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Controlled Release of Interleukin-2 from Chitosan Microspheres
Controlled Release of Interleukin-2 from Chitosan Microspheres ˘ A, CENK ARAL ¨ ZBAS¸ -TURAN, JULIDE AKBUG SUNA O Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Marmara, 81010 Haydarpasa, Istanbul, Turkey
Received 13 November 2000; revised 18 December 2001; accepted 4 January 2002
ABSTRACT: Chitosan microspheres were evaluated for sustained-release of recombinant human interleukin-2 (rIL-2) in this study. In addition, the effects of different formulation factors, such as chitosan and protein concentrations, the volume of sodium sulfate solution, addition technique of rIL-2, and presence of glutaraldehyde during the encapsulation process, on microsphere characteristics were investigated. Chitosan microspheres containing rIL-2 were prepared by using the precipitation technique. The average diameter of microspheres was between 1.11–1.59 mm. Recombinant IL-2 encapsulation efficiency in these micropheres was high (75–98%). Formulation factors had no effect on the microsphere size. Recombinant IL-2 had been released from chitosan microspheres over a period of 3 months. The encapsulated rIL-2 remained biologically active and could be completely recovered from the release medium. Briefly, rIL-2 was released from chitosan microspheres in a sustained manner. The efficacy of rIL-2 loaded chitosan microspheres was studied using two model cells, HeLa and L-strain cell lines. Chitosan microspheres were added to the cells at different concentrations, and the amount of rIL-2 was assayed using the ELISA kit. Cell culture studies indicated that microspheres were uptaken by cells, and rIL-2 was released from the microspheres. Cellular uptake of rIL-2-loaded microspheres was dose dependent. It can be said that chitosan microsphere is a suitable carrier for rIL-2 delivery. ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:1245–1251, 2002
Keywords:
interleukin-2; chitosan microspheres; precipitation technique
INTRODUCTION Human interleukin-2 (hIL-2) is a lymphotropic hormone produced by T-cells, which play a major role in immunological events. It is being investigated for its therapeutic benefits in various cancers and immunotherapies.1–3 Human interleukin-2 has a short half-life in vivo; therefore, frequent high-dose injections of IL-2 are necessary for the antitumor efficacy.4 The primary limitation with the use of high-dose IL-2 therapy Correspondence to: Julide Akbug˘a (Telephone: 90-216-41429-65;Fax:90-216-345-29-52;E-mail:[email protected]) Journal of Pharmaceutical Sciences, Vol. 91, 1245–1251 (2002) ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
is the associated toxicity.5 To achieve a sustained therapeutic level of IL-2, continuous infusion of this protein has been employed,6 but the use of high-dose IL-2 may also result in serious side effects such as coma or dehydration. An approach to overcome these side effects is to use a controlled release system. Therefore, several controlled release systems have been described for IL-2.7–12 In earlier studies, the poloxamer 407 gel matrix system7,10 delivered IL-2 for up to 2 days; gelatinadded IL-2 formulations or IL-2-loaded gelatin microspheres were used to prolong the serum levels of this protein and to obtain a sustainedrelease effect.13 Liposomes9,14,15 and supramolecular biovectors16 were reported for enhancement of IL-2 bioactivity.
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Moreover, biodegradable poly(DL-lactide-coglycolide)8 and poly(lactic acid)3 microspheres were evaluated for the controlled release of IL-2 and its modified forms,8 but with the poly(DLlactide-co-glycolide) microspheres, only 1–3% of the loaded IL-2 could be recovered form the in vitro release medium. Liu et al.,12 prepared alginate/chitosan porous microspheres for the controlled release of IL-2, and indicated that IL-2 release from these microspheres occurred over 5 days. On the other hand, chitosan is a nontoxic biodegradable, biocompatible polycationic polymer. It has been used as a matrix for controlled release microsphere formulations.17,18 Chitosan has also biostimulating activity on the immune system.19 In consideration of the problems associated with IL-2 therapy, chitosan microspheres were developed for sustained-release of IL-2. In addition, the effect of different formulation factors such as chitosan and protein concentrations, the volume of sodium sulfate solution, addition technique of IL-2, and use of glutaraldehyde during the preparation, on microsphere characteristics was investigated. Uptake of rIL-2-loaded microspheres into the cells was also studied.
EXPERIMENTAL Materials Human rIL-2 (200 I U/mL in PBS, Roche, USA), chitosan (Mr: ca. 400 kDa, viscosity: ca. 200 mPa in
Table 1.
Code C-9 C-10 C-11 C-13 C-15 C-16 C-17 C-18 C-19 A-4c
1% acetic acid at 208C, Fluka, Germany), sodium sulfate (Carlo Erba, Italy), minimal essential medium (MEM), L-glutamine, and fetal bovine serum (FBS) were from Gibco, USA. Other substances were of pharmaceutical or molecular grades.
