CN112641757B

CN112641757B - Carrier for transmembrane delivery of molecules and preparation method thereof

Info

Publication number: CN112641757B
Application number: CN201910965853.1A
Authority: CN
Inventors: 周卫; 丁宇; 王翘楚
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2019-10-10
Filing date: 2019-10-10
Publication date: 2023-07-28
Anticipated expiration: 2039-10-10
Also published as: CN112641757A

Abstract

The invention discloses a carrier for transmembrane delivery molecules and a preparation method thereof, and particularly provides an amphiphilic small molecule carrier material: cholesterol phosphate conjugate. The carrier provided by the invention takes cholesterol as a shell, a phosphate group as an inner core, and an additional soluble calcium salt takes the phosphate group in the inner core as a calcified binding site, the adjacent phosphate groups are combined through calcium, and a calcified layer is formed on the surface of the phosphate group of the inner core, so that the carrier structure is firmer. The carrier has high encapsulation efficiency, high drug loading capacity and good stability in the delivery process; meanwhile, the method has higher transmembrane efficiency, and can realize high-efficiency delivery of molecules.

Description

Carrier for transmembrane delivery of molecules and preparation method thereof

Technical field:

the invention belongs to the field of pharmaceutical preparations, relates to a carrier for transmembrane delivery molecules and a preparation method thereof, and in particular relates to an amphiphilic small molecular compound cholesterol phosphate conjugate for preparing the carrier.

The background technology is as follows:

compared with small molecular chemical drugs, the biological macromolecular drugs mainly represented by proteins, polypeptides, antibodies, vaccines, nucleic acids and the like have the advantages of high selectivity, high targeting, high curative effect and the like, play an extremely important role in preventing and treating serious epidemic diseases, and are one of the fields with the most development prospect in drug development in the 21 st century. However, the biomacromolecule has large molecular weight, complex structure, poor in-vivo and in-vitro stability, difficulty in effectively crossing in-vivo biological barriers (cell membranes, gastrointestinal tract barriers, blood brain barriers and the like), inability to effectively enter target sites, and a plurality of challenges and barriers to be solved in-vivo delivery still exist. Moreover, the administration route of the biological macromolecule medicine is mainly intravenous injection or intramuscular injection, the administration mode is inconvenient, and the long-term injection can bring more side effects, so that the compliance of patients is extremely poor. Therefore, by the drug delivery technology, the pharmacological activity of the biomacromolecule drug is greatly reserved, the biomacromolecule drug is efficiently delivered to a target site and released, and the realization of the efficient delivery of the biomacromolecule drug is a hot problem in the current research. In addition, the administration route of biological macromolecules is enriched, and the development of non-injection preparations mainly represented by oral preparations has great clinical application value and market development potential.

The micro-nano drug delivery system such as liposome, microsphere, micelle, microemulsion, vesicle and the like can effectively encapsulate and protect the pharmacological activity of the drug, change the in-vivo distribution condition of the drug, improve the stability and bioavailability of the drug and the like, and is excellent in each link of drug delivery and widely studied. Encapsulation of biomacromolecule drugs in a carrier for delivery is one of the effective strategies to solve biomacromolecule delivery problems. However, there are still a number of challenges and obstacles to the study of high-efficiency delivery vehicles for biomacromolecules.

Hydrophilic drugs represented by biological macromolecules have low entrapment efficiency. Drug delivery vehicles are often used for entrapment and delivery of hydrophobic drugs, and low encapsulation efficiency for hydrophilic drugs, particularly biomacromolecule drugs, is a common problem. For example, micelles can only entrap hydrophobic drugs, and liposomes can entrap hydrophobic and hydrophilic drugs, but the entrapment efficiency for hydrophilic drugs is generally low. Therefore, the construction of the hydrophilic drug, especially the biomacromolecule drug, can effectively encapsulate the hydrophilic drug, and is the basis for realizing the high-efficiency delivery of biomacromolecules.

Stability of the carrier during delivery is an important aspect of efficient delivery of biomacromolecules. The biomacromolecule has poor in vivo stability, and is easily degraded and inactivated by substances such as enzymes in a delivery environment if released in the delivery process. Especially with oral administration, the complex physiological environment of the gastrointestinal tract greatly limits the oral absorption of the biomacromolecule drug, resulting in oral delivery of biomacromolecules currently not being achieved. Liposomes, micelles, microemulsions, and the like, although having good stability in neutral physiological environments, have poor stability when the drug is delivered orally in a severe intragastric acidic environment. Therefore, the construction of a novel carrier capable of efficiently delivering and protecting active drugs has great research value and clinical application value.

Drug passage across cell membranes and other physiological barriers (e.g., gastrointestinal tract barrier, blood brain barrier, blood-fetal barrier, skin barrier) are key points in drug delivery systems. The biological macromolecule medicine has large molecular weight and large polarity, is difficult to cross the biological barrier autonomously, is difficult to enter the target part effectively, prevents the biological macromolecule from exerting the curative effect, and is a technical barrier which is not yet overcome in the biological macromolecule delivery link. The biomacromolecule needs to be transported by a carrier in a transmembrane way so as to improve the transmembrane transport efficiency. Therefore, improving the efficiency of the carrier across the membrane or across the barrier will greatly improve the systemic delivery efficiency of the drug, and constructing a carrier that can promote the absorption of the drug across the membrane or across the barrier can effectively solve this problem.

Currently, the route of administration of biomacromolecule drugs is mostly injection due to limitations of drug delivery technology. For chronic patients (such as diabetics), frequent administration is required for a long time, and side effects such as infection, subcutaneous blood stasis, unstable blood sugar level and the like are easy to occur, so that the compliance of the patients is poor. Oral delivery of biological macromolecules such as insulin is effective in solving the above problems and improving patient compliance. However, the physiological environment of the gastrointestinal tract is complex and severe, the biomacromolecule is easily destroyed and inactivated, and the bioavailability is extremely low due to the characteristics of large molecular weight, strong hydrophilicity and the like, which are difficult to cross the intestinal epithelial barrier, so far, the oral delivery of the biomacromolecule is still a technical barrier which is difficult to overcome. Therefore, the construction of a carrier capable of orally delivering biological macromolecules has great clinical application value and can provide more administration routes for biological macromolecules.

