A Biomimetic Multi-Component Subunit Vaccine via Ratiometric Loading of Hierarchical Hydrogels.

The development of subunit vaccines that mimic the molecular complexity of attenuated vaccines has been limited by the difficulty of intracellular co-delivery of multiple chemically diverse payloads at controllable concentrations. We report on hierarchical hydrogel depots employing simple poly(propylene sulfone) homopolymers to enable ratiometric loading of a protein antigen and four physicochemically distinct adjuvants in a hierarchical manner. The optimized vaccine consisted of immunostimulants either adsorbed to or encapsulated within nanogels, which were capable of noncovalent anchoring to subcutaneous tissues. These 5-component nanogel vaccines demonstrated enhanced humoral and cell-mediated immune responses compared to formulations with standard single adjuvant and antigen pairing. The use of a single simple homopolymer capable of rapid and stable loading and intracellular delivery of diverse molecular cargoes holds promise for facile development and optimization of scalable subunit vaccines and complex therapeutic formulations for a wide range of biomedical applications.

Controlled adsorption of multiple bioactive proteins enables targeted mast cell nanotherapy.

Protein adsorption onto nanomaterials often results in denaturation and loss of bioactivity. Controlling the adsorption process to maintain the protein structure and function has potential for a range of applications. Here we report that self-assembled poly(propylene sulfone) (PPSU) nanoparticles support the controlled formation of multicomponent enzyme and antibody coatings and maintain their bioactivity. Simulations indicate that hydrophobic patches on protein surfaces induce a site-specific dipole relaxation of PPSU assemblies to non-covalently anchor the proteins without disrupting the protein hydrogen bonding or structure. As a proof of concept, a nanotherapy employing multiple mast-cell-targeted antibodies for preventing anaphylaxis is demonstrated in a humanized mouse model. PPSU nanoparticles displaying an optimized ratio of co-adsorbed anti-Siglec-6 and anti-FcεRIα antibodies effectively inhibit mast cell activation and degranulation, preventing anaphylaxis. Protein immobilization on PPSU surfaces provides a simple and rapid platform for the development of targeted protein nanomedicines.

Rational Engineering of Islet Tolerance via Biomaterial-Mediated Immune Modulation.

Type 1 diabetes (T1D) onset is characterized by an autoimmune attack on ß islet cells within the pancreas, preventing the insulin secretion required to maintain glucose homeostasis. Targeted modulation of key immunoregulatory cell populations is a promising strategy to restore tolerance to ß cells. This strategy can be used to prevent T1D onset or reverse T1D with transplanted islets. To this end, drug delivery systems can be employed to transport immunomodulatory cargo to specific cell populations that inhibit autoreactive T cell-mediated destruction of the ß cell mass. The rational engineering of biomaterials into nanoscale and microscale drug carriers can facilitate targeted interactions with immune cells. The physicochemical properties of the biomaterial, the delivered immunomodulatory agent, and the target cell populations are critical variables in the design of these delivery systems. In this review, we discuss recent biomaterials-based drug delivery approaches to induce islet tolerance and the need to consider both immune and metabolic markers of disease progression.

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

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

Engineering endosomolytic nanocarriers of diverse morphologies using confined impingement jet mixing.

The clinical translation of many biomolecular therapeutics has been hindered by undesirable pharmacokinetic (PK) properties, inadequate membrane permeability, poor endosomal escape and cytosolic delivery, and/or susceptibility to degradation. Overcoming these challenges merits the development of nanoscale drug carriers (nanocarriers) to improve the delivery of therapeutic cargo. Herein, we implement a flash nanoprecipitation (FNP) approach to produce nanocarriers of diverse vesicular morphologies by using various molecular weight PEG-bl-DEAEMA-co-BMA (PEG-DB) polymers. We demonstrated that FNP can produce uniform (PDI < 0.1) particles after 5 impingements, and that by varying the copolymer hydrophilic mass fraction, FNP enables access to a diverse variety of nanoarchitectures including micelles, unilamellar vesicles (polymersomes), and multi-compartment vesicles (MCVs). We synthesized a library of 2 kDa PEG block copolymers, with DEAEMA-co-BMA second block molecular weights of 3, 6, 12, 15, 20, and 30 kDa. All formulations were both pH responsive, endosomolytic, and capable of loading and cytosolically delivering small negatively charged molecules - albeit to different degrees. Using a B16.F10 melanoma model, we showcased the therapeutic potential of a lead FNP formulated PEG-DB nanocarrier, encapsulating the cyclic dinucleotide (CDN) cGAMP to activate the stimulator of interferon genes (STING) pathway in a therapeutically relevant context. Collectively, these data demonstrate that an FNP process can be used to formulate pH-responsive nanocarriers of diverse morphologies using a PEG-DB polymer system. As FNP is an industrially scalable process, these data address the critical translational challenge of producing PEG-DB nanoparticles at scale. Furthermore, the diverse morphologies produced may specialize in the delivery of distinct biomolecular cargos for other therapeutic applications, implicating the therapeutic potential of this platform in an array of disease applications.

