Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic synovial inflammation and progressive joint destruction. Methotrexate (MTX) remains the gold standard in RA therapy, yet systemic administration often provides suboptimal joint targeting and causes dose-limiting toxicity. This study investigates intra-articular (IA) administration of an MTX-loaded Microsponge (MTX-MSP) as a localized strategy to enhance drug retention while minimizing systemic exposure. MTX-MSP is synthesized using hyaluronic acid-based cross-linking and MTX loading. Drug release and carrier mass loss were evaluated in a pathological human synovial fluid (HSF), and rheological and Fourier transform infrared spectroscopy analyses assess viscoelastic behavior and drug-carrier interactions. Biocompatibility and anti-inflammatory activity are tested in primary RA fibroblast-like synoviocytes, while therapeutic efficacy is evaluated in a collagen-induced arthritis rat model. MTX-MSP provides sustained release in HSF for 14 days, minimizes burst effects, and preserves structural integrity. Rheological profiling confirms injectability and interaction with the pathological synovial fluid, enhancing the elastic response. In vitro, MTX-MSP reduces IL-6, TNF-α, and IL-1β expression at gene and protein levels, outperforming free MTX. In vivo, weekly IA injections improve histological scores in treated and contralateral joints, suggesting systemic immunomodulation. MTX-MSP thus achieves prolonged release, anti-inflammatory efficacy, and reduced systemic toxicity, representing a promising IA formulation for RA management.

PAS-based polypeptides, composed of uncharged Proline, Alanine, and Serine residues, are intrinsically disordered polymers used to enhance biologics' solubility and pharmacokinetics. Despite their biomedical relevance, their aggregation and phase separation behaviour remain underexplored. Here we combined spectroscopy, microscopy and molecular dynamics simulations to investigate the structure of PAS peptides of varying lengths (20-80 residues). We found that all peptides form nanoscale aggregates (50-350 nm) above their critical concentrations, yet only the longest PAS80 chains undergo liquid-liquid phase separation, yielding micrometre-scale condensates over a broad concentration range (0.15-6 mM), as revealed by the phase diagram. Monitoring the phase-separation process over time provided direct evidence that condensates undergo fusion with droplet sizes increasing from approximately 1 μm to 5 μm. Fluorescence recovery after photobleaching (FRAP) microscopy and nuclear magnetic resonance (NMR) spectroscopy revealed that PAS80 condensates retain a high degree of molecular mobility and exhibit characteristic liquid-like behaviour. Furthermore, treatment of the condensates with a hydrogen-bond-disrupting agent, an amphiphilic solvent, and heating demonstrated that weak interactions between hydrophobic amino acids and the entropic gain associated with the hydrophobic effect are the primary driving forces underlying the formation of condensates. In contrast, hydrogen bonding was found to play a secondary role, likely contributing to the further stabilization of the assembled structures. Molecular dynamics simulations of isolated and clustered PAS chains revealed that individual PAS peptides mainly exist as highly flexible but compact random-coil structures, and that the transition from extended to compact conformations is driven by entropic effects. Furthermore, PAS peptides rapidly self-assemble into clusters that maintain random-coil conformations and display substantial structural flexibility and configurational heterogeneity. These findings elucidate the molecular basis of PAS peptides phase behaviour and establish PAS peptides as promising building blocks for the rational design of bioengineered coacervates with potential applications in drug delivery and compartmentalized biocatalysis.

Zein (ZP) is the major storage protein of corn (maize). It is safe, biodegradable, edible, and characterized by unique self-assembly properties. These properties were exploited to prepare ZP microcapsules filled with soybean oil (SO) by ultrasound-assisted emulsification of oil-in-water (o/w) dispersions under optimal experimental conditions. The morphology and stability of o/w ZP/SO microcapsules were investigated by optical spectroscopy (electronic circular dichroism and fluorescence) and dynamic light scattering, as well as bright-field, laser confocal fluorescence, and scanning electron microscopies. The results showed that ZP formed a stable protein shell protecting the inner oily phase from diffusion of the confined compounds. It was also found that ZP/SO microcapsules, stored under suitable conditions, could be redissolved in water, maintaining their spherical morphology. Proof-of-principle studies on the inclusion and release of curcumin, a very active anti-inflammatory and nutraceutical substance, from ZP/SO microcapsules under temperature and pH stimuli are also reported.

