Oligoalanine-modified Pluronic-F127 nanocarriers for the delivery of curcumin with enhanced entrapment efficiency.

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Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20

Oligoalanine-modified PluronicF127 nanocarriers for the delivery of curcumin with enhanced entrapment efficiency a

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Sydney Peng , Wei-Lun Hung , Yu-Shiang Peng & I-Ming Chu

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Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan, ROC Published online: 16 Jun 2014.

Click for updates To cite this article: Sydney Peng, Wei-Lun Hung, Yu-Shiang Peng & I-Ming Chu (2014) Oligoalanine-modified Pluronic-F127 nanocarriers for the delivery of curcumin with enhanced entrapment efficiency, Journal of Biomaterials Science, Polymer Edition, 25:12, 1225-1239, DOI: 10.1080/09205063.2014.924059 To link to this article: http://dx.doi.org/10.1080/09205063.2014.924059

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Journal of Biomaterials Science, Polymer Edition, 2014 Vol. 25, No. 12, 1225–1239, http://dx.doi.org/10.1080/09205063.2014.924059

Oligoalanine-modified Pluronic-F127 nanocarriers for the delivery of curcumin with enhanced entrapment efficiency


Sydney Peng, Wei-Lun Hung, Yu-Shiang Peng and I-Ming Chu* Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan, ROC (Received 14 March 2014; accepted 9 May 2014) Curcumin is a naturally occurring compound that has been shown to have anti-oxidant, anti-inflammatory, and anti-carcinogenic activities. However, its pharmaceutical potential has been limited due to its low solubility in water. The use of amphiphilic nanocarriers is an attractive and simple method to solubilize curcumin. In this study, we modified Pluronic F-127 [poly(ethylene glycol)100-block-poly(propylene glycol)65-block-poly(ethylene glycol)100] (PF-127) with oligomers of alanine, an amino acid, to increase the drug entrapment efficiency of curcumin through core stabilization. Alanine-modified PF-127 exhibited lower critical micelle concentration and decreased molecular motion in both the hydrophilic and hydrophobic segments (1H NMR). Nanocarriers in the size range of 54.2–68.4 nm were observed. Entrapment efficiency of curcumin increased by at most 66% (from 25.3 to 91.3%) and the difference in solubility was clearly visualized by increased transparency of the nanocarrier solutions. Curcumin was released continuously up to 120 h from modified carriers, while drug release from unmodified carriers plateaued within 24 h. These modified nanocarriers exhibited no cytotoxicity and more efficiently delivered drugs to HeLa cells as confirmed by fluorescent microscopy. This study demonstrated that alanine modification of FDA-approved PF-127 affects copolymer nanoassembly and has a profound impact on curcumin loading and possibly on other hydrophobic drugs as well. Keywords: Pluronic; alanine; curcumin; nanocarrier; anti-carcinogenic

1. Introduction Curcumin is turmeric-derived natural substance with anti-inflammatory, anti-oxidant, and anti-carcinogenic properties.[1–3] However, pharmaceutical potential of curcumin is limited by its poor water solubility, short biological half-life, and low bioavailability. The solubilization of curcumin is often accomplished by the use of nanocarriers including liposomes and polymeric micelles.[4] The solubilization of curcumin with polymeric nanocarriers has been reported by several groups and is made possible by the partitioning of curcumin into the hydrophobic moiety of the carrier. Hydrogen bonding of the drug with nanocarriers through its dicarbonyl, phenolic, and ester groups also act to increase solubilization of curcumin.[5–7] Amphiphilic copolymers are capable of self-assembling into nanocarriers, which, in particular, have drawn attention due to its potential for drug and gene delivery.[8,9] Hydrophobic drugs, when partitioned into the hydrophobic core, becomes readily solubilized and show minimal degradation and increased *Corresponding author. Email: [email protected] © 2014 Taylor & Francis


