Hydrophobicity-enhanced ferritin nanoparticles for efficient encapsulation and targeted delivery of hydrophobic drugs to tumor cells.

Ferritin, a naturally occurring iron storage protein, has gained significant attention as a drug delivery platform due to its inherent biocompatibility and capacity to encapsulate therapeutic agents. In this study, we successfully genetically engineered human H ferritin by incorporating 4 or 6 tryptophan residues per subunit, strategically oriented towards the inner cavity of the nanoparticle. This modification aimed to enhance the encapsulation of hydrophobic drugs into the ferritin cage. Comprehensive characterization of the mutants revealed that only the variant carrying four tryptophan substitutions per subunit retained the ability to disassemble and reassemble properly. As a proof of concept, we evaluated the loading capacity of this mutant with ellipticine, a natural hydrophobic indole alkaloid with multimodal anticancer activity. Our data demonstrated that this specific mutant exhibited significantly higher efficiency in loading ellipticine compared to human H ferritin. Furthermore, to evaluate the versatility of this hydrophobicity-enhanced ferritin nanoparticle as a drug carrier, we conducted a comparative study by also encapsulating doxorubicin, a commonly used anticancer drug. Subsequently, we tested both ellipticine and doxorubicin-loaded nanoparticles on a promyelocytic leukemia cell line, demonstrating efficient uptake by these cells and resulting in the expected cytotoxic effect.

Ferritin-Coated SPIONs as New Cancer Cell Targeted Magnetic Nanocarrier.

Superparamagnetic iron oxide nanoparticles (SPIONs) may act as an excellent theragnostic tool if properly coated and stabilized in a biological environment, even more, if they have targeting properties towards a specific cellular target. Humanized Archaeoglobus fulgidus Ferritin (HumAfFt) is an engineered ferritin characterized by the peculiar salt-triggered assembly-disassembly of the hyperthermophile Archaeoglobus fulgidus ferritin and is successfully endowed with the human H homopolymer recognition sequence by the transferrin receptor (TfR1 or CD71), overexpressed in many cancer cells in response to the increased demand of iron. For this reason, HumAfFt was successfully used in this study as a coating material for 10 nm SPIONs, in order to produce a new magnetic nanocarrier able to discriminate cancer cells from normal cells and maintain the potential theragnostic properties of SPIONs. HumAfFt-SPIONs were exhaustively characterized in terms of size, morphology, composition, and cytotoxicity. The preferential uptake capacity of cancer cells toward HumAfFt-SPIONs was demonstrated in vitro on human breast adenocarcinoma (MCF7) versus normal human dermal fibroblast (NHDF) cell lines.

Engineered Ferritin with Eu3+ as a Bright Nanovector: A Photoluminescence Study.

Ferritin nanoparticles play many important roles in theranostic and bioengineering applications and have been successfully used as nanovectors for the targeted delivery of drugs due to their ability to specifically bind the transferrin receptor (TfR1, or CD71). They can be either genetically or chemically modified for encapsulating therapeutics or probes in their inner cavity. Here, we analyzed a new engineered ferritin nanoparticle, made of the H chain mouse ferritin (HFt) fused with a specific lanthanide binding tag (LBT). The HFt-LBT has one high affinity lanthanide binding site per each of the 24 subunits and a tryptophane residue within the tag that acts as an antenna able to transfer the energy to the lanthanide ions via a LRET process. In this study, among lanthanides, we selected europium for its red emission that allows to reduce overlap with tissue auto-fluorescence. Steady state emission measurements and time-resolved emission spectroscopy have been employed to investigate the interaction between the HFt-LBT and the Eu3+ ions. This allowed us to identify the Eu3+ energy states involved in the process and to pave the way for the future use of HFt-LBT Eu3+ complex in theranostics.

Design of protein-binding peptides with controlled binding affinity: the case of SARS-CoV-2 receptor binding domain and angiotensin-converting enzyme 2 derived peptides.

