KR101401798B1 - Development of alginate microcapsules comprising perfluorodecalin micell for anti-hypoxia - Google Patents

Abstract

The present invention relates to a microcapsule composition, and more particularly, to an alginate microcapsule composition for pancreatic transplantation containing perfluorodecalin microparticles, a method for producing the microcapsule composition, and the like. According to the present invention, alginic acid capsules can be made stronger without additional coating process by using only barium which is not calcium. In addition, perfluorodecalin is made into microcapsules using only Fluonic F68 and inserted into the microcapsules so that a large amount of perfluorodecalin can be uniformly present in the alginate microcapsules. That is, according to the present invention, the manufacturing process is simplified and the antigenic substance is minimized, so that the survival rate of the pancreatic islet is higher, and the manufacturing cost can be reduced. Therefore, the microencapsulated islets according to the present invention are expected to broaden the selection of donors in the islet transplantation environment requiring two donors per beneficiary, and to inhibit the islet-induced islet hypoxia, thereby contributing to the treatment of insulin-dependent diabetes mellitus do.

Description

TECHNICAL FIELD The present invention relates to alginic acid microcapsule composition containing perfluorodecalin microparticles and to a process for preparing the same,

TECHNICAL FIELD The present invention relates to a microcapsule composition, and more particularly, to an alginate microcapsule composition for pancreas transplantation containing perfluorodecalin microparticles, a method for producing the microcapsule composition, and the like.

Diabetes is divided into type 1 diabetes due to autoimmune diseases of insulin-secreting beta cells and type 2 diabetes due to insulin resistance and insulin relative insufficiency. Type 2 diabetes also eventually requires insulin therapy like type 1 diabetes. Insulin is the central hormone that regulates blood glucose and is formed only in the beta cells of pancreatic islets. Therefore, type 1 diabetes represented by childhood diabetes and the basic treatment of type 2 diabetes entering the insulin treatment stage is the transplantation of pancreas or isolated islets of insulin-forming organs. However, since it is very difficult to obtain a donor pancreas with minimal immune rejection, most patients are treated with injections of insulin externally.

On the other hand, in organ transplantation such as pancreatic islet cells, immunosuppressive agents can be used to protect the organs to be transplanted from the immune rejection reaction. Such a method may have side effects causing damage to recipient organs and donor organs. Thus, in a newly designed method, a method of isolating donor organs from the recipient's immune system by forming an immune barrier has been studied. A representative material constituting the immune barrier is alginate, which was first attempted using an islet in 1981 . Currently, alginic acid is used as an immune barrier by attaching other substances or using other substances to form capsules, and it is proving its effect by implanting various species of islets into other species.

Alginic acid is a polymer composed of β-1,4-D-mannuronic acid and β-1,4-L-glucuronic acid. It is extracted from brown algae and has excellent biocompatibility. Pure alginic acid is solidified by divalent cations except Al ⁺⁺ . But spending a lot of Ca ⁺⁺ in order to solidify the representative, using the Ca ⁺⁺ is small and robustness have a problem. Therefore, a method of once coating the outside with poly-L-lysine (PLL) has also been proposed and used. However, poly-L-lysine has the side effect of accelerating the secretion of cytokines, so that the islets inside the capsule are destroyed by the cytokine. When poly-L-lysine is once coated on alginate capsules, macrophage, helper T cell (CD4 + T cell), monocyte (monocyte) Inflammatory cells secrete cytokines and complement, or they accumulate thickly, blocking the movement of oxygen and nutrients between the capsule and the outside, causing severe damage to the pancreatic islets inside the capsule. Accordingly, methods of forming capsules using various materials or coating other membranes on the outer membrane of alginic acid have been suggested. In the preceding study, chitosan overcomes this problem (Application No. 10-2009-0003173). However, in the case where a process such as coating of an additional membrane is added, the islet has to withstand a longer process, so that a simplification of the manufacturing process has been demanded.

In addition, inflammatory cell deposition as described above occurs only after a period of at least one week after transplantation, but oxygen deficiency in the capsule is a fatal effect on the survival of the pancreatic islet after transplantation. At present, attempts have been made to overcome these problems by reducing the size of islet microcapsules and transplanting them into the portal vein.

