- Email: [email protected]
Optimization of a microencapsulated liposome system for enzymatically controlled release of macromolecules
Journal of Controlled Release, 14 (1990) Elsevier Science Publishers
OPTIMIZATION ENZYMATICALLY Yasutaka
263
263-267
B.V., Amsterdam
OF A MICROENCAPSULATED LIPOSOME SYSTEM CONTROLLED RELEASE OF MACROMOLECULES
Igari’,4,t,
FOR
Paul G. Kibat2ttt and Robert Langerlm4,*
‘Division of Health Sciences and Technology, *Department of Chemical Engineering, 3 Whitaker College of Health Sciences, Technology, and Management, 4Department
MIT, Cambridge, MA 02 139 (U.S.A.)
of Surgery, Children’s Hospital Medical Center Boston, MA (U.S.
A .)
(Received March 5, 1990; accepted in revised form July 9, 1990) Keywords: phospholipase; microencapsulated
liposomes; enzymatically controlled release; shelf-life; macromolecules
To create microencapsulated liposomes, liposomes containing a biologically active agent are coated with phospholipase A2 and then embedded in alginate. Release of agents from the system are influenced by a number of factors. In this paper, concentration of alginate and the volume ratio of alginate solution to liposomes were shown to affect the enzymatically triggered release profile of macromolecules both in terms of release rate and the extent of cumulative release. The nature of the phospholipase also affects the release profile. Whereas phospholipase A2 is known to cause a pulsatile release, phospholipase C caused a zero order release of macromolecules from microencapsulated liposomes to occur and phospholipase D had little effect on the release process. Storage of the microencapsulated liposomes with phospholipase A2 at 10” C for 30 days kept the system latent with no release occurring indicating that the system has a reasonable shelf life. The system could be activated simply by exposure to 37” C.
INTRODUCTION Recently, a new combination of liposomes and an alginate microcapsule, microencapsulated liposomes, has been proposed as a novel drug delivery system (DDS ) to maximize the advantageous properties of both these vehicles and to overcome their drawbacks [ 1 J. In this system, drugs or polypeptides are entrapped within liposomes which are then embedded in ‘Present
address: Research
& Development
Division, Tak-
eda Chemical Industries, Ltd., Osaka, Japan. ‘+Present address: Pharma Research, Hoechst
Aktienge-
sellschaft, Frankfurt a.M., Germany. *To whom correspondence about this paper should be addressed at the Chem. Eng. Dept.
0168-3659/90/$03.50
0 1990 -
a hydrogel matrix of Ca alginate to produce microcapsules of an injectable size. This system can also employ phospholipase A2 as an internal trigger for timing the onset of a burst of drugs or polypeptides in order to achieve a pulsatile release. Phospholipase A, removes an acyl group from the 2-position of phospholipids and produces lysolecithin, which destabilizes the phospholipid bilayer membrane of the liposomes as soon as a critical concentration is reached [ 21. The system functioned effectively in vitro with bovine serum albumin (BSA) and in vivo with horse-radish peroxidase (HRP), retaining its biological activity. A number of factors were shown to govern the pulsatile release profile of macromolecules from the mi-
Elsevier Science Publishers
B.V.
264
croencapsulated liposomes [ 1 ] : the number of units of phospholipase A,, the molecular weight of poly (L-lysine) and the species of phospholipid. In the current study, we have examined how the volume and concentration of alginate and the species of phospholipase affected release profiles. In addition, we have studied the shelf-life of microencapsulated liposomes over a one month period.