Preparation of rIL-2-Loaded Microspheres Recombinant interleukin-2-loaded chitosan microspheres were prepared according to Berthold et al.20 with some modifications. Recombinant IL-2 was added to the sodium sulfate solution (20% w/v) immediately before the dropping. Sodium sulfate solution containing rIL-2 was dropped into the acidic chitosan solution (maintained at þ 48C) and stirred (Ika-Werk, Germany) at 500 rpm for 1 h. The microspheres were separated by centrifugation (Sigma, Germany) for 15 min at 10,000 rpm, freeze dried (Lyovac, Leybold Heraeus, Germany), and stored at þ 48C. Different formulations were prepared study the effect of various formulation factors on microsphere properties (Table 1). Protein loading of chitosan microspheres was made by using two different methods; rIL-2 was added to the sodium sulfate solution during the preparation, or it was adsorbed onto the surface of empty microspheres. The shape and size of the microspheres were characterized by optical microscopy (Olympus BX, Japan).
Codes, Formulations and Properties of rIL-2 Loaded Chitosan Microspheres
Chitosan Conc. (%)
Volume of Na2SO4 Solution (mL)
Volume of rIL-2 solution (mL)
Glutaraldehydea (mL)
Efficiency (%)
Particle Size (mm SD)
0.25 0.50 0.25 0.50 0.25 0.25 0.50 0.70 0.50
20 20 20 50 20 50 50 50 50
500 200 500 500 1000 1000 1000 1000 1000
þb þ þ þ
92.17 74.27 94.96 90.97 95.02 97.61 94.07 90.45 96.67 98.63
1.11 0.2 1.13 0.3 1.25 0.6 1.44 0.4 1.48 0.5 1.57 0.5 1.54 0.4 1.59 0.5 1.58 0.5
a
1 mL gluataraldehyde solution (12.5%, v/v) was used except C-9. 2 mL glutaraldehyde solution. Adsorption of rIL-2 on the empty formulation of C-16.
b c
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Determination of Protein Content The amount of rIL-2 entrapped in microspheres was determined by measuring the difference between the total amount of protein incorporated in the microparticle preparation medium and the amount of nonentrapped protein remaining in the aqueous medium after the encapsulation process. Protein content in the supernatant was spectrophotometrically determined by Bradford’s method.21 Protein Integrity Integrity of the encapsulated protein after the release study was analyzed by SDS-PAGE. Released samples were removed and lyophilized for concentrating the protein. Then the protein samples were dissolved in PBS containing 0.1% bromophenol blue and 60% glycerol and loaded into SDS-PAGE. Electrophoresis was performed at 100 V, 50 mA by using a vertical gel electrophoresis system (Sigma, USA). After running, the gel was stained with Comassie blue R-250 solution (Merck, Germany) to visualize the protein, and the protein was determined using a gel image system (KODAK, DC290, Electrophoresis Documentation and Analysis System, USA). In Vitro Release Studies Protein release from chitosan microspheres was determined as follows; 10 mg of the chitosan microspheres were placed in 2 mL of the PBS (BP 1998) and shaken in a water bath at 37 0.58C. The samples were periodically withdrawn, and fresh medium was added to the microspheres. The amount of total protein released was spectrophotometrically (Shimadzu 2100, Japan) determined according to Bradford21 at 595 nm. The rIL-2 concentration in the samples was also determined using an IL-2 bioassay kit as mentioned below. The mean of three determinations is given. Recombinant IL-2 Assay The concentration of rIL-2 was determined using an ELISA kit (Roche, USA). In brief, the rIL-2 assay was a solid-phase enzyme immunoassay, which employed a multiple antibody sandwich principle. Recombinant IL-2 was simultaneously bound by the biotin-labeled capture antibody and the peroxidase-conjugated detection
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antibody. This complex bound via the biotinlabeled antibody to the streptavidin-coated 96wells microtiter plate. Following the washing step, the peroxidase bound in the complex was developed by the substrate tetramethylbenzidine, the reaction was stopped by acidification, and rIL2 amount was measured at 450 nm in the reader (Bio-Tek, USA). Uptake Studies in Cell Culture Cell culture studies were carried out to determine the penetration of microspheres into cells. HeLa (ATCC CCL-2) and L-strain cell lines (ATCC CCL1) were generously supplied by Dr. Turan from Istanbul University. Cells were cultured in MEM supplemented with 0.03% L-glutamine, fetal bovine serum, and 0.1% antibiotic solutions (penicillin, streptomycin, and amphotericin) in humidified atmosphere (5% CO2, 95% air) at 378C (Heto-Holten, Denmark). The cells, which formed a single layer, were trypsinized for 5 min, collected by centrifugation at 3000 rpm for 3 min (Hettich, Germany), and were resuspended in the medium. The cells (5 104 cells/well) were seeded in 24-well plates and incubated for 18–24 h. In this experiment, C16 and C18 were used (Table 1). Microspheres (100 and 500 mg) were suspended in the medium and added to the cells and incubated for 24 or 48 h. Then the cells were washed with PBS to remove all the unadsorbed microspheres from the medium and fresh medium was added and incubated for additional 6 hours. To detach the cells, medium was removed, and the cells were washed with ice-cold PBS. The cells were collected by centrifugation and then were lysed by three freezing–thawing cycles. After centrifugation at 5000 rpm, the supernatant was removed, and IL-2 levels were determined by ELISA as described above. Statistical Analysis Results were compared for significance using the Mann-Whitney rank sum test. A probability p < 0.05 was considered significant.
