Administration sequence- and formation-dependent vaccination using acid-degradable polymeric nanoparticles with high antigen encapsulation capability.

Vaccines aim to efficiently and specifically activate the immune system via a cascade of antigen uptake, processing, and presentation by antigen-presenting cells (APCs) to CD4 and CD8 T cells, which in turn drive humoral and cellular immune responses. The specific formulation of vaccine carriers can not only shield the antigens from premature sequestering before reaching APCs but also favorably promote intracellular antigen presentation and processing. This study compares two different acid-degradable polymeric nanoparticles that are capable of encapsulating a moderately immunogenic antigen, GFP, at nearly full efficacy via electrostatic interactions or molecular affinity between His tag and Ni-NTA-conjugated monomners. This resulted in GFP-encapsulating NPs composed of ketal monomers and crosslinkers (KMX/GFP NPs) and NTA-conjugated ketal monomers and crosslinkers (NKMX/GFP NPs), respectively. Encapsulated GFP was found to be released more rapidly from NKMX/GFP NPs (electrostatic encapsulation) than from KMX/GFP NPs (affinity-driven encapsulation). In vivo vaccination studies demonstrated that while repeated injections of either NP formulation resulted in poorer generation of anti-GFP antibodies than injections of the GFP antigen itself, sequential injections of NPs and GFP as prime and booster vaccines, respectively, restored the humoral response. We proposed that NPs primarily assist APCs in antigen presentation by T cells, and B cells need to be further stimulated by free protein antigens to produce antibodies. The findings of this study suggest that the immune response can be modulated by varying the chemistry of vaccine carriers and the sequences of vaccination with free antigens and antigen-encapsulating NPs.

Beyond nanoparticle-based oral drug delivery: transporter-mediated absorption and disease targeting.

Various strategies at the microscale/nanoscale have been developed to improve oral absorption of therapeutics. Among them, gastrointestinal (GI)-transporter/receptor-mediated nanosized drug delivery systems (NDDSs) have drawn attention due to their many benefits, such as improved water solubility, improved chemical/physical stability, improved oral absorption, and improved targetability of their payloads. Their therapeutic potential in disease animal models (e.g., solid tumors, virus-infected lungs, metastasis, diabetes, and so on) has been investigated, and could be expanded to disease targeting after systemic/lymphatic circulation, although the detailed paths and mechanisms of endocytosis, endosomal escape, intracellular trafficking, and exocytosis through the epithelial cell lining in the GI tract are still unclear. Thus, this review summarizes and discusses potential GI transporters/receptors, their absorption and distribution, in vivo studies, and potential sequential targeting (e.g., oral absorption and disease targeting in organs/tissues).

Cell-free, Dendritic Cell-mimicking Extracellular Blebs for Molecularly Controlled Vaccination.

Dendritic cells (DCs) are prime targets for vaccination and immunotherapy. However, limited control over antigen presentation at a desired maturation status in these plastic materials remains a fundamental challenge in efficiently orchestrating a controlled immune response. DC-derived extracellular vesicles (EVs) can overcome some of these issues, but have significant production challenges. Herein, we employ a unique chemically-induced method for production of DC-derived extracellular blebs (DC-EBs) that overcome the barriers of DC and DC-derived EV vaccines. DC-EBs are molecular snapshots of DCs in time, cell-like particles with fixed stimulatory profiles for controlled immune signalling. DC-EBs were produced an order of magnitude more quickly and efficiently than conventional EVs and displayed stable structural integrity and antigen presentation compared to live DCs. Multi-omic analysis confirmed DC-EBs are majorly pure plasma membrane vesicles that are homogeneous at the single-vesicle level, critical for safe and effective vaccination. Immature vs. mature molecular profiles on DC-EBs exhibited molecularly modulated immune responses compared to live DCs, improving remission and survival of tumor-challenged mice via generation of antigen-specific T cells. For the first time, DC-EBs make their case for use in vaccines and for their potential in modulating other immune responses, potentially in combination with other immunotherapeutics.

