CN111249259A - Preparation method of mixed type biological synthetic cellulose membrane - Google Patents

Preparation method of mixed type biological synthetic cellulose membrane Download PDF

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CN111249259A
CN111249259A CN202010268106.5A CN202010268106A CN111249259A CN 111249259 A CN111249259 A CN 111249259A CN 202010268106 A CN202010268106 A CN 202010268106A CN 111249259 A CN111249259 A CN 111249259A
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cellulose membrane
mixed type
biosynthetic
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biosynthetic cellulose
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伊芙.菜
泰莎.里贾纳.斯顿夫
帕特里克.福涅尔
李通达
曹旭东
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Tai ShaLijianaSidunfu
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A61K9/7007—Drug-containing films, membranes or sheets
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
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    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof

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Abstract

The invention discloses a preparation method of a mixed type biosynthetic cellulose membrane, and particularly relates to the field of biosynthesis and medical materials, wherein the preparation method comprises the following steps: inoculating the strain A into a culture medium, performing static culture, and extracting to obtain an original biosynthetic cellulose membrane. And transferring the original biosynthetic cellulose membrane into a sodium hydroxide solution, stirring and washing to obtain a primary pure biosynthetic cellulose membrane. Washing the preliminary pure biological synthetic cellulose membrane by distilled water to obtain the biological synthetic cellulose membrane. Taking a biological synthetic cellulose membrane, adding distilled water, mixing and crushing to obtain paper pulp. And filtering the paper pulp by using a Buchner porous disc filter for 35min to obtain the mixed type biosynthetic cellulose membrane. And freeze-drying the obtained mixed type biosynthetic cellulose membrane to obtain the mixed type biosynthetic cellulose membrane. The biological cellulose membrane prepared by the method provided by the invention can be used as a substrate material in a drug release carrier system.

