Patent Application
20240125775
Purifying contaminated solutions is a pivotal challenge in many aspect. For example, contaminated water is a huge challenge in various parts of the world. Furthermore, infected blood leads to sepsis in human patients. Sepsis is one of the most common diseases worldwide and is associated with a very high death rate. Infections leading to pyrogenic reactions or even sepsis are usually caused by bacteria and in some cases also by fungal or viral pathogens. Pathogenic microorganisms like gram negative-bacteria cause sepsis not only by their direct presence but especially through pathogen components such as lipopolysaccharides (LPS) that are located in their outer cell membrane. Sepsis can be either caused by the pathogens themselves and/or by pathogen components such as endotoxins (e.g. LPS). In response to the pathogenic insult , a huge amount of cytokines are released possibly resulting in multiple organ dysfunctions and death despite treatment. It is often not possible to manage the cytokine and the coagulation cascade to a safe level without lethal outcome. Also in products like food, contaminated water or therapeutic agents, pathogens and/or pathogen components are present. This contamination can also influence the medical condition of healthy people and can worsen the condition of ill people. Thus, the reduction of pathogen and/or pathogen component concentration in aqueous solutions and body fluid samples is a real challenge at present and in future.
The current treatment, of e.g. sepsis, consists in the administration of a combination of broad-spectrum antibiotics and simultaneous fluid therapy, vasoactive drugs, respiratory and metabolic support, as well as the administration of corticoids, blood transfusions and dialysis to assist kidney function. Besides a few new approaches, classic treatment options like fluid therapy and hemodynamic support of antibiotics application are still the gold standard. The application of antibiotics has been widely discussed over the last years as more and more resistances occur. Nevertheless, one of the biggest issues delaying a proper identification of the pathogen causing a sepsis is the often very low pathogen concentration in the blood of the patient. Due to this low titer, the pathogens have to be amplified in vitro. This is time consuming but currently the only way to identify securely identify the pathogen itself but a prerequisite to be able to identify possible antibiotic resistances. Additionally, many patients are already under antibiotic treatment, when the sepsis diagnosis is intended. This is another obstacle for the rapid identification of the sepsis causing pathogen so that even new techniques like nanopore sequencing, often struggle to identify the pathogens, when present in the solution at a low concentration (Derrington et al. 2010). However, a fast identification of the pathogen is pivotal for a tailored treatment (e.g. antibiotic treatment of the patient).
New systems implemented in dialysis systems using special adsorbent filters are trying to filter cytokines out of the patients' blood to stop the uncontrolled reaction of the body (Zhang et al. 2005). At the moment, the effect of different inflammatory and anti-inflammatory substances or in what amount they are needed to fight the infection are discussed. However, for example LPS is known to play an important role, besides starting the cytokine cascade, in the patients' survival rate as it can also influence blood clotting. To detect LPS or other causes of sepsis, new biomarkers are currently being developed to improve diagnoses, but are very expensive and are specific to only one type of pathogen. Recent clearance methods like particle based endotoxin reduction using antibodies bound to the particles are too selective in most cases, as sepsis is caused by multiple and often unidentified, disease causing factors. Peptides that are similar to the ones found in human saliva, have been shown to absorb a broad spectrum of pathogens.
In addition, extracorporeal filtration systems have also been used, which are aimed at lowering various inflammation mediators such as cytokines. Less frequently used but clinically approved are specific filtrations of pathogens using polymyxin B columns (Toraymyxin™, Kunitomo et al. 2001). However, these columns have various limitations in a clinical setup. There are research approaches to use particles for the filtration of pathogens using e.g. specific antibodies. However, antibody-based systems have several disadvantages as they are often highly specific—possibly too specific—for particular pathogen components such as LPS and also very expensive (Kang et al. 2014). With regard to unspecific extracorporeal adsorbers, there are systems with polymer coatings that are able to bind LPS. However, these unspecific adsorbers merely bind LPS in a concentration range, which is considerably higher than the clinically relevant range of less than 10 EU/ml (EU=endotoxin unit).
In a previous pilot study, the use of iron oxide particles functionalized with a peptide to reduce the amount of LPS was shown (Karawacka et al. 2018). Hydroxyapatite and iron oxide coated particles have gained interest as they can be used in many biomedical fields and are also considered to be used in the reduction of pathogenic biofilms.
Thus, there is a need in the art to improve the purification of aqueous solutions and human body fluids. In this way, the concentration of pathogens and/or pathogen components in an aqueous or body fluid sample would be reduced.
The approach of the present invention uses superparamagnetic iron-based particles for reducing the concentration of pathogens and/or pathogen components in an aqueous or body fluid sample. The present disclosure provides several key advantages over the prior art. Compared to Karawacka et al. (2018), which first described iron-based particles linked to a pathogen binding peptide, the superparamagnetic iron-based particles as disclosed herein have been substantially improved. Specifically, the superparamagnetic iron-based particles as disclosed are magnetically attractable so that at least 65% of the particles can be immobilized when a magnetic field, of e.g. 310 mT, is applied. As compared and contrasted in Example 2 (
In particular, a method for reducing the concentration of pathogens and/or pathogen components in an aqueous or body fluid sample is described herein, wherein the method comprises the steps of:
whereby a reduced concentration of pathogens, and/or pathogen components in the sample is obtained, as further defined in the claims.
Furthermore, a method for identifying pathogens in an aqueous or body fluid sample is disclosed, the method comprising the steps of
Specifically, the magnetic attractability of the superparamagnetic iron-based particles may be characterized by a reduction of the superparamagnetic iron-based particle concentration in Ringer solution by 65% to 99.95% when applying a magnetic field of 0.31 Tesla for three minutes in static condition, as defined by the claims.
In another aspect, a use of superparamagnetic iron-based particles for reducing the concentration of pathogens and/or pathogen components in an aqueous or body fluid sample is disclosed, as defined in the claims. Furthermore, a use of superparamagnetic iron-based particles for identifying pathogens in an aqueous or body fluid sample is disclosed, wherein the superparamagnetic iron-based particles are linked to a target binding peptide, wherein the target is a pathogen, as defined in the claims.
Finally, superparamagnetic ironoxide nanoparticles (SPIONs) are disclosed, wherein the SPIONs are linked to a target binding peptide, wherein the target is a pathogen, and/or a pathogen component, as further defined in the claims.
In a first aspect, a method for reducing the concentration of pathogens and/or pathogen components in an aqueous or body fluid sample is described herein, wherein the method comprises the steps of:
In general, “superparamagnetic iron-based particles” as used herein are particles, which are magnetically attractable. The skilled person is aware that the magnetic behaviour of materials can be classified into five major groups: ferro-, ferri-, para-, antiferro-, and diamagnetism. Ferro- and ferrimagnetism are the basic mechanisms in permanent magnets. Permanent magnets can be magnetized by external magnetic fields and remain magnetized after the removal of the fields. Materials such as Fe (iron), Co, Ni, and their alloys, and some compounds of rare earth elements are ferromagnetic (Wu et al. 2019). Superparamagnetism is a form of magnetism, which appears in small ferromagnetic or ferrimagnetic particles. “Particles” as used herein are defined as particles of matter that are from 1 nm (nanometer) to 2 micrometers (μm) in diameter. The inventors contemplate that a particle as used herein can be formed of several individual crystallites forming an agglomerate while retaining superparamagnetism. Furthermore, the skilled person is aware of iron (Fe) based particles, such as ironoxide based particles, which have been used in biological applications (Wu et al. 2019). The skilled person is also aware of various iron-based particles. For example, the particles may be iron oxide, iron (Fe), iron-cobalt, alnico or permalloy particles. In other words, the superparamagnetic iron-based particles may be selected from iron oxide, iron (Fe), iron-cobalt, alnico, and permalloy particles.
The superparamagnetic iron-based particles may be superparamagnetic ironoxide nanoparticles (SPIONs). A “nanoparticle”, as used herein, is a particle of matter that is between 1 nm and 1 μm in diameter. Furthermore, the iron oxide may be selected from Fe3O4, γ-Fe2O3, CoFe2O4 or a mixture thereof. In a strongly preferred embodiment, the iron oxide is Fe3O4.
The “magnetic attractability” of the superparamagnetic iron-based particles, as disclosed herein, can be measured. Specifically, the magnetic attractability of the particles as disclosed herein may be characterized by a reduction of the superparamagnetic iron-based particle concentration in Ringer solution by at least 65% when applying a magnetic field of 310 Millitesla for three minutes in static condition. In other words, the skilled person can compare the iron content, in e.g. Ringer solution, before and after applying a magnetic field in the supernatant. Upon magnetic field application, the superparamagnetic iron-based particles are immobilised and the iron concentration in the supernatant is thereby reduced. The iron content in a solution may be for example determined with Atomic Emission Spectroscopy (AES). The skilled person is also aware of other methods in order to determine the iron content in a sample. For example, the concentration of iron in a sample can also be determined photometrically. However, a determination of the iron concentration by AES is preferred. In Example 1, exemplary AES results are presented herein.
Furthermore, Example 2 specifically compares magnetically attractable particles as disclosed herein with the particles as obtained by following the protocol of Karawacka et al. (2018). As exemplified by
Similarly to the static condition, the magnetic attractability may also be assessed in a flowing sample. In a preferred embodiment, the magnetic attractability is characterized by a reduction of the superparamagnetic iron-based particle concentration in water by at least 65% when applying a magnetic field of 0.8 Tesla at a flow speed of 0.057 m/s. The skilled person is able to assess the iron content in a supernatant solution, e.g. by AES, as described above. In a particularly preferred embodiment, the concentration of the superparamagnetic iron-based particles is reduced by 65% to 99.95%, preferably 70% to 99.95%, more preferably 75% to 99.95%, more preferably 80% to 99.95%, more preferably 90% to 99.95%, more preferably 91% to 99.95%, more preferably 92% to 99.95%, more preferably 93% to 99.95%, more preferably 94% to 99.95%, more preferably 95% to 99.95%, more preferably 96% to 99.95%, more preferably 97% to 99.95%, more preferably 98% to 99.95%, more preferably 99% to 99.95%, more preferably 99.1% to 99.95%, and even more preferably 99.2% to 99.95%. Said especially preferred ranges apply to the magnetic attractability when assessed in static condition and/or in flow condition. The exact ranges can be independently selected for the static condition and the flow condition.
The superparamagnetic iron-based particles may be superparamagnetic ironoxide nanoparticles (SPIONs). Of course, the skilled person is aware that iron oxides are chemical compounds composed of iron and oxygen. There are several known iron oxides and oxyhydroxides. In a strongly preferred embodiment, the iron oxide is selected from Fe3O4, γ-Fe2O3, CoFe2O4 or a mixture thereof, and even more preferably wherein the iron oxide is Fe3O4. Exemplary SPIONs and a method for the production of SPIONs is described in Example 1 and 2.
