CN115887678A - Liposome drug delivery system and application thereof - Google Patents

Abstract

The invention discloses a liposome drug delivery system and application thereof. The liposome drug delivery system comprises a liposome and an active drug encapsulated in the liposome, wherein the raw material of the liposome at least contains phospholipid with maleimide groups, the surface of the liposome is modified with a functional targeting molecule, the target of the functional targeting molecule is the same as that of the active drug, and the functional targeting molecule has sulfydryl capable of being coupled with the maleimide groups. The liposome drug delivery system can realize accurate targeted delivery of the active drug, and greatly improve the treatment effect of the active drug.

Description

Liposome drug delivery system and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a liposome drug delivery system and application thereof.

Background

Acute Myeloid Leukemia (AML), the leading type of acute leukemia, is on the rise in morbidity and mortality in our country. At present, the domestic clinical treatment schemes for AML mainly comprise two types: 1. hematopoietic stem cell transplantation, which is safe and effective, but has high treatment cost and limited matching bone marrow sources, which prevent the clinical popularization of hematopoietic stem cell transplantation; 2. chemotherapy, the treatment cost of chemotherapy is relatively low, but the chemotherapy drug has weak targeting property, so that the side effect of chemotherapy is large.

In order to solve the above problems, the prior art improves the targeting property of the drug by the following means:

1. the liposome drug delivery System is adopted to wrap chemotherapeutic drugs, gene drugs, diagnostic drugs (radioactive, paramagnetic and other substances) and the like, when the liposome drug delivery System wrapped with the drugs enters a human body, the liposome drug delivery System is identified as an invader, at the moment, the human body can start an immune mechanism, and a Mononuclear Phagocyte System (MPS) is utilized to phagocytize the liposome drug delivery System, so that the drugs wrapped in the liposome drug delivery System are concentrated in tissues such as liver, spleen, lung, bone marrow and the like in a targeted manner, and the wrapped drugs are released at the concentration position to play a therapeutic function.

For example, vyxeos (CPX-351), which is obtained by liposome-encapsulated cytarabine (Ara-C) and Daunorubicin (DNR) using a molar ratio of Ara-C to DNR of 5:1, liposomes are composed of 70mol% distearoyl phosphatidylcholine (DSPC), 20mol% distearoyl phosphatidyl glycerol sodium salt (DSPG) and 10mol% cholesterol (Chol).

However, the liposome delivery system can be targeted because of the active delivery of the human immune system, and the liposome delivery system exerts its passive targeting potential and cannot selectively target spinal cord and AML tumor cells, which results in insufficient targeting.

2. The drug or prodrug is directly coupled with functional targeting molecules, for example, chinese patent with publication No. CN1795009B discloses an immunoconjugate of anti-CD33 antibody and maytansinoid, which utilizes the specific combination of anti-CD33 antibody and CD33 to deliver the maytansinoid to AML tumor cells in a targeted manner; this is because CD33 is a myeloid cell differentiation antigen, which is highly expressed in more than 90% of AML patient tumor cells, while the expression level in hematopoietic stem cells, mature granulocytes, and other tissues is very low; when AML therapeutics are conjugated to anti-CD33 antibodies, the anti-CD33 antibodies will target the maytansinoids to AML tumor cells.

However, the coupling of the anti-CD33 antibody to the AML therapeutic drug must be via a linking group such as a disulfide group, a thioether group, an acid labile group, a photolabile group, a peptidase labile group, an esterase labile group, etc., but not all AML therapeutic drugs have the above linking group (such as cytarabine, daunorubicin, etc.), or even if the above linking group is present or is artificially added to the molecular structure of the AML therapeutic drug, it is unknown whether the coupling of the linking group to the anti-CD33 antibody or the artificial addition of the linking group affects the properties of the drug.

Disclosure of Invention

The invention aims to provide a liposome drug delivery system and application thereof, wherein the liposome drug delivery system can realize active targeted delivery of encapsulated active drugs and improve the treatment effect of the active drugs.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

a liposome drug delivery system comprises a liposome and an active drug encapsulated in the liposome, wherein the surface of the liposome is provided with a maleimide group, a sulfhydryl group or a carboxyl group, at least part of the maleimide group, the sulfhydryl group or the carboxyl group is coupled with a functional targeting molecule, and the target of the functional targeting molecule is the same as that of the active drug.

The active drug is encapsulated in the liposome, and then the surface of the liposome is modified, so that on one hand, the functional targeting molecule is not directly connected with the active drug, the function of the active drug is not influenced, the type of the delivered active drug is not required, any active drug can be encapsulated and delivered, and the application range is wide; on the other hand, compared with screening active drugs having a linking group capable of coupling with a functional targeting molecule, or artificially adding a linking group capable of coupling with a functional targeting molecule on an active drug, it is easier to arrange a linking group (such as a maleimide group, a thiol group, or a carboxyl group) capable of coupling with a functional targeting molecule in a liposome; therefore, under the dual targeting action of the passive targeting of the liposome and the active targeting of the functional targeting molecule, the liposome drug delivery system can realize the accurate targeted delivery of the active drug, and greatly improve the treatment effect of the active drug.

The invention has no special requirements on the types of active drugs and functional targeting molecules, as long as the targets of the functional targeting molecules and the active drugs are ensured to be the same. However, in view of the passive targeting potential of the liposomes themselves, the functional targeting molecules and active agents described above are preferably targeted to tissues such as liver, spleen, lung and bone marrow.

As a specific example of the embodiment, in the above liposome delivery system, the active drug is an acute myelogenous leukemia therapeutic drug, and the functional targeting molecule includes at least one of a protein and a polypeptide. The thiol groups (derived from cysteine) contained in proteins and polypeptides can be conveniently coupled to maleimide groups, thiol groups or carboxyl groups on the surface of liposomes.

As an example of a specific embodiment, in the above liposome delivery system, the functional targeting molecule is an anti-CD33 antibody, and the C-terminal of the heavy chain of the anti-CD33 antibody contains a cysteine providing a thiol group coupled to a maleimide group, a thiol group or a carboxyl group. Since the antigen recognition region of the antibody is at the N-terminus of the heavy chain and the light chain, in order to prevent the coupling of the antibody to the liposome from affecting the recognition of the antibody to the antigen, the C-terminus of the heavy chain of the anti-CD33 antibody of the present invention contains a cysteine to provide a thiol group coupled to a maleimide group, a thiol group, or a carboxyl group.

More preferably, in the liposome delivery system, the light chain of the anti-CD33 antibody has an amino acid sequence shown in SEQ ID No.1, and the heavy chain of the anti-CD33 antibody has an amino acid sequence shown in SEQ ID No. 2.

The anti-CD33 antibody is obtained by performing humanized modification on a murine CD33 monoclonal antibody M195, after the humanized modification, the anti-CD33 antibody not only has reduced immunogenicity, prolonged half-life and reduced ability to trigger complement effect, but also has a cysteine introduced into the C-terminal end of the heavy chain, and is connected with a liposome through the C-terminal end of the heavy chain, namely, the Fc fragment of the antibody is anchored with the liposome, while the Fab fragment of the antibody is not influenced at all, so that the recognition function of CD33 of AML tumor cells can be retained to the maximum extent.

Because the antibody is a macromolecule with a certain nano scale, the particle size of the liposome is 100-200 nm, and the steric hindrance is larger, in order to couple the antibody on the surface of the liposome as much as possible to improve the active targeting property, preferably, in the liposome drug delivery system, the maleimide group, the sulfhydryl group or the carboxyl group is connected with the liposome through a polyethylene glycol chain. The polyethylene glycol chain can bring maleimide groups, sulfydryl or carboxyl to a position far away from the surface of the liposome, so that the steric hindrance of the antibody is reduced, and the coupling amount of the antibody is increased.

Moreover, the polar polyethylene glycol group can enhance the hydrophilicity of a liposome membrane, reduce the interaction between opsonin in blood and the liposome membrane, and reduce the rapid phagocytosis or uptake of the liposome by a mononuclear macrophage system, thereby prolonging the in vivo circulation time of the liposome, facilitating the slow release of an active medicament and improving the curative effect of the liposome.

More preferably, in the liposome delivery system, the raw material of the liposome at least contains maleimide-polyethylene glycol-distearoylphosphatidylethanolamine (Mal-PEG-DSPE). The Mal-PEG-DSPE has maleimide group modification and polyethylene glycol group modification, and achieves two purposes at one stroke; and compared with other groups (sulfydryl or carboxyl), the coupling of the maleimide group and the sulfydryl on the antibody is more specific and convenient.

Preferably, in the liposome delivery system, the molar ratio of the functional targeting molecule to the maleimide group (i.e., maleimide group-polyethylene glycol-distearoylphosphatidylethanolamine) is 1: (36-200).

