AU2021101350A4 - Oral mscs liquid-filled capsule for treating inflammatory bowel diseases and other intestinal diseases - Google Patents
Oral mscs liquid-filled capsule for treating inflammatory bowel diseases and other intestinal diseases Download PDFInfo
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Abstract
An oral MSCs liquid-filled capsule includes a three-layered capsule shell and capsule contents.
The capsule shell includes, from inside to outside, a calcium carbonate coating for neutralizing
gastric acid, a water-soluble gelatin-hydroxypropyl methylcellulose gel and a Eudragit S100 coating,
and the capsule contents are MSCs microspheres suspended in a sodium alginate hydrogel culture
medium. The present invention analyzes the characteristics of colon oral drug delivery system,
combines the properties of biomaterials, and constructs an MSCs-loaded liquid-filled oral
microcapsule through high-precision biological 3D printing technology to realize oral delivery of
MSCs to colon and alleviate intestinal diseases. The new method is simple and convenient to operate.
MSCs in the microspheres can exert anti-inflammatory and immunoregulatory effects for extended
periods through paracrine to relieve symptoms of colonic-related diseases, thereby the cell-based oral
capsules of MSCs and other cells can reach the minimum cell volume during treatment in vivo.
Description
The present invention relates to the technical field of medicine for treating intestinal diseases,
and in particular to an oral MSCs liquid-filled capsule for treating inflammatory bowel diseases and
other intestinal diseases.
Mesenchymal stem cells (MSCs) are pluripotent stem cells with immunomodulatory and
anti-inflammatory functions. In recent years, a large number of studies have shown that MSCs have
the clinical potential to treat various inflammatory diseases, such as complex anal fistula and
inflammatory bowel diseases. It is reported that MSCs can navigate to the damaged inflammatory
intestinal site after transplantation, which can effectively alleviate the course of colitis. Therefore, the
method of MSCs transplantation is a primary factor in determining the therapeutic effect.
The main obstacle of traditional intravenous administration of MSCs is that the first pass effect
of lung leads to low navigating efficiency, large dose demand and treatment difference. Rectal
administration is complicated in operation, moreover, a large number of intestinal contents will
reduce the survival rate of cells. Additionally, the delivery site is limited to the rectum and
descending colon, and cannot reach major ulcer areas such as the transverse colon and ascending
colon, which affects the efficacy of MSCs. Oral colon targeted drug delivery can protect the targeted
release of drugs in the colon, fully improve the efficacy of drugs, reduce the dose, as well as reduce
the adverse effects of drugs on human body, which is an ideal way of drug delivery. Currently, most
of the drugs on the market or in clinical research are encapsulated with pharmaceutical gelatin and
coating materials, such as sulfasalazine colon-soluble capsules and Changweining capsules.
Therefore, in the present invention, we mix MSCs microspheres into hydrogel culture medium,
and encapsulate them in colon-soluble soft capsules with cell protection function through traditional
soft capsule technology or 3D printing technology. After oral administration, MSCs microspheres
begin to release after reaching the colon, and play a dual role in slow-release and colon positioning.
The oral MSCs liquid-filled capsule is easy to administer, safe and effective, which improves the
treatment efficiency. The successful application of oral MSCs liquid-filled capsule in preclinical
studies such as autoimmunity or inflammation opens up the way for clinical trials, and provides
guiding significance for the development of capsules with oral safety, feasibility and effectiveness.
Therefore, the present invention has application prospects and feasibility for the treatment research
of oral MSCs, and is worthy of attention.
In order to solve the disadvantages of low navigation rate, large infusion dose and unstable
treatment effect of traditional MSCs in the treatment of chronic intestinal diseases, the present
invention provides an oral MSCs liquid-filled biomimetic capsule and an application thereof in the
treatment of colitis.
To achieve the above objective, the technical solution provided by the present invention is as
follows.
An oral MSCs liquid-filled capsule, including a three-layered capsule shell and capsule contents.