RESULTS AND DISCUSSION Characterization of Microspheres Biological response modifiers, such as IL-2, have therapeutic potential for use in human JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002
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malignancies especially when combined with various cellular therapies. But there are some problems related to IL-2 therapy. Several attempts have been made to sustain the blood levels of rIL-2 for increasing the therapeutic efficacy. By this preparation method, spherical microspheres whose diameters ranging from 1.11 to 1.59 mm were obtained (Table 1). The microspheres had a narrow size distribution in contrast to the earlier reports about IL-2-loaded alginate microspheres.12 Higher amounts of rIL-2 could be encapsulated into the chitosan microspheres (between 75 and 98%). Excellent encapsulation capacity of chitosan nanoparticles for the association of proteins was previously reported by Calvo et al.22 Effect of Formulation Factors on Size and Encapsulation Efficiency of Recombinant IL-2 Microspheres No information is available on the formulation properties of rIL-2 microspheres. In this study different parameters such as amount and addition method of protein, concentration of chitosan, volume of sodium sulfate solution, and incorporation or not of the glutaraldehyde were investigated, as shown in Table 1. Ten microsphere formulations were made for evaluation. Although using aldehydes during the microencapsulation process can be inadequate for proteins, in this study, the effect of glutaraldehyde was investigated and no adverse effect was observed on the integrity of rIL-2 (data not shown). Recombinant IL-2 was very efficiently encapsulated in the chitosan microspheres. Encapsulation efficiency of rIL-2 was not affected by the chitosan concentration (C16–C18) and the volume of sodium sulfate solution (C15, C16). Moreover, glutaraldehyde concentration did not seem to affect the protein encapsulation efficiency (C17, C19), as reported previously.23 But with an increase in initial protein amount, a corresponding increase in the encapsulated amount of rIL-2 was found (C13 and C17). As summarized in Table 1, the size of rIL-2-loaded microspheres did not change with the formulation variables (p > 0.05). Effect of Formulation Factors on rIL-2 Release In vitro release profiles of rIL-2 microspheres are given in Figures 1–5. In general, the rIL-2 release profiles were biphasic, characterized by an initial protein burst followed by slow release (Fig. 1). In JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002
Figure 1. Effect of chitosan concentrations on rIL-2 release from microspheres. Inset: Release profiles of C16 and C17 according to bioassay of rIL-2.
our experiments, burst effect was observed on the release patterns of many formulations except A4. This result is consistent with the release data of rIL-2-loaded chitosan-treated alginate porous microspheres.12 But, on the first day, a very small amount of rIL-2 release was reported by Hora et al.8 Liu et al.12 have explained this difference with the porous structure of alginate microspheres. In their study, IL-2 release from the chitosan-treated alginate microspheres occurred over 5 days; but in our experiments protein release had continued for 200 days. Hora et al.8 have reported that only 3% of encapsulated IL-2 was released from poly(lactideco-glycolide) microspheres but that coencapsulation of IL-2 and human serum albumin (HSA) in the same carrier could increase the recovery rate of IL-2 to about 10%. This might be due to the porous structure of microspheres after the addition of HSA.8 Among the different formulation parameters, chitosan concentrations (C16–C18) and addition of glutaraldehyde (C17, C19) had no effect on the protein release characteristics of chitosan microspheres (p > 0.05) (Figs. 1 and 2). But the rIL-2 amount of the microspheres changed the in vitro release characteristic of microspheres. As the protein concentration increased, in vitro release significantly decreased (Fig. 3) (p ¼ 0.001) (C10, C13, and C17). On the other hand, the addition technique of rIL-2 significantly affected the release properties
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Figure 2. Effect of the amount (C9; C11) and addition (C17; C19) of glutaraldehyde, on protein release from chitosan microspheres.
Figure 4. Effect of rIL-2 addition technique (A-4) on the release properties of the protein from chitosan microspheres.
of microspheres ( p ¼ 0.0045). As mentioned in the Experimental section, rIL-2 was adsorbed onto the manufactured empty microspheres or it was added to sodium sulfate solution during the encapsulation process. Adsorption of rIL-2 onto the empty chitosan microspheres caused a remarkable delay on the release of protein (A4) (Fig. 4). Moreover, the volume of sodium sulfate solution used in the microsphere preparations af-
fected the release pattern of protein from chitosan microspheres ( p < 0.0001) (Fig. 5). Recombinant IL-2 was released rapidly from chitosan microspheres when larger volume of sodium sulfate solution (C15 and C16) was used during the preparation. As seen in Figure 2, rIL-2 release had also changed with the volume of glutaraldehyde solution ( p ¼ 0.001) (C9 and C11).