MATERIALS
AND METHODS
Materials
Sodium alginate (Kelcogel LV ) was a gift from Kelco. Hydrogenated soy lecithin (Phospholipon 100-H) was a gift from American Lecithin Co. Cholesterol, poly (L-lysine), vitamin B,, and fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) were obtained from Sigma. Phospholipase A, (PLA,) from bovine pancreas was obtained from either Sigma or ICN Biochemicals. Phospholipase C (Type I) (PLC) and phospholipase D (Type III) (PLD) were obtained from Sigma. Other chemicals were analytical grade or better. Liposome preparation
Liposomes were prepared by a modification [ 1 ] of the reverse phase evaporation technique [ 31. Briefly, 264 pmol of lipid were dissolved in 12 ml of chloroform-diisopropyl ether (2 : 1 v/
v) in a 100 ml round bottom flask. To this organic phase an aliquot of 5 ml of 25 mg/ml FITC-BSA or vitamin B,, in isotonic HEPES buffered saline (pH 7.4) was added and emulsified by sonication at 37’ C with a bath sonicator (Laboratory Supplies) for l-2 min. The organic phase was evaporated (Rotavapor RllO, Brinkman) until a gel formed, and the evaporation was continued until the liposome formation was complete and no foaming occurred. The unentrapped macromolecules were removed by repeated centrifugation (Sorvall RC-
5B, Du Pont ) and finally resuspended in 20 ml of the buffer. Microencapsulation
Microencapsulation of liposomes was performed according to a previously reported method [ 11.Briefly, 3 ml of the liposomes was centrifuged. To the pellet 50 ,ul of an enzyme (containing 10 units of PLA2, PLC or PLD ) in HEPES buffer or 50 ,ul of the HEPES buffer was added and incubated at 20’ C for 15 min with frequent mixing. PLAZ from ICN Biochemicals was used unless otherwise specified. The liposome pellet containing FITC-BSA was resuspended by vortexing in 4.5, 3 or 1.5 ml of a 1.5% (w/v) solution of Na alginate in isotonic HEPES buffered saline (pH 7.4 ) . In a separate set of experiments, the liposome pellet was resuspended in 3 ml of either a 1% or a 0.5% (w/ v) solution of Na alginate in HEPES buffer. The dispersed suspension was sprayed into a solution of 1.3% CaCl, in 13 mA4HEPES buffer with a jet head equipped with a 25 gauge needle (Beckton Dickinson & Co.), which was operated at an air flow rate of 5 l/min. Upon contact with the CaCl, solution, microspheres were formed. The spheres were coated with 0.06% (w/v) poly (L-lysine) of molecular weight 60,000 for 10 min followed by washing 3 times with 50 ml of isotonic HEPES buffered saline (pH 7.4). They were then coated with 0.06% Na alginate for 10 min followed by rinsing 3 times with 50 ml of the HEPES buffer. Finally they were resuspended in 10 ml of the HEPES buffer solution containing 3 mM CaCl, and 0.02% of gentamicin sulfate. The liposome pellet containing vitamin B,, was resuspended by vortexing in 3 ml of a 1.5% (w/v) solution of Na alginate after treatment with 50 ~1 of a soln. of 1 unit of Sigma PLA, in the HEPES buffer or 50 ~1 of the HEPES buffer. This liposome suspension was then treated using the same procedure as described above except that poly (L-lysine) of molecular weight 20,000 was used. The microencapsulated liposomes were
265
stored in a refrigerator until used (usually a few days). The particle sizes of the microencapsulated liposomes were around 500-800 pm as determined by using a light microscope (Micro Star, American Optical). In vitro release experiments
The resuspended microencapsulated liposomes were placed in an air gravity incubator (Imperial II, Lab Line) with a rotating shaker (Clinical Rotator, Thomas Scientific) at 37’ C. At specified times the HEPES buffer solution containing 3 mM CaCl, and 0.02% of gentamitin sulfate was filtered through an Econo Column (Bio-Rad) and replaced by fresh buffer. The collected buffer solution was centrifuged at 25,000 g for 30 min. Absorption at 495 nm for FITC-BSA or at 555 nm for vitamin Blz in the supernatant was determined with a spectrophotometer (Perkin-Elmer, 553 Fast Scan ). The initial dose of substance contained within the microencapsulated liposomes was determined in a separate set of experiments where either FITC-BSA or vitamin B,, was extracted from the microencapsulated liposomes using a probe type sonicator (Vibra Cell, Sonics and Material, Inc.) in the presence of EDTA and Triton X-100 [l] followed by centrifugation at 25,000 g for 30 min. Absorption at 495 nm for FITC-BSA or at 555 nm for vitamin B,, in the supernatant was then determined.