Nanomedicine in kidney disease.

PURPOSE OF REVIEW: The pathophysiological understanding of kidney-related disorders has profoundly increased; however, tissue-specific and cell-specific treatments in this field remain scarce. Advances in nanomedicine enable alteration of pharmacokinetics and targeted treatments improving efficiency and reducing toxicity. This review addresses recent developments of nanocarriers used for various purposes in the broad field of kidney disease, which may pave a path to new therapeutic and diagnostic solutions employing nanomedicine. RECENT FINDINGS: Controlled delivery of antiproliferative medications enables improved treatment of polycystic kidney disease and fibrosis. Directed anti-inflammatory treatment mitigated glomerulonephritis and tubulointerstitial nephritis. Multiple injury pathways in AKI have been targeted, with therapeutic solutions for oxidative stress, mitochondrial dysfunction, local inflammation and improving self-repair mechanisms. In addition to such treatment development, noninvasive early detection methods (minutes after ischemic insult) have been demonstrated as well. Sustained release of therapies that reduce ischemia-reperfusion injury as well as new aspects for immunosuppression bring hope to improving kidney transplant outcomes. The latest breakthroughs in gene therapy are made achievable by engineering the targeted delivery of nucleic acids for new treatments of kidney disease. SUMMARY: Recent advances in nanotechnology and pathophysiological understanding of kidney diseases show potential for translatable therapeutic and diagnostic interventions in multiple etiologies of kidney disease.

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

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

Drug delivery targets and strategies to address mast cell diseases.

INTRODUCTION: Current and developing mast cell therapeutics are reliant on small molecule drugs and biologics, but few are truly selective for mast cells. Most have cellular and disease-specific limitations that require innovation to overcome longstanding challenges to selectively targeting and modulating mast cell behavior. This review is designed to serve as a frame of reference for new approaches that utilize nanotechnology or combine different drugs to increase mast cell selectivity and therapeutic efficacy. AREAS COVERED: Mast cell diseases include allergy and related conditions as well as malignancies. Here, we discuss the targets of existing and developing therapies used to treat these disease pathologies, classifying them into cell surface, intracellular, and extracellular categories. For each target discussed, we discuss drugs that are either the current standard of care, under development, or have indications for potential use. Finally, we discuss how novel technologies and tools can be used to take existing therapeutics to a new level of selectivity and potency against mast cells. EXPERT OPINION: There are many broadly and very few selectively targeted therapeutics for mast cells in allergy and malignant disease. Combining existing targeting strategies with technology like nanoparticles will provide novel platforms to treat mast cell disease more selectively.

In vitro assessment of varying peptide surface density on the suppression of angiogenesis by micelles displaying αvß3 blocking peptides.