The carrier material is the basis for preparing the carrier and needs to have the following characteristics: safe and nontoxic, and has good biocompatibility and biodegradability; the carrier has certain molecular weight or rigidity to ensure the necessary strength of the carrier; can maintain good stability and other characteristics in preparation, storage and other links. Many polymeric materials have the above properties and are currently the main drug carrier materials. However, the polymer material has a complex structure, the structure after functional modification is more difficult to confirm, and the preparation reproducibility is poor. Some non-endogenous polymer materials, although having good biocompatibility, are foreign matters for organisms, and rejection phenomena generally occur due to instinctive self-protection, and the degree of rejection is different due to individual differences. Therefore, the non-endogenous high polymer material has biocompatibility problems, and the biocompatibility is subject to individual difference, and the problems affect the clinical application and the subsequent development of the drug carrier using the high polymer as the material. Therefore, the structure is simple and clear; the synthesis process is simple, the reproducibility is good, and the novel material which can meet the application requirement of the carrier is very valuable.

Cholesterol is an endogenous substance, vital to vital activities, and is mainly synthesized by the human body and absorbed by the small intestine. The human intestinal tract is responsible for the absorption of large amounts of dietary cholesterol, about 0.5g/d, per day; meanwhile, the small intestine absorbs cholesterol with an efficiency of 50% or more, which indicates that cholesterol is excellent in trans-intestinal epithelial barrier. Furthermore, cholesterol is an important component of cell membranes, some cells (e.g., cardiomyocytes) are rich in cholesterol and have a high affinity for cholesterol, and the cellular uptake of cholesterol-modified vectors or drugs is significantly increased. Thus, cholesterol has a unique potential in either trans-intestinal epithelial barriers or trans-cell membranes. Cholesterol has extremely high hydrophobicity and a rigid structure, can not be independently used as a drug carrier material, and is widely used for regulating the fluidity of liposome membranes. At present, a carrier material with cholesterol modification basically comprises cholesterol serving as a hydrophobic segment, and a hydrophilic segment of a macromolecule is coupled to form an amphiphilic conjugate, wherein the cholesterol plays a role in hydrophobic cohesion in carrier construction. The support material constructed in this way has several problems: most of cholesterol in the material is wrapped in the core part of the carrier, the amount of cholesterol exposed on the surface of the carrier is very small, and the high-efficiency transmembrane property of the cholesterol cannot be realized well; cholesterol is used as an inner core part, and has limited capacity for hydrophilic drugs, and other drug entrapment methods, such as electrostatic adsorption, must be added. Thus, vectors constructed with the highly efficient transmembrane properties of cholesterol have not achieved good results. Currently, few studies have been conducted to distribute cholesterol as much as possible on the surface of a carrier to take advantage of the highly efficient transmembrane properties of cholesterol. The main reasons are as follows: firstly, there are studies showing that excessive intake of cholesterol affects physical health, so that studies on efficient transmembrane characteristics of cholesterol, particularly efficient absorption of cholesterol in the intestinal tract, mainly focus on inhibiting cholesterol absorption, and few studies on promotion of drug, particularly transmembrane absorption of biomacromolecules, by virtue of efficient absorption capacity of cholesterol in the intestinal tract; secondly, cholesterol has extremely strong hydrophobicity, and when the cholesterol is distributed on the surface of the carrier to promote transmembrane or barrier-crossing absorption, the carrier mainly shows hydrophobicity, so that the carrier has poor dispersibility in water and is extremely easy to adhere; at the same time, there is no suitable way to distribute cholesterol on the carrier surface. Therefore, research to facilitate carrier transmembrane delivery by means of efficient transmembrane absorption properties of cholesterol is blank and of great research value.

Phosphate groups are important components of nucleic acids, exist in a large amount in human bodies, and are an endogenous substance harmless to human bodies. The phosphate group has extremely strong hydrophilicity and polarity, and can obviously improve the solubility of the original medicine after being modified in medicine molecules; the modification on the surface of the material can endow the material with an extremely strong polar segment, which is beneficial to the construction of the amphiphilic material. In addition, the phosphate group is modified at the tail end of the molecule, and can form a calcium phosphate structure with soluble calcium salt, so that the calcium phosphate has good adsorption and stabilization effects on proteins.

The invention comprises the following steps:

in order to overcome the defects in the prior art, the invention prepares the cholesterol phosphate conjugate and constructs a preparation method which takes cholesterol as a shell and a phosphate group as a core. In the inner core, calcium ions take phosphate groups as calcified binding sites, adjacent phosphate groups are combined through calcium, and a calcified layer is formed on the surface of the phosphate groups of the inner core, so that the structure of the carrier is firmer, and the stability of the carrier in the delivery process is improved. The carrier has higher encapsulation efficiency and drug loading capacity; the stability of the delivery process is good, and the medicine can be effectively delivered to the target site; the efficiency of the carrier crossing the membrane or the barrier is improved and the availability of the medicine is improved through the structural characteristics of the carrier.

The use of biological endogenous substances as raw materials for preparing carrier materials is an important design concept for obtaining biocompatible materials. Cholesterol is an endogenous substance that is critical to vital activities. Phosphate groups are important components of nucleic acids, exist in a large amount in human bodies, and are an endogenous substance harmless to human bodies. The cholesterol and the phosphate groups are coupled through different connecting groups to form the cholesterol phosphate conjugate with an amphipathic structure. The carrier material is safe and nontoxic, and has good biocompatibility; the structure is simple and clear, the synthesis process is simple, the repeatability is good, and the feasibility of subsequent development is realized.

