CN111265674A - Contrast agent, preparation method and application thereof - Google Patents

Abstract

The invention provides a contrast agent, a preparation method and application thereof, wherein erythrocytes in blood are taken as a matrix, and gadolinium chelates are loaded on the erythrocytes by a hypotonic dialysis method, a novel electroporation method, a drug induction method and a drug induction method under the hypotonic condition, so that the volume of the gadolinium chelates is increased, the gadolinium chelates are blocked from migrating to the outside of the blood vessel, the technical problem that the existing magnetic resonance contrast agent is easy to migrate to the clearance outside the blood vessel is solved, and the magnetic resonance contrast agent has wide application in the fields of magnetic resonance imaging and imaging.

Description

Contrast agent, preparation method and application thereof

Technical Field

The invention relates to a contrast agent, a preparation method and application thereof, and belongs to the field of medical contrast agents.

Background

In recent years, cardiac magnetic resonance imaging (CMR) has been widely used and focused on diagnosing heart diseases as a non-invasive image analysis means. By using the CMR technology, the blood flow conditions of the left ventricle, the right ventricle, the myocardial structure and valve tissue of the heart can be clearly observed, and a powerful image basis is provided for discovering and treating heart diseases. In the clinical practice of CMR, it is often necessary to inject a contrast agent to improve image contrast in order to obtain high-definition images. The most used contrast agent is gadolinium chelate at present, because gadolinium ions have the advantage of large magnetic moment and the enhancement effect of T1 is obvious. However, gadolinium chelates are small molecule structures that are free to pass through the capillary network into the extravascular space. When the intravascular diseases such as ischemic heart disease are examined, the gadolinium chelate has the defects of low relaxation rate, short in vivo circulation time and the like due to the small molecular weight, so that the imaging time is short, and the disease condition cannot be effectively diagnosed.

For nuclear magnetic resonance diagnosis of diseases originating in the heart vessels, there are two main ways: magnetic resonance vascular imaging and magnetic resonance perfusion imaging. According to the characteristics of the analyzed tissues, magnetic resonance blood vessel imaging is mainly magnetic resonance coronary artery imaging, and magnetic resonance perfusion imaging is mainly magnetic resonance myocardial perfusion imaging. By combining magnetic resonance vascular imaging and magnetic resonance perfusion imaging, the overall condition of the heart can be analyzed and diagnosed. However, to obtain both imaging results, the patient needs to inject two contrast agents one after the other and perform two magnetic resonance examinations. Such an operation not only lengthens the examination time, but also imposes a physical and economic burden on the patient.

① gadolinium chelate is combined with albumin, the purpose of the method is to increase the size of gadolinium chelate, thus blocking gadolinium chelate from diffusing out of blood vessel, the disadvantage is that the combination of the two substances is unstable, only 30% of the combination remains in blood vessel, the rest is still diffused to the space outside blood vessel, ② gadolinium chelate is encapsulated in liposome, the encapsulated gadolinium chelate has long circulation time in blood vessel, and can achieve better enhancement effect, but also has the disadvantages of complex preparation process, high cost and biological toxicity, ③ prepares superparamagnetic iron oxide, although the oxide can not diffuse to the space outside blood vessel, but is easy to be phagocytized by reticuloendothelial system and enriched in organs such as liver and spleen, therefore, the oxide is mainly used for enhancing imaging of organs such as liver and spleen.

Therefore, there is a need for improvements in existing contrast agents for cardiac magnetic resonance applications.

Disclosure of Invention

The heart muscle and the coronary artery are tissues through which blood flows in the heart structure, and diseases related to the heart muscle and the coronary artery are very common heart diseases, such as myocardial ischemia, myocardial infarction, coronary heart disease and the like. When a gadolinium chelate is used as a contrast agent in examining the cardiac muscle and coronary artery of a patient using the CMR technique, the small-molecule gadolinium chelate rapidly diffuses into the extravascular space immediately after being injected into the blood, resulting in a rapid decrease in the content of the gadolinium chelate in the blood. At this time, the CMR imaging result cannot truly reflect the structural conditions of the myocardium and coronary artery, so that the disease cannot be diagnosed in time. That is, gadolinium chelates with short circulation half-lives cannot meet the requirements of magnetic resonance vascular imaging and magnetic resonance perfusion imaging, and further cannot realize one-stop diagnosis of ischemic heart diseases. The invention aims to prepare the cardiac magnetic resonance contrast agent which has long circulation half-life period, does not diffuse to extravascular space and has good biological safety.