Immunological barrier formation is a good method for heterologous pancreatic islet transplantation, but the conventional immune barriers have the problems as described above. Accordingly, the present inventors have developed an improved alginate pancreatic microcapsule containing perfluorodecaline colloid particles capable of forming an alginate capsule while simplifying the manufacturing process and overcoming a significant oxygen deficiency at the initial stage after the transplantation.

The present invention relates to a method for producing an alginate microcapsule which protects an islet from an immunological rejection reaction occurring in organ transplantation by using only barium which does not require an additional coating material, It is an object of the present invention to provide a microcapsule of alginic acid and a method for producing the same, wherein the manufacturing process is simplified while the strength is strong and the damage of the islets is minimized.

In order to effectively overcome hypoxia in the capsules, perfluorocarbons, which can be clinically applicable among perfluorocarbons whose oxygen content is higher than hemoglobin, are made into colloidal particles using only Fluonic F68 and put into alginic acid microcapsules, It is an object of the present invention to provide an alginate microcapsule composition for pancreatic islet transplantation and a method for producing the same, which are capable of uniformly containing perfluorodecalin particles in a large amount without causing a problem such as deforming the shape.

One aspect of the present invention is a pharmaceutical composition comprising perfluorodecalin, fluorionic F68, alginic acid and barium, wherein the alginic acid is cross-linked using barium to form a semi-permeable capsule, wherein the perfluorodecalin is There is provided an alginic acid microcapsule composition which is gelled in a semipermeable capsule.

According to an embodiment of the present invention, the mixing ratio of the perfluorodecalin and the Fluonic F68 may be 3: 7 to 7: 3, and the mixture ratio of the perfluorodecalin particles and the alginic acid present in the mixture may be 1: 9 to 5: 5.

Another aspect of the present invention provides alginate microcapsules further comprising an islet cell in the alginate microcapsule composition, which can be used for islet transplantation.

In another aspect of the present invention, there is provided a method for preparing a fluoroquinolone compound, comprising the steps of: (1) mixing a solution of Fluonic F68 dissolved in a perfluorodecalin and a Kreps Ringer HEPES buffer at a concentration of 1.5 to 2.5%; (2) stirring the mixed solution; And (3) mixing alginic acid dissolved in a concentration of 1 to 3% in the Kreps Ringer HEPES buffer.

According to an embodiment of the present invention, the mixing ratio of the perfluorodecalin and the Fluonic F68 may be 3: 7 to 7: 3, and the mixing ratio of the perfluorodecalin particles and the alginic acid May be from 1: 9 to 5: 5.

According to another embodiment of the present invention, in the step (3), the islet cells may be further mixed to be used for the islet transplantation. (4) diluting the mixture further mixed with the pancreatic islet cells and injecting the diluted mixture into a syringe; (5) injecting carbon dioxide while pushing the pump of the syringe; (6) allowing the mixture to fall into a solution containing barium.

