removal of unloaded FITC-BSA by three rounds of centrifugation and resuspension in phosphate-buffered saline, FITC-BSA-loaded PPSU20 vesicular nanogels were subjected to digestion with trypsin protease (2 μg/mL; Trypsin Gold, Promega) for 24 h at 37° C., 80 rpm. Undigested FITC-BSA-loaded PPSU20 vesicular nanogels (i.e. no protease treatment) were included as a control. After 24 h incubation with trypsin, FITC fluorescence (Ex=490 nm, Em=525 nm) was quantified in triplicate using a SpectraMax M3 microplate reader (Molecular Devices). The percentage increase in FITC fluorescence (i.e., 100×(F(+)−F(−))/F(−)) after proteolysis was calculated, where F(+) is the fluorescence after protease treatment and F(−) is referred to the fluorescence in the absence of protease treatment.
Materials and Methods
Chemical Reagents
All chemical reagents were purchased from Sigma-Aldrich St. Louis, Mo., USA, unless stated otherwise. Recombinant A. victoria GFP protein (from the Jewett Lab), FITC-RNA and FITC-DNA conjugates (from the Mirkin Lab) were obtained as generous gifts from other Labs at Northwestern University.
Polymer Synthesis
PPSU was synthesized from complete oxidation of the corresponding PPS. Mixing PPS with 30% of hydrogen peroxide (1 g PPS per 100 mL of H2O2 solution) and shaking the mixtures led to a homogeneous solution overnight. Lyophilization of the obtained solution resulted in white shiny solids of PPSU without requirement for further purification. It is worthwhile to note that an oxidation intermediate (sulfone/sulfoxide mixtures) can be collected by precipitation the homogeneous solution in THF. Apparent differences were discovered for PPSU20 and the random copolymers of sulfoxides and sulfones. First, PPSU20 powders are demonstrated by WAXD to be crystalline while the sulfoxides/sulfone mixtures are mostly amorphous. Second, the sulfoxides/sulfone mixtures are readily dissolved by water while PPSU20 solids are completely insoluble in water.
All-Atom Explicit Solvent Molecular Dynamics Simulations
Classical all-atom molecular dynamics simulations were performed using the CHARMM 36m force field (31). The recommended CHARMM TIP3P water model (32) was applied with the structures constrained using the SETTLE algorithm (33). The simulations were performed using the package GROMACS (version 2016.3) (34). In all the simulations, the degree of polymerization (DP) of 20 was employed for the polymer chains, the same as that PPSU20 in the experiments. The polymer chains were created in the extended form. Six extended PPSU20 chains were randomly dissolved in each of the three water boxes initially. Whereas in the DMSO systems, the initially extended PPSU20 chains were equilibrated in a vacuum, forming coiled configurations, and were then dissolved in the DMSO boxes. The initial configurations are provided in
The system potential energy was first minimized using the steepest descent algorithm, followed by the equilibration of 1 ps in the NVT ensemble (constant number of particles, volume and temperature). Subsequently the NPT ensemble (constant number of particles, pressure and temperature) was applied. An equilibration of 10 ps using the time step of 1 fs was followed by another equilibration of 0.1 ns using a time step of 2 fs in the DMSO system, or 2.5 fs in the aqueous system. Subsequently long equilibration simulations were performed. The periodic boundary conditions were applied in all three dimensions. The neighbor searching was performed up to a cut-off distance of 1.2 nm by means of the Verlet particle-based approach and was updated every 20 time steps. The potential-switch method was applied for the short-range Lennard-Jones (LJ) 12-6 interactions from 1 nm to 1.2 nm. The short-range electrostatic interactions were calculated up to 1.2 nm, and the long-range electrostatic interactions were calculated by means of the Particle Mesh Ewald algorithm (35). A time step of 2 fs (2.5 fs) was employed by constraining all the covalent bonds using the LINCS algorithm (36) in the DMSO system (water system). The temperatures of the PPSU20 solute and the solvent molecules were separately coupled using the Nosé-Hover algorithm (reference temperature 298 K, characteristic time 1 ps). The isotropic Parrinello-Rahman barostat was utilized with the reference pressure of 1 bar, the characteristic time 4 ps and the compressibility 4.5×10−5 bar−1. Each of the simulations run 200 ns, with the last 50 ns employed for the data collection and analysis. The final simulation snapshots are presented in
