CN101254207A - Iron supplement liposomal iron and preparation method thereof - Google Patents

Abstract

The invention prepares liposomal iron by a rotating thin film-ultrasonic method, and obtains a novel iron supplement. Lecithin and cholesterol ethanol solution are prepared by dispersing into PBS iron solution under specific conditions, and its composition is expressed as: heme iron and/or inorganic iron: cholesterol: lecithin=(0.1～ 200): (1-10): (10-100). The iron supplement liposome iron of the invention is non-toxic and non-immunogenic, and as a drug carrier, it has targeting properties. Clinical use can reduce drug dosage, improve absorption efficiency, reduce toxicity, greatly increase the solubility and stability of heme iron, and improve bioavailability. The rotating thin-film ultrasonic method adopted in the invention can form stable liposomes with high encapsulation efficiency, the method can effectively remove the residue of organic matter (ethanol), the process conditions are easy to control, and the operation is simple and convenient.

Description

Iron supplement liposomal iron and preparation method thereof

technical field

The invention relates to a new iron supplement, in particular liposomal iron and a preparation method thereof, belonging to the technical field of biological science.

Background technique

Iron-deficiency anemia is one of the most common diseases in my country and the world. There are various kinds of medicines and nutrients for iron supplementation. However, since the absorption mechanism of intestinal iron has not been elucidated, people have developed a variety of Iron supplements have a great blindness, the effect is not ideal, and some are also very toxic, poor in taste and irritating to the digestive tract. With the understanding of intestinal iron absorption mechanism in recent years, we know the intestinal iron absorption mechanism and regulation pathway, which provides a theoretical basis for the development of new iron supplements. Studies have shown that heme iron is a kind of biological iron, which has a higher bioavailability than other iron supplements, can be directly absorbed by intestinal mucosal cells, and has mild digestive tract irritation symptoms. However, because it is composed of porphyrin and a molecule of ferrous iron to form a porphyrin compound, its structure is unstable, easy to oxidize, insoluble in water, and has a special smell. More importantly, if the gene for heme iron absorption is mutated or regulated by low expression, it will prevent and inhibit the absorption of heme iron. These characteristics greatly limit its application in industries such as food and health products, especially as Oral health products, the above characteristics make their development and application subject to certain restrictions. Other inorganic iron supplements such as ferrous sulfate and ferric citrate can stimulate the digestive tract, and improper use can also cause symptoms of poisoning, and have no relief effect on inflammatory anemia and anemia caused by genetic causes.

In recent years, liposomes have become a new type of biopharmaceutical carrier. Liposome is a closed vesicle composed of a lipid bilayer with an aqueous phase inside, ranging in size from tens of nanometers to tens of microns. Various substances can be wrapped in the aqueous phase and membrane of the liposome. For liposomes composed of natural membrane components, the bilayer structure of the liposome membrane is in principle the same as that of the natural cell membrane, forming multilayered vesicles when dispersed in water, and each layer is a lipid bilayer, each layer Separated by water, it is suitable for absorption and degradation in organisms. Liposomes are non-toxic and non-immunogenic, and as drug carriers, they have targeting properties. Clinical use has the advantages of reducing drug dosage, improving absorption efficiency, and reducing toxicity. Although liposome has the above excellent properties as a drug carrier, no related reports about iron supplement liposomal iron have been found in the existing literature after searching.

Contents of the invention

The purpose of the present invention is to provide a new type of iron supplement liposomal iron (or called iron liposome) aiming at the limitations of current various iron supplement preparations in practical application and the advantages of liposome as a drug carrier.

The object of the present invention is also to provide a preparation method of iron supplement liposome iron.

The technical scheme that realizes the object of the present invention to take is as follows:

The composition of the iron supplement liposomal iron of the present invention is expressed as:

Heme iron and/or inorganic iron: cholesterol: lecithin = (0.1-200): (1-10): (10-100)

The iron supplement liposomal iron of the present invention is composed of a lipid bilayer, and the inside is a closed large unicellular vesicle in the water phase of heme iron and/or inorganic iron PBS solution, with a particle size of 1.2-0.2 μm, wherein the After filtration through a 0.8 μm filter membrane, the average particle size is 0.59±0.05 μm, and the maximum encapsulation efficiency is 36%.