The magnetic attractability of such SPIONs may be characterized as a reduction of the SPION concentration to a range of 0.01 to 12 ug Fe/ml when incubating the SPIONs at a concentration of 300 ug Fe/ml in water and applying 0.31 Tesla for three minutes in static condition. In particular, the magnetic attractability may be characterized by a reduction of the SPION concentration to a range of 0.01 to 12 ug Fe/ml when incubating the SPIONs at a concentration of 17.5 ug Fe/ml in water and applying 0.8 Tesla at a flow speed of 0.057 m/s. Especially, the SPION concentration may be reduced to a concentration from 0.01 to 10 ug Fe/ml, preferably from 0.01 to 8 ug Fe/ml, preferably from 0.01 to 7 ug Fe/ml, more preferably from 0.01 to 6 ug Fe/ml, preferably from 0.01 to 5 ug Fe/ml, more preferably from 0.01 to 4 ug Fe/ml, and even more preferably from 0.01 to 3 ug Fe/ml.
The “sample”, as used herein is an aqueous of body fluid sample. In general, an “aqueous solution” is a solution in which the solvent is water. In other words, an aqueous solution is water soluble and the skilled person is aware of aqueous solutions. In addition, the method as disclosed herein reduces the concentration of pathogens or pathogen components in said samples. Exemplary aqueous solutions are waste water, ground water, or drinking water. In aqueous solutions such as waste water, the reduction of the concentration of pathogens or pathogen components, may support the regeneration of waste water. In aqueous solutions such as ground water or drinking water, the reduction of the concentration of pathogens or pathogen components may make the ground water or drinking water suitable for human consumption.
In general, a reduction of pathogens or pathogen components in aqueous of “body fluid sample” may have the advantage that the immune system of a human may be supported e.g. in fighting a pathogen infection. Furthermore, a “body fluid” is any fluid, which occurs in or is excrete from a body. Especially preferred are samples from a human body. In particular, the sample may be a body fluid sample, which may be selected from blood, serum, plasma, lymph, urine, liquor, saliva and sputum, even more preferably wherein the human body fluid is blood or serum, and even more preferably wherein the human body fluid is blood. In an especially preferred embodiment, the blood is from a septic patient. If the sample is blood of a body, it is preferred that the blood sample is a mammalian blood sample, more preferably a primate blood sample, and even more preferably a human blood sample.
According to the method as disclosed herein, the concentration of pathogens, and/or pathogen components in the sample of step (c) may be reduced by at least 20%, preferably at least 25%, more preferably at least 30%, more preferably at least 35%, even more preferably at least 40%, even more preferably at least 45%, even more preferably at least 50%, even more preferably at least 55%, even more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, and even more preferably at least 75% compared to the sample of step (a). The skilled person is able to determine the concentration of pathogens by standard experimentation. For example, the skilled person may measure the concentration of bacteria by counting the colony forming units (CFUs) on a growth plate. Alternatively, the skilled person may quantify the concentration of a pathogen by quantitative PCR, genome sequencing, ELISA, or mass spectrometry, which is also known in the art. The concentration of pathogen components, such as LPS or LTA, may be assessed as described in Example 3. However, the skilled person may also use other techniques in order to assess the concentration of pathogens and/or pathogen components.
In a preferred embodiment, the sample is incubated in step (b) with a concentration from 0.002 mg Fe/ml to 5 mg Fe/ml superparamagnetic iron-based particles per volume of sample, preferably from 0.004 mg Fe/ml to 3 mg Fe/ml superparamagnetic iron-based particles, more preferably from 0.007 mg Fe/ml to 2 mg Fe/ml superparamagnetic iron-based particles, even more preferably from 0.01 mg Fe/ml to 1 mg Fe/ml superparamagnetic iron-based particles, and even more preferably from 0.01 mg Fe/ml to 0.15625 mg Fe/ml superparamagnetic iron-based particles. As described above, the skilled person may assess the concentration of Fe/ml by AES. Strongly preferred concentrations are also disclosed in the Example section.
In light of the present disclosure, the skilled person is able to determine suitable time periods for the duration of step (b) (see e.g.
A “pathogen”, as used herein, is any organism that can produce a disease. A pathogen may also be referred to as an infectious agent. In the context of the present invention, “pathogens” includes viruses, prokaryotes like gram-positive and gram-negative bacteria, cyanobacteria, Mollicutes and unicellular eukaryotes (protozoa) like certain genera of fungi, algae and parasites (e.g. sporozoa). Pathogens often possess on their surface sulphated and phosphorylated structures and molecules, which can be bound by a target binding peptide as described in more detail below. Examples sulphated and phosphorylated structures include e.g. lipoteichoic acid (LTA) or lipopolysaccharide.
As the target binding peptide is not specific to a particular pathogen, as described below, the concentration of any pathogen can ideally be reduced. The present disclosure provides experimental data for several pathogens, such as gram-positive and gram-negative bacteria (
“Pathogen components” as used herein refers to any part of a pathogen. For example, a pathogen component may be a part of a cell wall or membrane or part of a viral envelope or capsid. For example, the pathogen components may comprise endotoxins and/or pathogenic cell wall components. Endotoxins are pathogen components of gram-negative bacteria and endotoxins are large molecules consisting of a lipid and a polysaccharide composed of O-antigen, outer core, and inner core joined by a covalent bond. Endotoxins are also referred to as lipopolysaccharides (LPSs). The O-antigen is a repetitive glycan polymer contained within an endotoxin. The O-antigen may also be referred to as an O-polysaccharide, or O-side-chain of the bacteria The outer core contains an oligosaccharide component that attaches directly to lipid A and commonly contains sugars such as heptose and 3-Deoxy-D-manno-oct-2-ulosonic acid. Lipid A is usually a phosphorylated glucosamine disaccharide decorated with multiple fatty acids. These hydrophobic fatty acid chains (Lipid A) anchor the LPS to the bacterial membrane, and the rest of the LPS projects from the cell surface. Put simply, LPS is typically found in the outer membrane of Gram-negative bacteria. However, LPS, as well as other pathogen components, are often remainders in an aqueous or body fluid sample. Such pathogen parts themselves can easily trigger an immune response in a human body. In patients, LPS can cause a huge immune response and can lead to septic shock. Consequently, a reduction of pathogen components, such as LPS, in body fluids has the advantage of cleaning the sample, which may reduce the immune response in the human body. Ideally, a patient can recover from the pathogenic attack more quickly, when then amount of pathogens and pathogen components is reduced. Furthermore, the reduction of pathogen components in aqueous samples can also lead to a reduction in immune response, when the aqueous sample is for example consumed as drinking water.
Typically the endotoxins are derived from gram-negative bacteria, more preferably wherein the endotoxins are derived from of one or more of Klebsiella spp., Escherichia coli (E. coli), and Pseudomonas aeruginosa, more preferably wherein the endotoxins is LPS, preferably wherein the endotoxins comprise E. coli LPS, and more preferably wherein LPS is selected from one or more of LPS O55:B5, LPS O26: B6, and LPS O111:B4.
In general, “pathogenic cell wall components” are parts the cell wall of any pathogen having a cell wall. Preferably the cell wall is the outer layer of the pathogen. For example, in gram-positive bacteria and in fungi the cell wall forms the outer layer of the pathogen. In a preferred embodiments, the pathogenic cell wall component is derived from one or more of gram-positive bacteria and fungi, preferably wherein the cell wall component is derived from gram-positive bacteria, more preferably wherein the cell wall component is Lipoteichoic acid (LTA), teichoic acid and/or a peptidoglycan. Lipoteichoic acid (LTA) is a major constituent of the cell wall of gram-positive bacteria. Gram-positive bacteria have an inner (or cytoplasmic) membrane and, external to it, a thick (up to 80 nanometer) peptidoglycan layer. The structure of LTA varies between the different species of Gram-positive bacteria and may contain long chains of ribitol or glycerol phosphate. LTA is anchored to the cell membrane via a diacylglycerol. Teichoic acids are bacterial copolymers of glycerol phosphate or ribitol phosphate and carbohydrates linked via phosphodiester bonds. Teichoic acids are found within the cell wall of most Gram-positive bacteria such as species in the genera Staphylococcus, Streptococcus, Bacillus, Clostridium, Corynebacterium, and Listeria, and appear to extend to the surface of the peptidoglycan layer. They can be covalently linked to N-acetylmuramic acid or a terminal D-alanine in the tetrapeptide crosslinkage between N-acetylmuramic acid units of the peptidoglycan layer, or they can be anchored in the cytoplasmic membrane with a lipid anchor. Peptidoglycan (also called murein) is a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of most bacteria, forming the cell wall. The sugar component consists of alternating residues of β-(1,4) linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Peptidoglycan serves a structural role in the bacterial cell wall, giving structural strength, as well as counteracting the osmotic pressure of the cytoplasm. In a strongly preferred embodiment, the pathogenic cell wall component is LTA, even more preferably wherein the pathogenic cell wall component is LTA of Staphylococcus aureus and/or Streptococcus pyogenes. Experimental data for the reduction of LTA after immobilisation with superparamagnetic iron-based particles is provided in
The “target binding peptide” as used herein is an amino acid sequence binding to a target, wherein the target is a pathogen or a pathogen component. A ‘peptide’ as used herein is an amino acid sequence of at most 100 amino acids, preferably at most 80 amino acids, more preferably at most 60 amino acids. Said amino acid sequence may have the more common L-configuration. However, the amino acids may also have the less common D-configuration. The D-configuration may have the advantage that natural proteases may digest the peptide less efficiently than the natural L-configuration. Binding of a peptide to a target can be measured by standard methods in the art such as an agglutination assay, ELISA, fluorescent polarization or FRET. In case the target comprises a nucleic acid, other electrophoretic shift assays may also be used for assessing the binding of target binding peptide to the target. Generally, the skilled person can use any target binding peptide, which binds to a pathogen or a pathogen component.
A “motif”, as used herein refers to a short amino acid sequence motif. Specifically, a motif is an amino-acid sequence pattern. A sequence motif shall be distinguished from a structural motif, which is formed by a three-dimensional structure of amino acids. In the present case, that motif is typically important for the pathogen or pathogen component binding. In a preferred embodiment, the target binding peptide has a sequence comprising 12-30 amino acids having one motif selected from SEQ ID NO: 5 (VEVLxxxxW), SEQ ID NO: 6 (VEILxxxxW), SEQ ID NO: 7 (VEIYxxxxW) and SEQ ID NO: 8 (VEVYxxxxW). The motifs of SEQ ID Nos: 5-8 are pathogen or pathogen component binding motifs, wherein the first four amino acids and the tryptophan are key for binding. In an even more preferred embodiment, the target binding peptide has a sequence comprising 12-30 amino acids having one motif selected from SEQ ID NO: 21 (VEILxxxxWGTV), SEQ ID NO: 22 (VEVLxxxxWGTV), and SEQ ID NO: 23 (VEIYxxxxWGTV), even more preferably from SEQ ID NO: 24 (VEILYRGSWGTV), SEQ ID NO: 25 (VEVLYRGSWGTV), and SEQ ID NO: 26 (VEIYHGGTWGTV). In another embodiment, the target binding peptide has a sequence comprising 12-30 amino acids having one motif selected from SEQ ID NO: 27 (VEVLxQ), SEQ ID NO: 28 (VEIFxxxxDSSL), SEQ ID NO: 29 (VVVLxxxxW), SEQ ID NO: 30 (VEVRxxxxW), SEQ ID NO: 31 (VEISxxxxW), SEQ ID NO: 32 (VGVLxxxxW), SEQ ID NO: 33 (VEVSxxxxW), SEQ ID NO: 34 (VEVFxxxxW), SEQ ID NO 35 (VELRxxxxW), SEQ ID NO: 36 (VELFxxxxW), SEQ ID NO: 37 (VEVHxxxxW), SEQ ID NO: 38 (VELFxxxxW), SEQ ID NO: 39 (VEILxxxxW), SEQ ID NO: 40 (VEIFxxxxW, SEQ ID NO: 41 (VEFFxxxxW), SEQ ID NO: 42 (VELHxxxxW), SEQ ID NO: 43 (LEVRxxxxW), SEQ ID NO: 44 (VEVQxxxxW), SEQ ID NO: 45 (VELLxxxxW), SEQ ID NO: 46 (VELYxxxxPPAL), and SEQ ID NO: 47 (LEVFxxxxW).