Figure 3. Effect of rIL-2 concentration on protein release from chitosan microspheres (codes are given in Table 1).
Figure 5. Effect of the volume of sodium sulfate solution on rIL-2 release characteristics of the microspheres. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002
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Protein Integrity SDS-polyacrylamide gel electrophoresis of released protein from rIL-2-loaded chitosan microspheres showed that rIL-2 remained biologically unchanged (Fig. 6). As indicated in this figure, the encapsulation process did not affect the structural integrity of rIL-2. Uptake Studies in Cell Culture Previously, the use of PLA microspheres to deliver rIL-2 to the tumor environment was suggested as a safe and simple alternative to gene therapy protocols in the treatment of cancer.3 On the other hand, the cell permeability increasing effect of chitosan has been shown in various in vivo models.24,25 In this part of this study, the efficacy and cell internalization of rIL-2-loaded chitosan microspheres (C16 and C18) were studied using two cell lines. Microsphere suspension (0.1 and 0.5 mg) was added into the medium, then the amount of rIL-2 into the cells was assayed. As seen in Figure 7, a high amount of rIL-2 was measured in both cells; however, the rIL-2 uptake amount was different between the cells. A higher rIL-2 amount was found when a high concentration of microspheres (0.5 mg/mL) was added to the cells. As seen in this figure, between the studied cells the rIL-2 amount was higher in the L-strain cells than HeLa. The effect of chitosan content of the microspheres on the cellular uptake was not seen clearly (C16 and C18).
Figure 6. SDS-polyacrylamide gel electrophoresis of rIL-2. Lane A: bovine serum albumin standard (66.2 kDa); lane B: ovalbumin standard (45.0 kDa); lane C: rIL-2 standard (13.0 kDa); lanes D–G: released samples of rIL-2 from the microspheres. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002
Figure 7. Cellular uptake of rIL-2-loaded chitosan microspheres.
In conclusion, chitosan microspheres are a suitable carrier for controlled release of rIL-2. They are also useful systems for the transport of rIL-2 into the cells. Some formulation factors must be taken into consideration during the microsphere production. Additional experiments are currently being performed to evaluate in vivo properties of these microspheres.
REFERENCES 1. Rosenberg SA, Spiess P, Lafreniere R. 1986. A new approach to the adaptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 233: 1318–1321. 2. Ettinghausen SE, Rosenberg SA. 1986. Immunotherapies of murine sarcomas using lymphokine activated killer cells: Optimization of the schedule and route of administration of recombinant interleukin-2. Cancer Res 46:2784–2792. 3. Eg˘ilmez NK, Jong YS, Iwanumo Y, Jacob JS, Santos CA, Chen FA, Mathiowitz E, Bankert RB. 1998. Cytokine immunotherapy of cancer with controlled release biodegredable microspheres in a human tumor xenograft/SCID mouse model. Cancer Immunol Immunother 46:21–24. 4. Lotze MT, Frana LW, Sharrow SO, Robb RJ, Rosenberg SA. 1985. In vivo administration of purified human interleukin-2. Half life and immunologic effects of Jurkat cell-line derived interleukin-2. J Immunol 134:157–166. 5. Richards JM. 1989. Therapeutic uses of interleukin-2 and lymphokine-activated killer (LAK) cells. Blood Rev 3:110–119. 6. Cheever MA, Thompson JA, Kern DE, Greenberg PD. 1985. Interleukin-2 (IL-2) administration in vivo: Influence of IL-2 route and timing on T cell growth. J Immunol 134:3895–3900. 7. Morikawa N, Okada F, Hosokawa M, Kobayashi H. 1987. Enhancement of therapeutic effects of recombinant interleukin-2 on a transplantable rat fibrosarcoma by the use of a sustained release vehicle pluronic gel. Cancer Res 47:37–41.
