Protease-Mediated T1 Contrast Enhancement of Multilayered Magneto-Gadolinium Nanostructures for Imaging and Magnetic Hyperthermia.

In this work, we constructed a multifunctional composite nanostructure for combined magnetic hyperthermia therapy and magnetic resonance imaging based on T1 and T2 signals. First, iron oxide nanocubes with a benchmark heating efficiency for magnetic hyperthermia were assembled within an amphiphilic polymer to form magnetic nanobeads. Next, poly(acrylic acid)-coated inorganic sodium gadolinium fluoride nanoparticles were electrostatically loaded onto the magnetic nanobead surface via a layer-by-layer approach by employing a positively charged enzymatic-cleavable biopolymer. The positive-negative multilayering process was validated through the changes occurring in surface ζ-potential values and structural characterization by transmission electron microscopy (TEM) imaging. These nanostructures exhibit an efficient heating profile, in terms of the specific absorption rates under clinically accepted magnetic field conditions. The addition of protease enzyme mediates the degradation of the surface layers of the nanostructures with the detachment of gadolinium nanoparticles from the magnetic beads and exposure to the aqueous environment. Such a process is associated with changes in the T1 relaxation time and contrast and a parallel decrease in the T2 signal. These structures are also nontoxic when tested on glioblastoma tumor cells up to a maximum gadolinium dose of 125 µg mL-1, which also corresponds to a iron dose of 52 µg mL-1. Nontoxic nanostructures with such enzyme-triggered release mechanisms and T1 signal enhancement are desirable for tracking tumor microenvironment release with remote T1-guidance and magnetic hyperthermia therapy actuation to be done at the diseased site upon verification of magnetic resonance imaging (MRI)-guided release.

Elucidating the Innate Immunological Effects of Mild Magnetic Hyperthermia on U87 Human Glioblastoma Cells: An In Vitro Study.

Cancer immunotherapies have been approved as standard second-line or in some cases even as first-line treatment for a wide range of cancers. However, immunotherapy has not shown clinically relevant success in glioblastoma (GBM). This is principally due to the brain's "immune-privileged" status and the peculiar tumor microenvironment (TME) of GBM characterized by a lack of tumor-infiltrating lymphocytes and the establishment of immunosuppressive mechanisms. Herein, we explore a local mild thermal treatment, generated via cubic-shaped iron oxide magnetic nanoparticles (size ~17 nm) when exposed to an external alternating magnetic field (AMF), to induce immunogenic cell death (ICD) in U87 glioblastoma cells. In accordance with what has been observed with other tumor types, we found that mild magnetic hyperthermia (MHT) modulates the immunological profile of U87 glioblastoma cells by inducing stress-associated signals leading to enhanced phagocytosis and killing of U87 cells by macrophages. At the same time, we demonstrated that mild magnetic hyperthermia on U87 cells has a modulatory effect on the expression of inhibitory and activating NK cell ligands. Interestingly, this alteration in the expression of NK ligands in U87 cells upon MHT treatment increased their susceptibility to NK cell killing and enhanced NK cell functionality. The overall findings demonstrate that mild MHT stimulates ICD and sensitizes GBM cells to NK-mediated killing by inducing the upregulation of specific stress ligands, providing a novel immunotherapeutic approach for GBM treatment, with potential to synergize with existing NK cell-based therapies thus improving their therapeutic outcomes.

Magnetic Nanostructures as Emerging Therapeutic Tools to Boost Anti-Tumour Immunity.

Cancer immunotherapy has shown remarkable results in various cancer types through a range of immunotherapeutic approaches, including chimeric antigen receptor-T cell (CAR-T) therapy, immune checkpoint blockade (ICB), and therapeutic vaccines. Despite the enormous potential of cancer immunotherapy, its application in various clinical settings has been limited by immune evasion and immune suppressive mechanisms occurring locally or systemically, low durable response rates, and severe side effects. In the last decades, the rapid advancement of nanotechnology has been aiming at the development of novel synthetic nanocarriers enabling precise and enhanced delivery of immunotherapeutics, while improving drug stability and effectiveness. Magnetic nanostructured formulations are particularly intriguing because of their easy surface functionalization, low cost, and robust manufacturing procedures, together with their suitability for the implementation of magnetically-guided and heat-based therapeutic strategies. Here, we summarize and discuss the unique features of magnetic-based nanostructures, which can be opportunely designed to potentiate classic immunotherapies, such as therapeutic vaccines, ICB, adoptive cell therapy (ACT), and in situ vaccination. Finally, we focus on how multifunctional magnetic delivery systems can facilitate the anti-tumour therapies relying on multiple immunotherapies and/or other therapeutic modalities. Combinatorial magnetic-based therapies are indeed offering the possibility to overcome current challenges in cancer immunotherapy.

Nano-immunotherapy: Overcoming tumour immune evasion.

