A genetically engineered neuronal membrane-based nanotoxoid elicits protective immunity against neurotoxins.

Given their dangerous effects on the nervous system, neurotoxins represent a significant threat to public health. Various therapeutic approaches, including chelating agents, receptor decoys, and toxin-neutralizing antibodies, have been explored. While prophylactic vaccines are desirable, it is oftentimes difficult to effectively balance their safety and efficacy given the highly dangerous nature of neurotoxins. To address this, we report here on a nanovaccine against neurotoxins that leverages the detoxifying properties of cell membrane-coated nanoparticles. A genetically modified cell line with constitutive overexpression of the α7 nicotinic acetylcholine receptor is developed as a membrane source to generate biomimetic nanoparticles that can effectively and irreversibly bind to α-bungarotoxin, a model neurotoxin. This abrogates the biological activity of the toxin, enabling the resulting nanotoxoid to be safely delivered into the body and processed by the immune system. When co-administered with an immunological adjuvant, a strong humoral response against α-bungarotoxin is generated that protects vaccinated mice against a lethal dose of the toxin. Overall, this work highlights the potential of using genetic modification strategies to develop nanotoxoid formulations against various biological threats.

Platelet Membrane-Derived Nanodiscs for Neutralization of Endogenous Autoantibodies and Exogenous Virulence Factors.

The multifaceted functions of platelets in various physiological processes have long inspired the development of therapeutic nanoparticles that mimic specific platelet features for disease treatment. Here, the development and characterization of platelet membrane-derived nanodiscs (PLT-NDs) as platelet decoys for biological neutralization is reported. In one application, PLT-NDs effectively bind with anti-platelet autoantibodies, thus blocking them from interacting with platelets. In a mouse model of thrombocytopenia, PLT-NDs successfully neutralize pathological anti-platelet antibodies, preventing platelet depletion and maintaining hemostasis. In another application, PLT-NDs effectively neutralize the cytotoxicity of bacterial virulence factors secreted by methicillin-resistant Staphylococcus aureus (MRSA). In a mouse model of MRSA infection, treatment with PLT-NDs leads to significant survival benefits for the infected mice. Additionally, PLT-NDs show good biocompatibility and biosafety, as demonstrated in acute toxicity studies conducted in mice. These findings underscore the potential of PLT-NDs as a promising platelet mimicry for neutralizing various biological agents that target platelets. Overall, this work expands the repertoire of platelet-mimicking nanomedicine by creating a unique disc-like nanostructure made of natural platelet membranes.

Glycan-modified cellular nanosponges for enhanced neutralization of botulinum toxin.

Botulinum toxin (BoNT) is a potent neurotoxin that poses a significant threat as a biowarfare weapon and a potential bioterrorist tool. Currently, there is a lack of effective countermeasures to combat BoNT intoxication in the event of a biological attack. Here, we report on a novel solution by combining cell metabolic engineering with cell membrane coating nanotechnology, resulting in the development of glycan-modified cellular nanosponges that serve as a biomimetic and broad-spectrum BoNT detoxification strategy. Specifically, we increase the expression levels of gangliosides on THP-1 cells through metabolic engineering, and then collect the modified THP-1 cell membrane and coat it onto synthetic polymeric cores, creating cellular nanosponges that closely mimic host cells. Our findings demonstrate that higher levels of gangliosides on the cellular nanosponges result in greater binding capacities with BoNT. The glycan-modified cellular nanosponges exhibit superior efficacy in neutralizing BoNT cytotoxicity in vitro when compared to their unmodified counterparts. In a mouse model of BoNT intoxication, the glycan-modified cellular nanosponges show more pronounced survival benefits when administered both as a treatment and a preventative regimen. These results highlight the potential of cellular nanosponges, especially when modified with glycans, as a promising countermeasure platform against BoNT and related clostridial toxins.

Cancer Cell Membrane Nanodiscs for Antitumor Vaccination.

