Patent Application
20220162232
The present invention relates to functionalized supports and the use thereof for the diagnosis of diseases.
Cellular microvesicles are vesicles released into the extracellular matrix by budding of a cell on activation following a stress. The microvesicles released into biological fluids (blood, urine, tears, etc.) contain, and/or carry on their surface, constituents of the parent cell (lipids, proteins and RNA, or mitochondrial DNA) and can therefore be regarded as early markers of a pathological state of a tissue.
A method described in international application PCT/FR2012/050610 employs a synthetic ligand for capturing phosphatidylserine-positive microvesicles.
The present invention relates to an improvement of the means and methods of capture presented in application PCT/FR2012/050610. It also relates to the identification of biomarkers of diseases of interest by means of functionalized supports according to the embodiments presented hereunder.
The invention relates to a functionalized solid support, useful for the capture, detection and/or characterization of cellular microvesicles. The support according to the invention is also useful in the medical field for carrying out methods of diagnosis and/or prognosis, in particular for early diagnosis of a disease.
The support according to the invention is a solid support, in particular polymeric, metallic or ceramic, functionalized by means of a compound of formula (I) or (II) as described below. According to a particular embodiment, the support is a polymeric support, in particular a support made of poly(vinyl chloride) (or PVC), poly(ethylene terephthalate) (or PET), or polystyrene (or PS), functionalized by means of a compound of formula (I) or (II) as described below.
The invention also relates to a method for the diagnosis of a disease, comprising detection of the presence or absence of specific markers identified by the inventors.
This detection may in particular be carried out after capturing microvesicles according to the embodiments presented in the present application.
According to a first aspect, the invention relates to a solid support, characterized in that it comprises, grafted on its surface, a compound of the following formula (I) or (II):
in which
According to the present invention, the compound of formula (I) or (II) is grafted covalently on the solid support. The method described in application PCT/FR2012/050610 does not teach covalent grafting of the complexes to the surface of the supports. Rather, it describes noncovalent adsorption of polyglutaraldehyde on a support and then immobilization of the complex on the polyglutaraldehyde via the NH2 functions of said complex. Advantageously, thanks to the method of the present invention, the complex is more accessible for phosphatidylserine. Moreover, the method according to the present invention gives better reproducibility of the functionalization. The method according to the present invention thus allows precise control of the density of complexes immobilized covalently, and in particular has the following advantages:
The compounds of formula (I) or (II) are cationic, and their counterion may be selected, for example, from the anions tosylate, nitrate, sulfate, sulfonate, thiosulfate, halide, hexafluorophosphate, tetraphenylborate, tetrafluoroborate, perchlorate, etc., in particular the perchlorate, nitrate, sulfate, halide and carbonate anions.
According to a particular embodiment, M is selected from Zn, Cu, Mn, Co, Ni and Fe, Zn or Cu being preferred, more particularly Zn.
According to a particular embodiment, Y represents H.
According to other particular embodiments:
According to a particular embodiment, the grafted compound is a compound of formula (I).
According to another particular embodiment, the compound of formula (I) is selected from:
It is to be understood that the present application also describes the compounds of formula (Ia), (Ib), (Ic) and (Id) associated with a counterion different from perchlorate, in particular selected from the anions tosylate, nitrate, sulfate, sulfonate, thiosulfate, halide, hexafluorophosphate, tetraphenylborate and tetrafluoroborate, more particularly from the nitrate, sulfate, halide and carbonate anions. Moreover, the salt used for generating the counterion may in particular be a zinc salt, a copper salt, a manganese salt, a cobalt salt, a nickel salt or an iron salt, more particularly a zinc salt, in particular zinc perchlorate, zinc nitrate, zinc sulfate, a zinc halide or a zinc carbonate.
According to a particular embodiment, the compound is a compound of formula:
it being understood that the counterion may be different from perchlorate, and in particular selected from the anions tosylate, nitrate, sulfate, sulfonate, thiosulfate, halide, hexafluorophosphate, tetraphenylborate and tetrafluoroborate, more particularly from the nitrate, sulfate, halide and carbonate anions. According to a particular embodiment, the counterion is a perchlorate anion. Moreover, the salt used for generating the counterion may in particular be a zinc salt, a copper salt, a manganese salt, a cobalt salt, a nickel salt or an iron salt, more particularly a zinc salt, in particular zinc perchlorate, zinc nitrate, zinc sulfate, a zinc halide or a zinc carbonate.
The compounds of formula (I) or (II) may be prepared according to the embodiments presented in application PCT/FR2012/050610.
The solid support used in the context of the invention may in particular be a microtiter plate, a sheet, a cone, a tube, a well, a bead, a particle, a strip, a film, a thread, a screw or a needle. Generally, the solid support according to the invention may be used for making any type of material of variable geometry and porosity.
In a particular embodiment, the solid support is a polymeric, metallic or ceramic support, functionalized by means of a compound of formula (I) or (II). According to a particular embodiment, the support is a polymeric support, in particular a support made of poly(vinyl chloride) (or PVC), poly(ethylene terephthalate) (or PET), or polystyrene (or PS).
In the context of the invention, “grafted to its surface” means, in reference to the grafting of the compound of formula (I) or (II) on the support, a covalent bond between the support and the compound of formula (I) or (II). As described below, the compound of formula (I) or (II) may be bound covalently to the support, directly or indirectly. In the case of an indirect covalent bond, the compound of formula (I) or (II) is bound covalently to a reactive function supplied to the surface of the support by a prefunctionalization agent, which has also been bound covalently to the support.
The solid support may be pretreated before grafting the compound of formula (I) or (II). The pretreatment may in particular have the aim of breaking undesirable functions on the surface of the support, in particular ester functions, increasing the density of desired functions, and/or prefunctionalizing the support by making it more electrophilic. In the context of the present invention, when the support is prefunctionalized, said prefunctionalization is carried out covalently by means of a prefunctionalization agent. According to a particular embodiment, the support used is prefunctionalized. The support may thus be prefunctionalized by means of glutaraldehyde, N-hydroxysuccinimide (NHS) or N-oxysuccinimide (NOS), for example. According to a representative embodiment in which the support is in particular made of PET, pretreatment may in particular comprise hydrolysis of the ester functions present on the surface of the support, increase in the density of COOH functions, in particular by oxidation of the support, in particular by means of potassium permanganate, and increase of the electrophilic character of this surface by modification with an attracting group and a good leaving group such as the NHS group. According to a particular embodiment, the PET used (Oxidized PET) comprises a density of COOH functions on its surface comprised between 1×1012 COOH/cm2 and 1×1018 COOH/cm2, in particular between 1×1013 and 1×1017, more particularly between 1.2×1015 and 1.2×1017, as measured by means of oxidized toluidine blue (TBO). According to one variant, the support is made of PET and comprises between 5×1015 and 5×1016 COOH functions/cm2 on its surface before prefunctionalization, more particularly 1.26×1016±10% COOH functions/cm2, in particular before prefunctionalization thereof by means of an NHS group.