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8. Hora MS, Rana RK, Nunberg JH, Tice TR, Gilley RM, Hudson ME. 1990. Controlled release of interleukin-2 from biodegredable microspheres. Biotechnology 8:755–758. 9. Joffret ML, Morgeaux S, Leclerc C, Oth D, Zanetti C, Sureau P, Perrin D. 1990. Enhancement of interleukin-2 activity by liposomes. Vaccine 8:385–389. 10. Wang PL, Johnston TP. 1995. Sustained release interleukin-2 following intramuscular injection in rats. Int J Pharm 113:73–81. 11. Ohno T, Liu SQ, Liu LS. 1996. Prolonged activity of interleukin-2 conjugated to macromolecular polysaccharides. Proceed Int Symp Control Rel Bioact Mater 23:873–874. 12. Liu LS, Liu SQ, Ng SY, Froix M, Ohno T, Heller J. 1997. Controlled release of interleukin-2 for tumor immunotherapy using alginate/chitosan porous microspheres. J Control Rel 43:65–74. 13. Sills AK, Zhao Z, vanWessen K, Crongeyer J, Tyler B, Ewend MG, Leong K, Brem H. 1996. Intracranial delivery of interleukin-2 by microspheres in the treatment of brain tumours. Proceed Int Symp Control Rel Bioact Mater 23:855–856. 14. Storm G, Koppenhagen FJ, Steeranberg PA, den Otter W, Crommelin DJA. 1996. Liposomal delivery of interleukin-2 in locoregional anticancer immunotherapy. Proceed Intern Sym Control Rel Bioactive Mater 23:95–96. 15. Khanna C, Hasz DE, Klausner JS, Anderson PM. 1996. Aerosol delivery of interleukin-2 liposomes is non-toxic and biologically effective: Canine studies. Clin Cancer Res 2(4):721–734. 16. Castignolles N, Betbeder D, Ioualalen K, Merten O, Leclerc C, Samain D, Perrin P. 1994. Stabilization and enhancement of interleukin-2 in vitro bioactivity by new carriers: Supramolecular biovectors. Vaccine 15:1413–1418.
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17. Akbug˘a J, Durmaz G. 1994. Preparation and evaluation of cross-linked chitosan microspheres containing furosemid. Int J Pharm III:217–222. 18. Akbug˘a J, Bergis¸adi N. 1999. Effect of formulation variables on cis-platin loaded chitosan microsphere properties. J Microencapsulat 16:697–703. 19. Suzuki K, Tokoro A, Okawa Y, Suzuki S, Suzuki M. 1986. Effect of N-acetyl-chito-oligosaccharides on activation of phagocytes. Microbiol Immunol 30: 777–787. 20. Berthold A, Cremer K, Kreuter J. 1996. Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model for anti-inflammatory drugs. J Control Rel 39:17–25. 21. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the priciple of protein-dye binding. Anal Biochem 72:248–254. 22. Calvo P, Remunan-Lopez C, Vila-Jato JL, Alonso MJ. 1997. Chitosan and chitosan/ethylene oxidepropylene oxide block copolymer nanoparticles as novel carriers for proteins and vaccines. Pharm Res 14:1431–1436. 23. Genta I, Constantini M, Montanari L. 1996. Chitosan microspheres for nasal delivery: Effect of cross-linking agents on release characteristics. Proceed Intern Symp Control Rel Bioact Mater 23:377. 24. Illum L, Farraj NF, Davis SS. 1994. Chitosan as a novel nasal delivery system for peptide drugs. Pharm Res 11:1186–1189. 25. Mooren FC, Berthold A, Domschko W, Kreuter J. 1998. Influence of chitosan microspheres on the transport of prednisolone sodium phosphate across HT-29 cell monolayers. Pharm Res 15: 58–65.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002
Received 13 November 2000; revised 18 December 2001; accepted 4 January 2002
ABSTRACT: Chitosan microspheres were evaluated for sustained-release of recombinant human interleukin-2 (rIL-2) in this study. In addition, the effects of different formulation factors, such as chitosan and protein concentrations, the volume of sodium sulfate solution, addition technique of rIL-2, and presence of glutaraldehyde during the encapsulation process, on microsphere characteristics were investigated. Chitosan microspheres containing rIL-2 were prepared by using the precipitation technique. The average diameter of microspheres was between 1.11–1.59 mm. Recombinant IL-2 encapsulation efficiency in these micropheres was high (75–98%). Formulation factors had no effect on the microsphere size. Recombinant IL-2 had been released from chitosan microspheres over a period of 3 months. The encapsulated rIL-2 remained biologically active and could be completely recovered from the release medium. Briefly, rIL-2 was released from chitosan microspheres in a sustained manner. The efficacy of rIL-2 loaded chitosan microspheres was studied using two model cells, HeLa and L-strain cell lines. Chitosan microspheres were added to the cells at different concentrations, and the amount of rIL-2 was assayed using the ELISA kit. Cell culture studies indicated that microspheres were uptaken by cells, and rIL-2 was released from the microspheres. Cellular uptake of rIL-2-loaded microspheres was dose dependent. It can be said that chitosan microsphere is a suitable carrier for rIL-2 delivery. ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:1245–1251, 2002
Keywords:
interleukin-2; chitosan microspheres; precipitation technique
INTRODUCTION Human interleukin-2 (hIL-2) is a lymphotropic hormone produced by T-cells, which play a major role in immunological events. It is being investigated for its therapeutic benefits in various cancers and immunotherapies.1–3 Human interleukin-2 has a short half-life in vivo; therefore, frequent high-dose injections of IL-2 are necessary for the antitumor efficacy.4 The primary limitation with the use of high-dose IL-2 therapy Correspondence to: Julide Akbug˘a (Telephone: 90-216-41429-65;Fax:90-216-345-29-52;E-mail:[email protected]) Journal of Pharmaceutical Sciences, Vol. 91, 1245–1251 (2002) ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
is the associated toxicity.5 To achieve a sustained therapeutic level of IL-2, continuous infusion of this protein has been employed,6 but the use of high-dose IL-2 may also result in serious side effects such as coma or dehydration. An approach to overcome these side effects is to use a controlled release system. Therefore, several controlled release systems have been described for IL-2.7–12 In earlier studies, the poloxamer 407 gel matrix system7,10 delivered IL-2 for up to 2 days; gelatinadded IL-2 formulations or IL-2-loaded gelatin microspheres were used to prolong the serum levels of this protein and to obtain a sustainedrelease effect.13 Liposomes9,14,15 and supramolecular biovectors16 were reported for enhancement of IL-2 bioactivity.
