High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4.

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods may have important implications in drug development and discovery. Here, we present the first approach that employs computational high-throughput virtual screening and molecular docking to search for small molecules in chemical libraries that function as mechano-regulators of the stability of human cluster of differentiation 4, receptor of HIV-1. Using single-molecule force spectroscopy, we prove that these small molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, that is, mechano-regulation, paving the way toward mechanopharmacology.

LGR5 as a Therapeutic Target of Antibody-Functionalized Biomimetic Magnetoliposomes for Colon Cancer Therapy.

Purpose: The lack of specificity of conventional chemotherapy is one of the main difficulties to be solved in cancer therapy. Biomimetic magnetoliposomes are successful chemotherapy controlled-release systems, hyperthermia, and active targeting agents by functionalization of their surface with monoclonal antibodies. The membrane receptor Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) stands out as colorectal cancer (CRC) biomarker and appears to be related to treatment resistance and the development of metastasis. The aim of this study was to assess the effectiveness and safety of LGR5-targeted biomimetic magnetoliposomes loaded with oxaliplatin (OXA) or 5-fluorouracil (5-FU) in the selective treatment of CRC and their possible application in hyperthermia. Methods: Synthesis, characterization and determination of heating capacity of magnetoliposomes transporting OXA or 5-FU (with and without LGR5 functionalization) were conducted. In vitro antitumoral activity was assayed in multiple colorectal cell lines at different times of exposition. In addition to this, cell internalization was studied by Prussian Blue staining, flow cytometry and fluorescence microscopy. In vivo acute toxicity of magnetoliposomes was performed to evaluate iron-related toxicity. Results: OXA and 5-FU loaded magnetoliposomes functionalized with LGR5 antibody showed higher cellular uptake than non-targeted nanoformulation with a reduction of the percentage of proliferation in colon cancer cell lines up to 3.2-fold of the IC50 value compared to that of free drug. The differences between non-targeted and targeted nanoformulations were more evident after short exposure times (4 and 8 hours). Interestingly, assays in the MC38 transduced cells with reduced LGR5 expression (MC38-L(-)), showed lower cell internalization of LGR5-targeted magnetoliposomes compared to non-transduced MC38 cell line. In addition, magnetoliposomes showed an in vitro favorable heating response under magnetic excitation and great iron-related biocompatibility data in vivo. Conclusion: Drug-loaded magnetoliposomes functionalized with anti-LGR5 antibodies could be a promising CRC treatment strategy for LGR5+ targeted chemotherapy, magnetic hyperthermia, and both in combination.

mAb-Functionalized Biomimetic MamC-Mediated-Magnetoliposomes as Drug Delivery Systems for Cancer Therapy.

In cancer therapy, new therapeutic nanoformulations able to mediate targeted chemotherapy are required. Recently, biomimetic magnetic nanoparticles (BMNPs) mediated by MamC, a magnetosome protein from Magnetococcus marinus MC-1, have proven, in vitro and in vivo, to be effective drug nanocarriers (following the application of an external gradient magnetic field) and to allow combination with hyperthermia. However, these nanoassemblies require further optimization to improve cytocompatibility, stability and active targeting ability. Herein, we describe the production of the magnetoliposomes (LP) embedding BMNPs functionalized (or not) with doxorubicin (DOXO), [LP(+/-DOXO-BMNPs)], and their surface modification with the DO-24 mAb, which targets the human Met/HGF receptor's ectodomain (overexpressed in many cancers). Nanoformulations were extensively characterized using TEM, DLS, FTIR and when tested in vitro, the lipid coating increased the colloidal stability and their biocompatibility, favoring the cellular uptake in cells overexpressing the cognate receptor. Indeed, the magnetoliposomes mAb-LP(+/-DOXO-BMNPs) exerted a specific active targeting ability by the presence of the mAb that preserved its immunocompetence. Both LP(BMNPs) and mAb-LP(BMNPs) were not toxic to cells, while +/-mAb-LP(DOXO-BMNPs) nanoformulations were indeed cytotoxic. Therefore, this study represents a proof of concept for the development of promising drug carriers for cancer therapy based on local chemotherapy directed by mAbs.