The capsule shell includes, from inside to outside, a calcium carbonate coating for neutralizing
gastric acid, a water-soluble gelatin-hydroxypropyl methylcellulose gel and a Eudragit S100 coating,
and the capsule contents are MSCs microspheres suspended in a sodium alginate hydrogel culture
medium.
A preparation method of the oral MSCs liquid-filled capsule includes the following steps:
(1) preparation of soft capsule shell:
preparation of calcium carbonate solution: adding a certain amount of calcium carbonate into
double distilled water to disperse and fully dissolve at room temperature;
preparation of gelatin-hydroxypropyl methylcellulose gel: adding a certain amount of gelatin
and hydroxypropyl methylcellulose into a suitable amount of double distilled water respectively to
dissolve, after cooling to room temperature, mixing them according to a certain ratio, stirring
thoroughly until uniform, and putting in a refrigerator to form a homogeneous mixed gel; a total
concentration of the gelatin and the hydroxypropyl methylcellulose in the final mixed gel is 0.5-10%,
of which a concentration of the gelatin is 0.05-5.0%; preparation of Eudragit S100 coating solution: preparing a 1-3% w/v of Eudragit S100 ethanol solution;
(2) preparation of MSCs microspheres-loaded alginate hydrogel:
mixing umbilical cord MSCs evenly in sodium alginate hydrogel at a certain density, spraying
the mixed solution into a calcium chloride solution under a high voltage by a microfluidic
electrospray technology to obtain cell-encapsulated microgel droplets; a size of the microspheres
could be regulated by a voltage and a flow rate;
(3) preparation of MSCs oral capsule by 3D printing
collecting biological information of the soft capsule, and inputting the biological information
into a computer for biomimetic modeling, loading the three-layered soft capsule shell and the MSCs
microspheres-loaded alginate hydrogel with predetermined amounts into a prepared extruded 3D
printing silo, and performing 3D printing after setting printing parameters; performing a
continuously circulated perfusion culture on the printed liquid-filled capsule in a biomimetic culture
medium to realize a pre-forming of the printed liquid-filled capsule.
In step (1), the calcium carbonate is dissolved at room temperature for 20-40 min, and a
concentration of the calcium carbonate is 5-30% w/v; the gelatin and hydroxypropyl methylcellulose
are added into the suitable amount of double distilled water respectively at a reaction temperature of
°C, for a stirring time of 1-4 h to be fully dissolved, and the two aqueous solutions are mixed and
placed in a 4W refrigerator for more than 24 h to form the homogeneous mixed gel.
In step (1), viscosity of the hydroxypropyl methylcellulose is 100-10,000 mPa-s; composition of
the Eudragit S100 is methacrylate: methacrylic acid = 1:2 (mass ratio).
The umbilical cord MSCs are selected from the second to fourth generations of human
umbilical cord mesenchymal stem cells with a cell-seeding density of 106 cells/mL.
The electrospray parameters of the microfluidic electrospray technology are set as follows: the
voltage is 5 kV and the flow rate is 30 L/min.
The sodium alginate hydrogel culture medium is prepared by an F12/DMEM complete culture
medium.
The MSCs-loaded sodium alginate microspheres prepared by the microfluidic electrospray
technology are 143 ±5.2 pm in size and suspended in 1% w/v sodium alginate hydrogel.
In step (3), the 3D printing is conducted with a 3D printer by repetier host V.0.5 software; the biomimetic culture medium is a MSCs culture medium containing F12/DMEM + 10% fetal bovine serum + antibiotics.
The present invention also provides an application of the oral MSCs liquid-filled capsule as a
drug for treating intestinal diseases.
The intestinal diseases include inflammatory bowel diseases.
Compared with the prior art, the present invention has the following advantages.
(1) The present invention analyzes the characteristics of colon oral drug delivery system,
combines the properties of biomaterials, and constructs an MSCs-loaded liquid-filled oral
microcapsule through high-precision biological 3D printing technology to realize oral delivery of
MSCs to colon and alleviate intestinal diseases. The new method is simple and convenient to
operate.