For example, Leito et al. (2008) describes the motif VEVLxxxxW (SEQ ID NO:5) and its binding to various bacteria in detail. Briefly, various pathogens have been shown to bind to a 16-mer peptide (QGRVEVLYRGSWGTVC, SEQ ID NO: 19) located within the SRCR domains of e.g. DMBT1. An exemplary SRCR domain is disclosed herein as SEQ ID NO: 11. For different bacteria, there was a highly significant correlation between adhesion to DMBT1 and adhesion to SRCR peptide 2 suggesting that SRCR peptide 2 is the major bacteria binding site. Leito et al. (2008) also disclose that 8 amino acids were involved in binding (xRVEVLYxxSWxxxx) by performing an alanine substitution scan. Said motif is derived from DMBT1 (SEQ ID NO: 4), which is a member of the scavenger receptor cysteine-rich (SRCR) superfamily. DMBT1 is secreted protein mainly expressed by epithelia and glands. DMBT1 is also known as salivary agglutinin or glycoprotein-340 (gp340). DMBT1 is a member of the scavenger receptor cysteine-rich (SRCR) superfamily, i.e. proteins that have one or more SRCR domains. Furthermore, DMBT1 is known to selectively interact with some bacterial pathogens (e.g. Prakobphol et al., 2000,) and viruses (e.g. HIV (Wu et al., 2003,) and influenza A viruses (Hartshorn et al., 2003)), and thus inhibits the infectivity of these pathogens. For example, WO 2005/079834 also describes the key elements of the motif and derivatives thereof in detail. In various species the SRCR domains as well as the motifs have slightly varying motifs. As disclosed in SEQ ID NOs: 13-18, DMBT1 from mouse, rat and pig have slightly varying sequences. Variants of the motif that the skilled person might expect, on the basis of reviewed or predicted occurrence in animals such as large and small mammals living on land, in fresh or salt water, reptiles such as crocodiles, snakes and lizards, birds, fish and invertebrates such as the barnacle and liver fluke, will result in a similar binding behaviour to that of the human variants of the binding sequence. This refers to both previously published sequences as they are currently listed under protein sequence databases, such as Uniprot or Genebank with the query DMBT1 or its homologues, or sequences that are published or listed or can be derived from the genome sequencing of further species or other scientific methods. For example, the skilled person can access sequences of DMBT1 with the following identifiers:
In a preferred embodiment, the motif, which is important for the pathogen or pathogen component interaction, can be selected from one of SEQ ID NOs: 5-8 or 21-47, preferably SEQ ID NOs: 5-8.
The target binding peptide may have a sequence comprising 12-30 amino acids having one motif selected from SEQ ID NO: 5-8 or 27-47, preferably the target binding peptide may have a sequence comprising 12-30 amino acids having one motif selected from SEQ ID Nos:5-8, more preferably the target binding peptide may have a sequence comprising 15-25 amino acids having one motif selected from SEQ ID Nos:5-8, more preferably the target binding peptide may have a sequence comprising 16-20 amino acids having one motif selected from SEQ ID Nos: 5-8, more preferably the target binding peptide may have a sequence comprising 17-19 amino acids having one motif selected from SEQ ID Nos: 5-8, even more preferably the target binding peptide may have a sequence comprising 17-19 amino acids having the motif selected from SEQ ID NO:5, even more preferably the target binding peptide may have a sequence comprising 17-19 amino acids having the motif selected from SEQ ID NO:5 and the motif is N-terminally preceded by 4-6 amino acids, most preferably the target binding peptide may have a sequence comprising 17-19 amino acids having the motif selected from SEQ ID NO:5 and the motif is N-terminally preceded by SEQ ID NO:9 (RCQGR). For example, the target binding peptide may have a sequence consisting of 12-30 amino acids having one motif selected from SEQ ID NO: 5-8.
“SRCR domains” are found in eukaryotes and these domains are present on the cell membrane and have a role in binding to specific ligands. The structure contains a six stranded beta-sheet and one alpha-helix. In a preferred embodiment, the target binding peptide shows at least 70% sequence identity to a sequence comprised within a scavenger receptor cysteine-rich (SRCR) protein domain, preferably wherein the target binding peptide shows at least 70% sequence identity to a sequence comprised within a SRCR protein domain of DMBT-1 (GP340). The skilled person can readily assess the identity of amino acids sequence by alignment, e.g. by online sequence alignment tools such as ClustalW2.
The skilled person is able to identify SRCR domain e.g. in the protein data bank (PDB). An exemplary SRCR domain is disclosed in SEQ ID NO: 11. It is even more preferred that the target binding peptide is comprised within one of the SRCR protein domain of DMBT-1 (GP340). All 14 SRCR domains within DMBT1 are disclosed below:
In the above table, the individual peptide sequences binding to the pathogen or pathogen binding component are underlined. Furthermore, the binding motif is shaded in grey. Based on these sequences as well as the gene identifiers of the 46 exemplary animals above, the skilled person will readily identify suitable sequences, which are pathogen or pathogen component binding.
It is preferred that the target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and wherein the target binding peptide comprises a sequence having at least 60% identity to the sequence of SEQ ID NO: 1, preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the target binding peptide comprises a sequence having at least 65% identity to the sequence of SEQ ID NO: 1, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47and the target binding peptide comprises a sequence having at least 70% identity to the sequence of SEQ ID NO: 1, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47and the target binding peptide comprises a sequence having at least 75% identity to the sequence of SEQ ID NO: 1, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the target binding peptide comprises a sequence having at least 80% identity to the sequence of SEQ ID NO:1, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the target binding peptide comprises a sequence having at least 85% identity to the sequence of SEQ ID NO: 1, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the target binding peptide comprises a sequence having at least 90% identity to the sequence of SEQ ID NO:1, and even more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the target binding peptide comprises a sequence having at least 95% identity to the sequence of SEQ ID NO:1.
Moreover, the target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the target binding peptide comprises a sequence having at least 60% identity to the sequence of SEQ ID NO:2, preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the target binding peptide comprises a sequence having at least 65% identity to the sequence of SEQ ID NO:2, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the target binding peptide comprises a sequence having at least 70% identity to the sequence of SEQ ID NO:2, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the target binding peptide comprises a sequence having at least 75% identity to the sequence of SEQ ID NO:2, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the target binding peptide comprises a sequence having at least 80% identity to the sequence of SEQ ID NO:2, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the target binding peptide comprises a sequence having at least 85% identity to the sequence of SEQ ID NO:2, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the target binding peptide comprises a sequence having at least 90% identity to the sequence of SEQ ID NO:2, and even more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the target binding peptide comprises a sequence having at least 95% identity to the sequence of SEQ ID NO:2.
In particular, the target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and wherein the target binding peptide comprises a sequence having at least 60% identity to the sequence of SEQ ID NO: 1, preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 65% identity to the sequence of SEQ ID NO: 1, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 70% identity to the sequence of SEQ ID NO:1, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 75% identity to the sequence of SEQ ID NO: 1, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 80% identity to the sequence of SEQ ID NO: 1, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 85% identity to the sequence of SEQ ID NO:1, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 1, and even more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 95% identity to the sequence of SEQ ID NO:1.
Furthermore, the target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 60% identity to the sequence of SEQ ID NO:2, preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 65% identity to the sequence of SEQ ID NO:2, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 70% identity to the sequence of SEQ ID NO:2, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 75% identity to the sequence of SEQ ID NO:2, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 80% identity to the sequence of SEQ ID NO:2, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 85% identity to the sequence of SEQ ID NO:2, more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 90% identity to the sequence of SEQ ID NO:2, and even more preferably target binding peptide may have a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the target binding peptide comprises a sequence having at least 95% identity to the sequence of SEQ ID NO:2.
For example, the target binding protein may have a sequence from 12 to 30 amino acids, preferably the target binding protein may have a sequence from 13 to 25 amino acids, more preferably the target binding protein may have a sequence from 14 to 20 amino acids, more preferably the target binding protein may have a sequence from 15 to 19 amino acids, even more preferably the target binding protein may have a sequence from 16 to 18 amino acids.
The target binding peptide may be a pathogen component binding peptide, preferably the target binding peptide binds to a poly-sulfated and/or poly-phosphorylated ligand located on the surface of the pathogen. For example, End et al. (2009) demonstrates with competition experiments and ELISA studies that the SRCR domains of DMBT1 specifically bind poly-sulfated and poly-phosphorylated structures, such as heparansulfate, LPS, and lipoteichoic acid. As demonstrated in End et al. (2009), the skilled person is able to determine whether a peptide sequence binds to poly-sulfated and poly-phosphorylated structures such as LPS. According to the present disclosure, the target binding peptide may be linked to superparamagnetic iron-based particles and a marked reduction of LPS may be measured upon immobilisation of the LPS-bound particles (
The superparamagnetic iron-based particles and the target binding peptide are linked. Said linkage is sufficiently stable throughout the performance of the method to not break when the particles are used to immobilise e.g. a pathogen. The superparamagnetic iron-based particles and the target binding peptide may be linked by any suitable linkage to stably connect the superparamagnetic iron-based particles and the target binding peptide. Numerous suitable linkages are known in the art. The skilled person can also test whether the linkage is suitably stable by assessing the amount of linked peptide per iron-based particles using UV spectrometer absorption (280 nm). For example, the amount of peptide linked to the superparamagnetic iron-based particles was assessed in Example 8. Specifically, the superparamagnetic iron-based particles and the target binding peptide may be suitably linked when the concentration of target binding peptide before and after carrying out a method as disclosed herein reduces by at most 20%, preferably at most 17.5%, more preferably at most 15%, even more preferably at most 12.5%, even more preferably at most 10%. The target binding peptide concentration can, for example, be assessed by UV absorption. However, any other methods known in the art to assess a protein concentration may be used.