Magnetite Mineralization inside Cross-Linked Protein Crystals.

Crystallization in confined spaces is a widespread process in nature that also has important implications for the stability and durability of many man-made materials. It has been reported that confinement can alter essential crystallization events, such as nucleation and growth and, thus, have an impact on crystal size, polymorphism, morphology, and stability. Therefore, the study of nucleation in confined spaces can help us understand similar events that occur in nature, such as biomineralization, design new methods to control crystallization, and expand our knowledge in the field of crystallography. Although the fundamental interest is clear, basic models at the laboratory scale are scarce mainly due to the difficulty in obtaining well-defined confined spaces allowing a simultaneous study of the mineralization process outside and inside the cavities. Herein, we have studied magnetite precipitation in the channels of cross-linked protein crystals (CLPCs) with different channel pore sizes, as a model of crystallization in confined spaces. Our results show that nucleation of an Fe-rich phase occurs inside the protein channels in all cases, but, by a combination of chemical and physical effects, the channel diameter of CLPCs exerted a precise control on the size and stability of those Fe-rich nanoparticles. The small diameters of protein channels restrain the growth of metastable intermediates to around 2 nm and stabilize them over time. At larger pore diameters, recrystallization of the Fe-rich precursors into more stable phases was observed. This study highlights the impact that crystallization in confined spaces can have on the physicochemical properties of the resulting crystals and shows that CLPCs can be interesting substrates to study this process.

Nanoassemblies of acetylcholinesterase and ß-lactamase immobilized on magnetic nanoparticles as biosensors to detect pollutants in water.

The use of enzymes immobilized on magnetic nanoparticles to detect contaminants in aqueous samples has gained interest, since it allows the magnetic control, concentration and reuse of the enzymes. In this work, the detection of trace amounts of organophosphate pesticides (chlorpyrifos) and antibiotics (penicillin G) in water was attained by developing a nanoassembly formed by either inorganic or biomimetic magnetic nanoparticles used as substrates to immobilize acetylcholinesterase (AChE) and ß-lactamase (BL). Other than the substrate, the optimization of the nanoassembly was done by testing enzyme immobilization both through electrostatic interaction (also reinforced with glutaraldehyde) and covalent bonds (by carbodiimide chemistry). Temperature (25 °C), ionic strength (150 mM NaCl) and pH (7) were set to ensure enzymatic stability and to allow both the nanoparticles and the enzymes to present ionic charges that would allow electrostatic interaction. Under these conditions, the enzyme load on the nanoparticles was ⁓0.1 mg enzyme per mg nanoparticles, and the preserved activity after immobilization was 50-60% of the specific activity of the free enzyme, being covalent bonding the one which yielded better results. Covalent nanoassemblies could detect trace concentrations of pollutants down to 1.43 nM chlorpyrifos and 0.28 nM penicillin G. They even permitted the quantification of 14.3 µM chlorpyrifos and 2.8 µM penicillin G. Also, immobilization conferred higher stability to AChE (⁓94% activity after 20 days storage at 4 °C) and allowed to reuse the BL up to 12 cycles.

Effects of Magnetic Nanoparticles on the Functional Activity of Human Monocytes and Dendritic Cells.

The use of nanoparticles in medicine is sometimes hampered by their potential to activate immune cells, eliciting inflammation or allergy. We investigated whether magnetic nanoparticles (MNPs) or biomimetic magnetic nanoparticles (BMNPs) affect relevant activities of human monocytes. We found that the nanoparticles neither elicited the production of pro-inflammatory mediators IL-6 and TNFα by resting monocytes (when BMNP dose < 300 µg/mL) nor enhanced their secretion induced by R848, a molecule engaging virus-recognizing receptors, or bacterial lipopolysaccharide (LPS). MNPs and BMNPs neither induced the generation of reactive oxygen species (ROS), nor affected the ROS production elicited by the NADPH oxidase activator phorbol myristate acetate (PMA) or the fungal derivative ß-glucan. BMNPs, but not MNPs, caused an up-regulation of the maturation markers CD80, CD83, and CD86 in immature monocyte-derived dendritic cells (DCs), whereas both nanoparticles did not affect the LPS-induced expression of these markers. Moreover, the nanoparticles were greedily ingested by monocytes and DCs without altering their viability. Therefore, these nanoparticles are candidates for medical applications because they do not activate pro-inflammatory activities of monocytes. Furthermore, their ability to stimulate DC maturation could be used for the design of vaccines. Moreover, harmlessly engulfed nanoparticles could be vehicles to carry molecules inside the immune cells to regulate the immune response.

