CN107929321B - Immune cell gel preparation capable of being preserved for long time and preparation method thereof - Google Patents

Immune cell gel preparation capable of being preserved for long time and preparation method thereof Download PDF

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CN107929321B
CN107929321B CN201711387344.2A CN201711387344A CN107929321B CN 107929321 B CN107929321 B CN 107929321B CN 201711387344 A CN201711387344 A CN 201711387344A CN 107929321 B CN107929321 B CN 107929321B
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曹毓琳
刘俊江
时兆田
白志惠
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Beijing Tangyihuikang Biomedical Technology Co ltd
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Abstract

The invention provides an immune cell gel preparation capable of being stored for a long time and a preparation method thereof, wherein the gel preparation consists of immune cells and a gel matrix, and the gel matrix mainly consists of the following components: deionized water, dextran, herba Visci leaf extract, propylene glycol alginate, oleyl alcohol, and guaiac; the method comprises the following steps: preparing mistletoe leaf extract; dissolving herba Visci leaf extract in deionized water, standing overnight, adding propylene glycol alginate, oleyl alcohol and guaiac, stirring, adding dextran, stirring, preparing gel matrix, adding immunocyte into the gel matrix, and stirring. The invention can ensure the cell activity of immune cells in the long-term storage process, and simultaneously, the invention not only can improve the stability of the immune cell gel preparation and ensure the drug effect of the preparation stored for a long time, but also can improve the drug release efficiency, prevent mildew and has strong practicability.

Description

Immune cell gel preparation capable of being preserved for long time and preparation method thereof
Technical Field
The invention belongs to the technical field of gel preparations, and particularly relates to an immune cell gel preparation capable of being stored for a long time and a preparation method thereof.
Background
At present, the diagnosis and treatment of diseases have entered molecular and cellular age, and stem cells and immune cells have great therapeutic potential, and the immune cells refer to cells involved in or associated with immune response, and the immune cells generally include lymphocytes, dendritic cells, monocytes/macrophages, granulocytes, mast cells, and the like. Immune cells can be classified into various types, and various immune cells play an important role in the human body. At present, the application of immune cells is limited, and no immune cell preparation is seen at home and abroad.
The gel preparation is a new medicament form gel preparation which is developed in recent years in China, and is a thick liquid or semisolid preparation of solution, suspension or emulsion type prepared by medicaments and auxiliary materials capable of forming gel, a common matrix for preparing the gel preparation is generally composed of water, glycerol or propylene glycol, cellulose derivatives, carbomer, alginate and other high polymer materials, and the gel preparation is hydrogel, has no greasy feeling and is easy to coat and remove, but has slow drug release, is easy to lose water and mildew, and seriously influences the quality and various properties of the gel preparation.
Disclosure of Invention
In order to solve the problems of the existing gel preparation, the invention provides the immune cell gel preparation which has good stability, quick drug release, difficult water loss and difficult mildew and can be stored for a long time.
The specific technical scheme of the invention is as follows:
the invention provides an immune cell gel preparation capable of being preserved for a long time, which consists of immune cells and a gel matrix, wherein each g of the gel matrix contains 0.3 multiplied by 1011-1.2×1011The gel matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000021
the gel preparation is added with the dextran and the mistletoe leaf extract to serve as a gel matrix, provides nutrient components for the survival of immune cells and ensures the survival rate of the immune cells, in addition, the addition of the propylene glycol alginate, the oleyl alcohol and the guaiac gum can improve the stability of the gel matrix and can be used for storing the immune cells for a long time, and the added propylene glycol alginate, the oleyl alcohol and the guaiac gum can also improve the drug release efficiency of the gel preparation, prevent mildew and have strong practicability.
Further, the immune cell is selected from one of T lymphocyte, B lymphocyte, K lymphocyte, NK lymphocyte and CIK cell.
Preferably, the extraction method of the mistletoe leaf extract is as follows:
cleaning mistletoe leaf, freeze drying, and grinding into powder;
Adding the powder into a test tube, adding 10ml of absolute ethyl alcohol into each g of the powder, performing ultrasonic extraction for 30-60 min, centrifuging, and transferring the supernatant into another test tube; extracting the residue for 2 times, and mixing the supernatants;
concentrating the supernatant obtained in the step S2 under reduced pressure to obtain a concentrated solution with the total volume less than 5 mL;
and fully dissolving the concentrated solution by using deionized water, and fixing the volume to 50mL to obtain the mistletoe leaf extract.
The mistletoe leaf extract prepared by the method and special parameter setting in the method can improve the stress resistance of cells, reduce the activity loss of immune cells in the preservation process and improve stable and suitable environment for the preservation of the cells.
