Disclosure of Invention
The invention aims to provide a preparation device of anthracycline liposome, which can improve encapsulation efficiency.
Based on the same inventive concept, another object of the present invention is to provide a method for preparing anthracycline liposomes using the aforementioned apparatus.
In particular, the method comprises the steps of,
an apparatus for preparing anthracycline liposome, comprising: an inner water phase pipe and an oil phase channel parallel to the inner water phase pipe; the outlet end of the inner water phase pipe penetrates from the outlet end of the oil phase channel, and a space forming a converging channel is arranged between the outer wall of the inner water phase pipe and the inner wall of the oil phase channel; the outlet end of the oil phase channel is positioned in the composite chamber; the composite chamber is provided with an external water phase inlet and a discharge outlet, and an external water phase flow passage pointing to the discharge outlet from the external water phase inlet covers the outlet end of the oil phase passage.
The working principle of the preparation device of the anthracycline liposome disclosed by the invention is as follows: continuously injecting the inner water phase dissolved with the anthracycline into an inner water phase pipe; dissolving lipid in polar organic solvent to obtain oil phase, and continuously injecting the oil phase into the oil phase channel; the external aqueous phase is injected into the compounding chamber through an external aqueous phase inlet. Adjusting the relative flow velocity of the inner water phase and the oil phase, so that when the inner water phase overflows from the outlet end of the inner water phase pipe, the inner water phase is wrapped and clamped by the extrusion of the oil phase which is forthcoming in the oil phase channel, and is dispersed in the oil phase; meanwhile, in the dispersing process, the polar organic solvent in the oil phase can diffuse to the internal water phase at the junction, so that the concentration of the polar organic solvent at the junction is reduced, the lipid is separated out, the internal water phase dispersed in the oil phase can be surrounded by the lipid, and discontinuous and dispersed blisters are formed in the oil phase. The relative flow speed of the oil phase and the outer water phase is regulated, so that when the oil phase wrapped with the water bubble overflows from the outlet end of the oil phase channel, the oil phase is wrapped and clamped by the outer water phase flowing from the inlet of the outer water phase to the discharge port and is dispersed in the outer water phase; in the dispersing process, the polar organic solvent in the oil phase diffuses to the outer water phase at the junction, so that the concentration of the polar organic solvent at the junction is reduced, lipids are separated out, and further, oil bubbles wrapping water bubbles are formed in the outer water phase, namely the anthracycline liposome.
Compared with the prior art, the preparation device that this application provided's beneficial effect is: the inner water phase dissolved with the anthracycline forms dispersed tiny water bubbles in the oil phase, and then the oil phase wrapped with the water bubbles is separated in the outer water phase to form anthracycline liposome; thus, the anthracycline can be fully encapsulated, and the encapsulation efficiency is improved.
Optionally, the external water phase inlet and the discharge port are arranged at two sides of the composite chamber opposite to each other; the oil phase channel is perpendicular to the outer water phase flow channel; the confluence channel is positioned at one side of the inner water phase pipe corresponding to the outer water phase inlet.
Optionally, the cross sections of the inner water phase pipe and the oil phase channel are fan-shaped, and the corresponding central angle is smaller than or equal to 180 degrees; the inner water phase pipe comprises a plane pipe wall and an arc surface pipe wall, and the oil phase channel comprises a plane inner wall and an arc surface inner wall; the plane pipe wall is attached to the plane inner wall, and a space for forming a converging channel is arranged between the cambered surface pipe wall and the cambered surface inner wall.
Optionally, the inner water phase pipe and the oil phase channel are arranged in a plurality of rows along the outer water phase channel, and the inner water phase pipe corresponds to the oil phase channel one by one; the inner water phase pipes of each row are staggered with the inner water phase pipes of the adjacent rows.
Optionally, an oil phase chamber is arranged at the top of the composite chamber, and the oil phase channel is communicated with the oil phase chamber and the composite chamber up and down; an inner water phase chamber is arranged at the bottom of the composite chamber, and the inner water phase pipe is vertically communicated with the oil phase channel and the inner water phase chamber; the oil phase chamber is provided with an oil phase inlet, and the inner water phase chamber is provided with an inner water phase inlet.
