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AIAA 92-01 18 A Novel Method for Producing Microspheres with Semipermeable Polymer Membranes K.C. Lin and T.G. Wang Center for Microgravity Research and Applications Vande rbi I t U n ive rs i ty Nashville, TN

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30th Aerospace Sciences Meeting & Exhibit

January 6-9,1992 / Reno, NV u

For permission to copy or republish, contact the American Institute of Aeronautics and Astronautics 370 L'Enfant Promenade, S.W., Washington, D.C. 20024

A NOVEL METHOD FOR PRODUCING MICROSPHERES WITH SEMIPERMEABLE POLYMER MEMBRANES

K.C. Lin* and T.G. Wang"

Center for Microgravity Research and Applications Department of Materials Science and Engineering

Vanderbilt University, Nashville, TN 37235

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A new and systematic approach for producing polymer microspheres has been demonstrated. The membrane of the microsphere is formed by immersing the polyanionic droplet into a collapsing annular sheet, which is made of another polycation polymer solution. This method minimizes the impact force during the time when the chemical reaction takes place, hence eliminating the shorlcomings of the current encapsulation techniques. The results of this study show the feasibility of this method for mass production of microcapsules.

btroduct ion

The production of microspheres with a semipermeable membrane has been considered for various medical and biological applications. One of the most important research goals is the transplantation of microspheres which encapsulate islet cells within a semipermeable polymeric membrane to treat diabetes patients. The porosity of the membrane is controlled such that the membrane is permeable to nutrients and to insulin but not to the antibody size molecules. Le., the membrane acts as an immuno-isolation system. This technique for diabetes treatment has been sought as an alternative to whole-organ pancreas transplants. Studies on rats have shown12 that the choice of proper materials results in highly biocompatible membranes which maintain appropriate porosity. As the methods for islet cell isolation become more efficient,3.4 this type of treatment may be a solution for insulin- dependent diabetes patients. Other applications for semipermeable microspheres are being developed for the controlled release of drugs and chemicals.

The common technique for producing polymer membranes is through the ionic interaction between polycation and polyanion polymers. By controlling the concentration of the polymer solutions and the number of membrane layers, the permeability of the resulting membrane can be altered. Currently, the most widely used methods for forming the polymer microspheres may be described as follow$: the liquid droplets from one polyanion polymer solution are formed by means of a drop generator; these droplets are then immersed either directly into another polycation polymer solution to produce the polymer spheres5 or first into some chemical solution to harden the droplets, and the hardened

polymer solution to form the desired membrane!.

that there were some inherent difficulties associated with the production of microspheres through these methods. In order to eliminate these problems, a quantitative study on the effect of degradation of the polymer used for microencapsulation was done and a new approach is proposed to minimize the difficulties in producing the microspheres.

Fvaluatlon of Curre nt Productio n Methods

encapsulation has been regarded as one of the most successful methods for microencapsulation of human cells. The materials used show good biocompatibility with living cells and the process is simple. In general, the cells to be encapsulated are suspended In 1.5% sodium alginate solution (polyanionic). Droplets of this solution are formed by using a syringe pump with a syringe connected to 3 controlled air jet and are impacted into 1 .l% calcium chloride solution to harden. The gel

droplets then react with another polycation V

However, preliminary investigation showed

The technique developed by Sunl.2 for cell

'Research Assistam Professor "Centennial Professor and Director, Member AlAA CopyrigM 0 1592 by the American Institute of Aeronautics and Astronautics. IN. All rights resewed.

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droplets (Le. calcium alginate) are then immersed into polylysine solution (polycationic) to form the membrane.

Although successful results have been shown, there are some drawbacks with this method. One is the harmful effect of calcium chloride on the islet cells which results in a lower yield rate. The other is that the impact of the droplets into the calcium chloride solution decentralizes the islet cells due to the deceleration, the cells are pushed onto the boundary of the droplet, and usually a bump will form on the hardened droplet. The imperfect surface of the droplet provides the chance for fibroblastic growth to occur at the point of surface discontinuity and induces an inflammatory reaction. It is obvious that these obstacles must be overcome before mass production can be achieved.

