CN110898007A

CN110898007A - Preparation of targeting antitumor uronic acid carboxyl free polysaccharide derivative micelle

Info

Publication number: CN110898007A
Application number: CN201911105399.9A
Authority: CN
Inventors: 尹鸿萍; 缪应花; 王苏妍
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2019-11-13
Filing date: 2019-11-13
Publication date: 2020-03-24

Abstract

Preparation of uronic acid carboxyl free polysaccharide derivative micelles is carried out by green and environment-friendly acylation reaction, wherein uronic acid carboxyl free polysaccharide with good biocompatibility and high safety is used as a hydrophilic end, a diamino compound is used as a connecting bond, and a connecting fatty acid is used as a hydrophobic end, so as to obtain the amphoteric micelles. The micelle can form particles with the particle size of less than 200nrn in an aqueous solution, and can realize passive targeting on tumors through the EPR effect of tumor tissues; after the micelle enters tumor tissues, the nanospheres can be degraded by the characteristics of tumor microacid. In addition, the micelle can entrap the photosensitizer, so that the photosensitizer can stably exist and is not easy to aggregate and precipitate; moreover, by controlling the illumination, tumor cells can be killed at fixed points and fixed time, and strong antitumor is realized due to the synergistic effect of the polyunsaturated fatty acid and the photosensitizer; in conclusion, the nano material can resist tumors in a targeted, fixed-point, timed and powerful manner.

Description

Preparation of targeting antitumor uronic acid carboxyl free polysaccharide derivative micelle

Technical Field

The invention belongs to the field of material science, and particularly relates to an uronic acid carboxyl free polysaccharide derivative which can form nanospheres in an aqueous solution, can entrap a photosensitizer and can play a targeted anti-tumor role.

Background

The photodynamic therapy is a minimally invasive therapy, and the mechanism is that under the irradiation of exciting light with a certain wavelength (usually near infrared light), singlet active oxygen is generated by combining with oxygen at a pathological change part, so that the toxic action of cells is generated, and then the obvious inhibition effect is generated on a tumor part. The traditional Chinese medicine composition is clinically used for treating skin diseases, and has good biological safety and high acceptability; however, the excitation wavelength is close to sunlight, and the illumination transmission depth is limited, so the administration needs to be protected from light. The photosensitizer is a large-plane conjugated compound, has low water solubility, is administered by taking castor oil as an auxiliary solvent, and is easy to generate systemic anaphylactic reaction; secondly, most photosensitizers do not have focal targeting; the energy of the absorbed exciting light is easy to be emitted in the form of heat energy, so that the yield of the singlet oxygen is reduced. Based on the defects of the existing photosensitizer, researchers mainly have the following aspects from all aspects: a. the photosensitizer structure is modified, so that the exciting light of the photosensitizer structure is red-shifted, the transmission depth is increased, the damage to normal tissues is reduced, and the photosensitizer structure does not need to be protected from light after treatment; b. in order to improve the water solubility, on one hand, a polar functional group is increased through chemical modification, and on the other hand, the photosensitizer is wrapped to the core through a nano material, so that the water solubility and the stability of the photosensitizer are increased.

Nanomaterial refers to a material having at least one dimension in three-dimensional space in the nanometer size or composed of them as basic units. The particle size of the nano material has great influence on the application of the nano drug carrier, the maximum limit of the particle size is the diameter of the smallest capillary, and the nano particle size is generally considered to be more than 1.5 mu m, so that the nano material is not suitable for intravenous administration, the venous circulation is influenced, even the capillary is blocked, and the nano particles with the particle size of more than 200nm can be filtered by the spleen, so that the drug concentration in the blood circulation is reduced. For tumor targeting, the particle size is more suitable to be smaller than 250nm, and the smaller particle size can ensure that the nano-drug-loaded micelle can penetrate through the gaps of vascular endothelial cells, thereby achieving tumor cell tissues.