The development of methods able to modulate the binding affinity between proteins and peptides is of paramount biotechnological interest in view of a vast range of applications that imply designed polypeptides capable to impair or favour Protein-Protein Interactions. Here, we applied a peptide design algorithm based on shape complementarity optimization and electrostatic compatibility and provided the first experimental in vitro proof of the efficacy of the design algorithm. Focusing on the interaction between the SARS-CoV-2 Spike Receptor-Binding Domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) receptor, we extracted a 23-residues long peptide that structurally mimics the major interacting portion of the ACE2 receptor and designed in silico five mutants of such a peptide with a modulated affinity. Remarkably, experimental KD measurements, conducted using biolayer interferometry, matched the in silico predictions. Moreover, we investigated the molecular determinants that govern the variation in binding affinity through molecular dynamics simulation, by identifying the mechanisms driving the different values of binding affinity at a single residue level. Finally, the peptide sequence with the highest affinity, in comparison with the wild type peptide, was expressed as a fusion protein with human H ferritin (HFt) 24-mer. Solution measurements performed on the latter constructs confirmed that peptides still exhibited the expected trend, thereby enhancing their efficacy in RBD binding. Altogether, these results indicate the high potentiality of this general method in developing potent high-affinity vectors for hindering/enhancing protein-protein associations.

Immobilization of Lathyrus cicera Amine Oxidase on Magnetic Microparticles for Biocatalytic Applications.

Amine oxidases are enzymes belonging to the class of oxidoreductases that are widespread, from bacteria to humans. The amine oxidase from Lathyrus cicera has recently appeared in the landscape of biocatalysis, showing good potential in the green synthesis of aldehydes. This enzyme catalyzes the oxidative deamination of a wide range of primary amines into the corresponding aldehydes but its use as a biocatalyst is challenging due to the possible inactivation that might occur at high product concentrations. Here, we show that the enzyme's performance can be greatly improved by immobilization on solid supports. The best results are achieved using amino-functionalized magnetic microparticles: the immobilized enzyme retains its activity, greatly improves its thermostability (4 h at 75 °C), and can be recycled up to 8 times with a set of aromatic ethylamines. After the last reaction cycle, the overall conversion is about 90% for all tested substrates, with an aldehyde production ranging between 100 and 270 mg depending on the substrate used. As a proof concept, one of the aldehydes thus produced was successfully used for the biomimetic synthesis of a non-natural benzylisoquinoline alkaloid.

Biocatalytic Production of Aldehydes: Exploring the Potential of Lathyrus cicera Amine Oxidase.

Aldehydes are a class of carbonyl compounds widely used as intermediates in the pharmaceutical, cosmetic and food industries. To date, there are few fully enzymatic methods for synthesizing these highly reactive chemicals. In the present work, we explore the biocatalytic potential of an amino oxidase extracted from the etiolated shoots of Lathyrus cicera for the synthesis of value-added aldehydes, starting from the corresponding primary amines. In this frame, we have developed a completely chromatography-free purification protocol based on crossflow ultrafiltration, which makes the production of this enzyme easily scalable. Furthermore, we determined the kinetic parameters of the amine oxidase toward 20 differently substituted aliphatic and aromatic primary amines, and we developed a biocatalytic process for their conversion into the corresponding aldehydes. The reaction occurs in aqueous media at neutral pH in the presence of catalase, which removes the hydrogen peroxide produced during the reaction itself, contributing to the recycling of oxygen. A high conversion (>95%) was achieved within 3 h for all the tested compounds.

Self-assembling ferritin-dendrimer nanoparticles for targeted delivery of nucleic acids to myeloid leukemia cells.