Thus, according to one embodiment, the support is a support made of PET, in particular of prefunctionalized PET, in particular by means of NHS functions. According to a particular embodiment, the prefunctionalized PET comprises a density of COOH functions on its surface comprised between 1×1013 COOH/cm2 and 1×1018 COOH/cm2, in particular between 1×1014 and 1×1017, more particularly between 8.1×1014 and 8.2×1016. According to one variant, the support made of prefunctionalized PET comprises between 5×1015 and 1×1016 COOH functions/cm2 on its surface, more particularly 8.18×1015±10% COOH functions/cm2.
According to another particular embodiment, the support is made of PVC, in particular of prefunctionalized PVC, in particular by means of glutaraldehyde.
According to a particular embodiment, the solid support is a support made of PS, in particular of prefunctionalized PS, in particular by means of N-oxysuccinimide (NOS) functions. In particular, the density of NOS functions of the support made of PS thus prefunctionalized may be comprised between 1013 and 1016 NOS/cm2, in particular between 1014 and 1015 NOS/cm2, the density being more particularly of about 68×1014 NOS/cm2.
The compound of formula (I) or (II) is immobilized covalently on the solid support, optionally prefunctionalized, by introducing said support into a solution comprising the compound of formula (I) or (II) to be grafted. According to the embodiment in which the support is prefunctionalized, the prefunctionalization agent is bound covalently to the support. According to a particular embodiment, the solution comprises between 10−5 and 10−2 M of compound of formula (I) or (II), in particular between 10−4 and 5×10−2 M, more particularly between 5×10−4 and 5×10−3 M. The compound of formula (I) or (II) may in particular be at a concentration of about 10−3 M in the solution. The immobilization time may vary widely. However, according to a particular embodiment, the support is brought into contact with the compound of formula (I) or (II) for between 1 h and 72 h, in particular between 5 h and 48 h, more particularly between 10 h and 24 h, and even more particularly for about 16 h.
The invention further relates to a support comprising, grafted on its surface, a compound of formula (I) or (II), said support being included in a kit intended for the detection or characterization of cellular microvesicles. Such a kit may advantageously be used, in particular in the medical analytical laboratory, for the diagnosis of diseases of interest. The kit according to the invention comprises a support according to the invention, and may optionally comprise any means usable for carrying out the method according to the invention. Thus, the kit may in particular comprise means for carrying out a preliminary purification of the microvesicles, if this should prove useful or necessary. The kit may also comprise buffers usable during execution of the method according to the invention, in particular buffers for suspending the microvesicles, washing buffers, or buffers for storage. Means for detecting or quantifying one or more markers that may be present on or in the microvesicles, in particular means for detecting or quantifying protein markers or nucleic acids may be included in the kit. To this end, the kit according to the invention may in particular comprise:
Moreover, the kit may also comprise the means for detecting or quantifying a normalization marker, in particular a normalization marker selected from annexin-A5 and beta-actin.
Thus, according to a particular embodiment, the kit according to the invention may comprise:
According to another particular embodiment, the invention relates to a kit comprising:
According to another particular embodiment, the invention relates to a kit comprising:
According to another particular embodiment, the invention relates to a kit comprising:
“Means for detecting or quantifying” means any means known by a person skilled in the art for detecting or quantifying a marker. Of course, the means employed will depend on the nature of the marker, a protein marker in particular being detectable by immunologic techniques (in particular ELISA and Western blot) and nucleic acid markers being detectable in particular by means of specific amplification techniques, which may be qualitative or quantitative (in particular PCR/qPCR or RT-PCR/RT-qPCR, or sequencing). Other means include chromogenic assays, depending on the nature of the biomarker being detected.
Moreover, the kit according to the invention may comprise a leaflet providing the user with instructions for carrying out the method according to the invention by means of the kit.
The invention therefore also relates to the use of the support according to the invention for capturing microvesicles present in a sample of biological fluid from a subject. The data presented by the inventors show that it is possible to perform detection of biomarkers in various types of biological samples, in particular in urine or blood, more particularly in plasma, by means of the method presented in the present application. The method according to the invention is therefore capable of providing detection of microvesicles in an extensive panel of samples of biological fluids. The biological fluid may be, in particular, a sample of blood, serum, plasma, saliva, tears, urine, lymphatic fluid, cerebrospinal fluid, or semen. The subject is a mammal, in particular a human being, of any age, sex or condition. According to a particular embodiment, the subject is an individual with a suspected pathological state, in particular on the basis of bioassays or medical consultations carried out beforehand. In another embodiment, the subject has not undergone a bioassay or prior medical consultation.
The invention therefore relates to a method for capturing microvesicles, comprising bringing a sample of biological fluid that may contain said vesicles into contact with a support according to the invention. The microvesicles thus captured may then be characterized. According to one embodiment, prior to capture with the support according to the invention, the microvesicles are first purified or isolated from the sample of biological fluid by procedures known by a person skilled in the art. However, the experimental data presented hereunder show that the microvesicles may advantageously be captured directly in a sample of biological fluid by means of the support according to the invention. Thus, in a particular embodiment, the microvesicles are captured directly from the sample of biological fluid, in particular directly from a sample of blood, serum, plasma, saliva, tears, urine, lymphatic fluid, cerebrospinal fluid, or semen, more particularly from plasma or urine.
According to a particular embodiment, characterization of the microvesicles captured allows diagnosis of a disease, evaluation of the risk of developing a disease, prognosis of a disease, differential diagnosis of a disease, monitoring the progression of a disease, or monitoring the efficacy of a therapeutic treatment of a disease. As mentioned above, the composition of the cellular microvesicles, in particular the lipid and protein composition, and the contents of the cellular microvesicles (e.g. proteins and genetic material, in particular RNA or mitochondrial DNA), may vary depending on the type of cell from which they originated, and the state of the cell. The microvesicles may therefore allow detection of the state of the cells from which they originated, and may therefore represent a tool of choice for the early detection of a pathological state. Thus, according to one aspect, the invention relates to the use of a support according to the invention in a method for diagnosis, differential diagnosis, evaluation of the risk, prognosis, monitoring of the progression or monitoring of the efficacy of a therapeutic treatment of various diseases, in particular thrombotic, inflammatory and/or metabolic, or of cardiovascular or neurovascular diseases or accidents, or of diseases such as diabetes, cancer, Alzheimer's disease, Parkinson's disease or any other diseases.
The invention therefore also relates to a method of diagnosis, differential diagnosis, risk evaluation, prognosis, monitoring of the progression or monitoring of the efficacy of a therapeutic treatment of various diseases, in particular thrombotic, inflammatory and/or metabolic, or of cardiovascular or neurovascular diseases or accidents, or of diseases such as diabetes, cancer, Alzheimer's disease, Parkinson's disease or any other disease. We may thus mention diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, multiple sclerosis, vasospasm after rupture of an aneurysm, Parkinson's disease or derivatives thereof. Depending on the specific aim, the result of the characterization of the sample tested will be compared against the result of the characterization carried out on a sample of a control biological fluid.
Advantageously, the control biological fluid is a biological fluid identical to that assayed, but coming from a subject considered to be healthy. Alternatively, the control biological fluid is from the same individual as the biological fluid assayed, but results from a previous sample-taking. A control according to this alternative may allow monitoring of the progression of the disease or of the treatment thereof.