The nano drug-loaded system is characterized in that a target drug is wrapped to a nano material hydrophobic core through a hydrophobic effect, and passive targeting is realized through an EPR effect (the delivery of a tumor tissue vascular wall is not tight and lymphatic return is absent) of a tumor tissue blood vessel; the surface of the nano material can also be modified, and the folic acid or the hyaluronic acid and the like which can be combined with the folic acid receptor or the CD44 receptor and the like on the surface of the tumor cell are covalently coupled, so that the nano material has an active targeting function; in addition, the spatial structure of the nano material can be designed, the chemical structure composition of the nano material can be reasonably designed, the nano material is endowed with photosensitivity, pH sensitivity, redox property, enzyme degradability and the like, the nano material can release the medicine at fixed points and fixed time, and the experiment can be controlled and actively targeted.

The exertion of the targeting effect of the nano drug-carrying system is also closely related to the surface charge. On the one hand, repulsion between like charges increases their stability in blood circulation; in addition, since the self-protein and other components in blood have negative charges, the negative charges on the surface of the nano material are more beneficial to the circulation of the nano material in the blood, the circulation period is prolonged, and the negative charges can reduce the elimination of reticuloendothelial cells on the nano material and increase the drug accumulation concentration in tumor tissues. However, the ECM on the surface of the tumor cell has many negatively charged substances such as heparan sulfate, etc., and the positively charged nanomaterial is more favorable for endocytosis of the nanomaterial by the cell. Positive and negative charges have both advantages and disadvantages.

The design and preparation of the nano material are changed with different drug effects and purposes. The nano materials reported in the literature at present are various, but the general principle is not changed. Except inorganic nano materials such as nano gold, nano silver, ferroferric oxide, silicon oxide and the like, the rest of the structure consists of a hydrophobic end, a linker and a hydrophilic end, and the nano gold, the nano silver, the ferroferric oxide, the silicon oxide and the like can be dissolved in a water phase or an oil phase. Under normal physiological conditions, the nanomaterial is in an oil-in-water state, the hydrophobic ends aggregate as hydrophobic centers, and the hydrophilic ends extend into interstitial fluid.

Fatty acids are common hydrophobic substances and can be used as a hydrophobic center of a nano material, wherein the fatty acid refers to a long aliphatic hydrocarbon chain with one end containing a carboxyl group, the saturated and unsaturated hydrocarbon chains can be divided into 3 types according to saturation and unsaturation, namely, saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids, wherein the saturated fatty acids comprise caprylic acid, lauric oil, myristic acid, stearic acid, arachidic acid and the like, the monounsaturated fatty acids comprise myristic acid, palmitoleic acid, oleic acid, ricinoleic acid and the like, and the polyunsaturated fatty acids α -linolenic acid, gamma-linolenic acid, linoleic acid and the like.

Polyunsaturated fatty acids (PUFAs) have two metabolic pathways in cells, namely COX and LOX, and the activation states of the two metabolic pathways are different between tumor cells and normal cells, so that the relative amounts of metabolic products prostaglandin (PGE 2) and LAX4 are different, the COX is activated in the tumor cells, the LOX pathway is inhibited, the PGE 2 is more, and the opposite is true in the normal cells, for example, when α -linolenic acid is supplied from the outside, the ingestion of PUFAs in the normal cells promotes the generation of Arachidonic Acid (AA), is beneficial to the metabolism and repair of the cells, protects the normal cells, can reduce cholesterol and the like, and the COX-2 pathway is mainly used for promoting the cell proliferation in the tumor cells, and when PUFAs are supplied too much, the occurrence of fenton reaction can be promoted, and the induced iron death of the tumor cells can be induced.

Iron death, which means that the content of iron ions in tumor cells is too high, and the iron death is used as a catalyst to catalyze the decomposition (Fenton reaction) of intracellular hydrogen peroxide and the like to form oxygen radicals and the like, and the free radicals can destroy phospholipid bilayers such as mitochondrial membranes and the like, so that the cells are killed; tumor cell death due to free Radicals (ROS), collectively referred to as iron death. In addition to unsaturated fatty acids which cause "iron death" in tumor cells, photosensitizers may also cause "iron death".

Photosensitizers are substances essential for photodynamic therapy, and absorb energy after being irradiated by light with a certain wavelength, and electrons are converted from a ground state to an excited state; when the electrons return to the ground state from the excited state, energy is released into the solvent, and a large number of free Radicals (ROS) such as superoxide anion (type I) and highly-lethal singlet oxygen (1O2) (type II) are formed, which can damage the membrane and cell membrane of the organelle, causing the death of the tumor cell.

