Ultrasonic Transformation of Antibiotic Molecules into a Selective Chemotherapeutic Nanodrug.

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.

Supramolecular Polyphenol-DNA Microparticles for In Vivo Adjuvant and Antigen Co-Delivery and Immune Stimulation.

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.

Formulation matters! A spectroscopic and molecular dynamics investigation on the peptide CIGB552 as itself and in its therapeutical formulation.

Synthetic therapeutic peptides (STP) are intensively studied as new-generation drugs, characterized by high purity, biocompatibility, selectivity and stereochemical control. However, most of the studies are focussed on the bioactivity of STP without considering how the formulation actually used for therapy administration could alter the physico-chemical properties of the active principle. The aggregation properties of a 20-mer STP (Ac-His-Ala-Arg-Ile-Lys-D-Pro-Thr-Phe-Arg-Arg-D-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe-Trp-NH2 ), showing antitumor activity, were investigated by optical spectroscopy and atomic force microscopy imaging, as itself (CIGB552) and in its therapeutic formulation (CIGB552TF). It has found that the therapeutic formulation deeply affects the aggregation properties of the investigated peptide and the morphology of the aggregates formed on mica by deposition of CIGB552 and CIGB552TF millimolar solutions. Molecular dynamics simulations studied the first steps of CIGB552 aggregation under physiological ionic strength conditions (NaCl 150 mM), showing that peptide oligomers, from dimers to tetramers, are preferentially formed in this environment. Interestingly, cell viability assays performed on H-460 cell lines indicate a major antiproliferative activity of the peptide in its therapeutic formulation with respect to the peptide aqueous solution.

Ultrasound-Assisted Microencapsulation of Soybean Oil and Vitamin D Using Bare Glycogen Nanoparticles.

Ultrasonically synthesized core-shell microcapsules can be made of synthetic polymers or natural biopolymers, such as proteins and polysaccharides, and have found applications in food, drug delivery and cosmetics. This study reports on the ultrasonic synthesis of microcapsules using unmodified (natural) and biodegradable glycogen nanoparticles derived from various sources, such as rabbit and bovine liver, oyster and sweet corn, for the encapsulation of soybean oil and vitamin D. Depending on their source, glycogen nanoparticles exhibited differences in size and 'bound' proteins. We optimized various synthetic parameters, such as ultrasonic power, time and concentration of glycogens and the oil phase to obtain stable core-shell microcapsules. Particularly, under ultrasound-induced emulsification conditions (sonication time 45 s and sonication power 160 W), native glycogens formed microcapsules with diameter between 0.3 µm and 8 µm. It was found that the size of glycogen as well as the protein component play an important role in stabilizing the Pickering emulsion and the microcapsules shell. This study highlights that native glycogen nanoparticles without any further tedious chemical modification steps can be successfully used for the encapsulation of nutrients.

Template-Mediated Assembly of DNA into Microcapsules for Immunological Modulation.

There is a need for effective vaccine delivery systems and vaccine adjuvants without extraneous excipients that can compromise or minimize their efficacy. Vaccine adjuvants cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) can effectively activate immune responses to secrete cytokines. However, CpG ODNs are not stable in serum due to enzymatic cleavage and are difficult to transport through cell membranes. Herein, DNA microcapsules made of CpG ODNs arranged into 3D nanostructures are developed to improve the serum stability and immunostimulatory effect of CpG. The DNA microcapsules allow encapsulation and co-delivery of cargoes, including glycogen. The DNA capsules, with >4 million copies of CpG motifs per capsule, are internalized in cells and accumulate in endosomes, where the Toll-like receptor 9 is engaged by CpG. The capsules induce up to 10-fold and 20-fold increases in tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion, respectively, in RAW264.7 cells compared with CpG ODNs. Furthermore, the microcapsules stimulate TNF-α and IL-6 secretion in a concentration- and time-dependent manner. The immunostimulatory activity of the capsules correlates to their intracellular trafficking, endosomal confinement, and degradation, assessed by confocal and super-resolution microscopy. These DNA capsules can serve as both adjuvants to stimulate an immune reaction and vehicles to encapsulate vaccine peptides/genes to achieve synergistic immune effects.