In order to solve the above problems, the present invention provides a contrast agent. The contrast agent comprises gadolinium-loaded red blood cells. The gadolinium-loaded red blood cell takes red blood cells in blood as a matrix, and small-molecule gadolinium chelates are loaded to form the large-molecule gadolinium-loaded red blood cell. The macromolecular gadolinium-loaded red blood cells are used as a magnetic resonance contrast agent, and the characteristics of red blood cell biological safety and strong contrast of gadolinium chelates are combined, so that the gadolinium-loaded red blood cells can be used for diagnosing heart diseases related to blood vessels. The gadolinium-loaded red blood cell has good dispersibility, stability, water solubility and biocompatibility, the loaded gadolinium chelate can keep the characteristics of chemical properties, and the gadolinium-loaded red blood cell is used as a contrast agent, so that the definition and contrast of an image are obviously improved.

In the preferred technical scheme of the invention, the concentration range of the gadolinium-loaded red blood cells in the contrast agent is 60-80%. The concentration of gadolinium-loaded red blood cells in the contrast agent has great influence on the magnetic resonance imaging result. The higher the concentration of the contrast agent, the clearer the imaging result and the more detail can be displayed. However, if the concentration of the contrast agent is too high, agglomeration between particles of the contrast agent may occur, resulting in a decrease in stability. And the gadolinium chelate complex with too high concentration can cause certain harm to human body. In the invention, the concentration range of the gadolinium-loaded red blood cells is set to be 60-80%, so that better imaging can be ensured, and the body can not be injured.

In a preferred embodiment of the invention, the concentration of gadolinium-loaded red blood cells is 75%. At this time, gadolinium-loaded erythrocytes exist stably in the blood environment, and aggregation between erythrocytes hardly occurs. The gadolinium-loaded red blood cells with the concentration of 75% enable images of magnetic resonance imaging to be clear and high in contrast. In addition, the damage to the human body is small, and the safety is high.

In the preferred technical scheme of the invention, the concentration range of gadolinium ions of the gadolinium-loaded red blood cells is 30-50%. Gadolinium chelates are used as contrast agents, where the substance playing a critical role is gadolinium ion. If the gadolinium ion load is high, the beneficial effects of higher relaxation rate and clearer nuclear magnetic resonance image are generated. However, the concentration of free gadolinium ions reaches a certain value, which may also cause damage to the human nervous system. Therefore, the gadolinium ion concentration needs to be determined within a suitable range.

In a preferred embodiment of the present invention, the concentration of gadolinium ions is 40%. Gadolinium ions at a concentration of 40% sufficiently ensure formation of a high-definition nuclear magnetic resonance image without causing irreversible damage to the human body.

In a preferred embodiment of the invention, the contrast agent can be used in a cardiac magnetic resonance examination. Especially, the method can be widely applied to the fields of magnetic resonance perfusion imaging, blood vessel imaging and the like, and the application effect of the magnetic resonance imaging in the fields is obviously improved. After the novel contrast agent is used for cardiac magnetic resonance imaging, the one-stop comprehensive assessment on the coronary artery stenosis degree and the myocardial ischemia condition is realized, and the examination which needs to be carried out twice before is finished once by utilizing the characteristics of long half-life period of the gadolinium-loaded erythrocyte contrast agent in vivo and the like aiming at ischemic heart disease, so that great convenience is brought to patients.

In a preferred embodiment of the present invention, the preparation of gadolinium loaded red blood cells using an improved low permeability assay comprises the steps of: s1, use from 10mM C₈H₁₈N₂O₄S, 154mM NaCl, 5mM glucose, pH7.4 HEPES buffer solution to wash the red blood cells for 3 times, to obtain a suspension with a hematocrit Hct of at least 70%;

s2, placing 1ml of the suspension in a 12-14kDa ultrafiltration tube, and adding 4ml of Gd-HP-DO₃A. 50ml osmotic pressure 64mOsm and is prepared from 10mM NaHCO₃、10mM NaH₂PO₄20mM glucose, 4mM MgCl₂Composed of dialyzate of pH7.4, 2mM MATP and 3mM reduced glutathione, and incubating for 75min, wherein Gd-HP-DO is added₃The concentration of a may be one of 50, 100, 150, 200, 250, 300 or 350 mM;