(2) Through the four-layer structure accurate 3D printing technology, the present invention
accurately regulates and controls the parameters of each layer structure of the liquid-filled capsule,
highly optimizes the structural characteristics of each layer of the soft capsule shell, highlights the
functions of each layer, and solves the problems of uneven coating thickness, simple structure and
large difference in the traditional soft capsule process. The present invention proposes a multi-layer
structure printing soft capsule to wrap the MSCs contents to form an oral liquid-filled capsule with
anti-acid and anti-digestion functions (preventing gastric acid and digestive juice from penetrating
into the cell layer) and colon-oriented release.
(3) The present invention solves the problem of long-term survival of oral MSCs in digestive
tract. The outermost layer of the soft capsule shell in the present invention is colon soluble material.
After oral administration, the colon soluble material is not dissolved in stomach and small intestine,
so as to protect the capsule from degradation by gastrointestinal protease. The middle layer is
water-soluble material (water-soluble gelatin-hydroxypropyl methylcellulose gel), which plays a role
in isolating the contents from the digestive tract. The innermost layer is calcium carbonate layer,
which neutralizes the infiltrated hydrogen ions and buffers the effect of gastric acid on cell activity.
After the capsule passes through the stomach and small intestine and enters the colon intestinal tract,
the outer layer Eudragit Si00 coating is first dissolved, and then the middle layer water-soluble
material is dissolved to release the sodium alginate hydrogel containing MSCs microspheres. Under
the protection of hydrogel that easily adheres to the intestinal tract, MSCs in the microspheres can exert anti-inflammatory and immunoregulatory effects for a long time through paracrine to relieve symptoms of colonic-related diseases, which are highly practical.
(4) The present invention provides an oral administration route of MSCs and related medical
applications, which alleviate the problem of cell-based oral capsules being destroyed by proteases
and acids in the digestive tract, ensure the long-term survival and stable biological functions of
MSCs in colon intestinal tract, thereby the cell-based oral capsules of MSCs and other cells can
reach the minimum cell volume during treatment in vivo.
FIG. 1 is a schematic diagram showing the structure of the MSCs oral liquid-filled capsule of
the present invention, of which 1 is Eudragit S100 coating material layer, 2 is calcium carbonate
coating, 3 is water-soluble gelatin-hydroxypropyl methylcellulose gel, 4 is sodium alginate
microsphere, 5 is MSCs, and 6 is sodium alginate hydrogel;
FIG. 2 is a diagram showing mice colons after the treatment of dextran sulfate sodium
(DSS)-induced inflammatory disease by the MSCs oral liquid-filled capsule;
FIG. 3 is a diagram showing hematoxylin and eosin (H&E) stained pathological slides of
DSS-induced mice colons after the treatment by the MSCs oral liquid-filled capsule; and
FIGS. 4A and 4B are diagrams showing the efficacy assessment of the MSCs oral liquid-filled
capsule in the treatment of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced inflammatory disease
in mice, of which FIG. 4A is a diagram showing the weight change curve of TNBS-induced mice
after the treatment by the MSCs oral liquid-filled capsule, and FIG. 4B is a diagram showing the
effect of the MSCs oral liquid-filled capsule on TNBS-induced disease activity index.
The present invention will be further described in combination with specific examples.
Example 1
An oral MSCs liquid-filled capsule, including a three-layered capsule shell and capsule contents.
The capsule shell includes, from inside to outside, a calcium carbonate coating for neutralizing
gastric acid, a water-soluble gelatin-hydroxypropyl methylcellulose gel and a Eudragit S100 coating, and the capsule contents are MSCs microspheres suspended in a sodium alginate hydrogel culture medium. The preparation method of the oral MSCs liquid-filled capsule includes the following steps.
(1) Preparation of soft capsule shell
Preparation of calcium carbonate solution: a certain amount of calcium carbonate is added into
double distilled water to disperse and fully dissolve at room temperature.