The characteristics of the carrier material of the cholesterol phosphate conjugate are mainly derived from the characteristics of the main constituent elements. Cholesterol is a conjugate of cyclopentane polyhydrophenanthrene, has a hydrophobic rigid cyclic backbone, is a typical van der Waals force group, and is introduced into a carrier material, so that van der Waals force will become one of main driving forces for carrier assembly. Cholesterol has a melting point of 148.5 ℃ and is higher than that of common linear alkyl alcohol, even exceeds polyethylene, and the hydrophobic force between molecules is strong. Therefore, cholesterol can be used as a hydrophobic part to effectively improve the aggregation stability of amphiphilic molecules in the solution. Meanwhile, due to the unique structural characteristics of cholesterol, space dislocation exists in the molecular stacking process, a gel structure can be formed in a proper solvent environment, and the cholesterol is taken as a shell, so that the problem that medicines leak from an inner core in the preparation and application processes can be effectively reduced. The phosphate group is a group with great polarity, is modified on the surface of a medicine or a material, can improve the hydrophilicity of the original medicine or the material, can endow the raw material with an extremely strong polar segment, is beneficial to the construction of an amphiphilic material, and endows the material with the capability of being assembled into a carrier. In addition, modification of the phosphate groups at the ends of the molecule provides binding sites for initial calcification of the soluble calcium salt, binding of adjacent phosphate groups by calcium, forming (CaHPO ₄ ) ₂ Dimer, further coagulates to form Ca ₉ (PO ₄ ) ₆ Forming a calcified layer on the surface of the nuclear phosphate group, wherein the calcified layer is formed to make the carrier structure more stable。

Cholesterol phosphate conjugate takes cholesterol as a hydrophobic end (small polarity), a phosphate group is taken as a hydrophilic end (large polarity), linear molecules are utilized to couple the cholesterol and the phosphate group to form an amphiphilic small molecular compound with large difference level of hydrophilic and hydrophobic (polarity) at two ends, in the process of preparation by adopting an inverse emulsion method, the hydrophobic (small polarity) cholesterol is positioned in an oil phase to form a shell, the hydrophilic (large polarity) phosphate group is positioned in a water phase to form an inner core, and a carrier with a shell-core structure and cholesterol as a shell and the phosphate group in the inner core is determined. The additional soluble calcium salt takes the phosphate groups in the core as calcified binding sites, and the adjacent phosphate groups are bound by calcium to form (CaHPO ₄ ) ₂ Dimer, further coagulates to form Ca ₉ (PO ₄ ) ₆ And forming a calcified layer on the surface of the nuclear phosphate group, wherein the formation of the calcified layer enables the carrier structure to be more stable. The carrier inner core structure takes a calcified layer formed by combining a phosphate group and phosphoric acid-calcium as a main component, the phosphate group provides a hydrophilic environment, the calcified layer has adsorption and stabilization effects on proteins, and the phosphate group and the calcified layer provide a friendly storage environment for biological macromolecular medicaments together, so that the medicaments can stably exist in the inner core. Meanwhile, cholesterol is used as a shell to form a layer of extremely hydrophobic barrier, and the barrier is formed by interlacing rigid structures of cholesterol to form a rigid shell which cannot be formed by chain segment materials with the same molecular weight, so that the carrier has higher encapsulation rate and good delivery stability.

In order to solve the problems, the invention adopts the following technical scheme:

the invention provides a carrier for transmembrane delivery of molecules, which is characterized in that the basic composition and proportion of the carrier are as follows:

(a) Cholesterol phosphate conjugate: 49-99%, the specific molecular structure is as follows:

(b) Soluble calcium salt: 0 to 30 percent,

(c) Biologically active molecules: 0.1 to 30 percent,

(d) pH regulator: a proper amount of the components;

the cholesterol phosphoric acid conjugate (a) has a molecular structure in which a connecting group R group is The m and n range from 0 to 18, and the R ₁ ，R ₂ ，R ₃ ，R ₄ Can be one or a combination of a plurality of hydrogen atoms, hydroxyl, carboxyl, amino, halogen, alkyl with 1-4 carbon atoms, alkoxy with 1-4 carbon atoms, nitryl and acyl with 1-3 carbon atoms.

The preparation process of the carrier is as follows:

1) Dissolving the cholesterol phosphate conjugate in chloroform to form an oil phase;

2) Dissolving bioactive molecules in an aqueous solution to form an inner aqueous phase 1; dissolving soluble calcium salt in water solution to form an inner water phase 2, and regulating the pH of the inner water phase by using a pH regulator;

3) Mixing the inner water phase 1 and the inner water phase 2 with the oil phase to form water-in-oil emulsion, removing chloroform, and drying to remove residual water to form a carrier A.

A carrier for transmembrane delivery of molecules, wherein the carrier is further optimised for its basic composition and percentages of the components as follows:

(a) Cholesterol phosphate conjugate: 49 to 98 percent,

(b) Soluble calcium salt: 0 to 30 percent,

(c) Biologically active molecules: 0.1 to 30 percent,

(d) pH regulator: a proper amount of the water-soluble polymer,

(e) Biological dispersing agent: 0 to 51 percent,

(f) Lyoprotectant: 0-10%;

the preparation process of the carrier is as follows:

2) Dissolving bioactive molecules in an aqueous solution to form an inner water phase 1, dissolving soluble calcium salt in the aqueous solution to form an inner water phase 2, and regulating the pH of the inner water phase by using a pH regulator;

3) Mixing the inner water phase 1 and the inner water phase 2 with the oil phase to form water-in-oil emulsion, removing chloroform, and drying to remove residual water to obtain a carrier A;

4) Dispersing the carrier A in chloroform, dissolving a biological dispersing agent in an organic solvent or water, mixing with the chloroform solution of the carrier A and the external water to form an oil-in-water emulsion, and removing the organic reagent to form a carrier B;

5) Adding proper amount of freeze-drying protective agent, and freeze-drying to obtain the solid preparation.

