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Preparation and characterization of hyaluronic acid-based hydrogel nanoparticles
ARTICLE IN PRESS
Journal of Physics and Chemistry of Solids 69 (2008) 1591–1595 www.elsevier.com/locate/jpcs
Preparation and characterization of hyaluronic acid-based hydrogel nanoparticles Ki Young Choia,b, Seulki Leeb, Kyeongsoon Parkb, Kwangmeyung Kimb,1, Jae Hyung Parka,c,, Ick Chan Kwonb, Seo Young Jeonga a
Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea b Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea c Department of Advanced Polymer and Fiber Materials, Kyung Hee University, Gyeonggi-do 449-701, Republic of Korea Received 1 July 2007; received in revised form 1 October 2007; accepted 30 October 2007
Abstract Novel polymeric amphiphilic hydrogel nanoparticles have been prepared by covalent attachment of hydrophobic tetradecylamine (TDA) to hyaluronic acid (HA) in the presence of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide. Their chemical structure and self-association behavior in an aqueous solution were investigated by using 1H NMR, dynamic light scattering, fluorescence spectroscopy, and transmission electron microscopy (TEM). Owing to their amphiphilic characteristics, the resulting conjugates could form self-assembled hydrogel nanoparticles in an aqueous phase via the intra- or intermolecular association of hydrophobic moieties conjugated to the backbone of HA. From the TEM results, it was observed that the hydrogel nanoparticles were spherical in shape. The particle sizes and critical aggregation concentrations (CACs) of the conjugates were significantly dependent on the degree of substitution (DS) of TDA. The CACs of the conjugates were as low as 50–180 mg/mL; this suggests that the conjugates can form nanoparticles in diluted aqueous media such as body fluids. The mean diameters of the nanoparticles decreased as the DS of TDA increased, indicating that a larger amount of hydrophobic moieties in the conjugate allows formation of compact hydrophobic inner cores. It is anticipated that this HA-based nanoparticles can be used as new drug carriers for biomedical applications. r 2007 Elsevier Ltd. All rights reserved. Keywords: A. Polymers; B. Chemical synthesis; C. Electron microscopy
1. Introduction During the recent few decades, amphiphilic polymers have received a lot of attention because of their unique physical and chemical properties. Amphiphilic polymers consist of hydrophobic and hydrophilic fragments and they are able to form self-assemblies in aqueous media. The properties of self-assemblies in solution depend on the architecture of the polymers and on the balance between hydrophilic and hydrophobic segments. The self-assembling behavior strongly correlates with the balance of Corresponding author at: Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea. Tel.: +82 31 201 3256; fax: +82 31 204 8114. E-mail addresses: [email protected] (K. Kim), [email protected] (J.H. Park). 1 Also for correspondence. Tel.: +82 2 958 5916; fax: +82 2 958 5909.
0022-3697/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpcs.2007.10.052
hydrophilicity and hydrophobicity, therefore, the properties of the amphiphiles can be varied over a broad range by changing the polymers, nature and content of hydrophobic segments. Both the inherent and modifiable properties of amphiphilic polymers make them particularly well suited for biomedical and pharmaceutical applications [1–4]. Among the various applications, amphiphilic polymers have been extensively studied as drug carriers because they can form self-assembled nanoparticles capable of imbibing hydrophobic drugs. When such nanoparticles are systemically administrated, they are known to prolong the blood circulation time, release the drug in a controlled manner, and selectively accumulate at the target site such as tumor tissues [5,6]. Overall, nanoparticles systems can minimize the side effects of the drugs and thereby improving patient compliance.
