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The targeting of natural tolerogenic liver sinusoidal endothelial cells (LSEC) by nanoparticles (NPs), decorated with a stabilin receptor ligand, is capable of generating regulatory T-cells (Tregs), which can suppress antigen-specific immune responses, including to ovalbumin (OVA), a possible food allergen. In this regard, we have previously demonstrated that OVA-encapsulating poly(lactic-co-glycolic acid) (PLGA) nanoparticles eliminate allergic airway inflammation in OVA-sensitized mice, prophylactically and therapeutically. A competing approach is a nanocarrier platform that incorporates pharmaceutical agents interfering in mTOR (rapamycin) or NF-κB (curcumin) pathways, with the ability to induce a tolerogenic state in nontargeted antigen-presenting cells system-wide. First, we compared OVA-encapsulating, LSEC-targeting tolerogenic nanoparticles (TNPs) with nontargeted NPs incorporating curcumin and rapamycin (Rapa) in a murine eosinophilic airway inflammation model, which is Treg-sensitive. This demonstrated roughly similar tolerogenic effects on allergic airway inflammation by stabilin-targeting NPOVA versus nontargeted NPs delivering OVA plus Rapa. Reduction in eosinophilic inflammation and TH2-mediated immune responses in the lung was accompanied by increased Foxp3+ Treg recruitment and TGF-β production in both platforms. As OVA incorporates IgE-binding as well as non-IgE-binding epitopes, the next experiment explored the possibility of obtaining immune tolerance by non-anaphylactic T-cell epitopes. This was accomplished by incorporating OVA323-339 and OVA257-264 epitopes in liver-targeting NPs to assess the prophylactic and therapeutic impact on allergic inflammation in transgenic OT-II mice. Importantly, we demonstrated that the major histocompatibility complex (MHC)-II binding (former) but not the MHC-I binding (latter) epitope interfered in allergic airway inflammation, improving TNPOVA efficacy. The epitope-specific effect was transduced by TGF-β-producing Tregs. In the final phase of experimentation, we used an OVA-induced anaphylaxis model to demonstrate that targeted delivery of OVA and its MHC-II epitope could significantly suppress the anaphylaxis symptom score, mast cell release, and the late-phase inflammatory response. In summary, these results demonstrate comparable efficacy of LSEC-targeting versus pharmaceutical PLGA nanoparticles, as well as the ability of T-cell epitopes to achieve response outcomes similar to those of the intact allergens.

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.

The principle cause of cardiovascular disease (CVD) is atherosclerosis, a chronic inflammatory condition characterized by immunologically complex fatty lesions within the intima of arterial vessel walls. Dendritic cells (DCs) are key regulators of atherosclerotic inflammation, with mature DCs generating pro-inflammatory signals within vascular lesions and tolerogenic DCs eliciting atheroprotective cytokine profiles and regulatory T cell (Treg) activation. Here, we engineered the surface chemistry and morphology of synthetic nanocarriers composed of poly(ethylene glycol)-b-poly(propylene sulfide) copolymers to selectively target and modulate DCs by transporting the anti-inflammatory agent 1, 25-Dihydroxyvitamin D3 (aVD) and ApoB-100 derived antigenic peptide P210. Polymersomes decorated with an optimized surface display and density for a lipid construct of the P-D2 peptide, which binds CD11c on the DC surface, significantly enhanced the cytosolic delivery and resulting immunomodulatory capacity of aVD in vitro. Intravenous administration of the optimized polymersomes achieved selective targeting of DCs in atheroma and spleen compared to all other cell populations, including both immune and CD45(−) cells, and locally increased the presence of tolerogenic DCs and cytokines. aVD-loaded polymersomes significantly inhibited atherosclerotic lesion development in high fat diet-fed ApoE(−/−) mice following 8 weeks of administration. Incorporation of the P210 peptide generated the largest reductions in vascular lesion area (~33%, p