s3, adding 0.1ml of a mixture of 5mM adenine, 100mM inosine, 2mM ATP, 100mM glucose, 100mM C to 1ml of red blood cells₃H₃NaO₃、4mM MgCl₂、194mM NaCl、1.606M KCl、35mM NaH₂PO₄A PIGPA solution with the pH value of 7.4 is formed;

s4, culturing PIGPA solution at 37 deg.C for 45min, settling erythrocytes at 400g for 20min, and discarding supernatant to remove free Gd-HP-DO₃And obtaining WEr, and washing WEr with HEPES buffer solution for 4 times to obtain the gadolinium-loaded red blood cells.

The preparation method is easy to control, the raw materials are easy to obtain, and large-scale industrial production is easy to realize.

In a preferred technical scheme of the invention, the gadolinium-loaded red blood cells are prepared by using a novel electroporation method, and the method comprises the following steps of: s1, mixing 1ml of red blood cells with Gd-HP-DO diluted by PBS buffer solution₃Mixing the suspension A, and performing electroporation at 4 deg.C with DC electric pulse with voltage of 300 to 1000V, and the pulse width is 1ms, 2ms, 100 mus, 20 mus or 10 mus respectively;

s2, perforating, and promoting erythrocyte repair by water bath at 37 ℃;

s3, settling the erythrocytes for 20min with a centrifugal acceleration of 400g, discarding the supernatant to remove the free Gd-HP-DO₃And obtaining WEr, and washing WEr in HEPES buffer solution for 4 times to obtain the gadolinium-loaded red blood cells.

In a preferred technical scheme of the invention, a preparation method of a contrast agent is provided, which comprises the following steps:

s1, mixing and shaking the erythrocytes and the isotonic solution evenly, adding chlorpromazine for cultivation, wherein the concentration of the chlorpromazine can be 50, 100 or 200uM, the cultivation temperature can be 4 ℃ or 37 ℃, and the cultivation time can be 30min, 60min or 90 min;

s2, centrifuging the solution obtained in the step S1 at 1500rpm for 5min, and sufficiently removing a supernatant to obtain WEr;

s3, mixing WEr and Gd-HP-DO-containing solution₃Mixing the diluted suspension of the A, culturing, adding physiological saline until the system reaches isotonic, shaking up, standing for 20min, and then incubating for 60min in a constant-temperature water bath;

s4, followed by sedimentation for WEr20min at a centrifugal acceleration of 400g, the supernatant was discarded to remove free Gd-HP-DO₃A, WEr was washed 4 times with HEPES buffer to obtain gadolinium-loaded erythrocytes.

The prepared gadolinium-loaded red blood cell has the advantages of large particle size, good water solubility, high relaxation rate and the like. The preparation method has simple process and can further meet the requirements of production and application.

In the preferred technical scheme of the invention, the preparation of the gadolinium-loaded red blood cells by using a drug induction method under the hypotonic condition comprises the following steps: s1, use from 10mM C₈H₁₈N₂O₄S, 154mM NaCl, 5mM glucose, pH7.4 HEPES buffer solution to wash the red blood cells for 3 times, to obtain a suspension with a hematocrit Hct of at least 70%;

s2, placing 1ml of the suspension in a 12-14kDa ultrafiltration tube, and adding 4ml of 250mM Gd-HP-DO₃A. 50ml osmotic pressure 64mOsm and is prepared from 10mM NaHCO₃、10mM NaH₂PO₄20mM glucose, 4mM MgCl₂Composed of dialyzate of pH7.4, 2mM ATP, 3mM reduced glutathione and chlorpromazine, and culturing for 60 min;

s3, adding 0.1ml of a mixture of 5mM adenine, 100mM inosine, 2mM ATP, 100mM glucose, 100mM C to 1ml of red blood cells₃H₃NaO₃、4mM MgCl₂、194mM NaCl、1.606M KCl、35mM NaH₂PO₄A PIGPA solution with the pH value of 7.4 is formed;

s4, incubating at 37 deg.C for 45min, settling erythrocytes with 400g centrifugal acceleration for 20min, and discarding supernatant to remove free Gd-HP-DO₃A, WEr obtained was washed 4 times with HEPES buffer solution to obtain gadolinium-loaded erythrocytes.

The preparation method is simple and easy to operate, and is expected to be popularized on a large scale.

