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Preparation, characterization and release of microencapsulated bromodeoxyuridine
Life Sciences, Vol. 54, pp. 27-34 Printed in the USA
Pergamon Press
PREPARATION, CHARACTERIZATION AND RELEASE OF MICROENCAPSULATED BROMODEOXYURIDINE
D. Maysinger, J. Filipovid-Grrir, T. Alebid-Kolbah McGill University, Department of Pharmacology and Therapeutics (DM, JF-G), Department of Oncology-Pharmacokinetics Division (TA-K), 3655 Drummond Street, Montreal H3G 1Y6, Canada. (Received in final form October 21, 1993) Summary Biodegradable and biocompatible microspheres with bromodeoxyuridine (BrUrd) have been prepared, characterized and tested in vitro and in vivo. Scanning electron microscopy and image analysis revealed regular spherical shapes and an average size -I- SD of 2.47 _ 0.59/xm. Total content of BrUrd as determined by HPLC was within the range of 0.2 - 1.5%. Kinetic analyses of two different preparations showed similar release half-times (approx. 12 hrs), kinetic constants -0.0556, -0.0564, and -0.0557, -0.0597 [h-l], and correlation coefficients of 0.998, and 0.999 when fitted to the first order or biphasic first order kinetics, respectively. Preliminary data from immunocytochemical studies revealed efficient incorporation of BrUrd delivered from these microcapsules into nuclei of proliferating cells surrounding brain lesions in rats. 5'-Bromo-2-deoxyuridine (BrUrd) is a thymidine analog which is incorporated into the DNA of cells as they pass through the S phase of the cell cycle, and like thymidine, it is permanently retained in the cell. BrUrd has been increasingly used as a marker for cell cycle traverse studies with the introduction of monoclonal antibodies (mAb) against BrUrd that had been incorporated into cellular DNA (1-3). In earlier studies on cell proliferation, much of the information was collected from autoradiographic studies using tritiated thymidine ([3H]dT). Although this technique has yielded valuable data for toxicologic and carcinogenic studies as well as information regarding the proliferation, migration and the time of origin of cells in the central nervous system (CNS) (4, 5) it has several limitations: [a] the possibility of contamination from the radioactive materials, [b] problems of waste disposal, [c] lengthy exposure times for adequate autoradiographic signal and [d] sometimes, a limited resolution. All of these problems can be overcome by using BrUrd instead of [3H]dT. However, use of BrUrd poses a similar problem to that of [3H]dT in that administration for prolonged periods of time is required. Most studies have focused on the intervals between the time of single and multiple injections of [3H]dT or BrUrd and the time of sacrifice. Such manipulations provide insight into the dynamic qualities of developing structures but they allow for a relatively "narrow window" of time within the system. For example, if DNA synthesis does not occur between the time of injection and time of sacrifice, mitotic events can be missed. In an attempt to expand the time for labelling newly synthesized DNA, two different
Corresponding author: D. Maysinger, Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Montreal, PQ, H3G 1Y6, Canada. Phone (514)398-1264, fax (514)398-6690. 0024-3205/94 $6.00 + .0o Copyright © 1993 Pergamon Press Ltd All rights reserved.
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Microencapsulation of Bromodeoxyuridine
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attempts have been made so far: [3H]dT and BrUrd were delivered via osmotic minipumps (6, 7) and via slow-release pellets (6). Both means of administrations were found to be superior to single injection in obtaining stronger labelling signal. This finding prompted the present studies, aimed at developing a more suitable delivery system for BrUrd administration in the brains of animals, with various cortical lesions. Our objective was to establish a prolonged drug release via the microencapsulation of BrUrd. This could eventually provide a means of delivery which involves less cumbersome surgical manipulation than implantation of permanent cannulae.