Antimicrobial defenses of table eggs: Importance of antibacterial proteins in egg white as a function of hen age in an extended production cycle.

The importance of egg natural defences to prevent bacterial contamination and their relation with hen age in extended production cycles were evaluated. Egg-white from eggs of different hen age groups (up 100-weeks-old) and lines (Hy-Line white and brown) were inoculated with Gram-positive Staphylococcus aureus or Gram-negative Salmonella Typhimurium, ranging from 103-106 CFU/mL. Our results show that concentrations of egg-white lysozyme and, particularly, ovotransferrin are important to modulate bacterial survival in a dose-dependent matter. Depending on protein concentration, their effect ranges from bactericidal to bacteriostatic, with a threshold for bacterial contamination that depends also on hen age and line. The concentrations of lysozyme and ovotransferrin increased with hen age (up to 2 and 22 w/w% of total protein, respectively), and eggs laid by older hens exhibited the greatest potential to prevent the growth of the highest Salmonella inoculum (106 CFU/mL). Salmonella-penetration experiments demonstrated that non-contaminated eggs display significantly higher concentrations of antimicrobial proteins. However, eggs from older hens needed a higher concentration of these proteins (>20% ovotransferrin) to prevent bacterial contamination, showing that antimicrobial protein concentrations in egg-whites was not the only factor influencing bacterial contamination. Finally, this study demonstrated that egg-white of eggs produced by old hens are less prone to contamination by Salmonella.

Biogeochemical fingerprinting of magnetotactic bacterial magnetite.

Biominerals are important archives of the presence of life and environmental processes in the geological record. However, ascribing a clear biogenic nature to minerals with nanometer-sized dimensions has proven challenging. Identifying hallmark features of biologically controlled mineralization is particularly important for the case of magnetite crystals, resembling those produced by magnetotactic bacteria (MTB), which have been used as evidence of early prokaryotic life on Earth and in meteorites. We show here that magnetite produced by MTB displays a clear coupled C-N signal that is absent in abiogenic and/or biomimetic (protein-mediated) nanometer-sized magnetite. We attribute the presence of this signal to intracrystalline organic components associated with proteins involved in magnetosome formation by MTB. These results demonstrate that we can assign a biogenic origin to nanometer-sized magnetite crystals, and potentially other biominerals of similar dimensions, using unique geochemical signatures directly measured at the nanoscale. This finding is significant for searching for the earliest presence of life in the Earth's geological record and prokaryotic life on other planets.

Enhanced Cytotoxic Effect of TAT-PLGA-Embedded DOXO Carried by Biomimetic Magnetic Nanoparticles upon Combination with Magnetic Hyperthermia and Photothermia.

The synergy between directed chemotherapy and thermal therapy (both magnetic hyperthermia and photothermia) mediated by a nanoassembly composed of functionalized biomimetic magnetic nanoparticles (BMNPs) with the chemotherapeutic drug doxorubicin (DOXO) covered by the polymer poly(lactic-co-glycolic acid) (PLGA), decorated with TAT peptide (here referred to as TAT-PLGA(DOXO-BMNPs)) is explored in the present study. The rationale behind this nanoassembly lies in an optimization of the nanoformulation DOXO-BMNPs, already demonstrated to be more efficient against tumor cells, both in vitro and in vivo, than systemic traditional therapies. By embedding DOXO-BMNPs into PLGA, which is further functionalized with the cell-penetrating TAT peptide, the resulting nanoassembly is able to mediate drug transport (using DOXO as a drug model) and behaves as a hyperthermic agent (induced by an alternating magnetic field (AMF) or by laser irradiation with a laser power density of 2 W/cm2). Our results obtained using the HepG2 cell line show that there is a synergy between chemotherapy and thermal therapy that results in a stronger cytotoxic effect when compared to that caused by the soluble DOXO. This is probably due to the enhanced DOXO release occurring upon the application of the thermal therapy, as well as the induced local temperature rise mediated by BMNPs in the nanoassembly following exposition to AMF or to near-infrared (NIR) laser irradiation. These results represent a proof of concept demonstrating that TAT-PLGA(DOXO-BMNPs) can be used to efficiently combine therapies against tumor cells, which is a step forward in the transition from systemic to local treatments.