Ligand targeted therapy (LTT) is a precision medicine strategy that can selectively target diseased cells while minimizing off-target effects on healthy cells. Integrin-targeted LTT has been developed recently for angiogenesis-related diseases. However, the clinical success is based on the optimal design of the nanoparticles for inducing receptor clustering within the cell membrane. The current study focused on determining the surface density of Ser-Asp-Val containing anti-integrin heptapeptide on poly (ethylene glycol)-b-poly(propylene sulfide) micelles (MC) required for anti-angiogenic effects on HUVECs. Varying peptide density on PEG-b-PPS/Pep-PA MCs (Pep-PA-Peptide-palmitoleic acid) was used in comparison to a random peptide (SGV) and cRGD (cyclic-Arginine-Glycine-Aspartic acid) construct at 5%-density on MCs. Immunocytochemistry using CD51/CD31 antibody was performed to study the integrin blocking by MCs. In addition, the expression of VWF and PECAM-1, cell migration and tube formation was evaluated in the presence of PEG-b-PPS/Pep-PA MCs. The results show PEG-b-PPS/SDV-PA MCs with 5%-peptide density to achieve significantly higher αvß3 blocking compared to random peptide as well as cRGD. In addition, αvß3 blocking via MCs further reduced the expression of vWF and PECAM-1 angiogenesis protein expression in HUVECs. Although a significant level of integrin blocking was observed for 1%-peptide density on MCs, the cell migration and tube formation were not significantly affected. In conclusion, the results of this study demonstrate that the peptide surface density on PEG-b-PPS/Pep-PA MCs has a significant impact in integrin blocking as well as inhibiting angiogenesis during LTT. The outcomes of this study provides insight into the design of ligand targeted nanocarriers for various disease conditions.

Leishmaniasis and Chagas disease: Is there hope in nanotechnology to fight neglected tropical diseases?

Nanotechnology is revolutionizing many sectors of science, from food preservation to healthcare to energy applications. Since 1995, when the first nanomedicines started being commercialized, drug developers have relied on nanotechnology to improve the pharmacokinetic properties of bioactive molecules. The development of advanced nanomaterials has greatly enhanced drug discovery through improved pharmacotherapeutic effects and reduction of toxicity and side effects. Therefore, highly toxic treatments such as cancer chemotherapy, have benefited from nanotechnology. Considering the toxicity of the few therapeutic options to treat neglected tropical diseases, such as leishmaniasis and Chagas disease, nanotechnology has also been explored as a potential innovation to treat these diseases. However, despite the significant research progress over the years, the benefits of nanotechnology for both diseases are still limited to preliminary animal studies, raising the question about the clinical utility of nanomedicines in this field. From this perspective, this review aims to discuss recent nanotechnological developments, the advantages of nanoformulations over current leishmanicidal and trypanocidal drugs, limitations of nano-based drugs, and research gaps that still must be filled to make these novel drug delivery systems a reality for leishmaniasis and Chagas disease treatment.

Scalable Generation of Multiphoton Entangled States by Active Feed-Forward and Multiplexing.

Multiphoton entangled quantum states are key to advancing quantum technologies such as multiparty quantum communications, quantum sensing, or quantum computation. Their scalable generation, however, remains an experimental challenge. Current methods for generating these states rely on stitching together photons from probabilistic sources, and state generation rates drop exponentially in the number of photons. Here, we implement a system based on active feed-forward and multiplexing that addresses this challenge. We demonstrate the scalable generation of four-photon and six-photon Greenberger-Horne-Zeilinger states, increasing generation rates by factors of 9 and 35, respectively. This is consistent with the exponential enhancement compared to the standard nonmultiplexed approach that is predicted by our theory. These results facilitate the realization of practical multiphoton protocols for photonic quantum technologies.

Shaping Neonatal Immunization by Tuning the Delivery of Synergistic Adjuvants via Nanocarriers.

Adjuvanted nanocarrier-based vaccines hold substantial potential for applications in novel early-life immunization strategies. Here, via mouse and human age-specific in vitro modeling, we identified the combination of a small-molecule STING agonist (2'3'-cyclic GMP-AMP, cGAMP) and a TLR7/8 agonist (CL075) to drive the synergistic activation of neonatal dendritic cells and precision CD4 T-helper (Th) cell expansion via the IL-12/IFNγ axis. We further demonstrate that the vaccination of neonatal mice with quadrivalent influenza recombinant hemagglutinin (rHA) and an admixture of two polymersome (PS) nanocarriers separately encapsulating cGAMP (cGAMP-PS) and CL075 (CL075-PS) drove robust Th1 bias, high frequency of T follicular helper (TFH) cells, and germinal center (GC) B cells along with the IgG2c-skewed humoral response in vivo. Dual-loaded cGAMP/CL075-PSs did not outperform admixed cGAMP-PS and CL075-PS in vivo. These data validate an optimally designed adjuvantation system via age-selected small-molecule synergy and a multicomponent nanocarrier formulation as an effective approach to induce type 1 immune responses in early life.

Dendritic peptide-conjugated polymeric nanovectors for non-toxic delivery of plasmid DNA and enhanced non-viral transfection of immune cells.

