ART. CELLS, BLOOD SUBS., AND IMMOB. BIOTECH., 23(2), 143-151 (1995)

IN VITRO SLOW RELEASE PROFILE OF ENDOTHELIAL CELL GROWTH FACTOR IMMOBILIZED WITHIN CALCIUM ALGINATE MICROBEADS

Clifford KO,' Vivek Dixit: William Shaw,' and Gary Gitnick2 'Division of Plastic Surgery and 2Division of Digestive Diseases

UCLA School of Medicine, Los Angeles, California 90024-70 19, USA

ABSTRACT

Although a variety of angiogenic growth factors have been isolated, its appropriate in vivo delivery remains problematic due to nonspecific, uncontrolled delivery by conventional methods. We have investigated calcium alginate microbeads as a vehicle for the controlled slow-release of endothelial cell growth factor (ECGF). Three different microbead compositions, dependent on ECGF amount and alginate percentage were studied. Microbeads were incubated in a 1.5% calcium chloride solution and release of ECGF into solution was measured spectrophotometrically at specific timepoints. Our results show release rate and amount released after the first 2 hours are dependent on initial concentration of ECGF and independent of alginate concentration. There was an initial quick delivery of ECGF in the first 2 hours after which a sustained controlled release occurred for 4-5 days. Beyond this point, release at a slower rate was noted for at least approximately 2 weeks. Calcium alginate microbeads demonstrated a controlled and predictable rate of release and that the amount of ECGF delivered can be varied by varying the initial concentration of ECGF in the microbeads. Based on these observations we conclude that calcium alginate microbeads are a convenient and practical vehicle for sustained ECGF delivery.

Address correspondence and reprint requests to: Vivek Dixit, Ph.D., Department of Medicine, Division of Digestive Diseases, 675 Circle Drive South, MRL Room 1240, Los Angeles, CA 90024-7019, USA.

143

Copyright 0 1995 by Marcel Dekker, Inc.

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144

INTRODUCTION

KO ET AL.

Previous investigations have demonstrated accelerated vascularization with the use of several different angiogenic growth factors (1 -4). Delivery of these growth factors, however, remains a problem as the half life of these growth factors in vivo is only several hours (5 , 6). Moreover, the introduction of these factors into the body by such methods as topical application and needle injection is nonspecific and uncontrolled. A number of delivery systems have been developed to obtain a controlled, localized, slow-release of growth factor. Such vehicles include gelfoam, collagen gels, multilamellar liposomes, polyethylene glycol, RBC ghosts, Elvax and Hydron (slow release polymers) (1, 7-10). No single vehicle has been shown to be optimal.

Recently, calcium alginate microbeads have been used as a growth factor release vehicle (1 1). Previous findings have shown that these microbeads can release growth factor in vitro (12) as well as induce neovascularization in vivo when incorporated with angiogenic growth factor (13). While important, these studies have not investigated the fundamental release rates for calcium alginate microbeads. In order for in vivo angiogenesis to be optimized or even improved, the release characteristics as well as factors which affect them should be established. The present study was performed to determine the rates of release from calcium alginate microbeads as well as how the concentration of alginate and loading of substrate affect growth factor release.

MATERIALS AND METHODS

Alpinate Preoaration; One and 4% solutions of sodium alginate (Keltone LV, Kelco, San Diego, CA) were prepared in sterile 0.9% NaCI. The alginate, derived primarily from Macrocystis pyrifera, contained approximately 6 1 % mannuronic acid and 39% guluronic acid. The solution was centrifuged at 20,OOOg for 3 hours to remove all particulate debris. The sodium alginate solution was then aliquotted into sterile tubes and left over night under a UV light to ensure complete sterilization.

Endothelial Cell Growth Factor (ECGF) Irnrnobili Z ation Wi 'thin Calci 'urn Alpinat e Microbeads : ECGF (Organon Teknika Corp Biotechnology Research Institute, Rockville, MD) was weighed fresh and dissolved in 0.5 ml sterile 0.9% saline. This was then mixed in 4 ml sodium alginate to obtain a final concentration of 6 mg ECGF/ml and 12 mg ECGF/ml sodium alginate. The mixture was extruded through a droplet-generating apparatus to form microdroplets of approximately 300-700 pm diameter. The aqueous sodium alginate

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ECGF RELEASE FROM ALGINATE MICROBEADS 145

6 mg ECGF/mI in 4% alginatc

6 mg ECGF/tnl in 1% n1gin;iic

12 mg ECGF/mI in 4% alginae

__ _-__ - ___---_

I I

I I I I I I 4 12 24

Time (hrs.)