The characterization of the microvesicles may be carried out by any means known by a person skilled in the art, the nature of the characterization marker being taken into account, as was mentioned above. Thus, in the case of a protein marker, immunological techniques may be employed, using specific antibodies of said protein marker. We may mention in particular the ELISA or Western blot techniques. The detection and characterization of nucleic acids, in particular of mitochondrial DNA or of RNA (in particular messenger RNA or microRNA) will comprise the use of methods of detection of specific nucleic acids such as PCR/qPCR, RT-PCR/RT-qPCR, and sequencing. Other assays, such as chromogenic assays, may be used depending on the nature of the biomarker to be detected. Advantageously, normalization may be carried out based on quantification of a marker present on or in the microvesicles. The normalization marker may in particular be selected from annexin-A5 and beta-actin. For example, the experimental data presented below show that the markers podocalyxin, alpha-synuclein and CD41 may be used for the diagnosis of specific diseases. According to a particular embodiment, quantification of these markers will be carried out on a relative basis by parallel quantification of a normalization marker such as annexin-A5 and beta-actin. Thus, according to a particular embodiment, the method according to the invention comprises detection of the amount of a biomarker present in or on the microvesicles captured and detection of the amount of a normalization marker. The quantities normalized are then compared for determining a diagnosis, a differential diagnosis, for evaluating the risk, the prognosis, monitoring of the progression or monitoring of the efficacy of a therapeutic treatment of various diseases.
According to a second aspect, the invention relates to the use of the biomarkers allowing detection of the presence or absence of a specific disease. The characterization biomarker will be selected in relation to the disease of interest.
According to one embodiment, the invention relates to a method of diagnosis, in particular of early diagnosis, of a nephropathy in a subject, comprising detection of the presence or absence of podocalyxin, or measurement of the level of podocalyxin in a sample of biological fluid from said subject. According to a particular embodiment, the method comprises measurement of the ratio of podocalyxin to the normalization marker (in particular annexin-A5 or beta-actin). More particularly, the method according to the invention may in particular comprise detection of the presence or absence of cellular microvesicles comprising the marker podocalyxin, according to the embodiments described above comprising the use of a support according to the invention. The invention also relates to the evaluation of the risk of development of a nephropathy, prognosis of a nephropathy, monitoring the progression of a nephropathy, or monitoring the efficacy of a therapeutic treatment of a nephropathy comprising detection of the presence or absence of podocalyxin, or measurement of the level of podocalyxin in a sample of biological fluid from a subject, in particular of podocalyxin comprised in cellular microvesicles characterized according to the embodiments disclosed above. According to a particular embodiment, the method according to the invention comprises quantifying the podocalyxin/annexin-A5 ratio or podocalyxin/beta-actin ratio, more particularly the podocalyxin/annexin-A5 ratio, for detecting nephropathic complications in the microvesicles derived from a sample of biological fluid from a patient, more particularly a urine sample. This ratio, when it is statistically higher than that calculated in a healthy subject, is indicative of a nephropathy. Moreover, if this ratio increases or decreases relative to the ratio calculated in the past for the same patient, it is evidence of progression or of regression of the nephropathy, respectively.
Moreover, the invention relates to a method of diagnosis of Parkinson's disease in a subject, comprising detection of the presence or absence of alpha-synuclein, or measurement of the level of alpha-synuclein in a sample of biological fluid from said subject. According to a particular embodiment, the method comprises measurement of the ratio of alpha-synuclein to a normalization marker (in particular annexin-A5 or beta-actin). More particularly, the method according to the invention may in particular comprise detection of the presence or absence of cellular microvesicles comprising the marker alpha-synuclein, according to the embodiments described above comprising the use of a support according to the invention. The invention also relates to evaluation of the risk of developing Parkinson's disease, prognosis of a Parkinson's disease, monitoring the progression of a Parkinson's disease, or monitoring the efficacy of a therapeutic treatment of Parkinson's disease, comprising detection of the presence or absence of alpha-synuclein, or measurement of the level of alpha-synuclein in a sample of biological fluid from a subject, in particular of alpha-synuclein comprised in cellular microvesicles or in the membranes of cellular microvesicles characterized according to the embodiments disclosed above. According to a particular embodiment, the method according to the invention comprises quantifying the alpha-synuclein/annexin-A5 ratio or alpha-synuclein/beta-actin ratio, more particularly the alpha-synuclein/annexin-A5 ratio, for detecting these neurological disorders (for example, Parkinson's disease) in the microvesicles derived from a sample of biological fluid from a patient. When this ratio is statistically higher than that calculated for a healthy subject, it is indicative of a Parkinson's disease. Moreover, if this ratio increases or decreases relative to the ratio calculated in the past for the same patient, it is evidence of progression or of regression of Parkinson's disease, respectively.
The invention further relates to a method of diagnosis, in particular of early diagnosis, of a diabetic nephropathy in a subject. According to a particular embodiment, the method according to the invention comprises detection of the presence or absence of the platelet marker CD41, or measurement of the level of CD41 in a sample of biological fluid from said subject, in particular a sample of urine or of plasma, more particularly a plasma sample. According to a particular embodiment, the method comprises measurement of the ratio of CD41 to a normalization marker (in particular annexin-A5 or beta-actin). More particularly, the method according to the invention may in particular comprise detection of the presence or absence of cellular microvesicles comprising the marker CD41, according to the embodiments described above comprising the use of a support according to the invention. According to a particular embodiment, the method according to the invention comprises quantifying the CD41/annexin-A5 ratio or CD41/beta-actin ratio, more particularly the CD41/annexin-A5 ratio, for detecting the nephropathic complications of diabetes in the microvesicles derived from a sample of biological fluid from a patient, in particular a sample of urine or of plasma. When this ratio is statistically higher than that calculated for a healthy subject, it is indicative of a diabetic nephropathy. Moreover, if this ratio increases or decreases relative to the ratio calculated in the past for the same patient, it is evidence of progression or of regression of the diabetic nephropathy, respectively.
Detection of the stage of nephropathy may in particular comprise comparing the result of characterization obtained from the test sample of biological fluid with the result of characterization of one or more control samples characteristic of different stages of the disease (in particular obtained from diabetic patients with nephropathy at the normoalbuminuric stage (Normo), microalbuminuric stage (Micro) or macroalbuminuric stage (Macro), these stages corresponding to the severity of the disease, the Macro stage being the most advanced). According to a particular embodiment, the method according to the invention thus allows monitoring and early detection of diabetes and of the potential complications thereof, in particular the renal or neuropathic complications thereof.
The invention will now be illustrated by the following nonlimiting examples.
DCM: dichloromethane
DMSO: dimethylsulfoxide
Eq: equivalent
EtOAc: ethyl acetate
MES: 2-(N-morpholino)ethanesulfonic acid
MilliQ: water purified with a Milli-Q water purification system
MV: microvesicle
NHS: N-hydroxysuccinimide
NOS: N-oxysuccinimide
Cplx: complex
PVC: poly(vinyl chloride)
PS: phosphatidylserine
Pht: phthalimide
MeCN: acetonitrile
XPS: X-ray photoelectron spectroscopy
EDA: ethylenediamine
HUVEC: Human Umbilical Vein Endothelial Cells
The complexes used in this experimental section were synthesized according to the embodiments presented in application PCT/FR2012/050610, in particular the complexes C1, C2 and C4 of formulas:
Polyethylene terephthalate (PET) was selected for covalent grafting of the complexes, this grafting is taking place owing to their primary amine function reacting with the carboxylic acids present on the surface.
