CN113456612A - Albumin drug delivery system for treating sepsis myocardial injury - Google Patents

Abstract

The invention provides a nano-medicine for treating sepsis myocardial injury, which is characterized in that: bovine serum albumin nanoparticles loaded with curcumin molecules. Wherein, the bovine serum albumin is subjected to disulfide bond cross-linking, thereby improving the bioavailability of the curcumin, and being capable of being phagocytized by cells more effectively, thereby achieving better treatment effect on the myocardial damage caused by the sepsis.

Description

Albumin drug delivery system for treating sepsis myocardial injury

Technical Field

The invention provides a drug delivery system, and particularly relates to an albumin drug delivery system for treating sepsis myocardial injury.

Background

Sepsis is the pathophysiological response of the body to severe infections that can lead to life-threatening organ dysfunction. The damage to the heart muscle caused by sepsis is particularly prominent in organ dysfunction caused by sepsis. Early stages of sepsis can manifest significant hemodynamic changes, including vascular paralysis and even myocardial depression. Inappropriate fluid resuscitation may result in increased cardiac load and vessel wall pressure, which may exacerbate myocardial cell injury, further lead to dysfunction of other organs, and result in a vicious circle. How to alleviate the adverse effects caused by the myocardial damage of the sepsis and improve the treatment effect and the life quality of patients with the myocardial damage of the sepsis is a key and hot point problem for treating organ dysfunction of the sepsis. Over-activation of inflammatory factors plays an important role in the development of septic sepsis myocardial injury.

A large number of animal experiments and clinical researches find that the application of the anti-inflammatory drug can effectively relieve sepsis inflammatory factor storm and improve the cardiac function index of sepsis patients. Curcumin is a natural compound with anti-inflammatory and anti-oxidation effects, plays a considerable role in treatment in various disease models, but has extremely low solubility, cannot be effectively utilized by organisms and greatly limits the clinical application of curcumin.

Disclosure of Invention

The invention aims to overcome the defects, solves the problem that curcumin is poor in solubility and cannot be used by the body, constructs a nano-carrier for treating myocardial damage caused by sepsis by combining high biocompatibility of albumin nano-particles and anti-inflammatory capability of curcumin, and can effectively improve the solubility and bioavailability of curcumin so as to play an anti-inflammatory role of curcumin in systemic and local damaged myocardium.

The invention provides an albumin drug delivery system, which is characterized in that: the albumin drug-loading system is serum albumin nanoparticles loaded with curcumin molecules.

Further, the albumin drug delivery system provided by the invention is characterized in that: the curcumin is loaded after the serum albumin is reduced by adopting a reducing agent.

Further, the albumin drug delivery system provided by the invention is characterized in that: the specific preparation method is as follows:

s1, dissolving serum albumin, alkyl sulfate and a reducing agent in deionized water, and stirring at 85-95 ℃ for 1-3 hours;

the serum albumin can be selected from bovine serum albumin, ovalbumin OVA, human serum albumin, and the like;

the alkyl sulfate is preferably long-chain alkyl sulfate compounds;

the reducing agent is preferably a sulfur-containing reducing agent, and more preferably a thiol-based reducing agent.

S2, dripping the curcumin solution into the solution of S1, and stirring for 4-5 hours at the temperature of 37 +/-1 ℃;

curcumin solution herein generally refers to dissolving curcumin in an organic solvent, for example: dissolving in alcohol solvent such as ethanol to make its concentration within 1-10 mg/ml.

And S3, ultrafiltering the synthesized sample for more than 2 times, and centrifugally collecting the upper-layer liquid of the ultrafiltration tube.

Specifically, in the above reduction process: the disulfide bonds of the serum albumin are broken and reduced into polypeptide fragments containing sulfhydryl-SH, the polypeptide fragments are reformed into disulfide bonds in the process of loading curcumin, a plurality of hydrophobic cavities are generated in the process, and the curcumin is hydrophobic and can be wrapped by the reassembled serum albumin particles by utilizing the hydrophobic force.

Further, the albumin drug delivery system provided by the invention is characterized in that: the mass ratio of the serum albumin to the alkyl sulfate to the reducing agent is 1: 0.1-1: 0.03-0.1.