Furthermore, the linkage between the superparamagnetic iron-based particles and the target binding peptide may be covalent or non-covalent. In case the linkage is non-covalent, the linkage is preferred to be electrostatic. Said electrostatic interaction may be an ionic interaction, arising from an electrostatic attraction between two groups of opposite charge. For example, Zainol Abidin et al. (2019) shows that iron oxide-based nanoparticles bind phosphate from aqueous solutions with high affinity and most probably due to electrostatic interaction. Furthermore, said electrostatic interaction is pH-insensitive in a range from pH 2-13 making such electrostatic interactions equally suitable for linkage of the target binding peptide to the superparamagnetic iron-based particle as covalent linkages. In a particularly preferred embodiment, the superparamagnetic iron-based particles are bound to the target binding peptide via a bond between a phosphate group on the target binding peptide and the superparamagnetic iron-based particle, preferably the phosphate group is part of a phosphorylated C-terminal serine of the target binding peptide. Of course, other amino acids may also be phosphorylated. In particular, phosphorylated serines, threonines, tyrosines and histidines may be linked to the superparamagnetic iron-based nanoparticle. Phosphorylated amino acids are known in the art and can be commercially purchased (e.g. peptide SPP04-pS herein) has been purchased from GeneCast (or Proteogenix). The inventors provide experimental data for said linkage of superparamagnetic iron-based particles to a target binding peptide in Example 8.
Advantageously, the superparamagnetic iron-based particles may be covalently linked to the target binding peptide via a connecting module. Said connecting module may be capable of covalently linking the superparamagnetic iron-based particles and the target binding peptide. For example, the connecting module may be selected from hydroxyapatite or a serine-derived aldehyde. In case the connecting module is a serine derived aldehyde, said serine-derived aldehyde may be at the terminus of the target binding peptide, preferably the serine-derived aldehyde may be at the C-terminus of the target binding peptide. In case the connecting module is hydroxyapatite, hydroxyapatite may link to an acidic amino acid (e.g. aspartic acid or glutamic acid) at a terminus of the target binding peptide, preferably the C-terminus.
The connecting module may consist of an ‘anchor unit’ and optionally a ‘linker unit’. Thus, the connecting module may be formed of (i) an anchor unit or of (ii) an anchor and linker unit. It is preferred that the anchor unit is covalently linked to the superparamagnetic iron-based particle and to the target binding peptide or to the linker unit. In other words, the anchor unit is a molecule, which has been covalently anchored/linked to the superparamagnetic iron-based particle and the target binding peptide has been be covalently linked to the anchor unit -directly or via a linker unit. In case an anchor and a linker unit is present, the anchor and the linker unit are covalently linked, the anchor unit is covalently linked to the superparamagnetic iron-based particle, and the linker unit is covalently linked to the target binding peptide. In another preferred embodiment, the anchor unit is covalently linked to the superparamagnetic iron-based particle and the target binding peptide. A possible covalent interaction between the superparamagnetic iron-based particle and the anchor unit can be achieved by a silanization reaction. The present disclosure provides guidance on silanization reactions in Example 1. Furthermore, Liu et al. (2013) discloses a silanization reaction of superparamagnetic iron oxide nanoparticles and suitable reaction conditions. In a preferred embodiment, the anchor unit is a molecule comprising an amino group and a silane, which are covalently bound to the target binding peptide or linker unit and the superparamagnetic iron-based particles, respectively. Prior to anchoring the anchoring unit to the superparamagnetic iron-based particle, the anchoring unit is capable of covalently binding to the superparamagnetic iron-based particle and to the target binding peptide or the linker unit. It is even more preferred that the anchor unit is an aminosilane. Exemplary aminosilanes can be independently selected from (3-aminopropyl)-triethoxysilane (APTES), (3-aminopropyl)-diethoxy-methylsilane (APDEMS), (3-aminopropyl)-dimethyl-ethoxysilane (APDMES), and (3-aminopropyl)-trimethoxysilane (APTMS). In a strongly preferred embodiment, the aminosilane is (3-Aminopropyl)triethoxysilan (APTES). For example, Karawacka et al. 2018 discloses possible anchor and linker units.
In a strongly preferred embodiment, the connecting module is formed by an anchor unit and a linker unit. Said linker unit may be any linker unit suitable to enhance the covalent binding of target binding peptide to the anchor unit. In particular, said linker unit may be linked to the anchor unit and the target binding peptide by a covalent linkage. In an even stronger preferred embodiment, the linker unit is N-succinimidyl bromoacetate (SBA) or succinimidyl 3-(2-pyridyldithio)propionate) (SPDP). In an especially preferred embodiment linker unit is SBA. However, any suitable linker unit may be used to enhance the concentration of target binding peptide on the surface of superparamagnetic iron-based particles, e.g. thiol selective linkers such as thiol-maleimide (Northrop et al. 2015). The enhanced concentration of target binding peptide per amount of iron may be assess by UV absorption (280 nm). For example,
“Immobilization”, as used herein, means that something is made immobile. In the present case, immobilization occurs by the influence of a magnetic field. This means that the magnetically attractable particles are immobilised by the magnet field. In step (c), the superparamagnetic iron-based particles are immobilised in a magnetic field. A suitable magnetic field can be determined by the skilled person. For example, a suitable magnetic field is capable of immobilizing the superparamagnetic iron based particles. Immobilization can be assessed e.g. by assessing the iron concentration in the supernatant or flow through sample, e.g. by AES, as exemplary shown in
Immobilization of the superparamagnetic iron-based particles increases with increasing time periods. In light of the present disclosure, and in particular
The method as disclosed herein may be carried out in a flow through set-up. A flow-through set-up may for example be set up like a dialysis system. It is preferred that the method is carried out using an extracorporeal blood-cleansing device. The general concept of extracorporeal blood-cleansing device has been disclosed in the art (e.g. Kang et al.(2014)). However, to the inventors knowledge, said extracorporeal blood-cleansing has not been carried out with the superparamagnetic iron-based particles linked to target binding peptides as disclosed herein and further defined in the claims. In a flow-through set up the flow speed may be at most 3 m/s, preferably at most 2.5 m/s, more preferably at most 2 m/s, even more preferably at most 1.5 m/s, even more preferably at most 1 m/s, even more preferably at most 0.9 m/s, even more preferably at most 0.8 m/s, even more preferably at most 0.7 m/s, even more preferably at most 0.6 m/s, even more preferably at most 0.5 m/s, even more preferably at most 0.4 m/s, even more preferably at most 0.35 m/s, even more preferably at most 0.3 m/s, even more preferably at most 0.25 m/s, even more preferably at most 0.2 m/s, even more preferably at most 0.15 m/s, even more preferably at most 0.1 m/s, even more preferably at most 0.09 m/s, even more preferably at most 0.08 m/s, even more preferably at most 0.07 m/s, and most preferably at most 0.06 m/s. Similarly, the flow speed may be at least 0.008 m/s, preferably at least 0.01 m/s, more preferably at least 0.02 m/s, even more preferably at least 0.025 m/s, even more preferably at least 0.03 m/s, even more preferably at least 0.035 m/s, even more preferably at least 0.04 m/s, even more preferably at least 0.045 m/s, even more preferably at least 0.05 m/s, and most preferably at least 0.055 m/s. Of course, the skilled person can freely combine any flow speed upper and/or lower limits freely. Flow speed can be assessed by standard methods in the art. For example, the flow speed can be assessed by determining volume output per time unit at a given tubing diameter.
The method as disclosed herein comprises superparamagnetic iron based particles linked to a target binding peptide. In a preferred embodiment, SPIONs are linked to a target binding peptide. Said SPIONs linked to a target binding peptide can show several characteristics when assessing the capability of reducing the concentration of pathogens and/or pathogen components in a sample. In a preferred embodiment, the SPIONs linked to a target binding peptide are capable of reducing the concentration of LPS in step (c) by at least 70%, preferably 80%, more preferably 90%, and even more preferably 95%, compared to the sample of step (a) comprising a LPS concentration of 10 EU/ml, wherein the reduction is determined after incubating the SPIONs linked to a target binding peptide at a concentration of 1 mg Fe/ml in Ringer solution followed by immobilising the SPIONs linked to a target binding peptide in a magnetic field of 0.31 Tesla for a time period of 30 seconds by using an assay based on the detection of recombinant factor C. For example,
Furthermore, the SPIONs linked to a target binding peptide may be capable of reducing the concentration of LPS of step (c) by at least 60%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90%, compared to the sample of step (a) comprising a LPS concentration of 10 EU/ml, wherein the reduction is determined after incubating the SPIONs linked to a target binding peptide at a concentration of 1 mg Fe/ml in Ringer solution followed by immobilising the SPIONs linked to a target binding peptide in a magnetic field of 0.31 Tesla for a time period of 3 seconds by using an assay based on the detection of recombinant factor C. Exemplary
In addition, the SPIONs linked to a target binding peptide may be capable of reducing the concentration of LPS of step (c) by at least 20%, preferably at least 30%, more preferably at least 35%, and even more preferably at least 40%, compared to the sample of step (a) comprising a LPS concentration of 10 EU/ml, wherein the reduction is determined after incubating the SPIONs linked to a target binding peptide at a concentration of 0.01 mg Fe/ml in Ringer solution followed by immobilising the SPIONs linked to a target binding peptide in a magnetic field of 0.31 Tesla for a time period of 30 seconds by using an assay based on the detection of recombinant factor C.
Equally, the SPIONs linked to a target binding peptide may be capable of reducing the concentration of LPS of step (c) by at least 20%, preferably at least 25%, even more preferably at least 30%, and even more preferably at least 35%, compared to the sample of step (a) comprising a LPS at a concentration of 10 EU/ml, wherein the reduction is determined after incubating the SPIONs linked to a target binding peptide at a concentration of 0.01 mg Fe/ml in Ringer solution followed by immobilising the SPIONs linked to a target binding peptide in a magnetic field of 0.31 Tesla for a time period of 3 seconds by using an assay based on the detection of recombinant factor C.
The SPIONs linked to a target binding peptide as disclosed herein may also be capable of reducing the concentration of pathogens, such as bacteria, viruses and/or fungi. In an embodiment, the SPIONs linked to a target binding peptide are capable of reducing the concentration of gram negative bacteria, e.g. E. coli, of step (c) by at least 20%, preferably at least 25%, even more preferably at least 30%, even more preferably at least 35% even more preferably at least 40%, even more preferably at least 45%, and even more preferably at least 50%, compared to the sample of step (a) comprising E. coli at a concentration resulting in 650 colony forming units per ml of sample (CFU/ml), wherein the reduction is determined after incubating the SPIONs linked to a target binding peptide at a concentration of 0.1-1.0 mg Fe/ml in Ringer solution for a time period of 5 minutes followed by immobilising the SPIONs linked to a target binding peptide in a magnetic field of 0.31 Tesla for a time period of 3 min. For example,
Advantageously, the SPIONs linked to a target binding peptide may be capable of reducing the concentration of gram positive bacteria, e.g. S.aureus, of step (c) by at least 50%, preferably at least 65%, even more preferably at least 70%, even more preferably at least 75% even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and even more preferably at least 95%, compared to the sample of step (a) comprising S. aureus at a concentration resulting in around 1400 CFU/ml, wherein the reduction is determined after incubating the SPIONs linked to a target binding peptide at a concentration of 0.3 mg Fe/ml in Ringer solution for a time period of 5 minutes followed by immobilising the SPIONs linked to a target binding peptide in a magnetic field of 0.31 Tesla for a time period of 3 min. Exemplary
Favourably, the SPIONs linked to a target binding peptide are capable of reducing the concentration of E. coli of step (c) by at least 20%, preferably at least 25%, even more preferably at least 30%, even more preferably at least 35% even more preferably at least 40%, even more preferably at least 45%, and even more preferably at least 50%, compared to the sample of step (a) comprising E. coli at a concentration resulting in around 1900 CFU/ml, wherein the reduction is determined after incubating the SPIONs linked to a target binding peptide at a concentration of 0.3 mg Fe/ml in human blood for a time period of 5 minutes followed by immobilising the SPIONs linked to a target binding peptide in a magnetic field of 0.31 Tesla for a time period of 3 min. Exemplary
In another aspect, the present disclosure also describes a method for identifying pathogens in an aqueous or body fluid sample, the method comprising the steps of
For the individual terms as used in said method, the definitions and embodiments as described above equally apply.