Additionally, control simulations were performed. In the control simulations, initially extended polymer chains were employed for both DMSO and water solvent systems. The annealing simulations were performed to speed up the convergence of the equilibrations (37). In the annealing simulations, the temperatures of polymer and solvent (DMSO or water) were separately coupled. The temperatures started at 298 K initially, which increased to 353 K within 1 ns. They stayed at 353 K for 9 ns, then dropped to 298 K within 1 ns. Finally, the temperatures stayed at 298 K for another 9 ns. Therefore, each annealing cycle lasted 20 ns. 5 annealing cycles were performed for the DMSO systems (100 ns in total), and 8 cycles (160 ns in total) for the water systems. Subsequently, the production simulations were performed for 40 ns each. Agreements were found in regards with the polymer chain distribution (molecularly dissolved in DMSO and aggregated in water), the polymer structures (end-to-end distance and persistence length) and the radial distribution function of the polymer sulfur atoms.
Calculation of the Dipolar Energy Between Neighbor Charge-Neutral Units from Atomistic Molecular Dynamics Simulations
In all the atomistic simulations, the PPSU repeat units and the solvent (DMSO or water) molecules are charge-neutral. The monopole interactions between them could be reasonably expected negligible. By following a previous work (38), we calculated the dipolar interactions between the neighbor units. Each PPSU repeat unit is defined as one charge-neutral unit, as well as one DMSO molecule and one H2O molecule. The dipolar interaction energy between the charge-neutral units is calculated as below:
1) The sulfur atoms of PPSU repeat units, the sulfur atoms of DMSO and the oxygen atoms of water molecules are employed as the center of the corresponding units. The radial distribution functions between these atoms are calculated (
2) The dipole moment of PPSU repeat units, DMSO molecules and water molecule were calculated via
{right arrow over (μ)}=Σi[({right arrow over (xl)}−{right arrow over (xcenter)})·qi] (S1)
{right arrow over (xl)} denotes the coordinate of atom i with the atomic partial charge qi. {right arrow over (xcenter)} stands for the coordinate of the center atom of S(PPSU), S(DMSO) or O(H2O). The average dipole moments were calculated to be 2.347 D for the CHARMM TIP3P water model, the same as the reported value of 2.347 D (39). The dipole moment of DMSO was calculated to be 5.22 D, in consistent with the reported value of 5.11 D in the original literature where the DMSO CHARMM force field were originally presented, and around 20% larger than the experimental value (40). The dipole moment was calculated to be 6.534 D for PPSU repeat units.
3) The dipolar interaction energy between units i and j could be thus obtained by
where {right arrow over (μl)}, {right arrow over (μJ)} is the dipole moment of units i and j, respectively; {circumflex over (r)} stands for the unit vector between the centers of the two units. Here the units refer to PPSU repeat units, DMSO molecules and water molecules. The cutoff distances were defined in step 1.
4) The steps 2 to 3 were performed for all the dipole-dipole interactions of PPSU-PPSU and PPSU-DMSO in the DMSO solution, and PPSU—PPSU and PPSU-water in the aqueous solution. The distribution of the calculated dipolar energies is provided in
Humidity Induced-Aggregation for DMSO Solutions of PPSU20
DMSO solutions of PPSU20 were exposed to humidity in air and the phase transition was tracked. Sol-to-gel phase transition was observed for a highly concentrated solution (200 mg/mL) overnight and a low concentration solution (25 mg/mL) became cloudy in 3 days. As the cloudy solution was allowed to age further (110 days), fluffy precipitates (can be observed in 7 days) were obtained by centrifugation. The fluffy precipitates were demonstrated by WAXD to be mostly amorphous (
CryoTEM Imaging
Samples were prepared by applying 3 μL of sample (5 mg/mL) on pretreated holey or lacey carbon 400 mesh TEM copper grids (Electron Microscopy Sciences). Following a 3 s blot, samples were plunge-frozen (Gatan Cryoplunge 3 freezer). Images of samples entrapped in vitreous ice were acquired using a field emission transmission electron microscope (JEOL 3200FS) operating at 300 keV with magnification ranging from 2000× to 12,000× nominal magnification. Digital Micrograph software (Gatan) was used to align the individual frames of each micrograph to compensate for stage and beam-induced drift. Any further image processing conducted on the aligned frames was completed in ImageJ.