The carrier B takes the whole carrier A as an inner core, and the biological dispersing agent is distributed on the periphery of the carrier A to form a hydrophilic outer layer, so that the aim of good dispersion in aqueous solution is fulfilled.

The carrier B may be a liquid preparation using water as a dispersion medium as a final preparation, or may be a lyophilized preparation as a final preparation.

(a) Cholesterol phosphate conjugate: 49 to 98 percent,

(b) Soluble calcium salt: 0 to 30 percent,

(c) Phosphate: 0 to 30 percent,

(d) Biologically active molecules: 0.1 to 30 percent,

(e) pH regulator: a proper amount of the water-soluble polymer,

(f) Biological dispersing agent: 0 to 51 percent,

(g) Lyoprotectant: 0-10%;

the (c) phosphate is added in the form of an internal aqueous phase.

A carrier for transmembrane delivery of molecules, characterized in that the soluble calcium salt is one or a combination of several of calcium chloride and calcium nitrate; the phosphate is one or a combination of more of phosphoric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, polyphosphoric acid and sodium phosphate;

the soluble calcium salt takes phosphate groups in the inner core as calcified binding sites, and adjacent phosphate groups are combined through calcium to form (CaHPO ₄ ) ₂ Dimer, further coagulates to form Ca ₉ (PO ₄ ) ₆ Forming a calcified layer on the surface of the nuclear phosphate group; the structure of the fixed layer can be further adjusted to one or a combination structure of a plurality of octacalcium phosphate, alpha-calcium triphosphate, beta-calcium triphosphate, amorphous calcium phosphate and hydroxyapatite.

The phosphate can accelerate the formation of the calcified layer of the inner core, so that the calcified layer is firmer.

A carrier for transmembrane delivery of molecules, wherein the bioactive molecule is a hydrophilic bioactive molecule, including a biomacromolecule drug, a small molecule drug, preferably a biomacromolecule drug;

the biological macromolecule medicine is selected from one or a combination of several of the following medicines but not limited to: polypeptide protein drugs selected from one or more of octreotide, somatostatin, leuprorelin acetate, calcitonin, insulin, thymopentin, tecokinin acetate, buserelin, exenatide, triptorelin acetate, salmon calcitonin, bovine serum albumin, ovalbumin, parathyroid hormone, prolactin, oxytocin, calcitonin, human growth hormone, bovine growth hormone, porcine growth hormone, ghrelin, GLP-1, PYY36, oxyntomodulin, GLP-2, glucagon, epidermal growth factor, interferon alpha; a genetic drug selected from one or a combination of a plurality of plasmid DNA, antisense oligonucleotide, small interfering RNA and Bei Faxi Ni; a cloned antibody drug selected from one or a combination of several of bevacizumab, ranibizumab and ramucirumab;

the insulin includes, but is not limited to, one or a combination of several of normal insulin, recombinant insulin, protamine insulin, insulin aspart, insulin glargine, insulin lispro, and insulin deglutition.

A carrier for transmembrane delivery of molecules, characterized in that the pH-modifying agent is one or a combination of several of hydrochloric acid, acetic acid, phosphoric acid, amino acids, sodium hydroxide, triethanolamine, ethanolamine;

the functions of the pH regulator include: adjusting the pH of the inner water phase 1 to dissolve bioactive molecules; the inner water phase 1 and the inner water phase 2 are regulated to obtain proper pH of the inner core, so that the environment of the inner core is more beneficial to calcification of calcium and phosphate groups, the formation of calcified layers is facilitated, and the stability of the carrier is enhanced; the pH range of the inner core is 2-10.

A carrier for transmembrane delivery of molecules, wherein the biodispersant is selected from one or more of phospholipids, cholate, polyvinyl alcohol, poloxamer, tween, hyaluronic acid, chitosan, sodium polyglutamate, vitamin E polyethylene glycol succinate;

the phospholipid may be a natural phospholipid or a synthetic phospholipid, wherein the natural phospholipid comprises one or a combination of several of Phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidic Acid (PA), phosphatidylethanolamine (PE), phosphatidylserine (PS) and Phosphatidylinositol (PI); synthetic phospholipids include, but are not limited to: dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), dipalmitoyl phosphatidylserine (DPPS), distearoyl lecithin (DSPC), dilauroyl Lecithin (DLPC), dimyristoyl lecithin (DMPC), dioleoyl phosphatidylserine (DOPS), diterucyl lecithin (DEPC), didecanoyl lecithin (DDPC), distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG 2000), dipalmitoyl phosphatidylethanolamine-methoxypolyethylene glycol 2000 (DPPE-MPEG 2000), dipalmitoyl phosphatidylethanolamine-methoxypolyethylene glycol 5000 (DPPE-MPEG 5000), or a combination of one or more thereof;

the cholate comprises one or a combination of several of sodium cholate, deoxysodium cholate, sodium taurocholate and sodium glycocholate;

the polyvinyl alcohol is preferably PV17-88;

the poloxamer is selected from one or a combination of a plurality of F68, F127 and F87;

the Tween is selected from one or more of Tween-20, tween-40, tween 65, tween 80 and Tween 85;

the hyaluronic acid is selected from one or a combination of several of 10 kDa-400 kDa;

the chitosan is selected from one or a combination of more than 80 percent of deacetylation degree and molecular weight of 5 kDa-200 kDa.

A carrier for transmembrane delivery of molecules, characterized in that the lyoprotectant is selected from one or a combination of several of mannitol, glucose, lactose, sucrose, trehalose, maltose.

Preferably, the lyoprotectant is selected from one or a combination of a plurality of trehalose and mannitol.