The preparation method of iron supplement liposome iron of the present invention comprises the following steps:

(1) Cholesterol and lecithin with mass ratio (1～10):(10～100) are dissolved in ethanol solution at 25～38°C;

(2) Put the cholesterol-lecithin ethanol solution into a pear-shaped bottle, rotate it at 80-140r/min under the condition of a constant temperature water bath at 25-38°C, and depressurize (0.08-0.1) MPa to volatilize the organic solvent. A lipid film forms on the wall;

(3) Weigh heme iron and/or inorganic iron according to the mass ratio of heme iron and/or inorganic iron: cholesterol: lecithin with (0.1～200): (1～10): (10～100), press Lecithin: PBS solid-liquid ratio is 1:15, and heme iron and/or inorganic iron are dissolved in PBS to obtain PBS iron solution;

(4) Fill the pear-shaped bottle treated in step (2) with N ₂ for 120-200 minutes to completely remove the solvent, add an appropriate amount of 45-55°C PBS iron solution prepared in step (3), and add 0.05% of the total volume % Tween 80 or add 0.5% to 1% of the total volume of pentaglyceride, and at the same time add glass beads with a particle size of 3 to 4mm, and spin to wash the membrane to hydrate the formed lipid film into a uniform milky lipid Plastid suspension;

(5) Disperse the obtained liposome suspension with a probe-type ultrasound of 200-300W in a cold water bath for 10-15 minutes, let it stand for 1-2 hours, and then repeatedly filter it 5-35 times with filters of different pore sizes to obtain Put liposomal iron with a diameter of 0.2-1 μm into a brown bottle, let it stand for 1-2 hours, fill it with N ₂ and seal it, and store it at 4°C to prepare a liposomal iron suspension.

In the preparation method of the present invention, the pH value of the PBS in step (3) is 6.0-7.5.

The liposome iron suspension that aforesaid method is made is observed with common biological microscope, transmission electron microscope respectively, and common biological microscope observes, and liposome outline is clear, and is spherical capsule-shaped particle (seeing accompanying drawing 1); Transmission electron microscope Negative staining with phosphotungstic acid was used, and the liposome was a typical single chamber, and its bimolecular membrane was regular, uniform and continuous, and it was in the form of a round microsphere (see Figure 2).

Take the liposomal iron suspension prepared by the above method, pass through filter membranes with different pore sizes, the particle size is 1.2-0.2 μm, among them, after filtering through a filter membrane of 0.8 μm, after diluting with the corresponding hydration medium, use JI- Model 1155 laser scattering particle size analyzer measures the particle size and distribution of liposomes, and the effective particle size is 0.59 ± 0.05 μm (see accompanying drawing 3).

Take 2ml of liposomal iron in a dialysis bag, dialyze to remove excess iron in PBS, take it out, put it in a 10ml centrifuge tube, add the corresponding volume of ether, mix well and then centrifuge. Centrifugation conditions were 25°C, 3500g, 20 minutes, and then 4000g, 10 minutes. The supernatant was discarded, and the lower part was iron solution, which was measured by spectrophotometry to obtain the iron content in the liposome, and then calculate the encapsulation efficiency of liposome iron to be 30-36%.

Differential scanning calorimetry (differential scanning calorimetry, DSC) namely DSC curve (also known as DSC thermal analysis phase diagram) investigates the change of material structure, the interaction of medicine and liposome in the preparation method process of liposome iron of the present invention , Effect of surfactants on liposome flexibility.

