pubs.acs.org/Langmuir © 2009 American Chemical Society
Photoreactive Polylactide Nanoparticles by the Terminal Conjugation of Biobased Caffeic Acid Tran Hang Thi,†,‡ Michiya Matsusaki,† and Mitsuru Akashi*,† †
Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita 565-0871, Japan, and ‡Faculty of Technology of Chemistry, College of Chemistry, Ministry of Industry and Trade, Tien Kien, Lam Thao, Phu Tho, Viet Nam Received April 17, 2009. Revised Manuscript Received August 3, 2009
Photoreactive 3,4-diacetoxycinnamic acid- (DACA-) terminally conjugated poly(L-lactide) (PLLA) (DACA-PLLA) nanoparticles with different diameters were prepared by mixing acetonitrile as a good solvent and water as a poor solvent. The average diameters and morphologies of the nanoparticles prepared under various conditions were evaluated by dynamic light scattering (DLS) measurement and scanning electron microscopic (SEM) observation. The particle size was controllable from about 100 to 300 nm by altering the composition ratio of the mixed solvent, the concentration of the polymers, or the L-lactic acid (LLA) unit number. The particle size increased upon decreasing the LLA unit number or increasing the polymer concentration and water content in the mixed solution. Interestingly, the DACA-PLLA nanoparticles showed different size change properties following UV irradiation at λ > 280 nm before or after particle formation. When UV irradiation was performed after particle formation, only a few percent (ca. 3%) size decrement was observed, although a significant size decrement (ca. 30%) was detected after UV irradiation before particle formation. The DACA-PLLA nanoparticles showed weight decrement during accelerated hydrolysis experiments (pH = 10.0, 37 °C) for 5 days, whereas the particle diameters were unchanged completely. DACA-PLLA nanoparticles have great potential as a photoreactive PLLA nanoparticles in the various applications.
Introduction Biodegradable polymer systems have received considerable attention as a medical device (implants,1 sutures,2 prosthetic,3 orthopedic repair materials,4 and dental materials5) and for the controlled release of a broad range of substances for a wide range of applications (veterinary,6 pesticide,7 and pharmaceutical8). Within these systems, biodegradable micro- and nanoparticles have been extensively investigated as drug delivery technologies for the sustained/controlled release of drugs.9,10 Aliphatic polyesters, especially poly(lactic acid), and its derivatives have been extensively studied in this regard because of their biocompatibility and biodegradability. Photoreactive biopolymers are interesting because of the molecular mechanisms of photochemistry in biological materials and processes, and thus have received increasing attention due to their broad applications as new formulation systems.11 We have previously reported cinnamic acid derivative, which are found in *Author to whom correspondence should be addressed. Telephone: þ81-66879-7356. Fax: þ81-6-6879-7359. E-mail:
[email protected].
(1) Kulkarni, R.; Pani, K.; Newman, C.; Leonard, F. Arch. Surg. 1966, 93, 839– 843. (2) Cutright, D. E.; Beasley, J. D.; Perez, B. Oral. Surg. 1971, 232, 165–173. (3) Hoffman, A. S. J. Appl. Polym. Sci.: Appl. Polym. Symp. 1977, 31, 313–334. (4) Lenslag, J. W.; Pennons, A. J.; Bos, R. R. M.; Rozema, F. R.; Boering, G. Biomaterials 1987, 8, 70–73. (5) Zislis, T.; Mark, D. E.; Cerbas, E. L.; Hollinger, J. O. J. Oral. Implantol. 1989, 15, 160–167. (6) Chien, Y. W. Characteristics and Biomedical Applications, Juliano, R. L., Ed.; Oxford University Press: New York, 1980; pp 11-83. (7) Langer, R. Chem. Eng. Commun. 1980, 6, 1–48. (8) Deasy, P. B. Microencapsulation and Related Drug Processes; Marcel Dekker: New York, 1984; pp 219-240. (9) Couvreur, P.; Puisieux, F. Adv. Drug Delivery Rev. 1993, 10, 141–162. (10) Okada, H.; Toguchi, H. Crit. Rev. Ther. Drug Carrier Syst. 1995, 12, 1–99. (11) Ohkawa, K.; Shoumura, K.; Yamada, M.; Nishida, A.; Shirai, H.; Yamamoto, H. Macromol. Biosci. 2001, 1, 149–156. (12) Ricarda, N.; Anthony, J. M.; Cathie, M. Nat. Biotechnol. 2004, 22, 746–754.