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To address the problem of toxic effect of calcium chloride on the islet cells, Kendall, Chang and Wang5 suggest that the intermediate step of the gel hardening process may be omitted. Instead of directing the droplets into the calcium chloride solution, the droplets are introduced into the chitosan solution. A polymer membrane forms when the droplet penetrates the liquid surface. As the chitosan solution is not harmful to the living cells, there will be no damage done to the encapsulant. However, further investigation reveals that new difficulties arise when the

properties of the calcium chloride solution are totally different from that of the chitosan solution. A 0.2% chitosan solution is about ten times as viscous as the water-like calcium chloride solution and the surface tension may also be changed. The experiments show that it is difficult for the submillimeter-size droplets to penetrate the liquid surface without excess deformation. Higher velocity is required and the penetration time is increased. The high impact velocity results in highly distorted microspheres, while the increased penetration time results in the collision of the second droplet on the penetrating one and the droplets stick together. Thus, a high quality of microspheres is hard to obtain and the impact force on the cells is even greater.

W proposed method is applied. First of all, the

ExDerlments The maior problems fac'ng the production

of good spherical microcapsules can be attributed to tne formation process, i.e.. control of tne oroplet format'on process so that the drop s'ze can be controlled, preparation of the solution so that the chemical reaction can oe controlled and spherica shells obta'ned. and m'nim'zat'on 01 the cell decentering problem as the droplets penetrate

into a stationary liquid surface while the chemical reaction takes place. To improve the yield rate, all the problems mentioned above must be controlled. Therefore, a new and systematic approach is needed for mass production.

sodium-alginate (Sigma Chemical Co., St. Louis, MO), which is used by Sun1 because it is highly compatible with living cells, CaC12, and chitosan (Protan Inc., Commack, NY), which is used by Changs.

A. Apparatus for Droplet Generation

The method developed by Sunl2for droplet generation is suitable for producing a small quantity of microspheres at a time. In considering the practice of mass production, another approach is used. The design of the droplet generator is based on the Rayleigh instability6. A liquid column is unstable and will spontaneously break up into droplets due to the disturbance from either the jet itself or external excitation. In general, a wide distribution of drop sizes usually appears when a liquid jet disrupts. This tendency can be suppressed by perturbation of the jet at a single frequency near the most unstable frequency of the jet. Rayleighs showed that the maximum instability occurs when the wave-length of the disturbance is given as:

In this study, the test materials selected are

h=4 .51*2a

where h is the wave length, and a is the radius of the jet.

The excitation frequency can then be calculated by the following equation:

f = V I h

where f is the most unstable frequency, and v is the jet velocity.

For a 0.22 mm diameter liquid jet with a velocity of 2 mlsec, the desired frequency is about 2 kHz. The droplet diameter produced by this method will be about 1.8 times that of the column diameter. By varying the nozzle diameter, jet velocity, excitation frequency and amplitude, the droplet size as well as the drop formation may be studied under the stroboscopic illumination. The rate of formation is the same as the frequency of the excitation, typically 2 - 4 kHz. Figure 1 shows the general scheme for the drop generator.

E. Sodium Alginate Properties and Reproducibility

Alginic acid is a polyuronide found in brown seaweeds'. This natural polymer contains two

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0 0 0

Figure 1. Schematic Diagram of Drop Generator.

monomers, mainly mannuronic and guluronic acids. The chain arrangements are

-M.M-M-M-M- -G-G.G-G-G. -M.G-M.G-M.G.

In general, the proportions of these three components greatly depend on the source of the seaweeds, hence the properties of sodium alginate vary among different manufacturers. The sodium alginate used in this research is from Macrocystis pyrifern (Kelp), which is constituted of 40.6% polymannuronan, 17.7% polyguluronan and 41.7% alternating7. One of the difficulties in dealing with the polymer is the tendency for the polymer to degrade under high temperature, under mechanical work, and through time. Hence, the properties of the sodium-alginate solution greatly depend on the preparation conditions. Experiments show that these variations have profound effects on the quality of microspheres. To ensure consistent experimental results, tests were done to study the effect of property variations on the quality of mlcrospheres.

The degree of degradation of the polymer solution may be quantified by measuring its viscosity. When the long molecular chain breaks into shorter chains, the viscosity of the solution is decreased. By observing the change of viscosity along with the results of experiments, a suitable range may be defined such that consistent products can be obtained. In this study, ten samples of 0.8% NaCl and 1.4% sodium-alginate solution were used to test the reproducibility. They were prepared under similar conditions: at room temperature; mixing for about an hour and filtered with 30 - 40 pm size filter. Figure 2 shows the viscosity measurements of these samples one hour after preparation. The viscosity difference from sample to sample is no more than +7%. Four samples were used to study the degradation with

10 "L 0 0 2 4 6 8 10 12

sample

Figure 2. Viscosity of 1.4% S-A & 0.8% NaCl Solution.