Due to the large steric hindrance of the antibody, it is difficult to perform a 1. Tests show that the administration molar ratio of the maleimide group-polyethylene glycol-distearoyl phosphatidyl ethanolamine in the antibody/liposome is 1/36.89, which is the maximum ratio of antibody modification, and if the dosage of the antibody is increased, the antibody can not be further modified to the surface of the liposome.

However, since the antibody recognition target protein needs to overcome a certain steric hindrance, if the antibody density on the liposome surface is too high, the steric hindrance of the antibody is too large, which is not favorable for the antibody to function; if the antibody density on the liposome surface is too low, the probability of the antibody contacting the target protein becomes low, and the targeting efficiency of the antibody is affected. After the maximum antibody modification amount is obtained by screening, the optimal antibody modification ratio is determined by the targeting efficiency of the antibody coupled liposome, and the prepared liposome has the best targeting efficiency when the dosage ratio (molar ratio) of the antibody/maleimide group-polyethylene glycol-distearoyl phosphatidyl ethanolamine is 1/147.56.

As a further preferred, in the above liposome delivery system, the raw materials of the liposome comprise, in mole percent: 40-60mol% hydrogenated soy phosphatidylcholine, 35-55mol% cholesterol, 0-5mol% polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE) and 0-5 (0 excluded) mol% maleimido-polyethylene glycol-distearoylphosphatidylethanolamine.

The invention selects hydrogenated soybean phosphatidylcholine as phospholipid material, the phase transition temperature of the hydrogenated soybean phosphatidylcholine is 50 ℃, and the prepared liposome still has good stability at normal physiological temperature. The mPEG-DSPE and the Mal-PEG-DSPE can PEG the prepared liposome, thereby prolonging the in vivo circulation time of the liposome, and is easy to artificially synthesize in a large quantity, high in purity and low in cost.

Preferably, the length of the polyethylene glycol chain in the maleimido-polyethylene glycol-distearoylphosphatidylethanolamine is greater than the length of the polyethylene glycol chain in the polyethylene glycol-distearoylphosphatidylethanolamine. For example, mPEG is adopted as mPEG-DSPE ₂₀₀₀ -DSPE, mal-PEG-DSPE adopted Mal-PEG ₃₄₀₀ DSPE, thus can further reduce the steric hindrance of the antibody connected with Mal-PEG-DSPE, and facilitate the recognition of the antibody and target protein.

Most preferably, in the liposome delivery system described above, the liposome comprises, in mole percent, the following: 50mol% hydrogenated soy phosphatidylcholine, 45mol% cholesterol, 2mol% polyethylene glycol-distearoylphosphatidylethanolamine and 3mol% maleimido-polyethylene glycol-distearoylphosphatidylethanolamine.

The invention also provides application of the liposome drug delivery system in preparation of a preparation for treating acute myeloid leukemia, wherein the functional targeting molecule is an anti-CD33 antibody, the active drug is a mixture of cytarabine and daunorubicin, and the molar ratio of the cytarabine to the daunorubicin is 5.

Compared with the prior art, the invention has the beneficial effects that:

(1) The invention encapsulates the active drug in the liposome, and then carries out surface modification on the liposome, on one hand, the functional targeting molecule is not directly connected with the active drug, which can not affect the function of the active drug, and has no requirement on the type of the delivered active drug, any active drug can be encapsulated and delivered, and the application range is wide; on the other hand, compared with the screening of active drugs with connecting groups capable of being coupled with functional targeting molecules or the artificial addition of connecting groups capable of being coupled with functional targeting molecules on the active drugs, the arrangement of the connecting groups capable of being coupled with the functional targeting molecules in the liposome is easier; under the dual targeting action of the passive targeting of the liposome and the active targeting of the functional targeting molecule, the liposome drug delivery system can realize the accurate targeted delivery of the active drug, and greatly improve the treatment effect of the active drug.

(2) The anti-CD33 antibody adopted by the invention not only has reduced immunogenicity, prolonged half-life and reduced ability of triggering complement effect, but also introduces a cysteine at the C terminal of the heavy chain, and is connected with the liposome by the C terminal of the heavy chain, namely, the Fc fragment of the antibody is anchored with the liposome, and the Fab fragment of the antibody is not influenced at all, thus the recognition function of the CD33 of AML tumor cells can be retained to the maximum extent.

(3) In the invention, the maleimide group is connected with the liposome through the polyethylene glycol chain, and the polyethylene glycol chain can bring the maleimide group to a position far away from the surface of the liposome, thereby reducing the steric hindrance of the antibody and improving the coupling amount of the antibody on the liposome; moreover, the polar polyethylene glycol group can enhance the hydrophilicity of a liposome membrane, reduce the interaction between opsonin in blood and the liposome membrane, and reduce the rapid phagocytosis or uptake of the liposome by a mononuclear macrophage system, thereby prolonging the in vivo circulation time of the liposome, facilitating the slow release of an active medicament and improving the curative effect of the liposome.

(4) In the invention, the liposome raw material contains Mal-PEG-DSPE and mPEG-DSPE, wherein the mPEG-DSPE and Mal-PEG-DSPE can both pegylate the prepared liposome, thereby prolonging the in vivo circulation time of the liposome, and being easy to artificially synthesize in large quantity, high in purity and low in cost; and Mal-PEG-DSPE has maleimide group modification and polyethylene glycol group modification, thereby achieving two purposes.

(5) In the present invention, the length of the polyethylene glycol chain in the maleimide-polyethylene glycol-distearoylphosphatidylethanolamine is greater than the length of the polyethylene glycol chain in the polyethylene glycol-distearoylphosphatidylethanolamine. For example, mPEG is adopted as mPEG-DSPE ₂₀₀₀ -DSPE, mal-PEG-DSPE adopts Mal-PEG ₃₄₀₀ DSPE, thus can further reduce the steric hindrance of the antibody connected with Mal-PEG-DSPE, and facilitate the recognition of the antibody and target protein.

Drawings

FIG. 1 is a diagram showing the steps of humanizing a murine M195 antibody to obtain a humanized anti-CD33 antibody;

FIG. 2 is an elution profile of humanized anti-CD33 antibody;

wherein the abscissa is elution time, and the ordinate is ultraviolet absorbance at 280 nm;

FIG. 3 is a SDS-PAGE band diagram of the humanized anti-CD33 antibody;

wherein Marker represents standard molecular weight protein, no transfer represents cell culture supernatant without transfection plasmid, super represents cell culture supernatant, through flow represents transudate, water represents Elution waste liquid, elution represents eluent, and dialysis represents protein component after dialysis;

FIG. 4 shows HPLC-MS characterization purity and molecular weight of humanized anti-CD33 antibody;

FIG. 5 is a SDS-PAGE band diagram of the liposomal delivery system of the invention;

wherein Marker represents a protein standard, antibody represents an Antibody, ab-lip @ Ara-C/DNR represents a liposome having an Ara-C/DNR mixture encapsulated therein and having an Antibody coupled to the surface thereof, lip @ Ara-C/DNR represents a liposome having an Ara-C/DNR mixture encapsulated therein, the same applies hereinafter;

FIG. 6 is a graph of the results of a particle size analysis of a liposomal delivery system of the present invention;

wherein Size (d.nm) represents Size (particle Size, nm) and Intensity (Percent) represents Intensity;

FIG. 7 is a standard curve fitted to quantify Ara-C by HPLC;

wherein Concentration (mg/mL) indicates the Concentration (mg/mL), and Peak area indicates the Peak; the same applies below;

FIG. 8 is a standard curve fitted to quantify DNR by HPLC;

FIG. 9 is a HPLC analysis of Ara-C, DNR and liposome delivery systems;

wherein Time (min) represents retention Time (min), UV Absorbance at 254nm (mAU) represents UV Absorbance at 254nm (milliabsorbance units);

FIG. 10 is a HPLC analysis profile in a liposome delivery system at different Ara-C/DNR entrapment ratios;

wherein, ara-C/DNR dosage ratio represents the dosage ratio of Ara-C/DNR, and Mass concentration ratio of Ara-C/DNR represents the Mass concentration ratio of Ara-C to DNR;

FIG. 11 is a flow cytometric map of the entry of a liposomal drug delivery system into HL-60 cells at different antibody/liposome coupling ratios;

FIG. 12 is a flow cytometric map of the entry of a liposomal delivery system into MOLM-13 cells at different antibody/liposome coupling ratios;

figure 13 is a flow cytometric map of liposomal delivery system into SUP-B15 cells at different antibody/liposome coupling ratios;