Description

Preparation method of mixed type biological synthetic cellulose membrane
Technical Field
The invention relates to the field of biosynthesis and medical materials, in particular to a preparation method of a mixed type biosynthetic cellulose membrane.
Background
Currently, many natural or synthetic biomaterials have been widely used in tissue engineering applications. Recently, naturally occurring materials such as cellulose, chitosan, hyaluronic acid and collagen have been widely used in tissue engineering and have attracted great interest1. Among these materials, cellulose is the most widely distributed natural biomaterial on earth2It is an important structural component of the cell wall of green plants, and some species of bacteria can also produce cellulose (i.e., biosynthetic cellulose). The advantage of biosynthetic cellulose is that it requires chemical treatment to obtain pure cellulose, unlike plant cellulose3. In addition, unmodified biosynthetic cellulose has unique physical and mechanical properties not found in other biomaterials, such as high purity, ultrafine fiber network structures, and pore structures. The biological synthetic cellulose has water absorbing capacity capable of adsorbing water 100 times higher than its own weight, high crystallinity (crystallinity reaches 84-89%), wide physical and chemical modification capacity and plasticity.
However, biosynthetic cellulose has not been fully utilized as a potential biomaterial for use in the field of tissue engineering. Most of the current research and development efforts directed to the biosynthesis of cellulose are focused on the improvement of the properties of the biosynthesis of cellulose, such as controlling the porosity of the cultured biosynthesis cellulose scaffold by optimizing the bacterial culture conditions, introducing functional groups on the biosynthesis cellulose matrix, and increasing the degradation rate of the biosynthesis cellulose, but there is no related research on the shaping process and treatment of the biosynthesis cellulose itself. For example, the drug loading capacity of the biosynthetic cellulose as the biomaterial is limited by the critical step of sterilization and disinfection, so that the biological activity of the loaded drugs, especially various protein-based growth factors, cannot be well maintained, and the development and application of the biosynthetic cellulose as the drug release carrier material are greatly limited. Therefore, how to prepare a drug delivery carrier using biosynthetic cellulose as a matrix material, which can solve the above-mentioned drawbacks, is a problem that the skilled person needs to solve.
Disclosure of Invention
The invention provides a preparation method of a biosynthetic cellulose membrane, which can be used for preparing the biosynthetic cellulose membrane used as a drug carrier.
In order to achieve the purpose, the invention provides the following technical scheme:
a preparation method of a mixed type biological synthetic cellulose membrane comprises the following steps:
s01, inoculating the strain A into a Hestrin-Schram culture medium, statically culturing for 7 days at the temperature of 26 ℃, and extracting to obtain the original biosynthetic cellulose membrane.
S02, transferring the obtained original biosynthetic cellulose membrane to a sodium hydroxide solution with the temperature of 50 ℃ and the concentration of 0.1M, and stirring and washing to obtain a primary pure biosynthetic cellulose membrane.
S03, washing the preliminary pure biological synthetic cellulose membrane obtained by the steps by distilled water to obtain the biological synthetic cellulose membrane.
S04, taking the obtained biological synthetic cellulose membrane, adding distilled water, mixing and crushing to obtain paper pulp.
S05, filtering the paper pulp by a Buchner porous disc filter for 40 minutes to obtain the mixed type biological synthetic cellulose membrane.
S06, freezing the obtained mixed type biological synthetic cellulose membrane for 24 hours at the temperature of-20 ℃, and obtaining the final mixed type biological synthetic cellulose membrane after freeze drying, wherein the final mixed type biological synthetic cellulose membrane can be placed in a vacuum drying dish for storage.
Preferably, after step S04, the preparation method further includes the steps of:
s04.1, weighing quantitative paper pulp, and performing suction filtration for 40 minutes to obtain the mass of an initial sample;
s04.2, setting the mass of the initial sample as M, MxRepresents the initial sample mass measured in the x-th experiment, x is 1,2,3 …;
let the mass of the mixed type biosynthetic cellulose film obtained in step S06 be N, NxRepresents the mass of the mixed type biosynthetic cellulose membrane measured in the x-th experiment, wherein x is 1,2,3 …;
by mixing MxCorresponding to NxPerforming data statistics and regression to obtain the following relational expression:
Nx＝Mx*0.1518；
s04.3, substituting the mass of the initial sample to be detected into the relational expression to calculate to obtain the mass (M) of the paper pulp required by the production of the required mixed type biosynthetic cellulose membrane (N);
s04.4, weighing the pulp mass calculated in the step S04.3, namely the pulp in the step S05, and carrying out the step S05.
Preferably, the strain A is gluconacetobacter hancei.
Preferably, in step S01, the culture temperature is 26 ℃ and the static culture time is 7 days.
Preferably, in step S04, the mass ratio of the biosynthetic cellulose membrane to distilled water is 1 g: 15 ml.
Preferably, in step S05, the filtration time is 40 minutes.
Preferably, the mixed type biosynthetic cellulose membrane is frozen at-20 ℃ for 24 hours.
The invention also provides a preparation method of the mixed type biological synthetic cellulose membrane, and the mixed type biological synthetic cellulose membrane is sterilized at high temperature and high pressure to prepare a drug release carrier system taking the biological cellulose membrane as a substrate.
