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High efficient encapsulation of plasmid DNA in PLGA microparticles by organic phase self-emulsification
J. Biochem. Biophys. Methods 52 (2002) 169 – 178 www.elsevier.com/locate/jbbm
High efficient encapsulation of plasmid DNA in PLGA microparticles by organic phase self-emulsification F.F. Zhuang, R. Liang, C.T. Zou, H. Ma, C.X. Zheng, M.X. Duan * Department of Biological Science and Biotechnology, Tsinghua University, 100084 Beijing, PR China Received 8 February 2002; received in revised form 27 May 2002; accepted 2 August 2002
Abstract To overcome the drawbacks of encapsulating plasmid DNA (pDNA) in poly (D,L-lactic-coglycolic acid) (PLGA) by water-in-oil-in-water double-emulsion solvent-evaporation method, we have developed a novel procedure for encapsulating pDNA in PLGA microparticles called DNA organic phase self-emulsification (DOPSM). This method was based on both the extraction plasmid DNA from aqueous phase into organic phase and the spontaneous emulsification DNA in organic phase by solvent diffusion method. The efficiency of extraction plasmid DNA into organic phase is 99% and the concentration of pDNA in organic phase is up to 2.4 mg/ml. The efficiency of microencapsulation of plasmid DNA in PLGA is up to 76% and can be enhanced by lowering the pH of aqueous solution of emulsion. The microparticles size of PLGA of pDNA is in a narrow range of 1 – 2 Am. This procedure does not involve the high mechanical energy to emulsify which may damage the integrity of pDNA. This method can be applied to encapsulate the pDNA into microparticles of other biocompatible polymers with high efficiency. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Poly (D,L-lactic-co-glycolic acid); DNA organic phase; Self-emulsification; Microencapsulation
1. Introduction Although significant progress has been made in gene therapy and DNA vaccine technology [1– 4], a common problem has been encountered whereby DNA is subjected
*
Corresponding author. Tel.: +86-10-62773255; fax: +86-10-62773255. E-mail address: [email protected] (M.X. Duan).
0165-022X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X ( 0 2 ) 0 0 0 7 3 - 8
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to degradation [5,6] during DNA delivery. This problem is particularly severe when the plasmid DNA is delivered through the mucosal route which has been chosen as a desirable site of inoculation for DNA vaccine to protect some pathogens, since many pathogens such as HIV infect the body from mucosa as their entry sites. It is therefore essential to develop a delivery system that can protect plasmid DNA from degradation. Carrier systems such as microspheres and liposomes have been proved to be able to provide an effective way of delivering plasmid DNA under protection through the mucosal route [7 –10]. PLGA-based microspheres have the potential to act as DNA delivery systems to protect against biological degradation by DNA nuclease. PLGA has a long history of safe use in veterinary and medical applications and is known to be biocompatible. Its degradation gives the natural products lactic acid and glycolic acid [11]. Microspheres of PLGA encapsulating plasmid DNA can induce an immunization response by oral administration [12,13]. One of the most common techniques for preparation of PLGA encapsulating plasmid DNA is water-in-oil-in-water double-emulsion solvent-evaporation method [14,15]. However the encapsulation efficiency of DNA into the hydrophobic matrix of PLGA is low, about 20%. The incorporation of plasmid DNA is about 1 –2 Ag/mg. Moreover, the high energy involved in emulsifying aqueous phase of DNA into organic phase can result in the denaturation of pDNA into a nicked, linear or even broken state, which may damage the bioactivity of the plasmid. Shuichi Ando has modified this encapsulation process by a freezing preparation before second emulsification [16]. The efficiency of encapsulation of DNA was increased from 23% to 85%, and the supercoiled DNA content in microspheres of PLGA was increased from 39% to 85%. In this study, a novel process of microencapsulating plasmid DNA in PLGA called DNA organic phase self-emulsification (DOPSM) has been developed. Since the encapsulation efficiency of a hydrophobic drug into a polymer such as PLGA would be higher than that of a hydrophilic drug, the hydrophobic interactions between plasmid DNA and cationic lipid should make it possible to dissolve the DNA into organic phase [17] and therefore could enhance the encapsulation efficiency of pDNA in PLGA microparticles. The spontaneous emulsification of PLGA into microparticles by solvent diffusion method can avoid the mechanical forces of emulsification, which would break the plasmid DNA [18]. Our DOPSM method, combining these two methods, can encapsulate 76% plasmid DNA into microspheres and the DNA content is up to 3.6 Ag/mg. This high encapsulation efficiency, together with its mild treatment of the DNA sample, makes it a significantly improved method of preparing the PLGA pDNA microspheres.