RESULTS
lease rate is achieved. The release rates decreased from 14% to 10% to 1% of dose per day with decreasing concentrations of Na alginate from 1.5% to 1% to 0.5% respectively (Fig. 1A); in addition, the extents of the cumulative release after 30 days of incubation became lower, from 66% to 45% to 23% of the dose with decreasing concentrations of Na alginate form 1.5% to 1% to0.5% respectively (Fig. 1B). When the alginate concentration was over 1.5%, the viscosity of the solution became so high that it was difficult to handle the solution. When the volume of 1.5% Na alginate solution for the preparation of the microencapsulated liposomes containing FITC-BSA was reduced from 4.5 ml to 3 ml to 1.5 ml, the release rates of FITC-BSA after the onset of a burst from the system made with either 4.5 ml or 3
0.0
0.5 Concentration
1 .o of Na
1.5 algtnale
2.0 (Oo)
AND DISCUSSION
Enzymatically activated microencapsulated liposomes usually display a sigmoid-type release curve with an initial lag-time [ 11. Maximum release rate is achieved after an initial slow release rate and is followed by a terminal slow release rate. The maximum release rate lasts for a defined time period during which the majority of FITC-BSA is released. The release rates discussed below were estimated in the linear portion of the release curve where maximum re-
01 u
0.0
I
0.5 Concentration
I
1 .o of Na
I
1.5 alginale
2.0 (“a)
Fig. 1A. Effect of concentration of Na alginate in the liposome suspension on the rate of release of FITC-BSA after the onset of a burst from microencapsulated liposomes treated with 10 units of PLA,. Fig. 1B. Effect of concentration of Na a&ate on the extent of cumulative release of FITC-BSA after 30 days of incubation. Each point was derived from the same set of experiments as in Fig. 1A.
266
ml in vitro were almost equal (14-E% of dose per day), while the release rate slowed down to 5% of dose per day for the system made with 1.5 ml (Fig. 2A). The extents of cumulative release after 30 days of incubation decreased from 77% to 66% to 45% of the total dose with decreasing volumes of 1.5% Na alginate from 4.5 ml to 3 ml to 1.5 ml, respectively (Fig. 2B). There was no significant difference in size distribution of the microencapsulated liposomes shown in Figs. 1 and 2. As shown elsewhere [ 11, the molecular weight of the poly (L-lysine) used in the coating has an important effect on the release profile of FITC-BSA. The lower the molecular weight of the poly (L-lysine) , the longer the lag time for the onset of pulsatile release. Since poly ( L-lysine) could penetrate further into the interior of microcapsule through the alginate gel with inf
16 -
t 8
12 -
z
10 -
$$ 0 ?2 t I
:
a6 14 :A :/4 2
I
0, 1
1
Fig.2A.
Effect
,
I
.