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the biosynthetic pathway of plants that synthesize lignin12 and in several photosynthetic bacteria as one of the protein components,13 homopolymers and its copolymers, which possessed photoreactivity, degradability and cell compatibility.14-18 Copolymers composed of 4-hydroxycinnamic acid and 3,4-dihydroxycinnamic acid formed photoreactive nanoparticles, and the particle size could be controlled by UV irradiation.19,20 However, that particle could not disperse in water, and thus it was difficult to use the nanoparticle for biomedical or environment applications. In a previous study, we successfully performed a terminal conjugation of 3,4-diacetoxycinnamic acid (DACA) to poly(Llactides) (PLLAs), and obtained DACA-terminally conjugated PLLAs (DACA-PLLAs) and their copolymers.21,22 The thermal properties of these photoreactive DACA-PLLAs were significantly improved; in particular, the 10% weight-loss temperature showed an increase of over 100 °C as compared to PLLAs with the same molecular weight. Furthermore, the crystallinities and solubility of the PLLAs were well-maintained after conjugation (13) Kyndt, J. A.; Meyer, T. E.; Cusanovich, M. A.; Van Beeumen, J. J. FEBS Lett. 2002, 512, 240. (14) Kaneko, T.; Tran, H. T.; Shi, D. J.; Akashi, M. Nat. Mater. 2006, 5, 966– 970. (15) Tran, H. T.; Matsusaki, M.; Shi, D. J.; Kaneko, T.; Akashi, M. J. Biomater. Sci. Polym. Edn. 2008, 19, 75–85. (16) Matsusaki, M.; Kishida, A.; Stainton, N.; Ansell, C. W. G.; Akashi, M. J. Appl. Polym. Sci. 2001, 82, 2357–2364. (17) Kaneko, T.; Matsusaki, M.; Tran, H. T.; Akashi, M. Macromol. Rapid Commun. 2004, 25, 673–677. (18) Matsusaki, M.; Tran, H. T.; Kaneko, T.; Akashi, M. Biomaterials 2005, 26, 6263–6270. (19) Shi, D. J.; Kaneko, T.; Akashi, M. Langmuir 2007, 23, 3485–3488. (20) Shi, D. J.; Matsusaki, M.; Akashi, M. Macromolecules 2008, 41, 8167–7172. (21) Tran, H. T.; Matsusaki, M.; Akashi, M. Chem. Commun. 2008, 33, 3918– 3920. (22) Tran, H. T.; Matsusaki, M.; Akashi, M. Biomacromolecules 2009, 10, 766– 772.
Published on Web 08/20/2009
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Thi et al. Table 1. Synthetic Conditions of PLLA50a and Various DACA-PLLAsb infeed c
sample
d
PLLA (mmol)
DACCe (mmol)
Mnf
Mw/Mnf
LLA/DACAg (unit)
PLLA50 3570 3.0 100/0 DACA-PLLA16 4.2 16.8 1440 2.1 16/1 DACA-PLLA38 5.5 22.0 2090 2.1 38/1 DACA-PLLA49 3.7 14.9 3690 2.8 49/1 DACA-PLLA79 1.9 7.6 6010 2.8 79/1 a The polymerization of PLLAs was carried out in the presence of SnOct2 as a catalyst (0.3 wt % of monomer) with mechanical stirring at 160 °C for 4 h. b The conjugation of DACA into the PLLA end chains was carried out at 0 °C for 2 h, and at room temperature for 24 h. c The numbers in the sample name are the unit numbers of LLA. d LLA unit number in PLLA corresponds to that in DACA-PLLA. e DACC is 3,4-diacetoxycinnamoyl chloride. f The molecular weights were estimated by GPC in THF with polystyrene standards. g The composition ratio of LLA and DACA was estimated by 1H NMR spectroscopy
with DACA. DACA-PLLAs have a carboxyl group as the hydrophilic group and a hydrophobic DACA moiety at the chain end. Therefore, it is expected that a photoreactive and thermally stable nanoparticle based on PLLA can be obtained. In this study, we prepared novel photoreactive DACA-PLLA nanoparticles and evaluated their photoreactivity and hydrolysis properties. These DACA-PLLA nanoparticles would be useful for various applications.
