CN116492448A - PEG-EPO and mesenchymal stem cell-loaded composition, medicament and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of biopharmaceuticals, and in particular relates to a composition loaded with PEG-EPO and mesenchymal stem cells, a medicine and a preparation method thereof. The present invention uses end-modified PEG to modify EPO molecules to obtain PEG-EPO, and the PEG modification can not only reduce the immunogenicity of the protein molecule EPO, but also reduce its allergenicity and increase its water solubility and stability. More importantly, PEG modification reduces the hydrolysis of EPO and prolongs the half-life in vivo. At the same time, when the present invention co-loads PEG-EPO, mesenchymal stem cells and islets for islet transplantation, it can greatly improve the biological efficacy of EPO and promote blood vessel reconstruction after transplantation.

Description

Composition, medicine and preparation method for loading PEG-EPO and mesenchymal stem cells

technical field

The invention belongs to the field of biopharmaceuticals, and in particular relates to a composition loaded with PEG-EPO and mesenchymal stem cells, a medicine and a preparation method thereof.

Background technique

Diabetes is one of the major diseases that seriously threaten human health. According to a report by the International Diabetes Federation (IDF), approximately 463 million adults aged 20-79 worldwide suffer from diabetes in 2019. Islet transplantation is the only treatment that can stably control blood sugar in a minimally invasive way. Erythropoietin (EPO), the master regulator of erythropoiesis, has been shown to promote angiogenesis. The main prerequisite for the survival of transplanted islets is rapid revascularization after transplantation; thus, EPO can improve the revascularization of transplanted islets.

Contents of the invention

The first object of the present invention is to provide a cell complex and its application in the preparation of medicines.

A cell complex, the active ingredients of which are protein linear PEG-EPO, pancreatic islet cells and mesenchymal stem cells.

The preparation method of the protein linear PEG-EPO in the aforementioned cell complex is as follows: the linear PEG is combined with the EPON terminal amino group and the lysine ε amino group through a stable covalent bond; after purification, a single-substituted PEGylated glycoprotein is obtained; Through synthesis, hydrophobicity, desalting, ion exchange, and ultrafiltration; the protein linear chain PEG-EPO is obtained.

More specifically, the preparation method of protein linear PEG-EPO is:

Linear PEG is combined with EPON terminal amino group and lysine ε amino group through a stable covalent bond; take EPO stock solution, add appropriate amount of pH 7.0±0.5 PBS solution, make EPO solution, and shake well;

Weigh PEG according to the mass ratio EPO:PEG of 0.07±0.03:1, add pH 3.0±0.5PBS solution to prepare a PEG solution, shake well; add the PEG solution to the EPO solution for reaction, and take a sample for SEC-HPLC detection after the reaction. Single substitution rate ≥ 40%;

After gradient elution with Buffer A and Buffer B at pH 7.0±1.0, the target peak is obtained, then desalted and eluted with Buffer C at pH 3.5±1.0, collected, and finally obtained by gradient elution with Buffer D at pH 3.5±1.0 For the target protein, the cut-off molecular weight is 10kd to obtain protein linear PEG-EPO.

The second object of the present invention is a water-soluble drug slow-release microsphere and its application in the preparation of drugs.

A water-soluble drug sustained-release microsphere, comprising:

The internal nuclear region, the internal nuclear region is composed of water-soluble drugs encapsulated in an isolated oil phase; the water-soluble drugs are protein linear PEG-EPO, pancreatic islet cells and mesenchymal stem cells;

The outer diffusion area is composed of medically acceptable polymer auxiliary materials.

The preparation method of the aforementioned water-soluble drug sustained-release microspheres, islet cells, mesenchymal stem cells and protein linear PEG-EPO are dissolved in a solution containing polyvinyl alcohol\carbomer\gum arabic\polyvinylpyrrolidone to form an internal water phase W1 ;

Add W1 to the acetone\ethyl acetate\dichloromethane organic solvent dissolved in PLGA and Span to form an oil phase\dispersion medium O;

Micro jet homogeneous treatment to form W1/O primary emulsion;

Then the primary emulsion is dispersed in polyvinyl alcohol solution containing Tween to form the external water phase W2, and the micro-jet homogeneous treatment is used to obtain the W1/O/W2 double emulsion;

The compound emulsion is injected into the polyvinyl alcohol solution and stirred, and the organic solvent is volatilized to form microspheres; the suspension liquid dispersed with the microspheres is centrifuged, filtered, washed, dried and collected to obtain the product.