I
2 Volume
of
3 Na
alginate
4 (ml)
2 Volume
of
3 Na alginate
4 (ml)
5
creasing time of incubation and decreasing molecular weight of poly (L-lysine), the enzymatically triggered release profile of macromolecles might be manipulated by changing poly ( L-lysine) coating time and, possibly, by changing the concentration in addition to the molecular weight of poly (L-lysine). PLA, has been shown to produce a pulsatile release of macromolecules from microencapsulated liposomes [ 11. The effects of other phospholipases, phospholipase C (PLC ) and phospholipase D (PLD ), which cleave on either side of the phosphodiester linkage, on the release profile of FITC-BSA from microencapsulated liposomes were examined in vitro (Fig. 3 ) . Ten units of PLC caused a zero order release of FITC-BSA from the microencapsulated liposomes, in contrast to the pulsatile release achieved with 10 units of PLAz [ 11. Ten units of PLD induced little release of FITC-BSA from the microencapsulated liposomes. Without the phospholipases, little release of FITC-BSA occurred from the microencapsulated liposomes [ 11. PLC is known, unlike other phospholipases, to render mammalian cells permeable to proteins [ 41. To conduct a preliminary examination of shelf-life and to explore appropriate storage conditions, microencapsulated liposomes containing FITC-BSA with 10 units of PLA, were placed in a refrigerator at 10’ C for 30 days and then transferred to an air gravity incubator at
5
of volume of Na alginate in the liposome
suspension on the release rate of FITC-BSA after the onset of a burst from microencapsulated liposomes treated with 10 units of PLA,. Fig. 2B. Effect of the volume of Na alginate on the extent of cumulative release of FITC-BSA after 30 days of incubation. Each point was derived from the same set of experiments as in Fig. 2A.
0
10
20
30
40
50
TIME(days)
Fig. 3. Time course of release of FITC-BSA from microencapsulated liposomes treated with 10 units of PLC (0 ) and 10 units of PLD (0 ).
267
37°C (Fig. 4). Little release of FITC-BSA was observed at 10’ C. The PLAz became active once the microencapsulated liposomes were exposed to a temperature of 37°C and FITC-BSA was released from the microencapsulated liposomes. This indicates that the system might be stored for a reasonable length of time in a cold place, and temperature might safely be used for activating the system when needed.
0
10
20
30
40
50
TIME(days)
Fig. 4. Retardation of PLA, enzyme reaction by lowering temperature. Microencapsulated liposomes were stored at 10°C for 30 days and then the incubation was started at 37°C.
The system with PLA, was also tested for a pulsatile release of a smaller hydrophobic compound, vitamin Blz (Fig. 5 ). The control system without PLA, showed slow and steady release of vitamin Blz; about 40% of the initial dose was released over 40 days. In contrast, 1 unit of PLAz caused a pulsatile release of vitamin B1z after a lag time of 20 days. The current study has added practical information on the design of enzymatically triggered microencapsulated liposomes. In conjunction with information previously reported [ 11, the system may, with further study, be optimized to meet demands for applications such as vaccination, time dependent reagents for diagnostics, cyclically produced hormones, release of agents at desired times in foods, and agrochemicals that might be optimally administered in a pulsatile manner. Since phospholipases are available from a variety of sources, including those of human origin, a range of triggering agents for such applications could be envisaged.
ACKNOWLEDGEMENT This work was supported by NIH Grants Nos. AI 25901 and AI 24764.
100
REFERENCES
.; n 4 z
80
60
t 1e
40
f ‘Z 4
20
; 2
0 0
9 0
10
20
30
40
60
TIME(days)
Fig. 5. Time course of release of vitamin Bi2 from microencapsulated liposomes with 1 unit of PLA, ( 0 ) and 0 units of PLA* (0 ) in vitro at 37” C. Sigma PLA, was used in this experiment.
P.G. Kibat, Y. Igari, M. Wheatley, H. Eisen and R. Langer, Microencapsulated liposomes: a system for enzymatically controlled pulsatile release of biologically active substances, FASEB J., 4 f 1990) 2533-2539. P.G. Kibat and H. Stricker, Lagerungsstabilitit von Liposom-dispersionen aus Sojalecithinen, Pharm. Ind., 48 (1986) 1184-1189. F. Szoka, Jr., and D. Papahadjopoulos, Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation, Proc. Natl. Sci. U.S.A., 75 (1978) 4194-4198. L. Carrasco, M.J. Otero and J.L. Castrillo, Modification of membrane permeability by animal viruses, Pharmacol. Ther., 40 (1989) 171-212.