Further, the albumin drug delivery system provided by the invention is characterized in that: the stirring speed in the S2 is 1.5-2.5 times of that in the S1.

Further, the albumin drug delivery system provided by the invention is characterized in that: the mass concentration ratio of the serum albumin to the curcumin is 10-200: 1.

in addition, the invention also provides the application of the albumin drug-loading system, which is characterized in that: can be used for treating sepsis myocardial injury.

Further, the invention also provides other uses of the albumin drug delivery system: namely, the application of the compound in preparing the medicine for treating and relieving the myocardial cell apoptosis caused by the sepsis.

Further, the invention also provides the albumin drug delivery system, which further has at least one of the following uses:

use A. for the preparation of a medicament for inhibiting glutamate pyruvate transaminase;

use B, in the preparation of a medicament for inhibiting glutamic-oxalacetic transaminase;

use C for the preparation of a medicament for inhibiting interleukin-1 beta;

use D, in the preparation of a medicament for inhibiting tumor necrosis factor TNF-alpha.

Drawings

FIG. 1 is a schematic diagram of the synthesis route of an albumin drug delivery system for the treatment of septic myocardial injury provided in this example;

wherein r-BSA is a reduced BSA fragment formed by breaking the disulfide bond of Bovine Serum Albumin (BSA), and CUR is a curcumin molecule.

FIG. 2 shows the product characteristics of an albumin drug delivery system for the treatment of septic myocardial injury provided in this example;

wherein, fig. 2a is an appearance diagram of the product, the left side is nanoparticles synthesized by pure BSA, and the right side is BSA-CUR particles synthesized by adding curcumin;

FIG. 2b is a graph of the particle size of the product;

FIG. 2c is an electron microscope image of the product.

FIG. 3 is a graph showing the results of the inflammation index of a sepsis mouse using an albumin drug delivery system for treating sepsis myocardial injury provided in this example;

wherein Ctrl is a normal control group, LPS is a sepsis mouse group induced by LPS, BSA-CUR is a sepsis mouse group treated by the curcumin-carried albumin nano-drug, and statistical analysis: n is 6, p <0.05, p <0.01, and p < 0.001.

FIG. 4 is a fluorescence control map of an albumin drug delivery system for the treatment of septic myocardial injury as provided in this example;

wherein Ctrl is a normal control group, LPS is a sepsis mouse group induced by LPS, and BSA-CUR is a sepsis mouse group treated by the curcumin-carried albumin nano-drug.

Detailed Description

As shown in fig. 1, the preparation method of albumin nanocarriers for treating myocardial damage caused by sepsis provided in this embodiment is characterized by comprising the following steps:

step 1, reducing bovine serum albumin: 80mg of Bovine Serum Albumin (BSA), 40mg of Sodium Dodecyl Sulfate (SDS) and 2.96mg of Dithiothreitol (DTT) were weighed out and dissolved in 2ml of deionized water, and stirred at 90 ℃ for 2 hours at a rotation speed of 180 r/h. The concentration of the reduced BSA solution was 40 mg/ml.

According to the difference of the actually selected raw materials and the product properties, the mass ratio of the serum albumin (for example, the bovine serum albumin is replaced by ovalbumin OVA, human serum albumin and the like), the alkyl sulfate and the reducing agent can be selected from 1: 0.1-1: any proportional relation in the range of 0.03-0.1.

Step 2, synthesizing the curcumin-loaded albumin nano-drug:

preparing 1mg/ml ethanol solution of curcumin, and respectively preparing the following components in percentage by weight of BSA: the mass concentration ratio of the curcumin is 10: 1. curcumin solution was slowly dropped into the BSA solution at ratios of 20:1, 50:1, 100:1, and 200: 1.

According to the experimental results, when BSA is used, BSA: the mass concentration ratio of the curcumin is 10: 1 is most preferred.

② stirring for 4.5 hours at 37 ℃ and 350r/h

③ ultrafiltering the sample synthesized in the step II for 3 times, 5000r/h, 15min and 4 ℃. The liquid on the upper layer of the ultrafiltration tube was collected (shape shown in figure 2 a).