ARTICLE IN PRESS K.Y. Choi et al. / Journal of Physics and Chemistry of Solids 69 (2008) 1591–1595
1592
Many attempts have been made to design various amphiphilic polymers as drug carriers that include polymer–drug conjugates [5], amphiphilic block copolymers [7], and hydrophobically modified water-soluble polymers [2–6]. We previously reported glycol chitosan-based polymeric amphiphiles form self-assembled hydrogel nanoparticles (150–300 nm in diameter) and hold a quantity of anticancer drugs such as doxorubicin [5] and paclitaxel [6]. In accordance with these substantiated observations that hydrophobically modified polysaccharides can be used as efficient drug carriers, we have now developed hydrogel nanoparticles based on hyaluronic acid (HA). HA is one of the main components of the extracellular matrix in the body. Since HA is a naturally occurring biopolymer, it is biocompatible and biodegradable. In this study, we prepared and characterized hydrophobically modified hyaluronic acid (HMHs) as a drug carrier which could self-assemble in an aqueous condition. 2. Experimental section 2.1. Materials HA (MW ¼ 300,000) was kindly provided by Amorepacific Co., Korea. Tetradecylamine (TDA), 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC), and Nhydroxysulfosuccinimide (sulfo-NHS) were purchased from Sigma. The water, used for synthesis and characterization, was purified by distillation, deionization, and
reverse osmosis (Milli-Q Plus). All other chemicals were analytical grade and used as received. 2.2. Preparation of HMHs HA (180 mg) was dissolved in 30 mL of distilled water containing EDC (18.2–182.9 mg) and sulfo-NHS (20.6– 206 mg). TDA (5.1–50.6 mg) in 1-methyl-2-pyrrolidinone (30–60 mL) was added and the pH was adjusted to 7.4 by addition of 0.1 N NaOH. After 24 h, the reaction mixture was extensively dialyzed against the excess amount of water/methanol (1v/3v–1v/1v) and distilled water for 3 days, followed by lyophilization. The TDA content in the conjugates was determined using 1H NMR. 2.3. Preparation and characterization of self-assembled hydrogel nanoparticles The HMHs were dissolved in a phosphate buffer (PB, pH 7.4), and each solution was sonicated for 2 min using a probe-type sonifier (VCX-750, Sonics & materials) at 90 W, in which the pulse was turned off for 1 s with an interval of 5 s. The solution passed through a syringe type membrane filter (pore size: 0.45 mm, Millipore). To determine the particle size and size distribution of the nanoparticles, dynamic light scattering (DLS) measurements were performed using helium ion laser system (Spectra Physics Laser Model 127–35) which operated at 633 nm and 2570.1 1C. The scattered light was measured at an angle of 901 and collected with BI-9000AT
a
b
CH2CH2(CH2)11CH3
EDC/sulfo-NHS HO
OH NHCOCH3
O
O
O
O OH
CH2OH
C O
O
O OH
HN
CH2OH
COOH
OH
O OH
TDA (Tetradecylamine)
O
OH
n
HA
HMH
n
NH CH3
c
c b a HMH
HA
4
3
2
1
ppm Fig. 1. Synthesis scheme of HA/TDA conjugates and 1H NMR spectra of native HA in D2O and HMH in D2O/CD3OD (1v/1v) in the presence of NaOD.
ARTICLE IN PRESS K.Y. Choi et al. / Journal of Physics and Chemistry of Solids 69 (2008) 1591–1595
autocorrelator. The concentration of self-aggregates was kept constant at 1 mg/mL. The morphology of nanoparticles was observed using a transmission electron microscopy (TEM, Philips CM 30) which was operated at an accelerating voltage of 200 keV.
Table 1 Effect of feed ratio on the degree of substitution (DS) of self-aggregates Samplea
Feed ratiob (%)
DS (%)
c
d
Yield (%)
HMH4 HMH5 HMH9 HMH12 HMH27
5 10 15 25 50
4.08 5.41 8.64 12.25 27.17
300,871 300,668 301,066 301,512 303,353
0.002 0.004 0.006 0.009 0.019
80.57 79.36 78.06 79.32 77.08
Mn
X
1593
2.4. Measurement of fluorescence spectroscopy A pyrene solution (3.0 102 M in acetone), which had been stored at 4 1C, was added to the distilled water to give a pyrene concentration of 12.0 107 M, and acetone was removed using a rotary evaporator at 60 1C for 1 h. This solution was mixed with the solution of HMHs to obtain a polymer concentration 1.0 104–1.0 mg/mL, resulting in a pyrene concentration of 6.0 107 M. Pyrene fluorescence spectra were obtained by using fluorescence spectrophotometer (F-5000, Hitachi). The excitation (lex) and emission (lem) wavelengths were 336 and 390 nm, respectively.
a
HMH, in which the number indicates the DS of TDA. Mole ratio of TDA to sugar residues of HA. c Number-average molecular weight, estimated from NMR. d Weight fraction of TDA. b
300 HMH 280
3. Results and discussion Diameter (nm)
260
3.1. Synthesis and characterization of HMHs 240
220
200
0
5
10 15 20 Degree of Substitution (%)
25
30
120
120
100
100
100
80
80
80
60 40 20
Intensity (a.u.)