The COVID-19 pandemic highlights the urgent need for rapid, accessible, and cost-effective diagnostic technologies. Traditional diagnostic methods, although effective, often require high-cost equipment and lengthy processing times. Herein, the development of an electrochemical immunosensor based on fluorine-doped tin oxide (FTO) electrodes modified with zinc oxide nanorods (ZnONRs) for detecting the receptor-binding domain (RBD) of SARS-CoV-2 in saliva is presented. ZnONRs offer a favorable platform due to their large surface area, low production cost, and efficient electron transport properties. To improve selectivity and sensitivity, ZnONRs are functionalized with monoclonal antibodies (mAbs) conjugated to gold nanoparticles (AuNPs). Four murine anti-RBD mAbs (2B9F9, 3E5G8, 4B1D3, and 4H4A2) are evaluated by ELISA and electrochemical methods. While all mAbs demonstrate recognition of the RBD in ELISA, only the 4B1D3 mAb produces a measurable electrochemical signal, achieving a detection limit of 1.7 mu g mL-1 and exhibiting recognition of both the original Wuhan-Hu-1 strain and the Omicron variant. The immunosensor demonstrates excellent performance in tests with real human saliva samples, reinforcing its potential as a noninvasive, rapid, and scalable platform for point-of-care viral diagnostics.

Targeted delivery of therapeutics to lymph nodes (LNs) via minimally invasive subcutaneous injection offers promise to treat B and T cell malignancies, latent HIV-1 reservoirs, and cancer metastasis. However, achieving therapeutic drug levels in subcutaneous tissues and LNs is challenging because of the poor retention and accumulation of soluble drugs. Engineered nanoparticles (NPs) provide a platform for prolonging drug stability and retention in the subcutaneous space and LNs, acting as reservoirs for the sustained release of drugs through the lymphatic system. Yet, their clinical translation for LN drug delivery faces limitations owing to the potential immunogenicity and toxicity of NP material components. Herein, we show that glycogen, a natural and biodegradable polysaccharide NP can be tailored by controlling its size, surface charge, and functional groups to enable the delivery of small and large bioactive molecules in LNs. Upon subcutaneous injection, positively charged glycogen NPs primarily accumulate in the liver and kidneys, whereas negatively charged glycogen NPs accumulate in the liver and lungs, irrespective of their size. Small and positively charged (19 ± 2 nm in diameter; 53 ± 4 mV) glycogen NPs accumulate more efficiently in LNs than the large and positively charged (57 ± 2 nm in diameter; 54 ± 9 mV) or small and negatively charged (13 ± 4 nm in diameter; -27 ± 1 mV) glycogen NPs. Moreover, smaller and slightly positively charged glycogen NPs (16 ± 4 nm in diameter; 10 ± 6 mV) enable the loading and delivery of Cre-recombinase mRNA, small organic molecules and antibodies at the site of injection and LNs. The glycogen NPs are associated with macrophages lining the subcapsular sinus and medullary regions of the LNs but are also detected within the paracortex at the T cell zone. This suggests that the glycogen NPs can overcome the phagocytic cell barrier by saturating the phagocytic capacity of the macrophages at the subcapsular sinus and spread to deeper parts of the paracortex region, providing an avenue for their application in the treatment of lymphatic diseases.