SAXS Measurements
SAXS measurements were performed at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) beamline at Argonne National Laboratory's Advanced Photon Source (Argonne, Ill., USA) with 10 keV (wavelength λ=1.24 Å) collimated X-rays. All the samples (5 mg/mL) were analyzed in the q-range (0.001-0.5 Å−1), with a sample-to-detector distance of approximately 7.5 m and an exposure time of 1 s. The diffraction patterns of silver behenate were utilized to calibrate the q-range. The momentum transfer vector q is defined as q=4π sin θ/λ, where θ is the scattering angle. Data reduction, consisting of the removal of solvent/buffer scattering from the acquired sample scattering, was completed using PRIMUS 2.8.2 software while model fitting was completed using SasView 4.0.1 software package. The core-shell cylinder, vesicle and core-shell sphere models were utilized to analyze the data.
Transmission Electron Microscopy of Negatively Stained PPSU20 Structures
1.5% uranyl formate (UF) was prepared in ultrapure water. The pH was adjusted to 4.5 by addition of 10 N KOH. 4 μL of nanostructures were applied to glow discharged (25 W, 10 s) formvar carbon film copper grids (400 mesh; Electron Microscopy Sciences, Inc.). Samples were gently washed twice via passage through ultrapure water, and were negatively stained by passage through two 30 μL volumes of 1.5% UF. Excess stained was removed by blotting each sample with Whatman filter paper. After this procedure, ˜0.5 μL stain remains on the grid with an activity of 2.55×10−5 μCi/grid. Images were acquired at 30,000× on a JOEL 1400 Transmission Electron Microscope operating at 120 kV.
Energy Dispersive X-Ray Spectroscopy
1 μL of PPSU20 nanobundles (5 mg/mL in water) were mixed thoroughly with 1 μL of 2% methyl cellulose and 3 μL of water. 5 μL of the mixture was deposited on Formvar/Carbon 200 Mesh Copper TEM grids from Electron Microscopy Sciences that were glow discharged to create a hydrophilic surface. After resting for 15 s, the grid was incubated in 1.5% UF for 15 s. Excess stain was wicked away with filter paper. Scanning transmission electron microscopy images were taken with high angle annular dark field (HAADF) mode where bright contrast is indicative of a species with a higher atomic number. In this case, uranium from UF provides the contrast, and the absence of that contrast shows the structure of the embedded sample. EDS maps were taken on a Hitachi HD-2300A dedicated STEM equipped with dual EDS detectors and Thermo Fischer Scientific NSS software. Operating at 200 kV with a 58 μA emission current and an aperture with 75 μm diameter, 90 frames of a 512×384 pixel EDS map were collected in EDX Mode. The overall frame time was 10 s with a pixel dwell time of 50 μs. Data was processed by binning with a 17×17 kernel size.
Adsorption of FITC-BSA by Preformed PPSU20 Vesicular Nanogels
PPSU20 vesicular nanogels were prepared by mixing 100 μL of water or 100 μL of aqueous FITC-BSA (1.0 mg/mL) solution with 100 μL of PPSU20 solution (25 mg/mL in DMSO) under vortex, followed by the addition of 1000 μL of water. Nanogels were collected by centrifugation and purified by 2 rounds of resuspension and centrifugation in 1000 L of water. The preformed blank vesicular nanogels were further incubated with 100 μL of aqueous FITC-BSA (1.0 mg/mL) solution overnight, giving FITC-BSA-adsorbed nanogels after removal of free FITC-BSA by three rounds of centrifugation and resuspension in water. Blank nanogels, FITC-BSA-loaded nanogels, and FITC-BSA-adsorbed nanogels were resuspened in 1000 μL of water for fluorescence measurements or as the stocking solutions for fluorescence quantitative analysis in sodium hydroxide solution (0.2 N).