2. Materials and methods 2.1. Materials Poly (D,L-lactic-co-glycolic acid) (PLGA, lactic – glycolic acid ratio = 50:50) was purchased from Sigma (USA). PVA 124 (polyvinyl alcohol) was purchased from Beijing Chemical (China). Cationic lipid Cetyldimethylethylammonium bromide (CDAB) was obtained from Sangon (China). A 5.6 kbps plasmid pGFP-HbsAg in which HBsAg
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antigen was inserted into pGFPC1 (Clontech, USA) was constructed by our laboratory. The plasmid was amplified in Escherichia coli DH5a strain and prepared by the method of alkali lysis [19]. 2.2. Extraction plasmid DNA from water phase into organic phase An amount of 1 Amol CDAB was dissolved in chloroform and mixed with ethanol. Then, 100 Ag DNA (solved in TE buffer) was added into the mixture and immediately vortexed at moderate rate on a vortexer. The volume ratio of chloroform/ethanol/water is 1:2:1. The mixture was stored at room temperature for 30 min, then added to 1/4 volume of chloroform and 1/4 volume of water, and vortexed for a few seconds. The resulting mixture was centrifuged at 10,000 rpm at room temperature for 10 min. The aqueous phase and organic phase were collected separately for the next stage of analysis. The DNA concentration was determined by UV absorption at 260 nm with water phase blanked against TE buffer and organic phase blanked against chloroform. The pDNA organic phase without CDAB was prepared as control using the above procedure. To recover the DNA from organic phase, 50 Al organic phase of DNA and 100 Al ddH2O were added into 1 ml tube. The tube was gently inverted several times and the lip was opened at 37 jC for 30 min to allow the evaporation of the organic solvent. The plasmid DNA sample recovered from organic phase and the aqueous phase was analyzed using agarose gel electrophoresis. Different ratios of CDAB/DNA (Amol/Ag) and different ratios of chloroform/ethanol/water were prepared to optimize the condition of formulation of DNA organic solution. The maximum dissolution of pDNA in organic phase was determined by monitoring the absorption at 600 nm after the organic solvent is removed by helium flow. 2.3. Effect of pH on recovering pDNA from oil phase to aqueous phase The oil phase of DNA was prepared by adding 0.5 ml organic phase of pDNA into 1 ml un-evaporated oil diacylphosphatidylcholine. Then the organic solvent is removed by rotary evaporators for 4 h. A total of 0.3 ml oil formation of pDNA (300 Ag) is separately added into 3 ml different pH buffers: 0.05 M pH = 2.2 Glycine –HCl buffer (50 ml 0.2 M Glycine, 44 ml 0.2 N HCL, add water to 200 ml); pH = 7.4 PBS (NaCl 8.5 g, Na2HPO4 2.2 g, NaH2PO4 0.2 g, dissolving into 1000 ml water); and pH = 10.83 Na2CO3 –NaHCO3 buffer (9 ml 0.1 M Na2CO3 + 1 ml 0.1 M NaHCO3). The mixtures were then emulsified by vortexing, stored in room temperature for 8 h and vortexed several times in this period. The mixtures were centrifuged at 10,000 g for 10 min before the pH was regulated to about 7.0. The aqueous phases were collected for analysis of agarose gel electrophoresis. 2.4. Preparation of microparticles of pDNA in PLGA The pDNA organic solution was prepared by the method described above but the ratio of chloroform/ethanol/water used was 1:0.9:1. Then, 0.6 ml pDNA (500 Ag) organic solution was mixed with 15 ml solution of PLGA (150 mg) in acetone. The resulting organic solution was dropped into 50 ml aqueous PVA solution (1%, W/V, pH = 5.0) with
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magnetic stirring at room temperature for 8 h to allow the evaporation of the organic solvent. The microparticles were collected by centrifugation (34,000 g for 15 min), washed three times with distilled water and freeze-dried for 1 day. 2.5. Determination of pDNA content in microparticles of PLGA The method of determination of pDNA content was similar to the method described by Tinsley-bown et al. [15]. In brief, aliquots of microparticles (5– 15 mg) were vortexed with 1 ml 0.2 N NaOH and incubated at 100 jC for 20 min. The aqueous phase was recovered by centrifugation at 10,000 g for 10 min and the DNA concentration was determined by UV absorption at 260 nm blanked against 0.2 N NaOH. The encapsulation efficiency was defined as the amount of DNA recovered from the microparticels relative to the initial amount of DNA used (encapsulation efficiency = 100 (recovered DNA/initial DNA). 2.6. Particle size analysis The morphological examination of microparticles was performed using a transmission electron microscope (TEM) (H-800, HITACHI, Japan). The particle size distribution of the microspheres was analyzed using the monochromatic laser ray diffusion counter (Brookhaven Instruments), and the effective diameter was determined.
3. Results and discussion 3.1. The formation of pDNA organic phase The pDNA was extracted from aqueous phase into organic phase by cationic lipid and the organic liquid of pDNA was very clear. Because the absorption of UV 260 in organic phase is higher than that in aqueous phase (Table 1), the extraction efficiency was defined as the amount of DNA in aqueous phase in relation to the total amount of DNA (Extraction efficiency = 100% (amount of DNA in organic phase/amount of DNA in aqueous phase + amount of DNA in organic phase). The extraction efficiency was 98.95% with CDAB was added. In contrast, the extraction efficiency was only 14.73% without CDAB. When the organic solvent is removed, a certain amount of DNA can be recovered into Table 1 Extraction efficiency in CDAB + and CDAB
A260 absorption (1:100) pDNA concentration (Ag/ml) pDNA amount (Ag) Extraction efficiency (%) a
Not to be diluted.