A carrier for transmembrane delivery of molecules, characterized in that the organic reagent in preparation process (4) may be one or a combination of several of chloroform, dichloromethane, diethyl ether, petroleum ether, ethyl acetate, methanol, ethanol, cyclohexane.

A carrier for transmembrane delivery of molecules, wherein the carrier a has a core-shell structure, wherein cholesterol stacked on each other is used as a shell, a phosphate group, a soluble calcium salt, a phosphate, a bioactive molecule and the like are used as cores, and wherein the phosphate group in the cores is used as a calcified binding site for the soluble calcium salt, and adjacent phosphate groups are bound by calcium to form (CaHPO ₄ ) ₂ Dimer, further coagulates to form Ca ₉ (PO ₄ ) ₆ Forming a calcified layer on the surface of the nuclear phosphate group, wherein the calcified layer is formed to enable the carrier structure to be more stable, so that the stability of the carrier in the delivery process is improved; the addition of phosphate can accelerate the formation of the calcified layer of the inner core, so that the calcified layer is firmer.

The carrier B takes the carrier A as an inner core, the biological dispersing agent is dispersed on the periphery of the carrier A to form a hydrophilic outer layer, and the dispersibility in aqueous solution is good.

The one carrier for transmembrane delivery molecules may be present in the form of carrier a or in the form of carrier B.

The carrier A is mainly used for oral drug delivery, and is dispersed into micro-nano particles to be absorbed under the action of an emulsifier in the gastrointestinal tract; the carrier B may be used for oral, intravenous, subcutaneous or transdermal drug delivery.

A carrier for transmembrane delivery of molecules, characterized in that the carrier has a particle size in the range of 1nm to 2000nm.

Preferably, the particle size of the carrier ranges from 50nm to 500nm.

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

1. the cholesterol phosphate conjugate synthesized by the invention takes endogenous substances cholesterol and phosphate groups as raw materials, has high safety and good biocompatibility, has no toxic or side effect after long-term application, and has great development potential.

2. The cholesterol phosphate conjugate synthesized by the invention has the advantages of simple and definite structure, simple synthesis process, good reproducibility and easy industrial transformation.

3. The cholesterol phosphate conjugate is used as a drug carrier for the first time to successfully prepare the carrier with a shell-core structure, the carrier takes cholesterol as a shell, phosphate groups, soluble calcium salt, bioactive molecules and the like as an inner core, the phosphate groups at the tail ends of carrier materials in the inner core and the soluble calcium salt can form a calcium phosphate structure, and the formation of the calcium phosphate structure in the inner core can enable the structure of the carrier to be firmer, so that the stability of the carrier in the delivery process is improved.

4. The carrier provided by the invention can effectively encapsulate and deliver hydrophilic drugs, especially biomacromolecule drugs, and has higher encapsulation efficiency and drug loading capacity.

5. The carrier provided by the invention has better stability in a delivery environment, and can effectively protect the drug from reaching a target part in the form of an integral carrier. Particularly in the gastric acid environment, the drug is hardly released, which is a significant advantage for oral drug delivery.

6. The carrier provided by the invention effectively utilizes the in-vivo absorption mode of cholesterol, has higher cell uptake efficiency compared with free medicine, and can effectively improve the medicine absorption efficiency.

Drawings

FIG. 1 shows the Tyndall phenomenon in chloroform for the support material synthesized in example 2 and the support prepared in example 3 (A: the material synthesized in example 2; B: the support prepared in example 3).

FIG. 2 is a view showing the microstructure of the support prepared in example 3 after being dispersed in chloroform by using a transmission electron microscope.

FIG. 3 is an observation of microstructure of the aqueous carrier solution prepared in example 6 using a transmission electron microscope.

FIG. 4 shows cytotoxicity of the vectors prepared in example 7 at different vector B concentrations in caco-2 cells.

Figure 5 is an in vitro release profile study of vector a in a gastrointestinal pH environment.

FIG. 6 is an in vitro release profile study of vector B in the pH environment of the gastrointestinal tract.

FIG. 7 is a graph showing the uptake of vector B on caco-2 cells using flow cytometry.

FIG. 8 is a graph showing the uptake of vector B on caco-2 cells using confocal microscopy.

Detailed Description

The foregoing of the invention will be further illustrated by the following examples, which are provided to illustrate the invention and not to limit the scope thereof, and the techniques implemented based on the foregoing of the invention are within the scope of the invention.

Example 1

By diethylene glycolAs a linking group, cholesterol and phosphate groups are coupled to form a new carrier material, having the structure:

the specific synthesis process is as follows: after 10g of cholesterol and 8g of p-toluenesulfonyl chloride are dissolved in 50 ml of pyridine, stirring reaction is carried out for 24 hours at room temperature, the reaction is tracked by using a thin layer chromatography, and a product A is obtained through separation and purification. Taking 5g of a product A, 20g of diethylene glycol, dissolving the product A with 90mL of epoxy hexacyclic ring, heating and refluxing the mixture at 120 ℃ for reaction for 10 hours, tracking the reaction by using a thin layer chromatography, and separating and purifying the product A to obtain a product B. 1.6g of a product B is taken, 6g of tetrabutyl disodium hydrogen phosphate is added, after the product B is dissolved by 16mL of chloroform, 2mL of trichloroacetonitrile is added under the condition of ice bath stirring, after the reaction is carried out for 30min, the final product is obtained through separation and purification, and the product is a white solid.

The linking group of example 1 was replaced with: glycine, lysine, and the like, the synthesis of the support material can still be carried out according to the synthesis procedure of example 1, obtaining the product.

