rapid-scalable-assembly-loading-bioactive-proteins-immunostimulants

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Northwestern University

Atherosclerosis is an inflammatory disorder with a pathophysiology driven by both innate and adaptive immunity and a primary cause of cardiovascular disease (CVD) worldwide. Vascular inflammation and accumulation of foam cells and their products induce maturation of atheromas, or plaques, which can rupture by metalloprotease action, leading to ischemic stroke or myocardial infarction. Diverse immune cell populations participate in all stages of plaque maturation, many of which directly influence plaque stability and rupture via inflammatory mechanisms. Current clinical treatments for atherosclerosis focus on lowering serum levels of low-density lipoprotein (LDL) using therapeutics such as statins, administration of antithrombotic drugs, and surgical intervention. Strategies that address cell-mediated inflammation are lacking, and consequently have recently become an area of considerable research focus. Nanomaterials have emerged as highly advantageous tools for these studies, as they can be engineered to target specific inflammatory cell populations, deliver therapeutics of wide-ranging solubilities and enhance analytical methods that include imaging and proteomics. Furthermore, the highly phagocytic nature of antigen presenting cells (APCs), a diverse cell population central to the initiation of immune responses and inflammation, make them particularly amenable to targeting and modulation by nanoscale particulates. Nanomaterials have therefore become essential components of vaccine formulations and treatments for inflammation-driven pathologies like autoimmunity, and present novel opportunities for immunotherapeutic treatments of CVD. Here, we review recent progress in the design and use of nanomaterials for therapeutic assessment and treatment of atherosclerosis. We will focus on promising new approaches that utilize nanomaterials for cell-specific imaging, gene therapy and immunomodulation.

Engineering nanomaterials to address cell-mediated inflammation in atherosclerosis.

Atherosclerosis, a leading cause of heart disease, results from chronic vascular inflammation that is driven by diverse immune cell populations. Nanomaterials may function as powerful platforms for diagnostic imaging and controlled delivery of therapeutics to inflammatory cells in atherosclerosis, but efficacy is limited by nonspecific uptake by cells of the mononuclear phagocytes system (MPS). MPS cells located in the liver, spleen, blood, lymph nodes, and kidney remove from circulation the vast majority of intravenously administered nanomaterials regardless of surface functionalization or conjugation of targeting ligands. Here, we report that nanostructure morphology alone can be engineered for selective uptake by dendritic cells (DCs), which are critical mediators of atherosclerotic inflammation. Employing near-infrared fluorescence imaging and flow cytometry as a multimodal approach, we compared organ and cellular level biodistributions of micelles, vesicles (i.e., polymersomes), and filomicelles, all assembled from poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) block copolymers with identical surface chemistries. While micelles and filomicelles were respectively found to associate with liver macrophages and blood-resident phagocytes, polymersomes were exceptionally efficient at targeting splenic DCs (up to 85% of plasmacytoid DCs) and demonstrated significantly lower uptake by other cells of the MPS. In a mouse model of atherosclerosis, polymersomes demonstrated superior specificity for DCs (p < 0.005) in atherosclerotic lesions. Furthermore, significant differences in polymersome cellular biodistributions were observed in atherosclerotic compared to naïve mice, including impaired targeting of phagocytes in lymph nodes. These results present avenues for immunotherapies in cardiovascular disease and demonstrate that nanostructure morphology can be tailored to enhance targeting specificity.

Tailoring Nanostructure Morphology for Enhanced Targeting of Dendritic Cells in Atherosclerosis.

The immune system is governed by an immensely complex network of cells and both intracellular and extracellular molecular factors. It must respond to an ever-growing number of biochemical and biophysical inputs by eliciting appropriate and specific responses in order to maintain homeostasis. But as with any complex system, a plethora of false positives and false negatives can occur to generate dysregulated responses. Dysregulated immune responses are essential components of diverse inflammation-driven pathologies, including cancer, heart disease, and autoimmune disorders. Nanoscale biomaterials (i.e., nanobiomaterials) have emerged as highly customizable platforms that can be engineered to interact with and direct immune responses, holding potential for the design of novel and targeted approaches to redirect or inhibit inflammation. Here, we present recent developments of nanobiomaterials that were rationally designed to target and modulate inflammatory cells and biochemical pathways for the treatment of immune dysregulation.

Overcoming Immune Dysregulation with Immunoengineered Nanobiomaterials.