Organic phase (CDAB + ) (0.7 ml)
Aqueous phase (CDAB + ) (0.8 ml)
Organic phase (CDAB ) (0.7 ml)
Aqueous phase (CDAB ) (0.8 ml)
0.027 138.5 96.92 98.95
0.026a 1.300 1.040
0.091a 4.550 14.49 14.73
0.020 98.40 83.64
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Fig. 1. Agarose gel of pDNA recovered from organic phase. Lanes 1 and 6 show the control pDNA (2.5 and 5 Ag/ ml pDNA in TE buffer). Lanes 2 and 3 show the aqueous phase of resulting preparation. Lanes 4 and 5 show the pDNA recovered from the organic phase.
water (Fig. 1). The recovered pDNA shows less fluorescent intensity than the same amount of DNA in TE buffer, probably because the interaction between the pDNA and cationic lipid affect the strain of dye EB on pDNA. The interaction of the pDNA and cationic lipid was also shown by the lag of pDNA under agarose gel electrophoresis (Fig. 1). The maximum dissolution of pDNA in organic phase is up to 2.4 mg/ml as determined by the 600-nm absorption of DNA organic phase when the organic solvent was removed. The cationic lipid acts as an intermediary molecule between the hydrophilic anion pDNA and the hydrophobic organic solvent, and the complex of pDNA/cationic lipid that presents the hydrophobic surface mediates pDNA to dissolve into organic phase. Without the cationic lipid CDAB, most of the DNA remained in the aqueous phase (Table 1). The efficiency of extraction is dependent on the ratio of CDAB to DNA (Fig. 2), and CDAB/ DNA (Amol/Ag) = 0.6:1 is the critical ratio of extraction by which most of the anions in the DNA molecule are neutralized by the cation of CDAB. Besides CDAB, other cationic
Fig. 2. Effect of the ratio of pDNA/CDAB on the efficiency of extraction pDNA from aqueous phase into organic phase. pDNA remained in the aqueous phase was measured by absorption at 260 nm.
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lipids such as DOTMA, which is used as a cationic liposome for in vitro cell DNA transfer [22], also have the capability to extract the DNA from aqueous phase into organic phase [17]. So for further DNA delivery experiments in vivo or in vitro, less toxic cationic lipids than CDAB should be applied in the preparation of DNA organic phase [23]. The mixture of chloroform, ethanol and water in some proper ratio can form a monophase. The monophase of mixture during extraction is important since it provides the interaction surface between the cationic lipid and anions of both DNA and the organic solvent. Experimental results show that when the ratio of ethanol to both chloroform and water was lower than 0.8:1, the DNA could not efficiently dissolve into organic phase. Since pDNA/cationic lipid complex surface is characteristically hydrophobic in nature, the state of pDNA in organic phase would be different from the condensation of pDNA/ cationic liposome complex in aqueous environment. Dorothy L.Reimer demonstrated that pDNA within the complex in the organic phase presents some degree of relaxed state by examining the ability of the DNA to bind a small fluorescent probe TOPRO-1 [17]. The relaxed state of pDNA in organic phase may affect the formulation of PLGA microparticles and the state of pDNA in microparticles of PLGA. 3.2. Encapsulation of pDNA into PLGA The transmission electron micrograph of the PLGA microparticles showed that the morphology of them were spherical (Fig. 3A). The microparticle size distribution is rather narrow, ranging from 1.0 to 2.0 Am (Fig. 3B) and the effective diameter is 1380 nm. The microparticles can easily pass through a membrane filter with a 2.0-Am pore size. The monophase spontaneous emulsification by solvent diffusion method can produce 200– 300 nm nanoparticles of PLGA [18], and previous studies by Bloomfield [20] demonstrated that pDNA can condense into small toroid or rod-shaped structures of 40– 60 nm size when the DNA phosphate charge is at least 90% neutralized in aqueous
Fig. 3. (A) The transmission electron micrograph of PLGA microparticles encapsulated in plasmid DNA. (B) Multimodal size distribution of PLGA microparticles encapsulated in plasmid DNA by the monochromatic laser ray diffusion counter.
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environment. So the microparticles of PLGA encapsulating pDNA could be smaller under optimum conditions. The relaxed state of the pDNA/cationic lipid complex in organic phase may affect the nanometer-sized emulsification of the organic solvent droplets by rapid diffusion of acetone. As a result, it is possible to prepare the smaller particle of PLGA encapsulating pDNA by changing the state of pDNA in organic phase during spontaneous emulsification. The encapsulation efficiency of plasmid DNA in PLGA microparticles is up to 76% when the aqueous phase pH of emulsification is 5.0. The loading DNA in PLGA microparticles is 3.6 F 0.2 Ag/mg. The high encapsulation can also be shown by the little leakage of pDNA from organic phase into aqueous phase (Fig. 4). The high encapsulation efficiency results from the hydrophobicity of the DNA/cationic lipid complex, which has a proclivity for remaining in the organic droplet under thermodynamic interaction. But the encapsulation efficiency is lower, 63%, when the PVA solution pH = 7.0. This may be due to the low pH in the aqueous phase which may prevent pDNA leaking into PVA solution. Agarose gel electrophoresis shows that a little DNA can leak from hydrophobic phase into PVA solution when pH = 7.0 (Fig. 4). Analysis of the effects of the pH on recovering pDNA from hydrophobic phase to aqueous phase shows that low pH or high pH inhibits the dissociation of the complex of DNA and cationic lipid (Fig. 5). The dissociation constant (pK) is essential to dissociation of DNA/cationic lipid complex. It is the stabilization of the DNA/cationic lipid complex that assists the encapsulation of DNA into PLGA microparticles. We observed that the higher pDNA content in microparticles of PLGA was discouraged since the microparticles of PLGA may agglomerate. The high concentration of pDNA organic solution may prevent the emulsification of organic phase, and since the state of pDNA in microparticles may be cross-linking with the PLGA, the fragment of pDNA exposed on the surface of the microparticle may initiate the agglomeration of micropaticles. But the pDNA content in our microparticle is suitable for transfection of cell by pDNA microparticles.