OPTIMIZATION ENZYMATICALLY Yasutaka
263
263-267
B.V., Amsterdam
OF A MICROENCAPSULATED LIPOSOME SYSTEM CONTROLLED RELEASE OF MACROMOLECULES
Igari’,4,t,
FOR
Paul G. Kibat2ttt and Robert Langerlm4,*
‘Division of Health Sciences and Technology, *Department of Chemical Engineering, 3 Whitaker College of Health Sciences, Technology, and Management, 4Department
MIT, Cambridge, MA 02 139 (U.S.A.)
of Surgery, Children’s Hospital Medical Center Boston, MA (U.S.
A .)
(Received March 5, 1990; accepted in revised form July 9, 1990) Keywords: phospholipase; microencapsulated
liposomes; enzymatically controlled release; shelf-life; macromolecules
To create microencapsulated liposomes, liposomes containing a biologically active agent are coated with phospholipase A2 and then embedded in alginate. Release of agents from the system are influenced by a number of factors. In this paper, concentration of alginate and the volume ratio of alginate solution to liposomes were shown to affect the enzymatically triggered release profile of macromolecules both in terms of release rate and the extent of cumulative release. The nature of the phospholipase also affects the release profile. Whereas phospholipase A2 is known to cause a pulsatile release, phospholipase C caused a zero order release of macromolecules from microencapsulated liposomes to occur and phospholipase D had little effect on the release process. Storage of the microencapsulated liposomes with phospholipase A2 at 10” C for 30 days kept the system latent with no release occurring indicating that the system has a reasonable shelf life. The system could be activated simply by exposure to 37” C.
INTRODUCTION Recently, a new combination of liposomes and an alginate microcapsule, microencapsulated liposomes, has been proposed as a novel drug delivery system (DDS ) to maximize the advantageous properties of both these vehicles and to overcome their drawbacks [ 1 J. In this system, drugs or polypeptides are entrapped within liposomes which are then embedded in ‘Present
address: Research
& Development
Division, Tak-
eda Chemical Industries, Ltd., Osaka, Japan. ‘+Present address: Pharma Research, Hoechst
Aktienge-
sellschaft, Frankfurt a.M., Germany. *To whom correspondence about this paper should be addressed at the Chem. Eng. Dept.
0168-3659/90/$03.50
0 1990 -
a hydrogel matrix of Ca alginate to produce microcapsules of an injectable size. This system can also employ phospholipase A2 as an internal trigger for timing the onset of a burst of drugs or polypeptides in order to achieve a pulsatile release. Phospholipase A, removes an acyl group from the 2-position of phospholipids and produces lysolecithin, which destabilizes the phospholipid bilayer membrane of the liposomes as soon as a critical concentration is reached [ 21. The system functioned effectively in vitro with bovine serum albumin (BSA) and in vivo with horse-radish peroxidase (HRP), retaining its biological activity. A number of factors were shown to govern the pulsatile release profile of macromolecules from the mi-
Elsevier Science Publishers
B.V.
264
croencapsulated liposomes [ 1 ] : the number of units of phospholipase A,, the molecular weight of poly (L-lysine) and the species of phospholipid. In the current study, we have examined how the volume and concentration of alginate and the species of phospholipase affected release profiles. In addition, we have studied the shelf-life of microencapsulated liposomes over a one month period.