Drawings

FIG. 1 is a graph showing the encapsulation efficiency of gadolinium-loaded red blood cells as a function of reaction temperature at a concentration of 250mM of gadoteridol added and a reaction time of 40 minutes;

FIG. 2 is a graph showing the encapsulation efficiency of gadolinium-loaded red blood cells as a function of reaction time at an incubation temperature of 36.5 ℃ with an addition concentration of gadoteridol of 250 mM;

FIG. 3 is a graph showing the gadolinium-loaded erythrocyte encapsulation efficiency as a function of the addition amount of gadoteridol under the conditions of an incubation temperature of 37 ℃ and a reaction time of 35 minutes;

FIG. 4 is a graph showing the encapsulation efficiency of gadolinium-loaded red blood cells as a function of the amount of gadoteridol added under the conditions of voltage 500V and pulse width 2 ms;

FIG. 5 is a graph showing the encapsulation efficiency of gadolinium-loaded red blood cells as a function of voltage under the condition that the amount of gadolinium (gadoteridol) added is 4ml and 200mM, and the pulse width is 2 ms;

FIG. 6 is a graph showing the encapsulation efficiency of gadolinium-loaded red blood cells as a function of pulse width under the condition that the addition amount of gadoteridol is 4ml and 200mM and the voltage is 300V;

FIG. 7 is a graph showing the encapsulation efficiency of gadolinium-loaded red blood cells as a function of the amount of gadoteridol added and the incubation temperature under the condition of the amount of chlorpromazine of 100 μm;

FIG. 8 is a graph showing the encapsulation efficiency of gadolinium-loaded red blood cells as a function of the amount of chlorpromazine pretreatment and incubation time at an incubation temperature of 37 ℃;

FIG. 9 is a graph showing the encapsulation efficiency of gadolinium-loaded red blood cells as a function of the amount of chlorpromazine pretreatment at an incubation temperature of 37 ℃;

FIG. 10 shows a comparison of normal red blood cells and gadolinium-loaded red blood cells under a transmission electron microscope.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to examples. The embodiments of the present invention are not limited to the following examples, and various alternatives and modifications are also included within the scope of the technical idea of the present invention.

Sources of experimental materials:

centrifuge (Ptima-L80XP, Beckman, USA);

automatic cell analyzer (adiia 2120, siemens);

automated biochemical analyzers (CleverChem380Plus, CleverChem);

horizontal electrophoresis tanks (EP1201-1EA, Sigma-Aldrich);

transmission electron microscopy (JEM-2100Plus, Japan Electron);

atomic absorption spectroscopy (AA800, PerkinElmer);

a superconducting magnetic resonance spectrometer (DMX-500);

all experimental animals were sourced from the experimental animal center of university of Zhejiang.

The main reagents are as follows:

gadoteridol (20 ml: 5.586g, prowsonia).

Preparation and character characterization of gadolinium-loaded red blood cells

1. Experimental animal erythrocyte collection and treatment

Approximately 5ml of blood (rat is not restricted to blood type) of sodium citrate anticoagulated SD rat was taken, washed packed erythrocytic cells (washing-packed erythrocytic, WEr) were prepared, and further the amount of red blood cells, the average hemoglobin concentration and the like were measured.

2. Preparation of gadolinium-loaded erythrocytes

2.1 modified hypotonic dialysis

Using a mixture of 10mM C₈H₁₈N₂O₄S, 154mM NaCl, 5mM glucose pH7.4 HEPES buffer to wash the red blood cells 3 times to obtain WEr, obtain WEr suspension of 70% hematocrit Hct. 1ml of this suspension was placed in a 12-14kDa ultrafiltration tube and 4ml of gadoteridol Gd-HP-DO was added₃A. 50ml osmotic pressure 64mOsm and is prepared from 10mM NaHCO₃、10mMNaH₂PO₄20mM glucose, 4mM MgCl₂Composed of dialyzate of pH7.4, 2mM ATP and 3mM reduced glutathione, wherein, the gadolinium specific alcohol Gd-HP-DO₃The concentration of A is 50, 100, 150, 200, 250, 300 or 350mM, respectively.

