An Up-to-date Review on Protein-based Nanocarriers in the Management of Cancer.

BACKGROUND: A big health issue facing the world's population is cancer. An alarming increase in cancer patients was anticipated by worldwide demographic statistics, which showed that the number of patients with different malignancies was rapidly increasing. By 2025, probably 420 million cases were projected to be achieved. The most common cancers diagnosed are breast, colorectal, prostate, and lung. Conventional treatments, such as surgery, chemotherapy, and radiation therapy, have been practiced. OBJECTIVE: In recent years, the area of cancer therapy has changed dramatically with expanded studies on the molecular-level detection and treatment of cancer. Recent advances in cancer research have seen significant advances in therapies such as chemotherapy and immunotherapy, although both have limitations in effectiveness and toxicity. METHODS: The development of nanotechnology for anticancer drug delivery has developed several potentials as nanocarriers, which may boost the pharmacokinetic and pharmacodynamic effects of the drug product and substantially reduce the side effects. RESULTS: The advancement in non-viral to viral-based protein-based nanocarriers for treating cancer has earned further recognition in this respect. Many scientific breakthroughs have relied on protein-based nanocarriers, and proteins are essential organic macromolecules for life. It allows targeted delivery of passive or active tumors using non-viral-based protein-based nanocarriers to viral-based protein nanocarriers. When targeting cancer cells, both animal and plant proteins may be used in a formulation process to create self-assembled viruses and platforms that can successfully eradicate metastatic cancer cells. CONCLUSION: This review, therefore, explores in depth the applications of non-viral to viral proteinbased noncarriers with a specific focus on intracellular drug delivery and anti-cancer drug targeting ability.

The role of polysaccharides in improving the functionality of zein coated nanocarriers: Implications for colloidal stability under environmental stresses.

Zein has gained popularity over the past few years as an incredible food and bio-based materials. The potential functions and health benefits of zein microcapsules or micro-/nanoparticles in bioactive components delivery, structured emulsion, etc., have received great attention. However, the development has been limited by colloidal destabilization, especially when thermal processing is involved. There is a recent trend in developing zein-polysaccharide complexes (ZPCs), which has tremendously improved the performance of zein-based colloidal carrier systems or emulsions. Increasing our understanding of zein interactions and their contribution to the structure of various macromolecules can help us to develop novel biomaterials that can be used in food, agriculture, biomedicine, and cosmetics. In addition, these nanocarriers are suitable for the encapsulation and delivery of bioactive compounds which have positive perspective in food industry. Therefore, this article aimed to review recent advances in the ZPCs that can be applied to functional or health-promoting foods, with a focus on the characteristics of different ZPCs, factors and mechanisms affecting the stability (especially thermal stability) of these complexes, and their application in food industry as a carrier for BCs. Further, the stability of ZPCs based emulsions under processing and physiological environments, as well some typical effective methods are introduced. Also, the principal challenges and prospects were enumerated and discussed.

Introduction to Optical Tweezers: Background, System Designs, and Applications.

Optical tweezers are a means to manipulate objects with light. With the technique, microscopically small objects can be held and steered, allowing for accurate measurement of the forces applied to these objects. Optical tweezers can typically obtain a nanometer spatial resolution, a picoNewton force resolution, and a millisecond time resolution, which makes the technique well suited for the study of biological processes from the single-cell down to the single-molecule level. In this chapter, we aim to provide an introduction to the use of optical tweezers for single-molecule analyses. We start from the basic principles and methodology involved in optical trapping, force calibration, and force measurements. Next, we describe the components of an optical tweezers setup and their experimental relevance. Finally, we will provide an overview of the broad applications in context of biological research, with the emphasis on the measurement modes, experimental assays, and possible combinations with fluorescence microscopy techniques.

Functionalized DNA nanoplatform for multi-target simultaneous imaging: Establish the atlas of cancer cell species.

Detection and imaging of cell membrane receptor proteins have gained widespread interest in recent years. However, recognition based on a single biomarker can induce false positive feedback, including off-target phenomenon caused by the absence of tumor-specific antigens. In addition, nucleic acid probes often cause nonspecific and undesired cell internalization during cell imaging. In this work, we constructed a logic gate DNA nano-platform (LGDP) for single-molecule imaging of cell membrane proteins to synergistically diagnose cancer cells. The traffic light-like color response of LGDP facilitates the precise discrimination among different cell lines. Combined with single molecule technology, the target proteins were qualitatively and quantitatively analyzed synergistically. Logic-gated recognition integrated in aptamer-functionalized molecular machines will prompt fast cells analysis, laying the foundation of cancer early diagnosis and treatment.