Vaccination against SARS-CoV-2 using extracellular blebs derived from spike protein-expressing dendritic cells.

COVID-19 has caused significant morbidity and mortality worldwide but also accelerated the clinical use of emerging vaccine formulations. To address the current shortcomings in the prevention and treatment of SARS-CoV-2 infection, this study developed a novel vaccine platform that closely mimics dendritic cells (DCs) in antigen presentation and T-cell stimulation in a cell-free and tunable manner. Genetically engineered DCs that express the SARS-CoV-2 spike protein (S) were chemically converted into extracellular blebs (EBs). The resulting EBs elicited potentially protective humoral immunity in vivo, indicated by the production of antibodies that potently neutralized S-pseudotyped virus, presenting EBs as a promising and safe vaccine.

Nanocomplexes of doxorubicin and DNA fragments for efficient and safe cancer chemotherapy.

Cancer-targeted therapy by a chemotherapeutic agent formulated in a nanoscale platform has been challenged by complex and inefficient manufacturing, low drug loading, difficult characterization, and marginally improved therapeutic efficacy. This study investigated facile-to-produce nanocomplexes of doxorubicin (DOX), a widely used cancer drug, and clinically approved DNA fragments that are extracted from a natural source. DOX was found to self-assemble DNA fragments into relatively monodispersed nanocomplexes with a diameter of ∼70 nm at 14.3% (w/w) drug loading by simple and scalable mixing. The resulting DOX/DNA nanocomplexes showed sustained DOX release, unlike overly stable Doxil®, cellular uptake via multiple endocytosis pathways, and high hematological and immunological compatibility. DOX/DNA nanocomplexes eradicated EL4 T lymphoma cells in a time-dependent manner, eventually surpassing free DOX. Extended circulation of DOX/DNA nanocomplexes, while avoiding off-target accumulation in the lung and being cleared from the liver, resulted in rapid accumulation in tumor and lowered cardio toxicity. Finally, tumor growth of EL4-challenged C57BL/6 mice (syngeneic model) and OPM2-challenged NSG mice (human xenograft model) were efficiently inhibited by DOX/DNA nanocomplexes with enhanced overall survival, in comparison with free DOX and Doxil®, especially upon repeated administrations. DOX/DNA nanocomplexes are a promising chemotherapeutics delivery platform for their ease of manufacturing, high biocompatibility, desired drug release and accumulation, efficient tumor eradication with improved safety, and further engineering versatility for extended therapeutic applications.

Extracellular vesicles with high dual drug loading for safe and efficient combination chemo-phototherapy.

Extracellular vesicles (EVs) have emerged as biocompatible nanocarriers for efficient delivery of various therapeutic agents, with intrinsic long-term blood circulatory capability and low immunogenicity. Here, indocyanine green (ICG)- and paclitaxel (PTX)-loaded EVs [EV(ICG/PTX)] were developed as a biocompatible nanoplatform for safe and efficient cancer treatment through near-infrared (NIR) light-triggered combination chemo/photothermal/photodynamic therapy. High dual drug encapsulation in EVs was achieved for both the hydrophilic ICG and hydrophobic PTX by simple incubation. The EVs substantially improved the photostability and cellular internalization of ICG, thereby augmenting the photothermal effects and reactive oxygen species production in breast cancer cells upon NIR light irradiation. Hence, ICG-loaded EVs activated by NIR light irradiation showed greater cytotoxic effects than free ICG. EV(ICG/PTX) showed the highest anticancer activity owing to the simultaneous chemo/photothermal/photodynamic therapy when compared with EV(ICG) and free ICG. In vivo study revealed that EV(ICG/PTX) had higher accumulation in tumors and improved pharmacokinetics compared to free ICG and PTX. In addition, a single intravenous administration of EV(ICG/PTX) exhibited a considerable inhibition of tumor proliferation with negligible systemic toxicity. Thus, this study demonstrates the potential of EV(ICG/PTX) for clinical translation of combination chemo-phototherapy.

Chemically Tuned Intracellular Gene Delivery by Core-Shell Nanoparticles: Effects of Proton Buffering, Acid Degradability, and Membrane Disruption.