Upon exposure to blood, a corona of proteins adsorbs to nanocarrier surfaces to confer a biological identity that interfaces with the immune system. While the nanocarrier surface chemistry has long been the focus of protein corona formation, the influence of nanostructure has remained unclear despite established influences on biodistribution, clearance, and inflammation. Here, combinations of nanocarrier morphology and surface chemistry are engineered to i) achieve compositionally distinct protein coatings in human blood and ii) control protein-mediated interactions with the immune system. A library of nine PEGylated nanocarriers differing in their combination of morphology (spheres, vesicles, and cylinders) and surface chemistry (methoxy, hydroxyl, and phosphate) are synthesized to represent properties of therapeutic and biomimetic delivery vehicles. Analysis by quantitative label-free proteomic techniques reveal that specific surface chemistry and morphology combinations adsorb unique protein signatures from human blood, resulting in differential complement activation and elicitation of distinct proinflammatory cytokine responses. Furthermore, nanocarrier morphology is shown to primarily influence uptake and clearance by human monocytes, macrophages, and dendritic cells. This comprehensive analysis provides mechanistic insights into rational design choices that impact the immunological identity of nanocarriers in human blood, which can be leveraged to engineer drug delivery vehicles for precision medicine and immunotherapy.

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.

Oral rapamycin administration rapamycin is plagued by poor bioavailability and wide biodistribution. Thus, this pleotropic mTOR inhibitor has a narrow therapeutic window, numerous side effects and provides inadequate transplantation protection. Parental formulation was not possible due to rapamycin’s hydrophobicity (log P 4.3). Here, we demonstrate that subcutaneous rapamycin delivery via poly(ethylene glycol)-b-poly(propylene sulfide)(PEG-b-PPS) polymersome (PS) nanocarriers modulates cellular biodistribution of rapamycin to change its immunosuppressive mechanism for enhanced efficacy while minimizing side effects. While oral rapamycin inhibits naïve T cell proliferation directly, subcutaneously administered rapamycin-loaded polymersomes (rPS) instead modulated Ly-6Clow monocytes and tolerogenic semi-mature dendritic cells, with immunosuppression mediated by CD8+ Tregs and rare CD4+ CD8+ double-positive T cells. As PEG-b-PPS PS are uniquely non-inflammatory, background immunostimulation from the vehicle was avoided, allowing immunomodulation to be primarily attributed to rapamycin’s cellular biodistribution. Repurposing mTOR inhibition significantly improved maintenance of normoglycemia in a clinically relevant, MHC-mismatched, allogeneic, intraportal (liver) islet transplantation model. These results demonstrate the ability of engineered nanocarriers to repurpose drugs for alternate routes of administration by rationally controlling cellular biodistribution.

Standard oral rapamycin (that is, Rapamune) administration is plagued by poor bioavailability and broad biodistribution. Thus, this pleotropic mammalian target of rapamycin (mTOR) inhibitor has a narrow therapeutic window and numerous side effects and provides inadequate protection to transplanted cells and tissues. Furthermore, the hydrophobicity of rapamycin limits its use in parenteral formulations. Here, we demonstrate that subcutaneous delivery via poly(ethylene glycol)-b-poly(propylene sulfide) polymersome nanocarriers significantly alters rapamycin's cellular biodistribution to repurpose its mechanism of action for tolerance, instead of immunosuppression, and minimize side effects. While oral rapamycin inhibits T cell proliferation directly, subcutaneously administered rapamycin-loaded polymersomes modulate antigen presenting cells in lieu of T cells, significantly improving maintenance of normoglycemia in a clinically relevant, major histocompatibility complex-mismatched, allogeneic, intraportal (liver) islet transplantation model. These results demonstrate the ability of a rationally designed nanocarrier to re-engineer the immunosuppressive mechanism of a drug by controlling cellular biodistribution.

Oral rapamycin administration rapamycin is plagued by poor bioavailability and wide biodistribution. Thus, this pleotropic mTOR inhibitor has a narrow therapeutic window, numerous side effects and provides inadequate transplantation protection. Parental formulation was not possible due to rapamycin’s hydrophobicity (log P 4.3). Here, we demonstrate that subcutaneous rapamycin delivery via poly(ethylene glycol)-b-poly(propylene sulfide)(PEG-b-PPS) polymersome (PS) nanocarriers modulates cellular biodistribution of rapamycin to change its immunosuppressive mechanism for enhanced efficacy while minimizing side effects. While oral rapamycin inhibits naïve T cell proliferation directly, subcutaneously administered rapamycin-loaded polymersomes (rPS) instead modulated Ly-6Clow monocytes and tolerogenic semi-mature dendritic cells, with immunosuppression mediated by CD8+ Tregs and rare CD4+ CD8+ double-positive T cells. As PEG-b-PPS PS are uniquely non-inflammatory, background immunostimulation from the vehicle was avoided, allowing immunomodulation to be primarily attributed to rapamycin’s cellular biodistribution. Repurposing mTOR inhibition significantly improved maintenance of normoglycemia in a clinically relevant, MHC-mismatched, allogeneic, intraportal (liver) islet transplantation model. These results demonstrate the ability of engineered nanocarriers to repurpose drugs for alternate routes of administration by rationally controlling cellular biodistribution.