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bioavailability.[10–13] Assembled carriers are usually smaller than 100 nm and are stabilized by the hydrophilic shell in an aqueous environment. These characteristics allow efficient loading of hydrophobic drugs, extended circulation, and sustained release. It is important to note that crucial prerequisites for sustained release include high initial drug loading and stable polymer–drug interactions.[14] Sustain-release nanocarrier formulations have been developed for the delivery of paclitaxel,[15,16] doxorubicin,[17] and common NSAIDS.[18] Pluronic F-127 is a FDA-approved triblock copolymer consisting of poly(propylene) glycol as the central block, flanked by poly(ethylene) oxide. (PEO100–PPO65– PEO100) (PF-127). PF-127 exhibits low toxicity and undergoes micellization in aqueous solutions at low concentrations due to their amphiphilic character. Self-assembly of PF127 is driven by hydrophobic interactions of the PPO blocks as they assemble into the core, while hydrophilic PEOs form the outer swollen corona. An extensive list of hydrophobic drugs encapsulated by Pluronic copolymers include doxorubicin,[19,20] ibuprofen,[21,22] and indomethacin.[23] The presence of terminal hydroxyl groups allows modification of Pluronic in several ways to confer desired properties. Hydroxyls may be activated by N,N-disuccinimidyl carbonate and then reacted with a bioadhesive end group to confer mucoadhesive properties.[24] Pluronic end-capped with carboxylic acid via an oligolactide spacer displayed pH and temperature-dependent sol–gel property, which is attributed to change in micellar packing.[25] End-capping Pluronic with oligocaprolactone changed the micelle structure into a vesicle exhibiting extensive hydrophobic associations and results from the same study suggest that the aqueous solubility of hydrophobic drugs may be improved by such modification.[26] In this study, we prepared end-capped PF-127 with three different molecular weights of alanine and deduce that the introduction of amide bonds will increase the stability of nanocarriers through core hydrogen bonding, which also improves solubilization of the hydrophobic drug curcumin. High entrapment efficiency and strong association of polymer with drug confer a sustain-release character to the nanocarriers. Herein, we use curcumin as the model drug due to its many pharmaceutical potentials and its phenolic component that is capable of participating in hydrogen bonding thus further associating with the copolymer. The results show both significant increase in entrapment efficiency and continuous release of curcumin up to 6 days, making this system an excellent candidate for hydrophobic drug delivery. 2. Materials and methods 2.1. Materials Pluronic F-127, L-alanine, triphosgene, triethylamine, and methanesulfonyl chloride were purchased from Sigma (St. Louis, MO, USA). Curcumin was purchased from Fluka Chemie (Buchs, Switzerland). Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), and trypsin were purchased from Gibco (Rockville, MD, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Merck KGaA (Darmstadt, Germany). All other reagents and solvents were of analytical grade and purchased from commercial suppliers.

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2.2. Synthesis of oligoalanine-modified PF-127 Hydroxyl-terminated PF-127 was first converted into a tosylate with triethylamine and methanesulfonyl chloride, followed by an amination reaction with ammonium hydroxide as the nucleophile.[27] Briefly, 10 g of PF-127 was dissolved in 150 mL of dichloromethane (DCM) over ice. Then, 3.6 mL of triethylamine and 1.96 mL of methanesulfonyl chloride were added dropwise. The reaction was carried out for 24 h under N2 atmosphere. After filtration and concentration by rotary evaporation, the product was precipitated in ice-cold ether and dried in vacuum overnight. To the tosylated product, 100 mL of 31% ammonium hydroxide solution was added and the mixture was stirred vigorously for 3 days. The solution was extracted using portions of dichloromethane and concentrated. The amine-terminated product was precipitated with iced ether, recrystallized in ethanol, and dried. The preparation of alanine-NCA was carried out according to literature.[28] Briefly, triphosgene (19 g, 64 mmol) was dispersed in tetrahydrofuran (THF) and added dropwise to finely grounded L-alanine (3 g, 33 mmol). The reaction was carried out for 12 h and the resulting product was concentrated and precipitated in ice-cold hexane. Ring-opening polymerization of alanine-NCA with amine-terminated PF-127 was carried out in dimethylformamide/chloroform (DMF/CHCl3 = 1/3) for 3 days. Briefly, the described solvent was added to amine-terminated PF-127 (2.5 g, 0.0002 mol) and alanine-NCA (0.2 g, 0.00175 mol). The amount of alanine-NCA added was varied to obtain different lengths of alanine. The final product was precipitated, dissolved in dimethyl sulfoxide (DMSO) for dialysis (mw cut-off 2000), and lyophilized. 2.3. Characterization of copolymers Nuclear magnetic resonance (1H NMR) spectra were recorded using a Varian Unity Inova 500 instrument with deuterated trifluoroacetic acid as solvent and tetramethylsilane (TMS) as the internal standard. Number average molecular weight and polydispersity (PDI) were determined by gel permeation chromatography (GPC) with THF as the eluent at a flow rate of 1 mL/min and PEG (mw 4000–20,000) as the standard. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was conducted on a Perkin Elmer FTIR system for chemical structure verification. Differentiated scanning calorimetry (DSC) was carried out using a Perkin Elmer Diamond DSC to determine the melting point of modified and unmodified polymers over the range of 10–120 °C at a heating rate of 10 °C/min. A Rigaku Ultima IV X-ray diffractometer was used to determine the crystallinity of copolymers. Data were collected over the range of 5–50o (2θ) and calculated using a deconvolution program PeakFit® (SPSS, Inc., Chicago, IL, USA), assuming Gaussian functions. Percent crystallinity was calculated by dividing the sum of sharp crystalline peaks by total area of crystalline and amorphous peaks. 2.4. Preparation of empty and curcumin-loaded nanocarriers Ten milligrams of curcumin and 100 mg of the copolymer (1:10 w/w) were dissolved in 1 mL of THF in a glass vial protected from light. Four milliliters of phosphate buffer saline (PBS) containing 0.5% polyvinyl acid (PVA) and 1% sucrose as dispersant were added and the resulting mixture was stirred overnight. The prepared solutions were lyophilized and stored at −20 °C in the dark prior to use. Empty carriers were prepared in similar fashion without curcumin.