General Protocol:
First, functionalization steps (
“Control” samples required for characterization of the materials were prepared.
These are samples of virgin PET without hydrolysis or oxidation and without EDC/NHS activation. Steps 1), 7) and 8) are carried out but without steps 2), 3), 4), 5) and 6).
Characterization of the Materials
Quantification of the Density of Carboxylic Acid on the Surface for all the Functionalization Steps:
The method used for quantifying the density of COOH groups present on the surface of the polymer is the method using Toluidine Blue O, as this cationic chemical reagent reacts on the surface with the COO− groups. Indeed, a basic pH (for example pH=10) is necessary for deprotonating the carboxylic acid groups so that they react with TBO to form a noncovalent bond. For resorbing the TBO fixed on the surface of the material, acetic acid is added to reprotonate the COO− groups and thus break the ionic bond [COO−. . . TBO].
A calibration curve is constructed beforehand, with different concentrations of TBO.
The optical density at 630 nm read for the samples was reported, and the calibration curve enabled us to find the surface concentration of COOH.
From the calibration curve, in the case of virgin PET (PET as delivered by the supplier and washed with ethanol and then dried), we obtain a density of COOH functions of 1.5 μmol/mm2.
In the same way, the surface density of COOH functions could be evaluated on the PET surfaces at the different steps of the treatment. We get:
Determination of Atomic Percentages on the Surface of PET at the Different Steps of the Treatment by Photoelectron Spectroscopy
These analyses were carried out with the “VG ESCALAB 220i-XL” spectrometer. Atomic quantification of the Si, S, C, N, O or Zn atoms was carried out by X-ray photoelectron spectroscopy (XPS), in the solvent DMSO/H2O 10/90.
The quantifications of the atomic percentages make it possible to state that the complex has indeed been immobilized on the surface of the PET. Indeed, relative to the activated PET that does not contain any metal, PET+C1 contains an atomic percentage of Zn. Moreover, we may note an increase in the percentage of nitrogen expected theoretically since the complex contains 6 nitrogen atoms at the level of the ligand. This result is supported by the ratios such as COO/C—O which changes from 0.93 (PET-NHS) to 0.65-0.68 (PET+C1) and NCO/COO which changes from 0.06 (PET-NHS) to 0.17-0.25 (PET+C1).
XPS Analysis of Functionalized Vs Unfunctionalized Supports, and/or Grafted with the Complexes C1, C2 or C4
These analyses were carried out with the “VG ESCALAB 220i-XL” spectrometer. 9 samples were prepared for X-ray photoelectron spectroscopy analysis: Virgin PET cleaned (Smp1), oxidized PET (Smp2), PET-NHS or activated PET (i.e. PET hydrolyzed, oxidized and then activated with EDC/NHS; Smp3), activated PET+C1 (Smp4), activated PET+C2 (Smp5), activated PET+C4 (Smp6), Virgin PET+C1 (Smp7), Virgin PET+C2 (Smp8) and Virgin PET+C4 (Smp9).
The high-resolution spectra O1s obtained by XPS show that the complexes are grafted on the surface of the material based on the appearance of peaks for “Zn—O” and “Cu—O” at 530.5-530.7 eV starting from sample 4. At the level of the area ratios Zn—O (or Cu—O)/C═O, the value is 0.15 for Smp4, 0.25 for Smp5, 0.11 for Smp6 and the latter decreases without the activation step, 0.08 for Smp7, 0.15 for Smp8 and 0.14 for Smp9. These results can be explained by the fact that without activation (Smp 7, 8 and 9), the complexes are adsorbed and not grafted covalently to the surface of the support. Thus, after several rinsings, the complexes are removed and are almost no longer detected.
When covalent grafting takes place, a peak at 400 eV (high-resolution spectrum N1s) corresponding to the amide bond N—C═O is easily observed by XPS, which means that the bond between the NH2 of the complex and the carbonyl of the material is indeed produced. Furthermore, when EDC/NHS activation is not carried out and the complexes are brought into contact with the surface (Smp 7, 8 and 9), a different peak than that at 400 eV is observed. This peak corresponds to the free NH2 borne by group X of the complex, which could be correlated with adsorption and not with a covalent bond between the complex and the material.
Optimization of the Density of the Complexes Grafted on the Surface
So as to use only the necessary amount of complexes on the surface, to promote better accessibility of the complexes by the phosphatidylserine present on the microvesicles, in a variant, the support may be prepared following the general protocol in which steps 2 and 3 are not carried out or in which steps 2, 3, 4 and 5 are not carried out.
Indeed, by omitting the steps of hydrolysis and/or of oxidation of the PET, it is possible to modulate the quantity of COOH groups on the surface of the PET and therefore modulate the quantity of complexes immobilized on the surface of the PET. In the proposed variant, virgin PETs are cleaned and then functionalized without prior oxidation or without prior oxidation and hydrolysis. With the proposed variant, only the —COOH functions present on the surface of the support are activated/functionalized with a view to the grafting of complexes. Advantageously, as the PET that has not undergone an oxidation step contains a smaller quantity of carboxylic acid groups, the grafting density obtained will therefore be lower (d1)
General Protocol:
The protocol employed will be described referring to
1) A PVC plate with 10 squares of 1 cm2 was cleaned by sonication for 1 h in 40 mL of MilliQ water in a 50 mL tube. Then the plate was immersed in a solution comprising 2 mL of 97% EDA and 38 mL of MilliQ water, for 2 h at 30° C. Finally, the plate of 10 squares was rinsed 3 times in 10 mL of MilliQ water, sonication being carried out at each rinsing step.
2) The plate was immersed in a solution comprising 4 mL of 50% glutaraldehyde and 30 mL of MilliQ water at pH 9.5, for 2 h at 50° C. Then the plate was rinsed 3 times in 10 mL of MilliQ water, sonication being carried out at each rinsing step.
3) Finally, the complex is grafted by immersing a square from each plate treated according to the above steps in a solution comprising 10−3 M of complex, 0.1 mL of DMSO and 1.9 mL of citrate phosphate buffer (0.5 M citric acid, 0.5 M disodium sodium phosphate) at pH4, for 16 h at room temperature with stirring. Then the PVC square was rinsed 3 times in 2 mL of MilliQ water, sonication being carried out at each of these rinsing steps. The treated PVC square was then rinsed 3 times in 2 mL of MilliQ water over 2 days.