Following intravenous administration, an adsorbed corona of blood proteins immediately forms on the surfaces of nanocarriers to confer a distinct biological identity that dictates interactions with the immune system. While the nanocarrier surface chemistry has long been the focus of protein corona formation, the influence of the nanocarrier structure has remained unclear despite well-documented influences on biodistribution, clearance and inflammation. Here, we present design rules for the combined engineering of both nanocarrier structure and surface chemistry derived from a comprehensive proteomic analysis of protein corona formation in human blood. A library of nine soft PEGylated nanocarriers that differ in their combination of morphology (spheres, vesicles, and cylinders) and surface chemistry (methoxy, hydroxyl, and phosphate) were synthesized to represent properties of commonly employed drug delivery vehicles. Using label-free proteomics and high-throughput techniques, we examined the relationship between physicochemical properties and the resulting nanocarrier biological identity, including dynamic changes in protein corona composition, differential immunostimulation and uptake by relevant immune cell populations. In human blood, non-polar spherical micelles developed a similar biological identity to polar vesicles, whereas the identities of polar spheres and cylinders resembled that of non-polar vesicles. The formed protein coronas were compositionally dynamic and morphology-dependent, and these time-dependent fingerprints altered nanocarrier complement activation as well as their uptake by human monocytes, macrophages, and dendritic cells. This comprehensive analysis provides mechanistic insights into rational design choices that impact nanocarrier fate in human blood.One Sentence SummaryWe demonstrate that not only the surface chemistry, but the combined chemical and structural properties of soft drug delivery vehicles impact the composition of blood proteins that adsorb to their surfaces, and these differences specify their interactions with and modulation of human immune cells.

Vesicular nanocarrier formulations confer the ability to deliver hydrophobic and hydrophilic cargos simultaneously to cells of interest in vivo. While liposomal formulations reached the clinic long ago, younger technologies such as polymeric vesicles (polymersomes) have yet to make the transition to clinical approval and use, in part due to difficulties in ensuring their safe and scalable production. In this work, we demonstrate the scalable production of poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-bl-PPS) polymersomes via flash nanoprecipitation, and further show the safe administration of these nanocarriers to mice and non-human primates. In mice, PEG-bl-PPS polymersomes were found to be well tolerated at up to 200 mg/(kg·week). Following the administration of a more relevant 20 mg/(kg·week) dosage in non-human primates, polymersomes were found to associate with numerous phagocytic immune cell populations, including a remarkable 68% of plasmacytoid dendritic cells and > 95% of macrophages in the spleen, while showing no toxicity or abnormalities in the liver, kidney, spleen, or blood. Despite the presence of a dense PEG corona, neither anti-PEG antibodies nor complement activation were detected. This work provides evidence of the translatability of PEG-bl-PPS polymersomes into the clinic for therapeutic applications in humans.

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., Nanocarrier administration is often performed via intermittent bolus injection while sustained delivery platforms are rarely reported. Here the authors demonstrate that the cylinder-to-sphere transitions of self-assembled filomicelle scaffolds can be used for sustained delivery with improved resorptive capacity and biocompatibility.

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 amphi-philic 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) (PEG(17)-bl-PPS(75)), 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 oval-bumin 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.