Further, the gel matrix also comprises the following components in parts by weight:
starch sodium phosphate 0.5-1
Calcium carboxymethyl cellulose 0.1-1.4
0.1-0.6 of hydroxyethyl methyl cellulose.
The invention can obviously improve the drug release speed of the gel matrix by adding the starch sodium phosphate, the carboxymethyl cellulose calcium and the hydroxyethyl methyl cellulose into the gel matrix, has better transdermal absorption performance, can also improve the dispersibility of immune cells in the gel matrix, and the added starch sodium phosphate, the carboxymethyl cellulose calcium and the hydroxyethyl methyl cellulose can not generate interference and adsorption on the immune cells and can not influence the activity of the immune cells.
Further, the gel matrix also comprises the following components in parts by weight:
0.2-1.2 parts of propylene glycol diethyl ester
0.1-1.5 of lauric acid isopropanol amide.
According to the invention, the moisture retention of the gel matrix can be obviously improved by adding the propylene glycol diethyl ester and the lauric acid isopropanol amide into the gel matrix, so that the gel preparation is more comfortable to contact with the skin, the adhesion of the gel preparation and the skin is ensured, and the irritation of the gel preparation to the skin can be obviously reduced.
Further, the gel matrix also comprises the following components in parts by weight:
0.1-0.5 parts of chlorobenzyl ethylamine
0.1-1% of isopropanol
0.1-0.8 of poly (vitamin A) iodone.
The gel preparation provided by the invention is added with the mixed components of the chloramine, the isopropanol and the povidone iodine, so that the gel preparation can effectively play roles of antisepsis and sterilization, provides a foundation for the long-term preservation of immune cells, effectively reduces the survival rate of the immune cells, ensures the activity of the cells and reduces the activity loss.
Preferably, the gel matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000041
the gel matrix provided by the invention is an optimal matrix component, and through mixing the components, the storage life of immune cells can be prolonged, the activity of the immune cells in a long-term storage process can be ensured, and meanwhile, the gel matrix has the effects of moisture preservation and corrosion prevention, is quick in drug release and has strong practicability.
The invention also provides a preparation method of the immune cell gel preparation capable of being preserved for a long time, which comprises the following steps:
s1: preparing mistletoe leaf extract;
s2, dissolving herba Visci leaf extract in deionized water, standing overnight, adding propylene glycol alginate, oleyl alcohol and guaiac, stirring for 10min, and transferringAdding dextran at 3500 r/min, stirring for 13min at 500 r/min, and preparing gel matrix by adding 0.3 × 10 gel matrix per g gel matrix11-1.2×1011Mixing the immune cells uniformly.
Preferably, in step S1, the mistletoe leaf extract is prepared by the following steps:
s1: cleaning mistletoe leaf, freeze drying, and grinding into powder;
s2: adding the powder into a test tube, adding 10ml of absolute ethyl alcohol into each g of the powder, performing ultrasonic extraction for 30-60 min, centrifuging, and transferring the supernatant into another test tube; extracting the residue for 2 times, and mixing the supernatants;
s3: concentrating the supernatant obtained in the step S2 under reduced pressure to obtain a concentrated solution with the total volume less than 5 mL;
s4: and fully dissolving the concentrated solution by using deionized water, and fixing the volume to 50mL to obtain the mistletoe leaf extract.
Further, the immune cell is selected from one of T lymphocyte, B lymphocyte, K lymphocyte, NK lymphocyte and CIK cell.
The invention has the following beneficial effects: the dextran and the mistletoe leaf extract added into the immune cell gel preparation capable of being stored for a long time can ensure the cell activity of immune cells in the long-term storage process, and in addition, the propylene glycol alginate, the oleyl alcohol and the guaiac added into the gel matrix can improve the stability of the immune cell gel preparation, ensure the drug effect of the preparation stored for a long time, improve the drug release efficiency, prevent mildew and have strong practicability.
Detailed Description
The present invention will be described in further detail with reference to the following examples.
Example 1
An immunocyte gel preparation capable of being preserved for a long time, wherein the gel preparation comprises immunocytes and a gel matrix, and each g of the gel matrix contains 0.3 × 1011An immune cell, said gelThe matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000061
wherein the immune cells are T lymphocytes.
Example 2
An immunocyte gel preparation capable of being preserved for a long time, wherein the gel preparation comprises immunocytes and a gel matrix, and each g of the gel matrix contains 0.8 × 10 11The gel matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000062
wherein the immune cell is a B lymphocyte.
