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Nanoprecipitation and Spectroscopic Characterization of Curcumin-Encapsulated Polyester Nanoparticles Mandy H. M. Leung, Takaaki Harada, Sheng Dai, and Tak W. Kee Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b02773 • Publication Date (Web): 06 Oct 2015 Downloaded from http://pubs.acs.org on October 11, 2015
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Nanoprecipitation and Spectroscopic Characterization of Curcumin-Encapsulated Polyester Nanoparticles Mandy H. M. Leung,† Takaaki Harada,† Sheng Dai,‡ and Tak W. Kee⇤,† Department of Chemistry, The University of Adelaide, Adelaide, South Australia, 5005, Australia, and School of Chemical Engineering, The University of Adelaide, Adelaide, South Australia, 5005, Australia E-mail:
[email protected] Phone: +61 (0)8 8313 5039. Fax: +61 (0)8 8313 4358
To whom correspondence should be addressed Department of Chemistry, The University of Adelaide ‡ School of Chemical Engineering, The University of Adelaide
⇤ †
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Abstract Curcumin-encapsulated polyester nanoparticles (Cur-polyester NPs) of approximately 100 nm in diameter with a negatively charged surface were prepared using a onestep nanoprecipitation method. The Cur-polyester NPs were prepared using polylactic acid, poly(D,L-lactic-co-glycolic acid) and poly(✏-caprolactone) without any emulsifier or surfactant. The encapsulation of curcumin in these polyester NPs greatly suppresses curcumin degradation in the aqueous environment due to its segregation from water. In addition, the fluorescence of curcumin in polyester NPs has a quantum yield of 4 to 5 %, which is higher than that of curcumin in micellar systems and comparable to those in organic solvents, further supporting that the polyester NPs are capable of excluding water from curcumin. Furthermore, the results from femtosecond fluorescence upconversion spectroscopy reveal that there is a decrease in the signal amplitude corresponding to solvent reorganization of excited state curcumin in the polyester NPs compared with curcumin in micellar systems. The Cur-polyester NPs also show a lack of deuterium isotope effect in the fluorescence lifetime. These results indicate that the interaction between curcumin and water in the polyester NPs is significantly weaker than that in micelles. Therefore, the aqueous stability of curcumin is greatly improved due to highly effective segregation from water. The overall outcome suggests that the polyester NPs prepared using the method reported herein are an attractive system for encapsulating and stabilizing curcumin in the aqueous environment.
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Introduction Curcumin is a hydrophobic polyphenol that consists of a keto-enol moiety and the major component of the pigments found in turmeric. 1–3 Curcumin exhibits a number of medicinal benefits including anti-cancer, anti-Alzheimer’s and anti-inflammatory properties. 4–7 Poor aqueous solubility and stability, however, hinder its bioavailability. Curcumin has a poor solubility in the aqueous environment (⇠ 10 µg mL 1 ) due to its hydrophobicity. 8 In contrast, it is highly soluble and stable in polar organic solvents, namely methanol, acetone and tetrahydrofuran (THF). Payton et al. showed that the majority of curcumin exists in a range of solvents as the keto-enol tautomer, as shown in Figure 1. 9 The intramolecular hydrogen bond in the keto-enol moiety and the ⇡-conjugation in curcumin are also shown in Figure 1. The ⇡-conjugation gives rise to a strongly allowed ⇡ ⇡ ⇤ transition, resulting in an intense absorption band centered around 420 nm. 10,11 In addition, curcumin exhibits fluorescence in a range of organic solvents. 10,12,13 It is, however, non-fluorescent in water due to efficient fluorescence quenching by water molecules and self-quenching as a result of aggregation in the aqueous environment. The fluorescence and other photophysical processes of curcumin in polar organic solvents and micelles have been investigated by a number of research groups. 14–17 In particular, previous work has demonstrated that solvent reorganization and excited-state intramolecular hydrogen atom transfer (ESIHT) in the keto-enol moiety of curcumin are major relaxation pathways of its excited state. 14–17 The ESIHT of curcumin exhibits a deuterium isotope effect while solvation remains unaffected. 14,15 In addition to having a low solubility in water, curcumin undergoes rapid degradation. 18,19 The degradation of curcumin has been also studied in a pH 7.4 buffer and more than 50 % of curcumin is degraded within 30 min. 18,20 Furthermore, hydrolysis of curcumin has been linked to its degradation mechanism, with trans-6-(4’-hydroxy-3’-methoxyphenyl)-2,4-dioxo5-hexenal as the major product. 18 Molecular fragmentation of this species produces other minor products including vanillin, ferulic acid and feruloyl methane. 18 In order to suppress the degradation of curcumin in water, a number of encapsulating systems including cyclodex3
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O
H
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O
H3CO
OCH3
HO
OH
Curcumin O
O O
HO
H
O
HO
x O PLGA
n PLA
O
H y
O
HO
O
H n
PCL
Figure 1: Chemical structure of curcumin, polylactic acid (PLA), poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(✏-caprolactone) (PCL). trins, micellar systems and polymer nanoparticles (NPs) have been used. 11,12,20–24 The two main features that enable a system to encapsulate and stabilize curcumin are a hydrophobic core and a hydrophilic exterior. The hydrophobic regions encapsulate curcumin to segregate it from water, improving the aqueous stability by suppressing hydrolysis of curcumin. In addition, the hydrophilic outer layer interacts with water, which enables the curcuminencapsulating agent complex to remain suspended in water. The use of polyester NPs as encapsulating and delivery agents has been investigated for their ability to form a stable suspension in water and biocompatibility. 25–28 It has been shown that polyester NPs, in particular poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(✏caprolactone) (PCL), undergo hydrolysis of their ester bonds to form smaller units, which are further metabolized by cells, 29,30 releasing the encapsulated molecules as a function of time. Although cyclodextrins and micellar systems are able to encapsulate curcumin effectively, their inability to release curcumin in a controlled manner is an issue. Therefore, polyester NPs are an attractive system to encapsulate and deliver curcumin. Recent studies have demonstrated delivery of curcumin by biodegradable polyester NPs and their medicinal effects against cancer, cystic fibrosis and wound healing. 27,31–36 The approaches in these 4