120
Intensity (a.u.)
Intensity (a.u.)
Fig. 2. Particle size of the self-aggregates as a function of the degree of substitution of TDA on the HA back bone measured by using dynamic light scattering.
The backbone of HA was hydrophobically modified by chemical attachment of hydrophobic TDA as shown in Fig. 1. The carboxylic acid group of HA was activated with equal amounts (4 equiv./[TDA]) of EDC and sulfo-NHS to form amide linkage by the reaction with primary amino groups in TDA. The detailed characteristics of HMHs are summarized in Table 1. By varying the feed ratio of TDA to HA, five different self-associated HMH nano-sized particles (197–285 nm in diameter) were obtained. The amount of conjugated TDA in HMHs was quantitatively characterized from the 1H NMR spectra by using the integration method. The characteristic peaks of N-acetyl group of HA (d/ppm ¼ 2 [t, 3H, –COCH3–]) and TDA (d/ ppm ¼ 0.9 [t, 3H, CH3–(CH2)11–]; 1.3–1.4 [m, 22H,
60 40 20
0 100
200 300 Diameter (nm)
400
500
40 20
0 0
60
0 0
100
200 300 Diameter (nm)
400
500
0
100
200 300 Diameter (nm)
Fig. 3. Size distribution of (a) HMH4, (b) HMH9, and (c) HMH27 measured by using dynamic light scattering.
400
500
ARTICLE IN PRESS K.Y. Choi et al. / Journal of Physics and Chemistry of Solids 69 (2008) 1591–1595
–CH3(CH2)11–]) were used for integration (Fig. 1). Depending on the feed ratio of TDA to HA, the degree of substitution (DS) of TDA ranged from 4 to 27. When the feed ratio was higher than 50, the resulting conjugates were insoluble in water because of their hydrophobicity due to the excess amount of conjugated TDA. Since the self-assembled hydrogel nanoparticles are formed based on an appropriate balance of hydrophilicity and hydrophobicity, the water-insoluble conjugates that might have high DS (430) were not investigated in this study. 3.2. Characterization of self-assembled hydrogel nanoparticles The self-associated amphiphilic characteristics of HMHs composed of hydrophilic HA and hydrophobic TDA in an aqueous solution was demonstrated by using particle size measurement obtained from dynamic light scattering, as summarized in Table 1. The particle size of hydrogel nanoparticles was dependent on the DS of TDA (Figs. 2 and 3). Water soluble native HA did not show size distribution in solution. The figure shows that HMHs with higher DSs of the TDA formed smaller nanoparticles. This is because as the DS increases, (i) the inner core of the aggregates with hydrophobic chain association gets more and more compact, (ii) more amphiphilic HA get to the surface of the solution, and (iii) thereby HMHs form smaller in size in the solution. However, due to the imbalance between hydrophilic and hydrophobic properties of HA and TDA, this phenomena was not observed in the case of HMSs with high DS (430). On the other hands, the size of nanoparticles was scarcely affected by the concentration of HMHs in the range 0.5–5 mg/mL. This indicates that the interparticle interaction between nanoparticles is almost negligible. Also, TEM showed that HA hydrogel nanoparticles were well-dispersed with spherical shape (Fig. 4). The critical aggregation concentrations (CACs) of HMHs were determined using fluorescence spectroscopy in the presence of pyrene molecules. Pyrene molecules are
known to preferably locate inside or close to the hydrophobic microdomains of hydrogel nanoparticles rather than the aqueous phase, resulting in different photophysical characteristics. Fig. 5 shows the intensity ratio I340/I334 of the pyrene excitation spectra versus the logarithm of the HMH concentration. The CAC was determined from the threshold concentration of self-assembled nanoparticle formation by intra- or intermolecular association. The results exhibited that the CAC values of HMHs were in the range 50–180 mg/mL, which was significantly lower than that of low molecular weight surfactant (e.g., 2.3 mg/mL for sodium dodecyl sulfate in water). Such low CAC values suggest that hydrogel nanoparticles can be formed at highly diluted conditions and can maintain its micellar structure. This is of high importance for their use as a drug 2.5 HMH27 HMH12 HMH9
2.0
1.5 I340 / I334
1594
1.0
0.5
0.0 -3
-2
-1 Log C (mg/ml)
0
1
Fig. 5. Intensity ratio I340/I334 from pyrene excitation spectra as a function of polymer concentration in phosphate buffered solution (pH 7.4).