Distribution of Particles in Human Stem Cell-Derived 3D Neuronal Cell Models: Effect of Particle Size, Charge, and Density.

Neurodegenerative diseases are generally characterized by a progressive loss of neuronal subpopulations, with no available cure to date. One of the main reasons for the limited clinical outcomes of new drug formulations is the lack of appropriate in vitro human cell models for research and validation. Stem cell technologies provide an opportunity to address this challenge by using patient-derived cells as a platform to test various drug formulations, including particle-based drug carriers. The therapeutic efficacy of drug delivery systems relies on efficient cellular uptake of the carrier and can be dependent on its size, shape, and surface chemistry. Although considerable efforts have been made to understand the effects of the physiochemical properties of particles on two-dimensional cell culture models, little is known of their effect in three-dimensional (3D) cell models of neurodegenerative diseases. Herein, we investigated the role of particle size (235-1000 nm), charge (cationic and anionic), and density (1.05 and 1.8 g cm-3) on the interactions of particles with human embryonic stem cell-derived 3D cell cultures of sensory neurons, called sensory neurospheres (sNSP). Templated layer-by-layer particles, with silica or polystyrene cores, and self-assembled glycogen/DNA polyplexes were used. Particles with sizes <280 nm effectively penetrated sNSP. Additionally, effective plasmid DNA delivery was observed up to 6 days post-transfection with glycogen/DNA polyplexes. The findings provide guidance in nanoparticle design for therapies aimed at neurodegenerative diseases, in particular Friedreich's ataxia, whereby sensory neurons are predominantly affected. They also demonstrate the application of 3D models of human sensory neurons in preclinical drug development.

Modular Assembly of Host-Guest Metal-Phenolic Networks Using Macrocyclic Building Blocks.

The manipulation of interfacial properties has broad implications for the development of high-performance coatings. Metal-phenolic networks (MPNs) are an emerging class of responsive, adherent materials. Herein, host-guest chemistry is integrated with MPNs to modulate their surface chemistry and interfacial properties. Macrocyclic cyclodextrins (host) are conjugated to catechol or galloyl groups and subsequently used as components for the assembly of functional MPNs. The assembled cyclodextrin-based MPNs are highly permeable (even to high molecular weight polymers: 250-500 kDa), yet they specifically and noncovalently interact with various functional guests (including small molecules, polymers, and carbon nanomaterials), allowing for modular and reversible control over interfacial properties. Specifically, by using either hydrophobic or hydrophilic guest molecules, the wettability of the MPNs can be readily tuned between superrepellency (>150°) and superwetting (ca. 0°).

Exploring New Applications of Lysozyme-Shelled Microbubbles.

This feature article provides a review of recent work on the synthesis of biopolymer-shelled microbubbles using various techniques with a particular focus on ultrasonic methodology that offers advantages over other conventional methods for tuning their physical and functional properties. A detailed discussion on the role of surface chemistry in fabricating functional lysozyme-shelled microbubbles has also been presented. Highlights on the applications of lysozyme-shelled microbubbles, particularly recent findings on their use for potential theranostic applications in lung diseases, have also been presented.

Self-Assembled Metal-Phenolic Networks on Emulsions as Low-Fouling and pH-Responsive Particles.