FIG. 14 is the mean fluorescence intensity for different cell uptake of liposomal drug delivery systems with different antibody/liposome coupling ratios;

FIG. 15 is the median fluorescence intensity for different cell uptake of liposomal drug delivery systems with different antibody/liposome coupling ratios;

FIG. 16 is the number of positive cells for different cell uptake of liposomal delivery systems with different antibody/liposome coupling ratios;

FIG. 17 is a graph showing the test of the surface recognition ability of the liposome delivery system Ab-lip @ Ara-C/DNR of the present invention on MOLM-13 cells;

wherein DNR intensity represents DNR signal intensity, count represents cell number, and Control represents Control; the same applies below;

FIG. 18 is a graph showing the test of the surface recognition ability of Ab-lip @ Ara-C/DNR of the liposome delivery system of the present invention on HL-7702 cells;

FIG. 19 is the early apoptosis of MOLM-13 cells under Ab-lip @ Ara-C/DNR treatment with the liposomal delivery system of the present invention;

wherein Annexin-FITC intensity represents Annexin-FITC signal intensity, the same applies below;

FIG. 20 is an early apoptotic picture of HL-7702 cells treated with the liposomal delivery system Ab-lip @ Ara-C/DNR of the present invention;

FIG. 21 shows the 24h toxicity evaluation results of the liposome delivery system of the present invention Ab-Lip @ Ara-C/DNR on MOLM-13 cells;

wherein Log [ μ M ]/Ara-C represents the Log of concentration of Ara-C, cell Viability (%) represents Cell Viability (percentage), the same applies below;

FIG. 22 shows the results of 24h toxicity evaluation of Ara-C on MOLM-13 cells;

wherein, IC ₅₀ Half inhibitory concentration; the same applies below;

FIG. 23 shows the results of 24h toxicity evaluation of DNR on MOLM-13 cells;

wherein Log [ μ M ]/DNR represents the concentration logarithm of DNR;

FIG. 24 is a statistical result of synergistic therapeutic index of Ara-C and DNR administered to MOLM-13 cells for 24 h;

wherein Fa represents cell growth rate, CI represents combination index, i.e. synergistic therapeutic index, CI <1 represents synergistic effect, CI =1 represents additive effect, CI >1 represents antagonistic effect; the same applies below;

FIG. 25 is the results of 72h toxicity evaluation of Ab-lip @ Ara-C/DNR for MOLM-13 cells for the liposomal delivery system of the present invention;

FIG. 26 shows the results of 72h toxicity evaluation of Ara-C on MOLM-13 cells;

FIG. 27 is the results of 72h toxicity evaluation of DNR on MOLM-13 cells;

FIG. 28 is a statistical result of synergistic therapeutic index of Ara-C and DNR administered to MOLM-13 cells for 72 h;

FIG. 29 is a flow chart of treatment and bioluminescence detection of mice using the liposome delivery system of the present invention Ab-lip @ Ara-C/DNR;

wherein 3.8million MOLM-13-luc indicates the number of 3.8million MOLM-13-luc cells injected into mice, AML transplant indicates AML cell transplantation, grouping indicates growth (of AML cells), 2.5;

FIG. 30 is a fluorescent detection plot of mice at different treatment times for the liposome delivery system Ab-lip @ Ara-C/DNR;

wherein Ventral represents the abdomen of the mouse, dorsal represents the back of the mouse, and the same applies below;

FIG. 31 is a graph of fluorescence intensity in the back of mice treated with the liposomal delivery system of the present invention Ab-lip @ Ara-C/DNR;

wherein, days represents the treatment Days, avg Radiance [ p/s/cm [ ] ² /sr]Represents the fluorescence brightness, saline represents the Saline group, the same applies below;

FIG. 32 is a graph of fluorescence intensity measurements of the abdomen of mice after treatment with the liposomal delivery system of the invention Ab-lip @ Ara-C/DNR;

FIG. 33 is a flow chart of the treatment and survival detection of mice using the liposome delivery system Ab-lip @ Ara-C/DNR of the present invention;

wherein, the observing every day represents the detection;

FIG. 34 is a graph of the change in body weight of mice after treatment with the liposomal delivery system of the invention Ab-lip @ Ara-C/DNR;

wherein Relative body weight (%) indicates Relative body weight (percentage);

FIG. 35 is a graph showing the survival time of mice treated with the liposome delivery system Ab-lip @ Ara-C/DNR of the present invention;

wherein subjects at risk represents the number of surviving test subjects, and Percent (%) represents the survival rate;

FIG. 36 is a graph showing the effect of Ab-lip @ Ara-C/DNR of the liposome delivery system of the present invention on the survival rate of mice;

wherein Percent increase in life span (% ILS) represents the growth rate of life cycle;

FIG. 37 is a confocal microscope observation of TUNEL staining of mouse bone marrow cells after treatment of mice with the liposome delivery system Ab-lip @ Ara-C/DNR of the present invention;

FIG. 38 is a graph of the effect of Ab-lip @ Ara-C/DNR treatment of mice with the liposomal delivery system of the present invention on TUNEL positive cell count of mouse bone marrow cells;

wherein, positive areas (pixels) represent Positive areas (pixels), the same applies below;

FIG. 39 is the result of confocal microscope observation of immunofluorescence of mouse myeloid cell CD33 antibody after treatment of mice with the liposome delivery system Ab-lip @ Ara-C/DNR of the present invention;

FIG. 40 is a graph showing the effect of Ab-lip @ Ara-C/DNR treatment of mice with the liposomal delivery system of the present invention on the CD 33-positive cell count of mouse bone marrow cells;

FIG. 41 is the results of Rui's-Giemsa staining of mouse bone marrow cells following treatment of mice with the liposomal delivery system Ab-lip @ Ara-C/DNR of the present invention;

wherein Blank represents a Blank control group; the same applies below;

FIG. 42 is a graph of the effect of Ab-lip @ Ara-C/DNR treatment of mice with the liposomal delivery system of the present invention on the number of leukocytes in the blood of mice;

among them, white blood cell count (10) ⁹ L) represents the number of leukocytes (10) ⁹ /L)；

FIG. 43 is a graph of the effect of Ab-lip @ Ara-C/DNR treatment of mice with the liposomal delivery system of the present invention on the number of lymphocytes in the blood of the mice;

among them, the lymphoma count (10) ⁹ L) number of lymphocytes (10) ⁹ /L)；

FIG. 44 is a graph of the effect of Ab-lip @ Ara-C/DNR treatment of mice with the liposomal delivery system of the present invention on the number of monocytes in the blood of the mice;

wherein, monocyte count (10) ⁹ /L) represents the number of monocytes (10) ⁹ L); the same applies below;

FIG. 45 is a graph of the effect of Ab-lip @ Ara-C/DNR treatment of mice with the liposomal delivery system of the present invention on the number of neutrophils in the blood of the mice;

wherein, neutrophil count (10) ⁹ /L) represents the number of neutrophils (10) ⁹ /L)；

FIG. 46 is a graph of the effect of Ab-lip @ Ara-C/DNR treatment of mice with the liposomal delivery system of the present invention on the proportion of lymphocytes in blood;

wherein Lymphocyte percentage (%) represents the proportion (percentage) of lymphocytes to leukocytes;

FIG. 47 is a graph of the effect of Ab-lip @ Ara-C/DNR treatment of mice with the liposomal delivery system of the present invention on the proportion of monocytes in the blood of the mice;

wherein, monocyte percent (%) indicates the proportion (percentage) of monocytes to leukocytes

FIG. 48 is a graph showing the effect of Ab-lip @ Ara-C/DNR treatment of mice with the liposomal delivery system of the present invention on the proportion of neutrophils in the blood of the mice;

wherein neutrophile percent (%) represents the proportion (percentage) of neutrophils in leukocytes;

FIG. 49 is a graph of the toxicity evaluation of Ab-lip @ Ara-C/DNR treated mice on mouse heart tissue in a liposome delivery system of the present invention;

FIG. 50 is a graph showing the toxicity evaluation of Ab-lip @ Ara-C/DNR-treated mice on mouse liver in the liposome delivery system of the present invention;

FIG. 51 is a graph showing the toxicity evaluation of Ab-lip @ Ara-C/DNR-treated mice to the spleen of mice in the liposome delivery system of the present invention;

FIG. 52 is a graph showing toxicity evaluation of Ab-lip @ Ara-C/DNR-treated mice to mice lungs by the liposome delivery system of the present invention;

FIG. 53 is a graph showing the toxicity evaluation of Ab-lip @ Ara-C/DNR-treated mice on mouse kidneys, in a liposome delivery system of the present invention;

FIG. 54 is a graph showing toxicity evaluation of Ab-lip @ Ara-C/DNR-treated mice on mouse brain tissue in the liposome delivery system of the present invention.