According to the present invention, alginic acid capsules are cross-linked by using barium rather than calcium to form capsules, thereby making it possible to simplify the manufacturing process of capsules, . Further, the present invention can improve the survival of pancreatic islet cells in the alginate capsule, which is a hypoxic environment, by supplying oxygen to the pancreatic cells existing in the alginate capsule by containing the perfluorodecalin colloidal particles. Perfluorocarbons that supply oxygen are very strong hydrophobic substances and have a very low mixing ratio with alginic acid which is hydrophilic. However, they use a surfactant (Fluonic F68) and undergo agitation to remove perfluorodecalin It can be made into a colloidal particle and exist as a more effective colloidal particle in the oxygen supply inside the alginic acid capsule. In other words, the gelling of perfluorodecalin makes perfluorodecalne, which is difficult to mix with alginic acid, uniformly exist in the preparation of alginic acid microcapsules, and not only allows the perfluorodecalin to enter the alginic acid microcapsules even more, but also permits perfluorodecalin to be introduced into the outer membrane of alginic acid Thereby preventing the shape from being deformed. On the other hand, Fluonic F68 used as a surfactant in the present invention is particularly suitable for pancreatic transplantation capsules because other materials are not required additionally to minimize consumed materials and proven clinical stability. Therefore, the microencapsulated islets according to the present invention broaden the selection of the donor through the maximum elimination of the immune rejection response in the current pancreatic islet transplantation environment in which two donors per recipient are required, while suppressing the damage of the islet by initial hypoxia Are expected to contribute to the treatment of insulin-dependent diabetes mellitus.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram showing a method for producing microcapsules of alginic acid containing perfluorodecalin colloid particles and an islet. Fig.
Figure 2 is a schematic diagram of microscopic findings and structure in the form of perfluorodecalin containing microcapsules. Non micell refers to perfluorodecalin-containing microcapsules that are not colloidal.
FIG. 3 is a graph showing a comparative analysis of the survival rates of islets for each microcapsule after preparation of microcapsules. The name of the graph of the present invention is designated Alginate micell PFD.
FIG. 4 is a graph showing the fluorescence microscopic findings of survival rates of the alginate microcapsule types and the presence or absence of perfluorodecalin contained in the SD-rat islets collected in the hypoxic chamber after 3 hours and 6 hours, respectively (green: Color: non-viable cells) and a graph comparing and analyzing them. The name of the graph of the present invention is designated Alginate micell PFD.
FIG. 5 shows fluorescence microscopic findings of islet survival according to the presence or absence of perfluorodecalin in alginate microcapsules containing SD-rat islets collected before and at 6 hours and 24 hours after transplantation in homologous SD-rats (green: Survival cells, red: non-viable cells), and graphs comparing and analyzing the same.

The present invention relates to a method for the treatment of islet cell transplantation comprising administering to a subject an effective amount of at least one compound selected from the group consisting of a first type of diabetes represented by childhood diabetes and an insulin- To an immunocompromised microcapsule composition which is immune-rejected and which is capable of normal functioning.

In order to protect the pancreatic islets from the immunodeficiency reaction, the present inventors tried to form the final microcapsules by cross-linking alginic acid as a basic capsule, but solidifying it using barium rather than calcium.

In the case of using calcium as in the prior art, the strength of the microcapsule is weakened and the capsule is easily broken, and the damage of the pancreatic islet can not be avoided. Therefore, a poly-L-lysine (PLL) coating and / or an additional alginate coating should be added to improve the strength of the microcapsules. The islet should withstand a longer process so that the survival rate of the islets is lowered. . In addition, the use of poly-L-ornithine or the addition of an additional substance to alginic acid for the purpose of enhancing the strength results in an increase in the manufacturing process and an increase in the manufacturing cost due to the addition of additional substances. According to the present invention, such conventional problems as described above can be solved.

The present inventors have also found that perfluorodecalin micell particles approved for clinical use among perfluorocarbons for oxygen supply for minimizing the oxygen deficiency in the capsules which are important in the early stage after transplantation can be applied to clinically applicable Fluonic F68 Pluronic F68) and then added to the alginate microcapsules together with the pancreatic islets to overcome the immune rejection and hypoxia in the capsule.

That is, the present invention includes perfluorodecalin, a surfactant (e.g., Fluonic F68), alginic acid, and barium, wherein the alginic acid is cross-linked with barium to form a semi-permeable capsule, Onic F68 results in an alginic acid microcapsule composition that is present in the semi- permeable capsule in gelled form.

'Alginic acid' microcapsules can be used to protect the pancreatic islets from the immune rejection reaction that is problematic in transplantation of pancreatic islets. Alginic acid is a polysaccharide having a positive charge and binds strongly to divalent cations (Ca, Ba) to form a minimum area spherical shape. It is excellent in biocompatibility and is used for making capsules used for organ transplantation. The microcapsules made with alginic acid easily pass low molecular weight molecules such as insulin, oxygen and glucose, but do not pass through polymers such as immunoglobulin, thus greatly reducing the possibility of attack from immune action. Alginic acid can be used by separating it from seaweed, and it can be purified and processed to have high purity. However, it has the disadvantage that endotoxin contained in purified alginic acid exists and causes inflammation. In addition, when inflammatory cells grow on the outer membrane, the supply of oxygen and nutrients is interrupted to form a deadly environment inside the capsule, and cytokine and the like move freely within the capsule as a low molecule, so that the death of the capsule islets can be induced .