Example 2

With 1, 6-hexanediolAs a linking group, cholesterol and phosphate groups are coupled to form a new carrier material, having the structure:

the specific synthesis steps are as follows: 5g of cholesterol and 4g of p-toluenesulfonyl chloride are taken, dissolved in 30 ml of pyridine, stirred at room temperature for reaction for 24 hours, tracked by thin layer chromatography, and separated and purified to obtain a product A. Taking 5g of a product A, 20g of hexanediol, dissolving the product A with 90mL of epoxy hexacyclic ring, heating and refluxing the mixture at 120 ℃ for reaction for 10 hours, tracking the reaction by using a thin layer chromatography, and separating and purifying the product A to obtain a product B. 0.9g of a product B is taken, 6g of tetrabutyl disodium hydrogen phosphate is added, after being dissolved by 8mL of chloroform, 1.1mL of trichloroacetonitrile is added under the condition of ice bath stirring, after 30min of reaction, the final product is obtained by separation and purification, and the product is a white solid.

The linking group of example 2 was replaced with: the synthesis of the support material can still be carried out according to the synthesis procedure of example 4, obtaining the product.

Example 3

100mg of the final product of example 2 was weighed and dissolved in 16mL of chloroform to form an oil phase; weighing 20mg of triptorelin acetate, and dissolving in 1200 mu L of purified water to form an inner water phase 1; 600. Mu.L of 200mM calcium nitrate solution, pH 5.0, formed an internal aqueous phase 2; and simultaneously adding the inner water phase 1 and the inner water phase 2 into the oil phase, forming water-in-oil emulsion by ultrasonic probe, removing chloroform under reduced pressure, and drying to remove residual water to form the carrier.

The preparation is analyzed by high performance liquid chromatography, and the drug loading rate and the encapsulation efficiency of the preparation are respectively monitored according to the following formulas. The drug loading and encapsulation efficiency of the carriers prepared in example 3 are shown in table 1.

Table 1 drug loading and encapsulation efficiency for example 3

Example 4

Based on the preparation procedure of example 3, carrier A was prepared by varying several prescription parameters such as drug variety, oil-water ratio, drug amount, calcium ion concentration, etc., with encapsulation rate > 85% as the preferred prescription, and the prescription composition of the specific examples is shown in Table 2.

Table 2 list of prescriptions

Example 5

The carriers prepared in example 3 and example 4 are the carrier A of the present invention, wherein the carrier A uses cholesterol stacked on each other as a shell, uses a phosphate group, a soluble calcium salt, a bioactive molecule and the like as a core, and is a typical shell-core structure. The soluble calcium salt takes the phosphate groups in the core as calcified binding sites, and the adjacent phosphate groups are combined by calcium to form (CaHPO ₄ ) ₂ Dimer, further coagulates to form Ca ₉ (PO ₄ ) ₆ Forming a calcified layer on the surface of the nuclear phosphate group, wherein the calcified layer is formed to enable the carrier structure to be more stable, so that the stability of the carrier in the delivery process is improved; the structure of the calcified layer can be further adjusted to one or a combination structure of a plurality of octacalcium phosphate, alpha-calcium triphosphate, beta-calcium triphosphate, amorphous calcium phosphate and hydroxyapatite; phosphate is added into the inner water phase according to the requirement, so that the formation of the inner core calcified layer can be quickened, and the calcified layer is firmer.

Example 5 was primarily aimed at characterizing the support A. Judging the structure formation of the carrier A by using the Tyndall phenomenon; measuring the particle size and distribution of the carrier A by using a dynamic light scattering method; the microstructure of the support a was observed using a transmission electron microscope. Dissolving 10mg of the final product synthesized in the example 2 in 10mL of chloroform, and placing a proper amount of the solution into a penicillin bottle, wherein the mark is A; 10mg of the carrier prepared in example 3 was dispersed in 10mL of chloroform solution, and an appropriate amount of the solution was taken in a penicillin bottle and labeled B. The laser pen is used for passing the laser beam through the liquid, the bottle A has no Tyndall phenomenon, the bottle B has the Tyndall phenomenon, and the result is shown in figure 1. The tyndall phenomenon has the effect of distinguishing true solution from colloidal solution, and bottle B has tyndall phenomenon, indicating the structural formation of carrier a.

The particle size of the carrier prepared in example 3 was measured to be 90.99 in chloroform by a dynamic light scattering method; the PDI was 0.244.

The carrier prepared in example 3 was dispersed in chloroform, the chloroform solution of the carrier was dropped on a copper mesh with a pure carbon film attached thereto, the chloroform solution was rapidly absorbed to prevent the carbon film from being excessively dissolved, the excessive chloroform solution was absorbed by filter paper, the negative dyeing was performed with 0.5% phosphotungstic acid, the excessive dye solution was gently absorbed by filter paper, and the natural volatilizing was performed. The appearance of the carrier a was observed by a Transmission Electron Microscope (TEM), and the result is shown in fig. 2. The results showed that carrier a was dispersible in chloroform and spherical in shape.

Example 6

50mg of the support material synthesized in example 2 was dissolved in 6mL of chloroform to form an oil phase; dissolving 5mg of liraglutide in 800 μl of purified water to form an inner aqueous phase 1; 200. Mu.L of calcium chloride (250 mM, pH 6.0) was dissolved in the aqueous solution to form an inner aqueous phase 2; adding the inner water phase 1 and the inner water phase 2 into chloroform solution in batches to form water-in-oil emulsion, removing chloroform under reduced pressure, and drying to remove residual water to obtain a carrier A; 20mg of carrier A was dispersed in 3mL of chloroform, 20mg of egg yolk lecithin was added thereto and dissolved, and purified water was added dropwise to the above chloroform solution to form an oil-in-water emulsion, and chloroform was removed under reduced pressure to form carrier B. The particle size of the obtained product is between 100 and 200nm, the particle size distribution of the particles is narrow and is not sticky, the particle size is detected by a dynamic light scattering method, the particle size is 112.2nm, and the PDI is 0.219.