Preparation of gelatin-hydroxypropyl methylcellulose gel: a certain amount of gelatin and
hydroxypropyl methylcellulose, are added into a suitable amount of double distilled water
respectively to dissolve, after being cooled to room temperature, the two are mixed according to a
certain ratio, stirred thoroughly until uniform, and put in a refrigerator to form a homogeneous mixed
gel. A total concentration of the gelatin and the hydroxypropyl methylcellulose in the final mixed gel
is 2%, of which a concentration of the gelatin is 4%.
Preparation of Eudragit S100 coating solution: a 2% w/v of Eudragit S100 ethanol solution is
prepared.
(2) Preparation of MSCs microspheres-loaded alginate hydrogel
Umbilical cord MSCs are mixed evenly in sodium alginate hydrogel at a certain density, the
mixed solution is sprayed into a calcium chloride solution under a high voltage by a microfluidic
electrospray technology to obtain cell-encapsulated microgel droplets, a size of the microspheres
could be regulated by a voltage and a flow rate, and the MSCs-encapsulated microspheres are
suspended in the sodium alginate hydrogel.
(3) Preparation of MSCs oral capsule by 3D printing
Biological information of the soft capsule is collected and input into a computer for biomimetic
modeling, the three-layered soft capsule shell and the MSCs microspheres-loaded alginate hydrogel
with predetermined amounts are loaded into a prepared extruded 3D printing silo for 3D printing
after printing parameters are set. A continuously circulated perfusion culture is performed on the
printed liquid-filled capsule in a biomimetic culture medium to realize a pre-forming of the printed
liquid-filled capsule. The 3D printing is conducted with a 3D printer by repetier host Vi.0.5
software.
In step (1), the calcium carbonate is dissolved at room temperature for 40 min, and a
concentration of the calcium carbonate is 10% w/v; the gelatin and hydroxypropyl methylcellulose
are added into the suitable amount of double distilled water, respectively, at a reaction temperature of
°C, for a stirring time of 1-4 h to be fully dissolved, and mixed and placed in a 4 refrigerator for more than 24 h to form the homogeneous mixed gel. Viscosity of the hydroxypropyl methylcellulose is 4,000 mPa-s, and composition of the Eudragit S100 is methacrylate: methacrylic acid = 1:2. The umbilical cord MSCs are selected from the second to fourth generations of human umbilical cord mesenchymal stem cells with a cell-seeding density of 106 cells/mL. The electrospray parameters of the microfluidic electrospray technology are set as follows: the voltage is 5 kV and the flow rate is 30 L/min. The sodium alginate hydrogel culture medium is prepared by an F12/DMEM complete culture medium. The MSCs-loaded sodium alginate microspheres prepared by the microfluidic electrospray technology are 143 ±5.2 pm in size and suspended in 1% w/v sodium alginate hydrogel. Example 2 An oral MSCs liquid-filled capsule, including a three-layered capsule shell and capsule contents. The capsule shell includes, from inside to outside, a calcium carbonate coating for neutralizing gastric acid, a water-soluble gelatin-hydroxypropyl methylcellulose gel and a Eudragit S100 coating, and the capsule contents are MSCs microspheres suspended in a sodium alginate hydrogel culture medium. The preparation method of the oral MSCs liquid-filled capsule includes the following steps. (1) Preparation of soft capsule shell Preparation of calcium carbonate solution: a certain amount of calcium carbonate is added into double distilled water to disperse and fully dissolve at room temperature. Preparation of gelatin-hydroxypropyl methylcellulose gel: a certain amount of gelatin and hydroxypropyl methylcellulose, are added into a suitable amount of double distilled water respectively to dissolve, after being cooled to room temperature, the two are mixed according to a certain ratio, stirred thoroughly until uniform, and put in a refrigerator to form a homogeneous mixed gel; a total concentration of the gelatin and the hydroxypropyl methylcellulose in the final mixed gel is 5%, of which a concentration of the gelatin is 2%.