Detailed Description

The technical scheme of the invention is further explained in detail by combining the attached drawings and the detailed description.

EXAMPLE 1 preparation of Liposomal delivery System

1. Preparation and characterization of anti-CD33 antibodies

According to the experimental requirements, a humanized modification scheme of the anti-CD33 antibody is established, pCDNA3.1+ eukaryotic expression vectors respectively carrying humanized anti-CD33 antibody light chain and heavy chain coding sequences are successfully constructed by means of molecular cloning, then the expression vectors of the antibody are amplified by competent DH5 alpha to obtain a large amount of endotoxin-free plasmid vectors, and then the plasmids are introduced into a eukaryotic cell expression system through a transfection reagent to express the antibody.

The method comprises the following specific steps:

(1) Humanization engineering protocols for M195 antibodies

The M195 antibody is a murine IgG2 antibody obtained by immunizing a mouse with a CD33 antigen, and can specifically recognize the surface antigen CD33 of human acute myeloid leukemia cells; in the early 90 s of the last century, researchers of Caron et al have attempted to use this antibody in the clinical treatment of AML. Although phase I clinical trial results showed that the M195 antibody, when used in acute promyelocytic patients after complete remission, was able to significantly reduce leukemic cells of peripheral blood and myeloid species and did not elicit toxicity beyond hematopoietic tissues, the incidence of human anti-mouse antibody response (HAMA) was as high as 37% due to the murine origin of M195.

To reduce the immunogenicity of the M195 antibody, this example first humanized the M195 antibody: the variable region amino acid sequences on the light and heavy chains of the M195 antibody were spliced to the constant region sequences on the light and heavy chains, respectively, of human IgG. It is well known that human IgG comprises 4 subtypes, igG1, igG2, igG3, igG4; the structures of the subtypes are approximately the same, but there are also subtle differences that also make them slightly different in function and stability. Wherein, igG1 is the most subtype in human body, can combine with various Fc receptors, activate complement and initiate ADCC, and has the strongest opsonization; igG2 has the weakest affinity for Fc receptors, the weaker ability to cause ADCC and opsonization, but the higher ability to activate complement than IgG4, and is relatively stable to protease cleavage, thus having the longest blood circulation time; the IgG3 has the strongest binding capacity with an Fc receptor and the strongest capacity of inducing ADCC; igG4 binds weakly to Fc receptors and cannot mediate complement effects and ADCC, but the half-life is the shortest of the four subtypes. Considering the characteristics of each IgG subtype, human IgG2 with the longest half-life and relatively weak ability to trigger complement efficacy was selected as the antibody constant region framework.

It is well known that the variable regions of antibody heavy and light chains do not completely bind to epitopes on the antigen, and that only the portions called Complementarity Determining Regions (CDRs), also called antigen recognition regions, bind to epitopes on the antigen. Therefore, in the process of humanizing the M195 antibody, the amino acid sequences of the variable regions of both the light and heavy chains that are not CDRs but belong to the framework region of the antibody are humanized by substitution with a reference IgG 2.

To further reduce the complement effect that humanized M195 antibodies can elicit in humans, we mutated the key sites of complement effect on the IgG2 constant region with reference to IgG4, i.e.: sequentially mutating H at 268, V at 309, A at 330 and P at 331 on an IgG2 heavy chain into Q268, L309, S330 and S331 of IgG4; the obtained hybrid IgG2m4 has greatly reduced binding capacity to various Fc receptors compared with IgG 2.

Finally, since the conjugation between the liposome and the functional targeting molecule in the liposome delivery system of the present invention is based on the electron coordination between the thiol group on the cysteine side chain in the protein or polypeptide and the nanocrystal (i.e. liposome) surface, and IgG2m4 itself has no free thiol group, we add a cysteine (Cys, C) to the C-terminus of the heavy chain of IgG2m4, so that the antibody can be conjugated to the liposome through the C-terminus of the heavy chain, and maximally ensure that the recognition region of the antibody is not affected by steric hindrance.

As shown in FIG. 1, through the above four steps, we finally designed and obtained the amino acid sequence of the humanized M195 antibody of IgG2 hybrid IgG4 with cysteine at the end of the heavy chain, i.e., finally obtained the anti-CD33 antibody required by the present invention, wherein the amino acid sequence of the antibody light chain is shown as SEQ ID No.1, and the amino acid sequence of the antibody heavy chain is shown as SEQ ID No. 2.

(2) Preparation and characterization of anti-CD33 antibodies

1) Enzyme digestion reaction

The synthesis of antibody heavy chain DNA vector, antibody light chain DNA vector, and XbaI and HindIII (New england biolabs) double digestion of the synthesized antibody heavy chain DNA vector, light chain vector and pCDNA3.1 vector were delegated to obtain the desired fragments. For the cleavage reaction, about 2. Mu.g of DNA was taken, 1. Mu.L each of restriction enzymes XbaI and HindIII and 10XNEB2.1 cleavage buffer (New england biolabs) were added, and finally an appropriate amount of double distilled water was added to make the total volume to 20. Mu.L. Incubate at 37 ℃ for 1.5h.

2) Agarose gel electrophoresis

Agarose gel electrophoresis is used to separate DNA molecules according to fragment size. To prepare the agarose gel, a 1% agarose solution (Biowest) was prepared first with about 30mL of 1 XTAE buffer, shaken and then heated in a microwave oven until completely dissolved. When the mixture is cooled to below 60 ℃,30 mu L of 0.5mg/mL ethidium bromide (Solarbio) is added, mixed evenly and poured into a rubber plate, a proper comb is inserted, and the mixture is kept stand at room temperature until the mixture is completely solidified. During electrophoresis experiments, the gel is placed in an electrophoresis tank, 1 XTAE buffer solution is added, and a comb is pulled out. And sucking the DNA sample mixed with the loading buffer solution in advance by using a pipettor, and adding the DNA sample into the spot sample hole. The voltage was adjusted to 80V and the electrophoretic separation time was about 40 minutes. After the electrophoretic separation is finished, the gel is taken out and placed in an ultraviolet transilluminator, and a DNA band with fluorescence is observed.

The formulation of 50 XTAE buffer is: 242g Tris,57.1mL glacial acetic acid, 0.5mol/L EDTA, pH 8.0;

the formulation of 6 × loading buffer is: 0.25% of bromophenol blue, 0.25% of xylene cyanide and 30% of glycerol.

3) DNA agarose gel recovery

The experimental procedure used a DNA gel recovery kit (Bio-engineering, inc.). The agarose gel containing the desired fragment was first cut out in an ultraviolet transilluminator and placed in a 1.5mL centrifuge tube. After weighing, add the equivalent of 3 gel volumes of QG buffer and heat to 50oC until the gel is completely dissolved. The obtained solution containing the target DNA fragment was then applied to an adsorption column, centrifuged at 13000rpm for 1 minute, and the liquid was discarded. After two-step PE buffer washing, the DNA fragment of interest was eluted by centrifugation for 1 minute with about 50. Mu.L of elution buffer (10 mM Tris.HCl, ph8.0).

4) DNA fragment ligation reaction

Carrying out enzyme digestion and purification on the obtained antibody DNA fragment (the nucleotide sequence of the antibody light chain DNA is shown in SEQ ID No.3, and the nucleotide sequence of the antibody heavy chain DNA is shown in SEQ ID No. 4) and the pCDNA3.1 vector in a molar ratio of 7:1, then 1. Mu.L of T4 ligase and 2. Mu.L of 10xT4 ligase buffer (New england biolabs) were added, and finally an appropriate volume of double distilled water was added to 20. Mu.L, and the mixture was incubated at 16 ℃ for about 8 hours.

5) Preparation of DH5 alpha competence

In principle, the competence is prepared by using CaCl ₂ The solution allows for an increased permeability of the DH 5. Alpha. Strain so that macromolecules such as DNA can enter the interior of the bacterium.

The DH 5. Alpha. Strain was first streaked on LB solid plates and cultured overnight at 37 ℃. Subsequently, a single colony of the bacterium was picked and transferred to 2mL of LB liquidThe medium was cultured in suspension at 37 ℃ and 220rpm for about 8 hours. The bacterial liquid was mixed according to a ratio of about 1:100 was diluted with LB medium, incubated at 37 ℃ for about 2-3 hours at 220rpm, and the absorbance (OD 600) at 600nm of the wavelength was monitored to about 0.4-0.6 (the bacteria were in logarithmic growth phase). The obtained cell suspension was placed on ice for about 20 minutes, and then centrifuged at 300rpm at 4 ℃ for 10 minutes to remove the supernatant and retain the cell pellet. 200mL of precooled 0.1M CaCl ₂ The solution was resuspended in the cells and allowed to stand on ice for about 30 minutes. Then centrifuged again at 3000rpm for 10 minutes at 4 ℃. The supernatant was removed, the cells were retained and finally the bacteria were resuspended in 50mL of pre-cooled 0.1M CaCl ₂ 10% glycerol solution. Subpackaging and storing at-80 ℃.