BACKGROUND: In recent years, the use of ferritins as nano-vehicles for drug delivery is taking center stage. Compared to other similar nanocarriers, Archaeoglobus fulgidus ferritin is particularly interesting due to its unique ability to assemble-disassemble under very mild conditions. Recently this ferritin was engineered to get a chimeric protein targeted to human CD71 receptor, typically overexpressed in cancer cells. RESULTS: Archaeoglobus fulgidus chimeric ferritin was used to generate a self-assembling hybrid nanoparticle hosting an aminic dendrimer together with a small nucleic acid. The positively charged dendrimer can indeed establish electrostatic interactions with the chimeric ferritin internal surface, allowing the formation of a protein-dendrimer binary system. The 4 large triangular openings on the ferritin shell represent a gate for negatively charged small RNAs, which access the internal cavity attracted by the dense positive charge of the dendrimer. This ternary protein-dendrimer-RNA system is efficiently uptaken by acute myeloid leukemia cells, typically difficult to transfect. As a proof of concept, we used a microRNA whose cellular delivery and induced phenotypic effects can be easily detected. In this article we have demonstrated that this hybrid nanoparticle successfully delivers a pre-miRNA to leukemia cells. Once delivered, the nucleic acid is released into the cytosol and processed to mature miRNA, thus eliciting phenotypic effects and morphological changes similar to the initial stages of granulocyte differentiation. CONCLUSION: The results here presented pave the way for the design of a new family of protein-based transfecting agents that can specifically target a wide range of diseased cells.

Exciplex Formation in Lipid-bound Escherichia coli Flavohemoglobin.

Flavohemoglobins have the particular capability of binding unsaturated and cyclopropanated fatty acids as free acids or phospholipids. Fatty acid binding to the ferric heme results in a weak but direct bonding interaction. Ferrous and ferric protein, in presence or absence of a bound lipid molecule, have been characterized by transient absorption spectroscopy. Measurements have been also carried out both on the ferrous deoxygenated and on the CO bound protein to investigate possible long-range interaction between the lipid acyl chain moiety and the ferrous heme. After excitation of the deoxygenated derivatives the relaxation process reveals a slow dynamics (350 ps) in lipid-bound protein but is not observed in the lipid-free protein. The latter feature and the presence of an extra contribution in the absorption spectrum, indicates that the interaction of iron heme with the acyl chain moiety occurs only in the excited electronic state and not in the ground electronic state. Data analysis highlights the formation of a charge-transfer complex in which the iron ion of the lipid-bound protein in the expanded electronic excited state, possibly represented by a high spin Fe III intermediate, is able to bind to the sixth coordination ligand placed at a distance of at 3.5 Šfrom the iron. A very small nanosecond geminate rebinding is observed for CO adduct in lipid-free but not in the lipid-bound protein. The presence of the lipid thus appears to inhibit the mobility of CO in the heme pocket.

Ethylchloroformate Derivatization for GC-MS Analysis of Resveratrol Isomers in Red Wine.

Resveratrol (3,5,4'-trihydroxystilbene) is a natural compound that can be found in high concentrations in red wine and in many typical foods found in human diet. Over the past decades, resveratrol has been widely investigated for its potential beneficial effects on human health. At the same time, numerous analytical methods have been developed for the quantitative determination of resveratrol isomers in oenological and food matrices. In the present work, we developed a very fast and sensitive GC-MS method for the determination of resveratrol in red wine based on ethylchloroformate derivatization. Since this reaction occurs directly in the water phase during the extraction process itself, it has the advantage of significantly reducing the overall processing time for the sample. This method presents low limits of quantification (LOQ) (25 ng/mL and 50 ng/mL for cis- and trans-resveratrol, respectively) and excellent accuracy and precision. Ethylchloroformate derivatization was successfully applied to the analysis of resveratrol isomers in a selection of 15 commercial Italian red wines, providing concentration values comparable to those reported in other studies. As this method can be easily extended to other classes of molecules present in red wine, it allows further development of new GC-MS methods for the molecular profiling of oenological matrices.

Biodistribution PET/CT Study of Hemoglobin-DFO-89Zr Complex in Healthy and Lung Tumor-Bearing Mice.