In order to solve the above-mentioned problem, it is possible to further include perfluorodecalin colloidal particles supplying oxygen into the capsules. The gelling of perfluorodecalin causes perfluorodecalin, which is difficult to mix with alginic acid, to be homogeneous and more enriched in the production of alginate microcapsules. In addition, the form in which perfluorodecalin is directly added to alginic acid causes problems such as deforming the capsule shape after being formed on the outer membrane of alginic acid after forming the capsules, but this problem is prevented when the granular particles are formed.

Perfluorocarbons are very strong hydrophobic materials and they have very low mixing ratio with alginic acid which is hydrophilic. Therefore, it is necessary to make hydrophilic and mixing with a surfactant. There are many types of surfactants, but only a limited number of types are available for clinical applications.

According to one embodiment of the present invention, colloidal particles can be made using Fluionic F68, a surfactant. As the surfactant, a liposome and the like can be used in addition to the fluorionic surfactant. However, the liposomal system has many additional kinds of substances required for the formation, but Fluonic F68 has an advantage in that it does not need any other substance. In addition, Fluonic F68 has been used in Fluosol-DA, an artificial blood that has been clinically approved in Green Cross of Japan, and its clinical stability has been proved. That is, according to the present invention, there is an effect of simplifying the manufacturing process, reducing the cost, and minimizing the antigenic substance.

Most of the size of the colloidal particles is about 10 μm, but most of the time, the colloidal particles bind to each other and have a diameter of 100 μm or more. Therefore, the colloidal particles can be shaken to a size of about 10 μm by using a stirrer. In the alginate capsule, the colloidal particles can not move freely, so they are mostly unbound.

According to another embodiment of the present invention, the mixing ratio of perfluorodecalin and 1.5 to 2.5% fluoronic F68 can be 3: 7 to 7: 3, and the mixing ratio of the perfluorodecalin particles and alginic acid May be 1: 9 to 5: 5.

Also, the present invention provides alginic acid microcapsules characterized by further comprising an islet cell in an alginic acid microcapsule composition comprising the perfluorodecalin colloidal particle and alginic acid.

In addition, the present invention relates to (1) mixing perfluorodecalin and a solution of Fluonic F68 dissolved in a Kreps Ringer HEPES buffer at a concentration of 1.5 to 2.5%; (2) stirring the mixed solution; And (3) mixing alginic acid dissolved in a concentration of 1 to 3% in the Kreps Ringer HEPES buffer.

According to an embodiment of the present invention, the mixing ratio of perfluorodecalin to pluronic F68 may be 3: 7 to 7: 3 in the step (1), and the concentration of Fluonic F68 dissolved in the KRH buffer may be 2% .

According to another embodiment of the present invention, in the step (2), the mixture can be stirred at room temperature for about 5 minutes using a stirrer.

According to another embodiment of the present invention, the mixing ratio of the perfluorodecalin particles and alginic acid present in the step (3) may be 1: 9 to 5: 5, preferably 2: 8 .

Also, the concentration of alginic acid dissolved in KRH buffer (Kreps Ringer HEPES buffer) is preferably 2%. In the step (3), the pancreatic islets may be further mixed to be used for pancreatic transplantation. (4) diluting the mixture further mixed with the pancreatic islet cells and injecting the diluted mixture into a syringe; (5) injecting carbon dioxide while pushing the pump of the syringe; (6) allowing the mixture to fall into a solution containing barium.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  1. Rats Islet  Cell separation

Male SD-rats averaging 200 g were anesthetized using a rumun ketamine mixture. After opening the abdominal cavity, the pancreatic duct connected to the duodenum was ligated, and the pancreatic duct connected to the liver was opened, and the PE-50 tube was connected to the incision. 8 to 9 ml of a solution of 1 mg of collagenase P per 1 ml of prepared Media 199 was introduced into the pancreas via PolyEthylene-50 (PE-50). The pancreas was isolated and digested by shaking in a 37 ° C water bath at 60 rpm for 25 minutes. After washing three times with Media 199 (+ 10% fetal bovine serum: FBS), the pancreas was removed by filtration through a sieve with a pore size of 500 μm. In order to increase the purity of the pancreatic islet, the pancreatic islet was isolated by Histopaque 1077 using the density difference method. The isolated islets were washed with Media 199 (+ 10% FBS) and the islets were cultured for 1 day in RPMI 1640 (+ 10% FBS) medium.