Improving the Cellular Uptake of Biomimetic Magnetic Nanoparticles.

Magnetococcus marinus magnetosome-associated protein MamC, expressed as recombinant, has been proven to mediate the formation of novel biomimetic magnetic nanoparticles (BMNPs) that are successful drug nanocarriers for targeted chemotherapy and hyperthermia agents. These BMNPs present several advantages over inorganic magnetic nanoparticles, such as larger sizes that allow the former to have larger magnetic moment per particle, and an isoelectric point at acidic pH values, which allows both the stable functionalization of BMNPs at physiological pH value and the molecule release at acidic (tumor) environments, simply based on electrostatic interactions. However, difficulties for BMNPs cell internalization still hold back the efficiency of these nanoparticles as drug nanocarriers and hyperthermia agents. In the present study we explore the enhanced BMNPs internalization following upon their encapsulation by poly (lactic-co-glycolic) acid (PLGA), a Food and Drug Administration (FDA) approved molecule. Internalization is further optimized by the functionalization of the nanoformulation with the cell-penetrating TAT peptide (TATp). Our results evidence that cells treated with the nanoformulation [TAT-PLGA(BMNPs)] show up to 80% more iron internalized (after 72 h) compared to that of cells treated with BMNPs (40%), without any significant decrease in cell viability. This nanoformulation showing optimal internalization is further characterized. In particular, the present manuscript demonstrates that neither its magnetic properties nor its performance as a hyperthermia agent are significantly altered due to the encapsulation. In vitro experiments demonstrate that, following upon the application of an alternating magnetic field on U87MG cells treated with BMNPs and TAT-PLGA(BMNPs), the cytotoxic effect of BMNPs was not affected by the TAT-PLGA enveloping. Based on that, difficulties shown in previous studies related to poor cell uptake of BMNPs can be overcome by the novel nanoassembly described here.

Synergistic Photothermal-Chemotherapy Based on the Use of Biomimetic Magnetic Nanoparticles.

MamC-mediated biomimetic magnetic nanoparticles (BMNPs) have emerged as one of the most promising nanomaterials due to their magnetic features (superparamagnetic character and large magnetic moment per particle), their novel surface properties determined by MamC, their biocompatibility and their ability as magnetic hyperthermia agents. However, the current clinical application of magnetic hyperthermia is limited due to the fact that, in order to be able to reach an effective temperature at the target site, relatively high nanoparticle concentration, as well as high magnetic field strength and/or AC frequency are needed. In the present study, the potential of BMNPs to increase the temperature upon irradiation of a laser beam in the near infrared, at a wavelength at which tissues become partially transparent, is explored. Moreover, our results also demonstrate the synergy between photothermia and chemotherapy in terms of drug release and cytotoxicity, by using BMNPs functionalized with doxorubicin, and the effectiveness of this combination therapy against tumor cells in in vitro experiments. Therefore, the findings of the present study open the possibility of a novel, alternative approach to fight localized tumors.

Enzyme Storage and Recycling: Nanoassemblies of α-Amylase and Xylanase Immobilized on Biomimetic Magnetic Nanoparticles.