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Moreover, biodegradable poly(DL-lactide-coglycolide)8 and poly(lactic acid)3 microspheres were evaluated for the controlled release of IL-2 and its modified forms,8 but with the poly(DLlactide-co-glycolide) microspheres, only 1–3% of the loaded IL-2 could be recovered form the in vitro release medium. Liu et al.,12 prepared alginate/chitosan porous microspheres for the controlled release of IL-2, and indicated that IL-2 release from these microspheres occurred over 5 days. On the other hand, chitosan is a nontoxic biodegradable, biocompatible polycationic polymer. It has been used as a matrix for controlled release microsphere formulations.17,18 Chitosan has also biostimulating activity on the immune system.19 In consideration of the problems associated with IL-2 therapy, chitosan microspheres were developed for sustained-release of IL-2. In addition, the effect of different formulation factors such as chitosan and protein concentrations, the volume of sodium sulfate solution, addition technique of IL-2, and use of glutaraldehyde during the preparation, on microsphere characteristics was investigated. Uptake of rIL-2-loaded microspheres into the cells was also studied.
EXPERIMENTAL Materials Human rIL-2 (200 I U/mL in PBS, Roche, USA), chitosan (Mr: ca. 400 kDa, viscosity: ca. 200 mPa in
Table 1.
Code C-9 C-10 C-11 C-13 C-15 C-16 C-17 C-18 C-19 A-4c
1% acetic acid at 208C, Fluka, Germany), sodium sulfate (Carlo Erba, Italy), minimal essential medium (MEM), L-glutamine, and fetal bovine serum (FBS) were from Gibco, USA. Other substances were of pharmaceutical or molecular grades.
Preparation of rIL-2-Loaded Microspheres Recombinant interleukin-2-loaded chitosan microspheres were prepared according to Berthold et al.20 with some modifications. Recombinant IL-2 was added to the sodium sulfate solution (20% w/v) immediately before the dropping. Sodium sulfate solution containing rIL-2 was dropped into the acidic chitosan solution (maintained at þ 48C) and stirred (Ika-Werk, Germany) at 500 rpm for 1 h. The microspheres were separated by centrifugation (Sigma, Germany) for 15 min at 10,000 rpm, freeze dried (Lyovac, Leybold Heraeus, Germany), and stored at þ 48C. Different formulations were prepared study the effect of various formulation factors on microsphere properties (Table 1). Protein loading of chitosan microspheres was made by using two different methods; rIL-2 was added to the sodium sulfate solution during the preparation, or it was adsorbed onto the surface of empty microspheres. The shape and size of the microspheres were characterized by optical microscopy (Olympus BX, Japan).
Codes, Formulations and Properties of rIL-2 Loaded Chitosan Microspheres
Chitosan Conc. (%)
Volume of Na2SO4 Solution (mL)
Volume of rIL-2 solution (mL)
Glutaraldehydea (mL)
Efficiency (%)
Particle Size (mm SD)
0.25 0.50 0.25 0.50 0.25 0.25 0.50 0.70 0.50
20 20 20 50 20 50 50 50 50
500 200 500 500 1000 1000 1000 1000 1000
þb þ þ þ
92.17 74.27 94.96 90.97 95.02 97.61 94.07 90.45 96.67 98.63
1.11 0.2 1.13 0.3 1.25 0.6 1.44 0.4 1.48 0.5 1.57 0.5 1.54 0.4 1.59 0.5 1.58 0.5
a
1 mL gluataraldehyde solution (12.5%, v/v) was used except C-9. 2 mL glutaraldehyde solution. Adsorption of rIL-2 on the empty formulation of C-16.
