One-Pot Aqueous Synthesis of Fluorescent Ag-In-Zn-S Quantum Dot/Polymer Bioconjugates for Multiplex Optical Bioimaging of Glioblastoma Cells

Cancer research has experienced astonishing advances recently, but cancer remains a major threat because it is one of the leading causes of death worldwide. Glioblastoma (GBM) is the most malignant brain tumor, where the early diagnosis is vital for longer survival. Thus, this study reports the synthesis of novel water-dispersible ternary AgInS2 (AIS) and quaternary AgInS2-ZnS (ZAIS) fluorescent quantum dots using carboxymethylcellulose (CMC) as ligand for multiplexed bioimaging of malignant glioma cells (U-87 MG). Firstly, AgInS2 core was prepared using a one-pot aqueous synthesis stabilized by CMC at room temperature and physiological pH. Then, an outer layer of ZnS was grown and thermally annealed to improve their optical properties and split the emission range, leading to core-shell alloyed nanostructures. Their physicochemical and optical properties were characterized, demonstrating that luminescent monodispersed AIS and ZAIS QDs were produced with average sizes of 2.2 nm and 4.3 nm, respectively. Moreover, the results evidenced that they were cytocompatible using in vitro cell viability assays towards human embryonic kidney cell line (HEK 293T) and U-87 MG cells. These AIS and ZAIS successfully behaved as fluorescent nanoprobes (red and green, resp.) allowing multiplexed bioimaging and biolabeling of costained glioma cells using confocal microscopy

Comparison of the Effect of Sol-Gel and Coprecipitation Routes on the Properties and Behavior of Nanocomposite Chitosan-Bioactive Glass Membranes for Bone Tissue Engineering

Recent studies in tissue engineering have highlighted the importance of the development of composite materials based on biodegradable polymers containing bioactive glasses, in particular, composites for high load support and excellent cell viability for potential application in bone regeneration. In this work, hybrid composite films were obtained by combining chitosan with bioactive glass in solution form and in nanoparticle dispersion form obtained by the two different synthesis routes: the sol-gel method and coprecipitation. The bioactive glass served both as a mechanical reinforcing agent and as a triggering agent with high bioactivity. The results of in vitro assays with simulated body fluid demonstrated the formation of a significant layer of fibrils on the surface of the film, with a typical morphology of carbonated hydroxyapatite, reflecting induction of a favorable bioactivity. Maximum tensile stress increased from 42 to 80 MPa to the sample with 5% wt bioactive glass. In addition, samples containing 5% and 10% wt bioactive glass showed a significant increase in cell viability, 18 and 30% increase compared to the control group. The samples showed significant response, indicating that they could be a potential material for use in bone regeneration through tissue engineering

Niobium-Doped Hydroxyapatite Bioceramics: Synthesis, Characterization and In Vitro Cytocompatibility

Doping calcium phosphates with ionic species can play an important role in biological responses promoting alkaline phosphatase activity, and, therefore inducing the generation of new bone. Thus, in this study, the synthesis of niobium-doped hydroxyapatite (Nb-HA) nanosize particles obtained by the precipitation process in aqueous media followed by thermal treatment is presented. The bioceramics were extensively characterized by X-ray diffraction, wavelength dispersive X-ray fluorescence spectrometry, Fourier transform infrared spectroscopy, scanning electron microscopy/energy dispersive X-ray spectroscopy analysis, transmission electron microscopy, atomic force microscopy and thermal analysis regarding their chemical composition, structure and morphology. The results showed that the precipitate dried at 110 °C was composed of amorphous calcium phosphate and HA, with polidisperse particles ranging from micro to nano dimensions. After the thermal treatment at 900 °C, the bioceramic system evolved predominantly to HA crystalline phase, with evident features of particle sintering and reduction of surface area. Moreover, the addition of 10 mol% of niobium salt precursor during the synthesis indicated the complete incorporation of the Nb(V) species in the HA crystals with detectable changes in the original lattice parameters. Furthermore, the incorporation of Nb ions caused a significant refinement on the average particle size of HA. Finally, the preliminary cytocompatibility response of the biomaterials was accessed by human osteoblast cell culture using MTT and resazurin assays, which demonstrated no cytotoxicity of the Nb-alloyed hydroxyapatite. Thus, these findings seem promising for developing innovative Nb-doped calcium phosphates as artificial biomaterials for potential use in bone replacements and repair

Nanozymes with Peroxidase-like Activity for Ferroptosis-Driven Biocatalytic Nanotherapeutics of Glioblastoma Cancer: 2D and 3D Spheroids Models