Characterization of the Surfaces by Photoelectron Spectroscopy (XPS)
To characterize the surface of the materials, analyses by XPS spectroscopy on PVC were carried out at each step of their functionalization with complex C1. The materials are respectively shown schematically in
Results
The spectra and deconvolutions of the spectra as well as the atomic percentages (Table 3) of the various elements present on the surface of the materials show that, after functionalization with complex C1, zinc (Zn) is present on the surface of the materials at a level of 0.25% for PVC. Zinc being the metal in the composition of complex C1, its presence tells us that complex C1 is fixed on the surface of the materials. The functionalization is therefore effective on PVC. Moreover, the atomic percentages determined at five different points of the surface (4 corners+1 center) show low standard deviations (Table 3), demonstrating uniformity of the treatment and reproducibility of the results. From this, we deduce that the functionalization is uniform on PVC.
General Protocol
The complexes were immobilized on DNA-BIND® commercial 96-well plates from Costar. This plate is of polystyrene prefunctionalized with N-oxysuccinimide (NOS) functions. This plate contains 68×1014 NOS/cm2. This surface density of NOS is comparable to the density of COOH functions that we had on polyethylene terephthalate (PET) (8.18×1015 COOH/cm2)
1) A solution is prepared (Vtotal=2 mL) with −1.78 g of Complex −0.2 mL DMSO −1.8 mL of MilliQ water. 200 μL of this solution is deposited in each well (10 wells). The grafting reaction is carried out for 16 h at room temperature away from the light.
2) After this reaction time, the solution is removed from the wells and the wells are rinsed by 3 flushes with MilliQ water. Each well is then incubated with fresh MilliQ water and is rinsed 3 times/day for 48 h.
Characterization of the Surfaces by Photoelectron Spectroscopy
To characterize the surface of the materials, XPS spectroscopy analyses on “DNA-BIND®” were carried out at each step of their functionalization with complex C1. The materials are respectively shown schematically in
Results
The spectra and deconvolutions of the spectra as well as the atomic percentages (Table 4) of the various elements present on the surface of the materials show that, after functionalization with complex C1, zinc (Zn) is present on the surface of the materials, at a level of 0.41% for “DNA-BIND®”. As zinc is the metal in the composition of complex C1, its presence tells us that complex 1 is immobilized on the surface of the materials. The functionalization is therefore effective on DNA-BIND®. Moreover, the atomic percentages determined at five different points of the surface (4 corners+1 center) show low standard deviations (Table 4), demonstrating uniformity of the treatment and reproducibility of the results. From this it is deduced that the functionalization is uniform on the two materials: PVC and DNA-BIND®.
Materials and Methods
a/ Sampling of Biological Fluids from Diabetic Patients
Samples of urine and plasma were obtained from diabetic patients in the diabetology unit of Pr Vincent Rigalleau. All the patients signed a free and informed consent that was explained in accordance with the Declaration of Helsinki and was approved by the ethics committee. The clinical picture, including the level of albuminuria in the urine, is established for each diabetic patient. The patients were divided into three groups: Normoalbuminuric patients or Normo (stage 1 of the complications of the disease), Microalbuminuric patients or Micro (stage 2) and Macroalbuminuric patients or Macro (stage 3). Urine samples were also collected from healthy donors, with no known nephropathic complication. All the samples are stored at −80° C. until used.
b/ Cell model: Human Umbilical Vein Endothelial Cells, HUVECs
The endothelial cells, Human Umbilical Vein Endothelial Cells (HUVECs) (Promocell, C-12208) are cultured in Endothelial Cell Growth Medium 2 Kit (Promocell, C-22111) consisting of a basal medium and a mixture of growth factors without antibiotics.
c/ Isolation of Model Microvesicles from Cell Culture (Human Umbilical Vein Endothelial Cells or HUVECs)
For activation of the production of model microvesicles, the HUVECs are rinsed twice with PBS buffer and are stimulated for 24 h in culture medium with 100 ng/mL of TNF-alpha (Peprotech, ref. 300-01A). The culture media of the cells are recovered after 24 h of stimulation and are centrifuged in a 50 mL tube at 12000 g for 2 min at 4° C. in order to remove the cells, cellular debris and apoptotic bodies. The supernatants are transferred to clean tubes and centrifuged at 20000 g for 90 min at 4° C. in order to sediment the microvesicles. The pellet of model microvesicles obtained is washed by resuspension in 1.5 mL of cold PBS buffer, transferred to a 1.5 mL tube and then centrifuged at 20000 g for 90 min at 4° C. This washing step is repeated once. The pellet of model microvesicles is then resuspended depending on the size of the pellet in cold PBS (100 to 500 μL). The amount of corresponding proteins is determined by absorbance at 280 nm using a nanodrop spectrophotometer. The sample of model microvesicles is stored at −80° C. until used.
d/ Purification of Microvesicles from Human Urine Samples
The urine from healthy donors and from patients is thawed and the microvesicles are purified by differential centrifugations: a first centrifugation is carried out at 12000 g for 2 min at 4° C. in order to remove the cells, cellular debris and apoptotic bodies. The supernatant is centrifuged at 20000 g for 90 min at 4° C. in order to sediment the microvesicles. The pellet of microvesicles obtained is washed by resuspension in 1.5 mL of cold PBS buffer, transferred to a 1.5 mL tube and then centrifuged at 20000 g for 90 min at 4° C. This washing step is repeated once. The sample is stored at −80° C. until used.
e/ Purification of Microvesicles from Human Blood Samples
The blood collected in a sodium citrate tube is subjected to a first centrifugation at 1500 g. The upper phase corresponding to the plasma is carefully removed and is centrifuged at 12000 g for 2 minutes in order to remove the platelets. The supernatant corresponding to the platelet-free plasma, PFP, is then centrifuged at 20000 g at 4° C. in order to sediment the microvesicles. The pellet of microvesicles obtained is washed by resuspension in 1.5 mL of cold PBS buffer, transferred to a 1.5 mL tube and then centrifuged at 20000 g for 90 min at 4° C. This washing step is repeated once. The sample is stored at −80° C. until used.
f/ Detection of Protein Biomarkers
Enzymatic Method: The model microvesicles produced by activations of the HUVEC cells have on their surface plasminogen activators, which activate plasminogen (present on the surface of the microvesicles) into plasmin; the activity of this plasm in can be detected using a chromogenic substrate, methylmalonyl-hydroxypropyl-arginyl-paranitroanilide, CBS0065. The chromophore is cleaved in the presence of plasmin into a yellow colored product, whose absorbance can be measured using a spectrophotometer at a wavelength of 450 nm. The microvesicles resuspended in PBS buffer after isolation from HUVECs are deposited in the wells of the 96-well polystyrene plate (DNA-BIND® Costar) grafted with the dinuclear metal complex I, called complex I. A total amount of 2.5 μg of microvesicles (determined by the A280 nm nanodrop technique) in 25 μL is incubated for 1 h at room temperature. After three rinsings, the enzymatic activity of each well is determined. Certain wells are not rinsed and represent the control 100% of enzymatic activity. First, a standard range is obtained by serial dilution with the following concentrations of plasminogen activator in IU/mL: 0, 0.00078125, 0.0015625, 0.003125, 0.00625, 0.0125, 0.025 and 0.05. Secondly, a plasminogen solution at 4 μM and a solution of the chromogenic substrate CBS0065 at 3 mM are prepared and then mixed volume by volume. A volume of 25 μL of this mixture is added to each well. The plate is covered with cling film and placed in the spectrophotometer at 37° C. for 18 h for reading the absorbance simultaneously at 405 nm and at 450 nm in each well. This kinetic study enables us to calculate the average rate of appearance of the product, reflecting the enzymatic activity of each well; it is expressed in mOD/min.