b c
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Determination of Protein Content The amount of rIL-2 entrapped in microspheres was determined by measuring the difference between the total amount of protein incorporated in the microparticle preparation medium and the amount of nonentrapped protein remaining in the aqueous medium after the encapsulation process. Protein content in the supernatant was spectrophotometrically determined by Bradford’s method.21 Protein Integrity Integrity of the encapsulated protein after the release study was analyzed by SDS-PAGE. Released samples were removed and lyophilized for concentrating the protein. Then the protein samples were dissolved in PBS containing 0.1% bromophenol blue and 60% glycerol and loaded into SDS-PAGE. Electrophoresis was performed at 100 V, 50 mA by using a vertical gel electrophoresis system (Sigma, USA). After running, the gel was stained with Comassie blue R-250 solution (Merck, Germany) to visualize the protein, and the protein was determined using a gel image system (KODAK, DC290, Electrophoresis Documentation and Analysis System, USA). In Vitro Release Studies Protein release from chitosan microspheres was determined as follows; 10 mg of the chitosan microspheres were placed in 2 mL of the PBS (BP 1998) and shaken in a water bath at 37 0.58C. The samples were periodically withdrawn, and fresh medium was added to the microspheres. The amount of total protein released was spectrophotometrically (Shimadzu 2100, Japan) determined according to Bradford21 at 595 nm. The rIL-2 concentration in the samples was also determined using an IL-2 bioassay kit as mentioned below. The mean of three determinations is given. Recombinant IL-2 Assay The concentration of rIL-2 was determined using an ELISA kit (Roche, USA). In brief, the rIL-2 assay was a solid-phase enzyme immunoassay, which employed a multiple antibody sandwich principle. Recombinant IL-2 was simultaneously bound by the biotin-labeled capture antibody and the peroxidase-conjugated detection
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antibody. This complex bound via the biotinlabeled antibody to the streptavidin-coated 96wells microtiter plate. Following the washing step, the peroxidase bound in the complex was developed by the substrate tetramethylbenzidine, the reaction was stopped by acidification, and rIL2 amount was measured at 450 nm in the reader (Bio-Tek, USA). Uptake Studies in Cell Culture Cell culture studies were carried out to determine the penetration of microspheres into cells. HeLa (ATCC CCL-2) and L-strain cell lines (ATCC CCL1) were generously supplied by Dr. Turan from Istanbul University. Cells were cultured in MEM supplemented with 0.03% L-glutamine, fetal bovine serum, and 0.1% antibiotic solutions (penicillin, streptomycin, and amphotericin) in humidified atmosphere (5% CO2, 95% air) at 378C (Heto-Holten, Denmark). The cells, which formed a single layer, were trypsinized for 5 min, collected by centrifugation at 3000 rpm for 3 min (Hettich, Germany), and were resuspended in the medium. The cells (5 104 cells/well) were seeded in 24-well plates and incubated for 18–24 h. In this experiment, C16 and C18 were used (Table 1). Microspheres (100 and 500 mg) were suspended in the medium and added to the cells and incubated for 24 or 48 h. Then the cells were washed with PBS to remove all the unadsorbed microspheres from the medium and fresh medium was added and incubated for additional 6 hours. To detach the cells, medium was removed, and the cells were washed with ice-cold PBS. The cells were collected by centrifugation and then were lysed by three freezing–thawing cycles. After centrifugation at 5000 rpm, the supernatant was removed, and IL-2 levels were determined by ELISA as described above. Statistical Analysis Results were compared for significance using the Mann-Whitney rank sum test. A probability p < 0.05 was considered significant.
RESULTS AND DISCUSSION Characterization of Microspheres Biological response modifiers, such as IL-2, have therapeutic potential for use in human JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002
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malignancies especially when combined with various cellular therapies. But there are some problems related to IL-2 therapy. Several attempts have been made to sustain the blood levels of rIL-2 for increasing the therapeutic efficacy. By this preparation method, spherical microspheres whose diameters ranging from 1.11 to 1.59 mm were obtained (Table 1). The microspheres had a narrow size distribution in contrast to the earlier reports about IL-2-loaded alginate microspheres.12 Higher amounts of rIL-2 could be encapsulated into the chitosan microspheres (between 75 and 98%). Excellent encapsulation capacity of chitosan nanoparticles for the association of proteins was previously reported by Calvo et al.22 Effect of Formulation Factors on Size and Encapsulation Efficiency of Recombinant IL-2 Microspheres No information is available on the formulation properties of rIL-2 microspheres. In this study different parameters such as amount and addition method of protein, concentration of chitosan, volume of sodium sulfate solution, and incorporation or not of the glutaraldehyde were investigated, as shown in Table 1. Ten microsphere formulations were made for evaluation. Although using aldehydes during the microencapsulation process can be inadequate for proteins, in this study, the effect of glutaraldehyde was investigated and no adverse effect was observed on the integrity of rIL-2 (data not shown). Recombinant IL-2 was very efficiently encapsulated in the chitosan microspheres. Encapsulation efficiency of rIL-2 was not affected by the chitosan concentration (C16–C18) and the volume of sodium sulfate solution (C15, C16). Moreover, glutaraldehyde concentration did not seem to affect the protein encapsulation efficiency (C17, C19), as reported previously.23 But with an increase in initial protein amount, a corresponding increase in the encapsulated amount of rIL-2 was found (C13 and C17). As summarized in Table 1, the size of rIL-2-loaded microspheres did not change with the formulation variables (p > 0.05). Effect of Formulation Factors on rIL-2 Release In vitro release profiles of rIL-2 microspheres are given in Figures 1–5. In general, the rIL-2 release profiles were biphasic, characterized by an initial protein burst followed by slow release (Fig. 1). In JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002
Figure 1. Effect of chitosan concentrations on rIL-2 release from microspheres. Inset: Release profiles of C16 and C17 according to bioassay of rIL-2.