Optionally, the composite chamber comprises a dish part and a top cover; the dish part, the inner water phase pipe and the inner water phase chamber are of an integrated structure, and the bottom of the dish part forms the top of the inner water phase chamber; the top cover, the oil phase channel and the oil phase chamber are of an integrated structure, and the top cover forms the bottom of the oil phase chamber.
The application also provides a preparation method of the anthracycline liposome, which adopts the preparation device and comprises the following steps:
s1, preparing an oil phase: dissolving phospholipid and cholesterol in absolute ethyl alcohol to prepare an oil phase with the phospholipid concentration of 30-50 g/L and the cholesterol concentration of 10-20 g/L;
s2, preparing an inner water phase: dissolving hydrochloric acid and anthracycline in water for injection to prepare an inner aqueous phase with pH of 4 and anthracycline concentration of 6-12 g/L;
s3, preparing an outer water phase: dissolving hydrochloric acid and monosaccharide in water for injection to prepare an external water phase with pH of 4 and sugar concentration of 600-1200 mmol/L;
s4, preparing liposome: injecting the inner water phase into the inner water phase pipe, injecting the oil phase into the oil phase channel, and injecting the outer water phase into the composite chamber through the outer water phase inlet; and the relative flow rates of the inner water phase, the oil phase and the outer water phase are regulated by controlling the injection speed; when the inner water phase overflows from the outlet end of the inner water phase pipe, the inner water phase is squeezed and wrapped by the oil phase coming from the oil phase channel, so that the oil phase wrapped by the water bubble is formed in the converging channel; when the oil phase wrapped with the water bubble overflows from the outlet end of the oil phase channel, the oil phase is wrapped and clamped by the outer water phase flowing from the outer water phase inlet to the discharge port, so that the oil phase is dispersed in the outer water phase, and the oil bubble wrapped with the water bubble, namely the anthracycline liposome is formed.
Optionally, the monosaccharide is glucose or fructose.
Optionally, when the inner water phase, the oil phase and the outer water phase are injected, the ratio of unit flow rates of the inner water phase, the oil phase and the outer water phase is 1: 2-3:16.
Optionally, the height and width of the external water phase flow channel are correspondingly adapted to the height and width of the external water phase inlet; the interval between the bottom of the oil phase channel and the bottom of the composite chamber is less than or equal to the height of the external water phase flow channel and less than twice the interval between the bottom of the oil phase channel and the bottom of the composite chamber; the ratio of the sum of the inner diameter sectional areas of the inner water phase pipes, the sum of the inner diameter sectional areas of the oil phase channels and the inner diameter sectional area of the outer water phase inlet is 2:3:3.
The working principle of the preparation method of the anthracycline liposome disclosed by the invention is as follows: the relative flow rates of the inner water phase and the oil phase are regulated by controlling the injection speed; when the inner aqueous phase in which the anthracycline is dissolved overflows from the outlet end of the inner aqueous phase tube, the inner aqueous phase is dispersed into the oil phase under extrusion wrapping of the oil phase which is forthcoming, so that tiny blisters are formed. Adjusting the relative flow rate of the oil phase and the external water phase by controlling the injection speed; when the oil phase overflows from the outlet end of the oil phase channel, the oil phase is dispersed into the outer water phase under the extrusion wrapping of the outer water phase, and the oil bubbles wrapping the water bubbles, namely the anthracycline liposome, are formed.