Plasmid DNA (pDNA) transfection is advantageous for gene therapies requiring larger genetic elements, including "all-in-one" CRISPR/Cas9 plasmids, but is limited by toxicity as well as poor intracellular release and transfection efficiency in immune cell populations. Here, we developed a synthetic non-viral gene delivery platform composed of poly(ethylene glycol)-b-poly(propylene sulfide) copolymers linked to a cationic dendritic peptide (DP) via a reduceable bond, PEG-b-PPS-ss-DP (PPDP). A library of self-assembling PPDP polymers was synthesized and screened to identify optimal constructs capable of transfecting macrophages with small (pCMV-DsRed, 4.6 kb) and large (pL-CRISPR.EFS.tRFP, 11.7 kb) plasmids. The optimized PPDP construct transfected macrophages, fibroblasts, dendritic cells, and T cells more efficiently and with less toxicity than a commercial Lipo2K reagent, regardless of pDNA size and under standard culture conditions in the presence of serum. The PPDP technology described herein is a stimuli-responsive polymeric nanovector that can be leveraged to meet diverse challenges in gene delivery.

Leveraging self-assembled nanobiomaterials for improved cancer immunotherapy.

Nanomaterials and targeted drug delivery vehicles improve the therapeutic index of drugs and permit greater control over their pharmacokinetics, biodistribution, and bioavailability. Here, nanotechnologies applied to cancer immunotherapy are discussed with a focus on current and next generation self-assembling drug delivery systems composed of lipids and/or polymers. Topics covered include the fundamental design, suitability, and inherent properties of nanomaterials that induce anti-tumor immune responses and support anti-cancer vaccination. Established active and passive targeting strategies as well as newer "indirect" methods are presented together with insights into how nanocarrier structure and surface chemistry can be leveraged for controlled delivery to the tumor microenvironment while minimizing off-target effects.

Nanotherapeutics in transplantation: How do we get to clinical implementation?

Patients undergoing organ transplantation transition from one life-altering issue (organ dysfunction) to a lifelong commitment-immunosuppression. Regimens of immunosuppressive agents (ISAs) come with significant side effects and comorbidities. Recently, the use of nanoparticles (NPs) as a solution to the problems associated with the long-term and systemic use of ISAs in transplantation has emerged. This minireview describes the role of NPs in organ transplantation and discusses obstacles to clinical implementation and pathways to clinical translation.

Subcutaneous nanotherapy repurposes the immunosuppressive mechanism of rapamycin to enhance allogeneic islet graft viability.

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.

Clinical Relevance of Pre-Existing and Treatment-Induced Anti-Poly(Ethylene Glycol) Antibodies.

Abstract: Poly(ethylene glycol) (PEG) is a nontoxic, hydrophilic polymer that is often covalently attached to proteins, drugs, tissues, or materials; a procedure commonly referred to as PEGylation. PEGylation improves solubility, circulation time, and reduces immunogenicity of therapeutic molecules. Currently, there are 21 PEGylated drugs approved by the Food and Drug Administration (FDA), and more in the developmental stage. In addition to the polymer's applications in the clinic, PEG is widely used as a solvent and emulsifying agent in the formulation of cosmetics, cleaning, and personal care products. Due to the ubiquitous presence of the polymer in everyday products, patients can develop antibodies against PEG (αPEG Abs) that can be problematic when a PEGylated drug is administered. These αPEG Abs can provoke hypersensitivity reactions, accelerated drug clearance, and decreased therapeutic efficacy. Herein, we review how the prevalence of PEG in everyday products has induced αPEG Abs within the general public as well as the effect of these Abs on the performance of PEGylated therapeutics. We will focus on clinical manifestations following the administration of PEGylated drugs. Lay Summary: Poly(ethylene glycol) (PEG) is a polymer found in products including cosmetics, personal care products, cleaning agents, medicine, and food. Due to the prevalence of PEG, people can develop antibodies (αPEG Abs) against the polymer, which recognize PEG as foreign. Of note, PEG is frequently incorporated into drug formulations to improve therapeutic efficacy. Complications can arise when a patient receiving a PEGylated drug has previously developed αPEG Abs from interactions with PEG in everyday products. The presence of high concentrations of αPEG Abs in blood can result in decreased treatment efficacy and allergic reactions to a wide range of therapeutics.