Experimental Section Materials. L-Lactide (LLA; Tokyo Chemical Industry (TCI), Japan) was recrystallized from ethyl acetate, and then dried in vacuo at room temperature for 24 h. L-Lactic acid (Tokyo Chemical Industry (TCI), Japan) was used as received. Thionyl chloride (SOCl2), 3,4-dihydroxycinnamic acid (DHCA), acetic anhydride (Ac2O), sodium hydroxide, sodium hydrogen carbonate (Wako Pure Chemical Industries, Ltd., Japan) and acetonitrile (Kishida Chemical Industry, Japan) were used as received. Preparation of DACA-PLLA Nanoparticles. DACA-PLLAs with varying LLA unit numbers were synthesized as described in a previous report.21 Table 1 summarizes the synthetic conditions of DACA-PLLAs used in this study. For particle formation, 5.5 mg of DACA-PLLA49 (49 is the LLA unit number) was dissolved in 1.57 mL of acetonitrile, and then dropped into 3.92 mL of water at room temperature followed by stirring. Other particles were prepared in an analogous procedure. The average particle size was determined by DLS measurement using a Zetasizer Nano ZS (Malvern Instruments, U.K.), and a morphological evaluation of the particle was performed with a JEOL JSM-6701F (JEOL, Japan) scanning electron microscope (SEM). The surface charge (ζ-potential) of the obtained particles in phosphate buffered saline (PBS) was measured by a Zetasizer Nano ZS (Malvern Instruments, U.K.). Photoreaction of DACA-PLLA Nanoparticles. UV Irradiation after Particle Formation. The obtained particle
solution was UV-irradiated at λ > 280 nm using a Supercure 352S UV light source (SAN-EI ELECTRIC Co., Ltd., Japan) for 3, 10, 30, and 90 min, respectively. The UV absorbance change of the particle solution at predetermined times was measured by UV-visible spectroscopy using a U3010 spectrophotometer (HITACHI, Japan). The particle size change after UV irradiation was determined by DLS measurement, and the morphology of the particle was observed by SEM. UV Irradiation before Particle Formation. Samples dissolved in acetonitrile at a 11 mg/mL concentration were UVirradiated at λ > 280 nm for 3, 10, 30, and 90 min. The UV absorbance change of the solution at predetermined times was measured by UV-visible spectroscopy. The UV irradiated solution was dropped into water to form particles. The average particle size was determined by DLS measurements, and the morphology of the particle was observed by SEM. Hydrolysis of DACA-PLLA Nanoparticles. The obtained DACA-PLLA nanoparticles in 1/5 v/v of acetonitrile/ 10568 DOI: 10.1021/la901353e
water at a 1 mg/mL concentration were dialyzed in distilled water for over 3 days to remove the acetonitrile. A NaOH-NaHCO3 buffer solution was then added to adjust the pH to 10, and the hydrolysis experiments were performed at 37 °C for the predetermined times. After the prescribed time, the particles were collected by centrifugation, washed with distilled water, dried in vacuo for 2 days, and then the weight of the samples was measured. The experimental weight values represent the average from two samples. The weight remaining percentage of the hydrolyzed particles (Wremaining) was calculated using the particle weight before (Wbefore) and after (Wafter) hydrolysis: W remaining ðwt%Þ ¼ 100 ðW after =W before Þ The average diameter and morphology of the particles before and after degradation were determined by DLS measurement and SEM observation, respectively. The composition ratios of LLA and DACA before and after hydrolysis were determined by 1 H NMR spectroscopy using a JNM-GSX-400 spectrometer (400 MHz; JEOL, Japan) in chloroform-d.