Preparation of Eudragit S100 coating solution: a 2% w/v of Eudragit S100 ethanol solution is prepared. (2) Preparation of MSCs microspheres-loaded alginate hydrogel Umbilical cord MSCs are mixed evenly in sodium alginate hydrogel at a certain density, the mixed solution is sprayed into a calcium chloride solution under a high voltage by a microfluidic electrospray technology to obtain cell-encapsulated microgel droplets, a size of the microspheres could be regulated by a voltage and a flow rate, and the MSCs-encapsulated microspheres are suspended in the sodium alginate hydrogel.
(3) Preparation of MSCs oral capsule by 3D printing
Biological information of the soft capsule is collected and input into a computer for biomimetic
modeling, the three-layered soft capsule shell and the MSCs microspheres-loaded alginate hydrogel
with predetermined amounts are loaded into a prepared extruded 3D printing silo for 3D printing
after printing parameters are set. A continuously circulated perfusion culture is performed on the
printed liquid-filled capsule in a biomimetic culture medium to realize a pre-forming of the printed
liquid-filled capsule. The 3D printing is conducted with a 3D printer by repetier host V.0.5
software.
In step (1), the calcium carbonate is dissolved at room temperature for 20-40 min, and a
concentration of the calcium carbonate is 5-30% w/v; the gelatin and hydroxypropyl methylcellulose
are added into the suitable amount of double distilled water respectively at a reaction temperature of
°C, for a stirring time of 1-4 h to be fully dissolved, and mixed and placed in a 4 refrigerator for
more than 24 h to form the homogeneous mixed gel. Viscosity of the hydroxypropyl methylcellulose
is 1,000 mPa-s, and composition of the Eudragit S100 is methacrylate: methacrylic acid = 1:2.
The umbilical cord MSCs are selected from the second to fourth generations of human
umbilical cord mesenchymal stem cells with a cell-seeding density of 106 cells/mL. The electrospray
parameters of the microfluidic electrospray technology are set as follows: the voltage is 6 kV and the
flow rate is 50 L/min. The sodium alginate hydrogel culture medium is prepared by an F12/DMEM
complete culture medium. The MSCs-loaded sodium alginate microspheres prepared by the
microfluidic electrospray technology are 123 ±6.1 pm in size and suspended in 1% w/v sodium
alginate hydrogel.
Example 3
An oral MSCs liquid-filled capsule, including a three-layered capsule shell and capsule contents.
The capsule shell includes, from inside to outside, a calcium carbonate coating for neutralizing
gastric acid, a water-soluble gelatin-hydroxypropyl methylcellulose gel and a Eudragit S100 coating,
and the capsule contents are MSCs microspheres suspended in a sodium alginate hydrogel culture
medium. The preparation method of the oral MSCs liquid-filled capsule includes the following steps.
(1) Preparation of soft capsule shell
Preparation of calcium carbonate solution: a certain amount of calcium carbonate is added into
double distilled water to disperse and fully dissolve at room temperature.
Preparation of gelatin-hydroxypropyl methylcellulose gel: a certain amount of gelatin and
hydroxypropyl methylcellulose, are added into a suitable amount of double distilled water
respectively to dissolve, after being cooled to room temperature, the two are mixed according to a
certain ratio, stirred thoroughly until uniform, and put in a refrigerator to form a homogeneous mixed
gel; a total concentration of the gelatin and the hydroxypropyl methylcellulose in the final mixed gel
is 6%, of which a concentration of the gelatin is 2%.
Preparation of Eudragit Si00 coating solution: a 3% w/v of Eudragit Si00 ethanol solution is
prepared.
(2) Preparation of MSCs microspheres-loaded alginate hydrogel
Umbilical cord MSCs are mixed evenly in sodium alginate hydrogel at a certain density, the
mixed solution is sprayed into a calcium chloride solution under a high voltage by a microfluidic
electrospray technology to obtain cell-encapsulated microgel droplets, a size of the microspheres
could be regulated by a voltage and a flow rate, and the MSCs-encapsulated microspheres are
suspended in the sodium alginate hydrogel.