Materials and Methods Chemicals. BrUrd and monoclonal antibodies against it bound to the single strain of DNA were purchased from Boehringer (Montreal, Canada), polymer (poly-(L-lactide): co- glycolide (PLGA) (70:30), [PLGA], from Polyscience (Warrington, PA, USA). All the chemicals were of analytical grade purity. Solvents, as well as water used were of HPLC quality and filtered through 0.22 #m Millipore type GV filters. Encapsulation Procedure. Phase separation by emulsification and subsequent organic solvent evaporation technique was used: the inner aqueous phase consisted of BrUrd (65 mg) in distilled water (1 ml) and gelatine (100 mg). The oil phase consisted of PLGA (70:30), (500 rag), dissolved in methylene chloride (2.5 ml). The oil phase was gradually poured into the inner water phase under vigorous stirring using ultrasonicator (Vibra Cell, VC 50; Sonics and Materials, Danbury, USA) over 3 minutes to make a microfine w/o emulsion. The emulsion was poured into an aqueous 0.5% polyvinyl alcohol (30 ml), (PVA, Anachemia, Montreal, Canada). The mixture was sonicated (Vibra Cell, VC 50; Sonics and Materials, Danbury, USA) for 3 minutes to make a (w/o)/w emulsion. Methylene chloride was evaporated (2 hours under no reduced pressure; the first 30 minutes by stirring at 30 °C, then left at ambient temperature), and the hardened microspheres were centrifuged, rinsed several times with distilled water and lyophilized. Cortical Lesions. Eight rats were subjected to a unilateral left-sided cortical devascularizing lesion, as previously described (8). Briefly, a bone flap was removed to expose the dura mater over a wide area (0.8 cm x 1.0 cm) of the dorsal brain surface, the dura was opened, and the terminal pia-arachnoid vessels were disrupted with the aid of a surgical needle. Following the lesion, the subcortical blood supply remained intact. Capsules were placed onto the surface of the lesioned cortex. All surgeries and preoperative handling were carried out in accordance with the NIH guidelines for care and use of laboratory animals and the McGill University Animal Care Committee. Preparation of Gelatin Films. Sterile gelatin (7.5 g; Sigma, St. Louis, MO, USA) was dissolved in double-distilled (dd) water (36.5 ml) with gentle heating and ultra pure glycerol (5 g; Bethesda Research Laboratories, MD, USA) was added. BrUrd (10 mg) was incorporated in the gel-film (0.8 x 1.0 cm). The pure crystalline or microencapsulated BrUrd was dissolved/dispersed in 1.5 ml of dd water and added to the gelatin solution with gentle stirring to avoid air bubbles. The warm solution was poured onto a flat glass surface to produce a uniform layer approximately 1 mm thick. After hardening of the gelatin, rectangular-shaped pieces were cut and stored in a sterile container at 4°C until use. Morphometric Analysis of Microspheres. Surface and shape characteristics of microspheres were
Vol. 54, No. 1, 1994
Microencapsulation of Bromodeoxyuridine
29
analyzed by a scanning electron microscope (JSM T-200, Pulp and Paper, Point Claire, Canada). Dry samples were mounted onto stubs using double side adhesive tape and were vacuum coated
2.~ 2OO i
b
150 100 SO 0
1
2
3
4 5 6 7 8 9 1 0
Fig. 1 PLGA microspheres with BrUrd. Panel a: scanning electron micrograph showing shapes and sizes of microspheres. Scale bar = 10 /~m, magnification 2,000 x. Panel b: frequency distribution of 625 spheres analyzed by an image analysis system.
o
a J,
o
B
~ i
o
e
15:
-1.3
ram
16:
-1.8
mm
o
f
~
"*
Fig. 2 Panel a: schematic representations of the rat brain at two different levels from bregma (-1.3 mm and -1.8 mm) with indicated sites of devascularizing cortical lesion and administration of PLGA microspheres with incorporated BrUrd. Panel b: nuclei stained with anti-BrUrd antibodies (Boehringer, Montreal, Canada) raised against BrUrd bound to a single stranded DNA. Immunocytochemical procedure was as described in Methods.
with gold film (approx. 30 ~m thick, engineering ion coater 1B type; 0.1 torr, high voltage 8001500 V and 8mA). The size of the microspheres were determined by image analysis using the M1 system (MCID, M1 Imaging Record Inc). The average diameter + SEM of randomly
30
Microencapsulation of Bromodeoxyuridine
measured microspheres diagram.
Vol. 54, No. 1, 1994
was estimated and plotted as a frequency or cumulative frequency
Immunocytochemistry. Eight rats (2 groups, n = 4 per group) were perfused with 4% paraformaldehyde. Protocol by Boehringer was used. All steps were carried out at the room temperature using PBS containing 0.2% Triton-X 100 (pH 7.4) for washes, dilutions of antibodies and 3,3'-diaminobenzedine tetrahydrochloride (DAB) reaction. Peroxidase-antiperoxidase (PAP; 1:30; Medicorp, Montreal, Canada) was used as a secondary antibody. Relative densities of BrUrd-positive nuclei in the area surrounding the lesion were determined, using a Quantimet image analysis system (Cambridge Instruments, Cambridge, England). In Vitro Release of BrUrd. The weighed amounts of microspheres (5-15 mg) were dispersed in a phosphate buffered saline (PBS). Samples were maintained at 37 0(7 and at the scheduled times between 1 day and 5 weeks, the suspension was centrifuged, release medium removed and content of the drug determined in lyophilized reconstituted aliquots. BrUrd content in the spheres and gels was determined by HPLC. Determination of BrUrd. The HPLC system consisted of a Beckman l l0B solvent pump (Beckman, San Ramon, CA, USA), 7125 Rheodyne injector (Rheodyne, Cotati, CA, USA) with a 20 #1 sample loop, an ABI 783 Programmable Absorbance Detector (ABI Kratos, Ramsey, N J, USA) and a DataJet integrator (Spectra-Physics, San