Immobilization of enzymes has been extensively required in a wide variety of industrial applications as a way to ensure functionality and the potential of enzyme recycling after use. In particular, enzyme immobilization on magnetic nanoparticles (MNPs) could offer reusability by means of magnetic recovery and concentration, along with increased stability and robust activity of the enzyme under different physicochemical conditions. In the present work, microbial α-amylase (AmyKS) and xylanase (XAn11) were both immobilized on different types of MNPs [MamC-mediated biomimetic MNPs (BMNPs) and inorganic MNPs] by using two different strategies (electrostatic interaction and covalent bond). AmyKS immobilization was successful using electrostatic interaction with BMNPs. Instead, the best strategy to immobilize XAn11 was using MNPs through the hetero-crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The immobilization protocols were optimized by varying glutaraldehyde (GA) concentration, enzyme quantity, and reaction time. Under optimal conditions, 92% of AmyKS and 87% of XAn11 were immobilized on BMNPs and MNPs-E/N, respectively (here referred as AmyKS-BMNPs and XAn11-MNPs nanoassemblies). The results show that the immobilization of the enzymes did not extensively alter their functionality and increased enzyme stability compared to that of the free enzyme upon storage at 4 and 20 °C. Interestingly, the immobilized amylase and xylanase were reused for 15 and 8 cycles, respectively, without significant loss of activity upon magnetic recovery of the nanoassemblies. The results suggest the great potential of these nanoassemblies in bioindustry applications.

Biomimetic Magnetite Nanoparticles as Targeted Drug Nanocarriers and Mediators of Hyperthermia in an Experimental Cancer Model.

Biomimetic magnetic nanoparticles mediated by magnetosome proteins (BMNPs) are potential innovative tools for cancer therapy since, besides being multifunctional platforms, they can be manipulated by an external gradient magnetic field (GMF) and/or an alternating magnetic field (AMF), mediating targeting and hyperthermia, respectively. We evaluated the cytocompatibility/cytotoxicity of BMNPs and Doxorubicin (DOXO)-BMNPs in the presence/absence of GMF in 4T1 and MCF-7 cells as well as their cellular uptake. We analyzed the biocompatibility and in vivo distribution of BMNPs as well as the effect of DOXO-BMNPs in BALB/c mice bearing 4T1 induced mammary carcinomas after applying GMF and AMF. Results: GMF enhanced the cell uptake of both BMNPs and DOXO-BMNPs and the cytotoxicity of DOXO-BMNPs. BMNPs were biocompatible when injected intravenously in BALB/c mice. The application of GMF on 4T1 tumors after each of the repeated (6×) iv administrations of DOXO-BMNPs enhanced tumor growth inhibition when compared to any other treatment, including that with soluble DOXO. Moreover, injection of DOXO-BMNPs in the tumor combined with application of an AMF resulted in a significant tumor weight reduction. These promising results show the suitability of BMNPs as magnetic nanocarriers for local targeted chemotherapy and as local agents for hyperthermia.

Nanoformulation Design Including MamC-Mediated Biomimetic Nanoparticles Allows the Simultaneous Application of Targeted Drug Delivery and Magnetic Hyperthermia.

The design of novel nanomaterials that can be used as multifunctional platforms allowing the combination of therapies is gaining increased interest. Moreover, if this nanomaterial is intended for a targeted drug delivery, the use of several guidance methods to increase guidance efficiency is also crucial. Magnetic nanoparticles (MNPs) allow this combination of therapies and guidance strategies. In fact, MNPs can be used simultaneously as drug nanocarriers and magnetic hyperthermia agents and, moreover, they can be guided toward the target by an external magnetic field and by their functionalization with a specific probe. However, it is difficult to find a system based on MNPs that exhibits optimal conditions as a drug nanocarrier and as a magnetic hyperthermia agent. In this work, a novel nanoformulation is proposed to be used as a multifunctional platform that also allows dual complementary guidance. This nanoformulation is based on mixtures of inorganic magnetic nanoparticles (M) that have been shown to be optimal hyperthermia agents, and biomimetic magnetic nanoparticles (BM), that have been shown to be highly efficient drug nanocarriers. The presence of the magnetosome protein MamC at the surface of BM confers novel surface properties that allow for the efficient and stable functionalization of these nanoparticles without the need of further coating, with the release of the relevant molecule being pH-dependent, improved by magnetic hyperthermia. The BM are functionalized with Doxorubicin (DOXO) as a model drug and with an antibody that allows for dual guidance based on a magnetic field and on an antibody. The present study represents a proof of concept to optimize the nanoformulation composition in order to provide the best performance in terms of the magnetic hyperthermia agent and drug nanocarrier.

Reactive oxygen species (ROS) production in HepG2 cancer cell line through the application of localized alternating magnetic field.