Resealing of erythrocytes: adding 0.1ml of a mixture of 5mM adenine, 100mM inosine, 2mM ATP, 100mM glucose, 100mM C to 1ml of red blood cells₃H₃NaO₃、4mM MgCl₂、194mM NaCl、1.606M KCl、35mM NaH₂PO₄Thus, a PIPPA solution with pH of 7.4 was obtained. Incubating at 37 deg.C for 45min, centrifuging at 400g for 20min to settle the red blood cells, discarding the supernatant to remove free gadoteridol, and washing the settled red blood cells with HEPES buffer solution for 4 times to obtain gadolinium-loaded red blood cells.

And selecting the optimal gadoteridol proportion parameter, reaction temperature, reaction time and the like according to the results of gadoteridol encapsulation efficiency, erythrocyte integrity and the like. Through exploration under various conditions, the gadolinium-loaded red blood cell obtained under the conditions that cultivation is carried out for 45min at 37 ℃ and 4ml of 250mM gadoteridol is added has the highest encapsulation efficiency and relatively complete red blood cells.

2.2 novel electroporation method

1ml of WER was mixed with a suspension of gadoteridol diluted in PBS buffer and subjected to electroporation at 4 ℃ using direct current pulses at a voltage of 300 to 1000V and a pulse width of 1ms, 2ms, 100. mu.s, 20. mu.s or 10. mu.s, respectively. After perforation, water bath at 37 ℃ promotes erythrocyte repair. The red blood cells were sedimented by centrifugation at 400g for 20min, the supernatant was discarded to remove free gadoteridol, and the sedimented red blood cells were washed 4 times with HEPES buffer to obtain gadolinium-loaded red blood cells. Through a plurality of experimental explorations, the result shows that the obtained gadoteridol has the highest encapsulation efficiency and relatively complete red blood cells under the conditions that the addition amount of the gadoteridol is 4ml and 200mM, the voltage is 800V and the pulse width is 2 ms.

2.3 drug Induction method

Mixing erythrocytes with hypotonic solution, shaking, adding chlorpromazine for culturing at 4 deg.C or 37 deg.C for 30min, 60min or 90min, wherein the concentration of chlorpromazine can be 50, 100 or 200 uM. Centrifuging at 1500rpm for 5min, and removing supernatant to obtain packed red blood cells. Mixing packed red blood cells with the diluted suspension of the gadoteridol plasma with different concentrations, adding a proper amount of physiological saline after culturing for a proper time to enable the system to reach the isotonic state, shaking up, standing for 20min, and then incubating for 60min in a constant-temperature water bath. The erythrocytes were then sedimented by centrifugation at 400g for 20min, the supernatant was discarded to remove free gadoteridol, and the sedimented erythrocytes were washed 4 times with HEPES buffer to give gadolinium-loaded erythrocytes. Through multiple experiments, the results show that the obtained gadolinium-loaded red blood cell has the highest encapsulation efficiency and relatively complete shape under the conditions that the addition amount of the gadoteridol is 4ml and 250mM, the cultivation temperature is 37 ℃, the cultivation time is 60min, and the chlorpromazine pretreatment concentration is 100 uM.

2.4 drug Induction under hypotonic conditions

Using a mixture of 10mM C₈H₁₈N₂O₄S, 154mM NaCl, 5mM glucose, and ph7.4 HEPES buffer WEr was washed 3 times, and suspended in the HEPES buffer so that Hct became 70%. Placing 1ml RBCs suspension in 12-14kDa ultrafiltration tube, adding 4ml gadolinium special alcohol Gd-HP-DO with concentration of 250mM₃A. 50ml osmotic pressure 64mOsm and is prepared from 10mM NaHCO₃、10mMNaH₂PO₄20mM glucose, 4mM MgCl₂The constituted dialysate, pH7.4, was incubated for 60min with 2mM ATP, 3mM reduced glutathione and varying concentrations of chlorpromazine.

Resealing of erythrocytes: adding 0.1ml of a mixture of 5mM adenine, 100mM inosine, 2mM ATP, 100mM glucose, 100mM C to 1ml of red blood cells₃H₃NaO₃、4mM MgCl₂、194mM NaCl、1.606M KCl、35mM NaH₂PO₄Thus, a PIPPA solution with pH of 7.4 was obtained. Culturing at 37 deg.CCulturing for 45min, centrifuging at 400g for 20min to settle erythrocyte, discarding supernatant to remove free gadoteridol, and washing the settled erythrocyte with HEPES buffer solution 4 times to obtain gadolinium-loaded erythrocyte. As a result, on the basis of improved hypotonic dialysis test parameters, 100mg of chlorpromazine is added, so that the gadolinium-loaded red blood cell encapsulation efficiency is highest, and red blood cells are relatively complete.