- Sample 1 - Sample3 - Sample2 - sample4

-a 20 . , . , . , . , . , . , . , . I . , . ,

Day

0 2 4 6 8 10 1 2 1 4 16 18 20

Figure 3. S-A Solution Viscosity V.S. Time.

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respect to time under room temperature storage (i.e. 20 - 23°C). The results are shown in Figure 3. It shows that even under similar conditions, the degradation curves are quite different. However, there exists a period of at least two days before any rapid degradation begins. The effect of the degraded polymer solution on the quality of microspheres is illustrated in Figures 4 and 5.

The sodium-alginate droplets were made from the drop generator shown in Figure 1, an electrical field was used to assist the process, and the reaction was completed by impacting these liquid droplets into CaCI2 solution. In Figure 4, the microspheres were produced by using freshly prepared sodium alginate solution reacted with 1 .O% CaCIz, In Figure 5, the microspheres were gerierated from the degraded sodium alginate solution. These pictures show that inconsistent results will occur if the degradation of the polymer is not taken into account In the production

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Figure 4. Microspheres (400pm) Produced from Fresh Prepared S-A Solution.

Figure 5. Distorted Microspheres (400pm) Produced From Degraded S-A Solution

process. It is believed that the breaking of the long molecular chain into shorter links changes the reaction time between sodium alginate and Ca as well as the diffusion time of Ca through the droplet. Further investigation is needed to confirm this hypothesis. In general, good quality spheres can be obtained by using freshly prepared solution, providing that the mass degradation of sodium alginate is prevented, i.e., the mixing temperature should be less than 30°C and the stirring time should be kept to a minimum. For a

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properly prepared solution, consistent results can be accomplished if the experiment is done before the rapid degradation begins. The delayed period, shown in Figure 3 for 1.4% sodium alginate solution, before any appreciable change of viscosity is the most suitable range for producing calcium alginate droplets through the impact method. However, if another source of sodium alginate is used, tests should be done to ensure the validity of the conclusion made above.

alginate by measuring its viscosity enables the characteristics of the polymer to be identified. By controlling the preparation process, the experimental results may be easily reproduced.

C. Impact Studies

The dynamic behavior of the impact process relating to encapsulation technology has been studied by Kendall, Chang and Wangs. The impact process was done by dropping a single droplet of 2.2 mm size into a chitosan solution. Excess deformation occurred during penetration and the deceleration was estimated on the order of log. However, no data relative to the viscosity of the alginate droplet was mentioned in their experiments. Since the properties of the alginate solution change with respect to the degradation, a study of this change on the impact and chemical dynamics is essential.

examined to take into account the effect of viscosity, which is used as an indication for measuring the degree of degradation. A high speed video camera was used to record the penetration process of 2.8 mm alginate droplets into CaC12 solution. The single droplet was produced through a fine syringe needle. The choice of a larger droplet size gives a good depth of field through the magnified lens of the camera. In order to have similar fluid dynamic responses, the Weber number, which is the ratio of kinetic energy and surface energy, was used to ensure that the larger droplet would have about the same impact response as the smaller one.

The study of the properties of sodium

In this research, the impact process was re-

According to the measurement done by Kendall, Chang and Wangs, the surface tension of the alginate solution is close to that of water. Assuming the degradation does not affect the surface tension, the Weber number can be computed. In general, the droplets used for producing microspheres are in the range of 350 bm to 450 pm with an impact speed between 2 m/sec and 4 m/sec. Because of the size difference, the impact speed of the 2.8 mm droplets was reduced to 1 m/sec to keep the ratio of the Weber number between the small droplets and the larger ones in the range of 0.5 to 2.

The droplets were made 01 1.4 % sodium alginate + 0.8 % NaCl solution. Experiments were done for two different viscosities: one with the viscosity of the alginate solution measured as 54 cp and the other 19 cp. The pictures shown in Figures 6 and 7 indicate different responses fof different viscosities. The shapes of the droplets during submersion are quite different from those shown by Kendall. The causes for these unusual shapes are uncertain. It may be caused by the non-Newtonian behavior of the alginate polymer, or by the viscosity, or by both. The final shapes are shown in Figures 8 and 9. Figure 8 shows that the 54 cp droplet rebounded back to the spherical shape, while the 19 cp droplet did not. It suggests that the chemical reaction time of the 54 cp droplet is much slower than that of the fluid dynamic response. However, for the 19 cp droplet, Figure 9 indicates that the chemical reaction time is ComDarable to that of the fluid dynamic response

3 ms

I O ms

5 1 ms

Figure 6. Impact Response of 54 cp 1.4% S-A Droplet (2.8 mm) Entering CaC12 Solution.