The microstructure of the particles prepared in example 6 was observed by means of a transmission electron microscope. The carrier solution of example 6 was dropped on a copper mesh, after 3min, the excess solution was blotted with filter paper, and the negative dyeing was performed with 0.5% phosphotungstic acid, and the excess dye solution was gently blotted with filter paper and naturally volatilized. As a result, as shown in FIG. 3, the particle size of the sample was about 100nm, and the sample was uniformly distributed in the form of a sphere.

The above-mentioned biodispersants may be replaced with single or mixed components as provided in Table 3, and also a preparation having good water dispersibility may be obtained.

TABLE 3 composition and ratio of biological dispersants

Example 7

50mg of the support material of example 2 was dissolved in 6mL of chloroform to form an oil phase; 5mg of insulin was dissolved in 800 μl of aqueous hydrochloric acid (ph=2) to form an inner aqueous phase 1; 200. Mu.L of calcium chloride (250 mM, pH 8.0) was dissolved in the aqueous solution to form an inner aqueous phase 2; adding the inner water phase 1 and the inner water phase 2 into chloroform solution in batches to form water-in-oil emulsion, removing chloroform by reduced pressure rotary evaporation, and removing residual water by freeze drying to obtain a carrier A; 20mg of carrier A was dispersed in 3mL of chloroform, 20mg of egg yolk lecithin was added thereto and dissolved, 10mg of sodium glycocholate was dissolved in 100. Mu.L of methanol, and the three organic solutions were thoroughly mixed and then mixed with purified water to form an oil-in-water emulsion, and after removing the organic reagent, carrier B was formed.

Cytotoxicity test. MTT experiments were used to investigate the cytotoxicity of different concentrations of vector B in caco-2 cells. Caco-2 cells were grown at 1X 10 ⁴ Density of individuals/wells was seeded in 96-well plates; at 37℃with 5.0% CO ₂ After 1 day of culture in an incubator of (C). Each well was washed 2 times with PBS, and after incubation for 12 hours, carrier B prepared in example 9 was added containing 1, 10, 100 and 1000. Mu.g/mL, and after removal of the preparation, 100. Mu.L of MTT solution (0.5 mg/mL) was added and incubated in an incubator for 4 hours. The MTT solution was then removed and 100. Mu.L of DMSO was added to dissolve the resulting formazan crystals. The absorbance A of the samples was measured at 490nm using an enzyme-labeled instrument, cells treated with a blank medium were used as a control, no-cell wells were used as a blank, and the number of repetitions was set to 5 for each sample. Cell activity was calculated according to the following formula:

as a result, as shown in FIG. 4, the carrier B was found to have good safety.

Example 8

Following the procedure of example 3, using insulin as a model drug, a sample was prepared and examined for release behavior in the gastrointestinal pH environment, which sample was carrier a. The specific method is as follows: about 10mg of the preparation is weighed and placed in a penicillin bottle, 10mL of hydrochloric acid solution with pH of 1.2 and HEPES buffer with pH of 6.8 are respectively added, the pH 1.2 solution group is sampled at 1 hour and 2 hours and the same amount of blank release medium is added under the shaking condition of 50rpm at 37 ℃, and the pH6.8 solution group is sampled at 0.5 hour, 1 hour, 2 hour, 4 hour and 6 hours and the same amount of blank release medium is added. Samples were collected by centrifugation and monitored by HPLC to give a cumulative release profile of drug as shown in figure 5. From the results, the preparation can be kept stable under the pH environment of the gastrointestinal tract, the leakage amount of the medicine is low, the medicine can be effectively protected, and the delivery efficiency is improved.

Example 9

A sample was prepared as in example 7 and examined for its release behavior in the pH environment of the gastrointestinal tract, this sample being carrier B. The specific method is as follows: taking a proper amount of preparation, placing the preparation into a penicillin bottle, respectively dispersing the preparation into a hydrochloric acid solution with the pH of 1.2 and a HEPES buffer with the pH of 6.8, and taking samples of the solution group with the pH of 1.2 at 1 and 2 hours at the timing and taking samples of the solution group with the pH of 6.8 at 0.5, 1, 2, 4 and 6 hours at the timing under the shaking condition of 50rpm at 37 ℃. Samples were collected by centrifugation and monitored by HPLC, the results of which are shown in FIG. 6. As a result, it was found that the carrier a was sufficiently dispersed with a biological dispersant such as phospholipid or cholate, and the stability of the preparation in the pH environment of the gastrointestinal tract was not affected, and the inner core structure of the carrier a was not destroyed.

Example 10

Fluorescein isothiocyanate-labeled insulin (FITC-INS) was used as a model drug for the evaluation of vector cell levels, and a sample was prepared as in example 7 and designated NCs as vector B. The ability of the vector to cross cell membranes was examined using a cell uptake assay.

Inoculating caco-2 cells in a logarithmic growth phase into a 24-pore plate, wherein the number of the caco-2 cells is 5 ten thousand per pore; at 37℃with 5.0% CO ₂ Is cultured in an incubator for 3 days. The culture solution is firstly sucked off before the experiment, washed twice by PBS, and added with free FITC-INS and drug carrier with the concentration corresponding to the free drugThe preparation was washed three times with PBS after 3h incubation in an incubator, cells in the well plate were sufficiently digested with pancreatin, the cells were collected with PBS, and then the uptake of the drug was measured using flow cytometry, and as a result, as shown in FIG. 7, the cell uptake of the preparation group was significantly increased over that of the free drug.

Cell uptake was observed using confocal microscopy. Inoculating caco-2 cells in a logarithmic growth phase into a 24-pore plate, wherein the number of the caco-2 cells is 5 ten thousand per pore; at 37℃with 5.O% CO ₂ Is cultured in an incubator for 3 days. The culture solution was first aspirated, washed twice with PBS, free FITC-INS and drug-loaded formulation corresponding to the concentration of free drug were added, incubated in an incubator for 3h, washed three times with PBS, then fixed with 4% paraformaldehyde for 15min, discarded and washed three times with PBS, finally stained with DAPI for 15min, and cell uptake was observed under confocal microscopy, as shown in FIG. 8.