Interfacial self-assembly is a powerful organizational force for fabricating functional nanomaterials, including nanocarriers, for imaging and drug delivery. Herein, the interfacial self-assembly of pH-responsive metal-phenolic networks (MPNs) on the liquid-liquid interface of oil-in-water emulsions is reported. Oleic acid emulsions of 100-250 nm in diameter are generated by ultrasonication, to which poly(ethylene glycol) (PEG)-based polyphenolic ligands are assembled with simultaneous crosslinking by metal ions, thus forming an interfacial MPN. PEG provides a protective barrier on the emulsion phase and renders the emulsion low fouling. The MPN-coated emulsions have a similar size and dispersity, but an enhanced stability when compared with the uncoated emulsions, and exhibit a low cell association in vitro, a blood circulation half-life of ≈50 min in vivo, and are nontoxic to healthy mice. Furthermore, a model anticancer drug, doxorubicin, can be encapsulated within the emulsion phase at a high loading capacity (≈5 fg of doxorubicin per emulsion particle). The MPN coating imparts pH-responsiveness to the drug-loaded emulsions, leading to drug release at cell internalization pH and a potent cell cytotoxicity. The results highlight a straightforward strategy for the interfacial nanofabrication of pH-responsive emulsion-MPN systems with potential use in biomedical applications.

Redox-Sensitive PEG-Polypeptide Nanoporous Particles for Survivin Silencing in Prostate Cancer Cells.

We report the engineering of intracellular redox-responsive nanoporous poly(ethylene glycol)-poly(l-lysine) particles (NPEG-PLLs). The obtained particles exhibit no toxicity while maintaining the capability to deliver a small interfering RNA sequence (siRNA) targeting the anti-apoptotic factor, survivin, in prostate cancer cells. The redox-mediated cleavage of the disulfide bonds stabilizing the NPEG-PLL-siRNA complex results in the release of bioactive siRNA into the cytosol of prostate cancer PC-3 cells, which, in turn, leads to the effective silencing (∼59 ± 8%) of the target gene. These findings, obtained under optimal conditions, indicate that NPEG-PLLs may protect the therapeutic nucleic acid in the extracellular and intracellular environments, thus preventing the occurrence of competitive interactions with serum and cytosolic proteins as well as degradation by RNase. The intracellular trafficking and final fate of the NPEG-PLLs were investigated by a combination of deconvolution microscopy, fluorescence lifetime imaging microscopy, and super-resolution structured illumination microscopy. A significant impairment of cell survival was observed in cells concomitantly exposed to paclitaxel and siRNA-loaded NPEG-PLLs. Overall, our findings indicate that NPEG-PLLs represent a highly loaded depot for the delivery of therapeutic nucleic acids to cancer cells.

Ultrasound driven assembly of lignin into microcapsules for storage and delivery of hydrophobic molecules.

Oil-filled microcapsules of kraft lignin were synthesized by first creating an oil in water emulsion followed by a high-intensity, ultrasound-assisted cross-linking of lignin at the water/oil interface. The rationale behind our approach is based on promoting documented lignin hydrophobic interactions within the oil phase, followed by locking the resulting spherical microsystems by covalent cross-linking using a high intensity ultrasound treatment. As further evidence in support of our rationale, confocal and optical microscopies demonstrated the uniformly spherical morphology of the created lignin microparticles. The detailed elucidation of the cross-linking processes was carried out using gel permeation chromatography (GPC) and quantitative (31)P NMR analyses. The ability of lignin microcapsules to incorporate and release Coumarin-6 was evaluated in detail. In vitro studies and confocal laser scanning microscopy analysis were carried out to assess the internalization of capsules into Chinese hamster ovary (CHO) cells. This part of our work demonstrated that the lignin microcapsules are not cytotoxic and readily incorporated in the CHO cells.

Emerging Strategies for Immunotherapy of Solid Tumors Using Lipid-Based Nanoparticles.

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.

YAP Signaling Regulates the Cellular Uptake and Therapeutic Effect of Nanoparticles.

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.

Electrochemical investigation of the interaction between lysozyme-shelled microbubbles and vitamin C.