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2.5. Characterization of micelles Critical micelle concentration (CMC) was determined in the range 0.0002–1% (w/v) by the 1,6-diphenyl-1,3,5-hexatriene (DPH) method using a Biotek Synergy HT microplate reader equipped with fluorescence detection according to literature.[29] DPH concentration of 4x10−6 M was used and the CMC was taken as the midpoint of discontinuity. Molecular movement of polymer segments after hydrophobic self-association was studied in modified and unmodified PF-127 by 1H NMR of a 0.2% (w/v) copolymer solution dispersed in deuterium oxide. Transmission electron microscopy (TEM) was conducted with a Hitachi H-7500 TEM. Nanocarrier solutions (0.1%) were casted on a copper grid and dried at room temperature prior to visualization. Particle size and polydipersity of 0.05 wt% solution of curcumin-loaded carriers were measured by dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS instrument at 25 °C and 90o scattering angle. Results from 10 runs were averaged. Absorbance spectra of DMSO-solubilized curcumin and curcumin encapsulated in unmodified and modified carriers (at 1% w/v) were recorded by a spectrophotometer in the range of 300–600 nm. Endothermic curves of empty and curcumin-containing nanocarriers after lyophilization were studied with DSC at temperature range of 100–200 °C at 10 °C/min. 2.6. Entrapment efficiency, drug loading, and cumulative release of curcumin Ten milligrams of lyophilized nanocarrier powder was dispersed in 5 mL of PBS, and all processes were performed and protected from light. The dispersed solution was centrifuged at 13,000 rpm for 5 min to separate the drug precipitate from nanocarriers. Drug concentration was determined after dilution in acetonitrile: H2O (4:1) by a microplate reader at 450 nm. Entrapment efficiency (EE%) and drug loading (DL%) were calculated as follows: EE% ¼ DL% ¼

Curcumin carrier ðwtÞ Total curcumin (wt)

Curcumin incarrier ðwtÞ Total polymer (wt)

Drug release study was carried out to evaluate the delayed release of entrapped curcumin. Prepared micelles were incubated at 37 °C and the samples were taken after a specified period for evaluation of drug loading and cumulative release. Cumulative release was calculated by taking into account the total drug (without centrifugation) and entrapped drug (after centrifugation) at each time point. Drug loading was calculated at each time point according to the above provided equation. 2.7. Cytotoxicity Cytotoxicity tests for nanocarriers with and without curcumin were determined by the MTT assay. Hela cells were seeded at a density of 1 × 104/well into 96-well plates and incubated overnight at 37 °C to reach 80% confluency. Nanocarrier solutions were added at a concentration range of 0–0.1 mg/mL and incubated for eight hours prior to the addition of MTT reagent. After four hours, the reagent was removed and the formazen crystals were dissolved with DMSO. Absorbance was read at 570 nm.


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2.8. Delivery of curcumin into HeLa cell The delivery of curcumin into HeLa cell was visualized using a fluorescent microscope (Leica DMI6000 B) and quantified by a plate reader equipped for fluorescence reading. Briefly, 4x104 cells/wells were seeded into 12-well plates and incubated overnight. Medium was replaced by solutions containing empty or 0.01 mg/mL curcumin-loaded nanocarriers, and incubated for one hour. Solution was removed and the cells were washed three times with PBS to ensure removal of carriers and free curcumin prior to observation.


3. Results and discussion 3.1. Synthesis and characterization of oligoalanine-modified copolymers The modification of linear poly(ethylene glycol) (PEG) with variable functionalities has been reported and can be similarly applied to PF-127.[30] Pluronic–alanine copolymers were synthesized by ring-opening polymerization as shown in Scheme 1. The 1H NMR spectra of PF-127 and PF-127-alanine copolymers are shown in Figure 1. The appearance of methyl protons at 1.23 ppm (peak b) in addition to methyl protons of PF-127 (peak a) confirmed the addition of alanine at terminal ends of the copolymer. Molecular weight was calculated by the ratio of methyl protons on alanine to methyl protons on PF-127. Chemical shift at 3.7 ppm is characteristic of the methylene group in the PEO segment of PF-127. 1 H NMR and GPC were used for the calculation of the molecular weights of synthesized copolymers. Molecular weights of the copolymers are summarized in Table 1 and the number after PLU-A in the sample name denotes the number of alanine attached as calculated by 1H NMR. Number average molecular weight calculated from GPC was 11,340, 11,323, and 12,195, respectively, for PLU-A3, PLU-A12, and PLUA15. Increase in number average molecular weight as determined by GPC further confirmed the modification of PF-127. Polydispersity in the range of 1.04–1.15 was observed in all copolymers. ATR-FTIR spectroscopy provided separate mean of verification of alanine addition and is shown in Figure 2. Amide I vibration at 1640 cm−1 and amide II vibration at 1550 cm−1 of modified copolymers confirmed the ring-opening of alanine-NCA and the formation of peptide bond between PF-127 and alanine.