The combined effect of linolenic acid and photosensitizer, isoqu, will kill tumor cells with half the effort.

The material is novel, and the amphoteric micelle is obtained by green and environment-friendly acylation reaction, wherein uronic acid carboxyl free polysaccharide with good biocompatibility and high safety is used as a hydrophilic end, a diamino compound is used as a connecting bond, and connecting fatty acid is used as a hydrophobic end. The micelle can form particles with the particle size of less than 200nm in an aqueous solution, and can realize passive targeting of tumors through the tumor retention Effect (EPR) effect of tumor tissues; after the micelle enters tumor tissues, the nanospheres can be degraded by the characteristics of tumor microacid. In addition, the micelle can entrap the photosensitizer, so that the photosensitizer can stably exist and is not easy to aggregate and precipitate; by controlling the illumination, the tumor cells can be killed and killed at fixed points and fixed time; and because of the synergistic effect of polyunsaturated fatty acid and photosensitizer, realize the strong antitumor; in conclusion, the nano material can resist tumors in a targeted, fixed-point, timed and powerful manner.

Disclosure of Invention

The invention relates to a preparation method of a targeted antitumor uronic acid carboxyl free polysaccharide derivative micelle.

Drawings

FIG. 1; reaction formula for synthesizing amphoteric micelle of heparin precursor derivative

FIG. 2, ultraviolet full-wavelength scan, HE, LY, lysine ethyl ester, LA, α -linolenical, HL10, the product of coupling HE and LY, HLA, heparin derivative micelle, znpc, free znpc, HLA-znpc, the product of encapsulation of zinc phthalocyanine in heparin derivative micelle

FIG. 3: heparosan infrared spectrum

FIG. 4: HLA infrared map

FIG. 5: HLA-znpc infrared spectra

FIG. 6: nuclear magnetic hydrogen spectrum of Heparasan and HL10 heavy water

FIG. 7: nuclear magnetic hydrogen spectrum of HLA in DMSO-d6

FIG. 8: tyndall effect

FIG. 9: empty carrier and particle size of encapsulated nano material

FIG. 10: stability of different znpc-loaded HLA in PBS (pH7.4) + 10% FBS

FIG. 11: stability study of different znpc-loading HLA in 1640 medium + 10% FBS

FIG. 12: mouse fluorescence imaging

Detailed Description

The invention will now be described with reference to an embodiment. The following examples are merely illustrative of the present invention and are not intended to limit the present invention.

Case name: preparation of heparin precursor polysaccharide derived amphoteric micelle and targeting anti-tumor effect thereof

The Heparosan polysaccharide derivative micelle mainly comprises four parts, namely hydrophilic end-Heparosan (Heparosan), Linker-lysine ethyl ester (lysine ethyl ester), hydrophobic end-linolenic acid (α -linolenic acid) and photosensitizer zinc phthalocyanine.

1. Preparation of heparosan polysaccharide derivative micelles:

(1) preparation of empty vector (see fig. 1):

① dissolving appropriate amount of Heparosan in 50-100mM MES buffer solution with final concentration of 2-5mg/mL, adding 2-10 times of acylating agent EDCI and NHS, stirring at room temperature (20-30 deg.C) for 30-60min in the dark, adjusting pH to neutral (7-8), adding lysine ethyl ester, reacting for 24h, dialyzing in dialysis bag for 3 days, and lyophilizing.

② dissolving appropriate amount of linolenic acid in DMSO, adding 2-10 times of acylation reagent EDCI and NHS, stirring at room temperature (20-30 deg.C) for 2-4 h in dark to obtain linolenic acid activation solution.

③ taking ① to obtain a proper amount of product, stirring and dissolving the product in FA/DMSO (1: 1) until the final concentration is 2-5mg/mL, dropwise adding a proper amount of linolenic acid activation solution (in proportion to the connected lysine ethyl ester) while stirring, continuing to react for 24h, after the reaction is finished, dialyzing the product in a 3500Da dialysis bag for 1-3 days by using DMSO, then replacing the bag with water for dialysis for 3 days, freeze-drying the product to obtain three empty carriers of linolenic acid and lysine ethyl ester, wherein the empty carriers are not loaded with photosensitizer, and the empty carriers are selected to be less than 200nm by particle size screening.