3. Characterization of gadolinium-loaded erythrocytes

3.1 in vitro characterization

(1) Cell integrity assay: taking 2ml of each of the gadolinium-loaded red blood cells obtained under the condition of optimal encapsulation rate by each method and red blood cells of a control group, and measuring indexes such as red blood cell number (RBC), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCHC) and hematocrit (Hct) by an automatic cell analyzer. The recovery was counted according to the number of erythrocytes before and after gadolinium loading.

TABLE 1 physiological indices of rat erythrocytes by different treatment methods

(2) Measurement of osmotic fragility of gadolinium-loaded erythrocytes: taking 0.3ml of each of the gadolinium-loaded red blood cells and the red blood cells of the control group obtained by the various methods under the condition of the optimal encapsulation efficiency, carrying out centrifugal washing three times by using PBS buffer solution at room temperature at 1500rpm for 5min, fully removing supernatant, and fully and uniformly mixing the whole blood Hct and autologous plasma to prepare plasma suspension of the carrier red blood cells. And taking the washed packed red blood cells, and fully and uniformly mixing the whole blood Hct and the autologous plasma to prepare the plasma suspension for washing the red blood cells. Hypotonic saline solutions were prepared in a series of concentrations of 0.4-0.6% (0.02% apart) in distilled water according to the whole blood erythrocyte osmometry assay. And (3) respectively adding 0.1ml of each group of erythrocyte suspension specimen into hypotonic saline with different concentrations, standing for 2 hours at room temperature, and observing the result, wherein when the result is judged, the upper layer solution starts to appear transparent red to start blood vessel lysis, and the solution is transparent red to completely disappear red erythrocytes at the tube bottom to obtain a complete hemolysis tube. The hypotonic saline concentrations at which hemolysis was initiated were recorded separately, with higher concentration values than normal indicating increased osmotic fragility and conversely decreased. The results of the osmotic fragility of the obtained gadolinium loaded red blood cells of each group are shown in the following table. The results show that the penetration brittleness of the gadolinium-loaded red blood cells prepared by the hypotonic dialysis method and the electroporation method is higher than normal, and the penetration brittleness of the gadolinium-loaded red blood cells prepared by the chlorpromazine pretreatment method is similar to that of normal red blood cells.

TABLE 2 osmotic fragility of different groups of gadolinium-loaded erythrocytes

(3) And (3) determining the encapsulation efficiency: 500ul of each of the gadolinium-loaded erythrocytes and the control erythrocytes are taken, 37% HCl 4500ul is added to decompose the gadolinium-loaded erythrocytes and the decomposition liquid is diluted properly and then the gadolinium ion concentration of the gadolinium-loaded erythrocytes and the control erythrocytes is measured by an atomic absorption spectrometer. The encapsulation efficiency is (gadolinium concentration in gadolinium-loaded erythrocytes-control erythrocyte gadolinium concentration)/gadolinium concentration initially added. Different methods the results of gadolinium red cell encapsulation efficiency under different conditions are shown in fig. 1 to 9.

Based on the comparison of the results of the integrity, the penetration brittleness, the encapsulation efficiency and the like of the gadolinium-loaded erythrocyte, the gadolinium-loaded erythrocyte obtained by the chlorpromazine pretreatment group has the highest encapsulation efficiency, the integrity, the penetration brittleness and the like of the gadolinium-loaded erythrocyte are closer to the normal, and the gadolinium-loaded erythrocyte contrast agent is more in line with the application requirement. On the basis of hypotonic dialysis, proper chlorpromazine is added, so that the encapsulation efficiency is higher, the integrity is good, and the osmotic fragility is relatively poor.

(4) Taking chlorpromazine pretreatment group-carried gadolinium red cells to perform morphological observation under an electron microscope: 2ml of each of the gadolinium-loaded erythrocytes and the control erythrocytes were washed 3 times with 0.1mol/L phosphate buffer (pH 7.3), and then centrifuged at 600g for 10min, the supernatant was discarded, and the washed erythrocytes were immediately fixed with 2.5% glutaraldehyde phosphate buffer for 1 h. Then fixing with 1% osmium tetroxide stationary solution for about 2h, rinsing with 0.1mol/L phosphate buffer solution or distilled water for 3 times, 5min each time; gradient ethanol dehydration, wherein the sequence is 30%, 50%, 70% and 90% ethanol respectively for 10-15min each time; 1:1 acetone: soaking the embedding agent for 1-2 h; polymerizing for 12 hours at 37 ℃ and for 24-36 hours at 60 ℃; dyeing the slices with saturated uranium acetate for 30min, and dyeing with lead citrate for 5-8 min; the observation and photography by a transmission electron microscope show that the transmission electron microscope results are shown in FIG. 10.

