Targeting PD-L1 in cholangiocarcinoma using nanovesicle-based immunotherapy.

This study demonstrates the potential of using biological nanoparticles to deliver RNA therapeutics targeting programmed death-ligand 1 (PD-L1) as a treatment strategy for cholangiocarcinoma (CCA). RNA therapeutics offer prospects for intracellular immune modulation, but effective clinical translation requires appropriate delivery strategies. Milk-derived nanovesicles were decorated with epithelial cellular adhesion molecule (EpCAM) aptamers and used to deliver PD-L1 small interfering RNA (siRNA) or Cas9 ribonucleoproteins directly to CCA cells. In vitro, nanovesicle treatments reduced PD-L1 expression in CCA cells while increasing degranulation, cytokine release, and tumor cell cytotoxicity when tumor cells were co-cultured with T cells or natural killer cells. Similarly, immunomodulation was observed in multicellular spheroids that mimicked the tumor microenvironment. Combining targeted therapeutic vesicles loaded with siRNA to PD-L1 with gemcitabine effectively reduced tumor burden in an immunocompetent mouse CCA model compared with controls. This proof-of-concept study demonstrates the potential of engineered targeted nanovesicle platforms for delivering therapeutic RNA cargoes to tumors, as well as their use in generating effective targeted immunomodulatory therapies for difficult-to-treat cancers such as CCA.

Biosynthesis of photostable CdS quantum dots by UV-resistant psychrotolerant bacteria isolated from Union Glacier, Antarctica.

BACKGROUND: Quantum Dots (QDs) are fluorescent nanoparticles with exceptional optical and optoelectronic properties, finding widespread utility in diverse industrial applications. Presently, chemically synthesized QDs are employed in solar cells, bioimaging, and various technological domains. However, many applications demand QDs with prolonged lifespans under conditions of high-energy radiation. Over the past decade, microbial biosynthesis of nanomaterials has emerged as a sustainable and cost-effective process. In this context, the utilization of extremophile microorganisms for synthesizing QDs with unique properties has recently been reported. RESULTS: In this study, UV-resistant bacteria were isolated from one of the most extreme environments in Antarctica, Union Glacier at the Ellsworth Mountains. Bacterial isolates, identified through 16 S sequencing, belong to the genera Rhodococcus, Pseudarthrobacter, and Arthrobacter. Notably, Rhodococcus sp. (EXRC-4 A-4), Pseudarthrobacter sp. (RC-2-3), and Arthrobacter sp. (EH-1B-1) tolerate UV-C radiation doses ≥ 120 J/m². Isolated UV-resistant bacteria biosynthesized CdS QDs with fluorescence intensities 4 to 8 times higher than those biosynthesized by E. coli, a mesophilic organism tolerating low doses of UV radiation. Transmission electron microscopy (TEM) analysis determined QD sizes ranging from 6 to 23 nm, and Fourier-transform infrared (FTIR) analysis demonstrated the presence of biomolecules. QDs produced by UV-resistant Antarctic bacteria exhibit high photostability after exposure to UV-B radiation, particularly in comparison to those biosynthesized by E. coli. Interestingly, red fluorescence-emitting QDs biosynthesized by Rhodococcus sp. (EXRC-4 A-4) and Arthrobacter sp. (EH-1B-1) increased their fluorescence emission after irradiation. Analysis of methylene blue degradation after exposure to irradiated QDs biosynthesized by UV-resistant bacteria, indicates that the QDs transfer their electrons to O2 for the formation of reactive oxygen species (ROS) at different levels. CONCLUSIONS: UV-resistant Antarctic bacteria represent a novel alternative for the sustainable generation of nanostructures with increased radiation tolerance-two characteristics favoring their potential application in technologies requiring continuous exposure to high-energy radiation.

Biological nanoparticles: Relevance as novel target drug delivery systems and leading chromatographic isolation approaches.