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17 ms 1 ms

3 ms 58 ms

Figure 7. Impact Response of 19 cp 1.4% S-A Droplet (2.8 mm) Entering CaC12 Solution.

11 ms

Figure 8. Final Shape of 54 cp 1.4% S-A Droplet.

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Figure 9. Final Shape of 19 cp 1.4% S-A Droplet.

In general, the dynamics of droplet formation through a liquid column is different if the encapsulants are present in the liquid. For most of the cases, the size of the spheres containing seeds are non-uniform and are larger than the spheres without seeds. Although by controlling the amplitude and the frequency of the drop induction vibration uniform droplets may be obtained through a liquid jet, further study is needed to attack this problem. Another problem is the position of the encapsulant in the droplet. It IS

desirable to have the seed located as close to the center of the capsule as possible. It has been demonstrated0 that under induced oscillation, the centering mechanism will occur for a drop containing a comparable size of seed. The theoretical study of the centering effect of a liquid thin shell is done by Lees However, a systematic study of this centering phenomenon is yet to be completed.

Whereas high quality empty microspheres may be produced easily by means of the impact method, difficulty arises when there is encapsulant inside the droplet. As shown in Figure 6, the impact reduces the velocity of the droplet to almost zero within a couple of milliseconds. The kinetic energy of the droplet is absorbed through the deformation of the droplet's shape, the liquid surface, and the shear flow inside and outside the droplet. The whole impact process is too complicated to be analyzed even for a simple droplet. To understand the response of the encapsulant inside a droplet during impact, a much simDler model has to be assumed.

From the photography study of the impact process, the chemical reaction time is about an order or two slower than that of the fluid dynamic response. Except for the immediate contacl surface between droplet and the CaCI2 solution. one may safely assume that there is no chemical process taking place inside the droplet during the first few milliseconds of impact. Furthermore, the impact process may be approximated by assuming that the droplet is stopped immediately at the time of impact, i.e. the droplet hits a flat, rigid surface. The integrity of the droplet is maintained by both the drop's surface tension and by the membrane formed on the contact area. The dynamic behavior of the encapsulant may be modeled as a simple fluid problem. Initially, the seed has the same velocity as the droplet. Upon impact. the droplet is stopped at once, however, the seed, due to density difference, will continue to move until the viscosity of the liquid inside the droplet stops it at a position away from center.

By using the physical picture above, the problem may be simplified as: A small rigid sphere is immersed in a stationary viscous liquid; the sphere is given an initial velocity; find the motion of the sphere. The differential equations of motion are given as followslo:

dyidt = V

dvidt = g Ap/p,, - (314) Cd p vz / (p,d)

where g is the gravitational acceleration, p is the density of the liquid, ps is the density of the sphere, Ap = ps - p. Cd is the coefficient of drag, d is the sphere diameter, v IS the sphere speed, y is the distance moved, and t is time.

The drag coefficient Cd depends on the Reynolds number (Re) and is readily available11 Note that the use of steady-state drag correlations is an approximation. In accelerated motion, there will be "added mass" and "history" terms which have been neglected. The results of calculation for a 0.15 mm sphere with initial velocity 2misec and density 1.06, liquid density 1.01 and viscosity 60 cp, are shown in Figures 10 and 11. Figure 10 indicates that extremely high deceleration occurs within the first 100 psec and the sphere is virtually stopped after 200 psec. The distance travelled by the sphere is about 40 pm.

The numerical results demonstrate the effect of the decentering force due to the impact. Figure 6 shows that the dimension of the droplet in the direction of impact is flattened to less than one half of the droplet diameter. For a 400 pm droplet, the flattened side will be less than 200 pm. If the droplet initially contains a 150 pm encapsulant in the center, the distance moved by the seed during

impact is comparable to that of the space between the boundary of the droplet and the encapsulant. The movement of the seed essentially pushes the liquid against the droplet's boundary, hence a small bump on the outer surface is produced. This non-perfect surface is then "frozen" by the chemical reaction of sodium alginate and CaC12 solution.