6) Transformation of DH 5. Alpha. Competence

Transformation of DH 5. Alpha. Competes with E.coli by introducing the DNA vector into E.coli by heat shock to amplify the DNA vector.

First, a tube of approximately 200. Mu.L of DH 5. Alpha. Was taken out and thawed on ice, and approximately 50ng of DNA was added, mixed well and placed on ice for 30 minutes. The mixture of competent and DNA was then placed in a 42 ℃ water bath for 60 seconds and quickly replaced on ice for about 2 minutes. Approximately 800. Mu.L of LB medium was added to the susceptibilities after the heat shock, and incubated at 37 ℃ and 220rpm for approximately 1 hour, which was intended to allow adequate resuscitation of the bacteria. Finally, the recovered bacteria are coated on an LB solid culture plate containing antibiotics corresponding to the DNA carrier resistance. The plates were inverted and incubated at 37 ℃ for about 12 hours.

7) DNA plasmid preparation

The method for extracting DNA plasmids from bacteria mainly comprises three basic steps: culturing the bacteria such that the plasmid is amplified; collecting and cracking thalli; and (3) separating and purifying plasmid DNA.

For the extraction of DNA plasmid molecules, a plasmid miniprep kit (Biotechnology engineering Co., ltd.) was used. First, 5-10mL of the bacterial suspension was cultured overnight at 37 ℃. The bacterial liquid was centrifuged at 8000rpm for 10 minutes in a centrifuge tube, and the supernatant was discarded to retain the cells. To the cells was added 250. Mu.L of RNAse A containing solution I (5 mM glucose, 25mM Tris pH8.0, 10mM EDTA) and the bacteria were resuspended thoroughly. Then 250. Mu.L of solution II (0.2mM NaOH,10g/L SDS) was added thereto, and the mixture was gently tumbled 6 to 8 times. Additional 350. Mu.L of solution II (5M sodium acetate, 60mL glacial acetic acid, 28.5mL double distilled water) was added to allow lysis of the bacteria and after gentle tumbling for 6-8 times, a white precipitate appeared in the centrifuge tube. The resulting bacterial lysate was centrifuged at 12000rpm for 10 minutes, and the supernatant was retained and transferred to an adsorption column. The column was centrifuged at 12000rpm for 1 minute, the liquid was discarded, and the column was washed twice with 600. Mu.L of wash buffer (wash buffer), centrifuged at 12000rpm for 1 minute, and the liquid was discarded. Finally, the adsorption column was placed in a new collection tube, and the DNA plasmid was eluted with about 50 to 100. Mu.L of the elution buffer.

The DNA plasmids used to transiently transfect cells to express proteins are critical in both concentration and purity, and therefore require the use of endotoxin-free plasmid bulk preparation kits (Qiagen). The principle is the same as for plasmid minipreparations.

First, 100-250mL of the bacterial solution was cultured overnight. After obtaining the thalli by centrifugation at 4 ℃, resuspending the bacteria by using 10mL of solution I, adding the solution II, overturning the centrifuge tube for 6-8 times, and standing for 5 minutes at room temperature. Then, the bacterial lysate was filtered with a filter and placed in a new centrifuge tube, and 2.5mL of an Endotoxin Remover (endo Remover) was added thereto, and after being sufficiently mixed, it was allowed to stand on ice for 30 minutes. Then, the liquid is put into an adsorption column, and the liquid penetrates through the resin and passes out of the adsorption column by virtue of gravity. The adsorption column was washed with 2x30 mL of washing buffer, and then the DNA was eluted with 15mL of eluent. 10.5mL of isopropyl alcohol (national institute of medicine, ltd.) was added to the eluate, mixed well to precipitate DNA, and centrifuged at a rotation speed of more than 15000 Xg for 30 minutes. After centrifugation, the supernatant was carefully decanted and the DNA precipitate was allowed to stand at room temperature until the liquid was completely evaporated. Finally, the DNA plasmid was reconstituted with the appropriate volume (about 2 mL) of endotoxin-free TE buffer.

The plasmid concentration was determined by measuring the absorbance of the sample at a wavelength of 260nm using a micro ultraviolet spectrophotometer (Implen), and OD260=1 corresponds to a double-stranded DNA concentration of 50. Mu.g/ml. OD260/OD280 should be close to 1.8, with protein contamination common in samples less than 1.6 and RNA contamination greater than 1.9.

8) Antibody heavy chain expression vector end site-directed mutagenesis

This example uses PCR site-directed mutagenesis to introduce an additional cysteine residue at the C-terminus of the antibody heavy chain. The first step of site-directed mutagenesis is to design a PCR primer (Biotechnology engineering Co., ltd.) capable of introducing a mutated sequence, specifically:

in the pCDNA3.1 vector inserted with antibody heavy chain DNA sequence, respectively taking 10-20 base sequences before and after the site to be introduced with mutation, adding a cysteine codon sequence in the middle of the base sequences, wherein the obtained sequence is a forward primer, and the reverse complementary sequence is a reverse primer; then, using the synthesized PCR primers, using the vector inserted with the antibody heavy chain DNA sequence as a template, 50. Mu.L of a reaction system (5. Mu.L of reaction buffer 10X, 2. Mu.L of template about 50ng, 1. Mu.L of forward and reverse primers 10nM, 2. Mu.L of dNTP (New england biolabs) mixture each 2.5mM, 1. Mu.L of pfu DNA polymerase (New england biolabs), 39. Mu.L of H ₂ O) carrying out PCR reaction, wherein the PCR reaction conditions are as follows: the denaturation temperature is 95 ℃,30s, the annealing temperature is 65 ℃ for 1min, the extension temperature is 72 ℃ for 6min, and the cycle time is 20.

Then, 1. Mu.L of DpnI enzyme (New england biolabs) was added to the resulting PCR product in order to remove the DNA template without mutation because the template plasmid DNA is methylated and can be cleaved by the DpnI enzyme. And finally, transforming DH5 alpha competence by using the reaction product, picking a single clone, and verifying whether the cysteine residue DNA sequence is successfully inserted or not by sequencing.

9) Cell culture

Expi293FTM cells (Thermo Fisher) were cultured in suspension in Expi293TM medium

Expression Medium serum-free Medium (Thermo Fisher). Placing the cells in a conical flask, and culturing on a horizontal rotary shaking table in a carbon dioxide incubator at 37 deg.C and 8% CO ₂ Rotation speed of 125 rpm; the cell density should be maintained at 0.3 × 10 ⁶ -3×10 ⁶ cell/mL, and the proportion of viable cells is maintained at 95% or more.

10 Transfection of cells

Transient transfection of Expi293FTM suspension cells was used for the bulk expression of anti-CD33 antibody. In thatExpi293FTM cells should be cultured to a density greater than 3X 10 prior to transfection ⁷ cells/mL, cell viability not less than 95%. Cells were first diluted to 2.9X 10 for transfection ⁷ cells/mL, volume 25.5mL. In centrifuge tube I, 15. Mu.g each of the antibody heavy and light chain expression vectors was mixed with 1.5mL of opti-MEM (Gibco). mu.L of Expifeacylamine (TM) transfection reagent (Thermo Fisher) was diluted in 1.5mL opti-MEM medium in centrifuge tube II and carefully mixed with a pipette. After standing at room temperature for 5 minutes, the mixture in centrifuge tube I was added to centrifuge tube II, carefully mixed, and allowed to stand at room temperature for 20 minutes. Finally, the transfection mixture is added dropwise to the cell culture system and cultured under appropriate conditions. And 150. Mu.L of Expifeacylamine (TM) 293Transfection Enhancer I and 1.5mL of Expifeacylamine (TM) 293Transfection Enhancer II were added 16 to 24 hours after Transfection. After 7 days of continuous culture, culture supernatants were collected by centrifugation for detection and purification of anti-CD33 antibody.

11 Purification of antibodies

The Anti-CD33 antibody is separated and purified by a Protein A antibody affinity chromatography column (Sechidaceae). The protein A/G is derived from the bacterial surface, both of which are capable of interacting with the Fc portion of human immunoglobulin IgG. When the antibody is purified. The column was first equilibrated with 10 column volumes of loading buffer (sample buffer: PBS) while using loading buffer at a rate of 1:1 and then the cell fluid was passed through the affinity column at a rate of 1 column volume per minute to allow loading of the antibody on the column. The column was washed with 20 column volumes of loading buffer. Finally, the antibody was eluted with an acidic pH of an elution buffer (elution buffer:100mM pH =3.0 Gly-HCl) and collected. The elution pattern of the Anti-CD33 antibody is shown in FIG. 2.