The preparation method of the gel preparation comprises the following steps:
s1: preparing mistletoe leaf extract;
s2, dissolving the mistletoe leaf extract in deionized water, standing overnight, adding propylene glycol alginate, oleyl alcohol and guaiac, stirring for 10min at 3500 rpm, adding dextran, stirring for 13min at 500 rpm, preparing gel matrix, and adding 0.8 × 10 gel matrix per g gel matrix11Mixing the immune cells uniformly.
In step S1, the preparation method of the mistletoe leaf extract is as follows:
s1: cleaning mistletoe leaf, freeze drying, and grinding into powder;
s2: adding the powder into a test tube, adding 10ml of absolute ethyl alcohol into each g of the powder, performing ultrasonic extraction for 50min, centrifuging, and transferring the supernatant into another test tube; extracting the residue for 2 times, and mixing the supernatants;
s3: concentrating the supernatant obtained in the step S2 under reduced pressure to obtain a concentrated solution with the total volume less than 5 mL;
s4: and fully dissolving the concentrated solution by using deionized water, and fixing the volume to 50mL to obtain the mistletoe leaf extract.
Example 3
An immunocyte gel preparation capable of being preserved for a long time, wherein the gel preparation comprises immunocytes and a gel matrix, and each g of the gel matrix contains 1.2 x 1011The gel matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000071
wherein the immune cell is a K lymphocyte.
Example 4
An immunocyte gel preparation capable of being preserved for a long time, wherein the gel preparation comprises immunocytes and a gel matrix, and each g of the gel matrix contains 0.5 × 1011The gel matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000081
wherein the immune cells are NK lymphocytes.
Example 5
An immunocyte gel preparation capable of being preserved for a long time, wherein the gel preparation comprises immunocytes and a gel matrix, and each g of the gel matrix contains 0.3 × 1011The gel matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000082
wherein the immune cells are CIK cells.
Example 6
An immunocyte gel formulation for long term storage, said gelThe gel preparation comprises immunocyte and gel matrix, and each g of the gel matrix contains 1.2 × 1011The gel matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000091
Wherein the immune cells are CIK cells.
Example 7
An immunocyte gel preparation capable of being preserved for a long time, wherein the gel preparation comprises immunocytes and a gel matrix, and each g of the gel matrix contains 0.6 x 1011The gel matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000092
Figure BDA0001516871770000101
wherein the immune cells are T lymphocytes.
Example 8
An immunocyte gel preparation capable of being preserved for a long time, wherein the gel preparation comprises immunocytes and a gel matrix, and each g of the gel matrix contains 1.2 x 1011The gel matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000102
Figure BDA0001516871770000111
wherein the immune cell is a K lymphocyte.
Example 9
An immunocyte gel preparation capable of being preserved for a long time, wherein the gel preparation comprises immunocytes and a gel matrix, and each g of the gel matrix contains 0.3 × 1011The gel matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000112
wherein the immune cells are NK lymphocytes.
Comparative example 1
An immunocyte gel preparation capable of being preserved for a long time, wherein the gel preparation comprises immunocytes and a gel matrix, and each g of the gel matrix contains 0.3 × 10 11The gel matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000121
wherein the immune cells are T lymphocytes.
Comparative example 2
An immunocyte gel preparation capable of being preserved for a long time, wherein the gel preparation comprises immunocytes and a gel matrix, and each g of the gel matrix contains 1.2 x 1011The gel matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000122
wherein the immune cell is a K lymphocyte.
Comparative example 3
An immunocyte gel preparation capable of being preserved for a long time, wherein the gel preparation comprises immunocytes and a gel matrix, and each g of the gel matrix contains 0.3 × 1011The gel matrix mainly comprises the following components in parts by weightConsists of the following components:
Figure BDA0001516871770000123
Figure BDA0001516871770000131
wherein the immune cells are CIK cells.
Comparative example 4
An immunocyte gel preparation capable of being preserved for a long time, wherein the gel preparation comprises immunocytes and a gel matrix, and each g of the gel matrix contains 0.6 x 1011The gel matrix mainly comprises the following components in parts by weight:
Figure BDA0001516871770000132
wherein the immune cells are T lymphocytes.
Experiment 1, detection of immune cell viability after cryopreservation of immune cell gel preparation
Gel preparations of the present invention according to examples 1, 2, 3, 5, 7, 9 and comparative examples 1 to 4 were taken, placed in liquid nitrogen, taken out of the liquid nitrogen after being frozen for 1 month, 2 months, 3 months, 6 months, 9 months and 12 months, respectively, placed in a water bath at 37 ℃ for resuscitation for 1 to 2min, subjected to cell counting by trypan blue staining, and the survival rate of exosomes was calculated, and the detection results are shown in table 1.