MATERIALS
AND METHODS
Materials
Sodium alginate (Kelcogel LV ) was a gift from Kelco. Hydrogenated soy lecithin (Phospholipon 100-H) was a gift from American Lecithin Co. Cholesterol, poly (L-lysine), vitamin B,, and fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) were obtained from Sigma. Phospholipase A, (PLA,) from bovine pancreas was obtained from either Sigma or ICN Biochemicals. Phospholipase C (Type I) (PLC) and phospholipase D (Type III) (PLD) were obtained from Sigma. Other chemicals were analytical grade or better. Liposome preparation
Liposomes were prepared by a modification [ 1 ] of the reverse phase evaporation technique [ 31. Briefly, 264 pmol of lipid were dissolved in 12 ml of chloroform-diisopropyl ether (2 : 1 v/
v) in a 100 ml round bottom flask. To this organic phase an aliquot of 5 ml of 25 mg/ml FITC-BSA or vitamin B,, in isotonic HEPES buffered saline (pH 7.4) was added and emulsified by sonication at 37’ C with a bath sonicator (Laboratory Supplies) for l-2 min. The organic phase was evaporated (Rotavapor RllO, Brinkman) until a gel formed, and the evaporation was continued until the liposome formation was complete and no foaming occurred. The unentrapped macromolecules were removed by repeated centrifugation (Sorvall RC-
5B, Du Pont ) and finally resuspended in 20 ml of the buffer. Microencapsulation
Microencapsulation of liposomes was performed according to a previously reported method [ 11.Briefly, 3 ml of the liposomes was centrifuged. To the pellet 50 ,ul of an enzyme (containing 10 units of PLA2, PLC or PLD ) in HEPES buffer or 50 ,ul of the HEPES buffer was added and incubated at 20’ C for 15 min with frequent mixing. PLAZ from ICN Biochemicals was used unless otherwise specified. The liposome pellet containing FITC-BSA was resuspended by vortexing in 4.5, 3 or 1.5 ml of a 1.5% (w/v) solution of Na alginate in isotonic HEPES buffered saline (pH 7.4 ) . In a separate set of experiments, the liposome pellet was resuspended in 3 ml of either a 1% or a 0.5% (w/ v) solution of Na alginate in HEPES buffer. The dispersed suspension was sprayed into a solution of 1.3% CaCl, in 13 mA4HEPES buffer with a jet head equipped with a 25 gauge needle (Beckton Dickinson & Co.), which was operated at an air flow rate of 5 l/min. Upon contact with the CaCl, solution, microspheres were formed. The spheres were coated with 0.06% (w/v) poly (L-lysine) of molecular weight 60,000 for 10 min followed by washing 3 times with 50 ml of isotonic HEPES buffered saline (pH 7.4). They were then coated with 0.06% Na alginate for 10 min followed by rinsing 3 times with 50 ml of the HEPES buffer. Finally they were resuspended in 10 ml of the HEPES buffer solution containing 3 mM CaCl, and 0.02% of gentamicin sulfate. The liposome pellet containing vitamin B,, was resuspended by vortexing in 3 ml of a 1.5% (w/v) solution of Na alginate after treatment with 50 ~1 of a soln. of 1 unit of Sigma PLA, in the HEPES buffer or 50 ~1 of the HEPES buffer. This liposome suspension was then treated using the same procedure as described above except that poly (L-lysine) of molecular weight 20,000 was used. The microencapsulated liposomes were
265
stored in a refrigerator until used (usually a few days). The particle sizes of the microencapsulated liposomes were around 500-800 pm as determined by using a light microscope (Micro Star, American Optical). In vitro release experiments
The resuspended microencapsulated liposomes were placed in an air gravity incubator (Imperial II, Lab Line) with a rotating shaker (Clinical Rotator, Thomas Scientific) at 37’ C. At specified times the HEPES buffer solution containing 3 mM CaCl, and 0.02% of gentamitin sulfate was filtered through an Econo Column (Bio-Rad) and replaced by fresh buffer. The collected buffer solution was centrifuged at 25,000 g for 30 min. Absorption at 495 nm for FITC-BSA or at 555 nm for vitamin Blz in the supernatant was determined with a spectrophotometer (Perkin-Elmer, 553 Fast Scan ). The initial dose of substance contained within the microencapsulated liposomes was determined in a separate set of experiments where either FITC-BSA or vitamin B,, was extracted from the microencapsulated liposomes using a probe type sonicator (Vibra Cell, Sonics and Material, Inc.) in the presence of EDTA and Triton X-100 [l] followed by centrifugation at 25,000 g for 30 min. Absorption at 495 nm for FITC-BSA or at 555 nm for vitamin B,, in the supernatant was then determined.