Cell membrane-based nanovaccines have demonstrated attractive features due to their inherently multiantigenic nature and ability to be formulated with adjuvants. Here, we report on cellular nanodiscs fabricated from cancer cell membranes and incorporated with a lipid-based adjuvant for antitumor vaccination. The cellular nanodiscs, with their small size and discoidal shape, are readily taken up by antigen-presenting cells and drain efficiently to the lymph nodes. Due to its highly immunostimulatory properties, the nanodisc vaccine effectively stimulates the immune system and promotes tumor-specific immunity. Using a murine colorectal cancer model, strong control of tumor growth is achieved in both prophylactic and therapeutic settings, particularly in combination with checkpoint blockades. Considerable therapeutic efficacy is also observed in treating a weakly immunogenic metastatic melanoma model. This work presents a new paradigm for the design of multiantigenic nanovaccines that can effectively activate antitumor immune responses and may be applicable to a wide range of cancers.

Immunostimulatory DNA Hydrogel Enhances Protective Efficacy of Nanotoxoids against Bacterial Infection.

While vaccines have been highly successful in protecting against various infections, there are still many high-priority pathogens for which there are no clinically approved formulations. To overcome this challenge, researchers have explored the use of nanoparticulate strategies for more effective antigen delivery to the immune system. Along these lines, nanotoxoids are a promising biomimetic platform that leverages cell membrane coating technology to safely deliver otherwise toxic bacterial antigens in their native form for antivirulence vaccination. Here, in order to further boost their immunogenicity, nanotoxoids formulated against staphylococcal α-hemolysin are embedded into a DNA-based hydrogel with immunostimulatory CpG motifs. The resulting nanoparticle-hydrogel composite is injectable and improves the in vivo delivery of vaccine antigens while simultaneously stimulating nearby immune cells. This leads to elevated antibody production and stronger antigen-specific cellular immune responses. In murine models of pneumonia and skin infection caused by methicillin-resistant Staphylococcus aureus, mice vaccinated with the hybrid vaccine formulation are well-protected. This work highlights the benefits of combining nanoparticulate antigen delivery systems with immunostimulatory hydrogels into a single platform, and the approach can be readily generalized to a wide range of infectious diseases.

Synthesis of Erythrocyte Nanodiscs for Bacterial Toxin Neutralization.

Nanodiscs are a compelling nanomedicine platform due to their ultrasmall size and distinct disc shape. Current nanodisc formulations are made primarily with synthetic lipid bilayers and proteins. Here, we report a cellular nanodisc made with human red blood cell (RBC) membrane (denoted "RBC-ND") and show its effective neutralization against bacterial toxins. In vitro, RBC-ND neutralizes the hemolytic activity and cytotoxicity caused by purified α-toxin or complex whole secreted proteins (wSP) from methicillin-resistant Staphylococcus aureus bacteria. In vivo, RBC-ND confers significant survival benefits for mice intoxicated with α-toxin or wSP in both therapeutic and prevention regimens. Moreover, RBC-ND shows good biocompatibility and biosafety in vivo. Overall, RBC-ND distinguishes itself by inheriting the biological functions of the source cell membrane for bioactivity. The design strategy of RBC-ND can be generalized to other types of cell membranes for broad applications.

Cellular Nanodiscs Made from Bacterial Outer Membrane as a Platform for Antibacterial Vaccination.

Vaccination has become an increasingly attractive strategy for protecting against antibiotic-resistant infections. Nanovaccines based on the outer membrane from Gram-negative bacteria are appealing due to their multiantigenic nature and inherent immunogenicity. Here, we develop cellular nanodiscs made of bacterial outer membrane (OM-NDs), as a platform for antibacterial vaccination. Using Pseudomonas aeruginosa as a model pathogen, the resulting OM-NDs can effectively interact with antigen-presenting cells, exhibiting accelerated uptake and an improved capacity for immune stimulation. With their small size, the OM-NDs are also capable of efficiently transporting to the lymph nodes after in vivo administration. As a result, the nanovaccine is effective at eliciting potent humoral and cellular immune responses against P. aeruginosa. In a murine model of pneumonia, immunization with OM-NDs confers strong protection against subsequent lung infection, resulting in improved survival, reduced bacterial loads, and alleviation of immune overactivation. Overall, this report illustrates the advantages of cellular nanodiscs, which can be readily generalized to other pathogens and may be applied toward other biomedical applications.

Systematic Screening and Therapeutic Evaluation of Glyconanoparticles with Differential Cancer Affinities for Targeted Cancer Therapy.