Immunotherapy is emerging as a groundbreaking cancer treatment, offering the unprecedented opportunity to effectively treat and in several cases, even cure previously untreatable malignancies. Anti-tumour immunotherapies designed to amplify T cell responses against defined tumour antigens have long been considered effective approaches for cancer treatment. Despite a clear rationale behind such immunotherapies, extensive past efforts were unsuccessful in mediating clinically relevant anti-tumour activity in humans. This is mainly because tumours adopt specific mechanisms to circumvent the host´s immunity. Emerging data suggest that the full potential of cancer immunotherapy will be only achieved by combining immunotherapies designed to generate or amplify anti-tumour T cell responses with strategies able to impair key tumour immune-evasion mechanisms. However, many approaches aimed to re-shape the tumour immune microenvironment (TIME) are commonly associated with severe systemic toxicity, require frequent administration, and only show modest efficacy in clinical settings. The use of nanodelivery systems is revealing as a valid means to overcome these limitations by improving the targeting efficiency, minimising systemic exposure of immunomodulatory agents, and enabling the development of novel combinatorial immunotherapies. In this review, we examine the emerging field of therapeutic modulation of TIME by the use of nanoparticle-based immunomodulators and potential future directions for TIME-targeting nanotherapies.

Advances in Lipid Nanoparticles for mRNA-Based Cancer Immunotherapy.

Over the past decade, messenger RNA (mRNA) has emerged as potent and flexible platform for the development of novel effective cancer immunotherapies. Advances in non-viral gene delivery technologies, especially the tremendous progress in lipid nanoparticles' manufacturing, have made possible the implementation of mRNA-based antitumor treatments. Several mRNA-based immunotherapies have demonstrated antitumor effect in preclinical and clinical studies, and marked successes have been achieved most notably by its implementation in therapeutic vaccines, cytokines therapies, checkpoint blockade and chimeric antigen receptor (CAR) cell therapy. In this review, we summarize recent advances in the development of lipid nanoparticles for mRNA-based immunotherapies and their applications in cancer treatment. Finally, we also highlight the variety of immunotherapeutic approaches through mRNA delivery and discuss the main factors affecting transfection efficiency and tropism of mRNA-loaded lipid nanoparticles in vivo.

Codelivery of mRNA with α-Galactosylceramide Using a New Lipopolyplex Formulation Induces a Strong Antitumor Response upon Intravenous Administration.

Recently, the use of mRNA-based vaccines for cancer immunotherapy has gained growing attention. Several studies have shown that mRNA delivered in a vectorized format can generate a robust and efficient immune response. In this work, a new lipopolyplex vector (multi-LP), incorporating the immune adjuvant α-galactosylceramide (α-GalCer) and a multivalent cationic lipid, was proposed for the in vivo delivery of mRNA into antigen-presenting cells. We demonstrate that dendritic cells (DCs) can be targeted in vivo by intravenous administration of a α-GalCer-/mRNA-loaded multi-LP vector, without the need for its functionalization with cell-specific antibodies or ligands. The multi-LP nanoparticles loaded with a reporter mRNA efficiently led to high expression of the enhanced green fluorescence protein in DCs both in vitro and in vivo, exhibiting an intrinsic selectivity for DCs. Finally, the TRP2-mRNA/α-GalCer-based multi-LP vaccine induced a significant therapeutic effect against a highly malignant B16-F10 melanoma tumor. This study provides the first evidence that a combination of antigen-mRNA and α-GalCer can be used as an effective antitumor vaccine, inducing strong innate and adaptive immune responses.

Lipid-Based Vectors for Therapeutic mRNA-Based Anti-Cancer Vaccines.

Cancer vaccines have been widely explored as a key tool for effective cancer immunotherapy. Despite a convincing rationale behind cancer vaccines, extensive past efforts were unsuccessful in mediating significantly relevant anti-tumor activity in clinical studies. One of the major reasons for such poor outcome, among others, is the low immunogenicity of more traditional vaccines, such as peptide-, protein- and DNA- based vaccines. Recently, mRNA emerged as a promising alternative to traditional vaccine strategies due to its high immunogenicity, suitability for large-scale and low-cost production, and superior safety profile. However, the clinical application of mRNA-based anti-cancer vaccines has been limited by their instability and inefficient in vivo delivery. Recent technological advances have now largely overcome these issues and lipid-based vectors have demonstrated encouraging results as mRNA vaccine platforms against several types of cancers. This review intends to provide a detailed overview of lipid-based vectors for the development of therapeutic mRNA-based anti-tumor vaccines.