Scheme 1.

Synthesis scheme of Pluronic–alanine from Pluronic-F127 and alanine-NCA.


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Figure 1. TFA-d. Table 1. Sample PLU PLU-A3 PLU-A12 PLU-A15

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1

H NMR spectra of unmodified Pluronic-F127 and alanine-modified copolymers in

Characterization of synthesized Pluronic–alanine copolymers. # Alanine

Mw*

Mn**

PDI*

Tm (oC)

Crystallinity (%)

0 3 12 15

12,500–0 12,500–229 12,500–868 12,500–1081

10,292 11,340 11,323 12,195

1.04 1.15 1.12 1.12

58.4 54.8 54.3 56.0

62.1 40.7 38.7 36.8

*GPC. **NMR.

Figure 2.

FTIR-ATR spectra of unmodified Pluronic-F127 and alanine-modified copolymers.

Thermal characteristics of the copolymers were determined by DSC. (Figure 3) Melting temperature of the polymer decreased 2–3 °C upon alanine addition. Decrease in the melting temperature suggested the decrease in crystallinity of PF-127 after conjugation of alanine, a phenomenon often observed after polymer conjugation.[31]



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Figure 3. DSC thermogram of unmodified Pluronic-F127 and alanine-modified copolymers (solid line: lyophilized micelles only, dashed line: lyophilized micelles encapsulating curcumin).

Xie et al. observed a similar trend in mPEG polycaprolactone copolymer, where increasing the PCL length hindered the crystallization ability of mPEG.[32] Changes in crystallization character of copolymers were studied through X-ray diffraction (XRD). Characteristic diffraction peaks of PF-127 were observed at 19.15o and 23.30o. Crystalline nature of PF-127 remained unchanged, yet percent crystallinity decreased from 62.1 to 36.8% as the length of alanine segment increased. This is in agreement with DSC results and confirmed that the incorporation of alanine reduced the crystallinity of PF-127. Crystallinity of linear block copolymers was reported to be tunable by modulating constituents in amphiphilic copolymers.[33,34] This result is concurrent with recently reported study of decreased crystallinity of hydroxyapatite with increasing poly-γ-benzyl-L-glutamates content.[35] The introduction of poly(caprolactone) (PCL) to PF-127 also resulted in the decrease in crystallinity in a contentdependent manner.[36] 3.2. Characterization of nanocarriers CMC is well associated with hydrophilic and hydrophobic block lengths and interactions. CMC was found to decrease with increasing alanine content as shown in Table 2. The CMC of PLU-A12 and PLU-A15 were significantly lower than that of PLU and PLU-A3, implying dynamic stability. In addition to increased hydrophobic interactions, the C=O and N–H on the alanine backbone theoretically allow hydrogen bonding

Table 2. Sample PLU PLU-A3 PLU-A12 PLU-A15

Characterization of prepared micelles from Pluronic–alanine copolymers. CMC (mM) 0.132 0.065 0.039 0.018

Particle size (nm) (PI) 54.2 68.4 60.0 55.6

(0.258) (0.255) (0.127) (0.275)

EE%

DL%

25.3 ± 2.2 69.3 ± 5.5 80.2 ± 7.4 91.3 ± 8.6

3.1 ± 0.3 8.6 ± 0.7 10.5 ± 0.7 11.3 ± 1.1


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between neighboring alanine segments, thereby stabilizing the carrier core and decreasing the CMC. These temporary crosslinks allow poly(alanine) with appropriate chain lengths to assemble into β-sheet structure, which is known to confer rigidity to silk.[37] Particle size and polydispersity were determined by DLS. Particle sizes ranging between 50 and 60 nm were observed. Nanoparticles in this range are capable of selectively accumulating in tumor tissues via the enhanced permeation and retention effect. It was noted that particle size decreased with increasing alanine content. Extensive hydrophobic interactions in the carrier core may elicit contraction of the core, thereby leading to a decrease in particle size, as described in literature.[38] In addition, a comparison study between Pluronic P123, Puronic P84, and Pluronic P65 showed that as hydrophobic PPO units increased, the micellar core became more compact.[39] Therefore, contraction is mainly due to hydrophobic and polymer interactions. Broadening of the methyl proton peak in 1H NMR spectra represents decreased motion of methyl protons. (Figure 4(a)) The extent of peak broadening corresponded to alanine chain length, indicating increased chain motion restriction in longer alanine segments. An upfield shift was also observed in the CH3 signal, representing the reduction of hydration in alanine-containing carriers. Similarly, broadening of the peak corresponding to PEG proton is an indication of the restricted motion of PEG upon formation of vesicle (Figure 4(b)).