Fig. 4. DNA leaking into PVA solution under different pH conditions during encapsulation of PLGA. The organic mixture of pDNA, PLGA and acetone was dripped into 50ml aqueous PVA solution. After the organic solvent is removed, the PVA solution is concentrated to 5 ml and then agarose gel electrophoresis analysis. Lane 1: the PVA solution pH = 5.0. Lane 2: the PVA solution pH = 7.0. Lane 3: the control pDNA.
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Fig. 5. pDNA recovered from hydrophobic phase to aqueous phase of different pH Buffer. Lane 1: Glycine – HCl buffer pH = 2.2. Lane 2: PBS buffer pH = 7.4. Lane 3: Na2CO3 – NaHCO3 buffer pH = 10.8. Lane 4: the Control pDNA.
The microparticles of PLGA were prepared by the spontaneous emulsification of pDNA organic phase using solvent diffusion method. The whole procedure did not require high mechanical energy, which may damage the DNA. The rapid diffusion of acetone into the aqueous phase caused a remarkable decrease of the interfacial tension between the organic and aqueous phase that result in formation of fine droplets of organic solution without strong mechanical agitation. After solvent evaporation, polymer droplets form microparticles and encapsulate the pDNA/cationic lipid complex into the microparticles. The pDNA encapsulated into the microparticles of PLGA is assumed to maintain its integrity not only because no high mechanical energy was applied but also because the pDNA/ cationic lipid complex may have some extent protection on pDNA. However, there is some difficulty in analyzing the integrity of pDNA since the recovered pDNA is in complex with the cationic lipid instead naked pDNA. The distinctness of this encapsulation procedure is that pDNA can be dissolved in organic phase with polymer PLGA. This means that other biodegradable and biocompatible polymers, for example poly-a`-alkylcyanoacrylate and poly (FA:SA) [8,21], can encapsulate pDNA with high efficiency. After forming the pDNA organic phase with polymer, alternate methods to emulsify the organic phase could be adopted, for example by using a high speed homogenizer or ultrasonic. We have prepared approximately 500 nm pDNA microparticles of PLGA by ultrasonic emulsification. But the balance of particle size and the damage of pDNA should be well considered. The pDNA in our microparticles of PLGA may be cross-linked with the polymer of PLGA. Instead, the pDNA in microcapsules which are produced by double-emulsion solvent-evaporation method are deposited in hollow inner of microcapsules with a thin polymer wall [15]. So the release behavior of pDNA from microparticles of PLGA, which may differ from the microcapsules of PLGA, should be analyzed in further experiments. Since pDNA may expose some of its fragments on the surface of microparticles, further experiments would demonstrate whether this cross-linked state between pDNA and polymer would provide enough protection of DNA in microparticles. Furthermore, the transfection of cells with our pDNA microparticles of PLGA in vitro or in vivo should be performed to evaluate the bioactivity of pDNA encapsulated in these microparticles.
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4. Conclusion We have developed a novel procedure to encapsulate pDNA into PLGA microparticles. The efficiency of encapsulation is up to 76%, and the microparticles size is 1 –2 Am. This method of encapsulation is based on the organic solution of pDNA. Applying the spontaneous emulsification solvent diffusion method, the pDNA avoids the high mechanical energy and maintains its integrity. Using this methodology, pDNA can be encapsulated into other biocompatible polymers with high pDNA content and efficiency of encapsulation without damage of pDNA. Acknowledgements This work was supported by the grants from Bioengineering Development Center of China (96-C01-04-01) and 985 Project of Tsinghua University. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
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Friedmann T. Progress toward human gene therapy. Science 1989;244:1275 – 81. Mulligan RC. The basic science of gene therapy. Science 1993;260:926 – 32. Wilson JM. Vehicles for gene therapy. Nature 1993;365:691 – 2. Ulmer JB, Sadoff JC, Liu MA. DNA vaccines. Curr Opin Immunol 1996;8:531 – 6. Jong YS, Jacob JS, Yip KP, Graham G, Ellen S, Marsha W. Controlled release of plasmid DNA. J Control Release 1997;47:123 – 34. Pachuk CJ, McCallus DE, Weiner DB, Satishchandran C. DNA vaccines—challenges in delivery. Curr Opin Mol Ther 2000;2:188 – 98. Morris W, Steinhoff MC, Russel PK. Potential of polymer microencapsulation technology for vaccine innovation. Vaccine 1994;12:5 – 11. Mathiowitz E, Jacob JS, Jong YS, Carino GP, Chickering DE, Chaturvedi P, et al. Biologically erodable microspheres as potential oral drug delivery. Nature 1997;386:410 – 4. Smith T, Zhang Y, Niven R. Toward development of a non-viral gene therapeutic. Adv Drug Deliv Rev 1997;26:135 – 50. Klavinshis LS, Gao L, Barnfield C, Lehne Tr, Parker S. Mucosal immunization with DNA – liposome complexes. Vaccine 1997;15:818 – 20. Visscher GE, Robison RL, Maulding HV, Fong JW, Pearson JE, Argentieri GJ. Biodegradation and tissue reaction to 50:50 poly (d,l-lactide-co-glycilide) microcapsules. J Biomed Mater Res 1985;19:349 – 65. Jones DH, Corris S, McDonald S, Clegg JCS, Farrar GH. Poly (d,l-lactide-co-glycolide)-encapsulated plasmid DNA elicited systemic and mucosal antibody response to encoded protein after administration. Vaccine 1997;15:814 – 7. Hedley ML, Curley J, Urban R. Microspheres containing plasmid-encoded antigen elicit cytotoxic T-cell response. Nat Med 1998;4:365 – 8. Wang D, Robinson DR, Kwon GS, Samuel J. Encapsulation of plasmid DNA in biodegradable poly(d,llactic-co-glycolic acid) microspheres as novel approach for immunogene delivery. J Control Release 1999;57:9 – 18. Tinsley-bown AM, Fretwelll R, Dowsett AB, Davis SL, Farrar GH. Formulation of poly(d,l-lactic-coglycolic acid) microparticles for rapide plasmid DNA delivery. J Control Release 2000;66:229 – 41. Ando S, Putnam D, W.Pac Dk, Langer R. PLGA microspheres containing plasmid DNA: preservation of superolied DNA via cryopreparation and carbohydrate stabilization. J Pharm Sci 1999;88:126 – 30. Reimer DL, Zhang YP, Kong S, Wheeler JJ, Grabam RW, Bally MB. Formation of novel hydrophobic complexes between cationic lipids and plasmid DNA. Biochemistry 1995;34:12877 – 83.