A Brief Introduction to Single-Molecule Fluorescence Methods.

One of the most popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we will review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We will end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, single-molecule localization super-resolution microscopy, to distance measurements with Förster resonance energy transfer and orientation measurements with fluorescence polarization.

Fluorescence Microscopy of Nanochannel-Confined DNA.

Stretching of DNA in nanoscale confinement allows for several important studies. The genetic contents of the DNA can be visualized on the single DNA molecule level, and the polymer physics of confined DNA and also DNA/protein and other DNA/DNA-binding molecule interactions can be explored. This chapter describes the basic steps to fabricate the nanostructures, perform the experiments, and analyze the data.

Unfolding and Refolding Proteins Using Single-Molecule AFM.

Single-molecule atomic force microscopy (AFM) allows capturing the conformational dynamics of an individual molecule under force. In this chapter, we describe a protocol for conducting a protein nanomechanical experiment using AFM, covering both the force-extension and force-clamp modes. Combined, these experiments provide an integrated vista of the molecular mechanisms-and their associated kinetics-underpinning the mechanical unfolding and refolding of individual proteins when exposed to mechanical load.

Single-Molecule FRET X.

Fluorescence resonance energy transfer (FRET) is a photophysical phenomenon that has been repurposed as a biophysical tool to measure nanometer distances. With FRET by DNA eXchange, or FRET X, many points of interest (POIs) in a single object can be probed, overcoming a major limitation of conventional single-molecule FRET. In FRET X, short fluorescently labeled DNA imager strands specifically and transiently bind their complementary docking strands on a target molecule, such that at most a single FRET pair is formed at each point in time and multiple POIs on a single molecule can be readily probed. Here, we describe the sample preparation, image acquisition, and data analysis for structural analysis of DNA nanostructures with FRET X.

Single-Cell Measurements Using Acoustic Force Spectroscopy (AFS).

Single-molecule force spectroscopy is a powerful tool to investigate the forces and motions related to interactions of biological molecules. Acoustic force spectroscopy (AFS) is a developed measurement tool to study single molecules or cells making use of acoustic standing waves. AFS permits high experimental throughput because many individual molecules can be manipulated and tracked in parallel. Moreover, a wide range of forces can be applied as well as a force loading rate with range of six orders of magnitude. At the same time, AFS stands out because of its simplicity and the compactness of the experimental setup. Even though the AFS setup is simple, it can still be challenging to perform high-quality measurements. Here we describe, in detail, how to setup, perform, and analyze an AFS measurement to determine cell adhesion.

Correlated Single-Molecule Magnetic Tweezers and Fluorescence Measurements of DNA-Enzyme Interactions.

Combining force spectroscopy and fluorescence microscopy provides a substantial improvement to the single-molecule toolbox by allowing simultaneous manipulation and orthogonal characterizations of the conformations, interactions, and activity of biomolecular complexes. Here, we describe a combined magnetic tweezers and total internal reflection fluorescence microscopy setup to carry out correlated single-molecule fluorescence spectroscopy and force/twisting experiments. We apply the setup to reveal the DNA interactions of the CRISPR-Cas surveillance complex Cascade. Single-molecule fluorescence of a labeled Cascade allows to follow the DNA association and dissociation of the protein. Simultaneously, the magnetic tweezers probe the DNA unwinding during R-loop formation by the bound Cascade complexes. Furthermore, the setup supports observation of 1D diffusion of protein complexes on stretched DNA molecules. This technique can be applied to study a vast range of protein-DNA interactions.

DNA Origami-Based Single-Molecule Force Spectroscopy and Applications.

Over the last years, single-molecule force spectroscopy provided insights into the intricate connection between mechanical stimuli and biochemical signaling. The underlying molecular mechanisms were uncovered and explored using techniques such as atomic force microscopy and force spectroscopy using optical or magnetic tweezers. These experimental approaches are limited by thermal noise resulting from a physical connection of the studied biological system to the macroscopic world. To overcome this limitation, we recently introduced the DNA origami force clamp (FC) which is a freely diffusing nanodevice that generates piconewton forces on a DNA sequence of interest. Binding of a protein to the DNA under tension can be detected employing fluorescence resonance energy transfer (FRET) as a sensitive readout.This protocol introduces the reader to the working principles of the FC and provides instructions to design and generate a DNA origami FC customized for a protein of interest. Molecular cloning techniques are employed to modify, produce, and purify a custom DNA scaffold. A fluorescently labeled DNA suitable to detect protein binding via FRET is generated via enzymatic ligation of commercial DNA oligonucleotides. After thermal annealing of all components, the DNA origami FC is purified using agarose gel electrophoresis. The final section covers the interrogation of the FC using confocal single-molecule FRET measurements and subsequent data analysis to quantify the binding of a DNA-binding protein to its cognate recognition site under a range of forces. Using this approach, force-dependent DNA-protein interactions can be studied on the single-molecule level on thousands of molecules in a parallelized fashion.