Nanoparticles consisting of a condensed nucleic acid core surrounded by protective layers which aid to overcome extracellular and intracellular hurdles to gene delivery (i. e., core-shell nanoparticles, CSNPs) synthetically mimic viruses. The outer shells shield the core and are particularly designed to enable facilitated release of the gene payload into the cytoplasm, the major limiting step in intracellular gene delivery. The hypothetical proton sponge effect and degradability in response to a stimulus (i. e., mildly acidic pH in the endosome) are two prevailing, although contested, principles in designing effective carriers for intracellular gene delivery via endosomal escape. Utilizing the highly flexible chemical-tuning of the polymeric shell via surface-initiated photo-polymerization of the various monomers at different molecular ratios, the effects of proton buffering capacity, acid-degradability, and endosomal membrane-lysis property on intracellular delivery of plasmid DNA by CSNPs were investigated. This study demonstrated the equivalently critical roles of proton buffering and acid-degradability in achieving efficient intracellular gene delivery, independent of cellular uptake. Extended proton buffering resulted in further improved transfection as long as the core structure was not compromised. The results of the study present a promising synthetic strategy to the development of an efficient, chemically-tunable gene delivery carrier.

Cancer nanotechnology: current status and perspectives.

Modern medicine has been waging a war on cancer for nearly a century with no tangible end in sight. Cancer treatments have significantly progressed, but the need to increase specificity and decrease systemic toxicities remains. Early diagnosis holds a key to improving prognostic outlook and patient quality of life, and diagnostic tools are on the cusp of a technological revolution. Nanotechnology has steadily expanded into the reaches of cancer chemotherapy, radiotherapy, diagnostics, and imaging, demonstrating the capacity to augment each and advance patient care. Nanomaterials provide an abundance of versatility, functionality, and applications to engineer specifically targeted cancer medicine, accurate early-detection devices, robust imaging modalities, and enhanced radiotherapy adjuvants. This review provides insights into the current clinical and pre-clinical nanotechnological applications for cancer drug therapy, diagnostics, imaging, and radiation therapy.

Temperature and pH-responsive in situ hydrogels of gelatin derivatives to prevent the reoccurrence of brain tumor.

Glioblastoma multiforme (GBM) is a grade IV malignant brain tumor with a median survival time of approximately 12-16 months. Because of its highly aggressive and heterogeneous nature it is very difficult to remove by surgical resection. Herein we have reported dual stimuli-responsive and biodegradable in situ hydrogels of oligosulfamethazine-grafted gelatin and loaded with anticancer drug paclitaxel (PTX) for preventing the progress of Glioblastoma. The oligosulfamethazine (OSM) introduced to the gelatin backbone for the formation of definite and stable in situ hydrogel. The hydrogels transformed from a sol to a gel state upon changes in stimuli. pH and temperature and retained a distinct shape after subcutaneous administration in BALB/c mice. The viscosity of the sol state hydrogels was tuned by varying the feed molar ratio between gelatin and OSM. The porosity of the hydrogels was confirmed to be lower in higher degree OSM by SEM. Sustained release of PTX from hydrogels in physiological environments (pH 7.4) was further retarded up to 63% in 9th days in tumor environments (pH 6.5). While the empty hydrogels were non-toxic in cultured cells, the hydrogels loaded with PTX showed antitumor efficacy in orthotopic-GBM xenograft mice. Collectively, the gelatin-OSM formed porous hydrogels and released the cargo in a sustained manner in tumor environments efficiently suppressing the progress of GBM. Thus, gelatin-OSM hydrogels are a potential candidate for the direct delivery of therapeutics to the local areas in brain diseases.

Discovery of New Imidazo[2,1-b]thiazole Derivatives as Potent Pan-RAF Inhibitors with Promising In Vitro and In Vivo Anti-melanoma Activity.