Glioblastoma (GBM) is the most common primary brain cancer in adults. Despite the remarkable advancements in recent years in the realm of cancer diagnosis and therapy, regrettably, GBM remains the most lethal form of brain cancer. In this view, the fascinating area of nanotechnology has emerged as an innovative strategy for developing novel nanomaterials for cancer nanomedicine, such as artificial enzymes, termed nanozymes, with intrinsic enzyme-like activities. Therefore, this study reports for the first time the design, synthesis, and extensive characterization of innovative colloidal nanostructures made of cobalt-doped iron oxide nanoparticles chemically stabilized by a carboxymethylcellulose capping ligand (i.e., Co-MION), creating a peroxidase-like (POD) nanozyme for biocatalytically killing GBM cancer cells. These nanoconjugates were produced using a strictly green aqueous process under mild conditions to create non-toxic bioengineered nanotherapeutics against GBM cells. The nanozyme (Co-MION) showed a magnetite inorganic crystalline core with a uniform spherical morphology (diameter, 2R = 6–7 nm) stabilized by the CMC biopolymer, producing a hydrodynamic diameter (HD) of 41–52 nm and a negatively charged surface (ZP~−50 mV). Thus, we created supramolecular water-dispersible colloidal nanostructures composed of an inorganic core (Cox-MION) and a surrounding biopolymer shell (CMC). The nanozymes confirmed the cytotoxicity evaluated by an MTT bioassay using a 2D culture in vitro of U87 brain cancer cells, which was concentration-dependent and boosted by increasing the cobalt-doping content in the nanosystems. Additionally, the results confirmed that the lethality of U87 brain cancer cells was predominantly caused by the production of toxic cell-damaging reactive oxygen species (ROS) through the in situ generation of hydroxyl radicals (·OH) by the peroxidase-like activity displayed by nanozymes. Thus, the nanozymes induced apoptosis (i.e., programmed cell death) and ferroptosis (i.e., lipid peroxidation) pathways by intracellular biocatalytic enzyme-like activity. More importantly, based on the 3D spheroids model, these nanozymes inhibited tumor growth and remarkably reduced the malignant tumor volume after the nanotherapeutic treatment (ΔV~40%). The kinetics of the anticancer activity of these novel nanotherapeutic agents decreased with the time of incubation of the GBM 3D models, indicating a similar trend commonly observed in tumor microenvironments (TMEs). Furthermore, the results demonstrated that the 2D in vitro model overestimated the relative efficiency of the anticancer agents (i.e., nanozymes and the DOX drug) compared to the 3D spheroid models. These findings are notable as they evidenced that the 3D spheroid model resembles more precisely the TME of “real” brain cancer tumors in patients than 2D cell cultures. Thus, based on our groundwork, 3D tumor spheroid models might be able to offer transitional systems between conventional 2D cell cultures and complex biological in vivo models for evaluating anticancer agents more precisely. These nanotherapeutics offer a wide avenue of opportunities to develop innovative nanomedicines for fighting against cancerous tumors and reducing the frequency of severe side effects in conventionally applied chemotherapy-based treatments

Bioengineered Carboxymethylcellulose–Peptide Hybrid Nanozyme Cascade for Targeted Intracellular Biocatalytic–Magnetothermal Therapy of Brain Cancer Cells

Glioblastoma remains the most lethal form of brain cancer, where hybrid nanomaterials biofunctionalized with polysaccharide peptides offer disruptive strategies relying on passive/active targeting and multimodal therapy for killing cancer cells. Thus, in this research, we report for the first time the rational design and synthesis of novel hybrid colloidal nanostructures composed of gold nanoparticles stabilized by trisodium citrate (AuNP@TSC) as the oxidase-like nanozyme, coupled with cobalt-doped superparamagnetic iron oxide nanoparticles stabilized by carboxymethylcellulose ligands (Co-MION@CMC) as the peroxidase-like nanozyme. They formed inorganic–inorganic dual-nanozyme systems functionalized by a carboxymethylcellulose biopolymer organic shell, which can trigger a biocatalytic cascade reaction in the cancer tumor microenvironment for the combination of magnetothermal–chemodynamic therapy. These nanoassemblies were produced through a green aqueous process under mild conditions and chemically biofunctionalized with integrin-targeting peptide (iRDG), creating bioengineered nanocarriers. The results demonstrated that the oxidase-like nanozyme (AuNP) was produced with a crystalline face-centered cubic nanostructure, spherical morphology (diameter = 16 ± 3 nm), zeta potential (ZP) of −50 ± 5 mV, and hydrodynamic diameter (DH) of 15 ± 1 nm. The peroxide-like nanostructure (POD, Co-MION@CMC) contained an inorganic crystalline core of magnetite and had a uniform spherical shape (2R = 7 ± 1 nm) which, summed to the contribution of the CMC shell, rendered a hydrodynamic diameter of 45 ± 4 nm and a negative surface charge (ZP = −41 ± 5 mV). Upon coupling both nanozymes, water-dispersible colloidal supramolecular vesicle-like organic–inorganic nanostructures were produced (AuNP//Co-MION@CMC, ZP = −45 ± 4 mV and DH = 28 ± 3 nm). They confirmed dual-nanozyme cascade biocatalytic activity targeted by polymer–peptide conjugates (AuNP//Co-MION@CMC_iRGD, ZP = −29 ± 3 mV and DH = 60 ± 4 nm) to kill brain cancer cells (i.e., bioenergy “starvation” by glucose deprivation and oxidative stress through reactive oxygen species generation), which was boosted by the magneto-hyperthermotherapy effect when submitted to the alternating magnetic field (i.e., induced local thermal stress by “nanoheaters”). This groundwork offers a wide avenue of opportunities to develop innovative theranostic nanoplatforms with multiple integrated functionalities for fighting cancer and reducing the harsh side effects of conventional chemotherapy