We report loading of vitamin C (ascorbic acid) on to lysozyme-shelled microbubbles. The interaction between lysozyme-shelled microbubbles and vitamin C was studied by use of cyclic and differential pulse voltammetry, zeta potential measurements, and scanning electron microscopy. The effect of microbubbles on electrochemical measurement of ascorbic acid was evaluated. The linear range for ascorbic acid obtained for differential pulse measurement in the presence of 1 mg mL(-1) microbubbles was 1-50 µmol L(-1) (y = 0.067x + 0.130, r(2) = 0.995), with a detection limit of 0.5 µmol L(-1). The experimental conditions, i.e., pH and ionic strength, were optimized to improve the interaction between ascorbic acid and lysozyme-shelled microbubbles. The results were satisfactory when the interaction was performed for 1 h in aqueous solution at pH 6. The amount of vitamin C loaded on the microbubbles (90% of the analyte added, RSD(inter-expt.) = 3%, n = 6) and the stability of microbubbles-ascorbic acid complex (until 72 h at 25 °C) were also evaluated by use of differential pulse voltammetry and zeta potential measurements.

Alginate Microsponges as a Scaffold for Delivery of a Therapeutic Peptide against Rheumatoid Arthritis.

The quest for biocompatible drug-delivery devices that could be able to open new administration routes is at the frontier of biomedical research. In this contribution, porous polysaccharide-based microsponges based on crosslinked alginate polymers were developed and characterized by optical spectroscopy and nanoscopic microscopy techniques. We show that macropores with a size distribution ranging from 50 to 120 nm enabled efficient loading and delivery of a therapeutic peptide (CIGB814), presently under a phase 3 clinical trial for the treatment of rheumatoid arthritis. Alginate microsponges showed 80% loading capacity and sustained peptide release over a few hours through a diffusional mechanism favored by partial erosion of the polymer scaffold. The edible and biocompatible nature of alginate polymers open promising perspectives for developing a new generation of polysaccharide-based carriers for the controlled delivery of peptide drugs, exploiting alternative routes with respect to intravenous administration.

An Engineered Nanosugar Enables Rapid and Sustained Glucose-Responsive Insulin Delivery in Diabetic Mice.

Glucose-responsive insulin-delivery platforms that are sensitive to dynamic glucose concentration fluctuations and provide both rapid and prolonged insulin release have great potential to control hyperglycemia and avoid hypoglycemia diabetes. Here, biodegradable and charge-switchable phytoglycogen nanoparticles capable of glucose-stimulated insulin release are engineered. The nanoparticles are "nanosugars" bearing glucose-sensitive phenylboronic acid groups and amine moieties that allow effective complexation with insulin (≈95% loading capacity) to form nanocomplexes. A single subcutaneous injection of nanocomplexes shows a rapid and efficient response to a glucose challenge in two distinct diabetic mouse models, resulting in optimal blood glucose levels (below 200 mg dL-1 ) for up to 13 h. The morphology of the nanocomplexes is found to be key to controlling rapid and extended glucose-regulated insulin delivery in vivo. These studies reveal that the injected nanocomplexes enabled efficient insulin release in the mouse, with optimal bioavailability, pharmacokinetics, and safety profiles. These results highlight a promising strategy for the development of a glucose-responsive insulin delivery system based on a natural and biodegradable nanosugar.

Molecular properties of lysozyme-microbubbles: towards the protein and nucleic acid delivery.

Microbubbles (MBs) have specific acoustic properties that make them useful as contrast agents in ultrasound imaging. The use of the MBs in clinical practice led to the development of more sensitive imaging techniques both in cardiology and radiology. Protein-MBs are typically obtained by dispersing a gas phase in the protein solution and the protein deposited/cross-linked on the gas-liquid interface stabilizes the gas core. Innovative applications of protein-MBs prompt the investigation on the properties of MBs obtained using different proteins that are able to confer them specific properties and functionality. Recently, we have synthesized stable air-filled lysozyme-MBs (LysMBs) using high-intensity ultrasound-induced emulsification of a partly reduced lysozyme in aqueous solutions. The stability of LysMBs suspension allows for post-synthetic modification of MBs surface. In the present work, the protein folded state and the biodegradability property of LysMBs were investigated by limited proteolysis. Moreover, LysMBs were coated and functionalized with a number of biomacromolecules (proteins, polysaccharides, nucleic acids). Remarkably, LysMBs show a high DNA-binding ability and protective effects of the nucleic acids from nucleases and, further, the ability to transform the bacteria cells. These results highlight on the possibility of using LysMBs for delivery of proteins and nucleic acids in prophylactic and therapeutic applications.