Jose, CA, USA). The chromatographic column used was an Altex Ultrasphere-ODS (25 cm x 4.6 mm I.D., 5 /x) (Beckman, Berkeley, CA, USA). The mobile phase used consisted of 85% sodium phosphate buffer (0.1 M, pH 6.8) and 15% methanol, (v/v). The flow was kept at 1.0 ml/min. The uv absorbance was measured at 277 nm. Reconstituted (with 0.2 ml sodium phosphate buffer, 0.1 M, pH 7.47) lyophilised samples were injected directly into the HPLC and the peak area and height compared with those of injected BrUrd standards. Linear detector response was observed in the BrUrd concentration range from 10 ng/ml (32 nM) to 1.0 mg/ml (3.2 mM). A typical chromatogram showing BrUrd peak at 9.05 rain ( k ' = 1.66) is given in Figure 3. To determine the BrUrd content of prepared microspheres, 0.3 ml of methylene chloride was added to 15 mg of microspheres in an Eppendorf test-tube, and the test-tube shaken on Vortex-shaker for 20 min. BrUrd was reextracted into aqueous phase by addition of 0.2 ml sodium phosphate buffer (0.1 M, pH 7.4). The mixture was vortex-shaken for 10 min, spun at 15,000 x g for 10 min, and the aqueous layer injected directly into HPLC. The reextraction into aqueous layer was repeated up to 6 times, until no more BrUrd was recovered. Statistics. The data were assessed by analysis of variance (ANOVA) and significance between groups was determined by post-hoc Newman-Keul's test. A level of p < 0.05 was considered statistically significant.
Results PLGA microspheres containing BrUrd were prepared, characterized and tested in vitro. Figure la illustrates scanning electron micrographs of spheres with encapsulated BrUrd in biodegradable copolymer PLGA at the magnification 2,000x. Surfaces and shapes are slightly irregular but the sizes are fairly uniform with an average diameter _ SD of 2.47 + 0.59/,m (number of spheres analyzed = 625), Figure lb. Total yield was 54.6%, and the total content of BrUrd was found to be 0.2 %-1.5 % depending on amounts of gelatin co-dissolved with BrUrd in the aqueous phase.
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Microencapsulation of Bromodeoxyuridine
31
The lowest content of BrUrd (0.2%) was found in PLGA preparations devoid of gelatin. The highest amount (1.5%) of BrUrd was incorporated in spheres when relatively large amount of gelatin (20% w/w of the PLGA) was co-encapsulated. Variation in internal pH (7.40-11.00) failed to affect BrUrd incorporation under the conditions employed. In vivo experiments showed that BrUrd can be efficiently incorporated into the cell nuclei in the lesioned brain. The intensity of immunostaining was most remarkable in the regions surrounding the devascularizing lesion (50% in comparison with contralateral side) and subventricular zone (Figure 2a,b). Although we did not perform double staining in these experiments, our previous studies revealed intense glial reaction in these regions, suggesting that BrUrd was most likely incorporated in dividing/proliferating glia. The kinetic profiles of two different BrUrd preparations were determined by HPLC (Figure 3). The release of BrUrd from preparations No. 1 and No. 2 was followed for two weeks and 48 days, respectively. In both cases the experimental values are best fitted to both the first order and biphasic first order kinetics. The correlation coefficients (r2), kinetic constants (k, [hl]) and tv2 [h] are summarized in Table I. The kinetic profile of one BrUrd preparation, followed for 48 days, is illustrated in Figure 4. In addition to microsphere preparations we also prepared several types of biodegradable gels. The total contents of BrUrd in these preparations were close to 100%. However, release of BrUrd was considerably faster (4 hours up to 2 days) than from the spheres.
TABLE I Kinetic Parameters for Release of BrUrd from PLGA Microspheres
Preparation No. correlation coefficient, ta kinetic constant, k [h~] t,,~ [h]
first order kinetics 1 (14 days) 2 (48 days) 0.998 -0.0556 12.38
0.998 -0.0564 12.29
biphasic first order kinetics 1 (14 days) 2 (48 days) 0.999 -0.0557 12.12
0.999 -0.0597 11.61
Discussion This study describes the development of a novel delivery system for BrUrd which can be applied into the study of infrequent mitotic events in the brain and in other organs. BrUrd is of particular interest because it can be used as an alternative marker for the examination of the proliferation, migration and time of origin of cells in the CNS. It does not appear to be toxic and it labels comparable number of cells when compared with [3H]dT (9). BrUrd has several advantages over [SH]dT, however, and therefore the development of a delivery system for its chronic administration would be useful. Previously described systems for continuous administration such as permanently installed cannulae and non-biodegradable pellets have provided useful information
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Microencapsulation of Bromodeoxyuridine
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on rates of DNA synthesis in different organs including brain (6, 7, 10). Mitogenic growth factors, such as b FGF, EGF and many others (11) may participate in neurogenesis in the mammalian central nervous system. To conduct such studies, new experimental ways are needed for the administration of BrUrd, trophic factors and/or other DNA markers which will allow their continuous delivery in fairly large quantities. A number of approaches to this problem have been CHANNEL A
INJECT
_
12/28/92
12:12:08
STORED TO BIN
II o ,05
DATA
SAVED
TO B1N ~
2.0
Fig. 3 HPLC chromatogram showing peak of BrUrd at 9.05 rain. BrUrd was released from PLGA microspheres. See details in experimental section.
