Opportunities for nanomaterials in enzyme therapy.

In recent years, enzyme therapy strategies have rapidly evolved to catalyze essential biochemical reactions with therapeutic potential. These approaches hold particular promise in addressing rare genetic disorders, cancer treatment, neurodegenerative conditions, wound healing, inflammation management, and infectious disease control, among others. There are several primary reasons for the utilization of enzymes as therapeutics: their substrate specificity, their biological compatibility, and their ability to generate a high number of product molecules per enzyme unit. These features have encouraged their application in enzyme replacement therapy where the enzyme serves as the therapeutic agent to rectify abnormal metabolic and physiological processes, enzyme prodrug therapy where the enzyme initiates a clinical effect by activating prodrugs, and enzyme dynamic or starving therapy where the enzyme acts upon host substrate molecules. Currently, there are >20 commercialized products based on therapeutic enzymes, but approval rates are considerably lower than other biologicals. This has stimulated nanobiotechnology in the last years to develop nanoparticle-based solutions that integrate therapeutic enzymes. This approach aims to enhance stability, prevent rapid clearance, reduce immunogenicity, and even enable spatio-temporal activation of the therapeutic catalyst. This comprehensive review delves into emerging trends in the application of therapeutic enzymes, with a particular emphasis on the synergistic opportunities presented by incorporating enzymes into nanomaterials. Such integration holds the promise of enhancing existing therapies or even paving the way for innovative nanotherapeutic approaches.

Biophysical evaluation of antiparallel triplexes for biosensing and biomedical applications.

Polypyrimidine sequences can be targeted by antiparallel clamps forming triplex structures either for biosensing or therapeutic purposes. Despite its successful implementation, their biophysical properties remain to be elusive. In this work, PAGE, circular dichroism and multivariate analysis were used to evaluate the properties of PPRHs directed to SARS-CoV-2 genome. Several PPRHs designed to target various polypyrimidine sites within the viral genome were synthesized. These PPRHs displayed varying binding affinities, influenced by factors such as the length of the PPRH and its GC content. The number and position of pyrimidine interruptions relative to the 4 T loop of the PPRH was found a critical factor, affecting the binding affinity with the corresponding target. Moreover, these factors also showed to affect in the intramolecular and intermolecular equilibria of PPRHs alone and when hybridized to their corresponding targets, highlighting the polymorphic nature of these systems. Finally, the functionality of the PPRHs was evaluated in a thermal lateral flow sensing device showing a good correspondence between their biophysical properties and detection limits. These comprehensive studies contribute to the understanding of the critical factors involved in the design of PPRHs for effective targeting of biologically relevant genomes through the formation of triplex structures under neutral conditions.

A Simple and Versatile Strategy for Oriented Immobilization of His-Tagged Proteins on Magnetic Nanoparticles.

Oriented and covalent immobilization of proteins on magnetic nanoparticles (MNPs) is particularly challenging as it requires both the functionality of the protein and the colloidal stability of the MNPs to be preserved. Here, we describe a simple, straightforward, and efficient strategy for MNP functionalization with proteins using metal affinity binding. Our method involves a single-step process where MNPs are functionalized using a preformed, ready-to-use nitrilotriacetic acid-divalent metal cation (NTA-M2+) complex and polyethylene glycol (PEG) molecules. As a proof-of-concept, we demonstrate the oriented immobilization of a recombinant cadherin fragment engineered with a hexahistidine tag (6His-tag) onto the MNPs. Our developed methodology is simple and direct, enabling the oriented bioconjugation of His-tagged cadherins to MNPs while preserving protein functionality and the colloidal stability of the MNPs, and could be extended to other proteins expressing a polyhistidine tag. When compared to the traditional method where NTA is first conjugated to the MNPs and afterward free metal ions are added to form the complex, this novel strategy results in a higher functionalization efficiency while avoiding MNP aggregation. Additionally, our method allows for covalent bonding of the cadherin fragments to the MNP surface while preserving functionality, making it highly versatile. Finally, our strategy not only ensures the correct orientation of the protein fragments on the MNPs but also allows for the precise control of their density. This feature enables the selective targeting of E-cadherin-expressing cells only when MNPs are decorated with a high density of cadherin fragments.

Extracellular vesicles as a source of prostate cancer biomarkers in liquid biopsies: a decade of research.