Nanotechnology is one of the most promising technologies of the 21st century, and it is now presenting an enormous impact on target drug delivery. In this context, the recent use of natural vesicle-like nanoparticles such as extracellular vesicles (i.e., exosomes, microvesicles, and apoptotic bodies) and virus-like particles is rendering encouraging results mostly because these delivery systems present cargo versatility, favorable body circulating advantages, biocompatibility, immunogenicity, and the capacity to be modified superficially to increase their affinity to a certain target or to control their entrance to the cell. However, some of the biggest challenges toward their clinical implementation are poorly standardized processing operations due to their inherent heterogeneity and expensive, long-lasting, and difficult to scale isolation procedures that can also affect the stability of the particles. Under these circumstances, chromatographic procedures represent an attractive and favorable alternative to overcome their downstream processing. Moreover, even when standardized chromatographic purification protocols are still in development, great achievements have been made using size exclusion, ionic exchange, hydrophobic interaction, and affinity protocols, mostly because of the correct harnessing of the nanovesicle membrane properties. In this sense, this review focuses on presenting the current understanding on the most promising therapeutic biological nanoparticles and the chromatographic isolation approaches employed in their recovery, providing at the same time recent findings and a general overview of the aspects that might impact the outcome of chromatographic techniques for this application.

Combination of fluconazole with silver nanoparticles produced by Fusarium oxysporum improves antifungal effect against planktonic cells and biofilm of drug-resistant Candida albicans.

Silver nanoparticles (AgNPs) have been extensively studied because of their anti-microbial potential. Here, we evaluated the effect of biologically synthesized silver nanoparticles (AgNPbio) alone and in combination with fluconazole (FLC) against planktonic cells and biofilms of FLC-resistant Candida albicans AgNPbio exhibited a fungicidal effect, with a minimal inhibitory concentration (MIC) and fungicidal concentration ranging from 2.17 to 4.35 µg/ml. The combination of AgNPbio and FLC reduced the MIC of FLC around 16 to 64 times against planktonic cells of allC. albicans There was no significant inhibitory effect of AgNPbio on biofilm cells. However, FLC combined with AgNPbio caused a significant dose-dependent decrease in the viability of both initial and mature biofilm. All concentrations of AgNPbio, alone or in combination with FLC, were not cytotoxic to mammalian cells.The results highlight the effectiveness of the combination of AgNPbio with FLC against FLC-resistant C. albicans.

Application of surface plasmon resonance imaging technique for the detection of single spherical biological submicrometer particles.

Recent proof-of-principle studies demonstrated the suitability of the surface plasmon resonance imaging (SPRi) technique for the detection of individual submicrometer and nanoparticles in solutions. In the current study, we used the SPRi technique for visualization of the binding of round-shaped viruses (inactivated influenza A virus) and virus-like particles (human immunodeficiency virus (HIV)-based virus-like particles) to the functionalized sensor surface. We show the applicability of the SPRi technique for the detection of individual virus-like particles in buffers without serum as well as in buffers containing different concentrations of serum. Furthermore, we prove the specificity of visualized binding events using two different pseudotypes of HIV virus-like particles. We also demonstrate the applicability of the SPRi technique for the determination of relative particle concentrations in solutions. Moreover, we suggest a technical approach, which allows enhancing the magnitude of binding signals. Our studies indicate that the SPRi technique represents an efficient research tool for quantification and characterization of biological submicrometer objects such as viruses or virus-like particles, for example.

Low density lipoprotein bionanoparticles: From cholesterol transport to delivery of anti-cancer drugs.

In this review article, we highlight the importance of low-density lipoprotein (LDL) and its implications in the field of drug delivery to cancer cells. LDL is naturally occurring bionanoparticles (BNP) with a size of 18-25 nm. These BNPs specifically transport cholesterol to cells expressing the LDL receptors (LDLRs). Several tumors overexpress LDLRs, presumably to provide cholesterol for sustaining a high rate of membrane synthesis. LDL BNPs are biocompatible and biodegradable, favorably bind hydrophobic and amphiphilic drugs, are taken up by a receptor-mediated mechanism, have a half-life of 2-4 days, and can be rerouted. Drugs can be loaded onto LDL BNPs by surface loading, core loading, and apoprotein interaction. LDL may be used as a drug carrier for treatment of atherosclerosis, cancer, and in photodynamic therapies.

[Detection and analysis technologies for single biological nanoparticles].