g/ Western Blot
The first step consists of lysis of the microvesicles using RIPA buffer+protease inhibitors and of phosphatases (990 μL of RIPA buffer+10 μL of the cocktail of protease inhibitors and of phosphatases inhibitors (100λ)). Add a volume of RIPA buffer+inhibitors as a function of the size of the pellet of microvesicles obtained in the last step of isolation before adding PBS (volume of RIPA about 4× greater than the volume of the pellet). The pellet is resuspended by pipetting up and down and is left in ice for 15 minutes. Then the lysate is centrifuged at 10000×g for 10 minutes at 4° C. to remove the debris. The supernatant is transferred to a clean tube. The amount of proteins present in the lysate of microvesicles is measured by the BiCinchoninic acid Assay (BCA) technique. The proteins are then denatured using the buffer Laemmli 4X+reducing agent (DTT, dithiothreitol). The denatured lysate can be stored at −80° C. before use. The denatured lysates of microvesicles are deposited on acrylamide gel in SDS-PAGE denaturing conditions at a concentration gradient of 4-12%. Migration is carried out at a constant voltage of 120 V for about 1.5 h (until the migration front reaches the end of the bottom of the gel). The gel is transferred onto a PVDF membrane. After saturation of the membrane for 1 h at room temperature with a 5% milk solution in TBS-Tween 0.1% buffer, the 3 primary antibodies diluted in the 5% milk solution in TBS-Tween 0.1% are incubated overnight at 4° C. The following dilutions are used: antibody against podocalyxin (Santacruz, Sc-23904) 1/2000 or against CD41 (Novus, MAB 7616) at 1/1000; plus antibody against beta-actin (SIGMA, A1978-100UL) 1/10000; plus antibody against annexin-A5 (SIGMA, A8604-100UL) 1/2000. After two washing operations in TBS-Tween 0.1% buffer, the secondary antibodies coupled to the enzyme horseradish peroxidase, HRP, and at a dilution of 1/5000, are incubated for 1 h at room temperature in the 5% milk solution in TBS-Tween 0.1%. Detection by chemiluminescence is carried out after two washing operations in TBS-tween 0.1% buffer. The signal is captured by a CCD camera and then the intensity of the latter is analyzed using the free software ImageJ.
h/ Statistical Analysis
For validation of the biomarker in the microvesicles obtained from the urine of diabetic patients, statistical analysis of the Western blot results was performed with the GraphPad Prism software version 7.
i/ ELISA
The microvesicles resuspended in PBS buffer after isolation from urine or plasma from patients or from healthy individuals are deposited in the wells of the 96-well polystyrene plate (DNA-BIND® Costar) grafted with the dinuclear metal complex I, called complex I. Incubation overnight at 4° C. with gentle horizontal agitation was observed. After washing twice in PBS, the saturated solution consisting of 5% of “bovine serum albumin” (BSA) protein is deposited in all the wells of the plate and incubated for 2 h at room temperature, stirring gently. Then the primary antibodies directed either against podocalyxin (Santacruz, Sc-23904) at 1/500, or against the CD41 platelet marker (Novus, MAB 7616) at 1/500, are brought into contact with the microvesicles captured in the wells for 2 h at room temperature with gentle horizontal agitation. After two washing operations, the secondary antibodies coupled to the enzyme HRP, at 1/5000 dilution, are incubated for 1 h at room temperature with gentle horizontal agitation. After six washing operations, 100 μL of chromogenic developer TMB (3,3′,5,5′-tetramethylbenzidine) is deposited in the wells for a duration of 30 minutes at room temperature with gentle horizontal agitation. The reaction is stopped by adding 100 μL of sulfuric acid, and the absorbance of each well is measured using a spectrophotometer at 450 nm.
Results
a/ Identification of Protein Markers Carried by Microvesicles
Validation of the Biomarker Podocalyxin in the Microvesicles Obtained from the Urine of Diabetic Patients According to the Stage of Nephropathic Complication by the Western Blot Technique
Podocalyxin is a transmembrane protein expressed by podocytes, glomerular cells that are only present in the kidney; it is described in the literature as a marker of cellular lesion of the podocytes.
The protein beta-actin is a protein of the cytoskeleton expressed in all cells; this stable, universal expression means that it is often used in Western blot as a reference protein when it is necessary to quantify the level of a protein whose expression may vary as a function of the disease.
The protein annexin-A5 is localized near the plasma membrane in all cells; like beta-actin, it is also used as a reference protein in Western blot. Accordingly, analysis of the expression of the protein podocalyxin by Western blot in the microvesicles obtained from the urine of healthy donors or of diabetic patients will therefore rather be expressed in the form of a ratio: podocalyxin/beta-actin ratio and podocalyxin/annexin-A5 ratio.
In conclusion, it may be envisaged that the podocalyxin/annexin-A5 ratio could be used in the context of a kit for detecting nephropathic complications in the microvesicles derived from patients' urine. When this ratio increases, this is evidence of a cellular lesion of the podocytes.
Detection of the Platelet Marker, CD41, in the Microvesicles Derived from the Plasma of Diabetic Patients According to the Stage of Nephropathic Complication by the Western Blot Technique
The purpose of our experiments in this part is to show that we are able to detect a marker in the microvesicles purified by centrifugation obtained from human plasma. For this, we developed detection of CD41, a protein localized on the surface of the membranes of the platelets, by the Western blot technique. The microvesicles that originate from the budding of the membranes of the platelets express CD41. Thus, we analyzed the CD41/annexin-A5 expression ratio in the microvesicles derived from the plasma of a Normoalbuminuric diabetic patient and of a Macroalbuminuric diabetic patient (
Conclusion
We have shown that we are able to quantify protein biomarkers in the microvesicles isolated from different biological fluids: urine and plasma.
Moreover, the podocalyxin/annexin-A5 ratio could be used in a diagnostic kit for detecting a nephropathic complication.
b/ Validation of the Surface of DNA-BIND® Polystyrene Grafted with Complex C1
Capture of Model Microvesicles and Enzymatic Detection of Biomarkers Carried by these Microvesicles
Microvesicles carry biological material such as enzymes, proteins with biological activity. We employed the enzymatic technique described in patent PCT/FR2012/050610 to validate grafting of complex C1 on the surface of DNA-BIND® polystyrene. The model microvesicles are captured by the material with an amount of 2.5 μg of total microvesicles (protein equivalent) in 25 μL for 1 h at room temperature. The enzymatic activity present in each experimental condition is analyzed by enzyme kinetics. That is, the rate of appearance of a chromogenic product, CBS0065, catalyzed by the enzyme will be monitored for 18 h. The experimental conditions are as follows:
Unrinsed condition 100%: this is the control condition, the model microvesicles are left in the wells for 1 h and the enzymatic activity is determined directly without rinsing the wells. The enzymatic activity detected is the maximum activity of the extract of model microvesicles. This condition therefore represents the positive control, 100%. Rinsed condition without complex: the model microvesicles are incubated for 1 h in the wells of the plate that has not been grafted with complex C1, then rinsed three times and the enzymatic activity is analyzed. This condition represents the negative control; the enzymatic activity obtained is evidence of nonspecific interaction of the model microvesicles.