Figure 1 : ECGF release during hours 1-3

microdroplets immediately formed solid calcium alginate microbeads upon contact with 1.5% calcium chloride solution (14). In this reaction free-flowing sodium alginate forms a solid calcium alginate hydrogel microbead upon exposure to the calcium chloride solution. The microbeads were rinsed once and then placed into 15 ml of calcium chloride. Three different compositions of calcium alginate microbeads were prepared as follows: 1) 4% alginate containing 6 mg ECGF; 2) 1% alginate containing 6 mg ECGF; and 3) 4% alginate containing 12 mg ECGF. Control calcium alginate microbeads were prepared using no ECGF.

,!?xuerimentd Protocol; Approximately 5 ml of ECGF-immobilized with calcium alginate microbeads were incubated with 1.5% calcium chloride for a period of 24 days with constant agitation. The incubation periods were as follows: Period 1: 0-4 hours; period 2: 1-10 days; period 3: 10-16 days; period 4: 16-24 days. Each period began with a fresh solution of CaC12 containing no ECGF. The bathing solution was replaced with fresh CaC12 solution when the release profile of ECGF plateaued, i.e., reached a state of equilibrium with the bathing solution. There were a total of 3 solution changes. At specific timepoints, ECGF concentration was determined spectrophotometrically by measuring its absorbance at 2 18nm. ECGF in calcium chloride solution was initially scanned in the spectrophotometer

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146 KO ET AL.

- -- -+-- - ___-___

6 mg ECGF /ml in 4 % zdginate

6 my ECGF/mI in I % alginate

12 mg ECGFhnl in 4% alginate

3

0 8 Fresh solution exchange cn

aJ

u w

0 - 1 2 4 6 8 10

Time (days)

Figure 2: ECGF release during days 1 - 10

6 mg ECG/ml in 4% alginzw

6 ing ECGl rnl in 1% alginate

12 mg ECGF/ml in 4% alginate

- -- -a__ - -----__

Figure 3: ECGF release during days 10- 16

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ECGF RELEASE FROM ALGINATE MICROBEADS 147

6 mg ECGF/inl in 4%$ alginate

6 mg ECGF/ml in I % alginate

12 mg ECGF/ml in 4% alginate

-- -+.-- - - - - - - - -

U 5 6 - Fresh solution exchnnge a2 -

16 18 20 22 24

Time (days)

Figure 4: ECGF release during days 16-24

(Beckman), and 218 nm was determined to be the optimal wavelength for quantifying concentration. Absorbance readings were converted to ECGF amounts (mg/ml) based on the appropriate standard curves. Five solution standards containing predetermined amounts of ECGF were used to make the standard curve on day 1 . These original ECGF solutions standards were used for preparing standard curves on each of the subsequent days of ECGF measurements to correct for any denaturation of the ECGF in solution.

RESULTS

There were 4 separate periods of release based upon the three solution changes. Period 1: 0-4 hours; period 2: 1-10 days; period 3: 10-16 days; period 4: 16-24 days. Each period began with fresh solution containing no ECGF.

Figures 1-4 show the amounts of ECGF released from the 4 periods, respectively. We observed a significant burst release during the first 1-2 hours of incubation after which it reached an equilibrium state (plateau) in solution (Figure 1). Subsequently, in period 2 (Figure 2), the ECGF release was more gradual reaching a plateau after day 4. During the final two periods, 3 and 4, ECGF release remained approximately constant with detectable

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148 KO ET AL.

- -- _*__ - ___-___

6 mg ECGF/ml in 4% zdginate

6 mg ECGF/ml in 1% alginate

12 m g ECGF/ml in 4% alginate

Time (days)

Figure 5: ECGF release rates

amounts still being measured after 24 days. Depending on the amount of ECGF loaded into the microbeads the release of ECGF at the conclusion of the experiment ranged from 60-300

FgfdaY.