Recent studies have shown the potential of magnetic hyperthermia in cancer treatments. However, the underlying mechanisms involved have not been yet fully described. In particular, the cell death related to magnetic hyperthermia observed in cultures incubated with low concentration of magnetic nanoparticles and under a low intensity alternating magnetic field, in which a macroscopic temperature rise is not observed, is still not understood. In the present study, we investigate the production of intracellular Reactive Oxygen Species (ROS) as a mechanism to induce cell death under these conditions. In this study, the production and influence of ROS on the viability of HepG2 human hepatoma cells (used as a model cell line) are analyzed under the application of variable magnetic fields using hyperthermia agents, such as biomimetic magnetic nanoparticles (BMNPs) mediated by magnetosome MamC protein from Magnetococcus marinus MC-1. The results show that intracellular ROS production increases up to ∼90% following upon the exposure of AMF to HepG2 cells containing BMNPs, which could determine the loss of cell viability (up to ∼40% reduction) without a significant rise in temperature. Such ROS production is linked to mitochondrial dysfunction caused by the application of AMF to cells containing BMNPs.

Biomimetic Magnetoliposomes as Oxaliplatin Nanocarriers: In Vitro Study for Potential Application in Colon Cancer.

Current chemotherapy for colorectal cancer (CRC) includes the use of oxaliplatin (Oxa), a first-line cytotoxic drug which, in combination with irinotecan/5-fluorouracil or biologic agents, increases the survival rate of patients. However, the administration of this drug induces side effects that limit its application in patients, making it necessary to develop new tools for targeted chemotherapy. MamC-mediated biomimetic magnetic nanoparticles coupled with Oxa (Oxa-BMNPs) have been previously demonstrated to efficiently reduce the IC50 compared to that of soluble Oxa. However, their strong interaction with the macrophages revealed toxicity and possibility of aggregation. In this scenario, a further improvement of this nanoassembly was necessary. In the present study, Oxa-BMNPs nanoassemblies were enveloped in phosphatidylcholine unilamellar liposomes (both pegylated and non-pegylated). Our results demonstrate that the addition of both a lipid cover and further pegylation improves the biocompatibility and cellular uptake of the Oxa-BMNPs nanoassemblies without significantly reducing their cytotoxic activity in colon cancer cells. In particular, with the pegylated magnetoliposome nanoformulation (a) hemolysis was reduced from 5% to 2%, being now hematocompatibles, (b) red blood cell agglutination was reduced, (c) toxicity in white blood cells was eliminated. This study represents a truly stepforward in this area as describes the production of one of the very few existing nanoformulations that could be used for a local chemotherapy to treat CRC.

Development and characterization of magnetic eggshell membranes for lead removal from wastewater.

An increasing concern for natural resources preservation and environmental safety is the removal of heavy metals from contaminated water. It is essential to develop simple procedures that use ecofriendly materials with high removal capacities. In this context, we have synthesized a new hybrid material in which eggshell membranes (ESMs) act as nucleation sites for magnetite nanoparticles (MNPs) precipitation in the presence of an external magnetic field. As a result, ESM was transformed into a magnetic biomaterial (MESM) in order to combine the Pb adsorption abilities of both MNPs and ESM and to facilitate collection of the bioadsorbant using an external magnetic field. This green co-precipitation method produced long strands of bead-like 50 nm superparamagnetic MNPs decorating the ESM fibers. When MESM were incubated in Pb(NO3)2 solutions, the hybrid material displayed a 2.5-fold increase in binding constant with respect to that of ESM alone, and a 10-fold increased capacity to remove Pb ions from aqueous solution. The manufactured MESMs present a maximum loading capacity of 0.066 ± 0.009 mg Pb/mg MNPs at 25 °C, which is increased up to 0.15 ± 0.05 mg Pb/mg MNPs at 45 °C. Moreover, the MESM system is very stable, since incubation in 1% HCl solution resulted in rapid Pb desorption, while MNP release from the MESM during the same period was negligible. Altogether, these results suggest that MESM could be utilized as an efficient nanoremediation agent for separation/removal of heavy metal ions or other charged pollutants from contaminated waters, with facile recovery for recycling.

Protein corona formation and its influence on biomimetic magnetite nanoparticles.