Rinsed condition with complex: the model microvesicles are incubated for 1 h in the wells of the plate grafted with complex C1, then rinsed three times and the enzymatic activity is analyzed. This condition represents capture of the microvesicles by the complex; the enzymatic activity obtained is evidence of specific interaction of the model microvesicles.
Capture of the Microvesicles Isolated from Human Urine and ELISA Detection of a Podocyte Marker Carried by these Microvesicles
We tested for the presence of podocalyxin and detection thereof by the ELISA immunologic method to validate specific capture of the microvesicles isolated from urine and to demonstrate the capacity for quantification of a biomarker after capture on our material. To do this, we incubated the microvesicles isolated by centrifugation from urine overnight at 4° C. with gentle agitation on the DNA-BIND® Costar 96-well polystyrene plate grafted entirely with complex C1. We deposited a maximum quantity of microvesicles equivalent to 20 μg of proteins assayed by spectrophotometry (i.e. 100 ng/μL in the 200 μL of final reaction). After several washing operations, we detected the presence of the protein podocalyxin using an antibody specifically directed against it. A secondary antibody coupled to the enzyme horseradish peroxidase is added in order to amplify and detect a signal by spectrophotometry at 450 nm in the presence of a substrate with chromogenic properties, TMB. The results are reported in
In order to demonstrate that ELISA detection of the biomarker of the protein podocalyxin in the microvesicles derived from human urine after capture on materials can be regarded as semiquantitative, we carried out a range of deposits of the microvesicles comprised between 0.5 μg and 20 μg, i.e. at concentrations comprised between 2.5 ng/μL and 100 ng/μL in 200 μL of final reaction. The results of the measurements of absorbances as a function of the different quantities of microvesicles are presented in
Thus, the material (complex C1 grafted on the DNA-BIND® polystyrene plate from Costar) not only allows specific capture of the microvesicles isolated from urine, but also makes it possible to quantify the presence of podocalyxin, by the ELISA technique with a threshold of detection of 2.5 ng/μL of microvesicles deposited.
Capture of the Microvesicles Isolated from Human Plasma and ELISA Detection of the CD41 Platelet Marker Carried by these Circulating Microvesicles
We wanted to show that the material (complex C1 grafted on the DNA-BIND® polystyrene plate from Costar) is also capable of capturing the circulating microvesicles (i.e. isolated from human plasma) isolated beforehand by centrifugation and that it is possible to quantify, by the ELISA technique, the presence of a protein on the captured microvesicles. For this, we selected the protein CD41 that is present on the surface of the platelets and is therefore found on the surface of the microvesicles derived from their membrane budding. The circulating microvesicles isolated beforehand by centrifugation from plasma from normoalbuminuric diabetic patients were deposited in the polystyrene plate functionalized with complex C1 and incubated overnight at 4° C., with gentle agitation. A quantity of microvesicles isolated from 1.5 mL of plasma was deposited per well. After several washing operations, we detected the presence of the protein CD41 by means of an antibody specifically directed against it. A secondary antibody coupled to the enzyme horseradish peroxidase is added in order to amplify and detect a signal by spectrophotometry at 450 nm in the presence of a substrate with chromogenic properties, TMB. The results are reported in
Capture of the Microvesicles Contained in Human Plasma and Detection of the Platelet Protein Biomarker, CD41, by ELISA
Finally, we wanted to show that the material (complex C1 grafted on the DNA-BIND® polystyrene plate from Costar) is capable of capturing the unisolated microvesicles. Therefore in this case the sample is not microvesicles isolated beforehand by centrifugation, but is obtained directly from human plasma containing microvesicles. We tried to quantify the presence of the protein CD41 carried by the microvesicles of the plasma sample, using the ELISA technique. For this, we incubated 300 μL of plasma from normoalbuminuric diabetic patients overnight at 4° C. with gentle agitation, on the DNA-BIND® polystyrene plate grafted with complex C1. After several washing operations, we detected the presence of the protein CD41 by means of an antibody specifically directed against it. A secondary antibody coupled to the enzyme horseradish peroxidase is added in order to amplify and detect a signal by spectrophotometry at 450 nm in the presence of a substrate with chromogenic properties, TMB. The results are reported in
Material and Methods
Human urine samples from healthy subjects: The human urine samples were collected at the Institute of Chemistry and Biology of Membranes and Nano-objects (CBMN) at Pessac from healthy subjects during the day in conventional urine pots without adding additives. The start of urination was not collected, and the samples were stored at −80° C. until used.
Human urine samples from diabetic patients: The donors are diabetic patients with nephropathy at the normoalbuminuric stage (Normo), microalbuminuric stage (Micro) or macroalbuminuric stage (Macro). These stages correspond to the severity of the disease, the Macro stage being the most advanced. The urine samples were collected during the day in conventional urine pots without adding additives. Moreover, the start of urination was not collected.
Purification of the Microvesicles
Microvesicles derived from a cell model: The human umbilical vein endothelial cells, HUVECs (Promocell; ref. C-12208), were cultured in complete medium (Promocell; Endothelial Cell Growth Medium 2 Kit; ref. C-22111) in accordance with the supplier's recommendations. Between passages 3 and 6, the cells were rinsed twice in PBS 1×, then activated with a solution of TNF-alpha (Peprotech; ref. 300-01A) prepared at 100 ng/mL in complete medium for 24 h at 37° C. in order to produce microvesicles. Then the supernatant containing the microvesicles was transferred to a 50 mL tube. First, the cellular debris and apoptotic bodies were sedimented by centrifugation at 12 000 g for 2 min at 4° C. (SIGMA; thermostatically controlled centrifuge 3-18KHS) in order to remove them. Secondly, the supernatant containing the microvesicles was transferred to a new 50 mL tube and the microvesicles were sedimented by centrifugation at 20 000 g for 1.5 h at 4° C. The supernatant was removed and the pellet was rinsed with 1 mL of buffer solution (HEPES 10 mM; NaCl 0.15 M; pH 7.4). The solution of microvesicles was transferred to a new 1.5 mL microtube and centrifuged at 20 000 g for 1.5 h at 4° C. The supernatant was removed and the pellet of microvesicles was rinsed again and then centrifuged at 20 000 g for 1.5 h at 4° C. This last pellet of microvesicles was finally resuspended in buffer solution (HEPES 10 mM; NaCl 0.15 M; pH 7.4) and this solution was stored at −80° C. until used.