Lipopolyplex potentiates anti-tumor immunity of mRNA-based vaccination.

mRNA-based vaccines have the benefit of triggering robust anti-cancer immunity without the potential danger of genome integration from DNA vaccines or the limitation of antigen selection from peptide vaccines. Yet, a conventional mRNA vaccine comprising of condensed mRNA molecules in a positively charged protein core structure is not effectively internalized by the antigen-presenting cells. It cannot offer sufficient protection for mRNA molecules from degradation by plasma and tissue enzymes either. Here, we have developed a lipopolyplex mRNA vaccine that consists of a poly-(ß-amino ester) polymer mRNA core encapsulated into a 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine/1,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 (EDOPC/DOPE/DSPE-PEG) lipid shell. This core-shell structured mRNA vaccine enters dendritic cells through macropinocytosis. It displayed intrinsic adjuvant activity by potently stimulating interferon-ß and interleukin-12 expression in dendritic cells through Toll-like receptor 7/8 signaling. Dendritic cells treated with the mRNA vaccine displayed enhanced antigen presentation capability. Mice bearing lung metastatic B16-OVA tumors expressing the ovalbumin antigen were treated with the lipopolyplex mRNA, and over 90% reduction of tumor nodules was observed. Collectively, this core-shell structure offers a promising platform for mRNA vaccine development.

Strategies for improving drug delivery: nanocarriers and microenvironmental priming.

INTRODUCTION: The ultimate goal in the field of drug delivery is to exclusively direct therapeutic agents to pathological tissues in order to increase therapeutic efficacy and eliminate side effects. This goal is challenging due to multiple transport obstacles in the body. Strategies that improve drug transport exploit differences in the characteristics of normal and pathological tissues. Within the field of oncology, these concepts have laid the groundwork for a new discipline termed transport oncophysics. Areas covered: Efforts to improve drug biodistribution have mainly focused on nanocarriers that enable preferential accumulation of drugs in diseased tissues. A less common approach to enhance drug transport involves priming strategies that modulate the biological environment in ways that favor localized drug delivery. This review discusses a variety of priming and nanoparticle design strategies that have been used for drug delivery. Expert opinion: Combinations of priming agents and nanocarriers are likely to yield optimal drug distribution profiles. Although priming strategies have yet to be widely implemented, they represent promising solutions for overcoming biological transport barriers. In fact, such strategies are not restricted to priming the tumor microenvironment but can also be directed toward healthy tissue in order to reduce nanoparticle uptake.

Label-Free Isothermal Amplification Assay for Specific and Highly Sensitive Colorimetric miRNA Detection.

We describe a new method for the detection of miRNA in biological samples. This technology is based on the isothermal nicking enzyme amplification reaction and subsequent hybridization of the amplification product with gold nanoparticles and magnetic microparticles (barcode system) to achieve naked-eye colorimetric detection. This platform was used to detect a specific miRNA (miRNA-10b) associated with breast cancer, and attomolar sensitivity was demonstrated. The assay was validated in cell culture lysates from breast cancer cells and in serum from a mouse model of breast cancer.

A pyruvate decarboxylase-mediated therapeutic strategy for mimicking yeast metabolism in cancer cells.

Cancer cells have high rates of glycolysis and lactic acid fermentation in order to fuel accelerated rates of cell division (Warburg effect). Here, we present a strategy for merging cancer and yeast metabolism to remove pyruvate, a key intermediate of cancer cell metabolism, and produce the toxic compound acetaldehyde. This approach was achieved by administering the yeast enzyme pyruvate decarboxylase to triple negative breast cancer cells. To overcome the challenges of protein delivery, a nanoparticle-based system consisting of cationic lipids and porous silicon were employed to obtain efficient intracellular uptake. The results demonstrate that the enzyme therapy decreases cancer cell viability through production of acetaldehyde and reduction of lactic acid fermentation.

A hybrid chimeric system for versatile and ultra-sensitive RNase detection.

We developed a new versatile strategy that allows the detection of several classes of RNases (i.e., targeting ss- or ds-RNA, DNA/RNA hetero-hybrid or junctions) with higher sensitivity than existing assays. Our two-step approach consists of a DNA-RNA-DNA chimeric Hairpin Probe (cHP) conjugated to magnetic microparticles and containing a DNAzyme sequence in its terminal region, and molecular beacons for fluorescence signal generation. In the first step, the digestion of the RNA portion of the cHP sequences in presence of RNases leads to the release of multiple copies of the DNAzyme in solution. Then, after magnetic washing, each DNAzyme molecule elicits the catalytic cleavage of numerous molecular beacons, providing a strong amplification of the overall sensitivity of the assay. We successfully applied our approach to detect very low concentrations of RNase A, E. coli RNase I, and RNase H. Furthermore, we analyzed the effect of two antibiotics (penicillin and streptomycin) on RNase H activity, demonstrating the applicability of our strategy for the screening of inhibitors. Finally, we exploited our system to detect RNase activity directly in crude biological samples (i.e., blood and saliva) and in cell culture medium, highlighting its suitability as cheap and sensitive tool for the detection of RNase levels.

Colorimetric detection of human papilloma virus by double isothermal amplification.

We developed a polymerase reaction free, low-cost and sensitive assay for the colorimetric detection of Human Papilloma Virus (HPV), based on the use of a smart design exploiting magnetic microbeads, chimeric RNA/DNAzyme oligonucleotides, and double signal amplification. This method allows obtaining a fast response with a detection limit of 10 pM, avoiding the amplification of the target via traditional PCR.