In recent years, nanoscale detection has played an increasingly important role in the research on viruses, exosomes, small bacteria, and organelles. The small size and complex biological natures of these particles, with the smallest known virus particle measuring only 17 nm in diameter and exosomes ranging from 30 nm to 150 nm in size, pose challenges to the classical large-scale (typically micron-scale) characterization methods, which has become a major obstacle in the research. The emergence of nanoscale detection and analysis technologies has filled the gap of optical microscopy, a conventional technique in this field. These technologies enable the sensitive and robust detection of objects that exceed the lower limit of optical detection, revealing the molecular composition and biological roles simultaneously. Currently, several commercialized instruments based on nanotechnology have emerged, providing complete single-particle detection solutions and achieving unique functionality based on their respective technological advantages. However, it is inevitable that these technologies have limitations in terms of application and detection capabilities, as they continue to evolve. This paper offers a thorough overview of the principles, advantages, limitations, and future development trends of several mainstream commercial instruments, aiming to serve researchers in selecting and utilizing these technologies.

Cell Membrane-Derived Nanoparticles as Biomimetic Nanotherapeutics to Alleviate Fatty Liver Disease.

The prevalence of metabolic dysfunction associated-steatotic liver disease (MASLD) (formerly known as nonalcoholic fatty liver disease; NAFLD) is estimated at around 32% of the world's population, resulting in a major healthcare concern in recent times. Current pharmaceutical methods lack efficacy for the treatment of the disease because of suboptimal pharmacokinetic parameters including poor bioavailability, short half-life, and premature clearance. Designing an efficient drug delivery system that provides a protective environment is critical for addressing these challenges. Such a system should aim to enhance the cellular uptake of drugs, improve their bioavailability, and reduce the chances of rapid clearance. Here, we developed nanoengineered natural cell membrane-derived nanoparticles (CMNs) incorporated with a model drug, rosuvastatin, in the bilayer assembly of CMNs to reduce the accumulation of lipids in hepatocytes, a hallmark of MASLD. We used a cell extrusion technique to develop self-assembled CMNs with precise size control compared to the cell shearing method. Interestingly, the prepared CMNs were found to be nonphagocytic, representing around 1.13% of phosphatidylserine receptors on healthy cells, which allows the possibility of their use as stealth nanoparticles for drug delivery. Furthermore, CMNs exhibit higher drug-loading efficiency, excellent cytocompatibility, and enhanced cellular internalization capabilities. Moreover, we show that the delivery of rosuvastatin-loaded CMNs in the in vitro MASLD model efficiently reduced hepatocyte lipid accumulation, including total cholesterol (26.8 ± 3.1%) and triglycerides (11.8 ± 0.8%), compared to the negative control. Taken together, the nanoengineered biomimetic CMNs enhance the drug's bioactivity in hepatic cells, establishing a foundation for further investigation of this drug delivery system in treating MASLD.

Engineering Cell-Derived Nanovesicles for Targeted Immunomodulation.

Extracellular vesicles (EVs) show promise for targeted drug delivery but face production challenges with low yields. Cell-derived nanovesicles (CDNVs) made by reconstituting cell membranes could serve as EV substitutes. In this study, CDNVs were generated from mesenchymal stem cells by extrusion. Their proteomic composition, in vitro and in vivo toxicity, and capacity for loading RNA or proteins were assessed. Compared with EVs, CDNVs were produced at higher yields, were comprised of a broader range of proteins, and showed no detrimental effects on cell proliferation, DNA damage, or nitric oxide production in vitro or on developmental toxicity in vivo. CDNVs could be efficiently loaded with RNA and engineered to modify surface proteins. The feasibility of generating immunomodulatory CDNVs was demonstrated by preparing CDNVs with enhanced surface expression of PD1, which could bind to PD-L1 expressing tumor cells, enhance NK and T cell degranulation, and increase immune-mediated tumor cell death. These findings demonstrate the adaptability and therapeutic promise of CDNVs as promising substitutes for natural EVs that can be engineered to enhance immunomodulation.

Tailoring the Inherent Properties of Biobased Nanoparticles for Nanomedicine.

Biobased nanoparticles are at the leading edge of the rapidly developing field of nanomedicine and biotherapeutics. Their unique size, shape, and biophysical properties make them attractive tools for biomedical research, including vaccination, targeted drug delivery, and immune therapy. These nanoparticles are engineered to present native cell receptors and proteins on their surfaces, providing a biomimicking camouflage for therapeutic cargo to evade rapid degradation, immune rejection, inflammation, and clearance. Despite showing promising clinical relevance, commercial implementation of these biobased nanoparticles is yet to be fully realized. In this perspective, we discuss advanced biobased nanoparticle designs used in medical applications, such as cell membrane nanoparticles, exosomes, and synthetic lipid-derived nanoparticles, and highlight their benefits and potential challenges. Moreover, we critically assess the future of preparing such particles using artificial intelligence and machine learning. These advanced computational tools will be able to predict the functional composition and behavior of the proteins and cell receptors present on the nanoparticle surfaces. With more advancement in designing new biobased nanoparticles, this field of research could play a key role in dictating the future rational design of drug transporters, thereby ultimately improving overall therapeutic outcomes.