Specifically, the magnetic attractability of the particles as disclosed herein may be characterized by a reduction of the superparamagnetic iron-based particle concentration in Ringer solution by at least 65% when applying a magnetic field of 310 Millitesla for three minutes in static condition. Similarly to the static condition, the magnetic attractability can also be assessed in a flowing sample. In a preferred embodiment, the magnetic attractability is characterized by a reduction of the superparamagnetic iron-based particle concentration in water by at least 65% when applying a magnetic field of 0.8 Tesla at a flow speed of 0.057 m/s.
In a preferred embodiment the pathogens are concentrated in step (c) and thereby the identification of the separated pathogens is facilitated. For example,
Furthermore, an aqueous sample, such as drinking water, may be contaminated with a particular pathogen, such as a bacterium. Thus, an improved and/or faster identification of the pathogen may also be highly advantageous, as the aqueous sample may be specifically treated.
In a preferred embodiment, the separated pathogens are still viable, preferably the viability is determined by multiplying the pathogens under suitable conditions, more preferably by plating of the pathogens on a suitable growth plate, even more preferably wherein at least one colony forming unit (CFU) is obtained by plating.
It is also preferred that the pathogens are multiplied under suitable condition in an additional step after step (c) and before step (d). The multiplication has the advantage that the pathogens are present in an even higher concentration. Thus, the identification by standard methods, as described below, is facilitated. It is more preferred that the pathogens are plated on suitable growth plates. For example, the pathogens may be identified by methods, such as PCR, next generation sequencing, mass spectrometry, and/or ELISA. Of course a combination of techniques may also be used.
As already stated above, the definitions and the embodiments regarding the superparamagnetic iron-based particles, in particular the SPIONs, the magnetic attractability, the sample, the duration of step (b) and/or (c), the pathogens, and/or the magnetic field equally apply to all methods as disclosed herein. Particularly preferred embodiments are referred to in embodiment 60 below. Specifically, the pathogen binding peptide may show the same characteristics as the target binding peptide, wherein the target is a pathogen. All embodiments regarding the linkage between the superparamagnetic iron-based particle and the target binding peptide equally apply to the linkage between the superparamagnetic iron-based particle and the pathogen binding peptide.
For completeness, the pathogen binding peptide may have a sequence comprising 12-30 amino acids having one motif selected from SEQ ID NO: 5-8 or 27-47 , preferably the pathogen binding peptide may have a sequence comprising 12-30 amino acids having one motif selected from SEQ ID NO: 5-8, more preferably the pathogen binding peptide may have a sequence comprising 15-25 amino acids having one motif selected from SEQ ID Nos:5-8, more preferably the pathogen binding peptide may have a sequence comprising 16-20 amino acids having one motif selected from SEQ ID Nos: 5-8, more preferably the pathogen binding peptide may have a sequence comprising 17-19 amino acids having one motif selected from SEQ ID Nos: 5-8, even more preferably the pathogen binding peptide may have a sequence comprising 17-19 amino acids having the motif of SEQ ID NO:5, even more preferably the pathogen binding peptide may have a sequence comprising 17-19 amino acids having the motif of SEQ ID NO:5 and the motif is N-terminally preceded by 4-6 amino acids, most preferably the pathogen binding peptide may have a sequence comprising 17-19 amino acids having the motif of SEQ ID NO:5 and wherein the motif is N-terminally preceded by SEQ ID NO:9 (RCQGR). It is strongly preferred that the pathogen binding peptide shows at least 70% sequence identity to a sequence comprised within a scavenger receptor cysteine-rich (SRCR) protein domain, preferably the pathogen binding peptide shows at least 70% sequence identity to a sequence comprised within a SRCR protein domain of DMBT-1 (GP340), and more preferably the pathogen binding peptide is comprised within the SRCR protein domain of DMBT-1 (GP340).
In an embodiment, the pathogen binding peptide has a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the pathogen binding peptide comprises a sequence having at least 60% identity to the sequence of SEQ ID NO: 1 or 2, preferably pathogen binding peptide has a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the pathogen binding peptide comprises a sequence having at least 65% identity to the sequence of SEQ ID NO:1 or 2.
It is even more preferred that the pathogen binding peptide has a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the pathogen binding peptide comprises a sequence having at least 70% identity to the sequence of SEQ ID NO: 1 or 2, more preferably the pathogen binding peptide has a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the pathogen binding peptide comprises a sequence having at least 75% identity to the sequence of SEQ ID NO:1 or 2, more preferably pathogen binding peptide has a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the pathogen binding peptide comprises a sequence having at least 80% identity to the sequence of SEQ ID NO:1 or 2, more preferably pathogen binding peptide has a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the pathogen binding peptide comprises a sequence having at least 85% identity to the sequence of SEQ ID NO:1 or 2, more preferably pathogen binding peptide has a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the pathogen binding peptide comprises a sequence having at least 90% identity to the sequence of SEQ ID NO:1 or 2, and even more preferably pathogen binding peptide has a sequence comprising one motif selected from SEQ ID Nos: 5-8 or 27-47 and the pathogen binding peptide comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 1 or 2. It is even more preferred that the pathogen binding peptide has a sequence comprising one motif selected from SEQ ID Nos: 5-8 and the pathogen binding peptide comprises a sequence having at least 60%, preferably 65%, more preferably 70%, even more preferably 75%, even more preferably 80%, even more preferably 85%, even more preferably 90% and most preferably at least 90% identity to the sequence of SEQ ID NO:1 or 2
The method for identifying pathogens may be carried out in a flow through set-up. In a preferred embodiment, the flow speed of such a flow though set-up may be independently selected from minimal and/or maximal flow speed as disclosed above.
In a further aspect, a use of superparamagnetic iron-based particles for reducing the concentration of pathogens, and/or pathogen components in an aqueous or body fluid sample is disclosed. Of course, the definitions and the embodiments equally apply to the use as to the method for reducing the concentration of pathogens and/or pathogen components. In particular the definitions and embodiments regarding the superparamagnetic iron-based particles, in particular the SPIONs, the magnetic attractability, the sample, the pathogens and/or pathogen components, the target binding peptide, the linkage between the superparamagnetic iron-based particles and the target binding peptide and/or the magnetic field equally apply to the use as to the method. Particularly preferred embodiments are referred to in embodiment 69 below.
In another aspect, a use of superparamagnetic iron-based particles for identifying pathogens in a blood sample is disclosed, wherein the superparamagnetic iron-based particles are linked to a target binding peptide, wherein the target is a pathogen. Of course, the definitions and the embodiments equally apply to the use as to the method for identifying pathogens. In particular the definitions and embodiments regarding superparamagnetic iron-based particles, in particular the SPIONs, the magnetic attractability, the sample, the pathogens, and/or the magnetic field equally apply to the methods and the uses as disclosed herein. Specifically, the pathogen binding peptide may show the same characteristics as the target binding peptide, wherein the target is a pathogen. All embodiments regarding the linkage between the superparamagnetic iron-based particle and the target binding peptide equally apply to the linkage between the superparamagnetic iron-based particle and the pathogen binding peptide. Particularly preferred embodiments are referred to in embodiment 72 below.
Finally, superparamagnetic ironoxide nanoparticles (SPIONs) are disclosed herein, wherein the SPIONs are linked to a target binding peptide, wherein the target is a pathogen and/or a pathogen component. Of course, the definitions and the embodiments as part of a method also apply to the SPIONs. In particular the definitions and embodiments regarding the SPIONs, the magnetic attractability, the pathogens and/or pathogen components, the target binding peptide, the linkage between the superparamagnetic iron-based particles and/or the magnetic field equally apply to the SPIONs. In a very strongly preferred embodiment, the SPIONs are magnetically attractable and the magnetic attractability is defined as a reduction of the SPION concentration in water by 65% to 99.95% when applying a magnetic field of 0.31 Tesla for three minutes in static condition. For example
The inventors further tested the specificity of the methods as disclosed herein. Specifically, the inventors assessed whether the concentration of pathogens and/or pathogen components in the sample is also reduced, when the target binding peptide is replaced with an anti-binding peptide. An anti-binding peptide, as used herein, does not bind to a pathogen and/or pathogen components. It is preferred that said anti-binding peptide does not comprise any of the motifs as disclosed herein, such as any motif as disclosed in SEQ ID Nos: 5-8 or 27-47. As used herein, an anti-binding protein does not bind to pathogens and/or pathogen components. Example 9 provides data and demonstrates that the target binding peptide, e.g. comprising a motif as disclosed in SEQ ID Nos: 5-8 or 27-47, is specific for pathogens, e.g. C. albicans, and/or pathogen components. In these experiments, the SPIONS are linked to either a target binding peptide or an anti-binding peptide and were tested side-by-side. Only the SPIONs linked to the target binding peptide showed a significant reduction in the pathogen concentration, wherein the SPIONs linked to the anti-binding peptide did not show a significant reduction in the pathogen concentration. Hence, when carrying out any of the methods as disclosed herein, the concentration of pathogens and/or pathogen components in the sample of step (c) is ideally reduced by at least 20%, preferably at least 25%, more preferably at least 30%, more preferably at least 35%, even more preferably at least 40%, even more preferably at least 45%, and even more preferably at least 50%. Said reduction in pathogen and/or pathogen component concentration can be compared to the concentration of pathogens and/or pathogen components in a second sample, wherein said second sample is incubated with superparamagnetic iron-based particles being linked to an anti-binding peptide and steps (a) to (c) of the methods as disclosed herein are otherwise carried out in the same way.
In a preferred embodiment, the anti-binding peptide comprises one motif selected from SEQ ID Nos: 5-8 or 27-47 and positions 1 and 3 in any of SEQ ID Nos: 5-8 or 27-47 are substituted. It is even more preferred that the positions 1 and 3 are substituted to alanine. However, any other suitable substitution is possible such as a mutation to glycine or to a charged amino acid such as lysine, arginine, glutamate or aspartate. In addition, the skilled person is aware of suitable mutations.
Furthermore, the SPIONs, which are linked to a target binding peptide, are ideally capable of reducing the concentration of C. albicans of step (c) by at least 20%, preferably at least 25%, even more preferably at least 30%, even more preferably at least 35% even more preferably at least 40%, even more preferably at least 45%, and even more preferably at least 50%, compared to the sample of step (a) comprising C. albicans at a concentration of an optical density of 1 measured at 600nm, wherein the reduction is determined after incubating the SPIONs linked to a target binding peptide at a concentration of 0.1-1.0 mg Fe/ml in Ringer solution for a time period of 5 minutes followed by immobilising the SPIONs linked to a target binding peptide in a magnetic field of 0.31 Tesla for a time period of 3 min. Further information on the experiment is provided in Example 9 herein and the material and methods paragraphs.