Cell Experiments
RAW 264.7 cells (murine macrophage cell line) were acquired from American Type Culture Collection (ATCC, Rockville, Md., USA) and used for cell culture experiments. This cell line was cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL) and streptomycin (100 μg/mL) at 37° C. with CO2 (5%).
Cell viability: Both MTT assay and flow cytometric assessment were performed to investigate cell viability upon nanostructures treatment. In MTT assay, RAW 264.7 cells (3×105 cells/mL, 100 μL) were plated in each well of a 96-well plate and left overnight in the incubator for adherence. The adhered cells were treated with different concentrations of PPSU20 nanogels (1, 0.5, 0.25 and 0.125 mg/mL). After incubation for 24 h, all wells were added with MTT (5 mg/mL in PBS, 10 μL) and further incubated for 4 h. The resultant formazan crystal deposition in each well was dissolved in DMSO (200 μL) and the absorbance was measured at 560 nm. All the samples were analyzed in quadruplicates. The percentage cell viability was calculated as: % cell viability=(OD of treated sample/OD of untreated sample)×100. Macrophage cell viability following nanostructure treatment was also determined using Zombie Aqua fixable cell viability dye. Following differentiation, cells were plated at 2.5×105 cells/mL (200 μL; 50,000 cells/well) in 96-well tissue culture treated plates in DMEM cell culture media. RAW 264.7 cells received matched volume treatments of either PBS or PPSU20 nanostructures (bundle-like or vesicle like nanostructures with concentrations of 1, 0.5, 0.25 and 0.125 mg/mL). After incubation for 24 h, cells were collected and transferred to 1.2 mL microtiter tubes prior to staining with Zombie Aqua fixable viability dye (Biolegend) for 15 min. Cells were washed with cell staining buffer and fixed with intracellular (IC) cell fixation buffer (Biosciences). Flow cytometry data was acquired on an LSR Fortessa analyzer (BD Biosciences) and analyzed using FlowJo.
Cellular uptake studies: RAW 264.7 cells (2.5×105 cells/mL, 400 μL) were seeded in each well of a 48-well plate and left overnight in the incubator for adherence. The adhered cells were treated with 0.25 mg/mL of FITC-dextran-loaded PPSU20 nanogels (bundle or vesicular morphology) and incubated for 1 h or 4 h. Cells were washed two times with PBS and incubated with 50 μL Zombie Aqua (1:100) fixable cell viability dye diluted in cell staining buffer for 15 min at 4° C. Then the cells were washed with 500 μL PBS, resuspended in cell staining buffer and immediately analyzed using a BD Fortessa flow cytometer. The cellular uptake was measured as % of FITC positive cells.
Analysis/imaging of cells: RAW 264.7 cells (1×105 cells/mL, 300 μL) were seeded in each well of an 8-well Chamber slide (Thermo Fischer Scientific) and left overnight in the incubator for adherence. The adhered cells were treated with 0.25 mg/mL of FITC-dextran-loaded PPSU20 nanostructures (bundle or vesicular morphology). After incubation for 4 h, cells were washed two times with PBS and incubated with LysoTracker™ Red DND-99 (1:5000 dilution, 300 μL DMEM) for 30 min. Then the cells were washed twice with PBS, added with 300 μL PBS and incubated with NucBlue™ Live ReadyProbes™ Reagent (nuclei stain, 1 drop) for 15 min in the dark. Images were acquired on a Leica TCS SP8 confocal microscope with a 63× oil immersion objective.