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[18] Niwa T, Takeuchi H, Hino T, Kunou N, Kawashima Y. In vitro drug release behavior of d,l-lactide/glycolide copolymer (PLGA) nanospheres with nafarelin acetate prepared by a novel spontaneous emulsification solvent diffusion method. J Pharm Sci 1994;83:727 – 32. [19] Sambrook J. Large-scale preparations of plasmid DNA. In: Fritsch EF, Maniatics T, editors. Molecular Cloning. 2nd ed. USA: Cold Spring Harbor Laboratory Press, 1989. p. 1.33 – 9. [20] Bloomfield VA. Condensation of DNA by multivalent cations: considerations on mechanism. Biopolymers 1991;31:1471 – 81. [21] Fallouh NAK, Treupel LR, Fessi H, Devissaguest JP, Puisieux F. Development of a new process for the manufacture of polyisobutylcyanoacrylate nanocapsulates. Int J Pharm 1986;28:125 – 32. [22] Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, et al. A highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A 1987;84:7413 – 7. [23] Scheule RK, George JASt, Bagley RG, Marshall J, Kapla JM, Akita GY, et al. Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to th mammalian lung. Hum Gene Ther 1997;8: 689 – 707.
High efficient encapsulation of plasmid DNA in PLGA microparticles by organic phase self-emulsification F.F. Zhuang, R. Liang, C.T. Zou, H. Ma, C.X. Zheng, M.X. Duan * Department of Biological Science and Biotechnology, Tsinghua University, 100084 Beijing, PR China Received 8 February 2002; received in revised form 27 May 2002; accepted 2 August 2002
Abstract To overcome the drawbacks of encapsulating plasmid DNA (pDNA) in poly (D,L-lactic-coglycolic acid) (PLGA) by water-in-oil-in-water double-emulsion solvent-evaporation method, we have developed a novel procedure for encapsulating pDNA in PLGA microparticles called DNA organic phase self-emulsification (DOPSM). This method was based on both the extraction plasmid DNA from aqueous phase into organic phase and the spontaneous emulsification DNA in organic phase by solvent diffusion method. The efficiency of extraction plasmid DNA into organic phase is 99% and the concentration of pDNA in organic phase is up to 2.4 mg/ml. The efficiency of microencapsulation of plasmid DNA in PLGA is up to 76% and can be enhanced by lowering the pH of aqueous solution of emulsion. The microparticles size of PLGA of pDNA is in a narrow range of 1 – 2 Am. This procedure does not involve the high mechanical energy to emulsify which may damage the integrity of pDNA. This method can be applied to encapsulate the pDNA into microparticles of other biocompatible polymers with high efficiency. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Poly (D,L-lactic-co-glycolic acid); DNA organic phase; Self-emulsification; Microencapsulation
1. Introduction Although significant progress has been made in gene therapy and DNA vaccine technology [1– 4], a common problem has been encountered whereby DNA is subjected
*
Corresponding author. Tel.: +86-10-62773255; fax: +86-10-62773255. E-mail address: [email protected] (M.X. Duan).
0165-022X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X ( 0 2 ) 0 0 0 7 3 - 8
170
F.F. Zhuang et al. / J. Biochem. Biophys. Methods 52 (2002) 169–178
to degradation [5,6] during DNA delivery. This problem is particularly severe when the plasmid DNA is delivered through the mucosal route which has been chosen as a desirable site of inoculation for DNA vaccine to protect some pathogens, since many pathogens such as HIV infect the body from mucosa as their entry sites. It is therefore essential to develop a delivery system that can protect plasmid DNA from degradation. Carrier systems such as microspheres and liposomes have been proved to be able to provide an effective way of delivering plasmid DNA under protection through the mucosal route [7 –10]. PLGA-based microspheres have the potential to act as DNA delivery systems to protect against biological degradation by DNA nuclease. PLGA has a long history of safe use in veterinary and medical applications and is known to be biocompatible. Its degradation gives the natural products lactic acid and glycolic acid [11]. Microspheres of PLGA encapsulating plasmid DNA can induce an immunization response by oral administration [12,13]. One of the most common techniques for preparation of PLGA encapsulating plasmid DNA is water-in-oil-in-water double-emulsion solvent-evaporation method [14,15]. However the encapsulation efficiency of DNA into the hydrophobic matrix of PLGA is low, about 20%. The incorporation of plasmid DNA is about 1 –2 Ag/mg. Moreover, the high energy involved in emulsifying aqueous phase of DNA into organic phase can result in the denaturation of pDNA into a nicked, linear or even broken state, which may damage the bioactivity of the plasmid. Shuichi Ando has modified this encapsulation process by a freezing preparation before second emulsification [16]. The efficiency of encapsulation of DNA was increased from 23% to 85%, and the supercoiled DNA content in microspheres of PLGA was increased from 39% to 85%. In this study, a novel process of microencapsulating plasmid DNA in PLGA called DNA organic phase self-emulsification (DOPSM) has been developed. Since the encapsulation efficiency of a hydrophobic drug into a polymer such as PLGA would be higher than that of a hydrophilic drug, the hydrophobic interactions between plasmid DNA and cationic lipid should make it possible to dissolve the DNA into organic phase [17] and therefore could enhance the encapsulation efficiency of pDNA in PLGA microparticles. The spontaneous emulsification of PLGA into microparticles by solvent diffusion method can avoid the mechanical forces of emulsification, which would break the plasmid DNA [18]. Our DOPSM method, combining these two methods, can encapsulate 76% plasmid DNA into microspheres and the DNA content is up to 3.6 Ag/mg. This high encapsulation efficiency, together with its mild treatment of the DNA sample, makes it a significantly improved method of preparing the PLGA pDNA microspheres.