As also shown in Example 9 herein, the concentration of C. albicans has also been reduced in blood samples. For example, the SPIONs linked to a target binding peptide are capable of reducing the concentration of C. albicans of step (c) by at least 20%, preferably at least 25%, even more preferably at least 30%, even more preferably at least 35% even more preferably at least 40%, even more preferably at least 45%, and even more preferably at least 50%, compared to the sample of step (a) comprising C. albicans at a concentration resulting in around 3000 CFU/ml, wherein the reduction is determined after incubating the SPIONs linked to a target binding peptide at a concentration of 0.3 mg Fe/ml in human blood for a time period of 5 minutes followed by immobilising the SPIONs linked to a target binding peptide in a magnetic field of 0.31 Tesla for a time period of 3 min. Further information on the experiment is provided in Example 9 herein and the material and methods paragraphs.
The invention is further described by the following embodiments:
whereby a reduced concentration of pathogens and/or pathogen components in the sample is obtained.
preferably wherein the target binding peptide shows at least 70% sequence identity to a sequence comprised within a SRCR protein domain of DMBT-1 (GP340), and
more preferably wherein the target binding peptide is comprised within the SRCR protein domain of DMBT-1 (GP340).
58. The method of any of embodiments 53-57, wherein the pathogens are multiplied under suitable condition in an additional step after step (c) and before step (d), preferably wherein the pathogen are plated on suitable growth plates.
preferably wherein the pathogen binding peptide shows at least 70% sequence identity to a sequence comprised within a SRCR protein domain of DMBT-1 (GP340), and
more preferably wherein the pathogen binding peptide is comprised within the SRCR protein domain of DMBT-1 (GP340).
Pathogenic microorganisms, such as bacteria, can contaminate drinking water and cause sepsis by their presence, and also through the presence of toxins, e.g. lipopolysaccharides (LPS) derived from the bacteria's outer cell membrane. Consequently, huge amount of cytokines are released, often resulting in multiple organ dysfunctions and death despite treatment. A novel experimental approach is to remove pathogens and/or pathogen components by using magnetic particles. For this purpose, the inventors synthesized iron-based particles, especially superparamagnetic ironoxide particles (SPION). In one example, the SPIONs were coated with an (3-Aminopropyl)-triethoxysilan (APTES) layer using a co-precipitation method. As a linker for the APTES-coated superparamagnetic iron oxide (SPIONAPTES), N-succinimidyl bromoacetate (SBA) was used to bind short peptide sequences to amino groups on these particles. The results showed a quantitative reduction of pathogens, such as Gram-positive and Gram-negative bacteria as well as a reduction of pathogen components, such as LPS and LTA by using a peptide-functionalized SPIONAPTES (SPIONAPTES-SBA-SP04 and SPIONAPTES-SBA-SP19). In the light of these results, the inventors aim to use these particles for example in extracorporeal clearance, as well as the removing of the pathogens from other liquid sables such as drinking water.
Example 1—Generation of Superparamagnetic Iron Oxide (SPION) Particles with Linked Peptide (SPIONAPTES-SBA-SPP04)—a Physiochemical Characterization
As a fast and reliable method of synthesizing particles, the commonly used co-precipitation method was carried out. With this technique it was possible to reproduce the synthesis of different particles with minimal changes in the overall parameters. An in-situ coating process was used for SPIONAPTES as described before. For SPIONHAp a two-step procedure, with intermediate citrate coating of iron oxide particles (SPIONCit) was used. SPIONCit have a stable coating with citrate showing no visible agglomeration and good storage capabilities. This can be an advantage to prevent agglomeration in the second step, especially if they are compared to bare iron oxide particles.
The hydrodynamic diameter (HD), polydispersity index (PDI), Z potential as well as the susceptibility are listed in Table 1. The mean hydrodynamic diameter of SPIONAPTES was found to be around 153 nm and around 175 nm for SPIONHAp. The PDI of the used batches was below 0.2 which indicates a narrow size distribution. In biomedical applications the size of the particles has to be considered very carefully before releasing particles into e.g. human blood. In this approach, the separation of pathogens and pathogen components is supposed to be taking place in e.g. an extracorporeal (in vitro) blood stream comparable to dialysis setups. Therefore, particles shall not to be too small in order to remove them very efficiently by magnetic means.
Scanning electron microscopy (SEM) was applied to examine the morphology of the particles. Because of aggregation during the process of drying, the HD differs from the one seen in the SEM-images. It is also clear that the HD is not the physical diameter of the particles, but includes also the surrounding hydration layer. In case of SPIONCit and SPIONAPTES, the SEM-diameter is smaller compared to their HD (see
Furthermore, Energy-dispersive X-ray spectroscopy (EDX) analysis was used for the elemental analysis and chemical characterization of a) SPIONAPTES b) SPIONHAp and c) SPIONCit (
X-ray diffraction (XRD) patterns (see
The Fourier transform infrared (FTIR) spectra of SPIONAPTES, SPIONCit and SPIONHAp can be seen in
In a next step, the inventors assessed the efficiency of covalently linking a peptide to the particles. The efficiency to bind peptide to the particles can vary greatly based on the chemoselective linker used. One possible candidate is SBA, which has been used in a previous study (Karawacka et al (2018)).
As shown in
Considering that the amount of bound peptide was significantly higher on the SPIONAPTES-SBA than on SPIONAPTES or SPIONHAp, it can be assumed that the linker increased the amount of peptide bound to the particles. Consequently, the chemoselective linker SBA improves the covalent linkage between the SPION and the peptide, however, the chemoselective linker is not necessary.
In summary, all particles as disclosed herein had a high magnetic susceptibility. Differences were related to the individual synthesis, affecting the core size, crystallinity, or the size of the coating layer. SPIONAPTES (and the functionalized SPIONAPTES-SBA-SPP) and SPIONHAp could be easily magnetically separated from water at a pH of 7.4, which was key for the subsequent experiments. The results show a good size distribution and stability for the different particle systems. The susceptibility Xv for the different particles is comparable for SPIONHAp and SPIONAPTES (see Table 1) and both can be magnetically separated.
Base on the SPION particles described in Karawacka et al. (2018), the SPION preparation has been further improved so that the SPIONs show a better magnetic attractability/separability and are more metastable in solution. The synthesis described by Karawacka et al. was based on a synthesis of Zaloga et al. 2014. The improved synthesis as disclosed herein uses the same stoichiometric ratios. However, the preparation volume of the synthesis was changed from 10 ml to 50 ml. In addition, the speed of mixing the components and the setup volume was changed that the stirring was constant at 450 rpm during synthesis. Furthermore, the subsequent clean-up was performed by multiple washing with DI water with direct dispersion in DI water and was not performed by dialysis or after drying.
Compared to Karawacka et al. (2018) the amount of Si on the particles could be increased by 25%. This results in an increase of the reactive, i.e. available —NH2 groups for the subsequent functionalization with succinimidyl bromine acetate (SBA). This leads to a higher degree of functionalization with the peptides.
The educt quantity of the SBA was increased by a factor of four and the time of functionalization was doubled compared to Karawacka et al. (2018). In the subsequent binding of the peptides (SPP04 and SPP19), an increase in the binding to peptide and thus an increase in the peptide content from 0.06 μmol peptides/mg Fe for Karawacka et al. (2018) to over 0.085 μmol peptides/mg Fe for SPP04 and 0.082 μmol peptides/mg Fe for SPP19 was achieved by doubling the reaction time. This improvement in the method resulted in an increase in peptide binding of 37% and 42%, respectively. Furthermore, this improvements was consistently at around 40% and thus it is expected that a functionalization with another peptide is expected to also show a similar increase in functionalization by changing the reaction conditions as described above.
Furthermore, the particle size increased in comparison to the SPIONs as disclosed by Karawacka et al. (2018). It is notes that the size of the particles before and after functionalization must be distinguished. Karawacka et. al. (2018) synthesized SPIONAPTES with a diameter between 100-190 nm. SPIONAPTES as disclosed herein have a diameter between 180-250 nm. Karawacka et al. (2018) is silent on the particle size (diameter) after functionalization with peptide. SPIONAPTES-SBA-SPP04/SPIONAPTES-SBA-SPP19 as disclosed herein are between 1.0 and 2.7 μm depending on pH. In neutral pH (e.g. around 7), a diameter of 2.54 μm is achieved for SPIONAPTES-SBA-SPP04. A magnetic separation of the particles could not be achieved with the particles produced by Karawacka et al. (2018). This is why the particles in
For testing and comparing the depletion efficiency of the SPIONs as disclosed herein and the SPIONs of Karawacka et al. (2018), the inventors performed atomic emission spectrometry (AES) measurements of the supernatants after the different separation experiments.
In order to evaluate general parameters of the concentration and incubation time with SPIONs, the inventors analysed conditions that are representative for these parameters. A high and a low particle concentration, as well as two exposure conditions were tested (agitation for 30 sec and 3 sec; particle concentration of 0.01 mg Fe/ml and 1 mg Fe/ml) were tested with the peptide exposing SPIONAPTES-SBA-SPP04 and non-peptide exposing SPIONHAP. The concentration of peptide for the functionalized particles was assumed to be 0.08 μmol peptide/mg Fe, as assessed above. As other particles have been reported to bind certain amounts of LPS unspecifically, SPIONHAp are interesting for our experiments concerning this concern as a control.
An amount of 10 EU/ml for the different types of LPS was used, which is equal to an endotoxin amount of 1 ng/ml. Interestingly, studies have shown that the amount of endotoxin found in plasma is higher in patients that show a sepsis than in healthy patients. Said amount is ranging from 2 pg/ml to about 5 ng/ml for healthy individuals and patients with sepsis, respectively. In case of a severe septic shock it can be 1000 to 10000 times higher than in healthy patients (Jeong et al (2019); Reich et al. (2016); Rhodes et al. (2017)). The median level of endotoxin for non-survivors was found to be about 0.5 ng/ml and 0.2 ng/ml for survivors. Therefore, the reduction of endotoxin should be as efficient as possible.
The efficiency of the separation is shown in
By reducing the amount of particles to 0.01 mg Fe/ml and therefore the amount of peptide in the same extend to a 100-fold lower concentration the binding of LPS was reduced to about 50% for SPIONAPTES-SBA-SPP04 (
In summary, the separation efficiency of LPS is illustrated
The binding of a further relevant toxin (from gram-positive bacteria) was also investigated with the improved particles as disclosed herein, which was not described in Karawacka et al. (2018). These experiments were performed in aqueous Ringer' solution.
Example 4—SPIONAPTES-SBA-SPP04 Specifically Separates Artificial Gram-Negative Bacteria (MicromerLPS)
For a better understanding of the separation process, the inventors tried to remove artificial micromers to simulate bacteria. For this, LPS coated polystyrene/polymethacrylate Micromer® particles (micromerLPS) were used and removed from a water suspension instead of living bacteria. MicromerLPS had a size of about 1 μm corresponding to the size of some bacteria e.g. S. aureus. This experiment was performed by optical density measurements at 620 nm, as it is done for actual bacteria.