BRAF is an important component of MAPK cascade. Mutation of BRAF, in particular V600E, leads to hyperactivation of the MAPK pathway and uncontrolled cellular growth. Resistance to selective inhibitors of mutated BRAF is a major obstacle against treatment of many cancer types. In this work, a series of new (imidazo[2,1-b]thiazol-5-yl)pyrimidine derivatives possessing a terminal sulfonamide moiety were synthesized. Pan-RAF inhibitory effect of the new series was investigated, and structure-activity relationship is discussed. Antiproliferative activity of the target compounds was tested against the NCI-60 cell line panel. The most active compounds were further tested to obtain their IC50 values against cancer cells. Compound 27c with terminal open chain sulfonamide and 38a with a cyclic sulfamide moiety showed the highest activity in enzymatic and cellular assay, and both compounds were able to inhibit phosphorylation of MEK and ERK. Compound 38a was selected for testing its in vivo activity against melanoma. Cellular and animal activities are reported.

Eradication of Intracellular Salmonella Typhimurium by Polyplexes of Acid-Transforming Chitosan and Fragment DNA.

Antibiotics are highly successful against microbial infections. However, current challenges include rising antibiotic resistance rates and limited efficacy against intracellular pathogens. A novel form of a nanomaterial-based antimicrobial agent is investigated for efficient treatment of an intracellular Salmonella enterica sv Typhimurium infection. A known antimicrobial polysaccharide, chitosan, is engineered to be readily soluble under neutral aqueous conditions for systemic administration. The modified biologic, named acid-transforming chitosan (ATC), transforms into an insoluble, antimicrobial compound in the mildly acidic intracellular compartment. In cell culture experiments, ATC is confirmed to have antimicrobial activity against intracellular S. Typhimurium in a concentration- and pH-dependent manner, without affecting the host cells, RAW264.7 macrophages. For improved cellular uptake and pharmacokinetic/pharmacodynamic properties, ATC is further complexed with fragment DNA (fDNA), to form nano-sized spherical polyplexes. The resulting ATC/fDNA polyplexes efficiently eradicated S. Typhimurium from RAW264.7 macrophages. ATC/fDNA polyplexes may bind with microbial wall and membrane components. Consistent with this expectation, transposon insertion sequencing of a complex random mutant S. Typhimurium library incubated with ATC does not reveal specific genomic target regions of the antimicrobial. This study demonstrates the utility of a molecularly engineered nanomaterial as an efficient and safe antimicrobial agent, particularly against an intracellular pathogen.

COVID-19 vaccines: The status and perspectives in delivery points of view.

Due to the high prevalence and long incubation periods often without symptoms, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected millions of individuals globally, causing the coronavirus disease 2019 (COVID-19) pandemic. Even with the recent approval of the anti-viral drug, remdesivir, and Emergency Use Authorization of monoclonal antibodies against S protein, bamlanivimab and casirimab/imdevimab, efficient and safe COVID-19 vaccines are still desperately demanded not only to prevent its spread but also to restore social and economic activities via generating mass immunization. Recent Emergency Use Authorization of Pfizer and BioNTech's mRNA vaccine may provide a pathway forward, but monitoring of long-term immunity is still required, and diverse candidates are still under development. As the knowledge of SARS-CoV-2 pathogenesis and interactions with the immune system continues to evolve, a variety of drug candidates are under investigation and in clinical trials. Potential vaccines and therapeutics against COVID-19 include repurposed drugs, monoclonal antibodies, antiviral and antigenic proteins, peptides, and genetically engineered viruses. This paper reviews the virology and immunology of SARS-CoV-2, alternative therapies for COVID-19 to vaccination, principles and design considerations in COVID-19 vaccine development, and the promises and roles of vaccine carriers in addressing the unique immunopathological challenges presented by the disease.

Synthetically Engineered Adeno-Associated Virus for Efficient, Safe, and Versatile Gene Therapy Applications.