(2) And (3) carrying the photosensitizer zinc phthalocyanine by an empty carrier:

① the first method comprises placing an appropriate amount of empty carrier in DMSO to final concentration of 2.5mg/mL, stirring for 6-12 h to dissolve and disperse completely, adding znpc DMSO solutions with different amounts by weight, stirring for 30min or performing ultrasound for 10min, placing in 3500Da dialysis bag, dialyzing with water for 3 days, and lyophilizing to obtain drug-loaded nanomaterial with different loading amounts of znpc (5%, 1%, 0.5%, 0.1%, 0%).

② the second method comprises dissolving appropriate amount of hepiosan and lysine ethyl ester in FA/DMSO (1: 1) under stirring to final concentration of 2-5mg/mL, adding appropriate amount of linolenic acid activating solution (in proportion to connected lysine ethyl ester) dropwise under stirring, continuing reaction for 24h, dialyzing in 3500Da dialysis bag with DMSO for 1-3 days, collecting dialysate, adding znpc DMSO solutions in different proportions, ultrasonic treating for 10min to mix thoroughly, replacing dialysis bag, and dialyzing with water for 3 days to obtain drug-loaded nanomaterial with znpc in different loading amounts (5%, 1%, 0.5%, 0.1%, 0%).

2. Structural identification of nanomaterials

(1) Ultraviolet full wavelength scanning (see FIG. 2)

Dissolving 5mg heparosan polysaccharide derivative in 1mL water, and performing ultraviolet scanning at an interval of 10nm within the range of 300nm-800nm after full dissolution.

(2) Infrared spectroscopic analysis (see FIG. 3, FIG. 4, FIG. 5)

Mixing 1-2mg heparosan polysaccharide derivatives with potassium bromide, and tabletting at 400cm^-1-4000cm^-1Spectral measurements were performed in the range.

(3) Nuclear magnetic resonance (see fig. 6 and 7)

Respectively dissolving 10-20mg of heparosan or heparosan polysaccharide derivative in heavy water (D2O) or deuterated dimethyl sulfoxide (DMSO-D6), and measuring with 500MHZ nuclear magnetic resonance apparatus.

3. Investigation of physical Properties

(1) Tyndall effect (see FIG. 8)

10mg of each heparosan derivative micelle was dissolved in 10mL of water, transmitted with a red laser pen, and distilled water was used as a control.

4. Particle size and potential (see FIG. 9)

1mg of heparin precursor derivative micelle is dissolved in distilled water, and 800. mu.L of the solution is absorbed and placed in a plastic cuvette for measurement in a dynamic light scattering system (DLS).

5. Stability (see fig. 10 and 11)

(1)PBS(pH7.4)+10％FBS

1mg of heparin precursor derivative micelle was dissolved in a solution (PBS (pH7.4) + 10% FBS), and 800. mu.L of the solution was pipetted and placed in a plastic cuvette and measured in a dynamic light scattering system (DLS) for 3 days.

(2)1640 medium + 10% FBS

1mg of heparin precursor derivative micelle was dissolved in a solution (1640 medium + 10% FBS), and 800. mu.L of the solution was pipetted and placed in a plastic cuvette and measured in a dynamic light scattering system (DLS) for 3 days.

5. In vivo targeting tumor validation experiment (see FIG. 12)

Kunming mice (more than or equal to 18g (6-8 weeks old)) are selected and injected with melanoma cells through the armpit to establish a melanoma animal model. Melanoma mice were divided into groups containing photosensitizer micelle administration and physiological saline. After grouping, the health condition and the weight of each group of mice are counted, and after no statistical difference exists, subsequent tests can be carried out. The photosensitizer micelle administration group was administered 100mg/kg of photosensitizer micelle PBS solution daily, and the control group was administered physiological saline in equal volume. After 1 week of continuous injection, the mice were coma treated with ether and the distribution of fluorescence intensity was observed under an animal imager.

Claims

1. The invention relates to a preparation method of a targeted antitumor uronic acid carboxyl free polysaccharide derivative micelle.