Prostate cancer is a global cancer burden and considerable effort has been made through the years to identify biomarkers for the disease. Approximately a decade ago, the potential of analysing extracellular vesicles in liquid biopsies started to be envisaged. This was the beginning of a new exciting area of research investigating the rich molecular treasure found in extracellular vesicles to identify biomarkers for a variety of diseases. Vesicles released from prostate cancer cells and cells of the tumour microenvironment carry molecular information about the disease that can be analysed in several biological fluids. Numerous studies document the interest of researchers in this field of research. However, methodological issues such as the isolation of vesicles have been challenging. Remarkably, novel technologies, including those based on nanotechnology, show promise for the further development and clinical use of extracellular vesicles as liquid biomarkers. Development of biomarkers is a long and complicated process, and there are still not many biomarkers based on extracellular vesicles in clinical use. However, the knowledge acquired during the last decade constitutes a solid basis for the future development of liquid biopsy tests for prostate cancer. These are urgently needed to bring prostate cancer treatment to the next level in precision medicine.

A tumor microenvironment responsive biodegradable CaCO3/MnO2- based nanoplatform for the enhanced photodynamic therapy and improved PD-L1 immunotherapy.

The low efficiency of photodynamic therapy (PDT) is caused by tumor hypoxia and the adaptive immune resistance/evasion of tumor cells, while the currently emerging immune checkpoint therapy restores the intrinsic immune capacities but can't directly attack the tumor cells. Methods: Herein we report an integrated nanoplatform that combines PDT with immunotherapy to enhance photodynamic therapeutic effects and simultaneously inhibit tumor cells resistance/evasion. To achieve this, we fabricated Mn@CaCO3/ICG nanoparticles and loaded them with PD-L1-targeting siRNA. Results: Thanks to the protection of CaCO3 on the loaded ICG and the oxygen produced by MnO2, an enhanced photodynamic therapeutic effect in vitro was observed. In vivo experiments demonstrated that the nanoplatform could efficiently deliver the loaded drug to the tumor tissues and significantly improve tumor hypoxia, which further contributes to the therapeutic effect of PDT in vivo. Moreover, the synergistic benefits derived from the siRNA, which silenced the checkpoint gene PD-L1 that mediates the immune resistance/evasion, resulted in a surprising therapeutic effect to rouse the immune system. Conclusions: The combination treatment strategy has great potential to be developed as a new and robust method for enhanced PDT therapy with high efficiency and a powerful antitumor immune response based on PD-L1 blockade.

Mn-Doping level dependence on the magnetic response of MnxFe3-xO4 ferrite nanoparticles.

Manganese/iron ferrite nanoparticles with different Mn2+/3+ doping grades have been prepared by a thermal decomposition optimized approach so as to ascertain the doping effect on magnetic properties and, especially, on the magnetic hyperthermia response. The oxidation state and interstitial position of Mn in the spinel structure is found to be critical. The particle size effect has also been studied by growing one of the prepared samples (from 10 to 15 nm in diameter) by a seed mediated growth mechanism. After analyzing the main structural and chemical parameters such as the Mn/Fe rate, crystalline structure, particle diameter, shape and organic coating, some Mn doping induced changes have been observed, such as the insertion of Mn2+ cations yielded more anisotropic shapes. Magnetic characterization, carried out by DC magnetometry (M(H), M(T)) and electron magnetic resonance (EMR) techniques, has shown interesting differences between samples with varying compositions. Lower Mn doping levels lead to larger saturation magnetization values, while an increase of the Mn content causes the decrease of the effective magnetic anisotropy constant at low T. The homogeneous magnetic response under applied magnetic fields, together with the great effect of nanoparticle size and shape in such a response, has been confirmed by the EMR analysis. Finally, a detailed magnetic hyperthermia analysis has demonstrated the large influence of NP size and shape on the magnetic hyperthermia response. The optimized Mn0.13Fe2.87O4_G sample with a diameter of 15 nm and slightly truncated octahedral shape is presented as an interesting candidate for future magnetic hyperthermia mediated biomedical treatments.

Triggering antitumoural drug release and gene expression by magnetic hyperthermia.

Magnetic nanoparticles (MNPs) are promising tools for a wide array of biomedical applications. One of their most outstanding properties is the ability to generate heat when exposed to alternating magnetic fields, usually exploited in magnetic hyperthermia therapy of cancer. In this contribution, we provide a critical review of the use of MNPs and magnetic hyperthermia as drug release and gene expression triggers for cancer therapy. Several strategies for the release of chemotherapeutic drugs from thermo-responsive matrices are discussed, providing representative examples of their application at different levels (from proof of concept to in vivo applications). The potential of magnetic hyperthermia to promote in situ expression of therapeutic genes using vectors that contain heat-responsive promoters is also reviewed in the context of cancer gene therapy.