Cancer-targeting ligands used for nanomedicines have been limited mostly to antibodies, peptides, aptamers, and small molecules thus far. Here, a library of glycocalyx-mimicking nanoparticles as a platform to enable screening and identification of cancer-targeting nanomedicines is reported. Specifically, a library of 31 artificial glycopolymers composed of either homogeneous or heterogeneous display of five different sugar moieties (ß-glucose, ß-galactose, α-mannose, ß-N-acetyl glucosamine, and ß-N-acetyl galactosamine) is converted to a library of glyconanoparticles (GlyNPs). GlyNPs optimal for targeting CT26, DU145, A549, and PC3 tumors are systematically screened and identified. The cypate-conjugated GlyNP displaying α-mannose and ß-N-acetyl glucosamine show selective targeting and potent photothermal therapeutic efficacy against A549 human lung tumors. The docetaxel-contained GlyNP displaying ß-glucose, ß-galactose, and α-mannose demonstrate targeted chemotherapy against DU145 human prostate tumors. The results presented herein collectively demonstrate that the GlyNP library is a versatile platform enabling the identification of cancer-targeting glyconanoparticles and suggest its potential applicability for targeting various diseased cells beyond cancer.

Targeting the tumor microenvironment with amphiphilic near-infrared cyanine nanoparticles for potentiated photothermal immunotherapy.

Despite the potential of photothermal therapy (PTT) for cancer treatments, PTT alone has limitations in treating metastatic tumors and preventing tumor recurrence, highlighting the need to combine PTT with immunotherapy. This study reports tumor microenvironment (TME)-targeting, near-infrared (NIR) dye derivative-based nanomedicine for effective combined PTT-immunotherapy. Amphiphilic NIR dye cyanine derivatives are used not only for constructing the nanoparticle mass, but also for creating a stable complex with CpG adjuvant; a peptide specific to fibronectin extra domain B (APTEDB) is also introduced as a TME-targeting ligand, yielding the TME-targeting nanomedicine, APTEDB-cyNP@CpG. APTEDB-cyNP@CpG shows cancer-targeting ability in EDB-overexpressing CT26 colon tumor-bearing mice. When combined with laser irradiation, it induces immunogenic cell death (ICD) and subsequently leads to significant increase in CD8+ T cell population in the tumor, resulting in greater antitumor therapeutic efficacy than does cyNP@CpG lacking the TME-targeting ligand. Moreover, the combination of APTEDB-cyNP@CpG-based PTT and an immune checkpoint blockade (ICB) antibody leads to remarkable antitumor efficacy against the laser-irradiated primary tumor as well as distant tumor through potentiation of systemic cancer cell-specific T cell immunity. Furthermore, the PTT-immunotherapy combination regimen is highly effective in inhibiting tumor recurrence and metastasis.

Immunogenic Cell Death Inducing Fluorinated Mitochondria-Disrupting Helical Polypeptide Synergizes with PD-L1 Immune Checkpoint Blockade.

Immunogenic cell death (ICD) is distinguished by the release of tumor-associated antigens (TAAs) and danger-associated molecular patterns (DAMPs). This cell death has been studied in the field of cancer immunotherapy due to the ability of ICD to induce antitumor immunity. Herein, endoplasmic reticulum (ER) stress-mediated ICD inducing fluorinated mitochondria-disrupting helical polypeptides (MDHPs) are reported. The fluorination of the polypeptide provides a high helical structure and potent anticancer ability. This helical polypeptide destabilizes the mitochondrial outer membrane, leading to the overproduction of intracellular reactive oxygen species (ROS) and apoptosis. In addition, this oxidative stress triggers ER stress-mediated ICD. The in vivo results show that cotreatment of fluorinated MDHP and antiprogrammed death-ligand 1 antibodies (αPD-L1) significantly regresses tumor growth and prevents metastasis to the lungs by activating the cytotoxic T cell response and alleviating the immunosuppressive tumor microenvironment. These results indicate that fluorinated MDHP synergizes with the immune checkpoint blockade therapy to eliminate established tumors and to elicit antitumor immune responses.

