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.

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.

Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR-Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.

Embedding Biomimetic Magnetic Nanoparticles Coupled with Peptide AS-48 into PLGA to Treat Intracellular Pathogens.

Among the strategies employed to overcome the development of multidrug-resistant bacteria, directed chemotherapy combined with local therapies (e.g., magnetic hyperthermia) has gained great interest. A nano-assembly coupling the antimicrobial peptide AS-48 to biomimetic magnetic nanoparticles (AS-48-BMNPs) was demonstrated to have potent bactericidal effects on both Gram-positive and Gram-negative bacteria when the antimicrobial activity of the peptide was combined with magnetic hyperthermia. Nevertheless, intracellular pathogens remain challenging due to the difficulty of the drug reaching the bacterium. Thus, improving the cellular uptake of the nanocarrier is crucial for the success of the treatment. In the present study, we demonstrate the embedding cellular uptake of the original nano-assembly into THP-1, reducing the toxicity of AS-48 toward healthy THP-1 cells. We optimized the design of PLGA[AS-48-BMNPs] in terms of size, colloidal stability, and hyperthermia activity (either magnetic or photothermal). The stability of the nano-formulation at physiological pH values was evaluated by studying the AS-48 release at this pH value. The influence of pH and hyperthermia on the AS-48 release from the nano-formulation was also studied. These results show a slower AS-48 release from PLGA[AS-48-BMNPs] compared to previous nano-formulations, which could make this new nano-formulation suitable for longer extended treatments of intracellular pathogens. PLGA[AS-48-BMNPs] are internalized in THP-1 cells where AS-48 is liberated slowly, which may be useful to treat diseases and prevent infection caused by intracellular pathogens. The treatment will be more efficient combined with hyperthermia or photothermia.

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.

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.

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.

Oxaliplatin-Biomimetic Magnetic Nanoparticle Assemblies for Colon Cancer-Targeted Chemotherapy: An In Vitro Study.

Conventional chemotherapy against colorectal cancer (CRC), the third most common cancer in the world, includes oxaliplatin (Oxa) which induces serious unwanted side effects that limit the efficiency of treatment. Therefore, alternative therapeutic approaches are urgently required. In this work, biomimetic magnetic nanoparticles (BMNPs) mediated by MamC were coupled to Oxa to evaluate the potential of the Oxa-BMNP nanoassembly for directed local delivery of the drug as a proof of concept for the future development of targeted chemotherapy against CRC. Electrostatic interactions between Oxa and BMNPs trigger the formation of the nanoassembly and keep it stable at physiological pH. When the BMNPs become neutral at acidic pH values, the Oxa is released, and such a release is greatly potentiated by hyperthermia. The coupling of the drug with the BMNPs improves its toxicity to even higher levels than the soluble drug, probably because of the fast internalization of the nanoassembly by tumor cells through endocytosis. In addition, the BMNPs are cytocompatible and non-hemolytic, providing positive feedback as a proof of concept for the nanoassembly. Our study clearly demonstrates the applicability of Oxa-BMNP in colon cancer and offers a promising nanoassembly for targeted chemotherapy against this type of tumor.

Magnetoliposomes of mixed biomimetic and inorganic magnetic nanoparticles as enhanced hyperthermia agents.

Recently, liposomes have been explored as a potential solution to improve the biocompatibility and the colloidal stability of magnetic nanoparticles. Protocols have been developed for producing magnetoliposomes of magnetite nanoparticles obtained inorganically (MNPs). However, the biomimetic synthesis of magnetite using heterologous proteins from magnetotactic bacteria has become a real alternative to produce novel biomimetic magnetic nanoparticles (BMNPs). Among these, the BMNPs obtained in presence of MamC protein from Magnetococcus marinus MC-1 have been proposed as excellent candidates to be potentially used as drug nanocarriers and as hyperthermia agents. However, their colloidal stability still needs to be improved while maintaining their magnetic properties intact. One possibility explored in this manuscript is to form magnetoliposomes that contain BMNPs. Indeed, the protocols developed for producing magnetoliposomes of MNPs need to be tested and modified to be able to include BMNPs. In this context, a protocol has been developed to produce both magnetoliposomes filled with MNPs and/or BMNPs and their potential as hyperthermia agents was tested. In fact, for the first time, these two types of nanoparticles were mixed in different proportions to test the composition that would optimize such as behaviour as hyperthermia agents. Interestingly, it was observed that the hyperthermia behaviour of the magnetoliposomes greatly improved if they were filled with a mixture of MNPs and BMNPs. These results indicate that these magnetoliposomes display optimal characteristics to become a potential agent for hyperthermia and that the opening of those liposomes could be externally controlled by applying an alternate magnetic field.

Enhancement of Magnetic Hyperthermia by Mixing Synthetic Inorganic and Biomimetic Magnetic Nanoparticles.

In this work we report on the synthesis and characterization of magnetic nanoparticles of two distinct origins, one inorganic (MNPs) and the other biomimetic (BMNPs), the latter based on a process of bacterial synthesis. Each of these two kinds of particles has its own advantages when used separately with biomedical purposes. Thus, BMNPs present an isoelectric point below neutrality (around pH 4.4), while MNPs show a zero-zeta potential at pH 7, and appear to be excellent agents for magnetic hyperthermia. This means that the biomimetic particles are better suited to be loaded with drug molecules positively charged at neutral pH (notably, doxorubicin, for instance) and releasing it at the acidic tumor environment. In turn, MNPs may provide their transport capabilities under a magnetic field. In this study it is proposed to use a mixture of both kinds of particles at two different concentrations, trying to get the best from each of them. We study which mixture performs better from different points of view, like stability and magnetic hyperthermia response, while keeping suitable drug transport capabilities. This composite system is proposed as a close to ideal drug vehicle with added enhanced hyperthermia response.

Tuning properties of biomimetic magnetic nanoparticles by combining magnetosome associated proteins.

The role of magnetosome associated proteins on the in vitro synthesis of magnetite nanoparticles has gained interest, both to obtain a better understanding of the magnetosome biomineralization process and to be able to produce novel magnetosome-like biomimetic nanoparticles. Up to now, only one recombinant protein has been used at the time to in vitro form biomimetic magnetite precipitates, being that a scenario far enough from what probably occurs in the magnetosome. In the present study, both Mms6 and MamC from Magnetococcus marinus MC-1 have been used to in vitro form biomimetic magnetites. Our results show that MamC and Mms6 have different, but complementary, effects on in vitro magnetite nucleation and growth. MamC seems to control the kinetics of magnetite nucleation while Mms6 seems to preferably control the kinetics for crystal growth. Our results from the present study also indicate that it is possible to combine both proteins to tune the properties of the resulting biomimetic magnetites. In particular, by changing the relative ratio of these proteins, better faceted and/or larger magnetite crystals with, consequently, different magnetic moment per particle could be obtained. This study provides with tools to obtain new biomimetic nanoparticles with a potential utility for biotechnological applications.