made, but all suffer from one or another defect. Thus pulse injections seriously jeopardize the possibility of identifying dividing cells outside the "window" during which CNS concentrations of [3H]dT or BrUrd reach maximal concentrations. BrUrd administration from slow releasing pellets, another approach, appears not to be reliable for chronic studies. It was hypothesized by Weghorst et al. (6), that release took place in uneven bursts causing toxic effects in hepatic and nonhepatic cell populations and that there was a possible "fainting" of the signal after four days due to the decrease/dilution in nuclear labelling at later times (7 days) compared to earlier periods (4 days). Finally, the continuous infusion of high concentrations of BrUrd and/or [3H]dT directly into the cerebrospinal fluid over a period of several days certainly enables maintenance of their steady and necessary levels. For example, intracerebroventricular administration of [3H]dT enabled electronmicroscopic demonstration of neurogenesis in the CNS following brain injury (10). In general, however, techniques such as the use of stainless steel cannulae result in considerable mechanical damage to the brain tissue in the vicinity of the needle. Against this background, we made an attempt to develop an alternative, less invasive system for BrUrd delivery into the CNS of animal models with different brain lesions, e.g. cortical, striatal, fimbria-fornix lesions (8, 10, 12) Results from this study show that we can microencapsulate BrUrd or incorporate it in the biodegradable film and place it over the lesioned cortex. Microencapsulated BrUrd can be administered directly onto the site of the lesion and secured with a piece of a sterile gel foam. Alternatively, a biocompatible film with a precise dose of the encapsulated drug can be placed at the same site. Depending on the polymer used, shape and size
Vol. 54, No. 1, 1994
Microencapsulation of Bromodeoxyuridine
33
of the film, one can modify the kinetics of the drug release. Gelatin film degrades relatively rapidly. Other polymers or matrix materials, e.g. collagen, could provide longer sustained release of BrUrd. Depending on the conditions of the microencapsulation procedure, we have produced several preparations with different BrUrd contents but similar kinetic profiles. Although we were able to ensure continuous release of relatively high concentrations for 48-50 hours, even longer times might be more suitable for neurogenesis studies. We are currently testing several biodegradable polymers for microencapsulation and film formation. Finally, several HPLC methods have been published for determination of BrUrd in serum and plasma (13-15) as well as in hamster V79 cells (16), either by using uv absorption (13, 14), or fluorescence (15, 16) for detection. Our HPLC method for determination of BrUrd content is very
I
I
I
[
I
T
20
30
40
50
"---
1.3 17
F --
1.i
"o
0.9
0.7 rn 0.5 I
I
0
10
Time
[days]
Fig. 4 Kinetic profile of BrUrd release from PLGA microspheres (Preparation No. 2) followed for 48 days (see details in Methods); first order = solid line, biphasic first order = dashed line.
simple, fast and reliable. It is particularly convenient for testing the amount of BrUrd incorporated in microspheres before using them in "in vivo" experiments because no sample purification is required. In summary, microencapsulated BrUrd could well be a viable alternative to intracerebroventricular administration of BrUrd to CNS in the study of developmental and regenerative changes in the brain following injury and trophic factor therapy.
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Microencapsulation of Bromodeoxyuridine
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Acknowledgements This work was supported by the Canadian Centre of Excellence for Neuronal Repair and Functional Recovery and MRC Canada. Editorial assistance of Dr. T. Keng and photographic reproductions by A. Forster are gratefully acknowledged.
References .
2. 3. 4.
5. . 7.
8. .
10. 11. 12. 13. 14. 15. 16.
