Aqueous-Soluble, Acid-Transforming Chitosan for Efficient and Stimuli-Responsive Gene Silencing.

Despite its promises for biomedical applications, the lack of solubility in a physiological solution, the limited molecular interactions with nucleic acids due to the rigid backbone, and the inefficient intracellular release limit the use of chitosan, a natural cationic polysaccharide, for gene delivery. In this study, a flexible, aqueous-soluble aminoethoxy branch was conjugated to the primary hydroxyl group of chitosan via an acid-cleavable ketal linkage, resulting in acid-transforming chitosan (ATC) with greatly increased aqueous solubility, improved siRNA complexation, and degradability in response to an acidic pH. Acid-hydrolysis of ketal linkages, which triggers the loss of the flexible, cationic aminoethoxy branch, transforms ATC to the native form of chitosan with low water solubility, reduces molecular interaction with siRNA, and cooperatively facilitates the cytosolic release of siRNA. The siRNA complexation by ATC resulted in stable polyplexes under a neutral physiological condition, rapid cytosolic siRNA release from the mildly acidic endosome/lysosome, and substantial silencing of GFP expression in cells, notably with minimal cytotoxicity. This study demonstrates a molecularly engineered natural polymer for a biomedical application.

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.

Stimuli-disassembling gold nanoclusters for diagnosis of early stage oral cancer by optical coherence tomography.

A key design consideration in developing contrast agents is obtaining distinct, multiple signal changes in diseased tissue. Plasmonic gold nanoparticles (Au NPs) have been developed as contrast agents due to their strong surface plasmon resonance (SPR). This study aims to demonstrate that stimuli-responsive plasmonic Au nanoclusters (Au NCs) can be used as a contrast agent for optical coherence tomography (OCT) in detecting early-stage cancer. Au NPs were clustered via acid-cleavable linkers to synthesize Au NCs that disassemble under mildly acidic conditions into individual Au NPs, simultaneously diminishing SPR effect (quantified by scattering intensity) and increasing Brownian motion (quantified by Doppler variance). The acid-triggered morphological and accompanying optico-physical property changes of the acid-disassembling Au NCs were confirmed by TEM, DLS, UV/Vis, and OCT. Stimuli-responsive Au NCs were applied in a hamster check pouch model carrying early-stage squamous carcinoma tissue. The tissue was visualized by OCT imaging, which showed reduced scattering intensity and increased Doppler variance in the dysplastic tissue. This study demonstrates the promise of diagnosing early-stage cancer using molecularly programmable, inorganic nanomaterial-based contrast agents that are capable of generating multiple, stimuli-triggered diagnostic signals in early-stage cancer.

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.

Good things come in small packages: Overcoming challenges to harness extracellular vesicles for therapeutic delivery.

Extracellular vesicles (EVs) hold great promise as potential therapeutic carriers. EVs are biologically active, intrinsically transporting cargo between cells. Moreover, they can be loaded with specific cargo for distribution and/or engineered to achieve enhanced uptake. Although studies have already demonstrated therapeutic delivery using EVs, various challenges must be overcome before EV technology is ready for the clinic. Since the properties of EVs are dependent upon their cell of origin and the conditions of their formation, establishing clear characterization practices is essential to ensuring reproducibility and safety. Identifying methods for mass production of EVs is crucial for achieving high EV yields necessary for clinical trials. This review introduces current theory behind EV formation and function, describes the latest methods for characterization and mass production, and discusses future opportunities for extracellular vesicles in therapeutic delivery.

Viral/Nonviral Chimeric Nanoparticles To Synergistically Suppress Leukemia Proliferation via Simultaneous Gene Transduction and Silencing.

Single modal cancer therapy that targets one pathological pathway often turns out to be inefficient. For example, relapse of chronic myelogenous leukemia (CML) after inhibiting BCR-ABL fusion protein using tyrosine kinase inhibitors (TKI) (e.g., Imatinib) is of significant clinical concern. This study developed a dual modal gene therapy that simultaneously tackles two key BCR-ABL-linked pathways using viral/nonviral chimeric nanoparticles (ChNPs). Consisting of an adeno-associated virus (AAV) core and an acid-degradable polymeric shell, the ChNPs were designed to simultaneously induce pro-apoptotic BIM expression by the AAV core and silence pro-survival MCL-1 by the small interfering RNA (siRNA) encapsulated in the shell. The resulting BIM/MCL-1 ChNPs were able to efficiently suppress the proliferation of BCR-ABL+ K562 and FL5.12/p190 cells in vitro and in vivo via simultaneously expressing BIM and silencing MCL-1. Interestingly, the synergistic antileukemic effects generated by BIM/MCL-1 ChNPs were specific to BCR-ABL+ cells and independent of a proliferative cytokine, IL-3. The AAV core of ChNPs was efficiently shielded from inactivation by anti-AAV serum and avoided the generation of anti-AAV serum, without acute toxicity. This study demonstrates the development of a synergistically efficient, specific, and safe therapy for leukemia using gene carriers that simultaneously manipulate multiple and interlinked pathological pathways.