2. Materials and methods 2.1. Materials Poly (D,L-lactic-co-glycolic acid) (PLGA, lactic – glycolic acid ratio = 50:50) was purchased from Sigma (USA). PVA 124 (polyvinyl alcohol) was purchased from Beijing Chemical (China). Cationic lipid Cetyldimethylethylammonium bromide (CDAB) was obtained from Sangon (China). A 5.6 kbps plasmid pGFP-HbsAg in which HBsAg
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antigen was inserted into pGFPC1 (Clontech, USA) was constructed by our laboratory. The plasmid was amplified in Escherichia coli DH5a strain and prepared by the method of alkali lysis [19]. 2.2. Extraction plasmid DNA from water phase into organic phase An amount of 1 Amol CDAB was dissolved in chloroform and mixed with ethanol. Then, 100 Ag DNA (solved in TE buffer) was added into the mixture and immediately vortexed at moderate rate on a vortexer. The volume ratio of chloroform/ethanol/water is 1:2:1. The mixture was stored at room temperature for 30 min, then added to 1/4 volume of chloroform and 1/4 volume of water, and vortexed for a few seconds. The resulting mixture was centrifuged at 10,000 rpm at room temperature for 10 min. The aqueous phase and organic phase were collected separately for the next stage of analysis. The DNA concentration was determined by UV absorption at 260 nm with water phase blanked against TE buffer and organic phase blanked against chloroform. The pDNA organic phase without CDAB was prepared as control using the above procedure. To recover the DNA from organic phase, 50 Al organic phase of DNA and 100 Al ddH2O were added into 1 ml tube. The tube was gently inverted several times and the lip was opened at 37 jC for 30 min to allow the evaporation of the organic solvent. The plasmid DNA sample recovered from organic phase and the aqueous phase was analyzed using agarose gel electrophoresis. Different ratios of CDAB/DNA (Amol/Ag) and different ratios of chloroform/ethanol/water were prepared to optimize the condition of formulation of DNA organic solution. The maximum dissolution of pDNA in organic phase was determined by monitoring the absorption at 600 nm after the organic solvent is removed by helium flow. 2.3. Effect of pH on recovering pDNA from oil phase to aqueous phase The oil phase of DNA was prepared by adding 0.5 ml organic phase of pDNA into 1 ml un-evaporated oil diacylphosphatidylcholine. Then the organic solvent is removed by rotary evaporators for 4 h. A total of 0.3 ml oil formation of pDNA (300 Ag) is separately added into 3 ml different pH buffers: 0.05 M pH = 2.2 Glycine –HCl buffer (50 ml 0.2 M Glycine, 44 ml 0.2 N HCL, add water to 200 ml); pH = 7.4 PBS (NaCl 8.5 g, Na2HPO4 2.2 g, NaH2PO4 0.2 g, dissolving into 1000 ml water); and pH = 10.83 Na2CO3 –NaHCO3 buffer (9 ml 0.1 M Na2CO3 + 1 ml 0.1 M NaHCO3). The mixtures were then emulsified by vortexing, stored in room temperature for 8 h and vortexed several times in this period. The mixtures were centrifuged at 10,000 g for 10 min before the pH was regulated to about 7.0. The aqueous phases were collected for analysis of agarose gel electrophoresis. 2.4. Preparation of microparticles of pDNA in PLGA The pDNA organic solution was prepared by the method described above but the ratio of chloroform/ethanol/water used was 1:0.9:1. Then, 0.6 ml pDNA (500 Ag) organic solution was mixed with 15 ml solution of PLGA (150 mg) in acetone. The resulting organic solution was dropped into 50 ml aqueous PVA solution (1%, W/V, pH = 5.0) with
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magnetic stirring at room temperature for 8 h to allow the evaporation of the organic solvent. The microparticles were collected by centrifugation (34,000 g for 15 min), washed three times with distilled water and freeze-dried for 1 day. 2.5. Determination of pDNA content in microparticles of PLGA The method of determination of pDNA content was similar to the method described by Tinsley-bown et al. [15]. In brief, aliquots of microparticles (5– 15 mg) were vortexed with 1 ml 0.2 N NaOH and incubated at 100 jC for 20 min. The aqueous phase was recovered by centrifugation at 10,000 g for 10 min and the DNA concentration was determined by UV absorption at 260 nm blanked against 0.2 N NaOH. The encapsulation efficiency was defined as the amount of DNA recovered from the microparticels relative to the initial amount of DNA used (encapsulation efficiency = 100 (recovered DNA/initial DNA). 2.6. Particle size analysis The morphological examination of microparticles was performed using a transmission electron microscope (TEM) (H-800, HITACHI, Japan). The particle size distribution of the microspheres was analyzed using the monochromatic laser ray diffusion counter (Brookhaven Instruments), and the effective diameter was determined.