RGD-Functionalized Fe3O4 nanoparticles for magnetic hyperthermia.

To improve the selectivity of magnetic nanoparticles for tumor treatment by hyperthermia, Fe3O4 nanoparticles have been functionalized with a peptide of the type arginine-glycine-aspartate (RGD) following a "click" chemistry approach. The RGD peptide was linked onto the previously coated nanoparticles in order to target αvß3 integrin receptors over-expressed in angiogenic cancer cells. Different coatings have been analyzed to enhance the biocompatibility of magnetic nanoparticles. Monodispersed and homogeneous magnetite nanoparticles have been synthesized by the seed growth method and have been characterized using X-ray diffraction, thermogravimetric analysis, infrared spectroscopy, transmission electron microscopy and magnetic measurements. The magnetic hyperthermia efficiency of the nanoparticles has also been investigated and cytotoxicity assays have been perfomed for functionalized nanoparticles.

Effect of Surface Chemistry and Associated Protein Corona on the Long-Term Biodegradation of Iron Oxide Nanoparticles In Vivo.

The protein corona formed on the surface of a nanoparticle in a biological medium determines its behavior in vivo. Herein, iron oxide nanoparticles containing the same core and shell, but bearing two different surface coatings, either glucose or poly(ethylene glycol), were evaluated. The nanoparticles' protein adsorption, in vitro degradation, and in vivo biodistribution and biotransformation over four months were investigated. Although both types of nanoparticles bound similar amounts of proteins in vitro, the differences in the protein corona composition correlated to the nanoparticles biodistribution in vivo. Interestingly, in vitro degradation studies demonstrated faster degradation for nanoparticles functionalized with glucose, whereas the in vivo results were opposite with accelerated biodegradation and clearance of the nanoparticles functionalized with poly(ethylene glycol). Therefore, the variation in the degradation rate observed in vivo could be related not only to the molecules attached to the surface, but also with the associated protein corona, as the key role of the adsorbed proteins on the magnetic core degradation has been demonstrated in vitro.

Nanoparticles for multi-modality cancer diagnosis: Simple protocol for self-assembly of gold nanoclusters mediated by gadolinium ions.

It is essential to develop a simple synthetic strategy to improve the quality of multifunctional contrast agents for cancer diagnosis. Herein, we report a time-saving method for gadolinium (Gd3+) ions-mediated self-assembly of gold nanoclusters (GNCs) into monodisperse spherical nanoparticles (GNCNs) under mild conditions. The monodisperse, regular and colloidal stable GNCNs were formed via selectively inducing electrostatic interactions between negatively-charged carboxylic groups of gold nanoclusters and trivalent cations of gadolinium in aqueous solution. In this way, the Gd3+ ions were chelated into GNCNs without the use of molecular gadolinium chelates. With the co-existence of GNCs and Gd3+ ions, the formed GNCNs exhibit significant luminescence intensity enhancement for near-infrared fluorescence (NIRF) imaging, high X-ray attenuation for computed tomography (CT) imaging and reasonable r1 relaxivity for magnetic resonance (MR) imaging. The excellent biocompatibility of the GNCNs was proved both in vitro and in vivo. Meanwhile, the GNCNs also possess unique NIRF/CT/MR imaging ability in A549 tumor-bearing mice. In a nutshell, the simple and safe GNCNs hold great potential for tumor multi-modality clinical diagnosis.

A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification).

pH-Sensitive self-assembling nanoparticles for tumor near-infrared fluorescence imaging and chemo-photodynamic combination therapy.

The development of visual tumor theranostic nanoparticles has become a great challenge. In this study, d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) was conjugated to acid-sensitive cis-aconitic anhydride-modified doxorubicin (CAD) to obtain pH-sensitive anti-tumor prodrug nanoparticles (TCAD NPs) via self-assembling. Subsequently, the photosensitizer chlorin e6 (Ce6) was loaded into the resulting prodrug nanoparticles to prepare a novel tumor near-infrared fluorescence imaging and chemo-photodynamic combination therapy system (TCAD@Ce6 NPs). An accelerated release of doxorubicin (DOX) and chlorin e6 (Ce6) from the TCAD@Ce6 NPs could be achieved due to the hydrolysis of the acid-sensitive amide linker under mild acidic conditions (pH = 5.5). An in vitro experiment showed that A549 lung cancer cells exhibited a significantly higher uptake of DOX and Ce6 by using our delivery system than the free form of DOX and Ce6. An in vivo experiment showed that TCAD@Ce6 NPs displayed better tumor targeting gathering through the enhanced permeability and retention (EPR) effect than free Ce6, thus improving fluorescence imaging. Moreover, the chemo-photodynamic combination therapy of TCAD@Ce6 NPs combined with near-infrared laser irradiation was confirmed to be capable of inducing high apoptosis and necrosis of tumor cells (A549) in vitro and to display a significantly higher tumor growth suppression in the A549 lung cancer-bearing mice model. Furthermore, compared with exclusive chemotreatment (DOX) or photodynamic treatment (Ce6), our system showed enhanced therapeutic effects both in vitro and in vivo. In conclusion, the high performance TCAD@Ce6 NPs can be used as a promising NIR fluorescence imaging and highly effective chemo-photodynamic system for theranostics of lung cancer, etc. in the near future.