Proteins, as a major component of organisms, are considered the preferred biomaterials for drug delivery vehicles. Hemoglobin (Hb) has been recently rediscovered as a potential drug carrier, but its use for biomedical applications still lacks extensive investigation. To further explore the possibility of utilizing Hb as a potential tumor targeting drug carrier, we examined and compared the biodistribution of Hb in healthy and lung tumor-bearing mice, using for the first time 89Zr labelled Hb in a positron emission tomography (PET) measurement. Hb displays a very high conjugation yield in its fast and selective reaction with the maleimide-deferoxamine (DFO) bifunctional chelator. The high-resolution X-ray structure of the Hb-DFO complex demonstrated that cysteine ß93 is the sole attachment moiety to the αß-protomer of Hb. The Hb-DFO complex shows quantitative uptake of 89Zr in solution as determined by radiochromatography. Injection of 0.03 mg of Hb-DFO-89Zr complex in healthy mice indicates very high radioactivity in liver, followed by spleen and lungs, whereas a threefold increased dosage results in intensification of PET signal in kidneys and decreased signal in liver and spleen. No difference in biodistribution pattern is observed between naïve and tumor-bearing mice. Interestingly, the liver Hb uptake did not decrease upon clodronate-mediated macrophage depletion, indicating that other immune cells contribute to Hb clearance. This finding is of particular interest for rapidly developing clinical immunology and projects aiming to target, label or specifically deliver agents to immune cells.

Ferritin Nanocages for Protein Delivery to Tumor Cells.

The delivery of therapeutic proteins is one of the greatest challenges in the treatment of human diseases. In this frame, ferritins occupy a very special place. Thanks to their hollow spherical structure, they are used as modular nanocages for the delivery of anticancer drugs. More recently, the possibility of encapsulating even small proteins with enzymatic or cytotoxic activity is emerging. Among all ferritins, particular interest is paid to the Archaeoglobus fulgidus one, due to its peculiar ability to associate/dissociate in physiological conditions. This protein has also been engineered to allow recognition of human receptors and used in vitro for the delivery of cytotoxic proteins with extremely promising results.

Gas Chromatographic-Mass Spectrometric Method for the Simultaneous Determination of Resveratrol Isomers and 2,4,6-Trihydroxyphenanthrene in Red Wines Exposed to UV-Light.

Resveratrol (3,5,4'-trihydroxystilbene) is one of the most abundant polyphenols in red grapes, and red wine represents one of the most important dietary sources of this compound. Although its beneficial properties on human health have been widely investigated over the last 30 years, very little is known about its derivatives. Resveratrol can indeed undergo glycosylation, oligomerization and, upon UV-light exposure, it can isomerize from the trans-to the cis-isomer, which can further cyclize to 2,4,6-trihydroxyphenanthrene (THP). Although the effects of THP on human health are not yet known, being a polycyclic aromatic hydrocarbon, it can be potentially harmful. Because no data about THP occurrence in plant food and beverages are available, a simple procedure based on liquid-liquid extraction and gas chromatography-mass spectrometry has been developed and validated for the simultaneous qualitative and quantitative analysis of trans-resveratrol, cis-resveratrol, and THP in red wine, before and after UV-light exposure.

Ferritin nanovehicle for targeted delivery of cytochrome C to cancer cells.

In this work, we have exploited the unique properties of a chimeric archaeal-human ferritin to encapsulate, deliver and release cytochrome c and induce apoptosis in a myeloid leukemia cell line. The chimeric protein combines the versatility in 24-meric assembly and cargo incorporation capability of Archaeglobus fulgidus ferritin with specific binding of human H ferritin to CD71, the "heavy duty" carrier responsible for transferrin-iron uptake. Delivery of ferritin-encapsulated cytochrome C to the Acute Promyelocytic Leukemia (APL) NB4 cell line, highly resistant to transfection by conventional methods, was successfully achieved in vitro. The effective liberation of cytochrome C within the cytosolic environment, demonstrated by double fluorescent labelling, induced apoptosis in the cancer cells.

Application of crossflow ultrafiltration for scaling up the purification of a recombinant ferritin.

Ferritin proteins are taking center stage as smart nanocarriers for drug delivery due to their hollow cage-like structures and their unique 24-meric assembly. Among all ferritins, the chimeric Archaeoglobus ferritin (HumFt) is able assemble/disassemble varying the ionic strength of the medium while recognizing human TfR1 receptor overexpressed in cancer cells. In this paper we present a highly efficient, large scale purification protocol mainly based on crossflow ultrafiltration, starting from fermented bacterial paste. This procedure allows one to obtain about 2 g of purified protein starting from 100 g of fermented bacterial paste. The current procedure can easily remove contaminant proteins as well as DNA molecules in the absence of expensive and time consuming chromatographic steps.