While the foregoing is directed to embodiments of the present invention, other and further details of the invention may be had by the present invention, it should be understood that the foregoing description is merely illustrative of the present invention and that no limitations are intended to the scope of the invention, except insofar as modifications, equivalents, and improvements may be made within the spirit and principles of the invention.

Claims

1. A carrier for transmembrane delivery of molecules, wherein the carrier comprises the following basic components in percentage by weight:

(b) Soluble calcium salt: 0 to 30 percent, but not 0 percent,

(c) Biologically active molecules: 0.1 to 30 percent,

(d) pH regulator: a proper amount of the components;

the cholesterol phosphate conjugate (a) has a linking group R in the molecular structureThe radicals being The ranges of m and n are 0 to 18, but neither m nor n is 0, R is ₁ 、R ₂ 、R ₃ 、R ₄ Is one or the combination of a plurality of hydrogen atoms, hydroxyl, carboxyl, amino, halogen, alkyl with 1-4 carbon atoms, alkoxy with 1-4 carbon atoms and acyl with 1-3 carbon atoms;

the preparation process of the carrier is as follows:

2. A carrier for transmembrane delivery of molecules according to claim 1, wherein the carrier comprises the following composition and percentages of the components:

(a) Cholesterol phosphate conjugate: 49 to 98 percent,

(b) Soluble calcium salt: 0 to 30 percent, but not 0 percent,

(c) Biologically active molecules: 0.1 to 30 percent,

(d) pH regulator: a proper amount of the water-soluble polymer,

(e) Biological dispersing agent: 0-51% of biological dispersing agent selected from one or two of phospholipid and cholate;

(f) Lyoprotectant: 0-10%;

the preparation process of the carrier is as follows:

4) Dispersing the carrier A in chloroform, dissolving a biological dispersing agent in an organic solvent or water, mixing with the carrier A chloroform solution and external water to form an oil-in-water emulsion, and removing the organic reagent to form a carrier B;

3. A carrier for transmembrane delivery of molecules according to claim 2, wherein the carrier comprises the following composition and percentages of the components:

(a) Cholesterol phosphate conjugate: 49 to 98 percent,

(b) Soluble calcium salt: 0 to 30 percent, but not 0 percent,

(c) Phosphate: 0 to 30 percent,

(d) Biologically active molecules: 0.1 to 30 percent,

(e) pH regulator: a proper amount of the water-soluble polymer,

(f) Biological dispersing agent: 0 to 51 percent,

(g) Lyoprotectant: 0-10%;

the (c) phosphate is added in the form of an internal aqueous phase 1.

4. A carrier for transmembrane delivery molecules according to claim 3, wherein the soluble calcium salt is one or a combination of calcium chloride and calcium nitrate; the phosphate is one or a combination of several of disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate and sodium phosphate.

5. The carrier for transmembrane delivery of molecules of claim 1, wherein the bioactive molecule is a hydrophilic bioactive molecule, including a biomacromolecule drug, a small molecule drug;

the biological macromolecule medicine is selected from one or a combination of several of the following medicines: polypeptide protein drugs selected from octreotide, somatostatin, leuprorelin acetate, calcitonin, insulin, thyme pentapeptide, tecatide acetate, buserelin, exenatide, triptorelin acetate, bovine serum albumin, ovalbumin, parathyroid hormone, prolactin, oxytocin, human growth hormone, bovine growth hormone, porcine growth hormone, ghrelin, GLP-1, PYY36, oxyntomodulin, GLP-2, glucagon, epidermal growth factor, interferon alpha; a genetic drug selected from one or a combination of several of plasmid DNA, antisense oligonucleotide and small interfering RNA; a monoclonal antibody drug selected from one or a combination of several of bevacizumab, ranibizumab and ramucirumab;

the insulin comprises one or more of common insulin, recombinant insulin, protamine insulin, insulin aspart, insulin glargine, insulin lispro and insulin deglutition.

6. A carrier for transmembrane delivery molecules according to claim 2, wherein the pH modifier is one or a combination of several of hydrochloric acid, acetic acid, phosphoric acid, amino acids, sodium hydroxide, triethanolamine, ethanolamine.

7. A carrier for transmembrane delivery of molecules according to claim 2,

the phospholipid is natural phospholipid or synthetic phospholipid, wherein the natural phospholipid comprises one or more of Phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidic Acid (PA), phosphatidylethanolamine (PE), phosphatidylserine (PS) and Phosphatidylinositol (PI); synthetic phospholipids include one or more of dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), dipalmitoyl phosphatidylserine (DPPS), distearoyl lecithin (DSPC), dilauroyl Lecithin (DLPC), dimyristoyl lecithin (DMPC), dioleoyl phosphatidylserine (DOPS), dithiinyl lecithin (DEPC), didecyl lecithin (DDPC), distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG 2000), dipalmitoyl phosphatidylethanolamine-methoxypolyethylene glycol 2000 (DPPE-MPEG 2000), dipalmitoyl phosphatidylethanolamine-methoxypolyethylene glycol 5000 (DPPE-MPEG 5000);

the cholate is one or a combination of more of sodium cholate, sodium deoxycholate, sodium taurocholate and sodium glycocholate.

8. A carrier for transmembrane delivery of molecules according to claim 2, wherein the lyoprotectant is selected from one or a combination of several of mannitol, glucose, lactose, sucrose, trehalose, maltose.

9. A carrier for transmembrane delivery of molecules according to claim 2, wherein the organic agent in preparation 4) is one or a combination of several of chloroform, dichloromethane, diethyl ether, petroleum ether, ethyl acetate, methanol, ethanol, cyclohexane.