CN112359009A - Chitin-based gel material cell carrier - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/72—Chitin, chitosan
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a chitin-based gel material cell carrier, which is prepared by the following steps: preparing chitin, dissolving raw materials for preparing chitin in a treatment solution, stirring, adding an extracting solution during stirring, finally adding a chitin aqueous solution, continuing to react, dialyzing the reaction solution, and freeze-drying to obtain chitin; mixing paclitaxel solution with chitin prepared in step one, adding penetrating fluid, performing ultrasonic treatment, and measuring pH value of the mixed solution; and (3) preparing the nanogel into a wafer shape, standing the solution treated in the step two, removing the upper colorless transparent liquid, depositing the lower layer on the wafer prepared from the nanogel, and drying and freezing to obtain the cell carrier. The preparation method is simple, has strong operability and simple material acquisition, is beneficial to reducing the production cost of the cell carrier, improves the survival probability and the production time of cells on the basis of chitin, and is beneficial to the transportation and the culture of the cells.
Description
Technical Field
The invention relates to the technical field of cell preservation, in particular to a chitin-based gel material cell carrier.
Background
The vector refers to a self-replicating DNA molecule for transferring a DNA fragment (target gene) to a recipient cell in a recombinant DNA technology by genetic engineering, and three most commonly used vectors are bacterial plasmids, bacteriophages and animal and plant viruses. The ideal drug carrier not only has proper particle size and shape, higher drug loading rate and encapsulation rate, but also is more importantly biodegradable, low in toxicity or even no toxicity, so that the drug carrier is required to use a biodegradable high polymer material, the biodegradable material becomes the focus of attention of researchers at home and abroad, and is a large research hotspot in the field of high polymer materials in recent decades, and the biodegradable material drug carrier can be divided into an artificially synthesized high polymer system and a natural high polymer system according to the source classification, such as Polylactide (PLA), polyglycolide/polylactide copolymer (PLGA), Polyglycolide (PGA), Polycaprolactone (PCL), polylactide-Polyhexamide (PLCL) and the like which belong to artificially synthesized high polymer; the natural polymer materials can be classified into polysaccharides, proteins and other types, the polysaccharides used for the drug delivery system mainly comprise chitosan, starch, alginate and the like, and the proteins mainly comprise gelatin, albumin and the like. According to the form and type, the drug-loading form of the biodegradable material carrier can be divided into sol and micelle, nanocapsule and vesicle, nanoparticle and nanosphere and the like. In addition, according to the in vivo degradation and metabolism pathway, biodegradable material carriers can be divided into two types, namely biodegradation and non-biodegradation, and the biodegradable carrier materials can control the release of the carried drugs or gene polypeptides by regulating the degradation rate, so that more and more attention and research are paid. In addition, multifunctional composite nanocarriers have also appeared in recent years, and their compositions include nanocarriers (polymers), active ingredients (therapeutic drugs), targeting molecules (antibodies), polyethylene glycol-modified ingredients, and the like. For example, gold nanoparticles connected by polypeptide, Farokhzad and the like are connected by a prostate cancer specific membrane antibody, PLGA nanoparticles are coated with docetaxel and the like.
Chitin, also known as chitin, is a structural homopolysaccharide formed by polymerizing N-acetylglucosamine through beta linkage, widely exists in shells of crustaceans, carapace of insects and cell walls of fungi, and also exists in some green algae, chitin is a N-amino acetylated polymeric compound mainly derived from shells of animals and plants in nature such as crabs and shrimps, chitin and derivatives thereof are widely used, chitin usually represents some chemical or physical characteristics of the shells according to the N-amino acetylation degree and/or the polymerization degree of sugar unit structures, DA represents the number of free amino groups on a certain chitin skeleton, the larger the value of DA, the larger the percentage of acetylated free amino groups on the chitin skeleton, the lower the percentage of free amino groups; DP represents the size of a chitin molecule, and the larger the value of DP, the more monosaccharide molecules constitute the chitin molecule, and the larger the molecular weight; the higher the DP of chitin, the lower the number of chitin molecules it contains at a given mass, and thus DA and DP affect the physicochemical and biological activity of chitin to some extent. In response to this situation, a cell carrier based on a gel-like material of chitin has been proposed to reduce the production cost of the cell carrier and to improve the safety of administration of the cell carrier.
Disclosure of Invention
The invention aims to provide a chitin-based gelatinous material cell carrier, which has the advantages of simple preparation method, strong operability, simple material acquisition and contribution to reducing the production cost of the cell carrier, improves the survival probability and the production time of cells and is beneficial to the transportation and the culture of the cells due to the chitin-based cell carrier, has small probability of immunological rejection reaction of the cell carrier and avoids the failure of the cell carrier in the cell culture.