As shown in
In addition to the removal of LPS and artificial bacteria, the inventors also set out to remove actual bacteria from aqueous solutions. Karawacka et. al. (2018) did not perform separations of whole bacteria. The step of plating out the particles after the separation is of high diagnostic importance, because it allows extract a high concentration of bacteria and thus an improvement of the pathogen diagnosis. The identification of the bacterial is of great therapeutic value as an anti-bacterial therapy can be tailored after the assessment of the (mixture of) bacteria, which infected the blood.
Based on the values from the experiment shown in
For testing the separation of S. aureus from a sample, particles were functionalized with the peptides SPP04 and SPP19 for this experiment. The bacterial suspension was adjusted to an OD (measured at 600 nm) of 0.13 and then diluted 1:800 with Ringer's solution. Both functionalized particles showed a high depletion of the bacteria and thus a subsequent plating was possible. It could also be shown that the particles have no toxic effect on the bacteria. For Staphylococcus aureus (gram+) a reduction from 1393 CFU/ml to 27 CFU/ml for SPIONAPTES-SBA-SPP04 and 73 CFU/ml for SPIONAPTES-SBA-SPP19 could be achieved by separation as shown in
When plating the bacteria loaded particles (also called re-plating), CFU values in the range of the positive control were found for SPIONAPTES-SBA-SPP04 and SPIONAPTES-SBA-SPP19. In the case of SPIONAPTES-SBA-SPP04, the number of CFU is even increased. Analogous observations are shown in
Example 6 - SPIONAPTES-SBA-SPP04 Separates E. coli From Blood
In further experiments, the inventors demonstrate herein that it is possible to separate and extract E. coli from blood and additionally to plate out the bacteria-bound particles again. For this experiment, E. coli was added to blood and then 0.3 mg Fe/ml particles were used to extract/separate the bacteria. Thus, the remaining bacterial load in the blood was tested as a first aspect of this experiment. Furthermore, citrate and EDTA-stabilized blood was used and compared, as EDTA is expected to hinder the binding of the peptide to the bacteria. Therefore, the depletion of bacteria is expected to be lower in EDTA-stabilized blood compared to citrate-stabilized blood. The overall bacterial load in citrate stabilized blood amounted to 1913 colony forming units per milliliter (CFU/ml) (
In a second aspect of the experiment, bacteria bound to the particles were recovered from the blood and (re-)plated in order to assess the plating efficiency. The plating efficiency also demonstrates that the separated bacteria from blood are still viable after separation. This is particularly valuable as this experiment can be used to assess the bacterial identity in blood. After having identified the bacterial identity of a patient's blood sample, the treatment can be tailored towards said specific bacteria. In EDTA- and citrate-stabilized blood, the bacteria were still viable and could be plated. In citrate stabilized blood, 913 CFU/ml for SPIONAPTES-SBA-SPP04 resulted from the plating experiment.
Furthermore, bacteria could also be recovered by re-plating in EDTA-stabilized blood (1066 CFU/ml for SPIONAPTES-SBA-SPP04)
In summary, the separation efficiency is highest in citrate-stabilized blood when using SPIONAPTES-SBA-SP04. Consequently, the SPIONs as disclosed herein are capable of separating pathogens such as gram-negative bacteria from blood, when functionalized with a pathogen binding peptide.
In this experiment, an approach as used with common blood cultures in order to identify pathogens was chosen. The total volume was set to 10 ml. The bacterial suspension was adjusted to an OD (measured at 600 nm) of 0.13 with Ringer's solution analogous to the previous experiments. A sample, which was not treated with SPIONs, served as a control. A concentration of 0.3 mg Fe/ml SPIONAPTES-SBA-SPP04 was used. Incubation was performed for 30 minutes at room temperature on a rolling incubator. The particles were then magnetically separated for 3 minutes at 310 mT. 0.5 ml Ringer's solution was added to the particle pellets and vortexed.
In previous experiments, the peptide was bound to the C-terminal cysteine of the peptide via the SBA linker. In this experiment, the peptide was bound to the SPION via an alternative binding process. The peptide had a C-terminal phorphoserine group (pS). With the alternative procedure, the reaction step via the SBA linker can be omitted, which makes the entire synthesis much quicker and more efficient. It was possible to bind 0.03 μmol/mg Fe of the peptide SPPpS (SEQ ID NO: 3 SSP04-pS, in comparison to SPP04, wherein the C-terminal cysteine replaced with phosphoserine) to SPIONHAp, as also disclosed herein. The reaction was performed in borate buffer pH 8.5 for 24h at 700 rpm and 25° C. Supernatants and wash steps were quantified in the UV spectrometer to calculate the amount of peptide on the particles. In conclusion an alternative method to covalently link the peptide to the SPION has been tested and it is expected that said particles can be used equally well to immobilize pathogens or pathogen components from aqueous solution and body fluids.
According to the protocol used for the separation with bacteria, as described above, the inventors carried out separation experiments with Candida albicans [SC ADH AGA4A #1509]. For the overall separation efficiency, the OD of the samples was set to 1.0 at 600 nm. Separation experiments were carried out using 100 μg Fe/mL and 300 μg Fe/mL of SPION-APTES-Pep (RKQGRVEVLYRASWGTVC; SEQ ID NO.1) and SPION-APTES-anti-binding Peptide (antibindingPep; RKQGRAEALYRASWGTVC; SEQ ID NO: 48) as seen in
In this Example, the removal efficiency was tested. Hence, a strong reduction in the OD value means that a lot of Candida albicans hase been removed from the sample. When the sample was incubated with 100 μg Fe/mL of SPION-APTES-Pep, a reduction to around 0.4 OD was achieved. Even more so, when the sample was incubated with 300 μg Fe/mL of SPION-APTES-Pep, a reduction to around 0.16 OD was achieved. Hence, a reduction by 60% and 84%, respectively, was obtained. Furthermore, it can be excluded that the removal efficiency is based on non-specific binding of the peptide to the pathogens, as a reduction in the C. albicans concentration could not be detected, when the SPIONS were functionalized to the anti-binding peptide, which has a mutated ‘VxV’ motif. In other words, the anti-binding peptide did not remove Candida albicans even when incubated with 300 μg Fe/mL.
In summary, a high removal efficiencies were detected for both samples so that the removal was efficient for both concentrations of SPION-APTES-Pep.
To evaluate the separation in more complex media, platelet-poor plasma isolated from citrate stabilized blood was used. The amount of CFU was set to be 3000 CFU/mL as seen in
Consistent with the previous results, the number of CFU could only be significantly reduced by using the SPION-APTES-Pep as the binding peptide. In contrast when using the anti-binding peptide, the reduction of the CFUs was not significant according to a Student's t-test. There were 3145 CFUs in the untreated sample. Furthermore, there were 1977 CFUs detected for the suspension sample SPION-APTES-Pep; there were 2455 CFUs detected for the on beads sample SPIONS-APTES-Pep; there were 2902 CFUs detected for the suspension sample SPION-APTES-antibindingPEP; and there were 400 CFUs detected for the on beads sample SPION-APTES-antibindingPep. Hence a significant reduction using SPION-APTES-Pep by around 37% was achieved (suspension sample SPION-APTES-Pep compared to the untreated sample; reduction of CFUs (1168) divided by CFUs of the untreated sample (3145)).
It was possible to plate out the particles and cultivate Candida albicans on plates. Hence, the pathogen is easily identifiable, if this was necessary due to an unknown pathogen being present the sample.
Iron(II) chloride tetrahydrate, iron(III) chloride hexahydrate, sodium sulphate anhydrous, n-hexane and sodium dihydrogenphosphate monohydrate were purchased from Merck KGaA, Germany. 3-mercaptopropionic acid, dichloromethane, N-hydroxysuccinimide, N,N′-dicyclohexylcarbodiimide, anhydrous N,N′-dimethylformamide and sodium citrate were obtained from Sigma-Aldrich Chemie GmbH, Germany. 25% (v/v) Ammonia, (3-aminoprpyl)triethoxysilane, methanol, citric acid, sodium hydroxide, 1N hydrochloric acid, 32% (v/v) hydrochloric acid, acetonitrile, ethanol, sodium bicarbonate anhydrous, 35% (v/v) hydrogen peroxide, chloroform, ethyl acetate, disodium hydrogenphosphate anhydrous, boric acid, 1,4-dithiothreitol, Triton X-100, calcium chloride dihydrate, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide-hydrochloride, calcium nitrate tetrahydrate and di-Ammonium hydrogen phosphate were purchased from Carl Roth GmbH & Co.- KG, Germany. Minisart NML syringe filters (0.2 and 0.8 jam pore size); Peptide RKQGRVEVLYRASWGTVC (SEQ ID NO:1) and RKQGRVEILYRGSWGTVC (SEQ ID NO:2) were synthesised by Gene Cust, Luxemburg. E. coli LPS O55:B5 (LPS 1) and E. coli LPS O26:B6 (LPS 3) was provided by SigmaAldrich. E. coli LPS O111:B4 (LPS 2) was provided by Mikrobiologisches Institut—Klinische Mikrobiologie, Immunologie und Hygiene Universitätsklinikum Erlangen und Friedrich-Alexander-Universität. Micromer® Particles (50 mg/ml) have been purchased from micromod Partikeltechnologie GmbH, NdFeB magnet provided by supermangnete.de. The water used for all experiments came from a Milli-Q system (Merck KGaA, Darmstadt, Germany). E. coli was provided by Mikrobiologisches Institut—Klinische Mikrobiologie, Immunologie und Hygiene Universitätsklinikum Erlangen und Friedrich-Alexander-Universität. Ringer solution was provided by Fresenius GmbH.
A modified protocol to synthesize citrate coated iron oxide particles was used. Briefly, 1 g of iron (II) chloride and 2 g iron (III) chloride (ratio 1:2) were dissolved in 50 ml water and stirred with 250 rpm under an argon atmosphere to prevent oxidation. Iron oxide was precipitated by addition of ammonia solution. After ten minutes of stirring, a solution bearing 4.4 g of sodium citrate in 10 ml was added and the mixture stirred for 30 minutes at 90° C. and 450 rpm. To remove excess sodium citrate, the cooled SPIONs were sterile filtered through a 0.2 pm pore diameter syringe filter and dialysed against water with 100 kDa Spectra/Por® dialysis-tubes from Spectrum Laboratories INC. Any other 100 kDa dialysis-tubing could have been also used. The dialysed particles were kept in water at 4° C. until further use.
This synthesis was carried out according to Mondal et al. (2013 and 2017). SPIONCit containing 160 mg Fe were dispersed in water and 60 mg of calcium nitrate tetrahydrate were added to the dispersion. The pH of the dispersion at 40° C. was adjusted to 9.1 by addition of NaOH (1 N). After 10 min, 30 mg of di-ammonium hydrogen phosphate were dissolved in 5 ml water and then added to the dispersion. The dispersion was kept at 40° C.for 1 hour and 250 rpm, before it was cooled to room temperature and magnetically washed with water for three times. The final dispersion was stored at 4° C.until further use.