(3) Preparation of MSCs oral capsule by 3D printing
Biological information of the soft capsule is collected and input into a computer for biomimetic
modeling, the three-layered soft capsule shell and the MSCs microspheres-loaded alginate hydrogel
with predetermined amounts are loaded into a prepared extruded 3D printing silo for 3D printing
after printing parameters are set. A continuously circulated perfusion culture is performed on the
printed liquid-filled capsule in a biomimetic culture medium to realize a pre-forming of the printed
liquid-filled capsule. The 3D printing is conducted with a 3D printer by repetier host V.0.5
software.
In step (1), the calcium carbonate is dissolved at room temperature for 40 min, and a
concentration of the calcium carbonate is 20% w/v; the gelatin and hydroxypropyl methylcellulose
are added into the suitable amount of double distilled water respectively at a reaction temperature of
°C, for a stirring time of 1-4 h to be fully dissolved, and mixed and placed in a 4 refrigerator for
more than 24 h to form the homogeneous mixed gel. Viscosity of the hydroxypropyl methylcellulose is 1,000 mPa-s, and composition of the Eudragit S100 is methacrylate: methacrylic acid = 1:2.
The umbilical cord MSCs are selected from the second to fourth generations of human
umbilical cord mesenchymal stem cells with a cell-seeding density of 106 cells/mL. The electrospray
parameters of the microfluidic electrospray technology are set as follows: the voltage is 7 kV and the
flow rate is 50 L/min. The sodium alginate hydrogel culture medium is prepared by an F12/DMEM
complete culture medium. The MSCs-loaded sodium alginate microspheres prepared by the
microfluidic electrospray technology are 101 ±3.7 m in size and suspended in 1% w/v sodium
alginate hydrogel.
Example 4
An application of the oral MSCs liquid-filled capsule in the treatment of DSS-induced enteritis
model.
The present invention adopts an intragastric administration method of the oral MSCs
liquid-filled capsule to give an appropriate dose. The effective amount of the capsule is the amount
that can significantly reduce the symptoms of DSS enteritis.
a. C57BL/6 male mice are given 3% DSS drinking water, and the treatment group is given
different doses of MSCs liquid-filled capsules prepared in advance, which are injected into the
stomach of mice by intragastric administration.
b. In DSS model, the mouse is given drugs continuously for a week. After intragastric
administration, the hair condition, fecal characteristics and weight of mice are observed and recorded
in detail every day.
c. When the colitis is the most serious, the mice in each group are killed. The colon tissues are
observed and photographed. Part of the colons is fixed and embedded for pathological analysis such
as H&E staining.
The normal control group is set up, the enteritis model group is given DSS free drinking water,
and the treatment group is given capsules by intragastric administration on the 1", 3 'd and 5 th day
after modeling. Eight (8) days later, the mice are killed and the colons are taken out. The photo
results are shown in FIG. 2. It can be seen from FIG. 2 that the colon length of the control mouse is
longer than that of other groups, there is no redness and swelling phenomenon, and no obvious other
inflammatory symptoms. The inflammatory symptoms are obvious in the DSS group, with redness
and swelling, and the colon becomes shorter. There is no significant difference in inflammatory symptoms between the MSCs suspension group and the model group, which may be due to the inactivation of unprotected MSCs in gastric acid and digestive juice. MSCs capsule group is similar to the normal group without obvious redness and swelling. It can be seen that MSCs capsule can alleviate DSS-induced colitis in mice after oral administration. The H&E staining pictures after the treatment with MSCs liquid-filled capsules are shown in FIG. 3. The result shows that compared with
DSS model group and MSCs group, only a small amount of inflammatory cells infiltration is found
in the capsule treatment group, and the colon structure is relatively complete. It can be seen that the
MSCs liquid-filled capsule can effectively alleviate DSS-induced intestinal inflammation.
Example 5
An application of the oral MSCs liquid-filled capsule in the treatment of TNBS-induced
ulcerative colitis in mice.
The present invention adopts an intragastric administration method of the oral MSCs
liquid-filled capsule to give an appropriate dose. The effective amount of the capsule is the amount
that can significantly reduce the symptoms of inflammatory bowel diseases.