FIG. 2 shows the elution profile of humanized CD33 antibody on Protein A antibody affinity chromatography column. After the eukaryotic cell Protein expression system produces the antibody, collecting the cell culture solution, loading the cell culture solution on a Protein A antibody affinity chromatography column (loading buffer solution: PBS), eluting by using 100mM pH =3.0Gly-HCl, and eluting the antibody in about 300 th column volume.

12 SDS-PAGE detection of antibody expression

Polyacrylamide gel electrophoresis (SDS-PAGE) is a commonly used experimental means for protein analysis. The protein can be separated into different strips according to molecular weight under the denaturation condition in an electric field.

The first step in SDS-PAGE detection is the preparation of samples. 2X 10. Mu.L of each fraction of the liquid (passage solution, eluent) was collected during antibody purification, and 2.5. Mu.L of a 5 XSDS-PAGE loading buffer (Hist History Biotech) with or without β -mercaptoethanol was added thereto. After mixing, heating at 100 ℃ for 10 minutes. Preparing 10% of SDS-containing polyacrylamide gel and 3% of compression gel, adding the prepared sample into a loading hole, compressing at 80V for about 15 minutes, adjusting the voltage to 120V, and continuing to separate for 35 minutes. After electrophoresis, the gel was stained in Coomassie Brilliant blue for about two hours, and then destained with destaining solution until the protein bands were clear. The SDS-PAGE bands of Anti-CD33 antibody are shown in FIG. 3.

Figure 3 shows SDS-PAGE characterization of antibody purity and molecular weight. After the antibody is reduced by a loading buffer solution (containing beta-mercaptoethanol), SDS-PAGE shows that the heavy chain is positioned at about 55kDa, and the light chain is positioned at about 25kDa, which is consistent with the theoretical molecular weight and has no other obvious bands.

13 HPLC-MS determination of antibody molecular weight

Dialyzing the prepared antibody with pure water, freeze-drying, dissolving the solid in a certain amount of pure water, and detecting the molecular weight of the solid by using HPLC-MS (Agilent 6230 LC/TOF); an additional amount of DTT (Sigma) was added to reduce the antibody, and the reduced antibody heavy and light chains were injected into HPLC-MS to confirm its molecular weight.

FIG. 4 shows HPLC-MS characterization of molecular weight. As can be seen from FIG. 4, the antibody molecular weight 145681.58, the antibody heavy chain molecular weight 50208, and the antibody light chain molecular weight 23758.

The chromatographic method comprises the following steps: chromatography column (Agilent, C4): 150X 2.1mm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B:70% isopropanol +20% acetonitrile +10% water (containing 0.1% trifluoroacetic acid); elution procedure: 2-10min 100% B; flow rate: 0.7mL/min; column temperature: 25 ℃; and (3) detection: UV 280nm, consistent with the theoretical molecular weight.

2. Preparation of liposomal delivery systems

(1) Preparation of liposome delivery systems

The humanized CD33 antibody prepared above was used to prepare the liposome delivery system of this example, the preparation method comprising the following steps:

1) Weighing hydrogenated soybean phosphatidylcholine (HSPC for short, purchased from Avanti), cholesterol (Chol for short, purchased from Sigma), and polyethylene glycol-distearoyl phosphatidylethanolamine (mPEG for short) according to a molar ratio of 50 ₂₀₀₀ DSPE from Laysan Bio and Maleimide-polyethylene glycol-distearoylphosphatidylethanolamine (Mal-PEG for short) ₃₄₀₀ -DSPE, laysan Bio), added to a rotary evaporation flask, after dissolution with chloroform, rotary evaporated to film formation;

2) Adding 1mL buffer solution with neutral pH into the rotary evaporation bottle to hydrate the lipid auxiliary material; the buffer contained 100mM copper gluconate, 110mM TEA and 400mM Ara-C; and sonicating to form a homogeneous liposome liquid;

3) Adding the liposome liquid into a Sephadex G-50 molecular sieve gel column, passing the column by taking pH neutral SPE buffer (sucrose phosphate EDTA buffer) as a mobile phase, collecting the liposome, and removing excessive non-entrapped copper gluconate and Ara-C;

4) To the collected liposome fractions was added a volume of DNR (2.5 mg/mL) dissolved SPE buffer to provide a DNR to Ara-C molar ratio of 1; after 1h incubation at room temperature, excess non-entrapped DNR was removed using Sephadex G-50 molecular sieves gel column with mobile phase PB buffer (10 mM, pH = 6.5), and the eluate containing lip @ Ara-C/DNR was collected;

5) The humanized anti-CD33 antibody was incubated with tris (2-carboxyethyl) phosphine (Tcep) at room temperature for 1h at a molar ratio of 1;

6) The reduced humanized anti-CD33 antibody and lip @ Ara-C/DNR were mixed at an antibody/Mal-PEG-DSPE molar ratio of 1.

The humanized anti-CD33 antibody, lip @ Ara-C/DNR and Ab-lip @ Ara-C/DNR were subjected to SDS-PAGE analysis and particle size analysis, and the results are shown in FIGS. 5 and 6, respectively.

As seen from the SDS-PAGE pattern of FIG. 5, two bands of light chain and heavy chain were detected in the humanized anti-CD33 antibody, and the light chain band of Ab-Lip @ Ara-C/DNR was shifted unchanged and the heavy chain band was shifted upward, whereas the antibody band was not detected by Lip @ Ara-C/DNR, as compared with the humanized anti-CD33 antibody.

As can be seen from the results of particle size analysis in FIG. 6, the particle size of lip @ Ara-C/DNR was 92.81nm, and PDI was 0.144; the particle size of Ab-lip @ Ara-C/DNR was 107.2nm, and the PDI was 0.160.

The above test results all indicate that the humanized anti-CD33 antibody has been successfully coupled to the surface of the liposome, and the liposome drug delivery system Ab-lip @ Ara-C/DNR is successfully constructed.

(2) Optimization of Ara-C/DNR dosing mass ratio

In order to achieve a fixed 5 molar ratio of Ara-C to DNR entrapped in liposomes with reproducible entrapment ratios, it was determined at which ratio of Ara-C to DNR was entrapped.

Now by means of the HPLC method, a means of quantification of the two drugs Ara-C and DNR was obtained, as shown in FIG. 7, FIG. 8 and FIG. 9. The HPLC chromatographic conditions are as follows:

liquid phase column: a C18 column;

mobile phase A:10mM pH =7.0PB;

and (3) mobile phase B: acetonitrile;

gradient elution: 0.7mL/min,5% -80% B in 30min.

As shown in FIGS. 7 and 8, the standard curves fitted to HPLC measurements of Ara-C and DNR concentrations had better linear regression coefficients, indicating that the method is suitable for Ara-C and DNR quantification. As shown in FIG. 9, after Ara-C and DNR are entrapped in the liposome, the peak areas of the two drugs can be accurately determined by HPLC, and the peak areas of the two drugs are not influenced with each other.

To determine the optimal dose ratio of Ara-C/DNR, the drug ratios of Ara-C/DNR in the upper, middle and lower groups were set to 3.09.

As can be seen from fig. 10, the Ara-C/DNR encapsulation ratio is almost identical to the dosage ratio, indicating that DNR is almost 100% encapsulated, if the Ara-C/DNR final drug molar ratio is 5, the Ara-C/DNR initial dosage molar ratio is 5, i.e. the mass ratio is 2.27.

(3) Antibody to liposome coupling ratio optimization

Preparation of a liposome delivery system Ab-lip @ DiD (1) - (3) carrying a Red fluorescent Probe DiD with low (1/590.24), medium (1/147.56) and high (1/36.89) antibody/liposome administration ratios, respectively, ab-lip @ DiD (1) - (3) was diluted to 1.5. Mu.M in cell culture medium, applied to AML cells HL-60 (CD 33 +), MOLM-13 (CD 33 +), and SUP-B15 (CD 33-), incubated at 37 ℃ for 2 hours, centrifuged at 1000rpm for 5min, cells were collected, washed once with PBS, and the fluorescence signals of the cells were quantified using a flow cytometer (BD Biosciences, FACS CantotM), and the results are shown in FIGS. 11 to 16.