The present invention uses end-modified PEG to modify EPO molecules to obtain PEG-EPO, and the PEG modification can not only reduce the immunogenicity of the protein molecule EPO, but also reduce its allergenicity and increase its water solubility and stability. More importantly, PEG modification reduces the hydrolysis of EPO and prolongs the half-life in vivo. At the same time, when the present invention co-loads PEG-EPO, mesenchymal stem cells and islets for islet transplantation, the biological efficacy of EPO can be greatly improved, and blood vessel reconstruction after transplantation can be promoted.

The microsphere preparation process of the present invention is stable and feasible, and the microsphere has round shape, smooth surface, good fluidity and uniform particle size distribution.

Sustained-release microspheres can prolong drug action time, avoid frequent injections, and improve patient compliance; blood drug concentration is stable, peak-to-valley fluctuations are small, and adverse reactions are reduced; liver first-pass effects of drugs are avoided; the therapeutic effect is good, and clinical preparations are reduced. , the workload of taking medicine and monitoring, the total treatment cost is low, the microsphere encapsulation material can be degraded, and it is non-toxic and harmless to human body.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Figure 1 shows the results of mouse islet cell staining to identify its activity.

Figure 2 shows the results of insulin release experiments to demonstrate islet cells.

Figure 3 is a diagram of the transplantation process of the mouse kidney capsule.

Figure 4 is a comparison between the control group and the experimental group (MSCs+PEG-EPO) diabetic mice maintaining normal blood sugar.

Figure 5 is a liquid chromatogram of the prepared protein linear PEG-EPO.

Figure 6 and Figure 7 are the electron microscope pictures of the prepared water-soluble drug slow-release microspheres.

Figure 8 shows the average erythrocyte HB content of mice after administration of the prepared water-soluble drug sustained-release microspheres with EPO and PEG-EPO.

Detailed ways

The present invention will be described in further detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only used to illustrate the present invention. The experimental methods used in the following examples, if there is no special instructions, the experimental methods that do not indicate the specific conditions in the examples, generally according to the conventional conditions, the materials, reagents, etc. used in the following examples, if there are no special instructions, all Commercially available.

In specific embodiments, steps, material selections, and numerical parameters that are not described in detail are conventional selections in the prior art, or any prior art disclosed in the prior art.

The animal experiment of embodiment 1 cell complex

(1) Isolation, purification and digestion of mouse islets

1) BALB/c mice (donors) were used to absorb 2.0 mL of collagenase solution with a 2.5 mL syringe to digest the pancreas.

1) Digest in a 37°C water bath until the pancreas appears milky or quicksand, then stop digestion;

2) Filter it into a 50mL centrifuge tube with a 450μm sieve equipped with a glass funnel;

3) Purify the islet cells with Histopaque solution and HBSS solution with densities of 1.119, 1.083 and 1.077.

(2) Islet identification, islet equivalent and purity calculation

1) Stain with DTZ for 3 minutes at room temperature, and calculate the number of islets.

2) Calculate the islet equivalent (IEQ) according to the method proposed by Lembert et al.

(3) Establishment of mouse-mouse islet allograft model and sample collection

1) Take the isolated mouse islet cell suspension and drop it into a counting dish, count under a microscope and count the islet equivalent.

2) 300 islets isolated from BALB/c mice (donor) were prepared for transplantation into the kidney capsule of a C57BL/6 diabetic mouse (recipient).

3) Put the transplanted kidney back in place and suture the wound.

(4) Preparation of PEG-EPO

PEG-EPO injection is made from PEG-EPO obtained after EPO undergoes PEG synthesis reaction and highly purified.

PEG-EPO is produced by combining straight-chain PEG with EPO N-terminal amino group and lysine ε amino group through stable covalent bonds. The main binding sites are N-terminal amino acid, 52-position and 45-position lysine, highly purified Afterwards, the single-substituted PEGylated glycoprotein is obtained, and the target protein linear PEG-EPO is obtained through synthesis, hydrophobicity, desalination, ion exchange, and ultrafiltration.