Microvesicles derived from human urine samples: In order to isolate the microvesicles, the urine was thawed overnight at 4° C. and 40 mL was centrifuged at 1500 g for 15 min at 4° C. The supernatant was transferred to a new tube and centrifuged at 12 000 g for 2 min at 4° C. to remove the cellular debris and apoptotic bodies. The supernatant was then transferred to a new tube and centrifuged at 20 000 g for 1.5 h at 4° C. in order to sediment the microvesicles. The supernatant was removed and the pellet was rinsed in 1 mL of buffer (HEPES 10 mM; NaCl 0.15 M; pH 7.4). The solution of microvesicles was transferred to a 1.5 mL microtube and then centrifuged at 20 000 g for 1.5 h at 4° C. The supernatant was removed and the pellet of microvesicles was rinsed again and then centrifuged at 20 000 g for 1.5 h at 4° C. This last pellet of microvesicles was finally resuspended in buffer solution (HEPES 10 mM; NaCl 0.15 M; pH 7.4) and this solution was stored at −80° C. until used.
CRYOMEB Observation
Capture of the microvesicles: a buffer solution (HEPES 10 mM; NaCl 0.15 M; pH 7.4) containing microvesicles (derived from cellular activation of the HUVEC cells or derived from a human urine sample from a healthy subject) was incubated overnight at room temperature, at a rate of 300 μL per material, and then removed by 10 successive rinsings with the buffer solution. The materials used are the 1 cm2 square of PVC+C1 and the “DNA-BIND®” plate well+C1.
CRYOMEB observation: The samples were observed with a “Quanta 250 FEG FEI” field-effect scanning electron microscope (SEM) equipped with a “Quorum PP3000T” cryo module. The samples were first glued to a support using a Tissue-Tek®/carbon mixture, and then cooled rapidly in pasty nitrogen. They were then put in the first chamber of the microscope to undergo sublimation for 20 min at −95° C. and then in the second chamber to undergo metallization by spraying platinum under argon at 10 mA for 1 min. Finally, the metallized samples were placed in the observation chamber for image acquisition.
Capture of the Microvesicles on DNA-BIND® Support
Assay of the proteins: The proteins were assayed by NanoDrop (ND1000; Thermo Fisher SCIENTIFIC) in order to determine the quantity of microvesicles present in each solution.
Capture of the microvesicles: the equipment used, also called “kit” in the present application, is the DNA-BIND® 96-well plate (COSTAR; ref. 2525) functionalized with complex C1. The experiment is divided into two parts: a part with capture of the microvesicles by the kit and a part with control microvesicles away from the plate and thus not captured by the kit. The “control” part informs us about the initial composition of the microvesicles without capture. A buffer solution (HEPES 10 mM; NaCl 0.15 M; pH 7.4) containing microvesicles is then separated into two equal parts: a part for capture and a part for the controls.
Part for capture of the microvesicles: The buffer solution (HEPES 10 mM; NaCl 0.15 M; pH 7.4) containing microvesicles was incubated on the materials for 1 h at 37° C. or overnight at 4° C. at a rate of 50 μL per well. The plate was covered with a film to prevent any evaporation. The buffer solution was then removed and the wells were rinsed three times for 10 min with the same buffer formula at a rate of 200 μL/well in order to isolate the captured microvesicles from the other objects present in solution and remove those that would be adsorbed nonspecifically on the material. The microvesicles were then lysed in 30 μL of lysis buffer (99% in RIPA buffer—1% of cocktail of protease inhibitors) per well for 30 min in ice in order to recover all the proteins from the microvesicles individually. After homogenization, the solution was transferred to a 1.5 mL microtube. The membrane debris was sedimented by centrifugation at 10 000 g for 10 min at 4° C. in order to remove it. The supernatant containing the proteins was recovered in a new microtube.
Part for control microvesicles: The buffer solution (HEPES 10 mM; NaCl 0.15 M; pH 7.4) containing microvesicles was incubated in a 1.5 mL microtube for 1 h at 37° C. or overnight at 4° C. at a rate of 50 μL per microtube. After homogenization of the solution, the microvesicles were sedimented by centrifugation at 20 000 g for 1.5 h at 4° C. The supernatant was removed by aspiration. The microvesicles were then resuspended and lysed in 30 μL of lysis buffer per microtube for 30 min in ice in order to recover all the proteins from the microvesicles individually. After homogenization of the solution, the membrane debris was sedimented by centrifugation at 10 000 g for 10 min at 4° C. in order to remove it. The supernatant containing the proteins was recovered in a new microtube.
Analysis by Western Blot:
Each sample (protein extract) was prepared in a microtube with loading buffer (Bolt™ LDS Sample Buffer 4X+Bolt™ Sample Reducing Agent 10X) at a rate of 19.5 μL of solution of proteins to 30 μL of final sample in the proportions indicated by the supplier. The samples were vortexed and quickly centrifuged with a benchtop centrifuge before being denatured at 95° C. for 5 min. The whole sample (30 μL) was deposited on acrylamide gel in denaturing conditions. A size marker was always added as reference. The samples migrated for 10 min at 50 V and then for 1.5 h at 120V. The proteins present in the gel were transferred onto PVDF membrane by semiliquid transfer for 15 min at constant 1.3 A. The nonspecific sites of the membrane were saturated in a bath of milk buffer (5% of skim milk in buffer Tris Buffer Saline (TBS)—Tween 0.1%) for 1 h at room temperature with stirring. After two rinsings of 10 min in TBS—Tween 0.1% buffer with stirring, the membrane was incubated in a solution of primary antibodies, prepared in milk buffer, overnight at 4° C. with stirring. The antibodies (Ab) used are Ab anti-podocalyxin (Santacruz, ref. Sc-23904; dilution 1/2000), Ab anti-beta-actin (Sigma, ref. A1978; dilution 1/5000) and Ab anti-annexin-A5 (Sigma, ref. A8604; dilution 1/2000). After two rinsings of 10 min in TBS—Tween 0.1% buffer with stirring, the membrane was incubated in a solution of secondary Ab, prepared in milk buffer, for 1 h at RT with stirring. The signal was detected by a gel imager (GeneGnome, SYNGENE) by means of a chemiluminescence reaction after incubation of the membrane with a developer in Pico for 5 min (ThermoFisher SCIENTIFIC, ref. 34580) or in Femto for 2 min (ThermoFisher SCIENTIFIC, ref. 34094) depending on the signal intensity. Image acquisition was carried out with the GeneSys software. An analysis, both qualitative and quantitative, was carried out using Image J. The qualitative aspect was assessed by the presence of bands, at the molecular weight specific to each marker of interest, as well as from the intensity of their signal. The quantitative aspect was determined by calculating a ratio: intensity of the signal of the pathological marker, podocalyxin, relative to the intensity of the signal of the reference marker of the microvesicles, annexin-A5. This ratio makes it possible to determine the quantity of pathological marker relative to the number of microvesicles.
Results
CRYOMEB Analysis
The images of capture of the microvesicles by PVC+C1 are shown in
Capture of the Microvesicles and Detection by Western Blot
First, capture of the microvesicles by the kit was demonstrated by detection of the reference marker annexin-A5 carried by model microvesicles, after incubation on the kit at two different doses. The results are shown in
The presence of the characteristic bands of podocalyxin and of annexin-A5 after capture on the kit, demonstrated in
The diagram in
| Number | Date | Country | Kind |
|---|---|---|---|
| 1902814 | Mar 2019 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2020/050593 | 3/18/2020 | WO | 00 |