The purpose of the invention can be realized by the following technical scheme:
a gel-like material cell carrier based on chitin is prepared by the following steps:
the method comprises the following steps: preparing chitin, dissolving raw materials for preparing chitin in a treatment solution, stirring, adding an extracting solution in the stirring process, reacting for 1-1.5 h at 20-45 ℃, finally adding a 1-3% chitin aqueous solution, continuing to react for 1-3 h, dialyzing the reaction solution, and freeze-drying to obtain chitin;
step two: mixing paclitaxel solution with chitin prepared in step one, adding penetrating fluid, performing ultrasonic treatment, and measuring pH value of the mixed solution;
step three: and (3) preparing the nanogel into a wafer shape, standing the solution treated in the step two, removing the upper colorless transparent liquid, depositing the lower layer on the wafer prepared from the nanogel, and drying and freezing to obtain the cell carrier.
Further, the treatment solution in the first step is a methanol solution, and the stirring speed is 1000-1500 rpm.
Further, the pH value is adjusted to 8.0-9.0 in the second step.
Further, the drying and freezing treatment in the third step is drying for 20-30 min by a freeze dryer (the temperature is between 50 ℃ below zero and 60 ℃ below zero and 18-20 pa).
The invention has the beneficial effects that:
1. the preparation method is simple, has strong operability and simple material acquisition, and is beneficial to reducing the production cost of the cell carrier;
2. the cell carrier is based on chitin, so that the survival probability and the production time of cells are improved, and the cell carrier is favorable for transferring and culturing the cells;
3. the cell carrier has small immune rejection probability, and avoids failure caused by the problem of the cell carrier in cell culture.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A gel-like material cell carrier based on chitin is prepared by the following steps:
the method comprises the following steps: preparing chitin, dissolving raw materials for preparing chitin in a treatment solution, stirring, adding an extracting solution in the stirring process, reacting for 1-1.5 h at 20-45 ℃, finally adding a 1-3% chitin aqueous solution, continuing to react for 1-3 h, dialyzing the reaction solution, and freeze-drying to obtain chitin;
step two: mixing paclitaxel solution with chitin prepared in step one, adding penetrating fluid, performing ultrasonic treatment, and measuring pH of the mixed solution.
Step three: preparing the nanogel into a wafer shape, standing the solution treated in the step two, removing the upper colorless transparent liquid, depositing the lower layer on the wafer prepared from the nanogel, and drying and freezing to obtain a cell carrier;
in the first step, the treatment solution is a methanol solution, and the stirring speed is 1000-1500 rpm.
And adjusting the pH value to 8.0-9.0 in the second step.
And drying and freezing for 20-30 min by using a freeze dryer (the temperature is between 50 and 60 ℃ below zero and 18-20 pa), so as to obtain the porous cell carrier particles.
Example 1
A gel-like material cell carrier based on chitin is prepared by the following steps:
the method comprises the following steps: preparing chitin, dissolving raw materials for preparing chitin in methanol solution at the stirring speed of 1000rpm, adding the extract during stirring, reacting at 20 ℃ for 1h, finally adding 1% chitin aqueous solution, continuing to react for 1h, dialyzing the reaction solution, and freeze-drying to obtain chitin;
step two: mixing paclitaxel solution with chitin prepared in step one, adding penetrating fluid, performing ultrasonic treatment, and measuring pH of the mixed solution to obtain pH of 8.0.
Step three: and (3) preparing the nanogel into a wafer shape, standing the solution treated in the step two, removing the upper colorless transparent liquid, depositing the lower layer on the wafer prepared from the nanogel, and drying for 30min at the temperature of 50 ℃ below zero and 20pa by using a freeze dryer to obtain the porous cell carrier particles.
Example 2
A gel-like material cell carrier based on chitin is prepared by the following steps:
the method comprises the following steps: preparing chitin, dissolving raw materials for preparing chitin in the treatment solution, stirring, adding the extract during stirring, reacting at 35 deg.C for 1.2h, adding 2% chitin water solution, continuing to react for 2h, dialyzing the reaction solution, and freeze-drying to obtain chitin;
step two: mixing paclitaxel solution with chitin prepared in step one, adding penetrating fluid, performing ultrasonic treatment, and measuring pH of the mixed solution.
Step three: and (3) preparing the nanogel into a wafer shape, standing the solution treated in the step two, removing the upper colorless transparent liquid, depositing the lower layer on the wafer prepared from the nanogel, and drying and freezing to obtain the cell carrier.
In the first step, the treatment solution is methanol solution, and the stirring speed is 1200 rpm. Adjusting the pH value to 9.0 in the second step, and drying for 25min by using a freeze dryer (-55 ℃, 19pa) in the third step to obtain the porous cell carrier particles.