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studies for preparing curcumin-encapsulated polyester NPs require either emulsifiers such as polyvinyl alcohol, 27,31–34 surfactants such as poloxamer 407, 35 or lengthy dialysis process to remove the organic phase of the solution. 36 One of the aims of this study is to use a simple, emulsifier- and surfactant-free method to prepare polyester NPs to encapsulate curcumin. We report a novel, one-step nanoprecipitation method for the preparation of nanometersized (⇠ 100 nm) curcumin-encapsulated, biodegradable polyester NPs (Cur-polyester NPs) using polylactic acid (PLA), PLGA and PCL. The aqueous stability of curcumin is enhanced in all three Cur-polyester NPs relative to micelles, plasma proteins and vesicles. 11,12,37 In particular, negligible degradation is observed for curcumin encapsulated in PLA NPs. The high aqueous stability is attributable to the lack of interaction between curcumin and water. The resulting Cur-polyester NPs exhibit a strong UV-visible absorption with a maximum around 420 nm and fluorescence around 500 nm. These spectroscopic characteristics indicate the presence of curcumin in an environment similar to the palisade layer of micelles. 12,15 Fluorescence spectroscopy was also used to offer insight into encapsulation of curcumin in the polyester NPs. Curcumin exhibits a higher fluorescence quantum yield when it is encapsulated by the polyester NPs than by micelles, implying weaker interactions between curcumin and water in the polyester NPs than in micelles. In addition, the low amplitude of the time-resolved fluorescence upconversion signals for solvent reorganization and the absence of a deuterium isotope effect in the fluorescence decays of the Cur-polyester NPs further support limited interactions between curcumin and water. The overall results show that the polyester NPs segregate curcumin from water effectively, suppressing the degradation of curcumin.
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Experimental section Materials Curcumin (purity > 98%) was purchased from LKT Laboratories. Polylactic acid (PLA, average Mw : 60 000 g mol 1 ), poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 lactic acid:glycolic acid, Mw : 40 000 to 75 000 g mol 1 ) and poly(✏-caprolactone) (PCL, average Mn : 45 000 g mol 1 ) were sourced from Sigma-Aldrich. Acetone and chloroform (AR grade) from Chem-Supply, methanol (isocratic HPLC grade, 254 nm) from Scharlau and D2 O (D, 99.9 %) from Cambridge Isotope Laboratories were used as received. Tetrahydrofuran (THF, HPLC grade, without stabilizer) was purchased from Scharlau and freshly distilled before used. Deionized water from a Millipore Milli-Q NANOpure system was used in all experiments.
Preparation of Curcumin-Polyester NPs The Cur-polyester NPs were prepared by a nanoprecipitation method as described previously with modifications. 34,36,38 For the preparation of curcumin-encapsulated PLA NPs (Cur-PLA NPs), stock solutions of 100 mg mL
1
PLA in chloroform and 1 mg mL
1
curcumin
in THF were prepared. A volume of 50-µL of the curcumin stock solution was diluted with 445 µL of THF. A 5-µL aliquot of the PLA stock solution was added to the diluted curcumin solution (THF) to yield a 500 µL mixed curcumin/PLA solution. For the preparation of curcumin-encapsulated PLGA or PCL nanoparticles (Cur-PLGA or Cur-PCL NPs), stock solutions of 4 mg mL
1
PLGA or PCL, and curcumin in acetone were prepared. A volume
of 125-µL PLGA or PCL stock solution was mixed with 25 µL of curcumin stock to yield a 150-µL curcumin/PLGA or curcumin/PCL solution. The ratio of curcumin:polyester in the resulting curcumin/polyester solutions was 1:5 by weight for curcumin:PLGA or PCL, and 1:10 for curcumin:PLA. For a 1:10 curcumin:polyester by weight solution of curcumin/PLGA or curcumin/PCL, 12.5µL of curcumin stock solution was mixed with 125 µL of polyester stock, which was followed by an addition of 12.5µL of acetone, resulting a 150-µL solution. 6
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All the experiments were done with a weight ratio of 1:10 for the Cur-PLA and 1:5 for Cur-PLGA or Cur-PCL NPs, except for the investigation on aqueous stability, where a 1:10 ratio for the Cur-PLGA and Cur-PCL NPs was used. The curcumin/polyester solution was then injected using a glass syringe into 40 mL of deionized water under moderate stirring. The solution was stirred for an additional 30 min in the dark. The overall %v/v of the organic solvent in the organic/water mixture was approximately 0.4 % for acetone (Cur-PLGA and Cur-PCL NPs); and about 1.3 % for THF/chloroform (Cur-PLA NPs). In particular, chloroform constituted merely about 0.02 %. The organic phase was then removed under reduced pressure at 40 C for 30 min. The volume of solution was reduced approximately by half. Additional water was used to adjust the evaporated solution to 20 mL. The resulting Cur-polyester NPs solution was filtered with a 0.45 µm hydrophilic syringe filter (Minisart) to remove large aggregates, which was followed by an additional filtration with a 0.2 µm hydrophilic syringe filter (Minisart). The concentration of the Cur-polyester NPs was estimated from the absorbance at 425 nm before and after filtration. The yield of the Cur-polyester NPs with a size of