The invention has the beneficial effects that:
in the invention, firstly, a biosynthetic cellulose membrane obtained by bacterial culture is mixed with water and crushed, and then the pulp obtained by crushing is subjected to suction filtration, freeze drying and high-temperature disinfection to obtain a final finished product. Due to the introduction of the paper pulp suction filtration forming process, the forming processing link of the biological cellulose membrane is skillfully separated from the drug loading link. Compared with the traditional preparation method of the cellulose membrane drug carrier (the traditional process usually combines a drug loading link and a cellulose membrane forming link, so that the inactivation of the loaded drug can be caused by the subsequent disinfection link), the new process well coordinates the relation between the disinfection link and the drug loading link, and particularly solves the possible contradiction between the loading process of some volatile and active drugs and the disinfection process thereof, so that the potential of the application of cellulose as the drug carrier is further improved. By applying the new process, after the mixed type biological cellulose membrane is sterilized, water-soluble growth factors (sterilized) are transferred to the surface of the prepared mixed type biological cellulose membrane (sterilized) through a liquid transfer gun, and then the growth factor water solution is completely absorbed by the mixed type biological cellulose membrane so as to complete medicine loading. Experimental data indicate that bovine serum albumin loaded in this way can achieve sustained release for up to twelve days. The prepared mixed type biosynthetic cellulose membrane is further proved to be easy to load the medicine.
Drawings
FIG. 1 is a graph showing the change in swelling ratio of the mixed type biosynthetic cellulose in test example 1.
FIG. 2 is a graph showing the mechanical properties of a sample of the mixed type biosynthetic cellulose film of test example 2. Wherein (A) represents the stress vs strain curve of sample BBC 1-5; (B) representative of the young's modulus of each sample; (C) represents the ultimate tensile strength of each sample; (D) representing the elongation at break of each sample.
FIG. 3 is a scanning electron microscope image of raw-state Biosynthetic Cellulose (BC) and mixed-type biological cellulose (BBC) in test example 3.
Fig. 4 is a drug release profile of the mixed type bio-cellulose film sample BBC5 in test example 4.
Detailed Description
Example 1
The embodiment provides a technical scheme:
a preparation method of a mixed type biological synthetic cellulose membrane comprises the following steps:
s01, inoculating the strain A into a Hestrin-Schram culture medium, statically culturing for 7 days at the temperature of 26 ℃, and extracting to obtain the original biosynthetic cellulose membrane.
S02, transferring the obtained original biosynthetic cellulose membrane to a sodium hydroxide solution with the temperature of 50 ℃ and the concentration of 0.1M, and stirring and washing to obtain a primary pure biosynthetic cellulose membrane.
S03, washing the preliminary pure biological synthetic cellulose membrane obtained by the steps by distilled water to obtain the biological synthetic cellulose membrane.
S04, taking the obtained biological synthetic cellulose membrane, adding distilled water, mixing and crushing to obtain paper pulp.
S05, weighing quantitative paper pulp, and performing suction filtration for 40 minutes to obtain the mass of the initial sample.
S06, setting the mass of the initial sample as M, MxRepresents the initial sample mass measured in the x-th experiment, wherein x is 1,2,3 ….
Let the mass of the mixed type biosynthetic cellulose membrane obtained in step S010 be N, NxRepresents the mass of the mixed type biosynthetic cellulose membrane measured in the x-th experiment, wherein x is 1,2,3 …;
by mixing MxCorresponding to NxPerforming data statistics and regression to obtain the following relational expression:
Nx＝Mx*0.1518。
and S07, substituting the mass of the initial sample to be measured into the relational expression to calculate to obtain the mass (M) of the paper pulp required by the production of the mixed type biosynthetic cellulose membrane (N).
S08, weighing the pulp mass M calculated in the step S07, namely the pulp in the step S05, and carrying out the step S05.
S09, filtering the paper pulp by a Buchner porous disc filter for 40 minutes to obtain the mixed type biological synthetic cellulose membrane.
S010, freezing the obtained mixed type biological synthetic cellulose membrane for 24 hours at the temperature of minus 20 ℃, and obtaining the final mixed type biological synthetic cellulose membrane after freeze drying, wherein the final mixed type biological synthetic cellulose membrane can be placed in a vacuum drying dish for storage.
Test example 1
As shown in fig. 1, the swelling performance of the mixed type biosynthetic cellulose film was tested in this test example, and the test included the following steps:
(1) preparing five mixed type biosynthetic cellulose membranes with different cellulose contents, sequentially increasing the mass,
designated samples BBC1, BBC2, BBC3, BBC4, and BBC5, respectively;
(2) the dried samples were first weighed (W)dry) Soaking in PBS buffer (pH7.4) at 37 deg.C; subsequently, at a specific different time point, the swollen cellulose film was taken out and weighed again (W)swo). The swelling ratio of the sample was calculated by the following formula;
Figure BDA0002442090720000061