Figure 4. 1H NMR spectra of polymeric micelles in D2O. (a) Characteristic peak at 1.14 ppm corresponds to methyl proton in the micelle core and (b) characteristic peak at 4.75 ppm corresponds to methylene proton of PEO.



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Figure 5. UV–visible absorbance spectra of curcumin in free form, dissolved in 1% DMSO, and solubilized with micelles.

3.3. Preparation of drug-loaded nanocarriers Nanocarriers were prepared at a drug/polymer ratio of 1/10 (w/w) via a solvent-switch method, where self-assembly was triggered by the addition of water into THF. As water was gradually added, the hydrophobic blocks and curcumin associate into a non-polar core, while solvated hydrophilic blocks assemble into the outer layer corona. The removal of THF was completed by lyophilization overnight and the redispersion of lyophilized nanocarriers in water yielded orange–yellow solutions. Opacity decreased as the alanine segment increased, an indication of improved solubilization of hydrophobic curcumin into alanine-containing core. Absorbance spectrum of drug-loaded nanocarrier solutions revealed an increase in absorbance at 420 nm after curcumin entrapment (Figure 5). Free curcumin and curcumin solubilized with 1% DMSO exhibited minimal absorption. Degree of increase in absorbance correlated with the entrapment efficiency as shown in Table 2. 3.4. Transmission electron microscopy TEM images of curcumin-loaded carriers are shown in Figure 6. The morphology of the carrier is spherical with no significant aggregation. Darker shaded cores observed in alanine-modified carriers indicate the increased interaction at the hydrophobic core. It should be noted that the average diameter of carriers decreased as alanine segment increased. Since alanine is conjugated to the hydrophilic segment of PF-127, it is likely that the alanine segments folded into the carrier core, thereby compacting the particle as a whole. Particle size decreased from approximately 100 nm to 50 nm as alanine segment increased. Since higher entrapment efficiency was obtained when more alanine was added, increased polymer–drug interaction and resultant stabilization of micelles could be responsible for the decrease in size.[40,41]

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Figure 6. TEM of 0.1 wt% solution of curcumin encapsulating (a) PLU, (b) PLU-A3, and (c) PLU-A15 micelles.

3.5. Differentiated scanning calorimetry As described in Section 3.1, the addition of alanine decreased the melting temperature of PF-127. The incorporation of curcumin decreased the melting temperature further and the characteristic endothermic peak at 170 °C was not present in the freeze-dried curcumin micelle samples. Characteristic melting point of curcumin was not present in the freeze-dried curcumin nanoparticle samples. This indicates that the drug is dispersed in the core and the incorporation of curcumin increased the amorphous character of the core, thereby decreasing chain mobility (Figure 7).[41] 3.6. Entrapment efficiency (EE%), drug loading (DL%), and cumulative release Oligoalanine-modified PF-127 significantly increased the entrapment efficiency from 25.3 to 91.3% under identical preparation conditions, as shown in Table 2. Potential for high entrapment efficiency is due to the large hydrophobic block, serving as cargo space, in PF-127. Increase in curcumin solubility may be visualized directly as PLU solution was turbid, while PLU-A12 and PLU-A15 samples were transparent. Loading content (%) of 3, 8, 10, and 12% were recorded for PLU, PLU-A3, PLU-A12, and PLU-A15, respectively. Polymer–drug interaction between a phenyl-containing drug, mTPP, and the alkyl-containing Pluronic micelle has been described in literature.[42] Furthermore, oligoalanine is capable of H-bonding with nearby molecules, including other alanine blocks and curcumin, thereby enhancing the entrapment of curcumin.[43,44]

Figure 7. DSC thermogram of free curcumin, curcumin mixed with Pluronic-F127, and PLU-A15 micelles with and without curcumin.



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Figure 8. Drug loading (%) and curcumin release (%) of curcumin encapsulated in unmodified and modified Pluronic-F127.