Parallel-Stacked Flexible Organic Light-Emitting Diodes for Wearable Photodynamic Therapeutics and Color-Tunable Optoelectronics.

Deformable organic light-emitting diode (OLED) based optoelectronic devices hold promise for various wearable applications including biomedical systems and displays, but current OLED technologies require high voltage and lack the power needed for wearable photodynamic therapy (PDT) applications and wearable displays. This paper presents a parallel-stacked OLED (PAOLED) with high power, more than 100 mW/cm2, at low voltage (<8 V). The current dispersion ratio can be tuned by optimizing the structure of the individual OLEDs stacked to create the PAOLED, allowing control of the PAOLED's wavelength shapes, current efficiency, and power. In this study, a fabricated PAOLED operated reliably for 100 h at a high power of 35 mW/cm2. Confirming its potential application to PDT, the measured singlet oxygen generation ratio of the PAOLED was found to be 3.8 times higher than the reference OLED. The high-power PAOLED achieved a 24% reduction in melanoma cancer cell viability after a short (0.5 h) irradiation. In addition, a white light PAOLED with color tuning was realized through OLED color combination, and a high brightness of over 30 000 cd/m2 was realized, below 8.5 V. In conclusion, the PAOLED was demonstrated to be suitable for a variety of low-voltage, high-power wearable optoelectronic applications.

Structure-inherent near-infrared bilayer nanovesicles for use as photoacoustic image-guided chemo-thermotherapy.

Image-guided therapy, combined with imaging and therapeutic action, forms an attractive system because it can induce outstanding effects at focused locations. However, the conventional liposomes-based system cannot figure in therapeutic or imaging roles themselves, thereby causing the disadvantage of their biological unavailability as a theragnosis tool. Herein, the structure-inherent near-infrared bilayer nanovesicles are fabricated with amphiphilic heptamethine cyanine dye, PEG conjugated heptamethine cyanine dye, and gemcitabine (NEPCG) is developed for the novel photoacoustic image-guided chemo-thermotherapy system. The organic structure-inherent near-infrared bilayer nanovesicles are self-assembled and exhibit a liposome-like bilayer structure. Furthermore, NEPCG showed the high photoacoustic signal (PA) due to the specific accumulation in the tumor site. Delivered NEPCG than displayed concurrent chemotherapy and photothermal therapy (PTT) effects against cancer, triggered by PA imaging with minimal side effects. In vitro and in vivo experiments show that NEPCG can be used as outstanding contrast agents and completely obliterate the tumor without reoccurrence under laser irradiation. Therefore, this work presents the potential for the realization of unprecedented structure-inherent near-infrared bilayer nanovesicles as highly accurate and effective theragnostic tools in clinical fields.

CD44-Mediated Methotrexate Delivery by Hyaluronan-Coated Nanoparticles Composed of a Branched Cell-Penetrating Peptide.

Branched polymers as drug delivery carriers have been widely attempted due to their outstanding drug loading capability and complex stability like branched polyethyleneimine (B-PEI). However, branched polymers without biodegradability may cause toxicity as they can accumulate in the body. Herein, we report branched modified nona-arginine (B-mR9) composed of redox-cleavable disulfide bonds to form stable complexes with methotrexate (MTX) as an anticancer agent, which is further coated with hyaluronic acid (HA). The HA-coated nanoparticles provide targetability for the CD44 cell surface receptor. The B-mR9-MTX/HA can effectively aid in intracellular MTX delivery to CD44 overexpressing cancer cells being degradable by the reducing environments of the cancer cells. The B-mR9-MTX/HA exhibits not only a glutathione-triggered degradability but also an outstanding CD44-mediated MTX delivery efficacy. In addition, its superior tumor inhibition capability was confirmed through an in vivo study. The results suggest that the HA-coated B-mR9 nanoparticle can be used as a drug delivery platform.

Self-Assembled Supramolecular Bilayer Nanoparticles Composed of Near-Infrared Dye as a Theranostic Nanoplatform To Encapsulate Hydrophilic Drugs Effectively.