Compared with the prior art, the preparation method provided by the application has the beneficial effects that: the inner water phase dissolved with the anthracycline forms dispersed tiny water bubbles in the oil phase, and then the oil phase wrapped with the water bubbles is separated in the outer water phase to form anthracycline liposome; thus, the anthracycline can be fully encapsulated, and the encapsulation efficiency is improved.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
As shown in fig. 1 to 6, the present invention provides a device for preparing anthracycline liposome. The preparation device comprises: an inner water phase pipe 1 and an oil phase channel 2 parallel to the inner water phase pipe 1; the outlet end of the inner water phase pipe 1 penetrates from the outlet end of the oil phase channel 2, and a space forming a confluence channel 7 is arranged between the outer wall of the inner water phase pipe 1 and the inner wall of the oil phase channel 2; the outlet end of the oil phase channel 2 is positioned in the composite chamber 3; the composite chamber 3 is provided with an external water phase inlet 4 and a discharge outlet 5, and an external water phase flow passage 6 which is directed to the discharge outlet 5 from the external water phase inlet 4 covers the outlet end of the oil phase channel 2. It will be appreciated that when the external aqueous phase flows from the external aqueous phase inlet 4 to the discharge port 5, it will sweep over the outlet end of the oil phase channel 2, thereby pinching the oil phase overflowing from the oil phase channel 2. The inner diameter of the inner water phase pipe 1 is usually equal to or less than 0.2mm, and the inner diameter of the oil phase channel 2 is usually equal to or less than 0.3mm. In addition, the indoor height of the composite chamber 3 can be compressed, so that the whole chamber of the composite chamber 3 forms an external water phase flow channel 6; the height of the external water phase flow channel 6 is basically the same as the height of the external water phase inlet 4, and the width of the external water phase flow channel 6 is basically the same as the width of the external water phase inlet 4; typically, the interval between the bottom of the oil phase channel 2 and the bottom of the complex chamber 3 is 1mm or less.
Specific embodiments of the preparation apparatus are described below: continuously injecting the inner water phase dissolved with the anthracycline into an inner water phase pipe 1; dissolving lipid in polar organic solvent to obtain oil phase, and continuously injecting the oil phase into the oil phase channel 2; the external aqueous phase is injected into the compounding chamber 3 through the external aqueous phase inlet 4. Adjusting the relative flow velocity of the inner water phase and the oil phase, so that when the inner water phase overflows from the outlet end of the inner water phase pipe 1, the inner water phase is wrapped by the extrusion of the oil phase coming from the oil phase channel 2, and is dispersed in the oil phase; meanwhile, in the dispersing process, the polar organic solvent in the oil phase can diffuse to the internal water phase at the junction, so that the concentration of the polar organic solvent at the junction is reduced, the lipid is separated out, the internal water phase dispersed in the oil phase can be surrounded by the lipid, and discontinuous and dispersed blisters are formed in the oil phase. And the relative flow speed of the oil phase and the external water phase is regulated, so that when the oil phase wrapped with the water bubbles overflows from the outlet end of the oil phase channel 2, the oil phase is wrapped and clamped by the external water phase flowing from the external water phase inlet 4 to the discharge port 5 and is dispersed in the external water phase; in the dispersing process, the polar organic solvent in the oil phase diffuses to the outer water phase at the junction, so that the concentration of the polar organic solvent at the junction is reduced, lipids are separated out, and further, oil bubbles wrapping water bubbles are formed in the outer water phase, namely the anthracycline liposome.
The preparation device is used for preparing the anthracycline liposome, so that the inner water phase dissolved with the anthracycline is firstly dispersed into tiny water bubbles in the oil phase, and then the oil phase wrapped with the water bubbles is separated in the outer water phase to form the anthracycline liposome; thus, the anthracycline can be fully encapsulated, and the encapsulation efficiency is improved. In addition, the relative flow rates of the inner water phase, the oil phase and the outer water phase can be regulated by controlling the injection speed, and the size of the prepared doxorubicin liposome can be regulated within a certain range. In addition, the preparation device disclosed by the application is also suitable for continuous production, and is beneficial to realizing industrial application.
Further, the external water phase inlet 4 and the discharge outlet 5 are arranged at two sides of the composite chamber 3 opposite to each other; the oil phase channel 2 is vertical to the outer water phase flow channel 6; the confluence passage 7 is located at a side of the inner aqueous phase pipe 1 corresponding to the outer aqueous phase inlet 4.