Investigation of cellular uptake mechanisms: RAW 264.7 macrophages were pre-treated with either cytochalasin D (5 μM; Cayman Chemical) for 2 h to inhibit macropinocytosis, or chlorpromazine (100 μM; Chlorpromazine HCl, Sigma Aldrich) for 30 min to inhibit clathrin-dependent endocytosis. PBS-treated cells were included as a control in these experiments. After this pre-treatment period, cells were incubated with 0.25 mg/mL FITC-dextran-loaded PPSU20 vesicular nanogels or nanobundles, and flow cytometry was performed. The median fluorescence intensity (MFI) was used to quantify the cellular internalization of PPSU20 nanogels and bundles. Sidak's multiple comparisons test was used to determine whether inhibitor pre-treatment significantly reduced the cellular uptake of PPSU20 nanostructures compared to PBS-treated control. In these experiments, a statistically significant reduction in nanostructure uptake indicates the inhibited endocytosis pathway contributes to internalization.
Develop Universal Molecular Encapsulation by PPSU as a Tool for the Optimization of a Model.
We present two novel encapsulation methods uniquely achievable by PPSU. First, collapsible networks of PPSU can encapsulate >4 payloads simultaneously, each at a controllable concentration (
This Example demonstrates that 1) PPSU vesicles can employ 2 distinct drug loading strategies: pre-assembly and post-assembly (
Standard Pre-Assembly Method of PPSU Nanogel Formation and Drug Loading:
The fabrication of 100 nm PPSU nanogel vesicles was performed utilizing a simple, one-step vortex mixing method. In brief, 2.5 mg of PPSU polymer (50 μL, 50 mg/mL in DMSO) was added with 100 μL of Milli-Q water and vortexed for 30 s. The resultant mixture was diluted with 500 μL Milli-Q water and then dialyzed (Thermo Scientific Slide-A-Lyzer MINI dialysis devices) against Milli-Q water/PBS to remove DMSO. The obtained nanostructures were utilized for further analysis. The antigen and adjuvants were loaded alone or simultaneously into PPSU nanogels via the method described above (
Novel Post-Assembly Method of PPSU Nanogel Formation and Drug Loading:
Interestingly, previously assembled and purified nanogels were found to stably capture diverse water soluble molecules when incubated in aqueous solutions (
A Proteomics-Based Assay for High Sensitivity Assessment of Protein Loading Stability.
Following loading of trypsin using either the pre- or post-assembly method, nanogels were incubated at room temperature for 48 h. PPSU vesicles were pelleted by centrifugation for isolation of free form trypsin in the supernatant that may have leaked out of the nanogels during the 48 h incubation. The supernatants were collected and mixed 1:1 with sinapinic acid matrix prepared in ultrapure water with 0.2% TFA. These mixtures were spotted onto a stainless steel MALDI disc (Bruker) and full-length Trypsin protein (˜23 kDa) was detected by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (
Trypsin Proteolysis Fluorescence-Based Assay.
This assay was designed to determine whether an enzyme encapsulated within PPSU nanogels could both retain enzymatic activity as well as access protein substrates in the surrounding solution (
MPLA will be employed as a nontoxic and clinically approved variant of LPS. To better mimic immunostimulation of an intracellular bacterium, STING agonist 2′3′-cGAMP will be incorporated and ovalbumin will be replaced with the tuberculosis vaccine candidate protein antigen Ag85B. Vaccine immunogenicity and efficacy will be assessed by both in vitro and in vivo assays that quantify changes in cytokine expression and T cell activation.
Assessment of PPSU NBM Biodistribution and Toxicity:
Preliminary assessment of vesicular PPSU nanogel biodistribution 48 h after subcutaneous (s.c.) injection (relevant for vaccination) was performed using near infrared fluorescence (NIRF) agent cardiogreen (ICG) as previously published by the Scott lab1. ICG was stably loaded into nanogels using the pre-assembly method, which shifted the biodistribution from heart, brain and kidneys to primarily liver and kidneys (
This application claims priority to U.S. Provisional Application No. 62/879,637, filed on Jul. 29, 2019, the contents of which are incorporated herein by reference.
This invention was made with government support under 1DP2HL 132390-01 awarded by the National Institutes of Health and DE-FG02-08ER46539 awarded by the Department of Energy. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62879637 | Jul 2019 | US |