The synthesis of SPIONAPTES was performed in a modified one step co-precipitation process (Friedrich et al. (2019)). An aqueous solution of FeCl2 and FeCl3 (ratio 1:2) was stirred and heated to 90° C. under argon atmosphere. Precipitation was initiated by the addition of 25% ammonia. Afterwards, the particles were stirred at 70° C. for 15 minutes and then (3-aminopropyl)triethoxysilane (APTES) was added to the mixture. The dispersion was stirred for another 3 h before it was cooled down to RT. The particles were magnetically separated and washed three times with water. Finally, the APTES coated particles were filtered through a 0.8 pm pore diameter syringe filter and stored at 4° C.until further use.
Linkage of SBA to SPIONAPTES (SPIONAPTES-SBA)
SBA was purchased commercially (TCS0852-100MG; VWR). Of course SBA could also be self-made. The binding of SBA to SPIONAPTES were performed according to a modified process as recently described (Karawacka et al. (2018)). In this first step, SPIONAPTES containing 1 mg Fe were diluted in 950 μl with borate buffer (0.05 M pH 8.5; prepared from boric acid and basified with 2 N NaOH). Afterwards, SBA stock solution was added (50 μl, 20 mM in dry DMF) and the dispersion was shaken at 1400 rpm and at RT for 1 h. Next, supernatants were magnetically decanted and washed with 1 ml of borate buffer.
Functionalized SPIONAPTES-SBA-SPP were prepared using SPIONAPTES-SBA. The final concentration in 1 ml of particle dispersion was 0.1 μmol peptide (as shown in SEQ ID NO: 1 or 2, SPP04 or SPP19) and 1 mg Fe/ml. The dispersions were shaken at RT with 1400 rpm for 2 h. Supernatants were collected as well as the supernatants of two washing steps (1 ml buffer) and the absorption was measured at 280 nm. A calibration curve was taken at concentrations of 0.01 to 0.1 μmol peptide/ml in borate buffer. The amount of particle bound peptide was calculated by subtraction of peptide detected in supernatants after functionalization and washing steps from the given peptide amount.
SPIONAPTES without SBA as a linker molecule were used as a control. UV-Vis measurements were taken with a Libra S22 instrument (Biochrom Ltd., Cambridge, UK). Borat buffer in different concentrations were used as a blank for each measurement.
For determination of the iron concentration, atomic emission spectroscopy (AES) was used with an Agilent 4200 MP-AES (Agilent Technologies, Santa Clara, CA, USA). A commercially available iron solution (1.000 mg/l, Bernd Kraft, Duisburg, Germany) served as external standard. Samples were diluted in a ratio of 1:20. To 20 μl diluted sample, 80 μl 65% nitric acid were added. The mixture was heated to 95° C. for 10 minutes and diluted with water to 2 ml afterwards. Measurements were done at a wavelength of 371.993 nm. These measurements were done in triplicate, the results were averaged.
The content of silicon, calcium and phosphorus were determined by means of Inductively Coupled Plasma Atom Emission Spectrometry (ICP-AES, Circo CCD, Spectro Analytical Instruments GmbH, Germany). Briefly, to 100 mg of lyophilized particles, 10 ml of a mixture containing hydrofluoric acid, nitric acid and hydrochloric acid (ratio 1:1:1) was added. Afterwards, samples were diluted to a total volume of 100 ml with water. Silicon was measured at a wavelength of 251.612 nm, calcium at 396.847 nm and phosphorus at 177.495 nm. These measurements were done in triplicate, the results were averaged.
Dynamic light scattering (DLS) was performed with a Zetasizer Nano ZS (Malvern Panalytical, Almelo, Netherlands) to determine the hydrodynamic particle size of the NP at 25° C.in water (refractive index 1.33; viscosity 0.8872 mPa-s; backscattering mode at 173°). The same device was used to measure the particle's aqueous zeta potential with 78.5 as dielectric constant. Measurements were done in triplicate at an iron concentration of 50 μg/ml and pH of 7.4, the results were averaged.
Scanning electron microscopy (SEM) images of the particles were taken with a Zeiss Auriga SEM (Carl Zeiss, Oberkochen, Germany) operated at an acceleration voltage of 3 kV. Samples were diluted to an iron concentration of 100 μg/ml, dropped on a silica wafer and lyophilized prior to the measurements.
Transmission electron microscopy (TEM) images were taken by a CM30 TEM/STEM (Philips, Netherlands) operating at 300 kV. Specimens were prepared by drop casting of diluted nanoparticle dispersions onto carbon-coated copper grids (Plano, Germany). The used CCD camera was Tietz Fast Scan-F114.
As an indicator for the SPIONs' magnetizability, the magnetic susceptibility at an iron concentration of 1 mg/ml was measured with a MS2G magnetic susceptibility meter (Bartington Instruments, Oxfordshire, UK).
The Rigaku MiniFlex 600 XRD was used to perform a 0/20-measurment to determine the phases and crystal structures present in the specimen. Powders were obtained from particle dispersions by lyophilisation at 1 mbar (Alpha 1-2 LDplus, Martin Christ Gefriertrocknungsanlagen). Powders were placed on a specimen holder and softly pressed. A Cu-Kα1 beam was used as the X-ray source (wavelength λ=1.54059 nm) and the angular range was 20° to 80° with a step size of 0.03°/second.
Fourier transform infrared (FTIR) measurements were recorded with an Alpha-P FTIR device (Bruker) equipped with an ATR crystal. Wavelength range was 400-4000 cm−1 with a resolution of 4 cm−1, 128 sample scans, 64 background scans. OPUS software (Bruker) was used for background subtraction and baseline correction.
The estimation of amino groups on iron-oxide based particles was done according to an existing protocol for the determination of amino group on silica-particles (Zaloga et al. 2014). Briefly, 20 μl of the sample (concentration 0.1 mg Fe/ml) was added to 190 μl of borate buffer (0.1 M, pH 8.0). An amount of 90 μl fluorescamine, dissolved in acetonitrile to a concentration of 1 mg/ml, was added to the dispersion. Calibration samples were prepared from fresh APTES in a concentration range of 0.004 to 0.1 mM. Measurements were performed at an excitation wavelength of 385 nm and an emission wavelength of 535 nm in a Filter-Max F5 Plate reader (Molecular Devices). SPIONHAp with no amino groups served as negative control. Significances are represented by asterisks (*p<0.05, **p<0.005).
For the experiment in static condition, 1 ml of particle solution was prepared in 2 ml Eppendorf tubes. All samples were analyzed for iron content using AES. Particle dispersions were adjusted so that the concentration was 0.3 mg Fe/ml for SPIONAPTES and SPIONAPTES-SBA-SPP04. As a control, an unseparated sample was measured by AES. The test samples were separated for three minutes using a permanent magnet with a strength 310 mT. The supernatants of the separated samples were removed and prepared for AES. 20 μl sample of sample was dissolved in and 80 μl nitric acid (HNO3) and then diluted and measured. Based on the results, the amount of iron originally present in the sample was calculated and then represented in
For the experiment under flow condition, 0.4 ml of particle solution (6 mg Fe/ml) was injected into a flow. As a control, an unseparated sample was measured by AES. The test samples were separated using a electromagnet with a strength of 0.8 T. Samples have been collected in a tube after separation and were analyzed using AES. 20 μl sample of sample was dissolved in and 80 μl nitric acid (HNO3) and then diluted and measured. Based on the results, the amount of iron originally present in the sample was calculated and then represented in
Binding studies of endotoxin were performed with EndoZyme Recombinant Factor C Assay kit (Hyglos, Germany). In this kit, LPS is detected by recombinant Factor C, thus excluding possible interferences by p-glucans. Calibration curves for the three different LPSs have been prepared in endotoxin free water. SPIONAPTES-SBA-SPP04 was used in this study as its efficacy has been described previously. For this experiment, we used two different iron concentrations (1 mg Fe/ml and 0.01 mg Fe/ml) and two incubation parameters (30 s at 1400 rpm and 37° C. as well as 3 s at RT). Three different LPSs were added to 200 μl of the different particle dispersions. The final concentration of LPS was set to 10 EU/ml in each tested sample. Spiking controls (8 and 5 EU/ml) were carried out to see if any interference occurred for all tested particles.
Supernatants were produced using a neodymium magnet (310 mT at separation side) for three minutes. 50 μl of each supernatant were mixed with 50 μl of the assay reagent (80% v/v assay buffer, 10% v/v substrate, 10% v/v enzyme) in black, endotoxin-free 96-well plates. The reaction was monitored for 90 minutes at 37° C. in 15 min intervals by recording the fluorescence at an excitation wavelength of 360 nm and an emission at 465 nm in a Filter-Max F5 Plate reader (Molecular Devices).
For this experiment micromer® particles (micromer) were incubated with endotoxin to receive endotoxin coated micromer particles (micromerLPS). Any other polymeric particles of similar size could have been used as an alternative. It has been reported, that endotoxins stick to surfaces and can be attached to polymeric particles. Therefore, particles bearing 1 mg particles/ml was incubated with 1 μg/ml endotoxin LPS3 for 2 h at 1400 rpm. After centrifugation the particles were washed several times.
SPIONAPTES-SBA-SPP04 and unmodified SPIONHAp with a Fe concentration of 1 mg/ml were incubated with 0.1 mg micromerLPS for 2 h at 1400 rpm and 37° ° C. SPIONAPTES served as a control. A calibration curve was prepared with different concentrations of micromerLPS. The calibration samples and the magnetically decanted supernatants were measured in the reader at 620 nm absorption wavelength like it is used for the OD measurements of bacteria (Brenner et al. 2005 and Chen et al. 2011). Significances of SPIONAPTES-SBA-SPP04 and SPIONHAp particles compared to control SPIONAPTES are represented by asterisks (*p<0.05, **p<0.005).
Separation of E. coli From Blood
In this experiment we used blood from health volunteers and added an amount of bacteria to it. A concentration of 0.3 mg Fe/ml was used for the particles. To 0.8 ml of blood, (citrate or EDTA stabilized) 0.1 bacteria suspension in Ringer's solution have been added. After 5 min. of incubation, 0.1 ml of particles (3 mg Fe/ml) in Ringer's solution have been added. Samples were incubated for 5 minutes on a shaker at 700 rpm. After incubation, the particles have been removed by placing a magnet (310 mT) behind the samples. After 3 min separation of the particles the supernatant were plated out. As a negative control only 0.2 ml Ringer's solution was added to blood. The positive control consisted of 0.8 ml blood and 0.1 ml bacteria suspension and another 0.1 ml Ringer's solution. In addition the particles were washed one time after separation and then redispersed in Ringer's solution and plated out.
After 16 h of incubation, the CFUs (colony forming units) on the plates have been counted. The samples have been compared to the amount found in the positive control.
For statistics, an Student's t-test was performed in MS Excel (Microsoft, Redmond, WA, USA). Asterisks refer to statistical significance of *p<0.05, **p<0.005.
taurus]
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| Number | Date | Country | Kind |
|---|---|---|---|
| 20210959.1 | Dec 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/082980 | 11/25/2021 | WO |