The evolution of drug resistance in tumor malignancies has necessitated advancements in anticancer drug therapy. Drug combination therapy, which can burden cancer progression at multiple target sites, has been used to address drug resistance and includes the coencapsulation of synergistic drugs within nanoparticle carriers. However, the use of organic and inorganic carriers can lead to additional material-induced safety concerns, including inflammation and antibody formation. Herein, we report an ultrasound-driven approach to combine synergistic anticancer drugs into carrier-free particles. Venetoclax (Vtx) (as a model anticancer drug) is combined with an anticancer anthracycline drug, doxorubicin (Dox), or a myeloid cell leukemia-1 inhibitor drug (S63845) to form spherical, submicrometer-sized (∼200-1000 nm in diameter) particles, consisting predominantly of the drug molecules stabilized by hydrophobic interactions. The coassembled particles, i.e., nanodrugs (NDs), display comparable and 2-fold higher anticancer activity than the free drugs and the monocomponent NDs, respectively, in Vtx-resistant SKOV-3 cells. The coassembled NDs containing Vtx and Dox increased the survival of SKOV-3 xenograft-bearing mice by at least 6 days in comparison with free Vtx or Vtx NDs and at least 10 days in comparison with saline-treated mice. Microscopy analysis of tumor tissues confirmed greater tissue damage and apoptosis induced by the NDs than those induced by the free drugs. The present findings highlight the potential of sono-driven assembled carrier-free systems in anticancer combination therapy, combining the advantages of a high surface area and slow-release particulate system with the synergistic action of multiple drugs to combat drug resistance.

Piezoelectric antimicrobial peptides are engineered to generate reactive oxygen species under ultrasound stimulation for treating spinal infections in goats.

Background/Objectives: This study aimed to develop a novel nanotechnological slow-release drug delivery platform based on hyaluronic acid Microsponge (MSP) for the subcutaneous administration of methotrexate (MTX) in the treatment of rheumatoid arthritis (RA). RA is a chronic autoimmune disease characterized by joint inflammation and damage, while MTX is a common disease-modifying antirheumatic drug (DMARD), the conventional use of which is limited by adverse effects and the lack of release control. Methods: MSP were synthesized as freeze-dried powder to increase their stability and allow for a facile reconstitution prior to administration and precise MTX dosing. Results: A highly stable and rounded-shaped micrometric MSP, characterized by an open porosity inner structure, achieved both a high MTX loading efficiency and a slow release of MTX after injection. Our drug release assays indeed demonstrated a characteristic drug release profile consisting of a very limited burst release in the first few hours, followed by a slow release of MTX sustained for over a month. By means of a preclinical rat model of RA, the administration of MTX-loaded MSP proved to nearly double the therapeutic efficacy compared to sole MTX, according to a steep reduction in arthritic score compared to control groups. The preclinical study was replicated twice to confirm this improvement in performance and the safety profile of the MSP. Conclusions: This study suggests that the MSP drug delivery platform holds significant potential for clinical use in improving RA therapy by enabling the sustained slow release of MTX, thereby enhancing therapeutic outcomes and minimizing side effects associated with conventional burst-release drug administration.

Zein protein (ZP) is the major storage protein of corn (maize). It is safe, biodegradable, edible, and characterized by unique amphiphilic and self-assembly properties. We exploited these properties to prepare stable ZP microcapsules by oil-in-water ultrasound-assisted emulsification. The opti-mal experimental conditions were determined in terms of the sonication frequency, applied acoustic power, sonication time, protein concentration and temperature. The morphology and stability of ZP microcapsules were characterized by optical spectroscopy and microscopy tech-niques. We found that a thick ZP outer layer protects the inner oily phase from internal/external diffusion, and enables the microencapsulation of Curcumin, a very active anti-inflammatory and nutraceutical agent.

The extracellular matrix (ECM)-and its mechanobiology-regulates key cellular functions that drive tumor growth and development. Accordingly, mechanotherapy is emerging as an effective approach to treat fibrotic diseases such as cancer. Through restoring the ECM to healthy-like conditions, this treatment aims to improve tissue perfusion, facilitating the delivery of chemotherapies. In particular, the manipulation of ECM is gaining interest as a valuable strategy for developing innovative treatments based on nanoparticles (NPs). However, further progress is required; for instance, it is known that the presence of a dense ECM, which hampers the penetration of NPs, primarily impacts the efficacy of nanomedicines. Furthermore, most 2D in vitro studies fail to recapitulate the physiological deposition of matrix components. To address these issues, a comprehensive understanding of the interactions between the ECM and NPs is needed. This review focuses on the main features of the ECM and its complex interplay with NPs. Recent advances in mechanotherapy are discussed and insights are offered into how its combination with nanomedicine can help improve nanomaterials design and advance their clinical translation.