Further, the cross sections of the inner water phase pipe 1 and the oil phase channel 2 are fan-shaped, and the corresponding central angle is smaller than or equal to 180 degrees; the inner water phase pipe 1 comprises a plane pipe wall and an arc surface pipe wall, and the oil phase channel 2 comprises a plane inner wall and an arc surface inner wall; the plane pipe wall is attached to the plane inner wall, and a space for forming a converging channel 7 is arranged between the cambered surface pipe wall and the cambered surface inner wall. It will be appreciated that the converging channel 7 is located on the side of the inner aqueous phase tube 1 facing the outer aqueous phase inlet 4. The outer water phase flowing from the outer water phase inlet 4 to the discharge port 5 is matched with the cambered surface pipe wall of the inner water phase pipe 1 to squeeze the oil phase overflowing from the converging channel 7; meanwhile, the outer water phase also wraps the oil phase and is separated from the edge of the cambered surface pipe wall, so that oil bubbles are formed in the outer water phase. The structure can make the outer water phase fully act on the oil phase overflowed from the confluence channel 7, fully extrude the oil phase, and is favorable for uniformly and discontinuously dispersing the oil phase in the outer water phase, thereby forming tiny oil bubbles in the outer water phase, namely the anthracycline liposome.
Further, the inner water phase pipe 1 and the oil phase channel 2 are arranged in a plurality of rows along the outer water phase channel 6, and the inner water phase pipe 1 corresponds to the oil phase channel 2 one by one; the inner water phase pipes 1 of each column are staggered with the inner water phase pipes 1 of the adjacent columns. It should be understood that the inner water phase tubes 1 of adjacent columns are staggered, so that the outer water phase can fully act on the oil phase overflowed from the confluence channel 7, and the oil phase is fully and effectively extruded, so that tiny and uniform oil bubbles, namely the anthracycline liposome, are obtained.
Further, an oil phase chamber 8 is arranged at the top of the composite chamber 3, and the oil phase channel 2 is communicated with the oil phase chamber 8 and the composite chamber 3 up and down; an inner water phase chamber 9 is arranged at the bottom of the composite chamber 3, and the inner water phase pipe 1 is communicated with the oil phase channel 2 and the inner water phase chamber 9 up and down; the oil phase chamber 8 is provided with an oil phase inlet, and the inner water phase chamber 9 is provided with an inner water phase inlet. It should be understood that the oil phase inlet communicates with the hose carrying the oil phase and is adapted to the peristaltic pump; the inner water phase inlet is communicated with a hose for conveying the inner water phase and is matched with a peristaltic pump; the external water phase inlet 4 is in communication with a hose for transporting the external water phase and is adapted to a peristaltic pump. In order to better control the hydraulic pressure difference of the preparation device in the working process, the discharge port 5 can be communicated with a discharge hose and is matched with a peristaltic pump. The preparation device is favorable for industrial production by arranging a plurality of inner water phase pipes 1 and oil phase channels 2 and arranging a converging inner water phase chamber 9 and an converging oil phase chamber 8. The peristaltic pumps of each phase are used for controlling the unit flow of upstream confluence, so that the relative flow rates of the inner water phase, the oil phase and the outer water phase can be controlled.
Further, the compound chamber 3 comprises a dish part 10 and a top cover 11; the dish part 10, the inner water phase pipe 1 and the inner water phase chamber 9 are of an integrated structure, and the bottom of the dish part 10 forms the top of the inner water phase chamber 9; the top cover 11, the oil phase channel 2 and the oil phase chamber 8 are of an integrated structure, and the top cover 11 forms the bottom of the oil phase chamber 8. It should be understood that dish portion 10, inner water phase tube 1, inner water phase chamber 9 may be directly formed by etching, photolithography, etc.; the top cover 11, the oil phase channel 2 and the oil phase chamber 8 can be directly processed and formed by means of etching, photoetching and the like. Finally, the dish part 10 and the top cover 11 are glued, so that the assembly of the preparation device can be completed, and the preparation device has the advantages of simple structure and convenience in assembly.