Cumulative release profile showed that 80% of the drug was released within the first 6 h from PLU as shown in Figure 8. Increasing alanine segment extended the release period of curcumin. For PLU-A3, rapid drug release was observed initially followed by moderate continuous release up to 144 h, when 85% of the total curcumin was released. The release of curcumin from prepared nanocarriers follows a two-step process. The initial burst release observed may be attributed to the quick release of surface tethered drugs or weak association with carrier, while the second phase is mainly associated with drug diffusion.[45] The hydrophobic core of alanine-modified curcumin consists of both PPO and alanine segments. The PPO contributes hydrophobic stabilizing force, while alanine peptides introduce H-bonding and other stabilizing forces to the core. Study has shown that stronger interaction between the a hydrophobic drug and the nanoparticle core led to slower release of drug.[46] Therefore, since curcumin is retained via dual forces, delayed release of entrapped drug may be a combinatorial effect of these factors. 3.7. Cytotoxicity against HeLa Cytotoxicity of the modified copolymers was tested against HeLa cells at polymer concentrations in the range of 0.050–1.000 mg/mL as shown in Figure 9(a). No significant difference was noted in cytotoxicity after alanine modification. Cytotoxicity of


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Figure 9. (a) Cytotoxicity of unmodified and modified copolymers and (b) cytotoxicity of curcumin dissolved with 1% DMSO and encapsulated in different micelle formulations.

curcumin-encapsulated nanocarriers was carried out in the range of 0.000–0.1 mg/mL as shown in Figure 9(b). Cytotoxicity was observed to be concentration dependent, and at dilute concentrations up to 0.05000 mg/mL, PLU-A15 formulation exhibited significantly greater toxicity against HeLa cells. The half-maximal inhibitory concentration (IC50) of curcumin-loaded carriers were 0.100, 0.067, 0.014, 0.007, and 0.005 mg/mL against HeLa cells for DMSO solubilized, PLU, PLU-A3, PLU-A12, and PLU-A15, respectively. Higher efficacy is likely the result of increased entrapment percentage. At low EE%, most of the hydrophobic drug is insoluble and incapable of cellular uptake.

Figure 10. Cellular uptake of curcumin by HeLa after entrapment in (a) PLU, (b) PLU-A3, and (c) PLU-A15.


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Therefore, it is crucial that the drug be stably incorporated and solubilized by a stable nanocarrier. These results show that curcumin encapsulated in modified copolymers are more efficient in inhibiting HeLa growth.


3.8. Visualization of drug uptake Curcumin is naturally fluorescent, which makes direct visualization of drug uptake by cells possible. Cell uptake of curcumin was confirmed for HeLa cells after 1 h incubation as shown in Figure 10. Greater fluorescent intensity corresponding to more efficient curcumin delivery was observed in PLU-A12 and PLU-A15 in comparison to unmodified PLU. (fluorescent readings of 2836 ± 286, 6133 ± 774, and 8407 ± 209 for PLU, PLUA12, and PLUA15, respectively) Increased drug delivery may be attributed to higher entrapment efficiency of curcumin with alanine-modified polymers, allowing greater amounts of solubilized curcumin to cross the cell membrane. 4. Conclusion In this study, alanine-modified Pluronic-F127 was prepared by ring-opening polymerization of alanine-NCA. Addition of oligoalanine stabilized the nanoparticle structure and confers H-bonding ability. In vitro cytotoxicity study revealed that the copolymers were non-toxic and safe. Loaded nanoparticles were smaller than 100 nm, thereby capable of escaping clearance by the reticuloendothelial system. Increased loading of curcumin may be attributed to the incorporation of amide moieties into the copolymer to stabilize the nanocarrier core, while providing H-bonding ability. These forces result in a significant increase in drug entrapment. The partitioning of a hydrophobic drug into the carrier is crucial because precipitation prevents effective delivery. Cytotoxicity of entrapped curcumin towards HeLa cells increased when larger oligoalanine was used. This system provides a dual force-driven nanocarrier that shows promising potential to increase the solubility of poorly soluble drugs. References [1] Ruby AJ, Kuttan G, Dinesh Babu KD, Rajasekharan KN, Kuttan R. Anti-tumour and antioxidant activity of natural curcuminoids. Cancer Lett. 1995;94:79–83. [2] Sharma OP. Antioxidant activity of curcumin and related compounds. Biochem. Pharmacol. 1976;25:1811–1812. [3] Srimal RC, Dhawan BN. Pharmacology of diferuloyl methane (curcumin), a non-steroidal anti-inflammatory agent. J. Pharm. Pharmacol. 1973;25:447–452. [4] Mohanty C, Das M, Sahoo SK. Emerging role of nanocarriers to increase the solubility and bioavailability of curcumin. Expert Opin. Drug Delivery. 2012;9:1347–1364. [5] Gou M, Men K, Shi H, Xiang M, Zhang J, Song J, Long J, Wan Y, Luo F, Zhao X, Qian Z. Curcumin-loaded biodegradable polymeric micelles for colon cancer therapy in vitro and in vivo. Nanoscale. 2011;3:1558–1567. [6] Mohanty C, Acharya S, Mohanty AK, Dilnawaz F, Sahoo SK. Curcumin-encapsulated MePEG/PCL diblock copolymeric micelles: a novel controlled delivery vehicle for cancer therapy. Nanomedicine. 2010;5:433–449. [7] Huang J, Wang K. Novel micelle formulation of curcumin for enhancing antitumor activity and inhibiting colorectal cancer stem cells. Int. J. Nanomed. 2012;7:4487–4497. [8] Allen C, Maysinger D, Eisenberg A. Nano-engineering block copolymer aggregates for drug delivery. Colloids Surf., B. 1999;16:3–27. [9] He C, Kim SW, Lee DS. In situ gelling stimuli-sensitive block copolymer hydrogels for drug delivery. J. Controlled Release. 2008;127:189–207.