Two endothermic peaks with different characteristics can be found on the DSC curve of liposomes formed by single component phospholipids. The former endothermic peak has a gentle peak shape and a small peak area, which is derived from the thermal movement of the polar end of the phospholipid molecule. The transformation of the bilayer structure of phospholipid Lβ into Pβ is called pre-phase transition. The combination of the polar regions of phospholipids with other molecules especially polar species can significantly affect the pre-phase transition. The endothermic peak that appears later is called the main phase transition, and the peak area is larger, which is derived from the melting of the hydrocarbon chain in the phospholipid molecule. The unsaturated bond in the structure will reduce the main Tm, and increasing the length of the carbon chain will increase the main Tm. Similarly, Binding of fat-soluble substances mainly affects the main Tm. The liposomes formed by multi-component phospholipids have different thermal transition characteristics from those of single-component liposomes. The homogeneous state of different components in the lipid bilayer can be reflected from the thermal analysis phase diagram. If the structures of the two components are quite different, the homogeneity of the system will be affected, the phase transition half-peak width (ΔT1/2) will be enlarged, and the peak shape will be asymmetric. If the main Tm disappears, it means that the liposome forms a state between the colloidal crystalline state and the liquid crystalline state. When the liposome membrane is composed of two or more components, they each have a specific Tm, and under certain circumstances they can simultaneously exist in different phases (ie, liquid crystal phase and colloidal crystal phase), which is called phase separation. Adding different substances in liposomes can induce block structure on the surface of lipid membranes, such as drugs, surfactants, etc., which may affect the Tm changes of liposome membranes.

The above results show that the liposomal iron obtained in the present invention is uniform and large single-chamber type, the effective particle size after passing through the 0.8 μm filter membrane is 0.59±0.05 μm, and the maximum encapsulation efficiency is 36%. This greatly increases the water solubility and stability of the heme iron and/or the inorganic iron, as well as the specificity of administration, and improves the bioavailability of the drug.

The beneficial effects that the present invention obtains are as follows:

Encapsulating organic iron and/or inorganic iron into liposomes not only has the characteristics of non-toxicity and non-immunogenicity, but also has good targeting. It can greatly reduce the dosage of drugs, improve absorption efficiency and reduce toxicity. The rotating thin-film ultrasonic method adopted in the invention can form stable liposomes with high encapsulation efficiency, the method can effectively remove the residue of organic matter (ethanol), the process conditions are easy to control, and the operation is simple and convenient.

Description of drawings

Figure 1 is a 400-fold magnified photo of heme liposome iron under a bio-optical microscope. It can be seen that liposomes have clear outlines, are spherical capsule-shaped particles, and are relatively uniformly distributed in the suspension.

Figure 2 is a 6000-fold magnified photo of heme liposome iron under a transmission electron microscope. It can be seen that the liposome bimolecular membrane is regular, uniform and continuous, and it is a round microsphere.

Fig. 3 is the particle size distribution result of testing heme liposomal iron with a laser scattering particle size analyzer. It can be seen from the figure that the average particle size of the liposomes passing through the 0.8 μm filter membrane is 0.59 μm. In addition, the liposomes are passed through the filter membranes of different pore sizes according to the needs to meet the needs of different applications.

Fig. 4 to Fig. 6 are the phase transition process of each component of heme liposome iron after differential scanning calorimetry (DSC) detection. It can be seen from the figure that the main phase transition temperatures of cholesterol, lecithin and heme iron are 147.78°C, 191°C and 203°C, respectively.

Figure 7 and Figure 8 are respectively the phase transition process of blank liposome and heme liposome iron after differential scanning calorimetry (DSC) detection. As shown in the figure, on the DSC curve of the blank liposome (only containing cholesterol and lecithin), its main phase transition temperature is 78.89°C, while the phase transition peaks of cholesterol and lecithin disappear. It is suggested that the structure of cholesterol and lecithin has changed, and the combination of the two leads to the change of the phase transition peak. On the DSC curve of heme liposomal iron, there is only one phase transition peak, and the main phase transition temperature is 62.83°C. The phase transition temperature, peak shape and peak position of the two are similar, indicating that after adding heme iron, the Formation of changes in the structure of the phospholipid bilayer. The above evidence fully proves that the liposome has been formed, which contains heme iron, and the purpose of the experiment has been achieved.

Detailed ways

The following examples serve to illustrate the invention.