The application also provides a preparation method of the anthracycline liposome, which comprises the following steps:
example 1
S1: dissolving phospholipid and cholesterol in absolute ethanol to obtain oil phase with phospholipid concentration of 50g/L and cholesterol concentration of 20 g/L.
S2: hydrochloric acid and doxorubicin were dissolved in water for injection to prepare an inner aqueous phase having a pH of 4 and a doxorubicin concentration of 10 g/L.
S3: hydrochloric acid and glucose are dissolved in water for injection to prepare an external water phase with pH of 4 and sugar concentration of 1200 mmol/L.
S4: injecting the inner water phase into the inner water phase pipe 1; injecting the oil phase into the oil phase channel 2; injecting the external water phase into the composite chamber 3 through the external water phase inlet 4; the ratio of the unit flow rates of the inner water phase, the oil phase and the outer water phase is kept at 1:3:16. The solution discharged from the discharge port 5 is collected and is the doxorubicin liposome solution.
Example 2
S1: dissolving phospholipid and cholesterol in absolute ethanol to obtain oil phase with phospholipid concentration of 50g/L and cholesterol concentration of 20 g/L.
S2: hydrochloric acid and epirubicin are dissolved in water for injection to prepare an inner water phase with pH of 4 and epirubicin concentration of 10 g/L.
S3: hydrochloric acid and glucose are dissolved in water for injection to prepare an external water phase with pH of 4 and sugar concentration of 1200 mmol/L.
S4: injecting the inner water phase into the inner water phase pipe 1; injecting the oil phase into the oil phase channel 2; injecting the external water phase into the composite chamber 3 through the external water phase inlet 4; the ratio of the unit flow rates of the inner water phase, the oil phase and the outer water phase is kept at 1:3:16. The solution discharged from the discharge port 5 was collected and was the epirubicin liposome solution.
Example 3
S1: dissolving phospholipid and cholesterol in absolute ethanol to obtain oil phase with phospholipid concentration of 30g/L and cholesterol concentration of 10 g/L.
S2: hydrochloric acid and daunorubicin are dissolved in water for injection to prepare an inner water phase with pH of 4 and daunorubicin concentration of 6 g/L.
S3: hydrochloric acid and fructose were dissolved in water for injection to prepare an external aqueous phase having a pH of 4 and a sugar concentration of 600 mmol/L.
S4: injecting the inner water phase into the inner water phase pipe 1; injecting the oil phase into the oil phase channel 2; injecting the external water phase into the composite chamber 3 through the external water phase inlet 4; the ratio of the unit flow rates of the inner water phase, the oil phase and the outer water phase is kept at 1:2:16. The solution discharged from the discharge port 5 is collected and is the daunorubicin liposome solution.
Example 4
S1: dissolving phospholipid and cholesterol in absolute ethanol to obtain oil phase with phospholipid concentration of 35g/L and cholesterol concentration of 16 g/L.
S2: hydrochloric acid and aclacinomycin are dissolved in water for injection to prepare an inner water phase with pH of 4 and aclacinomycin concentration of 7 g/L.
S3: hydrochloric acid and fructose were dissolved in water for injection to prepare an external aqueous phase having a pH of 4 and a sugar concentration of 800 mmol/L.
S4: injecting the inner water phase into the inner water phase pipe 1; injecting the oil phase into the oil phase channel 2; injecting the external water phase into the composite chamber 3 through the external water phase inlet 4; the ratio of the unit flow rates of the inner water phase, the oil phase and the outer water phase is kept at 1:2.4:16. The solution discharged from the discharge port 5 was collected and was the aclacinomycin liposome solution.
Comparative example 1
S1: dissolving phospholipid and cholesterol in absolute ethanol to obtain oil phase with phospholipid concentration of 50g/L and cholesterol concentration of 20 g/L.
S2: hydrochloric acid, doxorubicin and glucose were dissolved in water for injection to prepare an aqueous phase having a pH of 4, a doxorubicin concentration of 0.59g/L and a sugar concentration of 1129 mmol/L.