RESULTS
lease rate is achieved. The release rates decreased from 14% to 10% to 1% of dose per day with decreasing concentrations of Na alginate from 1.5% to 1% to 0.5% respectively (Fig. 1A); in addition, the extents of the cumulative release after 30 days of incubation became lower, from 66% to 45% to 23% of the dose with decreasing concentrations of Na alginate form 1.5% to 1% to0.5% respectively (Fig. 1B). When the alginate concentration was over 1.5%, the viscosity of the solution became so high that it was difficult to handle the solution. When the volume of 1.5% Na alginate solution for the preparation of the microencapsulated liposomes containing FITC-BSA was reduced from 4.5 ml to 3 ml to 1.5 ml, the release rates of FITC-BSA after the onset of a burst from the system made with either 4.5 ml or 3
0.0
0.5 Concentration
1 .o of Na
1.5 algtnale
2.0 (Oo)
AND DISCUSSION
Enzymatically activated microencapsulated liposomes usually display a sigmoid-type release curve with an initial lag-time [ 11. Maximum release rate is achieved after an initial slow release rate and is followed by a terminal slow release rate. The maximum release rate lasts for a defined time period during which the majority of FITC-BSA is released. The release rates discussed below were estimated in the linear portion of the release curve where maximum re-
01 u
0.0
I
0.5 Concentration
I
1 .o of Na
I
1.5 alginale
2.0 (“a)
Fig. 1A. Effect of concentration of Na alginate in the liposome suspension on the rate of release of FITC-BSA after the onset of a burst from microencapsulated liposomes treated with 10 units of PLA,. Fig. 1B. Effect of concentration of Na a&ate on the extent of cumulative release of FITC-BSA after 30 days of incubation. Each point was derived from the same set of experiments as in Fig. 1A.
266
ml in vitro were almost equal (14-E% of dose per day), while the release rate slowed down to 5% of dose per day for the system made with 1.5 ml (Fig. 2A). The extents of cumulative release after 30 days of incubation decreased from 77% to 66% to 45% of the total dose with decreasing volumes of 1.5% Na alginate from 4.5 ml to 3 ml to 1.5 ml, respectively (Fig. 2B). There was no significant difference in size distribution of the microencapsulated liposomes shown in Figs. 1 and 2. As shown elsewhere [ 11, the molecular weight of the poly (L-lysine) used in the coating has an important effect on the release profile of FITC-BSA. The lower the molecular weight of the poly (L-lysine) , the longer the lag time for the onset of pulsatile release. Since poly ( L-lysine) could penetrate further into the interior of microcapsule through the alginate gel with inf
16 -
t 8
12 -
z
10 -
$$ 0 ?2 t I
:
a6 14 :A :/4 2
I
0, 1
1
Fig.2A.
Effect
,
I
.