Specifically: the straight-chain PEG is combined with the N-terminal amino group of EPO and the ε-amino group of lysine through a stable covalent bond; take the EPO stock solution, add an appropriate amount of PBS solution with a pH of 7.0±0.5, prepare an EPO solution, and shake well;

Weigh PEG, add pH 3.0±0.5 PBS solution to prepare PEG solution, shake well; add PEG solution to EPO solution for reaction, after reaction, take samples for SEC-HPLC detection, single substitution rate ≥ 40%;

After gradient elution with Buffer A and Buffer B at pH 7.0±1.0, the target peak is obtained, then desalted and eluted with Buffer C at pH 3.5±1.0, collected, and finally eluted with Buffer D at pH 3.5±1.0 The target protein was obtained, and the cut-off molecular weight was 10kd to obtain protein linear PEG-EPO.

As shown in Fig. 5, it is a liquid chromatogram of protein linear PEG-EPO.

(5) Construction and performance of islet transplantation complex loaded with PEG-EPO and mesenchymal stem cells

Based on the optimized formulation process and the introduction of PEG-EPO, the islets and mesenchymal stem cells were finally wrapped into PEG-EPO, and the islets were transplanted after culturing at 37°C for 4 hours.

(6) Islet cell culture and insulin release experiment

1) The encapsulated islet cells were placed in RPMI 1640 medium and cultured for 2 hours under the stimulation of low glucose (concentration: 2.8mmol/L) and high glucose (concentration: 16.7mmol/L); The assay kit detects the insulin content in the supernatant.

(7) Detection of islet cell activity

The encapsulated islet cells were placed in 10 mL of RPMI 1640 medium, cultured in a 37°C CO ₂ incubator for 8 hours, and the activity of the islet cells was detected by the Calcein-AM/PI method.

(8) Experimental results

(1) After isolation and purification of mouse islets (as shown in part A in Figure 1, 50 times magnification), they were identified by DTZ (dithizone) staining (as shown in part B in Figure 1, the islet cells were stained with DTZ and showed Scarlet, oval or round, magnified 100 times). The activity of the islets was measured by Calcein-AM/PI staining method, and more than 90% of the islets showed green fluorescence, indicating that the islets had good activity (as shown in C and D in Figure 1, Calcein-AM/PI staining method Detect islet activity, green islet cells prove activity, 100 times magnification).

(2) The insulin release experiment of isolated mouse islet cells proved that the islet cells can secrete insulin, and are sensitive to high glucose stimulation, and can secrete more insulin. The insulin stimulation index reached 2.5 times on average (Figure 2, N=5).

(3) The islet transplantation complex loaded with PEG-EPO and mesenchymal stem cells was used for mouse renal capsule islet transplantation, as shown in Figure 3;

(4) Comparison of hypoglycemic function between the control group and the experimental group (mouse islet transplantation loaded with PEG-EPO and mesenchymal stem cells).

The results showed that syngeneic mouse islet transplantation loaded with PEG-EPO and mesenchymal stem cells could significantly reduce the blood sugar of diabetic C57BL/6 recipient mice to the normal range, and the time to maintain normal blood sugar was significantly longer than that of the control group (unloaded PEG-EPO and mouse islets of mesenchymal stem cells were used as grafts) (as shown in Figure 4).

Preparation and experiment of embodiment 2 water-soluble drug sustained-release microspheres

The protein linear PEG-EPO of embodiment 1

(1) Preparation of the inner aqueous phase

Islet cells, mesenchymal stem cells and protein straight-chain PEG-EPO are dissolved in a solution containing polyvinyl alcohol\carbomer\gum arabic\polyvinylpyrrolidone to form an internal aqueous phase W1;

(2) Preparation of oil phase

Add W1 to the acetone\ethyl acetate\dichloromethane organic solvent dissolved in PLGA and Span to form an oil phase\dispersion medium O;

(3) Preparation of primary emulsion

Micro jet homogeneous treatment to form W1/O primary emulsion;

(4) Preparation of double emulsion

Disperse W1/O primary emulsion in Tween-containing polyvinyl alcohol solution to form external water phase W2, and homogenize it with micro-jet to obtain W1/O/W2 double emulsion;

(5) Preparation of water-soluble drug sustained-release microspheres

The compound emulsion is injected into the polyvinyl alcohol solution and stirred, and the organic solvent is volatilized to form microspheres; the suspension liquid dispersed with the microspheres is centrifuged, filtered, washed, dried and collected to obtain the product.