(5) And (3) measurement of relaxation rate: 2ml of each of the chloropromazine-pretreated gadolinium-loaded erythrocytes and control erythrocytes were mixed with plasma to prepare blood having a hematocrit (Hct) of 40%. T1/T2 values were measured using a superconducting magnetic resonance spectrometer (DMX-500) under conditions of 4.7T magnetic field strength and 37 ℃. The sequence is as follows: the assay T1 used a 180 ° -90 ° inversion recovery sequence with a fixed relaxation decay time > 5T 1. The inversion time is determined based on the estimated value of T1. The measurement T2 was carried out by using the Carr-Purcell-Meiboom-Gill method, and the echo time was determined from the estimated T2 value. The relaxation rate R1 is 1/T1, and R2 is 1/T2. For multiple measurements, the T1 value of the gadolinium-loaded erythrocyte group is 458.2ms on average, and the T2 value is 69.5ms on average. While the mean T value of the control group was 731.3ms and the mean T2 was 221.2 ms. Compared with the T1 and T2 time of the gadolinium red cell, the T2 shortening effect is more obvious.

3.2 in vivo trait characterization

And (3) pharmacokinetic detection: 6-8 week old SD rats were divided on average into three groups: the first group is injected with gadolinium-loaded mouse red blood cells 20umol Fe/kg through tail vein; injecting the gadoteridol suspension with the same dosage into the second group through tail vein; the third group was injected with the same dose of control red blood cells via tail vein. Blood samples were taken from 5 rats per dose group at 0, 0.5, 1, 2, 4, 24, 48, 120 and 240 hours after injection of the contrast agent, and gadolinium content was determined by atomic absorption spectroscopy. The results show that the gadolinium content in blood samples collected at different times in the control group is basically constant, the gadolinium content in the gadoteridol suspension group is very high initially, but is rapidly reduced along with the time, and the gadolinium is rapidly cleared by tissues; the gadolinium content of the gadolinium-loaded erythrocyte group is maintained at a higher level for a longer time, and the gadolinium-loaded erythrocyte group shows that the gadolinium-loaded erythrocyte group is slowly cleared and has more stable concentration in vivo.

TABLE 3 variation of gadolinium content in blood with blood sampling time for different groups of rats

From the test results, compared with the improved hypotonic dialysis method, the novel electroporation method, the drug induction method and the drug induction method under the hypotonic condition, the drug induction method for chlorpromazine pretreatment can obtain gadolinium-loaded red blood cells with the highest encapsulation rate, and the integrity, the osmotic fragility and the like of the gadolinium-loaded red blood cells are closer to normal red blood cells. The gadolinium-loaded red blood cell is applied to nuclear magnetic resonance imaging, and a clear image result is further obtained. According to pharmacokinetic detection, the gadolinium content loaded by the gadolinium-loaded red blood cells can maintain high concentration for a long period of time, and the clinical requirements of the gadolinium-loaded red blood cells as contrast agents are met.

Claims (10)

1. A contrast agent comprising gadolinium loaded red blood cells.

2. The contrast agent according to claim 1, wherein the gadolinium loaded red blood cells in the contrast agent have an encapsulation efficiency ranging from 60% to 80%.

3. The contrast agent according to claim 1 or 2, wherein the concentration of said gadolinium loaded red blood cells in said contrast agent is 75%.

4. The contrast agent according to claim 3, wherein the gadolinium-loaded red blood cells have a concentration of gadolinium ions in the range of 30 to 50%.

5. The contrast agent according to claim 3, wherein said gadolinium loaded red blood cells have a concentration of gadolinium ions of 40%.