The strategy of co-loading therapeutic agents in a single nanocarrier is the most common method in theranostic cancer research. However, it is still challenging to encapsulate theranostic agents that have different physicochemical properties in a single nanocarrier system because of the immiscibility between the hydrophobic fluorescent molecule and the hydrophilic drug molecule. Thus, we report a novel concept of a theranostic nanoparticle (NP) consisting of an amphiphilic near-infrared (NIR) dye as a hydrophilic drug delivery carrier with enhanced NIR imaging capability. Unlike conventional nanocarrier systems, the newly designed amphiphilic NIR dyes (Cy-C dyes) function as both the drug delivery carrier and the fluorescent imaging agent. It can be utilized for therapy and diagnosis simultaneously by simply encapsulating the hydrophilic drug. This method is innovative not only due to formation of the theranostic nanoparticle for immiscible hydrophilic drug delivery but also because of generation of strong fluorescence signals due to the Cy-C dyes on the surfaces of the NPs. In this study, Cy-C (C = C3, C6, and C9) dyes were designed by conjugating the heptamethine cyanine dye with poly(ethylene glycol) (PEG5K) and polyethyleneimine 2000 (PEI2K). The result was self-assembled structures that effectively encapsulated a hydrophilic drug molecule (MTX) without self-quenching and scattered light interference. Among the Cy-C NPs encapsulating MTX (Cy-C/MTX NPs), Cy-C6/MTX and Cy-C9/MTX formed a concentric supramolecular bilayer (like liposomes in aqueous solution) and were capable of translocating hydrophilic drug molecules to their aqueous interior spaces. The supramolecular bilayer structure of Cy-C9/MTX provides better particle stability and drug delivery efficacy than does the supramolecular monolayer structure of Cy-C3/MTX. In addition, Cy-C9/MTX demonstrated excellent blood circulation and long-term tumor retention qualities in living mice. The effective tumor suppression ability of Cy-C9/MTX validated the concept that the amphiphilic Cy-C9 dye is the best nanoplatform for theranostics based on hydrophilic drug delivery.

Combination treatment with n-3 polyunsaturated fatty acids and ursodeoxycholic acid dissolves cholesterol gallstones in mice.

The increasing prevalence of cholesterol gallstone disease places an economic burden on the healthcare system. To identify novel therapeutics, we assessed the effects of n-3 polyunsaturated fatty acids (PUFA) in combination with UDCA in a mouse model of cholesterol gallstones. Gallstone dissolution, gallbladder wall thickness, mucin gene expression in the gallbladder, and levels of phospholipids, cholesterol, and bile acids in bile and serum were analysed. RNA was extracted from the liver for mRNA sequencing and gene expression profiling. Combination treatment resulted in greater gallstone dissolution compared with the control group, and PUFA and combination treatments reduced the thickness of the gallbladder wall. Expression levels of mucin genes were significantly lower in the UDCA, PUFA, and combination groups. Transcriptome analyses revealed that combination treatment modulated hepatic lipid metabolism. The PUFA and combination groups showed elevated bile phospholipid and bile acid levels and a lower cholesterol saturation index. Combination treatment with PUFA and UDCA dissolves cholesterol gallstones in mice by decreasing mucin production, increasing levels of phospholipids and bile acids in bile, and decreasing cholesterol saturation. Further studies of the therapeutic effects of combination PUFA and UDCA treatment in patients with cholesterol gallstones are warranted.

A Helical Polypeptide-Based Potassium Ionophore Induces Endoplasmic Reticulum Stress-Mediated Apoptosis by Perturbing Ion Homeostasis.

Perturbation of potassium homeostasis can affect various cell functions and lead to the onset of programmed cell death. Although ionophores have been intensively used as an ion homeostasis disturber, the mechanisms of cell death are unclear and the bioapplicability is limited. In this study, helical polypeptide-based potassium ionophores are developed to induce endoplasmic reticulum (ER) stress-mediated apoptosis. The polypeptide-based potassium ionophores disturb ion homeostasis and then induce prolonged ER stress in the cells. The ER stress results in oxidative environments that accelerate the activation of mitochondria-dependent apoptosis. Moreover, ER stress-mediated apoptosis is triggered in a tumor-bearing mouse model that suppresses tumor proliferation. This study provides the first evidence showing that helical polypeptide-based potassium ionophores trigger ER stress-mediated apoptosis by perturbation of potassium homeostasis.