1238

S. Peng et al.

[10] Rangel-Yagui CO, Pessoa A Jr, Tavares LC. Micellar solubilization of drugs. J. Pharm. Pharm. Sci. 2005;8:147–165. [11] Tang Y, Liu SY, Armes SP, Billingham NC. Solubilization and controlled release of a hydrophobic drug using novel micelle-forming ABC triblock copolymers. Biomacromolecules. 2003;4:1636–1645. [12] Basak R, Bandyopadhyay R. Encapsulation of hydrophobic drugs in Pluronic F127 micelles: effects of drug hydrophobicity, solution temperature, and pH. Langmuir. 2013;29: 4350–4356. [13] Kim Y, Dalhaimer P, Christian DA, Discher DE. Polymeric worm micelles as nano-carriers for drug delivery. Nanotechnology. 2005;16:S484–S491. [14] Natarajan JV, Darwitan A, Barathi VA, Ang M, Htoon HM, Boey F, Tam KC, Wong TT, Venkatraman SS. Sustained drug release in nanomedicine: a long-acting nanocarrier-based formulation for glaucoma. ACS Nano. 2014;8:419–429. [15] Li Y, Xu X, Shen Y, Qian C, Lu F, Guo S. Preparation and evaluation of copolymeric micelles with high paclitaxel contents and sustained drug release. Colloids Surf., A. 2013;429:12–18. [16] Wang Y, Li Y, Zhang L, Fang X. Pharmacokinetics and biodistribution of paclitaxel-loaded Pluronic P105 polymeric micelles. Arch. Pharmacal Res. 2008;31:530–538. [17] Han M, Diao YY, Fu YH, Hu YL, Jiang HL, Tsutsumi Y, Wei QC, Chen DW, Gao J-Q. Doxorubicin-loaded PEG-PCL copolymer micelles enhance cytotoxicity and intracellular accumulation of doxorubicin in adriamycin-resistant tumor cells. Int. J. Nanomed. 2011;6:1955–1962. [18] Chakraborty H, Banerjee R, Sarkar M. Incorporation of NSAIDs in micelles: implication of structural switchover in drug–membrane interaction. Biophys. Chem. 2003;104:315–325. [19] Batrakova EV, Li S, Brynskikh AM, Sharma AK, Li Y, Boska M, Gong N, Mosley RL, Alakhov VY, Gendelman HE, Kabanov AV. Effects of Pluronic and doxorubicin on drug uptake, cellular metabolism, apoptosis and tumor inhibition in animal models of MDR cancers. J. Controlled Release. 2010;143:290–301. [20] Ugarenko M, Chan C-K, Nudelman A, Rephaeli A, Cutts SM, Phillips DR. Development of Pluronic micelle-encapsulated doxorubicin and formaldehyde-releasing prodrugs for localized anticancer chemotherapy. Oncol. Res. 2009;17:283–299. [21] Foster B, Cosgrove T, Hammouda B. Pluronic triblock copolymer systems and their interactions with ibuprofen. Langmuir. 2009;25:6760–6766. [22] Wan D-H, Zheng O, Zhou Y, Wu L-Y. Solubilization of ibuprofen in Pluronic block copolymer F127 micelles. Acta Phys. Chim. Sin. 2010;26:3243–3248. [23] Park KM, Bae JW, Joung YK, Shin JW, Park KD. Nanoaggregate of thermosensitive chitosan-Pluronic for sustained release of hydrophobic drug. Colloids Surf., B. 2008;63:1–6. [24] Huang K, Lee BP, Ingram DR, Messersmith PB. Synthesis and characterization of selfassembling block copolymers containing bioadhesive end groups. Biomacromolecules. 2002;3:397–406. [25] Park SY, Lee Y, Bae KH, Ahn C-H, Park TG. Temperature/pH-sensitive hydrogels prepared from Pluronic copolymers end-capped with carboxylic acid groups via an oligolactide spacer. Macromol. Rapid Commun. 2007;28:1172–1176. [26] Jung YK, Park MH, Moon HJ, Shinde UP, Jeong B. Changes in nanoassembly of oligocaprolactone end-capped Pluronic F127 and the abnormal hydrophobicity trend of phase transition. Macromolecules. 2013;46:4215–4222. [27] Liu R, He B, Li D, Lai Y, Chang J, Tang JZ, Gu Z. Effects of pH-sensitive chain length on release of doxorubicin from mPEG-b-PH-b-PLLA nanoparticles. Int. J. Nanomed. 2012;7:4433–4446. [28] Habraken GJM, Peeters M, Dietz CHJT, Koning CE, Heise A. How controlled and versatile is N-carboxy anhydride (NCA) polymerization at 0 °C? Effect of temperature on homo-, block- and graft (co)polymerization. Polym. Chem. 2010;1:514–524. [29] Zhang X, Jackson JK, Burt HM. Determination of surfactant critical micelle concentration by a novel fluorescence depolarization technique. J. Biochem. Biophys. Methods. 1996;31:145–150. [30] Mahou R, Wandrey C. Versatile route to synthesize heterobifunctional poly(ethylene glycol) of variable functionality for subsequent pegylation. Polymers. 2012;4:561–589.