H.G. GRATZNER, Science 218 474-475 (1982). M. VANDERLAAN and C.B. THOMAS, Cytometry 6 501-504 (1985). F. DOLBEARE, H. GRATZNER, M. PALLAVICINI and J.W. GRAY, Proc. Natl. Acad. Sci. USA 80 5573-5579 (1983). C.P. LEBLOND, B. MESSIER and B. KOPRIWA, Lab. Invest. _8 276-306 (1959). R.L. SIDMAN, (1970) Contemporary Research Methods in Neuroanatomy, W.J.H. Nauta, and S.O.E. Ebbesson (Eds), 252-274, Springer, New York (1970). C.M. WEGHORST, J.R. HENNEMAN and J.M. WARD, J. Histochem. Cytochem. 39 177-184 (1991). C.M. MORSHEAD and D. VAN DER KOOY, J. Neurosci. 1__22249-256 (1992). P.H. STEPHENS, A.C. CUELLO and M.W. SOFRONIEW, J. Neurochem. 45 1021-1026 (1985). M.W. MILLER and R.S. NOWAKOWSKI, Brain Research 457 44-52 (1988). D.G. HERRERA, A. RIBEIRO DA SILVA and A.C. CUELLO, Proc. Nat. Acad. Sci. USA, (1993) submitted. Y.A. BARDE, Neuron 2 1525-1534 (1989). A.C. CUELLO, L. GAROFALO, R.L. KENIGSBERG and D. MAYSINGER, Proc. Natl. Acad. Sci. USA 86 2056-2060 (1989). D.A. GANES and J.G. WAGNER, J. Chromatogr. 432 233-242 (1988). P.G. MONTALDO and M. D'INCALCI, J. Chromatogr. 491 129-138 (1989). S. YOSHIDA, S. HIROSE and M. IWAMOTO, J. Chromatogr. 383 61-68 (1986). M.R.L. STRATFORD and M.F. DENNIS, Int. J. Radiation Oncology Biol. Phys. 22 485487 (1992).
Pergamon Press
PREPARATION, CHARACTERIZATION AND RELEASE OF MICROENCAPSULATED BROMODEOXYURIDINE
D. Maysinger, J. Filipovid-Grrir, T. Alebid-Kolbah McGill University, Department of Pharmacology and Therapeutics (DM, JF-G), Department of Oncology-Pharmacokinetics Division (TA-K), 3655 Drummond Street, Montreal H3G 1Y6, Canada. (Received in final form October 21, 1993) Summary Biodegradable and biocompatible microspheres with bromodeoxyuridine (BrUrd) have been prepared, characterized and tested in vitro and in vivo. Scanning electron microscopy and image analysis revealed regular spherical shapes and an average size -I- SD of 2.47 _ 0.59/xm. Total content of BrUrd as determined by HPLC was within the range of 0.2 - 1.5%. Kinetic analyses of two different preparations showed similar release half-times (approx. 12 hrs), kinetic constants -0.0556, -0.0564, and -0.0557, -0.0597 [h-l], and correlation coefficients of 0.998, and 0.999 when fitted to the first order or biphasic first order kinetics, respectively. Preliminary data from immunocytochemical studies revealed efficient incorporation of BrUrd delivered from these microcapsules into nuclei of proliferating cells surrounding brain lesions in rats. 5'-Bromo-2-deoxyuridine (BrUrd) is a thymidine analog which is incorporated into the DNA of cells as they pass through the S phase of the cell cycle, and like thymidine, it is permanently retained in the cell. BrUrd has been increasingly used as a marker for cell cycle traverse studies with the introduction of monoclonal antibodies (mAb) against BrUrd that had been incorporated into cellular DNA (1-3). In earlier studies on cell proliferation, much of the information was collected from autoradiographic studies using tritiated thymidine ([3H]dT). Although this technique has yielded valuable data for toxicologic and carcinogenic studies as well as information regarding the proliferation, migration and the time of origin of cells in the central nervous system (CNS) (4, 5) it has several limitations: [a] the possibility of contamination from the radioactive materials, [b] problems of waste disposal, [c] lengthy exposure times for adequate autoradiographic signal and [d] sometimes, a limited resolution. All of these problems can be overcome by using BrUrd instead of [3H]dT. However, use of BrUrd poses a similar problem to that of [3H]dT in that administration for prolonged periods of time is required. Most studies have focused on the intervals between the time of single and multiple injections of [3H]dT or BrUrd and the time of sacrifice. Such manipulations provide insight into the dynamic qualities of developing structures but they allow for a relatively "narrow window" of time within the system. For example, if DNA synthesis does not occur between the time of injection and time of sacrifice, mitotic events can be missed. In an attempt to expand the time for labelling newly synthesized DNA, two different
Corresponding author: D. Maysinger, Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Montreal, PQ, H3G 1Y6, Canada. Phone (514)398-1264, fax (514)398-6690. 0024-3205/94 $6.00 + .0o Copyright © 1993 Pergamon Press Ltd All rights reserved.
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Microencapsulation of Bromodeoxyuridine
Vol. 54, No. 1, 1994
attempts have been made so far: [3H]dT and BrUrd were delivered via osmotic minipumps (6, 7) and via slow-release pellets (6). Both means of administrations were found to be superior to single injection in obtaining stronger labelling signal. This finding prompted the present studies, aimed at developing a more suitable delivery system for BrUrd administration in the brains of animals, with various cortical lesions. Our objective was to establish a prolonged drug release via the microencapsulation of BrUrd. This could eventually provide a means of delivery which involves less cumbersome surgical manipulation than implantation of permanent cannulae.