Example 3
A gel-like material cell carrier based on chitin is prepared by the following steps:
the method comprises the following steps: preparing chitin, dissolving raw materials for preparing chitin in the treatment solution, stirring, adding the extract during stirring, reacting at 45 deg.C for 1.5h, adding 3% chitin water solution, continuing to react for 3h, dialyzing the reaction solution, and freeze-drying to obtain chitin;
step two: mixing paclitaxel solution with chitin prepared in step one, adding penetrating fluid, performing ultrasonic treatment, and measuring pH of the mixed solution.
Step three: and (3) preparing the nanogel into a wafer shape, standing the solution treated in the step two, removing the upper colorless transparent liquid, depositing the lower layer on the wafer prepared from the nanogel, and drying and freezing to obtain the cell carrier.
In the step one, the treatment solution is methanol solution, the stirring speed is 1500rpm, the pH value is adjusted to 8.0 in the step two, and in the step three, the drying and freezing treatment is drying for 30min by a freeze dryer (-60 ℃, 20pa), so that the porous cell carrier particles are obtained.
Example 3
A gel-like material cell carrier based on chitin is prepared by the following steps:
the method comprises the following steps: preparing chitin, dissolving raw materials for preparing chitin in the treatment solution, stirring, adding the extract during stirring, reacting at 25 deg.C for 1.5h, adding 3% chitin water solution, continuing to react for 3h, dialyzing the reaction solution, and freeze-drying to obtain chitin;
step two: mixing paclitaxel solution with chitin prepared in step one, adding penetrating fluid, performing ultrasonic treatment, and measuring pH of the mixed solution.
Step three: preparing the nanogel into a wafer shape, standing the solution treated in the step two, removing the upper colorless transparent liquid, depositing the lower layer on the wafer prepared from the nanogel, and drying and freezing to obtain a cell carrier;
and (3) in the first step, the treatment solution is a methanol solution, the stirring speed is 1100rpm, the pH value is adjusted to be 8.0 in the second step, the drying and freezing treatment in the third step is a freeze dryer-50, and the drying is carried out for 20-30 min at 20pa, so that the porous cell carrier particles are obtained.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Claims (4)
1. A gel-like material cell carrier based on chitin is characterized by being prepared by the following steps:
the method comprises the following steps: preparing chitin, dissolving raw materials for preparing chitin in a treatment solution, stirring, adding an extracting solution in the stirring process, reacting for 1-1.5 h at 20-45 ℃, finally adding a 1-3% chitin aqueous solution, continuing to react for 1-3 h, dialyzing the reaction solution, and freeze-drying to obtain chitin;
step two: mixing paclitaxel solution with chitin prepared in step one, adding penetrating fluid, performing ultrasonic treatment, and measuring pH value of the mixed solution;
step three: and (3) preparing the nanogel into a wafer shape, standing the solution treated in the step two, removing the upper colorless transparent liquid, depositing the lower layer on the wafer prepared from the nanogel, and drying and freezing to obtain the cell carrier.
2. The cell carrier of chitin-based gel-like material according to claim 1, wherein the treating solution in step one is methanol solution, and the stirring speed is 1000-1500 rpm.
3. The chitin-based gel-like material cell carrier according to claim 1, wherein the pH value is adjusted to 8.0-9.0 in step two.
4. The chitin-based gelatinous material cell carrier of claim 1, wherein the step three drying and freezing treatment is drying for 20-30 min by a freeze dryer (-50-60 ℃, 18-20 pa).
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115252829A (en) * | 2022-08-07 | 2022-11-01 | 山西省人民医院 | Preparation method of ultrasonic contrast agent capable of targeting kidney cancer administration |
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|---|---|---|---|---|
| WO1997003708A1 (en) * | 1995-07-17 | 1997-02-06 | Aber Technologies S.A. | Chitin gel dressing for chronic wounds, particularly sloughs |
| CN109988314A (en) * | 2019-04-04 | 2019-07-09 | 大连民族大学 | A kind of hyperbranched poly chitin and its preparation method and application |
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2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997003708A1 (en) * | 1995-07-17 | 1997-02-06 | Aber Technologies S.A. | Chitin gel dressing for chronic wounds, particularly sloughs |
| CN109988314A (en) * | 2019-04-04 | 2019-07-09 | 大连民族大学 | A kind of hyperbranched poly chitin and its preparation method and application |
Non-Patent Citations (2)
| Title |
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| K.T. SMITHA ET AL.: "In vitro evaluation of paclitaxel loaded amorphous chitin nanoparticles for colon cancer drug delivery", 《COLLOIDS AND SURFACES B: BIOINTERFACES》 * |
| SABITHA MANGALATHILLAM ET AL.: "Curcumin loaded chitin nanogels for skin cancer treatment via the transdermal route", 《NANOSCALE》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115252829A (en) * | 2022-08-07 | 2022-11-01 | 山西省人民医院 | Preparation method of ultrasonic contrast agent capable of targeting kidney cancer administration |
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