The application of the oral MSCs liquid-filled capsule in the treatment of TNBS-induced
enteritis model.
a. BALB/C mice are fasted for 24 h and given TNBS solution by intragastric administration.
b. 12 h after the establishment of TNBS mouse model, the prepared MSCs liquid-filled capsules
are injected into the colons of mice by intragastric administration.
c. After modeling, the mice are weighed every day to observe whether the mice have diarrhea,
hematochezia, fecal characteristics, etc., and the disease activity index (DAI) is recorded. DAI
(weight loss score + stool consistency score + occult/gross bleeding score)/3.
The effect of the MSCs oral liquid-filled capsule on TNBS-induced colitis is shown in FIG. 4.
According to the statistics of daily body weight of mice and the analysis of body weight change
curve (see FIG. 4A), MSCs capsule can effectively slow down the trend of TNBS-induced weight
loss of mice. After TNBS enema, diarrhea, significant weight loss and hematochezia are found in the
model mouse. The disease activity index can be used to evaluate the severity of colitis in
combination with the above index. MSCs liquid-filled capsule has a good protective effect on TNBS
model mouse, and has a better inhibition rate on disease activity index compared with blank control
group. DAI of MSCs liquid-filled capsule is significantly lower than that of model control group.
The scoring results are shown in FIG. 4B.
The above are only the preferred embodiments of the present invention, and do not limit the
present invention in any form. Any simple modification, equivalent replacement and improvement
made by any person familiar with the profession according to the technical essence of the present
invention without departing from the scope of the technical solution of the present invention shall
fall within the protection scope of the technical solution of the present invention.
Claims (10)
1. An oral MSCs liquid-filled capsule, comprising a three-layered capsule shell and capsule
contents, wherein the capsule shell comprises, from inside to outside, a calcium carbonate coating for
neutralizing gastric acid, a water-soluble gelatin-hydroxypropyl methylcellulose gel and a Eudragit
S100 coating, and the capsule contents are MSCs microspheres suspended in a sodium alginate
hydrogel culture medium;
a preparation method of the oral MSCs liquid-filled capsule comprises the following steps:
(1) preparation of soft capsule shell:
preparation of calcium carbonate solution: adding a predetermined amount of calcium carbonate
into double distilled water to disperse and fully dissolve at room temperature;
preparation of gelatin-hydroxypropyl methylcellulose gel: adding a predetermined amount of
gelatin and hydroxypropyl methylcellulose into a predetermined amount of double distilled water
respectively to dissolve, after cooling to room temperature, mixing them according to a
predetermined ratio, stirring thoroughly until uniform, and putting in a refrigerator to form a
homogeneous mixed gel; a total concentration of the gelatin and the hydroxypropyl methylcellulose
in the final mixed gel is 0.5-10%, wherein a concentration of the gelatin is 0.05-5.0%;
preparation of Eudragit SI00 coating solution: preparing a 1-3% w/v of Eudragit SI00 ethanol
solution;
(2) preparation of MSCs microspheres-loaded alginate hydrogel:
mixing umbilical cord MSCs evenly in sodium alginate hydrogel at a predetermined density,
spraying the mixed solution into a calcium chloride solution under a high voltage by a microfluidic
electrospray technology to obtain cell-encapsulated microgel droplets; a size of the microspheres
could be regulated by a voltage and a flow rate;
(3) preparation of MSCs oral capsule by 3D printing
collecting biological information of the soft capsule, and inputting the biological information
into a computer for a biomimetic modeling, loading the three-layered soft capsule shell and the
MSCs microspheres-loaded alginate hydrogel with predetermined amounts into a prepared extruded
3D printing silo, and performing 3D printing after setting printing parameters; performing a continuously circulated perfusion culture on the printed liquid-filled capsule in a biomimetic culture medium to realize a pre-forming of the printed liquid-filled capsule.
