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

Unlocking the treasure box: the role of HEPES buffer in disassembling an uncommon ferritin nanoparticle

Ferritins are ideal nanoparticles as drug delivery systems due to their hollow-sphere structure and the ability to target specific receptors on the cell surface. Here, we develop and characterize a new ferritin derived from the chimeric humanized A. fulgidus one, already designed to recognize the TfR1 receptor. Starting from the synthetic gene of this chimeric protein, we replaced two positively charged amino acids with two alanine residues to close the large triangular pores on its surface. These mutations make the protein nanoparticle suitable to incorporate even small therapeutics without leakage. Size-exclusion chromatography shows that the assembling/disassembling of this new protein cage can be easily fine-tuned by varying the HEPES buffer and MgCl2 concentration. The protein cage can be opened using 150 mM HEPES buffer without magnesium ions. Adding this divalent cation to the solution promotes the quick assembly of the ferritin as a 24-mer. The development of this new protein cage paves the way for encapsulation and delivery studies of small molecules for therapeutic and diagnostic purposes

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

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

Ethylchloroformate derivatization for GC-MS analysis of resveratrol isomers in red wine

Resveratrol (3,5,40-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

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. Graphic abstract: [Figure not available: see fulltext.]

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 time89 Zr 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 β93 is the sole attachment moiety to the αβ-protomer of Hb. The Hb-DFO complex shows quantitative uptake of89 Zr in solution as determined by radiochromatography. Injection of 0.03 mg of Hb-DFO-89 Zr 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

The structure of a class 3 nonsymbiotic plant haemoglobin from<i>Arabidopsis thaliana</i>reveals a novel N-terminal helical extension

Plant nonsymbiotic haemoglobins fall into three classes, each with distinct properties but all with largely unresolved physiological functions. Here, the first crystal structure of a class 3 nonsymbiotic plant haemoglobin, that fromArabidopsis thaliana, is reported to 1.77 Å resolution. The protein forms a homodimer, with each monomer containing a two-over-two α-helical domain similar to that observed in bacterial truncated haemoglobins. A novel N-terminal extension comprising two α-helices plays a major role in the dimer interface, which occupies the periphery of the dimer–dimer face, surrounding an open central cavity. The haem pocket contains a proximal histidine ligand and an open sixth iron-coordination site with potential for a ligand, in this structure hydroxide, to form hydrogen bonds to a tyrosine or a tryptophan residue. The haem pocket appears to be unusually open to the external environment, with another cavity spanning the entrance of the two haem pockets. The final 23 residues of the C-terminal domain are disordered in the structure; however, these domains in the functional dimer are adjacent and include the only two cysteine residues in the protein sequence. It is likely that these residues form disulfide bondsin vitroand it is conceivable that this C-terminal region may act in a putative complex with a partner moleculein vivo.</jats:p

Harnessing buffer solutions to modify the association state of Ferritin nanoparticles

Recent years have witnessed remarkable progress in the field of drug delivery driven by the pursuit of efficient and targeted delivery systems [1,2]. Among the promising candidates for such systems are ferritins, nanoparticles distinguished by their unique hollow sphere structure and the ability to specifically target cell surface receptors [3,4]. In a groundbreaking study, researchers developed a chimeric ferritin protein derived from both human and archaeal sources. This innovative design incorporates the BC loop of human H ferritin, endowing the chimeric ferritin with CD71 recognition capabilities [5]. Furthermore, the researchers introduced a novel modification to the ferritin protein, enhancing its drug delivery potential by utilizing buffer solutions to finely tune its assembly and disassembly properties [6]. These advancements open up new avenues for the study of encapsulation and delivery of small therapeutic and diagnostic molecules

Rapid and simultaneous determination of free aromatic carboxylic acids and phenols in commercial juices by GC-MS after ethyl chloroformate derivatization

Natural phenol and phenolic acids are widely distributed in the plant kingdom and the major dietary sources include fruits and beverages derived therefrom. Over the past decades, these compounds have been widely investigated for their beneficial effects on human health and, at the same time, several analytical methods have been developed for their determination in these matrices. In the present paper, 19 different aromatic carboxylic acids and phenols were characterized by GC-MS using ethyl chloroformate as the derivatizing agent. This procedure occurs quickly at room temperature and takes place in aqueous media simultaneously with the extraction step in the presence of ethanol using pyridine as a catalyst. The analytical method herein developed and validated presents excellent linearity in a wide concentration range (25–3000 ng/mL), low LOQ (in the range 25–100 ng/mL) and LOD (in the range 12.5–50 ng/mL), and good accuracy and precision. As a proof of concept, ethyl chloroformate derivatization was successfully applied to the analysis of a selection of commercial fruit juices (berries, grape, apple, pomegranate) particularly rich in phenolic compounds. Some of these juices are made up of a single fruit, whereas others are blends of several fruits. Our results show that among the juices analyzed, those containing cranberry have a total concentration of the free aromatic carboxylic acids and phenols tested up to 15 times higher than other juices