TABLE 1 examination results of the immune cell viability rate in the gel preparations of each example and comparative example
Figure BDA0001516871770000141
As can be seen from table 1, compared with comparative experiments 1 to 4, in examples 1, 3, 5, and 7 of the present invention, the gel preparation provided by the present invention has stable properties, can store immune cells for a long time, and after 12 months of storage, the survival rate of the immune cells exceeds 80.6%, and in addition, example 9 is the best example provided by the present invention, and the experiment shows that the survival rate of the immune cells can still reach 97.8% after 12 months of storage of the gel preparation provided by example 9;
in addition, the survival rate of the immune cells after the gel preparation provided in example 7 is stored for 12 months can reach 94.1%, which is significantly higher than that of examples 5, 3 and 1, and thus, the survival rate of the immune cells can be improved by adding the chloramine, the isopropanol and the povidone, and compared with the control example 4, it can be shown that any one of the three substances is absent, and the survival rate of the immune cells can be reduced.
The addition of propylene glycol diethyl ester and lauric acid isopropanolamide to the gel formulation provided in example 5 enables the survival rate of immune cells to be higher than that of example 3 and example 1, and for this reason, it can be shown that the addition of propylene glycol diethyl ester and lauric acid isopropanolamide can significantly improve the survival rate of immune cells, as compared with that of control 3, and the survival rate of cells is reduced by the absence of either of these two substances.
The gel preparation provided in example 3 was added with sodium starch phosphate, calcium carboxymethylcellulose and hydroxyethylmethylcellulose to increase the survival rate of immune cells compared to example 1, and thus, the survival rate of immune cells was improved by adding sodium starch phosphate, calcium carboxymethylcellulose and hydroxyethylmethylcellulose, and it was found that the survival rate was decreased in the absence of any one of these three substances as compared to comparative example 2.
Example 1 compared with example 2, the gel preparation prepared by the preparation method provided in example 2 preserved higher cell viability than example 1, and for this reason, it can be demonstrated that the preparation method can be used to preserve immune cells for a long period of time.
Compared with the comparative example 1, the survival rate of the cells is obviously higher than that of the comparative example 1, so that the gel preparation provided by the invention can be used for storing immune cells for a long time, and as can be seen from the survival rate of the cells stored in the gel preparation of the comparative example 1, the replacement and deletion of each component in the gel preparation can influence the cell viability of the frozen gel preparation and the freezing time.
Experiment 2, in vitro drug Release Rate
And (3) detecting the release degree of the pharmaceutical composition, namely referring to the examination of the in vitro drug release degree of the appendix XIXD of Chinese pharmacopoeia of 2010 edition.
The experimental method comprises the following steps: taking adult male rat, killing, peeling off abdominal skin, performing depilation treatment, placing in normal saline for use, adopting Franz diffusion cell, and having effective permeation area of 0.8cm2The receiving medium is normal saline, and the volume of the receiving pool is 5 mL.
The skin of the treated rat was evenly divided into 4 pieces of equal area, divided into test 1 group, test 2 group and control 1-2 group, the skin piece of the test 1 group was coated with the immunocytogel preparation of example 1 of the present invention, the skin piece of the test 2 group was coated with the immunocytogel preparation of example 3 of the present invention, and the skin pieces of the control 1-2 group were coated with the gel preparations of control 1-2, respectively; coating 0.2g of repairing gel on each piece of skin, placing the pieces of skin coated with gel preparation in a supply pool, performing transdermal test at 37 deg.C and 500r/min, removing all receiving liquid (simultaneously supplementing equal amount of physiological saline) at 0.5, 1, 2, 4, 6, 8, 10, and 12 hr, centrifuging at 8000r/min for 10min, collecting supernatant, calculating the number of exosomes by trypan blue staining method, and calculating the cumulative transdermal permeability Q per unit areanWith QnPlotting the time t and linearly regressing the linear portion of the curve to obtain the slope of the line, i.e., the steady state permeation rate Js (pieces/cm) 2H) the results are given in Table 2.
TABLE 2 in vitro release Rate test results (n ═ 3)
Figure BDA0001516871770000161
As can be seen from table 2, the permeability of the gel preparation provided in example 1 is much higher than that of comparative example 1, so that any deletion of one of the components provided in example 1 of the present invention will not achieve a good drug release effect, and the permeability will be reduced, and in addition, compared with example 3, the starch sodium phosphate, carboxymethyl cellulose calcium, hydroxyethyl methyl cellulose added in example 3 can significantly improve the skin permeability of the gel preparation, and the drug release speed is fast, and compared with comparative example 2, any absence or replacement of one of the three substances will not achieve a good effect.