Materials and Methods Chemicals. BrUrd and monoclonal antibodies against it bound to the single strain of DNA were purchased from Boehringer (Montreal, Canada), polymer (poly-(L-lactide): co- glycolide (PLGA) (70:30), [PLGA], from Polyscience (Warrington, PA, USA). All the chemicals were of analytical grade purity. Solvents, as well as water used were of HPLC quality and filtered through 0.22 #m Millipore type GV filters. Encapsulation Procedure. Phase separation by emulsification and subsequent organic solvent evaporation technique was used: the inner aqueous phase consisted of BrUrd (65 mg) in distilled water (1 ml) and gelatine (100 mg). The oil phase consisted of PLGA (70:30), (500 rag), dissolved in methylene chloride (2.5 ml). The oil phase was gradually poured into the inner water phase under vigorous stirring using ultrasonicator (Vibra Cell, VC 50; Sonics and Materials, Danbury, USA) over 3 minutes to make a microfine w/o emulsion. The emulsion was poured into an aqueous 0.5% polyvinyl alcohol (30 ml), (PVA, Anachemia, Montreal, Canada). The mixture was sonicated (Vibra Cell, VC 50; Sonics and Materials, Danbury, USA) for 3 minutes to make a (w/o)/w emulsion. Methylene chloride was evaporated (2 hours under no reduced pressure; the first 30 minutes by stirring at 30 °C, then left at ambient temperature), and the hardened microspheres were centrifuged, rinsed several times with distilled water and lyophilized. Cortical Lesions. Eight rats were subjected to a unilateral left-sided cortical devascularizing lesion, as previously described (8). Briefly, a bone flap was removed to expose the dura mater over a wide area (0.8 cm x 1.0 cm) of the dorsal brain surface, the dura was opened, and the terminal pia-arachnoid vessels were disrupted with the aid of a surgical needle. Following the lesion, the subcortical blood supply remained intact. Capsules were placed onto the surface of the lesioned cortex. All surgeries and preoperative handling were carried out in accordance with the NIH guidelines for care and use of laboratory animals and the McGill University Animal Care Committee. Preparation of Gelatin Films. Sterile gelatin (7.5 g; Sigma, St. Louis, MO, USA) was dissolved in double-distilled (dd) water (36.5 ml) with gentle heating and ultra pure glycerol (5 g; Bethesda Research Laboratories, MD, USA) was added. BrUrd (10 mg) was incorporated in the gel-film (0.8 x 1.0 cm). The pure crystalline or microencapsulated BrUrd was dissolved/dispersed in 1.5 ml of dd water and added to the gelatin solution with gentle stirring to avoid air bubbles. The warm solution was poured onto a flat glass surface to produce a uniform layer approximately 1 mm thick. After hardening of the gelatin, rectangular-shaped pieces were cut and stored in a sterile container at 4°C until use. Morphometric Analysis of Microspheres. Surface and shape characteristics of microspheres were
Vol. 54, No. 1, 1994
Microencapsulation of Bromodeoxyuridine
29
analyzed by a scanning electron microscope (JSM T-200, Pulp and Paper, Point Claire, Canada). Dry samples were mounted onto stubs using double side adhesive tape and were vacuum coated
2.~ 2OO i
b
150 100 SO 0
1
2
3
4 5 6 7 8 9 1 0
Fig. 1 PLGA microspheres with BrUrd. Panel a: scanning electron micrograph showing shapes and sizes of microspheres. Scale bar = 10 /~m, magnification 2,000 x. Panel b: frequency distribution of 625 spheres analyzed by an image analysis system.
o
a J,
o
B
~ i
o
e
15:
-1.3
ram
16:
-1.8
mm
o
f
~
"*
Fig. 2 Panel a: schematic representations of the rat brain at two different levels from bregma (-1.3 mm and -1.8 mm) with indicated sites of devascularizing cortical lesion and administration of PLGA microspheres with incorporated BrUrd. Panel b: nuclei stained with anti-BrUrd antibodies (Boehringer, Montreal, Canada) raised against BrUrd bound to a single stranded DNA. Immunocytochemical procedure was as described in Methods.
with gold film (approx. 30 ~m thick, engineering ion coater 1B type; 0.1 torr, high voltage 8001500 V and 8mA). The size of the microspheres were determined by image analysis using the M1 system (MCID, M1 Imaging Record Inc). The average diameter + SEM of randomly
30
Microencapsulation of Bromodeoxyuridine
measured microspheres diagram.