Example  2. Perfluorodecalin  Preparation of colloidal particles

99% of perfluorodecalin was mixed with Fluonic F68 solution in a Kreps Ringer HEPES buffer at a ratio of 5: 5, which was dissolved in 1.5 to 2.5%, and stirred at room temperature for 5 minutes using a stirrer to produce colloidal particles.

Example  3. Perfluorodecalin  Containing colloidal particles Islet  Microencapsulation

Alginic acid with high purity using activated carbon was dissolved in KRH buffer (Kreps Ringer HEPES buffer) at a concentration of 2%, and then filtered with a Hepa filter. 1000 isolated pancreatic cells were diluted in 1 ml of an alginate solution and a mixture of perfluorodecalin colloidal particles (PFD) in an 8: 2 ratio and placed in a 5 cc syringe. A 5cc syringe was attached to the syringe pump and connected to the microdrop maker. A carbon dioxide injection tube was connected to the side of the fine droplet generator as a prepared air inlet. Carbon dioxide was injected at a pressure of 6.0 kgf / cm ² while pushing the syringe pump at a rate of 3 ml / hr. Alginic acid and diluted pancreatic islets were allowed to drop into 10 mM Ba ⁺⁺ solution on magnetic stirrer. The mixture was agitated for 3 to 5 minutes and hardened. After stirring, the capsules were washed 2 ~ 3 times with HBSS solution and cultured in RPMI 1640 (+ 10% FBS) culture medium at 37 ° C in 5% CO _{2 for} one day. After the treatment of Acridine orange (survival cell stain) / Propidium Iodide (dead cell stain) (AO / PI), the survival rate of the islets after encapsulation was confirmed.

Comparative Example  One. Perfluorodecalin  Non-colloidal particles Islet  Microencapsulation

Islet microencapsulation was carried out in the same manner as in Example 3, except that the separated pancreatic cells were mixed with only the alginate solution with increased purity without mixing the perfluorodecalin particle (PFD).

Comparative Example  2. Perfluorodecalin  Containing comparative quality particles Islet  Microencapsulation

The microencapsulation of the pancreatic islets was carried out in the same manner as in Example 3, except that 20% of perfluorodecalin stock solution, which is comparative nitrification particles, was added to alginic acid with increased purity.

Comparative Example  3. Poly - lysine  Coating Method Microencapsulation

Only the alginic acid solution having increased purity without mixing the perfluorodecalin colloidal particles was mixed with the pancreatic cells and then the solution was treated with 100 mM Ca ⁺⁺ solution instead of 10 mM Ba ^{++ in} Example 3. [ The microcapsules were washed with HBSS solution and then coated with 0.1% Poly-L-lysine (PLL). After completion of the coating, the substrate was washed with HBSS solution. Subsequently, tertiary coating with 0.3% purity alginic acid was carried out, followed by washing with HBSS solution, followed by incubation.

As shown in FIG. 2, it can be seen that the difference in the presence or absence of colloidal particles (denoted as non micellization by non-micellization) shows a marked difference in the uniformity of the distribution of perfluorodecalin in the microcapsules. It was confirmed that some of the perfluorodecalin stock solution flowed out of the microcapsules when the microcapsules were prepared. This confirms the loss of perfluorodecalin. It was also confirmed that some of the perfluorodecalin was derived from the microcapsule outer membrane. This adversely affects the surface uniformity as well as the rigidity of the microcapsules.

As shown in FIG. 3, it was confirmed that the PLL coating (alginate PLL coating) is higher than other capsules in the damage of the pancreatic islets, and there was a statistically significant difference from the alginate micell PFD of the present invention. Compared to PLL coating, alginate only and alginate micell PFD, the base capsules of the present invention, are able to confirm a similar level of pancreatic injury. However, there was no statistically significant difference in islet damage during microencapsulation with or without colloidal particles.