I
2 Volume
of
3 Na
alginate
4 (ml)
2 Volume
of
3 Na alginate
4 (ml)
5
creasing time of incubation and decreasing molecular weight of poly (L-lysine), the enzymatically triggered release profile of macromolecles might be manipulated by changing poly ( L-lysine) coating time and, possibly, by changing the concentration in addition to the molecular weight of poly (L-lysine). PLA, has been shown to produce a pulsatile release of macromolecules from microencapsulated liposomes [ 11. The effects of other phospholipases, phospholipase C (PLC ) and phospholipase D (PLD ), which cleave on either side of the phosphodiester linkage, on the release profile of FITC-BSA from microencapsulated liposomes were examined in vitro (Fig. 3 ) . Ten units of PLC caused a zero order release of FITC-BSA from the microencapsulated liposomes, in contrast to the pulsatile release achieved with 10 units of PLAz [ 11. Ten units of PLD induced little release of FITC-BSA from the microencapsulated liposomes. Without the phospholipases, little release of FITC-BSA occurred from the microencapsulated liposomes [ 11. PLC is known, unlike other phospholipases, to render mammalian cells permeable to proteins [ 41. To conduct a preliminary examination of shelf-life and to explore appropriate storage conditions, microencapsulated liposomes containing FITC-BSA with 10 units of PLA, were placed in a refrigerator at 10’ C for 30 days and then transferred to an air gravity incubator at
5
of volume of Na alginate in the liposome
suspension on the release rate of FITC-BSA after the onset of a burst from microencapsulated liposomes treated with 10 units of PLA,. Fig. 2B. Effect of the volume of Na alginate on the extent of cumulative release of FITC-BSA after 30 days of incubation. Each point was derived from the same set of experiments as in Fig. 2A.
0
10
20
30
40
50
TIME(days)
Fig. 3. Time course of release of FITC-BSA from microencapsulated liposomes treated with 10 units of PLC (0 ) and 10 units of PLD (0 ).
267
37°C (Fig. 4). Little release of FITC-BSA was observed at 10’ C. The PLAz became active once the microencapsulated liposomes were exposed to a temperature of 37°C and FITC-BSA was released from the microencapsulated liposomes. This indicates that the system might be stored for a reasonable length of time in a cold place, and temperature might safely be used for activating the system when needed.
0
10
20
30
40
50
TIME(days)
Fig. 4. Retardation of PLA, enzyme reaction by lowering temperature. Microencapsulated liposomes were stored at 10°C for 30 days and then the incubation was started at 37°C.
The system with PLA, was also tested for a pulsatile release of a smaller hydrophobic compound, vitamin Blz (Fig. 5 ). The control system without PLA, showed slow and steady release of vitamin Blz; about 40% of the initial dose was released over 40 days. In contrast, 1 unit of PLAz caused a pulsatile release of vitamin B1z after a lag time of 20 days. The current study has added practical information on the design of enzymatically triggered microencapsulated liposomes. In conjunction with information previously reported [ 11, the system may, with further study, be optimized to meet demands for applications such as vaccination, time dependent reagents for diagnostics, cyclically produced hormones, release of agents at desired times in foods, and agrochemicals that might be optimally administered in a pulsatile manner. Since phospholipases are available from a variety of sources, including those of human origin, a range of triggering agents for such applications could be envisaged.
ACKNOWLEDGEMENT This work was supported by NIH Grants Nos. AI 25901 and AI 24764.
100
REFERENCES
.; n 4 z
80
60
t 1e
40
f ‘Z 4
20
; 2
0 0
9 0
10
20
30
40
60
TIME(days)
Fig. 5. Time course of release of vitamin Bi2 from microencapsulated liposomes with 1 unit of PLA, ( 0 ) and 0 units of PLA* (0 ) in vitro at 37” C. Sigma PLA, was used in this experiment.
P.G. Kibat, Y. Igari, M. Wheatley, H. Eisen and R. Langer, Microencapsulated liposomes: a system for enzymatically controlled pulsatile release of biologically active substances, FASEB J., 4 f 1990) 2533-2539. P.G. Kibat and H. Stricker, Lagerungsstabilitit von Liposom-dispersionen aus Sojalecithinen, Pharm. Ind., 48 (1986) 1184-1189. F. Szoka, Jr., and D. Papahadjopoulos, Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation, Proc. Natl. Sci. U.S.A., 75 (1978) 4194-4198. L. Carrasco, M.J. Otero and J.L. Castrillo, Modification of membrane permeability by animal viruses, Pharmacol. Ther., 40 (1989) 171-212.