B-cell lymphoma cells depend upon cholesterol to maintain pro-proliferation and pro-survival signaling via the B-cell receptor. Targeted cholesterol depletion of lymphoma cells is an attractive therapeutic strategy. We report here high-density lipoprotein mimicking magnetic nanostructures (HDL-MNSs) that can bind to the high-affinity HDL receptor, scavenger receptor type B1 (SR-B1), and interfere with cholesterol flux mechanisms in SR-B1 receptor positive lymphoma cells, causing cellular cholesterol depletion. In addition, the MNS core can be utilized for its ability to generate heat under an external radio frequency field. The thermal activation of MNS can lead to both innate and adaptive antitumor immune responses by inducing the expression of heat shock proteins that lead to activation of antigen presenting cells and finally lymphocyte trafficking. In the present study, we demonstrate SR-B1 receptor mediated binding and cellular uptake of HDL-MNS and prevention of phagolysosome formation by transmission electron microscopy, fluorescence microscopy, and ICP-MS analysis. The combinational therapeutics of cholesterol depletion and thermal activation significantly improves therapeutic efficacy in SR-B1 expressing lymphoma cells. HDL-MNS reduces the T2 relaxation time under magnetic resonance imaging (MRI) more effectively compared with a commercially available contrast agent, and the specificity of HDL-MNS toward the SR-B1 receptor leads to differential contrast between SR-B1 positive and negative cells suggesting its utility in diagnostic imaging. Overall, we have demonstrated that HDL-MNSs have cell specific targeting efficiency, can modulate cholesterol efflux, can induce thermal activation mediated antitumor immune response, and possess high contrast under MRI, making it a promising theranostic platform in lymphoma.

Prompted by a knowledge of the photoprotective mechanism operating in photosystem supercomplexes and bacterial antenna complexes by pigment binding proteins, we have appealed to a boxlike synthetic receptor (ExBox·4Cl) that binds a photosensitizer, 5,15-diphenylporphyrin (DPP), to provide photoprotection by regulating light energy. The hydrophilic ExBox4+ renders DPP soluble in water and modulates the phototoxicity of DPP by trapping it in its cavity and releasing it when required. While trapping removes access to the DPP triplet state, a pH-dependent release of diprotonated DPP (DPPH22+) restores the triplet deactivation pathway, thereby activating its ability to generate reactive oxygen species. We have employed the ExBox4+-bound DPP complex (ExBox4+⊃DPP) for the safe delivery of DPP into the lysosomes of cancer cells, imaging the cells by utilizing the fluorescence of the released DPPH22+ and regulating photodynamic therapy to kill cancer cells with high efficiency.

In this work, the hydrophobic small molecule NF-κB inhibitor celastrol was loaded into poly(ethylene glycol)-b-poly(propylene sulfide) (PEG-b-PPS) micelles. PEG-b-PPS micelles demonstrated high loading efficiency, low polydispersity, and no morphological changes upon loading with celastrol. Encapsulation of celastrol within these nanocarriers significantly reduced cytotoxicity compared to free celastrol, while simultaneously expanding the lower concentration range for effective inhibition of NF-κB signaling by nearly 50,000-fold. Furthermore, celastrol-loaded micelles successfully reduced TNF-α secretion after LPS stimulation of RAW 264.7 cells and reduced the number of neutrophils and inflammatory monocytes within atherosclerotic plaques of ldlr(−/−) mice. This reduction in inflammatory cells was matched by a reduction in plaque area, suggesting that celastrol-loaded nanocarriers may serve as an anti-inflammatory treatment for atherosclerosis.

Introduction: Atherosclerosis is a multi-stage disorder with an inflammatory immune component. We hypothesized that the targeted delivery of an anti-inflammatory small molecule to macrophages could modulate immune cells in the plaque and potentially reduce plaque progression. We employed celastrol as an inhibitor of NF-kB and used poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-bl-PPS) polymeric micelles (MCs) for controlled delivery. Methods: PEG-bl-PPS polymer was formulated into nanocarriers at a concentration of 15 mg/mL in 1xPBS, with or without 100 ng of celastrol (Cel-MC or Blank MC). Ldlr-/- mice were fed a high fat diet for 3 months prior to the beginning of nanocarrier treatment. 100 μL of each MC formulation was administered to mice via tail vein injection once per week for 12 weeks. Results: Cel-MC inhibited NF-kB at lower concentrations compared to free celastrol (Fig. 1a) . In vivo , Cel-MCs significantly decreased neutrophils and NK cells in the aortas of atherosclerotic mice compared to blank MC controls (Fig. 1b) . Cel-MCs reduced the plaque lesion area, as determined by Oil Red O staining of aorta histology sections (Fig. 1c) . Figure 1. Cel-MC treatment of atherosclerosis. (a) NF-kB inhibition and cell viability using RAW Blue cells, error bars = S.D., n = 4. (b) Heatmap of percent change in cell populations based on flow analysis of ldlr-/- mice after 12 weeks of Cel-MC treatment, compared to blank MC treatment, n=6/4 respectively. (c) Oil Red O staining fluorescence area of aorta histology slides, n=4 mice, 3-4 sections per mouse. P-value determined using Welch’s t test. Conclusions: Cel-MC reduced NF-kB signaling in vitro and reduced immune cells in the aorta in vivo. The treatment course was well-tolerated by the mice and resulted in a significant reduction in plaque lesion area. These results demonstrate both the utility of targeted modulation of immune cells contributing to vascular inflammation.