as can be seen from the data in FIG. 1, the swelling ratio of BBC1-5 decreases with increasing mass. For example, sample 1 exhibited the highest swelling ratio and sample 5 exhibited the lowest swelling ratio. It is worth mentioning that the swelling ratio of all samples rose rapidly within the first five hours and remained substantially constant after forty-eight hours. The embedding of the medicine is based on the adsorption principle of the mixed biological cellulose membrane to the water solution of the embedded medicine, which shows that the water-soluble medicine can be smoothly embedded in the prepared cellulose membrane.
Test example 2
As shown in FIG. 2, in this test example, in order to characterize the mechanical properties of the mixed type biosynthetic cellulose film, the cellulose film swollen in example 1 was subjected to a tensile test. The test comprises the following steps:
(1) five samples of example 1 were cut into a rectangle of uniform size (size: 25mmx 10 mm);
(2) the tested environmental parameters are 25 +/-2 ℃ and the relative humidity is 45 +/-5%;
(3) the samples tested were tested in Instron ElectroPulsTMTensile testing was performed on an E3000 All-Electric Dynamic Test Instrument (Norwood, MA) Instrument. The tensile test speed was set at 5mm/min and the load cell dynamic rate was set at 250N. Sample test data passage
Figure BDA0002442090720000071
Software (Norwood, MA) was processed to obtain the young's modulus, ultimate tensile strength and elongation at break of the samples as shown in fig. 2.
From the data shown in FIG. 2, it is understood that the Young's modulus and ultimate tensile strength of the cellulose film samples produced are improved as the mass of cellulose in the samples is increased. This trend also indicates that the mechanical strength of the mixed-type cellulose film can be achieved by adjusting the quality of cellulose in the cellulose film. For example, sample BBC5 has a Young's modulus of 0.37. + -. 0.02MPa and an ultimate tensile strength of 0.96. + -. 0.02 MPa; the values of these two indices are about 9 and 6 times higher than the indices of the corresponding BBC1 samples, respectively. However, the elongation at break of sample BBC5 was 3.90 ± 0.75%, which was not significantly different from the other samples.
Test example 3
As shown in fig. 3, in order to compare the microstructure difference between the biosynthetic cellulose membrane without the preparation process (i.e. as-is) and the mixed type biosynthetic cellulose membrane treated by the process, we observed the microstructure of the two membranes by a Scanning Electron Microscope (SEM), which specifically includes the following steps:
the freeze-vacuum dried biosynthetic cellulose film and the mixed type biosynthetic cellulose film were cut out to appropriate sizes, respectively, to expose cross sections, followed by observation with SEM (Phoenix, AZ). The cross section of the upper layer (the side in contact with air in the case of the original bacterial culture) and the lower layer (the side in contact with the medium in the case of the original bacterial culture) of the sample was selected and observed. The magnification of the upper layer and the lower layer is 300 times, and the magnification of the cross section is 2000 times.
From fig. 3 it can be seen that the original biosynthetic cellulose membrane consists of a highly entangled cellulose network and that the density of the cellulose fiber structure is higher on the membrane/air side than on the membrane/liquid side. In contrast, the hybrid biosynthetic cellulose membranes present a uniform cellulose network structure on both sides of the membrane. Furthermore, by comparing the SEM images of sample BBC1 with sample BBC5, it can be seen that as the cellulose content in the sample increases, the density of cellulose in the sample also increases and the cellulose structure in the resulting sample becomes denser. This trend also suggests that the microstructure of the hybrid biosynthetic cellulose membrane can be achieved by adjusting the quality of the cellulose in the membrane.
Test example 4
In order to investigate the possibility of using the mixed type biosynthetic cellulose membrane as a drug carrier, we performed drug release characterization on the mixed type biosynthetic cellulose membrane, which specifically includes the following steps:
(1) epidermal Growth Factor (EGF) and fibroblast growth factor-2 (FGF 2) were used as model drugs to investigate the possibility of mixed biosynthetic cellulose membranes as drug carriers.
(2) Sample BBC5 was selected as the drug carrier.
(3) The specific steps of loading the drug are: 70 microliters of PBS (pH7.4) solutions containing 430. + -.10 ng EGF and FGF2, respectively, were pipetted carefully onto the lyophilized and autoclaved BBC 5.
(4) The BBC5 sample loaded with EGF and FGF2 was left to stand in an ultraclean bench for two hours to ensure complete absorption of the EGF and FGF2 solutions by the BBC5 membrane;
(5) the sample BBC5 was then completely immersed in 2ml of PBS (pH7.4) release solution, and the temperature was maintained at 37 ℃.
(6) According to the preset sampling time points, 0.32ml of sample solution was carefully withdrawn from BBC5 release solution each time and stored at-80C, and then BBC5 release solution was supplemented with the same volume of PBS (pH7.4) release solution.
(7) The concentration of EGF and FGF2 in samples collected at different time points was determined by corresponding ELISA kits.
As can be seen from fig. 4, BBC5 has similar release profiles for EGF and FGF 2. Both growth factors exhibited a burst effect within the first day and exhibited a uniform release thereafter up to the tenth day; and after ten days the release profile began to flatten out. Our data indicate that the cumulative release of EGF and FGF2 over the first ten days is essentially the same, 397.4 + -6.9 ng and 393.6 + -9.2 ng, respectively, i.e., 92.4% and 91.5% of the original drug load, respectively. The above data fully demonstrate the potential of hybrid biosynthetic celluloses as carriers for sustained release drugs.