Evaluation of In Vivo Toxicity of Biological Nanoparticles.

Biologically derived nanoparticles such as extracellular vesicles are promising candidates for therapeutic applications. In vivo toxicity of biological nanoparticles can result in tissue or organ damage, immunological perturbations, or developmental effects but cannot be readily predicted from in vitro studies. Therefore, an essential component of the preclinical assessment of these particles for their use as therapeutics requires screening for adverse effects and detailed characterization of their toxicity in vivo. However, there are no standardized, comprehensive methods to evaluate the toxicity profile of nanoparticle treatment in a preclinical model. Here, we first describe a method to prepare bovine milk-derived nanovesicles (MNVs). These MNVs are inexpensive to isolate, have a scalable production platform, and can be modified to achieve a desired biological effect. We also describe two vertebrate animal models, mice and zebrafish, that can be employed to evaluate the toxicity profile of biologically derived nanoparticles, using MNVs as an example. Treatment-induced organ toxicity and immunological effects can be assessed in mice receiving systemic injections of MNVs, and developmental toxicity can be assessed in zebrafish embryos exposed to MNVs in embryo water. Utilizing these animal models provides opportunities to analyze the toxicity profiles of therapeutic extracellular vesicles in vivo. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of milk-derived nanovesicles Basic Protocol 2: In vivo screening for organ toxicity and immune cell profiling using mice Basic Protocol 3: In vivo developmental toxicity screening using zebrafish.

An Electrokinetically-Driven Microchip for Rapid Entrapment and Detection of Nanovesicles.

Electrical Impedance Spectroscopy (EIS) has been widely used as a label-free and rapid characterization method for the analysis of cells in clinical research. However, the related work on exosomes (40-150 nm) and the particles of similar size has not yet been reported. In this study, we developed a new Lab-on-a-Chip (LOC) device to rapidly entrap a cluster of sub-micron particles, including polystyrene beads, liposomes, and small extracellular vesicles (exosomes), utilizing an insulator-based dielectrophoresis (iDEP) scheme followed by measuring their impedance utilizing an integrated electrical impedance sensor. This technique provides a label-free, fast, and non-invasive tool for the detection of bionanoparticles based on their unique dielectric properties. In the future, this device could potentially be applied to the characterization of pathogenic exosomes and viruses of similar size, and thus, be evolved as a powerful tool for early disease diagnosis and prognosis.

Protein-Induced Dissociation of Biomolecular Assemblies.

Studies on protein adsorption on nanoparticles have attracted great interest over the past years due to the unique properties of the protein-immobilized nanoparticles. However, the effects of protein adsorption on the stability of nanoparticles and the role of hydrophobic interaction in the adsorption have not been fully understood. Herein, fundamental research on protein-induced dissociation of biomolecular assemblies based on hydrophobic interaction is reported. Bovine serum albumin (BSA) is used as a model protein, and cholesterol-glutathione bioconjugate (Ch-GSH) and cholesterol-terminated polyethylene glycol (Ch-PEG) are chosen as model amphiphilic biomolecules. Ch-GSH or Ch-PEG molecules are able to self-assemble into vesicles. The walls and the coronae of the assemblies are composed of hydrophobic Ch and hydrophilic GSH (or PEG), respectively. Upon addition of BSA into phosphate buffer saline solutions of the assemblies, vesicle structures are dissociated and small-sized aggregates composed of BSA, and amphiphilic biomolecules are formed. The dissociation temperatures of the vesicles can be determined by dynamic light scattering. Transmission electron microscopy and size exclusion chromatography are used to demonstrate the dissociation of the assemblies and the formation of aggregates. The hydrophobic interaction between hydrophobic patches on BSA molecules and Ch groups in the walls of the assemblies is responsible for the dissociation of the vesicles and the formation of the aggregates with smaller sizes.