Fig. 4. TEM images of (a) HMH4, (b) HMH9, and (C) HMH27.
ARTICLE IN PRESS K.Y. Choi et al. / Journal of Physics and Chemistry of Solids 69 (2008) 1591–1595
carrier because the drug-loaded nanoparticles would travel through the large volume of body fluids before they reach the target site. Based on these results, HMHs might be useful as a drug carrier because they can form selfassembled hydrogel nanoparticles bearing hydrophobic inner core that can play a role as container of poorly water-soluble drugs. Along this line of research, applications of HMHs as a drug carrier are currently under investigation. 4. Conclusion A novel type of polymeric amphiphiles composed of HA and TDA was prepared and characterized. The resulting HA conjugates could form self-assembled hydrogel nanoparicles with a size of 197–285 nm, which was significantly dependent on the DS of TDA. The hydrogel nanoparticles were able to form at highly diluted concentrations as low as 50–180 mg/mL, which might provide their potential for biomedical applications. Acknowledgment This work was supported by the Ministry of Health and Welfare (A062254B8150506N11C011B), BK21 BNT Scientist Renovating for Drug Development Coping with Aged
1595
Society and by Seoul R&BD Program (10524M0214852) in Korea. References [1] I. Brigger, C. Dubernt, P. Couvreur, Nanoparticles in cancer therapy and diagnosis, Adv. Drug Deli. Rev. 54 (2002) 631–651. [2] K. Kim, S. Kwon, J.H. Park, H. Chung, S.Y. Jeong, I.C. Kwon, Physicochemical characterizations of self-assembled nanoparticles of glycol chitosan-deoxycholic acid conjugates, Biomacromolecules 6 (2005) 1154–1158. [3] S. Kwon, J.H. Park, H. Chung, I.C. Kwon, S.Y. Jeong, Physicochemical characteristics of self-assembled nanoparticles based on glycol chitosan bearing 5aˆ-cholanic acid, Langmuir 19 (2003) 10188–10193. [4] K. Park, K. Kim, I.C. Kwon, S.K. Kim, S. Lee, D.Y. Lee, Y. Byun, Preparation and characteristics of self-assembled nanoparticles of heparin-deoxycholic acid, Langmuir 20 (2004) 11726–11731. [5] J.H. Park, S. Kwon, M. Lee, H. Chung, J.H. Kim, Y.S. Kim, R.W. Park, I.S. Kim, S.B. Seo, I.C. Kwon, S.Y. Jeong, Self-assembled nanoparticles based on glycol chitosan bearing hydrophobic moieties as carriers for doxorubicin: in vivo biodistribution and anti-tumor activity, Biomaterials 27 (2006) 119–126. [6] J.H. Kim, Y.S. Kim, S. Kim, J.H. Park, K. Kim, K. Choi, H. Chung, S.Y. Jeon, R.W. Park, I.S. Kim, I.C. Kwon, Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel, J. Contr. Rel. 111 (2006) 228–234. [7] S.C. Lee, Y. Chang, J.S. Yoon, C. Kim, I.C. Kwon, S.Y. Jeong, Syhthesis and micellar characterization of amphiphilic diblock copolymers based on poly(2-ethyl-2-oxazoline) and aliphatic polyesters, Macromolecules 32 (1999) 1847–1852.