Claims (8)

1. A preparation method of a mixed type biological synthetic cellulose membrane is characterized by comprising the following steps:
s01, inoculating the strain A into a Hestrin-Schram culture medium, statically culturing for 5-12 days at the temperature of 20-32 ℃, and extracting to obtain an original biosynthetic cellulose membrane;
s02, transferring the obtained original biosynthetic cellulose membrane to a sodium hydroxide solution with the temperature of 50 ℃ and the concentration of 0.1M, and stirring and washing to obtain a primary pure biosynthetic cellulose membrane;
s03, washing the preliminary pure biosynthetic cellulose membrane obtained in the previous step by distilled water to obtain the biosynthetic cellulose membrane;
s04, taking the obtained biological synthetic cellulose membrane, adding distilled water, mixing and crushing to obtain paper pulp;
s05, filtering the paper pulp by a Buchner porous disc filter for 30-45 minutes to obtain a mixed type biological synthetic cellulose membrane;
s06, freezing the obtained mixed type biological synthetic cellulose membrane for 24 hours at the temperature of-20 ℃, and obtaining the final mixed type biological synthetic cellulose membrane after freeze drying, wherein the final mixed type biological synthetic cellulose membrane can be placed in a vacuum drying dish for storage.
2. The method for preparing a biosynthetic cellulose membrane according to claim 1, further comprising, after step S04, the steps of:
s04.1, weighing quantitative paper pulp, and performing suction filtration in a porous glass filter for 40 minutes to obtain the mass of an initial sample;
s04.2, setting the mass of the initial sample as M, MxRepresents the initial sample mass measured in the x-th experiment, x is 1,2,3 …;
let the mass of the mixed type biosynthetic cellulose film obtained in step S06 be N, NxRepresents the mass of the mixed type biosynthetic cellulose membrane measured in the x-th experiment, wherein x is 1,2,3 …;
by mixing MxCorresponding to NxPerforming data statistics and regression to obtain the following relational expression:
Nx＝Mx*0.1518
s04.3, substituting the mass of the initial sample to be detected into the relational expression to calculate to obtain the mass (M) of the paper pulp required by the production of the mixed type biosynthetic cellulose membrane with certain required mass (N);
s04.4, weighing the pulp mass calculated in the step S04.3, namely the pulp in the step S05, and carrying out the step S05.
3. The method of claim 1, wherein the strain A is Gluconobacter hansenii.
4. The method for preparing a mixed type biosynthetic cellulose membrane of claim 1, wherein in step S01, the incubation temperature is 26 ℃ and the static incubation time is 7 days.
5. The method for preparing a mixed type biosynthetic cellulose membrane of claim 1, wherein in step S04, the mass ratio of the biosynthetic cellulose membrane to distilled water is 1 g: 15 ml.
6. The method for preparing a mixed type biosynthetic cellulose membrane of claim 1, wherein in step S05, the filtration time is 40 minutes.
7. The method according to claim 1, wherein the hybrid biosynthetic cellulose membrane is placed in a petri dish at-20 ℃ for 24 hours.
8. The method according to any of claims 1 to 7, wherein the mixed type biosynthetic cellulose membrane prepared by the method is sterilized at 121 ℃ under a pressure of 15 psi for 35 minutes, and then the drug delivery vehicle system based on the biological cellulose membrane is prepared.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113384752A (en) * 2021-06-11 2021-09-14 海南医学院 Exosome-based nerve dissociation repair material and preparation method thereof

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* Cited by examiner, † Cited by third party
Title
TAISA REGINA STUMPF等: "Design and evaluation of a biosynthesized cellulose drug releasing duraplasty", 《MATERIALS SCIENCE & ENGINEERING C》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113384752A (en) * 2021-06-11 2021-09-14 海南医学院 Exosome-based nerve dissociation repair material and preparation method thereof

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