6. The contrast agent as claimed in any of claims 1-2, 4-5, capable of use in cardiac magnetic resonance examinations.

7. A method for preparing a contrast agent, comprising the steps of:

s1, use from 10mM C₈H₁₈N₂O₄S, 154mM NaCl, 5mM glucose composed of HEPES buffer pH7.4 to red blood cells washing 3 times, obtained the hematocrit Hct of 70%A suspension;

s2, placing 1ml of the suspension in a 12-14kDa ultrafiltration tube, and adding 4ml of Gd-HP-DO₃A. 50ml osmotic pressure 64mOsm and is prepared from 10mM NaHCO₃、10mM NaH₂PO₄20mM glucose, 4mM MgCl₂Composed of dialyzate of pH7.4, 2mM ATP and 3mM reduced glutathione, and incubating for 75min, wherein Gd-HP-DO is added₃The concentration of a may be one of 50, 100, 150, 200, 250, 300 or 350 mM;

s3, adding 0.1ml of a mixture of 5mM adenine, 100mM inosine, 2mM ATP, 100mM glucose, 100mM C to 1ml of red blood cells₃H₃NaO₃、4mM MgCl₂、194mM NaCl、1.606M KCl、35mM NaH₂PO₄A PIGPA solution with the pH value of 7.4 is formed;

s4, culturing PIGPA solution at 37 deg.C for 45min, settling erythrocytes at 400g for 20min, and discarding supernatant to remove free Gd-HP-DO₃And washing the cells for 4 times by using HEPES buffer solution to obtain the gadolinium-loaded red blood cells.

8. A method for preparing a contrast agent, comprising the steps of:

s1, mixing 1ml packed red blood cells with Gd-HP-DO diluted by PBS buffer solution₃Mixing the suspension A, and performing electroporation at 4 ℃ by using direct current electric pulses, wherein the voltage of the direct current electric pulses is 300-1000V, and the pulse width is 1ms, 2ms, 100 mus, 20 mus or 10 mus respectively;

s2, punching, and promoting the repair of packed red blood cells by water bath at 37 ℃;

s3, settling the erythrocytes for 20min with a centrifugal acceleration of 400g, discarding the supernatant to remove the free Gd-HP-DO₃A was then washed 4 times in HEPES buffer to obtain gadolinium-loaded erythrocytes.

9. A method for preparing a contrast agent, comprising the steps of:

s1, mixing and shaking the erythrocytes and the hypotonic solution uniformly, and adding chlorpromazine to cultivate, wherein the concentration of the chlorpromazine can be 50, 100 or 200uM, the cultivation temperature can be 4 ℃ or 37 ℃, and the cultivation time can be 30min, 60min or 90 min;

s2, centrifuging the solution obtained in the step S1 at 1500rpm for 5min, and fully removing the supernatant;

s3, mixing the solution obtained in the step S2 with Gd-HP-DO₃Mixing the diluted suspension of the A, culturing, adding physiological saline until the system reaches isotonic, shaking up, standing for 20min, and then incubating for 60min in a constant-temperature water bath;

s4, and then sedimenting the erythrocytes for 20min with a centrifugal acceleration of 400g, discarding the supernatant to remove the free Gd-HP-DO₃And washing the cells with HEPES buffer solution for 4 times to obtain the gadolinium-loaded red blood cells.

10. A method for preparing a contrast agent, comprising the steps of:

s1, use from 10mM C₈H₁₈N₂O₄S, 154mM NaCl, 5mM glucose, pH7.4 HEPES buffer solution to wash the red blood cells for 3 times, to obtain a suspension with a hematocrit Hct of at least 70%;

s2, placing 1ml of the suspension in a 12-14kDa ultrafiltration tube, and adding 4ml of 250mM Gd-HP-DO₃A. 50ml osmotic pressure 64mOsm and is prepared from 10mM NaHCO₃、10mM NaH₂PO₄20mM glucose, 4mM MgCl₂Composed of dialyzate of pH7.4, 2mM ATP, 3mM reduced glutathione and chlorpromazine, and culturing for 60 min;

s3, adding 0.1ml of a mixture of 5mM adenine, 100mM inosine, 2mM ATP, 100mM glucose, 100mM C to 1ml of red blood cells₃H₃NaO₃、4mM MgCl₂、194mM NaCl、1.606M KCl、35mM NaH₂PO₄A PIGPA solution with the pH value of 7.4 is formed;

s4, incubating at 37 deg.C for 45min, settling erythrocytes with 400g centrifugal acceleration for 20min, and discarding supernatant to remove free Gd-HP-DO₃A, WEr obtained was washed 4 times with HEPES buffer solution to obtain gadolinium-loaded erythrocytes.

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