Journal of Physics and Chemistry of Solids 69 (2008) 1591–1595 www.elsevier.com/locate/jpcs
Preparation and characterization of hyaluronic acid-based hydrogel nanoparticles Ki Young Choia,b, Seulki Leeb, Kyeongsoon Parkb, Kwangmeyung Kimb,1, Jae Hyung Parka,c,, Ick Chan Kwonb, Seo Young Jeonga a
Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea b Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea c Department of Advanced Polymer and Fiber Materials, Kyung Hee University, Gyeonggi-do 449-701, Republic of Korea Received 1 July 2007; received in revised form 1 October 2007; accepted 30 October 2007
Abstract Novel polymeric amphiphilic hydrogel nanoparticles have been prepared by covalent attachment of hydrophobic tetradecylamine (TDA) to hyaluronic acid (HA) in the presence of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide. Their chemical structure and self-association behavior in an aqueous solution were investigated by using 1H NMR, dynamic light scattering, fluorescence spectroscopy, and transmission electron microscopy (TEM). Owing to their amphiphilic characteristics, the resulting conjugates could form self-assembled hydrogel nanoparticles in an aqueous phase via the intra- or intermolecular association of hydrophobic moieties conjugated to the backbone of HA. From the TEM results, it was observed that the hydrogel nanoparticles were spherical in shape. The particle sizes and critical aggregation concentrations (CACs) of the conjugates were significantly dependent on the degree of substitution (DS) of TDA. The CACs of the conjugates were as low as 50–180 mg/mL; this suggests that the conjugates can form nanoparticles in diluted aqueous media such as body fluids. The mean diameters of the nanoparticles decreased as the DS of TDA increased, indicating that a larger amount of hydrophobic moieties in the conjugate allows formation of compact hydrophobic inner cores. It is anticipated that this HA-based nanoparticles can be used as new drug carriers for biomedical applications. r 2007 Elsevier Ltd. All rights reserved. Keywords: A. Polymers; B. Chemical synthesis; C. Electron microscopy
1. Introduction During the recent few decades, amphiphilic polymers have received a lot of attention because of their unique physical and chemical properties. Amphiphilic polymers consist of hydrophobic and hydrophilic fragments and they are able to form self-assemblies in aqueous media. The properties of self-assemblies in solution depend on the architecture of the polymers and on the balance between hydrophilic and hydrophobic segments. The self-assembling behavior strongly correlates with the balance of Corresponding author at: Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea. Tel.: +82 31 201 3256; fax: +82 31 204 8114. E-mail addresses: [email protected] (K. Kim), [email protected] (J.H. Park). 1 Also for correspondence. Tel.: +82 2 958 5916; fax: +82 2 958 5909.
0022-3697/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpcs.2007.10.052
hydrophilicity and hydrophobicity, therefore, the properties of the amphiphiles can be varied over a broad range by changing the polymers, nature and content of hydrophobic segments. Both the inherent and modifiable properties of amphiphilic polymers make them particularly well suited for biomedical and pharmaceutical applications [1–4]. Among the various applications, amphiphilic polymers have been extensively studied as drug carriers because they can form self-assembled nanoparticles capable of imbibing hydrophobic drugs. When such nanoparticles are systemically administrated, they are known to prolong the blood circulation time, release the drug in a controlled manner, and selectively accumulate at the target site such as tumor tissues [5,6]. Overall, nanoparticles systems can minimize the side effects of the drugs and thereby improving patient compliance.
ARTICLE IN PRESS K.Y. Choi et al. / Journal of Physics and Chemistry of Solids 69 (2008) 1591–1595
1592
Many attempts have been made to design various amphiphilic polymers as drug carriers that include polymer–drug conjugates [5], amphiphilic block copolymers [7], and hydrophobically modified water-soluble polymers [2–6]. We previously reported glycol chitosan-based polymeric amphiphiles form self-assembled hydrogel nanoparticles (150–300 nm in diameter) and hold a quantity of anticancer drugs such as doxorubicin [5] and paclitaxel [6]. In accordance with these substantiated observations that hydrophobically modified polysaccharides can be used as efficient drug carriers, we have now developed hydrogel nanoparticles based on hyaluronic acid (HA). HA is one of the main components of the extracellular matrix in the body. Since HA is a naturally occurring biopolymer, it is biocompatible and biodegradable. In this study, we prepared and characterized hydrophobically modified hyaluronic acid (HMHs) as a drug carrier which could self-assemble in an aqueous condition. 2. Experimental section 2.1. Materials HA (MW ¼ 300,000) was kindly provided by Amorepacific Co., Korea. Tetradecylamine (TDA), 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC), and Nhydroxysulfosuccinimide (sulfo-NHS) were purchased from Sigma. The water, used for synthesis and characterization, was purified by distillation, deionization, and
reverse osmosis (Milli-Q Plus). All other chemicals were analytical grade and used as received. 2.2. Preparation of HMHs HA (180 mg) was dissolved in 30 mL of distilled water containing EDC (18.2–182.9 mg) and sulfo-NHS (20.6– 206 mg). TDA (5.1–50.6 mg) in 1-methyl-2-pyrrolidinone (30–60 mL) was added and the pH was adjusted to 7.4 by addition of 0.1 N NaOH. After 24 h, the reaction mixture was extensively dialyzed against the excess amount of water/methanol (1v/3v–1v/1v) and distilled water for 3 days, followed by lyophilization. The TDA content in the conjugates was determined using 1H NMR. 2.3. Preparation and characterization of self-assembled hydrogel nanoparticles The HMHs were dissolved in a phosphate buffer (PB, pH 7.4), and each solution was sonicated for 2 min using a probe-type sonifier (VCX-750, Sonics & materials) at 90 W, in which the pulse was turned off for 1 s with an interval of 5 s. The solution passed through a syringe type membrane filter (pore size: 0.45 mm, Millipore). To determine the particle size and size distribution of the nanoparticles, dynamic light scattering (DLS) measurements were performed using helium ion laser system (Spectra Physics Laser Model 127–35) which operated at 633 nm and 2570.1 1C. The scattered light was measured at an angle of 901 and collected with BI-9000AT
a
b
CH2CH2(CH2)11CH3
EDC/sulfo-NHS HO
OH NHCOCH3
O
O
O
O OH
CH2OH
C O
O
O OH
HN
CH2OH
COOH
OH
O OH
TDA (Tetradecylamine)
O
OH
n
HA
HMH
n
NH CH3
c
c b a HMH
HA
4
3
2
1
ppm Fig. 1. Synthesis scheme of HA/TDA conjugates and 1H NMR spectra of native HA in D2O and HMH in D2O/CD3OD (1v/1v) in the presence of NaOD.