Vol. 54, No. 1, 1994
was estimated and plotted as a frequency or cumulative frequency
Immunocytochemistry. Eight rats (2 groups, n = 4 per group) were perfused with 4% paraformaldehyde. Protocol by Boehringer was used. All steps were carried out at the room temperature using PBS containing 0.2% Triton-X 100 (pH 7.4) for washes, dilutions of antibodies and 3,3'-diaminobenzedine tetrahydrochloride (DAB) reaction. Peroxidase-antiperoxidase (PAP; 1:30; Medicorp, Montreal, Canada) was used as a secondary antibody. Relative densities of BrUrd-positive nuclei in the area surrounding the lesion were determined, using a Quantimet image analysis system (Cambridge Instruments, Cambridge, England). In Vitro Release of BrUrd. The weighed amounts of microspheres (5-15 mg) were dispersed in a phosphate buffered saline (PBS). Samples were maintained at 37 0(7 and at the scheduled times between 1 day and 5 weeks, the suspension was centrifuged, release medium removed and content of the drug determined in lyophilized reconstituted aliquots. BrUrd content in the spheres and gels was determined by HPLC. Determination of BrUrd. The HPLC system consisted of a Beckman l l0B solvent pump (Beckman, San Ramon, CA, USA), 7125 Rheodyne injector (Rheodyne, Cotati, CA, USA) with a 20 #1 sample loop, an ABI 783 Programmable Absorbance Detector (ABI Kratos, Ramsey, N J, USA) and a DataJet integrator (Spectra-Physics, San Jose, CA, USA). The chromatographic column used was an Altex Ultrasphere-ODS (25 cm x 4.6 mm I.D., 5 /x) (Beckman, Berkeley, CA, USA). The mobile phase used consisted of 85% sodium phosphate buffer (0.1 M, pH 6.8) and 15% methanol, (v/v). The flow was kept at 1.0 ml/min. The uv absorbance was measured at 277 nm. Reconstituted (with 0.2 ml sodium phosphate buffer, 0.1 M, pH 7.47) lyophilised samples were injected directly into the HPLC and the peak area and height compared with those of injected BrUrd standards. Linear detector response was observed in the BrUrd concentration range from 10 ng/ml (32 nM) to 1.0 mg/ml (3.2 mM). A typical chromatogram showing BrUrd peak at 9.05 rain ( k ' = 1.66) is given in Figure 3. To determine the BrUrd content of prepared microspheres, 0.3 ml of methylene chloride was added to 15 mg of microspheres in an Eppendorf test-tube, and the test-tube shaken on Vortex-shaker for 20 min. BrUrd was reextracted into aqueous phase by addition of 0.2 ml sodium phosphate buffer (0.1 M, pH 7.4). The mixture was vortex-shaken for 10 min, spun at 15,000 x g for 10 min, and the aqueous layer injected directly into HPLC. The reextraction into aqueous layer was repeated up to 6 times, until no more BrUrd was recovered. Statistics. The data were assessed by analysis of variance (ANOVA) and significance between groups was determined by post-hoc Newman-Keul's test. A level of p < 0.05 was considered statistically significant.
Results PLGA microspheres containing BrUrd were prepared, characterized and tested in vitro. Figure la illustrates scanning electron micrographs of spheres with encapsulated BrUrd in biodegradable copolymer PLGA at the magnification 2,000x. Surfaces and shapes are slightly irregular but the sizes are fairly uniform with an average diameter _ SD of 2.47 + 0.59/,m (number of spheres analyzed = 625), Figure lb. Total yield was 54.6%, and the total content of BrUrd was found to be 0.2 %-1.5 % depending on amounts of gelatin co-dissolved with BrUrd in the aqueous phase.
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The lowest content of BrUrd (0.2%) was found in PLGA preparations devoid of gelatin. The highest amount (1.5%) of BrUrd was incorporated in spheres when relatively large amount of gelatin (20% w/w of the PLGA) was co-encapsulated. Variation in internal pH (7.40-11.00) failed to affect BrUrd incorporation under the conditions employed. In vivo experiments showed that BrUrd can be efficiently incorporated into the cell nuclei in the lesioned brain. The intensity of immunostaining was most remarkable in the regions surrounding the devascularizing lesion (50% in comparison with contralateral side) and subventricular zone (Figure 2a,b). Although we did not perform double staining in these experiments, our previous studies revealed intense glial reaction in these regions, suggesting that BrUrd was most likely incorporated in dividing/proliferating glia. The kinetic profiles of two different BrUrd preparations were determined by HPLC (Figure 3). The release of BrUrd from preparations No. 1 and No. 2 was followed for two weeks and 48 days, respectively. In both cases the experimental values are best fitted to both the first order and biphasic first order kinetics. The correlation coefficients (r2), kinetic constants (k, [hl]) and tv2 [h] are summarized in Table I. The kinetic profile of one BrUrd preparation, followed for 48 days, is illustrated in Figure 4. In addition to microsphere preparations we also prepared several types of biodegradable gels. The total contents of BrUrd in these preparations were close to 100%. However, release of BrUrd was considerably faster (4 hours up to 2 days) than from the spheres.