And (3) putting the oil phase and the water phase into a homogenizer according to the volume ratio of 3:17 for homogenizing and mixing to obtain the doxorubicin liposome solution.
Comparative example 2
S1: dissolving phospholipid and cholesterol in absolute ethanol to obtain oil phase with phospholipid concentration of 50g/L and cholesterol concentration of 20 g/L.
S2: hydrochloric acid, epirubicin and glucose were dissolved in water for injection to prepare an aqueous phase having a pH of 4, a epirubicin concentration of 0.59g/L and a sugar concentration of 1129 mmol/L.
And (3) putting the oil phase and the water phase into a homogenizer according to the volume ratio of 3:17 for homogenizing and mixing to obtain the epirubicin liposome solution.
Comparative example 3
S1: dissolving phospholipid and cholesterol in absolute ethanol to obtain oil phase with phospholipid concentration of 30g/L and cholesterol concentration of 10 g/L.
S2: hydrochloric acid, daunorubicin and fructose are dissolved in water for injection to prepare an aqueous phase with pH of 4, daunorubicin concentration of 0.35g/L and sugar concentration of 565 mmol/L.
And (3) putting the oil phase and the water phase into a homogenizer according to the volume ratio of 2:17 for homogenizing and mixing to obtain daunorubicin liposome solution.
Comparative example 4
S1: dissolving phospholipid and cholesterol in absolute ethanol to obtain oil phase with phospholipid concentration of 35g/L and cholesterol concentration of 16 g/L.
S2: hydrochloric acid, aclacinomycin and fructose were dissolved in water for injection to prepare an aqueous phase having a pH of 4, aclacinomycin concentration of 0.41g/L and sugar concentration of 753 mmol/L.
And (3) putting the oil phase and the water phase into a homogenizer according to the volume ratio of 2.4:17 for homogenizing and mixing to obtain the aclacinomycin liposome solution.
Note 1: the anthracycline liposome solution prepared in examples 1 to 4 is separated and purified to remove impurities such as ethanol, and the like, so that the anthracycline liposome injection can be obtained.
And (2) injection: the device for preparing anthracycline liposome used in examples 1 to 4 has the same height and width of the outer water phase flow channel 6 as the height and width of the outer water phase inlet 4; the interval between the bottom of the oil phase channel 2 and the bottom of the compound chamber 3 is less than or equal to the height of the external water phase flow channel 6 and less than or equal to 2mm which is twice the interval between the bottom of the oil phase channel 2 and the bottom of the compound chamber 3; and the ratio of the sum of the inner diameter sectional areas of the inner water phase pipes 1, the sum of the inner diameter sectional areas of the oil phase channels 2 and the inner diameter sectional area of the outer water phase inlet 4 is 2:3:3.
Samples were obtained from the anthracycline liposome solutions prepared in examples 1 to 4 and comparative examples 1 to 4, and the anthracycline liposome and the free anthracycline were separated from each sample by dialysis. The content of the free anthracycline separated is measured by high performance liquid chromatography, and the total amount of the free anthracycline in each sample is calculated. The total amount of anthracyclines contained in each sample was calculated from the ingredients of examples 1 to 4 and comparative examples 1 to 4. Finally, the encapsulation efficiency can be calculated according to the ratio of the total amount of free anthracyclines to the total amount of anthracyclines in each sample.
Table 1: encapsulation efficiency of examples 1 to 4 and comparative examples 1 to 4
From Table 1, it can be seen. The encapsulation efficiency of examples 1-4 is significantly higher than that of comparative examples 1-4. This means that the inner aqueous phase in which the anthracycline is dissolved is dispersed in the oil phase to form fine vesicles; and dispersing the oil phase in the outer water phase to form oil bubbles wrapping the water bubbles. Can fully wrap the anthracycline, thereby improving the encapsulation efficiency.
While particular embodiments of the present invention have been described above, it will be understood by those skilled in the art that various changes and modifications may be made to these embodiments without departing from the spirit and scope of the invention.