Results and Discussion Preparation of Nanoparticles. DACA-PLLA nanoparticles were prepared by mixing acetonitrile as a good solvent and water as a poor solvent (Figure 1). These particles were stable in water, even after removing the acetonitrile, for several weeks. Figure 2 shows the DLS curve and SEM image of DACA-PLLA49 nanoparticles in 1/2.5 v/v of acetonitrile/water (A/W) at a 1 mg/mL concentration. The average diameter and polydispersity index (PDI) of the obtained particles were 158 nm and 0.07, respectively, and a spherical morphology was confirmed by SEM observation with a smooth surface. The mean diameter counted from the SEM image (50 particles) was ca. 100 nm, which was smaller than the results of the DLS measurement, probably due to shrinkage during the drying process. PLLAs and other DACA-PLLA nanoparticles with varying LLA unit numbers were successfully obtained, although a DACA particle was not obtained. The carboxyl group is one of the most important surface functional groups for particles to immobilize proteins or drugs intended for biomedical or diagnostic applications. We measured the surface charge, the ζ-potential of the particles in PBS (pH= 7.1). All particles showed around -37 mV of negative charge, suggesting the presence of carboxyl groups on the particle surface. In other words, the DACA units were arranged in the core of the particle probably due to their higher hydrophobicity than the LLA units. Effects of Polymer Composition. DACA-PLLAs with different composition ratios of DACA and LLA, which have a different hydrophilic-hydrophobic balance and chain lengths, will affect nanoparticle formation, diameter, and morphology. We investigated these effects on particles at varying LLA unit Langmuir 2009, 25(18), 10567–10574
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Figure 2. (a) DLS curve and (b) SEM image of DACA-PLLA49 nanoparticles formed in 1/2.5 v/v of A/W at a 1 mg/mL concentration.
Figure 1. (a) Chemical structure of DACA-PLLAs and (b) schematic illustration of nanoparticle formation using the solvent mixing method.
numbers from 16 to 79 in 1/2.5 v/v of A/W at a 1 mg/mL concentration. The average diameter decreased from 215 to 124 nm with an increasing LLA unit number, and the PDI also decreased from 0.18 to 0.07 in a similar fashion (Figure 3, parts a and b). Since the LLA units seem to act as hydrophilic polymers due to the terminal carboxyl group as compared to the more hydrophobic DACA unit, the particle diameter decreased with an increasing LLA unit number. It is known that increasing the hydrophilic unit number will induce a size decrement in the obtained particles.23-25 In contrast, the particle size of PLLAs was around 100 nm, independent of the LLA unit number. The difference in the structure between DACA-PLLA and PLLA is the terminal group; the -OH group of PLLA was substituted by hydrophobic DACA. The hydrophobic-hydrophilic balance between the hydrophobic DACA unit and LLA units with a hydrophilic carboxyl group seemed to affect the particle diameter. In other words, the influence of DACA decreased with increasing PLLA length. The trend of this diameter change estimated from SEM observation agreed well with the DLS measurement (Figure 3c). The surface morphologies of DACA-PLLA nanoparticles with over 38 unit numbers of LLA were smooth. These results indicated that the diameter of the DACA-PLLA nanoparticles was controllable by altering its LLA unit number. Effects of Polymer Concentration. The influence of the polymer concentration on particle size has been reported.26,27 Figure 4 shows the effects of the polymer concentration on the (23) Chen, M.-Q.; Serizawa, T.; Kishida, A.; Akashi, M. J. Polym. Sci. Part A Polym. Chem. 1996, 34, 2213–2220. (24) Kawaguchi, S.; Winnik, M. A.; Ito, K. Macromolecules 1995, 28, 1159– 1166. (25) Riza, M.; Tokura, S.; Kishida, A.; Akashi, M. New Polym. Mater. 1994, 4, 189–198. (26) Fujiwara, T.; Miyamoto, M.; Kimura, Y. Macromolecules 2001, 34, 4043– 4050. (27) Riley, T.; Stolnik, S.; Heald, C. R.; Xiong, C. D.; Garnett, M. C.; Illum, L.; Davis, S. S. Langmuir 2001, 17, 3168–3174.