Viabilidade celular de nanofibras de polímeros biodegradáveis e seus nanocompósitos com argila montmorilonita Cell viability of nanofibers from biodegradable polymers and their nanocomposites with montmorillonite

Mantas não tecidas de nanofibras de três polímeros biodegradáveis poli(ácido láctico), PDLLA, poli(Ε-caprolactona), PCL, e poli(butileno adipato-co-tereftalato), PBAT e seus nanocompósitos com uma nanoargila montmorilonita (MMT) foram produzidas por eletrofiação. A morfologia, o comportamento térmico e a estrutura interna das nanofibras foram analisados por microscopia eletrônica de varredura e transmissão, calorimetria diferencial de varredura e difração de raios X, respectivamente. Observou-se que as nanofibras dos nanocompósitos possuíam diâmetros menores do que os correspondentes polímeros puros e que as nanofibras de PBAT puro e de PBAT/MMT apresentavam a menor cristalinidade de todas as mantas. A viabilidade celular de todas as nanofibras foi analisada pela técnica de redução do sal de tetrazolium pelo complexo enzimático piruvato desidrogenase presente na matriz de mitocôndrias (teste MTT). Os resultados mostraram que nenhuma manta nanofibrílica apresentou toxicidade às células e que as nanofibras de PBAT puro e seu nanocompósito propiciaram ainda um ambiente mais favorável ao desenvolvimento celular de fibroblastos de cardiomiócitos do que as condições oferecidas pelo controles, provavelmente por apresentarem menores diâmetros e baixa cristalinidade em relação às demais nanofibras. Estes resultados mostram o potencial de uso destas mantas nanofibrílicas como suportes de crescimento celular.Non-woven mats of nanofibers of three biodegradable polymers, viz. poly(lactic acid), PDLLA, poly(Ε-caprolactone), PCL, and poly(butylene adipate-co-terephthalate), PBAT, and their nanocomposites with montmorillonite nanoclay (MMT) were produced by electrospinning. The morphology, thermal behavior and internal structure of the nanofibers were analyzed by scanning and transmission electron microscopy, differential scanning calorimetry and wide angle X-ray diffraction, respectively. The nanofibers of the nanocomposites had lower diameters than the nanofibers of the corresponding neat polymers, while the nanofibers from PBAT and PBAT/MMT were the least crystalline. The cell viability of all the nanofibers was analyzed by reduction of the tetrazolium salt by the pyruvate dehydrogenase enzymatic complex present in the mitochondria (MTT test). None of the nanofibers was toxic to the cells and the PBAT and PBAT/MMT nanofibers exhibited a more favorable environment for developing fibroblasts from cardiomyocytes than the control, probably due to their low crystallinity. These results demonstrated the potential use of nanofibers´ mats as scaffolds for cell growth

Design and Development of Polysaccharide-Doxorubicin-Peptide Bioconjugates for Dual Synergistic Effects of Integrin-Targeted and Cell-Penetrating Peptides for Cancer Chemotherapy

Polymer-drug conjugation is an attractive approach for target delivering insoluble and highly toxic drugs to tumor sites to overcome the side-effects caused by cancer chemotherapy. In this study we designed and synthesized novel polymer-drug-peptide conjugates for improved specificity on targeting cancer cells. Chemically modified polysaccharide, carboxymethylcellulose (CMC), was conjugated with doxorubicin (DOX) anticancer drug by amide bonds and dually biofunctionalized with integrin-target receptor tripeptide (RGD) and l-arginine (R) as cell-penetrating amino acid for synergistic targeting and enhancing internalization by cancer cells. These bioconjugates were tested as prodrugs against bone, breast, and brain cancer cell lines (SAOS, MCF7, and U87) and a normal cell line (HEK 293T, reference). The physicochemical characterization showed the formation of amide bonds between carboxylates (−RCOO^–) from CMC biopolymer and amino groups (−NH₂) from DOX and peptides (RGD or R). Moreover, these polymer-drug-peptide bioconjugates formed nanoparticulate colloidal structures and behaved as “smart” drug delivery systems (DDS) promoting remarkable reduction of the cytotoxicity toward normal cells (HEK 293T) while retaining high killing activity against cancer cells. Based on cell viability bioassays, DNA-staining, and confocal laser microscopy, this effect was assigned to the association of physicochemical aspects with the difference of the endocytic pathways and the drug release rates in live cells caused by the biofunctionalization of the macromolecule-drug systems with RGD and l-arginine. In addition, chick chorioallantoic membrane (CAM) assay was performed as an in vivo xenograft model test, which endorsed the in vitro results of anticancer activities of these polymer-drug systems. Thus, prodrug nanocarriers based on CMC-DOX-peptide bioconjugates were developed for simultaneously integrin-targeting and high killing efficacy against cancer cells, while preserving healthy cells with promising perspectives in cancer chemotherapy