The rates of release of ECGF illustrated in Figure 5 were derived from data presented in Figures 1-4. Only values measured prior to reaching equilibrium (for each period 1-4) were included for rate determination. The most substantial release rates were observed during the first 1-2 hours of period 1. During this period 48% of the total immobilized ECGF was released from the microbeads composed of 1 % alginate containing 6 mgml of ECGF.

Table 1 shows the percentage of ECGF remaining in the microbeads on specific days of incubation. It is interesting to note that even at the end of the study, there were substantial amounts of ECGF available for release from the microbeads

DISCUSSION

The use of calcium alginate microbeads as a vehicle for growth factor release is promising for a number of reasons. Calcium alginate is non-toxic, biocompatible and biodegradable. It is convenient to work with and the microbeads can be formed

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1 % Alginate Microbeads [6mg ECGF/ml]

52% 28%

18%

13% 9% 3%

2%

ECGF RELEASE FROM ALGINATE MICROBEADS

4% Alginate Microbeadr [ 12mg ECGF/ml]

62%

4Y%

38% 34%

30%

28% 25%

Table 1: Percentage of total immobilized ECGF remaining within microbeads

Time ( D a y s )

0.16

2 4 5

7 9 13

4% Alginate Microbeads [6mg ECGFlml]

62%

39%

30%

24%

19%

14% 12%

inexpensively. Most importantly, previous animal studies have shown calcium alginate microbeads incorporated with various growth factors have produced favorable clinical results (1 1,13).

Since these initial studies, there has been a need for further study of these microbeads and their basic release patterns of growth factor. As such, ours is the first study that investigates the fundamental release characteristics of growth factors from alginate microbeads as well as how different compositions of alginate and growth factor affect this release.

In our investigation we have observed the following: first, the rate of growth factor release in the first 96 hours as well as the amount released for at least the first 300 hours are both dependent on the initial concentration of ECGF in the microbeads. Second, except for an initial rate difference noted in the first 2 hours, the rate of release appears to be independent of the concentration of alginate used to synthesize the microbeads. Finally, the rate of release varies inversely with time as there appears to be an initial substantial release in

the first 2 hours, a controlled slow release for about 4-5 days, and then a much slower but detectable release for at least approximately 14 days. Of significance, the released amounts of ECGF detected at the conclusion of the experiment were in the same range as those used in previously published wound healing models (1,2,4,8).

Although it appears in table 1 that ECGF release may be influenced by the alginate concentration, this was only true during the first 2 hours of incubation. Thereafter, ECGF release was a function of the amount of ECGF loaded into the microbeads. We conclude alginate concentration does not affect the release characteristics of the immobilized growth

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150 KO ET AL.

factor. This is substantiated by a recent report by Tanaka et al. (12) which reached the same conclusion for the release of a variety of molecules from alginate microbeads.

Our results are promising in that they show alginate microbeads are consistent and predictable in their release characteristics for ECGF. While release is not substantially affected by the concentration of alginate utilized, the initial loading concentration of ECGF consistently and predictably affects the patterns and amounts released. Coupled to a previous report that alginate microbeads may provide a stabilizing environment which increases the length of biological activity ( I l,l3), the use of these microbeads as a vehicle for controlled and sustained growth factor delivery appears promising.

Future modifications of this delivery process include making a calcium alginate spaghetti as well as a calcium alginate sheet - both for delivery of growth factor. Such new shapes may be more appropriate for growth factor delivery to certain areas of the body. For example, ECGF immobilized within calcium alginate spaghetti may be wrapped around specific blood vessels to promote localized angiogenesis. Furthermore, we are in the process of attaching an additional polylysine/alginate coat onto the microbead which will not only increase the stability of the microbead, but also will likely be another factor which can be used to affect release.

We are currently conducting further studies for the in vivo delivery of specific growth factors for wound healing purposes and the development of organoids. Knowledge of the fundamental release patterns for angiogenic factors immobilized within calcium alginate, as well as the ways to modify these characteristics, should prove helpful in future investigations.

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ECGF RELEASE FROM ALGINATE MICROBEADS 151

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