In the invention, the preparation reaction formula of the micelle of the uronic acid carboxyl free polysaccharide derivative is as follows:

i is uronic acid carboxyl free polysaccharide;

II is a diamino compound;

III is a product of linking uronic acid carboxyl free polysaccharide and diamino compound, abbreviated as HL;

IV is carboxyl free fatty acid;

v is a connecting product of uronic acid carboxyl free polysaccharide, a diamino compound and fatty acid, and is called HLA for short;

VI is a photosensitizer;

HLA-PDT is a nano material formed after photosensitizer is encapsulated in HLA.

2. The present claim 1, wherein the I uronic acid carboxyl free polysaccharide comprises, but is not limited to, the following:

the uronic acid carboxyl free polysaccharide is water-soluble polysaccharide, such as heparin precursor polysaccharide, hyaluronic acid and the like, the polymerization degree n is between 10 and 500, and the molecular weight is between 1KDa and 70 KDa; the purity range is 90% -100%.

3. As described in claim 1, the fatty acid includes saturated fatty acids (caprylic acid, lauric oil, myristic acid, stearic acid, arachidic acid, etc.), monounsaturated fatty acids (myristoleic acid, palmitoleic acid, oleic acid, ricinoleic acid, etc.), polyunsaturated fatty acids (α -linolenic acid, γ -linolenic acid, linoleic acid, etc.).

4. Diamino compounds according to claim 1 comprising basic amino acids (lysine, arginine, histidine), basic amino acid esters (lysine ethyl ester, lysine methyl ester, etc.), amines (ethylenediamine, hexamethylenediamine, etc.).

5. As described in claim 1, the photosensitizers include first generation porphyrin photosensitizers such as hematoporphyrin derivatives, hematoporphyrin ethers and the like, second generation 5-aminolevulinic acid, m-tetrahydroxyphenyl dichloroporphyrin, phthalocyanines, hypericin, hematoporphyrin monomethyl ether and chlorophyll derivatives, pheophorbide a, dihydroporphine, pyropheophorbide a hexyl ether, mono-asparaginyl dihydroporphine and the like.

6. Uronic acid carboxyl free polysaccharide-diamino compound covalent coupling product (HL) according to claim 1, wherein the degree of substitution of the diamino compound is between 0.05 and 0.5; the purity is more than 90 percent; the structure includes but is not limited to the following:

a. b and c are natural numbers larger than zero; r1, R2 and R3 are amino donors; the following possible structures are illustrated by the example of lysine ethyl ester:

(1) r1 ═ OH, R2 ═ lysine ethyl ester, R3 ═ lysine ethyl ester

(2) R1 ═ lysine ethyl ester, R2 ═ OH, R3 ═ lysine ethyl ester

(3) R1 ═ lysine ethyl ester, R2 ═ lysine ethyl ester, R3 ═ OH

(4) R1 ═ OH, R2 ═ OH, R3 ═ lysine ethyl ester

(5) R1 ═ OH, R2 ═ lysine ethyl ester, R3 ═ OH

(6) R1 ═ lysine ethyl ester, R2 ═ OH, R3 ═ OH

The uronic acid carboxyl free polysaccharide-diamino compound covalently coupled product (HL) has a purity of not less than 80%, preferably 85%, particularly preferably 90%, even more preferably 95%. The product HL of the invention has the purity range of 90-98 percent.

7. A covalent coupling product (HLA) of an HL and a fatty acid as claimed in claim 1 wherein the degree of substitution of the fatty acid with respect to the diamino compound is from 0.01 to 1; the purity is more than 90 percent; the grain size of the nano-particles is between 100nm and 500nm, and the zeta potential is between 20mv and 0 mv; for targeting to anti-tumor; its structure includes but is not limited to the following:

a. b and c are natural numbers larger than zero.

8. The HLA photosensitizer-encapsulated product HLA-PDT of claim 1, wherein the photosensitizer encapsulation efficiency is between 20% and 98%, the encapsulation efficiency is between 0.01% and 5%, the nanoparticle size is between 100nm and 500nm, and the zeta potential is between-20 mv and 0 mv; can combine with the photodynamics and is used for targeting anti-tumor.

9. In the present invention, HLA-PDT can be formulated by any conventional method with one or several pharmaceutically acceptable carriers and/or excipients; it has anti-tumor effects, tumor types, including but not limited to the following: melanoma, liver cancer, breast cancer, prostate cancer, and the like.