Biomimetic magnetite nanoparticles (BMNPs) synthesized in the presence of MamC, a magnetosome-associated protein from Magnetoccus marinus MC-1, have gained interest for biomedical applications because of their unique magnetic properties. However, their behavior in biological systems, like their interaction with proteins, still has to be evaluated prior to their use in clinics. In this study, doxorubicin (DOXO) as a model drug was adsorbed onto BMNPs to form nanoassemblies. These were incubated with human plasma to trigger protein corona (PC) formation. Proteins from the human plasma stably attached to either BMNPs or DOXO-BMNP nanoassemblies. In particular, fibrinogen was detected as the main component in the PC of DOXO-BMNPs that potentially provides advantages, e.g. protecting the particles from phagocytosis, thus prolonging their circulation time. Adsorption of PC to the BMNPs did not alter their magnetic properties but improved their colloidal stability, thus reducing their toxicity in human macrophages. In addition, PC formation enhanced cellular internalization and did not interfere with DOXO activity. Overall, our data indicate that the adsorption of PC onto DOXO-BMNPs in biological environment even increases their efficiency as drug carrier systems.

Eu-Doped Citrate-Coated Carbonated Apatite Luminescent Nanoprobes for Drug Delivery.

In the field of Nanomedicine, there is an increasing demand for new inorganic nanophosphors with low cytotoxicity and efficient loading-release ability of drugs for applications in bioimaging and drug delivery. This work assesses the potentiality of matured Eu-doped citrate-coated carbonated apatite nanoparticles to be used as theranostic platforms, for bioimaging, as luminescent nanoprobes, and for drug delivery applications, using Doxorubicin as a model drug. The drug adsorption isotherm fits the Langmuir-Freundlich (LF) model, showing that the Eu:cit-cAp nanoparticles can carry a maximum of 0.29 ± 0.02 mg Doxo mg Eu:cit-cAp-1 (Qmax). The affinity constant KFL for this binding is 44 ± 2 mL mg-1, and the cooperativity coefficient r is 6 ± 1. The nanoparticle suspensions presented charge reversion from negative to positive after loading with Doxo as revealed by the ζ-potential versus pH characterization. The release of drug from the loaded nanoparticles was found to be strongly pH-dependent, being around 5 wt % at physiological pH 7.4 and 20 wt % at pH 5, in experiments lasting 24 h. Luminescence spectroscopic measurements of Doxo-loaded nanoparticles revealed the increase of luminescence with a decrease in the amount of adsorbed Doxo, due to the so-called inner filter effect. The nanoparticles free of Doxo were cytocompatible when interacted with two human cell lines derived respectively from a gastric carcinoma (GTL-16), and a hepatocarcinoma (Huh7), while Doxo-loaded nanoparticles displayed significant toxicity in a dose-dependent relationship. Therefore, the new nanoassemblies might have a dual function, as nanoprobes in bioimaging by detecting the fate of the nanoparticles in biological environments, and for monitoring the delivery of the drug in such environments, by measuring the rise of the luminescence provided by the desorption of Doxo.

Biomimetic Magnetic Nanocarriers Drive Choline Kinase Alpha Inhibitor inside Cancer Cells for Combined Chemo-Hyperthermia Therapy.

Choline kinase α1 (ChoKα1) has become an excellent antitumor target. Among all the inhibitors synthetized, the new compound Ff35 shows an excellent capacity to inhibit ChoKα1 activity. However, soluble Ff35 is also capable of inhibiting choline uptake, making the inhibitor not selective for ChoKα1. In this study, we designed a new protocol with the aim of disentangling whether the Ff35 biological action is due to the inhibition of the enzyme and/or to the choline uptake. Moreover, we offer an alternative to avoid the inhibition of choline uptake caused by Ff35, since the coupling of Ff35 to novel biomimetic magnetic nanoparticles (BMNPs) allows it to enter the cell through endocytosis without interacting with the choline transporter. This opens the possibility of a clinical use of Ff35. Our results indicate that Ff35-BMNPs nanoassemblies increase the selectivity of Ff35 and have an antiproliferative effect. Also, we demonstrate the effectiveness of the tandem Ff35-BMNPs and hyperthermia.