Checkpoint blocking antibodies that interfere in the PD-1/PD-L1 axis provide effective cancer immunotherapy for tumors that are immune inflamed or induced to become “hot”. It has also been demonstrated that a small molecule inhibitor of the signaling hub kinase GSK3 can interfere in the PD-1/PD-L1 axis in T-cells by suppressing PD-1 expression. This provides an alternative approach to intervening in the PD-1/PD-L1 axis to provide cancer immunotherapy. In this communication, we demonstrate the remote loading of GSK3 inhibitor AZD1080 into the porous interior of mesoporous silica nanoparticles coated with a lipid bilayer (a.k.a. silicasomes). In a MC38 colon cancer model, intravenous injection (IV) of silicasome-encapsulated AZD1080 significantly improved biodistribution and drug delivery to the tumor site. The improved drug delivery was accompanied by cytotoxic MC38 tumor cell killing by perforin-releasing CD8(+) T-cells, exhibiting reduced PD-1 expression. IV injection of encapsulated AZD1080 also resulted in significant tumor shrinkage in other syngeneic mouse tumor models, including another colorectal tumor (CT26), as well as pancreas (KPC) and lung (LLC) cancer models. Not only was the therapeutic efficacy of encapsulated AZD1080 similar or better than anti-PD-1 antibody, but the treatment was devoid of treatment toxicity. These results provide proof-of-principal demonstration of the feasibility of using encapsulated delivery of a GSK3 inhibitor to provide cancer immunotherapy, with the possibility to be used as a monotherapy or in combination with chemotherapy or other immunomodulatory agents.

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.

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.

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

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.

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.

Atherosclerosis is a chronic vascular inflammatory disease, in which several types of immune cells have been identified as playing important roles. Nanomaterials can function as powerful theranostic platforms for diagnostic imaging and controlled delivery of therapeutics in atherosclerosis. Here, we present a detailed investigation into the effects of morphology on the in vivo biodistribution of nanomaterials in naïve mice following intravenous injection. We applied these findings towards the targeting of diverse immune cells within the lesions of atherosclerotic mice. Three different nanostructures of the same surface chemistry were assembled from poly(ethylene glycol)- bl -poly(propylene sulfide) (PEG- bl -PPS) block copolymers: micelles (30 nm), vesicles (120 nm) and filomicelles (50 nm diameter by micron length). To assess the effects of the different morphologies, a multimodal approach was utilized that included 1) near infrared fluorescence (NIRF) imaging to quantify organ targeting, and 2) fluorescent polymer conjugation for subsequent flow cytometric analysis of uptake by immune cells. Of note, vesicles were exceptionally efficient at targeting the spleen and were associated with up to 85% of plasmacytoid dendritic cells. Micelles were associated with up to 90% of macrophages in the liver, and filomicelles were most effective at avoiding uptake by the cells of the mononuclear phagocyte system. Due to their enhanced uptake by dendritic cell subsets relative to other nanostructures, vesicles were selected for targeting cells within aortic lesions of atherosclerotic LDL -/- mice. In addition to associating with macrophages and eosinophils, vesicles were found to target significantly higher percentages of atheroma-resident dendritic cells (25%). In conclusion, differences in morphology can drastically change the biodistribution of nanomaterials at both the organ and cellular level. The ability to target or avoid phagocytic cell subsets will enhance current and future theranostic strategies. Furthermore, the targeting of dendritic cells by vesicular nanostructures within atherosclerotic lesions opens new avenues for immunotherapies in cardiovascular disease.