3. Results and discussion 3.1. The formation of pDNA organic phase The pDNA was extracted from aqueous phase into organic phase by cationic lipid and the organic liquid of pDNA was very clear. Because the absorption of UV 260 in organic phase is higher than that in aqueous phase (Table 1), the extraction efficiency was defined as the amount of DNA in aqueous phase in relation to the total amount of DNA (Extraction efficiency = 100% (amount of DNA in organic phase/amount of DNA in aqueous phase + amount of DNA in organic phase). The extraction efficiency was 98.95% with CDAB was added. In contrast, the extraction efficiency was only 14.73% without CDAB. When the organic solvent is removed, a certain amount of DNA can be recovered into Table 1 Extraction efficiency in CDAB + and CDAB
A260 absorption (1:100) pDNA concentration (Ag/ml) pDNA amount (Ag) Extraction efficiency (%) a
Not to be diluted.
Organic phase (CDAB + ) (0.7 ml)
Aqueous phase (CDAB + ) (0.8 ml)
Organic phase (CDAB ) (0.7 ml)
Aqueous phase (CDAB ) (0.8 ml)
0.027 138.5 96.92 98.95
0.026a 1.300 1.040
0.091a 4.550 14.49 14.73
0.020 98.40 83.64
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Fig. 1. Agarose gel of pDNA recovered from organic phase. Lanes 1 and 6 show the control pDNA (2.5 and 5 Ag/ ml pDNA in TE buffer). Lanes 2 and 3 show the aqueous phase of resulting preparation. Lanes 4 and 5 show the pDNA recovered from the organic phase.
water (Fig. 1). The recovered pDNA shows less fluorescent intensity than the same amount of DNA in TE buffer, probably because the interaction between the pDNA and cationic lipid affect the strain of dye EB on pDNA. The interaction of the pDNA and cationic lipid was also shown by the lag of pDNA under agarose gel electrophoresis (Fig. 1). The maximum dissolution of pDNA in organic phase is up to 2.4 mg/ml as determined by the 600-nm absorption of DNA organic phase when the organic solvent was removed. The cationic lipid acts as an intermediary molecule between the hydrophilic anion pDNA and the hydrophobic organic solvent, and the complex of pDNA/cationic lipid that presents the hydrophobic surface mediates pDNA to dissolve into organic phase. Without the cationic lipid CDAB, most of the DNA remained in the aqueous phase (Table 1). The efficiency of extraction is dependent on the ratio of CDAB to DNA (Fig. 2), and CDAB/ DNA (Amol/Ag) = 0.6:1 is the critical ratio of extraction by which most of the anions in the DNA molecule are neutralized by the cation of CDAB. Besides CDAB, other cationic
Fig. 2. Effect of the ratio of pDNA/CDAB on the efficiency of extraction pDNA from aqueous phase into organic phase. pDNA remained in the aqueous phase was measured by absorption at 260 nm.
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lipids such as DOTMA, which is used as a cationic liposome for in vitro cell DNA transfer [22], also have the capability to extract the DNA from aqueous phase into organic phase [17]. So for further DNA delivery experiments in vivo or in vitro, less toxic cationic lipids than CDAB should be applied in the preparation of DNA organic phase [23]. The mixture of chloroform, ethanol and water in some proper ratio can form a monophase. The monophase of mixture during extraction is important since it provides the interaction surface between the cationic lipid and anions of both DNA and the organic solvent. Experimental results show that when the ratio of ethanol to both chloroform and water was lower than 0.8:1, the DNA could not efficiently dissolve into organic phase. Since pDNA/cationic lipid complex surface is characteristically hydrophobic in nature, the state of pDNA in organic phase would be different from the condensation of pDNA/ cationic liposome complex in aqueous environment. Dorothy L.Reimer demonstrated that pDNA within the complex in the organic phase presents some degree of relaxed state by examining the ability of the DNA to bind a small fluorescent probe TOPRO-1 [17]. The relaxed state of pDNA in organic phase may affect the formulation of PLGA microparticles and the state of pDNA in microparticles of PLGA. 3.2. Encapsulation of pDNA into PLGA The transmission electron micrograph of the PLGA microparticles showed that the morphology of them were spherical (Fig. 3A). The microparticle size distribution is rather narrow, ranging from 1.0 to 2.0 Am (Fig. 3B) and the effective diameter is 1380 nm. The microparticles can easily pass through a membrane filter with a 2.0-Am pore size. The monophase spontaneous emulsification by solvent diffusion method can produce 200– 300 nm nanoparticles of PLGA [18], and previous studies by Bloomfield [20] demonstrated that pDNA can condense into small toroid or rod-shaped structures of 40– 60 nm size when the DNA phosphate charge is at least 90% neutralized in aqueous
Fig. 3. (A) The transmission electron micrograph of PLGA microparticles encapsulated in plasmid DNA. (B) Multimodal size distribution of PLGA microparticles encapsulated in plasmid DNA by the monochromatic laser ray diffusion counter.