Nanoparticle-mediated intracellular delivery of oligonucleotides is a complex phenomenon that depends on the architecture and the intracellular trafficking of the engineered nanoparticles. Unravelling the molecular arrangements of oligonucleotides within the nanoparticles as well as their intracellular behavior are essential for designing effective nucleic acid delivery systems. Herein, a simple and general strategy for probing the endosomal escape of nanoparticles carrying oligonucleotides in live cells is reported. A triplex-forming oligonucleotide probe is designed to target the transcription factor, kappa-light-chain-enhancer of activated B cells (NF-kappa B), in the cytosol of cells and to transduce the binding into a fluorescent Forster resonance energy transfer (FRET) signal. The combined use of the triplex-forming oligonucleotide probe and super-resolution microscopy enables the elucidation of the morphology, intracellular localization, and endosomal escape of the oligonucleotide-loaded nanoparticles on a molecular level and with nanoscale resolution. The co-delivery of the FRET probe and mRNA in cells via lipid- and polymer- based nanoparticles allow simultaneous correlation of the endosomal escape properties of nanoparticles and gene expression efficiency. A general strategy for tracking the endosomal escape of nanoparticles in cells is reported. A triplex-forming oligonucleotide is designed to target the transcription factor, NF-kappa B, in the cytosol of live cells and to transduce the recognition of the protein into a FRET signal. Oligonucleotide and super-resolution imaging enables probing of the intracellular pathways and endosomal escape of cationic lipid and phytoglycogen nanoparticles, in real-time and at the nanoscale.image

T cells play a major role in immune defense against viral infections and diseases such as cancer. Accordingly, developing nanoparticle (NP) systems to effectively deliver therapeutics to T cells is of interest. However, NP-mediated delivery of drugs to T cells is challenging because of the nonphagocytic nature of T cells. To engage T cells and induce cellular internalization, NPs are typically decorated with specific receptor-targeting antibodies, often using laborious and costly procedures. Herein, we report that natural glycogen NPs (i.e., nanosugars) with different sizes (20-80 nm) and surface charges (neutral and positively charged) engage Jurkat T cells, undergo intracellular trafficking, and release encapsulated drug without the use of receptor-targeting antibodies. Specifically, glycogen-resveratrol constructs are employed to reactivate HIV-1 latently infected Jurkat T cells (J-Lat A2) and trigger proviral expression. Both neutral and positively charged glycogen NPs engage with J-Lat A2 cells. Large (84 ± 29 nm) and positively charged (23 ± 5 mV) NPs, denoted phytoglycogen-ethylenediamine (PGEDA) NPs, readily associate with the cell membrane and are internalized (60%) in J-Lat A2 cells but remain confined in the endocytic vesicles, with moderate reactivation of latent HIV-1 (4.7 ± 0.5%). Conversely, small (21 ± 5 nm) and positively charged (10 ± 6 mV) NPs, bovine glycogen-EDA (BGEDA) NPs, associate slowly with T cells but show nearly 100% internalization and efficient endosomal escape properties, resulting in 1.5-fold higher reactivation of latent HIV-1 in T cells. PGEDA NPs and BGEDA NPs are also internalized by primary human T cells (>90% cell association) and enable the transfection of mRNA, with BGEDA NPs showing a 2-fold higher transfection than PGEDA NPs. This work highlights the potential of BGEDA NPs for the effective intracellular delivery of small-molecule drugs and mRNA in T cells.

Supramolecular assembly of polyphenols and biomacromolecules (proteins and nucleic acids) has emerged as a versatile and simple strategy to construct nanomaterials with biological activity. Here, we report a strategy to finely control the supramolecular assembly of tannic acid and oligonucleotides into uniform and stable nanoparticles by exploiting the thermal cycling of tannic acid. The equilibrium of complexation is investigated, and individual nanoparticles are resolved with nanoscale resolution by using stochastic optical reconstruction microscopy. The nanoparticles incorporating cytosine phosphoguanine (CpG) oligonucleotides are efficiently taken up by cells and trafficked via endo/lysosomal compartments and induce up to a 7-fold increase in tumor necrosis factor secretion in RAW 264.7 macrophage cells compared with naked CpG oligonucleotides. This work highlights the potential of this simple approach to engineer two-component tannic acid-oligonucleotide nanoparticles for the intracellular delivery of therapeutic nucleic acids.