our experiments, burst effect was observed on the release patterns of many formulations except A4. This result is consistent with the release data of rIL-2-loaded chitosan-treated alginate porous microspheres.12 But, on the first day, a very small amount of rIL-2 release was reported by Hora et al.8 Liu et al.12 have explained this difference with the porous structure of alginate microspheres. In their study, IL-2 release from the chitosan-treated alginate microspheres occurred over 5 days; but in our experiments protein release had continued for 200 days. Hora et al.8 have reported that only 3% of encapsulated IL-2 was released from poly(lactideco-glycolide) microspheres but that coencapsulation of IL-2 and human serum albumin (HSA) in the same carrier could increase the recovery rate of IL-2 to about 10%. This might be due to the porous structure of microspheres after the addition of HSA.8 Among the different formulation parameters, chitosan concentrations (C16–C18) and addition of glutaraldehyde (C17, C19) had no effect on the protein release characteristics of chitosan microspheres (p > 0.05) (Figs. 1 and 2). But the rIL-2 amount of the microspheres changed the in vitro release characteristic of microspheres. As the protein concentration increased, in vitro release significantly decreased (Fig. 3) (p ¼ 0.001) (C10, C13, and C17). On the other hand, the addition technique of rIL-2 significantly affected the release properties
CONTROLLED RELEASE OF IL-2 FROM CHITOSAN MICROSPHERES
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Figure 2. Effect of the amount (C9; C11) and addition (C17; C19) of glutaraldehyde, on protein release from chitosan microspheres.
Figure 4. Effect of rIL-2 addition technique (A-4) on the release properties of the protein from chitosan microspheres.
of microspheres ( p ¼ 0.0045). As mentioned in the Experimental section, rIL-2 was adsorbed onto the manufactured empty microspheres or it was added to sodium sulfate solution during the encapsulation process. Adsorption of rIL-2 onto the empty chitosan microspheres caused a remarkable delay on the release of protein (A4) (Fig. 4). Moreover, the volume of sodium sulfate solution used in the microsphere preparations af-
fected the release pattern of protein from chitosan microspheres ( p < 0.0001) (Fig. 5). Recombinant IL-2 was released rapidly from chitosan microspheres when larger volume of sodium sulfate solution (C15 and C16) was used during the preparation. As seen in Figure 2, rIL-2 release had also changed with the volume of glutaraldehyde solution ( p ¼ 0.001) (C9 and C11).
Figure 3. Effect of rIL-2 concentration on protein release from chitosan microspheres (codes are given in Table 1).
Figure 5. Effect of the volume of sodium sulfate solution on rIL-2 release characteristics of the microspheres. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002
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Protein Integrity SDS-polyacrylamide gel electrophoresis of released protein from rIL-2-loaded chitosan microspheres showed that rIL-2 remained biologically unchanged (Fig. 6). As indicated in this figure, the encapsulation process did not affect the structural integrity of rIL-2. Uptake Studies in Cell Culture Previously, the use of PLA microspheres to deliver rIL-2 to the tumor environment was suggested as a safe and simple alternative to gene therapy protocols in the treatment of cancer.3 On the other hand, the cell permeability increasing effect of chitosan has been shown in various in vivo models.24,25 In this part of this study, the efficacy and cell internalization of rIL-2-loaded chitosan microspheres (C16 and C18) were studied using two cell lines. Microsphere suspension (0.1 and 0.5 mg) was added into the medium, then the amount of rIL-2 into the cells was assayed. As seen in Figure 7, a high amount of rIL-2 was measured in both cells; however, the rIL-2 uptake amount was different between the cells. A higher rIL-2 amount was found when a high concentration of microspheres (0.5 mg/mL) was added to the cells. As seen in this figure, between the studied cells the rIL-2 amount was higher in the L-strain cells than HeLa. The effect of chitosan content of the microspheres on the cellular uptake was not seen clearly (C16 and C18).
Figure 6. SDS-polyacrylamide gel electrophoresis of rIL-2. Lane A: bovine serum albumin standard (66.2 kDa); lane B: ovalbumin standard (45.0 kDa); lane C: rIL-2 standard (13.0 kDa); lanes D–G: released samples of rIL-2 from the microspheres. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 5, MAY 2002
Figure 7. Cellular uptake of rIL-2-loaded chitosan microspheres.
In conclusion, chitosan microspheres are a suitable carrier for controlled release of rIL-2. They are also useful systems for the transport of rIL-2 into the cells. Some formulation factors must be taken into consideration during the microsphere production. Additional experiments are currently being performed to evaluate in vivo properties of these microspheres.
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