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obtained particle diameter. The average diameter of the DACA-PLLA nanoparticles ranged from 70 to 290 nm, depending on the concentration and the LLA unit number, and the PDI of the nanoparticles also ranged from 0.07 to 0.4, respectively (Figure 4, part a and b). Pure PLLA nanoparticles also showed the same phenomena related to the polymer concentration. These results are probably due to the increment of aggregation number of polymer chain with increasing polymer concentration. The trend of the diameter changes estimated from the SEM observation agreed well with the DLS measurement (Figure 4c). Effects of Solvent Composition. Particles composed of amphiphilic block copolymers such as polystyrene (PS)-b-poly(acrylic acid),28-30 PS-b-poly(ethylene glycol) (PEG)31,32 and PLLA-b-PEG26 were formed in a mixed solvent of dioxane/ THF/water or THF/water. When water as a poor solvent was added to the polymer solution, interactions of the hydrophobic moieties leads to self-aggregation, mixing polymer chains and subsequent particle formation. Therefore, the solvent composition is important in regard to the particle size.33 In this paper, the particle size changes were investigated in 1/10, 1/5, and 1/2.5 v/v of A/W at a 1 mg/mL concentration (Figure 5). The results of the DLS measurement showed the same diameter for PLLA nanoparticles regardless of the composition of the mixed solvent. However, the diameter of all DACA-PLLA nanoparticles increased with increasing water content, and the diameter and PDI ranged from 125 to 320 nm and from 0.07 to 0.2, respectively. Since an increment in water content causes higher interfacial tension, DACA-PLLA might show an increment in the diameter like an amphiphilic polymer (due to hydrophobic DACA unit and LLA unit with a hydrophilic carboxyl group). The SEM observation also suggested an increment in the diameter depending on the water content (Figure 5c). Effects of UV Irradiation. The cinnamoyl group is wellknown to undergo a [2 þ 2] photocycloaddition resulting in the (28) (29) (30) (31) (32) (33)
Zhang, L.; Eisenberg, A. Science 1995, 268, 1728–1731. Zhang, L.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118, 3168–3181. Shen, H.; Eisenberg, A. J. Phys. Chem. B 1999, 103, 9473–9487. Yu, K.; Eisenberg, A. Macromolecules 1996, 29, 6359–6361. Yu, K.; Eisenberg, A. Macromolecules 1998, 31, 3509–3518. Choucair, A.; Laviguer, C.; Eisenberg, A Langmuir 2004, 20, 3894–3900.
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Figure 3. (a) Average diameter change with increasing the LLA unit number in DACA-PLLAs (n=2) and (b) DLS curves and (c) SEM images of PLLA50 and various DACA-PLLA nanoparticles (the insert images are magnified) formed in 1/2.5 v/v of A/W at a 1 mg/mL concentration.
Figure 4. (a) Average diameter change of PLLA50 and various DACA-PLLA nanoparticles with polymer concentration (n=2) and (b) DLS curves and (c) SEM images of DACA-PLLA49 nanoparticles formed in 1/2.5 v/v of A/W at various concentrations.
formation of a cyclobutane ring (Figure 6a).14,21,22,34-37 1H NMR measurements after UV irradiation exhibited the formation of cyclobutane as a new peak appeared at 4.0-4.2 ppm. Fujiwara and co-workers reported that size of a PLLA/PEG/4-hydroxycinnamic acid copolymeric particle did not change with versus (34) Kawatsuki, N.; Matsuyoshi, K; Hayashi, M.; Takatsuka, H.; Yamamoto, T.; Sangen, O. Chem. Mater. 2000, 12, 1549–1555. (35) Gupta, P.; Trenor, S. R.; Long, T. E.; Wilkes, G. L. Macromolecules 2004, 37, 9211–9218. (36) Chae, B.; Lee, S. W.; Ree, M.; Jung, Y. M.; Kim, S. B. Langmuir 2003, 19, 687–695. (37) Ichimura, K.; Akita, Y.; Akiyama, H.; Kudo, K.; Hayashi, Y. Macromolecules 1997, 30, 903–911.
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without UV cross-linking.38 Jiang and co-workers reported little size decrement of an acrylic acid/PEG/coumarin copolymeric particle after UV irradiation at λ > 280 nm as compared to an un-cross-linked sample.39 In a previous paper, we confirmed high photoreactivities for DACA-PLLAs independent of the composition ratio of DACA and LLA.21 Therefore, we investigated the influence of UV irradiation on the DACA-PLLA nanoparticles via two methods: UV irradiation after particle formation (method 1) and before particle formation (method 2) (Figure 6b). (38) Fujiwara, T.; Iwata, T.; Kimura, Y. J. Polym. Sci., Part A: Polym. Chem. 2001, 39, 4249–4254. (39) Jiang, J.; Qi, B.; Lepage, M.; Zhao, Y. Macromolecules 2007, 40, 790–792.
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Figure 5. (a) Average diameter change of PLLA50 and various DACA-PLLA nanoparticles with the solvent composition ratio (n = 2) and (b) DLS curves and (c) SEM images of DACA-PLLA49 nanoparticles formed in varying solvent compositions at a 1 mg/mL concentration.
Figure 6. (a) Photoreaction scheme of the cinnamoyl group, (b) particle formation images using the two methods, and (c) UV absorbance change of a representative DACA-PLLA49 during 90 min of UV irradiation at λ > 280 nm for both methods.