(iv) measuring the synthesized particle size (as shown in FIG. 2 b), potential (-12mV), and electron microscope (as shown in FIG. 2 c).

For the albumin nanocarriers of the above examples, the following verification tests were also performed on the performance of the albumin nanocarriers in this example:

performance verification test I, inflammation indexes of sepsis mice: ELISA assay for alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), interleukin-1 beta, and tumor necrosis factor (TNF-alpha)

The ELISA detection process is as follows:

1. collecting samples: and (3) taking normal mice, sepsis mice induced by LPS and sepsis mouse peripheral blood samples injected with BSA-CUR through eye sockets by adopting an EDTA anticoagulant tube. The cell phone supernatant was collected by centrifugation at 1000g for 30 minutes for subsequent testing.

2. Reagent preparation-all reagents (synbiotics) and samples were returned to room temperature before testing. The following components are configured according to the requirements of the specification:

1 × washing solution: sucking 20 × concentrated lotion 50ml to 1L measuring cylinder, adding distilled water to 1000ml, and mixing gently to avoid foaming. Transfer to clean bottle.

1 × detection buffer solution: draw 10 Xthe assay buffer 5ml to 100ml graduated cylinder, add distilled water to 50ml, mix gently to avoid foaming.

Detecting the antibody: fully mixing the components before dilution. The concentrated detection antibody was diluted 1:100 with 1 × detection buffer, depending on the number of standards and samples to be tested.

Fourthly, horse radish peroxidase marked streptavidin: fully mixing the components before dilution. According to the number of the standard substance and the sample to be detected, the concentrated horseradish peroxidase-labeled streptavidin is diluted by 1 × detection buffer solution according to the ratio of 1: 100.

Preparing a standard product: centrifuging for a short time before opening the cover, re-dissolving the required standard substance with distilled water, and marking the re-dissolved volume on a label of the standard substance. Vortex gently to ensure thorough mixing, and the concentration of the standard after redissolving was 1000 pg/ml. Standing for 10-30 minutes after re-dissolving. Fully mixing the components before dilution.

3. Detection step

Soaking the enzyme label plate, adding 300ul of 1 Xwashing liquor, standing and soaking for 30 seconds. After discarding the wash solution, the microplate was patted dry on absorbent paper.

Adding standard substance, namely adding 100ul of 2-time diluted standard substance into a standard substance hole. 100ul of standard dilution was added to the blank wells.

And thirdly, adding samples, namely adding 90ul of 1 Xdetection buffer solution and 10ul of samples into the sample hole.

Adding detection antibody, namely adding 50ul of diluted detection antibody into each hole (1:100 dilution). A closure plate membrane is used to close the plate. Shaking at 300 rpm, and incubating at room temperature for 1.5 hours.

Fifthly, washing, namely discarding the liquid, adding 300ul of washing liquid into each hole to wash the plate, and washing for 6 times.

Sixthly, adding enzyme for incubation, namely adding 100ul of diluted streptavidin marked by horseradish peroxidase (diluted 1: 100) into each hole.

Seventhly, incubation, namely using a new sealing plate film sealing plate. Shaking at 300 rpm, and incubating at room temperature for 30 minutes. Washing is carried out 6 times.

Adding 100ul of color developing substrate TMB into each hole, keeping out of the sun, and incubating for 30 minutes at room temperature.

Adding 100ul of stop solution into each hole. The color changed from blue to yellow.

And reading in detection of the red (R) is that within 30 minutes, a dual-wavelength detection is carried out by using a microplate reader, and the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 570nm or 630nm is measured. The OD value after calibration was obtained by subtracting the measurement value at 570nm or 630nm from the measurement value at 450 nm.

As shown in FIG. 3, in LPS-induced mouse sepsis model, BSA-CUR particles significantly reduced the expression levels of ALT, AST and TNF- α, and the expression of IL-1 β was reduced but not statistically significant. The above results suggest that BSA-CUR can reduce inflammatory response caused by LPS and exert organ protective action.