Presentation of a nano-based tag for immunoassay, based on amine-modified bovine serum albumin nanoparticles.

The aim of this study was to evaluate four immunoassays, based on amine-modified bovine serum albumin nanoparticles (AMBSANPs). First, the capability of nitrate absorption by AMBSANPs under different conditions was evaluated. Then, serial concentrations of pure ßHCG were added to wells coated with ßHCG antibody for immunoassays 1 and 2, and wells coated with ßHCG aptamer for immunoassays 3 and 4. Next, AMBSANPs conjugated with ßHCG antibody was added for immunoassays 1 and 3, and AMBSANPs conjugated with ßHCG aptamer were added for immunoassays 2 and 4. Finally, the optical density (OD) of each well was read at 340 nm, and compared with controls. Moreover, the concentration of ßHCG in the clinical samples was quantified by immunoassays 1, 2, 3, 4 and ELISA, and then compared. The effect of some serum interferences on these immunoassay methods was evaluated. The authors observed that the amount of nitrate absorption by AMBSANPs increased with an increase in H+ ion concentration and temperature, and decreased with an increase in ion strength. The correlation (R2) between ELISA and immunoassays 1, 2, 3 and 4 were 0.97, 0.97, 0.98, 0.99, respectively. It was found that the increase in the serum interferences led to a decrease in the measured ßHCG concentration.

Silk fibroin nanoparticles constitute a vector for controlled release of resveratrol in an experimental model of inflammatory bowel disease in rats.

PURPOSE: We aimed to evaluate the intestinal anti-inflammatory properties of silk fibroin nanoparticles, around 100 nm in size, when loaded with the stilbene compound resveratrol, in an experimental model of rat colitis. METHODS: Nanoparticles were loaded with resveratrol by adsorption. The biological effects of the resveratrol-loaded nanoparticles were tested both in vitro, in a cell culture of RAW 264.7 cells (mouse macrophages), and in vivo, in the trinitrobenzenesulfonic acid model of rat colitis, when administered intracolonically. RESULTS: The resveratrol liberation in 1× phosphate-buffered saline (PBS; pH 7.4) was characterized by fast liberation, reaching the solubility limit in 3 hours, which was maintained over a period of 80 hours. The in vitro assays revealed immunomodulatory properties exerted by these resveratrol-loaded nanoparticles since they promoted macrophage activity in basal conditions and inhibited this activity when stimulated with lipopolysaccharide. The in vivo experiments showed that after evaluation of the macroscopic symptoms, inflammatory markers, and intestinal barrier function, the fibroin nanoparticles loaded with resveratrol had a better effect than the single treatments, being similar to that produced by the glucocorticoid dexamethasone. CONCLUSION: Silk fibroin nanoparticles constitute an attractive strategy for the controlled release of resveratrol, showing immunomodulatory properties and intestinal anti-inflammatory effects.

Photocrosslinking, micropatterning and cell adhesion studies of sodium hyaluronate with a trisdiazonium salt.

Chemically unmodified sodium hyaluronate has been crosslinked by photoinduced decomposition of a trifunctional diazonium salt to generate new biomaterials. In addition, the photocrosslinking process does not require a photoinitiator. Thin films of formulations of sodium hyaluronate and the photocrosslinker at different percentages have been processed. Cytotoxicity has been explored and toxicity was not observed with the selected cell lines. 2D patterns of controlled geometry have been generated by direct laser writing to perform cell adhesion studies. Different adhesion behavior of the cell lines, as assessed by vinculin immunostaining and scanning electron microscopy, has been observed in the polymeric films depending on the degree of photocrosslinking.

Paramagnetic Gd-based gold glyconanoparticles as probes for MRI: tuning relaxivities with sugars.

Combining sugar conjugates and DO3A-Gd complexes, paramagnetic gold glyconanoparticles (GNPs) with different relaxivity values were obtained and tested in vivo as MRI probes.