The photoreactivities in both methods were confirmed by UV-vis spectroscopy (Figure 6c). The lacking clear isosbestic points seem to suggest the formation of the mixed conformation of syn head-to-head, anti head-to-head, syn head-to-tail and anti head-to-head photodimers. The photoreaction speed of method 1 was faster than that of method 2, probably due to the morphology of the polymers. The DACA unit assembled as the core in the nanoparticles in method 1, and thus the [2 þ 2] photocycloaddition seemed to occur easily as compared to the soluble single Langmuir 2009, 25(18), 10567–10574
molecule condition in method 2. The size of final particle was determined by DLS measurement and SEM observation (Figure 7). In the case of method 1, the particle diameter was not strongly influenced by UV irradiation (the change was only 8 nm), although the diameter was dramatically changed from 140 to 112 nm depending on UV irradiation time in the solution condition (method 2) (Figure 7a and b). The diameter and conformation of the particles in method 1 were fixed during UV irradiation, and thus the size change was not strongly DOI: 10.1021/la901353e
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Figure 7. (a) Average diameter change with UV irradiation time (n=2), (b) DLS curves during UV irradiation at λ > 280 nm for both methods, and (c) SEM images of DACA-PLLA49 nanoparticles formed in 1/2.5 v/v of A/W at a 1 mg/mL concentration without or with UV irradiation.
Figure 8. The weight remaining percentage of (a) PLLA50 and various DACA-PLLA nanoparticles without UV irradiation and (b) DACA-PLLA49 nanoparticles without or with UV irradiation at λ > 280 nm for 90 min in a NaOH - NaHCO3 buffer solution at 37 °C (pH=10) (n=2).
influenced by UV irradiation at λ > 280 nm. In contrast, in method 2, the UV irradiation occurred under solution conditions, and therefore the solvation of the sample was changed during UV irradiation, leading to a diameter change with UV irradiation time at λ > 280 nm. The results of the SEM observation agreed with the results of DLS measurement (Figure 7c). Other DACA-PLLA nanoparticles with various concentrations or composition ratios of mixed solvents showed essentially the same phenomenon (data not shown). 10572 DOI: 10.1021/la901353e
Figure 9. Average diameter change of (a) PLLA50 and various DACA-PLA nanoparticles without UV irradiation, (b) DACA-PLLA49 nanoparticles without or with UV irradiation at λ > 280 nm for 90 min during degradation, and (c) DLS curves of DACA-PLLA49 nanoparticles without UV irradiation in a NaOH - NaHCO3 buffer solution at 37 °C (pH=10) (n = 2).
Hydrolytic Degradation. Thus far, many studies have reported the degradation of biocopolymer particles composed of Langmuir 2009, 25(18), 10567–10574
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Figure 10. SEM images of DACA-PLLA49 nanoparticles without UV irradiation at each degradation time point. The inserted images show the nanoparticle dispersion (left) and magnified SEM image (right).
Figure 12. Schematic diagram of the degradation behavior of DACA-PLLA nanoparticles.
Figure 11. 1H NMR spectra of remained DACA-PLLA38 nanoparticle without UV irradiation before (a) and after 3 days hydrolyzation in a NaOH-NaHCO3 buffer solution at 37 °C (pH=10).
LLA and other moieties as glycolic acid40 or pluronic F127.41 In this study, accelerated hydrolysis experiments on the DACA-PLLA nanoparticles were performed in a NaOH-NaHCO3 buffer solution at 37 °C (pH=10) with or without UV irradiation. (40) Park, T. G. Biomaterials 1995, 16, 1123–1130. (41) Xiong, X. Y.; Tam, K. C. Macromolecules 2004, 37, 3425–3430.