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environment. So the microparticles of PLGA encapsulating pDNA could be smaller under optimum conditions. The relaxed state of the pDNA/cationic lipid complex in organic phase may affect the nanometer-sized emulsification of the organic solvent droplets by rapid diffusion of acetone. As a result, it is possible to prepare the smaller particle of PLGA encapsulating pDNA by changing the state of pDNA in organic phase during spontaneous emulsification. The encapsulation efficiency of plasmid DNA in PLGA microparticles is up to 76% when the aqueous phase pH of emulsification is 5.0. The loading DNA in PLGA microparticles is 3.6 F 0.2 Ag/mg. The high encapsulation can also be shown by the little leakage of pDNA from organic phase into aqueous phase (Fig. 4). The high encapsulation efficiency results from the hydrophobicity of the DNA/cationic lipid complex, which has a proclivity for remaining in the organic droplet under thermodynamic interaction. But the encapsulation efficiency is lower, 63%, when the PVA solution pH = 7.0. This may be due to the low pH in the aqueous phase which may prevent pDNA leaking into PVA solution. Agarose gel electrophoresis shows that a little DNA can leak from hydrophobic phase into PVA solution when pH = 7.0 (Fig. 4). Analysis of the effects of the pH on recovering pDNA from hydrophobic phase to aqueous phase shows that low pH or high pH inhibits the dissociation of the complex of DNA and cationic lipid (Fig. 5). The dissociation constant (pK) is essential to dissociation of DNA/cationic lipid complex. It is the stabilization of the DNA/cationic lipid complex that assists the encapsulation of DNA into PLGA microparticles. We observed that the higher pDNA content in microparticles of PLGA was discouraged since the microparticles of PLGA may agglomerate. The high concentration of pDNA organic solution may prevent the emulsification of organic phase, and since the state of pDNA in microparticles may be cross-linking with the PLGA, the fragment of pDNA exposed on the surface of the microparticle may initiate the agglomeration of micropaticles. But the pDNA content in our microparticle is suitable for transfection of cell by pDNA microparticles.
Fig. 4. DNA leaking into PVA solution under different pH conditions during encapsulation of PLGA. The organic mixture of pDNA, PLGA and acetone was dripped into 50ml aqueous PVA solution. After the organic solvent is removed, the PVA solution is concentrated to 5 ml and then agarose gel electrophoresis analysis. Lane 1: the PVA solution pH = 5.0. Lane 2: the PVA solution pH = 7.0. Lane 3: the control pDNA.
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Fig. 5. pDNA recovered from hydrophobic phase to aqueous phase of different pH Buffer. Lane 1: Glycine – HCl buffer pH = 2.2. Lane 2: PBS buffer pH = 7.4. Lane 3: Na2CO3 – NaHCO3 buffer pH = 10.8. Lane 4: the Control pDNA.
The microparticles of PLGA were prepared by the spontaneous emulsification of pDNA organic phase using solvent diffusion method. The whole procedure did not require high mechanical energy, which may damage the DNA. The rapid diffusion of acetone into the aqueous phase caused a remarkable decrease of the interfacial tension between the organic and aqueous phase that result in formation of fine droplets of organic solution without strong mechanical agitation. After solvent evaporation, polymer droplets form microparticles and encapsulate the pDNA/cationic lipid complex into the microparticles. The pDNA encapsulated into the microparticles of PLGA is assumed to maintain its integrity not only because no high mechanical energy was applied but also because the pDNA/ cationic lipid complex may have some extent protection on pDNA. However, there is some difficulty in analyzing the integrity of pDNA since the recovered pDNA is in complex with the cationic lipid instead naked pDNA. The distinctness of this encapsulation procedure is that pDNA can be dissolved in organic phase with polymer PLGA. This means that other biodegradable and biocompatible polymers, for example poly-a`-alkylcyanoacrylate and poly (FA:SA) [8,21], can encapsulate pDNA with high efficiency. After forming the pDNA organic phase with polymer, alternate methods to emulsify the organic phase could be adopted, for example by using a high speed homogenizer or ultrasonic. We have prepared approximately 500 nm pDNA microparticles of PLGA by ultrasonic emulsification. But the balance of particle size and the damage of pDNA should be well considered. The pDNA in our microparticles of PLGA may be cross-linked with the polymer of PLGA. Instead, the pDNA in microcapsules which are produced by double-emulsion solvent-evaporation method are deposited in hollow inner of microcapsules with a thin polymer wall [15]. So the release behavior of pDNA from microparticles of PLGA, which may differ from the microcapsules of PLGA, should be analyzed in further experiments. Since pDNA may expose some of its fragments on the surface of microparticles, further experiments would demonstrate whether this cross-linked state between pDNA and polymer would provide enough protection of DNA in microparticles. Furthermore, the transfection of cells with our pDNA microparticles of PLGA in vitro or in vivo should be performed to evaluate the bioactivity of pDNA encapsulated in these microparticles.
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4. Conclusion We have developed a novel procedure to encapsulate pDNA into PLGA microparticles. The efficiency of encapsulation is up to 76%, and the microparticles size is 1 –2 Am. This method of encapsulation is based on the organic solution of pDNA. Applying the spontaneous emulsification solvent diffusion method, the pDNA avoids the high mechanical energy and maintains its integrity. Using this methodology, pDNA can be encapsulated into other biocompatible polymers with high pDNA content and efficiency of encapsulation without damage of pDNA. Acknowledgements This work was supported by the grants from Bioengineering Development Center of China (96-C01-04-01) and 985 Project of Tsinghua University. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
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