Gene therapy directly targets mutations causing disease, allowing for a specific treatment at a molecular level. Adeno-associated virus (AAV) has been of increasing interest as a gene delivery vehicle, as AAV vectors are safe, effective, and capable of eliciting a relatively contained immune response. With the recent FDA approval of two AAV drugs for treating rare genetic diseases, AAV vectors are now on the market and are being further explored for other therapies. While showing promise in immune privileged tissue, the use of AAV for systemic delivery is still limited due to the high prevalence of neutralizing antibodies (nAbs). To avoid nAb-mediated inactivation, engineered AAV vectors with modified protein capsids, materials tethered to the capsid surface, or fully encapsulated in a second, larger carrier have been explored. Many of these engineered AAVs have added benefits, including avoided immune response, overcoming the genome size limit, targeted and stimuli-responsive delivery, and multimodal therapy of two or more therapeutic modalities in one platform. Native and engineered AAV vectors have been tested to treat a broad range of diseases, including spinal muscular atrophy, retinal diseases, cancers, and tissue damage. This review will cover the benefits of AAV as a promising gene vector by itself, the progress and advantages of engineered AAV vectors, particularly synthetically engineered ones, and the current state of their clinical translation in therapy.

Solvent-driven, self-assembled acid-responsive poly(ketalized serine)/siRNA complexes for RNA interference.

Advances in bionanotechnology aim to develop smart nucleic acid delivery carriers with stimuli-responsive features to overcome challenges such as non-biodegradability, rapid clearance, immune response, and reaching intracellular targets. Peptide-based nanomaterials have become widely used in the field of gene and drug delivery due to their structural versatility and biomimetic properties. Particularly, polypeptide gene vectors that respond to biological stimuli, such as acidic intracellular environments, have promising applications in mediating efficient endosomal escape and drug release. Unfortunately, synthesis strategies for efficient polymerization of acid-labile peptides have been limited due to conditions that fail to preserve acid-degradable functional groups. Stable urethane derivatives of the acid-labile amino acid ketalized serine (kSer) were synthesized and polymerized to a high molecular weight under permissive conditions independent of elevated temperature, restrictive solvents, or an inert atmosphere. A new formulation strategy utilizing solvent-driven self-assembly of poly(kSer) peptides with small interfering RNA (siRNA) was developed, and the resulting poly(kSer)/siRNA complexes were further cross-linked for reinforced stability under physiological conditions. The complexes were highly monodisperse and precisely spherical in morphology, which has significant clinical implications in definitive biodistribution, cellular internalization, and intracellular trafficking patterns. Self-assembled, cross-linked poly(kSer)/siRNA complexes demonstrated efficient nucleic acid encapsulation, internalization, endosomal escape, and acid-triggered cargo release, tackling multiple hurdles in siRNA delivery. The acid-responsive polypeptides and solvent-driven self-assembly strategies demonstrated in this study could be applicable to developing other efficient and safe delivery systems for gene and drug delivery.

Biocompatible Chemotherapy for Leukemia by Acid-Cleavable, PEGylated FTY720.

Targeting the inability of cancerous cells to adapt to metabolic stress is a promising alternative to conventional cancer chemotherapy. FTY720 (Gilenya), an FDA-approved drug for the treatment of multiple sclerosis, has recently been shown to inhibit cancer progression through the down-regulation of essential nutrient transport proteins, selectively starving cancer cells to death. However, the clinical use of FTY720 for cancer therapy is prohibited because of its capability of inducing immunosuppression (lymphopenia) and bradycardia when phosphorylated upon administration. A prodrug to specifically prevent phosphorylation during circulation, hence avoiding bradycardia and lymphopenia, was synthesized by capping its hydroxyl groups with polyethylene glycol (PEG) via an acid-cleavable ketal linkage. Improved aqueous solubility was also accomplished by PEGylation. The prodrug reduces to fully potent FTY720 upon cellular uptake and induces metabolic stress in cancer cells. Enhanced release of FTY720 at a mildly acidic endosomal pH and the ability to substantially down-regulate cell-surface nutrient transporter proteins in leukemia cells only by an acid-cleaved drug were confirmed. Importantly, the prodrug demonstrated nearly identical efficacy to FTY720 in an animal model of BCR-Abl-driven leukemia without inducing bradycardia or lymphopenia in vivo, highlighting its potential clinical value. The prodrug formulation of FTY720 demonstrates the utility of precisely engineering a drug to avoid undesirable effects by tackling specific molecular mechanisms as well as a financially favorable alternative to new drug development. A multitude of existing cancer therapeutics may be explored for prodrug formulation to avoid specific side effects and preserve or enhance therapeutic efficacy.