Newborns display distinct immune responses, leaving them vulnerable to infections and impairing immunization. Targeting newborn dendritic cells (DCs), which integrate vaccine signals into adaptive immune responses, might enable development of age-specific vaccine formulations to overcome suboptimal immunization.

Toll-like receptor 8 agonist nanoparticles mimic immunomodulating effects of the live BCG vaccine and enhance neonatal innate and adaptive immune responses.

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.

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

Our data suggest that, after a myocardial infarction, integrin-associated protein CD47 on cardiac myocytes is elevated. In culture, increased CD47 on the surface of dying cardiomyocytes impairs phagocytic removal by immune cell macrophages. After myocardial ischemia and reperfusion, acute CD47 inhibition with blocking antibodies enhanced dead myocyte clearance by cardiac phagocytes and also improved the resolution of cardiac inflammation, reduced infarct size, and preserved cardiac contractile function. Early targeting of CD47 in the myocardium after reperfusion may be a new strategy to enhance wound repair in the ischemic heart.

Acute CD47 Blockade During Ischemic Myocardial Reperfusion Enhances Phagocytosis-Associated Cardiac Repair.

Intracellular delivery is a key step for many applications in medicine and for investigations into cellular function. This is particularly true for immunotherapy, which often requires controlled delivery of antigen and adjuvants to the cytoplasm of immune cells. Due to the complex responses generated by the stimulation of diverse immune cell populations, it is critical to monitor which cells are targeted during treatment. To address this issue, we have engineered an immunotheranostic polymersome delivery system that fluorescently marks immune cells following intracellular delivery.

Immunotheranostic Polymersomes Modularly Assembled from Tetrablock and Diblock Copolymers with Oxidation-Responsive Fluorescence.

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)-block-poly(propylene sulfide) (PEG-bl-PPS), 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.

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

Nanocarrier administration has primarily been restricted to intermittent bolus injections with limited available options for sustained delivery in vivo. Here, we demonstrate that cylinder-to-sphere transitions of self-assembled filomicelle (FM) scaffolds can be employed for sustained delivery of monodisperse micellar nanocarriers with improved bioresorptive capacity and modularity for customization. Modular assembly of FMs from diverse block copolymer (BCP) chemistries allows in situ gelation into hydrogel scaffolds following subcutaneous injection into mice. Upon photo-oxidation or physiological oxidation, molecular payloads within FMs transfer to micellar vehicles during the morphological transition, as verified in vitro by electron microscopy and in vivo by flow cytometry. FMs composed of multiple distinct BCP fluorescent conjugates permit multimodal analysis of the scaffold's non-inflammatory bioresorption and micellar delivery to immune cell populations for one month. These scaffolds exhibit highly efficient bioresorption wherein all components participate in retention and transport of therapeutics, presenting previously unexplored mechanisms for controlled nanocarrier delivery.

Sustained micellar delivery via inducible transitions in nanostructure morphology.

Increased stiffness of Schlemm's canal endothelial cells (SC cells) is a major contributing factor to the increased pressure characteristic of primary open-angle glaucoma. New treatments for glaucoma are being developed using actin depolymerizers and rho kinase inhibitors to address this increased stiffness. However, these agents have off-target effects and are not as potent as had been hoped. We have developed a micellar nanocarrier assembled from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers capable of encapsulating latrunculin A (Lat A) with the goal of modulating SC cell stiffness. Lat A-loaded nanocarriers were similar in size and morphology to unloaded poly (ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) micelles, loaded Lat A at 62% encapsulation efficiency, and retained loaded Lat A for at least 22 days. The continued functional activity of Lat A following encapsulation within micelles was verified in murine macrophages, which are known to display decreased endocytosis in response to Lat A-dependent cytoskeletal disruption. Endocytic inhibition remained unchanged when comparing equal concentrations of micelle-loaded versus free form Lat A. Uptake of Lat A-loaded micelles by human SC cells was verified in vitro with no sign of cytotoxicity, and modulation of SC cell stiffness was measured by atomic force microscopy. Lat A-loaded micelles significantly decreased SC cell stiffness, which resulted in visible changes in cell morphology as observed by confocal microscopy. Our results demonstrate that PEG-bl-PPS micelles represent a tunable platform for the controlled intracellular delivery of latrunculin. These self-assembled polymeric nanobiomaterials may support the rational design and engineering of delivery systems for the treatment of glaucoma. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1771-1779, 2018.

Modulation of Schlemm's canal endothelial cell stiffness via latrunculin loaded block copolymer micelles.