As seen from FIGS. 11 to 16, HL-60 and MOLM-13 were the highest in Ab-Lip @ DiD (2) and had very strong cell selectivity, while SUP-B15 was comparable to Ab-Lip @ DiD (1) - (3). The above results indicate that Ab-lip @ DiD (2) can exert the maximum targeting effect, and the corresponding antibody/liposome administration ratio is 1/147.56 optimal.

3. In vitro targeting analysis of liposomal drug delivery systems

This example analyzes the in vitro targeting of liposomal drug delivery systems by cell surface binding experiments. The analysis method is as follows:

incubating HL-7702 (CD 33-) and MOLM-13 (CD 33 +) of AML cells with 1% BSA/PBS at room temperature for 30min, centrifuging at 1000rpm for 5min, and removing the liquid; ab-lip @ Ara-C/DNR was diluted from the cell culture medium to an antibody concentration of 10. Mu.g/mL (control, ara-C/DNR (i.e., a mixture of Ara-C and DNR at a molar ratio of 5, 1), lip @ Ara-C/DNR, equal dilution), cells were added, incubated at 4 ℃ for 1h, centrifuged at 1000rpm for 5min, cells were collected, washed three times with PBS, and 2. Mu.g/mL Goat-Anti-Mouse (Goat Anti-Mouse) IgG (Alexa a @

488 1h at room temperature, centrifugation at 1000rpm for 5min, three washes with PBS, and quantitation of fine lines using a flow cytometerThe results of the fluorescence signals of the cells are shown in FIGS. 17 and 18.

As can be seen from FIGS. 17 and 18, ab-lip @ Ara-C/DNR has good recognition ability on MOLM-13 cell surface, but has no obvious combination with HL-7702 cells, indicating that Ab-lip @ Ara-C/DNR has good targeting performance on CD33+ cells.

4. In vitro pharmacodynamic evaluation of liposomal drug delivery systems

(1) Evaluation of apoptosis

Culturing AML cells HL-7702 (CD 33-) and MOLM-13 (CD 33 +) to 60-70%, respectively, and removing old culture solution; ab-lip @ Ara-C/DNR is diluted by a cell culture medium until the concentration of the antibody is 10 mu g/mL (control, ara-C/DNR, lip @ Ara-C/DNR and the like are diluted in proportion), the cells are added, the mixture is incubated for 1h at 4 ℃, the mixture is sucked out to a centrifuge tube, 200g of the mixture is centrifuged for 5min, supernatant is discarded, and 195 mu L of Annexin V-FITC binding solution is added to gently resuspend the cells; adding 5 μ L Annexin V-FITC, mixing, and incubating at room temperature in dark for 10min; centrifuging at 200g for 5min, discarding supernatant, and adding 190 μ L Annexin V-FITC conjugate to resuspend cells; and adding 10 mu L of propidium iodide staining solution, gently mixing, placing in ice bath and in dark place, and carrying out flow cytometry detection, wherein the detection result is shown in 19 and 20.

As can be seen from FIGS. 19 and 20, none of Ara-C/DNR, lip @ Ara-C/DNR and Ab-Lip @ Ara-C/DNR caused early apoptosis of cells, indicating no cytotoxicity.

(2) Evaluation of cytotoxicity

1) 24h cytotoxicity assessment

Ara-C/DNR, lip @ Ara-C/DNR and Ab-lip @ Ara-C/DNR were applied to MOLM-13 (CD 33 +) cells after being diluted with cell culture medium in a gradient manner, 20. Mu.L of CCK-8 was added to each well after incubation at 37 ℃ for 24 hours, incubation was continued at 37 ℃ for 2 hours, and then absorbance was measured at 450nm using a microplate reader, and cell viability was measured using CCK-8 (Dalian Meiren Biotechnology Co., ltd.), as shown in FIG. 21, FIG. 22, FIG. 23 and FIG. 24.

As can be seen from FIGS. 21 to 24, in vitro experiments, the free small molecule drugs in the Ara-C/DNR group entered the cells faster than the liposomes, and after 24h of incubation with the cells, the Ara-C/DNR group killed the cells most (IC) ₅₀ The lower contains 0.0401. Mu.M Ara-C and 0.0080. Mu.M DNR).

After 24h of co-incubation, ab-lip @ Ara-C/DNRLip @ Ara-C/DNR (IC) ₅₀ The killing effect of the mixture containing 0.0762. Mu. MAra-C and 0.0152. Mu.M DNR on MOLM-13 (CD 33 +) cells is better than that of Lip @ Ara-C/DNR (IC) ₅₀ The lower content of 0.0897 mu M Ara-C and 0.0179 mu M DNR) is good, which shows that the surface of the liposome is connected with the antibody and can effectively improve the targeting property.

2) 72h cytotoxicity evaluation

Ara-C/DNR, lip @ Ara-C/DNR and Ab-lip @ Ara-C/DNR were applied to MOLM-13 (CD 33 +) cells after being diluted with cell culture medium in a gradient, and after incubation at 37 ℃ for 72 hours, 20. Mu.L of CCK-8 was added to each well, and incubation was continued at 37 ℃ for 2 hours, and then absorbance was measured at 450nm using a microplate reader, and cell viability was measured using CCK-8 (Dalian Meiren Biotechnology Co., ltd.), as shown in FIG. 25, FIG. 26, FIG. 27 and FIG. 28.

As can be seen from FIGS. 25 to 28, in vitro experiments, the free small molecule drugs in the Ara-C/DNR group entered the cells faster than the liposomes, and after 24h of co-incubation with the cells, the Ara-C/DNR group killed the cells most (IC) ₅₀ The lower contains 0.0074. Mu.M Ara-C and 0.0015. Mu.M DNR).

After 72h of co-incubation, ab-lip @ Ara-C/DNR (IC) ₅₀ The killing effect of the lower containing 0.0142 μ M Ara-C and 0.0028 μ M MDNR) on MOLM-13 (CD 33 +) cells is better than that of lip @ Ara-C/DNR (IC) ₅₀ The lower content of 0.0155 mu M Ara-C and 0.0031 mu M DNR) shows that the surface of the liposome is connected with the antibody to effectively improve the targeting property.

5. In vivo efficacy evaluation of liposomal drug delivery systems

(1) Bioluminescence detection

Mice were harvested and injected with 3.8X 10 via the tail vein into mice as shown in FIG. 29 ⁶ MOLM-13-luc cells are grown for 2 days to construct an AML mouse model; the mice were then divided into four groups: saline group, ara-C + DNR group, lip @ Ara-C/DNR group and Ab-lip @ Ara-C/DNR group, and injecting corresponding drugs into each group of mice on days 3, 5, 7 and 9, with injection amount of 2.5:1.1 (Ara-C: DNR) mg/kg; bioluminescence assays were performed on days 4, 6, 8, and 10 and the results are shown in fig. 30, 31, and 32.

As can be seen from fig. 30, no fluorescence was detected in each group of mice on day 4 after model construction; fluorescence was detected on day 6 in both the Saline group and the Ara-C + DNR group, and should be mainly concentrated in the back of the Saline group, indicating that MOLM-13-luc cells were aggregated in the spinal cord; on day 8, the fluorescence of Ara-C + DNR and Saline groups spread almost over the entire back and abdomen of the mice, while the fluorescence luminosity of the Lip @ Ara-C/DNR group was relatively weak, mainly concentrated at the spinal location; whereas, only a small amount of fluorescence was observed in the Ab-lip @ Ara-C/DNR group, and the fluorescence gradually decreased on day 10.

As can be seen from FIGS. 31 and 32, the fluorescence intensity of the back and abdomen of mice in the Saline and Ara-C + DNR groups gradually increased with time, but the increase tendency of the Ara-C + DNR group was slow; the fluorescence brightness of the back and the abdomen of mice in the Lip @ Ara-C/DNR group also increases, but the increase is not obvious and is kept at a lower level all the time; while the fluorescence intensity in the back and abdomen of the Ab-lip @ Ara-C/DNR group mice remained at a very low level. The Ab-lip @ Ara-C/DNR can effectively inhibit the diffusion of MOLM-13-luc cells by MOL.

(2) Life cycle detection

As shown in fig. 33, an AML mouse model was constructed in the same manner as in the above section (1), with 5 doses starting on day 3; the body weight and survival status of the mice were checked daily after the dosing interval and the end of the dosing, and the results are shown in fig. 34 and fig. 35.

As can be seen in fig. 34, the body weight of each group of mice during the drug treatment generally tended to increase and then decrease, but the weight average of all the mice administered was higher than the initial body weight value, indicating that the dose administered was within the tolerance range.