Enhanced Photodynamic Cancer Treatment by Mitochondria-Targeting and Brominated Near-Infrared Fluorophores.

A noninvasive and selective therapy, photodynamic therapy (PDT) is widely researched in clinical fields; however, the lower efficiency of PDT can induce unexpected side effects. Mitochondria are extensively researched as target sites to maximize PDT effects because they play crucial roles in metabolism and can be used as cancer markers due to their high transmembrane potential. Here, a mitochondria targeting photodynamic therapeutic agent (MitDt) is developed. This photosensitizer is synthesized from heptamethine cyanine dyes, which are conjugated or modified as follows. The heptamethine meso-position is conjugated with a triphenylphosphonium derivative for mitochondrial targeting, the N-alkyl side chain is modified for regulation of charge balance and solubility, and the indolenine groups are brominated to enhance reactive oxygen species generation (ROS) after laser irradiation. The synthesized MitDt increases the cancer uptake efficiency due to the lipo-cationic properties of the triphenylphosphonium, and the PDT effects of MitDt are amplified after laser irradiation because mitochondria are susceptible to ROS, the response to which triggers an apoptotic anticancer effect. Consequently, these hypotheses are demonstrated by in vitro and in vivo studies, and the results indicate strong potential for use of MitDts as efficient single-molecule-based PDT agents for cancer treatment.

Conformation-switchable helical polypeptide eliciting selective pro-apoptotic activity for cancer therapy.

Artificial cationic helical peptides possess an enhanced cell-penetrating property. However, their cell-penetrability is not converted by cellular environmental changes resulting in nonspecific uptake. In this study, pH-sensitive anion-donating groups were added to a helical polypeptide to simultaneously achieve tumor targeting and pro-apoptotic activity. The mitochondria-destabilizing helical polypeptide undergoing pH-dependent conformational transitions selectively targeted cancer cells consequently disrupting mitochondrial membranes and subsequently inducing apoptosis. This work presents a promising peptide therapeutic system for cancer therapy.

pH-controllable cell-penetrating polypeptide that exhibits cancer targeting.

Helical peptides were naturally-occurring ordered conformations that mediated various biological functions essential for biotechnology. However, it was difficult for natural helical polypeptides to be applied in biomedical fields due to low bioavailability. To avoid these problems, synthetic alpha-helical polypeptides have recently been introduced by further modifying pendants in the side chain. In spite of an attractive biomimetic helical motif, these systems could not be tailored for targeted delivery mainly due to nonspecific binding events. To address these issues, we created a conformation-transformable polypeptide capable of eliciting a pH-activated cell-penetrating property solely at the cancer region. The developed novel polypeptide showed that the bare helical conformation had a function at physiological conditions while the pH-induced helical motif provided an active cell-penetrating characteristic at a tumor extracellular matrix pH. The unusual conformation-transformable system can elicit bioactive properties exclusively at mild acidic pH. STATEMENT OF SIGNIFICANCE: We developed pH-controllable cell-penetrating polypeptides (PCCPs) undergoing pH-induced conformational transitions. Unlike natural cell-penetrating peptides, PCCPs was capable of penetrating the plasma membranes dominantly at tumor pH, driven by pH-controlled helicity. The conformation of PCCPs at neutral pH showed low helical propensity because of dominant electrostatic attractions within the side chains. However, the helicity of PCCPs was considerably augmented by the balance of electrostatic interactions, thereby inducing selective cellular penetration. Three polypeptides undergoing different conformational transitions were prepared to verify the selective cellular uptake influenced by their structures. The PCCP undergoing low-to-high helical conformation provided the tumor specificity and enhanced uptake efficiency. pH-induced conformation-transformable polypeptide might provide a novel platform for stimuli-triggered targeting systems.

Correction: Stent containing CD44-targeting polymeric prodrug nanoparticles that release paclitaxel and gemcitabine in a time interval-controlled manner for synergistic human biliary cancer therapy.

Correction for 'Stent containing CD44-targeting polymeric prodrug nanoparticles that release paclitaxel and gemcitabine in a time interval-controlled manner for synergistic human biliary cancer therapy' by Dayeon Yun et al., J. Mater. Chem. B, 2017, 5, 6317-6324.