Experiment 3 stability test of immunocyte gel preparation provided by the invention
The gel formulations provided in examples 1, 3 and 5 and the gel formulations provided in comparative examples 1 and 3 were respectively stored at 25 ℃ ± 2 ℃ and a relative humidity of 10% ± 5% for 6 months, and were sampled once at the end of 1 month, 2 months, 3 months and 6 months during the test period, and the properties, the main component content (labeled amount%), and the water content of the gel formulations were measured, and as a result, it was found that the main component contents of examples 1, 3 and 5 and comparative example 3 did not significantly change, but the gel formulations provided in examples 1 and 3 were slightly shriveled and the viscosity was reduced, and the water content in the gel formulations provided in examples 1 and 3 was lower than that in example 5; while the main components in the gel preparations provided in comparative examples 1 and 3 are remarkably reduced, the preparations are shriveled and have no viscosity, and the gel preparations are remarkably deepened, which shows that the gel preparation provided by the invention has a certain moisturizing effect, compared with comparative example 3, the propylene glycol diethyl ester and the lauric acid isopropyl alcohol amide added in example 5 can have the moisturizing effect, and the effect is reduced due to the fact that one of the two components is absent.
Experiment 4 evaluation of safety of immunocyte gel preparation capable of being preserved for a long period
The gel formulations of examples 1, 3, 5 and 7 and comparative examples 1 and 4 were placed in a closed container at a temperature of 40 ℃. + -. 2 ℃ and a relative humidity of 60%. + -. 10% for 30 days, to accelerate the detection of the total number of bacteria in the gel formulations.
TABLE 3 evaluation of safety of gel formulations
Figure BDA0001516871770000171
As can be seen from the results in table 3, compared with the comparative example 1, the gel preparation provided in example 1 of the present invention has a certain bacteriostatic effect, and compared with examples 1, 3, and 5, the clobenzyl ethylamine, the isopropyl alcohol, and the povidone iodine added to the gel preparation provided in example 7 can effectively inhibit the growth of bacteria, so as to ensure the safe storage of the gel preparation, and compared with comparative example 4, in example 7, it can be seen that any one of the three components is absent or replaced, and the bacteriostatic effect cannot be achieved well.
The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone in the light of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as those of the present application, fall within the protection scope of the present invention.

Claims (2)

1. An immunocyte gel preparation capable of being preserved for a long time, which is characterized in that the gel preparation consists of immunocytes and a gel matrix, wherein each g of the gel matrix contains 0.3 multiplied by 1011-1.2×1011The gel matrix mainly comprises the following components in parts by weight:
60-85 parts of deionized water
Dextran 2-10
Mistletoe leaf extract 1-7
Propylene glycol alginate 1.5-8
Oleyl alcohol 1.5-10
1-5 parts of guaiac;
starch sodium phosphate 0.5-1
Calcium carboxymethyl cellulose 0.1-1.4
Hydroxyethyl methyl cellulose 0.1-0.6
0.2-1.2 parts of propylene glycol diethyl ester
Lauric acid isopropanol amide 0.1-1.5
0.1-0.5 parts of chlorobenzyl ethylamine
0.1-1% of isopropanol
0.1-0.8 of poly (vitamin A) iodone;
the immune cell is selected from one of T lymphocyte, B lymphocyte, K lymphocyte, NK lymphocyte and CIK cell;
the extraction method of the mistletoe leaf extract comprises the following steps:
s1: cleaning mistletoe leaf, freeze drying, and grinding into powder;
s2: adding the powder into a test tube, adding 10ml of absolute ethyl alcohol into each g of the powder, performing ultrasonic extraction for 30-60 min, centrifuging, and transferring the supernatant into another test tube; extracting the residue for 2 times, and mixing the supernatants;
S3: concentrating the supernatant obtained in the step S2 under reduced pressure to obtain a concentrated solution with the total volume less than 5 mL;
s4: and fully dissolving the concentrated solution by using deionized water, and fixing the volume to 50mL to obtain the mistletoe leaf extract.
2. The immunocytogel preparation according to claim 1, wherein said gel base comprises the following components in parts by weight:
deionized water 76.4
Dextran 5
Mistletoe leaf extract 2
Propylene glycol alginate 4
Oleyl alcohol 5
Guaiac 2
Starch sodium phosphate 0.8
Carboxymethyl cellulose calcium 1
Hydroxyethyl methyl cellulose 0.4
Propylene glycol diethyl ester 1
Lauric acid isopropanolamide 1
0.3 parts of chlorobenzyl ethylamine
0.6% of Isopropanol
0.5 of poly (vitamin A) iodone.
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