Novel Strategies Using Sagacious Targeting for Site-Specific Drug Delivery in Breast Cancer Treatment: Clinical Potential and Applications.

For more than a decade, researchers have been working to achieve new strategies and smart targeting drug delivery techniques and technologies to treat breast cancer (BC). Nanotechnology presents a hopeful strategy for targeted drug delivery into the building of new therapeutics using the properties of nanomaterials. Nanoparticles are of high regard in the field of diagnosis and the treatment of cancer. The use of these nanoparticles as an encouraging approach in the treatment of various cancers has drawn the interest of researchers in recent years. In order to achieve the maximum therapeutic effectiveness in the treatment of BC, combination therapy has also been adopted, leading to minimal side effects and thus an enhancement in the quality of life for patients. This review article compares, discusses and criticizes the approaches to treat BC using novel design strategies and smart targeting of site-specific drug delivery systems.

Aquasomal drug delivery system: A special emphasis on the formulation techniques and applications / Sistema de administración de fármacos Aquasomal: Especial énfasis en las técnicas de formulación y aplicaciones

Introducción: Aquasome es un sistema portador de nanopartículas autoensamblado con tres capas. El sistema se compone de un núcleo sólido nanocristalino interno recubierto de oligómero polihidroxilado. Adsorbidas en la capa recubierta se encuentran moléculas de fármacos o compuestos bioquímicamente activos. El autoensamblaje en este sentido se refiere a la formación independiente de moléculas en patrones organizados, de larga duración y con enlaces no covalentes. nueva tecnología de administración de fármacos. El artículo aborda principalmente los procesos de formulación utilizados para crear nanoestructuras autoensambladas y sus diversas aplicaciones posibles. Método: En la búsqueda bibliográfica se utilizaron varias bases de datos en línea, incluidas Science Direct, Medline, Web of Science, Google Scholar y Scopus. Se realizaron búsquedas en los conjuntos de datos en busca de entradas de estudios hasta julio de 2023. El documento de revisión aborda especialmente muchos elementos de la formación de aquasomas por parte de varios investigadores que emplean métodos/técnicas modificadas como la coprecipitación, la autoprecipitación, la pulverización catódica, etc. También ilustra una variedad de campos de terapia en los que se ha reconocido que el aquasoma tiene una gran influencia, como el oxígeno y el transporte de extractos. Resultados: El núcleo sólido es responsable de brindar estabilidad estructural, mientras que el recubrimiento oligomérico es crucial para proteger contra la deshidratación y estabilizar las moléculas bioactivas. Este vehículo de administración de fármacos biodegradable a escala nanométrica muestra una tendencia a acumularse en el hígado y los músculos. La no modificación de la adsorción del fármaco en la superficie del aquasoma facilita una respuesta farmacológica rápida al permitir el reconocimiento sin obstrucciones del receptor en el sitio de acción. (AU)

Introduction: Aquasome is a self-assembled nanoparticulate carrier system with three layers. The system is made up of a polyhydroxy oligomer-coated inner nanocrystalline solid core. Adsorbed on the coated layer are drug mole-cules or biochemically active compounds. Self-assembly in this sense refers to the independent formation of mole-cules into organised, long-lasting, and non-covalently bonded patterns.This paper gives an overview of aquasome formation, covering structural properties, formulation methodologies, and the benefits and drawbacks of this novel drug delivery technology. The article primarily addresses the formulation processes used to create self-assembled nanostructures and their various possible applications.Method: Several online databases, including Science Direct, Medline, Web of Science, Google Scholar and Scopus, were used in the literature search. The datasets were searched for entries of studies up to July, 2023. The review paper especially addresses many elements of aquasome formation by various researchers employing methods/modified techniques such as co-precipitation, self-precipitation, sputtering, and and so forth. It also illustrates a variety of fields of therapy in which aquasome has been recognised to have a major influence, such as oxygen and extract carrier.Results: The solid core is responsible for providing structural stability, while the oligomeric coating is crucial for safeguarding against dehydration and stabilising the bioactive molecules. This biodegradable drug delivery vehicle at the nanoscale level exhibits a tendency to accumulate in the liver and muscles. The non-modification of drug adsorption onto the aquasome surface facilitates prompt pharmacological response by allowing unobstructed re-ceptor recognition at the action site. (AU)