Interactions between living cells and nanoparticles are extensively studied to enhance the delivery of therapeutics. Nanoparticles size, shape, stiffness, and surface charge are regarded as the main features able to control the fate of cell-nanoparticle interactions. However, the clinical translation of nanotherapies has so far been limited, and there is a need to better understand the biology of cell-nanoparticle interactions. This study investigates the role of cellular mechanosensitive components in cell-nanoparticle interactions. It is demonstrated that the genetic and pharmacologic inhibition of yes-associated protein (YAP), a key component of cancer cell mechanosensing apparatus and Hippo pathway effector, improves nanoparticle internalization in triple-negative breast cancer cells regardless of nanoparticle properties or substrate characteristics. This process occurs through YAP-dependent regulation of endocytic pathways, cell mechanics, and membrane organization. Hence, the study proposes targeting YAP may sensitize triple-negative breast cancer cells to chemotherapy and increase the selectivity of nanotherapy.

DNA-based materials have attracted interest due to the tunable structure and encoded biological functionality of nucleic acids. A simple and general approach to synthesize DNA-based materials with fine control over morphology and bioactivity is important to expand their applications. Here, we report the synthesis of DNA-based particles via the supramolecular assembly of tannic acid (TA) and DNA. Uniform particles with different morphologies are obtained using a variety of DNA building blocks. The particles enable the co-delivery of cytosine-guanine adjuvant sequences and the antigen ovalbumin in model cells. Intramuscular injection of the particles in mice induces antigen-specific antibody production and T cell responses with no apparent toxicity. Protein expression in cells is shown using capsules assembled from TA and plasmid DNA. This work highlights the potential of TA as a universal material for directing the supramolecular assembly of DNA into gene and vaccine delivery platforms.

Ultrasound-based engineering of carrier-free nanodrugs by supramolecular self-assembly has recently emerged as an innovative and environmentally friendly synthetic approach. By applying high-frequency sound waves (490 kHz) in aqueous solutions, the transformation of small chemotherapeutic and antibiotic drug molecules into carrier-free nanodrugs with anticancer and antimicrobial activities was recently achieved. The transformation of the antibiotic drug molecules, i.e., doxycycline, into stable nanodrugs (~130 nm) with selective anticancer activity was achieved without requiring organic solvents, chemical agents, or surfactants. The obtained nanodrug exhibited reactive oxygen species (ROS)-mediated cytotoxicity on human breast cancer (MDA-MB 231 cells) but a negligible antiproliferative effect on healthy fibroblast cells. Imaging by super-resolution microscopy (STORM) provided insights into the intracellular trafficking and endosomal escape of the nanodrugs. Overall, these findings suggest that small antibiotic drugs can be transformed into chemotherapeutic nanodrugs with high selectivity against cancer cells.

The application of lipid-based nanoparticles for COVID-19 vaccines and transthyretin-mediated amyloidosis treatment have highlighted their potential for translation to cancer therapy. However, their use in delivering drugs to solid tumors is limited by ineffective targeting, heterogeneous organ distribution, systemic inflammatory responses, and insufficient drug accumulation at the tumor. Instead, the use of lipid-based nanoparticles to remotely activate immune system responses is an emerging effective strategy. Despite this approach showing potential for treating hematological cancers, its application to treat solid tumors is hampered by the selection of eligible targets, tumor heterogeneity, and ineffective penetration of activated T cells within the tumor. Notwithstanding, the use of lipid-based nanoparticles for immunotherapy is projected to revolutionize cancer therapy, with the ultimate goal of rendering cancer a chronic disease. However, the translational success is likely to depend on the use of predictive tumor models in preclinical studies, simulating the complexity of the tumor microenvironment (e.g., the fibrotic extracellular matrix that impairs therapeutic outcomes) and stimulating tumor progression. This review compiles recent advances in the field of antitumor lipid-based nanoparticles and highlights emerging therapeutic approaches (e.g., mechanotherapy) to modulate tumor stiffness and improve T cell infiltration, and the use of organoids to better guide therapeutic outcomes.