2. The oral MSCs liquid-filled capsule according to claim 1, wherein, in step (1), the calcium
carbonate is dissolved at room temperature for 20-40 min, and a concentration of the calcium
carbonate is 5-30% w/v; the gelatin and hydroxypropyl methylcellulose are added into the
predetermined amount of double distilled water respectively at a reaction temperature of 80°C, for a
stirring time of 1-4 h to be fully dissolved, and the two aqueous solutions are mixed and placed in a
4 W refrigerator for more than 24 h to form the homogeneous mixed gel.
3. The oral MSCs liquid-filled capsule according to claim 1, wherein, in step (1), viscosity of
the hydroxypropyl methylcellulose is 100-10,000 mPa-s; composition of the Eudragit S100 is
methacrylate: methacrylic acid = 1:2 (mass ratio).
4. The oral MSCs liquid-filled capsule according to claim 1, wherein the umbilical cord MSCs
are selected from the second to fourth generations of human umbilical cord mesenchymal stem cells
with a cell-seeding density of 106 cells/mL.
5. The oral MSCs liquid-filled capsule according to claim 1, wherein electrospray parameters of
the microfluidic electrospray technology are set as follows: the voltage is 5 kV and the flow rate is
pL/min.
6. The oral MSCs liquid-filled capsule according to claim 1, wherein the sodium alginate
hydrogel culture medium is prepared by an F12/DMEM complete culture medium.
7. The oral MSCs liquid-filled capsule according to claim 1, wherein the MSCs-loaded sodium
alginate microspheres prepared by the microfluidic electrospray technology are 143 ±5.2 pm in size
and suspended in 1% w/v sodium alginate hydrogel.
8. The oral MSCs liquid-filled capsule according to claim 1, wherein, in step (3), the 3D
printing is conducted with a 3D printer by repetier host V.0.5 software, and the biomimetic culture
medium is a MSCs culture medium containing F12/DMEM + 10% fetal bovine serum + antibiotics.
9. An application of the oral MSCs liquid-filled capsule according to any one of claims 1-8 as a
drug for treating intestinal diseases.
10. The application of the oral MSCs liquid-filled capsule as a drug for treating intestinal
diseases according to claim 9, wherein the intestinal diseases comprise inflammatory bowel diseases.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010310136.8 | 2020-04-20 | ||
| CN202010310136.8A CN111317727A (en) | 2020-04-20 | 2020-04-20 | Oral MSCs liquid-filled capsule for treating inflammatory bowel disease and other intestinal diseases |
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| AU2021101350A4 true AU2021101350A4 (en) | 2021-05-13 |
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| Country | Link |
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| CN (1) | CN111317727A (en) |
| AU (1) | AU2021101350A4 (en) |
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| CN111317727A (en) * | 2020-04-20 | 2020-06-23 | 南京鼓楼医院 | Oral MSCs liquid-filled capsule for treating inflammatory bowel disease and other intestinal diseases |
| CN114099459B (en) * | 2021-09-30 | 2023-02-24 | 国科温州研究院(温州生物材料与工程研究所) | Prebiotics and metazoan composite microcapsule for treating enteritis and preparation method thereof |
| CN114306901B (en) * | 2021-12-16 | 2023-03-21 | 浙江大学 | Medicine carrying capsule and implantable drug delivery device comprising same |
| CN115463153A (en) * | 2022-01-13 | 2022-12-13 | 南京中凯生物医药科技有限公司 | Preparation method of MSCs microsphere eye drops |
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| US10220003B2 (en) * | 2016-03-09 | 2019-03-05 | Incube Labs, Llc | Methods and articles for delivering viable cells into solid tissue |
| KR102124579B1 (en) * | 2018-12-04 | 2020-06-18 | 한국생산기술연구원 | Bio-ink composition for minimizing cell necrosis and method for manufacturing a biostructure using the same |
| CN110507637B (en) * | 2019-09-18 | 2021-12-21 | 吉林大学 | Intelligent capsule with gradual controlled release, preparation method and 3D printing system |
| CN111317727A (en) * | 2020-04-20 | 2020-06-23 | 南京鼓楼医院 | Oral MSCs liquid-filled capsule for treating inflammatory bowel disease and other intestinal diseases |
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| WO2021212678A1 (en) | 2021-10-28 |
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