Sonosynthesis of nanobiotics with antimicrobial and antioxidant properties.

Transforming small-molecule antibiotics into carrier-free nanoantibiotics represents an opportunity for developing new multifunctional therapeutic agents. In this study, we demonstrate that acoustic cavitation produced by high-frequency ultrasound transforms the antibiotic doxycycline into carrier-free nanobiotics. Upon sonication for 1 h at 10-15 W cm-3, doxycycline molecules underwent hydroxylation and dimerization processes to ultimately self-assemble into nanoparticles of ∼100-200 nm in size. Micrometer sized particles can be also obtained by increasing the acoustic power to 20 W cm-3. The nanodrugs exhibited antioxidant properties, along with antimicrobial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacterial strains. Our results highlight the feasibility of the ultrasound-based approach for engineering drug molecules into a nanosized formulation with controlled and multiple bio-functionalities.

Lysozyme microspheres incorporated with anisotropic gold nanorods for ultrasound activated drug delivery.

We report on the fabrication of lysozyme microspheres (LyMs) incorporated with gold nanorods (NRs) as a distinctive approach for the encapsulation and release of an anticancer drug, 5-Fluorouracil (5-FU). LyMs with an average size of 4.0 ± 1.0 µm were prepared by a sonochemical method and characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR). The LyMs were examined using hydrophobic (nile red) as well as hydrophilic (trypan blue) dyes under confocal laser scanning microscopy (CLSM) to obtain information about the preferential distribution of fluorescent molecules. Notably, the fluorescent molecules were accumulated in the inner lining of LyMs as the core was occupied with air. The encapsulation efficiency of 5-FU for LyMs-NR was found to be ∼64%. The drug release from control LyMs as well as LyMs incorporated with NRs was investigated under the influence of ultrasound (US) at 200 kHz. The total release for control LyMs and LyMs incorporated with gold NRs was found to be ∼70 and 95% after 1 h, respectively. The density difference caused by NR incorporation on the shell played a key role in rupturing the LyMs-NR under US irradiation. Furthermore, 5-FU loaded LyMs-NR exhibited excellent anti-cancer activity against the THP-1 cell line (∼90% cell death) when irradiated with US of 200 kHz. The enhanced anti-cancer activity of LyMs-NR was caused by the transfer of released 5-FU molecules from bulk to the interior of the cell via temporary pores formed on the surface of cancer cells, i.e., sonoporation. Thus, LyMs-NR demonstrated here has a high potential for use as carriers in the field of drug delivery, bio-imaging and therapy.

The Transdermal Delivery of Therapeutic Cannabinoids.

Recently, several studies have indicated an increased interest in the scientific community regarding the application of Cannabis sativa plants, and their extracts, for medicinal purposes. This plant of enormous medicinal potential has been legalised in an increasing number of countries globally. Due to the recent changes in therapeutic and recreational legislation, cannabis and cannabinoids are now frequently permitted for use in clinical settings. However, with their highly lipophilic features and very low aqueous solubility, cannabinoids are prone to degradation, specifically in solution, as they are light-, temperature-, and auto-oxidation-sensitive. Thus, plant-derived cannabinoids have been developed for oral, nasal-inhalation, intranasal, mucosal (sublingual and buccal), transcutaneous (transdermal), local (topical), and parenteral deliveries. Among these administrations routes, topical and transdermal products usually have a higher bioavailability rate with a prolonged steady-state plasma concentration. Additionally, these administrations have the potential to eliminate the psychotropic impacts of the drug by its diffusion into a nonreactive, dead stratum corneum. This modality avoids oral administration and, thus, the first-pass metabolism, leading to constant cannabinoid plasma levels. This review article investigates the practicality of delivering therapeutic cannabinoids via skin in accordance with existing literature.