As can be seen from fig. 35, on day 15, all mice in each group survived; on day 20, the Saline group had all died, while only one mouse survived in the Ara-C + DNR group, and all mice survived in the Lip @ Ara-C/DNR and Ab-Lip @ Ara-C/DNR groups; on day 25, only one mouse survived in the Lip @ Ara-C/DNR group, and all mice survived in the Ab-Lip @ Ara-C/DNR group; only one mouse survived in the Ab-lip @ Ara-C/DNR group on day 30, with a median survival of approximately 27.5 days, comparable to that of the Saline group (17 days) over 16.5 days following the end of dosing on day 11.

As can be seen from fig. 36,% ILS = (median survival in mice administered group-median survival in mice in saline group) × 100%/median survival in mice in saline group, which indicates the ability to improve survival in mice.

The free drug of Ara-C + DNR is co-administered to only improve the survival time of mice by about 8.8 percent, the survival time of mice can be improved by about 35.3 percent by lip @ Ara-C/DNR, and the survival time of mice can be improved by about 61.8 percent by Ab-lip @ Ara-C/DNR, thereby having obvious treatment effect.

The results show that the liposome coating is helpful for improving the drug effect of Ara-C/DNR, and the active targeting of the antibody and the liposome are more helpful for improving the drug effect of Ara-C/DNR, so that the antibody not only can kill tumor cells, but also can inhibit the growth rate of residual tumor cells; in addition, the drug effect can be further improved by prolonging the administration time interval and increasing the drug dose.

(3) Bone marrow cell TUNEL staining assay

The analysis method is as follows: after the 12 th day administration period, mice were sacrificed by cervical dislocation, their femurs and tibias were harvested, bone marrow cells were washed out using a 1mL syringe with cold D-PBS, filtered through 70 μm and 40 μm cell screens, respectively, counted, cell smeared, paraformaldehyde fixed, stained with TUNEL kit, stained with DAPI, and washed with pure water. The obtained sections were observed by confocal laser microscopy, and the obtained pictures were counted by ImageJ software to count TUNEL positive cells, and the results are shown in fig. 37 and 38.

As seen from FIGS. 37 and 38, the proportion of apoptotic cells was gradually increased in the Ara-C/DNR group, the lip @ Ara-C/DNR group and the Ab-lip @ Ara-C/DNR group, as compared with the Saline group, indicating that the therapeutic effect on AML cells was gradually enhanced.

(4) Bone marrow cell CD33 antibody immunofluorescence

The analysis method is as follows: on day 12, after the dosing period was complete, the mice were sacrificed by cervical dislocation, their femurs and tibias were removed, bone marrow cells were washed out using a 1mL syringe with cold D-PBS, filtered through 70 μm and 40 μm cell screens, respectively, cell counted, cell smeared, paraformaldehyde fixed, incubated with anti-CD33 antibody, and then stained with Cy 3-labeled anti-IgG secondary antibody, sections washed, stained DAPI, washed with pure water. The obtained sections were observed by confocal laser microscopy, and the number of CD33 positive cells was counted by calculating the obtained pictures by ImageJ software, and the results are shown in fig. 39 and 40.

As can be seen from FIGS. 39 and 40, the bone marrow cells of the non-tumorigenic Blank mice were substantially free of CD 33-positive tumor cells, and the bone marrow cells of the saline group mice were abundant in CD 33-positive tumor cells, while the bone marrow cells of the bone marrow were still abundant in Ara-C + DNR due to limited therapeutic effects; the lip @ Ara-C/DNR group and the Ab-lip @ Ara-C/DNR group can obviously kill the tumor cells in bone marrow, wherein the Ab-lip @ Ara-C/DNR has the least survival tumor cells, and the property of killing the tumor cells is optimal.

(5) Richter-Giemsa staining of bone marrow cells

The analysis method is as follows: after the 12 th day administration period, the mice were sacrificed by cervical dislocation, the femurs and tibias were harvested, bone marrow cells were washed out with cold D-PBS using a 1mL syringe, filtered through 70 μm and 40 μm cell screens, respectively, counted, smeared with 50% ethanol for 10min, stained with rui's-giemsa staining solution for 45min, washed with pure water several times, dried, and scanned using a section scanner, and the results were shown in fig. 41.

As can be seen from FIG. 41, no tumor cells with distinct morphology were found in bone marrow cells of non-tumorigenic Blank mice, whereas the leukocyte infiltration rate in bone marrow of salt group was 20-25%, ara-C + DNR treatment effect was limited, and only the leukocyte infiltration rate was reduced to 8-10%, and tumor cells in bone marrow could be significantly killed in Lip @ Ara-C/DNR group and Ab-Lip @ Ara-C/DNR group, and the leukocyte infiltration rate in Lip @ Ara-C/DNR group was reduced to 2.6%, while that in Ab-Lip @ Ara-C/DNR group was 0%.

(6) Cell number analysis

The analysis method is as follows: after the administration course on day 12 is finished, taking blood from the orbit of each group of mice, anticoagulating by using heparin sodium, carrying out conventional blood analysis on the blood by using a small animal blood cell instrument, and recording the number and the proportion of various types of blood cells; the results are shown in fig. 42, 43, 44, 45, 46, 47 and 48.

As can be seen from fig. 42 to 48, the numbers of leukocytes, granulocytes, lymphocytes and monocytes in the peripheral blood of the mice significantly increased as the course of acute myelogenous leukemia worsens; whereas the number and ratio of each cell was restored to normal levels by administration of Ab-Lip @ Ara-C/DNR.

(7) Tissue staining slice analysis

The analysis method is as follows: after the administration course on day 12, the mice were sacrificed by neck amputation, organs such as heart, liver, spleen, lung, kidney and brain were dissected out, fixed overnight in 4% paraformaldehyde, dehydrated with 30% sucrose, impregnated and embedded in wax block, the largest cross section was cut out by a pathological microtome, deparaffinized with xylene, hematoxylin-stained with cell nucleus, stained with eosin cytoplasm, dehydrated mounting, mounted with a cover glass, the cell morphology was observed using an inverted fluorescence microscope, the photographing record was taken, and the toxicity of organs was evaluated by reading the mounting, and the results are shown in fig. 49 to 54.

As seen from FIGS. 49 to 54, ab-lip @ Ara-C/DNR had some toxic side effects on the liver, lung and spleen, but had almost the same degree as the Ara-C + DNR group and the lip @ Ara-C/DNR, and no significant specific toxic side effects were observed.

Claims (10)

1. A liposome drug delivery system comprises a liposome and an active drug encapsulated in the liposome, and is characterized in that the liposome surface is provided with maleimide groups, sulfydryl or carboxyl, at least part of the maleimide groups, sulfydryl or carboxyl is coupled with a functional targeting molecule, and the functional targeting molecule and the target of the active drug are the same.

2. The liposomal delivery system of claim 1, wherein the active agent is an acute myeloid leukemia therapeutic agent and the functional targeting molecule comprises at least one of a protein and a polypeptide.

3. The liposomal delivery system of claim 2, wherein the functional targeting molecule is an anti-CD33 antibody having a cysteine at the C-terminus of the heavy chain providing a thiol group conjugated to a maleimide group, thiol group, or carboxyl group.

4. The liposomal delivery system of claim 3, wherein the light chain of the anti-CD33 antibody has the amino acid sequence shown in SEQ ID No.1 and the heavy chain of the anti-CD33 antibody has the amino acid sequence shown in SEQ ID No. 2.

5. The liposomal delivery system of any one of claims 1-4, wherein the maleimide group, thiol group, or carboxyl group is attached to the liposome via a polyethylene glycol chain.

6. The liposomal delivery system of claim 5, wherein the starting material of the liposomes comprises at least maleimido-polyethylene glycol-distearoylphosphatidylethanolamine.

7. The liposomal delivery system of claim 5, wherein the molar ratio of functional targeting molecule to maleimide group is 1: (36-200).

8. The liposomal delivery system of claim 6, wherein the starting materials for the liposomes comprise, in mole percent: 40-60mol% hydrogenated soy phosphatidylcholine, 35-55mol% cholesterol, 0-5mol% polyethylene glycol-distearoylphosphatidylethanolamine and 0-5 (excluding 0) mol% maleimido-polyethylene glycol-distearoylphosphatidylethanolamine.

9. The liposomal delivery system of claim 8, wherein the length of the polyethylene glycol chain in the maleimido-polyethylene glycol-distearoylphosphatidylethanolamine is greater than the length of the polyethylene glycol chain in the polyethylene glycol-distearoylphosphatidylethanolamine.

10. Use of a liposomal delivery system according to any one of claims 1 to 9 for the preparation of a therapeutic formulation for acute myeloid leukemia, wherein the functional targeting molecule is an anti-CD33 antibody, the active agent is a mixture of cytarabine and daunorubicin, and the molar ratio of cytarabine to daunorubicin is 5.

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