The prepared water-soluble drug slow-release microspheres are shown in Figure 6 and Figure 7 under the electron microscope. It can be seen that the shape of the microspheres is round, the surface is smooth, the fluidity is good, and the particle size distribution is uniform.

In order to compare the sustained-release effect of the water-soluble drug sustained-release microspheres, the same experimental method as in Example 1 was used to detect the average erythrocyte HB content in mice, and the results are shown in FIG. 8 .

In Fig. 8, EPO is continuous administration, once a week; PEG-EPO is continuous administration, once every 2 weeks; Slow-release PEG-EPO (i.e. water-soluble drug slow-release microspheres) is continuous administration, every 2 weeks once. It can be seen that the water-soluble drug sustained-release microspheres can better maintain the average erythrocyte hemoglobin level when the administration frequency is the same as that of PEG-EPO.

The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or changes that do not deviate from the present invention shall be included in the present invention. within the scope of the technical program.

Claims (7)

1. The cell complex is characterized in that the active ingredients are protein linear PEG-EPO, islet cells and mesenchymal stem cells.

2. The cell complex of claim 1, wherein the protein linear PEG-EPO is prepared by the process of: the linear PEG is combined with the EPON terminal amino group and the lysine epsilon amino group through stable covalent bonds; the monosubstituted PEG glycoprotein is obtained after purification; by synthesis, hydrophobic, desalting, ion exchange, ultrafiltration; obtaining the protein linear PEG-EPO.

3. The preparation method of the protein linear PEG-EPO is characterized by comprising the following steps:

the linear PEG is combined with the EPON terminal amino group and the lysine epsilon amino group through stable covalent bonds; taking an EPO stock solution, adding a proper amount of PBS solution with pH of 7.0+/-0.5 to prepare an EPO solution, and shaking uniformly;

weighing PEG according to the mass ratio of EPO to PEG of 0.07+/-0.03:1, adding PBS solution with pH of 3.0+/-0.5 to prepare PEG solution, and shaking uniformly; adding PEG solution into EPO solution for reaction, and sampling SEC-HPLC detection after reaction, wherein the single substitution rate is more than or equal to 40%;

obtaining a target peak after the gradient elution of BufferA and BufferB with the pH value of 7.0+/-1.0, desalting and eluting with BufferC with the pH value of 3.5+/-1.0, collecting, finally obtaining target protein by the gradient elution of BufferD with the pH value of 3.5+/-1.0, and obtaining the protein linear PEG-EPO with the cut-off molecular weight of 10 kd.

4. Use of the cell complex of claim 1 or 2 in the manufacture of a medicament.

5. A water-soluble drug sustained release microsphere, comprising:

an inner core region, the inner core region consisting of an isolated oil phase encapsulating a water-soluble drug; the water-soluble medicine is protein linear PEG-EPO, islet cells and mesenchymal stem cells;

and the external diffusion area is composed of medically acceptable high polymer auxiliary materials.

6. The method for preparing the water-soluble medicine sustained-release microsphere according to claim 5, wherein islet cells, mesenchymal stem cells and protein linear PEG-EPO are dissolved in a solution containing polyvinyl alcohol/carbomer/acacia/polyvinylpyrrolidone to form an inner water phase W1;

adding W1 into an acetone/ethyl acetate/dichloromethane organic solvent in which PLGA and span are dissolved to form an oil phase/dispersion medium O;

carrying out micro-jet homogenization treatment to form W1/O primary emulsion;

then dispersing the primary emulsion in a polyvinyl alcohol solution containing tween to form an external water phase W2, and carrying out micro-jet homogenization treatment to obtain W1/O/W2 compound emulsion;

injecting the complex emulsion into a polyvinyl alcohol solution, stirring, and volatilizing an organic solvent to form microspheres; centrifuging the suspension with the dispersed microspheres, filtering, washing, drying and collecting the microspheres.

7. The use of the water-soluble drug-eluting microsphere as defined in claim 5 in the preparation of a medicament.