Engineered ferritin for lanthanide binding.

Ferritin H-homopolymers have been extensively used as nanocarriers for diverse applications in the targeted delivery of drugs and imaging agents, due to their unique ability to bind the transferrin receptor (CD71), highly overexpressed in most tumor cells. In order to incorporate novel fluorescence imaging properties, we have fused a lanthanide binding tag (LBT) to the C-terminal end of mouse H-chain ferritin, HFt. The HFt-LBT possesses one high affinity Terbium binding site per each of the 24 subunits provided by six coordinating aminoacid side chains and a tryptophan residue in its close proximity and is thus endowed with strong FRET sensitization properties. Accordingly, the characteristic Terbium emission band at 544 nm for the HFt-LBT Tb(III) complex was detectable upon excitation of the tag enclosed at two order of magnitude higher intensity with respect to the wtHFt protein. X-ray data at 2.9 Å and cryo-EM at 7 Å resolution demonstrated that HFt-LBT is correctly assembled as a 24-mer both in crystal and in solution. On the basis of the intrinsic Tb(III) binding properties of the wt protein, 32 additional Tb(III) binding sites, located within the natural iron binding sites of the protein, were identified besides the 24 Tb(III) ions coordinated to the LBTs. HFt-LBT Tb(III) was demonstrated to be actively uptaken by selected tumor cell lines by confocal microscopy and FACS analysis of their FITC derivatives, although direct fluorescence from Terbium emission could not be singled out with conventional, 295-375 nm, fluorescence excitation.

Excimer based fluorescent pyrene-ferritin conjugate for protein oligomerization studies and imaging in living cells.

Ferritin self-assembly has been widely exploited for the synthesis of a variety of nanoparticles for drug-delivery and diagnostic applications. However, despite the crucial role of ferritin self-assembly mechanism for probes encapsulation, little is known about the principles behind the oligomerization mechanism. In the present work, the novel "humanized" chimeric Archaeal ferritin HumAfFt, displaying the transferrin receptor-1 (TfR1) recognition motif typical of human H homopolymer and the unique salt-triggered oligomerization properties of Archaeoglobus fulgidus ferritin (AfFt), was site-selectively labeled with N-(1-pyrenyl)maleimide on a topologically selected cysteine residue inside the protein cavity, next to the dimer interface. Pyrene characteristic fluorescence features were exploited to investigate the transition from a dimeric to a cage-like 24-meric state and to visualize the protein in vitro by two photon fluorescence microscopy. Indeed, pyrene fluorescence changes upon ferritin self-assembly allowed to establish, for the first time, the kinetic and thermodynamic details of the archaeal ferritins oligomerization mechanism. In particular, the magnesium induced oligomerization proved to be faster than the monovalent cation-triggered process, highly cooperative, complete at low MgCl2 concentrations, and reversed by treatment with EDTA. Moreover, pyrene intense excimer fluorescence was successfully visualized in vitro by two photon fluorescence microscopy as pyrene-labeled HumAfFt was actively uptaken into HeLa cells by human transferrin receptor TfR1 recognition, thus representing a unique nano-device building block for two photon fluorescence cell imaging.

Green Routes for the Production of Enantiopure Benzylisoquinoline Alkaloids.

Benzylisoquinoline alkaloids (BIAs) are among the most important plant secondary metabolites, in that they include a number of biologically active substances widely employed as pharmaceuticals. Isolation of BIAs from their natural sources is an expensive and time-consuming procedure as they accumulate in very low levels in plant. Moreover, total synthesis is challenging due to the presence of stereogenic centers. In view of these considerations, green and scalable methods for BIA synthesis using fully enzymatic approaches are getting more and more attention. The aim of this paper is to review fully enzymatic strategies for producing the benzylisoquinoline central precursor, (S)-norcoclaurine and its derivatives. Specifically, we will detail the current status of synthesis of BIAs in microbial hosts as well as using isolated and recombinant enzymes.

Humanized archaeal ferritin as a tool for cell targeted delivery.