1239

[31] Kajjari PB, Manjeshwar LS, Aminabhavi TM. Novel blend microspheres of poly(3-hydroxybutyrate) and Pluronic F68/127 for controlled release of 6-mercaptopurine. J. Appl. Polym. Sci. 2014:131. [32] Xie W, Zhu W, Shen Z. Synthesis, isothermal crystallization and micellization of mPEG– PCL diblock copolymers catalyzed by yttrium complex. Polymer. 2007;48:6791–6798. [33] Jia W, Gu Y, Gou M, Dai M, Li X, Kan B, Yang J, Song Q, Wei Y, Qian Z. Preparation of biodegradable polycaprolactone/poly (ethylene glycol)/polycaprolactone (PCEC) nanoparticles. Drug Delivery. 2008;15:409–416. [34] Huang M-H, Li S, Hutmacher DW, Schantz J-T, Vacanti CA, Braud C, Vert M. Degradation and cell culture studies on block copolymers prepared by ring opening polymerization of e-caprolactone in the presence of poly(ethylene glycol). J. Biomed. Mater. Res. A. 2004;69A:417–427. [35] Shan Y, Qin Y, Chuan Y, Li H, Yuan M. The synthesis and characterization of hydroxyapatite-β-alanine modified by grafting polymerization of γ-benzyl-L-glutamate-N-carboxyanhydride. Molecules. 2013;18:13979–13991. [36] Zhang Y, Zhao L, Chen M, Lang M. Synthesis and properties of Pluronic-based pentablock copolymers with pendant amino groups. Colloid Polym. Sci. 2013;291:1563–1571. [37] Rathore O, Sogah DY. Self-assembly of β-sheets into nanostructures by poly(alanine) segments incorporated in multiblock copolymers inspired by spider silk. J. Am. Chem. Soc. 2001;123:5231–5239. [38] Song MJ, Lee DS, Ahn JH, Kim DJ, Kim SC. Thermosensitive sol-gel transition behaviors of poly(ethylene oxide)/aliphatic polyester/poly(ethylene oxide) aqueous solutions. J. Polym. Sci., Part A: Polym. Chem. 2004;42:772–784. [39] Ma J, Guo C, Tang Y, Liu H. 1H NMR spectroscopic investigations on the micellization and gelation of PEO−PPO−PEO block copolymers in aqueous solutions. Langmuir. 2007;23:9596–9605. [40] Veeren A, Bhaw-Luximon A, Jhurry D. Polyvinylpyrrolidone–polycaprolactone block copolymer micelles as nanocarriers of anti-TB drugs. Eur. Polym. J. 2013;49:3034–3045. [41] Jeetah R, Bhaw-Luximon A, Jhurry D. New amphiphilic PEG-b-P(ester–ether) micelles as potential drug nanocarriers. J. Nanopart. Res. 2012;14:1–13. [42] Sezgin Z, Yuksel N, Baykara T. Preparation and characterization of polymeric micelles for solubilization of poorly soluble anticancer drugs. Eur. J. Pharm. Biopharm. 2006;64: 261–268. [43] Nardo L, Paderno R, Andreoni A, Másson M, Haukvik T, Tønnesen HH. Role of H-bond formation in the photoreactivity of curcumin. Spectroscopy. 2008;22:187–198. [44] Ghosh R, Mondal JA, Palit DK. Ultrafast dynamics of the excited states of curcumin in solution. J. Phys. Chem. B. 2010;114:12129–12143. [45] Fu Y, Kao WJ. Drug release kinetics and transport mechanisms of non-degradable and degradable polymeric delivery systems. Expert Opin. Drug Delivery. 2010;7:429–444. [46] Khoee S, Hassanzadeh S, Goliaie B. Effects of hydrophobic drug–polyesteric core interactions on drug loading and release properties of poly(ethylene glycol)–polyester–poly(ethylene glycol) triblock core–shell nanoparticles. Nanotechnology. 2007;18:175602.

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