Engineered extracellular vesicles and their mimetics for clinical translation.

Cells secrete extracellular vesicles (EVs) to external environments to achieve cellular homeostasis and cell-to-cell communication. Their therapeutic potential has been constantly spotlighted since they mirror both cytoplasmic and membranous components of parental cells. Meanwhile, growing evidence suggests that EV engineering could further promote EVs with a maximized capacity. In this review, a range of engineering techniques as well as upscaling approaches to exploit EVs and their mimetics are introduced. By laying out the pros and cons of each technique from different perspectives, we sought to provide an overview potentially helpful for understanding the current state of the art EV engineering and a guideline for choosing a suitable technique for engineering EVs. Furthermore, we envision that the advances in each technique will give rise to the combinatorial engineering of EVs, taking us a step closer to a clinical translation of EV-based therapeutics.

Extracellular blebs: Artificially-induced extracellular vesicles for facile production and clinical translation.

Extracellular vesicles (EVs) have emerged as promising biologic and comprehensive therapies for precision medicine. Despite their potential demonstrated at the benchtop, few EV formulations have made it to the clinic due to challenges in regulatory compliant scalable production; including purity, homogeneity, and reproducibility. For translation of this technology, there is a strong need for novel production methods that can meet clinical production criteria. Initial research aimed to address these challenges by taking advantage of natural pathways to increase EV yields. Such "conventional" approaches moderately increased yields but produced inhomogeneous EVs. Additionally, as there are currently no standard methods for isolation, characterization, or quantification, isolated EVs were often impure, contaminated with proteins and other biomacromolecules, and highly diverse in function. The use of shear stress and extrusion methods for EV-like vesicle production has also been investigated. While these processes can produce large EV-like vesicle yields nearly immediately, the harsh processes still result in inhomogeneous loading, and still suffer from poor purity. Chemically-induced membrane blebbing is a promising alternative production method that has the potential to overcome the previously insurmountable barriers of these current methods. This technique produces pure, and well defined EV-like vesicles, termed extracellular blebs (EBs), in clinically relevant scales over the course of minutes to hours. Furthermore, blebbing agents act on the cell in a way which locks the current surface properties and contents, preventing change, allowing for homogeneous EB production, and further preventing post-production changes. EBs may provide a promising pathway for clinical translation of EV technology.

Cancer Cell-Derived, Drug-Loaded Nanovesicles Induced by Sulfhydryl-Blocking for Effective and Safe Cancer Therapy.

Extracellular vesicles (EVs) pose great promise as therapeutic carriers due to their ideal size range and intrinsic biocompatibility. Limited scalability, poor quality control during production, and cumbersome isolation and purification processes have caused major setbacks in the progression of EV therapeutics to the clinic. Here, we overcome these setbacks by preparing cell-derived nanovesicles induced by sulfhydryl-blocking (NIbS), in the desirable size range for therapeutic delivery, that can be further loaded with the chemotherapeutic drug, doxorubicin (DOX), resulting in NIbS/DOX. Applicable to most cell types, this chemical blebbing approach enables efficient, quick, and simple harvest and purification as well as easily scalable production. Cellular uptake and intracellular release of DOX was improved using NIbS/DOX compared to a liposomal formulation. We also confirmed that in tumor-challenged C57BL/6 mice NIbS/DOX significantly slowed tumor growth and led to improved survival compared to treatment with free drug or liposomal drug. NIbS are a promising therapeutic carrier for improving cancer treatment outcomes since they are easy to prepare at a large scale, good candidates for drug loading, and capable of efficient administration of therapeutic agents with avoided nonspecific major distribution in vital organs. In addition, the utility of NIbS can be easily expanded to immunotherapy, gene therapy, and cell therapy when they are derived from applicable cell types.