Polymer vesicles, i.e. polymersomes (PS), present unique nanostructures with an interior aqueous core that can encapsulate multiple independent cargos concurrently. However, the sequential release of such co-loaded actives remains a challenge. Here, we report the rational design and synthesis of oxidation-responsive shell-crosslinked PS with capability for the controlled, sequential release of encapsulated hydrophilic molecules and hydrogels. Amphiphilic brush block copolymers poly(oligo(ethylene glycol) methyl ether methacrylate)-b-poly(oligo(propylene sulfide) methacrylate) (POEGMA-POPSMA) were prepared to fabricate PS via self-assembly in aqueous solution. As a type of unique drug delivery vehicle, the interior of the PS was co-loaded with hydrophilic molecules and water-soluble poly(N-isopropylacrylamide) (PNIPAM) conjugates. Due to the thermosensitivity of PNIPAM, PNIPAM conjugates within the PS aqueous interior underwent a phase transition to form hydrogels in situ when the temperature was raised above the lower critical solution temperature (LCST) of PNIPAM. Via control of the overall shell permeability by oxidation, we realized the sequential release of two water-soluble payloads based on the assumption that hydrogels have much smaller membrane permeability than that of molecular cargos. The ability to control the timing of release of molecular dyes and PNIPAM-based hydrogels was also observed within live cells. Furthermore, leakage of hydrogels from the PS was effectively alleviated in comparison to molecular cargos, which would facilitate intracellular accumulation and prolonged retention of hydrogels within the cell cytoplasm. Thus, we demonstrate that the integration of responsive hydrogels into PS with crosslinkable membranes provides a facile and versatile technique to control the stability and release of water-soluble cargos for drug delivery purposes.

Sequential intracellular release of water-soluble cargos from Shell-crosslinked polymersomes.

Nanomaterials provide numerous advantages for the administration of therapeutics, particularly as carriers of immunomodulatory agents targeting specific immune cell populations during immunotherapy. While the physicochemical characteristics of nanocarriers have long been linked to their therapeutic efficacy and applications, focus has primarily been placed on assessing influences of size and surface chemistry. In addition to these materials properties, the nanostructure morphology, in other words, shape and aspect ratio, has emerged as an equally important feature of nanocarriers that can dictate mechanisms of endocytosis, biodistribution and degree of cytotoxicity. In this review, we will highlight how the morphological features of nanostructures influence the immune responses elicited during therapeutic immunomodulation.

Article	Year
Engineering nanomaterials to address cell-mediated inflammation in atherosclerosis. Regenerative engineering and translational medicine\| PubMed ID: 27135051	2016
Tailoring Nanostructure Morphology for Enhanced Targeting of Dendritic Cells in Atherosclerosis. ACS nano\| PubMed ID: 27935698	2016
Overcoming Immune Dysregulation with Immunoengineered Nanobiomaterials. Annual review of biomedical engineering\| PubMed ID: 28226216	2017
Toll-like receptor 8 agonist nanoparticles mimic immunomodulating effects of the live BCG vaccine and enhance neonatal innate and adaptive immune responses. The Journal of allergy and clinical immunology\| PubMed ID: 28343701	2017
Facile assembly and loading of theranostic polymersomes via multi-impingement flash nanoprecipitation. Journal of controlled release : official journal of the Controlled Release Society\| PubMed ID: 28736263	2017
Acute CD47 Blockade During Ischemic Myocardial Reperfusion Enhances Phagocytosis-Associated Cardiac Repair. JACC. Basic to translational science\| PubMed ID: 28920097	2017
Immunotheranostic Polymersomes Modularly Assembled from Tetrablock and Diblock Copolymers with Oxidation-Responsive Fluorescence. Cellular and molecular bioengineering\| PubMed ID: 28989540	2017
Flash nanoprecipitation permits versatile assembly and loading of polymeric bicontinuous cubic nanospheres. Nanoscale\| PubMed ID: 29255814	2018
Sustained micellar delivery via inducible transitions in nanostructure morphology. Nature communications\| PubMed ID: 29434200	2018
Modulation of Schlemm's canal endothelial cell stiffness via latrunculin loaded block copolymer micelles. Journal of biomedical materials research. Part A\| PubMed ID: 29468812	2018
Sequential intracellular release of water-soluble cargos from Shell-crosslinked polymersomes. Journal of controlled release : official journal of the Controlled Release Society\| PubMed ID: 29601932	2018
Influences of nanocarrier morphology on therapeutic immunomodulation. Nanomedicine (London, England)\| PubMed ID: 30084296	2018

Evan Scott

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