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In addition to the non-smooth surface, there are two facts which may affect the survival of the encapsulant. First, the extremely high deceleration may cause irreversible damage to the encapsulant. Second, after the impact the seed is very close to the boundary and is immobilized by the effect of high liquid viscosity and subsequently the jelling process. As the CaC12 solution is toxic to the living cells, the closer the cells are to the boundary, the higher the chances for the cells to be damaged by the CaC12 solution.

Figure 10. Velocity History of Seed During Impact

Figure 11. Position History of Seed During Impact.

s for MlcrosDhere Production

It appears the drop size uniformity and chemical reaction can be controlled in a reasonably straight-forward manner. However, with current techniques, a cettain velocily is required for a droplet to overcome the surface tension of the stationary liquid, hence the distortion of the droplet is unavoidable. This limiting factor makes it hard to increase the yield rate. In this new approach, an annular jet is used for the purpose of reducing the impact force. The general scheme of an annular nozzle along with the drop generator is shown in Figure 12. When liquid emerges vetticaliy downward from an annular jet, a conical liquid sheet is formed due to the effects of surface tension, inertia and gravity, and the pressure differential between the inside and the outside of the sheet. Theoretical as well as experimental studies of the annular liquid jet may be found in references9.l2.i3.14. The alternative method proposed here for producing microspheres is that, instead of submerging the droplets into a stationary liquid surface, the stream of droplets is directed into a collapsing annular liquid sheet. By matching the speed of the liquid sheet to that of the droplets, impact may be minimized. The process is clearly illustrated in Figure 12.

sme wave l"D"f

"OlCI coli SWB*C

Figure 12. Apparatus Schematic Diagram for Producing Microspheres.

The impact response was studied by using an annular nozzle of 1 cm size with the thickness of the liquid sheet about 0.3 mm. The speed of the liquid sheet at the nozzle exit was measured by volume rate, which was controlled by the feeding pressure. The speed of the 2.8 mm single droplet was adjusted by changing the location of the

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syringe needle tip to match that of the CaCI2 liquid sheet at the annularjet exit. The resulting photos are shown in Figure 13. The speed used in this study was about 1.5 mlsec for both the droplet and the annular jet. The droplet was kept slightly off center to avoid the turbulence at the center of the collapsing sheet. Figure 13 shows that there is little distortion when the droplet is submerged into the liquid sheet and the spherical shape was maintained through the whole process.

To eliminate the toxic effect of CaCI2 solution, this new approach was used to produce microspheres directly from polyanion and polycation polymers. The tested materials were chosen as alginate droplets and chitosan solution. The size of the droplets from the drop generator was about 400 pm. The size of the annular nozzle is about 4.5 mm with the thickness of the liquid sheet about 0.15 mm. The free fall time of the droplet in the liquid column is kept such that the chemical reaction is completed before the liquid column is collected into a beaker. Note that the reaction between the alginate droplet and the chitosan solution only takes place on the surface of the droplet. The resulting microspheres with and without encapsulant are shown in Figure 14 and 15. In this study, plastic beads with density of 1.04 and sizes between 100 and 150 pm were used as encapsulants. The size of the microspheres is about 700pm. The forming of a membrane on the surface of the alginate droplet increases the drop size dramatically.

1 m5

2 m5

5 m5

8 m5

Figure 13. Impact Response of S-A Droplet Entering Annular Liquid Sheet.

Summary

The effect of the alginate properties on the quality of the microspheres produced from alginate and CaCI2 has been studied. By properly preparing and monitoring the viscosity of the alginate solution, the quality of Ca-Alginate microspheres produced through the impact method can be controlled. The impact dynamics study of the alginate drop reveals unusual shape deformation during the impact. Further investigation is needed to identify the causes. The study of the encapsulant decentering during impact based on a simplified model reveals the shortfalls of the impact method. A new approach for producing high quality microspheres is then proposed. The intermediate step of forming Ca- Alginate spheres is eliminated along with the high impact force. This method may be used for producing high quality microspheres from other types of polyanion and polycation polymers.

Figure 14. Empty Microspheres (700 pm).

Figure 15. Microspheres (700 pm) with Encapsulated Plastic Bead.

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Acltnowledament

The research described in this paper was carried out at the Center for Microgravity Research and Applications, Vanderbilt University, under a grant from the National Aeronautics and Space Administration's Office of Commercial Programs (NASA Code C).

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