Human ferritins have been extensively studied to be used as nanocarriers for diverse applications and could represent a convenient alternative for targeted delivery of anticancer drugs and imaging agents. However, the most relevant limitation to their applications is the need for highly acidic experimental conditions during the initial steps of particle/cargo assembly, a process that could affect both drug stability and the complete reassembly of the ferritin cage. To overcome this issue the unique assembly of Archaeoglobus fulgidus ferritin was genetically engineered by changing a surface exposed loop of 12 amino acids connecting B and C helices to mimic the sequence of the analogous human H-chain ferritin loop. This new chimeric protein was shown to maintain the unique, cation linked, association-dissociation properties of Archaeoglobus fulgidus ferritin occurring at neutral pH values, while exhibiting the typical human H-homopolymer recognition by the transferrin receptor TfR1. The chimeric protein was confirmed to be actively and specifically internalized by HeLa cells, thus representing a unique nanotechnological tool for cell-targeted delivery of possible payloads for diagnostic or therapeutic purposes. Moreover, it was demonstrated that the 12 amino acids' loop is necessary and sufficient for binding to the transferrin receptor. The three-dimensional structure of the humanized Archaeoglobus ferritin has been obtained both as crystals by X-ray diffraction and in solution by cryo-EM.

Probing bulky ligand entry in engineered archaeal ferritins.

BACKGROUND: A set of engineered ferritin mutants from Archaeoglobus fulgidus (Af-Ft) and Pyrococcus furiosus (Pf-Ft) bearing cysteine thiols in selected topological positions inside or outside the ferritin shell have been obtained. The two apo-proteins were taken as model systems for ferritin internal cavity accessibility in that Af-Ft is characterized by the presence of a 45Å wide aperture on the protein surface whereas Pf-Ft displays canonical (threefold) channels. METHODS: Thiol reactivity has been probed in kinetic experiments in order to assess the protein matrix permeation properties towards the bulky thiol reactive DTNB (5,5'-dithiobis-2-nitrobenzoic acid) molecule. RESULTS: Reaction of DTNB with thiols was observed in all ferritin mutants, including those bearing free cysteine thiols inside the ferritin cavity. As expected, a ferritin mutant from Pf-Ft, in which the cysteine thiol is on the outer surface displays the fastest binding kinetics. In turn, also the Pf-Ft mutant in which the cysteine thiol is placed within the internal cavity, is still capable of full stoichiometric DTNB binding albeit with an almost 200-fold slower rate. The behaviour of Af-Ft bearing a cysteine thiol in a topologically equivalent position in the internal cavity was intermediate among the two Pf-Ft mutants. CONCLUSIONS AND GENERAL SIGNIFICANCE: The data thus obtained indicate clearly that the protein matrix in archaea ferritins does not provide a significant barrier against bulky, negatively charged ligands such as DTNB, a finding of relevance in view of the multiple biotechnological applications of these ferritins that envisage ligand encapsulation within the internal cavity.

Interplay of the H-bond donor-acceptor role of the distal residues in hydroxyl ligand stabilization of Thermobifida fusca truncated hemoglobin.

The unique architecture of the active site of Thermobifida fusca truncated hemoglobin (Tf-trHb) and other globins belonging to the same family has stimulated extensive studies aimed at understanding the interplay between iron-bound ligands and distal amino acids. The behavior of the heme-bound hydroxyl, in particular, has generated much interest in view of the relationships between the spin-state equilibrium of the ferric iron atom and hydrogen-bonding capabilities (as either acceptor or donor) of the OH(-) group itself. The present investigation offers a detailed molecular dynamics and spectroscopic picture of the hydroxyl complexes of the WT protein and a combinatorial set of mutants, in which the distal polar residues, TrpG8, TyrCD1, and TyrB10, have been singly, doubly, or triply replaced by a Phe residue. Each mutant is characterized by a complex interplay of interactions in which the hydroxyl ligand may act both as a H-bond donor or acceptor. The resonance Raman stretching frequencies of the Fe-OH moiety, together with electron paramagnetic resonance spectra and MD simulations on each mutant, have enabled the identification of specific contributions to the unique ligand-inclusive H-bond network typical of this globin family.