TABLE I Kinetic Parameters for Release of BrUrd from PLGA Microspheres
Preparation No. correlation coefficient, ta kinetic constant, k [h~] t,,~ [h]
first order kinetics 1 (14 days) 2 (48 days) 0.998 -0.0556 12.38
0.998 -0.0564 12.29
biphasic first order kinetics 1 (14 days) 2 (48 days) 0.999 -0.0557 12.12
0.999 -0.0597 11.61
Discussion This study describes the development of a novel delivery system for BrUrd which can be applied into the study of infrequent mitotic events in the brain and in other organs. BrUrd is of particular interest because it can be used as an alternative marker for the examination of the proliferation, migration and time of origin of cells in the CNS. It does not appear to be toxic and it labels comparable number of cells when compared with [3H]dT (9). BrUrd has several advantages over [SH]dT, however, and therefore the development of a delivery system for its chronic administration would be useful. Previously described systems for continuous administration such as permanently installed cannulae and non-biodegradable pellets have provided useful information
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on rates of DNA synthesis in different organs including brain (6, 7, 10). Mitogenic growth factors, such as b FGF, EGF and many others (11) may participate in neurogenesis in the mammalian central nervous system. To conduct such studies, new experimental ways are needed for the administration of BrUrd, trophic factors and/or other DNA markers which will allow their continuous delivery in fairly large quantities. A number of approaches to this problem have been CHANNEL A
INJECT
_
12/28/92
12:12:08
STORED TO BIN
II o ,05
DATA
SAVED
TO B1N ~
2.0
Fig. 3 HPLC chromatogram showing peak of BrUrd at 9.05 rain. BrUrd was released from PLGA microspheres. See details in experimental section.
made, but all suffer from one or another defect. Thus pulse injections seriously jeopardize the possibility of identifying dividing cells outside the "window" during which CNS concentrations of [3H]dT or BrUrd reach maximal concentrations. BrUrd administration from slow releasing pellets, another approach, appears not to be reliable for chronic studies. It was hypothesized by Weghorst et al. (6), that release took place in uneven bursts causing toxic effects in hepatic and nonhepatic cell populations and that there was a possible "fainting" of the signal after four days due to the decrease/dilution in nuclear labelling at later times (7 days) compared to earlier periods (4 days). Finally, the continuous infusion of high concentrations of BrUrd and/or [3H]dT directly into the cerebrospinal fluid over a period of several days certainly enables maintenance of their steady and necessary levels. For example, intracerebroventricular administration of [3H]dT enabled electronmicroscopic demonstration of neurogenesis in the CNS following brain injury (10). In general, however, techniques such as the use of stainless steel cannulae result in considerable mechanical damage to the brain tissue in the vicinity of the needle. Against this background, we made an attempt to develop an alternative, less invasive system for BrUrd delivery into the CNS of animal models with different brain lesions, e.g. cortical, striatal, fimbria-fornix lesions (8, 10, 12) Results from this study show that we can microencapsulate BrUrd or incorporate it in the biodegradable film and place it over the lesioned cortex. Microencapsulated BrUrd can be administered directly onto the site of the lesion and secured with a piece of a sterile gel foam. Alternatively, a biocompatible film with a precise dose of the encapsulated drug can be placed at the same site. Depending on the polymer used, shape and size
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of the film, one can modify the kinetics of the drug release. Gelatin film degrades relatively rapidly. Other polymers or matrix materials, e.g. collagen, could provide longer sustained release of BrUrd. Depending on the conditions of the microencapsulation procedure, we have produced several preparations with different BrUrd contents but similar kinetic profiles. Although we were able to ensure continuous release of relatively high concentrations for 48-50 hours, even longer times might be more suitable for neurogenesis studies. We are currently testing several biodegradable polymers for microencapsulation and film formation. Finally, several HPLC methods have been published for determination of BrUrd in serum and plasma (13-15) as well as in hamster V79 cells (16), either by using uv absorption (13, 14), or fluorescence (15, 16) for detection. Our HPLC method for determination of BrUrd content is very
I
I
I
[
I
T
20
30
40
50
"---
1.3 17
F --
1.i
"o
0.9
0.7 rn 0.5 I
I
0
10
Time
[days]
Fig. 4 Kinetic profile of BrUrd release from PLGA microspheres (Preparation No. 2) followed for 48 days (see details in Methods); first order = solid line, biphasic first order = dashed line.
simple, fast and reliable. It is particularly convenient for testing the amount of BrUrd incorporated in microspheres before using them in "in vivo" experiments because no sample purification is required. In summary, microencapsulated BrUrd could well be a viable alternative to intracerebroventricular administration of BrUrd to CNS in the study of developmental and regenerative changes in the brain following injury and trophic factor therapy.
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Acknowledgements This work was supported by the Canadian Centre of Excellence for Neuronal Repair and Functional Recovery and MRC Canada. Editorial assistance of Dr. T. Keng and photographic reproductions by A. Forster are gratefully acknowledged.
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2. 3. 4.
5. . 7.
8. .
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