Composition of Iron Supplement Liposomal Iron:

The mass ratio of heme iron and/or inorganic iron: cholesterol: lecithin is (0.1-200): (1-10): (10-100)

Preparation:

Take by weighing an appropriate amount of cholesterol and lecithin, the mass ratio of the two is (1～10): (10～100), add a certain amount of organic solvent ethanol, after completely dissolving, place in a 250ml ground-mouthed pear-shaped flask, Then place the pear-shaped flask in a constant temperature water bath at 25-38°C, and use a rotary evaporator to volatilize the organic solvent under the conditions of a rotating speed of 80-140r/min and a reduced pressure (the pressure is maintained at 0.08-0.1MPa), so that phospholipids, etc., can form a film The material forms a uniform lipid film on the wall of the flask, and then fills the flask with N ₂ for 120-200 min to completely remove the solvent; at the same time, the mass ratio of heme iron and/or inorganic iron: cholesterol: lecithin is (0.1-200 ):(1～10):(10～100) Weigh heme iron and/or inorganic iron and dissolve them completely in PBS (lecithin:PBS solid-liquid ratio is 1:15, and the pH value of PBS is 6.5～7.5) , to obtain PBS iron solution; add an appropriate amount of temperature 45 ~ 55 ℃ PBS iron solution, Tween 80 (accounting for 0.05% of the total volume) or pentameric distearate (accounting for 0.5% of the overall volume) in the ground pear-shaped bottle ～1%), add the glass bead that diameter is 3～4mm at the same time, rotate and wash film with rotary evaporator under the same temperature condition, the lipoid film to be formed is hydrated and becomes milky liposome suspension; The liposome is ultrasonically dispersed with a probe-type ultrasonic pulverizer (200～300W, working 5s, gap 5s) for 10～15min to reduce the particle diameter of the liposome, while controlling the suspension temperature at 30°C under cold water bath conditions At about ℃, let stand for 1~2h, and then filter repeatedly 5~35 times with filter membranes of different pore sizes to obtain iron supplement liposomal iron with a diameter of 0.2~1μm, let stand for 1~2h, put it in a brown bottle, and fill it with N ₂ , airtight, store at 4°C.

Claims (3)

1. An iron supplement liposomal iron is characterized in that its composition is expressed as: Heme iron and/or inorganic iron: cholesterol: lecithin = (0.1-200): (1-10): (10-100). 2. A method for preparing the iron supplement liposomal iron as claimed in claim 1, characterized in that it comprises the following steps: (1) Cholesterol and lecithin with mass ratio (1～10):(10～100) are dissolved in ethanol solution at 25～38°C; (2) Put the cholesterol-lecithin ethanol solution into a pear-shaped bottle, rotate it at 80-140r/min under the condition of a constant temperature water bath at 25-38°C, and depressurize (0.08-0.1) MPa to volatilize the organic solvent. A lipid film forms on the wall; (3) Weigh heme iron and/or inorganic iron according to the mass ratio of heme iron and/or inorganic iron: cholesterol: lecithin with (0.1～200): (1～10): (10～100), press Lecithin: PBS solid-liquid ratio is 1:15, and heme iron and/or inorganic iron are dissolved in PBS to obtain PBS iron solution; (4) Fill the pear-shaped bottle treated in step (2) with N ₂ for 120-200 minutes to completely remove the solvent, add an appropriate amount of 45-55°C PBS iron solution prepared in step (3), and add 0.05% of the total volume % Tween 80 or add 0.5% to 1% of the total volume of pentaglyceride, and at the same time add glass beads with a particle size of 3 to 4mm, and spin to wash the membrane to hydrate the formed lipid film into a uniform milky lipid Plastid suspension; (5) Disperse the obtained liposome suspension with a probe-type ultrasound of 200-300W in a cold water bath for 10-15 minutes, let it stand for 1-2 hours, and then repeatedly filter it 5-35 times with filters of different pore sizes to obtain Put liposomal iron with a diameter of 0.2-1 μm into a brown bottle, let it stand for 1-2 hours, fill it with N ₂ and seal it, and store it at 4°C to prepare a liposomal iron suspension. 3. The preparation method according to claim 2, characterized in that the pH value of PBS in step (3) is 6.0-7.5.

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