Biosynthesis of gold nanoparticles by Penicillium rubens and catalytic detoxification of ochratoxin A and organic dye pollutants / Biosíntesis de nanopartículas de oro por Penicillium rubens y desintoxicación catalítica de ocratoxina A y contaminantes colorantes orgánicos

The environmental pollution caused by chemical dyes is a growing concern nowadays. Limitations of traditional methods opened the route for nanotechnology; owing to the versatile properties of nanomaterials, gold nanoparticles (AuNPs) became a potential strategy for different applications. In the present study, biosynthesis of gold nanoparticles (BioAuNPs) was carried out by reacting chloroauric acid (HAuCl4) with cell-free filtrate of Penicillium rubens sp. nov. NCIM 1937. The AuNPs were then characterized by UV–visible spectroscopy, HR-TEM, FTIR, and DLS analysis to further examine their efficacious biosynthesis and morphological properties including size, shape, and stability. The biogenic AuNPs are polydisperse in nature, with a mean size of 14.92 ± 5 nm. These AuNPs exhibited promising antimicrobial activity against Escherichia coli NCIM-2065, Bacillus subtilis NCIM-2010, and Penicillium verrucosum MTCC 4935. In vitro quantitative HPLC results revealed that BioAuNPs significantly inhibited the biosynthesis of ochratoxin A (OTA). Microbial fuel cells (MFCs) are intriguing for power generation and wastewater treatment since they can directly transform chemical energy stored in organic matter to electricity by extracellular electron transfer (EET) via membrane proteins. AuNPs also showed excellent potential for dye degradation of organic pollutants, viz., methylene blue (MB), phenol red (PR), bromothymol blue (BTB), Congo red (CR), and 4-nitrophenol (4-NP). All dye removal efficiencies were estimated and fitted to pseudo-first-order processes using kinetic rate constants (Ka).The present study reveals a simple, original, and eco-friendly method for the synthesis of multifunctional biogenic AuNPs that could be effective in OTA detoxification in food products and organic pollutant removal during wastewater treatment for a sustainable environment.(AU)

An aptamer-assisted nanopore strategy with a salt gradient for direct protein sensing.

Nanopore sensing is at the forefront of the technological revolution of the protein research field and has been widely used in molecular diagnosis and molecular dynamics, as well as for various sequencing applications. However, direct protein sensing with biological nanopores is still challenging owing to the large molecular size. Here, we propose an aptamer-assisted nanopore strategy for direct protein sensing and demonstrate its proof-of-concept utilities by experiments with SARS-Cov-2 nucleocapsid protein (NP), the most abundantly expressed viral protein, that is widely used in clinical diagnosis for COVID-19. NP binds with an oligonucleotide-tailed aptamer to form a protein-DNA complex which induces a discriminative two-level pattern of current blockades. We reveal the potential molecular interaction mechanism for the characteristic blockades and identify the salt gradient condition as the dominant factor of the phenomenon. Furthermore, we achieve a high sensitivity of 10 pM for NP detection within one hour and make a preliminary exploration on clinical diagnosis. This work promises a new platform for rapid and label-free protein detection.

An Integrated Multilevel Approach Unveils Complex Seed-Nanoparticle Interactions and Their Implications for Seed Priming.

Nanotechnology has the potential to revolutionize agriculture with the introduction of engineered nanomaterials. However, their use is hindered by high cost, marginal knowledge of their interactions with plants, and unpredictable effects related to massive use in crop cultivation. Nanopriming is an innovative seed priming technology able to match economic, agronomic, and environmental needs in agriculture. The present study was focused on unveiling, by a multilevel integrated approach, undisclosed aspects of seed priming mediated by iron oxide magnetic nanoparticles in pepper seeds (Capsicum annuum), one of the most economically important crops worldwide. Inductively coupled plasma atomic emission mass spectrometry and scanning electron microscopy were used to quantify the MNP uptake and assess seed surface changes. Magnetic resonance imaging mapped the distribution of MNPs prevalently in the seed coat. The application of MNPs significantly enhanced the root and vegetative growth of pepper plants, whereas seed priming with equivalent Fe concentrations supplied as FeCl3 did not yield these positive effects. Finally, global gene expression by RNA-sequencing identified more than 2,200 differentially expressed genes, most of them involved in plant developmental processes and defense mechanisms. Collectively, these data provide evidence on the link between structural seed changes and an extensive transcriptional reprogramming, which boosts the plant growth and primes the embryo to cope with environmental challenges that might occur during the subsequent developmental and growth stages.