Performance verification test two: curcumin-loaded albumin nanoparticles for relieving myocardial cell apoptosis caused by sepsis

The detection process is as follows:

1. preparing a frozen section:

firstly, material taking: taking a normal mouse, an LPS-induced sepsis mouse and a sepsis mouse heart injected with BSA-CUR, adding an OCT embedding medium to immerse tissues, quickly putting into liquid nitrogen, and forming a frozen tissue block after about 15 s.

Preparing a sample: putting the sample into a constant temperature freezing microtome, coating a layer of OCT embedding glue on a sample support, placing the quick-frozen tissue on the sample support, precooling the quick-frozen tissue for 5-10min at 4 ℃ in a refrigerator to allow the OCT glue to soak the tissue. The tissue is removed and placed on a tin foil or glass slide and the sample holder is quickly frozen. Placing the tissue on a sample holder, adding a layer of OCT gel on the sample holder to completely cover the tissue, and quickly freezing for 30min on a shelf.

Cutting into slices: the indoor temperature is preferably-15 to-20 ℃, the slices are cut at room temperature and placed for 30min, the slices with the thickness of 10um are cut, acetone with the temperature of 4 ℃ is placed for fixing for 10min, and the drying is carried out in an oven for 20 min. PBS wash 5min × 3. Performing antigen thermal restoration, and naturally cooling at room temperature.

Dyeing:

preparing Tunel staining solution (Biyuntian) according to the specification

② to room temperature cooling section adding 0.5% Triton X-100 PBS, room temperature incubation after 5 minutes, using PBS washing 2 times.

③ 50ul of TUNEL detection solution was added to the sections, incubated at 37 ℃ in the dark for 60 minutes, and washed 3 times with PBS.

And (4) sealing the plate by using an anti-fluorescence quenching sealing liquid and observing under a fluorescence microscope. The excitation wavelength of Cy3 was 550nm, and the emission wavelength was 570nm (red fluorescence).

As shown in figure 4, in the LPS-induced mouse sepsis heart dysfunction model, apoptotic cardiomyocytes (red) in the heart section of the mouse treated by BSA-CUR were significantly reduced compared with untreated mice, which proves that BSA-CUR can play a role in resisting cardiomyocyte apoptosis.

Claims (9)

1. An albumin drug delivery system, comprising: the albumin drug-loading system is serum albumin nanoparticles loaded with curcumin molecules.

2. The albumin drug delivery system of claim 1, wherein: the curcumin is loaded after the serum albumin is reduced by adopting a reducing agent.

3. The albumin drug delivery system of claim 1, wherein: the specific preparation method is as follows:

s1, dissolving serum albumin, alkyl sulfate and a reducing agent in deionized water, and stirring at 85-95 ℃ for 1-3 hours;

s2, dripping the curcumin solution into the solution of S1, and stirring for 4-5 hours at the temperature of 37 +/-1 ℃;

and S3, ultrafiltering the synthesized sample for more than 2 times, and centrifugally collecting the upper-layer liquid of the ultrafiltration tube.

4. The albumin drug delivery system of claim 1, wherein:

the mass ratio of the serum albumin to the alkyl sulfate to the reducing agent is 1: 0.1-1: 0.03-0.1.

5. The albumin drug delivery system of claim 1, wherein:

the stirring speed in the S2 is 1.5-2.5 times of that in the S1.

6. The albumin drug delivery system of claim 1, wherein:

the mass concentration ratio of the serum albumin to the curcumin is 10-200: 1.

7. the use of an albumin drug delivery system as claimed in any of claims 1 to 6, wherein:

is used for preparing the medicine for treating the myocardial damage caused by the sepsis.

8. The use of an albumin drug delivery system as claimed in any of claims 1 to 6, wherein:

is used for preparing medicines for treating and relieving myocardial cell apoptosis caused by sepsis.

9. The use of the albumin drug delivery system of any one of claims 1 to 6, further comprising at least one of the following:

use A. for the preparation of a medicament for inhibiting glutamate pyruvate transaminase;

use B, in the preparation of a medicament for inhibiting glutamic-oxalacetic transaminase;

use C for the preparation of a medicament for inhibiting interleukin-1 beta;

use D, in the preparation of a medicament for inhibiting tumor necrosis factor TNF-alpha.

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