ARTICLE IN PRESS K.Y. Choi et al. / Journal of Physics and Chemistry of Solids 69 (2008) 1591–1595
autocorrelator. The concentration of self-aggregates was kept constant at 1 mg/mL. The morphology of nanoparticles was observed using a transmission electron microscopy (TEM, Philips CM 30) which was operated at an accelerating voltage of 200 keV.
Table 1 Effect of feed ratio on the degree of substitution (DS) of self-aggregates Samplea
Feed ratiob (%)
DS (%)
c
d
Yield (%)
HMH4 HMH5 HMH9 HMH12 HMH27
5 10 15 25 50
4.08 5.41 8.64 12.25 27.17
300,871 300,668 301,066 301,512 303,353
0.002 0.004 0.006 0.009 0.019
80.57 79.36 78.06 79.32 77.08
Mn
X
1593
2.4. Measurement of fluorescence spectroscopy A pyrene solution (3.0 102 M in acetone), which had been stored at 4 1C, was added to the distilled water to give a pyrene concentration of 12.0 107 M, and acetone was removed using a rotary evaporator at 60 1C for 1 h. This solution was mixed with the solution of HMHs to obtain a polymer concentration 1.0 104–1.0 mg/mL, resulting in a pyrene concentration of 6.0 107 M. Pyrene fluorescence spectra were obtained by using fluorescence spectrophotometer (F-5000, Hitachi). The excitation (lex) and emission (lem) wavelengths were 336 and 390 nm, respectively.
a
HMH, in which the number indicates the DS of TDA. Mole ratio of TDA to sugar residues of HA. c Number-average molecular weight, estimated from NMR. d Weight fraction of TDA. b
300 HMH 280
3. Results and discussion Diameter (nm)
260
3.1. Synthesis and characterization of HMHs 240
220
200
0
5
10 15 20 Degree of Substitution (%)
25
30
120
120
100
100
100
80
80
80
60 40 20
Intensity (a.u.)
120
Intensity (a.u.)
Intensity (a.u.)
Fig. 2. Particle size of the self-aggregates as a function of the degree of substitution of TDA on the HA back bone measured by using dynamic light scattering.
The backbone of HA was hydrophobically modified by chemical attachment of hydrophobic TDA as shown in Fig. 1. The carboxylic acid group of HA was activated with equal amounts (4 equiv./[TDA]) of EDC and sulfo-NHS to form amide linkage by the reaction with primary amino groups in TDA. The detailed characteristics of HMHs are summarized in Table 1. By varying the feed ratio of TDA to HA, five different self-associated HMH nano-sized particles (197–285 nm in diameter) were obtained. The amount of conjugated TDA in HMHs was quantitatively characterized from the 1H NMR spectra by using the integration method. The characteristic peaks of N-acetyl group of HA (d/ppm ¼ 2 [t, 3H, –COCH3–]) and TDA (d/ ppm ¼ 0.9 [t, 3H, CH3–(CH2)11–]; 1.3–1.4 [m, 22H,
60 40 20
0 100
200 300 Diameter (nm)
400
500
40 20
0 0
60
0 0
100
200 300 Diameter (nm)
400
500
0
100
200 300 Diameter (nm)
Fig. 3. Size distribution of (a) HMH4, (b) HMH9, and (c) HMH27 measured by using dynamic light scattering.