Example  4. In the alginate microcapsule Islet  Cell survival rate experiment

In order to confirm the islet cell viability in a hypoxic environment, the alginate microcapsules prepared in Example 3 and Comparative Example 1 were stabilized for one day and then a part thereof was collected. The collected samples were placed in hypoxic chambers and cultured for 3 hours and 6 hours. The cells were collected and treated with Acridine orange / Propidium Iodide (dead cell stain) (AO / PI) Respectively. The purpose of this experiment was to confirm hypoxia rather than immunosuppressive efficacy. Therefore, we used islet of the same species and did not measure blood glucose which is not suitable for comparison of viability. Respectively. The results of the experiment are shown in FIG.

As shown in FIG. 3, the islet cells in the microcapsules containing perfluorodecalin were found to have a significantly lower percentage of dead PI-specific staining. Through this, it was confirmed that the survival rate was increased by adding perfluorodecalin.

As shown in FIG. 4, the inventive product containing perfluorodecalin (alginate micell PFD) showed statistically insignificantly lower islet injury rate than the control group except the comparative nitrification group (alginate non micell PFD). In particular, it was confirmed that the PLL coating (alginate PLL coating) has an islet damage rate at least twice that of the other groups.

Example  5. Islet  After transplantation of alginate microcapsules containing cells Islet  Cell survival rate experiment

The alginate microcapsules prepared in Example 3 and Comparative Example 1 were placed in 1 ml of physiological saline solution to which oxygen was injected 1 hour before implantation. Male SD-rats were anesthetized and incised to a side of 1 cm. SD-rat pancreatic islets were implanted by injecting 500 alginate microcapsules through the incision site. The microcapsules grafted at 6 and 24 hours after transplantation were collected and treated with Acridine orange / Propidium Iodide (AO / PI). The purpose of this experiment was to confirm hypoxia rather than the immunosuppressive effect. We used the islet of the same species and confirmed its efficacy by using AO / PI, which is not suitable for comparison of viability, as an index , And hypoxic damage was observed at the initial stage after transplantation. The results of the experiment are shown in Fig.

As shown in FIG. 5, it was confirmed that the survival rate of somatic cells in microcapsules containing perfluorodecalin was significantly high.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (11)

Perfluorodecalin, pluronic F68, alginic acid, and barium,
Wherein the alginic acid is crosslinked using barium to form a semipermeable capsule, and the perfluorodecalin is present in the semipermeable capsule due to the fluionic F68 being present in the microcapsule composition.
The method according to claim 1,
Wherein the mixing ratio of the perfluorodecalin to the Fluonic F68 is 30 ml: 70 ml to 70 ml: 30 ml based on 100 ml in total.
The method according to claim 1,
Wherein the mixing ratio of the perfluorodecalin particles and the alginic acid present in the gum is 10 ml: 90 ml to 50 ml: 50 ml based on 100 ml in total.
The method according to claim 1,
Wherein the alginic acid microcapsule composition is used for islet transplantation.
An alginic acid microcapsule characterized by further comprising an islet cell in the alginic acid microcapsule composition of claim 1.
A method for producing alginic acid microcapsules comprising the steps of:
(1) mixing a solution of peronic F68 dissolved in 1.5 to 2.5 ml per 100 ml of perfluorodecalin and KRR buffer (Kreps Ringer HEPES buffer);
(2) stirring the mixed solution; And
(3) Mixing alginic acid dissolved in 1 to 3 ml based on 100 ml of the above-mentioned crosslinking solution and KRR buffer (Kreps Ringer HEPES buffer).
The method according to claim 6,
Wherein the mixing ratio of the perfluorodecalin and the Fluonic F68 is 30 ml: 70 ml to 70 ml: 30 ml based on 100 ml in total.
The method according to claim 6,
Wherein the mixing ratio of the perfluorodecalin particles and the alginic acid which are gelled in the above-mentioned cross-linking solution is 10 ml: 90 ml to 50 ml: 50 ml based on the total 100 ml.
The method according to claim 6,
Wherein the alginic acid microcapsules are used for pancreatic islet transplantation.
The method according to claim 6,
Wherein the islet cell is further mixed with the microcapsule in the step (3).
11. The method of claim 10,
Diluting the islet mixture with the islet cells and injecting the diluted mixture into a syringe; Injecting carbon dioxide while pushing the pump of the syringe; And allowing the mixture to fall into a solution comprising barium. ≪ Desc / Clms Page number 20 >

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