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The diameter of the nanoparticles decreased a little after immersion in the buffer solution, probably due to a difference in ionic strength. Figure 8a shows the weight remaining percentage of the PLLA50 and various DACA-PLLA nanoparticles without UV irradiation. When hydrophobic DACA was conjugated into the PLLA end chain, their hydrolysis speed was slower than that of PLLA alone. However, the hydrolysis speed of the DACA-PLLAs was independent of their LLA unit number. It is known that there are many factors, which affect the degradation speed of the nanoparticles, such as the particle size, molecular weight and polymer composition. The hydrolysis speed of DACA-PLLA nanoparticles might be influenced by all of these factors. We also investigated the effects of UV cross-linking for 90 min at λ > 280 nm on the hydrolysis behavior of method 1 and method 2 DACA-PLLA49 nanoparticles (Figure 8b). The size of method 1 and method 2 nanoparticles in the buffer without UV irradiation was 168 and 132 nm, respectively. In the case of method 1, the hydrolysis speed was slower than that of the uncross-linked sample. This is probably due to the inhibition of water molecule diffusion into the particle core by the cross-linked cyclobutane networks. However, interestingly, the opposed result was obtained for the method 2; the hydrolysis speed was faster as compared to the un-cross-linked sample. The reasons for this difference might be attributed to the cross-linking percentages in method 1 (ca. 70%) and method 2 (ca. 60%) after 90 min of UV irradiation (Figure 6c). In addition, the difference in surface area related to the particle diameter would also be another reason. Although the weight remaining percent decreased with increasing hydrolysis time, the diameter of the PLLA and DACA-PLLA nanoparticles did not change during degradation (Figure 9). To clarify this phenomenon, SEM observation was performed (Figure 10). The SEM images revealed the same diameter of the hydrolyzed particle as compared to the original one, but a rougher surface including holes was observed after the hydrolysis, suggesting some degradation of the nanoparticles. It is known that hydrolysis occurs as two processes: surface and bulk degradation.42,43 Therefore, we confirmed the composition ratio of hydrolyzed nanoparticles by 1H NMR measurement (Table 2 and Figure 11). The LLA content before and after degradation was (42) Mathiowitz, E.; Jacob, J.; Pekarek, K.; Chickering, D. Macromolecules 1993, 26, 6756–6765. (43) Gopferich, A.; Tessmar, J. Adv. Drug Delivery Rev. 2002, 54, 911–931.
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Table 2. LLA Content of Various DACA-PLLAs before and after Degradationa LLA content duringb degradation time sample
0 day
1 day
2 days
3 days
4 days
5 days
DACA-PLLA16 16 26 43 53 81 109 DACA-PLLA38 38 45 48 56 57 79 DACA-PLLA49 49 70 80 84 90 96 DACA-PLLA79 79 137 141 193 206 224 c 49 98 137 170 174 195 DACA-PLLA49 49 162 169 209 216 210 DACA-PLLA49d a Hydrolysis was performed in NaOH-NaHCO3 buffer at 37 °C (pH = 10). b Estimated by 1H NMR spectra. c Photo-cross-linking by method 1 for 90 min at λ > 280 nm. d Photo-cross-linking by method 2 for 90 min at λ > 280 nm.
calculated by the peak area ratio of the vinylene proton at 6.2 ppm in DACA and the C-H proton at 5.2 ppm in PLLA. The LLA content of all nanoparticles increased with increasing degradation time, supporting the degradation of the DACA unit caused by bulk degradation, as shown in a schematic diagram in Figure 12, although the DACA unit has higher hydrophobicity than LLA unit. This might be caused that DACA has the electron attractive acetoxyl groups, lead to high positive charge density of the carbon in carbonyl group between DACA unit and PLLA unit. Accordingly, the ester group between DACA unit and PLLA polymer should be easily hydrolyzed as compared to the ester groups in
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PLLA polymers. Photo-cross-linking of the DACA-PLLA nanoparticles in both methods showed higher increasing the remaining LLA units as compared to un-cross-linked nanoparticles (Table 2), suggesting a rapid hydrolysis of the cyclobutane dimer structure as compared to the monomer structure of cinnamic acid. This phenomenon agrees well with our previous report.14
Conclusion We designed novel degradable nanoparticles composed of a photoreactive DACA moiety and LLA units, and the diameter of the DACA-PLLA nanoparticles could be controlled by altering various factors. When UV irradiation was performed after particle formation (method 1), only a few percent (ca. 3%) size decrement was observed, although a significant size decrement (ca. 30%) was detected following UV irradiation before particle formation (method 2). The DACA-PLLA nanoparticles showed degradability, and the particle size was maintained completely over 5 days of hydrolysis, even though almost 80% of the weight of the nanoparticles decreased. These photoreactive DACA-PLLA nanoparticles have great potential for various applications. Acknowledgment. This research was supported by the Osaka University 21st Century COE Program “Center for Integrated Cell and Tissue Regulation”.
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