400
500
ARTICLE IN PRESS K.Y. Choi et al. / Journal of Physics and Chemistry of Solids 69 (2008) 1591–1595
–CH3(CH2)11–]) were used for integration (Fig. 1). Depending on the feed ratio of TDA to HA, the degree of substitution (DS) of TDA ranged from 4 to 27. When the feed ratio was higher than 50, the resulting conjugates were insoluble in water because of their hydrophobicity due to the excess amount of conjugated TDA. Since the self-assembled hydrogel nanoparticles are formed based on an appropriate balance of hydrophilicity and hydrophobicity, the water-insoluble conjugates that might have high DS (430) were not investigated in this study. 3.2. Characterization of self-assembled hydrogel nanoparticles The self-associated amphiphilic characteristics of HMHs composed of hydrophilic HA and hydrophobic TDA in an aqueous solution was demonstrated by using particle size measurement obtained from dynamic light scattering, as summarized in Table 1. The particle size of hydrogel nanoparticles was dependent on the DS of TDA (Figs. 2 and 3). Water soluble native HA did not show size distribution in solution. The figure shows that HMHs with higher DSs of the TDA formed smaller nanoparticles. This is because as the DS increases, (i) the inner core of the aggregates with hydrophobic chain association gets more and more compact, (ii) more amphiphilic HA get to the surface of the solution, and (iii) thereby HMHs form smaller in size in the solution. However, due to the imbalance between hydrophilic and hydrophobic properties of HA and TDA, this phenomena was not observed in the case of HMSs with high DS (430). On the other hands, the size of nanoparticles was scarcely affected by the concentration of HMHs in the range 0.5–5 mg/mL. This indicates that the interparticle interaction between nanoparticles is almost negligible. Also, TEM showed that HA hydrogel nanoparticles were well-dispersed with spherical shape (Fig. 4). The critical aggregation concentrations (CACs) of HMHs were determined using fluorescence spectroscopy in the presence of pyrene molecules. Pyrene molecules are
known to preferably locate inside or close to the hydrophobic microdomains of hydrogel nanoparticles rather than the aqueous phase, resulting in different photophysical characteristics. Fig. 5 shows the intensity ratio I340/I334 of the pyrene excitation spectra versus the logarithm of the HMH concentration. The CAC was determined from the threshold concentration of self-assembled nanoparticle formation by intra- or intermolecular association. The results exhibited that the CAC values of HMHs were in the range 50–180 mg/mL, which was significantly lower than that of low molecular weight surfactant (e.g., 2.3 mg/mL for sodium dodecyl sulfate in water). Such low CAC values suggest that hydrogel nanoparticles can be formed at highly diluted conditions and can maintain its micellar structure. This is of high importance for their use as a drug 2.5 HMH27 HMH12 HMH9
2.0
1.5 I340 / I334
1594
1.0
0.5
0.0 -3
-2
-1 Log C (mg/ml)
0
1
Fig. 5. Intensity ratio I340/I334 from pyrene excitation spectra as a function of polymer concentration in phosphate buffered solution (pH 7.4).
Fig. 4. TEM images of (a) HMH4, (b) HMH9, and (C) HMH27.
ARTICLE IN PRESS K.Y. Choi et al. / Journal of Physics and Chemistry of Solids 69 (2008) 1591–1595
carrier because the drug-loaded nanoparticles would travel through the large volume of body fluids before they reach the target site. Based on these results, HMHs might be useful as a drug carrier because they can form selfassembled hydrogel nanoparticles bearing hydrophobic inner core that can play a role as container of poorly water-soluble drugs. Along this line of research, applications of HMHs as a drug carrier are currently under investigation. 4. Conclusion A novel type of polymeric amphiphiles composed of HA and TDA was prepared and characterized. The resulting HA conjugates could form self-assembled hydrogel nanoparicles with a size of 197–285 nm, which was significantly dependent on the DS of TDA. The hydrogel nanoparticles were able to form at highly diluted concentrations as low as 50–180 mg/mL, which might provide their potential for biomedical applications. Acknowledgment This work was supported by the Ministry of Health and Welfare (A062254B8150506N11C011B), BK21 BNT Scientist Renovating for Drug Development Coping with Aged
1595
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