Solid-State Nanopore Sensors with Enhanced Sensitivity through Nucleic Acid Amplification.

Solid-state nanopores have wide applications in DNA sequencing, energy conversion and storage, seawater desalination, sensors, and reactors due to their high stability, controllable geometry, and a variety of pore-forming materials. Solid-state nanopore sensors can be used for qualitative and quantitative analyses of ions, small molecules, proteins, and nucleic acids. The combination of nucleic acid amplification and solid-state nanopores to achieve trace detection of analytes is gradually attracting attention. This review outlines nucleic acid amplification strategies for enhancing the sensitivity of solid-state nanopore sensors by summarizing the articles published in the past 10 years. The future development prospects and challenges of nucleic acid amplification in solid-state nanopore sensors are discussed. This review helps readers better understand the field of solid-state nanopore sensors. We believe that solid-state nanopore sensors will break through the bottleneck of traditional detection and become a powerful single-molecule detection platform.

Tuning the Envelope Structure of Enzyme Nanoreactors for In Vivo Detoxification of Organophosphates.

Encapsulated phosphotriesterase nanoreactors show their efficacy in the prophylaxis and post-exposure treatment of poisoning by paraoxon. A new enzyme nanoreactor (E-nRs) containing an evolved multiple mutant (L72C/Y97F/Y99F/W263V/I280T) of Saccharolobus solfataricus phosphotriesterase (PTE) for in vivo detoxification of organophosphorous compounds (OP) was made. A comparison of nanoreactors made of three- and di-block copolymers was carried out. Two types of morphology nanoreactors made of di-block copolymers were prepared and characterized as spherical micelles and polymersomes with sizes of 40 nm and 100 nm, respectively. The polymer concentrations were varied from 0.1 to 0.5% (w/w) and enzyme concentrations were varied from 2.5 to 12.5 µM. In vivo experiments using E-nRs of diameter 106 nm, polydispersity 0.17, zeta-potential -8.3 mV, and loading capacity 15% showed that the detoxification efficacy against paraoxon was improved: the LD50 shift was 23.7xLD50 for prophylaxis and 8xLD50 for post-exposure treatment without behavioral alteration or functional physiological changes up to one month after injection. The pharmacokinetic profiles of i.v.-injected E-nRs made of three- and di-block copolymers were similar to the profiles of the injected free enzyme, suggesting partial enzyme encapsulation. Indeed, ELISA and Western blot analyses showed that animals developed an immune response against the enzyme. However, animals that received several injections did not develop iatrogenic symptoms.

Solid-State Nanopore Distinguishes Ferritin and Apo-Ferritin with Identical Exteriors through Amplified Flexibility at Single-Molecule Level.

Protein identification and discrimination at the single-molecule level are big challenges. Solid-state nanopores as a sensitive biosensor have been used for protein analysis, although it is difficult to discriminate proteins with similar structures in the traditional discrimination method based on the current blockage fraction. Here, we select ferritin and apo-ferritin as the model proteins that exhibit identical exterior and different interior structures and verify the practicability of their discrimination with flexibility features by the strategy of gradually decreasing the nanopore size. We show that the larger nanopore (relative to the protein size) has no obvious effect on discriminating two proteins. Then, the comparable-sized nanopore plays a key role in discriminating two proteins based on the dwell time and fraction distribution, and the conformational changes of both proteins are also studied with this nanopore. Finally, in the smaller nanopore, the protein molecules are trapped rather than translocated, where two proteins are obviously discriminated through the current fluctuation caused by the vibration of proteins. This strategy has potential in the discrimination of other important similar proteins.

The role of germanium in diseases: exploring its important biological effects.

With the development of organic germanium and nanotechnology, germanium serves multiple biological functions, and its potential value in biochemistry and medicine has increasingly captured the attention of researchers. In recent years, germanium has gradually gained significance as a material in the field of biomedicine and shows promising application prospects. However, there has been a limited amount of research conducted on the biological effects and mechanisms of germanium, and a systematic evaluation is still lacking. Therefore, the aim of this review is to systematically examine the application of germanium in the field of biomedicine and contribute new insights for future research on the functions and mechanisms of germanium in disease treatment. By conducting a comprehensive search on MEDLINE, EMBASE, and Web of Science databases, we systematically reviewed the relevant literature on the relationship between germanium and biomedicine. In this review, we will describe the biological activities of germanium in inflammation, immunity, and antioxidation. Furthermore, we will discuss its role in the treatment of neuroscience and oncology-related conditions. This comprehensive exploration of germanium provides a valuable foundation for the future application of this element in disease intervention, diagnosis, and prevention.